Novel imidazo[1,2,4] triazole derivatives: Synthesis, fluorescence, bioactivity for SHP1.

The Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1) is a convergent node for oncogenic cell-signaling cascades. Consequently, SHP1 represents a potential target for drug development in cancer treatment. The development of efficient methods for rapidly tracing and modulating the SHP1 activity in complex biological systems is of considerable significance for advancing the integration of diagnosis and treatment of the related disease. Thus, we designed and synthesized a series of imidazo[1,2,4] triazole derivatives containing salicylic acid to explore novel scaffolds with inhibitory activities and good fluorescence properties for SHP1. The photophysical properties and inhibitory activities of these imidazo[1,2,4] triazole derivatives (5a-5y) against SHP1PTP were thoroughly studied from the theoretical simulation and experimental application aspects. The representative compound 5p exhibited remarkable fluorescence response (P: 0.002) with fluorescence quantum yield (QY) of 0.37 and inhibitory rate of 85.21 ± 5.17% against SHP1PTP at the concentration of 100 µM. Furthermore, compound 5p showed obvious aggregation caused quenching (ACQ) effect and had high selectivity for Fe3+ ions, good anti-interference and relatively low detection limit (5.55 µM). Finally, the cellular imaging test of compound 5p also exhibited good biocompatibility and certain potential biological imaging application. This study provides a potential way to develop molecules with fluorescent properties and bioactivities for SHP1.

The regulating role of mutant IkappaBalpha in expression of TIMP-2 and MMP-9 in human glioblastoma multiform.

BACKGROUND: Our previous studies demonstrated that mutant IkappaBalpha (IkappaBalphaM) inhibited the occurrence, growth and angiogenesis of human glioblastoma multiform (GBM). However, the specific mechanism by which IkappaBalphaM regulates protein-degrading enzymes secreted from GBM to inhibit invasion and metastasis has remained unclear. The aim of the present study was to investigate the regulatory role and significance of IkappaBalphaM genes in the expression of tissue inhibitor of metalloproteinase (TIMP)-2 and matrix metalloproteinase (MMP)-9 in human GBM. METHODS: We established the following four GBM cell lines stably expressing IkappaBalphaM by plasmid construction, gene transfection and screening for IkappaBalphaM protein expression: mutant IkappaBalpha-transfected cells (G36Delta-M), wild-type IkappaBalpha-transfected cells (G36Delta-W), empty plasmid transfected cells (G36Delta-P) and untransfected cells (G36Delta). The TIMP-2 and MMP-9 expression was detected by RT-PCR and Western blotting. Tumor cells were then implanted subcutaneously into nude mice to establish an animal model of ectopic tumor growth, and TIMP-2 and MMP-9 expression was determined by immunohistochemical methods. RESULTS: The results showed that there was a significant increase in TIMP-2 expression and a significant decrease in MMP-9 expression in the G36Delta-M group at both the RNA and protein levels compared with the G36Delta-W group, G36Delta-P group and G36Delta group. Similar results were observed in the immunohistochemical staining analysis of tumor tissues. In the G36Delta-M group, TIMP-2 expression was significantly higher while MMP-9 expression was significantly lower than in the other three groups. CONCLUSIONS: Our findings indicate that IkappaBalphaM inhibits the activation of NF-kappaB. It significantly up-regulates TIMP-2 expression in human malignant glioma cells and down-regulates the expression of MMP-9. Thus, IkappaBalphaM maintains the integrity of the extracellular matrix and further inhibits the growth and metastasis of tumor tissues.

[Effect of shRNA-mediated silence of H-ras gene on proliferation of human SACC-M cells].

