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. 2018 Oct 6;9(21):3874-3885.
doi: 10.7150/jca.25581. eCollection 2018.

Ethanol Enhances Estrogen Mediated Angiogenesis in Breast Cancer

Rachana Maniyar  1 , Sanjukta Chakraborty  1 , Robert Suriano  1   2
Affiliations

Ethanol Enhances Estrogen Mediated Angiogenesis in Breast Cancer

Rachana Maniyar et al. J Cancer. .

Abstract

Angiogenesis, a highly regulated process, is exploited by tumors like breast cancer to ensure a constant supply of oxygen and nutrients and is key for tumor survival and progression. Estrogen and alcohol independently have been observed to contribute to angiogenesis in breast cancer but their combinatorial effects have never been evaluated. The exact mechanism by which estrogen and alcohol contribute to breast cancer angiogenesis remains to be elucidated. In this study, we defined the in vitro effects of the combination of estrogen and alcohol in breast cancer angiogenesis using the tubulogenesis and scratch wound assays. Conditioned media, generated by culturing the murine mammary cancer cell line, TG1-1, in estrogen and ethanol, enhanced tubule formation and migration as well as modulated the MAP Kinase pathway in the murine endothelial cell line, SVEC4-10. Additionally, estrogen and ethanol in combination enhanced the expression of the pro-angiogenic factors VEGF, MMP-9, and eNOS, and modulated Akt activation. These observations suggest that TG1-1 cells secrete pro-angiogenic molecules in response to the combination of estrogen and ethanol that modulate the morphological and migratory properties of endothelial cells. The data presented in this study, is the first in attempting to link the cooperative activity between estrogen and ethanol in breast cancer progression, underscoring correlations first made by epidemiological observations linking the two.

Keywords: alcohol; angiogenesis; breast cancer; estrogen; ethanol.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Enhanced Tubulogenesis in SVEC4-10 cells in presence of alcohol and estrogen A) Representative brightfield images (50X) of SVEC4-10 cells cultured in alcohol (Ethanol/EtOH) and/or estrogen (10-8M) treated TG1-1 conditioned media; B) Number of master segments, and C) Total Segment Length in pixels, analyzed using angiogenesis analyzer by Image J. (n=2) Error bars represent ±SEM.
Figure 2
Figure 2
Enhanced pro-angiogenic factor expression in TG1-1 breast cancer cells in presence of alcohol and estrogen Western blot analysis of whole cell lysates from TG1-1 cells treated with Ethanol and Estrogen. Whole cell lysates were assayed for expression of A) VEGF; B) eNOS and C) MMP-9. Expression was determined by normalizing to TG1-1 protein lysates treated with estrogen alone (n=3) Error bars represent ±SEM a Student T test was performed. *p<0.05, **p<0.01
Figure 3
Figure 3
Enhanced MEK Activation in SVEC4-10 cells in the presence of alcohol and estrogen Western blot analysis from whole cell lysates of SVEC4-10 cells cultured in conditioned media from ethanol and estrogen treated TG1-1 cells. Whole cell lysates were assayed for expression of A) MEK Activation; and B) MAPK Activation. Expression was determined by normalizing to SVEC4-10 protein lysates treated with TG1-1 conditioned media cultured in the presence of estrogen alone (n=2) Error bars represent ±SEM.
Figure 4
Figure 4
Alcohol and estrogen enhance expression of FAK in SVEC4-10 cells. Western blot analysis from whole cell lysates of SVEC4-10 cells cultured in conditioned media from ethanol and estrogen treated TG1-1 cells. Whole cell lysates were assayed for expression of FAK, a marker for migration. Expression was determined by normalizing to SVEC4-10 protein lysates treated with TG1-1 conditioned media cultured in the presence of estrogen alone (n=2) Error bars represent ±SEM.
Figure 5
Figure 5
SVEC4-10 cell migration is enhanced in the presence of alcohol and estrogen. A) Representative bright field (100X) image of a scratch wound assay using SVEC4-10 cells in presence of conditioned media from ethanol and estrogen treated TG1-1 cells for 24 hours, demonstrating enhanced migration in presence of ethanol and estrogen. (n=2) B) XTT assay using SVEC4-10 cells cultured in presence of conditioned media from ethanol and estrogen treated TG1-1 cells for 24 hours (n=3) Error bars represent ±SEM.
Figure 6
Figure 6
Enhanced pro-survival pathway activation in TG1-1 breast cancer cells in presence of ethanol and estrogen Western blot analysis using whole cell lysates from TG1-1 cells treated with Ethanol and Estrogen. Expression of activated Akt was determined by normalizing to protein lysates from TG1-1 cells treated with estrogen alone (n=3) Error bars represent ±SEM.
Figure 7
Figure 7
Proposed model TG1-1 cells, in presence of ethanol and estrogen, activate its pro-survival AKT pathway, leading to enhanced secretion of pro-angiogenic factors, such as VEGF, eNOS and MMP9. These proangiogenic factors in turn act on SVEC4-10 endothelial cells resulting in enhanced activation of MEK thus leading to increased endothelial cell migration and tubulogenesis. Overall, the events described consequently lead to enhanced breast cancer angiogenesis and progression.
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