Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Aug 21:9:820.
doi: 10.3389/fphar.2018.00820. eCollection 2018.

The Curcumin Analogs 2-Pyridyl Cyclohexanone Induce Apoptosis via Inhibition of the JAK2-STAT3 Pathway in Human Esophageal Squamous Cell Carcinoma Cells

Ying Wang  1   2 , Pengjun Zhou  1 , Shurong Qin  1 , Dandan Xu  3 , Yukun Liu  1 , Wuyu Fu  4 , Bibo Ruan  4 , Li Zhang  1 , Yi Zhang  5 , Xiao Wang  6 , Yuwei Pan  7 , Sheng Wang  1 , Haizhao Yan  8 , Jinhong Qin  1 , Xiaoyan Wang  1 , Qiuying Liu  1 , Zhiyun Du  9 , Zhong Liu  1 , Yifei Wang  1
Affiliations

The Curcumin Analogs 2-Pyridyl Cyclohexanone Induce Apoptosis via Inhibition of the JAK2-STAT3 Pathway in Human Esophageal Squamous Cell Carcinoma Cells

Ying Wang et al. Front Pharmacol. .

Abstract

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.

Keywords: 2-pyridyl cyclohexanone; Bcl-2; STAT3; apoptosis; human esophageal squamous cell carcinoma.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1
2-Pyridyl cyclohexanone inhibits esophageal cancer cell proliferation. (A) Effects of 2-pyridyl cyclohexanone on cell growth. (B) Micrographs of Eca109 and EC9706 cells after treatment with 2-pyridyl cyclohexanone. Eca109 and EC9706 cells were treated with the indicated concentrations of 2-pyridyl cyclohexanone for 48 h. Cell viability was quantified by MTT assay. Data are presented as mean ± SD of the results from at least three independent experiments.
FIGURE 2
FIGURE 2
2-Pyridyl cyclohexanone induces apoptosis of Eca109 and EC9706 cells. (A) Morphological changes of apoptotic Eca109 and EC9706 cells after treatment with 2-pyridyl cyclohexanone. Eca109 and EC9706 cells were treated with 3.2 and 1.6 μM 2-pyridyl cyclohexanone, respectively, for 48 h. Morphological changes were observed under a fluorescent microscope after DAPI staining. (B) Induction of apoptosis by 2-pyridyl cyclohexanone in Eca109 and EC9706 cells. Cells were treated with 0, 0.8, 1.6, or 3.2 μM 2-pyridyl cyclohexanone for 48 h. Apoptosis was assessed by flow cytometry after Annexin V-FITC/PI staining. The percentages of apoptotic cells are indicated as mean ± SD of the results from three independent experiments. Data were analyzed using GraphPad Prism 6.02 software (GraphPad Software Inc., La Jolla, CA, United States). ∗ indicates p < 0.05, whereas ∗∗ indicates p < 0.01. (C) Western blot analysis of the apoptosis-associated proteins PARP and caspase-3. GAPDH was used as the protein loading control. Data are presented for three independent experiments.
FIGURE 3
FIGURE 3
Effects of 2-pyridyl cyclohexanone on MMP. Flow cytometry analysis of MMP by JC-1 staining. Cells were treated with 0, 0.8, 1.6, or 3.2 μM 2-pyridyl cyclohexanone for 48 h, and cells with MMP loss were gated. Data are presented as mean ± SD of the results from three independent experiments. ∗ indicates p < 0.05, whereas ∗∗ indicates p < 0.01.
FIGURE 4
FIGURE 4
Effects of 2-pyridyl cyclohexanone on Bcl-2 family proteins. (A) Bcl-xL, Bcl-2, Bid, and Bax protein levels were assessed by western blotting in Eca109 and EC9706 cells after treatment for 48 h with 2-pyridyl cyclohexanone. GAPDH was used as an internal control. (B) Bax/Bcl-2 ratio in Eca109 and EC9706 cells. Data are presented for three independent experiments.∗ indicates p < 0.05, whereas ∗∗ indicates p < 0.01.
