Metformin enhances radiosensitivity in hepatocellular carcinoma by inhibition of specificity protein 1 and epithelial-to-mesenchymal transition

Objective: Radiotherapy becomes more and more important in hepatocellular carcinoma (HCC) due to the development of technology, especially in unresectable cases. Metformin has a synergistic benefit with radiotherapy in some cancers, but remains unclear in HCC. This study aims to investigate the effect of metformin on radiosensitivity of HCC cells and the roles of specificity protein 1 (Sp1) as a target of metformin. Methods: The SMMC-7721 cell line was exposed to various doses of γ-ray irradiation (0, 2, 4, 6, and 8 Gy) and with or without different concentrations of metformin (0, 1, 5, 10, and 20 mM) to measure the radiosensitivity using MTT assay. Flow cytometry was used to determine cell cycle by propidium iodide (PI) staining and apoptosis by Hoechst 33342/PI staining and Annexin V-FITC/PI staining. Real-time polymerase chain reaction and Western blotting were performed to analyze the Sp1 mRNA and protein expressions of Sp1 and epithelial-to-mesenchymal transition (EMT) marker E-cadherin and Vimentin. The invasion capability was measured by the Boyden chamber assay. Results: In SMMC-7721 cells exposed to irradiation, metformin reduced proliferation and survival cells at various concentrations (0, 1, 5, 10, and 20 mM) and induced cell cycle arrest, apoptosis, and inhibited invasion. In SMMC-7721 cells with irradiation, the mRNA and protein expressions of Sp1 were significantly decreased by metformin as well as a selective Sp1 inhibitor. Metformin attenuated transforming growth factor-β1 induced decrease of E-cadherin and increase of Vimentin proteins. Conclusion: Metformin demonstrated enhanced radiosensitivity and inhibition of EMT in HCC cells. Sp1 might be a target of metformin in radiosensitization

Regulation of c1orf109 gene by transcription factor MAZ in vitro / 中国病理生理杂志

AIM:To study the regulation of human chromosome 1 open reading frame 109(c1orf109)gene by transcription factor Myc-associated zinc-finger protein(MAZ)in vitro.METHODS:In vitro study,electrophoretic mobili-ty shift assay was performed to screen the binding sites of MAZ in the promoter region of c1orf109 gene.The HeLa cells were co-transfected with the enhanced green fluorescent protein reporter vector driven by c1orf109 promoter,and MAZ and transcription factor specificity protein 1(Sp1)expression plasmids.After 24 h,the transcriptional expression of c1orf109 gene in the co-transfected cells was determined by confocal scanning microscopy and flow cytometry.RESULTS:The c1orf109 promoter could bind to MAZ ,and shared the binding sites with Sp 1.MAZ and Sp1 both inhibited the transcrip-tional expression of c1orf109 gene and the inhibition effect of Sp1 was greater than MAZ(P<0.05).CONCLUSION:Both MAZ and Sp1 regulate the expression of c1orf109 gene in physiological and pathological conditions ,and this regulation is redundancy with the same direction.The existence of redundancy transcriptional regulation manner of this gene suggests that precise regulation of c1orf109 gene is vital important for cell biological processing.

Effect of retinoblastoma binding protein 4 (RBBP4)on Sp1-mediated transcription of HIV long terminal repeat in 293 T cells / 中华临床感染病杂志

Objective To investigate the effect of retinoblastoma binding protein 4 (RBBP4)in Sp1 -mediated HIV long terminal repeat(LTR)transcription.Methods RBBP4 expression vector and Sp1 expression vector were respectively co-transfected into 293 T cells with HIV promoter pHIV-LTR-Luc or Sp1 site mutated pHIV-LTR-sp1 -mut by liposome transfection,and the transfected cells were examined by dual luciferase reporter assay system.The effect of RBBP4 on the binding of Sp1 to LTR was further studied by chromatin immunoprecipitation (ChIP)and electrophoretic mobility shift assay (EMSA).Results The relative firefly luciferase activity activated by Sp1 was decreased from 62.5 to 16 at the dose of 500 ng of RBBP4 expression vector (t =14.52,P <0.01 ).When the Sp1 binding sites were mutated,the effects of 100,300 or 500 ng of RBBP4 expression vector on the firefly luciferase activity of HIV LTR were not statistically significance (t =1 .897,2.357 and 3.162,all P <0.05).ChIP results showed that when the binding of RBBP4 on HIV LTR increased,the binding of Sp1 on HIV LTR increased significantly (t =11 .93,P <0.01 ),while the reduced binding of RBBP4 on HIV LTR significantly attenuated the binding of Sp1 onto LTR(t =11 .38,P <0.01 ).The effect of RBBP4 on the binding of Sp1 to DNA in ChIP assays was further verified by EMSA assays.Conclusion RBBP4 can inhibit the Sp1 -mediated HIV LTR transcription in 293 T cells.

