Sep 1, Cannabidiol rather than Cannabis sativa extracts inhibit cell growth and induce apoptosis in cervical cancer cells. In this study we compare the anti- proliferative effects of crude extract of Cannabis sativa and its main. Sep 1, Keywords: Apoptosis, Cervical cancer, Cannabidiol, Cannabis sativa Media was changed and the cells were treated with Cannabis sativa plant extracts . Effect of Cannabis sativa extracts and cannabidiol on cell growth of. Representative cell viability bar graphs of cervical cancer cell lines. Cells were treated with IC 50 of Cannabis sativa extracts and cannabidiol for a period of 24 effects of crude extract of Cannabis sativa and its main compound cannabidiol .
cells of Effect Cannabis of on extracts growth cancer cannabidiol sativa and cervical cell
And so the study looked at effectiveness of CBD to induce apoptosis between normal thymocytes thymus gland cells and thymoma thymus cancer cells. Both cells had a marked increase in apoptosis based on concentration and time.
They found that CBD induced apoptosis in both types of cells. CBD affected apoptosis with more potency in the normal thymocytes than in the thymoma cells, but apoptosis occurred earlier in the thymoma cancer cells.
This study investigated the effects of CBD on the infection of endothelial cells with Kaposi sarcoma-associated herpesvirus KSHV — including growth and apoptosis of the cells.
The efficiency in which KSHV infected the cells was not changed by CBD introduction, but it did reduce proliferation spreading of the cells and induced apoptosis normal cell death of the KSHV infected cells.
The study concluded that evidence supports further examination of CBD as a treatment for Kaposi sarcoma. With respect to leukemia, this study wanted to further analyze the effect of cannabidiol on lymphoblastic disease post-treatment recovery, after the initial cancer cell destruction.
Cannabidiol was shown to reduce cell viability incompletely, and slowed the cell cycle. However, the proportion of viable cells that were gated as small, increased in response to cannabidiol and normally sized cells decreased. This proportion of small cells persisted in the recovery period and did not return to basal levels. Cells grown in normal oxygen levels were seen as more resistant to CBD.
Cannabidiol did cause a reduction in cell size and this persisted post-treatment. However, resistance to cannabidiol under normal oxygen conditions for these cells would imply that CBD may not be useful in the clinic as an anti-leukemic agent. Exposure of CBD showed reduction in cell viability and induction of apoptosis via cannabinoid receptor CB2. Possible crosstalk between natural and external normal-cell-death apoptotic pathways was suggested. Yet another possible destructive element was that cannabidiol exposure led to an increase in reactive oxygen species ROS production.
ROS production in cells has been shown to cause cell degradation and cell death. Of note in this study was that CBD induced apoptosis could be blocked by introduction of ROS scavengers — these would be the antioxidants we think of as beneficial — suggesting that antioxidants may slow down or block the effects of CBD as a cancer cell killer. The conclusion of this study was that cannabidiol may be a novel and highly selective treatment for leukemia.
Almost all of the CBD studies looking into cannabidiol take the anti-tumor properties of CBD as a given, because so much research has been done with results reaching this conclusion. All in all, the conclusion of this study was that it along with their previous study supported evidence showing CBD has anti-tumor properties. Tests were conducted using lung cancer cell lines and the reaction just described above was confirmed by this study.
Results showed that cannabidiol caused a profound inhibition of human lung cancer cell lines. As in other studies, there was noted some reversal of CBD effectiveness by introducing cannabinoid receptor antagonists proteins that bind to the receptors and block action.
However, the result was that data provided evidence for an unknown mechanism underlying the anti-invasive action of cannabidiol on human lung cancer cells. Anti-tumor and anti-inflammatory effects of CBD and CBG cannabigerol in fighting prostate cancer, and how this is accomplished, were investigated in this study. THC combinations tested were unclear. It was also noted that CBD may effectively inhibit spheroid formation in cancer stem cells which may contribute to its anti-cancer effect against prostate cancer and its ability to sensitize the cancer to chemotherapy.
This paper showed comprehensive evidence that cannabidiol CBD is a potent inhibitor of prostate carcinoma viability in vitro lab dish conditions. They also showed that the extract was active in vivo biological conditions , either alone or when administered with drugs commonly used to treat prostate cancer and explored the possible mechanisms behind these anti-tumor effects. The aim of this study was to see if the anti-cancer activity was linked to induction of phosphatases, an enzyme that removes a phosphate group from a protein and acts to modulate the activities of the proteins in a cell.
They examined the effects of the cannabinoids on prostate and colon cancer and measured cancer cell proliferation, apoptosis, phosphatase activity, and cannabinoid receptor activity. Only CBD caused cannabinoid receptor induced apoptosis cancer cell death.
