Expression and purification of human interferon alpha 2a (IFNα2a) in the methylotrophic yeast Pichia pastoris.

Human interferon alpha 2a (IFNα2a) is a secreted glycoprotein that exerts a wide spectrum of biological effects, such as triggering of antiviral, antitumor and immunosuppressive responses. IFNα2a is used as pharmaceutical polypeptide in chronic hepatitis C virus (HCV) infection, chronic myelogenous leukemia, advanced renal cell carcinoma, and metastatic malignant melanoma. So far, the pharmaceutical polypeptide of this cytokine is produced in prokaryotic expression systems (E. coli). Here we report the expression and purification of recombinant human IFNα2a in the methylotrophic yeast Pichia pastoris. The cDNA encoding for human IFNα2a, modified to bear the P. pastoris codon bias, was cloned into the pPinkα-HC vector in order to be expressed as a secreted protein upon induction. Proper expression and secretion of recombinant human IFNα2a (approximately 19 kDa) was confirmed by PCR-sequencing, SDS-PAGE and Western blot analysis following methanol-induced expression in a number of individual transformed strains. Purification of the recombinant protein was performed by affinity chromatography, achieving a robust yield of purified active form. The purified recombinant protein showed an impressive stability to thermal denaturation as observed by Differential Scanning Fluorimetry. The biological activity of the P. pastoris-produced IFNα2a was confirmed in A549 and HT29 cells by monitoring transcriptional up-regulation of a panel of known interferon-stimulated genes (ISGs). Our results document that the P. pastoris expression system is a suitable system for producing biologically functional IFNα2a in a secreted form.

Identification of a peptide ligand for human ALDH3A1 through peptide phage display: Prediction and characterization of protein interaction sites and inhibition of ALDH3A1 enzymatic activity.

Aldehyde dehydrogenase 3A1 (ALDH3A1) by oxidizing medium chain aldehydes to their corresponding carboxylic acids, is involved in the detoxification of toxic byproducts and is considered to play an important role in antioxidant cellular defense. ALDH3A1 has been implicated in various other functions such as cell proliferation, cell cycle regulation, and DNA damage response. Recently, it has been identified as a putative biomarker of prostate, gastric, and lung cancer stem cell phenotype. Although ALDH3A1 has multifaceted functions in both normal and cancer homeostasis, its modes of action are currently unknown. To this end, we utilized a random 12-mer peptide phage display library to identify efficiently human ALDH3A1-interacting peptides. One prevailing peptide (P1) was systematically demonstrated to interact with the protein of interest, which was further validated in vitro by peptide ELISA. Bioinformatic analysis indicated two putative P1 binding sites on the protein surface implying biomedical potential and potent inhibitory activity of the P1 peptide on hALDH3A1 activity was demonstrated by enzymatic studies. Furthermore, in search of potential hALDH3A1 interacting players, a BLASTp search demonstrated that no protein in the database includes the full-length amino acid sequence of P1, but identified a list of proteins containing parts of the P1 sequence, which may prove potential hALDH3A1 interacting partners. Among them, Protein Kinase C Binding Protein 1 and General Transcription Factor II-I are candidates of high interest due to their cellular localization and function. To conclude, this study identifies a novel peptide with potential biomedical applications and further suggests a list of protein candidates be explored as possible hALDH3A1-interacting partners in future studies.

Investigating the Functional Roles of Aldehyde Dehydrogenase 3A1 in Human Corneal Epithelial Cells.

Aldehyde dehydrogenase 3A1 (ALDH3A1) oxidizes medium-chain aldehydes to their corresponding carboxylic acids. It is expressed at high rates in the human cornea, where it has been characterized as a multi-functional protein displaying various cytoprotective modes of action. Previous studies identified its association with the DNA damage response (DDR) pathway. Here, we utilized a stable transfected HCE-2 (human corneal epithelium) cell line expressing ALDH3A1, to investigate the molecular mechanisms underlying the cytoprotective role(s) of ALDH3A1. Our data revealed morphological differences among the ALDH3A1-expressing and the mock-transfected HCE-2 cells accompanied by differential expression of E-cadherin. Similarly, the ALDH3A1/HCE-2 cells demonstrated higher mobility, reduced proliferation, upregulation of ZEB1, and downregulation of CDK3, and p57. The expression of ALDH3A1 also affected cell cycle progression by inducing the sequestration of HCE-2 cells at the G2/M phase. Following 16 h cell treatments with either H2O2 or etoposide, a significantly lower percentage of ALDH3A1/HCE-2 cells were apoptotic compared to the respective treated mock/HCE-2 cells. Interestingly, the protective effect of ALDH3A1 expression under these oxidative and genotoxic conditions was accompanied by a reduced formation of γ-H2AX foci and higher levels of total and phospho (Ser15) p53. Finally, ALDH3A1 was found to be localized both in the cytoplasm and the nucleus of transfected HCE-2 cells. Its cellular compartmentalization was not affected by oxidant treatment, while the mechanism by which ALDH3A1 translocates to the nucleus remains unknown. In conclusion, ALDH3A1 protects cells from both apoptosis and DNA damage by interacting with key homeostatic mechanisms associated with cellular morphology, cell cycle, and DDR.

