| BACKGROUND: Prior studies have shown that EST (estrogen replacement treatment) in postmenopausal women could significantly reduce theincidence of CRC (colorectal cancer). Further studies have found that estrogen and its receptors have a protective effect in the inhibition of CRC, but ERT will increase the incidence of vein thrombosis, breast cancer and endometrial cancer.Phytoestrogen is a kind of compounds extracted from contained in soybean,kudzuvine root and astragalus root and so on, which has estrogen-like compounds. its chemical structure similar to estrogen. Phytoestrogen could combined with ER and play some kind of estrogen effect or anti-estrogen effect.Phytoestrogen has rich source, low toxic and side effect. Therefore, the role and value of phytoestrogens in anti-tumor effects gradually attracted people's attention. Studies have found that phytoestrogens were the most active components from soybean sources and soy products can reduce the incidence of CRC, prostate cancer, breast cancer and other tumors in human. Further evidence showed that phytoestrogens could inhibit proliferation and induce apoptosis through combination with estrogen receptor or inhibit tyrosine kinase activity in some cancer cells, such as colon cancer, breast cancer, prostate cancer,cervical cancer and so on.Astragalus belongs to leguminous plants of the genus, and is a widely used traditional Chinese medicine in our country. The effective components of Astragalus mainly contains polysaccharide, triterpenes and flavonoids, and has a variety of biologically active on the cells, such as anti-inflammation, anti-virus,anti-tumor and resistance to mutations. Calycosin as one of the main effective ingredient can play an important role in antioxidant, protecting liver and anti-tumor effect. Study show that calycosin play an important role in anti-tumor through combine with ER receptor, such as inhibit cell metastasis and invasion in lung cancer, inhibit cell proliferation and induce apoptosis in breast cancer,ovarian cancer and melanoma, but But the effect of calycosin on CRC cells and its mechanism has not been reported.OBJECTIVE: This study mainly to investigate the effects of calycosin on CRC cells in vitro, to demonstrate that calycosin may interact with EGFR PI3K/AKT or EGFRIMAPKs pathway via ER? to inhibit CRC cells proliferation and induce cell apoptosis and to explore the potential anti-tumor mechanism of calycosin in CRC.METHODS: The HCT116 and HT29 cell lines were selected and treated with different concentrations of calycosin, MTT assay was used to detect the inhibitory effect of CRC cells. Hoechest33258 staining and flow cytometry were used to measure cells apoptosis rate. qRT-PCR was used to detect the mRNA level of ER?, EGFR and caspase3. Western blot was used to measure the expression level of EGFR, MAPKs (ERK1/2/p-ERK1/2, JNK/p-JNK,p38/p-p38), Akt/p-Akt, Bax/Bcl-2, Caspase3 and cleaved-Caspase3.RESULTS: MTT assay showed that calycosin can effectively inhibited cell proliferation of HCT116 and HT29 in a dose-dependent manner,Hoechest33258 staining and flow cytometry show that calycosin can inhibited the growth of CRC cells by inducing cell apoptosis. qRT-PCR show that calycosin can increases the mRNA level of ER? and Caspase-3 with the increase of concentration of calycosin, but the mRNA level of EGFR decreased with the increase of concentration. Western blot showed that calycosin can decreases the expression of EGFR and Bcl-2, but increases the apoptosis-related protein level of Bax, Caspase3 and cleaved-Caspase3 with the increases concentration of calycosin.Moreover, calycosin can inhibit the phosphorylation lvels of Akt,but up-regulate phosphorylation levels of JNK with the increases concentration of calycosin.CONCLUSION: Calycosin can effectively inhibit the proliferation and induce apoptosis on HT29 and HCT116 cells. Calycosin can inhibit cell proliferation and induce cell apoptosis by interact with EGFR PI3K/AKT or EGFR/MAPKs pathway via ER?. |
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