氨氯地平对小鼠肝癌H22体内外抗肿瘤作用研究
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摘要
钙离子作为细胞内的重要信使,参与细胞多种重要功能的调节。氨氯地平,属于二氢吡啶类选择性L型钙离子通道阻滞剂,大量研究表明,其通过阻断细胞膜上的L型钙通道,在心血管系统疾病的治疗方面具有良好疗效。近年来亦有研究发现氨氯地平在抗肿瘤增殖以及影响肿瘤细胞增殖相关蛋白表达方面具有作用[1,2]。本实验通过研究氨氯地平对小鼠肝癌H22细胞的体内外抗肿瘤作用,进一步探讨氨氯地平在抗肿瘤方面的作用机制,为以氨氯地平为代表的钙拮抗药物在抗肿瘤方面的临床应用提供一定的实验依据。
目的:探讨氨氯地平对小鼠肝癌H22的体内外抗肿瘤作用,并分析其可能的作用机制。
方法:采用MTT法,设立氨氯地平(28×10~(-3)、14×10~(-3)、7×10~(-3)、3.5×10~(-3)、1.75×10~(-3)mg/mL)五个浓度梯度组,分别作用24h、36h、48h,检测其对小鼠肝癌H22细胞不同作用时间和不同浓度下细胞增殖的影响;建立小鼠肝癌H22荷瘤小鼠模型,通过抽取腹水瘤小鼠体内肿瘤细胞接种至小鼠腋下的方式,将小鼠划分为四个不同药物作用小组(氨氯地平3 mg/kg.d组、氨氯地平10mg/kg.d组、5-FU 10 mg/kg.3d组、生理盐水0.1 ml/10g.d组),连续灌胃10 d处死小鼠,剥离肿瘤组织并称取瘤重,观察氨氯地平对小鼠体内肿瘤的抑制作用,并采用免疫组织化学方法检测肿瘤组织切片中细胞凋亡相关基因bcl-2和bax蛋白的表达水平;流式细胞仪(FCM)检测1/2IC_(50)氨氯地平组和IC_(50)氨氯地平组处理48h后的小鼠肝癌H22细胞周期的变化情况,观察氨氯地平对肿瘤细胞周期的阻滞作用;收集经不同浓度1/2IC_(50)氨氯地平浓度组,IC_(50)氨氯地平浓度组,空白组,5-FU 0.38μmol·L~(-1)组,作用48h后的小鼠肝癌H22细胞,通过免疫组化和半定量逆转录聚合酶链反应(RT-PCR)方法检测氨氯地平对小鼠肝癌H22细胞周期蛋白Cyclin B1、凋亡基因p53表达的影响。
结果: MTT法检测发现,氨氯地平对小鼠肝癌H22具有抗增殖作用,并呈剂量和时间依赖性。经中效方程计算得氨氯地平对小鼠肝癌H22细胞的半数抑制浓度(IC_(50))为13.4μmol·L~(-1)。
体内抗肿瘤实验研究发现,高剂量氨氯地平组(10mg/kg.d组)对小鼠肝癌H22有明显的抑制作用,以生理盐水组为对照,其抑瘤率高达40.5 %。
免疫组化学方法研究发现,氨氯地平对细胞凋亡重要调控基因bcl-2、bax蛋白表达存在明显影响,其中小鼠肝癌H22细胞bcl-2蛋白存在表达下降,而bax蛋白存在高表达现象,bcl-2/bax比例也有明显的下调趋势。
流式细胞仪检测发现,经氨氯地平作用48h,小鼠肝癌H22细胞被阻滞于细胞周期G2期,并剂量依赖性的发生凋亡。在此基础上通过免疫组化和RT-PCR方法检测发现,氨氯地平13.4μmol·L~(-1)组可显著提高凋亡基因p53表达,降低细胞周期蛋白Cyclin B1的表达。
结论:氨氯地平对小鼠肝癌H22细胞具有体内外抗增殖作用及促进细胞凋亡作用,此作用与上调凋亡相关基因和下调细胞周期相关蛋白表达有关。
Objective:to investigate the anti-tumor effect Of amlodipine on murine hepatic cancer H22 in vitro and in vivo and vitro ,and to analyze its possible mechanism.
