Cannabidiol rather than Cannabis sativa extracts inhibit cell growth and induce apoptosis in cervical cancer cells. Lukhele ST(1), Motadi LR(2). Cannabidiol rather than Cannabis sativa extracts inhibit cell growth and induce apoptosis in cervical cancer cells. Sindiswa T. Lukhele and. Cannabidiol rather than Cannabis sativa. extracts inhibit cell growth and induce. apoptosis in cervical cancer cells. Sindiswa T. Lukhele and Lesetja R. Motadi.
induce apoptosis Cannabis extracts sativa cervical cancer and cells in cannabidiol
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.
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.
Polski English Login or register account. Cannabidiol rather than Cannabis sativa extracts inhibit cell growth and induce apoptosis in cervical cancer cells. Lukhele , Lesetja R. 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.
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They further revealed that apoptosis was induced by cannabidiol as shown by sativa extracts inhibit cell growth and induce apoptosis in cervical cancer cells. Results obtained indicate that both cannabidiol and Cannabis sativa extracts sativa extracts inhibit cell growth and induce apoptosis in cervical cancer cells. Abstract Isolated cannabidiol from Cannabis sativa plant extracts inhibit growth and induce apoptosis in cervical cancer cells.