FIGURE 5
FIGURE 5
Effects of 2-pyridyl cyclohexanone on p38/ERK MAPK expression in Eca109 and EC9706 cells. Cells were treated with 0, 0.8, 1.6, or 3.2 μM 2-pyridyl cyclohexanone for 48 h and subjected to western blot analysis. GAPDH was used as the internal control. Data are presented for three independent experiments.
FIGURE 6
FIGURE 6
2-Pyridyl cyclohexanone inhibits the STAT3 signaling pathway. Eca109 and EC9706 cells were treated with 0, 0.8, 1.6, or 3.2 μM 2-pyridyl cyclohexanone for 48 h. The expression levels of STAT3, p-STAT3, JAK2, and p-JAK2 were determined by western blot analysis. GAPDH was used as the internal control. Data are presented for three independent experiments.
FIGURE 7
FIGURE 7
Effect of S3I-201 on Eca109 cells. Eca109 cells were treated with 50 or 100 μM S3I-201 for 48 h, after which the expression levels of p-STAT3, STAT3, and Bcl-2 were measured by western blot analysis. GAPDH was used as the internal control. Data are presented for three independent experiments.
FIGURE 8
FIGURE 8
STAT3 activates Bcl-2 transcription in Eca109 cells. (A) The binding of STAT3 to Bcl-2 promoter was analyzed in a ChIP assay. Input DNA was used as a positive control. (B) The transcription activities of Bcl-2 were analyzed following overexpression of STAT3 vector in Eca109 cells (∗∗ indicates p < 0.01). The cells were co-transfected with pcDNA3.1-STAT3 vector, and Renilla luciferase plasmid was used as a control. (C) The transcription activities of Bcl-2 were analyzed following treatment of the cells with 3.2 μM 2-pyridyl cyclohexanone and 100 μM S3I-201. S3I-201 was used as the positive control. All data are presented as mean ± SD of the results from three experiments. ∗ indicates p < 0.05, whereas ∗∗ indicates p < 0.01. (D) ECa109cells were co-transfected with pcDNA3.1-STAT3 vector. The effect of proliferation on ECa109 cells treated with 3.2 μM 2-pyridyl cyclohexanone for 48 h were to analyzed (∗∗ indicates p < 0.01).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abroun S., Saki N., Ahmadvand M., Asghari F., Salari F., Rahim F. (2015). STATs: an old story, yet mesmerizing. Cell J. 17 395–411. - PMC - PubMed
    1. Adachi M., Cui C., Dodge C. T., Bhayani M. K., Lai S. Y. (2012). Targeting STAT3 inhibits growth and enhances radiosensitivity in head and neck squamous cell carcinoma. Oral Oncol. 48 1220–1226. 10.1016/j.oraloncology.2012.06.006 - DOI - PMC - PubMed
    1. Aggarwal B. B., Kumar A., Bharti A. C. (2003). Anticancer potential of curcumin: preclinical and clinical studies. Anticancer Res. 23 363–398. - PubMed
    1. Assi H. H., Paran C., VanderVeen N., Savakus J., Doherty R., Petruzzella E., et al. (2014). Preclinical characterization of signal transducer and activator of transcription 3 small molecule inhibitors for primary and metastatic brain cancer therapy. J. Pharmacol. Exp. Ther. 349 458–469. 10.1124/jpet.114.214619 - DOI - PMC - PubMed
    1. Ball D. P., Lewis A. M., Williams D., Resetca D., Wilson D. J., Gunning P. T. (2016). Signal transducer and activator of transcription 3 (STAT3) inhibitor, S3I-201, acts as a potent and non-selective alkylating agent. Oncotarget 2016 20669–20679. 10.18632/oncotarget.7838 - DOI - PMC - PubMed