Cyclopamine is a novel Hedgehog signaling inhibitor with significant anti-proliferative, anti-invasive and anti-estrogenic potency in human breast cancer cells

Abstract

Stimulation of Hedgehog (Hh) signaling induces carcinogenesis or promotes cell survival in cancers of multiple organs. In epithelial cancer with aberrant Hedgehog activation, abrogation of Hedgehog signaling by cyclopamine, a naturally occurring Hedgehog-specific small-molecule inhibitor, causes profound inhibition of tumor growth. In the present study, cyclopamine displayed a significant potency in suppressing the proliferation of both estrogen-responsive (MCF-7) and estrogen-independent (MDA-MB-231) human breast cancer cells. Cyclopamine induced a robust G1 cell cycle arrest and elicited notable effects on the expression of cyclin D1 through modulation of the MAPK/ERK signaling pathway. Cyclopamine also inhibited the invasive ability of both breast cancer cell lines by suppressing the expression levels of NF-κB, MMP2 and MMP9 protein. Furthermore, in estrogen-responsive MCF-7 cells, cyclopamine significantly downregulated the production of estrogen receptor-α protein. Our results implicate cyclopamine as a novel, potent inhibitor of human breast cancer proliferation and estrogen responsiveness that could potentially be developed into a promising therapeutic agent for the treatment of breast cancer.

Keywords: MAPK/ERK; breast cancer; cyclopamine; estrogen receptor-α; invasion; proliferation.

Figure 1

Cyclopamine decreases the proliferation of human breast cancer cells. (A and B) Breast cancer cells were treated with or without cyclopamine for 6 days, and then counted at the indicated time. All samples were prepared in triplicate. The proliferation rate was measured as fold changes in cell growth. (C and D) Proliferation curve of breast cancer cells. Cells were treated with cyclopamine and counted after 5 and 10 days. Results are representative of three independent experiments.

Figure 2

Cyclopamine affects breast cancer cell cycle and invasive ability. (A) Cell cycle distribution was evaluated by propidium iodide labeling and analysed by flow cytometry. (B) Cells treated with or without cyclopamine were trypsinized and transferred to the upper compartment of the modified Transwell chambers. Following 24 h of incubation, the invasive cells attached to the lower surface of the Matrigel-coated filter were fixed, stained and photographed under a phase contrast microscope and then counted in 15 randomly selected microscopic fields.

Figure 3

Cyclopamine inhibits the expression of proliferation and invasion-related proteins. (A) Whole cell lysates were prepared, and 50 μg proteins were resolved using SDS-PAGE, followed by immunoblotting with the indicated specific antibodies against NF-κB, cyclin D1, MMP2 and MMP9. (B) The expression levels are displayed as fold changes in band density. ^*P<0.01 vs. the control group.

Figure 4

U0126 partly rescues cyclin D1 inhibition to cyclopamine treatment. (A and B) Comparison of cyclin D1 expression between cells treated with or without cyclopamine. (C) Statistical analysis was conducted using a Student’s t-test. The expression levels are displayed as fold changes in band density. ^*P<0.01 vs. the control group. (D) Dose-dependent effects of cyclopamine on expression of estrogen receptor-α (ER-α) protein. MCF-7 cells were treated with indicated concentrations of cyclopamine for 24 h, and the level of ER-α was determined by western blot analysis.