Effect of infertility treatment and pregnancy-related hormones on breast cell proliferation in vitro

Abstract

Background: Breast cancer development involves a series of mutations in a heterogeneous group of proto-oncogenes/tumor suppressor genes that alter mammary cells to create a microenvironment permissive to tumorigenesis. Exposure to hormones during infertility treatment may have a mutagenic effect on normal mammary epithelial cells, high-risk breast lesions and early-stage breast cancers. Our goal was to understand the association between infertility treatment and normal and cancerous breast cell proliferation.

Methods: MCF-10A normal mammary cells and the breast cancer cell lines MCF-7 [estrogen receptor (ER)-positive, well differentiated] and HCC 1937 (ER-negative, aggressive, BRCA1 mutation) were treated with the weak ER activator clomiphene citrate and hormones that are increased during infertility treatment. Direct effects of treatment on cell proliferation and colony growth were determined.

Results: While clomiphene citrate had no effect on MCF-10A cells or MCF-7 breast cancer cells, it decreased proliferation of HCC 1937 versus untreated cells (P= 0.003). Estrogen had no effect on either MCF-10A or HCC 1937 cells but, as expected, increased cell proliferation (20-100 nM; P≤0.002) and colony growth (10-30 nM; P< 0.0001) of MCF-7 cells versus control. Conversely, progesterone decreased both proliferation (P= 0.001) and colony growth (P= 0.01) of MCF-10A cells, inhibited colony size of MCF-7 cells (P= 0.01) and decreased proliferation of HCC 1937 cells (P= 0.008) versus control. hCG (100 mIU/ml) decreased both proliferation (P ≤ 0.01) and colony growth (P ≤ 0.002) of all three cell lines.

Conclusions: Although these data are preclinical, they support possible indirect estrogenic effects of infertility regimens on ER-positive breast cancer cells and validate the potential protective effect of pregnancy-related exposure to hCG.

Figure 1

Effects of clomiphene citrate and pregnancy hormones on MCF-10A cells. (A) MTS assay performed in 2D culture for MCF-10A cells; absorbance indicates number of viable cells at each time point as a measure of cell proliferation. Significant differences between treated and untreated values on Day 10 were determined by two-sided Student's t-test: estrogen, P= 0.34; clomiphene citrate, P= 0.16; progesterone, P= 0.001; 0.5 mIU/ml hCG, P= 0.001; 10 mIU/ml hCG, P= 0.001; 100 mIU/ml hCG, P< 0.0001. (B) Ratio of total area of colonies for treated versus untreated control cell 3D cultures (a measure of colony growth/size) on Day 4 and Day 10. Day 10 P-values are as follows: estrogen, P= 0.93; clomiphene citrate, P= 0.91; progesterone, P= 0.01; 0.5 mIU/ml hCG, P= 0.31; 10 mIU/ml hCG, P= 0.0001; 100 mIU/ml hCG, P< 0.0001. Asterisks indicate a significant difference between treated and untreated cells at each time point. All values are mean ± SEM for n = 3 experiments.

Figure 2

Effects of clomiphene citrate and pregnancy hormones on MCF-7 cells. (A) MTS assay performed in 2D culture for MCF-7 cells; absorbance indicates number of viable cells at each time point as a measure of cell proliferation. Significant differences between treated and untreated values on Day 10 were determined by two-sided Student's t-test: estrogen, P= 0.23; clomiphene citrate, P= 0.57; progesterone, P= 0.99; 0.5 mIU/ml hCG, P= 0.34; 10 mIU/ml hCG, P= 0.06; 100 mIU/ml hCG, P= 0.01. (B) Ratio of total area of colonies for treated versus untreated control cell 3D cultures on Day 4 and Day 10. Day 10 P-values are as follows: estrogen, P= 0.04; clomiphene citrate, P= 0.82; progesterone, P= 0.01; 0.5 mIU/ml hCG, P= 0.35; 10 mIU/ml hCG, P< 0.0001; 100 mIU/ml hCG, P< 0.0001. Asterisks indicate a significant difference between treated and untreated cells at each time point. All values are mean ± SEM for n = 3 experiments.

Figure 3

Effects of escalating doses of estrogen on MCF-7 cells. (A) MTS assay performed in 2D culture for MCF-7 cells; absorbance indicates number of viable cells at each time point as a measure of cell proliferation. Significant differences between treated and untreated values on Day 10 were determined by two-sided Student's t-test: 10 nM, P= 0.21; 20 nM, P= 0.002; 30 nM, P= 0.001; 60 nM, P= 0.0001; 100 nM, P= 0.0002. (B) Ratio of total area of colonies for treated versus untreated control cell 3D cultures on Day 4 and Day 10. Day 10 P-values are as follows: 10 nM, P< 0.0001; 20 nM, P< 0.0001; 30 nM, P< 0.0001. Asterisks indicate a significant difference between treated and untreated cells at each time point. All values are mean ± SEM for n = 3 experiments.

Figure 4

Effects of clomiphene citrate and pregnancy hormones on HCC-1937 cells. (A) MTS assay performed in 2D culture for HCC-1937 cells; absorbance indicates number of viable cells at each time point as a measure of cell proliferation. Significant differences between treated and untreated values on Day 10 were determined by two-sided Student's t-test: estrogen, P= 0.80; clomiphene citrate, P= 0.003; progesterone, P= 0.008; 0.5 mIU/ml hCG, P= 0.009; 10 mIU/ml hCG, P= 0.002; 100 mIU/ml hCG, P< 0.0001. (B) Ratio of total area for colonies in treated versus untreated control cell 3D cultures on Day 4 and Day 10. Day 10 P-values are as follows: estrogen, P= 0.70; clomiphene citrate, P= 0.03; progesterone, P= 0.06; 0.5 mIU/ml hCG, P= 0.59; 10 mIU/ml hCG, P= 0.41; 100 mIU/ml hCG, P= 0.002. Asterisks indicate a significant difference between treated and untreated cells at each time point. All values are mean ± SEM for n = 3 experiments.