Experimental Study On The Activation Of Mice NK Cells Killing Hepatic Carcinoma Cells By NDV-HN And GA Sulfonamide Derivative

Background:NDV is an oncolytic virus wihch has been shown to replicate selectively in human tumor cells,inducing tumor cells death.The selective antitumor activity of NDV make it as an oncolytic virus highly valuable in the field of tumor biotherapy.HN is the primary component of the large spike glycoprotein on the NDV envelopes,which plays a critical role in the anti-tumor effects.HN has both HA and NA activity.HA has the ability to agglutinate red blood cells,in addition to allowing the virus to host cells,in order to cause infection.In antitumor,HA binds to tumor cells,exerts antitumor effects by lysing cells.In other hands,HA can bind to immune cells,and activate immune cells,which exerts an anti-tumor immune response.The substrate of NA is the sialic acid at the end of the glycoprotein on the host cell.Its function is to remove sialic acid and expose tumor cell antigens on the host cells.HA also can be induced to attach to host cells and the subsequent membrane fusion is initiated.GA is a natural plant triphenol,previous studies have shown that these compounds have potent therapeutic properties,including antitumoral,antimicrobial and antiviral,as well as being a potent antioxidant.The sulfonamide family consists of a broad spectrum of synthetic bacteriostatic antibiotics and was commonly used in human and veterinary medicine for therapeutic and prophylactic purposes in last century due to their easy penetration through the membrane and into body fluids and tissues.Researcher on the antitumor by using NDV has been carried out by our team for more than 10 years.We found that NDV stimulation could trigger the tumoricidal activity of mouse spleen NK cells by TRAIL induction through the IFN-?pathway.In other hands,we found that the IFN-?pathway that mediated this upregulation was not completely inhibited by neutralizing IFN-?,suggesting that the existence of a IFN-?-independent signaling mechanism.To this end,we conducted a further study of NDV activation of NK cells via a"IFN-?independent pathway".In this study,NDV-HN was directly acted on NK cells,and induced NK cells to express anti-tumor molecule TRAIL.A scientific hypothesis"NDV-HN binding to NKp46activating Syk-NF-?B signalingup-regulating NK cells expressing TRAIL"was proposed.In the hypothesis,NDV-HN binds directly to NKp46 of NK cells(belonging to activated receptors on cells and is one of the surface markers unique to NK cells)and activates the Syk and nuclear NF-?B,and finally cause the up-regulation of anti-tumor molecules such as TRAIL.In addition to the secretion of TRAIL and TNF,NK cells can express GrB,Fas and FasL.The main topic of this research is NDV-HN directly triggers the upregulation of GrB,Fas and Fas L in murine NK cells after the activation of Syk and NF-?B,and the effect of activated NK cells on tumor cells.In addition,we selected compounds that have a cytotoxic effect on hepatocarcinoma cells(EJTC),to test whether there is an activation of mouse NK and its molecular events after activation.Through this study,we will further clarify the molecular mechanism of NDV-HN direct activation of NK cells.This has a positive meaning to NDV oncolytic virus combined with NK cells in the biological treatment of cancer.While NK cell killing tumor cells as a model,the anti-tumor effect of natural drugs through immunological perspective,lay the experimental foundation for the development of new drugs.Objects:1.To evaluate whether the tumoricidal activity of mouse IFN R^+/+NK cells is induced by NDV-HN.And to investigate what is the mechanism of them stimulated mouse IFN R^+/+NK cells to kill mouse hepatoma cell lines in vitro.2.To evaluate whether the tumoricidal activity of mouse IFN R^-/-NK cells is induced by NDV-HN,and whether this tumoricidal activity can be reversed by signal inhibitors.With or without signal inhibitors affected the expression of GrB and Fas/FasL in NDV-HN stimulated mouse IFN R^-/-NK cells were determined.3.To screen out the effective gallic acid sulfonamide derivatives with research value by observing the killing effect of different compounds on mouse hepatoma cells.4.To prove that tumoricidal activity of mouse IFN R^+/+NK cells is induced by EJTC and this anti-tumor activity can be produced by mechanism of immunomodulatory.Methods:1.The NK cells were stimulated with NDV and NDV-HN in vitro,CFSE/PI staining was used to detect the killing effect of mouse IFN R^+/+NK cells on mouse hepatoma cell lines by flow cytometry.ELISA was used to detect the concentration of GrB and Fas in supernatant of mouse IFN R^+/+NK cell culture medium.Western Blotting method was used to analyze the expression of GrB and FasL protein in mouse IFN R^+/+NK cells.2.The mouse IFN R^-/-NK cells were stimulated with