Effect Of Adenovirus-mediated HSulf-1Expression On Tumor Cell Cycle And Chemosensitivity

The hSulf-1(human sulfatase-1) gene locates on8q13.39, encodes anendosulfatase. The hSulf-1is regarded as a tumor suppressor, which results in reducedproliferation and migration of cancer cells, as well as increased sensitivity toapoptosis induced by chemotherapy. HSPGs are important components of theextracellular matrix, and play an important role in the interaction between cells andextracellular matrix glycoprotein. It directly modulates heparin-binding growthfactors and their special receptors. hSulf-1can desulfate HSPGs and block the bindingof growth factors and their receptors, and finally inhibit the activation of growthfactor signaling pathways, thereby inhibiting the cell proliferation, invasion, migration,angiogenesis and the formation of metastasis. Additionally, it also relates with celladhesion, cytoskeleton repair, coagulation function, and TGF-β1, Wnt/β-catenin,AKT/ERK signaling pathways and active signaling pathway of epithelialmesenchymal transformation. In this study, we constructed a series of adenovirusvectors (including non-proliferative adenovirus Ad5-hSulf1, tumor-selectiveproliferative oncolytic virus AdSurp-hSulf1and radiation-induced enhanced oncolyticvirus Ad.eSurp-hSulf1) and investigate the effect of hSulf-1gene on cell cycleprogression in hepatocellular carcinoma and chemosensitivity in breast cancer anddiscussed the molecular mechanism of hSulf-1anti-tumor effect. Our researchprovides a foundation for cancer gene therapy.Part I Construction and identification of adenovirus vectors【Objective】 To clone the hSulf-1gene and construct adenovirus vectorAd5-hSulf1, oncolytic adenovirus vector AdSurp-hSulf1, and radiation-inducibleenhanced adenovirus vectorAd5.eSurp-hSulf1and a series of their control adenovirus vectors carrying the enhanced green fluorescent protein gene (EGFP). includingAd5-EGFP, AdSurp-EGFP and Ad.eSurp-EGFP.【Methods】The plasmid pcDNA3.1-hSulf1was used to amplify the hSulf-1cDNA, the PCR product was digested with BamHI and SalI, inserted into theadenovirus plasmid pDC315to generate pDC315-hSulf1. Based the previouslyrecombined oncolytic adenovirus (AdSurp-P53), the hSulf-1gene was used to replacethe P53gene, and obtained the plasmid pAdSurp-hSulf1, the pAdSurp-hSulf1wasinserted the CArG regulatory element of the Egr-1gene as enhancer upstream of theSurp promoter to generate pAd.eSurp-hSulf1. The EGFP gene was used to replace thehSulf-1gene in pDC315-hSulf1, pAdSurp-hSulf1and pAd.eSurp-hSulf1to generatedpDC315-EGFP, pAdSurp-EGFP and pAd.eSurp-EGFP. After properly identification,the plasmids pDC315-hSulf1, pAdSurp-hSulf1, pAd.eSurp-hSulf1and the adenoviruspackaging plasmid pBHGloxdelE13cre were transfected into HEK293cells byLipofectamine2000transfection reagent, respectively. After a homologousrecombination in HEK293cells, we obtained adenoviruses named Ad5-hSulf1,oncolytic adenovirus AdSurp-hSulf1, radiation-inducible enhanced adenovirusAd5.eSurp-hSulf1, as well as to generate a series of control adenoviruses, includingAd5-EGFP,AdSurp-EGFP and Ad.eSurp-EGFP.【Results】 We successfully constructed the non-proliferative adenovirusAd5-hSulf1, oncolytic adenoviral AdSurp-hSulf1, radiation-induced enhancedoncolytic adenovirus Ad.eSurp-hSulf1and their corresponding negative controlviruses, which were demonstrated to express hSulf-1in eukaryotic cells.