Pretreatment with proton pump inhibitor increases sensitivity of human gastric adenocarcinoma cell line to antitumor drugs

Abstract

Objective To investigate whether pantoprazole (PPZ), a proton pump inhibitor,could reverse the transmember pH gradient by inhibiting vacuolar H+-ATPase so as to increase the sensitivity of human gastric adenocarcinoma cell line SGC7901 to antitumor drugs and to evaluate the optimal time of drug administration, dosage of PPZ and the possible mechanism. Methods Western blotting and immunofluorescent staining were used to determine the expression and intracellular distribution of vacuolar H+-ATPase in human gastric adenocarcinoma cell line SGC7901 with or without PPZ pretreatment. 2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM) fluorescent probe was used to measure the intracellular pH value of human gastric adenocarcinoma cell line SGC7901 which pretreated with different dose and time of PPI. Methyl thiazolyl tetrazolium (MTT) assay and annexin V-fluorescent isothiocyanate-propidium iodide double staining were performed to evaluate the cytotoxic effects and apoptosis of cells treated with antitumor drugs combined with PPZ. Adriamycin (ADR) was used as probe to estimate drug accumulation and retention with PPZ pretreatment. Results After 24 hours, the expression of vacuolar H+-ATPase in cells pretreated with PPZ of 10 μg/ml (1.19±0.03) or 100 μg/ml (0. 70±0.03) was significantly lower than that in blank control (1.53±0. 05), but this expression was increased by pretreatment with PPZ of 1 μg/ml (2.29 +0.06, P<0.05). The inhibitory effects of PPZ (10 μg/ml) on vacuolar H+-ATPase was observed at 6 hours (0.32±0.02)and 12 hours (0. 13±0.02). And it could alter the intracellular distribution of vacuolar H+-ATPase at 24-hours. The intracellular pH value in cells pretreated with PPZ of 10 μg/ml (7.44±0. 09 ) or 100 μg/ml (7.31 ± 0. 06) was significantly decreased in comparison with untreated cells (7.51±0.05, P< 0. 01). After administration of anti-tumor drugs, the viability in SGC7901 cells pretreated with PPZ for 24 hours (58.71%±1.18 %) was significantly lower than that in cells untreated with PPZ (74. 33% ± 1.77%, P<0.05), while thetotal and early apoptotic rates in former cells(80.81% ±1. 16% and 77.52 %±1.13 %, respectively) were significantly higher than those in later cells (26. 42%±1.19% and 23. 18% ±0.92%,respectively,P < 0. 01). And the ADR releasing index in cells treated with PPZ (20, 50 and 100 μg/ml) for 24 hours was obviously lower than that in the blank control (0. 164±0. 013, 0. 162±0.015, 0. 152±0. 012 vs 0. 277±0. 011, respectively, P<0. 01). Conclusion The sensitivity of human gastric adenocarcinoma cell line to antitumor drugs may be increased by pretreatment with PPZ. Key words: Vacuolar proton-translocationg ATPases; Pantoprazole; Antineoplastic agents