OBJECTIVE: To examine the effects of H-ras gene silence on cell cycle, proliferation and apoptosis of salivary adenoid cystic carcinoma -M (SACC-M) cell lines. METHODS: The plasmid H-ras-shRNA, containing the sequence of shRNA targeting H-ras, and HK-shRNA (without interfering effect) were constructed and transfected into SACC-M cells. The cell line with shRNA plasmid stable expression was isolated by G418. The expression levels of H-ras were detected by RT-PCR and protein immunofluorescent assay; cell cycle and cell apoptosis were analyzed by flow cytometry (FCM). The proliferation of cell was also determined by subcutaneous tumor formation in nude mice. RESULTS: After transfection of H-ras-shRNA plasmid, the mRNA expression of H-ras in SACC-M cells was down-regulated by 61.80% and protein expression of H-ras was inhibited by 62.76%; the cell proliferation was inhibited obviously; the G0G1 phase cells were increased. The cell apoptosis rate of H-ras-shRNA group was significantly higher than that of HK-shRNA group (P <0.05). The volume of subcutaneous tumor in nude mice was significantly smaller in Hras-shRNA group than in control group. CONCLUSIONS: The recombinant plasmid HRAS-shRNA could efficiently down-regulate the expression of H-ras gene and protein, induce apoptosis of SACC-M cells and simultaneously inhibit proliferation of these cells in vitro and in vivo.

[Construction of eukaryotic expression vector of short hairpin RNA targeting human xylosyltransferase-I gene].

OBJECTIVE: To design and construct the plasmids expressing short hairpin RNA (shRNA) targeting human xylosyltransferase- I (XT- I) which is the initiating enzyme in the biosynthesis of proteoglycans (PC). METHODS: Short chain oligonucleotides were designed according to the sequence of XT-I provided by GenBank. The DNA segments were gained through annealing after chemosynthesis, and were cloned into Pgenesil-1 vector. The recombinant XT- I shRNA expression vectors were identified by digestion and sequencing analysis. At last the constructed XT-I expression vectors were transfected into salivary adenoid cystic carcinoma cell line (ACC-M) by Lipofectomine 2000. The expression of green fluorescent protein (GFP) was detected by inverted fluorescent microscope and the rates of transfection were detected by flow cytometer. Semiquantitative RT-PCR was used to detect the expression of mRNA level of XT- I in transfected ACC-M cells and the protein expression of XT- I was detected by Western blot. RESULTS: The plasmids expressing shRNA targeting XT-I gene are called WJ1, WJ2, WJ3, WJ4, WJ5 and WJ6. Successful constructions were identified by digestion and sequencing. The mean rate of transfection was 50.26%. ACC-M cells transfected with WJ1-WJ6 expressed GFP successfully. And by RT-PCR and Western blot, WJ3 showed the most powerful RNAi gene silencing of inhibitory. The inhibition rate was 72.39% of mRNA level and 70.18% of protein level respectively. CONCLUSION: The XT-I shRNA expression vectors were constructed successfully which lays the foundation for RNAi study on the biosynthesis of PG in salivary gland tumors.

[Preparation and identification of anti-human polyclonal antibody against xylosyltransferase-I].

AIM: To prepare the rabbit polyclonal antibody against human xylosyltransferase-I (XT-I) protein and to identify its specificity. METHODS: The predominant epitope of XT-I gene was predicted by the DNAssist software. The DNA fragment of this epitope region was synthesized by PCR and cloned into the pGEX-4T-2 vector. The recombinant plasmid was transformed into E.coli ER2566 and the fusion protein GST-XT was induced and isolated. The purified fusion protein was used to immunize New Zealand rabbits. The antibody titer was determined by ELISA. Purified polyclonal antibody was obtained through affinity chromatography column and the specificity of the purified antibody was characterized by Western blot. RESULTS: The amino acid 175-205 of XT-I (QKHQPELAKKPPSRQK-ELLKRKLEQQEKGKG) was selected as an antigen epitope. The synthesized DNA fragment of XT-I was successfully inserted into pGEX-4T-2 vector and the protein GST-XT was expressed. The purified fusion protein GST-XT was used as the immunogen to immunize rabbits and the polyclonal antibody against XT-I protein was obtained. The result of ELISA showed that the antibody titer was 1:640 000. Western blot analysis showed that the antibody had a good specificity. CONCLUSION: The rabbit polyclonal antibody against human XT-I protein has been successfully prepared, which lays the foundation for further study on the biosynthesis of PG by neoplastic myoepithelial cells in salivary tumors.