金合欢素调节HIF-1α/VEGF信号通路对胆囊癌细胞恶性生物学行为的影响

Effect of acacetin on the malignant behaviors of gallbladder cancer cells by regulating the HIF-1α/VEGF signaling pathway

目的探讨金合欢素对胆囊癌细胞恶性生物学行为的影响及其与缺氧诱导因子1α(HIF-1α)/血管内皮生长因子(VEGF)信号通路的调节关系。方法采用CCK-8法检测经0、10、25、50、100、150μmol/L的金合欢素处理后的人胆囊癌细胞GBC-SD增殖率,筛选合适的金合欢素作用浓度。将体外培养的GBC-SD细胞随机分为对照组、金合欢素低剂量组、金合欢素高剂量组、金合欢素高剂量+空载组、金合欢素高剂量+HIF-1α过表达组,采用金合欢素和质粒分组处理后,采用实时荧光定量PCR和免疫印迹法检测各组的GBC-SD细胞HIF-1α/VEGF信号通路表达;采用CCK-8法和流式细胞术检测各组GBC-SD细胞增殖率、凋亡率;采用Transwell侵袭实验和细胞划痕实验检测各组GBC-SD细胞侵袭数、迁移率;提取GBC-SD细胞条件培养基,采用酶联免疫吸附反应(ELISA)检测其中VEGF水平。将体外培养的人脐静脉血管内皮细胞(HUVEC)以同样方法分组以上述提取的GBC-SD细胞条件培养基分组处理,采用小管形成实验检验各组HUVEC细胞成管长度。将分组处理后的各组GBC-SD细胞接种在裸鼠右腋皮下建立移植瘤模型,饲养3周后采用免疫组化染色检测各组裸鼠肿瘤组织微血管密度(CD31表达)及肿瘤体积。结果与对照组相比,金合欢素低、高剂量组GBC-SD细胞HIF-1α、VEGF mRNA与蛋白表达、增殖率、侵袭数、迁移率、GBC-SD细胞培养基中VEGF水平、HUVEC细胞成管长度、裸鼠肿瘤组织CD31阳性细胞比例及肿瘤体积均降低(P<0.05),凋亡率均升高(P<0.05);金合欢素高剂量组与金合欢素低剂量组相比,GBC-SD细胞HIF-1α、VEGF mRNA与蛋白表达、增殖率、侵袭数、迁移率、GBC-SD细胞培养基中VEGF水平、HUVEC细胞成管长度、裸鼠肿瘤组织CD31阳性细胞比例及肿瘤体积降低,凋亡率升高(P<0.05)。与金合欢素高剂量组相比,金合欢素高剂量+HIF-1α过表达组GBC-SD细胞HIF-1α、VEGF mRNA与蛋白表达、增殖率、侵袭数、迁移率、GBC-SD细胞培养基中VEGF水平、HUVEC细胞成管长度、裸鼠肿瘤组织CD31阳性细胞比例及肿瘤体积升高(P<0.05),凋亡率降低(P<0.05);金合欢素高剂量+空载组各指标无显著变化(P>0.05)。结论金合欢素通过下调HIF-1α/VEGF通路而抑制胆囊癌细胞增殖、迁移和侵袭,减弱其体内外血管生成,并延缓其体内肿瘤生长,最终改善其恶性生物学行为。

Objective To investigate the effect of acacetin on the malignant behaviors of gallbladder cancer cells and its regulatory effect on the hypoxia inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF)signaling pathway.Methods The Cell Counting Kit-8(CCK-8)assay was performed to detect the proliferative rate of GBC-SD cells treated with 0,10,25,50,100,150μmol/L acacetin,thus screening the optimal concentration of acacetin.GBC-SD cells cultured in vitro were treated with blank control,low-dose and high-dose acacetin,high-dose acacetin plus transfection of vector and high-dose acacetin plus transfection of HIF-1αoverexpression plasmid.Expression levels of HIF-1αand VEGF were detected by quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot.The proliferative rate,apoptotic rate,invasive cell number and migratory rate were measured by CCK-8 assay,flow cytometry,Transwell assay and wound healing assay,respectively.Serum VEGF in cell supernatant was measured by enzyme-linked immunosorbent assay(ELISA).The human umbilical vein endothelial cells(HUVEC)cultured in vitro were similarly treated as above mentioned.The tube length of HUVEC cells in each group was tested by tube formation assay.GBC-SD cells with different treatments were inoculated subcutaneously in the right armpit of nude mice to establish the transplanted tumor model,and after 3 weeks of feeding,the microvessel density(CD31 expression)and tumor volume of in each group were detected by immunohistochemical staining.Results Compared with those of the control group,GBC-SD cells treated with low-dose and high-dose acacetin presented lower mRNA and protein levels of HIF-1αand VEGF,proliferative rate,invasive cell number,migratory rate,serum VEGF level,tube length,CD31-positive cell ratio in nude mouse tumor tissues,and tumor volume,and significantly higher apoptotic rate(all P<0.05).Compared with those of low-dose acacetin group,GBC-SD cells treated with high-dose acacetin presented significantly lower mRNA and protein levels of HIF-1αand VEGF,proliferative rate,invasive cell number,migratory rate,serum VEGF level,tube length,CD31-positive cell ratio in nude mouse tumor tissues,and tumor volume,and significantly higher apoptotic rate(all P<0.05).Compared with those of high-dose acacetin group,GBC-SD cells treated with high-dose acacetin plus transfection of transfection of HIF-1αoverexpression plasmid significantly higher mRNA and protein levels of HIF-1αand VEGF,proliferative rate,invasive cell number,migratory rate,serum VEGF level,tube length,CD31-positive cell ratio in nude mouse tumor tissues,and tumor volume,and significantly lower apoptotic rate(all P<0.05).The above indexes were similar in GBC-SD cells treated with high-dose acacetin plus transfection of vector(P>0.05).Conclusion Acacetin alleviates malignant behaviors of gallbladder cancer cells by inhibiting in vitro proliferation,migration,invasion,and angiogenesis and in vivo tumor growth through inhibiting the HIF-1α/VEGF signaling pathway.