Establishment Of Methotrexate-resistant Human Choriocarcinoma Cell Line And Of It's Mechanisms Of Drug-resistance

Choriocarcinoma is one of solid tumors which are sensitive to chemotherapy. Methotrexate (MTX) is a first-line and essential component in combined chemotherapy in the treatment of choriocarcinoma. However acquired drug-resistance is common and serious in choriocarcinoma. It is estimated that about 30% of patients will develop drug-resistance if single MTX is used. Drug-resistance is an important reason that causes failure of treatment and results in death of patients .The mechanisms of MTX-resistance are complicated, including decrease of MTX intake, increase of DHFR activity and decrease of the infinity of MTX to DHFR. But these mechanisms can not explain cross-resistance to other chemotherapy drugs. Following the advance of studies on cell apoptosis, it is found that cell apoptosis is the common pathway of cell toxity of chemotherapy drugs. Therefore , we suposed that suppression of apoptosis maybe one of mechanisms of drug-resistance. The basic apoptotic pathway includes death receptor pathway and mitocondrial pathway. Little reports have been found on the roles of apoptosis in cytotoxicity of MTX and it's relationship to drug reisitance. In the part one of the present study , we established MTX-resistant cell line (JAR/MTX) which was derived from JAR cell line by exposed to MTX with intervally and progressively increasing concentration , and observed it's biological characters . In the part two of the study , we detected the rate of methotrexate-induced apoptosis and apoptotic relatedproteins .In the part three of the study . we investigated the apoptotic pathway involved in methotrexate-induced apoptosis by detecting the activity of Caspases-8, Caspases-9, and the change of mitochondrial membrane potential.Part I Establishment and biological characters of methotrexate-resistant human choriocarcinoma cell line(JAR/MTX)Objective: To establish a MTX-resistant choriocarcinoma cell line (JAR/MTX) and determine its biologic characteristics. Methods: JAR/MTX was derived from JAR cell line by exposed to MTX with intervally and progressively increasing concentration . Sensitivity to some chemotherapy drugs was detected by MTT. P-gp> GST-7I and PCNA expressions were detected by immunohistochemistry. Cells apoptosis was detected with flow cytometry (FCM) assessment of PI/Annexin V stain. Growth rates and human chorionic gonadotropin (hCG) were also detected. Results: JAR/MTX with stable drug resistance was established after one year. Its resistance index to MTX was 7.35. Besides resistance to MTX, it exhibited cross-resistant to TAX and VCR. Growth rates of JAR/MTX were slower than that of JAR. Expression level of PCNA in JAR/MTX was lower and GST-it expression level in JAR/MTX was higher than that in JAR. No statistical difference of expression level of P-gp existed between in the two cell lines. JAR/MTX secreted high hCG than JAR. The flow cytometry showed that the spontaneous apoptosis in JAR/MTX was significantly lower than that in JAR. Conclusions: JAR/MTX cell line presented stable resistant to MTX with different characteristics, including morphology, proliferation, apoptosis rate from it's parental line JAR. It is indicated that JAR/MTX may serve as an ideal model for studying the mechanisms of drug resistance in choriocarcinoma.Part II Methotrexate-induced apoptosis and expression of apoptotic-related proteins in JAR and JAR/MTX.Objective: To investigate MTX-induced apoptosis in human choriocarcinoma JAR and JAR/MTX cell line and the change of Bax/Bcl-2 expression during apoptosis. Methods: Cells apoptosis was detected by assessment of PI/Annexin V stain with FCM.Bcl-2 and Bax expressions were detected by immunocellchemistry. Results: JAR cells underwent apoptosis and cycle retardation when exposed to 0.1-1.0ug/ml MTX after 24 hours but not JAR/MTX cells. JAR cells presented necrotic death when exposed to 5.0ug/ml MTX,while JAR/MTX cells just started to present apoptosis. The H-score of Bcl-2 was decreased while H-score of Bax was increased when both kind of cells underwent apo...