Expression Of Multidrug Resistance Gene (mdr1) And Apoptosis-inhibited Genes During Arsenic Trioxide-mediated Reversal Of Apoptosis Resistance In Multidrug-resistant Human Leukemia Cells

Objective:To study the expression of multidrug resistance gene (mdr1) and apoptosis-inhibited genes during apoptosis induced by Arsenic trioxide and explore the molecule mechanism of reversal of apoptosis resistance by arsenic trioxide ( AS₂O₃) in multidrug-resistant human leukemia cells.Methods:Human multidrug-resistant leukemia cell line K562/ADM over-expressing mdrl gene was used as the target cells. The cell proliferating activity was assessed with a MTT assay. Cell morphology, DNA fragmentation, cell cycle analysis and AnnexinV/PI double staining were employed to detect apoptosis in K562/ADM cells. Expression of Bcl-2 protein and P-gp, Caspase-3 activity and cellular adriamycin (ADM) were measured with flow cytomery (FCM). The level of reactive oxygen species (ROS) was detected with confocal laser scanning microscope and FCM. The expression of mdrl, bcl-2, survivin and caspase-3 mRNA were examined with reverse transcription polymerase chain reaction (RT-PCR).Results:AS₂O₃ could inhibite growth of K562/ADM cells at a manner depending on both concentration and time (γ_24h=0.921 , γ_48h=0.939, γ_72h=0.865). We observed typical morphological changes of apoptosis in K562/ADM cells during the apoptosis-inductionof AS2O3, such as plasma membrance blebbing, chromatin condensation, and formation of apoptotic bodies. Agrose gel electrophoresis showed evident DNA fragmentation (DNA ladder). After treatment with 2 or 5 nmol/Lof As2O3 for 24 hours, the percentage of K562/ADM cells in the G2/M phases of cell cycle was much higer than untreated cells (26.4% and 36.5%, respectively compared with 13.1%), and treated with the similar AS2O3 for 72 hours, the percentage of Sub-Gl containing hypodiploid amounts of DNA was increased to 25.6% and 52.9%, respectively. The apoptosis rate of the cells by AnnexinV/PI staining was obviously increased. Constitutive expression of mdrl mRNA and P-gp was detected in K562/ADM cells using RT-PCR and FCM. Incubated with 2 and 5 (xmol/L As2O3 for 24 and 48 hours, K562/ADM cells exhibited decreased mdrl mRNA expression. There were no evident changes in positive rate of P-gp expression for 72h> but at 96h the expression of P-gp showed significant decrease, the positive rate from 97.2% to 96.0%-91.2% and MFI from 19.0 to 9.24-7.43. Simultaneously the function of P-gp was restrained so that the cellular content of ADM was obviously increased. By use of RT-PCR, We noted that the expression of survivin mRNA markedly decresed after application of 2 and 5 nmol/L AS2O3 for 24-48 hours. K562/ADM cells overexpessed Bcl-2 protein, after application of 2 and 5 jxmol/L AS2O3 for 24-96 hours, the expression of Bcl-2 protein significantly decreased depending on both concentration and time, and a 24-hour treatment of 2 and 5 ^mol/L AS2O3 could down-reregulate the expression of bcl-2 mRNA. It was shown that the caspase-3 could be gradually activated from 17.9% to 30.0 % to 47.4% during action of As2O3 for 48 hours, and similarly, caspase-3 mRNA could be up-regulated for 24 hours. Flow cytometry analysis demonstrated that the inherent level of ROS was very high in K562/ADM cells. There was no significant change in both positive rate and MFI of ROS after treatment with 2 nmol/L As2O3 for 12 to 72h, but the level of ROS was decreased from 94.4% to 40.5% after 5 jAmol/L As2O3 -treatment for 72h. Under confocal laser scanning microscopy, 2 to 5 pimol/L AS2O3 treatment from 24 to 48h showed morphological changes of apoptosis and cellular slack of fluorescence of ROS,which meaned the degradation of ROS in apoptotic K562/ADM cells.Conclusion:As2O3 inhibits the proliferation of multidrug-resistant K562/ADM cells and induces the cells to undergo apoptosis, this fact means that AS2O3 is able to overcome the apoptosis resistance in the drug-resistant cells by modulating the expression or activity of apoptotic key factors such as P-gp, bcl-2, survivin, capase-3 and ROS: I . AS2O3 down-regulates the expression of mdrl/P-gp and survivin to weaken P-gp- and suvivin-mediated suppression on caspase-3 activity to overcome the apoptosis resistance in the P-gp+cells mediated by overexpression of P-gp and survivin;II. AS2O3 inhibits the expression of bcl-2 and survivin to induce apoptosis of K562/ADM cells via activating mitochondria-dependent apoptosis pathway;III. AS2O3 reduces the cellular ROS level to remove the agumentation of ROS on mdrl/P-gp expression to reverse the P-gp-mediated phenomena of multidrug-resistance and apoptosis resistance in drug-resistant K562/ADM cells.