缝隙连接蛋白在钙离子介导的乳腺癌细胞转移和侵袭中的作用

目的 探讨缝隙接蛋白(Cx)在钙离子介导的乳腺癌细胞转移和侵袭中的作用. 方法 选用不影响细胞生长的缝隙连接蛋白阻断剂,辛醇100μmol/L处理高转移MDA-MB-231细胞和低转移MCF-7细胞.倒置相差显微镜下观察细胞形态,激光扫描共焦显微镜下观察Cx43的位置、微丝纤维的排列和细胞内钙离子浓度变化,划痕和侵袭实验观察细胞的转移情况. 结果 辛醇处理细胞后,细胞的生长状态由大面积片状生长转变为单个或少数几个团状的独立生长；Cx43蛋白的形成和表达位置虽无改变,但相邻细胞间微丝纤维排列的平行同向性显著性降低；MDA-MB-231细胞穿过基底膜成胶和Transwell小室基底面的细胞数显著低于对照组；此外,细胞内钙离子浓度强度显著性降低,钙离子螯合剂(EGTA)处理显著性加剧辛醇对细胞转移和侵袭能力的抑制.但是,上述现象在低转移MCF-7细胞中效果不明显.结论 缝隙连接蛋白阻断剂在干扰乳腺癌细胞Cx功能活性,而不影响Cx形成的情况下,能够显著性抑制高转移乳腺癌的恶性进展和侵袭转移,此机制与细胞内钙离子浓度降低有关.

Effects of the gap junctional connexin on the Ca2+ -mediated migration and invasion of breast cancer cells

Objective To explore effects of the connexin on the Ca SUP>2+/SUP> -mediated migration and invasion of breast cancer cells. Methods MDA-MB-231 and MCF-7 cells, with high and low metastatic potentials, respectively, were treated with a safe concentration of octanol (100μmol/L) for 24 hours. The morphology change was observed under an inverted phase contrast microscope.The location ofconnexin43(Cx43), arrangement of microfilament(MF)and concentration of intracellular Ca SUP>2+/SUP> were determined by confocal microscopy, and cell migration was checked by wound healing and Transwell chamber assays. Results Functional blockade of gap junctions during the formation of a cellular monolayer resulted in discordance of actin stress fibers between neighboring cells, even though whole-cell morphology of these cells did not change. Confocal microscopy revealed that immunoreactivity of Cx 43 was in the cell membrane，particularly at the region of cell-to-cell apposition regardless of the presence of gap junction inhibition. The number of MDA-MB-231 cells was significantly increased in migration assays, as compared with the control. The concentration of intracellular Ca SUP>2+/SUP> was significantly decreased. EGTA treatment enhanced the inhibition of cell migration and invasion which induced by octanol alone. However, inhibition had no effects on the migration and invasion of low metastatic potential MCF-7cells. Conclusion These data imply that gap junctional inhibitor does not interrupt the formation but rather the function of gap junctions, and the underlying mechanism may be related to the decrease of intracel