Mechanisms underlying differential response to estrogen-induced apoptosis in long-term estrogen-deprived breast cancer cells

Abstract

Models of long-term estrogen-deprived breast cancer cells are utilized in the laboratory to mimic clinical aromatase inhibitor-resistant breast cancer and serve as a tool to discover new therapeutic strategies. The MCF-7:5C and MCF-7:2A subclones were generated through long-term estrogen deprivation of estrogen receptor (ER)-positive MCF-7 cells, and represent anti-hormone‑resistant breast cancer. MCF-7:5C cells paradoxically undergo estrogen-induced apoptosis within seven days of estrogen (estradiol, E2) treatment; MCF-7:2A cells also experience E(2)-induced apoptosis but evade dramatic cell death until approximately 14 days of treatment. To discover and define the mechanisms by which MCF-7:2A cells survive two weeks of E(2) treatment, systematic experiments were performed in this study. The data suggest that MCF-7:2A cells employ stronger antioxidant defense mechanisms than do MCF-7:5C cells, and that oxidative stress is ultimately required for MCF-7:2A cells to die in response to E2 treatment. Tumor necrosis factor (TNF) family member activation is also essential for E(2)-induced apoptosis to occur in MCF-7:2A cells; upregulation of TNFα occurs simultaneously with oxidative stress activation. Although the unfolded protein response (UPR) signaling pattern is similar to that in MCF-7:5C cells, it is not sufficient to cause cell death in MCF-7:2A cells. Additionally, increased insulin-like growth factor receptor β (IGF-1Rβ) confers a mechanism of growth and anti-apoptotic advantage in MCF-7:2A cells.

Figure 1.

Network enrichment analysis for MCF-7:WS8, MCF-7:5C and MCF-7:2A cells. Global gene arrays were performed to compare activated gene networks associated with 1 nM E₂ treatment in the cell lines. Genes were analyzed after 2–24 and 24–96 h treatment.

Figure 2.

MCF-7:2A growth response to E₂. (A) DNA was measured from MCF-7:WS8, MCF-7:5C and MCF-7:2A cells after 7 or 14 days treatment with vehicle or 1 nM E₂. Values are normalized to vehicle-treated cells. Means represent samples in triplicate. (B) MAPK and AKT growth pathway protein levels were measured by western blot analysis after 24 h vehicle or 1 nM E₂ treatment. β-actin was used as a loading control. (C) Cell cycle analysis was performed after 24 h vehicle or 1 nM E₂ treatment.

Figure 2.

MCF-7:2A growth response to E₂. (A) DNA was measured from MCF-7:WS8, MCF-7:5C and MCF-7:2A cells after 7 or 14 days treatment with vehicle or 1 nM E₂. Values are normalized to vehicle-treated cells. Means represent samples in triplicate. (B) MAPK and AKT growth pathway protein levels were measured by western blot analysis after 24 h vehicle or 1 nM E₂ treatment. β-actin was used as a loading control. (C) Cell cycle analysis was performed after 24 h vehicle or 1 nM E₂ treatment.

Figure 2.

MCF-7:2A growth response to E₂. (A) DNA was measured from MCF-7:WS8, MCF-7:5C and MCF-7:2A cells after 7 or 14 days treatment with vehicle or 1 nM E₂. Values are normalized to vehicle-treated cells. Means represent samples in triplicate. (B) MAPK and AKT growth pathway protein levels were measured by western blot analysis after 24 h vehicle or 1 nM E₂ treatment. β-actin was used as a loading control. (C) Cell cycle analysis was performed after 24 h vehicle or 1 nM E₂ treatment.

Figure 3.

MCF-7:5C and MCF-7:2A UPR. Cell lines were probed for UPR-related proteins after treatment with vehicle or 1 nM E₂ for 24, 48 and 72 h. β-actin was used as a loading control.

Figure 4.

Apoptosis-related genes in MCF-7:5C (white bars) and MCF-7:2A (black bars) cells. (A) MCF-7:5C and MCF-7:2A cells were treated with vehicle or 1 nM E₂ for 24, 48 and 72 h. LTA, LTB, TNFα and BCL2L11 mRNA levels were measured using RT-PCR. 36B4 was used as an internal control. (B) MCF-7:2A cells were treated with vehicle or 1 nM E₂ for 3, 6, 9 and 12 days. TNFα and BCL2L11 mRNA levels were then measured using RT-PCR. 36B4 was used as an internal control. Means represent at 7 to 18 replicates.

Figure 4.

Apoptosis-related genes in MCF-7:5C (white bars) and MCF-7:2A (black bars) cells. (A) MCF-7:5C and MCF-7:2A cells were treated with vehicle or 1 nM E₂ for 24, 48 and 72 h. LTA, LTB, TNFα and BCL2L11 mRNA levels were measured using RT-PCR. 36B4 was used as an internal control. (B) MCF-7:2A cells were treated with vehicle or 1 nM E₂ for 3, 6, 9 and 12 days. TNFα and BCL2L11 mRNA levels were then measured using RT-PCR. 36B4 was used as an internal control. Means represent at 7 to 18 replicates.

