2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibits human ovarian cancer cell proliferation

Abstract

Purpose: The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, mediates a broad spectrum of biological processes, including ovarian growth and ovulation. Recently, we found that an endogenous AhR ligand (ITE) can inhibit ovarian cancer proliferation and migration via the AhR. Here, we tested whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, an exogenous AhR ligand) may exert similar anti-ovarian cancer activities using human ovarian cancer and non-cancerous human ovarian surface epithelial cells.

Methods: Two human ovarian cancer cell lines (SKOV-3 and OVCAR-3) and one human ovarian surface epithelial cell line (IOSE-385) were used. Cell proliferation and migration activities were determined using crystal violet and FluoroBlok insert system assays, respectively. AhR protein expression was assessed by Western blotting. Expression of cytochrome P450, family 1, member A1 (CYP1A1) and member B1 (CYP1B1) mRNA was assessed by qPCR. Small interfering RNAs (siRNAs) were used to knock down AhR expression.

Results: We found that TCDD dose-dependently suppressed OVCAR-3 cell proliferation, with a maximum effect (~70% reduction) at 100 nM. However, TCDD did not affect SKOV-3 and IOSE-385 cell proliferation and migration. The estimated IC50 of TCDD for inhibiting OVCAR-3 cell proliferation was 4.6 nM. At 10 nM, TCDD time-dependently decreased AhR protein levels, while it significantly increased CYP1A1 and CYP1B1 mRNA levels in SKOV-3, OVCAR-3 and IOSE-385 cells, indicating activation of AhR signaling. siRNA-mediated AhR knockdown readily blocked TCDD-mediated suppression of OVCAR-3 cell proliferation.

Conclusion: Our data indicate that TCDD can suppress human ovarian cancer cell proliferation via the AhR signaling pathway and that TCDD exhibits an anti-proliferative activity in at least a subset of human ovarian cancer cells.

Fig. 1

Effects of TCDD on SKOV-3, OVCAR-3 and IOSE-385 cell proliferation. Cells were treated without or with TCDD for 6 days, with a daily change of media containing TCDD or DMSO. Cell proliferation is expressed as means±SEM % of the control (n=3–4). *: Different from control at each corresponding day (p<0.05)

Fig. 2

Effects of TCDD on SKOV-3 and IOSE-358 cell migration. a SKOV-3 and IOSE-358 cells were treated with TCDD (10 nM) for 0, 2, 4 or 6 days, followed by b migration assay. Migrated cells were stained and counted. Cell numbers are expressed as means±SEM % of the day 0 control (n=3). *: Different from the day 0 control (p<0.05). Bars in (a): 200 μm

Fig. 3

Effect of TCDD on AhR protein levels in SKOV-3, OVCAR-3 and IOSE-385 cells. Cells were treated with a single dose of TCDD (10 nM) up to 48 h and protein extracts were subjected to Western blot analysis. Data are expressed as means±SEM fold of the time 0 control (n=3–5). *: Different from the time 0 control (p<0.05)

Fig. 4

Effects of TCDD on CYP1A1 mRNA expression in SKOV-3, OVCAR-3 and IOSE-385 cells as detected by qPCR. Cells were treated with a single dose of TCDD (10 nM) up to 48 h and total mRNA extracts were subjected to qPCR. Data normalized to β-actin are expressed as means±SEM fold of the time 0 control (n=3–4). *: Different from the time 0 control (p<0.05)

Fig. 5

Effects of AhR knockdown on OVCAR-3 cell proliferation. a Cells were transfected with vehicle control, scrambled siRNA (ssRNA) control or AhR-specific siRNA (siRNA) for 2, 4 or 6 days. Proteins were collected and subjected to Western blot analysis. Data from 20 nM siRNA treatment are presented and expressed as means±SEM fold of the vehicle control (n=3). b After siRNA transfection for 2 days, cells were treated with TCDD (10 nM) for an additional 4 days with a daily change of TCDD, followed by cell proliferation assays. Data are expressed as means±SEM % of the vehicle control (n=5–6). *: Different from the vehicle control (p<0.05)