J Breast Cancer.  2016 Jun;19(2):122-132. 10.4048/jbc.2016.19.2.122.

Role of STAT3 Phosphorylation in Ethanol-Mediated Proliferation of Breast Cancer Cells

Affiliations
  • 1Department of Biotechnology, Anna University, Chennai, India. lakshmibs@annauniv.edu
  • 2Department of Biochemistry, University of Madras, Chennai, India.
  • 3Centre for Food Technology, Department of Biotechnology, Anna University, Chennai, India.

Abstract

PURPOSE
In this study, we investigated the molecular mechanism involved in ethanol (EtOH)-mediated proliferation of breast cancer cells.
METHODS
EtOH concentration was optimized by studying its effect on cell proliferation in MCF-7 and MDA MB-231 cells. We used flow cytometry and immunoblot analysis to evaluate the increased proliferation caused by the optimized concentrations of EtOH. The mechanism of EtOH-mediated proliferation was determined using reactive oxygen species (ROS) release assay, reverse transcription polymerase chain reaction, and immunoblot studies. Gene silencing followed by quantitative real-time polymerase chain reaction studies and inhibitor studies indicated the involvement of signal transducer and activator of transcription 3 (STAT3) in EtOH-mediated breast cancer proliferation.
RESULTS
Exposure to EtOH caused an increase in cell proliferation and an accumulation of cells in S-phase in MCF-7 (347 µM EtOH) and MDA MB-231 (173 µM EtOH) cells. Additionally, increased release of ROS and the expression of pro-inflammatory cytokines, such as interleukin 6 and tumor necrosis factor α, confirmed that the proliferation was induced by the ROS-linked inflammatory response in breast cancer. The proinflammatory response was followed by phosphorylation of STAT3. The importance of STAT3 activation in EtOH-mediated proliferation was confirmed through the silencing of STAT3, followed by an investigation on the expression of cyclins and matrix metalloproteinases. Finally, studies using specific inhibitors indicated that the EtOH-mediated effect on STAT3 activation could be regulated by phosphoinositide-3-kinase and Janus kinase 2.
CONCLUSION
The study demonstrates the involvement of STAT3 signaling in EtOH-mediated breast cancer proliferation.

Keyword

Breast neoplasms; Cell proliferation; Ethanol; Inflammation

MeSH Terms

Breast Neoplasms*
Breast*
Cell Proliferation
Cyclins
Cytokines
Ethanol
Flow Cytometry
Gene Silencing
Inflammation
Interleukin-6
Janus Kinase 2
Matrix Metalloproteinases
Phosphorylation*
Polymerase Chain Reaction
Reactive Oxygen Species
Real-Time Polymerase Chain Reaction
Reverse Transcription
STAT3 Transcription Factor
Tumor Necrosis Factor-alpha
Cyclins
Cytokines
Ethanol
Interleukin-6
Janus Kinase 2
Matrix Metalloproteinases
Reactive Oxygen Species
STAT3 Transcription Factor
Tumor Necrosis Factor-alpha

Figure

  • Figure 1 Dose and time course effect of EtOH on proliferation in MCF-7 and MDA MB-231 cells. (A) MTT assay in MCF-7 cells. (B) MTT assay in MDA MB-231 cells. Cells were treated with 400–1,600 µg/dL of EtOH for 24, 48, 72, and 96 hours. (C) Thymidine incorporation assay in MCF-7 cells. (D) Thymidine incorporation assay in MDA MB-231 cells. Cells were treated with the optimized concentration of EtOH 1,600 and 800 µg/dL for 48 hours. Results are expressed as percentage of proliferation. Data expressed as mean±SD from triplicates of three independent experiments. CON=control; EtOH=ethanol. *p≤0.005; †p≤0.05.

  • Figure 2 EtOH induced G1-S phase transition at 48 hours. Results of flow cytometry analysis (A) MCF-7. (B) MDA MB-231. (C) Immunoblot analysis of cell cycle proteins. (D) Expression of cell cycle proteins Integrated density value (IDV) in MCF-7 and MDA MB-231 cells. Target protein expressions were measured as IDVs using Image J and was normalized with β-actin expression. Data expressed as mean±SD from triplicates of three independent experiments. CON=control; EtOH=ethanol. *p≤0.005; †p≤0.05.

  • Figure 3 (A) EtOH induced reactive oxygen species (ROS) accumulation at 48 hours in MCF-7 and MDA MB-231 cells. Results of nitro blue tetrazolium assay. (B) Effect of EtOH on the expression of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in MCF-7 and MDA MB-231 cells at 48 hours using reverse transcription polymerase chain reaction. (C) Expression of target genes normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression in MCF-7 and MDA MB-231 cells. Data were observed from three independent experiments performed in triplicates. CON=control; EtOH=ethanol; IDV=Integrated density value. *p≤0.005.

  • Figure 4 (A) EtOH induced phosphorylation of STAT3 in MCF-7 and MDA MB-231 cells at 48 hours. Results of immunoblot analysis. (B) Expression of target protein normalized with β-actin expression. (C) Gene silencing studies for optimising the maximum transfection efficiency in MCF-7 and MDA MB-231 cells using immunoblot analysis. (D) Expression of target protein normalized with β-actin expression in MCF-7 and MDA MB-231 cells. CON=control; EtOH=ethanol; siSTAT3=signal transducer and activator of transcription 3 silenced cells; IDV=Integrated density value.

  • Figure 5 Quantitative real-time polymerase chain reaction analysis for STAT3 targeted proteins (cyclins and MMPs) at 48 hours. (A) MCF-7. (B) MDA MB-231 cells. Results are expressed as mean of gene expression values±SD from duplicates of two independent experiments. MMPs=matrix metalloproteinases; CON=control; siSTAT3=signal transducer and activator of transcription 3 silenced cells; EtOH=ethanol; siSTAT3+EtOH=STAT3 silenced cells in presence of ethanol.

  • Figure 6 Effect of EtOH on the expression of total signal transducer and activator of transcription 3 (STAT3) in MCF-7 and MDA MB-231 cells at 48 hours. (A) Results of immunoblot analysis. (B) Expression of target protein normalized with β-actin expression in MCF-7 and MDA MB-231 cells. CON=control; EtOH=ethanol; IDV=Integrated density value.

  • Figure 7 Effect of PI3K and JAK2 inhibitors on EtOH-mediated proliferation at 48 hours. (A) MTT assay in MCF-7 and MDA MB-231 cells. (B) Effect of PI3K and JAK2 inhibitors on STAT3 and cyclin D1 expression using immunoblot analysis. Results were based on three independent experiments performed in triplicates. (C) Expression of target protein normalized with β-actin expression in MCF-7 and MDA MB-231 cells. CON=control; EtOH=ethanol; Wort=wortmannin; Wort+E=wortmannin in presence of ethanol; IDV=Integrated density value. *p≤0.005.

  • Figure 8 Schematic representation of the proposed mechanism of ethanol-mediated proliferation in MCF-7 (ER+) and MDA MB-231 (ER-) cells. ER=estrogen receptor; ROS=reactive oxygen species; IL-6=interleukin 6; TNF-α=tumor necrosis factor α; PI3K=phosphatidylinositol-3-kinases; JAK2=Janus kinase 2; STAT3=signal transducer and activator of transcription 3; MMP=matrix metalloproteinase.


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