Figure 5.

MCF-7:5C and MCF-7:2A HMOX1 regulation. (A) MCF-7:5C and MCF-7:2A cells were treated with vehicle or 1 nM E₂ for 24, 48 and 72 h; HMOX1 mRNA was measured using RT-PCR. 36B4 was used as an internal control. Mean represents 18 replicates. (B) MCF-7:2A cells were treated with vehicle or 1 nM E₂ for 3, 6, 9 and 12 days; HMOX1 mRNA was measured using RT-PCR. 36B4 was used as an internal control. Means represent at least 8 replicates.

Figure 5.

MCF-7:5C and MCF-7:2A HMOX1 regulation. (A) MCF-7:5C and MCF-7:2A cells were treated with vehicle or 1 nM E₂ for 24, 48 and 72 h; HMOX1 mRNA was measured using RT-PCR. 36B4 was used as an internal control. Mean represents 18 replicates. (B) MCF-7:2A cells were treated with vehicle or 1 nM E₂ for 3, 6, 9 and 12 days; HMOX1 mRNA was measured using RT-PCR. 36B4 was used as an internal control. Means represent at least 8 replicates.

Figure 6.

MCF-7:2A oxidative stress and glutathione. (A) Total basal glutathione (GSSG+GSH) levels were measured in MCF-7:WS8, MCF-7:5C and MCF-7:2A cells. Means represent samples in triplicate. (B) Total glutathione in MCF-7:5C and MCF-7:2A cells were quantified after 72 h treatment of vehicle or 100 μM BSO. Means represent samples in triplicate. (C) MCF-7:2A cells were treated for 24, 48 and 72 h with either vehicle, 1 nM E₂, 100 μM BSO or 1 nM E₂ + 100 μM BSO; HMOX1 mRNA was measured using RT-PCR. 36B4 was used as an internal control. Means represent at least 8 replicates. (D) MCF-7:2A were subjected to the aforementioned treatments for 5, 7, 9 and 12 days, and ROS levels were measured. Data are normalized to vehicle treatment. (E) MCF-7:2A cells were treated likewise, and DNA was harvested and quantified after two weeks. Means represent samples in triplicate. ^**P<0.01, ^***P<0.001.

Figure 6.

MCF-7:2A oxidative stress and glutathione. (A) Total basal glutathione (GSSG+GSH) levels were measured in MCF-7:WS8, MCF-7:5C and MCF-7:2A cells. Means represent samples in triplicate. (B) Total glutathione in MCF-7:5C and MCF-7:2A cells were quantified after 72 h treatment of vehicle or 100 μM BSO. Means represent samples in triplicate. (C) MCF-7:2A cells were treated for 24, 48 and 72 h with either vehicle, 1 nM E₂, 100 μM BSO or 1 nM E₂ + 100 μM BSO; HMOX1 mRNA was measured using RT-PCR. 36B4 was used as an internal control. Means represent at least 8 replicates. (D) MCF-7:2A were subjected to the aforementioned treatments for 5, 7, 9 and 12 days, and ROS levels were measured. Data are normalized to vehicle treatment. (E) MCF-7:2A cells were treated likewise, and DNA was harvested and quantified after two weeks. Means represent samples in triplicate. ^**P<0.01, ^***P<0.001.

Figure 6.

MCF-7:2A oxidative stress and glutathione. (A) Total basal glutathione (GSSG+GSH) levels were measured in MCF-7:WS8, MCF-7:5C and MCF-7:2A cells. Means represent samples in triplicate. (B) Total glutathione in MCF-7:5C and MCF-7:2A cells were quantified after 72 h treatment of vehicle or 100 μM BSO. Means represent samples in triplicate. (C) MCF-7:2A cells were treated for 24, 48 and 72 h with either vehicle, 1 nM E₂, 100 μM BSO or 1 nM E₂ + 100 μM BSO; HMOX1 mRNA was measured using RT-PCR. 36B4 was used as an internal control. Means represent at least 8 replicates. (D) MCF-7:2A were subjected to the aforementioned treatments for 5, 7, 9 and 12 days, and ROS levels were measured. Data are normalized to vehicle treatment. (E) MCF-7:2A cells were treated likewise, and DNA was harvested and quantified after two weeks. Means represent samples in triplicate. ^**P<0.01, ^***P<0.001.

Figure 6.

MCF-7:2A oxidative stress and glutathione. (A) Total basal glutathione (GSSG+GSH) levels were measured in MCF-7:WS8, MCF-7:5C and MCF-7:2A cells. Means represent samples in triplicate. (B) Total glutathione in MCF-7:5C and MCF-7:2A cells were quantified after 72 h treatment of vehicle or 100 μM BSO. Means represent samples in triplicate. (C) MCF-7:2A cells were treated for 24, 48 and 72 h with either vehicle, 1 nM E₂, 100 μM BSO or 1 nM E₂ + 100 μM BSO; HMOX1 mRNA was measured using RT-PCR. 36B4 was used as an internal control. Means represent at least 8 replicates. (D) MCF-7:2A were subjected to the aforementioned treatments for 5, 7, 9 and 12 days, and ROS levels were measured. Data are normalized to vehicle treatment. (E) MCF-7:2A cells were treated likewise, and DNA was harvested and quantified after two weeks. Means represent samples in triplicate. ^**P<0.01, ^***P<0.001.

