经典蛋白激酶C同工酶在胃癌细胞系及其耐药亚系中的表达与功能
Expression and function of classical protein kinase C isoenzymes in gastric cancer cell line and its drug-resistant sublines.
作者信息
Han Ying, Han Zhe-Yi, Zhou Xin-Min, Shi Ru, Zheng Yue, Shi Yong-Quan, Miao Ji-Yan, Pan Bo-Rong, Fan Dai-Ming
机构信息
Institute of Digestive Disease, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.
出版信息
World J Gastroenterol. 2002 Jun;8(3):441-5. doi: 10.3748/wjg.v8.i3.441.
AIM
To investigate the expression and function of classical protein kinase C (PKC) isoenzymes in inducing MDR phenotype in gastric cancer cells.
METHODS
Two cell lines were used in the study: gastric cancer cell SGC7901 and its drug-resistant cell SGC7901/VCR stepwise-selected by vincristine 0.3, 0.7 and 1.0 mg.L(-1), respectively. The expression of classical PKC (cPKC) isoenzymes in SGC7901 cells and SGC7901/VCR cells were detected using immunofluorescent cytochemistry, laser confocal scanning microscope and Western blot. The effects of anti-PKC isoenzymes antibody on adriamycin accumulation in SGC7901/VCR cells were determined using flow cytometric analysis.
RESULTS
(1)SGC7901 cells exhibited positive staining of PKC-alpha. SGC7901/VCR cells exhibited stronger staining of PKC-alpha than SGC7901 cells. The higher dosage vincristine selected, the much stronger staining of PKC-alpha was observed on SGC7901/VCR cells. (2)Both SGC7901 and SGC7901/VCR cells exhibited positive staining of PKC-beta I and PKC-beta II with no significant difference. (3) Compared with SGC7901, SGC7901/VCR cells had decreased adriamycin accumulation and retention. Accumulation of adriamycin in SGC7901 was 5.21+/-2.56 mg.L(-1),in SGC7901/VCR 0.3 was 0.85+/-0.29 mg.L(-1), in SGC7901/VCR 0.7 was 0.81+/-0.32 mg.L(-1), and in SGC7901/VCR 1.0 was 0.80+/-0.33 mg.L(-1); Retention of adriamycin in SGC 7901 was 2.51+/-1.23 mg.L(-1), in SGC7901/VCR 0.3 was 0.47+/-0.14 mg.L(-1), in SGC7901/VCR 0.7 was 0.44+/-0.15 mg.L(-1), and in SGC 7901/VCR 1.0 was 0.41+/-0.11 mg.L(-1). (4) Fluorescence intensity presented adriamycin accumulation in SGC7901/VCR cells was increased from 1.14+/-0.36 to 2.71+/-0.94 when cells were co-incubated with anti-PKC-alpha but not with anti-PKC-beta I PKC-beta II and PKCgamma antibodies.
CONCLUSION
PKC-alpha, but not PKC-beta I, PKC-beta II or PKCgamma, may play a role in multidrug resistance of gastric cancer cells SGC7901/VCR.
目的
研究经典蛋白激酶C（PKC）同工酶在诱导胃癌细胞多药耐药（MDR）表型中的表达及作用。
方法
本研究采用两种细胞系：胃癌细胞SGC7901及其分别用0.3、0.7和1.0 mg.L⁻¹长春新碱逐步筛选出的耐药细胞SGC7901/VCR。采用免疫荧光细胞化学、激光共聚焦扫描显微镜及蛋白质免疫印迹法检测SGC7901细胞和SGC7901/VCR细胞中经典PKC（cPKC）同工酶的表达。采用流式细胞术分析抗PKC同工酶抗体对阿霉素在SGC7901/VCR细胞中蓄积的影响。
结果
（1）SGC7901细胞PKC-α呈阳性染色。SGC7901/VCR细胞PKC-α染色强于SGC7901细胞。长春新碱选择剂量越高，SGC7901/VCR细胞PKC-α染色越强。（2）SGC7901和SGC7901/VCR细胞PKC-βⅠ和PKC-βⅡ均呈阳性染色，差异无统计学意义。（3）与SGC7901相比，SGC7901/VCR细胞阿霉素蓄积和滞留减少。SGC7901中阿霉素蓄积量为5.21±2.56 mg.L⁻¹，SGC7901/VCR 0.3中为0.85±0.29 mg.L⁻¹，SGC7901/VCR 0.7中为0.81±0.32 mg.L⁻¹，SGC7901/VCR 1.0中为0.80±0.33 mg.L⁻¹；SGC 7901中阿霉素滞留量为2.51±1.23 mg.L⁻¹，SGC7901/VCR 0.3中为0.47±0.14 mg.L⁻¹，SGC7901/VCR 0.7中为0.44±0.15 mg.L⁻¹，SGC 7901/VCR 1.0中为0.41±0.11 mg.L⁻¹。（4）当细胞与抗PKC-α抗体共同孵育时，SGC7901/VCR细胞中呈现阿霉素蓄积的荧光强度从1.14±0.36增加至2.71±0.94，而与抗PKC-βⅠ、PKC-βⅡ和PKCγ抗体共同孵育时无此现象。
结论
PKC-α而非PKC-βⅠ、PKC-βⅡ或PKCγ可能在胃癌细胞SGC7901/VCR多药耐药中起作用。
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