转移相关分子链Actin-CD44-MMP-2在乳腺癌转移实验中的改变

The coordinate alteration of actin cytoskeleton,CD44 and matrix metalloproteinase-2 in the metastasis of breast cancer cells

目的研究乳腺癌转移相关的分子机制及抑制体内外转移的作用和机制。方法选择高、低转移性乳腺癌细胞系BICR-H1和MCF-7,用明胶底物非变性电泳分析法、Western blot和免疫荧光染色等方法,观察肌动蛋白、CD44和基质金属蛋白酶2分子链（ACM）成员分子的定性与定位关系。利用荧光标记肿瘤鸡胚尿囊膜（CAM）模型,血管内注射顺铂或MMP-2 C末端PEX融合蛋白,利用荧光显微镜直接观察鸡胚肺转移抑制情况与癌细胞的ACM水平。结果高转移的BICR-H1细胞ACM分子链呈高表达,分子定位相互关联。低转移性的MCF-7细胞呈低表达或无表达。体外实验中,顺铂和PEX分别抑制了CD44和MMP-2的表达。体内实验中,5~30μg顺铂能够抑制BICR-H1和MCF-7肿瘤的体内生长,呈剂量依赖性。30μg顺铂使乳腺癌BICR-H1细胞体内转移克隆数下降到（8±6）个[对照组为（30±15）个],而同剂量PEX可以完全抑制其体内转移。结论 ACM分子链表达与乳腺癌的转移性密切相关。顺铂和PEX分别通过封闭ACM分子链的环节,使CD44或MMP-2表达抑制,从而抑制乳腺癌细胞的体内外生长和转移。

Objective To study the roles of actin and associated molecules in the control of human breast cancer cell malignant behaviors in vitro and in vivo.Methods A highly metastatic human breast cancer cell line BICR-H1 was compared with another breast cancer cell line MCF-7,which was well differentiated and non-metastatic.Western blot,immunofluorescence,gelatin zymography analysis and a chick embryonic chorioallantoic membrane（CAM） assay were used in this research.5～30 μg cisplatin or MMP-2 C terminal PEX domain were injected i.v.in CAM.Results BICR-H1 expressed high level of CD44,which was closely associated with actin aggregates at the bottom side of attached cells.It was also shown with MMP-2 activity.On the contrary,MCF-7 cells showed weak disruption of actin cytoskeleton structures and a few actin aggregates.It expressed low or minimal level of CD44 and MMP-2.The expression of CD44 was down-regulated in cisplatin-treated BICR-H1 cells,and the activity of MMP-2 was also decreased upon PEX treatment.Both cell lines could form tumors in CAM,but only BICR-H1 cells could metastasize to distant tissues.Cisplatin inhibited the growth of BICR-H1 and MCF-7 cells in a time and dose dependent manner in CAM.The lung metastatic foci of BICR-H1 cells treated with 30 μg cisplatin were reduced from 30±15/embryo（PBS group） to 8±6/embryo,and the same dose of PEX could completely inhibit BICR-H1 metastasis.Conclusion It is concluded that actin cytoskeleton,CD44 and MMP-2（ACM）molecular linkage is associated with breast cancer metastatic phenotypes,and both cisplatin and PEX can interfere with the ACM molecular linkage,resulting in the suppression of both tumor growth and metastasis.