仙鹤草-黄连通过调控分子伴侣介导自噬抑制结直肠癌的作用机制

Mechanism of Agrimoniae Herba and Coptidis Rhizoma in Inhibiting Colorectal Cancer by Regulating Chaperone-mediated Autophagy

目的:观察仙鹤草-黄连药对(XHC-HL)对结直肠肿瘤细胞增殖、迁移、侵袭、凋亡及对自噬的影响,并探讨XHC-HL通过调控分子伴侣介导自噬(CMA)抑制结直肠癌的作用机制。方法:制备XHC-HL含药血清,培养人结直肠癌细胞株HT29和LOVO,分为空白组(20%空白血清),XHC-HL组(5%、10%、20%含药血清),5-氟尿嘧啶(5-FU)组。用细胞增殖与活性检测法(CCK-8)检测细胞活力;5-乙炔基-2′-脱氧尿苷(EdU)染色法检测细胞增殖能力;划痕实验及Transwell迁移实验检测细胞迁移能力;Transwell侵袭实验检测细胞侵袭能力;流式细胞术检测细胞凋亡率;蛋白免疫印迹法(Western blot)检测XHC-HL对自噬关键分子酵母Atg6同系物-1(Beclin-1)、微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)及p62的影响;Western blot、实时荧光定量聚合酶链式反应(Real-time PCR)检测XHC-HL对CMA相关蛋白和mRNA影响。结果:与空白组比较,XHC-HL组和5-FU组均能抑制HT-29和LOVO细胞活力(P<0.01)。与空白组比较,XHC-HL组和5-FU组均能够明显抑制HT-29和LOVO细胞的增殖能力(P<0.05,P<0.01)。与空白组比较,XHC-HL组和5-FU组2种细胞迁移率均显著减弱(P<0.01),迁移到下室的数量均显著减少(P<0.01),细胞侵袭个数均降低(P<0.01),细胞凋亡增多(P<0.01)。Western blot结果显示,与空白组比较,XHC-HL组和5-FU组2种细胞中Beclin-1和LC3Ⅱ的表达明显增加(P<0.05,P<0.01),而p62的水平明显降低(P<0.05,P<0.01);与空白组比较,XHC-HL组和5-FU组2种细胞中溶酶体相关膜蛋白2A(LAMP2A)、热休克同源蛋白70(HSC70)、热休克蛋白90(HSP90)蛋白表达量均有不同程度的降低,甘油醛-3-磷酸脱氢酶(GAPDH)表达水平显著增高(P<0.01)。与空白组比较,XHC-HL组和5-FU组2种细胞中LAMP2A、HSC70、HSP90 mRNA水平均下调,GAPDH mRNA表达水平显著上调(P<0.01)。结论:XHC-HL可通过抑制CMA活性,从而抑制结直肠癌细胞的增殖、迁移、侵袭能力,诱导其自噬,并促进细胞凋亡。

Objective To observe the effects of Agrimoniae Herba and Coptidis Rhizoma(XHC-HL)on the proliferation,migration,invasion,apoptosis,and autophagy of colorectal tumor cells and explore the mechanism of Agrimoniae Herba and Coptidis Rhizoma in inhibiting colorectal cancer by regulating chaperone-mediated autophagy(CMA).Method XHC-HL-containing serum was prepared,and human colorectal cancer cell lines HT29 and LOVO were cultivated and divided into blank group(20%blank serum),low XHC-HL groups(5%,10%,20%drug-containing serum),and a positive control group using 5-fluorouracil(5-FU).Cell viability was measured using the cell counting kit-8(CCK-8)method.Cell proliferation ability was assessed through 5-ethynyl-2′-deoxyuridine(EdU)staining.Scratch assays and Transwell migration tests were conducted to evaluate cell migration ability,and Transwell invasion assays were used to assess cell invasion ability.Apoptosis rates were determined by flow cytometry,and the impact of XHC-HL on yeast Atg6 homologous 1(Beclin-1),microtubule-associated protein1 light chain3Ⅱ(LC3Ⅱ),and p62 was analyzed via Western blot.The influence of XHC-HL on CMA-related proteins and mRNA was examined using Western blot and real-time fluorescence quantitative polymerase chain reaction(Real-time PCR).Result Compared to that in the blank group,the vitality of colorectal cancer cells HT-29 and LOVO was significantly inhibited in XHC-HL groups and the 5-FU group(P<0.01).Compared to that in the blank group,the proliferation of colorectal cancer cells HT-29 and LOVO was significantly inhibited in XHC-HL groups and the 5-FU group(P<0.05,P<0.01).Compared to the blank group,there was a significant reduction in cell migration rates(P<0.01),the number of cells migrating to the lower chamber(P<0.01),and the number of invasive cells(P<0.01),as well as and an increase in cell apoptosis(P<0.01)in XHC-HL groups and the 5-FU group(P<0.01).Western blot results indicated that compared to that in the blank group,the expression levels of Beclin-1 and LC3Ⅱwere increased(P<0.05,P<0.01)in XHC-HL groups and the 5-FU group,while the p62 levels were decreased(P<0.05,P<0.01).Furthermore,the protein expression levels of lysosomal associated protein 2A(LAMP2A),heat shock cognate protein 70(HSC70),and heat-shock protein 90(HSP90)in XHC-HL groups and the 5-FU group were decreased to varying extents,whereas the expression levels of glyceraldehyde-3-phosphate dehydrogenase(GAPDH)were elevated(P<0.01).The Real-time PCR results showed that compared to those in the blank group,the mRNA levels of LAMP2A,HSC70,and HSP90 were downregulated in XHC-HL groups and the 5-FU group,and the mRNA expression level of GAPDH was upregulated(P<0.01).Conclusion XHC-HL can inhibit the proliferation,migration,and invasion of colorectal cancer cells by suppressing CMA activity,thus inducing autophagy and promoting apoptosis.