Discovery of thieno[2,3-d]pyrimidin-4(3H)-one derivatives as a new class of ROCK inhibitors.

Herein, we report the discovery of a series of thieno[2,3-d]pyrimidin-4(3H)-one derivatives as a new class of ROCK inhibitors. Structure-activity relationship studies of these compounds led to the identification of the most potent compound, 3-(3-methoxybenzyl)-6-(1H-pyrrolo[2,3-b]pyridin-4-yl)thieno[2,3-d]pyrimidin-4(3H)-one (8k), which showed IC50 values of 0.004 µM and 0.001 µM against ROCK Ⅰ and ROCK Ⅱ, respectively. In vitro, 8k significantly reduced the phosphorylation level of ROCK downstream signaling protein and induce changes in cell morphology and migration. Overall, this study provides a promising lead compound for drug discovery targeting ROCKs.

Research proceedings on amphibian model organisms.

Model organisms have long been important in biology and medicine due to their specific characteristics. Amphibians, especially Xenopus, play key roles in answering fundamental questions on developmental biology, regeneration, genetics, and toxicology due to their large and abundant eggs, as well as their versatile embryos, which can be readily manipulated and developed in vivo. Furthermore, amphibians have also proven to be of considerable benefit in human disease research due to their conserved cellular developmental and genomic organization. This review gives a brief introduction on the progress and limitations of these animal models in biology and human disease research, and discusses the potential and challenge of Microhyla fissipes as a new model organism.

[cDNA cloning and expression analysis of MSTN gene from Schizopygopsis pylzovi].

Myostatin (MSTN) is a member of the TGF-ß superfamily that acts as a negative regulator of skeletal muscle growth. A full-length, 2 180 bp, cDNA sequence of the myostatin gene from Schizopygopisis pylzovi was cloned with RT-PCR,5'-RACE and 3'-RACE and the cDNA clone included a 1 128 bp ORF, encoding a 375 amino acid peptide. Using PCR, we obtained the sequences of two introns of the MSTN gene and found that its structure in Schizopygopsis pylzovi was similar to that of other vertebrates, including three exons and two introns. Likewise, the putative MSTN peptide of Schizopygopsis pylzovi contains a conserved RXXR proteolytic cleavage domain, and 8 conserved cysteine residues in the C terminal of the protein, similar to other vertebrates. Phylogenetic analysis showed that the MSTN of Schizopygopsis pylzovi has high homology with other cyprinid fishes, but a low homology with mammals and birds. In the 9 examined tissues, the MSTN gene was highly expressed in heart, kidney, intestine and spermary, while weakly expressed in muscle, brain, fat, gill and hepatopancreas. Quantitative real-time PCR analysis showed that the expression of MSTN gene was different during embryo development, suggesting that the fish MSTN may not only play roles in muscle development but also contribute to other biological functions.

[Construction and expression of recombinant human serum albumin-EPO fusion protein].

UNLABELLED: OBJECTIVE To construct the recombinant plasmid pCI-HLE encoding human serum album-EPO (HSA-EPO) fusion protein and to express it in CHO cell. METHODS: The cDNA encoding human serum album and EPO were amplified by PCR, and then spliced with the synsitic DNA fragment encoding GS (GGGGS), by overlap PCR extension to form LEPO. After BamH I digestion, the HSA and LEPO was ligated to generate the fusion HSA-EPO gene and was then cloned into the expression vector pCI-neo to generate the recombinant plasmid pCI-HLE. The plasmid pCI-HLE was transfected into CHO cell by liposome protocol. Then, the recombinant cells were screened by G418 and identified by PCR and Western blot. Expression of fusion protein was evaluated by Enzyme Linked Immunosorbent Assay (ELISA). RESULTS: Restrictive enzymes digestion and DNA sequencing revealed that HSA-EPO fusion gene was cloned into expression vector pCI-neo successfully. PCR and Western blot analysis confirmed that the fusion gene was integrated in the genome of CHO cells and expressed successfully. The HSA-EPO production varied from 86 Iu/(mL x 10(6) x 72 h) to 637 IU/(mLx 10(6) x 72 h). CONCLUSION: The results confirmed that HSA-EPO fusion gene can be expressed in the CHO cells, with EPO immunogenicity, which could serve as foundation for the development of long-lasting recombinant HSA-EPO protein.

