Establish Of Cisplatin-resistant Human Laryngeal Cancer Cell Subline And Study On Drug Resistance Mechanism

BackgroundCisplatin(CDDP)is mainly used in laryngeal cancer patients diagnosed in advanced stage or with postoperative recurrence,However,only part of patients showed effective response to CDDP based chemotherapy.Drug resistance is one of the factors limiting the efficacy of larynx cancer patients;Epithelial mesenchymal transition(EMT)is a process in which tumor cells with a polar epithelial are translated into mesenchymal cells.Snail is a member of the zinc finger transcription factor family involved in epithelial mesenchymal transition,it can inhibit the expression of epithelial marker E-cadherin and then active EMT process;Experiments show that process of EMT,cancer stem cell(CSC),aggressive behaviors of invasion and metastasis are closely associated with drug resistance.However,the underlying mechanism of cisplatin resistance in laryngeal cancer and its malignant biological behavior induced by EMT is still not clear.Object1.To establish cisplatin-resistance cell subline of laryngeal cancer and study the malignant biological characteristics;2.To study the role of EMT in cisplatin resistant cells of laryngeal cancer and to explore the influence on cisplatin resistance by interfering Snail.Method Part 1: The establishment of cisplatin-resistant cell of human laryngeal cancer and evaluation of malignant biological characteristicsHep-2/CDDP cells were induced by increasing the concentration of cisplatin;Cell morphology was observed by inverted microscope;The half maximal inhibitory concentration(IC50)value and drug resistant index(RI)were detected by CCK-8 assay;Microsphere formation ability was detected by culturing in serum-free medium(SFM);The relative mRNA expression level of MDRl/P-gp、ABCG2、CD44、CD133 were detected by RT-qPCR method;The relative protein expression level of MDRl/P-gp、ABCG2、CD44、CD133 were detected by Western blot method.The invasion and migration biological behaviors were detected by transwell and scratch assay.Part 2:To study the role of EMT in cisplatin resistant cells of laryngeal cancer and to explore the influence on cisplatin resistance by interfering Snail.the expression of E-cadherin、Zo-1、Snail、Slug、Twist1、Vimentin on the mRNA level were detected by RT-qPCR and the protein level by Western blot.siRNA interference of Snail was transfected by small RNA interference technology;The interference efficiency on mRNA level were detected by RT-qPCR assay;The expression of Snail protein level was assessed by immunofluorescence;The inhibition ratio of different cisplatin concentration(0、1、2、4、8、16ug/ml)was detected by CCK-8 assay;The protein level of Snail、E-cadherin、MDR1were detected by Western Blot assay.ResultPart 1: The cisplatin resistant Hep-2/CDDP cells were successfully established in vitro after cisplatin induction of 12 months,compared with Hep-2 cells,cell morphology of Hep-2/CDDP was changed;The resistant ability was stable and RI value was 5.43;Microsphere formation test indicated higher formation ratio of Hep-2/CDDP cells;The relative mRNA expression level of resistant associated gene MDRl/P-gp、ABCG2 in Hep-2/CDDP cells were increased significantly;The relative protein expression level of stem cell associated gene CD44、CD133 in Hep-2/CDDP cells were increased significantly.Transwell and scratch assay show the invasion and migration behaviors were increased in hep-2/CDDP cells.Part 2: The epithelial marker E-cadherin and ZO1 were downregulated in hep-2/CDDP cells transcription factor Snail、Slug were up-regulated in mRNA and protein level while Twist1 had no significant changed in protein level(P =0.25).the expression of mesenchymal marker Vimentin was also increased in mRNA and protein levels in cisplatin resistant cells.It was confirmed that the hep-2/CDDP cells possessed EMT phenotypes.RT-qPCR assay show the expression of Snail on mRNA level was decreased to(67.85 ± 9.5)% after transfection in Hep-2/CDDP cell;Immunofluorescence show fluorescence intensity of si-Hep-2/CDDP group was reduced both in nucleus and cytoplasm;CCK-8 assay show the inhibitory ratio of transfected group was increased compared to negative control and Hep-2/CDDP group in different cisplatin concentration(0、1、2、4、8、16ug/ml);Western Blot assay show the protein expression of Snail and MDR1 were down-regulated in transfected Hep-2/CDDP cells while epithelial marker E-cadherin was up-regulated in protein level.ConclusionPart 1: Drug resistant protein P-gp is highly expressed in cisplatin resistant cells of human laryngeal carcinoma,and the drug-resistant cells have characteristics of stem cells,invasion and migration,which are more enhanced than their parental cells.Part 2: The EMT was observed of cisplatin resistant cells in human laryngeal carcinoma,and has up-regulated expression of Snail,interfering of Snail can enhance the sensitivity of resistant cells by increasing the expression of E-cadherin and decreasing the expression of P-gp.