Melatonin decreases breast cancer metastasis by modulating Rho-associated kinase protein-1 expression - PubMed (original) (raw)

doi: 10.1111/jpi.12270. Epub 2015 Oct 20.

Ali Syed Arbab 2, Gabriela Bottaro Gelaleti 1 3, Lívia Carvalho Ferreira 1 3, Marina Gobbe Moschetta 1, Bruna Victorasso Jardim-Perassi 1, A S M Iskander 2, Nadimpalli Ravi S Varma 4, Adarsh Shankar 2, Verena Benedick Coimbra 1, Vanessa Alves Fabri 1, Juliana Garcia de Oliveira 5, Debora Aparecida Pires de Campos Zuccari 1 3

Affiliations

Melatonin decreases breast cancer metastasis by modulating Rho-associated kinase protein-1 expression

Thaiz Ferraz Borin et al. J Pineal Res. 2016 Jan.

Abstract

The occurrence of metastasis, an important breast cancer prognostic factor, depends on cell migration/invasion mechanisms, which can be controlled by regulatory and effector molecules such as Rho-associated kinase protein (ROCK-1). Increased expression of this protein promotes tumor growth and metastasis, which can be restricted by ROCK-1 inhibitors. Melatonin has shown oncostatic, antimetastatic, and anti-angiogenic effects and can modulate ROCK-1 expression. Metastatic and nonmetastatic breast cancer cell lines were treated with melatonin as well as with specific ROCK-1 inhibitor (Y27632). Cell viability, cell migration/invasion, and ROCK-1 gene expression and protein expression were determined in vitro. In vivo lung metastasis study was performed using female athymic nude mice treated with either melatonin or Y27832 for 2 and 5 wk. The metastases were evaluated by X-ray computed tomography and single photon emission computed tomography (SPECT) and by immunohistochemistry for ROCK-1 and cytokeratin proteins. Melatonin and Y27632 treatments reduced cell viability and invasion/migration of both cell lines and decreased ROCK-1 gene expression in metastatic cells and protein expression in nonmetastatic cell line. The numbers of 'hot' spots (lung metastasis) identified by SPECT images were significantly lower in treated groups. ROCK-1 protein expression also was decreased in metastatic foci of treated groups. Melatonin has shown to be effective in controlling metastatic breast cancer in vitro and in vivo, not only via inhibition of the proliferation of tumor cells but also through direct antagonism of metastatic mechanism of cells rendered by ROCK-1 inhibition. When Y27632 was used, the effects were similar to those found with melatonin treatment.

Keywords: 99mTc-tetrofosmin; MCF-7 cells; MDA-MB-231 cells; Rho-associated kinase protein-1 inhibitor; breast cancer; lung metastasis; melatonin; single photon emission computed tomography.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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Conflict of interest statement

Conflict of interests The authors declare that they have no competing interests.

Figures

Fig. 1

Fig. 1

Evaluation of cell viability by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (A) MDA-MB-231 and (B) MCF-7 breast cancer cell lines after 48 hr of melatonin treatment; (C) MDA-MB-231 and (D) MCF-7 breast cancer cell lines after 24 hr of Y27632 treatment. Significant value in ANOVA followed by Bonferroni’s test (±S.E.M. *P < 0.05).

Fig 2

Fig 2

Analysis of migration and invasion rate after melatonin and Y27632 treatments. (A) MDA-MB-231 and (B) MCF-7 breast cancer cell lines. Significant value in ANOVA followed by Bonferroni’s test (±S.E.M. *P < 0.05, **P < 0.001).

Fig. 3

Fig. 3

Analysis of expression of Rho-associated kinase protein (ROCK)-1 gene and protein after 24 hr of treatment with melatonin and Y27632. (A) and (C) MDA-MB-231, and (B) and (D) MCF-7 breast cancer cell lines. The gene expression values were relative quantification (RQ) represented and protein expression values were represented in relative levels. ROCK-1 protein levels were normalized by α-tubulin protein, presented in the boxes. Significant value in ANOVA followed by Bonferroni’s test (±S.E.M. *P < 0.05, **P < 0.001, and ***P < 0.0001).

Fig. 4

Fig. 4

Lung metastasis after 2 and 5 wk of melatonin and Y27632 treatments. Semiquantitative analysis of radioactivity intensity by single photon emission computed tomography (SPECT) images following (A) 2 wk of treatment and following (C) 5 wk of treatment. SPECT images obtained after injection of the 99mTc-tetrofosmin showing lung metastasis following (B) 2 wk of treatment and following (D) 5 wk of treatment. (a) and (b) negative control, (c) and (d) positive control, (e) and (f) melatonin treatment, (g) and (h) Rho-associated kinase protein (ROCK)-1 inhibitor (Y27632) treatment. (E) High-resolution computed tomography images at 25-µm thickness. (a) Control animal without metastasis; (b) positive control animal without treatment showing high-density area (white arrow) indicating lung metastasis in animal after 6 wk following induction of lung metastasis. Significant value in ANOVA followed by Bonferroni’s test (±S.E.M. *P < 0.05; ***P < 0.0001).

Fig. 5

Fig. 5

Hematoxylin and eosin staining showing lung tissues in all groups. (A) Representative images from 2 wk of treatment and (B) from 5 wk of treatment. (a) and (e) negative control, (b) and (f) positive control, (c) and (g) melatonin treatment, (d) and (h) Y27632 treatment. Images were taken with 10× and 40× magnification. Arrows show the sites of metastasis. Note the sparsity of tumor cells in Y27632-treated group.

Fig. 6

Fig. 6

Cytokeratin protein expression analysis in lung metastasis. (A) and (C) Semiquantitative analysis from animals with 2 and 5 wk of treatment, respectively. (B) and (D) Representative images from (a) and (b) negative control, (c) and (d) positive control, (e) and (f) melatonin treatment, (g) and (h) Y27632 treatment. Images were taken with 10× and 40× magnification. Arrows show the sites of metastasis. The values of mean optical density and standard error for each group are shown in arbitrary units (au). (**) represents P ≤ 0.001 and (***) P ≤ 0.0001 by ANOVA followed by Bonferroni’s test.

Fig. 7

Fig. 7

Rho-associated kinase protein (ROCK)-1 protein expression analysis in lung metastasis. (A) and (C) Semiquantitative analysis from animals with 2 and 5 wk of treatment, respectively. (B) and (D) Representative images from (a) and (b) negative control, (c) and (d) positive control, (e) and (f) melatonin treatment, (g) and (h) Y27632 treatment. Images were taken with 10× and 40× magnification. Arrows show the sites of metastasis. The values of mean optical density and standard error for each group are shown in arbitrary units (au). (*) represents P ≤ 0.05 and (**) P ≤ 0.001 by ANOVA followed by Bonferroni’s test.

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