Method: for the effect of amlodipine on the growth of murine hepatic cancer H22 cells , five concentration gradients of amlodipine (28×10~(-3),14×10~(-3),7×10~(-3),3.5×10~(-3),1.75×10~(-3)mg/mL) were introduced. At the 12th、24th、36th、48 th h after the cells have exposed the drugs , the cells proliferation was evaluated by MTT assay; while the effect of amlodipine on the growth of murine hepatic cancer H22 in vivo,the KM mice bearing inoculum of murine hepatic cancer H22 were introduced by inoculated the tumor to mice armpit subcutaneously,The mice were divided into four groups with different drugs (amlodipine 3 mg / kg.d Ig. amlodipine 10mg/kg.d Ig. ,5-FU 10 mg/kg.3d Ig.and normal saline 0.1 ml/10g . d Ig. respectivly) and treated for 10 days, then,stripped tumors and measured the weight. The cell apoptosis-related genes bcl-2 and bax protein expression of murine hepatic cancer H22 were observed by immunohistochemistry in tumor tissue sections. The cell cycle of murine hepatic cancer H22 cells were detected by Flow cytometry (FCM) at the 48thh after treated by the drugs(1/2IC_(50) of amlodipine, IC_(50) of amlodipine, normal saline, and 0.38μmol·L~(-1) of 5-FU 0.38μmol·L~(-1) respectivly).
The effect of amlodipine on the expression of Cyclin B1 in murine hepatic cancer H22 cells was detected by immunohistochemistry, and The effect of amlodipine on expression of p53 gene murine hepatic cancer H22 cells was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).
Results: amlodipine exhibed a significant anti-proliferative effect on murine hepatic cancer H22 cells by dose and time dependently,with a 3.4μmol·L~(-1) of median inhibitory concentration (IC_(50)) .
high-dose amlodipine (amlodipine 10mg/kg. d ip.) showed significant anti tumor effect on the mice bearing murine hepatic cancer H22 with the inhibitory rate of 40.5%.
amlodipine decreased the expression of the apoptosis regulatory genes bcl-2, increased the expression of bax protein , and bcl-2/bax ratio was been downward obviously.
amlodipine could significantly arrest the cells cycle in the G2 phase, and could induce apoptosis in the tumor cells with a dose–dependent manner. And the mechanisim of the effect of amlodipine on the cell cycle and apoptosis were involved in the reduction of cell cycle protein Cyclin B1 expression and increase of p53 gene expression.
Conclusion: amlodipine exhibited a significant anti-tumor effect on murine hepatic cancer H22 both in vitro and invivo,and these effects were related to the cell cycle arresting and apoptosis activating by up cell cycle related gene Cyclin B1 and bax protein,p53 gene expression,down bax protein expression .
