| BACKGROUND & OBJECTIVEColorectal cancer (CRC) is a type of common malignant tumor, and it's incidence rate keeps increasing over the years. The therapeutic efficacy of CRC was not significantly improved in spite of great efferts having been made in many years. One of the main obstacles is the multidrug-resistance (MDR) property of CRC, which leads to the failure of chemotherapy. Researchs show that solid tumor is a three-dimensional cell cluster. The inter-communication among the cells results the complex drug resistance mechanisms. The signal transduction among the cells may directly involve in and contribute to the drug resistance. This may shade some new light on the study of the MDR mechanism.Glycogen synthase kinase 3β(GSK-3β), a multiFunctional serine/threonine kinase which has become a research focus, participates in a variety of important physiological processes, such as intracellular glucose metabolism, cell proliferation, differentiation and apoptosis. Study has shown that GSK-3β, as a major regulating enzyme to Wnt/β- catenin, nuclear factor-KB (NF-κB), RB/E2F-1 and other moleculars in many signaling pathways, participates in regulating cell proliferation, apoptosis and MDR of tumor by influence on downstream nuclear transcription factors. However, the role of GSK-3βand GSK-3βinhibitors, the influence of GSK-3P to the biological properties of tumor cell, and whether GSK-3βis the prime target during the tumor therapy remain controversial. The MDR regulation mechanism of GSK-3βin CRC is not clear. Thus, It's important to elucidate the role of of GSK-3p and the mechanism of MDR in CRC.MDR refers to the phenomenon that the tumor cells may simultaneously become cross-resistant to a wide variety of chemotherapeutic drug with different structures, Functions and cellular targets, when selected for resistance to a single cytotoxic agent. The mechanism of MDR is very complex. At present, some drug-resistant mechanisms are highly concerned:1) The change of apoptosis regulation gene or protein which results in tumor cells resistance or escape from apoptosis induced by varieties of chemotherapeutic drug, such asβ-catenin, P53, Bcl-2, etc; 2) ATP binding cassette(ABC) transmembrane transporter protein increases drug's efflux. There are 48 genes have been found in ABC translocator family, the ones which are associated with drug resistance as follow:ABCA2, ABCB1 (P-gp), ABCC1 (MRP1), ABCC2 (MRP2), ABCC3, ABCC4, ABCC5, ABCC6, ABCC11, ABCG2. Among of them, ABCB1 (P-gp) and ABCC2 (MRP2) have higher expression in colorectal tissues; 3) Activating drug's detoxification systems, such as the activation of glutathione-S-transferase (GST) system; 4) The changing of proteases which participate in DNA replication and repair, such as topoisomeraseⅡ(TOPOⅡ); 5) The changing of the key enzymes of DNA synthesis, such as thymidylate synthase (TS), TS is a key enzyme in the process of DNA synthesis and metabolism and it is a target enzyme of 5-Fu, which is an essential chemotherapy drug for colorectal cancer. The level of TS expression is an important factor in chemosensitivity of 5-Fu; 6) the mechanisms of cancer stem cell resistance to chemotherapy. cancer stem cells could survive cytotoxic or targeted therapies and enhanced ABC transporter protein expression.Recent experimental study on combination therapy of GSK-3βinhibitor with chemotherapy drugs found that GSK-3βinhibitors reduces some cell apoptosis induced by chemotherapy drug, however, the mechanism is still not clear. It has not yet known the effect of GSK-3βinhibitor on apoptosis induced by 5-Fu chemotherapy in colorectal cancer. In order to Further study the effects and regulatory mechanism of GSK-3βin drug resistance of colorectal cancer, the SW480 cells from primary lesion and the SW620 cells from lymph node metastases lesion, which originate from the same patient with colon cancer, were