Chronic oxidative stress increases growth and tumorigenic potential of MCF-7 breast cancer cells

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.

Figure 1. Bar graph representation of data from DCFH-DA assay on H₂O₂ treated MCF-7 cells.

Cell treatment and DCFH-DA assay was performed as described in materials and methods. Statistically significant (p<0.05) changes are indicated by symbol *.

Figure 2. Bar graph representation of cell growth data from cell count analysis (Figure 2A), and MTT assay (Figure 2B) of MCF-7 cells with acute and chronic exposure to H₂O₂.

Values for cell count and MTT assay were converted into percentage of control (control = 100%). The error bars represent the standard error of the mean (±SEM). Statistically significant (p<0.05) changes are indicated by symbol *.

Figure 3. Flow cytometry histograms representing MCF-7 cells population in G0/G1, S, and G2/M phases of cell cycle after acute (Figure 3A) and chronic (Figure 3B) exposure to H₂O₂.

Cells treated with H₂O₂ or untreated control were collected, fixed, and stained for cell cycle analysis by flow cytometry as described in Materials and Methods Section. Percentage of cells in G0/G1, S and G2/M phase of cell cycle from each histogram represent the average value from three independent experiments.

Figure 4. Real time quantitative reverse transcription PCR analysis of gene expression of cell growth and survival related genes.

Total RNA isolated from MCF-7 cells with acute and chronic exposure to H₂O₂ was used to perform one step real-time quantitative reverse transcription PCR as described in materials and methods. Cycle threshold value (Ct value) of each gene was normalized to the Ct value of housekeeping gene GAPDH obtained from the same sample. The gene expression in fold change was calculated and histogram was plotted using the means of triplicate values. Results of acute (Figure 4A) and chronic (Figure 4B) exposure were presented in separate histograms. Statistically significant change (p<0.05) in gene expression in treated groups as compared to the untreated control is indicated by symbol *.

Figure 5. Real time quantitative reverse transcription PCR analysis of gene expression of metastasis related genes.

Total RNA isolated from MCF-7 cells with acute and chronic exposure to H₂O₂ was used to perform one step real-time quantitative reverse transcription PCR as described in materials and methods. Cycle threshold value (Ct value) of each gene was normalized to the Ct value of housekeeping gene GAPDH obtained from the same sample. The gene expression in fold change was calculated and histogram was plotted using the means of triplicate values. Results of acute (Figure 5A) and chronic (Figure 5B) exposure were presented in separate histograms. Statistically significant change (p<0.05) in gene expression in treated groups as compared to the untreated control is indicated by symbol *.

Figure 6. Representative photographs (40X magnification) of soft agar grown colonies.

Actively growing MCF-7 cells with chronic exposure to H₂O₂ were harvested, plated, and grown in soft agar as described in materials and methods section. The representative images of colonies from control, cells chronically treated with 25 µM and 250 µM of H₂O₂ are given in panel A, B, and C respectively.

Figure 7. Histogram showing the effect of chronic exposure to H₂O₂ in MCF-7 on colony counts in soft agar assay.

Colony numbers were converted into percentage of control (control = 100%). The error bars represent the standard error of the mean (±SEM). Statistically significant changes (p<0.05) when compared to untreated control are indicated by symbol *.