The Concept of Cancer Stem Cells: Elaborating on ALDH1B1 as an Emerging Marker of Cancer Progression.

Cancer is a multifactorial, complex disease exhibiting extraordinary phenotypic plasticity and diversity. One of the greatest challenges in cancer treatment is intratumoral heterogeneity, which obstructs the efficient eradication of the tumor. Tumor heterogeneity is often associated with the presence of cancer stem cells (CSCs), a cancer cell sub-population possessing a panel of stem-like properties, such as a self-renewal ability and multipotency potential. CSCs are associated with enhanced chemoresistance due to the enhanced efflux of chemotherapeutic agents and the existence of powerful antioxidant and DNA damage repair mechanisms. The distinctive characteristics of CSCs make them ideal targets for clinical therapeutic approaches, and the identification of efficient and specific CSCs biomarkers is of utmost importance. Aldehyde dehydrogenases (ALDHs) comprise a wide superfamily of metabolic enzymes that, over the last years, have gained increasing attention due to their association with stem-related features in a wide panel of hematopoietic malignancies and solid cancers. Aldehyde dehydrogenase 1B1 (ALDH1B1) is an isoform that has been characterized as a marker of colon cancer progression, while various studies suggest its importance in additional malignancies. Here, we review the basic concepts related to CSCs and discuss the potential role of ALDH1B1 in cancer development and its contribution to the CSC phenotype.

Aldehyde Dehydrogenase 1B1 Is Implicated in DNA Damage Response in Human Colorectal Adenocarcinoma.

Aldehyde dehydrogenase 1B1 (ALDH1B1) has been correlated with colorectal tumorigenesis and is considered a potential biomarker for colon cancer. Its expression has been associated with attenuation of the cell cycle in the G2/M phase and resistance to DNA damaging agents. The present study examines the role of ALDH1B1 in DNA damage response (DDR) in human colorectal adenocarcinoma. To this end, we utilized an isogenic HT29 cell line pair differing in the expression of ALDH1B1. The overexpression of ALDH1B1 was related to the translational upregulation of the total and phosphorylated (at ser15) p53. Comet and apoptosis assays revealed that the expression of ALDH1B1 protected HT29 cells from etoposide-induced DNA damage as well as apoptosis, and its overexpression led to increased constitutive phosphorylation of H2AX (at ser139). Furthermore, the expression profile of a variety of DNA damage signaling (DDS)-related genes was investigated by utilizing the RT2 profiler™ PCR array. Our results demonstrated that ALDH1B1 triggered a transcriptional activation of several DNA repair-related genes (MRE11A, PMS1, RAD18 and UNG). Finally, Spearman's rank correlation coefficient analysis in 531 publicly available colorectal adenocarcinoma clinical samples indicated the statistically significant positive correlation between ALDH1B1 and DDR and repair genes or proteins, such as APEX1, FEN1, MPG, UNG, XRCC1, DDB1, XPC, CIB1, MRE11, PRKDC, RAD50, RAD21, TP53BP1, XRCC6 and H2AX. Collectively, our results suggest that ALDH1B1 may play an essential role in the DDR and DNA repair processes. Further studies on ALDH1B1 will elucidate its precise role in DDR.

Zn(II) 5,5-Diethylbarbiturate Complex Selectively Induces Apoptosis in Breast Cancer and Breast Cancer Stem-Like Cells.

