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.

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.

Toxicity Profiling of Biosurfactants Produced by Novel Marine Bacterial Strains.

Surface active agents (SAAs), currently used in modern industry, are synthetic chemicals produced from non-renewable sources, with potential toxic impacts on humans and the environment. Thus, there is an increased interest for the identification and utilization of natural derived SAAs. As such, the marine environment is considered a promising source of biosurfactants with low toxicity, environmental compatibility, and biodegradation compared to their synthetic counterparts. MARISURF is a Horizon 2020 EU-funded project aiming to identify and functionally characterize SAAs, derived from a unique marine bacterial collection, towards commercial exploitation. Specifically, rhamnolipids produced by Marinobacter MCTG107b and Pseudomonas MCTG214(3b1) strains were previously identified and characterized while currently their toxicity profile was assessed by utilizing well-established methodologies. Our results showed a lack of cytotoxicity in in vitro models of human skin and liver as indicated by alamar blue and propidium iodide assays. Additionally, the use of the single gel electrophoresis assay, under oxidative stress conditions, revealed absence of any significant mutagenic/anti-mutagenic potential. Finally, both 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) cell-free assays, revealed no significant anti-oxidant capacity for neither of the tested compounds. Consequently, the absence of significant cytotoxicity and/or mutagenicity justifies their commercial exploitation and potential development into industrial end-user applications as natural and environmentally friendly biosurfactants.

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.

Antioxidant and Cytoprotective Potential of the Essential Oil Pistacia lentiscus var. chia and Its Major Components Myrcene and α-Pinene.

The antioxidant, cytoprotective, and wound-healing potential of the essential oil from the resin of Pistacia lentiscus var. chia (mastic oil) was evaluated, along with that of its major components, myrcene and α-pinene. Antioxidant potential was monitored as: (i) direct antioxidant activity as assessed by 2,2-di-phenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and ABTS assays; (ii) DNA damage protection activity; and (iii) cytoprotective activity as assessed via induction of transcription of genes related to the antioxidant response in human keratinocyte cells (HaCaT). The cytoprotective potential of the test substances was further evaluated against ultraviolet radiation B (UVB)- or H2O2-induced oxidative damage, whereas their regenerative capability was accessed by monitoring the wound closure rate in HaCaT. Μastic oil and major components did not show significant direct antioxidant activity, however they increased the mRNA levels of antioxidant response genes, suggesting indirect antioxidant activity. Treatment of HaCaT with the test substances before and after UVB irradiation resulted in increased cell viability in the cases of pre-treatment with mastic oil or post-treatment with myrcene. Increased cytoprotection was also observed in the case of cell treatment with mastic oil or its major components prior to H2O2 exposure. Finally, mastic oil and myrcene demonstrated a favorable dose-dependent effect for cell migration and wound closure. Collectively, mastic essential oil may exert its promising cytoprotective properties through indirect antioxidant mechanisms.

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.

Aldehyde dehydrogenase 3A1 promotes multi-modality resistance and alters gene expression profile in human breast adenocarcinoma MCF-7 cells.

Aldehyde dehydrogenases participate in a variety of cellular homeostatic mechanisms like metabolism, proliferation, differentiation, apoptosis, whereas recently, they have been implicated in normal and cancer cell stemness. We explored roles for ALDH3A1 in conferring resistance to chemotherapeutics/radiation/oxidative stress and whether ectopic overexpression of ALDH3A1 could lead to alterations of gene expression profile associated with cancer stem cell-like phenotype. MCF-7 cells were stably transfected either with an empty vector (mock) or human aldehyde dehydrogenase 3A1 cDNA. The expression of aldehyde dehydrogenase 3A1 in MCF-7 cells was associated with altered cell proliferation rate and enhanced cell resistance against various chemotherapeutic drugs (4-hydroxyperoxycyclophosphamide, doxorubicin, etoposide, and 5-fluorouracil). Aldehyde dehydrogenase 3A1 expression also led to increased tolerance of MCF-7 cells to gamma radiation and hydrogen peroxide-induced stress. Furthermore, aldehyde dehydrogenase 3A1-expressing MCF-7 cells exhibited gene up-regulation of cyclins A, B1, B2, and down-regulation of cyclin D1 as well as transcription factors p21, CXR4, Notch1, SOX2, SOX4, OCT4, and JAG1. When compared to mock cells, no changes were observed in mRNA levels of ABCA2 and ABCB1 protein pumps with only a minor decrease of the ABCG2 pump in the aldehyde dehydrogenase 3A1-expressing cells. Also, the adhesion molecules EpCAM and CD49F were also found to be up-regulated in aldehyde dehydrogenase 3A1expressing cells. Taken together, ALDH3A1 confers a multi-modality resistance phenotype in MCF-7 cells associated with slower growth rate, increased clonogenic capacity, and altered gene expression profile, underlining its significance in cell homeostasis.

