| Tumor angiogenesis play an essential role in tumor cell growth, infiltration, andmetastasis. Since Folkman first raised the tumor angiogenesis theory in1971, it has beenwidely accepted the concept that tumor blood vessels are generated through angiogenesis,referring to that tumor generates its blood vessels through the migration of normal vascularendothelial cells into tumor tissue and formation of new vascular branches. Accordingly,many anti-angiogenic drugs have been developed. So far, five anti-angiogenic drugs havebeen applied to the clinics to treat malignant tumors. Among these, Avastin, a recombinantmonoclonal antibody against VEGF, was the first anti-angiogenic drug approved by theFDA. Current anti-angiogenic therapeutics gain promising in treatment of malignanttumors; however, recent studies indicated that the benefit of the therapy is difficult tosustain. Once the withdrawal of the drugs, tumors will easily recur and metastases. Inanti-tumor angiogenic drug research, we need to open mind to develop specific andeffective anti-angiogenic drugs. Recent studies have shown that not all the malignant tumorvessels are formed by vascular endothelial cells. In1999, Mary JC Hendrix and hiscolleagues revealed tumor cells directly formed blood vessels, named tumor vasculogenicmimicry, a distinct vasculogenic model completely different from the old one. It has beenpaid attention and accepted by more and more people.In this study, we applied the model of tumor cell-dominant vasculogenic mimicry onMatrigel and tested the anti-tumor vasculogenic effect of4monoclonal antibodies made byour laboratory. The results shows that the monoclonal antibody SZ117, which is initiallydesigned to target the fibronectin like domain of the matrix metalloproteinase-2(MMP2),could effectively inhibit vasculogenic mimicry in vitro by tumor endothelial cells3B11andhuman sarcoma cells MG63cells. Gelatin zymography showed that SZ117could inhibitMMP2activity, but not affect MMP9activity, indicating that SZ117executed itsanti-tumor vasculogenic effect through inhibiting the activity of MMP2. To our surprise, however, we could not detect the activity of MMP2in3B11by thegelatin zymography method, and MMP2protein could not be detected by Western blot,suggesting that the inhibitory effect of SZ117on3B11mediated vasculogenic mimicry invitro may not be due to the inhibition of MMP2. According to these data, we hypothesesthat SZ117may execute its anti-tumor vasculogenic effect through targeting othermolecular targets which play a role in tumor cell-mediated vasculogenic mimicry. Next, weused Western bolt, Immunoprecipitation and Mass spectrometry to identify the othermolecular targets, rather than MMP2, which could be recognized by SZ117. Ourresearches confirmed that SZ117could recognize a280kDa protein in many tumor celllines, the protein was separated by immunoprecipitation, purified by SDS-PAGE, andanalyzed by Mass spectrometry, and identified as filamin A. Cross-reaction experimentconfirmed it.Filamin A is a cytoskeleton protein which maintains the cytotostation throughinteraction with many proteins; it directly or indirectly participates in tumor cellproliferation, adhere, migration, invasion, signal transduction. Western blot result showedthat Filamin A was not only expressed in many tumor cells, but also existed in the serumfree supernatant of tumor cell culture. In serum free supernatant, SZ117could detect280kDa and53kDa protein bands which were confirmed to be filamin A and its fragmentusing the standard filamin A antibody. In addition, we also detected the filamin A and itsfragment in Matrigel (rich in matrix proteins); these data indicated that many tumor cellscould express filamin A and secrete filamin A and its fragment into extracellular matrix.We speculate that secreted filamin A in extracellular matrix may contribute toanti-angiogenesis, and SZ117could specially bind to filamin A and its fragment and inhibitthe vasculogenic mimicry in vitro.In addition,, the280kDa filamin A protein was detected by Western blot using SZ117in the peripheral blood samples and bone marrow samples of some leukemia patents, Bycontrast, but SZ117recognized the280kDa protein of filamin A could not detected bySZ117in all the volunteers., suggesting that SZ117could specially recognize tumorfilamin AAccording to the alignment between MMP2and normal filamin A, there are39animoacid shared by both MMP2fibronectin-like domain and filamin A11~14repeats(1300-1612), although we did not identify the epitope recognized by SZ117, that may the basement of why SZ117could recognize the protein of filamin A. In11~14repeats(1300-1612) of filamin A, we did not find any mutation in tumor cell line in this area atgene level.Considered that SZ117recognized filamin A protein may mutant at the level ofprotein translating or post translating, we performed the gelatin zymography experiment todetect the specific filamin A's function. The results show that the280kDa band coulddegrade gelatin in tumor cell lines. From this, it seems that280kDa band recognized bySZ117may have specific, and it may contributed to infiltration of tumor cell lines.In conclusion, SZ117inhibited tumor vasculogenic mimicry in vitro throughinhibiting the activity of MMP2; additionally, it also recognizes filamin A, suggesting thatSZ117has anti-tumor vasculogenic potential. This study revealed the role and mechanismof anti-tumor vasculogenic mimicry of monoclonal antibody SZ117and provided the newtool for studying the molecular mechanism of tumor neovascularization and set up a solidfoundation for novel anti-cancer drug discovery.
|
PDF Full Text Request