Study On The Effect Of Proton Pump Inhibitor On Leukemia Therapy In Vitro

Objective:1.The study was purposed to explore the effect of proton pump inhibitor(omeprazole) in promoting apoptosis of human leukemia cell line K562 in vitro and its mechanism;2.To explore the enhancement of omeprazole on the cytotoxic effects of doxorubicin on K562 cells in vitro and its mechanism;3.To explore the effect of omeprazole in reversaling the resistance of human leukemic multidrug resistance cell line K562/A02 in vitro and its mechanism.Methods:1.The first part of the experiment:(1)Tetrazolium dye (MTT) assay was used to investigate the cytotoxic effects of omeprazole on K562 ceils.The half inhibition ratio(IC₅₀),twenty percent inhibition ratio(IC₂₀) of omeprazole against K562 calls was calculated and less than the IC₂₀ dose would be chosen to carry out the following experiments; (2)Trypan blue coloring method was used to investigate the effects of omeprazole on K562 cells growth in different PH environment;(3)A pH meter was used to determinate extracellular pH(PHe) after pretreating K562 cells for 24 hours with 50μg/ml omeprazole;(4)Annexinâ…¤was used to test the apoptosis of K562 cells treated with different concentrations of omeprazole.2.The second part of the experiment: (1)After pretreating K562 cells for 0 or 24 hours with omeprazole,MTT assay was used to investigate the cytotoxic effects of doxorubicin on K562 cells by calculating IC₅₀ of doxorubicin against K562 cells;(2)Flow cytometry(FCM) was used to measure intracellular doxorubicin concentration of K562 cells after treated with 5.0μg/ml omeprazole for 24 hours at the uptake phase and the efflux phase.3.The third part of the experiment:(1)Trypan blue coloring method was used to investigate the effects of omeprazole on K562/A02 cells growth,and 5.0,10.0,20.0,40.0μg/ml omeprazole had no effects on K562/A02 cell growth,they would be choosen to carry out the following experiments which had no effect on K562/A02 cell growth;(2)After pretreating K562/A02 cells for 24 hours with omeprazole that its' final concentration was 5.0,10.0,20.0,40.0μg/ml,MTT assay was used to investigate the cytotoxic effects of doxorubicin on K562/A02 cells by calculating the half inhibition ratio (IC₅₀) of doxorubicin against K562/A02 cells;(3)Flow cytometry was complied to measure the expression level of P-gp after treated with 5.0,10.0,20.0,40.0μg/ml omeprazole for 24 hours;(4)Intracellular Rhodamine 123(Rho123) retention assay was applied to test the function of P-gp.Results:1.The half inhibition ratio(IC₅₀) of omeprazole against K562 cells was 127.031μg/ml,IC₂₀ of omeprazole against K562 cells was 59.315μg/ml,and we choosed 50μg/ml omeprazole to carry out the following experiments;2.K562 cells treated with 50μg/ml omeprazole cultured in media maintained at different pH including 7.0,6.5,6.0,5.5,5.0 for 24 hours were almost appeared blue-stained cells by trypan blue coloring method,and the lower of the PH the more blue-stained cells,while untreated-K562 cells were almost no blue-stained cells; 3.Extracellular pH of untreated-K562 cells were 7.133±0.057(n=3), extracellular pH of treated-K562 cells with 50μg/ml omeprazole were 7.300±0.100(n=3),the results indicated that PHe of K562 cells would be increased by treated with omeprazole(P＜0.05);4.The apoptosis rate of K562 cell treated with 180,240,300,400μg/ml omeprazole for 24 hours were 18.960±0.282%,49.367±1.233%,75.510±0.958%,90.657±0.398%, the apoptosis rate of untreated-K562 cells were 0.757±0.006%;compared omeprazole treated cells with untreated cells,the apoptosis rate significantly increased(P＜0.05).The apoptosis rate of K562 cell treated with 180,240,300,400μg/ml omeprazole for 48 hours were 