CAJ | 학술논문

目的探讨质子泵抑制剂(PPI)泮托拉唑(PPZ)是否通过抑制空泡型质子泵来逆转细胞内外pH梯度,从而增加肿瘤细胞化学治疗敏感性,并探讨PPZ最佳预处理时间、剂量及机制.方法免疫印迹及免疫荧光法比较PPZ处理前、后人胃低分化腺癌细胞株SGC7901空泡型质子泵的表达及胞内分布变化.用BCECF-AM荧光探针检测不同浓度PPZ作用不同时间对细胞内pH值的影响.用四甲基偶氮唑盐比色法和膜联蛋白V-异硫氰酸荧光素-碘化丙啶试剂盒检测化学治疗药物联合PPZ的细胞毒性和细胞凋亡变化.用阿霉素检测PPZ对细胞内药物蓄积和潴留量的影响.结果 10和100μg/ml PPZ作用24 h后,空泡型质子泵表达的相对吸光度值(1.19±0.03和0.70±0.03)明显低于空白对照组(1.53±0.05),而1μg/ml PPZ可促进其表达(2.29±0.06).在10μg/ml PPZ作用第6和12小时,可见其对空泡型质子泵表达的抑制作用(相对吸光度值分别为0.32±0.02和0.13±0.02),在作用24 h后可改变空泡型质子泵的细胞内分布.10和100 ttg/ml PPZ作用24 h可使细胞内pH值(7.44±0.09和7.31±0.06)明显低于空白对照组(7.51±0.05),10μg/ml及以上浓度的PPZ可逆转细胞内外pH梯度.PPZ预处理24 h后予化学药物治疗的细胞存活率(58.71%±1.18%)低于单用化学治疗药物(74.33%±1.77%,P<0.05),其总凋亡率(80.81%±1.16%)和早期凋亡率(77.52%±1.13%)显著高于单用化学治疗药物(26.42%±1.19%和23.18%±0.92%,P值均<0.01).20、50和100 μg/ml PPZ作用24 h后的阿霉素释放指数(0.164±0.013、0.162±0.015、0.152±0.012)低于空白对照组(0.277±0.011,P值均<0.01).结论 PPZ预处理可提高人胃腺癌细胞的化学治疗敏感性.
목적 탐토질자빙억제제(PPI)반탁랍서(PPZ)시부통과억제공포형질자빙래역전세포내외pH제도,종이증가종류세포화학치료민감성,병탐토PPZ최가예처리시간、제량급궤제.방법 면역인적급면역형광법비교PPZ처리전、후인위저분화선암세포주SGC7901공포형질자빙적표체급포내분포변화.용BCECF-AM형광탐침검측불동농도PPZ작용불동시간대세포내pH치적영향.용사갑기우담서염비색법화막련단백V-이류청산형광소-전화병정시제합검측화학치료약물연합PPZ적세포독성화세포조망변화.용아매소검측PPZ대세포내약물축적화저류량적영향.결과 10화100μg/ml PPZ작용24 h후,공포형질자빙표체적상대흡광도치(1.19±0.03화0.70±0.03)명현저우공백대조조(1.53±0.05),이1μg/ml PPZ가촉진기표체(2.29±0.06).재10μg/ml PPZ작용제6화12소시,가견기대공포형질자빙표체적억제작용(상대흡광도치분별위0.32±0.02화0.13±0.02),재작용24 h후가개변공포형질자빙적세포내분포.10화100 ttg/ml PPZ작용24 h가사세포내pH치(7.44±0.09화7.31±0.06)명현저우공백대조조(7.51±0.05),10μg/ml급이상농도적PPZ가역전세포내외pH제도.PPZ예처리24 h후여화학약물치료적세포존활솔(58.71%±1.18%)저우단용화학치료약물(74.33%±1.77%,P<0.05),기총조망솔(80.81%±1.16%)화조기조망솔(77.52%±1.13%)현저고우단용화학치료약물(26.42%±1.19%화23.18%±0.92%,P치균<0.01).20、50화100 μg/ml PPZ작용24 h후적아매소석방지수(0.164±0.013、0.162±0.015、0.152±0.012)저우공백대조조(0.277±0.011,P치균<0.01).결론 PPZ예처리가제고인위선암세포적화학치료민감성.
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.