| Background:Cantharidin(CTD)is an active compound from Chinese blister beetles in the families of Meloidae and Oedemeridae,which have been used in Chinese traditional medicine for over 2000 years.Recently,CTD has been identified as a potent inhibitor of protein phosphatases protein phosphatase 2A(PP2A).Clinically,CTD has been used to treat various skin-related diseases,mainly for the treatment of cutaneous warts.Numerous studies have shown that CTD induced cytotoxic effects on various types of cancer cells,such as hepatoma,lung cancer,gastric cancer,colorectal cancer,and bladder cancer.The anti-tumor mechanism includes blocking the cell cycle progression,inducing cells apoptosis,eventually inhibition cell proliferation and metastasis.Colorectal cancer(CRC)is a common malignant tumor.Despite recent advancement in treatments,existing therapies for CRC are of limited effectiveness once cancer has become invasive or metastatic.Metastasis is responsible for the greatest number of cancer deaths,almost a third of CRC patients have metastatic disease at diagnosis,half of the patients diagnosed and resected with early-stage disease subsequently develop metastasis.Thus,metastasis is one of the most important challenges in the treatment of CRC and accordingly,targeting metastasis has been considered a promising strategy for improving treatment for CRC.The invasion and metastasis of tumor cells requires not only the extracellular protease to degrade the surrounding tissues,but also the ability to migrate.Many aspects of these processes are linked to the dynamic movement of the cytoskeleton network.Therefore,the dynamic assembly of cytoskeleton proteins and the changes of adhesion with ECM play a decisive role in the motility of the cell.In this study,we investigated the effects of CTD on HCT116 cells,including cell cycle and apoptosis,the alterations in the cytoskeleton,cell adhesion,and migration to investigate whether they contribute to the anti-tumor effect on CTD.Objective:In this study,the effect of CTD on HCT116 tumor cells was studied by observing the morphological changes of the cell microfilament skeleton,the effects of CTD on cell adhesion and migration even of the mechanism of CTD inhibition of cell proliferation was investigated.Through the construction of RhoA overexpression vector,the relationship between the antitumor effect of CTD and the Rho GTPase is elucidated,which provides a theoretical basis and target for the development of a new antitumor drug.Methods:(1)Using the IncuCyte ZOOM system to analyze the proliferation effect of CTD inhibiting HCT116 cells.At the same time,the cloning experiment was applied to further verify the proliferation effect.(2)The application of flow cytometry instrument(BD II FACS Canto TM)to detect the HCT116 cell cycle and apoptosis after CTD processing.(3)Use adhesive detection kit to evaluate the effect of CTD on adhesion of HCT116 cells.(4)Use the FITC-phalloidin immunofluorescence staining method to evaluate the effect of CTD on F-actin in the cytoskeleton of HCT116 cells in a confocal microscope.(5)Use live cell workstation to observe the effect of CTD on the healing of HCT116 cells in real time and analyze its specific values.(6)The effect of RhoA on cell growth and cytoskeleton was observed by constructing RhoA overexpression vector and transfection of HCT116 cells.(7)CTD was applyed to HCT116 cells in wild type and overexpress RhoA,and the expression level of RhoA was detected by Western Blot,the inhibitory effect of CTD on RhoA expression was verified.(8)By using immunofluorescence staining,the expression of integrin and Zo-1 was detected after the action of CTD,the effect of CTD on cell attachment and adhesion was elucidated.(9)Use ROCK inhibitor Y27632 to act on HCT116 cells,observe the morphological changes of cells and explore the downstream target molecules of CTD.Results:(1)The inhibitory effect of CTD on cell proliferation:compared with the control group,the CTD treatment group proliferation was significantly reduced(**P<0.01).In addition,CTD can cause the change of cell shape,and the fusiform of the original adhesion state gradually becomes a circular shape of floating state.It is shown that CTD can not only inhibit cell proliferation,but also change the cell shape and adhesion to the base surface.(2)The effect of CTD on cell cycle and apoptosis:Annexin V/PI was used to detect cell apoptosis,the apoptosis could be detected at 24h,but can not be detected at 48h.The results of PI staining showed that CTD significantly retarded cell cycle in G2/M period(**P<0.01).Therefore,cell cycle arrest is a major factor in the growth inhibition induced by CTD compared with apoptosis.(3)The effect of CTD on the adhesion of cells:in real time,the HCT116 cells in the Incucyte system gradually became round and separated from the base of the medium.The ELISA test also found that the adhesion of HCT116 cells with collagen package is weakened(**P<0.01).In addition,after remove CTD,the cells gradually restored and base layer adhesion ability.This phenomenon fully demonstrates the significant deadhesion effect of CTD on HCT116 cells.(4)The effect of CTD on cell microfilament polymerization:immunofluorescence experiment results showed that a clear microfilament network structure was observed in the cytoplasm of the control group of HCT116 cells,and radiated from the periphery of the nucleus to the cell membrane.2.5μM CTD treatment caused cytoplasm decreased;with increased concentration of CTD,microfilament cytoskeleton depolymerized-refactoring phenomenon is significant,the aggregation state of microfilament significantly reduced,explain CTD caused HCT116 cell microfilament skeleton reconstruction significantly.(5)The effect of CTD on cell migration ability:scratch width after healing Incucyte system show that compared with the control group,CTD treatment group reduced tumor cell density,at the same time reduced the migration ability of tumor cells(**P<0.01).(6)CTD had effect on HCT116 with RhoA overexpression cells:FITC-phalloidin staining revealed that HCT116 RhoA overexpression cell pseudomonas increased,and the stress fibers increased significantly.CTD can reverse cell morphological changes after the action,indicating the success of RhoA construction,while CTD inhibits the expression of RhoA.(7)The effect of CTD on the expression of RhoA:Western Blot was used to detect the change of RhoA protein content in HCT116 of wild type and overexpressed RhoA,and it was proved that CTD could significantly inhibit the expression of RhoA protein(**P<0.01).(8)The effect of CTD on intercellular connection and adhesion:CTD significantly reduced the expression level of integrin in the HCT116 wild type and overexpressed RhoA cells,but had no effect on the expression of ZO-1,indicating that CTD only reduced adhesion to basal surface,and did not affect intercellular connections.(9)The effect of ROCK inhibitor Y27632 on cell morphology:with the increase of Y27632 concentration,the cell deformation and the antennae increase,unlike the morphology of the cells gradually turns round after the action of CTD,indicating that CTD may not play a role through the RhoA/ROCK pathway.Conclusion:(1)CTD can induce the adhesion of cells and basement by affecting the cytoskeleton structure of cells,and significantly inhibit the proliferation and migration of HCT116 cells by blocking the cell cycle in G2/M phase.(2)CTD can play a potential role in preventing and controlling tumor recurrence and metastasis by inhibiting the expression of RhoA,and playing a role in remodeling and inhibiting tumor cell invasion and metastasis.These results indicate that CTD can be used as a specific inhibitor of RhoA to reduce the invasion and metastasis ability of tumor cells,and provide experimental basis for future research and clinical application. |
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