| Objective:Cancer metastases caused by the circulating tumor cells(CTCs)account for 90%of cancer death,and the situation has changed little.Paradigm-shifting from traditional post-metastatic chemotherapy to pre-metastatic chemoprevention may revolutionize current cancer treatments.Enlightened by recent large epidemiological studies that oral contraceptives may reduce the risk of deaths from several common cancers with duration of treatment inducing greater effect,and considering about striking similarities present between the behavior of invasive placental cells and that of invasive HT29 cells,we hypothesized that a drug with the ability of anti-implantation may serve well for pre-metastatic chemo-prevention.By using the bioactivity-guided fast screen assay,ginsenoside Ro,an antifertility active saponion was isolated from the root of Achyranthes bidentata.Herein,we systematically investigated the anti-metastatic activity of ginsenoside Ro on human colorectal cancer HT29 cell line and uncovered its underlying mechanism.Methods:Dried A.bidentata roots were cut into pieces and extracted with 80%refluxing ethanol.The extraction was partitioned with petroleum ether and n-Bu-OH,respectively.And the residue from the n-Bu-OH was subjected to a porous polymer resin D101 column.Finally,saponions and sterones were prepared by semi-reparative HPLC on ODS.Mass spectrometry and 1H and 13C NMR spectroscopy were employed to elucidate their structures.Subsequently,they were screened using a high-throughput assay for the study of human embryo implantation.In the present study,cell survival,cell cycle,apoptosis,transwell and adhesion assay,were adopted to test the anti-metastatic activity of ginsenoside Ro.Based on the common pathway shared by embryo implantation and tumor metastasis,we further investigated its molecular mechanism by western-blotting,flow cytometric,and real time polymerase chain reaction at the protein and mRNA level in vitro.Finally,the anti-metastatic property was further validated in lung metastasis experiments.Results:In the high throughput spheroid attachment assay,ginsenoside Ro was able to induce a reproducible concentration-dependent inhibition,more significant compared with other four compounds.MTT assay revealed that treatment of gradient concentrations of ginsenoside Ro did not obviously inhibit the cell viability of HT29 cells after 24-h treatment.Additionally,a slight concentration-dependent increase in the percentage of apoptotic cells was observed when HT29 cells were incubated with ginsenoside Ro.In the current work,we demonstrated that ginsenoside Ro at high concentration(100 ?g/ml)significantly decreased the adhesive,motility and invasive capability of HT29 cells,and inhibition of cell-matrix adhesion and directional motility on a fibronectin matrix was up to fifty percent.Around 52%reduction in the number of cells passing through the membrane treated with ginsenoside Ro at the concentration of 20 ?g/ml.By flow cytometry,a variety of cellular adhesion molecules(CAMs)involved in migration and invasion of tumor cells and implantation of the placenta were tested for their protein expression under the treatment of ginsenoside Ro.Among those tested,only integrin ?v?6 expression was down-regulated significantly in the ginsenoside Ro treated groups than that in the control group.RT-PCR and western blot analysis revealed that ginsenoside Ro reduced the expression of MMP-2,-9,and p-ERK1/2 at the protein and mRNA level in vitro in a concentration-dependent manner.Experimental lung-metastasis model in vivo was used to examine the anti-metastasis effect of ginsenoside Ro.Obviously,ginsenoside Ro treatment(250 mg/kg)caused a dramatic decrease in the number of tumor nodules on the lung surface,yielding inhibition rates of 93%?In the meantime,histopathological H&E staining of various lung sections were analyzed,revealing that ginsenoside Ro produced significant decrease in HT29-induced metastatic lesion and tissue density,compared to that in the vehicle-treated group.And In agreement with in vitro data,the expression of integrin ?v?6 was reduced in tumors from ginsenoside Ro-treated mice compared to the vehicle-treated groups.Results:In the current work,we demonstrated that ginsenoside Ro,the active compound isolated from Achyranthes bidentata,decreased the adhesive,motility and invasive capability of HT29 cells by interfering ?v?6 associated ERK signal pathway that regulates metastatic properties,while exhibited low toxicity.Collectively,these altered parameters led to suppressing the tumor lung metastasis without significant side effect,indicating promising characteristics of the traditional contraceptives against this minimal residual disease. |
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