used as the research objects.. A small molecule ATP competitive GSK-3βinhibitor (2'Z,3'E) -6-bromoindirubin-3'-oxime (BIO) was used in colon cancer SW480 and SW620 cells, to compare the variation of multidrug resistence protein P-gp, MRP2, TS and the corresponding regulatory proteinβ-catenin, Bcl-2, E2F-1 in two cells before and after BIO was used. At the same time, the effects and mechanism of BIO on cancer cell's biological properties and apoptosis induced by 5-Fu were observed. Gene chips, Real-time PCR and Bioinformatics analysis method were applied to Further investigate the affect of BIO on the multidrug resistance-associated gene expression and regulation mechanism in both types of colon cancer cells.METHODS1. The effects of GSK-3βinhibitor BIO on biological properties of primary and metastatic colon cancer cells.The colon cancer SW480 and SW620 cells were treated with BIO in different concentrations. The growth of SW480 and SW620 cells was observed by inverted microscope and intracellular ATP concentration was detected by luminometer. Cell cycle distribution and apoptosis levels were detected by Flow Cytometry and morphology of Apoptosis cell was observated by Tunel staining. The expressions ofβ-catenin, E2F-1 and Bcl-2 protein were detected by Western blot. Theβ-catenin, Ki-67 and Cyclin D1 expression, and cellular localization were detected by Immunocytochemical. Cell morphology of SW480 and SW620 was observated using HE staining and light microscopy and cell ultrastructure was observated by electron microscopy before and after 24h of BIO treatment in different concentration.2. The effects of GSK-3βinhibitor BIO on expression of MDR-associated proteins in primary and metastatic colon cancer cell.The expressions of P-gp, MRP2, TS protein were detected by Western blot in SW480 and SW620 cells before and after 24h of BIO treatment in different concentration. P-catenin and P-gp, P-catenin and MRP2 were stained with double immunofluorescence, and the staining of P-catenin and P-gp, P-catenin and MRP2 were observed by confocal microscope, meanwhile, the ability of rhodamine 123 efflux was detected in SW480 and SW620 cells before and after 24h of BIO treatment.3. The effects of GSK-3P inhibitor BIO on the apoptosis induced by 5-Fu in primary and metastatic colon cancer cellThe Apoptosis induced by 5-Fu with different concentration was detected by flow cytometry in colon cancer SW480 cells and then appropriate 5-Fu concentration was selected. The cell apoptosis induced by 5-Fu and 5-Fu combined with BIO was detected with flow cytometry and Tunel staining in colon cancer cell SW480 and SW620. 4. Gene Chip detection and analysis of the effects of GSK-3βinhibitors BIO on expression of MDR-associated genes in primary and metastatic colon cancer cells Gene expression was detected with NimbleGen Human Gene Expression Microarrays(consist of 45,033 gene) in colon cancer SW480 and SW620 cells before and after BIO treatment. The changing of gene expression was validated by Real-time PCR, and analysed with Bioinformatics.RESULTS1. GSK-3βinhibitor BIO affects the biological characteristics of primary and metastatic colon cancer cellsCompared with those of untreated colon cancer SW480 and SW620 cells, the intracellular ATP concentration is slightly elevated in SW480 and SW620 cells after the GSK-3βinhibitor BIO treatment with different concentration. In SW480 cells, BIO significantly increases the expression ofβ-catenin protein and promotes nuclear translocation ofβ-catenin (F=33.250, P=0.000), and decreases the expressions of E2F-1 and Bcl-2 protein (F=317.869, P=0.001; F=12.389, P=0.002), also decreases apoptosis rate (F=11.114, P=0.003). BIO significantly increases the expression of Ki-67 protein and the cell quantity in S and G2/M phase. The cells showed a high proliferation state, while Cyclin Dl expression change is not obvious. In SW620 cells, BIO significantly increases the expression of P-catenin protein and promotes nuclear translocation of P-catenin protein (F=19.608 P=0.000), increases the expression of E2F-1 protein (F=22.630, P=0.000), slightly decreases the expression of Bcl-2 protein, and moderately decreases the cell apoptosis rate. BIO increases the expression of Ki-67 protein, but the change of cell quantity in S and G2/M phase, cell morphology and Cyclin D1 expression were not obvious. 