The biological activities of Zn(II) compounds have been extensively studied in recent years. In this study, the growth suppressive effect of Zn(II) 5,5-diethylbarbiturate complex on MCF-7 and MDA-MB-231 human breast cancer cells was determined by SRB and ATP viability assays and apoptosis-inducing effect by double staining method. Significant increase in cytokeratin 18 level, caspase 3/7 activity and annexin-V upregulation prove that Zn(II) complex has apoptotic effect in breast cancer cells. Intrinsic apoptosis pathway in MCF-7 cells and extrinsic apoptosis pathway in MDA-MB-231 cells was determined by Western blot (PARP, Cleave PARP, BAX, COX4, RIP, Caspase 8, Split Caspase 8, DR4 and B-Actin) and RT-PCR (PARP, Fas, Bcl-2, TNF10A, P53) analysis. No reduction of viability was found in MCF-710A healthy breast cells treated with Zn(II) complex. In breast cancer stem-like cells (MCF-7s), the Zn(II) complex was found to have a cytotoxic effect and to activate the apoptotic pathway. As a result, it was concluded that Zn(II) complex has anti-proliferative and apoptotic effects on breast cancer and breast cancer stem-like cells. Also this complex prevents the metastatic effect of cancer cells and does not effect to healthy cells so this complex has a specific effect on cancer cells. These findings might shed light on the discovery of new chemotherapeutic agents.

Profiling of Aldehyde Dehydrogenase Isoforms in In Vitro Formed Tumorspheres.

BACKGROUND/AIM: Aldehyde dehydrogenases (ALDHs) are considered as markers for normal and cancer stem cells (CSC) and are involved in cell metabolism, proliferation, differentiation, stemness, and retinoic acid (RA) biosynthesis. The aim of the present study was to identify the ALDH isoforms that are associated with the CSC phenotype in non-small cell lung and hepatocellular carcinomas. MATERIALS AND METHODS: We utilized lung (A549) and hepatocellular (HepG2) cancer cells and generated tumor spheres to isolate the CSC sub-population. RESULTS: The CSC enrichment was confirmed by the up-regulation of various CSC-related genes. Comparative qPCR analysis indicated the up-regulation of several ALDH isoforms in A549 and HepG2 spheres. Interestingly, cyclin D1 and Akt, down-stream targets of the RA signaling pathway, were also shown to be significantly up-regulated in both sphere populations. CONCLUSION: Specific ALDH isoforms appear to be important mediators for the acquisition of an CSC phenotype and thus, are potential promising targets for CSC-based therapeutic approaches in lung and hepatocellular carcinomas.

A New Controlled Release System for Propolis Polyphenols and Its Biochemical Activity for Skin Applications.

Propolis is a resinous substance produced by bees that exhibits antimicrobial, immunostimulatory and antioxidant activity. Its use is common in functional foods, cosmetics and traditional medicine despite the fact that it demonstrates low extraction yields and inconsistency in non-toxic solvents. In this work, a new encapsulation and delivery system consisting of liposomes and cyclodextrins incorporating propolis polyphenols has been developed and characterized. The antioxidant, antimutagenic and antiaging properties of the system under normal and UVB-induced oxidative stress conditions were investigated in cultured skin cells and/or reconstituted skin model. Furthermore, the transcript accumulation for an array of genes involved in many skin-related processes was studied. The system exhibits significant polyphenol encapsulation efficiency, physicochemical stability as well as controlled release rate in appropriate conditions. The delivery system can retain the anti-mutagenic, anti-oxidative and anti-ageing effects of propolis polyphenols to levels similar and comparable to those of propolis methanolic extracts, making the system ideal for applications where non-toxic solvents are required and controlled release of the polyphenol content is desired.

Aldehyde Dehydrogenase 1B1 Is Associated with Altered Cell Morphology, Proliferation, Migration and Chemosensitivity in Human Colorectal Adenocarcinoma Cells.

