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Exploring the Universe of Cannabinoids and Terpenes: Your Gateway to Understanding and Industry Insights

Welcome to GalaxyGroves.com, where we embark on an enlightening journey through the ever-expanding universe of cannabinoids and terpenes. Our Learning Center Blog is your trusted guide, illuminating the intricate world of these remarkable compounds. Here, you'll uncover the myriad benefits of different cannabinoids and terpenes, delve into their unique characteristics, and stay updated with the latest trends and news in the industry. Whether you're a curious newcomer or a seasoned connoisseur, our insights will enrich your understanding and enhance your experience in the vibrant cosmos of cannabis. Join us as we explore the depths of knowledge and bring the most exciting discoveries right to your screen!

LEARNING CENTER

THCA and THC: What’s the Difference?

THCA and THC: What’s the Difference?

Why we get high on THC and not THCA, how cannabinoids convert, and raw cannabis as a superfood

Surprise! You’re just not going to get high by eating that freshly picked weed. At all. When cannabis is harvested and raw, no matter how much potential resides within, there is practically none of marijuana’s most famous and intoxicating cannabinoid, delta-9-tetrahydrocannabinol (THC). There is, however, a wealth of tetrahydrocannabinolic acid (THCA), an inactive compound found within the trichomes of living cannabis plants. 

So, if someone ever asks you “what does THC stand for?” don’t confuse the two similar terms. As you’ll soon discover, they are vastly different in both chemical structure and how they interact with the human body.

THCA is a cannabinoid that until recently has been closely compared to THC. Though THCA doesn’t get one high and THC certainly does, there is a relation: THCA is the precursor to psychoactive THC effects.

So why does THC get us elevated and THCA doesn’t? The reason is due to the three-dimensional shape of the THCA molecule. It is a larger molecule that doesn’t fit into our cannabinoid receptors, specifically the CB1 receptors. A cannabinoid must fit into a body’s CB1 receptor in order to have an intoxicating effect at all.

The cannabis plant produces hundreds of cannabinoids, the chemical compounds responsible for the therapeutic and psychoactive effects of cannabis. Only a few cannabinoids contribute to the euphoric high that is unique to the cannabis plant, though. The most celebrated, researched, and sought after is THC.

It’s commonly assumed that during the marijuana plant’s growth process that it is ramping up THC levels until ripe for the picking, but the primary cannabinoid being produced is actually THCA. How does THCA become THC?

The simplified answer is through heat and light — or the process of decarboxylation. Heat removes a carboxylic acid group of atoms from THCA, converting it into a molecule and altering the THC chemical structure, thus becoming the perfect shape to fit into our endocannabinoid system (ECS) CB1 receptors that run throughout the central nervous system, producing the elevated experience.

The non-intoxicating effects of THCA are a big part of the reason that fresh, raw, unheated cannabis is a superfood. You may have heard of juicing cannabis or adding raw cannabis to smoothies for health enhancement. There’s good reason.

Like other superfoods, including avocados, kale, Greek yogurt, green tea, and garlic, raw cannabis has potential to ease arthritis, chronic pain, fibromyalgia, and other ailments. 

THCA is believed to offer an assortment of medicinal benefits, and is commonly used as a nutritional supplement and dietary enhancement for its:

  • Anti-inflammatory properties  – 2011 study published in the Biological and Pharmaceutical Bulletin suggested that, along with other cannabinoids, THCA demonstrated anti-inflammatory properties. 
  • Anti-proliferative properties  A 2013 study that analyzed cell cultures and animal models concluded that THCA could prevent the spread of prostate cancer cells.
  • Neuroprotective properties – In a 2012 preclinical study published in Phytomedicine, researchers found that THCA showed the ability to help protect against neurodegenerative diseases.  
  • Antiemetic properties (increasing appetite and decreasing nausea) – A  2013 study conducted by researchers at the University of Guelph in Ontario found that both THCA and CBDA were effective in reducing nausea and vomiting in rat models, even moreso than THC and CBD, respectively. 

Most cannabinoids, including cannabidiol (CBD), cannabigerol (CBG), and tetrahydrocannabivarin (THCV), are in the acidic form (CBDA, CBGA, and THCVA) when cannabis is harvested. The unactivated forms of THC and CBD, along with other cannabinoids, have benefits themselves that we are still learning about.

Decarboxylation of THCA

The human body is not capable of converting THCA into THC.

 

It’s only after these unactivated cannabinoid acids go through the decarboxylation process, though, that they become the cannabinoids we’re most familiar with and that most interact with our ECS.

The acidic precursors are considered “thermally unstable,” which is another way to emphasize that they will alter when exposed to heat. Because of this instability, the molecules lend themselves to several different methods of decarboxylation.

THCA vs. THC: Decarboxylation Process 

Here are the most common ways that weed is decarboxylated:

Sunlight conversion: THCA converts to THC in varying degrees through exposure to heat or light. If a cannabis plant sits in the warm sun for an extended period of time, its THCA molecules will slowly convert to THC.

Room temperature conversion: THCA also converts to THC when stored at room temperature for a long enough time. In olive oil, 22% of THCA will convert over the course of 10 days at 77 degrees Fahrenheit, or 25 degrees Celsius. Under the same conditions, 67% will convert in an ethanol extraction. And over time, cannabis stored at room temperature and with little light exposure, will convert 20% of its THCA into THC.

Smoking: When a flame is used to smoke dried, cured bud, a high degree of heat is applied in a short amount of time, resulting in the rapid conversion of THCA to THC. However, not all THCA will convert and, though smoking is the most common way to enjoy THC’s effects, it’s not the most efficient.

Vaporizing: This is perhaps the most efficient way of decarboxylating ground nugs. When heated at a low temperature, the cannabinoids are converted and released. Continuing to increase the heat with each pull or sesh will make sure that the prime amount of THCA is converted into THC and binds to CB1 receptors.

Vape pens: Even more efficient than vaporizing flowers is the use of already decarboxylated cannabis distillate found in preloaded vape pens. Since the THCA is already mostly converted to THC and the following vaporization takes care of even more, this is a good, efficient method of taking in intoxicating cannabis.  Be sure you’re using a reliable brand of vape pen, for safety’s sake, and do your best to purchase products that are recyclable.

Cannabis concentrates:  By isolating the THCA content from a cannabis plant, THCA crystalline can be extracted and consumed in dabs. Similar to vaporization, decarboxylation transpires rapidly when using the dabbing method, breaking down the THCA into active THC. In its pure form, THCA crystalline has little flavor or aroma, as most cannabis extractions aim to strip away the terpenes and flavonoids to isolate the cannabinoids. But many producers reintroduce cannabis-derived terpene blends back into the concentrate. Not only does the addition of terpenes improve the flavor, but these distinctively aromatic plant molecules also work together with cannabinoids to produce entourage effects that enhance the therapeutic potential of cannabis.   

Conventional oven: When making edibles, you’ll want to activate, or decarboxylate, the weed before adding it to the butter, oil, or other medium. When weed gets ground up, spread evenly across a baking sheet that’s lined with parchment paper, and is baked at 230 degrees Fahrenheit, or 110 degrees Celsius, for 30-90 minutes (depending on the bud’s moisture content), it slowly converts most THCA into THC.

Whether cannabis is smoked, eaten, vaped, or juiced raw, understanding the plant’s properties and how and why they interact with our bodies the way they do is crucial in achieving the desired effects and avoiding adverse side effects. Cannabis molecules each have their own benefits and as raw cannabis is further studied, we can rest easy knowing that it’s safe to integrate it into a healthful diet.


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CBDA, CBGA AND COVID

In follow-up virus neutralization assays, cannabigerolic acid and cannabidiolic acid prevented infection of human epithelial cells by a pseudovirus expressing the SARS-CoV-2 spike protein and prevented entry of live SARS-CoV-2 into cells. Importantly, cannabigerolic acid and cannabidiolic acid were equally effective against the SARS-CoV-2 alpha variant B.1.1.7 and the beta variant B.1.351.

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DELTA 8 THC

 

Delta 8 THC: Myths and Reality

 

Still, freaking out about the term Delta 8 THC! Let’s have a tour of some Revolutionary Facts of Delta 8 THC.

Some people still have some confusion about [1] Delta (∆) 8 THC. Delta (∆) 8 THC is an oxidized isomer of Delta (∆) 9 THC [2]. It generates almost similar psychometrics as Delta-9 THC [3]. What amazes people is that it shows a 200% more effective anti-emetic property! [3]. FDA also has found many outstanding medical benefits of cannabinoids [4][5]. Moreover, there is no such direct legal obligation upon Delta (∆) 8 THC. Delta-8 not only, legal but it has some astonishing health benefits.

 

Benefits of Delta (∆) 8 THC:

 

Delta (∆) 8 THC has become the game-changer among the cannabinoid family because of its Neuroprotective properties! It also has some remarkable medical benefits that why it’s used in various medical treatments. Such as:

 

  1. [6] Insomnia & Anxiety
  2. [7] Emotional Stress & Depression
  3. [8] Nausea
  4. [9] Epilepsy
  5. [10] Tumor Inhibit
  6. [11] [12] Cancer Prevention & Muscle Recovery
  7. [13] Increased Food Consumption, and Altered Neurotransmitter levels
  8. [14] Pain Management like Migraine & Headache

 

The Proper Way of Consumption Delta (∆) 8: 

 

  1. Direct application to the skin or infused with cosmetics, lotions, body butter, and rubs,
  2. Can be inhaled as a vapor,
  3. Can be consumed orally as oil, capsules or gummies;

 

 

Side Effects of Delta (∆) 8 THC:

 

There is not such harmful side effect found in Delta (∆) 8 THC yet. Delta 8 THC has a positive Psychoactive Reaction that won’t make anyone high! It also doesn’t show any Withdrawal Symptoms. Though some people claim about Slight Dehydration or Dry Mouth.

 

Legalization:

 

The Agricultural Improvement Act was published in 2018 by H.R.2, which is also known as Firm Bill 2018 now [15]  [16]. It says that cannabinoids extracted from Hemp contain Δ-9 THC less than 0.3% are legal to produce or industrial use [17]. Since then, Federal Law count only Δ-9 THC to be controlled. As a result, Δ-8 THC is free from control under Federal Law.

 

Though Delta-8 THC contains Tetrahydrocannabinol (THC), the percentage is lower (around 0.1%). So, there is no such direct legal obligation upon Delta-8 THC. That is why Delta-8 THC is federally legal and can be consumed legally by 40 states of the USA.

 

Keep in Mind If You are Planning to Purchase Delta (∆) 8 THC:

 

  1. Is that company is certified with a third-party lab tested from where you are buying it?
  2. Whether any added chemicals or ingredients?
  3. Safety and Quality.

 

Reference:

 

  1. https://pubchem.ncbi.nlm.nih.gov/compound/delta8-THC
  2. https://pubmed.ncbi.nlm.nih.gov/18205240/
  3. https://patents.google.com/patent/US20040143126A1/en
  4. https://www.fda.gov/news-events/congressional-testimony/researching-potential-medical-benefits-and-risks-marijuana-07122016-07122016
  5. https://www.fda.gov/news-events/public-health-focus/fda-regulation-cannabis-and-cannabis-derived-products-including-cannabidiol-cbd
  6. https://pubmed.ncbi.nlm.nih.gov/30624194/
  7. https://pubmed.ncbi.nlm.nih.gov/28268256/
  8. https://pubmed.ncbi.nlm.nih.gov/30209152/
  9. https://pubmed.ncbi.nlm.nih.gov/29124331/
  10. https://pubmed.ncbi.nlm.nih.gov/12514108/
  11. https://pubmed.ncbi.nlm.nih.gov/16501583/
  12. https://academic.oup.com/jnci/article/100/1/59/2567700?searchresult=1
  13. https://pubmed.ncbi.nlm.nih.gov/15099912/
  14. https://pubmed.ncbi.nlm.nih.gov/29450258/
  15. https://www.congress.gov/115/bills/hr2/BILLS-115hr2enr.pdf
  16. https://www.congress.gov/bill/115th-congress/house-bill/2/text
  17. https://www.nysenate.gov/legislation/laws/AGM/505

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HIGH CBD

Inflammation and oxidative stress are intimately involved in the genesis of many human diseases. Unraveling that relationship therapeutically has proven challenging, in part because inflammation and oxidative stress “feed off” each other. However, CBD would seem to be a promising starting point for further drug development given its anti-oxidant (although relatively modest) and anti-inflammatory actions on immune cells, such as macrophages and microglia.

Abstract

Oxidative stress with reactive oxygen species generation is a key weapon in the arsenal of the immune system for fighting invading pathogens and to initiate tissue repair. If excessive or unresolved, however, immune-related oxidative stress can initiate further increasing levels of oxidative stress that cause organ damage and dysfunction. Targeting oxidative stress in these various diseases therapeutically has proven more problematic than first anticipated given the complexities and perversity of both the underlying disease and the immune response. However, growing evidence suggests that the endocannabinoid system, which includes the CB1 and CB2 G protein-coupled receptors and their endogenous lipid ligands, may be an area that is ripe for therapeutic exploitation. In this context, the related nonpsychotropic cannabinoid cannabidiol, which may interact with the endocannabinoid system, but has actions that are distinct, offers promise as a prototype for anti-inflammatory drug development. This review discusses recent studies suggesting that cannabidiol may have utility in treating a number of human diseases and disorders now known to involve activation of the immune system and associated oxidative stress, as a contributor to their etiology and progression. These include rheumatoid arthritis, types I and II diabetes, atherosclerosis, Alzheimer’s disease, hypertension, the metabolic syndrome, ischemia-reperfusion injury, depression, and neuropathic pain.

Keywords: Inflammation, Oxidative Stress, Immune System, Metabolic Syndrome, Endocannabinoid

Introduction

(−)-Cannabidiol (CBD) is the major nonpsychotropic cannabinoid compound derived from the plant Cannabis sativa, commonly known as marijuana. CBD was first isolated in 1940 and its structure and stereochemistry determined in 1963 [,]. Interest in exploiting CBD therapeutically was initially focused on its interactions with the primary psychotropic ingredient of Cannabis, Δ9-THC and its sedative and antiepileptic effects, and later its antipsychotic and anxiolytic actions and utility in treating movement disorders []. As chronicled elsewhere [], the last several years have seen a renewed interest in CBD due to the discovery of its antioxidative, anti-inflammatory, and neuroprotective effects, actions that occur for the most part independently of the canonical cannabinoid CB1 and CB2 receptors [,]. CBD may prove to have therapeutic utility in a number of conditions involving both inflammation and oxidative stress, including Parkinson’s disease, diabetes, rheumatoid arthritis, Alzheimer’s disease, and ischemia-reperfusion injury.

