Primary Study On Mechanism And Targeting Reversal Of Multidrug Resistance Of High-differentiated Pancreatic Carcinoma

Ⅰ. The differential expressions of P-gp,MRP,LRP and IGF-ⅠR on high- differentiated pancreatic carcinoma tissue and its significanceⅡ. Evaluation of IGF-ⅠR antisense oligodeoxynucleotide on regulation of MRP expression of SW1990 pancreatic carcinoma cell lineⅢ. Effects of IGF-ⅠR antisense oligodeoxynucleotide on chemotherapeutic sensitivity of SW1990 pancreatic carcinoma cell lineObjective: To study the mechanism of multidrug resistance(MDR) of high- differentiated pancreatic carcinoma(HDPC) and its regulation from the point of membranous transport proteins, then to establish the targeting therapy for MDR of HDPC. To detect the effects on MRP expression as blocking the IGF-Ⅰ/IGF-ⅠR circular route of SW1990 pancreatic carcinoma cell line. To discuss the interventional effects of IGF-ⅠR antisense oligodeoxynucleotide on chemotherapeutic sensitivity of SW1990 pancreatic carcinoma cell line.Methods: Our study included three parts. In the first part, 26 paraffinembeded specimens, with identified diagnosis of high differentiated pancreatic carcinoma, were collected from four hospitals ranging 2000.4-2001.12, and immunohistochemical SP method was used to assess the expression of P-gp,MRP,LRP and IGF-ⅠR in tumor and adjacent non-tumor tissues, then comparative studies and related analysis were accessed. In the second part, we designed and synthesized a 20mer mixed-boned antisense oligodeoxynucleotide(ASODN) aiming at IGF-ⅠR mRNA, and the ASODN was transfected into the pancreatic carcinoma cell line SW1990 at the concentration of 250ng/ml mediated by non-liposomal transfection reagent FugeneTM6. Dynamic detection was managed on variety of IGF-ⅠR level at 12nd,24th,48th,72nd,96th hour postotransfectively to learn the effects of the all-renewed IGF-ⅠR ASODN on IGF-ⅠR; and immunocytochemistry, western-blot and RT-PCR were taken to test the expressions of MRP after transfection of IGF-ⅠR ASODN both in protein and mRNA levels. In the third part, to evaluate the influence of MRP down-regulation on MDR resulted in IGF-ⅠRASODN transfection, HPLC, MTT method, Flow cytometry and AO fluorescent stain were adopted to observe the changes of the drugs' concentration, killing effect, cell circle and apoptosis of SW1990 cell as combining different doses of IGF-ⅠR ASODN with various chemotherapeutic agents——5-Fu, MMC and Gemcitabine in different concentrations, and empty transfection, DMEM pseudotransfection and sense oligodeoxynucleotide(SODN) transfection were taken as the control groups.Results: 1.1,In the tumor tissue of HDPC, the three membranous transport proteins were demonstrated a distinctly positive expression rate: as MRP, the highest, with 76.9 percent, P-gp 42.3 percent and LRP only 7.69 percent; whereas, in the para-tumor tissue, the expression of P-gp was the highest with 34.6 percent, MRP 7.69 percent and no detection of LRP. The expression rate of MRP in tumor tissue was apparently higher than that of in para-tumor tissue(p<0.05), however, no statistical differentiation was found on either P-gp or LRP between tumor tissue and para-tumor tissue (p>0.05).1.2,IGF-ⅠR overexpressed in tumor tissue with positive rate of 73.1 percent in contrast to no appearance in para-tumor tissue(p<0.05), and the related analysis manifested the notably positive correlation between IGF-ⅠR and MRP in HDPC(rs=0.711, p<0.05).2.1,The pancreatic carcinoma cell line SW1990, presenting a typical growth characteristics of the epithelial cell, highly expressed both IGF-ⅠR and MRP. The immunocytochemistry showed that the positive stain was localized at cellular membrane and cytoplasm, someone in nuclear.2.2,The IGF-ⅠR level of SW1990 cell line, obviously decreased as comparing with that of the control groups after transfection with IGF-ⅠR ASODN (p<0.05): the positive expression rate of IGF-ⅠR protein was 84.2% in empty group, but 51.3%,22.6%,19.7%,38.8% and 62.4% at 12nd, 24th, 48th, 72nd, 96th hour after transfection measured with immunocytochemistry, and the RT-PCR outcomed that the relative...