Figure 6.

MCF-7:2A oxidative stress and glutathione. (A) Total basal glutathione (GSSG+GSH) levels were measured in MCF-7:WS8, MCF-7:5C and MCF-7:2A cells. Means represent samples in triplicate. (B) Total glutathione in MCF-7:5C and MCF-7:2A cells were quantified after 72 h treatment of vehicle or 100 μM BSO. Means represent samples in triplicate. (C) MCF-7:2A cells were treated for 24, 48 and 72 h with either vehicle, 1 nM E₂, 100 μM BSO or 1 nM E₂ + 100 μM BSO; HMOX1 mRNA was measured using RT-PCR. 36B4 was used as an internal control. Means represent at least 8 replicates. (D) MCF-7:2A were subjected to the aforementioned treatments for 5, 7, 9 and 12 days, and ROS levels were measured. Data are normalized to vehicle treatment. (E) MCF-7:2A cells were treated likewise, and DNA was harvested and quantified after two weeks. Means represent samples in triplicate. ^**P<0.01, ^***P<0.001.

Figure 7.

MCF-7:2A IGF-1Rβ. (A) Basal IGF-1Rβ mRNA was measured in MCF-7:5C cells and MCF-7:2A cells via RT-PCR. MCF-7:2A values are normalized to MCF-7:5C. 36B4 was used as an internal control. Means represent samples in triplicate. (B) Basal IGF-1Rβ protein levels were measured in MCF-7:5C and MCF-7:2A cells by western blot analysis. β-actin was used as a loading control. (C) MCF-7:2A cells were treated with vehicle, 1 nM E₂, 10 μM AG1024, or 1 nM E₂ + 10 μM AG1024. DNA was harvested and quantified after seven days. Means represent samples in triplicate. (D) MCF-7:2A cells were treated for 72 h with vehicle or 10 μM AG1024. Growth pathway protein levels were visualized via western blot analysis. Total MAPK and total AKT were used as loading controls. ^*P<0.05, ^***P<0.001.

Figure 7.

MCF-7:2A IGF-1Rβ. (A) Basal IGF-1Rβ mRNA was measured in MCF-7:5C cells and MCF-7:2A cells via RT-PCR. MCF-7:2A values are normalized to MCF-7:5C. 36B4 was used as an internal control. Means represent samples in triplicate. (B) Basal IGF-1Rβ protein levels were measured in MCF-7:5C and MCF-7:2A cells by western blot analysis. β-actin was used as a loading control. (C) MCF-7:2A cells were treated with vehicle, 1 nM E₂, 10 μM AG1024, or 1 nM E₂ + 10 μM AG1024. DNA was harvested and quantified after seven days. Means represent samples in triplicate. (D) MCF-7:2A cells were treated for 72 h with vehicle or 10 μM AG1024. Growth pathway protein levels were visualized via western blot analysis. Total MAPK and total AKT were used as loading controls. ^*P<0.05, ^***P<0.001.

Figure 7.

MCF-7:2A IGF-1Rβ. (A) Basal IGF-1Rβ mRNA was measured in MCF-7:5C cells and MCF-7:2A cells via RT-PCR. MCF-7:2A values are normalized to MCF-7:5C. 36B4 was used as an internal control. Means represent samples in triplicate. (B) Basal IGF-1Rβ protein levels were measured in MCF-7:5C and MCF-7:2A cells by western blot analysis. β-actin was used as a loading control. (C) MCF-7:2A cells were treated with vehicle, 1 nM E₂, 10 μM AG1024, or 1 nM E₂ + 10 μM AG1024. DNA was harvested and quantified after seven days. Means represent samples in triplicate. (D) MCF-7:2A cells were treated for 72 h with vehicle or 10 μM AG1024. Growth pathway protein levels were visualized via western blot analysis. Total MAPK and total AKT were used as loading controls. ^*P<0.05, ^***P<0.001.

Figure 7.

MCF-7:2A IGF-1Rβ. (A) Basal IGF-1Rβ mRNA was measured in MCF-7:5C cells and MCF-7:2A cells via RT-PCR. MCF-7:2A values are normalized to MCF-7:5C. 36B4 was used as an internal control. Means represent samples in triplicate. (B) Basal IGF-1Rβ protein levels were measured in MCF-7:5C and MCF-7:2A cells by western blot analysis. β-actin was used as a loading control. (C) MCF-7:2A cells were treated with vehicle, 1 nM E₂, 10 μM AG1024, or 1 nM E₂ + 10 μM AG1024. DNA was harvested and quantified after seven days. Means represent samples in triplicate. (D) MCF-7:2A cells were treated for 72 h with vehicle or 10 μM AG1024. Growth pathway protein levels were visualized via western blot analysis. Total MAPK and total AKT were used as loading controls. ^*P<0.05, ^***P<0.001.