| Colorectal cancer is the common gastrointestinal malignant tumor, the mortality rate occupy the third in the world. Currently, chemotherapy is one of the main methods of colon cancer treament. But it can bring with some side effect, lead to the patients benefit less from chemotherapy. Hence, finding low toxicity and efficient anti-cancer drugs is becoming the current focus.Evodiamine(EVO), an alkaloid isolated from the Evodia rutaecarpa Bentham (Rutaceae), have a potential anti-tumor activity in a number of human cancer cells. However, the effects of evodiamine in human colorectal cancer cells and the underlying molecular mechanisms have been poorly determined.Phosphoglucose isomerase (PGI) is a multifunctional enzyme, which catalyzes the isomerization between glucose-6-phosphate (G-6-P) and fructose-6-phosphate (F-6-P) in the glycolysis and gluconeogenesis pathways inside the cell. As AMF, the endogenic PGI with properties that include autocrine motility factor (AMF) regulating tumor cell motility, when it secreted outside of the tumor cell. AMF binds to AMFR on cell surface, causes proliferation and angiogenesis in tumor cell. Silenced the gene of AMF/PGI would result in the reduction of cell growth and metastasis.Matrix metalloproteinases (MMPs) are implicated in cancer cell metastasis. Wen et al. proposed that in MMPs family only MMP-3 gene, its transcription and translation were induced by AMF/PGI. In addition, it also reported that phosphorylated STAT3 directly bound to the MMP-3 promoter region and regulated MMP-3 gene in nuclear. Both PGI and STAT3 are related to MMP-3, whether PGI is a key driving force for communal migration?ObjectiveThe aim of this study is to explore the mechanisms of EVO on prolife ration and migration of HCT-116 cells. We provided the evidence that EV-O could reduce PGI, p-STAT3 and MMP-3 in HCT116 cells firstly.MethodsHuman colorectal cancer cell lines (HCT-116) was cultured for 24h in vitro, then cocultrued with Evodiamine at the concentration of 1.5,3.0,6.0 μmol·L-1 for 24,48 and 72 h, respectively. The proliferation of cells was detected by CCK-8; The cell cycle and apoptosis of cells treated with Evodiamine for 48 h was measured by flow cytometry; Changes in cell morphology was observed under inverted microscope and morphological changes of apoptotic cells were observed by fluorescence microscope after Hochest staining. The protein expression levels of apoptotic related proteins P53,Bax,Bcl-2,Cleaved caspase-3 and cell-cycle related protein cyclinAl were examined by Western Blot; Variations in AMF protein levels in cell supernatant which treated with different concentrations of EVO for 6 h were quantified by ELISA; Migration potential were assessed by Transwell assays after cells was treated by agents; Cells treated with 6 μmol·L-1 EVO for various lengths of time, and then analyzed for PGI, JAK2/STAT3 and MMP-3 protein levels; And we explored the relationship of PGI, STAT3 and MMP-3 by treating with AG490 and lentiviral vector transduction of siPGI.Results1. HCT-116 cells were treated with different concentration of Evodiamine after 24,48 and 72 h, the proliferation of cells was significantly inhibited by Evodiamine, in a time and dose-dependent manner.2. The percentage of cells in S phase was increased from (13.91±6.95)% to (39.70±11.74)% and the percentage of G2/M was increased from (10.01±1.28)% to (41.85±19.76)% when compared with control group, (P<0.05).3. The rate of apoptosis cells up-regulated from (4.25±2.97)% to (14.90±5.76)% when compared with control group, (P<0.05).4. Under the microscope, plasmatorrhexis and aberration were happened with the increase of drug concentration, and a large number of floating cells were emergenced.5. Hoechst staining revealed that Evodiamine could induce apoptosis and cells in each group which treated with Evodiamine had chromatin condensation gathered and typical apoptotic morphological changes.6. The expression of P53,Bax and Cleaved caspase-3 obviously increased, however the expression of Bcl-2 and cyclinAl protein significantly decreased.7. The secreted form of AMF was decreased after cells were incubated with different concentration of EVO treated for 6 h.8. Transwell assay suggested that EVO could significantly abolish PGI/AMF-induced cell motility.9. Western Blot suggested that EVO could inhibited PGI, p-STAT3 and MMP-3 significantly; the protein levels of P-STAT3 and MMP-3 were significantly down-regulated following treatment with AG490, while PGI remained unchanged.10. Western Blot suggested that the expression of MMP-3 and p-STAT3 significantly decrease in siPGI (HCT-116) cells, and it could further reduced by EVO.Conclusion1. Evodiamine induce the apoptosis of human colorectal cancer cells mainly through down-regulation the expression of CyclinA1 and activation of P53 signal pathway, broken the balance of Bcl-2/Bax, ultimately activated caspase-3.2.EV0-inhibited migration of HCT-116 cells mainly through down-re gulation the expression of PGI, reduction of JAK2/STAT3 signaling path way activity and inhibition of MMP3 production... |
PDF Full Text Request