The contribution of the endocannabinoid system to inflammation and regulation of the immune system is an area of intense study that is beyond the scope of this article, and the reader is referred to several recent excellent reviews []. However, a brief overview of the system is helpful in discussing CBD. The endocannabinoid system comprises the following: (1) the G protein-coupled cannabinoid receptors CB1 and CB2, which are located in both the central nervous system and periphery; (2) their arachidonate-based lipid ligands, e.g., 2-arachidonoylglycerol (2-AG) and anandamide (N-arachidonoylethanolamine, AEA) and (3) the enzymes that synthesize and degrade these ligands. The endocannabinoid system plays a role in a variety of physiological processes including appetite, pain sensation, and mood. Evidence indicates that both CB1 and CB2 are expressed by cells of the immune system and are upregulated in the activation state. Levels of CB2 appear to be higher than those of CB1 with decreasing amounts of CB2 in human B cells, NK cells, monocytes, polymorphonuclear neutrophils and T cells []. Macrophages and related cells, microglia and osteoclasts, express both cannabinoid receptors. CB2 activation of immune cells is associated with changes in cytokine release and migration [].

Biochemistry of Cannabidiol

CBD (Fig. 1) is a resorcinol-based compound that was shown to have direct, potent antioxidant properties by cyclic voltammetery and a spectrophotometric assay of oxidation in a Fenton reaction []. In an in vitro glutamate neuronal toxicity model, CBD was shown to be more protective than either α-tocopherol or vitamin C and comparable to butylated hydroxytoluene (BHT); although as noted by the authors, CBD unlike BHT does not seem to promote tumors []. CBD was also reported to act as an antioxidant at submicromolar concentrations in preventing serum-deprived cell death of cultured human B lymphoblastoid and mouse fibroblasts cells []. The antioxidant chemistry of CBD may have utility in vivo as well. The protective effects of CBD in a rat binge ethanol-induced brain injury model [] and a rat model of Parkinson’s disease [] were ascribed to its antioxidant properties. As will become clear from this review, however, the anti-oxidant actions ascribed to CBD in various in vivo models of human diseases likely exceed those attributable to its chemistry alone. Rather, the therapeutic anti-oxidant properties of CBD would seem to result in no small measure from its modulation of cell signaling events that underlie the self-sustaining cycle of inflammation and oxidative stress.

Chemical structure of cannabidiol (CBD).

Mechanisms of Action

Several interactions with relevance to the immune system and oxidative stress are discussed here. First, despite having low affinity for CB1 and CB2 receptors, CBD has been shown to antagonize the actions of cannabinoid CB1/CB2 receptor agonists in the low nanomolar range, consistent with non-competitive inhibition []. At 1–10 µM, CBD appears to function as an inverse agonist at both CB1 and CB2 receptors []. Second, CBD acts as an inhibitor (IC50 = 28 µM) of fatty acid amide hydrolase (FAAH), the major enzyme for endocannabinoid breakdown. Because FAAH activity correlates with gastrointestinal mobility, CBD may have utility in treating intestinal hypermotility associated with certain inflammatory diseases of the bowel [].

Third, CBD is a competitive inhibitor with an IC50 in the nanomolar range of adenosine uptake by the equilibrative nucleoside transporter 1 (ENT1) of macrophages and microglial cells, the resident macrophage-like immune cells of the brain. By increasing exogenous adenosine, which in turn activates the A2A adenosine receptor, CBD exerts immunosuppressive actions on macrophages and microglial cells as evidenced by decreased TNFα production after treatment with lipopolysaccharide (LPS) [,]. CBD may thus be of benefit in treating neueodegenerative diseases associated with hyperactivation of microglial, as well as retinal neuroinflammation seen in such conditions as uveitis, diabetic retinopathy, age-related macular degeneration, and glaucoma. Note, however, that adenosine activates other receptors besides A2A that often have opposing consequences on immune regulation and inflammation [,]. In several in vivo models of neurodegeneration or inflammation, moreover, the beneficial effects of CBD were demonstrated not to involve adenosine receptors.

Fourth, CBD has been shown to have potent actions in attenuating oxidative and nitrosative stress in several human disease models, although the exact mechanism is unclear. For instance, CBD pretreatment was found to attenuate high glucose-induced mitochondrial superoxide generation and NF-κB activation in human coronary artery endothelial cells, along with nitrotyrosine formation and expression of inducible nitric oxide synthase (iNOS) and adhesion molecules ICAM-1 and VCAM-1 []. Notably, high glucose-induced transendothelial migration of monocytes, monocyte-endothelial adhesion, and barrier disruption were attenuated as well. These findings lend support to the conclusion that that CBD may have therapeutic utility in treating diabetic complications and atherosclerosis. In another study, CBD was reported to reduce expression of reactive oxygen species (ROS) generating NADPH oxidases, as well as iNOS and nitrotyrosine generation in a cisplatin nephropathy model in vivo, consequently lessening cell death in the kidney and improving renal function []. From these studies, it is tempting to speculate that CBD may act directly at the level of the mitochondrion or nucleus to oppose oxidative/nitrosative stress.

Fifth, at low micromolar concentrations, CBD was found to inhibit indoleamine-2,3-dioxygenase activity thereby suppressing tryptophan degradation by mitogen-stimulated peripheral blood mononuclear cells and LPS-stimulated myelomonocytic THP-1 cells in vitro []. Based on this finding, CBD might be useful therapeutically to counter the increased risk of depression in diseases associated with immune activation and inflammation, which often lead to decreased tryptophan, the precursor of serotonin. Finally, CBD has been shown to act as an antagonist at G protein-coupled receptor 55 (GPR55) and as an antagonist or agonist at several transient receptor potential (TRP) channels; however, these observations are controversial and the pharmacophysiological significance of these interactions is not known [,].

Actions on Immune Cells

CBD has been shown to modulate the function of the immune system. Overall these actions may be nuanced and concentration-dependent, but in general include suppression of both cell-mediated and humoral immunity and involve inhibition of proliferation, maturation, and migration of immune cells, antigen presentation, and humoral response [,]. Key aspects are discussed here. In most in vivo models of inflammation, CBD attenuates inflammatory cell migration/infiltration (e.g. neutrophils) []. During neuroinflammation, activated microglial cells migrate towards the site of injury where they release pro-inflammatory cytokines and cytotoxic agents, including ROS. Although important in removal of cellular debris and fighting infection, activated microglial cells often exacerbate local cell damage. CBD was shown to inhibit activated microglial cell migration by antagonizing the abnormal-cannabidiol (Abn-CBD)-sensitive receptor at concentrations < 1 µM []. Evidence that the Abn-CBD receptor is the orphan G protein-coupled receptor GPR18 was recently reported []. CBD was also shown to block endotoxin-induced oxidative stress resulting from retinal microglial cell activation in uveitis []. CBD blocked the immediate activation of NADPH oxidase as well as a second wave of ROS formation and the associated TNFα secretion and p38 MAPK activation. The direct antioxidant property of CBD is unlikely to be the entire explanation for these actions as they occurred at a concentration of 1 µM. Inhibition of adenosine uptake as discussed previously may have been involved. However, a complete understanding of the anti-inflammatory actions of CBD on microglial cells is not yet available. Recently, through an unidentified mechanism, CBD was reported to suppress LPS-induced pro-inflammatory signaling in cultured microglial cells, including NF-κB and STAT1 activation, while enhancing STAT3-related anti-inflammatory signaling [].

CBD induces apoptosis of monocytes and certain normal and transformed lymphocytes, including thymocytes and splenocytes, through oxidative stress and increased ROS levels []. The basis for this action appears to be glutathione depletion due to adduct formation with the reactive metabolite of CBD, cannabidiol hydroxyquinone, thereby triggering cell death through caspase 8 activation and/or the intrinsic apoptotic pathway. Increased ROS from the upregulation of NADPH oxidases via an undefined mechanism may contribute to cell death as well []. A recent study assessed the impact of repeated administration of relatively low levels of CBD to adult male Wistar rats on peripheral blood lymphocyte subset distribution []. At 2.5 mg/kg/day for 14 days, CBD did not produce lymphopenia, but increased the total number of natural killer T (NKT) cells and percentage numbers of NKT and natural killer (NK) cells. A dose of 5 mg/kg/day did have a lymphopenic effect, but by reducing B, T, Tc and Th lymphocytes. Thus, CBD would appear to suppress specific immunity, while enhancing nonspecific antitumor and antiviral immune response. As discussed by the authors [], the lymphopenic effect of CBD was observed at a concentration shown to be efficacious in a number of animal models of neurodegenerative and inflammatory diseases, including blocking the progression of collagen-induced arthritis in a murine model of rheumatoid arthritis, decreasing damage to pancreatic islets in the NOD mouse model of type 1 diabetes, lessening hyperalgesia in rat models of neuropathic and inflammatory pain, and preventing cerebral ischemia in gerbils.

Pain

Neuropathic pain is associated with microglia activation in the spinal cord and brain and their subsequent release of pro-inflammatory cytokines, such as interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNFα) []. The etiology of neuropathic pain, which is common in cancer, diabetes, multiple sclerosis, and peripheral nerve injury, is poorly understood, but recent evidence indicates that increased ROS generation by microglial cells is the critical initiating factor []. The drug Sativex, which consists of Δ9-THC and CBD, is approved in several countries for treatment of central and peripheral neuropathic pain and for spasticity associated with multiple sclerosis []. In a mouse model of type I diabetic peripheral neuropathic pain, intranasal or intraperitoneal administration of a moderate-high dose of CBD attenuated tactile allodynia and thermal hypersensitivity without affecting the diabetic state []. The antinociceptive effects of CBD were associated with less of an increase in microglial density and p38 MAPK activity in the dorsal spinal cord. Finally, the anti-inflammatory and immunosuppressive actions of CBD may be of use in treating rheumatoid arthritis and the associated pain [,].

Diabetes and Diabetic Complications

CBD was shown to reduce either the initiation of diabetes or the development of overt or latent diabetes in non-obese diabetes-prone (NOD) mice by reducing insulitis [,]. This action was accompanied by a shift in the immune response from a dominant Th1 pattern with pro-inflammatory cytokines to a Th2 pattern with increased levels of the anti-inflammatory cytokine IL-10. Major effectors of β-cell death in type 1 diabetes are various free radicals and oxidant species, including NO, and infiltrating macrophages are one source of high concentrations of NO and inflammatory cytokines that further enhance NO and ROS formation []. CBD was also shown to be effective in blocking ROS-induced up-regulation of surface adhesion molecules on endothelial cells due to high glucose and in preserving endothelial barrier function [,]. Adhesion of monocytes followed by their transmigration into the subendothelial space is an early event in atherosclerosis, the most common macrovascular complication of diabetes, and may contribute as well to diabetic retinopathy [,,]. The anti-inflammatory actions of CBD may also protect retinal neurons in diabetes by attenuating activation and ROS generation by Müller glia, thus preventing tyrosine nitration and inhibition of Müller cell glutamine synthetase and the consequent accumulation of glutamate, which in turn leads to oxidative stress-induced death of retinal neuronal cells [].

In a mouse model of type I diabetic cardiomyopathy, both pre- and post-treatment with CBD attenuated cardiac fibrosis and cell death, myocardial dysfunction, inflammation, oxidative/nitrosative stress, and the activation of related signaling pathways []. CBD attenuated diabetes-induced activation in the heart of the key pro-inflammatory transcription factor, NF-κB and its consequences, e.g. expression of ICAM-1, iNOS, VCAM-1, and TNFα. These observations underscore the point that CBD likely attenuates inflammation far beyond its antioxidant properties per se. CBD also reduced high glucose-induced increases in both cytosolic and mitochondrial reactive oxygen and nitrogen species generation in primary human cardiac myocytes, which was accompanied by reduced NF-κB activation and cell death. These findings indicate that CBD may have great therapeutic potential in alleviating cardiac complications of diabetes.

Hypertension

Although CBD has not been considered for treating hypertension, a parallel between the role of microglia in diabetes and hypertension deserves mention. Activation of microglia within the paraventricular nucleus (PVN) was recently shown to contribute to neurogenic hypertension resulting from chronic angiotensin II infusion in the rat []. Microglia activation was associated with enhanced expression of pro-inflammatory cytokines, the acute administration of which into the left ventricle or PVN resulted in increased blood pressure. The hypertensive action of angiotensin II infusion could be blocked by overexpression of IL-10 in the PVN or intracerebroventricular infusion of minocycline, supporting the involvement of ROS.

The immune system contributes as well to systemic endothelial dysfunction observed in hypertension []. Local production of angiotensin II by activated leukocytes within the vessel wall is thought to reduce endothelial function and NO production, leading to attenuated vasodilation and increased blood pressure, through the production of inflammatory cytokines and ROS [,]. Interestingly, recent evidence has shown that the initial stimulus for peripheral leukocyte activation in angiogensin II-induced hypertension is the increase in blood pressure that results from stimulation of cells within the anteroventral third ventricle (AV3V) of the brain by angiotensin II [].

Ischemia Reperfusion Injury

Redox stress and ROS produced by ischemia-reperfusion of organs activates the immune system, which aids in repair by removing debris and stimulating remodeling. An excessive or prolonged inflammatory response, however, may prove detrimental to organ function by exacerbating ROS production and causing death of the parenchyma. Several hours after ischemia-reperfusion in the heart, a model of myocardial infarction, neutrophils accumulate in the myocardium []. Several lines of evidence suggest that this accumulation of neutrophils worsens injury to the myocardium []. In rats, treatment with CBD for 7 days following a 30 minute occlusion of the left anterior descending coronary artery markedly reduced infarct size, myocardial inflammation and IL-6 levels and preserved cardiac function []. In addition, the number of leukocytes infiltrating the border of the infarcted area was dramatically reduced. CBD has been shown to inhibit stimulated migration of neutrophils []. CBD treatment was also recently shown to reduce neutrophil migration in a rat model of periodontitis []. Hyperactive neutrophils exacerbate periodontal tissue injury and lead to tooth loss in part by excessive ROS formation in individuals with refractory periodontitis []. Finally, pre- or post-ischemic treatment with CBD was shown to have a prolonged and potent protective action in cerebral ischemia. The neuroprotective actions of CBD were attributed to reduced neutrophil accumulation and myeloperoxidase activity [], as well as decreased high mobility group box 1 (HMGB1) expression by microglia [].

Depression

CBD is reported to have anti-depressive actions, the basis for which is not established although activation of 5-HT1A receptors may be involved at least at higher concentrations [,,]. Growing evidence in recent years has implicated pro-inflammatory cytokines, free radical species, and oxidants in the etiology of depression [,]. One explanation is that the resultant oxidative stress adversely affects glial cell function and leads to neuron damage in the brain.

