A Novel Synthetic Agonist of P2Y1: Purinergic Receptor Acts as Anticancer Agent for Prostate Cancer Treatment

Prostate cancer (PCa) is one of the most common tumors and the fifth leading cause of death in men worldwide. The high incidence of PCa all over the world has thrown up the challenge of developing new drugs and/or drug combinations that target various signaling pathways, DNA repair mechanisms, and specific epigenetic mechanisms. Despite the wealth of research focused on investigating potential compounds to tackle PCa, its treatment still demands novel therapeutic approaches to overcome castration resistance and metastasis.

One of the most promising approaches to overcoming castration resistance and metastasis is the use of G protein-coupled receptors (GPCRs). These receptors are the largest family of cell surface receptors, and they respond to extracellular nucleotides. They also modulate various physiological functions during cancer progression and treatment. Among this eight-member receptor family, it has been observed that the activation of the purinergic receptor 1 (P2Y1R) induces apoptosis and cell death, not only in negative-androgen PCa cells but also in other cancer cells. This indicates that P2Y1 is a potential target for the treatment of PCa. Furthermore, a combination of MRS2365 and P2Y1R both inhibited cell proliferation and increased the apoptotic response through increased caspase 3 activity and higher lactate dehydrogenase levels in the PC3 cells. However, no detailed investigation of the molecular mechanisms occurring in P2Y1R and its agonists in negative-androgen PCa has yet been carried out.

Thus, this thesis focuses on investigating the molecular mechanisms that occur when potential ligands target P2Y1R, particularly those mechanisms that inhibit the growth and proliferation of PCa cells. Docking analysis of about 900 ligands as substituents of 1-indolinoalkyl 2-phenols with P2Y1R reveals 1-(2-Hydroxy-5- nitrophenyl) (4-hydroxyphenyl) methyl) indoline-4-carbonitrile) (HIC) and Methyl 4-((4-cuamoimdolin-1-yl) (2,5-dihydroxyphenyl) methyl) benzoate (MB) to be the two top ligands with the highest docking score. Both of these compounds increase the intracellular calcium level in two of the PCa cells, PC3 and DU145. They have thus been identified as potent agonists of P2Y1R. Although both ligands were able to suppress cell proliferation in a dose- and time-dependent manner, HIC effectively and selectively inhibits cell proliferation at lower concentrations and is thus selected for further analysis.

A molecular dynamics simulation specifically confirms the stable binding of HIC- P2Y1R, and this is validated through siRNA analysis. In addition, our results reveal that HIC could reduce and inhibit the cancer’s adherence properties, its ability to form colonies and cancer metastasis, and it could also interrupt the cell progression of both PC3 and DU145 cells at G1/S phase arrest. Gene expression analysis also indicates that HIC is able to induce DNA damage, the activation of p53 and p21signaling-mediated apoptosis, and cell cycle arrest in PCa cells. It achieves these effects by modulating the expression of significant genes like PARPs, BAX, CDKs, and MDMs. The data also indicates HIC to be a potent anti-cancer agent through its ability to inhibit mitochondrial membrane activity and the glutathione levels in PCa cells.

During the development of PCa therapy, combination chemotherapy has emerged as a mainstay in the treatment of cancer cells with a reduced risk of incurring drug resistance. The effect of combining HIC and abiraterone acetate (AA), a chemotherapeutic drug for PCa, was investigated for its anti-cancer effects on PCa cells. The synergistic effect of HIC and AA increases the cytotoxicity in PCa cells more effectively than each drug did on its own. Moreover, a combination of HIC and AA also induces apoptosis via p53 and p21 signaling, cell cycle arrest, caspase 3/7 activation, increased ROS levels, and the inhibition of migrated and invaded cancer cells. Thus, combination treatment with HIC and AA could contribute to the development of new strategies for PCa treatment.

HIC could be seen as multiple molecular targets through their inhibition of the MDMs and PARPs in PCa cells. The activation of P2Y1R might also benefit patients with lower AR expression levels and metastasis cancer. In addition, the synergy of HIC and AA could facilitate the targeted inhibition of PCa cells more effectively than monotherapy. All in all, the results shed light on the important role HIC-P2Y1 agonist play in inhibiting the development and progression of PCa through their ability to promote p53 and p21 signaling. However, further preclinical investigation on in-vivo models and patients’ samples should be carried out to evaluate the efficacy of adopting a combinatorial therapeutic approach with HIC in the future.