The Reversal Effect Of Raf-1/Mdr-1 SiRNAs On Human Oral Squamous Cell Carcinoma With Multidrug Resistence

Multidrug resistance(MDR) is a major barrier for chemotherapy of most patients with oral squamous cell carcinoma. MDR means that tumor cells develop the resistance to many kinds of chemotherapy agents with various structures and functions.The multidrug resistance is of a multisteps process and a very complex mechanism. The overexpression of P-glycoprotein(P-gp) encoded by mdr-1 gene is one of the most important reasons for MDR development..The human Pgp(Mdr-1) on cell membrane as efflux pump belongs to the ATP-binding cassette transporter expelling out antitumor agents from the cytoplasm , reducing intracellular drug concentrations in the cells to sublethal ,so that P-pg is a important target for reversing MDR. Besides,there are many other factors impacting the genesis of the MDR,for example, glutathlone S-transferase(GST) which acts by detoxification of some chemotherapeutic drugs,multidrug resistance-associated protein(MRP), transcription factor nuclear factor-kappa B(NF-κB) and so on. So many kinds of the agents such as antisense RNA and traditional medicines were released to have partly effect on reversing MDR of the tumor cells because MDR mechanism is too complex. we cannot attain ideal effect if we merely block one target or signaling pathway related to MDR. Researchers has adoptted many methods to reverse MDR,such as traditional Chinese drugs,anti-RNA, blockade of the gene expressions or signal transductions related to MDR.In recent years,RNA interference(RNAi) has widely used as an experimental tool to analyse the function of mammalian genes. Double- stranded RNA(dsRNA) reagents are used to bind to and promote the degradation of target RNAs,resulting in knockdown of the expression of specific genes.In the field of reversal of multidrug resistance,small interfering double-stranded RNAs (siRNA) were designed to target the factors such as P-gp or GST mRNA as a strategy to inhibit resistant gene expression at the mRNA level in order to restore sensitivity to chemotherapeutic drugs.Raf-1 is a cytoplasmic serine/threonine protein kinase that plays an important role in the tumorigenesis and inhibiton of apoptosis by modulating transcriptional activation and mitogenesis.Some experiments has indicated that Raf-1 kinase is associated with drug resistance in human breast cancer and prostate cancer and ectopic expression of Raf1 will increase the levels of the Mdr-1 drug pump.This increased expression of Mdr-1 most likely occurs by a transcriptional mechanism.The present research was to investigate the effect of Raf-1 on the multidrug resistance in multidrug resistant human oral squamous cell carcinoma (KBV) cells and detect the effect of Raf-1 siRNA, Mdr-1 siRNA and Raf-1/Mdr-1 siRNAs (Mdr-1 siRNA and Raf-1siRNA transfected into drug resistant KBV cells at the same time) on the reversal of multidrug resistance in KBV cells. We hoped that we might put forward a more effective method to reverse the multidrug resistance in KBV cells by the present study.In this study, we investigated Mdr-1 and Raf-1 genes expression and abilities to resist the chemotherapeutic drug vincristine (VCR) in six groups: Mdr-1 siRNA transfected KBV cells,Raf-1 siRNA transfected KBV cells,Mdr1 /Raf-1 two siRNAs transfected KBV cells, KBV, KB and Control siRNA as control. Using RT-PCR , western blotting assay and immuno- fluorescent method the changes of the RNA and protein levels of both Mdr-1 and Raf-1 genes were studied.Using tetrazolium-based chemosensitivity assay(MTT) and flow cytometry (FCM) the changes of chemosensitivity to VCR in six groups of