Role of Intracellular Glutathione in Cell Sensitivity to the Apoptosis Induced by Tumor Necrosis Factor α–Related Apoptosis-Inducing Ligand/Anticancer Drug Combinations

Abstract

We have recently shown that combination of tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL) with anticancer drugs induced an apoptotic cell death pathway involving both caspases and mitochondria. The present work further explores the role of intracellular reduced glutathione (GSH) level in cell sensitivity to this cell death pathway. Intracellular GSH level was measured by high-performance liquid chromatography. Cell death was detected by immunofluorescence after Hoechst 33342/propidium iodide staining. Reactive oxygen species production was evaluated by flow cytometry after dihydroethidium probe labeling. Western blot analysis was done to study stress-activated protein kinase/c-jun NH(2)-terminal kinase (SAPK/JNK) phosphorylation. The Student's t test was used to determine significance of the results. Three to six experiments were done. GSH depletion enhanced apoptosis induced by TRAIL/cisplatin (CDDP) or TRAIL/5-fluorouracil (5-FU) combinations in both human HT29 colon carcinoma and HepG2 hepatocarcinoma cells, whereas it enhanced cytotoxicity induced only by TRAIL/CDDP in human primary hepatocytes. Our results further suggested that GSH depletion enhanced SAPK/JNK phosphorylation upon TRAIL/5-FU exposure and likely reduced the detoxification mechanisms of CDDP in HT29 cells. Resistance of Bcl-2-expressing HT29 and HepG2 cells to combined treatment was not overcome by GSH depletion, thus indicating that Bcl-2-mediated antiapoptotic effect occurs independently of intracellular GSH level. GSH depletion could be useful to increase the therapeutic efficacy of cancer treatment by TRAIL/anticancer drug combinations. Furthermore, TRAIL/5-FU combination might be a potential anticancer treatment of human tumors, being ineffective on human primary hepatocytes and thus could be of interest in clinical cancer treatment. Nevertheless, Bcl-2 expression remains an important resistance factor.