To Down-regulate βⅢ-tubulin Expression And Increase The Drug Sensitivity Of A549/Taxol Cells Lines

Purpose and background: Lung cancer is the most common cancer in the worldwith>1million cases diagnosed every year, and remains the leading cause of cancerdeath in both men and women (1). Advanced non–small cell lung carcinoma (NSCLC)accounts for>80%of lung cancer cases. More than half of these patients havedeveloped metastasis at the time of diagnosis and chemotherapy remains the mosteffective treatment option. platinum-based doublets are considered to be the standardtreatments for NSCLC. However, its efficacy in the clinic has been hampered by theacquisition of drug resistance by cancer cells. The clinical effectiveness of thechemotherapeutic agents used in the treatment of NSCLC is often negated by drugresistance.Microtubules are multifunctional cytoskeletal proteins involved in manyessential cellular roles, including maintenance of cell shape, intracellular transport,and in mitosis, forming mitotic spindles to ensure proper chromosome segregationand cell division. Tubulin forms a heterodimer that consists of a-and b-tubulinsubunits that constitute the microtubule.Six distinct b-tubulin isotypes have beenidentified in mammalian cells.And βⅢ-tubulin is associated with resistance to Taxol.β-Tubulin is the cellular target of clinically important tubulin-binding agents (TBA) used in cancer therapy. It is clinically in many tumors including NSCLC,breast cancer, ovarian cancer and esophageal cancer, one of the most commonly usedchemotherapeutic drugs. Paclitaxel are known to promote cell death by inducing apotent mitotic block causing cells to accumulate at the G2-M phase of the cell cycle.Paclitaxel is an antimitotic drug that promotes the formation of stable microtubulesand prevents depolymerisation. The clinical effectiveness of the chemotherapeuticagents used in the treatment of NSCLC is often negated by drug resistance. AndβⅢ-tubulin is associated with resistance to Taxol.Taxol is an important anti-tumour agent that is particularly effective in thetreatment of ovarian and breast carcinomas, although its usefulness is often hamperedby the development of drug resistance. β-Tubulin is the cellular target of clinicallyimportant tubulin-binding agents (TBA) used in cancer therapy. Expression ofβⅢ-tubulin is associated with resistance to taxanes in a range of tumor types,including lung, ovarian, breast, gastric, and cancers of unknown origin.We transfect NSCLC Taxol-resistant A549cells(A549/Taxol) by Lipofectamine2000. Real-time fluorescence quantitative RT-PCR is used to detect βⅢ-tubulinmRNA level in he transfected cells.And we select the siRNA sequence which has thehighest transfection effiency.Then we measureβⅢ-tubulin protein level inA549/Taxol after transtection by western blot.The cell inhibition ration is determinedby MTT assay after transfection.At last,we detect cell apoptosis and cell cycle aftertransfection by flow cytometer. Thus we provide a theoretical basis for clinicalindividual chemotherapy.Materials and Methods: Design and synthesize3paires of siRNA, Lipofectamine2000is used for the transient transfection of A549/Taxol cell by siRNA.48h after thetransfection,total RNA and protein were isolated from transfected cell lines.βⅢ-tubulin mRNA were analyzed by quantitative RT-PCR with β-actin asendogenous control.MeanwhileβⅢ-tubulin protein was detected by Western blots.At last the cells which were transfected after48h were treated with Paclitaxel withdiffernent concentrations, inhibition ratio of Taxol were evaluated by MTT assay. Atlast,we detect cell apoptosis and cell cycle after transfection by flow cytometer.Results: βⅢ-tubulin mRNA levels in A549/Taxol after transfection decreaseanalyzed by quantitative RT-PCR than control groups.And βⅢ-tubulin siRNA-1sequence show the highest transfection efficiency,andβⅢ-tubulin mRNA oftransfected cells was reduced by89%compared to control cells Meanwhile, Wemeasured βⅢ-tubulin protein levels in A549/Taxol transfected byβⅢ-tubulinsiRNA-1by western blot and it shows that it were strongly down-regulated thancontrol groups.Then after48h by transfection,we detected chemotherapeuticsensitivity of Paclitaxel by MTT assay,which shows that the inhibition ratio ofA549/Taxol transfected by siRNA was higher than that of control cells at sameconcentrations. Cell Apoptosisanalysis of βⅢ-tubulin–depleted A549/Taxol cellstreated with Taxol showed that the percentage of apoptotic cells was significantlyhigher in βⅢ-tubulin siRNA-treated cells than in control siRNA-treated cells. cellcycle analysis consider that the βⅢ-tubulin–silenced cells and control siRNA-treatedcells had a higher G2-M content than other groups.And apoptotic cells in the former ishihgher than the controls,however it doesn’t show statistical difference(p>0.05).Conclusion:Down-regulation of βⅢ-tubulin using conventional small interferingRNA(siRNA) significantly sensitized NSCLC cells to Paclitaxel.It may relate to cellapoptosis.