It was concluded that cannabinoid receptor agonists substance that initiates a response induce phosphatases and phosphatase-dependent apoptosis in cancer cell lines. A pure form of CBD was also analyzed. Proliferation of cancer cells vs healthy cells was evaluated by inducing polyps, tumors with carcinogenic agents in mice. Xenografts of colon cancer in mice were also examined. Basis for the study was that cannabidiol had been shown to be an antioxidant and an intestinal anti-inflammatory.
This study looks at the effect of CBD on colon carcinogenesis and possible preventative effects on colon cancer. Using animal subjects, they examined polyp and tumor formation. It was shown that in colorectal carcinoma cell lines, cannabidiol protected DNA from oxidative damage.
Additionally, CBD increased endocannabinoid cannabinoids produced internally levels and reduced cancer cell proliferation through multiple mechanisms. Previously, this team had identified CBDA cannabidiolic acid as an inhibitor of the migration of metastatic breast cancer cells. This study digs in deeper to examine possible mechanisms by which this occurs involving the COX-2 gene. Results showed that CBDA inhibits migration of highly invasive human breast cancer cells.
This was apparently done through a mechanism involving inhibition of a protein kinase. It was established that activation of certain signaling pathway led to inhibition of the mobility of various cancer cells, including aggressive tumor cells.
This study investigated the exact molecular mechanism through which CBD combats tumor cell growth and migration. They were able to show that cannabidiol induced death of breast cancer cells without involving activation of cannabinoid receptors. Further investigation showed that CBD produces a chain of reactions within cancer cells to ultimately activate apoptosis normal cell death in breast cancer cells.
An intricate interplay was shown between cell death apoptosis and the normal process for the destruction of the cells autophagy in CBD-treated breast cancer cells. A conclusion was that continued investigation into the potential use of CBD as an antineoplastic anti-cancer agent was warranted.
This team had previously shown that cannabidiol downregulates Id-1 gene expression in breast cancer cells which inhibited tumor invasion and metastasis. They now look at how this happens and the pathways used. Using various assays, they were able to demonstrate those pathways, which inhibited human breast cancer cell proliferation and invasion through modulation of the extracellular signal-regulated pathways. Both of those pathways led to down-regulation of Id-1 expression.
The study was also able to demonstrate that CBD up-regulates Id All of this showed that treatment with CBD significantly reduces primary tumor mass as well as the size and number of lung metastatic foci in two models of metastasis.
The basis for this study was; since THC exhibited anti-tumor activity, do other cannabinoids also possess such anti-tumor qualities? Results indicated that pure CBD was the most potent of the cannabinoids in inhibiting tumor cell growth. With regards to potency ranking, cannabigerol and cannabichromene followed in the rank behind cannabidiol. Both cannabidiol and the cannabidiol-rich extract inhibited the growth of human breast carcinoma xenograft tumors in mice.
Also, growth of tumors were inhibited in thyroid epithelial cells and lung metastases using rat and mice subjects with cancerous implants. Experiments indicated that the CBD effect was due to its ability to induce apoptosis through activation of the CB 2 and vanilloid receptors. This breast cancer focused study focused on metastatic breast cancer cells and whether CBD would down-regulate Id-1 shown as a key regulator of breast cancer metastasis.
It was demonstrated that cannabidiol CBD did indeed down-regulate the Id-1 gene expression and showed that CBD represents the first nontoxic exogenous agent that can significantly decrease Id-1 expression in metastatic breast cancer cells leading to the reduction of tumor aggressiveness.
As a basis for this study, growing evidence suggested CBD, a non-psychotropic compound, has anti-tumor capabilities, including effectiveness against glioblastomas. Therefore, this study looked at CBD alone and CBD in combination with chemotherapy agents and their effectiveness in fighting human and mice glioblastoma cell lines. Along with this angle of testing, they also looked at whether CBD had any potential toxic effects within the nervous system, using mice cells in culture.
Results demonstrated that CBD induced a reduction in proliferation and viability of cancer cells in proportion to dose. When both CBD and chemotherapy drugs were used, they showed synergistic anti-proliferating and cell-killing responses in various concentrations.
In a press release from GW Pharmaceuticals, they announced that clinical trials had provided some positive results when using a THC: CBD proprietary blend in combination with dose-intensive temozolomide a chemotherapy drug.
The trial showed that patients with documented recurrent glioblastomas treated with THC: CBD had an 83 percent one year survival rate compared with 53 percent for patients using the placebo. Average survival for the THC: CBD group was greater than days compared with days in the placebo group. Keep in mind, they were treating a very aggressive form a brain cancer with very low prognosis for survival.
These promising results are of particular interest as the pharmacology of the THC: CBD product appears to be distinct from existing oncology medications and may offer a unique and possibly synergistic option for future glioma treatment.
