Roles Of Mitochondria During Apoptosis Induction Of Multidrug-resistant K562/ADM Cells

Objective:Mitochondria is a key factor in regulation of cells survival and death, the changes of mitochondria inner membrane potential and permeability play a central role in cellular apoptosis, but the alteration of mitochondrial morphology and structure involved in apoptotic procedure of drug-resistant leukemia cells remains unclear. This study was designed to investigate the structural and functional changes of mitochondria during Vitamin E succinate (VES)-induced apoptosis in K562/ADM cells, and to explore the possible molecular mechanisms of apoptosis of the multidrug-resistant leukemia cells induced by VES.Methods:Human multidrug-resistant leukemia cell line K562/ADM over-expressing mdr1 gene was used as the target cells. The cell proliferating activity was assessed with a MTT colorimetric assay. The apoptosis of K562/ADM cells was investigated with optical and electronic microscopic morphology, and double staining of FITC-Annexin V and propidium iodide (PI). Morphological structure of mitochondria was observed by electron microscope. The cells were stained with Rhodaminel23 (Rhl23) to analyze the changes in mitochondrial inner membrane potential (△ψm) by confocal laser scanning microscope and flow cytometry (FCM). Cytochrome c (Cyt c) in cytoplasm was assessed by confocal laser scanning microscope. The cells were stained withFITC-DEVD-FMK to measure active caspase-3 using FCM. The cell cycle distribution, P-glycoprotein (P-gp) expression and its function were measured by FCM. The expression of caspase-9, caspase-3 and mdr-1 mRNA was examined with reverse transcription polymerase chain reaction (RT-PCR).Results:VES obviously inhibited the proliferation of K562/ADM cells in a mannerdepending on both dose and time (P<0.01), 50% inhibitory concentration ofK562/ADM cells was ll^mol/L, 66.3^01/1., 49.7/anol/L and 39.9,umol/L,respectively for 24h, 48h, 72h and 96h. The cells treated with 20-40//mol/L VESshowed the typical apoptotic morphological changes such as plasma membraneblebbing, condensation and margination of chromatin, crescent structures of chromatinon the inner nuclear membrane, nuclear fragmentation and formation of apoptoticbodies. The apoptotic rate of K562/ADM cells by Annexin V/PI staining was greatlyincreased, from 1.39% to 11.02% and 31.12% after exposure to 20//mol/L and40//mol/L VES for 24 hours. Cell cycle analysis indicated the increased Sub-Giproportion as well as the apparent Gi phase arrest in VES-induced K562/ADM cells.Mitochondria became smaller and fragmentation, inner and outer membranes fusion,reduced and confused cristae at the concentration of 40//mol/L VES. The mitochondialinner membrane potential greatly decreased from 99.9% to 79.8% and 49.3% after12-hour incubation with 20/amol/L and 40jamol/L VES. The analysis with confocallaser scanning microscope showed an increase in the release of cytochrome c frommitochondria to cytoplasm. The RT-PCR analyzed that the expression of caspase-9mRNA was raised in proportion to the dose of VES added. The expression of mdrlmRNA and its product P-gp was markedly down-regulated in VES-inducedK562/ADM cells. The P-gp positive rate declined from 98.2% to 87.8%-75.1% afterapplication of 20/imol/L and 40//mol/L VES for 72 hours. The expression of caspase-3mRNA up-regulated and,caspase-3 activity improved from 5.31% to 25.0%-32.6%.VES significantly enhanced the sensitivity of K562/ADM cells to adriamycin.Conclusion:The apoptotic initiation of Vitamin E succinate on drug-resistant K562/ADM cells maybe associate with the destabilization of mitochondria, dissipation of the mitochondrial inner membrane potential, release of cytochrome c from mitochondria to cytoplasm, and the subsequent activation of caspase-9 and caspase-3. Meanwhile, VES markedly down-regulates the expression of mdrl and its product P-gp, weakens the suppression of P-gp on caspase-3 activity to overcome the P-gp-mediated apoptosis resistance and drug-resistance in multidrug-resistant K562/ADM cells.