Abstract B51: The pan deacetylase inhibitor panobinostat shows cytotoxic activity as monotherapy and synergism with topoisomerase inhibitors in cervical cancer cells
Authors: Wasim Lubna , Chopra Madhu 
Published: May 2016

Abstract
Cervical carcinoma a tumor related to high risk human papillomavirus (HPV) is one of the most common malignancies for women of reproductive age worldwide. It is established now that genetic alterations together with epigenetic changes are associated with cancer progression. Epigenetic alterations have been identified both in the genome of host cell and HPV during cancer progression. The use of epigenetic drugs, methylation inhibitors and/or histone deacetylases inhibitors (HDACi) for cancer treatment, therefore utilizes the reversible nature of epigenetic aberrations, which would re-expresses the silenced tumor suppressor genes and consequently exert their anticancer effects. Studies showing the aberrant recruitment and/or over-expression of HDACs in many human cancers, and promising anticancer results through induction of cell cycle arrest, apoptosis and autophagy with HDACi have been reported. Combination of HDACi with other anticancer drugs that target genomic DNA have shown synergistic effects and enhanced apoptosis because of the increased accessibility of these agents to the chromatin. Panobinosat (LBH589), is a non-selective HDACi, showing anticancer activity against both hematological and solid malignancies. However the anticancer effect of panobinostat in cervical cancer cells has not been tested. Therefore, in the present study, we investigated the effects of panobinostat on cell growth and death in human cervical cells alone and in combination with topoisomerase inhibitors. HeLa and SiHa cells were selected for this study, they represents useful cellular models for cervical carcinoma, as both lines contain an integrated form of HPV 18 and HPV 16, respectively. MTT assay was used to evaluate the effect of panobinostat, cells were incubated with escalating concentration of panobinostat for 24, 48 and 72 hours. The results showed time and dose dependent decrease in cell proliferation of both HeLa and SiHa cells. The data also indicate that panobinostat is more potent in SiHa than in HeLa cells for inhibiting cell proliferation. The IC50 at 72 h in SiHa cells is approximately 50 nM and for HeLa cells is approximately 150 nM. Cell cycle analysis, revealed G1 arrest in HeLa cells but G2-M arrest in SiHa cells after 24 h of treatment. However increase in sub-G1 population was significant in both cell lines. These results demonstrate that panobinostat exerts cytotoxic effects on cervical cancer cells. Next we determined the combined anticancer effect between panobinostat and topoisomerase I and II inhibitors (topotecan and etoposide) using Chou and Talalay combination index (CI). Three different treatment schedules were explored, either simultaneous or sequential (with 24 h delay between the two agents). It was found that simultaneous treatment was synergistic for panobinostat and topotecan/etoposide combination in both cell lines. Flow cytometry analysis showed that topoisomerase inhibitors induced G2+M arrest, and the sub-G1 population was markedly increased in combination in comparison to single agents. Annexin V-FITC and PI double staining and DNA fragmentation showed enhanced cell death in combination treatment and demonstrated that apoptosis was the main mechanism for the observed synergistic interaction. Induction of apoptosis was found to be correlated to enhanced ROS levels in combination treatment. Furthermore, coadministration of antioxidant L-NAC substantially blocked the panobinostat and topotecan/etoposide mediated ROS levels. In conclusion, to our knowledge, this study is the first to show, the antiproliferative effect of panobinostat, and a synergistic cytotoxicity effect between panobinostat and topoisomerase inhibitors in a cervical cancer cell model.
Citation Format: Lubna Wasim, Madhu Chopra. The pan deacetylase inhibitor panobinostat shows cytotoxic activity as monotherapy and synergism with topoisomerase inhibitors in cervical cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B51.