A Study Of The Mechanism Of Multi-drug Resistance In Mucoepidermoid Carcinoma

Mucoepidermoid carcinoma(MEC) is a common malignacy in salivary glands，itaccounts for about30%of malignant tumors in salivary glands and for5%to10%ofmalignant tumors in maxillofacial region in Chinese population. Anyhow,the main way ofits therapy is still surgery because chemotherapy is not very efficient to the tumor. The mainobstacle is the Multidrug resistance (MDR) effect after medication. Solving the problemof MDR may provide a new efficient way to treat MEC.MDR reduces the effectiveness of chemotherapy. MDR is characterized by crossresistance to antineoplastic drugs, and the members of the ATP-binding cassette (ABC)transporter superfamily, such as ABCB1(MDR1) and ABCC1(MRP1), which play themost importent role in MDR.Most biological functions of cells work by signal pathway. The bionomics of cellscan be revealed by studying the signal pathway. Interrupting these signal pathways relatedto illness may creat a new way to study and therapy.In previous study we established multi drug resistant cell line MC3/5-FU from theparental cell line MC3. Therefor, in this study, we designed several pieces of shRNA,constructed MDR1-shRNA expression vector and silenced MDR1in MC3/5-FU cells withstable transfection. Then we detected the reverse rate of MDR by RNAi. Furthermore, wedetected the NF-κB,Nrf2-ARE and MAPK/ERK signal pathways between MC3/5-FU andMC3to find the MDR related ones. We used RNAi and signal pathway depressor todeduce the relationship between MDR1,MRP1, signal pathways and multidrug resistancein mucoepidermoid carcinoma. The study includs four parts as follows.1Reversion of MDR in MC3/5-FU by RNAi.The plasmid expressing shRNA homologous to MDR1mRNA was constructed withshRNA expressing vector pRNAT-U-6.1/Neo based on the principle of siRNA design.Thecombinant plasmid was transfected stably into MC3/5-FU Cells using Lipofectamine2000.The shRNA efficiency was detected by real time-PCR analysis. The result showedMDR1-shRNA could effectively block the expression of MDR1in MC3/5-FU cells.RT-PCR analysis showed the expression of MDR1was silenced after stable transfection.The resistance index of MC3/5-FU was detected by MTT assay. The result showed thatthe multidrug resistance rates of MC3/5-FU cells to5-FU, BLM, VCR and VBL werereversed by70.6%,40.8%,48.6%and58.8%respectively.2Reversion of MDR in MC3/5-FU by inhibiting signal pathway.The expression of signal pathways between MC3/5-FU and MC3cells were detectedby western blot analysis. The results show that the activity of NF-κB,Nrf2-ARE andMAPK/ERK in MC3/5-FU was much higher than in MC3. The reversal of MDR wasdetected after the signal pathways was inhibited by the inhibitors. Inhibition of NF-κB，Nrf2-ARE and MAPK/ERK reversed MDR of MC3/5-FU cells to5-FU by67.7%,43.4%and54.3%respectively. MTT assay and soft agar cloning experiments showed thatinhibition of NF-κB resulted in proliferation inhibition of MC3/5-FU cells.3The relationship between signal pathway and MDR1gene inmucoepidermoid carcinoma.The relationship between signal pathway and MDR1gene in MC3/5-FU cell wereinvestigated by RT-PCR and western blot. Inhibition of NF-κB， Nrf2-ARE andMAPK/ERK inhibited MDR1mRNA expression in MC3/5-FU cells, inhibition of NF-κBshowed the strongest effects.While silence of MDR1gene by RNAi did not change theexpression of NF-κB，Nrf2-ARE and MAPK/ERK activity in MC3/5-FU cells.4The function of MRP1in MDR MRP1protein was found in surgical samples of MEC by immunohistochemistryexperiment. RT-PCR analysis showed the expression of MDR1in MC3/5-FU cells wasinhibited after MRP1was silenced by RNAi. The activity of each signal pathway wasdetected by western blot after MRP1was silenced by RNAi. The resuit showed onlyNrf2-ARE was inhibited.Conclusion1MDR1is an important factor of multi-drug resistance in mucoepidermoid carcinoma.2Silenc of MDR1by RNAi is effective in the reversal of MDR in mucoepidermoidcarcinoma.3NF-κB,Nrf2-ARE and MAPK/ERK signal pathways take part in the MDR inmucoepidermoid carcinoma.4NF-κB pathway has close relationship with the expression of MDR1in mucoepidermoidcarcinoma5MRP1may enhance the expression of MDR1by activating Nrf2-ARE rather than bymembrane protein as drug pump.