The Mechanism Of PP-22 And The Temperature Intervention On The Nasopharyngeal Carcinoma CNE-2 Cell Line

ObjectiveThis study investigates the inhibitory effect of PP-22 on CNE-2 cells and its relationship with cell apoptosis and autophagy,and the effects of using temperature intervention in combination with PP-22.MethodsCNE-2 cells were treated with different concentrations of PP-22(a monomer of Paris polyphylla),the MTT assay and EDU kit were used to investigate the profileration of cells following PP-22.Annexin V-FITC/PI staining assay was used to analyze the apoptosis rate of CNE-2 treated with PP-22.Hoechst 33258 staining method was used to detect the morphological changes of PP-22 in CNE-2 cells.Confocal microscopy immunofluorescence assay was used to observe LC3 puncta.And the expression levels of apoptosis related proteins,autophagy related proteins,p38 MAPK,ER-stress,Stat3 and their downstream proteins wethe r x wo detected by western blotting.Using temperature intervention and combining with PP-22 on CNE-2 cells,to detect the cells apoptosis effect and the influence of clone formation ability.ResultsMTT results showed that with the increase concentrations of PP-22,PP-22 had a cytotoxic effect on CNE-2 cells in a dosage and time-dependent way.Also the results of EDU test further illustrated that PP-22 inhibited the proliferation of CNE-2 cells.Cells were stained with Hoechst 33258,the cell morphology was observed.PP-22 induced the apoptosis and autophagy of CNE-2 cells.The expression of caspase family related proteins,Bcl-2 family related proteins,were changed in significant.the level of p-p38 was up-regulated while the p-ERK1/2 was down-regulated,and ERK,JNK,p-JNK and p38 were unchanged.The expression of ER-stress related proteins,Beclin-1 and LC3-Ⅱ increased but p62 decreased.p-stat3 and its downstream proteins were changed significantly.Cells were incubated in different temperature and combined with same concentrations of PP-22.The experiment results show that under the incubation in 39℃ and 34℃,the apoptosis rate is higher than incubation in 37℃.And when CNE-2 cells were incubated in 34℃,the cell clone formation ability was obviously lower than in 37℃,while compared 39℃ with 37℃,cell clone forming ability is no different in significantly.ConclusionPP-22 can block the Jak/Stat3 signal pathway,and activate p38 MAPK and induce apoptosis and autophagy events of CNE-2 cells.Beside,with different temperature,the apoptosis rate and clone forming ability were changed.