| Summary: | We previously reported that 5-[4-(4-fluorophenoxy) phenyl] methylene-3-{4-[3-(4-methylpiperazin-1-yl)propoxy]phenyl}-2-thioxo-4-thiazolidinone dihydrochloride (KSK05104) has potent, selective and metabolically stable IKKβ inhibitory activities. However, the apoptosis-inducing of KSK05104 and its underlying mechanism have not yet been elucidated in human colon cancer cells. We show that KSK05104 triggered apoptosis, as indicated by externalization of Annexin V-targeted phosphatidylserine residues in HT-29 and HCT-116 cells. KSK05104 induced the activation of caspase-8, -9, and -3, and the cleavage of poly (ADP ribose) polymerase-1 (PARP-1). KSK05104-induced apoptosis was significantly suppressed by pretreatment with z-VAD-fmk (a broad caspase inhibitor). KSK05104 also induced release of cytochrome <i>c</i> (Cyt <i>c</i>), apoptosis inducing factor (AIF), and endonuclease G (Endo G) by damaging mitochondria, resulting in caspase-dependent and -independent apoptotic cell death. KSK05104 triggered endoplasmic reticulum (ER) stress and changed the intracellular calcium level ([Ca<sup>2+</sup>]<sub>i</sub>). Interestingly, treatment with KSK05104 activated not only ER stress marker proteins including inositol-requiring enzyme 1-alpha (IRE-1α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK), but also μ-calpain, and caspase-12 in a time-dependent manner. KSK05104-induced apoptosis substantially decreased in the presence of BAPTA/AM (an intracellular calcium chelator). Taken together, these results suggest that mitochondrial dysfunction and ER stress contribute to KSK05104-induced apoptosis in human colon cancer cells.
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