Anti-proliferative activity of disulfiram through regulation of the AKT-FOXO axis: A proteomic study of molecular targets.

Due to its potent anti-tumor activity, well-investigated pharmacokinetic properties and safety profile, disulfiram (DSF) has emerged as a promising candidate for drug repurposing in cancer therapy. Although several molecular mechanisms have been proposed for its anti-cancer effects, the precise underlying mechanisms remain unclear. In the present study, we showed that DSF inhibited proliferation of cancer cells by inducing reactive oxygen species (ROS) production, a G1 cell cycle arrest and autophagy. Moreover, DSF triggered apoptosis via suppression of the anti-apoptotic protein survivin. To elucidate the mechanisms for the anti-proliferative activities of DSF, we applied a 2-DE combined with MALDI-TOF-MS/MS analysis to identify differentially expressed proteins in breast cancer cells upon treatment with DSF. Nine differentially expressed proteins were identified among which, three candidates including calmodulin (CaM), peroxiredoxin 1 (PRDX1) and collagen type I alpha 1 (COL1A1) are involved in the regulation of the AKT signaling pathway. The results of western blot analysis confirmed that DSF inhibited p-AKT, suggesting that DSF induces its anti-tumor effects via AKT blockade. Moreover, we found that DSF increased the mRNA levels of FOXO1, FOXO3 and FOXO4, and upregulated the expression of their target genes involved in G1 cell cycle arrest, apoptosis and autophagy. Finally, DSF potentiated the anti-proliferative effects of well-known chemotherapeutic agents such as arsenic trioxide (ATO), doxorubicin, paclitaxel and cisplatin. Altogether, these findings provide mechanistic insights into the anti-growth activities of DSF.

Cancer biomarkers in atherosclerotic plaque: Evidenced from structural and proteomic analyses.

Atherosclerosis and cancer are the leading causes of mortality around the world that share common pathogenic pathways. The aim of this study is the investigation of the protein profile of atherosclerotic plaque in order to find similar biomarker between cancer and atherosclerosis. The small pieces of human coronary artery containing advanced atherosclerotic plaque is obtained from patients during bypass surgery. Structural characterization of type V plaque, including fibrous connective tissue, necrotic lipid core, cholesterol clefts and calcium deposits are performed using high resolution transmission electron microscopy (HR-TEM). The protein profile of atherosclerosis plaque is also analyzed using 2-dimensional electrophoresis and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF). TEM analysis shows that vascular smooth muscle cells (VSMCs) exhibit different and uncommon morphologies in atherosclerotic plaque which is correlated to the proliferative state of the cells. The proteomics analysis reveals proteins related to atherosclerosis formation including Mimecan, Ras Suppressor Protein-1 (RSUP-1) and Cathepsin D which identified as biomarker of cancerous tumors. The expression of Mimecan and RSUP-1 is down-regulated in atherosclerotic plaque while the expression of Cathepsin D is up-regulated. These data support that atherosclerotic plaque presents some degree of tumorgenesis with the significant activity of VSMCs as the key player in atherogenesis.

Engineering E. coli cell surface in order to develop a one-step purification method for recombinant proteins.

Sortases are enzymes mostly found in Gram-positive bacteria which cleave proteins site-specifically. This feature makes them a promising tool in molecular biology and biotechnology. In this study, using bacterial surface display of recombinant proteins and ability of sortase A in site-specifically cleavage of the amino acid sequences, a novel method for one-step purification of recombinant proteins was developed. Using computational program tools, a chimeric protein containing a metallothionein (mt) and chitin binding domain (ChBD) was attached to the C-terminal domain of the truncated outer membrane protein A (Lpp'-ompA) using sortase recognition site (amino acid residues: LPQTG) as a separator. The structure of the chimeric protein was simulated using molecular dynamics to determine if the LPQTG motif is accessible to the sortase active site. The designed chimeric protein was expressed and purified. The purified chimeric protein was also displayed on the surface of E. coli cells. Both purified chimeric protein and the E. coli cells displaying Lpp'-ompA-mt-ChBD carrier protein were then treated with sortase to evaluate the efficiency of sortase-mediated cleavage of purified chimeric protein as well as surface displayed-chimeric protein. It is shown that mt-ChBD protein was successfully cleaved and dissociated from Lpp'-ompA carrier and released into the medium after treatment with sortase in both recombinant protein and surface displayed-chimeric protein. The experimental results confirmed the molecular dynamics analysis results. The presented method could be regarded as a novel strategy for one step expression and purification of recombinant proteins.

