An endocrine-disrupting chemical, fenvalerate, induces cell cycle progression and collagen type I expression in human uterine leiomyoma and myometrial cells

Abstract

Fenvalerate (Fen), widely used for its high insecticidal potency and low mammalian toxicity, is classified as an endocrine-disrupting chemical. Recently, Fen has received great attention for its adverse effects on human reproductive health. In this study, we found that Fen (10 microM) had a stimulatory effect on the growth of both cell lines at 24 h compared with controls by MTS (p < 0.01) and BrdU (p < 0.01) assays in hormonally responsive uterine leiomyoma (UtLM) cells and normal uterine smooth muscle cells (UtSMC). Flow cytometry results showed that Fen enhanced the escape of cells from the G(0)-G(1) checkpoint and promoted progression of both cell types into the S phase. An Annexin V assay showed that Fen had an anti-apoptotic effect on both cell types. By Real-time PCR, we found that collagen I mRNA expression increased (p < 0.05) in Fen-treated cells compared to controls, although it was greater in UtLM tumor cells. Accordingly, Fen increased (p < 0.05) collagen I protein levels in both cell lysate and supernatant when compared to controls. To further test the mechanism of Fen's effects, transactivation and competitive binding assays were done. The results showed Fen did not significantly stimulate luciferase activity at concentrations of 0.1 microM, 1.0 microM or 10.0 microM in either of the cell types. Competitive binding assays revealed that the affinity of Fen binding to estrogen receptors (ERs) was non-detectable compared to E(2). Our data show that Fen can stimulate the growth of both UtLM cells and UtSMC, which involves a combination of enhanced cell cycle progression and inhibition of apoptosis. Also this compound can increase collagen I expression, at both mRNA and protein levels. Interestingly, the ER is less likely involved in either the hyperplasia or extracellular matrix (ECM) overproduction induced by Fen. Our results indicate that Fen exposure could be considered a novel risk factor for uterine fibroids through molecular mechanisms that do not directly involve the ERs.

Figure 1. Cell morphology

Morphology of UtLM cells and UtSMCs after treatment with 0, 10 µM Fen and 0.1 µM E₂ at 24 h. (A) UtLM cells and (B) UtSMCs at 0 µM; (C) UtLM cells and (D) UtSMCs at 10 µM of Fen; (E) UtLM cells and (F) UtSMCs at 0.1 µM of E₂. Original Magnification ×200.

Figure 2. Cell proliferation assay with MTS

UtLM cells (A) and UtSMCs (B) were exposed to Fen for 24 h at concentrations ranging from 0.01 µM to 100 µM. E₂ at a concentration of 0.1 µM was used as a positive control. Compared with proliferation in vehicle controls, UtLM cell proliferation was increased at 10 µM to 100 µM Fen; while UtSMC cell proliferation was increased at 0.1 to 100 µM Fen. Error bars represent means ± SEM. *p<0.01, Student’s t test with vehicle control.

Figure 3. Cell proliferation assay with BrdU

UtLM cells (A) and UtSMCs (B) exposed to Fen for 24 h at concentrations ranging from 0.01 µM to 100 µM. E₂ at concentration of 0.1 µM was used as a positive control. Compared with proliferation in untreated controls, BrdU labeling in UtLM cells was increased at Fen concentrations in the 0.1 µM to 100 µM range. BrdU labeling in UtSMC cells was increased in the 1 µM to 100 µM range. Representative data were shown from two separate experiments. *p<0.01 compared with vehicle control cells.

Figure 4. Cell cycle analysis

UtLM cells (A) and UtSMCs (B) were treated with Fen at 10 µM for 24 h. E₂ at a concentration of 0.1 µM was used as a positive control. The values represent the number of cells in different phases of the cell cycle as a percentage (%) of the total cells observed. The data shown represent the average of three independent experiments. Representative data were shown from three separate experiments. Data were statistically analyzed by a two-way ANOVA with Bonferroni post-tests after arcsine transformation.

Figure 5. Analysis of apoptosis

UtLM cells (A) and UtSMCs (B) treated with Fen at 10 µM for 24 h. E₂ at a concentration of 0.1 µM was used as a positive control. After treatment, UtLM cells and UtSMCs were harvested, and stained with the apoptotic indicator Annexin V dye and were assayed by FACS flow cytometer. The values represent the percentage of apoptotic cells induced by Fen and E₂ to total gated cells. Error bars represent means ± SEM. *p<0.05, Student’s t test (n = 3).

Figure 6. Real-time PCR analysis of Collagen I in UtLM (A) and UtSMC (B) cells

After treatment with Fen (10 µM) for 0, 4, 8, 12 and 24 h, total RNA was isolated from treated and vehicle control cells and subjected to real-time RT-PCR. Bar graphs show the mean ± SEM of three independent cell cultures. *p<0.05 compared with vehicle controls.

Figure 7. Effects of Fen on Collagen Type I protein levels in UtLM cells and UtSMCs

Fen upregulates collagen type I protein levels in UtLM cells and UtSMCs. UtLM cells (A) and UtSMCs (B) were treated with Fen (0.01–10 µM) for 24 h. After treatment, cell lysate and supernatant samples were collected and analyzed by western blotting. E₂ (0.10 µM) was used as a positive control. Representative immunoblots and a graph showing protein levels of collagen I relative to HPRT are shown. Data represent mean ± SEM of three independent experiments.

Figure 8. Transient transfection and luciferase assay in UtLM cells and UtSMCs

Relative luciferase activity in UtLM cells and UtSMCs transfected with hERα (A, C), hERβ (B, D), and 3×-Vit-ERE-TATA-Luc plasmids that were treated with DMSO (vehicle control, VC), 10 nM E₂, or 0.1, 1.0, 10 µM Fen in the presence or absence of 1.0 µM ICI 182,780. Each value was obtained from three independent experiments performed in triplicate. *p< 0.05, **p<0.01, and ***p< 0.001, compared with VC.

Figure 9. Estrogen receptor α and β competitive binding assays

Competitive binding curve between ER/Fluormone ™ and increasing concentrations of E₂ and Fen to ERα (A) and ERβ (B), after 4 h incubation period. A 100% polarization value corresponds to no competition, due to the absence of the competitor. Results represent the means ± SEM. of three separate experiments. ND, non-detectable.