Preliminary Study Of Molecular Mechanisms On The Inhibitory Effect Of Orf Virus On The Growth Of Colorectal Cancer Cells

Orf virus(ORFV)is a prototype parapoxvirus that causes orf(contagious ecthyma,contagious pustular dermatitis,and scabby mouth).ORFV is also a candidate of oncolytic virus,because it has adventages of safety,re-infection in short time,immunoregulatory effect,and primary researches have reported the oncolytic effect of ORFV.Recently,oncolytic virus has been recognized as a promising new tool for tumor treatment.However,colorectal carcinoma is the common malignant tumor of digestive tract in the world,but it is short of effective therapeutic approach.In this study,we aim to the oncolytic effect of ORFV to colorectal carcinoma cell and explore the mechanism preliminarily.To explore the oncolytic effect of ORFV,this research contains three parts,including vitro experiments,animal trials and mechanism explorations.Vitro test:Primarily,we cultured several colorectal carcinoma cell lines with ORFV to observe their sensitivity to ORFV.Next,Virus replication was identified by virus mutant strain ORFV-GFP infection and the TCID50 test.Then,crystal violet staining and CCK-8 assay were performed to verify the oncolytic effect of ORFV.Finally,we performed wound healing assay to observe the effect of ORFV on migration of colorectal cancer cells.Animal trials:We preliminarily valuated safety of ORFV in Balb/c mice and test oncolytic effect by treating Balb/c mice with ORFV in the xenograft models,and explored effect of ORFV on migration of colorectal carcinoma cell in tumor metastasis models.Explorations in mechanism:Firstly,we detected the apoptosis of colorectal cancer cells infected with ORFV at 24h by FCM,and detected the expression of apoptosis-related protein post ORFV infection.Secondly,we detected the proteins about autophagy by western blot and immunofluorescence assay.The results show as follows:1)CCK-8 assay show that the survival rate of multiple colorectal cancer cell lines declined post ORFV infection,and LoVo cells are most sensitive.2)Virus replicate cruve indicated that the number of ORFV increased by times and ORFV replicated in colorectal cancer cells.3)In CCK-8 and crystal violet staining assays,the amount of tumor cells reduced after infected with ORFV.ORFV can prevent tumor cells form proliferating.4)ORFV prevent scratch healing post infection.5)ORFV do not damage vital organs of Balb/c mice and the blood-level was normal,but leukomonocyte increases after management with ORFV.6)In xenograft model,tumors of mice treat with ORFV were smaller in contrast to control group.Caspase-3 expression of tumors increased after treated with ORFV.Tumor metastasis focuses in the lung or liver in ORFV-treated group were less.ORFV inhibits colorectal cancer cell proliferation in vivo.7)In tumor metastasis model,the results show enlargement of spleens,reduction of tumor metastasis and increasing of caspase-3 expression in lung.ORFV inhibits colorectal cancer cell metasis in vivo.8)FCM results exhibited increasing of apoptosis cells after dealing with ORFV,and results of western blot show that expression of cleaved-caspase-3 and cleaved-parp increased by the times.ORFV promotes apoptosis in colorectal cancer cells.9)The results of immunofluorescent assay indicated expression of LC3 also increased post ORFV infection.The results of western blot also show the increasing of LCO Ⅱ,reduction of p62 and invariability of p-mTOR.ORFV induces autophagy in colorectal cancer cells.In this study,we found ORFV can infect and replicate in colorectal cancer cells,and ORFV prevent tumor cells from proliferating and migrating in vitro.Animal trials also verified oncolytic effect of ORFV in vivo and indicated that the host immunity system was activated by ORFV.Finally,we found ORFV induces colorectal cancer cells apoptosis and autophagy,which lay a foundation for further study of ORFV and even clinical application in the future.