葛根素对肾癌GRC-1细胞多药耐药的逆转作用
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摘要
目的探讨葛根素对肾癌GRC-1细胞多药耐药的逆转作用。方法体外培养GRC-1人肾细胞癌株,并分为空白对照组、阿霉素处理组(10mg·L-1ADM)、葛根素处理的对照组(0.48g·L-1Pur)、阿霉素联合葛根素处理组(10mg·L-1ADM+0.24g·L-1Pur或10mg·L-1ADM+0.48g·L-1Pur),药物处理24h后,采用细胞毒性实验检测GRC-1细胞的生存率,用流式细胞术(Annexin-V/PI双标记)及TUNEL法检测细胞的凋亡情况。结果以对照组细胞生存率为100%,ADM组细胞生存率为(69.7±0.04)%,显著低于对照组(P<0.05),经0.12、0.24和0.48g·L-1Pur联合用药后,细胞生存率分别为(70.1±0.03)%、(63.2±0.02)%和(42.1±0.04)%,均明显低于空白对照组(P<0.05),且0.24和0.48g·L-1Pur联合用药的细胞生存率显著低于ADM处理组(P<0.05);0.12和0.24g·L-1Pur单独用药的细胞生存率分别为(97.4±0.02)%和(90.1±0.01)%,与对照组比较差异无统计学意义(P>0.05),而0.48g·L-1Pur单独用药的细胞生存率为(75.0±0.03)%,显著低于空白对照组(P<0.05)。此外,与空白对照组相比,0.24和0.48g·L-1Pur处理细胞后,细胞凋亡率明显升高(P<0.05),且随Pur剂量增加而升高;而0.24和0.48g·L-1Pur联合用药组细胞凋亡率均明显高于ADM组(P<0.05)。结论葛根素可有效促进肾癌GRC-1细胞的凋亡,并可减轻GRC-1细胞对阿霉素的耐药性。
Objective To investigate the reversal effect of puerarin on mediated multidrug resistance in renal cell carcinoma line GRC-1. Methods GRC-1 cells were cultivated in vitro, and four groups were designed in this experiment: control group, adriamycin treatment group with 10 mg·L -1 ADM, puerarin control group with 0.48 g·L -1 Pur, combined treatment group in which 10 mg·L -1 ADM+0.24 g·L -1 Pur or 10 mg·L -1 ADM+0.48 g·L -1 Pur respectively. After treatment with drugs for 24 h,MTT assay was employed to measure cell viability. Flow cytometry with Annexin-V/PI double-labeled) and TUNEL method were used for detecting cell apoptosis. Results Compared with the control group in which the GRC-1 cell viability was 100%, cell viability in ADM group significantly decreased to (69.7±0.04)% (P<0.05). In the combined treatment group, in which 10 mg ·L -1 ADM, combined with 0.12,0.24 and 0.48 g·L -1 Pur was administered to GRC-1cells, the cell viabilities were (70.1±0.03)%, (63.2±0.02)% and (42.1±0.04)% respectively, which were obviously lower than that in the control group (P<0.05), The cells viabilities of two subgroups, in which 10 mg ·L -1 ADM was combined with 0.24 or 0.48 g·L -1 Pur, were much lower than that in the ADM group(P<0.05). The cell viabilities of the only 0.12 g ·L -1 or 0.24 g·L -1 Pur treated group were respectively (97.4±0.02)% and (90.1±0.01)%, having no significant difference from the control group (P>0.05). But after treated by 0.48 g ·L -1 Pur alone, the cell viability was (75.0±0.03)%, much lower than that in the control group (P<0.05). Compared with the control group, the apoptotic rates of GRC-1cells in 0.24 and 0.48 g ·L -1 puerarin groups significantly increased in a dose-dependent manner (P<0.05). The apoptotic rates of 10 mg ·L -1 adriamycin plus 0.24 g·L -1 puerarin or 0.48 g·L -1 puerarin groups were remarkablely higher than that of the ADM group (P<0.05). Conclusion Puerarin may effectively promote the apoptosis of GRC-1 cells and increase the anticarcinogenic effect of adriamycin by reversing multidrug resistance in GRC-1 cells.
Objective To investigate the reversal effect of puerarin on mediated multidrug resistance in renal cell carcinoma line GRC-1. Methods GRC-1 cells were cultivated in vitro, and four groups were designed in this experiment: control group, adriamycin treatment group with 10 mg·L -1 ADM, puerarin control group with 0.48 g·L -1 Pur, combined treatment group in which 10 mg·L -1 ADM+0.24 g·L -1 Pur or 10 mg·L -1 ADM+0.48 g·L -1 Pur respectively. After treatment with drugs for 24 h,MTT assay was employed to measure cell viability. Flow cytometry with Annexin-V/PI double-labeled) and TUNEL method were used for detecting cell apoptosis. Results Compared with the control group in which the GRC-1 cell viability was 100%, cell viability in ADM group significantly decreased to (69.7±0.04)% (P<0.05). In the combined treatment group, in which 10 mg ·L -1 ADM, combined with 0.12,0.24 and 0.48 g·L -1 Pur was administered to GRC-1cells, the cell viabilities were (70.1±0.03)%, (63.2±0.02)% and (42.1±0.04)% respectively, which were obviously lower than that in the control group (P<0.05), The cells viabilities of two subgroups, in which 10 mg ·L -1 ADM was combined with 0.24 or 0.48 g·L -1 Pur, were much lower than that in the ADM group(P<0.05). The cell viabilities of the only 0.12 g ·L -1 or 0.24 g·L -1 Pur treated group were respectively (97.4±0.02)% and (90.1±0.01)%, having no significant difference from the control group (P>0.05). But after treated by 0.48 g ·L -1 Pur alone, the cell viability was (75.0±0.03)%, much lower than that in the control group (P<0.05). Compared with the control group, the apoptotic rates of GRC-1cells in 0.24 and 0.48 g ·L -1 puerarin groups significantly increased in a dose-dependent manner (P<0.05). The apoptotic rates of 10 mg ·L -1 adriamycin plus 0.24 g·L -1 puerarin or 0.48 g·L -1 puerarin groups were remarkablely higher than that of the ADM group (P<0.05). Conclusion Puerarin may effectively promote the apoptosis of GRC-1 cells and increase the anticarcinogenic effect of adriamycin by reversing multidrug resistance in GRC-1 cells.
