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
. 2020 Aug 18;11(8):644.
doi: 10.1038/s41419-020-02906-y.

Melatonin suppresses chronic restraint stress-mediated metastasis of epithelial ovarian cancer via NE/AKT/β-catenin/SLUG axis

Shixia Bu  1   2   3 , Qian Wang  1   2   3 , Junyan Sun  1   2   3 , Xiao Li  4 , Tingting Gu  1   2   3 , Dongmei Lai  5   6   7
Affiliations

Melatonin suppresses chronic restraint stress-mediated metastasis of epithelial ovarian cancer via NE/AKT/β-catenin/SLUG axis

Shixia Bu et al. Cell Death Dis. .

Erratum in

Abstract

Chronic stress has been shown to facilitate progression of epithelial ovarian cancer (EOC), however, the neuro-endocranial mechanism participating in this process still remains unclear. Here, we reported that chronic restraint stress (CRS) promoted the abdominal implantation metastasis of EOC cells and the expression of epithelial-mesenchymal transition-related markers in tumor-bearing mouse model, including TWIST, SLUG, SNAIL, and β-catenin. We observed that β-catenin co-expressed with SLUG and norepinephrine (NE) in tumor tissues obtained from nude mice. Further ex vivo experiments revealed that NE promoted migration and invasion of ovarian cancer cells and SLUG expression through upregulating expression and improving transcriptional function of β-catenin in vitro. A human phosphor-kinase array suggested that NE activated various kinases in ovarian cancer cells, and we further confirmed that AKT inhibitor reduced NE-mediated pro-metastatic impacts and activation of the β-catenin/SLUG axis. Furthermore, the expression levels of NE and β-catenin were examined in ovarian tumor tissues by using tumor tissue arrays. Results showed that the expression levels of both NE and β-catenin were associated with poor clinical stage of serous EOC. Moreover, we found that melatonin (MLT) effectively reduced the abdominal tumor burden of ovarian cancer induced by CRS, which was partially related to the inhibition of the NE/AKT/β-catenin/SLUG axis. Collectively, these findings suggest a novel mechanism for CRS-mediated ovarian cancer metastasis and MLT has a potential therapeutic efficacy against ovarian cancer.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. CRS promotes metastasis of SK-OV-3 ovarian cancer cells in nude mouse models.
a The schematic graph of establishing chronic restraint stress (CRS) animal models in this study. b ELISA was used to evaluate the effects of CRS on nude mice by examining the concentration of serum corticosterone (n = 6). c Gross morphology of the abdominally implanted tumor burden of ovarian cancer cells in nude mice. Quantification of abdominal tumor nodules (d) and tumor weights (e) of the control group and the CRS group (n = 6). f Representative images of H&E staining (i, ii), IF staining detecting EMT-related transcription factors, including TWIST (iii, iv), SLUG (v, vi), SNAIL (vii, viii), and β-catenin (ix, x) in tumor tissues obtained from nude mouse models. *P < 0.05; ***P < 0.001. Scale bar, 100 μM.
Fig. 2
Fig. 2. CRS activates the β-catenin/SLUG axis in nude mouse models and in vitro.
Co-expression of β-catenin/SLUG (a), β-catenin/TWIST (b), and β-catenin/SNAIL (c) in the tumor tissues obtained from nude mouse models was evaluated by confocal laser scanning microscopy. d CCK-8 assay was used to test the effects of β-catenin agonist SKL2001 in different concentration on the viability of ovarian cancer cells at 24 h. Western blot (e) and double immunofluorescence staining (f) were used to examine the impacts of SKL2001 on the expression of β-catenin and SLUG in ovarian cancer cells at 24 h. Transwell assays were used to test the effects of SKL2001 on the migration (g) and invasion (h) ability of ovarian cancer cells in vitro at 24 h. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bar, 200 μM.
Fig. 3
Fig. 3. CRS-related NE promotes the motility of epithelial ovarian cancers via the β-catenin/SLUG axis in nude mouse models and in vitro.
a Content of NE in mouse serum and tumor tissues from the control group and the CRS group was evaluated by ELISA (n = 6). b Co-expression of β-catenin/NE in the tumor tissues obtained from animal models was examined by confocal laser scanning microscopy. CCK-8 assay was used to test the effects of different concentration of NE (c) and β-catenin inhibitor KYA1797K (e) on the viability of ovarian cancer cells at 24 h. d NE increased the expression of β-catenin and SLUG in ovarian cancer cells time-dependently. f Results from western blot showed that KYA inhibited NE-mediated upregulation of β-catenin and SLUG in ovarian cancer cells at 24 h. KYA reduced NE-induced pro-migration (g) and pro-invasion (h) effects on ovarian cancer cells at 24 h. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bar, 200 μM.
