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Multiple modifications to the structure of curcumin have been investigated with an aim to improve its potency and biochemical properties. Previously, we have synthesized a series of curcumin analogs. In the present study, the anticancer effect of 2-pyridyl cyclohexanone, one of the curcumin analogs, on esophageal carcinoma Eca109 and EC9706 cell lines and its molecular mechanisms were investigated. 2-Pyridyl cyclohexanone inhibited the proliferation of Eca109 and EC9706 cells by inducing apoptosis as indicated by morphological changes, membrane phospholipid phosphatidylserine ectropion, caspase 3 activation, and cleavage of poly(ADP-ribose) polymerase. Mechanistic studies indicated that 2-pyridyl cyclohexanone disrupted mitochondrial membrane potential, disturbed the balance of the Bcl-2 family proteins, and triggered apoptosis via the mitochondria-mediated intrinsic pathway. In 2-pyridine cyclohexanone-treated cells, the phosphorylation levels of JAK2 and STAT3 were dose-dependently decreased and p38 and p-ERK signals were notably activated in a dose-dependent manner. Moreover, we found that the addition of S3I-201, a STAT3 inhibitor, led to a decreased expression level of Bcl-2 in Eca109 cells. The chromatin immunoprecipitation assay demonstrated that STAT3 bound to the promoter of Bcl-2 in the Eca109 cells. Furthermore, the mutation of four STAT3 binding sites (−1733/−1723, −1627/−1617, −807/−797, and −134/−124) on the promote of Bcl-2 gene alone attenuated the transcriptional activation of STAT3. In addition, down-regulation of STAT3 resulted in less of transcriptional activity of STAT3 on Bcl-2 expression. These data provide a potential molecular mechanism of the apoptotic induction function of 2-pyridyl cyclohexanone, and emphasize its important roles as a therapeutic agent for esophageal squamous carcinoma. [ABSTRACT FROM AUTHOR]

Objective: To clone and express human octamer-binding protein-4 (Oct4) cDNA in Escherichia coli and HEK293T cells., Methods: Primers were designed based on the coding sequence for the human Oct4 cDNA. The fragment containing Oct4 gene, amplified by reverse transcription PCR (RT-PCR) from human HeLa cell cDNA as the template, was subcloned into pET-22b(+) and pcDNA3.1(+) vectors to construct the gene recombinant prokaryotic expression vector pET-22b(+)-Oct4 and eukaryotic expression vector pcDNA3.1(+)-his-Oct4, respectively. Thereafter, the double enzymes digestion and the target gene sequencing were performed for identification. The prokaryotic expression vector pET-22b(+)-Oct4 and eukaryotic expression vector pcDNA3.1(+)- his-Oct4 from the positive clones were respectively transformed into E.coli BL21 (DE3) and the embryo HEK293T cells for protein expression., Results: DNA fragments (1100 bp) containing the coding region of Oct4 gene were obtained from RT-PCR. The recombinant expression plasmids were confirmed by enzymes digestion identification and sequencing analysis, proving both of the expression vectors were successfully constructed. SDS-PAGE analysis showed that the molecular mass of OCT4 expressed in the host E.coli BL21 (DE3) was 38 000, which was consistent with the theoretical prediction. Western blotting confirmed the expression of pcDNA3.1(+)-his-Oct4 transfected into HEK293T., Conclusion: Human Oct4 cDNA was cloned and its protein was successfully expressed in E.coli and HEK293T cells.