cells were detected.This study includes four sections: (1) detection of relationships between Raf-1 and MDR in KBV cells; (2)the effect of Mdr-1 siRNA transfection on MDR in KBV cells; (3) the effect of Raf-1 siRNA transfection on MDR in KBV cells; (4) the effect of Mdr1 /Raf-1 siRNAs transfection on MDR in KBV cells. The first section was to detect the relationgships between Raf-1 and multidrug resistance in KBV cells.The changes of the mRNA and protein levels of both Mdr-1 and Raf-1 genes in KBV and KB cells were determined by RT-PCR ,western blotting assay and immunofluorescent method.By MTT and FCM, IC₅₀ ratios and apoptosis rates in both KBV and KB cells were detected.The findings indicated that expressions of both Raf-1 and Mdr1 mRNA and protein levels in KBV cells were lower than those in KB cells statistically (P<0.01). Immunofluorescent image showed that both Mdr1 and Raf-1 protein expressions in KBV cells were much stronger than those in KB cells.The IC₅₀ ratio in KB cells was 21.33±2.25μg/L and that in KBV cells was 352.68±12.36μg/L.The resistance Index (RI) was 16.5.The apoptosis rate in KB cells was 98.8±1.2% and that in KBV cells was 23.5±2.1%.These results showed that differences in the chemosensitivity to VCR between KBV cells and KB cells were significant statistically (P<0.01). This study suggested that Raf-1 might play an important role in the process of developing the multidrug resistance in the human oral squamous carcinoma.Mdr-1 gene overexpression was still closely related with MDR in KBV cells.The second section was to investigate the effect of Mdr-1 siRNA on MDR in KBV cells. Mdr-1 siRNA was transfected into KBV cells. The changes of the Mdr-1 and raf-1 gene expression at the mRNA and protein levels in Mdr-1 siRNA transfected KBV cells were determined by RT-PCR ,western blotting assay and immunofluorescent method. Using MTT and FCM IC₅₀ and apoptosis rates in Mdr-1 siRNA transfected KBV cells were determined. The findings suggested that the mRNA and protein levels of Mdr-1 genes in Mdr1 siRNA transfected KBV cells were much lower than those in KBV cells statistically (P<0.01). Immunofluorescent image showed that P-gp expression in Mdr-1 siRNA transfected KBV cells were much weaker than that in KBV cells.The IC₅₀ in Mdr-1 siRNA transfected KBV cells,KBV,KB and Control siRNA transfected KBV cells were respectively 130.68±6.35μg/L, 352.68±12.36μg/L, 21.33±2.25μg/L, 356.47±14.23μg/L. RI(Mdr-1 siRNA/KB) was 6.1 and RI(KBV/KB) was 16.5.The apoptosis rates in Mdr-1 siRNA transfected KBV cells was 67.2±3.7% ,24.3±1.6% in Control siRNA transfected KBV cells,98.8± 1.2% in KB cells and 23.5±2.1% in KBV cells.These results showed that the chemosensitivity to VCR in Mdr-1 siRNA transfected KBV cells was higher than that in KBV cells statistically (P<0.01). This study suggested that Mdr-1 siRNA was effective inhibitor of Mdr-1 gene expression to reverse the resistance of human oral squamous cell carcinoma.The third section was to explore the effect of Raf-1 siRNA on MDR in KBV cells. Raf-1 siRNA was transfected into KBV cells.The Mdr-1 and Raf-1 genes expression at the mRNA and protein levels in Raf-1 siRNA transfected KBV cells were determined using RT-PCR ,western blotting assay and immunofluorescent method. Using MTT and FCM IC₅₀ ratios and apoptosis rates in Raf-1 siRNA transfected KBV cells were determined. The findings suggested that the mRNA and protein levels of both Raf-1 and Mdr-1 genes in Raf-1 siRNA transfected KBV cells were lower than those in KBV cells statistically (P<0.01). Immunofluorescent image showed that P-gp and Raf-1 proteins expression in Raf-1 siRNA transfected KBV cells were much weaker than