A Comparative Cytotoxic Evaluation of Disulfiram Encapsulated PLGA Nanoparticles on MCF-7 Cells.

Background: Disulfiram is oral aldehyde dehydrogenase (ALDH) inhibitor that has been used in the treatment of alcoholism. Recent studies show that this drug has anticancer properties; however, its rapid degradation has limited its clinical application. Encapsulation of disulfiram polymeric nanoparticles (NPs) may improve its anticancer activities and protect rapid degradation of the drug. Materials andMethods: A poly (lactide-co-Glycolide) (PLGA) was developed for encapsulation of disulfiram and its delivery into breast cancer cells. Disulfiram encapsulated PLGA NPs were prepared by nanoprecipitation method and were characterized by Scanning Electron Microscopy (SEM). The loading and encapsulation efficiency of NPs were determined using UV-Visible spectroscopy. Cell cytotoxicity of free and encapsulated form of disulfiram is also determined using MTT assay. Results: Disulfiram encapsulated PLGA NPs had uniform size with 165 nm. Drug loading and entrapment efficiency were 5.35 ±0.03% and 58.85±1.01%. The results of MTT assay showed that disulfiram encapsulated PLGA NPs were more potent in induction of apoptosis compare to free disulfiram. Conclusion: Based on the results obtained in the present study it can be concluded that encapsulation of disulfiram with PLGA can protect its degradation in improve its cytotoxicity on breast cancer cells.

Combining NT3-overexpressing MSCs and PLGA microcarriers for brain tissue engineering: A potential tool for treatment of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that characterized by destruction of substantia nigrostriatal pathway due to the loss of dopaminergic (DA) neurons. Regardless of substantial efforts for treatment of PD in recent years, an effective therapeutic strategy is still missing. In a multidisciplinary approach, bone marrow derived mesenchymal stem cells (BMSCs) are genetically engineered to overexpress neurotrophin-3 (nt-3 gene) that protect central nervous system tissues and stimulates neuronal-like differentiation of BMSCs. Poly(lactic-co-glycolic acid) (PLGA) microcarriers are designed as an injectable scaffold and synthesized via double emulsion method. The surface of PLGA microcarriers are functionalized by collagen as a bioadhesive agent for improved cell attachment. The results demonstrate effective overexpression of NT-3. The expression of tyrosine hydroxylase (TH) in transfected BMSCs reveal that NT-3 promotes the intracellular signaling pathway of DA neuron differentiation. It is also shown that transfected BMSCs are successfully attached to the surface of microcarriers. The presence of dopamine in peripheral media of cell/microcarrier complex reveals that BMSCs are successfully differentiated into dopaminergic neuron. Our approach that sustains presence of growth factor can be suggested as a novel complementary therapeutic strategy for treatment of Parkinson disease.

The inhibitory effect of disulfiram encapsulated PLGA NPs on tumor growth: Different administration routes.

The strong anticancer activity of disulfiram is hindered by its rapid degradation in blood system. A novel folate-receptor-targeted poly (lactide-co-glycolide) (PLGA)-polyethylene glycol (PEG) nanoparticle (NP) is developed for encapsulation and delivery of disulfiram into breast cancer tumor using passive (EPR effect) and active (folate receptor) targeting. The anticancer activity of disulfiram and its effect on caspase-3 activity and cell cycle are studied. The administration of encapsulated PLGA NPs using intra-peritoneal, intravenous and intra-tumor routes is investigated using animal model. Disulfiram shows strong cytotoxicity against MCF7 cell line. The activity of caspase-3 inhibited with disulfiram via dose dependent manner while the drug causes cell cycle arrest in G0/G1 and S phase time-dependently. The encapsulated disulfiram shows higher activity in apoptosis induction as compared to free drug. In nontoxic dose of encapsulated disulfiram, the highest and lowest efficacy of NPs in tumor growth inhibition is observed for intravenous injection and intraperitoneal injection. It is suggested that administration of disulfiram by targeted PLGA nanoparticles using intravenous injection would present an alternative therapeutic approach for solid tumor treatment.