【Conclusion】 A series of recombinant adenoviruses were constructedsuccessfully, which lay a foundation for further research to explore the gene functionson tumor cell cycle and chemosensitivity and the application in cancer gene therapy. Part II Impacts of adenovirus-mediated hSulf-1expressionon tumor cell cycle【Objective】To investigate the changes of cell cycle distribution, after infectionof adenovirus carrying the hSulf-1gene in hepatocellular carcinoma and breastcancer.【Methods】The hSulf-1expression was examined in HCC cells and breastcancer cells by Western blotting, The changes of cell cycle distribution were detectedby flow cytometry.【Results】Western blotting analysis showed that the relative expression ofhSulf-1gene was low in hepatoma cell lines SMMC-7721, MHCC-97L and breastcancer cell line MCF-7. Flow cytometry results showed that in the hSulf-1genelow-expression tumor cell lines, the cell cycle was arrested at G2/M-phase afterinfectedAd5-hSulf1.【Conclusion】 Over-expression of hSulf-1gene can induce cell cycledistribution changes.Part III Adenovirus-mediated hSulf-1gene reversesbFGF-stimulated cell cycle progression and presents antitumorefficacy in hepatocellular carcinoma【Objective】To investigate the hSulf-1effect on the bFGF-stimulated cell cycleprogression and the antitumor efficacy in hepatocellular carcinoma(HCC).【Methods】 Hepatocellular carcinoma SMMC-7721cells and normal livercells WRL-68were treated with bFGF, and then infected with Ad5-hSulf1. The cellcycle was detected by flow cytometry, the relative expression levels of cellcycle-related proteins CDK4and E2F were measured by Western blotting. Theactivity of cell proliferation, invasion and migration was detected by MTT, transwell,wound healing experiments, respectively.【Results】 The flow cytometry results showed that, the hSulf-1gene can antagonizethe bFGF-induced stimulation of cell cycle distribution changes, inducedcell G2/M phase arrest. The western blotting analysis showed that the relativeexpression of CDK4and E2F was increased after stimulated by bFGF in SMMC-7721cells, meanwhile, the hSulf-1can significantly antagonize the bFGF effect and reducethe expression of these two proteins. The MTT assay showed that, the hSulf-1canreverses the bFGF induced proliferation of cancer cells. The transwell and woundhealing assays showed the hSulf-1can reverse the action of bFGF and inhibit theinvasion and migration of SMMC-7721cells.【Conclusion】 The hSulf-1gene reverses bFGF-stimulated cell cycleprogression and inhibits cell proliferation, invasion and migration in HCC cells.Part IV Adenovirus-mediated hSulf-1expression increasesthe sensitivity toAZD2281in human breast cancer MCF-7cells【Objective】 To investigate the potential mechanism of hSulf-1effect onsensitivity of MCF-7cells toAZD2281.【Methods】The MCF-7cells were treated with AZD2281and infected withAd5-hSulf1. Cell cycle was determined by flow cytometry (FCM); The expression ofp-AKT (phosphorylated protein kinase B) and CDK4was detected by Westernblotting; Colony-formation of MCF-7cells were assessed by soft agarose assay; Cellmigration ability was evaluated by Transwell; Cell viability was examined by MTTassay.【Results】 The inhibitory effect of AZD2281came to peak value atconcentration of7μM. When MCF-7cells was treated with AZD2281and Ad5-hSulf1,compared with the group of AZD2281used alone, cell cycle was arrested atG2/M-phase, and the expression of CDK4and p-AKT was down-regulated obviously.Meanwhile, the combination of AZD2281and Ad5-hSulf1also resulted in a decreaseof neoplastic capacity and capacity of proliferation of MCF-7cells.【Conclusion】 The expression of hSulf-1can increase MCF-7cell sensitivityto AZD2281, and result in cell cycle arrest at G2/M-phase and decrease of cell proliferation and migration. These results provided a new basis for clinical referencein the therapy of breast cancer.