Effects of multidrug resistance, antisense RNA on the chemosensitivity of hepatocellular carcinoma cells.

BACKGROUND: Multidrug resistance is a major obstacle in cancer chemotherapy. We examined whether the antisense RNA of multidrug resistance gene 1 (mdr1) could reverse multidrug resistance in the human hepatocellular carcinoma (HCC) cell line SMMC7721/ADM. METHODS: The recombinant adenoviruses pAdEasy-GFP-ASmdr1 product was produced by the adenoviral vector AdEasy system, which can express antisense RNA against the mdr1 gene. Following that, the recombinant adenovirus was transfected into the P-glycoprotein-producing multidrug resistance cell line, SMMC7721/ADM human HCC cells resistant to adriamycin (ADM) and daunorubicin (DNR). In order to investigate the reversal of multidrug resistance phenotype, we measured the expression of mdr1 mRNA by RT-PCR and the production of P-glycoprotein by flow cytometry. The sensitivities for ADM and DNR SMMC7721/ADM cells were examined by [3-(4, 5-dimethylthi-azol-2-yl)-2,5 diphenyl-terazolium bromide] (MTT) analysis. RESULTS: The low-level expression of mdr1 mRNA and P-glycoprotein production were observed in parental sensitive cells SMMC/7721 in addition to the overexpression of mdr1 mRNA and P-glycoprotein in SMMC7721/ADM cells. The transfection of antisense-RNA into SMMC7721/ADM cells resulted in decreases of mdr1 mRNA and P-glycoprotein, but increase of drug sensitivities. The sensitivities of transfected SMMC7721/ADM cells to ADM and DNR in IC50 reduced by 31.25% and 62.96% respectively. CONCLUSIONS: Mdr1 antisense RNA can increase the sensitivities of SMMC7721/ADM cells to anticancer drug by decreasing the expression of the mdr1 gene and inhibiting P-glycoprotein expression. This strategy may be applicable to cancer patients with P-glycoprotein mediated multidrug resistance.

Overexpression of TIMP-1 mediated by recombinant adenovirus in hepatocellular carcinoma cells inhibits proliferation and invasion in vitro.

BACKGROUND: Matrix metalloproteinases (MMPs) and its natural tissue inhibitors of metalloproteinases (TIMPs) are involved in cancer progression. This study was undertaken to determine the effects of overexpression of TIMP-1 on human hepatocellular carcinoma (HCC) cell growth, proliferation, and invasion. METHODS: Employing the efficient AdEasy(TM) system, recombinant adenovirus AdTIMP-1 containing full-length cDNA of TIMP-1 was generated by homologous recombination and amplified in 293 cells. Then, human HCC cell line (HepG2) underwent gene transfection to overexpress TIMP-1 (so-called HepG-T cells). The mRNA and protein expressions of TIMP-1 were detected with RT-PCR and Western blotting, respectively. The ultrastructure was observed with a transmission electron microscope and the proliferation of HepG-T cells was determined by MTT assay and growth curve. The potential of in vitro invasion was measured with Millicell Chamber. RESULTS: The resulting AdTIMP-1 and HepG-T cells were generated and the expression of TIMP-1 was detected in vitro. The cell proliferation curves and MTT assay showed HepG-T cells' growth, and proliferation were obviously inhibited. The invasion across Matrigel-coated filters was significantly decreased compared with controls. The suppression rate of HepG-2 cells with AdhTIMP-1 transfection was 50%, and AdhTIMP-1 transfection inhibited by more than 91.6% of the invasion into the Matrigel-coated filter (P<0.01). CONCLUSIONS: TIMP-1 overexpression results in the suppression of proliferative and invasive potential of HepG2 cells in vitro. This study demonstrates the potential role of TIMP-1 as a target for liver cancer gene therapy and has laid a foundation for further study on its anticancer function.

