(T1530-06-33) PAR-1 Selective Antagonist, SCH-79797 Dihydrochloride, Inhibits the Proliferation and Migration of the Metastatic Human Prostate Cancer (PC-3) Cells

Purpose: Prostate cancer is the most diagnosed and second-leading cause of cancer-related death in men in North America. Poor prognosis, delayed diagnosis, and higher metastasis are the major contributing factors towards increased mortality rate in prostate cancer.¹ Various treatment therapies such as chemotherapy, surgery, and androgen deprivation therapy although they cannot cure the cancer, can help delay the spread, improve the quality of life, and control its symptoms. Thus, the lack of curative therapies and limitations such as treatment resistance, and inconsistent therapeutic approaches have resulted in an unmet need to explore new therapeutic strategies for treating cancer.² Thus, a lot of research is being done to explore new target molecules and treatment therapies that can be used to provide better curative measures against prostate cancer. Protease-activated receptors (PARs) are a subfamily of G-protein-coupled receptors that play an important role in regulating normal biological processes, but also significantly contribute to cancer cell growth and survival. Protease-activated receptor 1 (PAR1) is overexpressed in various types of cancer and plays a crucial role in maintaining the tumor microenvironment.^3,4 It also activates signaling pathways that promote the growth, proliferation, and metastasis of cancer cells. Due to their significant role in cancer regulation, these receptors are being studied as potential therapeutic targets for cancer treatment. Hence, the objective of our study is 1) to evaluate the cytotoxic potential of SCH-79797 dihydrochloride, a selective PAR1-antagonist, on human metastatic prostate cancer (PC-3) cells and 2) to determine the mechanism by which it induces cancer cell death in metastatic prostate cancer cells.

Methods: Western blot analysis was performed to determine the expression levels of PAR-1 receptors in PC-3 and HPrEC cells with Hep G2 used as the positive control, samples were collected after the cells achieved 80% confluency. Cells were treated for 24 hours with SCH-79797 dihydrochloride in increasing concentration (0.25-10µM) with docetaxel being the positive control, and BrdU cell proliferation assay was performed to determine the effect of PAR-1 antagonist in inhibiting cell proliferation. Furthermore, an LDH cytotoxicity assay was performed after treating the cells for 24 hours to determine the cytotoxic potential of the PAR-1 antagonist against cancer and epithelial cells. Finally, to assess whether inhibiting the PAR-1 receptor activity will cancer cell migration, a Transwell migration assay was conducted with staurosporine used as a positive control.

Results: The Western blot data revealed that the PC-3 cells expressed significantly higher levels of PAR-1 receptors ~ 2 fold higher than normal prostate epithelial cells, indicating that PAR-1 receptors are overexpressed in this cancer cell line (Fig 1a). The BrdU cell proliferation data indicates that SCH-79797 significantly inhibited cell proliferation by more than 50 % (p < 0.05) in a concentration-dependent manner, in both cancer and normal cells, suggesting that inhibiting the PAR-1 receptors inhibits cell proliferation and in turn prevents growth and division of the cells. LDH assay data suggested that SCH-79797 dihydrochloride significantly induced cytotoxicity in both cancer (~ 6 fold higher) and normal prostate epithelial cells (~ 2 fold higher) compared to control (p < 0.01), respectively. However, the PAR-1 antagonist exhibits greater cytotoxicity towards the metastatic prostate cancer cells approximately 3 times higher compared to the normal prostate epithelial cells (Fig 2a and 2b). Furthermore, Transwell migration assay was performed and the data suggested that SCH-79797 significantly decreased the migration of cancer by almost 90% in a concentration-dependent manner compared to the control (p < 0.0001), suggesting that inhibition of PAR-1 can inhibit the metastatic potential of the prostate cancer cells. Currently, experiments are being performed to determine the mechanism by which SCH-79797 dihydrochloride induces cancer cell death.

Conclusion: SCH-79797 dihydrochloride inhibits cell proliferation and shows significant cytotoxic potential against metastatic prostate cancer cells. Additionally, SCH-79797 dihydrochloride impedes cancer cell migration, highlighting its potential as an anticancer agent for prostate cancer.

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2. Saranyutanon S, Srivastava SK, Pai S, Singh S, Singh AP. Therapies Targeted to Androgen Receptor Signaling Axis in Prostate Cancer: Progress, Challenges, and Hope. Cancers (Basel). Dec 23 2019;12(1)doi:10.3390/cancers12010051
3. Liu X, Yu J, Song S, Yue X, Li Q. Protease-activated receptor-1 (PAR-1): a promising molecular target for cancer. Oncotarget. Dec 5 2017;8(63):107334-107345. doi:10.18632/oncotarget.21015
4. Sekhoacha M, Riet K, Motloung P, Gumenku L, Adegoke A, Mashele S. Prostate Cancer Review: Genetics, Diagnosis, Treatment Options, and Alternative Approaches. Molecules. 2022;27(17). doi:10.3390/molecules27175730