Selective Inhibition Of Protein Tyrosine Phosphatase 1B And The Cellular Effects By Vanadium And Zinc Complexes

Protein tyrosine phosphatases(PTPs)are a large family of signaling enzymes which play important role in signal transduction and regulation of cellular processes.They remove phosphate moieties on tyrosine residues of the target proteins and widely affect cell process,which contains cell proliferation,differentiation,migration,apoptosis and immune process.Protein tyrosine phosphatase 1B(PTP1B),the first identified and characterized PTP,acts as a key negative regulator of insulin signal transduction by dephosphorylating the insulin receptor(IR)and insulin receptor substrate-1(IRS-1)in the regulatory domain.Several experiments have demonstrated that PTPIB-deficient mice exhibit the phenotypes of increased insulin sensitivity and grow normally.PTP1B is thus emerged as potential target for treatment of type 2 diabetes.Vanadium mimics the effect of insulin.Large number of studies demonstrated that vanadium complexes have insulin enhancing effect and can improve the symptoms of diabetes.The insulin mimetic effect of vanadium is partly related to its ability to inhibit of PTP IB.We investigated the selective inhibition of PTP IB and the effects on viability and apoptosis over cells by vanadium complexes 1 and 3,based on the results that vanadium complexes 1 and 3 could effectively and selectively inhibit recombinant PTP1B in vitro.Then we investigated cellular uptake of vanadium complexes by ICP-MS.Furthermore we initially investigated the activity of proteasome in MCF7 cells by vanadium complexes.Zinc is the second abundant trace element ranks only second to Iron.Zinc has insulin-like activity,so zinc complexes may also be effective PTP1B inhibitors.Selective inhibition of PTP1B by five new zinc complexes with multi-benzimidazole derivatives is reported in this paper.Main results were obtained as follows:1.Western blot detected the phosphorylation levels of PTP1B substrates indicated that vanadium complexes efficiently inhibited the activity of PTP1B in MCF7 and HepG2 cells,which is consistent with our previous study results.It is worth mentioned that 1 exhibited consistent selectivity in cells as for in vitro results,whereas 3 not.2.The viability of MCF7 and HepG2 cells was detected by MTT staining.The cell viability of both MCF7 and HepG2 decreased with time and concentration dependent models.1 exhibits evidentlyless cytotoxicity toward MCF7 and HepG2 cells compared with 3.When MCF7 cells were treated by 1 for 72 hours,the cell viability reached as high as 86%.The results indicated that the structures of vanadium complexes influence cell viability on different cell lines.Flow cytometry and Annexin V-PI double staining were used to detect the ability of MCF7 cells apoptosis induced by 1 and 3.The results showed that vanadium complexes 1 and 3 induced apoptosis with concentration dependent model,indicating that the vanadium complexes increased cytotoxicity over cells by apoptotic induction.Complexes 1 and 3 had different effects on cellular phosphorylation,cell viability and apoptosis on different cell lines,which may due to the different cellular permeability of the two complexes.We investigated the uptake of the vanadium complexes in MCF7 and HepG2 cells through ICP-MS.The results indicated that complexes 1 and 3 can enter cells,with sufficient permeability.The uptake of 1 is better than 3 in both the cell lines due to the small spatial structure.In addition,inorganic vanadium salts VOSO4 efficiently inhibited PTP1B in cells,but it is difficult to penetrate the cell membrane and result in poor bioavailability.3.Studies on apoptosis by flow cytometry showed 1 and 3 can induce apoptosis of MCF7 cells.It was known that PTPs widely affect cell proliferation and apoptosis process.So we presumed that the apoptosis of cells induction by vanadium complexes may due to the inhibition of PTPs.It was previously reported that metal complexes inhibit proteasome activity,resulting in apoptosis induction in cultured cells.The specific fluorogenic peptide substrate Suc-LLVY-AMC was used to monitor the chymotrypsin like activity of MCF7 treated by complexes 1 and 3,further to explore the way vanadium complexes induce apoptosis by.The results indicated that complexes 1 and 3 increased chymotrypsin-like activity.So apoptosis induction is not due to the inhibition of proteasome activity in cells but inhibition of PTPs by vanadium complexes,probably.However,the extracellular chymotrypsin-like activity was not obviously changed when cellular extract was treated directly with complexes 1 and 3 These results suggest that the promotion of intracellular proteasome activity by complexes 1 and 3 is not due to the direct activation of complexes 1 and 3 to chymotrypsin-like activity,maybe result from inhibition of PTPs,which influence cellular phosphorylation and the expression of proteasome.In short,with selective inhibition of PTP1B,low cytotoxicity and sufficient cell-permeable characteristics,complex 1 is regarded as a promising metal based antidiabetic drug candidate.4.Five zinc(?)complexes 6-10 with multi-benzimidazole derivatives had been synthesized and characterized.The five zinc(?)complexes potently inhibited PTP1B,but showed weaker inhibition against PTP-MEG2 and SHP-1 and almost no inhibition against SHP-2.It was interesting that complexes 7 and 9 exhibited weaker inhibition than 6,8 and 10 against TCPTP which is highly homologous with PTP1B.Therefore,7 and 9 displayed obvious selectivity against PTP1B.Kinetic analysis indicated that complexes 7 and 9 were reversible competitive inhibitors of PTP1B but nocompetive inhibitors for TCPTP.Fluorescence study on the interaction between complex 7 or 9 and PTP1B or TCPTP suggested that 7 and 9 stronger bind to PTP1B than to TCPTP,which is in accordance with their inhibition against the two enzymes.These results suggested that further study on inhibition of PTP1B by zinc complexes may help us to filter out efficient anti-diabetic drugs with high selectivity on PTP1B and low toxicity in vivo.