Epigenetic therapy as a novel approach in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of liver malignancy and one with high fatality. Its 5-year survival rate remains low and thus, there is a need for improvement of current treatment strategies as well as development of novel targeted methodologies in order to optimize existing therapeutic protocols. To this end, only recently, it was discovered that its pathophysiology also involves epigenetic alterations in DNA methylation, histone modifications and/or non-coding microRNA patterns. Unlike genetic events, epigenetic alterations are reversible and thus potentially considered to be an alternative option in cancer treatment protocols. In this review, we describe the general characteristics and resulted major alterations of the epigenetic machinery as well as current state of progress of epigenetic therapy (via different single or combinatorial experimental approaches) in HCC.

In-vitro assessment of Jurkat T-cells response to 1966 MHz electromagnetic fields in a GTEM cell.

This paper presents the experimental configuration and procedure as well as the in-vitro assessment of Jurkat T-cells response to 1966 MHz exposure of modulated and unmodulated electromagnetic signals within a Gigahertz Transverse Electro-Magnetic (GTEM) cell. Different combinations of electric field intensity, exposure duration and modulation schemes were applied. Exposures at continuous wave (CW) signal at low intensity levels (3 V/m) did not induce any significant DNA damage, but a slight increase was observed for extreme stress levels (76.4 V/m). On the other hand, the results indicate that, at both, low and high electric field intensity UMTS (Universal Mobile Telecommunications System) signal could be statistically related to DNA damage in-vitro. Nevertheless, further experiments are required, increasing the statistical number of samples and recruiting more DNA damage endpoints before conclusive statements are drawn.

Pleiotrophic effects of natural products in ROS-induced carcinogenesis: the role of plant-derived natural products in oral cancer chemoprevention.

Cancer is a multistage process where each stage involves different molecular, biochemical and cellular events all of which, however, contribute to malignant transformation. Over the last years, substantial scientific evidence has promoted the hypothesis that ROS-induced cellular damage underlies key steps during development of the malignant phenotype including evasion of apoptosis, limitless proliferation, angiogenesis, tissue invasion and metastasis, etc. On the other hand, natural products hold great promise as anti-cancer compounds in preventing against carcinogenesis both in vitro and in vivo. Throughout this article, we aim to review the evidence as to how some of these natural products exert their chemopreventive effects in human carcinogenesis. For this reason, we have placed particular emphasis on oral cancer where significant efforts have been made in alternative therapeutic strategies such as the use of plant-derived natural products. This is of paramount importance given the disease's high morbidity and mortality rates across the world and specifically in the geographic regions of India and South-East Asia where its incidence is increasing.

DNA damage induced by endogenous aldehydes: current state of knowledge.

DNA damage plays a major role in various pathophysiological conditions including carcinogenesis, aging, inflammation, diabetes and neurodegenerative diseases. Oxidative stress and cell processes such as lipid peroxidation and glycation induce the formation of highly reactive endogenous aldehydes that react directly with DNA, form aldehyde-derived DNA adducts and lead to DNA damage. In occasion of persistent conditions that influence the formation and accumulation of aldehyde-derived DNA adducts the resulting unrepaired DNA damage causes deregulation of cell homeostasis and thus significantly contributes to disease phenotype. Some of the most highly reactive aldehydes produced endogenously are 4-hydroxy-2-nonenal, malondialdehyde, acrolein, crotonaldehyde and methylglyoxal. The mutagenic and carcinogenic effects associated with the elevated levels of these reactive aldehydes, especially, under conditions of stress, are attributed to their capability of causing directly modification of DNA bases or yielding promutagenic exocyclic adducts. In this review, we discuss the current knowledge on DNA damage induced by endogenously produced reactive aldehydes in relation to the pathophysiology of human diseases.