Expression of soluble Flt-1 gene in Pichia pastoris and the effect of the product on multiple-myeloma cells in vitro.
VEGF (vascular endothelial growth factor), a potent angiogenic molecule specific for vascular endothelial cells, is overexpressed in most tumours including MM (multiple myeloma) and closely associated with tumour growth and prognosis. It has been shown that a soluble fragment of the VEGF receptor Flt-1 (Fms-like tyrosine kinase-1) [sFlt-1 (soluble Flt-1)] has antiangiogenic properties by way of its antagonist activity against VEGF. VEGF and its receptors have been shown to be targets for treating tumours. In the present study, sFlt-1 gene was expressed in Pichia pastoris and the product was applied for studying the effect on KM3 MM cells. sFlt-1 gene was inserted into the pPICZalphaA vector and the expressed product was analysed by SDS/PAGE, immunoblot and ELISA. The sFlt-1 protein was expressed by 0.5% (v/v) methanol induction and it accumulated up to 23% of total proteins in the supernatant. The product was further purified with metal-chelating resin [Ni-NTA (Ni(2+)-nitrilotriacetate)]. The functional analysis of the sFlt-1 protein was performed with HUVEC (human umbilical-vein endothelial cells) proliferation assay. We next showed that the sFlt-1 protein acted directly on MM cells and inhibited the VEGF-induced proliferation of MM cells with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] and (3)H uptake assay. The sFlt-1 protein blocked VEGF-induced ERK (extracellular-signal-regulated kinase) phosphorylation and inhibited the MAPK (mitogen-activated protein kinase) signalling cascades. The present study demonstrated that anti-MM activity of the sFlt-1 protein, coupled with its antiangiogenic effects, provides the basis for clinical trials of this agent to improve the outcome in MM.
Liu
Junru
J
Department of Hematology, The First Affiliated Hospital, SunYat-sen University, Guangzhou 510080, People's Republic of China.
Li
Juan
J
Su
Chang
C
Huang
Beihui
B
Luo
Shaokai
S
D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D009101:Multiple Myeloma / Q000473:pathology* / Q000503:physiopathology*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000008:administration & dosage* / Q000737:chemistry; D012995:Solubility; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics
D011994:Recombinant Proteins; C501162:FLT1 protein, human; D040281:Vascular Endothelial Growth Factor Receptor-1