Study Of The Relationship Between The Expression Of Neutral Glycosphingolipids And Multi-drug Resistance In Ovarian Cancer Cell Line SKOV₃/AdrR

ABSTRACTObjectives:To investigate the relationship between the expression of neutral glycosphingolipids (N-GSL) and multi-drug resistance, the multidrug resistant human ovarian cancer cell line SKOV3-Adriamycin-resistant (AdrR) and its parental SKOV3 wild type (drug-sensitive) cell line were studied. The expression of N-GSL , mdrl gene and P-glycoprotein and Rhodamine 123 retention in SKOV3 and SKOV3/AdrR were analyzed. Methods:SKOV3/AdrR cells were treated with PPMP (1-phenyl-2-palmitoylamino-3-. morpholino -l-propanol), a kind of glucosylceramide synthase inhibitor, in different acceptable concentration. SKOV3 and SKOV3/AdrR (before and after the treatment of PIPMIP) cells were collected and N-GSLs (neutral glycosphingolipids) of the cells was isolated and purified with the modified Hakamori's method, changes of CMII (Monohexosylceramide) content were analyzed by HPTLC (high performance thin layer chromatography). The alterations of chemosensitivity to ADM (Adriamycin) and VCR (Vincristine) were evaluated by MTT (the tetrazolium dye) assay. Expression of mdrl gene were analyzed by Reverse Transcriptase-Polymerase chain reaction (RT-PCR), determination of P-gp expression was performed by flow cytometry using UIC2 monoclonal antibody, characterization of P-gp function was determined by Rhodamine 123 retention, cells were also analyzaed by flow cytometry. Results: The levels of CMH in SKOV3/AdrR cells were much higher than those in SKOV3 cells. The synthesis of CMII in SKOV3/AdrR can be inhibited completely by 25 g mol/L PPMP treatment for 48h, 44% of synthesis were inhibited by 15 ~ molfL PPMP treatment for 48h, 40% of synthesis were inhibited by 5 .u molIL PPMP treatment for 48h. PPMP appears to enhance chemosensitivity of SKOV3/AdrR cells to ADM and VCR. PPMP was found to inhibit mdrl expression of SKOV3/AdrR at the mRNA level, this modulation of gene expression was concentration dependent and complete inhibition appeared at 25 ~imoIJL PPMP treatment for 48h. Flow cytometry showed that about 94.8% of SKOV3/AdrR cells-4-were P-gp positive. After treated with 5, 15, 25 g mol/L PPM1P for 48h, SKOV3/AdrR cell line had 73.8%, 41.8%, 17.7% P-gp positive cells, respectively. PPMP could increase intracellular Rh123 accumulation in resistant cell lines. This modulation of Rh123 accumulation was positive correlation with concentration of PPMP. Conclusion: PPMP can effectively inhibit CMII synthesis in ovarian cancer MDR cell line SKOV3/AdrR. When treated with PPMIP, SKOV3/AdrR cells can regain sensitivity to Adriamycin. In the mean time, PPMP could modulate mdrl expression at the mRNA level, inhibit expression of P-gp. These changes paralleled the decreases in P-glycoprotein pump function.