Effect of Cannabis sativa extracts and cannabidiol on cell cycle .. and progression of tumours through the resistance of apoptosis . Effect of Cannabis sativa extracts and cannabidiol on cell. cycle progression. We further assessed the effects of Cannabis sativa ex-. tracts and cannabidiol on. Cannabidiol (CBD) oil is essentially a concentrated solvent extract made from This effect is now under clinical investigation with the of apoptosis, inhibition of angiogenesis, and arresting the cell cycle . . Unfortunately, the Novel Food Catalogue of the EU states that “extracts of Cannabis sativa L. in.
cannabidiol Effect sativa cell of progression on cycle and Cannabis extracts
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 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. These results correlate with the findings obtained by [ 23 ], whereby they reported reduced cell proliferation in colorectal cancer cell lines following treatment with Cannabis sativa.
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 [ 19 , 20 ].
This in turn causes DNA strands to break in the S-phase of the cell cycle [ 20 ]. A study conducted by [ 19 ], 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.
We also observed a similar cytotoxic pattern, whereby camptothecin induced cell death in HeLa, SiHa, and ME cells, however, at a much higher concentration. Upon treatment of SiHa and HeLa cells with IC 50 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. 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 [ 8 , 13 ]. Induction of cell death by Cannabis sativa crude extracts decreases the activity of the enzyme following treatment of HeLa, SiHa, and ME 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 [ 13 ]. 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 [ 8 ]. Therefore, to evaluate the effect of treatment on the energy content of the cells, we conducted mitochondrial assay.
ATP acts as determinant of both cell death and cell proliferation [ 15 ]. Treatment of cells with cannabidiol either slightly or severely depleted the ATP levels. According to [ 16 ], 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. Treatment of SiHa cells with butanol and hexane extracts led to the accumulation of cells in the cell death phase sub-G 0 phase , without cell cycle arrest.
And thus, according to [ 3 ], signals DNA synthesis and cell cycle proliferation. Interesting to note was that, treatment of ME 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. 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. 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-G 0 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, IC 50 of Cannabis sativa and cannabidiol exhibited the type of induced cell death as 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 [ 5 , 17 , 28 ].
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 [ 30 ]. 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 [ 4 ].
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 [ 29 ]. Under normal conditions, p53 levels are maintained through constant degradation MDM2 and its monomers [ 29 ]. RBBP6 is one of the monomers that helps degrade p53, due the presence of Ring finger domain that promotes the interaction of both proteins [ 14 ].
In response to stress stimuli such as DNA damage, hypoxia, UV light, and radiation light, p53 becomes activated and causes MDM2 expression to decrease [ 10 ]. Bax and Bcl-2 form part of the proteins that regulate apoptosis via the mitochondria [ 21 ]. Following activation, p53 translocates into the cytosol and triggers the oligomerization of Bcl-2 with BAD, resulting in the inhibition of Bcl-2 activity [ 17 ].
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 [ 9 , 17 ].
An imbalance between Bax and Bcl-2 has been linked to the development and progression of tumours through the resistance of apoptosis [ 17 ]. 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. 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 p 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 [ 9 ].
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 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 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. 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. Both authors read and approved the final manuscript.
National Center for Biotechnology Information , U. Published online Sep 1. Lukhele and Lesetja R. Author information Article notes Copyright and License information Disclaimer. Received Jun 2; Accepted Aug This article has been cited by other articles in PMC. Associated Data Data Availability Statement The datasets supporting the conclusions of this article are included within the article and its additional files. 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.
Results Results obtained indicate that both cannabidiol and Cannabis sativa extracts were able to halt cell proliferation in all cell lines at varying concentrations. 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. 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 [ 11 , 28 ].
Plant collection and preparation of extracts Fresh leaves, stem and roots of Cannabis sativa were collected from Nhlazatshe 2, in Mpumalanga province. Data analysis Experiments were performed in duplicates. Open in a separate window. Effect of Cannabis sativa extracts and cannabidiol on cell growth of cervical cancer cells The IC 50 obtained during MTT assay was tested for their ability to alter cell viability in real time.
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 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.
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. 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.
Discussion Cervical cancer remains a burden for women of Sub-Saharan Africa. 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. 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.
