HIV-TAT-fused FHIT protein functions as a potential pro-apoptotic molecule in hepatocellular carcinoma cells.

Accumulating evidence has demonstrated that FHIT (fragile histidine triad) is a bona fide tumour suppressor gene in a large fraction of human tumours, including hepatocellular cancer. A virus-based delivery system has been developed to transfer the FHIT gene into many types of cancer cells to inhibit growth or even induce apoptosis. However, a protein-based replacement strategy for FHIT has not been performed in cancer cells. Here, we used HIV-TAT (transactivator of transcription)-derived peptide to transfer the purified FHIT protein into HCC (hepatocellular carcinoma) cells and determine the biological effect of this fusion protein in inducing apoptosis. Affinity chromatography was used to purify TAT peptide-fused human FHIT (TAT-FHIT) protein from BL21 Escherichia coli. Immunofluorescence staining and Western blot analysis were performed to identify the expression and internalization of TAT-FHIT in HCC cells compared with the purified FHIT protein. Our study showed that TAT-FHIT protein can translocate into cancer cells in 1 h after incubation at 37°C. Furthermore, the results of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay, Annexin-V staining and Western blotting demonstrated that TAT-FHIT can robustly inhibit growth and induce apoptosis of HCC cells in vitro. In addition, a mechanistic study showed that both exogenous and intrinsic apoptotic pathways were involved in TAT-FHIT-mediated apoptosis and this effect could be attenuated partially by a mitochondrial protector TAT-BH4, indicating that mitochondrion plays a critical role in TAT-FHIT-mediated pro-apoptotic effect in cancer cells. Taken together, our study suggests that TAT-FHIT is a potential pro-apoptotic molecule in HCC cells and strengthen the hypothesis of its therapeutic application against HCC.

[Expression and functional analysis of transcriptional factor JunB in hepatic cancer cells].

AIM: To construct the eukaryotic expression vector harboring transcriptional factor JunB and to observe the expression, localization and biological function of JunB in hepatic cancer cells. METHODS: The JunB gene was amplified by PCR from the human liver tissue cDNA library. After confirmed by DNA sequencing in the T-vector, the JunB gene was subcloned into pcDNA3.1(-) and pEGFP-C3 vectors, respectively. The recombinant vectors pcDNA-JunB and pEGFP-JunB were confirmed by Kpn I and BamH I restriction enzyme digestion. The recombinant vectors were transiently transfected into the hepatic cancer cells HepG2 using Lipofectamin2000. Western blotting was used to detect the expression of exogenous JunB and fluorescence microscopy was applied to observe the localization of JunB protein coupled with enhanced green fluorescent protein (EGFP) in hepatic cancer cells. Double luciferase reporter assay was performed to determine the effect of JunB on the transcriptional regulation of target genes. RESULTS: The recombinant eukaryotic expression vector carrying JunB or JunB-EGFP fusion gene was constructed and transiently transfected into HepG2 cells. Western blot analysis confirmed the exogenous expression of JunB. JunB-EGFP was observed uniquely in the nuclei. Double luciferase assay showed the transcriptional up-regulation of the VEGF in the presence of JunB. CONCLUSION: We constructed the recombinant eukaryotic expression vectors harboring JunB and JunB-EGFP successfully. The exogenous JunB is localized in the nuclei of transiently transfected HepG2 cells, suggesting JunB may function as a transcriptional factor. The result of reporter assay showed that JunB up-regulates the expression of VEGF, a tumor-angiogenesis associated molecule, at a transcriptional level. These demonstrate that JunB might be a novel target for an anti-angiogenesis treatment for hepatic cancers.