Fig. 4
Fig. 4. NE exerts upregulating effects on phosphorylation of various kinases in SK-OV-3 ovarian cancer cells.
a A human phosphor-kinase array was performed to evaluate the effects of NE on the phosphorylation level of kinases in SK-OV-3 ovarian cancer cells at 24 h. b The list of NE-increased top 20 kinases in SK-OV-3 ranked by the relative expression. The protein–protein interaction among top 20 upregulated kinases was shown by PPI network (c) and PPI score (d). GO enrichment analysis of these kinases based on following aspects: molecular function (e), biological process (f), cellular component (g), and Reactome pathway analysis (h). NE-enhanced protein domains were analyzed by InterPro (i), SMART (j), and Pfam database (k).
Fig. 5
Fig. 5. NE activates the β-catenin/SLUG axis through phosphorylating AKT.
a NE phosphorylates the Ser473 and Thr308 residues of AKT in ovarian cancer cells by using western blotting at 24 h. b CCK-8 assay was used to evaluate the effects of different concentration of AKT inhibitor AKTi VIII on the viability of ovarian cancer cells at 24 h. c Western blotting showed that AKTi VIII inhibited NE-mediated activation of the AKT/β-catenin/SLUG axis in ovarian cells at 24 h. AKTi VIII reduces NE-mediated pro-migration (d) and pro-invasion (e) influence on ovarian cancer cells at 24 h. **P < 0.01; ***P < 0.001. Scale bar, 200 μM.
Fig. 6
Fig. 6. The expression of NE and β-catenin and their clinicopathologic significance in ovarian cancer tissues.
Representative images of β-catenin (a) and NE (e) expression in benign ovarian disease and different FIGO stages of ovarian cancers. The IHC scores of β-catenin (b) and NE (f) in benign ovarian disease and different FIGO stages of ovarian serous cancer. The IHC scores of β-catenin (c) and NE (g) in cancerous tissue and paracancerous tissues. The IHC scores of β-catenin (d) and NE (h) in different pathological types of stages I–II ovarian cancer. The correlation analysis between the β-catenin expression and the NE content in benign ovarian disease (i) and ovarian serous cancer (j). *P < 0.05; **P < 0.01; ***P < 0.001. Scale bar, 200 μM.
Fig. 7
Fig. 7. Melatonin inhibits CRS-mediated metastasis-facilitating effects on EOC cells via downregulating the NE/AKT/β-catenin/SLUG axis in nude mouse models and in vitro.
a Gross morphology of the abdominally implanted tumor burden of ovarian cancer cells (n = 6). b Quantification of abdominal tumor nodules and tumor weights (n = 6). c The content of MLT in mouse serum and tumor tissues was evaluated by ELISA (n = 6). Representative images of IF staining detecting NE (d), β-catenin (e), and SLUG (f) in tumor tissues. g CCK-8 assay was conducted to test the effects of different concentration of MLT on the viability of ovarian cancer cells at 24 h. h Western blotting showed that MLT inhibited NE-induced activation of AKT/β-catenin/SLUG axis in ovarian cells at 24 h. MLT reduced the migration (i) and invasion (j) ability of NE-treated ovarian cancer cells at 24 h. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bar, 200 μM.
Fig. 8
Fig. 8. Melatonin suppresses β-catenin-mediated transcriptional regulation of SLUG expression in EOC cells.
Representative images of IF staining detecting the effects of NE, AKTi, KYA and melatonin on the SLUG expression (green), β-catenin expression (red), and nuclear localization of β-catenin (purple) in SK-OV-3 cells (a) and HO-8910pm cells (e) at 24 h. Quantification of the mean fluorescence integrated densities of SLUG (b, f) and β-catenin (c, g) in EOC cells (n = 3). The percentage of nuclear localization of β-catenin in SK-OV-3 cells (d) and HO-8910pm cells (h) (n = 3). i Dual luciferase reporter assays with the SLUG’s promoter reporter plasmid (pGL3-basic vector) were used to evaluate the effects of NE, AKTi, KYA and melatonin on the activity of SLUG’s promoter in EOC cells at 24 h (n = 3). **P < 0.01; ***P < 0.001. Scale bar, 200 μM.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jayson GC, Kohn EC, Kitchener HC, Ledermann JA. Ovarian cancer. Lancet. 2014;384:1376–1388. - PubMed
    1. Huang TY, et al. Depression and risk of epithelial ovarian cancer: results from two large prospective cohort studies. Gynecol. Oncol. 2015;139:481–486. - PMC - PubMed
    1. Armer JS, et al. Life stress as a risk factor for sustained anxiety and cortisol dysregulation during the first year of survivorship in ovarian cancer. Cancer. 2018;124:3401–3408. - PMC - PubMed
    1. Krizanova O, Babula P, Pacak K. Stress, catecholaminergic system and cancer. Stress. 2016;19:419–428. - PubMed
    1. Lutgendorf SK, et al. Stress-related mediators stimulate vascular endothelial growth factor secretion by two ovarian cancer cell lines. Clin. Cancer Res. 2003;9:4514–4521. - PubMed

Publication types

MeSH terms