Anticancer Effect And Mechanism Of Exogenously Expressed Marine Lectin Genes UPL1/AJHl1/HdSABL

Human health is still under threat of cancer. There exist many biological differencesbetween the cancer cells and the healthy cells. One of the most important changes is alteredglycosylation of proteins in cancer cells. Lectin is a type of sugar-binding protein, which canidentify particular glycosylation patterns and regulate cell growth, differentiation, signaltransduction, cell migration, apoptosis and other biological processes. At present, lectins havebeen used in immunology, cancers, cell biology, and other fields of studies.Our group has shown that mannose-binding lectin Pinellia pedatisecta agglutinin (PPA)induced cancer cell death through interacting with protein arginine methyltransferase5(PRMT5),suggesting that the lectin superfamily may harbor anticancer genes. Marine lectins containdiversified structure and carbohydrate recognition specificities. In this paper, we select threemarine lectins, N-acetyl glucosamine lectin (UPL1) from Ulva lectuca, sialic acid lectin(HdSABL) from Haliotis discus discus and galactose lectin (AJL1) in Anguilla japonica, tostudy the cytotoxicity of lectins through gene transferring.In this work, UPL1, HdSABL and AJL1genes were inserted into a eukaryotic expressionvector pEGFP-C1. Furthermore, three recombinant adenoviruses carrying UPL1, HdSABL andAJL1genes were constructed and the anti-proliferative effects on various cancer cells and theunderlying mechanisms were investigated. Results may provide a new method to investigatelectin-based marine drug and provide three new anti-oncogenes from marine organisms.Methods:(1) Construction of eukaryotic expression vectors pEGFP-GENE-C1.(2) Analyzing the cytotoxicity of lectins on cancer cells.(3) Subcellular localization of lectins in cells by laser scanning confocal microscope.(4) Packaging recombinant adenovirus AD-FLAG, AD-FLAG-AJL1, AD-FLAG-HdSABLand AD-FLAG-UPL1using Adeasy system.(5) Detecting the killing effect of constructed virus on a variety of malignant cancer celllines by MTT. (6) Cell morphological observation.(7) Detecting apoptosis of Hep3B induced by virus by the mathod of Annexin V/PI stainingand flow cytometry analysis.(8) Investigating the intracellular signaling pathway to study the underlying mechanism ofthe lectin induced cytotoxicity.Results:(1) Eukaryotic expression vector pEGFP-GENE-C1and the constructed adenovirusesincluding lectin AJL1, HdSABL and UPL1were constructed successfully.(2) The expression of AJL1, HdSABL and UPL1in cancer cell is poisonous to cell observedby fluorescence microscope.(3) AJL1, UPL1and HdSABL distribution in cytoplasm and some organelles was observedunder confocal laser scanning microscope. All three lectins entered into the nucleus in laterperiod.(4) MTT and morphological observation experiments showed that AJL1, HdSABL andUPL1genes have significant cytotoxicity in vitro.(5) Annexin V/PI flow cytometry showed that AJL1and HdSABL could induce apoptosis inHep3B cells.(6) Western blot revealed that HdSABL could induce apoptosis by down-regulatingapoptosis inhibiting factor Bcl-2.ConclusionIn conclusion, the expression of AJL1, HdSABL and UPL1in cancer cell can lead to celldeath. HdSABL can induce apoptosis by down-regulating apoptosis inhibiting factor Bcl-2.AJL1can also induce apoptosis of Hep3B cells. AJL1, HdSABL and UPL1genes may providenew therapeutic genes for cancer gene therapy.