| Protein tyrosine phosatases(PTPs),widely expressed in human tissues,regulate the balance of tyrosine protein phosphorylation level together with protein tyrosine kinases(PTK).Recently,numerous studies have shown that the abnormal expression of some PTPs is related to the development or metastasis of cancer in human Hence,these PTPs have become new targets for anticancer drug research.PRL-3(liver regeneration phosphatase),a member of the PTPs family,has attracted attention because of its high expression in colon cancer cells.PRL-3inhibitors are expected to be potential anti-colorectal cancer drug candidates.At present,the researches about PRL-3 inhibitors are gradually increasing.Most inhibitors are focus on organic compounds.However,few metal complexes inhibitors are reported.Considering the biological activity of metal ions and the importance of metal complexes in antitumor therapy,the inhibition of zinc complex against PRL-3 will be investigated in this paper.Main research contents are as follows:1.Six zinc complexes C1-C6,which contain four zinc complexes with terpyridyl/pyrazine derivatives ligands and two zinc complexes with2-(2-pyridinyl)-benzimidazole-5-carboxylic acid ligand,were synthesized.Their structures were characterized using elemental analysis,IR spectrometry,electrospray mass spectrometry.The results showed that these compounds were mononuclear zinc complexes with tridentate or didentate ligands.Moreover,the solution stability of the complex in DMSO and Tris-HCl buffer was studied using the UV-vis spectrometry.The results show that the complex can be stable in DMSO and buffer solution within the required time.2.PRL-3 was expressed and purified.The inhibition of PRL-3activity assay was explored through changing the concentration of the substrate,the concentration of the enzyme,the time of the interactionbetween the inhibitor and the enzyme,and incubation temperature.The best condition of the assay was chose.Then,ten zinc complexes,including six zinc complexes mentioned above and four zinc complexes with triazol Schiff base derivative ligands reported by my groups,were chose and their inhibitions against PRL-3 were determined.The results show that the zinc complexes can inhibit activity of PRL-3 with IC50 value range of 1.08 ~ 103 μM.The zinc complexes exhibit different inhibitory ability on PRL-3.Of them,three complexes C1,C3 and C4 can more efficient inhibit PRL-3 activity with lower IC50 values.To evaluate the selectivity of the three zinc complexes inhibiting PRL-3,their inhibitions against PTP1 B and TCPTP were determined.We found that complex C1 can efficiently and selectively inhibit PRL-3 activity.3.Because PRL-3 excessively expressed in colorectal cancer cells,SW480 cells belonging to colorectal cancer cells were chose and antitumor activities of the zinc complexes were studied.Cell morphology and MTT assays of SW480 cells in the absence and presence of zinc complexes were carried out.In the same experimental condition,the anti-proliferative effects of the zinc complexes on Hep G2 and Hela cells were performed.The results show that three zinc complexes can inhibit the proliferation of SW480,Hep G2 and Hela cells with the dependence of concentration and time.Complex C1 is the most efficient and selective anti-proliferative inhibitor on SW480 cells.The result is consistent with that of the inhibition of C1 against the recombinant PRL-3.In addition,flow cytometry with Annexin V/PI double staining was used to investigate the apoptosis of SW480 cells treated with different concentrations of C1 for 48 h.The results showed that it is not obvious for C1 to induce the apoptosis of SW480.4.The effects of different concentrations of zinc complexes C1 and C4 on the phosphorylation levels of PRL-3 and various substrates in cells were studied by western blotting.It was found that the complex C1 significantly reduced the expression of PRL-3 and increased thephosphorylation level of its specific binding substrate Thr353.The effect of C1 on the expression of PRL-3 and the phosphorylation level of its specific substrate is the most obvious when the concentration of C1 is10μM.We speculate that the higher concentration of the complex may begin to affect the activity or expression of other proteins in the cell being related to PRL-3,thus affecting the expression and activity of PRL-3.Complexes C4 can also reduce the expression of PRL-3 and increase phosphorylation levels of Thr353,but its effect is lower than C1.The results of this study show that the zinc complex C1 can effectively inhibit the expression and activity of PRL-3 in SW480 cells,thereby inhibiting the proliferation of SW480 cells,and has the potential to become a clinical anti-colon cancer drug. |
