Abstract 2950: Silencing of STAT3 and Src gene by RNA interference enhances the cytotoxic effect of cisplatin in cervical cancer cell lines (2010)

Abstract Purpose: Signal transducer and activator of transcription-3 (STAT3) is well known to regulate oncogenesis, angiogenesis and immunosuppression in cancer cells. The Src family consists of nonreceptor tyrosine kinases and participates in cell cycle progression, adhesion, spreading, migration, and differentiation. Activation of STAT3 by constitutively activated Src pathway in cancer cells results in cancer progression. We investigated the cytotoxic effect of cisplatin in cervical cancer cell lines according to the presence of RNA interference-mediated STAT3 and Src gene silencing. Experimental procedures: We prepared two cervical cancer cell lines of C4I and HeLa. First of all, reverse transcription with an RT-PCR kit was performed in order to confirm the increased STAT3 expression in cervical cancer cells. We synthesized STAT3-specific siRNA, using six different sequences of oligonucleotides encoding STAT3 siRNA. After ligation and transformation into bacteria, the positive colonies were selected by antibiotic resistance. HeLa cells were transfected with the plasmids with luciferase reagent according to the established methods. As the same methods, we obtained the HeLa cells transfected with c-Src and v-Src-targeting siRNAs. We checked the reduction rate of each mRNAs by RT-PCR after 48 hours. HeLa and C4I cells transfected with indicated siRNAs, which showed the highest reduction rate at silencing STAT3, c-Src and v-Src, were cultured and mock transfection without any DNA was always included as experimental controls. At 36 hours after starting culture, cisplatin was administered as indicated concentrations (0, 100, 250, 500, 1000 ng/mL). At 96 hours after starting culture, the viability of HeLa and C4I cells was determined by the MTT assay. Results: We confirmed the increased expression of STAT3 in HeLa cell lines. STAT3 mRNAs were reduced very effectively in all cases of STAT3-targeting siRNAs with different 6 sequences to HeLa (80.1%∼93.4%) and the following sequence of oligonucleotides encoding STAT3 siRNA showed the highest reduction rate; 5′-GCGTCCAGTTCACTACTAAAGTCAG-3′ (upper strand) and 5′-CTGACTTTAGTAGTGAACTGGACGC-3′ (lower strand). The reduction rate at silencing c-Src with different 5 sequences siRNAs was high (73.8%∼88.9%). The reduction rate at silencing v-Src with different 5 siRNAs was lower than other two cases (40.5%∼66.6%). The enhancement of cytotoxicity of cisplatin by gene silencing of STAT3 and c-Src, but not of v-Src, was confirmed in HeLa and C4I cells. Conclusion: In this study, we confirmed that STAT3/c-Src targeting siRNAs down-regulated the expression of STAT3 and c-Src and they could enhance the cytotoxic effect of cisplatin in cervical cancer cell lines. We will continue the upgraded study to investigate methods for enhancement of drug susceptibility, including the effect of co-silencing STAT3 and c-Src and their in vivo responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2950.