Interactions between IGF-I, estrogen receptor-α (ERα), and ERβ in regulating growth/apoptosis of MCF-7 human breast cancer cells

Abstract

Understanding of the interactions between estradiol (E₂) and IGF-I is still incomplete. Cell lines derived from the MCF-7 breast cancer cells were generated with suppressed expression of the IGF-I receptor (IGF-IR), termed IGF-IR.low cells, by stable transfection using small interfering RNA (siRNA) expression vector. Vector for control cells carried sequence generating noninterfering RNA. Concomitant with reduction in the IGF-IR levels, the IGF-IR.low cells also showed a reduction in estrogen receptor α (ERα) and progesterone receptor expressions, and an elevation in the expression of ERβ. The number of the IGF-IR.low cells was reduced in response to IGF-I and human GH plus epidermal growth factor, but E₂ did not cause an increase in the number of the IGF-IR.low cells compared to controls. The proliferation rate of IGF-IR.low cells was only reduced in response to E₂ compared to controls, whereas their basal and hormone-stimulated apoptosis rate was increased. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was increased in the IGF-IR.low cells after treatment with E₂, without affecting control cells. Furthermore, phosphorylation of the tumor suppressor protein p53 was elevated in the IGF-IR.low cells compared to the controls. In conclusion, suppressing IGF-IR expression decreased the level of ERα but increased the level of ERβ. Overall growth rate of the IGF-IR.low cells was reduced mostly through an increase in apoptosis without affecting proliferation substantially. We hypothesize that a decreased ERα:ERβ ratio triggered a rapid phosphorylation of p38 MAPK, which in turn phosphorylated the p53 tumor suppressor and accelerated apoptosis rate.

Figure 1

Western blot analysis showing expression of the insulin-like growth factor-I receptor (IGF-IR) and actin (internal control) in cloned MCF-7 cells stably transfected with a vector producing a small interfering RNA (siRNA) to the IGF-IR (IGF-IR.low), and control cells transfected with a vector producing an inert small RNA of the same size as the IGF-IR-siRNA. Cells were plated onto 100 mm culture dishes in DME/F12 medium containing 10% FBS and cultured for 24-h, when cells were harvested, lysed, total protein extracted and analyzed. For the controls, two different cloned cells lines are shown (control1,2), and three different IGF-IR.low cell lines (IGF-IR.low1, 2, 3) are shown (A). IGF-IR protein levels were quantified using densitometric analysis and the results are expressed as IGF-IR/β-βactin ratio. Each bar represents the mean ± SEM of 5 (controls) and 6 (IGF-IR.low) replicates. Asterisk indicate significant differences between controls and IGF-IR.low cells, P<0.05 (B).

Figure 2

Growth response of MCF-7 cells stably transfected with a vector producing a small interfering RNA (siRNA) to the insulin-like growth factor-I receptor (IGF-IR.low), and control cells transfected with a vector producing an inert small RNA of the same size as the IGF-IR-siRNA. The cells were plated onto 24-well plates in DME/F12 medium containing 10% FBS and incubated for 24-h. The media were then changed to serum-free and incubated for additional 24-h and then treated with (A) 50 ng/ml insulin-like growth factor-I (IGF-I), (B) 10⁻⁷ M 17β-estradiol (E2) and (C) 500 ng/ml human growth hormone (GH) plus 10 ng/ml epidermal growth factor (EGF). The cultures were continued for 10 days and media replenished every other day, at the same time of day. Four replicate wells were terminated at each time point to establish growth rate over the 10-day period. At the end of the culture period, total DNA was measured to assess cell number. Each bar represents mean ± SEM for four replicate wells. Asterisks indicate significant differences between controls and IGF-IR.low cells, P<0.05.

Figure 2

Growth response of MCF-7 cells stably transfected with a vector producing a small interfering RNA (siRNA) to the insulin-like growth factor-I receptor (IGF-IR.low), and control cells transfected with a vector producing an inert small RNA of the same size as the IGF-IR-siRNA. The cells were plated onto 24-well plates in DME/F12 medium containing 10% FBS and incubated for 24-h. The media were then changed to serum-free and incubated for additional 24-h and then treated with (A) 50 ng/ml insulin-like growth factor-I (IGF-I), (B) 10⁻⁷ M 17β-estradiol (E2) and (C) 500 ng/ml human growth hormone (GH) plus 10 ng/ml epidermal growth factor (EGF). The cultures were continued for 10 days and media replenished every other day, at the same time of day. Four replicate wells were terminated at each time point to establish growth rate over the 10-day period. At the end of the culture period, total DNA was measured to assess cell number. Each bar represents mean ± SEM for four replicate wells. Asterisks indicate significant differences between controls and IGF-IR.low cells, P<0.05.

Figure 2

Growth response of MCF-7 cells stably transfected with a vector producing a small interfering RNA (siRNA) to the insulin-like growth factor-I receptor (IGF-IR.low), and control cells transfected with a vector producing an inert small RNA of the same size as the IGF-IR-siRNA. The cells were plated onto 24-well plates in DME/F12 medium containing 10% FBS and incubated for 24-h. The media were then changed to serum-free and incubated for additional 24-h and then treated with (A) 50 ng/ml insulin-like growth factor-I (IGF-I), (B) 10⁻⁷ M 17β-estradiol (E2) and (C) 500 ng/ml human growth hormone (GH) plus 10 ng/ml epidermal growth factor (EGF). The cultures were continued for 10 days and media replenished every other day, at the same time of day. Four replicate wells were terminated at each time point to establish growth rate over the 10-day period. At the end of the culture period, total DNA was measured to assess cell number. Each bar represents mean ± SEM for four replicate wells. Asterisks indicate significant differences between controls and IGF-IR.low cells, P<0.05.

