Morous Alba, known as white mulberry contains many oxidative flavonoids, widely used in the treatment of many diseases like hyperglycemia, inflammation, fever and cancer. In the present study we investigated the interaction of extracted flavonoids from Iranian Morus Alba leaves with DNA as a main target for anticancer drugs. Various spectroscopic techniques (UV/Vis, CD and Fluorescence Spectroscopy) were used to detect the interaction. In vivo studies also were done to confirm the effectiveness of the extracted flavonoids. The spectroscopic results showed that the extracted flavonoids bind to DNA especially to the sugar-phosphate backbone and making DNA conformational changes upon this binding. Experiments on the cancerous mice with solid tumors indicated that the treatment of mice with these extracted flavonoids increased significantly the life span but they did not have any effects on the tumor size reduction. These data suggest that Morus Alba flavonoids may use as an effective natural anticancer drug in the near future.
DAB389IL‐2 (Denileukin diftitox) is considered an immunotoxin, and it is the first immunotoxin approved by Food and Drug Administration. It is used for the treatment of a cutaneous form of T‐cell lymphoma. This fusion protein has two disulfide bonds in its structure that play an essential role in toxicity and functionality of the immunotoxin. Escherichia coli (E. coli) strain BL21 (DE3) is not capable of making disulfide bonds in its reductive cytoplasm, but the E. coli strain Rosetta‐gami (DE3) is a proper strain for the correct expression of the protein due to mutations in glutaredoxin reductase and thioredoxin reductase. In this study, a pET21a vector with the His6‐tag fused at the N‐terminus of DAB389IL‐2 was used to express the soluble immunotoxin in E. coli Rosetta‐gami (DE3). After the purification of the soluble protein by two‐step column chromatographies, the structure of DAB389IL‐2 was analyzed using the Native‐PAGE and circular dichroism methods. In the following, the nuclease activity of soluble DAB389IL‐2 and its cytotoxicity activity were determined. It is concluded that the soluble recombinant protein expressed in the E. coli Rosetta‐gami (DE3) has an intact structure and also functional; hence, this form of immunotoxin could be competitive with its commercial counterparts.
Background: DAB389IL-2 (Denileukin diftitox) as an immunotoxin is a targeted pharmaceutical protein and is the first immunotoxin approved by FDA. It is used for the treatment of various kinds of cancer such as CTCL lymphoma, melanoma, and Leukemia but among all of these, treatment of CTCL has special importance. DAB389IL-2 consists of two distinct parts; the catalytic domain of Diphtheria Toxin (DT) that genetically fused to the whole IL-2. Deamidation is the most important reaction for chemical instability of proteins occurs during manufacture and storage. Deamidation of asparagine residues occurs at a higher rate than glutamine residues. The structure of proteins, temperature and pH are the most important factors that influence the rate of deamidation. Methods: Since there is not any information about deamidation of DAB389IL-2, we studied in silico deamidation by Molecular Dynamic (MD) simulations using GROMACS software. The 3D model of fusion protein DAB389IL-2 was used as a template for deamidation. Then, the stability of deamidated and native form of the drug was calculated. Results: The results of MD simulations were showed that the deamidated form of DAB389IL-2 is more unstable than the normal form. Also, deamidation was carried by incubating DAB389IL-2, 0.3 mg/ml in ammonium hydrogen carbonate for 24 h at 37o C in order to in vitro experiment. Conclusion: The results of in vitro experiment were confirmed outcomes of in silico study. In silico and in vitro experiments were demonstrated that DAB389IL-2 is unstable in deamidated form.
Background: DAB 389 IL-2 is considered a fusion immunotoxin and used for the treatment of Cutaneous T Cell Lymphoma (CTCL). It is composed of two distinct portions; the catalytic domain of diphtheria toxin and IL-2. Because of DAB 389 IL-2 free cysteine residue (Cys 513 in IL-2 part), it is prone to unwanted intramolecular and intermolecular disulfide bonds formation and aggregation problems. Aggregation is considered as the most common physical instability. PEGylation is a practical approach to increase the stability and half-life of therapeutic proteins. Materials and Methods: In this study, the PEGylation of recombinant DAB 389 IL-2 was performed by mPEG-vinylsulfone, through partial denaturation condition at 4° C for 24 h. To confirm the PEGylation reaction, SDS-PAGE and Dynamic Light Scattering (DLS) was used. The structure of DAB 389 IL-2 and its PEGylated immunotoxin form were analyzed using the Circular Dichroism (CD) and fluorescence methods. Also, the K562 cells line were treated with various concentrations of DAB 389 IL-2 and conjugated form. In the following, the nuclease activities of DAB 389 IL-2 and PEGylated form were determined. Results: The SDS-PAGE result confirmed the site-specific PEGylation of DAB 389 IL-2. Spectroscopy results exhibited that the PEGylation does not affect the protein original structure. Also, the cytotoxicity assay and nuclease activity test confirmed that PEGylated protein induces death in K562 cells line and DNA degradation, respectively. Conclusion: PEGylated immunotoxin DAB 389 IL-2 has a proper structure and function; thus, PEGylated immunotoxin requires more study because of its unique properties.
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