Abstract
Abstract Background: Cervical cancer (CxCa) remains the fourth leading cause of cancer related death among women worldwide. Cervical cancer is mainly (∼ 99.7%) derived from high risk Human papillomavirus (HR HPV). HPV E6/E7 oncoproteins interfere with p53 and pRb (retinoblastoma) cell cycle regulatory proteins and hinder their efficacy of controlling cell growth. Advanced stage cervical cancer is difficult to treat and patients diagnosed with metastatic disease have a poor survival rate of only 16%. Therefore, there is an urgent need to develop newer treatment modalities. Ormeloxifene is a non-hormonal, anti-estrogen, oral contraceptive for human use that effectively suppresses the proliferation of decidual cells in the endometrium to inhibit egg implantation. Growing evidences also suggest that ormeloxifene has anti-cancerous properties in breast, head and neck, and chronic myeloid leukemia cancers. Hence, considering its strong anti-proliferative/cancerous effect, we hypothesized that ormeloxifene may also inhibit the growth of cervical cancer cells and may suppress the expression of E6 and E7 HPV oncoproteins. Methods: We performed in vitro anti-cancer studies with ormeloxifene (dose range: 5 µM to 25 µM) in the HPV positive cervical cancer cell lines (Caski and SiHa) by utilizing MTS and colony formation assays. To further determine the effects of ormeloxifene on the cellular and molecular level, immunoblotting, flow cytometry and real time PCR analyses were performed using the Caski cell line model. Results: Ormeloxifene effectively inhibited cervical cancer cell growth as determined by proliferation and colony formation assays in a dose dependent manner. Ormeloxifene decreased mitochondrial membrane potential and induced apoptosis as shown by flow cytometry analysis of cells stained with TMRE (tetramethylrhodamine, ethyl ester) and 7AAD/Annexin V, respectively. Apoptosis was also confirmed by immunoblotting for cleaved PARP (Poly ADP ribose polymerase), Caspase 3 and Caspase 9. Additionally, ormeloxifene treatment arrested cell cycle in G1-S transition and decreased the expression of p-Akt while total Akt remained the same. Specifically relevant to cervical cancer, ormeloxifene decreased the expression of HPV E6/E7 at mRNA and protein levels as determined by quantitative PCR analysis and immunoblotting, respectively. Interestingly, ormeloxifene restored the levels of p53, Rb and PTPN 13 (Protein tyrosine Phosphatase non receptor type 13) tumor suppressor proteins as shown by immunoblotting. Conclusion: Our findings suggest that ormeloxifene inhibits cervical cancer cell growth and decreases mitochondrial membrane potential which clearly demonstrates that apoptosis is induced via intrinsic pathway. Importantly, ormeloxifene downregulates the expression of HPV E6/E7 oncoproteins. Thus, ormeloxifene appears to be a novel treatment modality for cervical cancer. Citation Format: Neeraj Chauhan, Mohd S. Zaman, Murali M. Yallapu, Diane M. Maher, Mara C. Ebeling, Subhash C. Chauhan, Meena Jaggi. Ormeloxifene inhibits cervical cancer cell growth through intrinsic apoptotic pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2753. doi:10.1158/1538-7445.AM2014-2753
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