Preliminary Study On Anti-Tumor Activity Of Sulforaphane And Heterelogous Expression Of Myrosinase Gene From Brassica Oleracea Var. Italica

Sulforaphane (SFN), which has been identified in broccoli as a product of enzymatic or acid hydrolysis of the corresponding glucoraphanin(one of glucosinolates), is viewed as a conceptually promising agent in cancer prevention and/or therapy. SFN was prepared by author laboratory and its purity was about 85% determined by HPLC and GC/MS. The effect of SFN on the proliferation of human glioma cell line A172, human intestinal adenocarcinoma cell lines A549 and H460, human hepatoma carcinoma cell HepG2 was investigated in vitro by MTT assay,Hoechst fluorescent staining,Annexin V /PI fluorescent staining and FCS Technique. The results show that SFN can induce A172 cells apoptosis and SFN-mediated apoptosis depend on dose and time. In vitro experiments with SFN Simultaneously indicated a pronounced role for cell cycle arrest in its anti-cancer properties; G2/M arrest is the predominant stage of cell cycle arrest induced by SFN which indicated that SFN could inhibit cell microtube formation. Apoptosis and cell cycle arrest both contribute to the cell proliferation inhibition and the latter is the dominance mechanism for cell line A172. to contrast with cell line A172, sulforaphene concentration for cell H460 proliferation inhibition are higher than it's concentration for cell line A172, and the chief mechanism for proliferation inhibition is due to apoptosis. So the mechanisms of the proliferation inhibition are different with regard to different tumor cell lines. And it's essential to approach the mechanisms for tumor cell line when we do rearch for drug anti-tumor experiment.The proliferation inhibition evoked by SFN was also observed in tumor cell lines A549 and HepG2. Combined with the results about proliferation inhibition of SFN on tumor cell lines HeLa, HL-60, CNE, PC3 and P388 which were got by author laboratory by MTT method, the conclusion was that SFN had anti-tumor activity and can act via several mechanisms to suppress cancer progression, such as modulate cell growth and cell death signals. In conclution, SFN has a promising potential as a new anti-tumor drug.Myrosinases, thioglucoside glucohydrolases (EC 3.2.3.1) are isoenzymes present in most organs of Brassicaceae species. They catalyse the degradation of glucosinolates, a group of low molecular weight compounds to form an aglucone and D-glucose. The aglucone is unstable and spontaneously decomposes into nitriles, thiocyanates, isothiocyanates or indoles depending on the nature of the side chain and the reaction conditions. In some Brassica vegetables such as cabbage, cauliflower, brussels sprouts and broccoli, glucosinolate degradation products, especially isothiocyanates have been shown to have anticarcinogenic properties. Myrosinase-glucosinolate system in plant is also an important prevention mechanism for responding to the attacks by herbivores, entomologica, pathogenic microorganisms et al. All plant myrosinases characterized to date are glycosylated and are probably transported via the secretory pathway to the myrosin grains present in idioblasts called myrosin cells. Only when the plant tissues are damaged by physical or biological factors and the physical separation is disappeared, glucosinolates can contact with myrosinases and be hydrolyzed by myrosinases.Myrosinases have been well characterized in biochemical and molecular biological studies. Myrosinase genes have been cloned fr om oilseed rape (B. napus), turnip (B. campestris), leaf mustard (B. juncea), white mustard (Sinapis alba), and thale cress (Arabidopsis thaliana) and classified into four subtypes, MA, MB, MC and TGG, on the basis of amino acid sequences. Studies of myrosinases in B. napus and Sinapis alba showed that they are encoded by large gene families, with up to 15-20 genes in B.napus. MB showed dominant expression in seeds, seedlings, young leaves, and other organs in the mature plant, whereas MA and MC were expressed only in developing seeds. Few reports were about the Myrosinase genes from Broccoli (Brassica oleracea var. italica). In this paper, a full-length cDNA encoding myrosinase from Broccoli are cloned and characterized. And the gene is further fusion expressed in E.coli BL21 and secretory expressed in Pichia pastoris. Moreover, fermentation research of the recombinant yeast for myrosinase secretion was carried on in the last chapter.Based on the reported plant myrosinase genes in GenBank, software Primer Premier 5 was used to design degenerate primers MyrF and MyrR for amplification gene consensus regions. And then, using 3'RACE and the corresponding 5'RACE technique, the full-length cDNA of Broccoli homologous gene of Brassicaceae myrosinase (BoMyr2, GenBank number: EU004075) was obtained. The 1830bp cDNA contained an ORF of 1641bp, which coded for a polypeptide of 548 amino acids. The deduced polypeptide