Objective To evaluate the long-term efficacy of recombinant human endostatin (rh-Endo) combined with FOLFOX4 as an adjuvant treatment for patients of stage Ⅱ and Ⅲ colorectal cancer.Methods Eligible patients were randomly assigned to receive FOLFOX4 or FOLFOX4 plus rh-Endo regimen in which patients receiving 7.5 mg/m2 Ⅳ on day 1-7,repeated every 2 weeks,to a total of 12 cycles in 6 months.Results A total of 197 eligible patients were accrued in this research with 105 patients in the control group and 92 patients in the experimental arm.Median follow-up period was 42 months.The baseline characteristics distributed were balanced by treatment.Rh-Endo combined with FOLFOX4 regimen resulted in significant improvement on DFS compared to FOLFOX4 regimen for patients with stage Ⅲ colon cancer (HR =0.19,95％ CI0.05-0.75,P =0.0124),and with a 34％ improvement on 3-year DFS and 81％ reduced recurrence.Although rh-Endo combined with FOLFOX4 regimen failed to make significant difference on DFS in the whole (HR =0.75,95％ CI 0.31-1.83,P =0.5589),it was also observed a 17％ improveiment on 3-year DFS.No statistical significant difference on DFS was observed in patients with stage Ⅱ disease.Conclusions Rh-Endo combined with FOLFOX4 regimen significantly improved the disease-free survival for patients with stage Ⅲ colorectal cancer,indicating that patients with stage Ⅲ disease,but not stage Ⅱ disease,can benefit from FOLFOX4 plus rh-Endo regimen in adjuvant treatment.

Objective To design and construct a new non-fusion soluble expression vector pTIG-mSUMO(small ubiq-uitin-related modifier) using the widely used solubility promoting protein SUMO and based on the translational coupling phenomenon in order to enable the non-fusion soluble expression of the broad-spectrum antiviral protein RA in Escherichia coli by pTIG-mSUMO.Methods The smt3 gene coding for SUMO protein was cloned from yeast genome DNA by PCR. After directed-site silent mutation to eliminate the EcoRⅠsite, the mutant mSUMO was inserted into pET-22b to obtain the translational coupling expression vector pTIG-mSUMO.The RA was subject to PCR amplification and cloned into the pTIG-mSUMO to obtain the expression plasmid pTIG-mSUMO/RA which was supposed to direct the soluble expression of RA by the translational coupling with mSUMO.Results A translational coupling expression vector pTIG-mSUMO which could di-rect/drive the SUMO and heterogonous protein non-fusion expression simultaneously was designed and constructed.The Western blotting result indicated that pTIG-mSUMO could direct the high-level expression of RA, around 40%of which was soluble.Conclusion A translational coupling expression vector pTIG-mSUMO is obtained.After coupling with SUMO, RA is highly expressed in E.coli and both the expression level and solubility are greatly improved.pTIG-mSUMO might contrib-ute to soluble expression of other proteins.

Objective To investigate the impact of shikonin(SHI)on the malignant biological activity of liver cancer cells by regulating Notch signaling pathway.Methods Western blot assay was used to detect the expression of Notch,Hairy mitosis-related enhancer-1(Hes1),hairy-related transcription factor-1(HEY1)protein in liver cancer tissue,paracancerous tissue,hepatoma cells(HepG2 cells,Hep3B cells,HCCLM3 cells,Huh-7 cells and SMMC-7721 cells)and normal liver cells(HL-7702 cells).Huh-7 cells were divided into the control group,the L-SHI group(1μmol/L SHI),the M-SHI group(2μmol/L SHI),the H-SHI group(4μmol/L SHI),the DAPT group(50μmol/L Notch signal inhibitor DAPT)and the H-SHI+VPA group[4μmol/L SHI and 8 mmol/L Notch pathway activator Valproic acid(VPA)].The proliferation of Huh-7 cells was detected by CCK-8 method and plate cloning test.The apoptosis and cell cycle of Huh-7 cells were detected by flow cytometry.Cell scratch test and Transwell invasion test were used to detect migration and invasion of Huh-7 cells.Western blot assay was used to detect the expression of epithelial-mesenchymal transformation(EMT)and apoptosis related proteins.Results The expression levels of Notch,HES1 and HEY1 were obviously increased in liver cancer tissue and cells,and Huh-7 cells showed the most obvious difference,therefore,Huh-7 cells were taken as the research object.Compared with the control group,the protein levels of Notch,HES1,HEY1 and Bcl-2 decreased,and the proportions of S phase and G2 phase cells,OD450 value,number of clones,migration rate,number of invasive cells and levels of N-cadherin and Vimentin decreased significantly in the L-SHI group,the M-SHI group,the H-SHI group and the DAPT group(P＜0.05).The proportion of G1/G0 phase cells,apoptosis rate and levels of Bax,cleaved Casase-3,and E-cadherin increased obviously(P＜0.05).The effect of SHI was dose-dependent.Compared with the H-SHI group,the above indexes showed the opposite trend in the H-SHI+VPA group.VPA attenuated the effect of SHI on reducing the malignant biological activity of liver cancer cells.Conclusion SHI may inhibit the proliferation,migration and invasion of Huh-7 cells and promote apoptosis of Huh-7 cells by inhibiting Notch signal pathway.