Effects Of ATPR Combined With Cox-2 Inhibitors On Biological Behavior And Molecular Mechanism Of Human Hypopharyngeal Carcinoma FaDu Cells

Objective Effects of ATPR(all-trans retinoic acid derivatives)combined with Cox-2inhibitors(Celecoxib and Nimesulide)on proliferation and apoptosis of hypopharyngeal cancer cells(Fa Du)and their specific molecular mechanismsMethods MTT and flow cytometry were used to detect the effects of Celecoxib and Nimesulide alone or in combination with ATPR on proliferation of Fa Du cells.Flow cytometry and TUNEL assays were used to detect the effect of combined administration on apoptosis of Fa Du cells.The effects of Celecoxib and Nimesulide on the expression of p53,Rb,cyclin-D1 and I?B? m RNA in Fa Du cells were detected by RT-q PCR method.Westernblot detected the expression of apoptotic-associated proteins(caspase family,Bcl-2,Bax,p53,p21)as well as MAPK,NF-?B signaling pathway-related proteins.Inhibitory effect of ATPR combined with Cox-2 inhibitor on tumorigenesis in nude mice.Results ATPR combined with Cox-2 inhibitor had obvious inhibitory effect on the proliferation of Fa Du cells.The results showed that ATPR,Celecoxib and Nimesulide had obvious inhibitory effect on the proliferation of Fa Du cells,and the inhibitory effect of combined treatment was stronger than that of ATPR,Celecoxib,Nimesulide alone.The results of flow cytometry cycle test showed that the proportion of cell cycle arrest in G1-S phase in combination group was much higher than that in control group and single drug groups.The inhibitory effect of the combined drug group was greater than that of the control group.Flow cytometry showed that combined drug and single drug could promote the apoptosis of Fa Du cells and increase the number of cells in early and late apoptosis,and the effect of combined treatment group was more obvious.RT-q PCR results showed that the expression of p53,I?B?,Rb were increased.The expression of cyclin-D1 was decreased.Western Blot results showed that the protein expression levels of p53,p21,caspase-3,caspase-8 and caspase-9were up-regulated with the treatment of ATPR and Cox-2 inhibitors alone or in combination.The phosphorylation level of p38 and JNK decreased,but the phosphorylation level of ERK did not change significantly.The addition of JNK/MAPK pathway inhibitor can inhibit the proliferation of Fa Du cells and promote the expression of apoptosis-related proteins.ATPR combined with Cox-2 inhibitors can inhibit the expression of p65 in NF-?B signaling pathway and promote the expression of I?B?,and the upregulation of caspase-8 inhibits the NF-?B signaling pathway.The tumor growth was inhibited by combination therapy in nude mice,and the effect of combination therapy was more obvious.Conclusions In the present study,we demonstrated that ATPR combined with Cox-2inhibitors induces cell cycle arrest and apoptosis in hypopharyngeal carcinoma Fa Du cells by wild-type p53.What's more,ATPR combined with Cox-2 inhibitors-induced cell apoptosis were depended on the activation of caspases which involved in intrinsic and extrinsic pathway.The suppression of JNK/MAPK and NF-?B signaling pathway contribute the apopotosis of Fa Du cells under the treatment of ATPR combined with Cox-2 inhibitors.