Curcumol Suppresses Breast Cancer Cell Metastasis And Proteomic Technique Analyzes Its Potential Protein Targets

Background Breast cancer is one of the most common cause of cancer related death for women in the world which is expected to account for 29% all new cancer diagnoses in women according to the data in 2015 published in Cancer Statistics of the year 2016. In china, the incidence of breast cancer was increased from 2009 to 2011. Breast cancer metastasis, which refers to the spread of cancer cells from the primary sites to distant sites and colonies, is the main cause of poor clinical outcome or death in the majority of breast cancer patients. Rhizoma Curcumae is traditionally used for the treatment of gynecologic tumors in China. Curcumol, a major bioactive component of Rhizoma Curcumae oil, has been reported to possess anti-tumor activity. Compared with many chemical antitumor drugs, curcumol has the advantages of high efficiency, low toxicity, safety, reliability and wide clinical application. However, its effect and mechanisms against tumor metastasis are still unclear. The aim of this study is to investigate the inhibitory effect of curcumol on breast cancer cell metastasis and elucidate the underlying molecular mechanisms.Methods Cell death was analyzed by sulforhodamine B assay and growth inhibition was determined by colony formation assay. Western blotting was used to assess the protein expression of related signaling proteins. The enzyme activity of matrix metalloproteinases(MMPs) was determined by zymography assay. Anti-metastasis activity was evaluated by ibidi wound healing assay, Boyden chamber migration and invasion assay and adhesion assay. Luciferase assays were performed using the Dual-Luciferase Reporter Assay System to detect the expression of transcriptional activity of nuclear factor κB(NF-κB). The two-dimensional gel electrophoresis-based proteomics was applied to investigate the differential protein profiles in MDA-MB-231 cells with or without the treatment of Curcumol. The different proteins were identified by MALDI-TOF/TOF mass spectrometry. GO and KEGG analysis were used to analized the function of the target and its related pathways. PPI analysis was used to analyze the protein interactions of the target proteins. RT-PCR was used to confirm the proteins.Results 1 Inhibition of migration, invasion, and adhesion by curcumol in breast cancer cells Our results showed that non-cytotoxicity was caused by curcumol within 10-40 μg/m L in highly metastatic MDA-MB-231 and 4T1 cells for 24 h, whereas sustained treatment with 40 μg/m L curcumol for 14 days significantly suppressed the clonogenic activity of MDA-MB-231 and 4T1 cells. The concentration of curcumol 10 μg /ml, 20 μg /ml, 40 μg /ml were select to further study on the inhibition of breast cancer cell migration, invasion, and adhesive ability. Importantly, curcumol at non-cytotoxic concentrations suppressed the migration ability of both MDA-MB-231 and 4T1 cells in the ibidi wound healing assay. Moreover, curcumol suppressed the migration and invasion of MDA-MB-231 cells in the Boyden chamber migration and invasion assay and inhibited the adhesion of MDA-MB-231 cells onto the matrigel. 2 Curcumol inhibited MMP-9, activation of JNK1/2 and Akt, and nuclear translocation of NF-kB p65 in MDA-MB-231 cells Further mechanistic investigation revealed that curcumol decreased the enzyme activity and protein expression of MMP-9 in MDA-MB-231 cells. Moreover, curcumol inhibited the activation of c-Jun N-terminal kinase(JNK) 1/2 and Akt by decreasing their phosphorylation levels. Meanwhile, curcumol also inhibited the nuclear translocation and transcriptional activity of nuclear factor κB(NF-κB). Furthermore, JNK inhibitor SP600125 and PI3K-Akt inhibitor LY294002 enhanced the inhibition of curcumol on NF-κB p65 nuclear translocation. Finally, supplementation with SP600125, LY294002 or NF-κB inhibitor PDTC significantly enhanced the inhibitory effect of curcumol on the MMP-9 expression and the cell migration, invasion and adhesion in MDA-MB-231 cells. Our findings provide evidence for the suppression of breast cancer cell metastasis by curcumol and suggest that the inhibition of MMP-9 via JNK1/2 and Akt dependent NF-κB signaling pathway may be the underlying mechanisms. 3 Application of proteomics technique analysis of target proteins induced by curcumol with differential proteomics Our results show that, 19 proteins affected by curcumol were seprated by 2-D gel electrophoresis-based proteomics. And 11 proteins were identified by MALDI-TOF mass spectrometry. Gene Ontology analysis showed that those proteins participated in several important biological processes and exhibited diverse molecular functions. KEGG analysis shows that 11 proteins involved in 76 signal pathways. In particular, four proteins(EEF1A1, NRAS, SUB1, ACAT2) are able to participate in the regulation of cell migration ability. PPI analysis was used to analize the protein interactions of the target proteins. The results of RT-PCR were further confirmed the curcumol was able to regulate these four proteins.Conclusion Curcumol suppresses breast cancer cell metastasis by inhibiting MMP-9 via JNK1/2 and Akt dependent NF-κB signaling pathway. EEF1A1, NRAS, SUB1, ACAT2 may be the potential targets of the inhibition of curcumol in breast cancer cell migration, invasion, and in vitro adhesion.