Anti-invasion And Anti-metastatic Effect Of RpNK-lysin By Inhibiting Fascin1 And Matrix Metalloproteinases In Hepatocellular Carcinoma Cell Lines

Objectives:The cancer therapeutics having ability to target cancer cells specifically and exhibiting nominal cytopathic effect on normal healthy cells are highly significant for cancer therapeutic applications.Recombinant porcine natural killer lysin(rpNK-lysin)is a peptide which displays potent cationic anti-bacterial and anti-tumor activity as indicated in literature.However,its potency and molecular mechanism of actions are not fully understood.Herein,we report anti-invasion and anti-metastasis effects of rpNK-lysin on hepatocellular carcinoma cells in vitro.Furthermore,in this study we investigated the hidden molecular mechanism involved in regulating Fascin1 and Wnt/?-catenin signaling pathway using three hepatocellular carcinoma cell lines.Methods:We first investigate the maximum non-toxic concentration(MNTC)of rpNK-lysin for the normal hepato cells(L-02)through MTT assay.Using MNTC concentration along with 2 fold dilution,control group(medium only)and PBS group(medium+PBS),we further explore anti-proliferative,anti-adhesive,anti-invasive and anti-metastatic effect of rpNK-lysin on three different hepatocellular carcinoma(HCC)cell lines(SMMC-7721,97-H and HepG2)through MTT,wound-healing,adhesion and invasion assays.The role of rpNK-lysin on MAPK pathway(ERK1/2,SRK2 and CREB1)in regulating Fascin 1 and its co-relation with Wnt/?-catenin signaling pathway(?-catenin,MMP2 and MMP9)was investigated by real-time PCR and Western blot.The enzymatic gelatin degradation ability of gelatinase A(MMP2)and gelatinase B(MMP9)treated with rpNK-lysin was also determined using the gelatin degradation assay(alternative to gelatin zymography).Results:1.The maximum non-toxic concentration(MNTC)of rpNK-lysin is the concentration which shows less than 20%cytotoxicity on normal hepato cells,which is calculated to be 90.8 ?g/mL.In the follow up study we focused on this MNTC value along with its 2 fold dilutions(45.4 ?g/mL and 22.7?g/mL)to check its effect on the HCC cell line.2.Compared with the control groups,morphological study shows that rpNK-lysin treated cells become rounded,and had obvious inter-cellular spaces,disintegration,and necrosis,which were both concentration and time dependent manner.3.The results of wound healing assay shows that compared with the untreated control groups(medium and PBS),the MNTC treated cells has significantly(P<0.001)inhibited the movements of the HCC cells both in time and dose dependent manner.Compared with the treated groups,gaps of the control groups are almost filled post 72 h.4.The migration ability of the cells in the MNTC rpNK-lysin treated group was significantly(P<0.001)decreased and in PBS control group(P>0.05)remained as same compared to the medium control group.5.Cell adhesion ability of rpNK-Lysin treated groups was compared with the adhesion ability of the control cells.The results shows that the adhesion ability of cells in different ability of the control groups,cell adhesion ability of treated groups especially of the MNTC groups were significantly(P<0.001)weakened.6.The results of cell invasion experiments showed that the invasive ability of different concentration of rpNK-Lysin treated cells was significant(P<0.001)decreased in dose dependent manner compared with the PBS group(P>0.05)7.The expression of Fascin1 along with the Wnt/?-catenin pathway genes(?-catenin,MMP2 and MMP9)were significantly down-regulated in dose dependent manner compared with the PBS group.The MNTC rpNK-lysin has strong inhibiting effect(P<0.001)on the expression of mRNA level as compared with that of the control group(P>0.05)8.The protein expression level of Fascin1,?-catenin,MMP2 and MMP9 of the rpNK-lysin treated groups compared with the control group was significantly down-regulated in dose dependent manner9.The gelatin degradation assay shows that rpNK-lysin has significantly reduced the enzymatic gelatin degradation activity of MMP2 and MMP910.The expression of MAPK pathway genes(ERK1 and 2,RSK2 and CREB1)were investigated and the results showed that this pathway is involved in the activation of Fascin1 which further regulate Wnt/?-catenin pathway.Moreover,rpNK-lysin has significantly down-regulated the MAPK pathway and Fascin1 compared with the untreated control groups11.The protein expression level showed that ERK1/2,its substrate RSK2 and transcriptional factor CREB1 were successfully activated by phosphorylation which causes over-expression of Fascin1 rpNK-lysin successfully suppressed the phosphorylation which in turn suppressed the expression of Fascin1.Conclusion:The investigated MNTC value of rpNK-Lysin(90.8 ?g/mL)has a significant inhibitory effect on the proliferation,adhesion,invasion and metastasis of HCC cell line(SMMC-7721,MHCC 97H and HepG2).This study demonstrated that Fascinlis activated by MAPK pathway in which ERK1/2 starts a series of phosphorylation cascade of RSK2 and its downstream transcription factor CREB1,which ultimately activate Fascin 1,resulted in filopodia formation.Fascin1 activate ?-catenin which regulates metastatic genes MMP2 and MMP9,responsible for ECM degradation to provide a path for filopodia and HCC invasion and metastasis.Furthermore,MNTC rpNK-lysin inhibited the expression of all these genes which results in the suppression of filopodia formation and ECM degradation needed for invasion and metastasis of HCC cells.This is an enormous revelation,which suggests that rpNK-lysin may be a candidate compound for HCC treatment,at the same time,insisting in the development of rpNK-lysin as anti-cancer drug.The findings from this study can inform the development of anticancer drugs in other metastatic cancers.