苦参碱对BBN诱发大鼠膀胱癌发生发展作用及其机制探讨
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摘要
目的研究苦参碱对N-丁基-N-(4-羟丁基)亚硝基胺(BBN)致大鼠膀胱癌发生发展的影响及其分子机制。方法雄性Sprague-Dawley大鼠,随机分为空白对照组、BBN组(模型组)、阳性药物对照组〔塞来昔布500mg/(kg·d)〕、苦参碱50、100和200mg/(kg·d)剂量组6组。采用BBN诱发大鼠膀胱癌,苦参碱灌胃给药35周,病理学观察膀胱组织形态,免疫组织化学法和蛋白质印迹法分别检测膀胱组织中表皮生长因子受体(epidermal growth factor receptor,EGFR)蛋白表达和细胞周期调控通路p16INK4a/Cyclin D1/CDK4各蛋白的表达。结果 BBN组、塞来昔布组、苦参碱组膀胱癌的发生率没有显著性变化,差异无统计学意义,P=0.068。在确诊的膀胱癌组织中,与BBN组相比,苦参碱组浸润性膀胱癌的发生率明显降低,χ2=6.065,P=0.014。免疫组织化学法检测结果显示,BBN组EGFR蛋白表达评分为3.845±0.972,与50mg/kg苦参碱的2.691±1.045(P=0.036)、100 mg/kg苦参碱的2.230±0.748(P=0.029)和200mg/kg苦参碱的2.739±0.814(P=0.041)比较,差异有统计学意义。蛋白质印迹法检测结果显示,大鼠膀胱组织p16INK4a蛋白平均表达量苦参碱200mg/kg组(0.448 6±0.195)较BBN组(0.303 8±0.183)上调,P=0.045;Cyclin D1蛋白的平均表达量苦参碱50mg/kg组(0.448 0±0.236)和200mg/kg组(0.389 2±0.242)较BBN组(0.630 2±0.221)下降,P值分别为0.018和0.010;CDK4蛋白的平均表达量苦参碱200mg/kg组(0.648 4±0.366)较BBN组(0.913 3±0.312)下降,P=0.041。结论苦参碱对BBN诱导的大鼠膀胱癌的发生无抑制作用,但可抑制其向浸润进展,其作用机制可能与抑制大鼠膀胱组织EGFR蛋白的表达及对p16INK4a/Cyclin D1/CDK4细胞周期调控通路的调控有关。
OBJECTIVE The present study was designed to investigate the effects and mechanisms of matrine on occurrence and development of urinary bladder cancers induced by N-butyl-N-(4-hydroxybutyl)nitrosamine(BBN)in rats.METHODS After 7days of acclimatization,male Sprague-Dawley rats were randomly divided into six groups:control,BBN,celecoxib and matrine[50,100,200mg/(kg·d)]groups.The rats were given BBN(200mg/0.5mL)twice a week for a period of 8weeks.Oral administration of matrine(50,100 and 200mg/kg)or celecoxib(500mg/kg)were started one week before BBN exposure for 35 weeks.Half of each bladder was analyzed histopathologically and immunohitochemically,and the otherhalf extracted for protein analysis by Western blot.RESULTS The incidence of urothelial carcinoma was not changed significantly in BBN,celecoxib and matrine group,P=0.068.However,the frequency of invasive tumors in matrine treatment group was significantly lower than those in BBN alone groups(2χ=6.065,P=0.014).The expression index of epidermal growth factor receptor(EGFR)in matrine(50,100 and 200mg/kg)group were(2.691±1.045),(2.230±0.748)and(2.739±0.814)respectively,which were significantly lower than in BBN group(3.845±0.972),P=0.036,P=0.029 and P=0.041.Compared with BBN group(0.303 8±0.183),the expressions of p16INK4 ain matrine(200 mg/kg)group(0.448 6±0.195)were increased,P=0.045.The expressions of Cyclin D1 in matrine(50,200mg/kg)group(0.448 0±0.236,0.389 2±0.242)were significantly lower than that in BBN group(0.630 2±0.221),P=0.018 and P=0.010.Compared with BBN group(0.913 3±0.312),Cyclin D1-dependent kinase 4(CDK4)in matrine(200mg/kg)group(0.648 4±0.366)also performed a significant decrease,P=0.041.CONCLUSION Our results suggest that matrine suppress BBN-induced bladder tumor invasion via modulation of bladder EGFR expression and regulation of p16INK4a/Cyclin D1/CDK4 pathway.
OBJECTIVE The present study was designed to investigate the effects and mechanisms of matrine on occurrence and development of urinary bladder cancers induced by N-butyl-N-(4-hydroxybutyl)nitrosamine(BBN)in rats.METHODS After 7days of acclimatization,male Sprague-Dawley rats were randomly divided into six groups:control,BBN,celecoxib and matrine[50,100,200mg/(kg·d)]groups.The rats were given BBN(200mg/0.5mL)twice a week for a period of 8weeks.Oral administration of matrine(50,100 and 200mg/kg)or celecoxib(500mg/kg)were started one week before BBN exposure for 35 weeks.Half of each bladder was analyzed histopathologically and immunohitochemically,and the otherhalf extracted for protein analysis by Western blot.RESULTS The incidence of urothelial carcinoma was not changed significantly in BBN,celecoxib and matrine group,P=0.068.However,the frequency of invasive tumors in matrine treatment group was significantly lower than those in BBN alone groups(2χ=6.065,P=0.014).The expression index of epidermal growth factor receptor(EGFR)in matrine(50,100 and 200mg/kg)group were(2.691±1.045),(2.230±0.748)and(2.739±0.814)respectively,which were significantly lower than in BBN group(3.845±0.972),P=0.036,P=0.029 and P=0.041.Compared with BBN group(0.303 8±0.183),the expressions of p16INK4 ain matrine(200 mg/kg)group(0.448 6±0.195)were increased,P=0.045.The expressions of Cyclin D1 in matrine(50,200mg/kg)group(0.448 0±0.236,0.389 2±0.242)were significantly lower than that in BBN group(0.630 2±0.221),P=0.018 and P=0.010.Compared with BBN group(0.913 3±0.312),Cyclin D1-dependent kinase 4(CDK4)in matrine(200mg/kg)group(0.648 4±0.366)also performed a significant decrease,P=0.041.CONCLUSION Our results suggest that matrine suppress BBN-induced bladder tumor invasion via modulation of bladder EGFR expression and regulation of p16INK4a/Cyclin D1/CDK4 pathway.
