Study Of Anticancer Effect And Mechanisms Of 2-pyridyl Cyclohexanone On Esophageal Carcinoma

Objective:Anti-cancer activity of 2-pyridyl cyclohexanone in esophageal cancer cell lines Eca109 and EC9706 were evaluated in vitro and in vivo.Methods:?1?The antitumor activity of curcumin analogues was evaluated by MTT assay.?2?Morphological analysis,DAPI staining and flow cytometry were used to detect the effect of2-pyridyl cyclohexanone on apoptosis of esophageal cancer cells.?3?Cell cycle and mitochondrial membrane potential of esophageal carcinoma cells treated with2-pyridylcyclohexanone were assayed by flow cytometry.?4?The effect of 2-pyridyl cyclohexanone mitochondrial Bcl-2 family proteins,MAPK,JAK2/STAT3 signal pathway was analyzed by Western blot.?5?Real-time PCR was used to detect the effect of 2-pyridyl cyclohexanone on m RNA expression level of STAT3 downstream target gene.?6?Chromatin immunoprecipitation,mutant vector,dual luciferase assay and western blot assay were used to investigate the transcriptional regulation mechanism of 2-pyridyl cyclohexanone on STAT3 in esophageal cancer cells.?7?The model of nude mice transplanted tumor was established.The anti-esophageal cancer activity of 2-pyridyl cyclohexanone was detected by H&E staining,TUNEL staining and immunohistochemistry.Results:A series of curcumin analogs exhibited inhibitory activity on the growth of Eca109and EC9706 cells;the effect of 2-pyridyl cyclohexanone was especially significant.After 48h,the IC₅₀of Eca109 and EC9706 cells were 0.75 and 0.61?M,respectively.It was found that2-pyridylcyclohexanone curcumin analogue induced apoptosis of esophageal cancer cells through mitochondrial pathway and arrested the cell cycle in G2/M phase,as incidated by chromatin condensation,membrane phosphatidylserine from lipid Membrane to medial to the lateral,mitochondrial membrane potential decreased,Caspase 3 protein activation and PARP protein cleavage.In addition,the 2-pyridyl cyclohexanone down-regulates the anti-apoptotic proteins Bcl-2,Bcl-x L in the mitochondrial-related Bcl-2 family,up-regulates pro-apoptotic proteins Bax,Bid,and promotes MAPK Activation of P38,ERK and JNK in the signaling pathway.On the other hand,2-pyridyl cyclohexanone can inhibit the phosphorylation of JAK2/STAT3 signaling pathway in esophageal cancer cells and down-regulate genes related to apoptosis and cycle?BCL2,BIRC5,CCDN1,and CDKN1A?.By detecting the effect of STAT3overexpression or inhibition on the expression of Bcl-2,the transcriptional regulation of STAT3on Bcl-2 in Eca109 cells was confirmed,and the interaction between STAT3 and Bcl-2 was further confirmed by chromatin immunoprecipitation.The results of dual luciferase reporter assay showed that STAT3 could act on the four predicted sites of Bcl-2 promoter?-1733/-1723,-1627/-1617,-807/-797 and-134/-124?.In vivo experiments showed that 2-pyridyl cyclohexanone of a curcumin analog in vivo no obvious toxicity.Conclusion:2-pyridyl cyclohexanone can inhibit the expression of STAT3 in JAK2-STAT3signaling pathway and inhibit the effect of STAT3 on Bcl-2,and it can induce apoptosis of esophageal carcinoma cell line mitochondrial pathway.TUNEL staining showed that 2-pyridyl cyclohexanone could induce cell apoptosis.Immunohistochemistry showed that2-pyridinocyclohexanone could decrease the expression of Bcl-2 protein.