Pirfenidone promotes cell cycle arrest and apoptosis of triple‑negative breast cancer cells by suppressing Hedgehog/GLI1 signaling

Abstract

Breast cancer is a common malignant tumor in women and triple-negative breast cancer (TNBC) is the most challenging type of breast cancer with poor prognosis. We aimed to elucidate the effects of pirfenidone, a FDA-approved oral anti-fibrotic drug which has recently shown antitumor potential, in the progression of TNBC and the underlying mechanisms. After TNBC cells were treated with pirfenidone, cell viability was evaluated using CCK-8 assay. The EDU staining was applied for reflecting the ability of cell proliferation. Additionally, cell cycle distribution and apoptotic rate of TNBC cells exposed to pirfenidone were determined by flow cytometry. The levels of proteins associated with cell cycle, apoptosis and Hedgehog/gliomaassociated oncogene homolog (GLI)1 signaling was examined through western blot. Then, GLI1 was upregulated to analyze the proliferation, cell cycle and apoptosis of TNBC cells in the presence of pirfenidone to reveal the regulatory mechanism. Pirfenidone dose-dependently decreased the viability and proliferation of MDA-MB-231 and HCC-1937 cells. Besides, the distribution of TNBC cells in G0/G1 phase was significantly elevated by pirfenidone, accompanied by downregulated levels of CyclinD1, CDK4 and CDK6. Simultaneously, pirfenidone caused remarkably increased apoptotic MDA-MB-231 and HCC-1937 cells, coupled with downregulated BCL2 expression as well as upregulated Bax, cleaved caspase3 and cleaved PARP expression. Expression of proteins related to Hedgehog/gliomaassociated oncogene homolog (GLI)1 signaling was tested through western blot. Particularly, GLI1 and PTCH1 levels were dose-dependently inhibited after pirfenidone exposure. Interestingly, GLI1 overexpression attenuated the influences of pirfenidone on the proliferation, cell cycle and apoptosis of TNBC cells. Collectively, pirfenidone arrests the cell cycle and promotes apoptosis of TNBC cells by suppressing Hedgehog/GLI1 signaling.

Keywords: Apoptosis; Cell cycle; Hedgehog/GLI1 signaling; Pirfenidone; Triple-negative breast cancer.