The expression of Sp1 and CEA and the correlation between the two factors in colon cancer / 国际检验医学杂志

Objective To study the expression of transcription factor Sp1 and CEA and the correlation between the two tran‐scription factors in colorectal cancer .Methods To detect expression Sp1 and CEA mRNA by Real‐Time PCR in 60 colon cancer tis‐sues and corresponding normal tissues and the results were compared with the clinical features and pathological characters .The re‐lationship between the expression of Sp1 mRNA and CEA mRNA in 60 colon cancer tissues was determined .Results The expres‐sion rates of Sp1 and CEA mRNA was detectable to highly expressed rates in colon cancer tissues than the matched normal tissues (P0 .05) . Sp1 and CEA mRNA was detectable to highly expressed in the different histological grade and Dukes stages .In addition ,a positive correlation was found between the expression of Sp1 mRNA and CEA mRNA(r=0 .706 ,P<0 .01) ,(0< r<1) .Conclusion Sp1 and CEA was detectable to highly expressed in colon cancer .Positively correlation occurred in Sp1 mRNA and CEA mRNA indica‐ted that Sp1 and CEA provide the new clues of genetic diagnosis and treatment .

Essential regulatory elements for NHE3 gene transcription in renal proximal tubule cells

The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5’-flanking sequence (5’FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5’FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5’FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5’FS are used as promoter, efficient transcription only occurs with 44 bp of 5’FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5’FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.

The Apoptotic Effect of the Hexane Extract of Rheum undulatum L. in Oral Cancer Cells through the Down-regulation of Specificity Protein 1 and Survivin / 한국실험동물학회지

The hexane extract of Rheum undulatum L. (HERL) has been shown to have anti-cancer activity in several cancers in vivo and in vitro. However, the anti-cancer activity of HERL and its molecular mechanism in human oral cancer cells has not been explored. Thus, the aim of this study was to elucidate the growth-inhibitory and apoptosis-inducing effects of HERL in HN22 and SCC15 oral cancer cell lines. This study shows that HERL inhibits oral cancer growth, decreases cell viability, and causes apoptotic cell death in HN22 and SCC15 cells, as characterized by morphological changes, nuclear condensation and fragmentation, the cleavage of PARP and the accumulation of cells in the sub-G1 phase. The treatment of oral cancer cells with HERL also resulted in decreased expression of specificity protein (Sp1) and its downstream protein, survivin. Therefore, our results suggest that the regulation of Sp1 and survivin plays a critical role in HERL-induced apoptosis in human oral cancer cells.

The expression of Sp1 and AP-2α and the correlation between the two transcription factors in colon cancer / 肿瘤研究与临床

Objective To study the expression of transcription factor specificity protein1 and activator protein-2α and the correlation between the two transcription factors in the process of occurrence and development of colorectal cancer. Methods To detect expression of Sp1 and AP-2α mRNA by Real-Time PCR in 60 colon cancer tissues and corresponding normal tissues and the results were compared with the clinical features and pathological characters. The relationship between the expression of Sp1 mRNA and AP-2α mRNA in 60 colon cancer tissues was determined. Results The expression rates of Sp1 mRNA was detectable to highly expressed rates in colon cancer tissues than the matched normal tissues (P ＜0.01),whereas AP-2α mRNA in the colon cancer tissues was significantly lower than that in the matched normal tissues (P ＜0.01). Sp1 mRNA and AP-2α mRNA expression rates had no significant difference between the clinical features (sex, age and tumor areas) respectively. Loss expression or down regulation expression of AP-2α mRNA was detected, whereas Sp1 mRNA was detectable to highly expressed in the different histological grade and Dukes stages. The expression of Spl mRNA and AP-2α mRNA were positively correlated with the histological grade in colon cancer. A significant correlation was found between the expression of Sp1 mRNA and AP-2α mRNA (r =-0.849, P ＜0.001). Conclusion Loss or down regulation expression of AP-2α mRNA,whereas Sp1 was detectable to highly expressed in colon cancer. Negative correlation occurred in Sp1 mRNA and AP-2α mRNA indicated that AP-2α and Sp1 provide the new clues of genetic diagnosis and treatment.

Enhancement of cisplatin on cytocidal effect of tineposide against small cell lung cancer cell line / 基础医学与临床

Objective To investigate the mechanism of cisplatin enhanceing the effect of tineposide killing small cell lung cancer cell line by combination of the two drug.Methods The microculture tetrazolium(MTT) assay was used to determine the inhibition rates of CDDP combined with VM-26.Acridine orange/ethidium bromide(AO/EB) fluorescent staining was used to show the cell vitality rates,DNA disruption ladder were applied to reveal cell apoptosis.The mRNA and protein level of topoisomerase Ⅱ(Topo Ⅱ) and transcript factor SP1 were measured by semi-quantitative RT-PCR and western blot.Results There is synergistic effect between the two drug.The cell vitality rates were decreased in combination group than that of CDDP or VM-26 used alone.the combination treatment group resulted in more serious DNA strand breakage.The expression of Topo Ⅱ?,? and SP1 mRNA and protein both increased in CDDP treatment group,while the Topo Ⅱ?,? expression decreased and(the SP1 expression) has no obviously change in VM-26 treatment group.In combination group,Topo Ⅱ? expressionwere decreased comparing with CDDP used alone,SP1 expression increased comparing with VM-26 used alone,but has no obviously change as comparing with CDDP used alone.Conclusion CDDP could enhance Topo Ⅱ expression through up-regulating SP1 expression,which offer more target for Topo Ⅱ inhibitor VM-26 to act on killing small cell lung cancer cell.