Abstract
Although murine embryonic fibroblasts (MEFs) with Bax or Bak deleted displayed no defect in apoptosis signaling, MEFs with Bax and Bak double knock-out (DKO) showed dramatic resistance to diverse apoptotic stimuli, suggesting that Bax and Bak are redundant but essential regulators for apoptosis signaling. Chelerythrine has recently been identified as a Bcl-xL inhibitor that is capable of triggering apoptosis via direct action on mitochondria. Here we report that in contrast to classic apoptotic stimuli, chelerythrine is fully competent in inducing apoptosis in the DKO MEFs. Wild-type and DKO MEFs are equally sensitive to chelerythrine-induced morphological and biochemical changes associated with apoptosis phenotype. Interestingly, chelerythrine-mediated release of cytochrome c is rapid and precedes Bax translocation and integration. Although the BH3 peptide of Bim is totally inactive in releasing cytochrome c from isolated mitochondria of DKO MEFs, chelerythrine maintains its potency and efficacy in inducing direct release of cytochrome c from these mitochondria. Furthermore, chelerythrine-mediated mitochondrial swelling and loss in mitochondrial membrane potential (DeltaPsi(m)) are inhibited by cyclosporine A, suggesting that mitochondrial permeability transition pore is involved in chelerythrine-induced apoptosis. Although certain apoptotic stimuli have been shown to elicit cytotoxic effect in the DKO MEFs through alternate death mechanisms, chelerythrine does not appear to engage necrotic or autophagic death mechanism to trigger cell death in the DKO MEFs. These results, thus, argue for the existence of an alternative Bax/Bak-independent apoptotic mechanism that involves cyclosporine A-sensitive mitochondrial membrane permeability.
Highlights
Chelerythrine Triggers Caspase-dependent Cytotoxicity in the Absence of Bax and Bak—In agreement with the published literature, double-knock-out mice (DKO) Murine embryonic fibroblasts (MEFs) were found to be resistant to a variety of apoptotic stimuli including etoposide, staurosporine, camptothecin, cisplatin, methotrexate, and UV irradiation (Fig. 1, A, panel c and d, and B; data not shown)
The appearance of sub-G1 DNA was seen in DKO MEFs treated with chelerythrine, but not etoposide, staurosporine, or camptothecin even though these compounds were fully effective in inducing sub-G1 DNA appearance in the WT MEFs (Fig. 1B; data not shown)
The appearance of sub-G1 DNA in DKO MEFs upon chelerythrine treatment was clearly caspase-dependent, as the effect was abolished by pretreatment with caspase inhibitor (Fig. 1E, upper panel) or overexpression of hXIAP (Fig. 1E, lower panel), a protein that is known to sequester and inhibit activated caspases [24]
Summary
Reagents and Cell Lines—SV40 T antigen-transformed WT and DKO MEFs were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics (100 g of streptomycin/ml and 100 IU of penicillin/ml, Invitrogen). Equal amounts of mitochondria were treated with the indicated compounds for 15 min at room temperature followed by centrifugation [20]. Reactive Oxygen Species (ROS) Measurement—Cells were treated with the indicated concentrations of compounds for 16 h After such treatment, cells were washed in PBS and loaded with 1 M 5-(and 6-)chloromethyl-2Ј,7Ј-dichlorodihydrofluorescein diacetate, acetyl ester dye (CM-H2DCFDA; Molecular Probes) in PBS for 30 min at 37 °C as described [23]. Cells were recovered by centrifugation at 1000 ϫ g for 5 min at 4 °C, washed, stained with 50 mg/ml of propidium iodide for 30 min at room temperature, and analyzed in a FACScan flow cytometer (BD Biosciences). The results presented are representative of at least three experiments
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