目的:探讨氨氯地平对小鼠肝癌H22的体内外抗肿瘤作用,并分析其可能的作用机制。
方法:采用MTT法,设立氨氯地平(28×10~(-3)、14×10~(-3)、7×10~(-3)、3.5×10~(-3)、1.75×10~(-3)mg/mL)五个浓度梯度组,分别作用24h、36h、48h,检测其对小鼠肝癌H22细胞不同作用时间和不同浓度下细胞增殖的影响;建立小鼠肝癌H22荷瘤小鼠模型,通过抽取腹水瘤小鼠体内肿瘤细胞接种至小鼠腋下的方式,将小鼠划分为四个不同药物作用小组(氨氯地平3 mg/kg.d组、氨氯地平10mg/kg.d组、5-FU 10 mg/kg.3d组、生理盐水0.1 ml/10g.d组),连续灌胃10 d处死小鼠,剥离肿瘤组织并称取瘤重,观察氨氯地平对小鼠体内肿瘤的抑制作用,并采用免疫组织化学方法检测肿瘤组织切片中细胞凋亡相关基因bcl-2和bax蛋白的表达水平;流式细胞仪(FCM)检测1/2IC_(50)氨氯地平组和IC_(50)氨氯地平组处理48h后的小鼠肝癌H22细胞周期的变化情况,观察氨氯地平对肿瘤细胞周期的阻滞作用;收集经不同浓度1/2IC_(50)氨氯地平浓度组,IC_(50)氨氯地平浓度组,空白组,5-FU 0.38μmol·L~(-1)组,作用48h后的小鼠肝癌H22细胞,通过免疫组化和半定量逆转录聚合酶链反应(RT-PCR)方法检测氨氯地平对小鼠肝癌H22细胞周期蛋白Cyclin B1、凋亡基因p53表达的影响。
结果: MTT法检测发现,氨氯地平对小鼠肝癌H22具有抗增殖作用,并呈剂量和时间依赖性。经中效方程计算得氨氯地平对小鼠肝癌H22细胞的半数抑制浓度(IC_(50))为13.4μmol·L~(-1)。
体内抗肿瘤实验研究发现,高剂量氨氯地平组(10mg/kg.d组)对小鼠肝癌H22有明显的抑制作用,以生理盐水组为对照,其抑瘤率高达40.5 %。
免疫组化学方法研究发现,氨氯地平对细胞凋亡重要调控基因bcl-2、bax蛋白表达存在明显影响,其中小鼠肝癌H22细胞bcl-2蛋白存在表达下降,而bax蛋白存在高表达现象,bcl-2/bax比例也有明显的下调趋势。
流式细胞仪检测发现,经氨氯地平作用48h,小鼠肝癌H22细胞被阻滞于细胞周期G2期,并剂量依赖性的发生凋亡。在此基础上通过免疫组化和RT-PCR方法检测发现,氨氯地平13.4μmol·L~(-1)组可显著提高凋亡基因p53表达,降低细胞周期蛋白Cyclin B1的表达。
结论:氨氯地平对小鼠肝癌H22细胞具有体内外抗增殖作用及促进细胞凋亡作用,此作用与上调凋亡相关基因和下调细胞周期相关蛋白表达有关。
Objective:to investigate the anti-tumor effect Of amlodipine on murine hepatic cancer H22 in vitro and in vivo and vitro ,and to analyze its possible mechanism.
Method: for the effect of amlodipine on the growth of murine hepatic cancer H22 cells , five concentration gradients of amlodipine (28×10~(-3),14×10~(-3),7×10~(-3),3.5×10~(-3),1.75×10~(-3)mg/mL) were introduced. At the 12th、24th、36th、48 th h after the cells have exposed the drugs , the cells proliferation was evaluated by MTT assay; while the effect of amlodipine on the growth of murine hepatic cancer H22 in vivo,the KM mice bearing inoculum of murine hepatic cancer H22 were introduced by inoculated the tumor to mice armpit subcutaneously,The mice were divided into four groups with different drugs (amlodipine 3 mg / kg.d Ig. amlodipine 10mg/kg.d Ig. ,5-FU 10 mg/kg.3d Ig.and normal saline 0.1 ml/10g . d Ig. respectivly) and treated for 10 days, then,stripped tumors and measured the weight. The cell apoptosis-related genes bcl-2 and bax protein expression of murine hepatic cancer H22 were observed by immunohistochemistry in tumor tissue sections. The cell cycle of murine hepatic cancer H22 cells were detected by Flow cytometry (FCM) at the 48thh after treated by the drugs(1/2IC_(50) of amlodipine, IC_(50) of amlodipine, normal saline, and 0.38μmol·L~(-1) of 5-FU 0.38μmol·L~(-1) respectivly).
