Inhibitory Effects And Mechanisms Of Paris Sponin ? On Migration And Invasion Of Colon Cancer Cells

Background:Colon cancer is one of the most common malignant tumors,with global incidence and mortality ranking the third and fourth,respectively.Invasion and migration are the main reasons for death in cancer patients.Thus,inhibition of cancer metastasis is an important way to improve the survival rate of colon cancer patients.Epithelial-mesenchymal transition(EMT)is a key mechanism in the early stage of metastasis of malignant tumors derived from epithelial cells.Paris Sponin ? is one of the active components of Rhizoma chrysanthemi.Previous study of our group has found that Rhizoma chrysanthemi had a significant inhibitory effect on the proliferation and migration of lung cancer cells,however,its mechanismswere not clear.The effectsand mechanismsof Paris Sponin ? on invasion and migration of colon cancer have not been reported yet.In this study,two kinds of human colon cancer cells were used to investigate the effects of Paris Sponin ? on the migration and invasion of colon cancer cells in vitro and in vivo,and to study whether theinhibitory effects are mediated by EMT mechanism.Objective:The aim of this study was to investigate the inhibitory effects of Paris Sponin ? on migration and invasion of human colon cancer cells in vitro and in vivo,and to explore its possible mechanisms.Methods:The half inhibitory concentration of Paris Sponin ? on HCT116 and SW620 colon cancer cells was obtained by MTT assay.Wound-healing assay and transwell migration experiment were used to observe the effects of Paris Sponin ? on cell migration ability,and transwell invasion experiment was used to observe the effects of Paris Sponin ? on cell invasion ability.Western Blot was used to examine the effects of Paris Sponin ? on the expression of EMT-associated proteins,such as E-cadherin,N-cadherin,Snail,Slug and Twist.Scanning electron microscope was used to observe the effects of Paris Sponin ? on cell morphological changes.Liver is the most common target for colon cancer metastasis.The experimental hepatic metastasis model of colon cancer in nude mice was established by injecting colon cancer cells in the spleen.The general status of nude mice was observed during the experimental period.On the 30 th day after modeling,the fluorescence intensity of nude mice was detected by IVIS imaging system in vivo,and the liver metastasis of colon cancer in nude mice and the effect of Paris Sponin ? were observed.On the 31 st day after modeling,we killed the nude mice and took out the liver,and then observed tumor formation on the liver surface of each group and inhibition rate of liver migration of colon cancer after administration.Histological section of metastatic tumors were prepared and the effect of Paris Sponin ? on the histopathological morphology of colon cancer cells were observed by HE staining.The effect of Paris Sponin ? on the expression of EMT related proteins(E-cadherin,N-cadherin,Snail,Slug and Twist)were detected by Western Blot.Results:The results of MTT showed that the Paris Sponin ? had inhibitory effect on proliferation of HCT116 cells and SW620 cells with half inhibitory concentration IC50 value of being(3.72 ± 0.09)?M and(3.46 ± 0.13)?M,respectively.The results of wound-healing assay showed that the migration distance of Paris Sponin ? 0.5 ?M,1.0 ?M and 2.0 ?M groups were shortened significantly compared with the control group(P<0.05 or P<0.01),suggesting that cell migration ability was significantly decreased.was especially obvious,The effects are dose-dependent,with Paris Sponin ? 2.0 ?M group being most effective.The results of Transwell migration assay and invasion assay showed that Paris Sponin ? inhibited cell migration and invasion of colon cancer cells in a dose-dependent manner(P<0.01).Western Blot showed that,compared with the control group,Paris Sponin ? 0.5 ?M?1.0 ?M?2.0 ?M group had higher expression of epithelial marker E-cadherin,while the mesenchymal marker proteins N-cadherin and EMT related transcription factors(Snail,Slug,Twist)were significantly downregulated,indicating that Paris Sponin ? inhibits EMT of the cells.The SEM results showed that compared with control group,the morphology of cells of Paris Sponin ? 0.5 ?M,1.0 ?M and 2.0 ?M groups changed.After administration the cell turned round from polygonal shape or fusiform.The parapodium of cells shortened and decreased.The connection between cells became loose and the gap increased.These effects aggravated with the increase of dose.These results suggest that Paris Sponin ? caused the morphological change of EMT.Thirty days after modeling,imaging detection results in vivo showed that the positive signal of liver area in the model control group was 100%,but in the 5-FU,2.5mg/kg Paris Sponin ? and 5.0mg/kg Paris Sponin ? treatment groups were 60%,40% and 20%,respectively.The results suggested that 5-FU and Paris Sponin ? could inhibit the metastasis of HCT116 cells to the liver at the early stage of inoculation,and the inhibitory effect of high-dose Paris Sponin ? was better than that of 5-FU.After the nude mice were killed,livers of the nude mice were taken out.White or gray-yellow metastatic tumors were observed at the edge of the liver.The incidence of liver metastases is equal to that of imaging reaults in vivo.The pathological sections of hepatic metastases of colon cancer showed a large number of tumor cells under the microscope.5-FU and Paris Sponin ? treatments caused varying degrees of necrosis in the central region of tumor tissues,of which 5.0 mg/kg Paris Sponin ? treatment group had the most severe necrosis in tumor tissues and showd large scale coagulation necrosis.Western Blot results showed that compared with the control group,the expression of epithelial marker E-cadherin increased significantly in 2.5 mg/kg Paris Sponin ? and 5.0 mg/kg Paris Sponin ? treatment groups,while the expression of mesenchymal marker N-cadherin and EMT related transcription factors(Snail,Slug and Twist)decreased significantly.Conclusion:Paris Sponin ? could inhibit migration and invasion of human colon cancer cells in vitro and in vivo,and the mechanism may be related to inhibiting the epithelial-mesenchymal transition of colon cancer cells.