2,4-dinitrophenol induces G1 phase arrest and apoptosis in human pulmonary adenocarcinoma Calu-6 cells.
2,4-dinitrophenol (DNP) is an uncoupler of oxidative phosphorylation in the mitochondria. Here, we investigated the effect of DNP on the growth of Calu-6 lung cancer cells in view of cell cycle, apoptosis, ROS production and GSH content. DNP dose-dependently decreased cell viability at 72 h (EC50 of about 200 microM) as measured by a MTT assay. The lower doses of DNP induced a G1 arrest of the cell cycle in Calu-6 cells. Analysis of the cell cycle regulatory proteins demonstrated that DNP decreased the steady-state levels of cyclin proteins and cyclin dependent kinase (CDK), but increased the protein levels of cyclin dependent kinase inhibitor (CDKI) p27. DNP also caused a marked increase in apoptosis, as evidenced by DNA fragmentation (sub-G1 DNA content), DAPI staining, the loss of mitochondrial membrane potential (DeltaPsim), externalization of phosphatidylserine (PS). In addition, DNP-treated cells significantly increased the intracellular H2O2 and O2.- levels. All of caspase inhibitors could markedly rescue Calu-6 cells from DNP-induced cell death and only pan-caspase inhibitor, Z-VAD-FMK, could slightly prevent the loss of mitochondrial membrane potential (DeltaPsim). However, none of the caspase inhibitors reduced the increased H2O2 levels, but the increased O2.- levels was slightly attenuated by pan-caspase inhibitor. In addition, the depletion of GSH content in DNP-treated cells was prevented by all of caspase inhibitors. In conclusion, DNP, which induced ROS and reduced GSH content, inhibited the growth of Calu-6 cells via cell cycle arrest at G1 phase and apoptosis.
Han
Yong Hwan
YH
Department of Physiology, Medical School, Institute for Medical Sciences, Centers for Healthcare Technology Development, Chonbuk National University, Jeonju 561-180, Republic of Korea.
Kim
Sang Wook
SW
Kim
Suhn Hee
SH
Kim
Sung Zoo
SZ
Park
Woo Hyun
WH
D019297:2,4-Dinitrophenol / Q000494:pharmacology*; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D005456:Fluorescent Dyes; D016193:G1 Phase / Q000187:drug effects*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007202:Indicators and Reagents; D007211:Indoles; D008564:Membrane Potentials / Q000187:drug effects; D051336:Mitochondrial Membranes / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D014475:Uncoupling Agents / Q000494:pharmacology*
D017304:Annexin A5; D016213:Cyclins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007211:Indoles; D017382:Reactive Oxygen Species; D014475:Uncoupling Agents; C007293:DAPI; D006861:Hydrogen Peroxide; D018844:Cyclin-Dependent Kinases; D005978:Glutathione; D019297:2,4-Dinitrophenol