An ROS generator, antimycin A, inhibits the growth of HeLa cells via apoptosis

Abstract

Antimycin A (AMA), an inhibitor of electron transport in mitochondria, has been used as a reactive oxygen species (ROS) generator in biological systems. Here, we investigated the in vitro effect of AMA on apoptosis in HeLa cells. AMA inhibited the growth of HeLa cells with an IC(50) of about 50 microM. AMA efficiently induced apoptosis, as evidenced by flow cytometric detection of sub-G1 DNA content, annexin V binding assay, and DAPI staining. This apoptotic process was accompanied by the loss of mitochondrial membrane potential (DeltaPsi(m)), Bcl-2 down-regulation, Bax up-regulation, and PARP degradation. All caspase inhibitors used in this experiment, especially pan-caspase inhibitor (Z-VAD), could rescue some HeLa cells from AMA-induced cell death. When we examined the changes of the ROS, H(2)O(2) or O(2) (.-), in AMA-treated cells, H(2)O(2) and O(2) (.-) were markedly increased. In addition, we detected the depletion of GSH content in AMA-treated cells. Pan-caspase inhibitor showing the efficient anti-apoptotic effect significantly reduced GSH depletion by AMA. Superoxide dismutase (SOD) and catalase did not reduce intracellular ROS, but these could strongly rescue the cells from apoptosis. However, these anti-apoptotic effects were not accompanied by the recovery of GSH depletion. Interestingly, catalase significantly decreased the CMF negative (GSH depletion) and propidium iodide (PI) positive cells, indicating that catalase strongly maintained the integrity of the cell membrane in CMF negative cells. Taken together, these results demonstrate that AMA potently generates ROS, induces the depletion of GSH content in HeLa cells, and strongly inhibits the growth of HeLa cells throughout apoptosis.

Glutathionylation State of Uncoupling Protein-2 and the Control of Glucose-stimulated Insulin Secretion

“…antimycin A or rotenone) to artificially enhance cell ROS levels and stimulate GSIS (15). In our opinion, however, use of electron transport chain inhibitors should be avoided when investigating mitochondrial ROS signaling (especially in ␤ cells) because these drugs also compromise ATP production and induce cell death quite rapidly through uncontrolled ROS production and impaired ATP genesis (41)(42)(43). Here, we demonstrate that low doses of H 2 O 2 amplify GSIS but can also induce proton leak through UCP2.…”

The effects of N-acetyl cysteine, buthionine sulfoximine, diethyldithiocarbamate or 3-amino-1,2,4-triazole on antimycin A-treated Calu-6 lung cells in relation to cell growth, reactive oxygen species and glutathione

“…Evidence indicates that either the presence of ROS or the collapse of MMP (Δae m ) opens the mitochondrial permeability transition pore, which is accompanied by the release of proapoptotic molecules such as cytochrome c into the cytoplasm (15,16). Because AMA acts directly on the mitochondria, AMA-induced apoptosis has been reported in many experiments (17)(18)(19)(20)(21)(22)(23)(24).…”

Anti-apoptotic effects of pan-caspase inhibitor (Z-VAD), SOD or catalase on antimycin A-induced HeLa cell death

“…This occurrence is accompanied by the release of proapoptotic molecules such as cytochrome c into the cytoplasm (11), triggering apoptosis. Since AMA acts directly on the mitochondria, AMA-induced apoptosis has been reported in many experiments, including our recent report on HeLa and As4.1 juxtaglomerular cells (12)(13)(14)(15)(16)(17)(18).…”