Neurodegenerative Diseases

Microglial hyperactivation is a common feature of a number of neurodegenerative diseases, including Parkinson’s, Alzheimer’s, Huntington’s, amyotrophic lateral sclerosis (ALS) and multiple sclerosis(MS) [,]. Activated microglia produce a number of pro- and anti-inflammatory cytokines, chemokines, glutamate, neurotrophic factors, prostanoids, and a variety of free radicals that together create a state of oxidative stress. Alzheimer’s disease, which is the most common form of dementia, is characterized by the deposition of “senile” plaques that are sites of microglia activation and inflammation. The resultant oxidative stress is a critical factor in the pathophysiology of Alzheimer’s []. The plaques are composed of insoluble aggregates of the beta-amyloid peptide (Aβ), which self-assembles as monomers, oligomers and finally fibrils. Recent evidence shows that the oligomeric form of beta-amyloid is the most neurotoxic species and is most effective as a chemotactic agent for microglia and stimulator of microglial oxidative stress [,]. Activated microglia are a major contributor of inflammatory factors in Alzheimer’s and secret a number of pro-inflammatory cytokines, which ironically further enhance Aβ production by neuronal cells []. In addition, an inflammatory state was shown to block the ability of microglia to phagocytize fibrillar Aβ []. Aging was also shown to negatively impact on the ability of microglia to internalize Aβ []. Microglia from aged mice were also shown to be less responsive to stimulation and to secrete greater amounts of IL-6 and TNFα compared to microglia of younger mice. Aged microglia also had lower levels of glutathione, suggesting an increased susceptibility to the harmful effects of oxidative stress. Finally, although controversial, evidence has been put forward suggesting that bone marrow-derived monocytic cells may somehow gain access to the diseased brain in Alzheimer’s and be better at phagocytosing amalyoid plaques than resident microglia [,].

Based on rather scant evidence, some have proposed that CBD might have utility in treating neurodegenerative diseases [,,]. CBD was shown to have a protective effect on cultured rat pheocromocytoma PC12 cells exposed to Aβ [,]. In a concentration-dependent manner, CBD increased cell survival, while decreasing ROS and nitrite production, lipid peroxidation, and iNOS protein expression. CBD was shown to have anti-inflammatory actions in vivo in a mouse model of Alzheimer’s neuroinflammation induced by injection of human Aβ into the hippocampus. CBD dose-dependently attenuated Aβ-induced glial fibrillary acidic protein (GFAP) mRNA, iNOS and IL-1β protein expression, and NO and IL-1β release []. In a recent study, CBD was found to protect against amphetamine-induced oxidative protein damage in a rat model of mania and to increase brain-derived neurotrophic factor (BDNF) expression levels in the reversal protocol []. Results of these preclinical studies are persuasive and support the need for double-blind placebo controlled trials to assess the therapeutic utility of CBD in patients with neurodegenerative diseases.

Obesity and the Metabolic Syndrome

Metabolic syndrome is a combination of medical disturbances including central obesity, glucose intolerance, hypertension, and dyslipidemia that increases the risk for developing cardiovascular diseases and type 2 diabetes. Adipocyte dysfunction leading to a low-grade chronic inflammatory state is thought to underpin the etiology of the metabolic syndrome []. Metabolic overload of adipocytes causes production of ROS, pro-inflammatory cytokines, and adipokines that activate inflammatory genes and stress kinases and interfere with insulin signaling [,]. Saturated fatty acids also activate toll-like receptors on adipocytes and macrophages, components of the innate immune system, to induce production of proinflammatory cytokines and chemokines. Enhanced mitochondrial flux together with relative hypoxia due to adipocyte tissue hypertrophy, endothelial cell apoptosis, and inflammation-impaired angiogenesis further enhances ROS generation. Enhanced rupture of adipocytes due to excessive hypertrophy attracts and activates macrophages that further exacerbate the inflammatory state through the production of inflammatory cytokines and ROS. The chronic inflammatory state compromises the ability of adipose tissue to absorb incoming fat leading to fat build up in other organs, including liver, heart, and skeletal muscle, and creating a local inflammatory state that progresses to insulin resistance in those organs as well. Increased ROS levels are thought to be the major contributing factor to insulin resistance [,].

Macrophages, both resident and to a greater extent bone marrow-derived, play a critical role in initiating adipose tissue dysregulation and inflammation in the metabolic syndrome and together with adipocytes constitute a paracrine loop that sustains the chronic inflammatory state []. Macrophages secrete TNFα which acts on hypertrophied adipocytes to downregulate adiponectin and induce pro-inflammatory cytokines and lipolysis. The released free fatty acids act in turn on the toll-like receptor 4 (TLR4) of macrophages to induce production of pro-inflammatory cytokines, including TNFα. Both macrophages and adipocytes secrete monocyte chemotactic protein 1 (MCP1), which serves to recruit more macrophages to the adipose tissue.

Recent evidence has revealed that most macrophages in obese adipose tissue are polarized towards the M1 or classically activated, pro-inflammatory state, as opposed to the M2 or alternatively activated, anti-inflammatory state [,]. Th1 cytokine interferon gamma (IFNγ), microbial byproducts (e.g., LPS), and free fatty acids from visceral adipose tissue promote polarization towards the M1 state, whereas Th2 cytokines IL-4 and IL-13 promote polarization towards the M2 phenotype. Ligand-dependent transcription factors peroxisome proliferator activated receptors (PPARs) play a key role in determining the M1/M2 phenotype [,]. Activation of PPARγ or PPARδ promotes differentiation towards the M2 phenotype, while PPARγ activation inhibits M2 to M1 phenotype switch and represses the M1 pro-inflammatory gene expression profile. Of interest, CBD as well as some other cannabinoids, has been shown to activate PPARγ, possibly through direct binding [,]. Although tonic activation of CB1 receptors by endocannabinoids is implicated in the development of abdominal obesity and CB1 antagonists and inverse agonist reduce obesity, their clinical use is problematic due to serious neuropsychiatric effects []. Given its anti-inflammatory actions and PPARγ agonism, CBD might serve as the basis for design of a new anti-obesity drug []. In this regard, a cautionary note regarding the PPARγ agonism associated with CBD should be sounded, although this was observed only in very high concentrations and only in vitro, which is that several PPARγ agonists have been retracted because of various problems [,].

Atherosclerosis

Atherosclerosis is an inflammatory disease in which monocytes/macrophages play a critical role in the initiation and progression, as well as rapture, of the atherosclerotic plaque []. Plaques form in the arterial wall at areas of disturbed flow and endothelial dysfunction (Fig. 2). The initiating event is the transcytosis of low density lipoprotein (LDL) into the subendothelial space where it is trapped by binding to proteoglycans of the extracellular matrix [,]. LDL is oxidized by various cells including macrophages, first to minimally modified LDL (mmLDL) and then extensively oxidized LDL (oxLDL). The former activates endothelial cells to secrete various factors that attract monocytes and to express adhesion molecules that support the binding and transmigration of monocytes into the subendothelial space. Once there, monocytes differentiate into macrophages under the influence of cytokines and oxLDL. Macrophages take up oxLDL and differentiate into foam cells that secrete a number of cytokines and growth factors that sustain the inflammatory response and stimulate migration of smooth muscle and endothelial cells into the intima. Continued oxLDL uptake by foam cells combined with impaired cholesterol efflux results in their apoptosis and exposure of thrombogenic lipids [,]. A number of events in monocyte/macrophage physiology may be potential therapeutic targets for dealing with atherosclerosis and are discussed in detail elsewhere [,].

Inflammation and oxidative stress in atherosclerotic plaque formation. Endothelial dysfunction causes monocyte activation and their binding to endothelial cells, via the production of MCP-1, its binding to CCR2 receptors, and the upregulation of adhesion molecules on endothelial cells (1). Monocytes cross the endothelium and differentiate into macrophages (2). Due to ROS, LDL that traverses the endothelium is converted to mmLDL and oxLDL. Macrophages accumulate oxLDL through scavenger receptors and are turned into foam cells (3). Along with T cells, foam cells produce inflammatory mediators that stimulate migration of smooth muscle and endothelial cells into the intima (4). Figure taken with permission from Reference .

ROS play a pivotal role in atheroma development and macrophages are the major source for ROS with NADPH oxidase, cyclooxygenases (COX), lipooxygenases (LOX), iNOS, and myeloperoxidase contributing [,]. ROS participate in atherosclerosis in part by causing LDL oxidation, activating stress signaling pathways, inducing apoptosis, and facilitating plaque rupture []. Based on their ability to inhibit 15-LOX, CBD and its mono- and dimethylated derivatives have been proposed as potentially useful in treating atherosclerosis []; however, the question of whether 15-LOX has a detrimental or beneficial role in atherosclerosis is unsettled []. Nevertheless, a growing body of evidence supports the utility of targeting endocannabinoid signaling, particularly that of macrophages, in the treatment of atherosclerosis []. Differentiation of human monocytes, including that induced by oxLDL, results in a change in their CB1 and CB2 expression profile such that CB1 becomes more prominent []. Activation of macrophage CB1 receptor was shown to upregulate the CD36 scavenger receptor and cholesterol accumulation by macrophages/foam cells []. CB1 receptor activation of human macrophages was linked to ROS generation via p38 MAPK activation, as well as production of TNFα and MCP1 []. In contrast, activation of the CB2 receptor was shown to attenuate the pro-inflammatory actions of the CB1 receptor through activation of the Ras family small G protein, Rap1 []. Consistent with these findings, a nonselective CB1/CB2 receptor agonist reduced oxLDL-induced ROS generation and TNFα secretion via the CB2 receptor of murine macrophage, which in contrast to human macrophages do not express much CB1 receptor [,]. Such tantalizing findings have fueled the idea that the endocannabinoid system may be a avenue for further drug development in dealing with atherosclerosis, likely involving a role for CBD as well [].

Opposing regulatory effects of CB1 and CB2 receptors on inflammation and oxidative/nitrative stress is a general theme that has significance in atherosclerosis, as well as other human maladies. CB2 activation in endothelial cells, which play a key role in development of early atherosclerosis and any inflammatory response, decreases activation and the inflammatory response [], while CB1 activation in human coronary artery endothelial cells was reported to induce ROS-dependent and -independent MAPK activation and cell death []. CB1 cannabinoid receptors promote oxidative stress and cell death in murine models of doxorubicin-induced cardiomyopathy and in human cardiac myocytes []. In contrast, CB2 activation was found to reduce oxidative stress and neutrophil infiltration in the infracted mouse myocardium []. In nephropathy, CB2 limits oxidative/nitrosative stress, inflammation, and cell death [], while activation of CB1 cannabinoid receptors promote oxidative/nitrosative stress, inflammation, and cell death [].

Conclusions

Inflammation and oxidative stress are intimately involved in the genesis of many human diseases. Unraveling that relationship therapeutically has proven challenging, in part because inflammation and oxidative stress “feed off” each other. However, CBD would seem to be a promising starting point for further drug development given its anti-oxidant (although relatively modest) and anti-inflammatory actions on immune cells, such as macrophages and microglia. CBD also has the advantage of not having psychotropic side effects. Studies on models of human diseases support the idea that CBD attenuates inflammation far beyond its antioxidant properties, for example, by targeting inflammation-related intracellular signaling events. The details on how CBD targets inflammatory signaling remain to be defined. The therapeutic utility of CBD is a relatively new area of investigation that portends new discoveries on the interplay between inflammation and oxidative stress, a relationship that underlies tissue and organ damage in many human diseases.

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Cannabidiol (CBD) rather than Cannabis sativa extracts inhibit cell growth and induce apoptosis in cervical cancer cells. In conclusion, these data suggest that cannabidiol rather than Cannabis sativa crude extracts prevent cell growth and induce cell death in cervical cancer cell lines.

Associated Data

Data Availability Statement

Abstract

Background

Cervical cancer remains a global health related issue among females of Sub-Saharan Africa, with over half a million new cases reported each year. Different therapeutic regimens have been suggested in various regions of Africa, however, over a quarter of a million women die of cervical cancer, annually. This makes it the most lethal cancer amongst black women and calls for urgent therapeutic strategies. In this study we compare the anti-proliferative effects of crude extract of Cannabis sativa and its main compound cannabidiol on different cervical cancer cell lines.

Methods

To achieve our aim, phytochemical screening, MTT assay, cell growth analysis, flow cytometry, morphology analysis, Western blot, caspase 3/7 assay, and ATP measurement assay were conducted.

Results

Results obtained indicate that both cannabidiol and Cannabis sativa extracts were able to halt cell proliferation in all cell lines at varying concentrations. They further revealed that apoptosis was induced by cannabidiol as shown by increased subG0/G1 and apoptosis through annexin V. Apoptosis was confirmed by overexpression of p53, caspase 3 and bax. Apoptosis induction was further confirmed by morphological changes, an increase in Caspase 3/7 and a decrease in the ATP levels.

Conclusions

In conclusion, these data suggest that cannabidiol rather than Cannabis sativa crude extracts prevent cell growth and induce cell death in cervical cancer cell lines.

Keywords: Apoptosis, Cervical cancer, Cannabidiol, Cannabis sativa

Background

Cannabis sativa is a dioecious plant that belongs to the Cannabaceae family and it originates from Central and Eastern Asia [, ]. It is widely distributed in countries including Morocco, South Africa, United States of America, Brazil, India, and parts of Europe [, ]. Cannabis sativa grows annually in tropical and warm regions around the world []. Different ethnic groups around the world use Cannabis sativa for smoking, preparing concoctions to treat diseases, and for various cultural purposes []. According to [], it is composed of chemical constituents including cannabinoids, nitrogenous compounds, flavonoid glycosides, steroids, terpenes, hydrocarbons, non-cannabinoid phenols, vitamins, amino acids, proteins, sugars and other related compounds. Cannabinoids are a family of naturally occurring compounds highly abundant in Cannabis sativa plant [, , , ]. Screening of Cannabis sativa has led to isolation of at least 66 types of cannabinoid compounds [, , ]. These compounds are almost structurally similar or possess identical pharmacological activities and offer various potential applications including the ability to inhibit cell growth, proliferation and inflammation []. One such compound is cannabidiol (CBD), which is among the top three most widely studied compounds, following delta-9-tetrahydrocannabinol (Δ9-THC) []. It has been found to be effective against a variety of disorders including neurodegerative disorders, autoimmune diseases, and cancer [, ]. In a research study conducted by [], it was found that CBD inhibited cell proliferation and induces apoptosis in a series of human breast cancer cell lines including MCF-10A, MDA-MB-231, MCF-7, SK-BR- 3, and ZR-7-1 and further studies found it to possess similar characteristics in PC-3 prostate cancer cell line []. However, to allow us to further our studies in clinical trials a range of cancers in vitro should be tested to give us a clear mechanism before we can proceed. Cannabis sativa in particular cannabidiol, we propose it plays important role in helping the body fight cancer through inhibition of pain and cell growth. Therefore, the aim of this study was to evaluate the cytotoxic and anti-proliferative properties of Cannabis sativa and its isolate, cannabidiol in cervical cancer cell lines.

