The Expression Changes Of HMLH1, HMSH2 With Resistance Development Of Cell Lines BGC-823, LoVo Induced By CDDP, 5-FU

Objective: Gastric and colorectal carcinomas are two of the most common malignant carcinomas in our country. Chemotherapy is one of the important therapies of them. However, frequently drug resistance, particularly multidrug resistance (MDR) of carcinoma cells resulted in failure of chemotherapy. While the mechanism of drug resistance of carcinoma cells have not been known. Now, it is known that the direct mechanism of drug resistance is up- regulation of some drug resistance associated proteins in carcinoma cells. These proteins, including P-glycoprotein(P-gp), multidrug resistance associated protein(MRP), lung resistance protein(LRP), glutathioneS-transFerase- pi(GST-pi) and so on, can eliminate drugs from carcinoma cells or decrease toxicity of drugs. But the causes inducing these proteins to express in carcinoma cells were seldom involved in researches. As normal cells, carcinoma cells also need gene repair systems so as to repair different gene damage. Mismatch repair system that is one of them can maintain the stability in the process of DNA synthesis by repairing mismathed basic groups. The dysfunction of this system might make mismatched basic groups not to be repaired , gene mutations to be collected and the expressions of genes to be disordered, which can cause the development of some carcinomas of instable cells, such as gastric carcinoma, colorectal carcinoma and endometrial cancer. Resently, some reseachers found that the dysfunction of mismatch repair also took part in the drug resistance.Then,is the expressions of drug resistance associated proteins due to the gene instability caused by the dysfunction of mismatch repair? In this study, the expression changes of drug resistance associated proteins P-gp, MRP, LRP, GST-πand mismatch repair proteins hMSH2, hMLH1 with resistance of gastric carcinoma cell line BGC-823 and colon carcinoma cell line LoVo induced by cDDP and 5-FU were detected, then the expression relationship of two kinds of proteins was studied, in order to explore themechanism of the drug resistance in gastrointestinal carcinomas.Methods: 1. To establish resistance cell lines BGC-823/cDDP, BGC-823/5-FU and LoVo/5-FU:①Cell culture and gradually increasing dose of cDDP and intermittent administration were used to establish resistance cell line BGC-823/ cDDP; cell culture and large dose, gradually increasing dose of 5-FU and intermittent administration were used to establish resistance cell line BGC-823/5-FU; cell culture and large dose of 5-FU and intermittent administration were used to establish resistance cell line LoVo /5-FU.②MTT assay was used to test RIs of resistance cell line BGC-823/cDDP, BGC-823/5-FU and LoVo/5-FU. 2. To detect the expressions of drug resistance associated proteins P-gp, MRP, LRP, GST-π: immunocytochemistry and Western·blot methods were used to detected the expressions of these proteins in cell line BGC-823 and its resistance cell lines BGC-823/cDDP, BGC-823/5-FU, cell line LoVo and its resistance cell line LoVo/5-FU; the expression relationship of these proteins between parent cell lines and their resistance cell lines was studied. 3. To detect the expressions of mismatch repair proteins hMSH2, hMLH1: immunocytochemistry method was used to detected the expressions of these proteins in cell line BGC-823 and its resistance cell lines BGC-823/cDDP, BGC-823/5-FU, cell line LoVo and its resistance cell line LoVo/5-FU; the expression relationship of these proteins between parent cell lines and their resistance cell lines was studied.Results: 1. Resistance cell lines and their RIs:①After cell line BGC-823 was cultured for 8 months, resistance cell line BGC-823/ cDDP was obtained with RI to cDDP being 3.93;②After cell line BGC-823 was cultured for 4 months, resistance cell line BGC-823/ 5-FU was obtained with RI to 5-FU being 8.21;③After cell line LoVo was cultured for 4 months, resistance cell line LoVo / 5-FU was obtained with RI to 5-FU being 10.26. 2. The expressions of drug resistance associated proteins P-gp, MRP, LRP, GST-π:①Cell line BGC-823 and its resistance cell line BGC-823/cDDP:the result of immunocytochemistry showed that the expressions of these proteins in resistance cell line were stronger than that in parent cell line.②Cell line BGC-823 and its resistance cell line BGC-823/5-FU: the results of immunocytochemistry and Western·blot showed that the expressions of these proteins in resistance cell line were stronger than that in parent cell line.③Cell line LoVo and its resistance cell line LoVo/5-FU: the results of immunocytochemistry and Western·blot showed that the expressions of these proteins in resistance cell line were stronger than that in parent cell line. 3. The expressions of mismatch repair proteins hMSH2, hMLH1:①Cell lineBGC-823 and its resistance cell line BGC-823/cDDP: the result of immunocytochemis- try showed that the expression of hMSH2 protein in resistance cell line was weaker than that in parent cell line, while the expressions of hMLH1 protein between two cell lines were not obviously different.②Cell line BGC-823 and its resistance cell line BGC-823/5-FU: the result of immunocytochemistry showed that the expression of hMSH2 protein in resistance cell line was weaker than that in parent cell line, while the expressions of hMLH1 protein between two cell lines were not obviously different.③Cell line LoVo and its resistance cell line LoVo/5-FU the result of immunocytoche- mistry showed that the expression of hMSH2 protein in resistance cell line was weaker than that in parent cell line, while the expressions of hMLH1 protein between two cell lines were not obviously different.Conclusion: 1. The expressions of P-gp, MRP, LRP and GST-πprotein are concerned with the natural resistance in gastrointestinal carcinoma. 2. The expressions of LRP and GST-πprotein are the mechanism of resistance of gastric carcinoma cells to cDDP. 3. The expressions of P-gp, MRP, LRP and GST-πprotein are the mechanism of resistance of gastrointestinal carcinoma to 5-FU. 4. There has down-regulation of hMSH2 protein expression in resistance cell lines BGC-823/cDDP, BGC-823/5-FU and LoVo/5-FU. This indicates that down-regulation of hMSH2 protein expression takes part in drug resistance in carcinoma cells. 5. The gene instability that results from the dysfunction of mismatch repair caused by drugs may be one of the reasons of the resistance development of carcinoma cells.