Delivery of disulfiram into breast cancer cells using folate-receptor-targeted PLGA-PEG nanoparticles: in vitro and in vivo investigations.

BACKGROUND: A folate-receptor-targeted poly (lactide-co-Glycolide) (PLGA)-Polyethylene glycol (PEG) nanoparticle is developed for encapsulation and delivery of disulfiram into breast cancer cells. After a comprehensive characterization of nanoparticles, cell cytotoxicity, apoptosis induction, cellular uptake and intracellular level of reactive oxygen species are analyzed. In vivo acute and chronic toxicity of nanoparticles and their efficacy on inhibition of breast cancer tumor growth is studied. RESULTS: The folate-receptor-targeted nanoparticles are internalized into the cells, induce reactive oxygen species formation, induce apoptosis and inhibit cell proliferation more efficiently compared to the untargeted nanoparticles. The acute and toxicity test show the maximum dose of disulfiram equivalent of nanoparticles for intra-venous injection is 6 mg/kg while show significant decrease in the breast cancer tumor growth rate. CONCLUSION: It is believed that the developed formulation could be used as a potential vehicle for successful delivery of disulfiram, an old and inexpensive drug, into breast cancer cells and other solid tumors.

Poly(ethyleneimine) functionalized carbon nanotubes as efficient nano-vector for transfecting mesenchymal stem cells.

For gene and drug delivery applications, carbon nanotubes (CNTs) have to be functionalized in order to become compatible with aqueous media and bind with genetic materials. In this study, combination of polyethyleneimine (PEI) grafted multi-walled carbon nanotubes (PEI-g-MWCNTs) and chitosan substrate is used as an efficient gene delivery system for transfection of hard-to-transfect bone marrow mesenchymal stem cells (BMSCs) with enhanced green fluorescent protein (EGFP) gene. Fourier transform infrared (FT-IR) spectra, dynamic light scattering (DLS) analysis and zeta potential measurements are used to characterize binding of PEI, particle size distribution and colloidal stability of the functionalized CNTs, respectively. DNA binding affinity, cellular uptake, transfection efficiency and possible cytotoxicity are also tested by agarose gel electrophoresis, flow cytometry, cytochemisty and MTT assay. The results demonstrate that cytotoxic effect of PEI-g-MWCNTs is negligible under optimal transfection condition. In consistency with high cellular uptake (>82%), PEI-g-MWCNTs give higher delivery of EGFP into the BMSCs which results in a more sustained expression of the model gene (EGFP) in short-term culture. These results suggest that PEI-g-MWCNTs in corporation with chitosan substrates would be a promising delivery system for BMSCs, a cell type with relevancy in the regenerative medicine and clinical applications.

Functional analysis of the sortase YhcS in Bacillus subtilis.

Sortases of Gram-positive bacteria catalyze the covalent C-terminal anchoring of proteins to the cell wall. Bacillus subtilis, a well-known host organism for protein production, contains two putative sortases named YhcS and YwpE. The present studies were aimed at investigating the possible sortase function of these proteins in B. subtilis. Proteomics analyses revealed that sortase-mutant cells released elevated levels of the putative sortase substrate YfkN into the culture medium upon phosphate starvation. The results indicate that YfkN required sortase activity of YhcS for retention in the cell wall. To analyze sortase function in more detail, we focused attention on the potential sortase substrate YhcR, which is co-expressed with the sortase YhcS. Our results showed that the sortase recognition and cell-wall-anchoring motif of YhcR is functional when fused to the Bacillus pumilus chitinase ChiS, a readily detectable reporter protein that is normally secreted. The ChiS fusion protein is displayed at the cell wall surface when YhcS is co-expressed. In the absence of YhcS, or when no cell-wall-anchoring motif is fused to ChiS, the ChiS accumulates predominately in the culture medium. Taken together, these novel findings show that B. subtilis has a functional sortase for anchoring proteins to the cell wall.