Aldehyde dehydrogenases (ALDHs) are NAD(P)+-dependent enzymes that catalyze the oxidation of endogenous and exogenous aldehydes to their corresponding carboxylic acids. ALDHs participate in a variety of cellular mechanisms, such as metabolism, cell proliferation and apoptosis, as well as differentiation and stemness. Over the last few years, ALDHs have emerged as cancer stem cell markers in a wide spectrum of solid tumors and hematological malignancies. In this study, the pathophysiological role of ALDH1B1 in human colorectal adenocarcinoma was investigated. Human colon cancer HT29 cells were stably transfected either with human green fluorescent protein (GFP)-tagged ALDH1B1 or with an empty lentiviral expression vector. The overexpression of ALDH1B1 was correlated with altered cell morphology, decreased proliferation rate and reduced clonogenic efficiency. Additionally, ALDH1B1 triggered a G2/M arrest at 24 h post-cell synchronization, probably through p53 and p21 upregulation. Furthermore, ALDH1B1-overexpressing HT29 cells exhibited enhanced resistance against doxorubicin, fluorouracil (5-FU) and etoposide. Finally, ALDH1B1 induced increased migratory potential and displayed epithelial-mesenchymal transition (EMT) through the upregulation of ZEB1 and vimentin and the consequent downregulation of E-cadherin. Taken together, ALDH1B1 confers alterations in the cell morphology, cell cycle progression and gene expression, accompanied by significant changes in the chemosensitivity and migratory potential of HT29 cells, underlying its potential significance in cancer progression.

Honey Extracts Exhibit Cytoprotective Properties against UVB-Induced Photodamage in Human Experimental Skin Models.

In the present study, we aimed to examine the antioxidant, antiaging and photoprotective properties of Greek honey samples of various botanical and geographical origin. Ethyl-acetate extracts were used and the and the total phenolic/flavonoid content and antioxidant capacity were evaluated. Honey extracts were then studied for their cytoprotective properties against UVB-induced photodamage using human immortalized keratinocytes (HaCaT) and/or reconstituted human skin tissue models. Specifically, the cytotoxicity, oxidative status, DNA damage and gene expression levels of specific matrix metalloproteinases (MMPs) were examined. Overall, the treatment of HaCaT cells with honey extracts resulted in lower levels of DNA strand breaks and attenuated the decrease in cell viability following UVB exposure. Additionally, honey extracts significantly decreased the total protein carbonyl content of the irradiated cells, however, they had no significant effect on their total antioxidant status. Finally, the extracts alleviated the UVB-induced up-regulation of MMPs-3, -7 and -9 in a model of reconstituted skin tissue. In conclusion, honey extracts exhibited significant photoprotective and antiaging properties under UVB exposure conditions and thus could be further exploited as promising agents for developing novel and naturally-based, antiaging cosmeceutical products.

Assessment of the Antimicrobial, Antioxidant, and Antiproliferative Potential of Sideritis raeseri subps. raeseri Essential Oil.

The aim of the present study was to investigate the antimicrobial potential of Sideritis raeseri subps. raeseri essential oil (EO) against common food spoilage and pathogenic microorganisms and evaluate its antioxidant and antiproliferative activity. The EO was isolated by steam distillation and analyzed by GC/MS. The main constituents identified were geranyl-p-cymene (25.08%), geranyl-γ-terpinene (15.17%), and geranyl-linalool (14.04%). Initially, its activity against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Listeria monocytogenes, Salmonella Enteritidis, Salmonella Typhimurium, Pseudomonas fragi, Saccharomyces cerevisiae, and Aspergillus niger was screened by the disk diffusion method. Subsequently, minimum inhibitory concentration (MIC), non-inhibitory concentration (NIC), and minimum lethal concentration (MLC) values were determined. Growth inhibition of all microorganisms tested was documented, although it was significantly lower compared to gentamycin, ciproxin, and voriconazole, which were used as positive controls. In a next step, its direct antioxidant properties were examined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, and the IC50 values were determined. The potential cytoprotective activity of the oil against H2O2-induced oxidative stress and DNA damage was studied in human immortalized keratinocyte (HaCaT) cells using the comet assay. Finally, the antiproliferative activity of the oil was evaluated against a panel of cancer cell lines including A375, Caco2, PC3, and DU145 and the non-cancerous HaCaT cell line using the sulforhodamine B (SRB) assay, and the EC50 values were determined. The oil demonstrated weak radical scavenging activity, noteworthy cytoprotective activity against H2O2-induced oxidative stress and DNA damage in HaCaT cells, and antiproliferative activity against all cell lines tested, being more sensitive against the in vitro model of skin melanoma.

Aldehyde dehydrogenase 3A1 confers oxidative stress resistance accompanied by altered DNA damage response in human corneal epithelial cells.