2. GSK-3βinhibitor BIO affects the expression of MDR-associated protein in primary and metastatic colon cancer cells.GSK-3βinhibitor BIO significantly upregulates the expressions of P-gp, MRP2 and TS protein in SW480 (F=29.600, P=0.000; F=11.555, P=0.003; F=32.996, P=0.000) and SW620 cells (F=26.792, P=0.000; F=17.657, P=0.001; F=92.953, P=0.000), and enhances the efflux ability of of rhodamine 123 in SW480 and SW620 cells (P=0.027; P=0.038). The P-gp, MRP2 andβ-catenin retains obvious co-localization expressing phenomenon in SW480 and SW620 cells.3. GSK-3βinhibitor BIO affects the apoptosis induced by 5-Fu in primary and metastatic colon cancer cellsThe optimum concentration of 5-Fu inducing cell apoptosis in SW480 cells is 50μmol /L. With this concentration, GSK-3βinhibitor BIO reduces apoptosis of SW480 and SW620 which induced by 5-Fu, and this is particularly proninent in SW480 cell (P=0.000)4. Gene Chip detection and analysis of GSK-3P inhibitor BIO affects the expression of MDR-associated genes in primary and metastatic colon cancer cellsThe differential expression genes with drug resistance between SW620 and SW480 cells were as follows:GSTM1, TOP2A, ABCB7, ABCB7, ABCC2, TOP1, ABCA11, ABCB9. The increased expression genes with drug resistance after GSK-3P inhibitor BIO treatment in SW480 cells was as follow:ABCB1; The increased expression genes with drug resistance after GSK-3βinhibitor BIO treatment in SW620 cells were as follows:ABCB1,ABCC2,ABCB10,TAP2,ABCE1. With the detection of Real-time PCR, GSK-3βinhibitor BIO significantly increases mRNA content of ABCB1 in SW480 cells (F=6.45,P=0.016) and increases mRNA content of ABCB1 and ABCC2 in SW620 cells (F=16.204,P=0.001; F=13.122, P=0.002).CONCLUSION1. GSK-3βinhibitor BIO enhance the drug-resistant phenomenon in SW480 and SW620 cells. GSK-3βinhibitor BIO significantly inhibited apoptosis induced by 5-Fu in SW480 cells, whereas the effect was slight in SW620 cells.2. The mechanisms of drug-resistant induced by GSK-3βinhibitor BIO in SW480 and SW620 cells including:(1) The enhancement of apoptosis resistance (2) The enhancement of expression and Function of P-gp and MRP2 transporter protein; (3) The increase of TS protein expression; (4) the increase of intracellular ATP level may enhance the transportion Function of ABC transmembrane transporter protein. The apoptosis resistance induced by GSK-3βinhibitor BIO was obvious in SW480 cells, whereas the effect was slight in SW620 cells.3.β-catenin, E2F-1 and Bcl-2 jointly participate in the cell proliferation, apoptosis and drug-resistant regulation induced by BIO in colon cancer SW480 and SW620 cells, and The P-catenin is probably a key factor in this process. The different expression of E2F-1 in SW480 and SW620 cells is likely one of the mechanisms leading to the different outcome in the two kinds of cells.4. BIO is able to increase the expressions of P-gp, MRP2 transporter protein and TS protein in SW480 and SW620 cells. The increase of P-gp and MRP2 expressions induced by BIO is bound up with the activation ofβ-catenin signal transduction pathways. The Increase of TS is likely related with E2F-1 or non E2F-1 pathways, and the mechanism needs to be made a Further studied.5. There are differences of gene expressions between SW480 cell and SW620 cell, which are the bases to bring different responses to GSK-3βinhibitor BIO and BIO combined with 5-Fu.
|
PDF Full Text Request