| BACKGROUND: The drug resistance of tumor cells is one of the most important factors that interfere with the effect of chemotherapy and prognosis of patients. The solid tumors in human being grow actually as three-dimensional aggregates that exist very complex mechanism in drug resistance. The previous studies on drug susceptibility were mostly based on two-dimensional monolayer cultured (2D) cells, which represented the characteristics of individual cells and ignored the intercellular signal system and also the special microenvironment where they existed. It is important to clarify the drug resistance of individual cells as well as interaction of cells. Therefore, the concept of multicellular resistance was raised. OBJECTIVE: In this study,the pulmonary adenocarcinoma cell line A549 cells were cuktured by 2D and 3D methods.The difference in drug resistance and possible mechanism of the difference between the two methods of culture.To clarify the modulating effects of E-cadherin and its relative PI3K signal transduction pathway in multicellular drug resistance.METHODS: Material: Pulmonary adenocarcinoma cell line A549 cells were employed in our study. Methods: (1) To establish the 3D cell culture system in vitro,rotation cultural bottle and homoiothermy oscillatore were employed and the cells were identified inverted microscope and scanning electron microscope. (2) Comparison of the expression of E-cadherin, drug susceptibility to cytotoxicity drugs (ADM), cell cycle, cell apoptosis and different expression of related proteins such as p27, Bcl-xl, Bad between 2D and 3D cultured cells before and after treatment with the antagonist of E-cadherin-SHE78-7 by means of western blotting, flow cytometry, immunohistochemistry and MTT. (3) To compare the expression of PI3K pathway related protein (such as pAkt, Akt) between 2D and 3D cells before and after treatment with SHE78-7, the antagonist of E-cadherin. (4) To compare the drug susceptibility to cytotoxitic drugs (ADM), cell cycle, cell apoptosis and the expression of related proteins ( Bcl-xl,Bad) after giving LY294002,the inhibitor ofPI3K.RESULTS: (1) The 3D culture model of A549 cells was confirmed by observing cultured cells under inverted microscope and scanning electron microscope.(2) There were marked differences in E-cad expression and drug susceptibility to ADM comparing 2D with 3D cells. Higher E-cad expression and lower drug susceptibility to ADM were found in 3D culture..(3) Cell cycle retardation (most obviously in G1 phase), lower cell apoptosis rate, higher expression of p27 and Bcl-xl as well as lower expression of Bad were found in 3D cells, these differences between 2D and 3D cells were statistically significant.(4) Comparing 2D with 3D cultured cells ,those differences decreased after giving SHE78-7 to block t the effect of adherence in A549 cells.(5) Application of LY294002, an inhibitor of PI3K transduction pathway, in 3D culture system could increase drug susceptibility to ADM ,cell apoptosis rate and the expression of Bad ,decrease the expression of Bcl-xl,has no change in cell cycle.(6) In 3D cells, the expression of PI3K related protein pAkt decreased after anti-adherence treatment with SHE78-7, while the Akt expression had no marked difference.CONCLUSIONS:(1) 3D cell culture system in vitro was successfully established, which simulated the situation of solid tumors in vivo, and was helpful to the exploration of the mechanism of drug resistance of tumor cells. (2) The significant differences in the expression of E-cad and drug susceptibility to ADM between 2D and 3D cells decreased after anti-adherence treatment.(3) Cell adherence could inhibit cell apoptosis, improve cell viability, which lead to the tolerance to chemotherapeutic drugs, by means of regulation of the p27 expression, up-regulation of antiapoptotic protein (such as Bcl-xl), down-regulation of proapoptotic protein (such as bad).(4) PI3K pathway played an important role in adherence-mediated multicellular drug resistance in tumor c...
|
PDF Full Text Request