Signal Transducer and Activator of Transcription 3 for the Differentiation of Hepatocellular Carcinoma from Cirrhosis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Overexpression and constitutive activation of signal transducer and activator of transcription (STAT) 3 have been suggested in the tumorigenesis of many human cancers, including multiple carcinomas, melanoma, and lymphoma. The diagnosis of hepatocellular carcinoma (HCC) in lobectomy specimens is usually straightforward, but distinguishing cirrhosis from well-differentiated HCC can be challenging in core biopsies. Our aims were to investigate the expression level of STAT3 and phosphorylated STAT3 (pSTAT3) in HCC and cirrhosis, and the application of STAT3 in the differential diagnosis of HCC and cirrhosis.</p><p><b>METHODS</b>Sixty cases were divided into three groups: patients with HCC only (Group 1), HCC and cirrhosis (Group 2), and cirrhosis only (Group 3). Formalin-fixed and paraffin-embedded tissue sections were stained immunohistochemically for STAT3, pSTAT3, and CD163. The values obtained from the tissue sections of each group were compared in statistical analysis.</p><p><b>RESULTS</b>STAT3 showed a high level in HCC and was a significant marker for differentiating HCC from cirrhosis (P < 0.0001). The odds ratio between HCC and cirrhosis increased 34.4 times when the intensity of STAT3 increased by 1 level. Spearman's correlation and Chi-square tests also demonstrated that expression level of STAT3 did not correlate with age, gender, or the presence of a cirrhotic background.</p><p><b>CONCLUSIONS</b>STAT3 staining differs significantly in HCC and cirrhosis. The findings reinforce the role of STAT3 in the tumorigenesis of HCC and provide a useful marker to differentiate HCC from cirrhosis in challenging liver biopsies.</p>

Inhibition of proliferation, adhesion and invasion ability of human lung carcinoma cell A549 by tumor necrosis factor-alpha converting enzyme (TACE) / 生物工程学报

We constructed prokaryotic expression vectors for different domains of TACE gene and expressed the fusion proteins, so as to explore their effects on the proliferation, adhesion and invasion potential of tumor cells in vitro. The total RNA was isolated from THP1 cell. TACE cDNA was amplified by RT-PCR and subcloned into pMD18-T vector to construct pMD-18T-TACE vector. The different cDNA fragment of TACE were amplified from plasmid pMD-18T-TACE and then cloned into pET-28a( + ) to construct expression vector pET28a( + )- 300, pET28a( + )-T800, and pET28a( + )-T1300, which respectively transformed into E. coli BL21 (DE3). The expression of His-tagged fusion proteins were induced with IPTG and purified through BBST NTA resin. The proliferation ability was examined by MTT assay. The adhesive and invasive ability were examined by plated adhesion model and Transwell assay. The protein pET28a( + )-T300 and pET28a( + )-T1300 can reduce the proliferation, adhesion and invasion ability of human lung carcinoma cell A549 in vitro, but otherwise the protein pET28a( + )-T800 had not shown the inhibitive function. The fusion protein of disintegrin domain of TACE have the similar biological function to other disintegrins, which can be used for further research on function of TACE in inflammation and tumor.

Screening of TACE peptide inhibitors from a phage display random 15-peptide library by recombinant TACE ecotodomain / 生物工程学报

Tumour necrosis factor-alpha converting enzyme (TACE) is the major protease responsible for processing proTNF from membrane-anchored precursor into secreted TNF-alpha. It was validated that TACE is involved in many diseases such as arthritis, multiple sclerosis and Alzheimers, therefore it represents a novel and significant target for therapeutic intervention in a variety of inflammatory and neuroimmunological diseases. To obtain the recombinant TACE ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coded for catalytic domain (T800) and full-length ectodomain (T1300) of TACE were amplified by RT-PCR, the expression plasmid was constructed by inserting T800/T1300 into plasmid pET-28a/pET-28c and transformed into E. coli BL21 (DE3). SDS-PAGE and Western blotting analysis revealed that T800/T1300 was highly expressed in the form of inclusion body being induced by IPTG. After Ni2+ -NTA resin affinity chromatography, the purity of the recombinant T800/T1300 protein was more than 90%. T800 and T1300 protein were used in the screening of TACE-binding peptides from the phage display random 15-peptide library. After four rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence-TRWLVYFSRPYLVAT was found and synthesized. The synthetic peptide was shown to bind to TACE and inhibit the TNF-alpha release from LPS-stimulated human peripheral blood mononuclear cells (PBMC) up to 60.3%. FACS analysis revealed that the peptide mediated the accumulation of TNF-alpha on LPS-stimulated PBMC surface. These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE and the deduced motif might be applied to molecular design of anti-inflammation drugs.