had a predicted molecular weight of 62.2kD, a pI of 8.71. And The predicted amino acid sequence include a signal peptide, nine N-glycosylation sites and a characteristic domain of the glycosyl hydrolases family 1 which include N-terminal signature(FiFGvAsSAYQiEgG) and active site(IYITENGFS). Homology model of the myrosinase from Broccoli was obtained with MOE on the base of the crystal structure of myrosinase from S. alba as template. Putative three-dimensional structures of the myrosinase showed a (β/α)8 barrel structure common to other glucosidases. Neighbor-joining tree constructed by MEGA 3.1 showed that BoMyr2 was belonged to myrosinase MB subfamily.The codon region of BoMyr2 was amplified using designed primers based on the RACE results and inserted into E.coli expression vector pGEX-4T-1.The recombinant plasmid was transformed into E.coli BL12 to produce expression strain which would express GST-myrosinase fusion protein. SDS-PAGE showed that the recombinant protein was highly expressed at 90KDa which is coincided with the designed protein. The protein was accumulated in the E.coli BL12 cells by IPTG induced. The product of fusion protein was fairly well after four hours IPTG induced and reached the maxium after six hours IPTG induced. Enzyme activity analysis found that the fusion protein was lack of myrosinase activity. This phenomenon maybe due to the fusion protein was deficient in modification or had a GST addition. The fusion protein presented mostly as inclusion body would also lead to enzyme deficient. So the further investigation, such as protein degeneration and regeneration, enzyme cleavage, is necessary for the aim to get enzymatic protein.Primary myrosinase has a signal peptide using different SignalP prediction models predicted. And all mature myrosinase which three dimension structure are mentioned and published in PDB database are glycosylated. Pichia pastoris expression system has glycosylation and other modification after protein translated. So it was also used to express recombinant myrosinase BoMyr2 in this paper. The interested BoMyr2 gene was got by digesting the pMD-18-BoMyr2 vector using restriction endonuclease, and then it was inserted into the secretory pPIC9K Pichia pastoris expression vector and transformed into E.coli. Positive recombinant plasmids were selected,sequenced and named pPIC9K-BoMyr2 and was linearized by Sac I, then the linear DNA transferred into Pichia pastoris KM71 by electroporation. The recombinant expression vector pPIC9K-BoMyr2 integrated into KM71 via homologous recombination between the transforming DNA and regions of homology within yeast genome. The positive transformants were screened by MD His- plates and colony PCR. The recombinants were induced expression with methanol and the expression products were tested by SDS-PAGE and enzymatic activity. The results showed that the BoMyr2 gene had been expressed successfully in Pichia pastoris and secreted in the culture medium. The expression product is about a 65 kDa protein and can degrade sinigrin in suitable condition. Six strains were selected in the experiment. Myrosinase activities in the culture supernatant of number 13 # and 15# reached 1.7-1.9U/ml. Determination of the special activity of myrosinase reflected that protein expression levels and enzyme activity had good correlation in culture supernatant. This is the first report on successful heterologous expression of a myrosinase in Pichia pastoris KM71 and provides an important tool for, e.g., further characterization of myrosinase by site-directed mutagenesis and for studying the interaction between myrosinase and myrosinase-binding proteins, myrosinase-associated proteins, and epithiospecifier proteins.Further fermentation research on number 13# strain were carried out using single factor analysis. The result show that the optimum pH and methanol concentration were respectively as 6.0 and 1.0% for myrosinase expression in BMMY medium.0.05% oleic acid or 0.1% tween-20 can also enhance myrosinase expression in small scale. Opportunity for recovering the fermentation culture was 36 hours after methanol inducing began. And myrosinase activity in the supernatant would come up to 3U/ml or so.The free, soluble form of myrosinase of the MA gene family has so far been intensively analyzed. In contrast, only limited information is available about the biochemical properties of the myrosinases encoded by the MB gene family for the reason of its low-level exression and complex form. the successful heterologous expression of recombinant BoMyr2 myrosinase from Broccoli in the yeast Pichia pastoris provides a new feasible procedure to produce free myrosinase homogeneity, facilitates studies on structure and function of the enzyme and will be a key for determining the consequences of complex formation with, e.g., myrosinase-binding proteins and myrosinaseassociated proteins, including their effection on myrosinase complex activity.