Sinensetin suppresses breast cancer cell progression via Wnt/β-catenin pathway inhibition

Abstract

Background: Although there are many treatments for breast cancer, such as surgery, radiotherapy, chemotherapy, estrogen receptor antagonists, immune checkpoint inhibitors and so on. However, safer and more effective therapeutic drugs for breast cancer are needed. Sinensetin, a safer therapeutic drugs, come from citrus species and medicinal plants used in traditional medicine, while its role and underlying mechanism in breast cancer remain unclear. Our study aimed to investigate the role and mechanism of sinensetin in breast cancer.

Methods: Cell Counting Kit-8 (CCK-8) was used to determine the safe concentration of sinensetin in MCF-10A, MCF7 and MDA-MB-231 cells; 120 μM sinensetin was used in subsequent experiments. Real time polymerase chain reaction (RT-PCR), Western blotting, Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay, Transwell invasion assay and Clone formation assay were used in this study to determine cell viability, mRNA expression, protein levels, apoptosis, proliferation, invasion and so on.

Results: Herein, our results showed that 120 μM sinensetin suppressed the cell viability and promoted apoptosis of MCF7 and MDA-MB-231 cells. Treatment with 120 µM sinensetin for 24 h showed no significant toxicity to normal mammary cells; 120 μM sinensetin decreased cell proliferation, invasion, and epithelial-mesenchymal transition (EMT), and downregulated β-catenin, lymphatic enhancing factor 1 (LEF1), T-cell factor (TCF) 1/TCF7, and TCF3/TCF7L1 expression in MCF7 and MDA-MB-231 cells. The Wnt agonist SKL2001 reversed the inhibitory effect of sinensetin on cell survival, metastasis, and EMT. Sinensetin-induced downregulation of β-catenin, LEF1, and TCF1/TCF7 expression were upregulated by SKL2001 in MCF7 and MDA-MB-231 cells.

Conclusions: In summary, sinensetin suppressed the metastasis of breast cancer cell via inhibition of Wnt/β-catenin pathway and there were no adverse effects on normal breast cells. Our study confirmed the role of sinensetin in breast cancer cells and provided a better understanding of the underlying mechanism.

Keywords: Breast cancer; Wnt; apoptosis; epithelial-mesenchymal transition (EMT); sinensetin.

Figure 1

Sinensetin reduces breast cancer cell viability and increases breast cancer apoptosis. (A) MCF7 and MDA-MB-231 cell viability was measured by CCK-8 after sinensetin treatment. (B) MCF-10A cell viability was measured by CCK-8 after 120 µM sinensetin treatment. (C) Caspase 9, Caspase 3, Cleaved-caspase 9, Cleaved-caspase 3, Bcl-2, Bax and β-actin protein expression in MCF7 and MDA-MB-231 cells were determined by Western blotting. Quantitative analysis for Cleaved-caspase 9, Cleaved-caspase 3 and Bcl-2/Bax protein expression in MCF7 and MDA-MB-231 cells. (D) MCF7 and MDA-MB-231 cell apoptosis were measured by TUNEL assay. Scale bar =50 µm. Briefly, cells were fixed with 4% paraformaldehyde, washed with PBS twice for 3 min, and then treated with 0.3% Triton X-100 for 10 min. Aliquots of 50 µL TUNEL solution were placed in coverslips and cells were cultured for 1 h. Coverslips were washed with TBST three times. Cells without any substance acted as control. Quantitative analysis for MCF7 and MDA-MB-231 cell apoptosis. ***, P<0.001; **, P<0.01. Sin, sinensetin; DAPI, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride; TUNEL, TdT-mediated dUTP Nick-End Labeling; CCK-8, Cell Counting Kit-8; PBS, phosphate buffered saline; TBST, tris buffered saline tween.

