Abstract 4656: Targeting Sp1 and survivin for potential ovarian cancer therapy: Comparative assessment of anti-cancer NSAID tolfenamic acid and cisplatin

Abstract

Introduction: Expression of specificity protein 1 (Sp1) transcription factor is negatively associated with survival in some cancer patients. Survivin, a member of Inhibitor of Apoptosis Protein family, causes resistance to chemo- and radiation therapy. Tolfenamic acid (TA), a NSAID, targets Sp1 and inhibits survivin expression in some cancer models. Our aims were to examine the expression of Sp1 and survivin in ovarian cancer (OC) specimens and compare the in vitro anticancer response of cisplatin and TA alone and in combination. Methods: Tissues from clinical specimens (16 advanced stage tumors, 3 normal) were used to isolate RNA and prepare protein extracts. Sp1 and survivin expression was measured using qPCR and Western blots. OC cell lines (ES-2, SKOV3-AF2) were used for in vitro assays. Anti-proliferative response of cisplatin and TA was measured at 24, 48 and 72 h, post-treatment. Cells were treated with DMSO (vehicle), TA (25/50/100 µM) or cisplatin (5/10/20/50 nM), and cell viability was assayed using CellTiter Glo kit. Subsequently, cells were treated with optimized doses of TA (50 µM) or cisplatin (20 nM) for 48 h. Caspase-3/7 activity was measured using Caspase-3/7 Glo kit and Sp1, survivin, and c-PARP expression was determined by Western blots. Cell viability, Sp1 expression, and caspase-3/7 activity were assessed following individual and combination treatment of TA and cisplatin. Results: qPCR revealed that all tumor samples have increased survivin expression (>5 fold) and the majority showed increased Sp1 expression (2-3 fold). Consistent with the qPCR data, Western blot confirmed that all (survivin) or majority (Sp1) of specimens have high expression levels. TA and cisplatin showed a dose- and time-dependent inhibition of cell viability. TA caused 75% (ES-2) and 25% (SKOV3-AF2) growth inhibition while cisplatin caused ∼20% inhibition in both cell lines. Combination TA and cisplatin treatment resulted in maximum inhibition (ES2: >90%; SKOV3-AF2: 75%), showing a synergistic effect. These data confirmed that both agents up-regulated c-PARP and caspase-3/7 indicating activation of apoptotic pathways. However, only TA caused a significant decrease in Sp1, survivin, and c-Met expression. Increased inhibition of cell growth following combination treatment is consistent with increased caspase-3/7 activity and decreased Sp1 expression. Conclusions: Sp1 and survivin expression in OC specimens confirmed that targeting these molecules/pathways would be therapeutically relevant. TA enhanced the response of cisplatin when used in combination. Sp1 regulates survivin expression and targeting survivin with TA might render the OC cells radiation-sensitive. TA is less toxic than cisplatin but causes greater anticancer activity over cisplatin. Further studies to evaluate the mechanism(s) using proteomic and molecular profiling approach are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4656. doi:1538-7445.AM2012-4656