Induction of Caspase Mediated Apoptosis and Down-Regulation of Nuclear Factor-κB and Akt Signaling are Involved in the Synergistic Antitumor Effect of Gemcitabine and the Histone Deacetylase Inhibitor Trichostatin A in Human Bladder Cancer Cells

Purpose: Previously we reported that the histone deacetylase inhibitor trichostatin A (Sigma (R)) synergistically potentiates the antitumor effects of cisplatin in human bladder cancer cells. In the current study we explored the synergistic interaction between trichostatin A and gemcitabine (Novartis Korea, Seoul, Korea), the other mainstay chemotherapeutic regimen for advanced bladder cancer. Materials and Methods: The bladder cancer cell lines HTB5, HTB9, T24, J82, UMUC14 and SW1710 (ATCC (R)) were exposed to gemcitabine and/or trichostatin A. Synergism between the 2 drugs was determined by the combination index based on the Cell Counting Kit-8 assay (Dojindo Molecular Technologies, Rockville, Maryland) and by a clonogenic assay. Flow cytometry was used to evaluate cell cycle distribution and apoptosis. The expression of cell cycle (p21(WAF1/CIP1), cyclin A, B1 and D1, p-CDC2C, CDC2C, p-CDC25C, CDC25C and pRb), apoptosis (caspase-3, 8 and 9, PARP, Bcl-2, Bad and Bax), NF-kappa B (NF-kappa B, p-I kappa B alpha, I kappa B alpha, p-IKK alpha, IKK alpha, cIAP1, cIAP2 and XIAP) and survival (p-Akt, Akt, p-mTOR, mTOR and PTEN) related proteins was analyzed by Western blot. Results: Isobolic analysis of the Cell Counting Kit-8 assay revealed strong synergism between gemcitabine and trichostatin A, which caused a 4.6 to 25.4-fold gemcitabine dose reduction and a 1.9 to 41.4-fold trichostatin A dose reduction while killing an estimated 90% of bladder cancer cells. The underlying mechanisms could be synergistic cell cycle arrest, induction of caspase mediated apoptosis, and down-regulation of the antiapoptotic NF-kappa B and Akt signaling pathways. Conclusions: Results show that trichostatin A may synergistically enhance gemcitabine mediated cell cycle arrest and apoptosis, suggesting the potential of using histone deacetylase inhibitors as combination agents to enhance the antitumor effect of gemcitabine for advanced bladder cancer.