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. 2020 Sep 1;11(8):713.
doi: 10.1038/s41419-020-02859-2.

Sulforaphene inhibits esophageal cancer progression via suppressing SCD and CDH3 expression, and activating the GADD45B-MAP2K3-p38-p53 feedback loop

Sichong Han  1 , Yandong Wang  1 , Jie Ma  2 , Zhe Wang  1 , Hui-Min David Wang  3   4   5   6   7 , Qipeng Yuan  8
Affiliations

Sulforaphene inhibits esophageal cancer progression via suppressing SCD and CDH3 expression, and activating the GADD45B-MAP2K3-p38-p53 feedback loop

Sichong Han et al. Cell Death Dis. .

Abstract

Esophageal cancer is one of the most common cancer with limited therapeutic strategies, thus it is important to develop more effective strategies to against it. Sulforaphene (SFE), an isothiocyanate isolated from radish seeds, was proved to inhibit esophageal cancer progression in the current study. Flow cytometric analysis showed SFE induced cell apoptosis and cycle arrest in G2/M phase. Also, scrape motility and transwell assays presented SFE reduced esophageal cancer cell metastasis. Microarray results showed the influence of SFE on esophageal cancer cells was related with stearoyl-CoA desaturase (SCD), cadherin 3 (CDH3), mitogen-activated protein kinase kinase 3 (MAP2K3) and growth arrest and DNA damage inducible beta (GADD45B). SCD and CDH3 could promote esophageal cancer metastasis via activating the Wnt pathway, while the latter one was involved in a positive feedback loop, GADD45B-MAP2K3-p38-p53, to suppress esophageal cancer growth. GADD45B was known to be the target gene of p53, and we proved in this study, it could increase the phosphorylation level of MAP2K3 in esophageal cancer cells, activating p38 and p53 in turn. SFE treatment elevated MAP2K3 and GADD45B expression and further stimulated this feedback loop to better exert antitumor effect. In summary, these results demonstrated that SFE had the potential for developing as a chemotherapeutic agent because of its inhibitory effects on esophageal cancer metastasis and proliferation.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. SFE inhibits esophageal cancer proliferation in vivo and in vitro.
a Mice body weight and tumor volume were measured every 3 days after injection of SFE or saline. b Image of tumor lumps removed from nude mice (n = 6) injected saline or SFE (75 mg/kg) and the weight of harvestes transplanted tumors. c Colony formation assay was carried out in SFE-treated EC109 and KYSE510 cells. d, e EC109 and KYSE510 cells were treated with gradient concentration of SFE for 24 h and 48 h, respectively, followed by using flow cytometry to asses the apoptotic rates (d) and cell cycle distribution (e). Data represent the mean ± s.d. of three independent experiments. The statistical significance was assessed by Student’s t-test. **P < 0.01 and ***P < 0.005. ns, not significant.
Fig. 2
Fig. 2. SFE suppresses esophageal cancer cell metastasis.
a, b Scrape motility assay (a) and transwell assay (b) were performed in SFE-treated EC109 and KYSE510 cells. c The RNA and protein levels of EMT markers in SFE-treated EC109 and KYSE510 cells were analyzed by qRT-PCR (upper panel) and western blotting (lower panel). Original magnification ×100. Scale bars = 100 μm. Data represent the mean ± s.d. of three independent experiments. The statistical significance was assessed by Student’s t-test. *P < 0.05 and **P < 0.01. ns, not significant.
Fig. 3
Fig. 3. SCD and CDH3 are the target genes of SFE in esophageal cancer cells.
a (upper) The top 30 upregulated and downregulated mRNAs in both EC109 and KYSE510 cell lines were selected for further experiments. (lower) qRT-PCR in EC109 and KYSE510 cells treated with 20 µM SFE or DMSO. b Western blotting showed the effect of SFE on SCD and CDH3 protein abundance in EC109 and KYSE510 cells. c Representative images of immunohistochemistry which demonstrated SCD and CDH3 protein levels in saline- or SFE-treated xenograft tumor lumps. d The relative expression of SCD and CDH3 in vivo was analyzed by qRT-PCR and western blotting, respectively. Original magnification ×400. Scale bars = 60 μm. Data represent the mean ± s.d. of three independent experiments. The statistical significance was assessed by Student’s t-test. *P < 0.05, **P < 0.01, and ***P < 0.005. ns, not significant.
Fig. 4
Fig. 4. SFE inactivates the Wnt pathway by downregulating SCD and CDH3 expression.
a, b Scratch motility assay (a) and transwell assay (b) in EC109 and KYSE510 cells. c Western blotting detection of EMT related gene expression in EC109 and KYSE510 cells. d qRT-PCR and western blotting were used to show the mRNA and protein levels of the Wnt pathway-related genes in SFE-treated EC109 and KYSE510 cells. e Western blotting detection of the Wnt pathway-related gene expression after overexpressing SCD and CDH3 in SFE-treated EC109 and KYSE510 cells. Original magnification ×100. Scale bars = 100 μm. Data represent the mean ± s.d. of three independent experiments. The statistical significance was assessed by Student’s t-test. *P < 0.05, **P < 0.01, and ***P < 0.005. ns, not significant.
Fig. 5
Fig. 5. MAP2K3 and GADD45B are the target genes of SFE in esophageal cancer cells.
a The relative expression of MAP2K3 and GADD45B in SFE-treated EC109 and KYSE510 cells was analyzed by qRT-PCR and western blotting, respectively. b Representative images of immunohistochemistry showed the MAP2K3 and GADD45B protein levels in saline-treated or SFE-treated xenograft tumor lumps. c The relative expression of MAP2K3 and GADD45B in vivo was analyzed by qRT-PCR and western blotting, respectively. d qRT-PCR and western blotting in EC109 and KYSE510 cells. Original magnification ×400. Scale bars = 60 μm. si-MAP2K3, equal mixed si-MAP2K3-1 and si-MAP2K3-2. NC: negative control RNA duplex, the control of siRNA. Data represent the mean ± s.d. of three independent experiments. The statistical significance was assessed by Student’s t-test. *P < 0.05 and **P < 0.01.
Fig. 6
Fig. 6. A GADD45B-MAP2K3-p38-p53 positive feedback loop in SFE-treated esophageal cancer cells.
a co-IP assay was carried out in EC109 cells with GADD45B overexpressing or SFE treatment (20 μM). b The phosphorylation level of MAP2K3 in EC109 and KYSE510 cells with GADD45B overexpression or expression decreased was detected by western blotting. c After decreasing MAP2K3 and GADD45B expression in SFE-treated EC109 and KYSE510 cells, the p38 pathway-related protein levels were detected by western blotting. si-MAP2K3, equal mixed si-MAP2K3-1 and si-MAP2K3-2. si-GADD45B, equal mixed si-GADD45B-1 and si-GADD45B-2. NC: negative control RNA duplex, the control of siRNA. Data represent the mean ± s.d. of three independent experiments. The statistical significance was assessed by Student’s t-test. *P < 0.05 and **P < 0.01. ns, not significant.
Fig. 7
Fig. 7. Schematic representation illustrates SFE modulation of esophageal cancer cell metastasis and proliferation.
It illustrates the mechanism by which SFE inhibits esophageal cancer metastasis and proliferation.
See this image and copyright information in PMC

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