those in Control siRNA transfected KBV cells.The IC₅₀ in Raf-1 siRNA transfected KBV cells ,KBV,KB and Control siRNA transfected KBV cells were respectively 201.38±7.13μg/L, 352.68±12.36μg/L, 21.33±2.25μg/L, 356.47±14.23μg/L.RI(Raf-1 siRNA/KB) was 9.5 and RI(KBV/KB) was 16.5.The apoptosis rate in Raf-1 siRNA transfected KBV cells was 49.2±3.2% , 24.3±1.6% in Control siRNA transfected KBV cells,98.8±1.2% in KB cells and 23.5±2.1% in KBV cells..These results showed that differences in the chemosensitivity to VCR between Raf-1 siRNA transfected KBV cells and KBV cells were significant statistically (P<0.01). This study suggested that Raf-1 siRNA was also effective inhibitor of Raf-1 and Mdr-1 gene expression to reverse the resistance of KBV cells.In multidrug resistant human oral squamous cell carcinoma, overexpression of Raf-1 could increase the levels of the Mdr-1 drug pump, which might be the reversal mechanism of Raf-1.The fourth section was to investigate the the effect of Mdr-1 /Raf-1 siRNAs transfection on MDR in KBV cells. Mdr-1 /Raf-1 siRNAs were transfected into KBV cells at the same time. The Mdr-1 and Raf-1 genes expression at the mRNA and protein levels were assayed by RT-PCR, western blotting assay and immunofluorescent method. By MTT and FCM IC₅₀ ratios and apoptosis rates in groups of cells were determined. The findings suggested that the mRNA and protein levels of Mdr-1 genes in Mdr-1 /Raf-1 siRNAs transfected KBV cells were much lower than those in Mdr-1 siRNA transfected KBV cells or Raf-1 siRNA transfected KBV cells statistically (P < 0.01). Immunofluorescent image showed that P-gp expression in Mdr-1 /Raf-1 siRNAs transfected KBV cells was much weaker than those in Raf-1 siRNA or Mdr-1 siRNA transfected KBV cells.The IC₅₀ in Mdr-1 /Raf-1 siRNAs transfected KBV cells,Raf-1 siRNA transfected KBV cells,Mdr-1 siRNA transfected KBV cells,KB,KBV and Control siRNA transfected KBV cells were respectively 58.76±3.27μg/L, 201.38±7.13μg/L, 130±6μg/L, 21.33±2.25μg/L, 352.68±12.36μg/L, 356.47±14.23μg/L.RI (KBV/KB) was 16.5, RI(Raf-1 siRNA/KB) was 9.5,RI(Mdr-1 siRNA/KB) was 6.1 and RI[(Raf-1 / Mdr-1 siRNAs)/KB] was 2.8.The apoptosis rate in Raf-1 / Mdr-1 siRNAs transfected KBV cells was 88.2%±4.3%, 49.2±3.2% in Raf-1 siRNA transfected KBV cells, 67.2%±3.7% in Mdr-1 siRNA transfected KBV cells,98.8±1.2% in KB cells;23.5±2.1% in KBV cells and 24.3±1.6% in Control siRNA transfected KBV cells respectively.These results showed that the chemosensitivity to VCR in Raf-1 / Mdr-1 siRNAs KBV cells was much higher than Raf-1 siRNA transfected KBV cells or Mdr-1 siRNA transfected KBV cells statistically (P< 0.01). This study suggested that Raf-1 / Mdr-1 siRNAs transfection that block Mdr-1 genes expression more effectively has proved to be highly beneficial in reversing the resistance of KBV cells,compared with Raf-1 siRNA or Mdr-1 siRNA single transfection.In sum, Raf-1 might play an important role in the process of developing the multidrug resistance in the human oral squamous carcinoma.Mdr-1 gene expression was still closely related to MDR in KBV cells. Mdr1 siRNA was effective inhibitor of Mdr-1 gene expression to reverse the resistance of human oral squamous cell carcinoma. Raf-1 siRNA could down regulated both Raf-1 and Mdr-1 genes expression and reverse the resistance of KBV cells. Raf1 could modulate the levels of the Mdr-1 drug pump in KBV cells, which might be the reversal mechanism of Raf1. Mdr1 is still a main target for therapeutic intervention. Raf-1 / Mdr-1 siRNAs transfection could block Mdr-1 gene expression and reverse the resistance of KBV cells more effectively compared with Raf-1 siRNA or Mdr-1 siRNA single transfection.