| Anoikis is a form of apoptosis triggered by disruption of cell-matrix contacts. It is often seen in normal epithelial and endothelial cells. The significance of anoikis is that it may prevent the accidentally detached cells from reattaching to new matrices and growing dysplastically. On the other hand, most tumor cells derived from epithelial tissues have lost this characteristic, especially those malignant tumors that are easy to metastasize. The cells detached from tumors do not undergo apoptosis and can colonize elsewhere and grow. It is an important mechanism of tumor's metastasis, which attributes to clinical death of most of tumor patients and is often seen in breast, lung, hepatic, gastric and prostate tumors. Therefore, it is important to elucidate the molecular mechanism of tumor cells' anoikis resistance. It will help us to understand the molecules and pathways related to this phenomenon and it has a practical value for developing new strategies to prevent tumor metastasis.To understand the molecular mechanism of tumor cell resistance of anoikis, we first analyzed several breast cancer cell lines, including Bcap-37, MCF-7, MDA-MB-231 and SK-BR-3, to determine their sensitivity to anoikis through DNA laddering assay, FCM and soft agar assay. The results showed that the MDCK cells, which are derived from normal canine kidney, are sensitive to anoikis. Fragmented DNA could be detected by agarose gelDepartment of Biochemistry and Molecular Biology, FMMU10electrophoresis. High proportion of cells was in Sub-Gl phase of eel] cycle and the cells could not grow in soft agar to form colonies. On the contrary, the three breast cancer cell lines, Bcap-37, MCF-7 and MDA-MB-231, are anoiMs resistant, as indicated by DNA laddering and FCM assays. Furthermore, they all grow in soft agar and form colonies. SK-BR-3 cells are also anoikis resistant, but it could not form colonies in soft agar. Therefore, detachment may have some effects on the growth of SK-BR-3 cell cultured in suspension.Next we tried to find out which signaling molecules in the selected three tumor cell lines are related to their resistance to anoikis. We first investigated the effects of specific inhibitors of several signaling molecules on the tumor cell resistance of anoikis. These are KER2 inhibitor AG825; PDGFR inhibitor AG17; P38MAPK inhibitor SB203580; PD-K inhibitor LY294002 and MAPKK (MEK) inhibitor PD98059. Agrose gel electrophoresis showed that these inhibitors failed to induce suspended tumor cells to undergo apoptosis in a short time (15h). But they had different effects on tumor cells grew in soft agar. AG17 could inhibit the growth of three tumor cell Lines apparently and PD98059 could inhibit MDA-MB-231 cell forming colonies partially. The rest had no apparent effect on cell growth. These results imply that PDGFR may be involved in anchorage-independent growth of the tumor cells. MAPK signaling pathway may play a role hi the process of MDA-MB-231 cell's resistance to anoikis. Because of the complexity of signal transduction networks, the importance of these two signaling pathways in tumor cells' resistance to anoikis are need to be further studied.The effect of the general protein tyrosine kinase inhibitor genistein on tumor cell anoikis resistance was also analyzed. Although the effect of genistein on anoikis was not apparent in a short time (less than 24h), as indicated by both agrose gel electrophoresis assay and FCM assay, genistein could inhibit the growth of the tumor cells in soft agar completely. The total levels of protein tyrosine phosphorylation in the three tumor cells upon genistein treatment were also decreased to some extent. These data imply that the tumor cell resistance to anoikis is mediated by protein tyrosine kinases.Department of Biochemistry and Molecular Biology, FMMUFurthermore, we analyzed the changes in expression and phosphorylation of key signaling molecules in several related signal transduction pathways. We found that two important survival signaling molecules, ERK1/2 and AKT, h...
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