ABSTRACT
BACKGROUND: Recent studies have shown that the high mortality of patients with colorectal cancer (CRC) is related to its ability to spread the surrounding tissues, thus there is a need for designing and developing new drugs. OBJECTIVE: Here, we proposed a combinational therapy strategy, an inhibitory peptide in combination with miRNA targeting, for modulating CRC metastasis. In this study, some of the recent patents were also reviewed. METHODS: After data analysis with GEO2R and gene annotation using DAVID server, regulatory interactions of differentially expressed genes (DEGs) were obtained from STRING, GeneMANIA, KEGG and TRED databases. In parallel, the corresponding validated microRNAs (miRNAs) were obtained from mirDIP web server and a miRNA-DEG regulatory network was also reconstructed. Clustering and topological analyses of the regulatory networks were performed using Cytoscape plug-ins. RESULTS: We found the HOXB family as the most important functional complex in DEG-derived regulatory network. Accordingly, an anti-HOXB7 peptide was designed based on the binding interface of its coactivator, PBX1. Topological analysis of miRNA-DEG network indicated that hsa-miR-222 is one of the most important oncomirs involved in regulation of DEGs activities. Thus, this miRNA, along with HOXB7, was also considered as the potential target for inhibiting CRC metastasis. Molecular docking studies exhibited that the designed peptide can bind to desired binding pocket of HOXB7 in a highaffinity manner. Further confirmations were also observed in Molecular dynamics (MD) simulations carried out by GROMACS v5.0.2 simulation package. CONCLUSION: In conclusion, our findings suggest that simultaneous targeting of key regulatory genes and miRNAs may be a useful strategy for prevention of CRC metastasis.
Subject(s)
Antineoplastic Agents/pharmacology , Colorectal Neoplasms/drug therapy , Drug Design , Homeodomain Proteins/antagonists & inhibitors , MicroRNAs/genetics , Peptides/pharmacology , Antineoplastic Agents/chemistry , Cluster Analysis , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Computer-Aided Design , Data Mining , Databases, Genetic , Gene Expression Regulation, Neoplastic/drug effects , Homeodomain Proteins/metabolism , Humans , MicroRNAs/metabolism , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Structure , Molecular Targeted Therapy , Neoplasm Metastasis , Peptides/chemistry , Protein Interaction Maps , Signal Transduction/drug effects , Structure-Activity RelationshipABSTRACT
Darbepoetin alfa is an engineered and hyperglycosylated analog of recombinant human erythropoietin (EPO) which is used as a drug in treating anemia in patients with chronic kidney failure and cancer. This study desribes the secretory expression of a codon-optimized recombinant form of darbepoetin alfa in Leishmania tarentolae T7-TR. Synthetic codon-optimized gene was amplified by PCR and cloned into the pLEXSY-I-blecherry3 vector. The resultant expression vector, pLEXSYDarbo, was purified, digested, and electroporated into the L. tarentolae. Expression of recombinant darbepoetin alfa was evaluated by ELISA, reverse-transcription PCR (RT-PCR), Western blotting, and biological activity. After codon optimization, codon adaptation index (CAI) of the gene raised from 0.50 to 0.99 and its GC% content changed from 56% to 58%. Expression analysis confirmed the presence of a protein band at 40 kDa. Furthermore, reticulocyte experiment results revealed that the activity of expressed darbepoetin alfa was similar to that of its equivalent expressed in Chinese hamster ovary (CHO) cells. These data suggested that the codon optimization and expression in L. tarentolae host provided an efficient approach for high level expression of darbepoetin alfa.
Subject(s)
Cloning, Molecular , Darbepoetin alfa/genetics , Leishmania/genetics , Protein Engineering/methods , Protozoan Proteins/genetics , Animals , CHO Cells , Codon/genetics , Codon/metabolism , Cricetinae , Cricetulus , Darbepoetin alfa/metabolism , Gene Expression , Protozoan Proteins/metabolismABSTRACT
Oxidoreductases are an important family of enzymes that are used in many biotechnological processes. An experimental design was applied to optimize partition and purification of two recombinant oxidoreductases, glucose dehydrogenase (GDH) from Bacillus subtilis and d-galactose dehydrogenase (GalDH) from Pseudomonas fluorescens AK92 in aqueous two-phase systems (ATPS). Response surface methodology (RSM) with a central composite rotatable design (CCRD) was performed to optimize critical factors like polyethylene glycol (PEG) concentration, concentration of salt and pH value. The best partitioning conditions was achieved in an ATPS composed of 12% PEG-6000, 15% K2HPO4 with pH 7.5 at 25°C, which ensured partition coefficient (KE) of 66.6 and 45.7 for GDH and GalDH, respectively. Under these experimental conditions, the activity of GDH and GalDH was 569.5U/ml and 673.7U/ml, respectively. It was found that these enzymes preferentially partitioned into the top PEG-rich phase and appeared as single bands on SDS-PAGE gel. Meanwhile the validity of the response model was confirmed by a good agreement between predicted and experimental results. Collectively, according to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of any enzyme from oxidoreductase family.
