Oscc Cell's Apoptotic Pathway Through Endoplasmic Reticulum Stress Induced By Hcpt

Objective:To study whether endoplasmic reticulum stress participates the apoptotic process of oral squamous cell carcinoma cells stimulated by hydroxycamptothecin (HCPT).Methods:Human OSCC(oral squamous cell carcinoma) cell line-KB cells in logarithmic phase were cultured in vitro and used in the study. OSCC cells were divided into different groups according to HCPT's density,and the HCPT's cytotoxic effect to KB cells was measured by MTT test, the growth inhibiting ratio and IC50 were calculated, the growth inhibiting curve was analysised. Hoechst 33258 staining and flow cytometry are used for qualitative and quantitative study of apoptosis, Western blot for the expression of following protein:Caspase-3 protein and Bcl-2 protein associated with apoptosis, GRP78 protein associated with ERS(endoplamic reticulum stress). Immunfluorescence cytochemistry detected the localization and transposition status of GRP78 protein in KB cells. The data was showed in the form of x±s, and analyzed in the method of one factor AN OVA using SPSS 10.0 software.Results:HCPT had cytotoxic effect to oral squamous cell carcinoma cells, the intensive cytotoxic effect was increased with the dose of HCPT. IC50 of KB cells treated by HCPT for 24h and 48h were 2.43μg/ml and 0.072μg/ml. Hoechst33258 staining showed: most nucleus of control group were round or ellipse, nucleus of experimental group show typical apoptotic nucleus which was the apperence of pyknosis or broken bits. Following the HCPT density step up, the apoptotic ratio of each group become higher, Western blot result also showed Caspase-3 protein and GRP78 protein overexpressed, but the downexpression of Bcl-2. There were signigicant difference between control group and experimental group (P< 0.05)'.Under normal condition, GRP78 protein located in cytoplasm of KB cells, after stimulated by HCPT, GRP78 ptotein is overexpression, then translocateed to perinucelus, and nucleus at last.Conclusion:1 HCPT has cytotoxicity to oral squamous cell carcinoma cells with time and dose dependent relationship.2 HCPT can induce KB cells to apoptosis with dose-dependent.3 The possible apoptotic mechanism of KB cells induced by HCPT,is to upregulate the expression of Caspase-3 protein and to dowmregulate the expression of Bcl-2 protein.4 ERS participates KB cell's apoptotic process induced by HCPT.5 The fuction of GRP78 was not clear in the process of apoptosis induced by ERS.