Research was conducted of all major medical study publishers up through the end of for the anti-tumoral effects of cannabinoids on gliomas. CBD was not singled out in this review.
Only 35 articles matched the criteria. All studies discovered were experimental except for one involving a clinical trial. In all of the experimental studies, cannabinoids were shown to have antitumoral activity and properties both in vitro and in vivo in lab dish and in animal subjects.
Cannabinoids are a family of naturally occurring compounds highly abundant in Cannabis sativa plant [ 1 , 6 , 14 , 24 ]. Screening of Cannabis sativa has led to isolation of at least 66 types of cannabinoid compounds [ 1 , 14 , 30 ]. 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 [ 22 ].
It has been found to be effective against a variety of disorders including neurodegerative disorders, autoimmune diseases, and cancer [ 24 , 25 ].
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. Fresh leaves, stem and roots of Cannabis sativa were collected from Nhlazatshe 2, in Mpumalanga province.
Extracts were filtered using Whatman filter paper and dried. 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. 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. A blank with media only was included to rule the possibility of media having a negative effect on the cells. Procedure was repeated for C. The plate was incubated overnight to allow the cells to attach. Cells were washed twice with PBS and once with 0. BX Olympus microscope Germany was used to visualize the cells.
The next day, cells were treated with 0. Protein content was measured by the BCA assay and equal amounts were electrophoresed in SDS polyacrylamide gel and then transferred onto nitrocellulose membranes. Experiments were performed in duplicates. Statistical analysis of the graphical data was expressed as the mean standard deviation. Camptothecin, as our positive control, significantly reduced cell viability in SiHa As shown in Fig.
Whereas ethanol exhibited between 7. Representative cell viability bar graphs of cervical cancer cell lines. The IC 50 obtained during MTT assay was tested for their ability to alter cell viability in real time. Cells were seeded in an E-plate and allowed to attach. 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.
Suggesting that cannabidiol is the most effective compound. BXfluorescence confocal microscopy was used to visualize the cells. Apoptosis assessment following treatment of cervical cancer cells with IC 50 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 c and f cells.
Flow cytometry revealed a significant increase in SiHa cells undergoing apoptosis during treatment with butanol from 2 to In HeLa cells, apoptosis was increased to A similar events was observed following treatment of ME cells with butanol extract were 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 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 IC 50 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. 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. Bar graphs representing changes in the ATP levels following treatment of cervical cancer cells with Cannabis sativa and cannabidiol. Untreated and camptothecin were included as controls for comparative purposes.
This was done in order to determine whether Cannabis sativa and cannabidiol deplete ATP levels in cervical cancer cells. In general, ATP depletion was cell type dependent. Whereas in ME there was no change between treatments and untreated.
Similar results were observed in cannabidiol treated cells. 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. There was no significant change in ME cells Figure. ME was fairly increased also to from RLU all increase were significant and in line with other increase in apoptosis as shown in Annexin V.
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 IC 50 concentrations of Cannabis sativa extracts and cannabidiol.
We further assessed the effects of Cannabis sativa extracts and cannabidiol on cell cycle progression using flow cytometry. In butanol, sub-G 0 phase was increased from 4. In ME there was insignificant increase in all cell cycle stages.
Each cell line responded differently to cannabidiol treatment. A similar trend was observed in HeLa cells but much lower sub-G 0 from 5. A similar event was observed during treatment of ME cells. Cannabidiol significantly increased sub-G0 in ME cells to From this data, we can conclude that cannabidiol induced cell death without cell cycle arrest.
A densitometry analysis SiHa protein was performed using ImageJ quantification software to measure the relative band intensity. 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. A densitometry analysis HeLa protein was performed using ImageJ quantification software to measure the relative band intensity.
A densitometry analysis ME protein was performed using ImageJ quantification software to measure the relative band intensity.
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. 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. 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 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 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 [ 12 ].
Currently, the recommended therapeutic regimens include chemotherapy, radiation therapy, and surgery. However, they present several limitations including side effects or ineffectiveness [ 2 ]. 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 [ 22 ].
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 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.
Cervical Cancer And Marijuana: What You Need To Know
Library of Cannabis Research Studies for Cervical Cancer by SATIVAisticated: A Besides palliative effects of cannabinoids used in cancer treatment, Cannabidiol Rather Than Cannabis Sativa Extracts Inhibit Cell Growth And Induce. Oct 6, Cannabis acted on the cancerous cells through apoptosis, a process of cell death . CBD (cannabidiol), taken from a Cannabis sativa extract, could hold causing only the cancerous cells to kill themselves, and inhibiting their growth. its main compound cannabidiol on different cervical cancer cell lines. BACKGROUND Cervical cancer remains a global health related issue In this study we compare the anti-proliferative effects of crude extract of Cannabis sativa and its extracts inhibit cell growth and induce apoptosis in cervical cancer cells.