[Recombinant adenovirus vectors carrying antisense matrix metalloproteinase-2 inhibits hepatocellular carcinoma growth in vivo].

OBJECTIVE: To investigate the inhibitory effect of a recombinant adenoviral vector carrying antisense matrix metalloproteinase-2(MMP2) on the growth of hepatocellular carcinoma(HCC) in vivo. METHODS: The recombinant adenoviral vector carrying antisense MMP2(Ad-MMP2(AS))which had been constructed by us in readiness was used to infect the human HCC cell line (Bel-7402). Then the invasiveness of the Bel-7402 cells was assayed in Matrigel, and the production of MMP2 in the Bel-7402 cells was detected with Western blot analysis and Gelatin zymography. After the Ad-MMP2(AS)-infected Bel-7402 cells being subcutaneously inoculated in nude mice, the production of tumors was under observation, and then Ad-MMP2(AS) was injected intratumorally into the pre-existing tumors. RESULTS: Compared with PBS or Ad-CMV-infected cells, infection of Bel-7402 cells with Ad-MMP2(AS) significantly reduced MMP2 enzyme activity, the invasiveness resulted in 52% reduction in Matrigel assays, and the tumor volume displayed a 4.3-fold reduction in nude mice. In addition, direct intratumoral injection of Ad-MMP2(AS) into pre-existing tumors significantly impaired the further expansion of the tumor mass and resulted in a 63% reduction in tumor cell growth. CONCLUSION: The recombinant adenovirus with antisense MMP2 can effectively inhibit the invasiveness and growth of Bel-7402 cells in vitro and in vivo, and has a therapeutic potential for HCC.

Construction of the recombinant adenovirus vector carrying antisense multidrug resistance gene.

BACKGROUND: Multidrug resistance proteins serve as transporters for chemical drugs in human malignancies. The objective of this study was to construct a homologous recombinant adenovirus carrying a reversal fragment of multidrug resistance gene 1 (mdr1) gene cDNA sequence. METHODS: The fragment of the mdr1 gene from the plasmid pHaMDR1-1 carrying the whole human mdr1 cDNA sequence was inserted reversely into the shuttle plasmid pAdTrack-CMV of adenoviral vector system AdEasy. The homologous recombination process was taken place in E. coli BJ5183 with the backbone plasmid pAdEasy-1. After packaging in 293 cells, recombinant adenoviral plasmid was generated. The recombinant adenoviral plasmid was identified by polymerase chain reaction (PCR), restriction endonucleases digest, DNA sequence analysis and fluorescence microscopic photograph, respectively. RESULTS: The recombinant adenovirus pAdEasy-GFP-ASmdr1 was successfully constructed and identified by PCR, restriction digest, and sequencing with strong green fluorescence expression in fluorescence microscopic photograph. CONCLUSIONS: The recombinant adenoviral mdr1 vector would introduce the antisense mdr1 gene into the human multidrug resistance hepatocellular cell line effectively, which would provide an experimental basis to study the multidrug resistance in human hepatocellular carcinoma.

[Construction of recombinant adeno-associated virus vectors carrying double gene of antisense multidrug resistance-associated protein and antisense multidrug resistance].

OBJECTIVE: To construct a recombinant adeno-associated virus vectors carrying double gene of antisense multidrug resistance-associated protein (MRP) and antisense multidrug resistance (MDR1) for use in studying the gene therapy to reverse the multidrug resistance (MDR) in hepatocellular carcinoma (HCC). METHODS: The 500 bp fragment (mrp) of MRP cDNA 5' region and the 600 bp fragment (mdr1) of MDR1 cDNA 5' region were amplified through polymerase chain reaction (PCR), and then they were linked to a combined gene fragment (mrp+mdrl) by overlapping technique. The combined gene fragment(mrp+mdrl) was cloned reversely into the multiple cloning site (MCS) of the expression plasmid pAAV-IRES-hrGFP in AAV Helper-Free System to construct the recombinant expression plasmid pAAV-IRES-hrGFP-(mrp + mdr1)AS. The packaging cell line (HEK 293 cell) was co-transfected with the pAAV-IRES-hrGFP-(mrp+mdr1)AS together with the control plasmid pAAV-RC and pHelper in AAV Helper-Free System by means of lipofectamine. The recombinant adeno-associated virus vector : rAAV2-(mrp+mdr1)AS carrying the double gene of antisense multidrug resistance-associated protein (MRP)and antisense multidrug resistance (MDR1) was packaged. Then the viral titer was checked by GFP. RESULTS: The recombinant adeno-associated virus vector : rAAV2-(mrp + mdr1)AS carrying antisense MRP and antisense MDR1 was constructed successfully, the strong green fluorescence was observed in HEK 293 cells under a fluorescence microscope. The viral titer was 2.5 X 10(6) efu/ml. CONCLUSION: The rAAV2-(mrp+mdr1)AS thus constructed could introduce the antisense MRP and antisense MDR1 into the human drug-resistant hepatocellular cell line effectively, which might provide a sound basis for the mechanisms and reversal methods of the multidrug resistance in HCC.