Methods

Materials

An aggressive HeLa, a metastatic ME-180 and a primary SiHa cell lines were purchased from ATCC (USA, MD). Camptothecin was supplied by Calbiochem® and cannabidiol was purchased from Sigma-Aldrich and used as a standard reference.

Plant collection and preparation of extracts

Fresh leaves, stem and roots of Cannabis sativa were collected from Nhlazatshe 2, in Mpumalanga province. Air dried C. sativa plant material was powdered and soaked for 3 days in n-hexane, ethanol and n-butanol, separately. Extracts were filtered using Whatman filter paper and dried. Dimethyl sulfoxide was added to dried extracts to give a final concentration of a 100 mg/ml. Different concentrations (50, 100, and 150 μg/ml) of C. sativa extracts were prepared from the stock and used in treating cells during MTT assay. HPLC-Mass spectrophotometry was performed to verify the presence of cannabidiol in our extracts. The plant was identified by forensic specialist in a forensic laboratory in Pretoria. The laboratory number 201213/2009 and the voucher number is CAS239/02/2009.

Cell culture

HeLa, ME-180 and SiHa were cultured in Dulbecco’s Modified Eagle Media (DMEM) supplemented with 10 % Fetal Bovine Serum (FBS) (Highveld biological,) and 1 % penicillin/streptomycin (Sigma, USA). Cells were maintained at 37 °C under 5 % of carbon dioxide (CO2) and 95 % relative humidity. After every third day of the week, old media was removed and replaced with fresh media, to promote growth until the cells reach a confluence of ~70–80 %.

Methods

MTT assay

Ninety microlitres of HeLa and SiHa cells were seeded into 96-well plates at 5×103 cells per well and incubated overnight at 37 °C under 5 % CO2 and 95 % relative humidity to promote cell attachment at the bottom of the plate. Media was changed and the cells were treated with Cannabis sativa plant extracts at various concentrations (0, 50, 100, and 150 μg/ml (w/v)) for 24 h. After 24 h, the cells were treated with 10 μl of (5 mg/ml) MTT reagent (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) for 4 h at 37 °C under 5 % CO2. Ninety microlitres of DMSO was added into each well including wells containing media only and serves as a blank, to dissolve formazan crystals. Camptothecin and DMSO were included as controls. Optical density was measured using a micro plate reader (Bio-Rad) at 570 nm to determine the percentage of viable cells and account for cell death induced according to the outlined equation below:

%Cellviability=AbsorbanceoftreatedcellsAbsorbanceofblankAbsorbanceofuntreatedcellsAbsorbanceofblank×100

 

Cell growth analysis

Before seeding cells, a 100 μl of media was added to the 16 well E-plate and placed in the incubator to record background readings. A blank with media only was included to rule the possibility of media having a negative effect on the cells. In each well of the E-plate, 1×104 cells were seeded and left in the incubator for 30 min to allow the cells to adhere to the bottom of the plate. The E-plate was placed and locked in the RTCA machine and experiment allowed to run for 22 h prior to the addition of the test compounds. Cells were treated with various concentrations (0, 50, 100, 150 μg/ml) of C. sativa hexane extract. Following treatment, the experiment was allowed to run for a further 22 h. Camptothecin (0.3 μM (v/v)) and DMSO (0.1 % (v/v)) were used as controls for comparative purposes. Procedure was repeated for C. sativa butanol extract.

Apoptosis assay

Cells were washed twice with 100 μl of cold Biolegend’s cell staining buffer followed by resuspension in 100 μl of Annexin V binding buffer. A 100 μl of cell suspension was transferred into 15 ml falcon tube and 5 μl of FITC Annexin V and 10 μl of Propidium iodide solution (PI) were added into untreated and treated cell suspension. The cells were gently vortexed and incubated at room temperature (25 °C) in the dark for 15 min. After 15 min, 400 μl of Annexin V binding buffer was added to the cells. The stained cells were analysed using FACSCalibur (BD Biosciences, USA).

Morphological analysis

Five hundred microliter of 1×104 cells was added onto a 6-well plate containing coverslips. The plate was incubated overnight to allow the cells to attach. Following attachment, media was removed and cells were washed twice with PBS, prior to incubation with IC50 of Cannabis sativa extracts for 24 h. After 24 h, media was removed and cells were washed twice with PBS. Four percent (4 %) was added into each well and the plate incubated for 20 min at room temperature, to allow efficient fixation of cells. Cells were washed twice with PBS and once with 0.1 % BSA wash buffer and further stained with DAPI and Annexin V/FITC for 5 min. BX-63 Olympus microscope (Germany) was used to visualize the cells.

Mitochondrial assay (ATP detection)

Twenty five microlitres of 1×104 cells per well were plated in a white 96-well luminometer plate overnight. Cells were treated with 25 μl of IC50 concentrations of Cannabis sativa crude extracts and cannabidiol dissolved in a glucose free media supplemented with 10 mM galactose. The plate was incubated at 37° in a humidified and CO2-supplemented incubator for a period of 24 h. Fifty microlitres of ATP detection reagent was added to each well and the plate further incubated for 30 min. Luminescence was measured using GLOMAX (Promega, USA). The assay was conducted in duplicates and ATP levels were reported as a mean of Relative Light Units (RLU). The following formula was used to calculate the ATP levels in RLU:

RLU Luminescence(sample) − Luminescence(blank)

 

Caspase 3/7 activity

A hundred microliters of 1×104 cells were plated overnight on a 96-well luminometer plate and allowed to attach overnight. The next day, cells were treated with 0.3 μM camptothecin and the IC50 concentrations of Cannabis sativa crude extracts and further incubated for a period of 24 h. Caspase-Glo 3/7 assay was performed according to manufacturer’s protocol (Promega, USA). Briefly, following treatment, media was replaced with caspase glo 3/7 reagent mixed with a substrate at a ratio of 1:1 v/v of DMEM: Caspase-glo 3/7 reagent and was incubated for 2 h at 37 °C in 5 % CO2. Luminescence was quantified using GLOMAX from Promega (USA). The assay was conducted in duplicates and caspase 3/7 activity was reported as a mean of Relative Light Units (RLU). The following formula was used to calculate caspase 3/7 activity in RLU:

RLU Luminiscence(sample) − Luminiscence(blank)

 

Cell cycle analysis

Cells were harvested with 2 ml of 0.05 % trypsin-EDTA. Ten millilitres of media was added to the cells to inactivate trypsin and the cell suspension was centrifuged at 1500 rpm for 10 min. The supernatant was discarded and pellet was re-suspended twice in 1 ml PBS. Cell suspension was centrifuged at 5000 rpm for 2–5 min and PBS was discarded. Seven hundred microlitres of pre-chilled absolute ethanol was added to the cell suspension followed by storage at −20 °C for 30 min, to allow efficient permeabilization and fixing of the cells. After 30 min, cells were centrifuged at 5000 rpm for 5 min to remove ethanol. The pellet was washed twice with PBS and centrifuged at 5000 rpm to remove PBS. Five hundred microlitres of FxCycle™ PI/RNase Staining solution (Life technologies, USA) was added to the cells and vortexed for 30 s (sec). The cells were analysed with FACSCalibur (BD Biosciences, USA).

Western blot

Following 24 h of treatment with IC50 concentrations, cells were lysed using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 % NP-40, 0.1 % SDS, 2 mM EDTA). Protein content was measured by the BCA assay and equal amounts were electrophoresed in SDS polyacrylamide gel and then transferred onto nitrocellulose membranes. Membranes were subsequently immunoblotted with Anti-mouse monoclonal p53, Bcl-2, Bax, RBBP6, Caspase-3 and -9 antibodies were used at 1:500–1000 dilutions as primary antibodies, while a goat anti-mouse horseradish peroxidise-conjugated horse IgG (Santa Cruz, USA) were used at a 1:2000 dilution as a secondary antibody. The membranes were developed using Chemiluminescence detection kit (Santa Cruz Biotechnology, CA). The membranes were imaged using a Biorad ChemiDoc MP.

Data analysis

Experiments were performed in duplicates. Statistical analysis of the graphical data was expressed as the mean standard deviation. The p-value was analysed in comparison to the untreated using Students t-Test wherein p < 0.05 was considered as significant.

Results

Effect of Cannabis sativa and cannabidiol on SiHa, HeLa, and ME-180 cells

To determine half maximal inhibitory concentration (IC50) for both Cannabis sativa and cannabidiol, MTT assay was used. Camptothecin, as our positive control, significantly reduced cell viability in SiHa (40.36 %), HeLa (47.19 %), and ME-180 cells (32.25 %), respectively. As shown in Fig. 1a and andd,d, the IC50 was cell type dependent rather than time dependent with SiHa at less than 50 μg/ml in both butanol (56 %) and hexane (48.9 %). Similarly IC50 in HeLa was at 100 μg/ml at p < 0.001 (Fig. 1b). while ME-180 cells treated with butanolic extract exhibited an IC50 of a 100 μg/ml, reducing viability to 48.6 % (Fig.1c) and hexane extracts IC50 was at 50 μg/ml with 54 % death (Fig. 1f). This was not the case in cannabidiol with SiHa (51 %) and HeLa (50) IC50 at a much lower dose (3.2 μg\ml) while ME-180 cells (56 %) at 1.5 μg\ml when compared to Cannabis sativa extracts (p < 0.001) (Fig. 1g–i). Dimethyl sulfoxide (DMSO) was included as a vehicle control and it inhibited between 4 and 11 %. Whereas ethanol exhibited between 7.3 and 7.8 % since cannabidiol was alcohol extracted.

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xCELLigence analysis of the cell growth pattern after treatment of cervical cancer cells with Cannabis sativa extracts and cannabidiol. SiHa (a, d, g), HeLa (b, e, h), and ME-180 (c, f, i) cells were seeded for a period of 22–24 h, followed by treatment with IC50 concentration of butanol (a, b, c), hexane (d, e, f), and cannabidiol (g, h, i)

 

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Representative cell viability bar graphs of cervical cancer cell lines. MTT assay was conducted to determine IC50 following incubation of SiHa, HeLa, and ME-180 cells with different doses of butanol extract (a, b, c), hexane extract (d, e, f), and cannabidiol extract (g, h, i) for a period of 24 h. Data was expressed as mean value ± standard deviation (SD). The level of significance was determined using Students t-Test with nsrepresenting p > 0.05, ***represents p < 0.001, **represents p < 0.01, and *represents p < 0.05

Effect of Cannabis sativa extracts and cannabidiol on cell growth of cervical cancer cells

The IC50 obtained during MTT assay was tested for their ability to alter cell viability in real time. An impedance based system was employed to evaluate the effect of Cannabis sativa and cannabidiol on SiHa, HeLa, and ME-180. Cells were seeded in an E-plate and allowed to attach. Cells were further treated with IC50 for a period of 22–24 h, depending on their doubling time. Continuous changes in the impedance were measured and displayed as cell index (CI). Little can be read from xCELLigence except that cannabidiol in all cell lines has shown to reduce cell index while the plant extract had mixed results sometimes showing reduction on the other hand remained unchanged (Fig. 3). Suggesting that cannabidiol is the most effective compound.

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Morphological analysis and assessment of apoptosis in SiHa cells stained with DAPI and Annexin V dye. Cells were incubated with IC50 of Cannabis sativa extracts for a period of 24 h. Cells were stained with Annexin V and counterstained with DAPI. BX63-fluorescence confocal microscopy was used to visualize the cells

 

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Morphological analysis and assessment of apoptosis in HeLa cells stained with DAPI and Annexin V dye. Cells were incubated with IC50 of Cannabis sativa extracts for a period of 24 h. Cells were stained with Annexin V and counterstained with DAPI. BX63-fluorescence confocal microscopy was used to visualize the cells

 

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Apoptosis assessment following treatment of cervical cancer cells with IC50 concentrations of Cannabis sativa extract and cannabidiol. These bar graphs are a representative of apoptosis induction in SiHa (a and d), HeLa (b and e), and ME-180 (c and f) cells. Cells were treated with IC50 of Cannabis sativa extracts and cannabidiol for a period of 24 h and further stained with Annexin-V/PI. Data represented as mean ± standard deviation with ***p < 0.001, **p < 0.01 and ns p > 0.05 representing the level of significance in comparison to the untreated

Cannabis sativa extracts and cannabidiol induce apoptosis in cervical cancer cells

Flow cytometry revealed a significant increase in SiHa cells undergoing apoptosis during treatment with butanol (from 2 to 28.5 %) and hexane (from 2 to 17.2 %) as compared to camptothecin with 30.4 %. In HeLa cells, apoptosis was increased to 31.9 % in butanol extract and only 15.3 % in hexane cells (Fig. 3b). A similar events was observed following treatment of ME-180 cells with butanol extract were 44.8 % apoptosis was recorded and 43.2 % in hexane treated cells (Fig. 3c). Cannabidiol was also tested for its ability to induce apoptosis in all three cell lines. The results further confirmed that the type of cell death induced was apoptosis. Figure shows that cannabidiol induced early apoptosis in all three cell lines. Cannabidiol was more effective in inducing apoptosis In comparison to both extracts of Cannabis sativa. In SiHa cells cannabidiol induced 51.3 % apoptosis (Fig. 3d), 43.3 % in HeLa and 28.6 % in ME-180 cell lines (Fig. 3f).

Effect of Cannabis sativa extracts and cannabidiol on the morphology of SiHa and HeLa cells

To characterise the cell death type following treatment with our test compounds, cell were stained with DAPI and Annixin V to show if apoptosis was taking place. Treatment of SiHa and HeLa cells with IC50 of both butanol and hexane extracts confirmed the type of cell death as apoptosis since they picked a green colour from Annexin V that bind on phosphotidyl molecules that appear in early stages of apoptosis. Similar results were also observed in cannabidiol treated cells. Another feature that is a representative of cell death is the change in morphology. Morphological appearance of live cells displayed a round blue nuclei following staining with DAPI. Exposure of SiHa and HeLa cells to IC50 of Cannabis sativa extracts caused a change in morphology coupled with an uptake of annexin V. Loss of shape, nuclear fragmentation, reduction in cell size and blebbing of the cell membrane were among the observed morphological features implicated to be associated with apoptosis (Fig. 6).

