ABSTRACT
Aggregation of recombinant proteins, a major problem in E. coli expression system, is improved by using EnBase culture system based on slow release of glucose. In the present study, to understand the intracellular mechanisms involved in increased solubility of the target recombinant protein through EnBase system, the effect of this system was investigated on E. coli cells proteome profile. The proteome profile of E. coli cells cultured in EnBase and conventional batch mode was analyzed by two-dimensional gel electrophoresis. The proteins with significant expressional changes were identified through MALDI-TOF/TOF mass spectrometry. In EnBase system, the expressions of carbon metabolism-related proteins, sugar transport system-related proteins, and amino acids metabolism-related proteins were significantly altered. Furthermore, the expression of Thioredoxin 1 as the facilitator of protein folding was up-regulated in EnBase system that could be related to the increased solubility of recombinant protein. The proteomics analysis of E. coli cells cultured in EnBase system revealed that Thioredoxin 1 can be a potential candidate for future studies aiming at increased anti-VEGF fab fragment solubility. Studying proteomics is a valuable tool for revealing the target proteins that play the central role in EnBase culture system for increasing the solubility.
Subject(s)
Escherichia coli/genetics , Immunoglobulin Fab Fragments/genetics , Proteomics/methods , Vascular Endothelial Growth Factor A/immunology , Electrophoresis, Gel, Two-Dimensional , Immunoglobulin Fab Fragments/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
PURPOSE: Resistance to gemcitabine in pancreatic cancer (PC) may account for the failure of conventional treatments. Recently, salinomycin (SAL) has been identified as selective inhibitor of cancer stem cells (CSCs). In our study, we aimed to deliver SAL to gemcitabine-resistant PC by the aid of poly ethylene glycol-b-poly lactic acid (PEG-b-PLA) polymeric micelles (PMs). METHODS: SAL-loaded PMs were prepared and investigated in terms of pharmaceutical properties. MTT and Annexin V/PI assays were used to study cell proliferation and apoptosis in AsPC-1 cells in response to treatment with SAL micellar formulations. Alterations in CSC phenotype, invasion strength, and mRNA expression of epithelial mesenchymal transition (EMT) markers were also determined in the treated cells. In vivo antitumor study was performed in Balb/c AsPC-1 xenograft mice. RESULTS: PM formulations of SAL were prepared in suitable size and loading traits. In gemcitabine-resistant AsPC-1 cells, SAL was found to significantly increase cell mortality and apoptosis. It was also observed that SAL micellar formulations inhibited invasion and harnessed EMT in spite of induced expression of Snail. The in vivo antitumor experiment showed significant tumor eradication and the highest survival probability in mice treated with SAL PMs. CONCLUSIONS: The obtained results showed the efficacy of SAL nano-formulation against PC tumor cells.
Subject(s)
Antineoplastic Agents/administration & dosage , Deoxycytidine/analogs & derivatives , Drug Carriers/chemistry , Drug Resistance, Neoplasm , Pancreatic Neoplasms/drug therapy , Polyethylene Glycols/chemistry , Pyrans/administration & dosage , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Chromatography, High Pressure Liquid , Deoxycytidine/administration & dosage , Deoxycytidine/pharmacokinetics , Deoxycytidine/therapeutic use , Drug Liberation , Flow Cytometry , Humans , Mice , Mice, Inbred BALB C , Micelles , NIH 3T3 Cells , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Particle Size , Pyrans/pharmacokinetics , Pyrans/therapeutic use , Solubility , Surface Properties , Xenograft Model Antitumor Assays , GemcitabineABSTRACT
CD22 marker is a highly internalizing antigen which is located on the surface of B-cells and is being used as a promising target for treatment of B cell malignancies. Monoclonal antibodies targeting CD22 have been introduced and some are currently under investigation in clinical trials. Building on the success of antibody drug conjugates, we developed a fusion protein consisting of a novel anti-CD22 scFv and apoptin and tested binding and therapeutic effects in lymphoma cells. The recombinant protein was expressed in E. coli and successfully purified and refolded. In vitro binding analysis by immunofluorescence and flow cytometry demonstrated that the recombinant protein specifically binds to CD22 positive Raji cells but not to CD22 negative Jurkat cells. The cytotoxic properties of scFv-apoptin were assessed by an MTT assay and Annexin V/PI flow cytometry analysis and showed that the recombinant protein induced apoptosis preferentially in Raji cells with no detectable effects in Jurkat cells. Our findings indicated that the recombinant anti-CD22 scFv-apoptin fusion protein could successfully cross the cell membrane and induce apoptosis with high specificity, make it as a promising molecule for immunotherapy of B-cell malignancies.
ABSTRACT
The overexpression of cyclooxygenase-2 (COX-2) enzyme has been strongly contributed to tumorigenesis. The efficacy of celecoxib as a selective COX-2 inhibitor has been shown in many studies, but the underlying mechanism as a chemopreventative agent has not yet been well known. For better understanding the chemopreventative molecular mechanisms, we used a comparative proteomics analysis of lipopolysaccharide (LPS) treated and untreated J774.A1 macrophage-like cell lines before and after treatment with celecoxib. Our findings define the contribution of several interesting proteins, including ferritin heavy chain, glyoxalase-1, cofilin, vimentin, and galectin-1, which could extend our understanding of the chemopreventative effects of celecoxib and provide new valuable tools for further anticancer research.