Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic response to Bortezomib, a novel proteasome inhibitor, in human H460 non-small cell lung cancer cells.
Bortezomib, a proteasome inhibitor, shows substantial anti-tumor activity in a variety of tumor cell lines, is in phase I, II, and III clinical trials and has recently been approved for the treatment of patients with multiple myeloma. The sequence of events leading to apoptosis following proteasome inhibition by bortezomib is unclear. Bortezomib effects on components of the mitochondrial apoptotic pathway were examined: generation of reactive oxygen species (ROS), alteration in the mitochondrial membrane potential (Delta psi m), and release of cytochrome c from mitochondria. With human H460 lung cancer cells, bortezomib exposure at 0.1 microM showed induction of apoptotic cell death starting at 24 h, with increasing effects after 48-72 h of treatment. After 3-6 h, an elevation in ROS generation, an increase in Delta psi m, and the release of cytochrome c into the cytosol, were observed in a time-dependent manner. Co-incubation with rotenone and antimycin A, inhibitors of mitochondrial electron transport chain complexes I and III, or with cyclosporine A, an inhibitor of mitochondrial permeability transition pore, resulted in inhibition of bortezomib-induced ROS generation, increase in Delta psi m, and cytochrome c release. Tiron, an antioxidant agent, blocked the bortezomib-induced ROS production, Delta psi m increase, and cytochrome c release. Tiron treatment also protected against the bortezomib-induced PARP protein cleavage and cell death. Benzyloxycarbonyl-VAD-fluoromethyl ketone, an inhibitor of pan-caspase, did not alter the bortezomib-induced ROS generation and increase in Delta psi m, although it prevented bortezomib-induced poly(ADP-ribose) polymerase cleavage and apoptotic death. In PC-3 prostate carcinoma cells (with overexpression of Bcl-2), a reduction of bortezomib-induced ROS generation, Delta psi m increase was correlated with cellular resistance to bortezomib and the attenuation of drug-induced apoptosis. The transient transfection of wild type p53 in p53 null H358 cells caused stimulation of the bortezomib-induced apoptosis but failed to enhance ROS generation and Delta psi m increase. Thus ROS generation plays a critical role in the initiation of the bortezomib-induced apoptotic cascade by mediation of the disruption of Delta psi m and the release of cytochrome c from mitochondria.
Ling
Yi-He
YH
Department of Oncology, Albert Einstein College of Medicine, Bronx, New York 10461, USA. New York University Cancer Institute, School of Medicine, New York University, New York, New York 10016, USA.
Liebes
Leonard
L
Zou
Yiyu
Y
Perez-Soler
Roman
R
D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis; D001897:Boronic Acids / Q000494:pharmacology*; D000069286:Bortezomib; D002289:Carcinoma, Non-Small-Cell Lung / Q000473:pathology*; D061945:Caspase Inhibitors; D002470:Cell Survival; D003546:Cysteine Endopeptidases; D003574:Cytochrome c Group / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007202:Indicators and Reagents / Q000494:pharmacology; D008175:Lung Neoplasms / Q000473:pathology*; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000473:pathology*; D008956:Models, Chemical; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011719:Pyrazines / Q000494:pharmacology*; D017382:Reactive Oxygen Species; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D000970:Antineoplastic Agents; D000975:Antioxidants; D001897:Boronic Acids; D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D011719:Pyrazines; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; D000069286:Bortezomib; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex