Membrane Associated Protein A3 And Preliminary Study Of Cancer Drug Resistance Correlation

Background and objectives:In the current therapy of gestational trophoblastic tumor, the most difficult problem to be solved is chemoresistance. The mechanism of tumor cells developing resistance to chemotherapeutic drugs is extremely complicated. In order to clarify mechanism of chemoresistance developed in GTN cells, two series of chemoresistant JeG-3sublines by intermittent-and consecutive-inducing methods were established in previous work of our research group. Each sery consists of cell sublines resistant to five different chemodrugs, including FUDR,5-Fu, MTX, KSM and VP16.Microarray technology has become a tool widely used in the biological sciences. Gene Chip is a high-throughput tool used to study gene expression which can greatly improve efficiency. The parent JeG-3cell line and chemoresistant cell line induced by FUDR were selected to construct two diffenerent pools in order to screen differential expressed genes between them which were thought to be related to drug-mediated resistance in our previous work. It was found that annexin A3(ANXA3) gene was significantly up-regulated in JeG-3/FUDR-resistant cell line. Based on this result and many other researches on annexin A3which suggested the function of this protein in genesis, malignant grades, prognosis and chemoresistance in many other tumors, we decided to do some research on expression of this protein in GTN cell lines and identify whether there was differential expression between the parent and chemoresistant JeG-3cell lines. Besides, until now there has been no report on annexin A3expression in choriocarcinoma cells.The technology of cell culture in vitro is the foundation of research on mechanism of chemoresistance in GTN. However, chemoresistance in vivo may be more complicated than in vitro because it is also regulated by micro-environment and many other kinds of cells. Besides, the fact that GTN is one of few solid tumors which can be cured by chemotherapy limits the enrollment of surgical specimen for study. Therefore, cell line-based human choriocarcinoma xenograft model should be established for the research on protein expression in tumor tissue and the chemoresistance of xenograft tumor in vivo can be also tested.Methods:1. Chemosensitivity of choriocarcinoma cell sublines resistant to FUDR, MTX, VP16, and KSM were tested18months after chemodrug induction using Cell Counting Kit-8(CCK8) assay. Intermittent induction using high-dose chemotherapeutic drugs were given in three cell sub-lines. The chemoresistance of these sublines were tested after six months of culture in medium without drugs.2. Expression of annexin A3were detected in parent JeG-3cell line and sublines of JeG-3/FUDR, JeG-3/MTX, JeG-3/VP16and JeG-3/KSM using western blot.3. Real-time quantitative reverse transcription-PCR (qRT-PCR) was used to detect the differential expression of annexin A3at the transcript levels between the chemo-resistant cell lines and the parent cell line.4. Cell line-based human choriocarcinoma xenograft nude model was established using JeG-3cell line and JeG-3/FUDR subline. Appropriate FUDR dosage in nude model was determined and chemosensitivity of xenograft tumor in vivo. was tested.5. Histology of choriocarcinoma xenograft and serum β-hCG level were evaluated to identify biological characteristics of choriocarcinoma. Expression of annexin A3was also evaluated in xenograft tumors by immunohistochemistry.Results:1. Resistance Index (RI) was caculated and most chemoresistant cell sublines previously constructed showed no significant change of chemoresistance after18months refrigeration. But after intermittent re-induction by high-dose chemotherapeutic drugs, RI showed trends of decline.2. Significant higher expression level of annexin A3was found in JeG-3/FUDR and JeG-3/MTX sublines compared with JeG-3parent cell line by semiquatitation of Western Blot.3. Differential expression of ANXA3at mRNA level showed by qRT-PCR was in exact cohenrence with that at protein level.4. Cell-line based subcutaneously implanted xenograft model was established in nude mice and appropriate dosage of FUDR for intraperitoneal injection was62.5ug/g/mice-weight. But the inhibition rate of FUDR towards xenograft tumor based on JeG-3and JeG-3/FUDR showed no difference in our experiment.5. Annexin A3was expressed in cytoplasma and cytomembrane in choriocarcinoma tissue of xenograft tumor. JeG-3/FUDR based xenograft showed differential expression of Annexin A3compared with JeG-3based xenograft which coincided with results at cell level. Conclusion:1. Chemoresistance of JeG-3chemoresistant sublines previously constructed were relatively stable. The ways of induction can influence chemoresistance.2.Annexin A3may be involved in the mechanism of chemoresistance of GTN and the relevance was probably drug-specific and accumulative.3. Cell-line based xenograft nude model mirrored the characteristic of choriocarcinoma and can be used for further research. Lots of factors can influence chemoresistance of tumor cells in vivo.