Sirna Study On Reversal Of Multidrug Resistance In Tumor In Vivo

Background and Purpose: MDR (multi-drug resistance, MDR) is oneof the major causes of failures of chemotherapy of human malignancy.MDR is the phenomenon in which cancer cells exposed to oneanticancer drug show resistance to various anticancer drugs that arestructurally and functionally different from the initial anticancer drug.The classical MDR is due mostly to increased efflux pumps in the cellmembrane of cells pumping anticancer drugs out of cells. The mosttypical efflux pump in the cell membrane is P-gp. RNAi (RNAinterference, RNAi) is a new molecular biology technique. RNAi refersto the introduction of homologous dsRNA (double stranded RNA,dsRNA) to specifically target a gene's product, resulting in null orhypomorphic phenotypes, and mediates the process of gene silencingafter transcription. We studied the possibility and specificity of siRNAon reversing multidrug-resistance in human glioma cells and in the nudemice tumor xenografts. Methods: Two different MDR1-targeted siRNAduplexes homologous to the P-gp-encoding MDR1 mRNA consensussequence (GeneBank accession number NM₀00927) were designed.One non-specific siRNA was used as a control. All of the siRNAduplexes were synthesized by Invitrogen Research, Inc. U251 cells inexponential phase of growth were plated in six-well plates at 1×10⁵ cells/well, grown for 24 h then transfected with siRNA usingoligofectamine and OPTI-MEM I reduced serum medium, according tothe manufacturer's protocol. Silencing was examined 24-48 h aftertransfection by reverse transcription-PCR (RT-PCR) and Western blot.Control cells were treated with oligofectamine and serum-reducedmedium (mock). To examine the sensitivity of tumor cells to cytotoxicagents, siRNA- and mock-transfected cells were treated with differentconcentrations of Vincristine, Becenun, Me-CCN, Pacilitaxel, Epirubicinand Vm-26. The inhibition of cell growth was evaluated by the MTTmethod. The absorbance in individual wells was determined at 570nmby a microplate Reader and the rate of inhibition (IR) was calculatedaccording to the formula: IR=(1-mean A value of experimentalgroup/mean A value of control group)×100%. To test whether siRNAcould be used to reverse MDR of tumor xenografts in vivo, transfectionand tumor growth suppression experiments were conducted in nude micebearing human NCIH460 solid tumors. Tumor xenografts weretransfected with different amounts of MDR1siRNAs by in vivoelectroporation combinated intro-tumor injection method(0.5nmol/150mm³,1nmol/150mm³,2nmol/150mm³,3nmol/150mm³).Silencing was examined 48h after transfection by reversetranscription-PCR (RT-PCR) and immunohistochemistry. To furtherdetermine whether introduction of MDR1siRNA could increase the inhibition of Vinorelbine Bitartrate to tumors transfected with siRNA,mice were treated with Vinorelbine Bitartrate (NVB) (5mg/kg/w, i.p.) 48h after siRNA transfected. The two perpendicular diameters of tumorswere measured every two days and tumor volume was estimatedaccording to the formula: V=лab2/6. The curve of tumor growth wasdrawn according to tumor volume and time of transfection. Two daysafter the last drug treatment, the tumor tissue was excised from mice andits weight was measured. The IR was calculated according to theformula: IR=(1-mean tumor weight of experimental group/mean tumorweight of control group)×100%. Results: To test whether siRNA couldbe used to modulate MDR and determine the effect of siRNA on targetprotein expression, we treated tumor cells with siRNA duplexs designedto target MDR1 mRNA. The results showed that as compared with mocktransfection, the siRNA duplexes significantly reduced the levels of theendogenous mRNA and the expressions of P-gp in tumor cells 24 and 48h after treatment, as determined by reverse transcription-PCR andWestern Blot. To assess whether siRNA-directed suppression of P-gpsensitized tumor cells to cytotoxic agents, we compared the drugsensitivity of the siRNA-treated to that of mock-treated cells using MTTcytotoxicity assay. The sensitivity of the tumor cells to Vincristine,Becenun, Me-CCN, Pacilitaxel, Epirubicin was increased significantlyby introduction of MDR1-targeted siRNAs. The sensitivity to Vm-26, a drug that is not transported by P-gp, was not affected by silencing ofP-gp expression. To test whether siRNA could be used to reverse MDRof tumor xenografts in vivo, tumors were treated with different amountsof MDR1siRNAs. The results showed that the inhibitory effect on thelevels of MDR1 mRNA and P-gp were clearly correlated with the doseof introducted siRNAs, as determined by reverse transcription-PCR andimmunohistochemistry. To further determine whether introduction ofMDR1siRNA could increase the inhibition of NVB to siRNA transfectedtumors, mice were treated with NVB (i.p.). The results demonstrated thatNVB inhibited the growth of MDR1siRNA transfected tumorsdramatically, but the same treatment didn't inhibit the growth ofnon-transfected tumors. Conclusions: Our data showed that theadministration of siRNA targeted MDR1 gene can effectively reverseMDR of tumors both in vitro and in vivo models.