Targeting tgfβ induced metastatic potential of prostate cancer cells using combinatorial approach

Abstract

Cancer occurs when the cells lose their capability of regulating the cell cycle and divide uncontrollably, paralyzing the affected organ. Prostate cancer is diagnosed as the most prevalent as well as the second most common cause of mortality due to cancer in males. Several treatments are available for prostate cancer, but all are associated with harmful side effects. Most cases are diagnosed at the late stages when cancer has metastasized, and cancer metastasis is also the predominant reason for death among cancer patients. Minimizing side effects, overcoming cancer drug resistance, and preventing cancer metastasis are of growing interest in current cancer therapeutics. Transforming growth factor-beta (TGFβ) plays a crucial role in the metastasis of prostate cancer and cancer progression by promoting epithelial to-mesenchymal transition (EMT). Phytochemicals are being deeply researched as they are protective to normal cells and have lesser side effects. Targeting epigenetic modifications that cause cancer is another class of upcoming therapeutics, as these changes are reversible. Global H3K27 trimethylation levels have been found to be decreased in invasive prostate adenocarcinomas. Combining a demethylase inhibitor and an anti-cancer phytochemical is a unique approach to target cancer to attain the aforementioned objectives. Combining two drugs might reduce individual drug toxicity and delay the onset of drug resistance. Hesperetin is well known for its antioxidant, anti-microbial, anti-fungal, and anti-cancer properties and good bioavailability, so we looked at its anti-cancer potential against TGFβ-induced EMT in prostate cancer. We used an H3K27 demethylase (JMJD3/KDM6B(Jumonji domain-containing protein-3/Lysine demethylase 6B)) inhibitor, GSK-J4, to study its effects on TGFβ-induced EMT in prostate cancer cells (PC3). We then gave a combined Hesperetin (a phytochemical) and GSK-J4 (inhibitor of H3K27 demethylase) treatment to the PC3 and the LNCaP cell lines. The outcomes were studied using techniques like western blot analysis, RT-qPCR (Real-Time Quantitative Polymerase Chain Reaction), wound healing assay, invasion assay, and flow cytometry. The Compusyn software was used to study drug synergism. It was found that hesperetin can significantly inhibit the cell proliferation of PC3 cells and arrest the cells in the S and G2M phases. The invasion and migration assay results decipher the inhibitory effect of hesperetin on the TGFβ-induced migratory and invasive potential of prostate cancer cells. The results confirmed that hesperetin also acts through the canonical signaling pathway, as we observed that there was a significant decrease in the phosphorylation of smad3 (target of canonical TGFβ signaling) in the PC3 cell line. In the LNCaP cell line, however, the non-canonical pathway involving the c-jun phosphorylation (target of non-canonical TGFβ signaling) was inhibited. Hesperetin can inhibit the TGFβ induced EMT by upregulating E-cadherin expression and downregulating N cadherin expression. Hesperetin could also modulate the TGFβ induced histone methylation marks. There was a dose-dependent increase in cytotoxicity and inhibition of TGFβ-induced migration and invasion of prostate cancer cells after GSK-J4 treatment. GSK-J4 did not affect the cell cycle of the PC3 cell line. It, however, inhibited the c-jun phosphorylation in the PC3 cell line and the phosphorylation of smad3 in the LNCaP cell line. GSK-J4 not only induced trimethylation of H3K27 but also induced trimethylation of H3K4 levels. Surprisingly there was a reduction in the H3K9me3 levels. We found that the combination of Hesperetin and GSK-J4 was synergistic as there was a drastic decrease in the individual doses of both compounds and inhibition of TGFβ induced EMT, invasion, and migration of PC3 and LNCaP cells. The combination treatment also inhibited both the canonical as well as non-canonical TGFβ signaling in both cell lines. GSK-J4 or Hesperetin alone and a combination of Hesperetin and GSK-J4 treatment effectively inhibit the important hallmarks of cancer, like cell proliferation, migration, and invasion, by altering the epigenetic landscape of cancer cells.