Effects Of Glycosylation Modification On Bioactivities Of Antitumor Drugs

With the development of glycobiology, carbohydrate drugs including saccharide, glycosylated protein, lipids and other chemical molecules, and substrates of certain enzymes are growing more and more important in new drug exploration. In order to investigate the effects of glycosylation on bioactivity of glycosylated protein drugs and chemical drugs, human recombinant interferonβ(rhIFNβ), monoclonal antibody ofα-Gal(Galα1, 3Gal) modified epidermal growth factor receptor HER2,α-Gal-Herceptin and a novel NO donor,β-galactosyl-pyrrolidinyl diazeniumdiolate (β-Gal-NONOate) were used to demonstrate the mechanism and meaning of glycosylation. These studies offered innovative thoughts and theories in drug design and reconstruction so as to achieve site-specific delivery, decreased or eliminated side-effects, declined dose and reduced production cost. What more important is to reach the optimal drug activity in vivo.1. High-level expression of recombinant human interferon-beta in dhfr^- -CHOcellsIFNβgene was amplified by PCR from pLG104R vector. The fragments were digested with restriction enzyme Xho I and Sma I, and were then cloned into the eukaryotic vector pCI-dhfr. Eukaryotic recombinant expression plasmid pCI-dhfr-ifnβwas transfected into dhfr^- -CHO cell by lipofectin, and cell strain highly expressing rhIFNβwas obtained by stressing selection of MTX. Antiviral activity of rhIFNβwas analyzed by virus-induced Cytopathic Effection Inhibition (CPEI). After characterization and purification with Blue Sepharose and CM Sepharose chromatography, dialysis and concentration with PEC 6000, the expression products were assayed with ELISA and west-blotting. As a result, eukaryotic vector pCI-dhfr- ifnβwas constituted successfully; the optimal concentration of MTX stressing selection was determined to be 500 nmol·L^-1; a stable cell line dhfr^- -CHO(9.3×10⁵ IU/10⁶cells·mL^-1)with high level rhIFNβexpression was acquired; bioactivity of rhIFNβwhich was purified and concentrated was tested to be 2.27×10⁷U·mg^-1. This study built foundation for the national CHO-expressed rhIFNβproduction on a large scale. 2. Bioactivities comparison of recombinant human interferon-beta from different expression systemsTo compare anti-virus and antitumor activities of rhIFNβproduced by E. coli, Yeast and CHO, respectively, with CPEI and MTT assay in vitro; to evaluate the therapeutic effect of rhIFNβfrom three expression systems to BALB/c mice which were transplanted with A549 tumor (human adenocarcinoma) through weight comparison of transplanted tumors. The results indicated that under the concentration of 100 ng·mL^-1, CHO-expressed rhIFNβpossessed the highest anti-virus and anti-proliferation activity; the tumor weight decrease percentages of mice treated with rhIFNβfrom E.coIi, Yeast and CHO, respectively, were 16%, 33.3% and 50.6%. Therefore, the order of rhIFNβbioactivity in vivo was CHO＞Yeast＞E.coli and the superiority of CHO-expressed glycosylated protein drug was obviously witnessed.3. Release of NO inside cells and antitumor effects of galactosylated NO donor conjugateβ-Gal-NONOateGlyeosylation is an effective way to promote drug's solubility, stability, specificity and bioactivity. This study first used C6/LacZ cells harboringβ-galactosidase activity and C6 cell withoutβ-galactosidase activity as in vitro experimental model to estimate the NO level fromβ-Gal-NONOate and NONOate with Griess assay and compare the anticancer effects ofβ-Gal-NONOate and NONOate with cell counting, trypan blue dye and MTT. The results demonstrated that the NO level fromβ-Gal-NONOate inside C6/LacZ cells was dose-dependent and apparently higher than NONOate(P＜0.01). However, in C6 cells without LacZ gene expression,β-Gal-NONOate was more stable. The toxicity to C6/LacZ cells ofβ-Gal-NONOate (IC₅₀=5 mmol·L^-1) is obviously more powerful than that of NONOate(IC₅₀=10 mmol·L^-1)(P＜0.05), but no evident toxicity to C6 cells was detected. While, there is no notable difference of NO level and antitumor activity of NONOate in C6/LacZ and C6 cells(P＞0.05). The results indicated that NO release and antitumor activity ofβ-Gal-NONOate depended onβ-galactosidase expressed by cells; that is to say,β-Gal-NONOate targeted cells expressingβ-galactosidase, but NONOate was independent ofβ-galactosidase activity. 4. Apoptosis effects of human uterine cervix cancer HeLa cells induced by beta-Gal-NONOateThrough cationic liposome-mediated transfection, the eukaryotic expression vector pcDNA3-LacZ was transferred into Hela cells; through G418 (400μg·mL^-1) screening and ONPG to detect galactosidase activity, HeLa ceil line steadily expressingβ-galactosidase was obtained; investigate the influence ofβ-Gal- NONOate on Hela cell apoptosis with LDH assay, DNA ladder electrophoresis and morphological analysis; MTT assay were used to estimate the toxicity ofβ-Gal-NONOate and NONOate to the two cell lines above, respectively. The results indicated thatβ-Gal-NONOate could induce apoptosis of Hela/LacZ cells and was dose-dependent within concentrations ranging from 0-5 mmol·L^-1; formation of apoptotic bodies was observed under fluorescent microscope and typical band of DNA ladder was also witnessed in Sepharose Electrophoresis; the results of MTT assay indicated that the cytotoxicity ofβ-Gal-NONOate toward Hela/LacZ cells is apparently higher than NONOate(P＜0.05), but no evident difference to Hela cells without transfected LacZ gene was detected(P＞0.05); the cytotoxicity of NONOate to both cell lines were similar (P＞0.05). The results showed that HeLa/LacZ cell apoptosis was induced byβ-Gal-NONOate through the expression ofβ-galactosidase intracellularly.5. Cytotoxicity enhancement of alpha-Gal modified Herceptin toward breast adenocarcinoma SK-BR-3 cellsThe binding efficiency ofα-Gal-Herceptin and SK-BR-3 cells was evaluated with flow cytometry assays; the cytotoxicity ofα-Gal-Herceptin and Herceptin, respectively, to SK-BR-3 cells was estimated through the comparison of cell survivals, activities of LDH and SDH.α-Gal antibodies in human serum guided byα-Gal-Herceptin could specifically target SK-BR-3 cells; with human serum as the source of anti-Gal and complement,α-Gal-Herceptin coated theα-Gal epitopes covered SK-BR-3 cells surface through recognition of HER2 on cancer cell surface with Herceptin, thus cancer cells became more sensitive to human serum. The cytotoxicity ofα-Gal-Herceptin was obviously higher than control and Herceptin. However, in FBS, cytotoxicity ofα-Gal-Herceptin to SK-BR-3 cells had no remarkable difference in comparison with Herceptin. In conclusion, rational glycosylation modification can not only increase bioactivities of drugs, but also improve site-specific delivery and stability of drugs. These studies offered innovative thoughts and theories in drug design and reconstruction.