Experimental Study On Inducting Of Cisplatin Resistance In Nude Mice Bearing Human Tongue Squamous Cell Carcinoma CAL-27 Cells

Objective: In this study,we used the intermittent induction method in vivo to construct a CAL-27/CDDP cell line stably expressing cisplatin-resistant characteristics,providing a cell model for the study of cisplatin-resistant mechanism of tongue squamous cell carcinoma.On this basis,we further explore the relationship between it and epithelial-mesenchymal transformation phenotype,and lay a theoretical foundation for fully elucidating the mutual regulatory mechanism of TSCC cisplatin resistance and EMT.Methods:(1)We cultured human tongue squamous cell carcinoma CAL-27 cells in vitro and inoculated them subcutaneously in the right armpit of nude mice to observe the morphology and histopathology of the tumor.(2)When the volume of transplanted tumor reaches about 0.5cm~3,the induction group was intermittently injected intraperitoneally with5mg/kg cisplatin,and the control group was given equal volume of normal saline once a week for 8 weeks,and the tumor growth curve was drawn to evaluate the drug resistance time.(3)We used primary culture to establish cisplatin-sensitive CAL-27 cells and cisplatinresistant CAL-27/CDDP cell lines,and detected their resistance index.(4)We used morphological observation and scratch healing experiment to prove the morphology and migration ability changes of cisplatin-sensitive CAL-27 cells and cisplatin-resistant CAL-27/CDDP cells.(5)We used real-time quantitative PCR and immunofluorescence assay to detect the expression of molecular markers related to EMT such as E-cadherin and Vimentin in cisplatin-sensitive CAL-27 cells and cisplatin-resistant CAL-27/CDDP cells.Results:(1)The morphology and histology of transplanted tumor in nude mice bearing tumor were consistent with the characteristics of human tongue squamous cell carcinoma CAL-27 cells.(2)After intermittent induction of cisplatin in vivo for 5 weeks,the transplanted tumor initially developed resistance to cisplatin.(3)We successfully established cisplatin-sensitive CAL-27 cells and cisplatin-resistant CAL-27/CDDP cell lines by primary culture method.And the drug resistance of cisplatin-resistant CAL-27/CDDP cells was 12.45 times higher than that of cisplatin-sensitive CAL-27 cells,and the difference between the two groups was statistically significant.(4)The cisplatin-sensitive CAL-27 cells showed epithelial cell morphology,while the cisplatin-resistant CAL-27/CDDP cells showed interstitial cell morphology,and their migration ability was significantly enhanced.The difference between the two groups was statistically significant.(5)Real-time quantitative PCR and immunofluorescence assay showed that compared with cisplatin-sensitive CAL-27 cells,the expression of E-cadherin in cisplatin-resistant CAL-27/CDDP cells was significantly downregulated,while the expression of Vimentin was significantly up-regulated.The difference between the two groups was statistically significant.Conclusions:(1)This study successfully induced cisplatin-resistant CAL-27/CDDP cells in nude mice bearing human tongue squamous cell carcinoma CAL-27 cells,providing an ideal research platform for studying the mechanism of TSCC cisplatin-resistance.(2)This study proved that cisplatin-resistant CAL-27/CDDP cells had EMT phenotype,which laid a foundation for clarifying the regulatory mechanism of cisplatin-resistance and EMT in TSCC.