Every year, millions of people across the United States contract HPV, also known as the human papillomavirus. HPV is a type of viral infection that results in the growth of warts along the skin and mucous membranes of the body. In some cases of HPV, but not all, these warts can lead to a variety of different cancers. In particular, HPV types 16 and 18 are associated with the development of cervical cancer in women — according to the American Cancer Society, about 70% of all cases of cervical cancer are thought to be caused by the human papillomavirus. However, HPV can also cause cancers in the genitals of men and women, as well as cancers of the anus, mouth, and the back of the throat.
Previously, some research had suggested that there may be a possible link between cannabis use and the development of human papillomavirus (HPV) associated oropharyngeal cancer. This report published in 1990, for example, theorized that there could be a connection between cannabis smoking and tongue cancer. However, a study published in 2006 by the
More recent research has shown similar findings: A 2018 study found that heavy cannabis use was strongly associated with periodontitis, but the researchers did not find sufficient evidence to suggest that using marijuana led to a higher risk of oral HPV infection. Another study, published in Infectious Agents and Cancer, found that there was no correlation between cannabis use and risk of cervical HPV. This study also found no evidence to suggest that cannabis aided in the formation of new cervical squamous intraepithelial lesions (SIL) among HIV-positive women.
Cannabis use by itself does not cause HPV, nor does it lead to the formation of HPV-related cancers. However, since HPV typically affects the mucous membranes in the body, including the lips and mouth, it is possible to contract HPV from sharing drinks, food, and even cosmetics with someone who is infected with HPV. By the same logic, it is possible that someone could contract HPV by sharing a joint or a vaporizer with someone who has HPV. For this reason, it is advised to use caution when consuming cannabis in a group setting.
For a number of years, it was thought that using cannabis may increase one’s risk of contracting HPV. However, current research has shown little evidence to suggest a direct connection between HPV and marijuana use itself. In fact, as I will explain below, cannabis may actually be effective in bringing relief to those living with pain caused by HPV and other conditions.
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 [9, 17]. 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.
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 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.
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
A hundred microliters of 1×10 4 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:
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
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.