Abstract 399: Molecular mechanism of ormeloxifene mediated chemo-sensitivity in hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is a lethal cancer with a dismal 5-year survival rate as the standard therapy available Sorafenib (SRB), is only effective in extending survival for a subset of patients. Resistance to apoptosis is a defining characteristic of cancer cells and a crucial factor in cancer recurrence and relapse. Although, anticancer potential of the FDA-approved compound Ormeloxifene (ORM), which has well-defined pharmacokinetic and pharmacodynamic properties, has been explored in a variety of cancers, its efficacy in HCC has remained unknown. Therefore, the purpose of this study was to investigate the role and molecular mechanisms underlying ORM in SRB-induced apoptosis in HCC. Methods: In this study, we investigated the efficacy of ORM in hepatocellular carcinoma cell (HepG2, Hep3B, C3a, and SKHep-1) lines alone, and in combination with SRB. Cell proliferation was determined by MTT and xCELLigence assay. Effect of ORM on clonogenic potential of HCC cells was examined by colony formation assay. Effect of ORM on apoptosis induction was performed by Annexin V assays. The cell migration assay was performed using Boyden chamber and cell invasion assay was done by Matrigel invasion chambers. Following treatment in hepatocellular carcinoma cells, Western blotting, quantitative polymerase chain reaction (qPCR), and immunofluorescence studies were carried out in order to investigate the effects of ORM, SRB alone, and/or combination on pro-apoptotic, anti-apoptotic, EMT, and associated signaling effector proteins. Results: By employing MTT and xCELLigence assays, our study showed that ORM induces dose- and time-dependent inhibition of cell proliferation in hepatocellular cancer (HepG2, Hep3B, C3a, and SKHep-1) cells at various concentrations (0, 5, 10, and 15 µM). The combination of ORM and SRB together had a significant synergistic inhibitory effect on HCC cells. Additionally, compared to ORM and SRB treatment alone, Annexin V staining showed that combined treatment of ORM and SRB significantly enhance the apoptosis induction in hepatocellular carcinoma cells. Mechanistically, the expression of anti-apoptotic markers like Mcl-1, Bcl-2, and Bcl-xl is significantly reduced when ORM and SRB are combined as evidenced by Western blotting. Similarly, the inhibition of colony formation, invasion, and migration of hepatocellular carcinoma cells following co-treatment was significantly higher as compared to either treatment alone. In addition, the expression of EMT markers like N-cadherin, snail, vimentin, and MMPs in HCC cells is down-regulated following combined treatment compared to ORM and SRB alone. Conclusion: Taken together, ORM displayed an effective chemo-therapeutic and chemo-sensitizing agents in treatment of hepatocellular carcinoma. Citation Format: Mohammed Sikander, Shabnam Malik, Anyssa Rodriguez, Daniel Zubieta, Sheema Khan, Fathi T. Halaweish, Murali M. Yallapu, Subhash C. Chauhan, Meena Jaggi. Molecular mechanism of ormeloxifene mediated chemo-sensitivity in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 399.