| Background:Choriocarcinoma (CC) is a form of gestational trophoblastic neoplasia (GTN). Because of its highly invasive and destructive nature, widespread metastases appear at comparatively early stages of pregnancy. Fortunately, CC is curable, and chemotherapy may be effective for 90% patients. However, drug-resistance is a main cause for the death of CC patients. Thus, understanding the mechanisms of chemo-resistance may lead to biomarkers screening, improved risk stratification and new treatment strategies.Much evidence has shown that the mechanisms responsible for chemo-resistance are multi-factorial, and analysis of a single protein as a factor determining chemo-resistance or as a biomarker of it is of limited use. Fortunately, the development of high-resolution techniques such as proteomics and progress in systems biology have made it possible to perform network investigations to identify panels of proteins involved in particular pathological conditions, as to facilitate the study of chemo-resistance mechanisms and biomarker identification.In order to clarify the mechanism of chemo-resistance of GTN, we generated chemo-resistant human CC JeG-3 sub-line with five different clinical chemo-reagents including (FUDR,5-FU, MTX, KSM and VP16) by sub-culturing JeG-3 cells with incremental and consecutive feeding methods respectively. Chemo-resistance related proteins were searched for the first time by using differential proteomics-based approaches in choriocarcinoma sensitive and resistance cell lines. Systems biology and bioinformatics were used to screen differentially expressed proteins identified by 2D-DIGE and MALDI-TOF-MS. The roles of the candidate proteins in chemo-resistance choriocarcinoma were further discussed.Methods:1. Starting from the chemo-sensitive JeG-3 human choriocarcinoma cell line, chemo-resistant variants were obtained by exposing parental cell line to stepwise increased related chemo-reagents concentrations by intermittent and consecutive feeding methods.2. The biological characteristics of all the sub-lines were examined under an inverted phase contrast microscope. IC50, cell cycle, hormone secretion and growth curve were detected in all the sub-lines. Cell Counting Kit-8 (CCK-8) assay was used to measure the inhibitory concentration 50%. qRT-PCR was used to measure dynamical profiles of DHFR, GST-π, MDR1, MRP, survivin, TopoⅡαand TS genes mRNA expression during the establishment of related chemo-resistant sub-lines.3.2D-DIGE and MALDI-TOF-MS approaches were used to identify the differential proteins in JeG-3 parent cell line and chemo-resistant sub-line. The differentially expressed proteins were screened by using systems biology and bioinformatics methods.4. The levels of the candidate proteins in chemo-resistant sub-lines were validated by western blot respectively. RNAi was used to knockdown the expression of CALR and/or PDIA3 respectively. The effects on growth and proliferation and IC50 of RNAi cells were detected.5. CALR, PDIA3 and GRP78 expression and sub-cellular location were also detected in different concentration of chemo-reagents-induced sub-lines by immunocytochemistry methods.6. qRT-PCR was used to detect the difference of the transcript levels of candidate proteins and ER function related pathway among the chemo-resistance sub-lines and parent cell line.Results:1. Five kinds of chemo-resistant sub-lines named JeG-3/FUDRA1,JeG-3/FUB1,JeG-3/MTXA1,JeG-3/KSMB1 and JeG-3/VPC1 were established by intermittent inducing method. In consecutive-inducing, three kinds of chemo-resistant sub-lines were established:JeG-3/FUDRC2,JeG-3/FUC2 and JeG-3/MTXC2. The RI of these chemo-resistant sub-lines ranged form 11.26-65.87, and had no significant change after half a year refrigeration.2. Many differences could be found among chemo-resistant sub-lines and parent JeG-3 cell line. The dynamical profile ofβ-HCG and P secretion suggested that the hormone secretions were stage-related, and had no relationship with the concentration of chemo-reagent exposure and RI.3. The transcript level of LRP seemed to be in coincidence with the concentration of FUDR-and 5-FU-exposure; the mRNA expression level of GST-πhad positive correlation with the concentration of 5-FU-and KSM-exposure and the RI. The transcription levels of other chemo-resistant related genes were all stage-related with the concentration of the drug exposure, and had no relationship with the RI.4. Forty-six proteins spots were found to be significantly different in spot intensity by statistical analysis between chemo-resistance sub-line and parent cell line, of which 31 proteins were identified by MALDI-TOF-MS. Comparing to the parent cell lines, CALR, PDIA3 and GRP78 were screened out finally. The expression folds in chemo-resistance sub-lines of these three proteins were 1.56,2.44 and 1.76 respectively.5. These three proteins were upregulated in all chemo-resistance sub-lines, and the expression levels of these three proteins seemed to enhance gradually in coincidence with the increased concentration of drug inducing and the increased RI. The RI decreased 1.52-4.22 in JeG-3/FUDRA1 and JeG-3/MTXA1 sub-lines by knockdown the CALR and/or PDIA3 expression respectively. There were no significant changes in morphology and cell growth after RNAi.6. All these three proteins were located in ER by confocal microscopy. Besides the high intensities of these three proteins were exhibited in chemo-resistance sub-lines, the translocation of CALR and PDIA3 were also found, especially in JeG-3/VPC1 sub-lines.7. The genes involved in ER protein fold-related pathway were changed to certain extention by comparing with parent cell line and chemo-resistance sub-lines.Conclusion:1. We established eight kinds of chemo-resistance choriocarcinoma sub-lines successfully.2. The disaccording profile ofβ-HCG secretion with the concentration of chemo-reagents exposure and RIs of related sub-lines indicated that the P-HCG could not be accurate for the prediction of chemo-resistance in chemo-therapy of choriocarcinoma.3. The transcript level of GST-πand LRP could serve as a reliable candidate biomarker for predicting chemo-resistance to 5-FU-based chemotherapy, and might also predict KSM-resistant and FUDR-resistant respectively.4. Three endoplasmic reticulum molecular chaperones were found to be up-regulated in all chemo-resistance sub-lines which indicated enhanced ER protein folding ability may be involved in the mechanism of chemo-resistance of GTN.5. ERS and UPR seemed to be activated in chemo-resistance choriocarcinoma cell lines, which indicated the related pathway of ER function might be the next target of chemo-resistance choriocarcinoma.6. The translocation of CALR and PDIA3 suggested that the immunogenicity of tumour cell death might participate in the mechanism of chemo-resistance. |
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