The Effect of Cannabidiol on the Biology and Epigenetics of Prostate Cancer

Prostate cancer, excluding non-melanoma skin cancers, is the 2nd most prominent cancer in men worldwide and 5th leading cause of cancer related deaths. This equated to over 390,000 deaths in 2022 and over 600 deaths per annum in Ireland. The 5-year survival rate of localised disease is almost 100%. In saying this, the hunt for new therapies to improve clinical outcomes persists. Cannabidiol (CBD) is a secondary metabolite of the cannabis sativa plant. It is one of over 120 cannabinoids, which show pharmacological promise. Cannabinoids, including CBD, have proven to reduce cancer cell proliferation, invasion and migration, and induce cell death. These are key cancer hallmarks, making cannabinoids a promising anti-cancer therapy. Moreover, few studies suggest that CBD can influence gene expression and the epigenetic landscape. However, studies investigating the effect of CBD on the cell biology, genetics, and epigenetics of cancer cells are lacking. As prostate cancer is an epigenetic-based disease, our hypothesis was that CBD has chemotherapeutic effects in prostate cancer and can modulate the prostate cancer transcriptome and epigenome. To test this, we investigated CBD’s chemotherapeutic effect in prostate cancer cell lines, to assess its effect on the prostate cancer transcriptome and epigenome. To accomplish this, we first investigated the effect of CBD on the viability and proliferation of prostate cancer cell lines (DU145, PC3, and LNCaP) and non-cancerous controls (PWR1E and HFFF2). CBD reduced the viability of all cell lines in a dose-dependent manner, with the cancerous cell lines being more sensitive. Sensitivity was influenced by the presence of foetal bovine serum. CBD reduced the proliferation of PC3 cells and not PWR1E or HFFF2. We investigated the effect of CBD on the efficacy of targeted therapies. CBD had a synergistic or additive effect on Talazoparib (PARP inhibitor) and GSK126 (EZH2 inhibitor), depending on concentration. The highest synergistic effect with GSK126 was seen in PC3, whilst this was in DU145 for Talazoparib. We investigated the effect of CBD on the prostate cancer transcriptome by measuring its effect on gene expression. CBD altered the expression (adj p ≤ 0.5, fold change +/- 2) of 353 genes in DU145, 3,337 genes in PC3, and 5,468 genes in LNCaP. Gene ontology analysis confirmed that many of the altered genes are involved in the cell cycle, and that CBD downregulated cell cycle genes, namely CDK1 and CDK2. We then investigated the effect of CBD on the prostate cancer. CBD increased total 5-methylcytosine levels in LNCaP and PWR1E cells, but not DU145 or PC3. CBD also altered DNA methylation patterns in the hormone-dependent LNCaP cells, but not the hormone-independent PC3 cells (adj p ≤ 0.5,  value +/- 0.2). CBD altered the gene expression of the DNA methyltransferase DNMT1 (greatest effect in PC3 and LNCaP cells), and both the gene and protein expression of EZH2 in all prostate cancer cell lines. However, CBD did not alter the levels of H3K27me3 in any of the three cell lines, indicating that CBD does not affect the histone methyltransferase activity of EZH2. Overall, this study provides novel insights into the effect of CBD on prostate cancer gene expression and epigenetic mechanisms. CBD reduced prostate cancer cell viability and proliferation, with the therapeutic potential to synergise with targeted therapies. Our data supports the concept that CBD may be exhibiting this effect through modulating, and potentially halting, cell cycle progression. Furthermore, in androgen-dependent prostate cancer cells, CBD modulates total 5-methylcytosine levels as well as specific DNA methylation patterns. CBD can also affect EZH2 levels in both androgen-dependent and independent cell lines, but does not alter H3K27me3 levels, indicating that CBD may influence EZH2s non-canonical functions instead. Thus, this study provides key novel insights on CBD as a chemotherapeutic agent.