The Mechanism That Nidus Vespae Protein [NVP(1)] Inhibis Proliferation Of HepG2 Hepatoma Cells

Wasp is a kind of insect with black body,yellow stria and binate speckle of the order Hymenoptera,family Vespidae and genus Polistes.Wasp is widely distributed in most of the mountain of China whose nest,Nidus Vespae contains a lot of anti-cancer component. It is significant to study Nidus Vespae protein in order to find a new nature anti-proliferation drug.However Nidus Vespae protein is difficult to be isolated and purified into single protein.Very few studies at present have been reported on this aspect because of its complicated compositions.A new protein was isolated from Nidus Vespae, which has the activity of inhibiting the proliferation of HepG2 hepatomcells.The mechanisms of its action were studied in the present study and the main results are as follows:1.A new protein named NVP(1) was isolated from Nidus Vespae after lixiviated in 4℃for 48 h,precipitated by ammonium sulfate,dialyzed by filtration membrane of 0.22μm,separated by Sephdex-G50 gel and reversed-phase HPLC method.2.The aim of the present study was to elucidate whether and how NVP(1) modulates the proliferation of HepG2 cells.NVP(1) at a concentration of 6.6μg/ml could arrest the cell cycle at stage G₁ and inhibit the mRNA expression of cyclin B,cyclin D1 and cyclin E.NVP(1) suppressed cdk2 protein expression,while increased p27 and p21 protein expression.On the other hand,NVP(1) did not alter p16 protein expression level.NVP(1) promoted apoptosis in HepG2 cells which was indicated by nuclear chromatin condensation.In addition,the ERK kinase signaling pathway was activated.Moreover,the p-ERK protein expression level was attenuated when the HepG2 cells were pretreated with ERK inhibitor PD98059.These results demonstrated that NVP(1) inhibits proliferation of HepG2 through ERK signaling pathway.NVP(1) could be a potential drug for liver cancer. 3.We investigated the effect of rSK3 and rIK anti-NVP(1) in inhibiting proliferation of Hek-293.NVP(1) could inhibit proliferation of Hek-293 cell.At the same time,the effect of rSK3 and rIK anti-NVP(1) on Hek-293 cells proliferation was investigated by cell count assay.Transfection and Southern-blot were used to detect the rSK3 and rIK gene.rSK3 and rIK gene could prompt proliferation and inhibit the effect of NVP(1). rSK3 and rIK channel could prompt proliferation of Hek-293.SK may be a novel target on cancer therapy.4.We found that NVP(1) could inhibit proliferation of bronchial smooth muscle cell. At the same time,we investigated the mechanism of NVP(1) in inhibiting proliferation of bronchial smooth muscle cell.The effect of NVP(1) on bronchial smooth muscle cells proliferation was assyed by primary cell culture and MTT method.The cell cycle was detected by flow cytometry.Western-blotting was used to examine p21,cdk2 protein expression.NVP(1)(6.4×10^-3 g/L) could arrest bronchial smooth muscle cell in cell cycle G1 and increase the expression level of p21.On the contrary,NVP(1) could inhibit proliferation of bronchial smooth muscle cell by down-regulating cdk2 protein expression. NVP(1) inhibits proliferation of bronchial smooth muscle cells and induces G1 cell cycle arrest.