Figure 3

Proliferation rates of the cultured MCF-7 cells stably transfected with a vector producing a small interfering RNA (siRNA) to the insulin-like growth factor-I receptor (IGF-IR.low) and controls were measured using an ELISA kit based on 5-bromo-2′-deoxyuridine (BrdU) incorporation. The cells were plated onto 96-well plates in 10% FBS and incubated for 24-h and then serum-starved for additional 24-h, followed by treatments with 50 ng/ml insulin- like growth factor-I (IGF-I), 10⁻⁷ M 17β-estradiol (E2) and, 500 ng/ml human growth hormone (GH) plus 10 ng/ml epidermal growth factor (EGF) or vehicle (untreated) and the cells were incubated overnight. The following morning, fresh treatment media containing 10 μM BrdU were added and cells were incubated for additional 90 min, when cultures were terminated and BrdU incorporation measured. Each bar represents mean ± SEM of eight replicate wells. (*) indicates significantly different from all hormone-treated cells, (#) indicates significantly lower than control cells receiving the same treatment, p<0.05.

Figure 4

Rate of apoptosis of the cultured MCF-7 cells stably transfected with a vector producing a small interfering RNA (siRNA) to the insulin-like growth factor-I receptor (IGF-IR.low) and control cells. ELISA kit based on neo-epitope exposure after caspase-cleaved cytokeratin 18 was used to measure the rate of apoptosis. The cells were plated onto 96-well plates in 10% FBS and incubated for 24-h and then serum-starved for additional 24-h. The medium was then changed to treatment media consisting of 50 ng/ml insulin-like growth factor-I (IGF-I), 10⁻⁷ M 17β-estradiol (E2), and 500 ng/ml human growth hormone (GH) plus 10 ng/ml epidermal growth factor (EGF) or vehicle (untreated) and the cells incubated overnight. The following morning, the cells were exposed to NP-40, final concentration 0.5%, and lysed for 5 min on a rotatory shaker. Assays of the cell lysates were carried out according to the manufacturer’s instructions. Each bar represents mean ± SEM for five to eight replicate wells. (*) indicates significantly different from control cells with the same treatment; (*) indicates significantly different from the same cells when untreated, p<0.05.

Figure 5

Western blot analysis showing expression of the estrogen receptor-α (ERα), the progesterone receptor (PR), and the ERβ in cloned MCF-7 cells stably transfected with a vector producing a small interfering RNA (siRNA) to the insulin-like growth factor-I receptor (IGF- IR.low), and control cells transfected with a vector producing an inert small RNA of the same size as the IGF-IR-siRNA. B-Actin was used as an internal control. Cells were plated onto 100 mm culture dishes in DME/F12 medium containing 10% FBS and cultured for 24-h, followed by a 24-h serum starvation. The cells were then harvesting, lysed, total protein extracted and total protein concentration of the lysate measured before western blot analyses (A). Densitometry was used for quantification of the receptor protein levels and the results are shown as ratios of β-actin expression. Each bar represents mean ± SEM of 4 (controls) and 6 (IGF-IR.low) replicates. Asterisks indicate significantly differences between controls and IGF-IR.low cells, P<0.05 (B).

Figure 6

The activity (phosphorylation) of p38 mitogen-activated protein kinase (p38 MAPK). MCF-7 cells stably transfected with a vector producing a small interfering RNA (siRNA) to the insulin-like growth factor-I receptor (IGF-IR.low), and control cells transfected with a vector producing an inert small RNA of the same size as the IGF-IR-siRNA were cultured in 60 mm culture plates in 10% FBS. After overnight serum starvation, the cells were either left untreated or treated with 10⁻⁷ M 17β-estradiol (E2) and incubation continued for 10 min, when the cells were washed in ice-cold PBS and then harvested by scraping in ice-cold PBS. After centrifugation for 5 min the cells were lysed and the assays carried out according to the manufacturer’s instructions. Total protein concentrations of the cell lysates were used for normalizing the assay results. Each bar represents mean ± SEM for three replicate plates. Asterisk indicates significantly higher than all other groups, p<0.05 (A). To determine the total basal protein levels of the p38 MAPK, western blot analysis was carried out on samples from identical cultures to that shown in (A) for untreated cells and the results quantified using densitometric analysis. Results are expressed as p38 MAPK/β-actin ratio. Each bar represents mean ± SEM of 4 (controls) and 5 (IGF-IR.low) replicates (B). The activity (phosphorylation) of the p53 tumor suppressor protein was measured using ELISA assay. The IGF-IR.low cells, and control cells were plated onto 60 mm culture plates in 10% FBS and incubated overnight, followed by 24-h serum starvation. Fresh serum free medium was then applied and incubation continued for 10 min, when the cells were washed in ice-cold PBS and then harvested by scraping in ice-cold PBS and assayed for p53 phosphorylation. Each bar represents mean ± SEM for three replicate plates. Asterisk indicates significantly different from the control cells, p<0.05 (C). The total protein expression levels of p53 were determined using western blot analysis and densitometric quantification from cultures identical to those described in C. The results are expressed as p53/β-actin ratio. Each bar represents mean ± SEM of 5 (controls) and 5 (IGF-IR.low) replicates (D).