28.073±0.445%,60.773±0.545%,83.257±0.635%,93.997±0.095%; compared omeprazole treated cells with untreated cells,the apoptosis rate significantly increased(P＜0.05).Likewise compared omeprazole treated cells for 48 hours with omeprazole treated cells for 24 hours at the same concentration,the apoptosis rate significantly increased(P＜0.05);5.The apoptosis rate of K562 cell treated with 180,240,300,400μg/ml omeprazole and 2μL Z-VAD-FMK of Caspase inhibitor for 24 hours were 18.843±0.075%,48.233±0.150%,74.073±0.825%,90.777±0.188%; compared omeprazole and Z-VAD-FMK total-treated cells with omeprazole only-treated cells for 24 hours at the same concentration,the apoptosis rate was no significant difference(P＞0.05);6.Pretreating K562 cells for 0 hour with 1.0,2.0,5.0,10.0μg/ml omeprazole,IC₅₀ of doxorubicin against K562 cells were 0.797±0.006μg/ml,0.800±0.026μg/ml,0.813±0.060μg/ml,0.740±0.010μg/ml,IC₅₀ of omeprazole untreated cells were 0.772±0.026μg/ml;compared omeprazole pretreated cells with untreated cells,the value of IC₅₀ was no significant difference(P＞0.05).Whereas pretreating K562 cells for 24 hours with 2.0,5.0,10.0μg/ml omeprazole,the value of IC₅₀ significantly decreased(P＜0.01);7.Compared 5μg/ml omeprazole pretreated cells with untreated cells,the concentration of doxorubicin intaked into the cells significantly increased by 48.973±1.643 to 55.043±3.117(P＜0.05),and the output at 18,24,40 hours significantly decreased(P＜0.05);8.Treating K562/A02 cells for 48 hours with 5.0＜10.0＜20.0＜40.0μg/ml omeprazole were almost no blue-stained cells by trypan blue coloring method; 9.Pretreating K562/A02 cells for 24 hours with 5.0,10.0,20.0,40.0μg/ml omeprazole,IC₅₀ of doxorubicin against K562/A02 cells were 5.665±0.007μg/ml,4.742±0.016μg/ml,2.889±0.010μg/ml,0.856±0.007μg/ml,IC₅₀ of omeprazole untreated cells were 8.704±0.036μg/ml,compared omeprazole pretreated cells with untreated cells,the value of IC₅₀ significant decreased(P＜0.05);10.Pretreating K562/A02 cells for 24 hours with 5.0,10.0,20.0,40.0μg/ml omeprazole, the expression level of P-gp were 89.237±0.610,77.437±0.539,63.730±1.026,48.393±0.413,the expression level of untreated cells was 101.163±0.847;compared omeprazole pretreated cells with untreated cells,the expression level of P-gp significant decreased(P＜0.05); 11.Correspondingly,pretreating K562/A02 cells with various omeprazole, the expression level of Rho123 were 8.553±0.055,12.740±0.060,20.640±0.181,26.607±0.368,the expression level of untreated cells was 3.063±0.032;compared omeprazole pretreated cells with untreated cells,the expression level of Rho123 significant increased(P＜0.05).Coclusions:1.Omeprazole in a certain concentration would promote apoptosis of K562 cells in a dose- and time-dependent manner; 2.Omeprazole-induced apoptosis in K562 cells was caspase-independent pathway;3.Pretreating K562 ceils with omeprazole,extracellular pH of K562 cells increased.The result suggested that omeprazole could inverse the acidification of the tumor microenvironment,which creating advantages for inhibiting proliferation and promoting apoptosis of tumor cells;4.Pretreating K562 cells with omeprazole could enhance the cytotoxic effects of doxorubicin on the K562 cells in vitro in a dose-dependent manner.These results also suggested that pretreating K562 cells with omeprazole not only increased intracellular doxorubicin concentration but also strongly inhibited the elimination of antitumor drugs through the secretory pathway;5.Pretreating K562/A02 cells with omeprazole,the level of P-gp expression decreased,and the function of P-gp also decreased.Pretreating K562/A02 cells with omeprazole could sensitize K562/A02 cells to cytotoxic drugs and inverse multidrug resistance in a dose-dependent manner.