Abstract
Accumulating evidence suggests that exposures to elevated levels of either endogenous estrogen or environmental estrogenic chemicals are associated with breast cancer development and progression. These natural or synthetic estrogens are known to produce reactive oxygen species (ROS) and increased ROS has been implicated in both cellular apoptosis and carcinogenesis. Though there are several studies on direct involvement of ROS in cellular apoptosis using short-term exposure model, there is no experimental evidence to directly implicate chronic exposure to ROS in increased growth and tumorigenicity of breast cancer cells. Therefore, the objective of this study was to evaluate the effects of chronic oxidative stress on growth, survival and tumorigenic potential of MCF-7 breast cancer cells. MCF-7 cells were exposed to exogenous hydrogen peroxide (H2O2) as a source of ROS at doses of 25 µM and 250 µM for acute (24 hours) and chronic period (3 months) and their effects on cell growth/survival and tumorigenic potential were evaluated. The results of cell count, MTT and cell cycle analysis showed that while acute exposure inhibits the growth of MCF-7 cells in a dose-dependent manner, the chronic exposure to H2O2-induced ROS leads to increased cell growth and survival of MCF-7 cells. This was further confirmed by gene expression analysis of cell cycle and cell survival related genes. Significant increase in number of soft agar colonies, up-regulation of pro-metastatic genes VEGF, WNT1 and CD44, whereas down-regulation of anti-metastatic gene E-Cadherin in H2O2 treated MCF-7 cells observed in this study further suggests that persistent exposure to oxidative stress increases tumorigenic and metastatic potential of MCF-7 cells. Since many chemotherapeutic drugs are known to induce their cytotoxicity by increasing ROS levels, the results of this study are also highly significant in understanding the mechanism for adaptation to ROS-induced toxicity leading to acquired chemotherapeutic resistance in breast cancer cells.
Highlights
Breast cancer is the most commonly diagnosed cancer in women worldwide and the leading cause of mortality in US women [1,2,3]
Cell culture and treatments MCF-7 cells were purchased from ATCC and were maintained in phenol red free Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotic and antimycotic solution
The same dose of H2O2 after chronic exposure for 3 months resulted in 60.11% increase in cell growth (Figure 2A).The exposure to higher dose of H2O2 (250 mM) resulted in statistically significant (p,0.05) decrease in growth of cells by 60.86% after acute exposure, chronic exposure (3 months) caused 92.85% growth of cells, suggesting thereby the increased adaptability of cells due to chronic exposure (Figure 2B)
Summary
Breast cancer is the most commonly diagnosed cancer in women worldwide and the leading cause of mortality in US women [1,2,3]. Evidence suggests that multiple intrinsic and extrinsic risk factors and their interactions are involved in breast cancer development and progression [4,5]. Long-term exposure to extrinsic or environmental factors has been attributed for more than 70% of sporadic breast cancers [7]. The accumulating evidence suggest a potential link between environmental chemicals and breast cancer risk [1]. Some of the well-established xenoestrogens such as Diethylstilbesterol [8], Polychlorinated biphenyls [1,9], Bisphenol [8], Organochlorine pesticides [9], have been linked with breast cancer. Because of the lipophilic nature, these xenoestrogens tends to bio-accumulate and persist in the body for longer time and increases the potential risk for breast cancer development [10]
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