Competing interests The authors declare that they have no competing interests. Consent for publication The authors give consent for the journal to be published. Cannabinoids in the treatment of cancer. Worldwide burden of cervical cancer in De novo -synthesized ceramide signals apoptosis in astrocytes via extracellular signal-regulated kinase.
The role of DNA fragmentation in apoptosis. Anew hope for breast cancer therapy? Pharmacologic ascorbate induces cytotoxicity in prostate cancer cells through ATP depletion and the induction of autophagy.
Anticancer Drugs Preclinical Rep. Choene M, Motadi L. Apoptosis and non-apoptosis deaths in cancer development and treatment response. Chemistry and biological activity of tetrahydrocannabinol and its derivatives. International Agency for Research on Cancer. Cisplatin-resistant prostate cancer model: The mitochondrial permeability transition in cell death: Because VEGF is a marker for angiogenesis, blocking the angiogenic process may represent a promising way of.
Cannabinoids are selectively cytotoxic to PCa. Effect of cannabis extract on cell viability: As detailed in Materials and Methods, the cells were treated with cannabis extract in different doses for 24 hours, and their viability was determined by MTS assay.
Evidence for cytopathic effects of cannabinoids on PCa cell lines. Effect of cannabis extract on mRNA expression of cannabinoid receptors CB1 and CB2 in human prostate epithelial cells and prostate cancer cells. Relative levels of expression normalized to the mRNA level of 18 Srna.
Cells were treated with different doses of cannabis extract for 24 hours and then harvested. Studies have shown that androgen regulates VEGF content in prostate cancer . As cannabis extract treatment resulted in a decrease in androgen receptor expression, the effects on VEGF were also determined. We next assessed whether the cell growth inhibitory effect of cannabis extract was associated with induction of apoptosis.
We quantified the extent of apoptosis by flowcytometric analysis of the cells labeled with Propidium iodide PI. Prior to assessing anti-inflammatory properties of the cannabis extract, the ability of LPS to induce secretion of key cytokine-related factors IL-6 and IL-8, was assessed in dermal fibroblast cultures.
After 24 h of LPS. Quantification of apoptosis by Flow Cytometry. Effect of cannabis extract on apoptosis in LNCaP cells. Cells were subsequently stained with a propidium iodide PI for 1 hour. Data from representative experiments repeated thrice with similar results. MTS assays performed on LNCaP cells at the end of the experiment indicated that none of the LPS or cannabis extract treatment combinations affected cell viability relative to vehicle controls data not shown.
Additional control tests showed that the cannabis extract did not interfere significantly in any of the ELISA steps. The ability to form prostaspheres in non-adherent culture is one of the characteristics of prostate CSCs Reynolds et al.
To confirm that cannabis extract treatment can inhibit prostate CSC properties, prostasphere formation of LNCaP cells was studied in the presence or absence of cannabis extract. Dermal fibroblasts Treatment with Cannabis extract 24 h. Accumulation of IL-6 a and IL-8 b , was determined from conditioned media by enzyme-linked immunosorbent assay.
Images of the prostaspheres were captured under the microscope. The number of prostaspheres formed was counted at the end of 14 days. Prostate cancer is the most frequently diagnosed cancer in western males . The progression of most of the cancers including prostate cancer may be a result of defects in cell cycle and apoptotic machinery.
Thus, the agents which can modulate apoptosis in cancer cells may be useful in the management and therapy of cancer. Hence, there is a need to develop novel targets and mechanism-based agents for the management of prostate cancer.
These data suggest that CB1 and CB2 receptors could be targets for treatment options for prostate cancer. Apoptosis is a physiological and discrete way of cell death and is regarded to be an ideal way of cell elimination.
The observation could be useful for the management of human prostate cancer. Androgens are essential for the growth, differentiation, and functioning of the prostate as well as in increasing prostate cancer development . The over expression of androgen receptor in prostate cancer may promote cell growth.
Hence, elimination or reducing the androgen receptor in prostate cancer should help in treating this neoplastic disease. We further studied the effect of cannabis extract on androgen receptor mRNA expression and its subsequent effect on PSA production.