The effect of amlodipine on the expression of Cyclin B1 in murine hepatic cancer H22 cells was detected by immunohistochemistry, and The effect of amlodipine on expression of p53 gene murine hepatic cancer H22 cells was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).
Results: amlodipine exhibed a significant anti-proliferative effect on murine hepatic cancer H22 cells by dose and time dependently,with a 3.4μmol·L~(-1) of median inhibitory concentration (IC_(50)) .
high-dose amlodipine (amlodipine 10mg/kg. d ip.) showed significant anti tumor effect on the mice bearing murine hepatic cancer H22 with the inhibitory rate of 40.5%.
amlodipine decreased the expression of the apoptosis regulatory genes bcl-2, increased the expression of bax protein , and bcl-2/bax ratio was been downward obviously.
amlodipine could significantly arrest the cells cycle in the G2 phase, and could induce apoptosis in the tumor cells with a dose–dependent manner. And the mechanisim of the effect of amlodipine on the cell cycle and apoptosis were involved in the reduction of cell cycle protein Cyclin B1 expression and increase of p53 gene expression.
Conclusion: amlodipine exhibited a significant anti-tumor effect on murine hepatic cancer H22 both in vitro and invivo,and these effects were related to the cell cycle arresting and apoptosis activating by up cell cycle related gene Cyclin B1 and bax protein,p53 gene expression,down bax protein expression .
引文
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[8]徐兴华,孙文娟.氨氯地平对人乳腺癌细胞株MDA-MB-231侵袭影响的体外实验[J].第三军医大学学报, 2008, 30(4):340-342.
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[2] YoshidaJ,Ishibashi,Nishio M.G1 cell cycle arrest by amlodipine,a dihydropyridine Ca2+ channel blocker,in human epidermoid carcinoma A431 cells[J]. Biochem Pharmacol,. 2007,73(7):973-53.
[3]郑晓克.抗肿瘤药物的研究进展[J].中山大学研究生学报, 2008, 29(4):2008.
[4]程春旭,高颜茹.抗肿瘤药物作用机制的研究进展[J].吉林医学,2009, 30(23):3080-3083.
[5] Chu-Xia Deng. BRCAI:cell cycle checkpoint,genetic instability,DNA damage response and cancer evolution[J].Nucleic Acids Research, 2006, 34(5):1416-1426.
[6] Cross BW, Kirby MG, Miller S, et al. A multicentre study of the safety and effecacy of anzlodipine in mild moderate hypertension[J]. Br J Clin Pract, 1993, 47(5):237-240.
[7]王富贵,王慧,李文华.左旋氨氯地平治疗老年高血压患者的疗效观察[J].中国实用医药, 2009, 4(22):21-22.
[8]徐兴华,孙文娟.氨氯地平对人乳腺癌细胞株MDA-MB-231侵袭影响的体外实验[J].第三军医大学学报, 2008, 30(4):340-342.
[9] Bruce E C,James MR.Cel1 cycle and cancer[J].J Nationa1 Cancer Institute, 1995,87:1499.
[10] Zernig G. widening potential for Ca2+antagonists:non-L-type Ca2+channel interaction[J].Trends Pharmacology, 1990, 11:38-44.
[11] jensen RL , Wurster RD.Calcium channel antagonists inhibit growth of subcutaneous xenograft meningiomas in nude mice[J].Surg Neurol 2001, 55:275-283.
[12] Hartwell LH,Kastan MB.Cell cycle control and cancer[J]. Science, 1994, 266(5792):1821-1828.
[13] Badie C,Bourhis J,Sobczak-Thepot J,et al. p53-dependent G2 arrest associated with a decrease in cyclins A2 and B1 levels in a human carcinoma cell line [J].Br J Cancer, 2000, 82(3) :642-650.
[14] Soria JC,Jang SJ,Khuri FR,et al. Overexpression of cyclin B1 in early-stage non-small cell lung cancer and its clinical implication [J].Cancer Res, 2000, 60(15):4000-4004.
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