NDV-HN,or with anti-HN Ab,Syk specific inhibitor Herbimycin A or NF-?B inhibitor PDTC.Quantify the cytotoxic activities of mouse IFN R^-/-NK cells against mouse hepatoma cells by flow cytometry.GrB and FasL concentrations in the supernatants of mouse IFN R^-/-NK cells medium were determined by specific ELISA assay.The expression of cell surface GrB and FasL was determined by Western blotting.3.The cytotoxicity of gallic acid and different gallic acid sulfonamide derivatives at various concentrations and time on mouse hepatocellular carcinoma cells was detected by CCK-8 method.4.The mouse IFN R^+/+NK cells were stimulated with gallic acid sulfonamide derivatives in vitro,CFSE/PI staining was used to detect the killing effect of mouse IFN R^+/+NK cells on mouse hepatoma cell lines by flow cytometry.5.Data are expressed as means±standard deviation of the mean of separate experiments(n?3).Student's t-test was applied for comparison of the means of2 groups,and ANOVA was used for the means of multiple groups.Values of P<0.05 were considered statistically significant.Results:1.NDV-HN stimulation enhanced tumoricidal activity of NK cells toward Hepa1-6 in vitro.NDV-HN had significant difference(P<0.05)compared with control group,1:5 and 1:10 groups(P<0.01),but NDV-HN group showed no significant difference compared with NDV positive group(P>0.05).NDV-HN induce the production of GrB and Fas/Fas L in mouse IFN R^+/+NK cells.NDV-HN significantly increased the concentration of GrB and Fas in the supernatant of mouse IFN R^+/+NK cells,P<0.01 compared with the control group,and no difference compared with the NDV positive control group(P>0.05).And significantly increased the expression of GrB,Fas and FasL protein in IFN R^+/+NK cells.Compared with the control group,P<0.05.There was no difference(P>0.05)compared with NDV positive group.2.NDV-HN stimulation enhanced tumoricidal activity of IFN R^-/-NK cells toward Hepa1-6 in vitro.NDV-HN had significant difference(P<0.05)compared with control group,1:5 and 1:10 groups(P<0.01),but NDV-HN group showed no significant difference compared with NDV positive group(P>0.05).NDV-HN significantly increased the concentration of GrB and Fas in the supernatant of mouse IFN R^-/-NK cells,and significantly increased the expression of GrB,Fas and FasL protein in IFN R^-/-NK cells.Compared with the control group,P<0.05.There was no difference(P>0.05)compared with NDV positive group of mouse IFN R^-/-NK cells.After treating with anti-HN Ab,Syk specific inhibitor Herbimycin A and NF-?B inhibitor PDTC the tumoricidal activity of NDV-HN stimulated mouse IFN R^-/-NK cells(P<0.05)was down-regulated.Moreover,significant suppressions in the production of GrB and Fas/FasL were observed in them stimulated mouse IFN R^-/-NK cells(P<0.05).3.The activity of compound of synthesized sulfonamido-based gallate was the strongest EJTC IC₅₀=0.032?M,second is HAMDL IC₅₀=0.064?M,and LDQN-C activity was poor,IC₅₀=0.14?M.The optimum stimulation time for each compound was 48 hours.Compared with the GA at 20?g/ml and 40?g/ml,the killing effect of gallic acid sulfonamide derivatives on mice hepatoma carcinoma cell was significantly improved(P<0.05).4.EJTC stimulation enhanced tumoricidal activity of mouse IFN R^+/+NK cells toward Hepa1-6 in vitro and the effect is same as NDV(P>0.05).And significantly increased the expression of GrB protein in IFN R^+/+NK cells.Compared with the control group,P<0.05.There was no difference(P>0.05)compared with NDV positive group.Conclusions:1.NDV-HN can enhance the anti-tumor activity of mouse IFN R^+/+NK cells by increasing the release of GrB and Fas from mouse IFN R^+/+NK cells and up-regulating the expression of GrB and FasL proteins in mouse IFN R^+/+NK.2.NDV-HN can enhance the anti-tumor activity of mouse IFN R^-/-NK cells by increasing the release of GrB and Fas from mouse IFN R^-/-NK cells and up-regulating the expression of GrB and Fas L proteins in mouse IFN R^-/-NK.We concluded that killer activation receptors pathway is involved in the IFN-?-independent GrB and Fas/FasL expression of NDV-HN stimulated mouse IFN R^-/-NK cells,and these are activated by Syk and NF-?B.3.The killing effect of synthesized sulfonamido-based gallate on mouse hepatoma cells is diversity due to its different structure,but the activity of each compound is improved compared with the gallic acid which is known to be active.It is suggested that the new compounds synthesized by introducing sulfonamide derivatives into gallic acid are structurally significant.EJTC has obvioued inhibitory effect on the activity of mouse hepatoma cells.4.EJTC can enhance the anti-tumor activity of IFN R^+/+NK the ability to mouse hepatoma cell lines in vitro,and up-regulating the expression of GrB proteins in mouse IFN R^+/+NK.Which indicated that it can produce anti-tumor effect through immunoregulation mechanism.