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Bar graphs representing changes in the ATP levels following treatment of cervical cancer cells with Cannabis sativa and cannabidiol. Cells were treated with IC50 of both Cannabis sativa extracts and cannabidiol for a period of 2–24 h. Untreated and camptothecin were included as controls for comparative purposes. The level of significance was determined using Students t-Test with ***p < 0.001, **p < 0.01, *p < 0.05, and ns p > 0.05 in comparison to the untreated

 

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Caspase 3/7 activity after treatment of SiHa, HeLa, and ME-180 cells with IC50 of Cannabis sativa extract and cannabidiol. Cells were treated with IC50 of Cannabis sativa and cannabidiol extracts for a period of 24 h. Caspase 3/7 reagent was added to the treated cells for 1 h. Luminescence–was measured using GLOMAX instrument in RLU. Data represented as mean ± standard deviation with ***p < 0.001, **p < 0.01, and *p < 0.05 representing the level of significance in comparison to the untreated

Effect of Cannabis sativa extracts and cannabidiol on the ATP levels

Since Adenosine 5’-triphosphate acts as a biomarker for cell proliferation and cell death, an ATP assay was conducted. This was done in order to determine whether Cannabis sativa and cannabidiol deplete ATP levels in cervical cancer cells. SiHa, HeLa, and ME-180 cells were treated at different time points, between 2 and 24 h. ATP levels were first detected after 2 h. In general, ATP depletion was cell type dependent. In HeLa cells treated with the crude extracts from butanol and hexane, ATP was significantly reduced by 74 % (from 627621 to 164208 RLU) and 78 % (from 627621 to 133693 RLU) respectively. While with SiHa there was reduction of 31 % (from 4719589 to 3221245 RLU) and 22.5 % (4719589 to 3655730 RLU) respectively (figure). Whereas in ME-180 there was no change between treatments and untreated. Similar results were observed in cannabidiol treated cells. At 2 h, treatment with IC50 led to a reduction in ATP levels by ~ 61 % (from 4704419 to 1802508 RLU), 93 % (from 627621 to 40371 RLU), and 8 % (from 798688 to 734039 RLU) in SiHa, HeLa, and ME-180 cells respectively (Figure). A prolonged incubation period (24 h) of cells with IC50 led to a further decrease in the ATP levels by ~66 % (from 4486150 to 1497648 RLU), 97 % (from 601694 to 13426 RLU), and 8.5 % (from 790757 to 723039 RLU) in SiHa, HeLa, and ME-180 cells respectively. This could mean that cannabidiol depletes ATP levels more than Cannabis sativa extracts and might be the main compound responsible for cell death in cancer cells treated with Cannabis sativa.

Effect of Cannabis sativa and cannabidiol on caspase 3/7 activity of SiHa, HeLa, and ME-180 cells

As shown in Fig. 8a, ,bb and andc,c, we observed an increase in caspase 3/7 activity all three cell lines following treatment with 0.3 μM of camptothecin. Similar results were observed in crude extract treated cells by 25 % (SiHa) and 40 % (HeLa) in butanol extract and 50 % (SiHa) and 100 % (HeLa) in hexane treated. There was no significant change in ME-180 cells (Figure). When cells were treated with cannabidiol, caspase 3/7 activity increased in all three cell lines. SiHa cells so an increase from 200000 RLU to 2500000 while HeLa increased to 900000 from 800000 RLU. ME-180 was fairly increased also to 230000 from 200000 RLU all increase were significant and in line with other increase in apoptosis as shown in Annexin V.

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Representative bar graph of the cervical cancer cell cycle before and after treatment with Cannabis sativa extracts and cannabidiol. Cells were harvested and treated with camptothecin and the IC50 concentrations of Cannabis sativa extracts and cannabidiol. Bar graph a and d represents SiHa cells, b and e represents HeLa cells, c and f represents ME-180 cells. Data represented as mean ± standard deviation with ***p < 0.001, **p < 0.01, *p < 0.05, and ns p > 0.05 representing the level of significance in comparison to the untreated

Effect of Cannabis sativa extracts and cannabidiol on cell cycle progression

We further assessed the effects of Cannabis sativa extracts and cannabidiol on cell cycle progression using flow cytometry. Flow cytometry showed that in the presence of Cannabis sativa crude extracts and camptothecin, SiHa cells exhibited a significant increase (p < 0.001) in sub-G0 population with a decrease in G0/G1, S, and G2/M phase. In butanol, sub-G0 phase was increased from 4.2 to 20.1 %, while decreasing the G0/G1 (from 64.0 to 48.7 %), S-phase (from 9.3 to 6.5 %), and G2/M (from 18.5 to 17 %) in SiHa population (Fig. 9a) while in hexane treated sub-G0 phase was at 39.1 % compared to 4.2 % in untreated, with a decrease in G0/G1 (from 64.0 to 30.4 %), S (from 9.3 to 6.5 %), and G2/M (from 18.5 to 13.8 %) population (Fig. 9a). In HeLa cells, butanol extracts reduced G0/G1 to 54.9 % while the S-phase and G2/M significantly increased to 18.4 and 25.7 % while with hexane there was increase in the G2/M phase (20.3 %) and a decrease in the S-phase (8.1 %). In ME-180 there was insignificant increase in all cell cycle stages. Each cell line responded differently to cannabidiol treatment. Almost 42.2 % of SiHa cells were observed in the sub-G0 (p < 0.001) while there was reduction in cells in the G0/G1 phase, from 57.9 to 42.8 % (Fig. 9d). A similar trend was observed in HeLa cells but much lower sub-G0 (from 5.1 to 17.4 %) and S phase (from 4.8 to 11.2 %) (Fig. 9e). A similar event was observed during treatment of ME-180 cells. Cannabidiol significantly increased sub-G0 in ME-180 cells to 34.3 % (Fig. 9f). From this data, we can conclude that cannabidiol induced cell death without cell cycle arrest.

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A densitometry analysis SiHa protein was performed using ImageJ quantification software to measure the relative band intensity. CPT represents camptothecin. Data represented as mean ± standard deviation with ***p < 0.001, **p < 0.01 and ns p > 0.05 representing the level of significance in comparison to the untreated represent the western blot analysis of SiHa and HeLa cells. The genes analyzed are p53 and RBBP6 including caspases. Equal amount of protein (conc) was loaded in each well. Note that the darker the bands increased expression of the gene

 

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A densitometry analysis HeLa protein was performed using ImageJ quantification software to measure the relative band intensity. CPT represents camptothecin. Data represented as mean ± standard deviation with ***p < 0.001, **p < 0.01 and ns p > 0.05 representing the level of significance in comparison to the untreated

 

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A densitometry analysis ME-180 protein was performed using ImageJ quantification software to measure the relative band intensity. CPT represents camptothecin. Data represented as mean ± standard deviation with ***p < 0.001, **p < 0.01 and ns p > 0.05 representing the level of significance in comparison to the untreated

 

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Western blot analysis of the protein expression before and after 24 h treatment with IC50 of Cannabis sativa extracts and cannabidiol. SiHa (a and d), HeLa (b and e), and ME-180 (c and f) cells were treated for a period of 24 h and protein lysates were separated using SDS-PAGE gel. Untreated protein was used as a control. Antibodies against pro-apoptotic proteins (p53 and Bax) and anti-apoptotic proteins (Bcl-2 and RBBP6), Initiator caspase-9 and effecter caspase-3 were included to elucidate apoptosis induction

Effect of Cannabis sativa extracts and cannabidiol on the expression of upstream and downstream target proteins

From the apoptosis experiments conducted, it is clear that the mode of cell death induced by cannabidiol and extract of Cannabis Savita was that of apoptosis. However, we needed to confirm whether the type of apoptosis induced is it p53 dependent or independent as it is well known that p53 is mutated in many cancers. Protein expression of RBBP6, Bcl-2, Bax and p53were performed and results recorded. In butanol extract p53 was significantly increased in SiHa and HeLa cells while remaining unchanged in ME-180. Similar results were observed in hexane treated cells. In all cell lines the level of p53 negative regulator in cancer development was reduced by all treatment.

Following treatment of cervical cancer cells, Bax protein was up-modulated and Bcl-2 was down-modulated. Western blot analysis revealed that cannabidiol effectively caused an increase in the expression of pro-apoptosis proteins, p53 and Bax, while simultaneously decreasing the anti-apoptosis proteins, RBBP6 and Bcl-2 in all three cervical cancer cell lines (SiHa, HeLa, and ME-180 cells). Caspases play an effective role in the execution of apoptosis, an effector caspase-9 and executor capsase-3 were included in our western blot to check if they played a role in inducing apoptosis. In all Cannabis sativa extracts, caspase-3 and caspase-9 were upregulated in all cell lines. Similar results were also observed in cannabidiol treated cells with upregulation of both caspase-3 and -9.

Discussion

Cervical cancer remains a burden for women of Sub-Saharan Africa. Half a million new cases of cervical cancer and a quarter of a million deaths are reported annually due to lack of effective treatment []. Currently, the recommended therapeutic regimens include chemotherapy, radiation therapy, and surgery. However, they present several limitations including side effects or ineffectiveness []. Therefore, it is important to search for new novel therapeutic agents that are naturally synthesized and cheaper, but still remain effective. Medicinal plants have been used for decades for health benefits and to treat several different diseases []. In South Africa, over 80 % of the population are still dependent on medicinal plants to maintain mental and physical health []. However, some of the medicinal plants used by these individuals are not known to be effective and their safety is still unclear. It is therefore important to scientifically evaluate and validate their efficacy and safety. In the present study, cervical cancer cell lines (SiHa, HeLa, and ME-180) were exposed to different concentrations of Cannabis sativa extracts and that of its compound, cannabidiol, with the aim of investigating their anti-proliferative activity.

We first determined whether Cannabis sativa extracts and cannabidiol possess anti-proliferative effects using MTT assay. MTT assay determines IC50, which represents the half maximal concentration that induces 50 % cell death. Cannabis sativa extracts were able to reduce cell viability and increase cell death in SiHa, HeLa, and ME-180 cells. These results correlate with the findings obtained by [], whereby they reported reduced cell proliferation in colorectal cancer cell lines following treatment with Cannabis sativa. According to [, , ] Cannabis sativa extracts rich in cannabidiol were able to induce cell death in prostate cancer cell lines LNCaP, DU145, and PC3 at low doses (20–70 μg/ml). It was suggested that cannabidiol might be responsible for the reported activities. Therefore, in this study, cannabidiol was included as a reference standard in order to determine whether the reported pharmacological activities displayed by Cannabis sativa extracts might have been due to the presence of this compound. For positive extract inhibitory activity, Camptothecin was included as a positive control. Camptothecin functions as an inhibitor of a topoisomerase I enzyme that regulates winding of DNA strands [, ]. This in turn causes DNA strands to break in the S-phase of the cell cycle []. A study conducted by [], exhibited the ability of camptothecin to be cytotoxic against MCF-7 breast cancer cell line and also induce apoptosis as a mode of cell death at 0.25 μM. We also observed a similar cytotoxic pattern, whereby camptothecin induced cell death in HeLa, SiHa, and ME-180 cells, however, at a much higher concentration.

xCELLigence continuously monitors cell growth, adhesion, and morphology in real-time in the presence of a toxic substance. Upon treatment of SiHa and HeLa cells with IC50 of butanol extract, we noted that there was little to no inhibitory effect observed on cell growth. The growth curve continued in its exponential growth in all cells including the treated, untreated and 0.1 % DM’SO. However, at a similar IC50 of 100 μg/ml, a reduction in cell viability was observed following treatment of HeLa cells with hexane extract. On the other hand, ME-180 cells responded after a period of 2 h following treatment with the IC50 of butanol and hexane extract. In comparison to butanol and hexane extracts, cannabidiol reduced the cell index of ME-180 cells after 2 h of treatment, signalling growth inhibition. Differences in the findings could be attributable to the fact that both methods have different principles and mechanism of action. MTT assay is an end-point method that is based on the reduction of tetrazolium salt into formazan crystals by mitochondrial succinate dehydrogenase enzyme. Mitochondrial succinate dehydrogenase is only active in live cells with an intact metabolism [, ]. Induction of cell death by Cannabis sativa crude extracts decreases the activity of the enzyme following treatment of HeLa, SiHa, and ME-180 cervical cancer cell lines. On the other hand, xCELLigence system is a continuous method that relies on the use of E-plates engraved with gold microelectrodes at the bottom of the plate. The xCELLigence system is based on the changes in impedance influenced by cell number, size and attachment []. Therefore, we concluded that it was possible that dead cells might have been attached at the bottom of the E-plate after treatment.

Cell death can be characterized by a decrease in the energy levels as a result of dysfunction of the mitochondria []. Therefore, to evaluate the effect of treatment on the energy content of the cells, we conducted mitochondrial assay. We only used IC50 as indicated by MTT assay only. ATP acts as determinant of both cell death and cell proliferation []. Exposure of SiHa, HeLa, and ME-180 cells to the IC50 of Cannabis sativa extracts caused a reduction in the ATP levels. Treatment of cells with cannabidiol either slightly or severely depleted the ATP levels. According to [], a reduction of the ATP levels compromises the status of cell and often leads to cell death either by apoptosis or necrosis, while an increase is indicative of cell proliferation. Therefore, we concluded that the reduction of ATP might have been as a result of cell death induction since the cells ATP production recovered.

Following confirmation that Cannabis sativa and cannabidiol have anti-proliferative activity, we had to verify whether both treatments have the ability to induce cell cycle arrest in all three cell lines. This method uses a PI stain and flow cytometry to measure the relative amount of DNA present in the cells. In this study, propidium iodide (PI) was used to stain cells. Propidium iodide can only intercalate into the DNA of fixed and permeabilized cells with a compromised plasma membrane or cells in the late stage of apoptosis. Viable cells with an intact plasma membrane cannot uptake the dye. The intensity of stained cells correlates with the amount of DNA within the cells. HeLa, SiHa, and ME-180 cervical cancer cells were stained with PI and analysed using flow cytometry. Treatment of SiHa cells with butanol and hexane extracts led to the accumulation of cells in the cell death phase (sub-G0 phase), without cell cycle arrest. When compared to the S-phase and G2/M phase of untreated cells, exposure of HeLa cells to Cannabis sativa butanol extract resulted in the accumulation of cells in the S-phase of the cell cycle and slight cell death induction. And thus, according to [], signals DNA synthesis and cell cycle proliferation. A decrease in the S-phase and an increase in the G2/M phase of HeLa cells following treatment with hexane extract, suggests a blockage of mitosis and an induction of cell cycle arrest. Interesting to note was that, treatment of ME-180 cells with both extracts led to an increase of cells coupled by an increase in the S-phase population which favours replication and duplication of DNA. This was not the case following treatment of cells with cannabidiol. Cannabidiol resulted in the accumulation of cells in the cell death phase of the cell cycle. SiHa, and HeLa, and ME-180 cells were committed to the cell death phase. In summary, Cannabis sativa induces cell death with or without cell cycle arrest while cannabidiol induces cell death without cell cycle arrest.

