Abstract
The inhibitory mechanism of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (CMEP-NQ) against apoptosis induced by the microtubule-damaging agents (MDAs), nocodazole (NOC) and 2-methoxyestradiol (2-MeO-E2), or a DNA-damaging agent (DDA), camptothecin (CPT) were investigated in human Jurkat T cell clones (J/Neo and J/BCL-XL cells). Treatment of J/Neo cells with NOC, 2-MeO-E2, or CPT caused cytotoxicity and apoptotic DNA fragmentation but these events were significantly attenuated in the presence of CMEP-NQ. Although not only MDA (NOC or 2-MeO-E2)-induced mitotic arrest, CDK1 activation, and BCL-2, BCL-XL and BIM phosphorylation, but also DDA (CPT)-induced S-phase arrest and ATM-CHK1/CHK2-p53 pathway activation were not or were barely affected in the presence of CMEP-NQ, the levels of anti-apoptotic BAG3 and MCL-1, which were markedly downregulated after MDA- or DDA-treatment, were rather elevated by CMEP-NQ. Under the same conditions, MDA- or DDA-induced mitochondrial apoptotic events including BAK activation, mitochondrial membrane potential (Δψm) loss, caspase-9 activation, and PARP cleavage were significantly inhibited by CMEP-NQ. While MDA- or DDA-induced sub-G1 peak and Δψm loss were abrogated in J/BCL-XL cells, MDA-induced mitotic arrest and DDA-induced S-arrest were more apparent in J/BCL-XL cells than in J/Neo cells. Simultaneously, the induced cell cycle arrest in J/BCL-XL cells was not significantly disturbed by CMEP-NQ. MDA- or DDA-treatment caused intracellular reactive oxygen species (ROS) production; however, MDA- or DDA-induced ROS production was almost completely abrogated in J/BCL-XL cells. MDA- or DDA-induced ROS production in J/Neo cells was significantly suppressed by CMEP-NQ, but the suppressive effect was hardly observed in J/BCL-XL cells. Together, these results show that CMEP-NQ efficiently protects Jurkat T cells from apoptotic cell death via the elevation of BAG3 and MCL-1 levels, which results in the inhibition of intrinsic BAK-dependent mitochondrial apoptosis pathway, as does the overexpression of BCL-XL.
Highlights
Mitochondria, double membrane-bound organelles, are present in most aerobic eukaryotic cells and play a key role in the generation of ATP via electron transport and oxidative phosphorylation
The results show that CMEP-NQ prevents mitochondrial damage via the blockade of BAK activation and caspase cascade activation through the upregulation of anti-apoptotic BCL-2-associated athanogene 3 (BAG3) and myeloid cell leukemia 1 (MCL-1) levels, which protects the cells from apoptotic cell death induced by microtubule-damaging agents (MDAs) or DNA-damaging agent (DDA) treatment
To examine whether CMEP-NQ protects cells from mitochondrial damage and apoptotic cell death, we evaluated the effect of CMEP-NQ on the cytotoxicity and apoptotic DNA fragmentation induced by NOC, 2-MeO-E2, and CPT in J/Neo cells, which were previously reported to occur through mitochondria-dependent caspase cascade activation [31,32,33]
Summary
Mitochondria, double membrane-bound organelles, are present in most aerobic eukaryotic cells and play a key role in the generation of ATP via electron transport and oxidative phosphorylation. The initial triggers provoking these apoptotic induction pathways are different, mitochondrial damage and the release of mitochondrial apoptosis inducers, such as cytochrome c, apoptosis-inducing factor (AIF), and reactive oxygen species (ROS), into the cytosol have been frequently associated with apoptosis induction caused by cellular stress and cytotoxic conditions. This clearly indicates a critical involvement of mitochondria in determining cell fate [3,16]
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