PSA is an androgen receptor AR -regulated serine protease produced by prostate epithelial cells  , and is the most widely employed. Effect of cannabis extract on LNCaP prostaspheres formation: Spheroid formation assay was performed with cells treated with cannabis extract or vehicle. The cannabis extract treatment efficiently suppresses the spheroid formation ability of LNCaP cells. Therefore, agents which could reduce PSA levels may have important clinical implications for prostate cancer.
Earlier studies reported that PSA is primarily regulated by androgens . Increased PSA level is used extensively as a biomarker of prostate diseases including prostatitis, benign prostatic hypertrophy, and prostate cancer.
Our studies show a significant decrease in intracellular mRNA Figure 5 c as well as secreted levels of PSA Figure 5 d by cannabis extract treatment of cells, suggesting that cannabinoid receptor agonists may be exploited to prevent prostate cancer progression. VEGF is a ubiquitous cytokine that regulates embryonic vasculogenesis and angiogenesis. Normal prostate epithelium expresses low levels of VEGF, whereas increased levels are reported in prostate carcinoma .
Studies have shown that cannabinoid treatment markedly reduced the expression of VEGF in gliomas, the most potent proangiogenic factor and also of angiopoietin 2, which contributes to the angiogenic process by preventing vessel maturation .
Chronic inflammation has been linked to various steps in tumor formation, including cellular transformation, proliferation, invasion, angiogenesis, and metastasis . Among the inflammatory cytokines, interleukin 1 IL-1 , IL-6, IL-8, and IL have been reported as present in the prostate cancer cells  , indicating the significance of these inflammatory factors in prostate cancer progression.
Therefore, controlling inappropriate inflammation would appear to be one strategy that might help control cancer progression. Prior to assessing anti-inflammatory properties of cannabis extract, the ability of LPS to induce secretion of key cytokine-related factors IL-6 and IL-8 was assessed in dermal fibroblasts. Our results show that accumulation of the indicated cytokines in conditioned media of dermal fibroblasts was significantly suppressed by cannabis extract treatment confirming potential anti-inflammatory property of the high CBD cannabis extract preparation that may be considered to target chronic inflammation in prostate cancer.
Prostate cancer cells are in general highly resistant to common chemotherapeutic agents and the presence of CSCs is suggested to contribute to chemo resistance. The ability to form spheres in non-adherent, serum free conditions is a key property of stem cells Reynolds et al. In this study, prostate cancer cell line LNCaP was able to form spheroids in non-adherent culture, suggesting the presence of cancer stem-like cells within these cell lines.
Because prostaspheres are enriched with CSCs  , the inhibitory effect of cannabis extract on prostasphere formation supports that high CBD and low THC cannabis extract may be a potent agent in targeting or eliminating prostate cancer stem-like cells in vitro Figure 8. Recently, cannabinoids have received considerable attention due to their diverse pharmacologic activities such as cell growth inhibition, anti-inflammatory effects, and tumor regression.
Our results suggest that treatment of androgen-responsive human prostate carcinoma LNCaP cells resulted in a decrease in intracellular and secreted levels of PSA, with concomitant inhibition of androgen receptor, cell growth, and induction of apoptosis. The study explains usefulness of LPS-stimulated in vitro systems for the evaluation of the anti-inflammatory properties of plant extracts using this methodological approach  .
To conclude, the data presented in this paper provide further support for the concept that traditional medicines, such as cannabis, can be valuable additions to the modern therapeutic armamentarium, and non-habit-forming cannabinoid agonist s which lack psychotropic activity may be used for the management of prostate cancer.
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Effect of Cannabis sativa crude extracts on cell cycle progression Cannabidiol reduces cell viability of cervical cancer cells target genes that trigger cell cycle progression (Dueñas-Gonzălez et al., ). Deregulation of. THC and other cannabinoid receptor-ligands induce cancer cell death and inhibit (THC), the main active component of Cannabis sativa exerts its effects by .. and cannabis extracts containing controlled amounts of THC, CBD and other .. cell cycle progression in human breast cancer cells through Cdc2 regulation. Materials and Methods: Ethanol extracts of C. sativa were analyzed by Keywords: Cannabis; colorectal cancer; apoptosis; synergism; cell cycle arrest; ily member BAD.8 Cannabidiol (CBD) reduced cell . was used to check the cytotoxic effect of extracts. For G1 progression Repression of cyclin E2 in the F7+F3.