Apoptosis plays a major role in determining cell survival. Annexin V/FITC and PI were used to stain the cells to be able to distinguish between viable, apoptotic and necrotic cells. Annexin V/ FITC can only bind to phosphatidylserine residues exposed on the surface of the cell membrane while PI intercalates into the nucleus and binds to the fragmented DNA. Viable cells cannot uptake both dyes due to the presence of an intact cell membrane. Since treatment caused the accumulation of cells in the sub-G0 phase, also known as the cell death phase, and the severe depletion of ATP levels by cannabidiol, we further conducted an apoptosis assay. Treatment of all three cell lines with camptothecin, IC50 of Cannabis sativa and cannabidiol exhibited the type of induced cell death as apoptosis. Sharma et al. [] also showed a similar pattern of cell death, whereby treatment of a prostate cancer cell lines with Cannabis sativa resulted in the induction of apoptosis.

Apoptosis is characterized by morphological changes and biochemical features which include condensation of chromatin, convolution of nuclear and cellular outlines, nuclear fragmentation, formation of apoptotic blebs within the plasma membrane, cell shrinkage due to the leakage of organelles in the cytoplasm as well as the presence of green stained cells at either late or early apoptosis [, , ]. Annexin V/FITC and DAPI were used to visualize the cells under a fluorescence confocal microscopy. According to [], an uptake of Annexin V/FITC suggests the induction of apoptosis, since it can only bind to externalized PS residues. This also proves that during cell growth analysis, SiHa and HeLa cells were undergoing cell death while still attached to the surface of the flask.

Apoptosis is known to occur via two pathways, the death receptor pathway and the mitochondrial pathway []. Cannabis sativa isolates including cannabidiol have been implicated in apoptosis induction via the death receptor pathway, by binding to Fas receptor or through an activated of Bax triggered by the synthesis of ceramide in the cells []. However, not much has been reported on the induction of apoptosis via activation of p53 by Cannabis sativa. Our focus in this study was also to identify downstream molecular effect of extracts. One such important gene is p53 which acts as a transcription factor for a number of target genes []. Under normal conditions, p53 levels are maintained through constant degradation MDM2 and its monomers []. RBBP6 is one of the monomers that helps degrade p53, due the presence of Ring finger domain that promotes the interaction of both proteins []. In response to stress stimuli such as DNA damage, hypoxia, UV light, and radiation light, p53 becomes activated and causes MDM2 expression to decrease []. Mutation of p53, implicated to be associated with 50 % of all human cancers, promote the tumorigenesis. Bax and Bcl-2 form part of the proteins that regulate apoptosis via the mitochondria []. Following activation, p53 translocates into the cytosol and triggers the oligomerization of Bcl-2 with BAD, resulting in the inhibition of Bcl-2 activity []. This in turn allows Bax protein to be translocated to the mitochondria and participate in the release of cytochrome c through poration of the outer mitochondrial membrane [, ]. An imbalance between Bax and Bcl-2 has been linked to the development and progression of tumours through the resistance of apoptosis []. It is therefore crucial to design drugs that would effectively target these genes involved in the execution of apoptosis via the mitochondrial pathway. Camptothecin, hexane extract, and cannabidiol effectively up-modulated the expression of p53 in all three cell lines, leading to a decrease in RBBP6 protein expression. Apart from SiHa and HeLa, butanol extract failed to up-modulate p53 in ME-180 cells. Interesting to note is that butanol extract reduced the expression of RBBP6 protein in ME-180 cells. The mechanism behind failure of butanol to up-modulate p53 while down-modulating RBBP6 is unclear. However, we came to a conclusion that butanol induces apoptosis independently of p53. We further demonstrated that Cannabis sativa extracts, cannabidiol, and camptothecin were able to down-modulate the expression of Bcl-2 protein and up-modulate Bax expression.

Caspases play an effective role in the execution of apoptosis either through the extrinsic or intrinsic pathway []. In this study, we wanted to validate whether caspase-9 and caspase-3 were involved in the initiation and execution of apoptosis. We demonstrated the ability of Cannabis sativa to initiate apoptosis by activating caspase-9. However, execution of apoptosis was either with or without the presence of capsase-3, depending on each cell line. Western blot revealed that Cannabis sativa hexane extract induced apoptosis via the activation of caspase-9 and caspase-3 when compared to untreated cells in all three cell lines. Similar results were obtained during treatment of all three cell lines with camptothecin. This was not the case with butanol. Butanol extracts up-modulated caspase-9 and caspase-3 in SiHa and HeLa cells only. Caspase-3 was not up-modulated in ME-180 cells. Caspase 3/7 activity assay revealed the up-modulation of caspase 3/7 following treatment of cervical cancer cells. However on the basis of the Western blot results, wherein butanol extract failed to up-modulate caspase-3, we can conclude that caspase-7 was responsible for the reported activity. Cannabidiol effectively up-modulated caspase-9 and caspase-3 in all three cell lines, when compared to the untreated and Cannabis sativa extract. From the results we can conclude that, apoptosis induction was caspase dependent.

Conclusions

The aim of this study was to evaluate for the anti-growth effects of Cannabis sativa extracts and to also determine the mode of cell death following treatment. The activity of Cannabis sativa extracts was compared to that of cannabidiol, in order to verify whether the reported results were due to the presence of the compound. The study showed that the activity of one of the extracts might have been due to the presence of cannabidiol. It further demonstrated the ability of Cannabis sativa to induce apoptosis with or without cell cycle arrest and via mitochondrial pathway. More research needs to be done elucidating the mechanism between the active ingredients and molecular targets involved in the regulation of the cell cycle.

Acknowledgements

Our gratitude goes to South African MRC for funding assistance.

Funding

The work was funded by MRC.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article and its additional files.

Authors’ contributions

STL was responsible for the experimental design and LRM prepared the manuscript. Both authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

The authors give consent for the journal to be published.

Ethics approval and consent to participate

This study was approved by the Human Research Ethics Committee (Medical): M140801.

Abbreviations

Bad Bcl-2-associated death promoter
Bak-1 Bcl2-antagonist/killer 1
Bax Bcl2-associated X protein
Bcl-2 B-cell lymphoma 2
BH Bcl-2 homology domain
Bid BH3 interacting-domain
Bik Bcl-2-interacting killer
DMEM Dulbecco’s modified eagle’s medium
DMSO Dimethyl sulfoxide
FITC Fluorescein isothiocyanate
P53 protein 53
HPLC High Perfomance Liquid Chromatography
RBBP6 Retinoblastoma binding protein 6

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CBDA / CBD COMBO

Cannabidiolic acid, a major cannabinoid in fiber-type cannabis, is an inhibitor of MDA-MB-231 breast cancer cell migration. Cannabidiol (CBD), a major non-psychotropic constituent of fiber-type cannabis plant, has been reported to possess diverse biological activities, including anti-proliferative effect on cancer cells. Although CBD is obtained from non-enzymatic decarboxylation of its parent molecule, cannabidiolic acid (CBDA), few studies have investigated whether CBDA itself is biologically active. Results of the current investigation revealed that CBDA inhibits migration of the highly invasive MDA-MB-231 human breast cancer cells, apparently through a mechanism involving inhibition of cAMP-dependent protein kinase A, coupled with an activation of the small GTPase, RhoA. It is established that activation of the RhoA signaling pathway leads to inhibition of the mobility of various cancer cells, including MDA-MB-231 cells. 

Abstract

Cannabidiol (CBD), a major non-psychotropic constituent of fiber-type cannabis plant, has been reported to possess diverse biological activities, including anti-proliferative effect on cancer cells. Although CBD is obtained from non-enzymatic decarboxylation of its parent molecule, cannabidiolic acid (CBDA), few studies have investigated whether CBDA itself is biologically active. Results of the current investigation revealed that CBDA inhibits migration of the highly invasive MDA-MB-231 human breast cancer cells, apparently through a mechanism involving inhibition of cAMP-dependent protein kinase A, coupled with an activation of the small GTPase, RhoA. It is established that activation of the RhoA signaling pathway leads to inhibition of the mobility of various cancer cells, including MDA-MB-231 cells. The data presented in this report suggest for the first time that as an active component in the cannabis plant, CBDA offers potential therapeutic modality in the abrogation of cancer cell migration, including aggressive breast cancers.

Keywords: Cannabidiolic acid, RhoA, Fiber-type cannabis plant, Cannabidiol, MDA-MB-231 cells

1. Introduction

It is well-established that Δ′-tetrahydrocannabinol (Δ′-THC), the major psychoactive constituent in the medicinal/drug-type variety of the cannabis plant, displays a number of biological activities including anti-atherosclerotic, anti-proliferative and endocrine disrupting properties (; ; , , ; ). There is also growing experimental evidence suggesting that cannabidiol (CBD), found principally in the fiber-type of cannabis, also possesses biological activities. For example, CBD has been reported as an inhibitor of human glioma cell migration and an inducer of programmed cell death in tumor cells, including breast cancer cells (; ; ). In fresh plants, the concentrations of neutral cannabinoids, including CBD, are much lower than those of cannabinoid acids that contain a carboxyl group (–COOH) in the structures. CBD is produced largely through non-enzymatic decarboxylation of its acidic precursor, cannabidiolic acid (CBDA), during extraction from the leaf materials of the plant (see Fig. 1) (). Although studies on the biological efficacies of CBD are ongoing, there is a relative lack of information regarding the potential biological activities of CBDA. We have reported that CBDA is a selective cyclooxygenase-2 (COX-2) inhibitor (, ), and it has been reported by others that CBDA is a necrosis-inducing factor for the leaf cells of cannabis plant (). However, no investigations have evaluated potential anti-migration effects of CBDA on human breast cancer cells. Among the available experimental human breast cancer cell lines, MDA-MB-231 cells have been established as a valuable mesenchymal breast cancer model (see Fig. 2A–b) for pre-clinical studies as they are highly aggressive, both in vitro and in vivo (; ; ).

Chemical structures of CBDA and CBD. In the fiber-type cannabis plant, the concentration of CBD is much lower than that of its precursor CBDA. CBD is formed artificially from CBDA by non-enzymatic decarboxylation during extraction step ().

Effect of CBDA on the vertical migration of highly aggressive human breast cancer MDA-MB-231 cells. (A) Morphologies of two human breast cancer cell lines; MCF-7 cells (a) and MDA-MB-231 cells (b). MCF-7 cells display epithelial morphology (collective) and MDA-MB-231 cells display single elongated morphology (mesenchymal). (B) MDA-MB-231 cells were exposed for 12 h to CBDA (5, 10, 25 μM) and CBD (5, 10, 25 μM). After the treatments, cell viability was measured according to the methods described in Section 2. Data are expressed as the percent of vehicle-treated group (indicated as Cont.), as mean ± S.D. (n = 6). *Significantly different (p < 0.05) from the vehicle-treated control. (C) Transwell migration assays (vertical migration) were performed to determine MDA-MB-231 cell migration 12 h after treatments with 5 μM, 10 μM, or 25 μM CBDA and 5 μM CBD. Data are expressed as the percent of vehicle-treated group (indicated as Cont.), as mean ± S.D. (n = 8). *Significantly different (p < 0.05) from the vehicle-treated control. N.D., not detectable due to complete inhibition of the migration.

It is well-recognized that Rho family small GTPases (~21 kDa) regulate actin cytoskeletal dynamics, thereby affecting multiple cellular functions including cell mobility and polarity (; ). In the GTP-bound state, Rho family small GTPases are active and able to interact with specific downstream effectors such as Rhotekin, which lead to the translocation and activation of the effectors, and induction of various intracellular responses. In these respects, altered Rho GTPase activity or expression is implicated in cancer progression (). Among the Rho subfamily members that include the isoforms RhoA, RhoB, and RhoC, RhoA has specifically been identified as an inhibitor of cancer cell mobility, including breast cancer cells (; ; ). Post-translational regulation of Rho activity has been demonstrated for RhoA. This Rho protein is phosphorylated in vitro and in vivo by kinases such as a cAMP-dependent protein kinase (PKA) on serine 188 (Ser188) (; ; ), and it is generally accepted that phosphorylation of this site is an important negative regulation of the RhoA activity, leading to termination of the signaling process.

In this study we demonstrate for the first time that (i) CBDA is an inhibitor of MDA-MB-231 breast cancer cell migration, and that (ii) the mechanism responsible for the inhibitory effects of CBDA likely involves activation of RhoA via inhibition of PKA.

2. Materials and methods

2.1. Reagents

CBD and CBDA were isolated from the fiber-type cannabis leaves according to the established methods (; ). The purity of CBD and CBDA was determined as >98% by gas chromatography (). SC-560 (purity: >98%), DuP-697 (purity: >96%), SR141716A (purity: >98%), and SR144528 (purity: >98%) were purchased from Cayman Chemicals (Ann Arbor, MI, USA). 2,4-Dihydroxybenzoic acid (β-resorcylic acid, purity: >95%) was purchased from Wako Pure Chemical (Osaka, Japan). Pertussis toxin was purchased from Tocris Bioscience (Ellisville, MO, USA). All other reagents were of analytical grade commercially available and used without further purification.

2.2. Cell cultures and proliferation assays

Cell culture conditions and methods were based on procedures described previously (, , ). Briefly, the human breast cancer cell lines, MDA-MB-231 and MCF-7 (obtained from the American Type Culture Collection, Rockville, MD, USA), were routinely grown in phenol red-containing minimum essential medium alpha (Invitrogen, Carlsbad, CA, USA), supplemented with 10 mM HEPES, 5% fetal bovine serum, 100 U/mL of penicillin, 100 μg/mL of streptomycin, at 37 °C in a 5% CO2–95% air-humidified incubator. Before chemical treatments, the medium was changed to phenol red-free minimum essential medium alpha (Invitrogen, Carlsbad, CA, USA) supplemented with 10 mM HEPES, 5% dextran-coated charcoal-treated serum (DCC-serum), 100 U/mL of penicillin, and 100 μg/mL of streptomycin. Cultures of approximately 60% confluence in a 100-mm Petri dish were used to seed for the proliferation experiments. In the proliferation studies, the cells were seeded into 96-well plates at a density of ~5000 cells/well, and test substances were introduced 4 h after plating. After the indicated periods of incubation (see Results), cell proliferation was analyzed using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (MTS reagent; Promega, Madison, WI, USA), according to the manufacturer’s instructions. Test chemicals were prepared in appropriate organic solvents including DMSO or ethanol. Control incubations contained equivalent additions of solvents with no measurable influence of vehicle observed on cell viability at the final concentrations used.

