Beschreibung:
Abstract Signal Transducer and Activator of Transcription 3 (STAT3) is constitutively activated in many cancer cell lines, leading to survival, proliferation, angiogenesis and metastasis. The inhibition of STAT3 by natural or synthetic compounds has been shown to sensitise cancer cell lines to the chemotherapy agent cisplatin, however the molecular mechanisms contributing to this chemosensitisation have not yet been fully elucidated. Therefore we investigated the effect of STAT3 inhibition on cisplatin-induced DNA damage and key DNA repair factors. STAT3 inhibitors stattic and curcumin were investigated in combination with cisplatin using the Suphorhodamine B cell growth inhibition assay in the DU145 prostate cancer and A549 non-small cell lung cancer cell lines. Combination treatments result in a significant (P<0.0001) reduction in cisplatin GI50 values. These combinations were synergistic as determined by combination index value analysis using the software Calcusyn, confirming that inhibition of STAT3 enhances the cytotoxic effect of cisplatin. Using a modification of the alkaline comet assay we observed that STAT3 inhibitors significantly prevent the unhooking step of repair of cisplatin induced interstrand crosslinks (ICLs). Treatment with cisplatin alone induces formation of ICLs which peak at 9 hours and unhook by 24 hours post cisplatin exposure in both cell lines. Pre-treating cells with 12μM stattic or 48μM curcumin followed by cisplatin results in an equivalent peak formation of ICLs at 9 hours, however 24 and 48 hours post cisplatin no unhooking is observed. Therefore both STAT3 inhibitors used in this study block the unhooking stage of ICL repair. Using RT-PCR and immunoblotting we show that treatment with STAT3 inhibitors dose-dependently down-regulates EME1, BRCA1 and FANCD2 mRNA and protein levels. These three DNA repair proteins have been shown to be involved in the early stages of ICL repair and therefore down-regulation of these genes may be responsible for the impairment of ICL unhooking seen with STAT3 inhibition. As the effects observed here have been replicated with two STAT3 inhibitors of differing chemical structure, this suggests that STAT3 is directly involved in the regulation of cisplatin-induced ICL repair through modulation of key ICL repair genes. We propose this as a contributing mechanism to the synergy reported between STAT3 inhibitors and cisplatin. The data presented here will inform the design of future drug combination strategies. Citation Format: Helen Valentine, Emily Dixon, John A. Hartley, Konstantinos Kiakos. Chemosensitisation to cisplatin by STAT3 inhibitors is mediated through the inhibition of DNA interstrand crosslink unhooking. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C51.