奈达铂对乳腺癌细胞增殖、侵袭转移、凋亡及TLR4NF-κB信号通路的影响

Effects of nedaplatin on proliferation,invasion and metastasis,apoptosis and TLR4/NF-κB signaling pathway in breast cancer cells

目的探讨奈达铂对乳腺癌MDA-MB-231细胞增殖、侵袭转移及凋亡的影响,并探讨相关作用机制。方法体外培养人乳腺癌MDA-MB-231细胞株,分为空白对照组、阳性对照组(1μmol/L多西紫杉醇)及奈达铂低、中、高剂量组(15、30、45μg/mL奈达铂)。MTT法检测MDA-MB-231细胞增殖能力;AnnexinV-FITC/PI双染法检测MDA-MB-231细胞细胞凋亡情况;Transwell实验、划痕实验分别检测细胞侵袭、迁移能力;蛋白免疫印迹法检测增殖细胞核抗原(PCNA)、凋亡相关蛋白[B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)]、侵袭迁移相关蛋白[E-钙粘附蛋白(E-cadherin)、N-钙粘附蛋白(N-cadherin)]及通路相关蛋白[Toll样受体4(TLR4)、髓样细胞分化蛋白88(MyD88)、核转录因子kappa B(NF-κB)、白细胞介素(IL-1β)、肿瘤坏死因子-α(TNF-α)]的表达变化。结果与空白对照组相比,24、48 h时奈达铂低、中、高剂量组MDA-MB-231细胞增殖抑制率显著升高(均P<0.05),呈剂量依赖性,且高剂量组与阳性对照组比较差异无统计学意义(P>0.05)。与空白对照组比较,奈达铂低、中、高剂量组MDA-MB-231细胞凋亡率、Bax、E-cadherin蛋白表达显著升高,侵袭细胞数、细胞迁移率及PCNA、Bcl-2、N-cadherin、TLR4、MyD88、NF-κB、IL-1β、TNF-α蛋白表达水平显著降低(均P<0.05),呈剂量依赖性,且高剂量组与阳性对照组无显著差异(P>0.05)。结论奈达铂可抑制乳腺癌MDA-MB-231细胞增殖、侵袭及迁移并促进细胞凋亡,可能是通过抑制TLR4/NF-κB信号通路活化实现的。

Objective To study the effect of nedaplatin on the proliferation,invasion,metastasis and apoptosis of breast cancer MDA-MB-231 cells,and explore the related mechanism.Methods Human breast cancer cell line MDA-MB-231 was cultured in vitro and divided into blank control group,positive control group(1μmol/L docetaxel)and nedaplatin low,medium and high dose groups(15,30,45μg/mL nedaplatin).The proliferation of MDA-MB-231 cells was detected by MTT assay.The apoptosis of MDA-MB-231 cells was detected by Annexin V-FITC/PI double staining.Transwell test and scratch test were used to detect cell invasion and migration;the changes of proliferating cell nuclear antigen(PCNA),B cell-lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),invasion migration-associated proteins(E-cadherin),N-cadherin(N-cadherin)and Toll-like receptor 4(TLR4),myeloid differentiation primary response gene 88(MyD88),nuclear transcription factor-kappa B(NF-κB),interleukin(IL-1β)and tumor necrosis factor-α(TNF-α)were detected by Western blot.Results Compared with that in the blank control group,the proliferation inhibition rate of MDA-MB-231 cells in low,medium and high dose nedaplatin groups was significantly increased at 24 and 48 h(P<0.05),in a dose-dependent manner,and there was no significant difference between the high-dose group and the positive control group(P>0.05).Compared with those in the blank control group,the apoptosis rate of MDA-MB-231 cells and Bcl-2,E-cadherin protein expression in low,medium and high dose nedaplatin groups were significantly increased(P<0.05),the invasive cell number,cell migration rate and the expression levels of PCNA,Bax,N-cadherin,TLR4,MyD88,NF-κB,IL-1βand TNF-αwere significantly decreased(P<0.05),in a dose-dependent manner,there was no significant difference between high dose group and positive control group(P>0.05).Conclusion Nedaplatin can inhibit the proliferation,invasion and migration of MDA-MB-231 cells and promote cell apoptosis,which may be realized by inhibiting the activation of TLR4/NF-b signaling pathway.