Stable suppression of MDR-1 gene using siRNA expression vector to reverse drug resistance in a human uterine sarcoma cell line.
OBJECTIVE
Chemotherapy is highly effective in treating a number of gynecologic malignancies; however, its effectiveness diminishes with repeated exposures due to the emergence of multi-drug resistance (MDR). The aim of this study was to establish a permanent MDR gene knockdown model via infection with the siRNA-hairpin expression vector. The impact of transfecting the RNAi upon MDR-1 mRNA and P-glycoprotein expression as well as resultant chemotherapy resistance was assessed.


METHODS
Multi-drug resistant cell line MES-SA/DX5 was transfected with the siRNA-hairpin expression vector (pSMDR-HYG) designed to target MDR-1 mRNA. A negative control was established utilizing a vector lacking the anti-sense component (pSCON-HYG). The LD(50) of doxorubicin for the stable transfectants was determined utilizing a cytotoxic MTT assay. The mRNA expression of MDR-1 gene among those cell lines was evaluated by semi-quantitative RT-PCR. The product of P-glycoprotein (P-gp) was examined by Western blotting hybridization and immunostaining.


RESULTS
Two stable transfected cell lines: MES-SA/DX5-M (with pSMDR-HYG) and MES-SA/DX5-C (with pSCON-HYG) were established. The cell line MES-SA/DX5-M was nearly 7 times more sensitive to doxorubicin than MES-SA/DX5-C and its parent cell line MES-SA/DX5 (P < 0.01). The mRNA expression of the MDR-1 gene in MES-SA/DX5-M was also statistically significantly lower than in the other 2 cell lines (P < 0.01) as assessed by semi-quantitative RT-PCR. A barely detectable signal for P-gp (170 kDa) was observed in MES-SA/DX5-M. The vast majority of MES-SA/DX5-M cells were immunohistochemically negative for P-gp.


CONCLUSIONS
Stable, sequence-specific MDR-1 gene silencing can be demonstrated by inducing the endogenous expression of hairpin siRNA. Hairpin-siRNA-based MDR-1 gene silencing correlated with decreased levels of MDR-1 mRNA and P-gp, thereby restoring permanent native chemosensitivity. This methodologic strategy may have significant clinical impact in reversing chemo-resistance, especially the multi-drug-resistant phenotype, in the treatment of gynecologic malignancies.
Hua
Jun
J
Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Washington University School of Medicine, Box 8064, 4911 Barnes Hospital Plaza, St. Louis, MO 63110, USA.
Mutch
David G
DG
Herzog
Thomas J
TJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D045744:Cell Line, Tumor; D002934:Cinnamates / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D019450:Genes, MDR; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006921:Hygromycin B / Q000031:analogs & derivatives* / Q000494:pharmacology; D008969:Molecular Sequence Data; D034622:RNA Interference; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D012509:Sarcoma / Q000188:drug therapy* / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014594:Uterine Neoplasms / Q000188:drug therapy* / Q000235:genetics* / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002934:Cinnamates; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D006921:Hygromycin B; C026273:hygromycin A; D004317:Doxorubicin