[Inhibition of adenovirus-mediated gene transfer of antisense matrix metalloproteinase-2 on hepatocellular carcinoma growth in vivo].

OBJECTIVE: To investigate if a recombinant adenoviral vector carrying antisense matrix metalloproteinase-2 (MMP2) gene would inhibit the growth of hepatocellular carcinoma (HCC) in vivo. METHODS: Using the recombinant adenoviral vector carrying antisense MMP2 gene (Ad-MMP2AS) which was constructed by us previously, to infect the human HCC cell line (Bel-7402). Then the invasiveness of the Bel-7402 cells was assayed in Matrigel, and the production of MMP2 in the Bel-7402 cells was detected with Western blotting analysis and Gelatin zymography. Then the Ad-MMP2AS-infected cells were subcutaneously inoculated in nude mice. After tumors developed, Ad-MMP2AS was injected intratumorally into pre-existing tumors. The tumors were removed, sectioned, and stained with H E. RESULTS: Compared with PBS or Ad-CMV-infected cells, the infected Bel-7402 cells with Ad-MMP2AS injections significantly reduced their MMP2 enzyme activity and invasiveness about 52.05% in Matrigel assays, and the tumor volumes in nude mice resulted in a 3.3-fold reduction. In addition, direct intratumoral injection of Ad-MMP2AS into pre-existing tumors significantly prevented further expansion of the tumor masses and resulted in a 63.06% reduction in tumor cell growth. CONCLUSION: The recombinant adenovirus with antisense MMP2 can effectively inhibit the invasiveness and growth of Bel-7402 cells in vitro and in vivo, and it has a therapeutic potential for HCC.

Construction of recombinant adenoviral vector carrying human tissue inhibitor of metalloproteinase-1 gene and its expression in vitro.

BACKGROUND: Overexpression of human tissue inhibitors of metalloproteinase-1 (TIMP-1) may play an antitumor role in inhibiting hepatocellular carcinoma (HCC) growth and progression. The aim of the study was to construct a recombinant adenovirus vector carrying hTIMP-1 cDNA for liver gene therapy and observe its expression in vitro. METHODS: The full-length cDNA of hTIMP-1 was positively cloned into the adenoviral shuttle vector pAdTrack-CMV, and then cotransformed into competent BJ5183 cells with the adenoviral backbone plasmid pAdEasy-1. Thus, a recombinant adenovirus AdhTIMP-1 containing full-length cDNA of hTIMP-1 was generated by homologous recombination in E. coli. AdhTIMP-1 was then packaged and amplified in adenoviral packaging cells, or human embryonic kidney 293 cells. The viral titer was checked by green fluorescent protein (GFP), and the expression of hTIMP-1 in vitro was detected by the techniques of Western blot and RT-PCR. RESULTS: The recombinant adenovirus vector carrying hTIMP-1 was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. The transcription of TIMP-1 mRNA in 293 cells was checked by RT-PCR and TIMP-1 protein could be detected in the culture by Western blot analysis. CONCLUSION: The successful construction of recombinant adenoviral vector carrying human TIMP-1 and the effective expression in vitro has laid a foundation for further study of its antitumor function and may pave the way for future application in liver gene therapy.