Figure 2

Sinensetin inhibits MCF7 and MDA-MB-231 cell proliferation, invasion and migration through EMT process. (A) MCF7 and MDA-MB-231 cell proliferation were determined by Clone formation assay. Scale bar =1,000 µm. Cells without any substance acted as control. Briefly, cells were fixed in 4% paraformaldehyde (P0099, Beyotime Biotechnology) and then washed three times. Cells were cultured with 0.1% crystal violet and washed with water. (B) MCF7 and MDA-MB-231 cell invasion were determined by Transwell invasion assay. Scale bar =100 µm. Cells below the membrane were fixed with 4% paraformaldehyde. Subsequently, cells were stained with 0.1% crystal violet for 15 min and photographs were taken to assess the relative number of invaded cells for each well. Cells without any substance acted as control. (C) Vimentin, E-cadherin, Slug, Snail, Twist and β-actin protein expression in MCF7 and MDA-MB-231 cells were determined by Western blotting. Quantitative analysis for Vimentin, E-cadherin, Slug, Snail and Twist protein expression in MCF7 and MDA-MB-231 cells. (D) Gene expression analysis to Vimentin, E-cadherin, Slug, Snail and Twist in MCF7 and MDA-MB-231 cells were performed by qRT-PCR. ***, P<0.001; **, P<0.01; *, P<0.05. Sin, sinensetin; EMT, epithelial-mesenchymal transition; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 3

Sinensetin reduces mRNA expression of EMT marker. (A) Zeb1, Zeb2, E47 and β-actin protein expression in MCF7 and MDA-MB-231 cells were determined by Western blotting. (B) Quantitative analysis for Zeb1, Zeb2 and E47 protein expression in MCF7 and MDA-MB-231 cells. (C) Gene expression analysis to Zeb1, Zeb2 and E47 in MCF7 and MDA-MB-231 cells were performed by qRT-PCR. ***, P<0.001; **, P<0.01. Sin, sinensetin; EMT, epithelial-mesenchymal transition; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 4

Sinensetin results in a decreased protein expression of Wnt/β-catenin signaling pathway. (A) β-catenin, LEF-1, TCF1/TCF7, TCF3/TCF7L1 and β-actin protein expression in MCF7 and MDA-MB-231 cells were determined by Western blotting. (B) Quantitative analysis for β-catenin, LEF1, TCF1/TCF7 and TCF3/TCF7L1 protein expression in MCF7 and MDA-MB-231 cells. (C) β-catenin, LEF-1, TCF1/TCF7, TCF3/TCF7L1 and β-actin protein expression in MCF-10A cells were determined by Western blotting. Quantitative analysis for β-catenin, LEF-1, TCF1/TCF7 and TCF3/TCF7L1 protein expression in MCF-10A cells. (D) Gene expression analysis to β-catenin, LEF-1, TCF1/TCF7 and TCF3/TCF7L1 in MCF7 and MDA-MB-231 cells were performed by qRT-PCR. ***, P<0.001; **, P<0.01. Sin, sinensetin; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 5

SKL20001 reverses the anti-cancer effect of sinensetin on breast cancer cells. (A) β-catenin, LEF-1, TCF1/TCF7 and β-actin were measured by Western blotting. Quantitative analysis for β-catenin, LEF-1 and TCF1/TCF7MCF7 in MCF cells. (B) Bcl-2, Bax and β-actin were measured by Western blotting. Quantitative analysis for Bcl-2/Bax in MCF cells. (C) MCF7 cell apoptosis were determined by TUNEL assay. scale bar =50 µm. Briefly, cells were fixed with 4% paraformaldehyde, washed with PBS twice for 3 min, and then treated with 0.3% Triton X-100 for 10 min. Aliquots of 50 µL TUNEL solution were placed in coverslips and cells were cultured for 1 h. Coverslips were washed with TBST three times. Cells without any substance acted as control. Quantitative analysis for MCF7 cell apoptosis. (D) MCF7 cell invasion were measured by Transwell invasion assay. Scale bar =100 µm. Cells below the membrane were fixed with 4% paraformaldehyde. Subsequently, cells were stained with 0.1% crystal violet for 15 min and photographs were taken to assess the relative number of invaded cells for each well. Cells without any substance acted as control. Quantitative analysis for MCF7 cell invasion. ***, P<0.001; **, P<0.01. Sin, sinensetin; DAPI, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride; TUNEL, TdT-mediated dUTP Nick-End Labeling; PBS, phosphate buffered saline; TBST, tris buffered saline tween.