Aldehyde dehydrogenase 3A1 is constitutively expressed in a taxon-specific manner in the cornea, where, due to its high abundance, it has been characterized as a corneal crystallin. ALDH3A1 has been proposed to be a multifaceted protein that protects cellular homeostasis through several modes of action. The present study examines the mechanisms by which ALDH3A1 exerts its cytoprotective role under conditions of oxidative stress. To this end, we have utilized an isogenic HCE-2 (human corneal epithelium) cell line pair differing in the expression of ALDH3A1. Single cell gel electrophoresis assay and H2DCFDA analysis revealed that the expression of ALDH3A1 protected HCE-2 cells from H2O2-, tert-butyl peroxide- and etoposide-induced oxidative and genotoxic effects. Furthermore, comparative qPCR analysis revealed that a panel of cell cycle (Cyclins B1, B2, D, E), apoptosis (p53, BAX, BCL-2, BCL-XL) and DNA damage response (DNA-PK, NBS1) genes were up-regulated in the ALDH3A1 expressing HCE-2 cells. Moreover, the expression profile of a variety of DNA damage signaling (DDS)-related genes, was investigated (under normal and oxidative stress conditions) by utilizing the RT2 profiler™ PCR array in both isogenic HCE-2 cell lines. Our results demonstrated that several genes associated with ATM/ATR signaling, cell cycle regulation, apoptosis and DNA damage repair were differentially expressed under all conditions tested. In conclusion, this study suggests that ALDH3A1 significantly contributes to the antioxidant defense of corneal homeostasis by maintaining DNA integrity possibly through altering the expression of specific DDS-related genes. Further studies will shed light on the precise role(s) of this multifunctional protein.

Propolis Extracts Inhibit UV-Induced Photodamage in Human Experimental In Vitro Skin Models.

The aim of this study was to assess the antioxidant, photoprotective, and antiaging effects of Greek propolis. Propolis was subjected to n-heptane or methanol extraction. Total phenolic/flavonoid content and antioxidant potential were determined in the extracts. Promising extracts were evaluated for their cytoprotective properties using human immortalized keratinocyte (HaCaT) or reconstituted human skin tissue following exposure to UVB. Assessment of cytotoxicity, DNA damage, oxidative status, and gene/protein expression levels of various matrix metalloproteinases (MMPs) were performed. The propolis methanolic fractions exhibited higher total phenolic and flavonoid contents and significant in vitro antioxidant activity. Incubation of HaCaT cells with certain methanolic extracts significantly decreased the formation of DNA strand breaks following exposure to UVB and attenuated UVB-induced decrease in cell viability. The extracts had no remarkable effect on the total antioxidant status, but significantly lowered total protein carbonyl content used as a marker for protein oxidation in HaCaT cells. MMP-1, -3, -7, and -9, monitored as endpoints of antiaging efficacy, were significantly reduced by propolis following UVB exposure in a model of reconstituted skin tissue. In conclusion, propolis protects against the oxidative and photodamaging effects of UVB and could be further explored as a promising agent for developing natural antiaging strategies.

Human aldehyde dehydrogenase 3A1 (ALDH3A1) exhibits chaperone-like function.

Aldehyde dehydrogenase 3A1 (ALDH3A1) is a metabolic enzyme that catalyzes the oxidation of various aldehydes. Certain types of epithelial tissues in mammals, especially those continually exposed to environmental stress (e.g., corneal epithelium), express ALDH3A1 at high levels and its abundance in such tissues is perceived to help to maintain cellular homeostasis under conditions of oxidative stress. Metabolic as well as non-metabolic roles for ALDH3A1 have been associated with its mediated resistance to cellular oxidative stress. In this study, we provide evidence that ALDH3A1 exhibits molecular chaperone-like activity further supporting its multifunctional role. Specifically, we expressed and purified the human ALDH3A1 in E. coli and used the recombinant protein to investigate its in vitro ability to protect SmaI and citrate synthase (from precipitation and/or deactivation) under thermal stress conditions. Our results indicate that recombinant ALDH3A1 exhibits significant chaperone function in vitro. Furthermore, over-expression of the fused histidine-tagged ALDH3A1 confers host E. coli cells with enhanced resistance to thermal shock, while ALDH3A1 over-expression in the human corneal cell line HCE-2 was sufficient for protecting them from the cytotoxic effects of both hydrogen peroxide and tert-butyl hydroperoxide. These results further support the chaperone-like function of human ALDH3A1. Taken together, ALDH3A1, in addition to its primary metabolic role in fundamental cellular detoxification processes, appears to play an essential role in protecting cellular proteins against aggregation under stress conditions.