引文
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[15]张林,刘伏友,彭佑铭,等.维生素E对顺铂所致的小鼠肾毒性的影响及其机制研究[J].肿瘤药学,2012,2(5):343-346,350.Zhang L,Liu FY,Peng YM,et al.The Effect of Vitamin E on Cisplatin Induced Nephrotoxicity in Mice and its Mech-anism[J].Anti-Tumor Pharm,2012,2(5):343-346,350.
[2]任华益,曾勇,谢仲秋,等.上皮间质转化与肿瘤干细胞在化疗耐药中作用的研究进展[J].肿瘤药学,2012,2(1):7-9.Ren HY,Zeng Y,Xie ZQ,et al.Research Progress on Effects of Epithelial-mesenchymal Transition and Cancer Stem Cell on Chemotherapy Resistance[J].Anti-Tumor Pharm,2012,2(1):7-9.
[3]付荣国,王中东,周琳.葛根素对肾缺血/再灌注时肾小管上皮细胞c-Fos蛋白表达的影响[J].西安交通大学学报:医学版,2005,26(5):454-456.Fu RG,Wang ZD,Zhou L.Effects of puerarin injection on c-Fos protein of renal tubule cells during ischemic-reperfusion in kidney[J].J X‘ian Jiaotong Univer:Med Sci,2005,26(5):454-456.
[4]Liu XJ,Zhao J,Gu XY.The effects of genistein and pu-erarin on the activation of nuclear factor-kappaB and the production of tumor necrosis factor-alpha in asthma pa-tients[J].Pharmazie,2010,65(2):127-131.
[5]Wang H,Liu Y,Zeng Z,et al.Study on HPLC chromatographic fingerprint of antitumor active site SSCE of Caulis spatholobi[J].Chin J Chin Mater Med,2012,36(18):2525-2529.
[6]Zhang Q,Huang WD,Lv XY,et al.Puerarin protects differentiated PC12cells from H2O2-induced apoptosis through the PI3K/Akt signalling pathway[J].Cell Biol Int,2012,36(5):419-426.
[7]Yu Z,Li W.Induction of apoptosis by puerarin in colon cancer HT-29cells[J].Cancer Lett,2006,238(1):53-60.
[8]Moriarty K,Kim KH,Bender JR.Estrogen receptor-mediated rapid signaling[J].Endocrinol,2006,147(12):5557-5563.
[9]Hien TT,Kim HG,Han EH,et al.Molecular mechanism of suppression of MDR1by puerarin from Pueraria lo-bata via NF-κB pathway and cAMP-responsive element transcriptional activity-dependent up-regulation of AMP-activated protein kinase in breast cancer MCF-7/adr cells[J].Mol Nutr Food Res,2010,54(7):918-928.
[10]Walsh N,Larkin A,Kennedy S,et al.Expression of multidrug resistance markers ABCB1(MDR-1/P-gp)and ABCC1(MRP-1)in renal cell carcinoma[J].BMC Urol,2009,(9):6.
[11]Soto-Vega E,Arroyo C,Richaud-Patin Y,et al.Pgly-coprotein activity in renal clear cell carcinoma[J].Urol Oncol,2009,27(4):363-366.
[12]HodorováI,RybárováS,Solár P,et al.Multidrug resistance proteins in renal cell carcinoma[J].Folia Biol(Praha),2008,54(6):187-192.
[13]Bilim V,Yuuki K,Itoi T,et al.Double inhibition of XIAP and Bcl-2axis is beneficial for retrieving sensitivity of renal cell cancer to apoptosis[J].Br J Cancer,2008,98(5):941-949.
[14]Mignogna C,Staibano S,Altieri V,et al.Prognostic sig-nificance of multidrug-resistance protein(MDR-1)in renal clear cell carcinomas:a five year follow-up analysis[J].BMC Cancer,2006,(6):293.
[15]张林,刘伏友,彭佑铭,等.维生素E对顺铂所致的小鼠肾毒性的影响及其机制研究[J].肿瘤药学,2012,2(5):343-346,350.Zhang L,Liu FY,Peng YM,et al.The Effect of Vitamin E on Cisplatin Induced Nephrotoxicity in Mice and its Mech-anism[J].Anti-Tumor Pharm,2012,2(5):343-346,350.