RET抑制剂通过下调神经胶质细胞来源神经营养因子表达水平抑制胰腺癌的机制研究

Mechanism of RET inhibitors inhibiting pancreatic cancer by down-regulating the expression level of glial cell-derived neurotrophic factor

目的 探索RET抑制剂RPI-1能否通过下调GDNF等神经胶质细胞来源神经营养因子表达水平,进而抑制胰腺癌肿瘤生长。方法 人胰腺癌细胞系ASPC-1,BxPC-3,CAPAN-1和T3M4,实时荧光定量PCR方法筛选出GDNF高表达的细胞株,构建荷瘤小鼠。将建立模型成功的荷瘤小鼠40只(移植瘤约4 mm×3 mm)随机分为4组,分别为对照组、低剂量用药组,中剂量用药组及高剂量用药组,每组10只。干预方法:对对照组小鼠腹腔注射PBS 0.1 mL/10 g,对低、中、高剂量组小鼠分别腹腔注射RET抑制剂RPI-1 50 mg/kg,100 mg/kg和200 mg/kg,各组小鼠每3 d给药1次,持续38 d。检测移植瘤体积,采用免疫组化法检测移植瘤组织中神经营养因子的表达水平,检测肿瘤组织微血管密度值(MVD),蛋白免疫印迹法(Western blotting)检测凋亡相关蛋白表达水平。结果 人胰腺癌细胞系CAPAN-1中GDNF表达量最高,采用CAPAN-1细胞构建荷瘤小鼠;低剂量RPI-1对小鼠瘤组织的生长干预不显著,中、高剂量RPI-1显著的降低了瘤组织的生长速度,抑制效率呈现浓度依赖(P<0.05);中、高剂量的RPI-1能显著抑制神经胶质细胞来源神经营养因子GDNF,PSPN、ARTN及NRTN的表达水平,下调程度呈浓度依赖(P<0.05)。同时,中、高剂量的RPI-1显著抑制了小鼠移植瘤的血管生成,抑制程度呈浓度依赖(P<0.05)。中、高剂量的RPI-1显著提升移植瘤中Bax表达量,降低BCl-2表达量(P<0.05),显著引起移植瘤组织中细胞形成凋亡。低剂量和对照组则差异无统计学意义(P>0.05)。结论 通过抑制剂RPI-1对RET通路进行调控,可以有效抑制胰腺癌移植瘤的生长,诱发癌细胞形成凋亡,机制同胰腺癌移植瘤的新生血管的生成与神经胶质细胞来源神经营养因子相关。

Objective To explore whether the RET inhibitor RPI-1 can inhibit pancreatic cancer tumour growth by down-regulating the expression levels of neurotrophic factors of glial cell origin such as GDNF.Methods The human pancreatic adenocarcinoma cell lines ASPC-1,BxPC-3,CAPAN-1 and T3M4 were screened for high GDNF expression by real-time fluorescence quantitative PCR,and tumor-bearing mice were constructed.Forty tumorbearing mice(transplanted tumors about 4 mm×3 mm) were randomLy divided into four groups:control group,low-dose drug group,medium-dose drug group and high-dose drug group,10 mice in each group.The mice in the control group were injected intraperitoneally with PBS 0.1 mL/10 g,and the mice in the low,medium and high dose groups were injected intraperitoneally with RET inhibitor RPI-1 50 mg/kg,100 mg/kg and 200 mg/kg,respectively,and the mice in each group were dosed once every 3 d until 38 d.The volume of transplanted tumors was measured,and the expression levels of neurotrophic factors in transplanted tumor tissues were detected by immunohistochemistry.The expression levels of neurotrophic factors in transplanted tumor tissues were measured by immunohistochemistry,microvascular density(MVD) in tumor tissues,and apoptosis-related proteins were detected by protein immunoblotting(Western blotting).Results The highest GDNF expression in the human pancreatic cancer cell line CAPAN-1,so the CAPAN-1cells were used to construct tumour-bearing mice model.The tumor volumes at different time of the low dose RPI-1group were not significantly different from those of the control group(all P>0.05),The expression levels of GDNF,PSPN,ARTN,and NRTN,and the expression level of MVD of the medium and high dose RPI-1 groups were all significantly lower than those of the low dose RPI-1 group(all P<0.05),and the degree of down-regulation was concentration-dependent(P<0.05).The expression level of Bax in the tumor of the medium and high dose of RPI-1group was significantly increased,and the expression level of BCl-2 of the medium and high dose of RPI-1 group was significantly reduced(both P<0.05).Conclusion Modulation of the RET pathway by the inhibitor RPI-1 can effectively inhibit the growth of transplanted pancreatic cancer and induce the formation of apoptosis in cancer cells by the same mechanism as generation of neovascularisation in the transplanted tumours associated with glial cell-derived neurotrophic factors.