2.3. Cell morphology studies

For morphological examination of the breast cancer cells, MDA-MB-231 and MCF-7, images were obtained using a Leica DMIL inverted microscope (Leica Microsystems, Wetzlar, Germany), and captured with a Pixera® Penguin 600CL Cooled CCD digital camera (Pixera Co., Los Gatos, CA, USA). Data were processed using Pixera Viewfinder 3.0 software (Pixera Co., Los Gatos, CA, USA). The breast cancer cells were plated in 6-well plates. Three areas with approximately equal cell densities were identified in each well and images of each of these areas were captured.

2.4. Cell migration assays

The cancer cell wound-healing assay was performed according to established methods (). Briefly, MDA-MB-231 cells were seeded into 24-well plates at a density of approximately 1 × 105 cells (1 mL/well) and incubated for 24 h. The formed monolayer was wounded by scratching lines with plastic tip. The wells were then washed one with PBS to remove cell debris, and photographed (i.e., designated as time 0). CBD, CBDA, DuP-697, or SC-560 were then added to test their respective effects on MDA-MB-231 cell migration. In these assays, the cells were incubated in the presence of 5% DCC-serum (chemoattractant) at 37 °C under 5% CO2 for 12 h, 24 h, or 48 h, after which the cells were imaged with photomicrosopy (i.e., designated as time 12 h, 24 h, or 48 h) and the migration distance measured (). The percent wounded area filled was calculated as follows; [(mean wound breadth − mean remained breadth)/mean wound breadth] × 100 (%). The cancer cell Transwell (Boyden Chamber) migration assay (i.e., vertical migration) was performed utilizing the CytoSelect 24-well cell migration and invasion assay in a colorimetric format (8 μm pore size) (Cell Biolabs, Inc., San Diego, CA, USA). The assay was performed exactly as specified by the manufacturer’s protocol. In brief, MDA-MB-231 cells (1.2 × 105 cells) suspended in serum free medium containing endotoxin-free bovine serum albumin (5 mg/mL) were added to the upper chamber of an insert, and the insert was placed in a 24-well plate containing medium with 5% DCC-serum (chemoattractant). When used, CBDA was added to the upper chamber. Migration assays were carried out for 12 h.

2.5. RhoA pull-down assay

To determine whether CBDA can modulate RhoA activities in MDA-MB-231 cells, the cells were treated with 25 μM CBDA or vehicle alone for 24 h or 48 h. RhoA pull-down assays with glutathione S-transferase-tagged Rho-binding domain of Rhotekin on glutathione-agarose beads was performed using a RhoA Activation Assay Biochem Kit (Cytoskeleton Inc., Denver, Co, USA). Active RhoA was normalized to the total RhoA by Western blot analysis (RhoA monoclonal antibody, Abcam, Cambridge, MA, USA; anti-mouse secondary antibody, Sigma–Aldrich, St. Louis, Mo, USA). Western blot analysis was performed based on procedures described previously (). MDA-MB-231 cell extract loaded with GTPγS was used as a positive control, and the same extract was used to determine the phosphorylation status of RhoA using an anti-RhoA antibody specific to RhoA phosphorylated on Ser188 (Abcam, Cambridge, MA, USA). In addition, equivalence of protein loading was assessed by performing Western blot analysis using an anti-β-actin antibody (Sigma–Aldrich, St. Louis, Mo, USA). Quantification of band intensity was performed using NIH Image 1 .61 software (http://rsb.info.nih.gov/nih-image/).

2.6. Determination of protein kinase A activity

cAMP-dependent PKA activities were assayed using the PepTag® Assay for Non-Radioactive Detection of PKA (Promega, Madison, WI, USA) which quantifies the phosphorylation of fluorescent-tagged PKA-specific peptides. MDA-MB-231 cell lysate for PKA protein was prepared using a hypotonic extraction buffer (25 mM Tris-HCl, pH 7.4, 5 mM EDTA, 0.5 mM EGTA, 10 mM β-mercaptoethanol, 1 mg/mL aprotinin, and 1 mg/mL leupeptin). Aliquots of the PKA preparation were incubated for 30 min at 30 °C in PepTag® PKA reaction buffer (100 mM Tris–HCl, pH 7.4, 50 mM MgCl2, and 5 mM ATP) and 0.4 mg/μL of the PKA-specific peptide substrate PepTag® A1 (L–R–R–A–S–L–G; Kemptide). For positive and negative controls, the reactions were performed in the presence or absence of PKA catalytic subunit, respectively. Reactions was stopped by heating for 10 min at 95 °C. Phosphorylated and non-phosphorylated PepTag® peptides were separated electrophoretically using a 0.8% agarose gel prepared in 50 mM Tris–HCl (pH 8.0). For the PKA assays, 20 μg of protein was applied. The phosphorylated peptides migrate toward the anode (+) and non-phosphorylated peptides migrate toward the cathode (−). The gel was then visualized and photographed. The band intensity was analyzed using NIH Image 1.61 software. The intensities of the phosphorylated peptide bands are proportional to the PKA activities.

2.7. Data analysis

IC50 values were determined using SigmaPlot 11® software (Systat Software, Inc., San Jose, CA, USA), according to analyses described previously (). Differences were considered significant when the p value was calculated as <0.05. Statistical differences between two groups were calculated by Student’s t test. Other statistical analyses were performed by Scheffe’s F test, a post hoc test for analyzing results of ANOVA testing. These calculations were performed using Statview 5.0 J software (SAS Institute Inc., Cary, NC, USA).

3. Results and discussion

3.1. Effects of CBDA on the migration of MDA-MB-231 cells

When compared with the epithelial-like morphology of MCF-7 cells that are capable of forming domes, with close contact between cells (collective, Fig. 2A–a), MDA-MB-231 cells exhibit a mesenchymal-like morphology (mesenchymal, Fig. 2A–b), a hallmark feature of tumor aggressiveness (; ; ; ; ). Thus, we selected the use of MDA-MB-231 cells to investigate whether CBDA can inhibit the cell migration under the incubation period and its concentration that do not affect overall MDA-MB-231 cell growth (Fig. 2B, see also Fig. 6B). In this study we could not directly compare the efficacy of CBDA and CBD-mediated inhibition of the migration of MDA-MB-231 cells because of CBD’s strong cytostatic effect (see also ); 10 μM and 25 μM CBD treatment for 12 h significantly reduced MDA-MB-231 cell viability (Fig. 2B). Thus, we compared 5 μM or 25 μM CBDA treatments with that of 5 μM CBD. As shown in Figs. 2C, 3A–a/b and B, CBDA inhibited migration of MDA-MB-231 cells in a concentration-dependent manner, whereas 5 μM CBD exhibited no significant inhibitory effects on migration. Futher, CBDA’s significant abrogation of cell migration was detected even 48 h after its exposure (Fig. 3B). It is noteworthy that CBDA-mediated attenuation of MDA-MB-231 cell migration was more pronounced when assessing vertical migration versus horizontal migration. Although in this study we could not detect CBD-mediated inhibition of the migration of MDA-MB-231 cells, it has been reported that low concentrations of CBD (i.e., 0.1–1.5 μM), treated once a day up to 72 h, suppress cell migration (). The discrepancy between the latter report and the result presented here may be attributed to the difference in the experimental conditions employed. In short, our data strongly suggest that CBDA is an effective inhibitor of breast cancer cell migration in vitro.

Effect of CBDA on the horizontal migration of MDA-MB-231 cells. (A) Wound-healing assays (horizontal migration) were performed to determine the effects of CBDA or CBD on MDA-MB-231 cell migration. (a) Representative images of the migrating cells were captured 12 h after vehicle (indicated as Cont.), 5 μM or 25 μM CBDA and 5 μM CBD treatments. (b) Migration data presented in panel (a) was assessed on the basis of percent wounded area filled in. Data are expressed as the percent of vehicle-treated group (indicated as Cont.), as mean ± S.D. (n = 8). (B) Migration data was assessed on the basis of percent wounded area filled in 12, 24, or 48 h after treatments with 25 μM CBDA. Data are expressed as the percent of vehicle-treated group (indicated as Cont.), as mean ± S.D. (n = 8) *Significantly different (p < 0.05) from the respective vehicle-treated controls.

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CBDA inhibits PKA activity. (A) (a) A representative photograph of phosphorylated bands and non-phosphorylated bands of PKA-specific substrate peptide in MDA-MB-231 cells incubated with vehicle (time 0, lane 3), vehicle (48 h, lane 4), and 25 μM CBDA (48 h, lane 5) was shown. For positive and negative controls, the reactions were performed in the presence (lane 1) or absence (lanes 2) of PKA catalytic subunit, respectively. An arrow in the image indicates wells that samples are applied. (b) The relative intensity of phosphorylated bands in MDA-MB-231 cells is shown. Data are expressed as fold change vs. non-CBDA-treated group (left panel; lanes 4 vs. 5), as mean ± S.D. (n = 3). *Significantly different (p < 0.05) from the vehicle-treated control. (B) MDA-MB-231 cells were treated with vehicle (indicated as Cont.) or 25 μM CBDA for 48, 72, or 96 h. After the treatments, cell viability was measured according to the methods described in Section 2. Data are expressed as the percent of vehicle-treated group, as mean ± S.D. (n = 6). *Significantly different (p < 0.05) from the vehicle-treated control. N.S., not significant. (C) A model of CBDA-mediated RhoA activation through PKA inhibition.

3.2. COX-2 inhibitory activity of CBDA is not critical in the attenuation of MDA-MB-231 cell migration

It has been reported that selective COX-2 inhibitors interfere with the migration of certain cancer cell types (; ). Previously, we reported that CBDA is an effective COX-2 inhibitor (, ). Therefore, we initially hypothesized that CBDA may exert its inhibitory effects on MDA-MB-231 cell migration via COX-2 inhibition. However, as demonstrated by the data shown in Fig. 4, vertical migration of MDA-MB-231 cells was not influenced by 25 μM DuP-697 (an established selective COX-2 inhibitor), nor SC-560 (an established selective COX-1 inhibitor), although the same concentration of CBDA completely inhibited cell migration. In addition, horizontal migration was not affected by DuP-697 (data not shown). These results indicate that COX-2 activity is not an essential factor for the migration of MDA-MB-231 cells, and that other pathway(s) are likely to be involved in the anti-migration effects of CBDA.

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COX-2 inhibitory activity of CBDA is not essential to attenuate MDA-MB-231 cell migration. Transwell migration assays were performed to determine the effects of COX-2 selective inhibitors (25 μM CBDA or 25 DuP-697) and COX-1selective inhibitor (25 μM SC-560) on MDA-MB-231 cell migration 12 h after their respective treatments. Data are expressed as the percent of vehicle-treated group (indicated as Cont.), as mean ± S.D. (n = 8). *Significantly different (p < 0.05) from the vehicle-treated control. N.D., not detectable due to complete inhibition of the migration.

3.3. CBDA-mediated stimulation of RhoA activity

In an effort to investigate CBDA’s mode of action, we focused on the Rho family of small GTPases, in particular the isoform RhoA, since active RhoA has been reported to inhibit breast cancer cell mobility in vitro (; ; ). We discovered that the active RhoA levels were remarkably stimulated by 25 μM CBDA, especially at 48 h post-treatments (Fig. 5A and B). In accordance with the RhoA activation time-course, horizontal migration of MDA-MB-231 cells was significantly inhibited by 25 μM CBDA at 48 h (see Fig. 3B). GTPγS, a hydrolysis-resistant GTP analog, was used as positive control for the experiment (Fig. 5A, left portion). Because RhoA can be regulated by post-transcriptional modification via phosphorylation on Ser188, leading to inactivation of the protein (; ; ), we also assessed the phosphorylation status of RhoA following 25 μM CBDA exposure. Although total RhoA levels were not changed, the phosphorylation levels at Ser188 on RhoA were reduced by CBDA in a time-dependent manner, as compared with vehicle-treated groups (Fig. 5A and B). Thus, there exists an apparently negative relationship between the levels of active RhoA and phosphorylated RhoA, suggesting that CBDA activates RhoA via an inhibition of its phosphorylation.

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CBDA stimulates RhoA activity. (A) RhoA affinity pull-down assays were used to determine the level of active RhoA according to the methods described in Section 2. Resulted pull-down samples were subjected to Western blot analyses using an anti-RhoA antibody. Active RhoA was increased by 25 μM CBDA (indicated as +) in a time-dependent manner. Western blot analyses were also performed using an anti-RhoA antibody specific to RhoA phosphorylated at Ser188 and a β-actin antibody. (B) Results are expressed as the ratio of active RhoA to total RhoA protein in each cell lysate. Data are expressed as the fold change vs. vehicle-treated group (indicated as −), as mean ± S.D. (n = 3). *Significantly different (p < 0.05) from the vehicle-treated control.

3.4. CBDA-mediated inhibition of PKA activity and implications for its anti-migration mechanism

It has been reported that the phosphorylation status of RhoA is modulated by cAMP-dependent PKA (; ; ). Therefore, we hypothesized that CBDA may interfere with PKA expressed in MDA-MB-231 cells, that in turn would result in activation of RhoA (see a model described in Fig. 6C). First, to confirm the efficacy of the PKA assay used in the present study, experiments were performed in the absence or presence of purified PKA catalytic subunit (Fig. 6A–a; lanes 1 and 2). A phosphorylated band that migrated toward the anode (+) was detected only in the presence of purified PKA (lane 2). As shown in Fig. 6A–a, PKA activity was substantially reduced by 25 μM CBDA at 48 h when compared with the vehicle-treated group (Fig. 5A–a; lanes 4 vs. 5), although PKA activity in the MDA-MB-231 cells was up-regulated following 48 h incubation (lane 4). Densitometric analysis of the phosphorylated band in the CBDA-treated sample indicated its decrease to ~50% of the levels detected in the vehicle-treated sample (Fig. 6A–b). Taken together with the results described in Fig. 5, the data suggest that CBDA-mediated anti-migration effects are primed by PKA inhibition, which in turn leads to decreased levels of phosphorylated RhoA. However, it is not yet clear mechanistically as to how CBDA inhibits PKA activity in MDA-MB-231 cells. Studies are ongoing to investigate these parameters with a possibility that some PKA specific inhibitors contain a β-resorcylic acid moiety in the structures as the moiety is present in CBDA (see Fig. 1A) (; ).

