The Anti-tumor And Anti-angiogenesis Activity Of Recombinant Human Disintegrin Domain Of ADAM15

ADAMs (A Disintegrin And Metalloproteinase), a family of transmembrane glycopro-teins that contain seven main functional domains, are expressed in many tissues and organsand have been implicated in a variety of physiological and pathological processes, such asmediating cell-cell signal transduction and shedding the growth factors. Over40ADAMshave been identified in mammalian and plant cells. ADAM15is the unique in the ADAMswhich has an RGD motif in the disintegrin domain. The recombinant human disintegrin do-main of ADAM15(rhddADAM15) was found to have the anti-tumor activity, but the struc-ture and mechanism of rhddADAM15is still unclear. In this study, the structure ofrhddADAM15was identified and the fusion protein HSA-rhddADAM15-His was expressedin Pichia pastoris yeast. The activity of rhddADAM15expressed by Escherichia coli (E. coli)on different tumor cell lines was determined in vitro and in zebrafish. The anti-proliferationmechanism of rhddADAM15was investigated. Further more, the anti-angiogenesis activity ofrhddADAM15in zebrafish was determined. The main results were described as follows:(1) Homology modeling simulations indicated that the disintegrin domain of ADAM15was loose and90%of the secondary structure of rhddADAM15was random coil analyzed bycircular dichroism. So disulfide cross-linking is the major factor to maintain its stable con-formation. Non-reducing SDS-PAGE and liquid chromatography mass spectrometry provedthat pured rhddADAM15expressed by E.coli was mixed product. There were three mainpeaks which had different retention time and similar molecular weight when the puredrhddADAM15was analyzed by HPLC, indicating that there were three different configura-tions in the product. Peak1and2formed six of disulfide bonds and peak3formed sevenpairs. The cause of the molecular weight of rhddADAM15was bigger than the theoreticalmolecular weight was the reaction of the separated mercapto with glutathione. After refoldedin vitro, rhddADAM15formed a single configuration as peak3in buffer8and9(the concen-tration of Guanidine hydrochloride, L-arginine, EDTA, GSH, GSSG was1.4,0.4-0.8,0.001,0.002,0.0002-0.0004mol L-1respective) and had a3times higher activity in inhibition ofproliferation and migration of MCF-7.(2) rhddADAM15is one of the seven main domains of ADAM15. The mixed productexpressed by E.coli indicated that the correct folding of rhddADAM15required co-expressedprotein. In this study, human serum albumin was used as the auxiliary folded molecule andHSA-rhddADAM15-His was expressed by Pichia pastoris yeast as soluble recombinant pro-tein in culture supernatant. The protein was purified with blue-sepharose, Ni2+sepharose andDEAE Sephadex, and the high purity protein was obtained. The fusion protein exhibited anti-genicity of disintegrin domain of ADAM15. HSA-rhddADAM15-His could inhibit the migra-tion of B16cells strongly and had a weak activity in inhibition of proliferation of B16cells. The activity of the fusion protein was2-3times higher than rhddADAM15expressed byE.coli in inhibiting migration of B16.(3) In vitro, rhddADAM15expressed by E. coli inhibited proliferation and migration ofcells of breast cancer, liver cancer, melanoma, pancreatic cancer, stomach cancer, cervicalcancer and the IC50were1.0-6.0μmol L-1. The liver cancer cells Bel-7402with an IC50of1.04μmol L-1was the most sensitive and the pancreatic cancer cells8988was the least sensi-tive with an IC505.77μmol L-1. In vivo, rhddADAM15(1pmol per fish) also inhibited thegrowth and metastasis of Bel-7402cell xenografts in zebrafish and a lower concentration (0.1pmol per fish) induced severe apoptosis in the somatic cells of zebrafish.(4) rhddADAM15inhibited the proliferation, migration and tube formation of the endo-thelial cells Eahy926.(10.96±1.40)%G0/G1arrest and (55.85±1.06)%apoptotic cells wasobserved when treated with rhddADAM15. rhddADAM15also inhibited the tube formationof pancreatic cancer cells8988. rhddADAM15significantly inhibited the angiogenesis of ze-brafish. With a maximum non-lethal dose of200ng per fish, rhddADAM15inhibited (72±1.26)%angiogenesis of subintestinal vessel and with a maximum non-lethal dose of50ng perfish,(48±2.92）%angiogenesis of intersegmental vessels.(5) Bel-7402cells had apoptotic amorphological nucleus changes when treated withrhddADAM15. The activity of caspases8,9and3in Bel-7402cells was all increased, indi-cating that the apoptosis induced by rhddADAM15was not aroused by a single pathway.Moreover, partial G2/S arrest was observed on Bel-7402cells. The level of CDC2wasdown-regulated and the phosphorylation of CDC-Tyr15was increased; the level of the totalSrc was not changed, but the activity was down-regulated, indicating that the inhibition ofproliferation and migration was involved in Src-associated signal pathways.