Abstract 1354: Relevance of Sp1 and survivin expression in epithelial ovarian cancer patients and their usefulness as potential therapeutic targets
Authors: Sankpal Umesh T. , Ingersoll Susan B. , Bhat Vadiraja B. , Ahmad Sarfraz , Holloway Robert W. , Basha Riyaz M. 
Published: May 2015

Objectives: Specificity protein 1 (Sp1) is a transcription factor that is over-expressed in several cancers and is inversely associated with survival. Survivin is known to cause resistance to chemo- and radiation therapy. Herein, we evaluated the correlation of Sp1 and survivin expression in epithelial ovarian cancer (EOC) patients and tested a pre-clinical strategy to target these candidates using tolfenamic acid (TA) for enhancing the efficacy of a chemotherapeutic agent, Cisplatin (Cis).

Methods: Expression of Sp1 and survivin in clinical specimens was determined by qPCR and Western blot analysis. Anti-proliferative response of EOC cell lines (ES2 and OVCAR-3) was assessed in the presence of Cis, TA or Cis+TA using CellTiter-Glo. Apoptosis was determined by flow cytometry using annexin-V staining, measuring the effector caspases activity using caspase3/7 Glo kit. Cell cycle analysis was carried out using flow cytometry. Matrigel coated ranswell chambers were used to assess cell migration/invasion. Proteomics nalysis was performed at Agilent Technologies, Inc.

Results: qPCR showed an increase in the expression of urvivin (∼5-fold) and Sp1 (∼2-fold) in clinical specimens, which was corroborated by Western blot analysis [Sp1 (&gt;2.6-fold) and survivin &gt;100-fold)]. TA and Cis showed a dose- and time-dependent inhibition of cell viability in EOC cell lines [50 µM TA caused 50% (ES2) and 40% (OVCAR-3) cell growth inhibition; Cis (5 µM) caused 60% (ES2) and 40% (OVCAR-3) inhibition at 48 h post-treatment]. Combination of TA (50 µM) and Cis (5 µM) resulted in a synergistic response causing higher growth inhibition (ES2: ∼80%, p&lt;0.001; OVCAR-3: 60%, p&lt;0.001) compared to single-agent. This is accompanied by cell-cycle arrest (G2/M phase), an increase in apoptosis, as determined by Caspase 3/7 activity, annexin-V staining, PARP cleavage, and inhibition of ES2 cell invasion and migration. Global proteomic profiling of ES2 cells indicated that the combination treatment up-regulated proteins associated with oxidative phosphorylation, apoptotic execution, and electron transport chain and down-regulated ribosomal proteins, translational actors, DNA damage and cell cycle proteins.

Conclusions: Elevated expression of Sp1 and survivin suggests a strong association of these two agents in EOC. Our pre-clinical results confirmed that targeting these candidates with small molecule (TA) potentially sensitizes EOC cells to chemotherapy.

Citation Format: Umesh T. Sankpal, Susan B. Ingersoll, Vadiraja B. Bhat, Sarfraz Ahmad, Robert W. Holloway, Riyaz M. Basha. Relevance of Sp1 and survivin expression in epithelial ovarian cancer patients and their usefulness as potential therapeutic targets. [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 1354. doi:10.1158/1538-7445.AM2014-1354