Recent experimental evidence, for example as expressed in prostate carcinoma cells, suggests the involvement of cannabinoid receptor CB receptors (CBs), and especially CB1, as specific agonist targets whose activation results in the abrogation of cell migration (). Further, it is known that CB1 activation results in inhibition of adenylate cyclase, coupled with inactivation of PKA. Thus, one anti-migration strategy for preventing cancer cell migration is an activation of CB1. However, efforts to develop therapies using CB1 agonists are hampered by associated adverse side effects, such as psychoactive effects, due to agonism at receptors within the central nervous system (). In these respects, it should be noted that non-psychoactive cannabinoid, CBD, does not exhibit affinity for either CB1 or CB2. Although at present we have not assessed whether CBDA directly interacts with these receptor subtypes, it was reported previously that CBDA exhibits no affinity for either CB1 or CB2 receptors (). Consistent with this finding, the results obtained here indicated that the anti-migration effects of CBDA were not affected by co-treatments with the established CB1/CB2 receptor antagonists, SR141717A and SR144528, respectively, or with pertussis toxin, a blocker of the coupling between CB receptors and G proteins (data not shown). Thus, it is suggested that CBDA does not require CBs to exert its anti-migration activity in MDA-MB-231 cells.

Based on the high consistency observed between time-course profiles of CBDA-mediated RhoA activation kinetics and inhibition of MDA-MB-231 cell migration, these data suggest that CBDA’s RhoA activation specifically mediates the inhibition of horizontal cell migration rather than that of vertical migration. Since a number of cancer cells, including the mesenchymal-like MDA-MB-231 cells, exhibit low RhoA activity, this condition likely facilitates a higher migration potential in the cells (; ; ). Cancer cell metastasis is triggered when migration of cancer cells is abnormally up-regulated (; ; ). Therefore, RhoA activators may represent useful pharmaceutical approaches for inhibiting cancer cell migration in cell types represented by the mesenchymal-like MDA-MB-231 cells. The data presented in this report support the view that CBDA is a biologically active component of the fiber-type cannabis plant with potential utility as an effective anti-migration agent.

HIGHLIGHTS

  • Cannabidiolic acid (CBDA) exists as a major component in the fiber-type cannabis.

  • CBDA is identified as an “active component”.

  • CBDA is an inhibitor of highly aggressive human breast cancer cell migration.

  • The mechanism responsible for the effects of CBDA involves activation of RhoA.

  • RhoA is an inhibitor of cancer cell mobility.

Acknowledgments

This study was supported in part by Grant-in-Aid for Young Scientists (B) [Research Nos. 20790149 and 22790176, (S.T.)] from the Ministry of Education, Culture, Sport, Science and Technology of Japan. This study was also supported by the donation from NEUES Corporation, Japan (H.A.). C.J.O. was supported by a USPHS award, ES016358.

Abbreviations

CBDA cannabidiolic acid
CBD cannabidiol
COX-2 cyclooxygenase-2
PKA cAMP-dependent protein kinase
Δ′-THC Δ′-tetrahydrocannabinol

Footnotes

 

Conflict of interest statement

The authors declare that there are no conflicts of interest in this study

 

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  • Takeda S, Matsuo K, Yaji K, Okajima-Miyazaki S, Harada M, Miyoshi H, Okamoto Y, Amamoto T, Shindo M, Omiecinski CJ, Aramaki H. (−)-Xanthatin selectively induces GADD45γ and stimulates caspase-independent cell death in human breast cancer MDA-MB-231 cells. Chemical Research in Toxicology. 2011b;24:855–865. [PMC free article] [PubMed] []
  • Vaccani A, Massi P, Colombo A, Rubino T, Parolaro D. Cannabidiol inhibits human glioma cell migration through a cannabinoid receptor-independent mechanism. British Journal of Pharmacology. 2005;144:1032–1036. [PMC free article] [PubMed] []
  • Vega FM, Fruhwirth G, Ng T, Ridley AJ. RhoA and RhoC have distinct roles in migration and invasion by acting through different targets. Journal of Cell Biology. 2011;193:655–665. [PMC free article] [PubMed] []
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  • Watanabe K, Motoya E, Matsuzawa N, Funahashi T, Kimura T, Matsunaga T, Arizono K, Yamamoto I. Marijuana extracts possess the effects like the endocrine disrupting chemicals. Toxicology. 2005;206:471–478. [PubMed] []
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  • Yamauchi T, Shoyama Y, Aramaki H, Azuma T, Nishioka I. Tetrahydro-cannabinolic acid a genuine substance of tetrahydrocannabinol. Chemical and Pharmaceutical Bulletin (Tokyo) 1967;15:1075–1076. [PubMed] []
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HIGH CBDA

CBDA to be “1,000 times more potent than CBD in reducing acute nausea” (6) and this raw compound also demonstrates remarkable COX-2 inhibition that could prove valuable in reducing inflammatory pain without the risk of gastrointestinal injury, as is seen with NSAID applications (7).

Oleksandrum/Adobe Stock
Dr. Mechoulam Does It Again
In an exciting mainstream media report, NBC news highlighted a groundbreaking new development from the lab of Dr. Raphael Mechoulam, the godfather of cannabis chemistry: cannabinoid acids.

September 27, 2019
Andrea Sparr-Jaswa
Breaking News International Medical Cannabis News
In an exciting mainstream media report yesterday, NBC News highlighted a groundbreaking new development from the lab of Dr. Raphael Mechoulam, the godfather of cannabis chemistry: cannabinoid acids.

Dr. Mechoulam, an organic chemist and professor of medicinal chemistry based at the Hebrew University in Jerusalem, led the first team of researchers to discover and synthesize the two primary medicinal compounds in cannabis, CBD and THC, back in 1963 and 1964, respectively (1). He began the first clinical trials on CBD and epilepsy in 1980, but it would be nearly 40 years before FDA approval, in the form of GW Pharmaceuticals’ Epidiolox, would grant access to patients seeking better treatment alternatives in 2018 (2).

On Monday, while presenting at this year’s CannMed Conference in Pasadena, Calif., Mechoulam and his team announced that they had “developed a process for creating synthetic, stable acids” ready to be licensed for drug development and subsequent research.

Until now, acidic cannabinoids, including THCA and CBDA, have been notoriously difficult to source and sustain. These raw-plant compounds are the primary constituents of the cannabis plant—with “activated” molecules only popping up in small numbers—and are readily converted into their neutral forms, THC and CBD, with the addition of heat and time, a process known as “decarboxylation,” making stable formulations easier said than done (3).

Cannabinoid acids have shown great therapeutic promise without risk of intoxication, in the case of THC, and with greater water solubility and bioavailability than their activated progeny (4, 5).

For example, as the NBC article pointed out, researchers at the University of Guelph have shown CBDA to be “1,000 times more potent than CBD in reducing acute nausea” (6) and this raw compound also demonstrates remarkable COX-2 inhibition that could prove valuable in reducing inflammatory pain without the risk of gastrointestinal injury, as is seen with NSAID applications (7).

One of the most beneficial aspects of THCA is its ability to act therapeutically without the intoxicating effects associated with THC, leaving more wiggle room in its therapeutic window. Additionally, THCA has demonstrated potent neuroprotective benefits in an in vitro model of Parkinson’s disease and exciting antineoplastic (anti-cancer) results in several prostate and breast cancer models (5).

According to the NBC article, Mechoulam’s research on acidic cannabinoids is in partnership with “a startup called EPM, six universities in Israel, the U.K. and Canada, the world’s largest topical cream company, and a publicly traded laboratory company”—the U.S. is still out of the picture due to restrictive research access and the prohibitory scheduling of cannabis compounds. As EPM co-founder, Reshef Swisa, expressed to NBC, he sees the first applications for the acidic compounds likely going towards topical products targeting symptoms of psoriasis with an eye on Phase 1 FDA testing within six to twelve months.

While this news is encouraging, FDA approval doesn’t move quickly and there are still many years ahead of us before we see a marketable product from the pharmaceutical industry making its way into the hands of the people who need it now. A good reminder that all you need is access to the raw plant and a juicer to find relief today.

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THCA

THCA and Whats the Difference

THCA and THC: What’s the Difference?

Why we get high on THC and not THCA, how cannabinoids convert, and raw cannabis as a superfood

Surprise! You’re just not going to get high by eating that freshly picked weed. At all. When cannabis is harvested and raw, no matter how much potential resides within, there is practically none of marijuana’s most famous and intoxicating cannabinoid, delta-9-tetrahydrocannabinol (THC). There is, however, a wealth of tetrahydrocannabinolic acid (THCA), an inactive compound found within the trichomes of living cannabis plants. 

So, if someone ever asks you “what does THC stand for?” don’t confuse the two similar terms. As you’ll soon discover, they are vastly different in both chemical structure and how they interact with the human body.

THCA is a cannabinoid that until recently has been closely compared to THC. Though THCA doesn’t get one high and THC certainly does, there is a relation: THCA is the precursor to psychoactive THC effects.

So why does THC get us elevated and THCA doesn’t? The reason is due to the three-dimensional shape of the THCA molecule. It is a larger molecule that doesn’t fit into our cannabinoid receptors, specifically the CB1 receptors. A cannabinoid must fit into a body’s CB1 receptor in order to have an intoxicating effect at all.

The cannabis plant produces hundreds of cannabinoids, the chemical compounds responsible for the therapeutic and psychoactive effects of cannabis. Only a few cannabinoids contribute to the euphoric high that is unique to the cannabis plant, though. The most celebrated, researched, and sought after is THC.

It’s commonly assumed that during the marijuana plant’s growth process that it is ramping up THC levels until ripe for the picking, but the primary cannabinoid being produced is actually THCA. How does THCA become THC?

The simplified answer is through heat and light — or the process of decarboxylation. Heat removes a carboxylic acid group of atoms from THCA, converting it into a molecule and altering the THC chemical structure, thus becoming the perfect shape to fit into our endocannabinoid system (ECS) CB1 receptors that run throughout the central nervous system, producing the elevated experience.

The non-intoxicating effects of THCA are a big part of the reason that fresh, raw, unheated cannabis is a superfood. You may have heard of juicing cannabis or adding raw cannabis to smoothies for health enhancement. There’s good reason.

Like other superfoods, including avocados, kale, Greek yogurt, green tea, and garlic, raw cannabis has potential to ease arthritis, chronic pain, fibromyalgia, and other ailments. 

THCA is believed to offer an assortment of medicinal benefits, and is commonly used as a nutritional supplement and dietary enhancement for its:

  • Anti-inflammatory properties  – 2011 study published in the Biological and Pharmaceutical Bulletin suggested that, along with other cannabinoids, THCA demonstrated anti-inflammatory properties. 
  • Anti-proliferative properties  A 2013 study that analyzed cell cultures and animal models concluded that THCA could prevent the spread of prostate cancer cells.
  • Neuroprotective properties – In a 2012 preclinical study published in Phytomedicine, researchers found that THCA showed the ability to help protect against neurodegenerative diseases.  
  • Antiemetic properties (increasing appetite and decreasing nausea) – A  2013 study conducted by researchers at the University of Guelph in Ontario found that both THCA and CBDA were effective in reducing nausea and vomiting in rat models, even moreso than THC and CBD, respectively. 

Most cannabinoids, including cannabidiol (CBD), cannabigerol (CBG), and tetrahydrocannabivarin (THCV), are in the acidic form (CBDA, CBGA, and THCVA) when cannabis is harvested. The unactivated forms of THC and CBD, along with other cannabinoids, have benefits themselves that we are still learning about.

The human body is not capable of converting THCA into THC.

It’s only after these unactivated cannabinoid acids go through the decarboxylation process, though, that they become the cannabinoids we’re most familiar with and that most interact with our ECS.

The acidic precursors are considered “thermally unstable,” which is another way to emphasize that they will alter when exposed to heat. Because of this instability, the molecules lend themselves to several different methods of decarboxylation.

THCA vs. THC: Decarboxylation Process 

Here are the most common ways that weed is decarboxylated:

Sunlight conversion: THCA converts to THC in varying degrees through exposure to heat or light. If a cannabis plant sits in the warm sun for an extended period of time, its THCA molecules will slowly convert to THC.

Room temperature conversion: THCA also converts to THC when stored at room temperature for a long enough time. In olive oil, 22% of THCA will convert over the course of 10 days at 77 degrees Fahrenheit, or 25 degrees Celsius. Under the same conditions, 67% will convert in an ethanol extraction. And over time, cannabis stored at room temperature and with little light exposure, will convert 20% of its THCA into THC.

Smoking: When a flame is used to smoke dried, cured bud, a high degree of heat is applied in a short amount of time, resulting in the rapid conversion of THCA to THC. However, not all THCA will convert and, though smoking is the most common way to enjoy THC’s effects, it’s not the most efficient.

Vaporizing: This is perhaps the most efficient way of decarboxylating ground nugs. When heated at a low temperature, the cannabinoids are converted and released. Continuing to increase the heat with each pull or sesh will make sure that the prime amount of THCA is converted into THC and binds to CB1 receptors.

Vape pens: Even more efficient than vaporizing flowers is the use of already decarboxylated cannabis distillate found in preloaded vape pens. Since the THCA is already mostly converted to THC and the following vaporization takes care of even more, this is a good, efficient method of taking in intoxicating cannabis.  Be sure you’re using a reliable brand of vape pen, for safety’s sake, and do your best to purchase products that are recyclable.

Cannabis concentrates:  By isolating the THCA content from a cannabis plant, THCA crystalline can be extracted and consumed in dabs. Similar to vaporization, decarboxylation transpires rapidly when using the dabbing method, breaking down the THCA into active THC. In its pure form, THCA crystalline has little flavor or aroma, as most cannabis extractions aim to strip away the terpenes and flavonoids to isolate the cannabinoids. But many producers reintroduce cannabis-derived terpene blends back into the concentrate. Not only does the addition of terpenes improve the flavor, but these distinctively aromatic plant molecules also work together with cannabinoids to produce entourage effects that enhance the therapeutic potential of cannabis.   

Conventional oven: When making edibles, you’ll want to activate, or decarboxylate, the weed before adding it to the butter, oil, or other medium. When weed gets ground up, spread evenly across a baking sheet that’s lined with parchment paper, and is baked at 230 degrees Fahrenheit, or 110 degrees Celsius, for 30-90 minutes (depending on the bud’s moisture content), it slowly converts most THCA into THC.

Whether cannabis is smoked, eaten, vaped, or juiced raw, understanding the plant’s properties and how and why they interact with our bodies the way they do is crucial in achieving the desired effects and avoiding adverse side effects. Cannabis molecules each have their own benefits and as raw cannabis is further studied, we can rest easy knowing that it’s safe to integrate it into a healthful diet.