美洲大蠊多肽PAE2逆转肝癌多药耐药性

目的:探讨美洲大蠊多肽PAE2体内逆转肝癌多药耐药(MDR)的作用及机制。方法:采用Balb/c-nude小鼠腋下接种HepG2和HepG2/ADM细胞分别建立肝癌敏感株动物模型和肝癌MDR动物模型,模型制备成功后,将裸鼠随机分为正常组,HepG2模型组,HepG2/ADM模型组,索拉菲尼组30 mg·kg^-1,灌胃(ig)],HepG2/ADM+PAE2静脉注射(iv)]低、中、高剂量组(50,100,200 mg·kg^-1),HepG2/ADM+PAE2ig低、中、高剂量组(50,100,200 mg·kg^-1),脱脂膏组(200 mg·kg^-1,ig),美洲大蠊前期粗提物CⅡ-3组(200 mg·kg^-1,ig),每2日测量1次裸鼠体质量及肿瘤体积,并记录,末次给药后,次日取裸鼠肿瘤组织,记录瘤质量,采用免疫组织化学(IHC)和实时荧光定量聚合酶链式反应(Real-time PCR)测定美洲大蠊多肽PAE2对肿瘤组织中P糖蛋白(P-gp),肺耐药相关蛋白(LRP),乳腺癌耐药蛋白(BCRP),蛋白激酶C(PKC),谷胱甘肽巯基转移酶-π(GST-π),拓扑异构酶Ⅱ(ToPoⅡ),MDR基因1(MDR1)及MDR相关蛋白1(MRP1)蛋白水平和基因水平表达的影响。另同时设置口服和静脉2种给药组别以研究美洲大蠊PAE2的基本药代动力学特征。结果:裸小鼠体质量方面,造模成功后,与正常组比较,裸小鼠体质量明显减轻(P<0.05),经各药物治疗后,体质量有一定回升,但仍然不及正常裸小鼠。抑瘤作用方面,与模型组比较,美洲大蠊多肽PAE2iv组中剂量组抑瘤效果最好(P<0.05),口服给药组抑瘤作用随剂量的增加而增加,高剂量组效果最好(P<0.05),美洲大蠊前期粗提物CⅡ-3无明显抑瘤作用,脱脂膏具有一定的抑瘤效果(P<0.05)。MDR相关蛋白和酶方面,美洲大蠊多肽PAE2在体内对MDR相关的蛋白和酶均有一定影响,主要可能通过抑制肿瘤组织内LRP,BCRP表达和不同程度影响上述相关蛋白、基因表达而抑制细胞内药物外排,从而促进肿瘤凋亡,且作用优于美洲大蠊前期粗提物CⅡ-3和脱脂膏。结论:美洲大蠊多肽PAE2可能通过影响介导MDR的相关蛋白和酶的表达,减少药物外排,促进细胞内药物累积、细胞凋亡,从而发挥逆转HepG2/ADM细胞Balb/c-nude小鼠移植瘤MDR的作用。

Study on Periplaneta americana Polypeptide PAE2 in Reversing Multidrug Resistance for Liver Cancer

Objective: To investigate the effect and mechanism of PAE2,a polypeptide of Periplaneta americana,in reversing multidrug resistance(MDR)for liver cancer in vivo. Method: Balb/c-nude mice were inoculated with HepG2 and HepG2/ADM cells under the armpits to establish animal models of liver cancer sensitive strains and animal models of MDR respectively. After successful modeling,the nude mice were randomly divided into normal group, HepG2 model group, HepG2/ADM model group, sorafenib group(positive drug control group,ig 30 mg·kg^-1),HepG2/ADM+PAE2(iv)low,medium and high dose groups(50, 100, 200 mg·kg^-1), HepG2/ADM+PAE2(ig) low, medium, and high dose groups(50, 100,200 mg·kg^-1),skim cream group(ig 200 mg·kg^-1),and CⅡ-3 group(ig 200 mg·kg^-1),all of which received corresponding drug treatment. The body weight and tumor volume of nude mice were measured and recorded every 2 days. The next day after the last administration,tumor tissues of nude mice were taken to record the tumor weight. The effect of P. americana polypeptide PAE2 on permeability-glycoprotein(P-gp),lung resistance protein(LRP),breast cancer resistance protein(BCRP),protein kinase C(PKC),glutathione S-transferase-π(GST-π),topo-isomerase type Ⅱ(ToPo Ⅱ),multidurg resistance gene 1(MDR1)and Multidrug resistanceassociated proteins(MRP1)of the protein level and gene level expression in tumor tissues were determined by immunohistochemistry(IHC)and real-time quantitative polymerase chain reaction(Real-time PCR). In addition,both oral and intravenous administration groups were set up at the same time for preliminary study on the basic pharmacokinetic characteristics of P. americana polypeptide PAE2. Result: After the successful modeling,the body weight of the nude mice was significantly lower than that in the normal mice(P<0.05). After treatment with corresponding drugs,the body weight increased to a certain extent,but it was still not as good as the normal nude mice. In iv administration,the medium-dose P. americana polypeptide PAE2 showed the best anti-tumor effect as compared with the model group(P<0.05),while in oral administration,the anti-effect increased with the increase of the dose,so the high-dose group showed the best effect(P<0.05). Preliminary crude extract CII-3 had no obvious anti-tumor effect,and skim cream showed a certain anti-tumor effect(P<0.05). P. americana polypeptide PAE2 had certain effects on MDR related proteins and enzymes in vivo,mainly by inhibiting the expression of LRP and BCRP in tumor tissues and affecting the expression of these related proteins and genes to different degrees to inhibit intracellular drugs outflow,thereby promoting tumor apoptosis,and the effect was superior to that of the P. americana crude extract C Ⅱ-3 and skim cream. Conclusion:P. americana polypeptide PAE2 may reduce the drug efflux, promote intracellular drug accumulation and apoptosis by affecting the expression of related proteins and enzymes that mediate multidrug resistance,thereby exerting a reverse effect on HepG2/ADM cells Balb/c MDR in nude mice.