引文
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[15]Liu JY,Qian D,He LR,et al.PinX1suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1pathway[J].Mol Cancer,2013,12(1):148.
[16]王新华,崔涌,孟宪杰.PTEN、p16、突变型p53在膀胱癌组织中的表达及意义[J].山东医药,2013,53(9):37-39.
[17]Kopparapu PK,Boorjian SA,Robinson BD,et al.Expression of cyclin d1and its association with disease characteristics in bladder cancer[J].Anticancer Res,2013,33(12):5235-5242.
[18]Ren B,Li W,Yang Y,et al.The impact of cyclin D1overexpression on the prognosis of bladder cancer:a meta-analysis[J].World J Surg Oncol,2014,12(1):55.
[19]Quentin T,Henke C,Korabiowska M,et al.Altered mRNA expression of the Rb and p16tumor suppressor genes and of CDK4in transitional cell carcinomas of the urinary bladder associated with tumor progression[J].Anticancer Res,2004,24(2B):1011-1023.
[2]Dai G,Cui L,Song L,et al.Bladder epithelial cell proliferation of rats induced by terephthalic acid-calculi[J].Food Chem Toxicol,2005,43(2):217-224.
[3]朱丹丹,姚树坤,闫建国,等.苦参碱对大鼠原发性肝癌防治作用及其机制的研究[J].中华肿瘤防治杂志,2010,17(4):241-244.
[4]王涌,刘雅辉,姜建帅,等.苦参碱对肝癌干细胞增殖及侵袭转移能力的抑制作用[J].中华实验外科杂志,2013,30(1):61-63.
[5]李海军,赵晓霞,白美玲,等.苦参碱对人乳腺癌细胞MCF-7增殖、凋亡的影响[J].江苏医药,2011,37(4):396-398.
[6]陈立波.苦参碱对宫颈癌Hela细胞增殖、凋亡及Survivin基因表达的影响[J].中国实验方剂学杂志,2013,19(15):235-238.
[7]Jacobs BL,Lee CT,Montie JE.Bladder cancer in 2010:how far have we come?[J].CA Cancer J Clin,2010,60(4):244-272.
[8]Burger M,Catto JW,Dalbagni G,et al.Epidemiology and risk factors of urothelial bladder cancer[J].Eur Urol,2013,63(2):234-241.
[9]姚玉霜,张婷婷,戚玉言,等.卵巢癌组织EGFR及下游信号分子的表达及其临床意义[J].中华肿瘤防治杂志,2011,18(3):212-215.
[10]Kim WJ,Kim SK,Jeong P,et al.A four-gene signature predicts disease progression in muscle invasive bladder cancer[J].Mol Med,2011,17(5-6):478-485.
[11]孙鸿燕,孙然,刘铁,等.结直肠癌组织EGFR表达与淋巴结及肝转移相关性的Meta分析[J].中华肿瘤防治杂志,2013,20(4):304-308.
[12]Grivas PD,Day KC,Karatsinides A,et al.Evaluation of the antitumor activity of dacomitinib in models of human bladder cancer[J].Mol Med,2013,19(1):367-376.
[13]Wülfing C,Michaels,Richel DJ,et al.A single-arm,multicenter,open-label phase 2study of lapatinib as the second-linetreatment of patients with locally advanced or metastatic transitional cell carcinoma[J].Cancer,2009,115(13):2881-2890.
[14]付子乾,黎玮.E-CD和p16在膀胱移行细胞癌组织中的表达及其临床意义[J].中华实验外科杂志,2011,28(2):284-285.
[15]Liu JY,Qian D,He LR,et al.PinX1suppresses bladder urothelial carcinoma cell proliferation via the inhibition of telomerase activity and p16/cyclin D1pathway[J].Mol Cancer,2013,12(1):148.
[16]王新华,崔涌,孟宪杰.PTEN、p16、突变型p53在膀胱癌组织中的表达及意义[J].山东医药,2013,53(9):37-39.
[17]Kopparapu PK,Boorjian SA,Robinson BD,et al.Expression of cyclin d1and its association with disease characteristics in bladder cancer[J].Anticancer Res,2013,33(12):5235-5242.
[18]Ren B,Li W,Yang Y,et al.The impact of cyclin D1overexpression on the prognosis of bladder cancer:a meta-analysis[J].World J Surg Oncol,2014,12(1):55.
[19]Quentin T,Henke C,Korabiowska M,et al.Altered mRNA expression of the Rb and p16tumor suppressor genes and of CDK4in transitional cell carcinomas of the urinary bladder associated with tumor progression[J].Anticancer Res,2004,24(2B):1011-1023.