LKB1通过增强间隙连接细胞通讯增加乳腺癌细胞对顺铂的敏感性

LKB1 enhances sensitivity of breast cancer cells to cisplatin through gap junction intercellular communication

关键词:  乳腺肿瘤  肝激酶B1  间隙连接蛋白  间隙连接  旁观者效应

Key words:  breast tumor  liver kinase B1  connexins  gap junction  bystander effect

摘要:

目的研究LKB1对乳腺癌细胞顺铂化学敏感性的作用及机制。方法构建LKB1高表达的稳定转染细胞MDA-MB-231/LKB1及其空载对照细胞MDA-MB-231/VEC。RT-PCR、Western印迹法检测细胞间隙连接蛋白Cx26、Cx32、Cx43和Cx50的核酸、蛋白水平表达;免疫荧光实验对Cx43进行胞内表达定位;染料传输实验评估间隙连接细胞通讯功能;细胞毒实验检测顺铂杀伤乳腺癌细胞的＂旁观者效应＂及计算顺铂的半数抑制浓度。结果LKB1增加了间隙连接蛋白Cx43的核酸及蛋白水平表达（P〈0.01）,但对Cx26、Cx32及Cx50的表达水平无明显影响。LKB1增加了细胞膜上Cx43的表达,增强了细胞传递荧光黄染料的能力,并使顺铂对细胞的旁观者杀伤效应增强。LKB1也增加了细胞对顺铂的敏感性,MDA-MB-231、MDA-MB-231/VEC和MDA-MB-231/LKB1的半数抑制浓度分别为22.46、24.72、6.55 nmol/L,差异有统计学意义（P〈0.01）。结论LKB1通过增强乳腺癌细胞的Cx43表达及间隙连接细胞通讯功能增强了顺铂的旁观者杀伤效应,并增加了细胞对顺铂的敏感性。

Abstract:

Objective To investigate the effect of LKB 1 on chemosensitivity of breast cancer cells to cisplatin and its mechanism. Methods LKB1 over-expressing MDA-MB-231/LKB1 cells and its mock transfected control cells MDA-MB-231/VEC were constructed. Reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting were used to detect the expression level of Cx26, Cx32, Cx43 and Cx50 in cells. Immunofluorescent assay was carried out to localize the expression of Cx43 in cells. Scrap-loading assay was employed to assess the function of Gap Junction Intercellular Communication （GJIC）. The bystander effect and half inhibition concentration （IC₅₀） of cisplatin were determined by cytotoxicity assay. Results Overexpression of LKB1 increased Cx43 mRNA and protein expression （P〈0.01 ）, but had no significant effect on expression of Cx26, Cx32 and Cx50 .More Cx43 was localized at cell membrane, Lucifer yellow was transmitted further to more cells and stronger cytotoxic bystander effect were observed after LKB1 transfection. Furthermore, LKB1 increased the sensitivity to cisplatin, the IC₅₀ of MDA-MB-231, MDA-MB-231/VEC and MDA-MB- 231/LKB1 were 22.46, 24.72 and 6.55 nmol/L, respectively （P〈0.01 ）. Conclusion LKB1 enhances the cytotoxic bystander effect of cisplatin and sensitized breast cancer cells to cisplatin, by up- regulating Cx43 expression and enhancing GJIC function.