Constructing interactive networks of functional genes and metabolites to uncover the cellular events related to colorectal cancer cell migration induced by arsenite.

Tumor cell migration induced by arsenite (iAsIII) is closely associated with cancer progression. However, transcriptomic and metabolic traits of migrative human cells exposed to iAsIII remain to be well characterized. Here, the combination of transcriptomics and metabolomics approaches were employed to construct interactive networks of functional genes and metabolites in human colorectal cancer (DLD-1) cells exposed to iAsIII. The number of DLD-1 cells passing through the Transwell membrane was at least 6 times greater in the iAsIII-treated groups than in controls. Following iAsIII treatment, the expression of ZEB1 and SLUG protein was significantly upregulated while the expression of CRB2 was downregulated (p < 0.05), indicating the onset of epithelial to mesenchymal transition (EMT). Meanwhile, integrin- and collagen-mediated biological adhesion were enhanced by SLUG under iAsIII treatment. The expression of matrix metallopeptidase (MMP) genes was fostered by iAsIII, which have the functions to degrade extracellular matrix. Glutamine metabolism could be considerably interfered by iAsIII, and in turn glutamine supplementation could effectively enhance DLD-1 cell movement. Overall, our results suggested that DLD-1 cell migration could be promoted by iAsIII via a series of cellular events, including EMT activation, altered cell adhesion, MMP-dependent matrix degradation, accompanying with a metabolic focus on glutamine.

Metabolomics analysis of the 3D L-02 cell cultures revealing the key role of metabolism of amino acids in ameliorating hepatotoxicity of perfluorooctanoic acid.

To simulate the real cell status and morphology in the living systems is substantial for using cell models to address the detrimental effects of toxic contaminants. In this study, the comparative profiles of metabolites in three-dimensional (3D) human normal liver (L-02) cell spheroids with perfluorooctanoic acid (PFOA) treatment were analyzed using a metabolomic approach. The uniform 3D cell spheroids were well formed in 3 days (e.g., sphericity index >0.9) and stably maintained over the subsequent 11 days. The cytotoxicity of PFOA to the 3D L-02 cell spheroids was highly dependent on both exposure concentration and duration. Comparative analysis of metabolomes showed that the number of differential metabolites in the 3D cell spheroids treated with 300 µM PFOA for 10 days (n = 59) was greater than those with a 4-day exposure to 300 µM PFOA (n = 17). Six metabolic pathways related to amino acids metabolism were only found in the 3D cell spheroids with a 10-day treatment of 300 µM PFOA, which could not be found in the 2D monolayer cells and those 3D cell spheroids with a 4-day exposure. The suppression of PFOA on glutamine metabolism substantially decreased glutathione (GSH) production and accordingly increased the level of reactive oxygen species in the 3D cell spheroids. On the contrary, the supplementation of glutamine increased GSH production and the viability of cell spheroids, indicating that glutamine metabolism played a critical role in the chronic toxic effects of PFOA. Our study strongly suggested that comprehensive toxicological methodologies based on the 3D cell models could currently be robust and suitable for addressing the chronic adverse effects of toxic contaminants.

Non-targeted metabolomics of multiple human cells revealing differential toxic effects of perfluorooctanoic acid.

Differences in toxic effects of contaminants among human cells are essential for evaluating their health risks to humans. In this study, non-targeted metabolomics of multiple human cell lines (A549 (lung), DLD-1 (intestine) and L-02 (liver) cells) was used to address the differential toxicity of perfluorooctanoic acid (PFOA). The number of differential metabolites (DMs) identified in the PFOA-treated A549 cells (67) was highest, followed by DLD-1 (12) and L-02 cells (10). The categorization of DMs was almost uniquely specific to each of cell lines. PFOA significantly promoted linoleic acid metabolism in L-02 cells whereas this metabolism was inhibited in the PFOA-treated A549 cells. The levels of interleukin (IL)-1ß, IL-6, IL-8 and IL-13 were about 1.5 times higher in the PFOA-treated A549 and L-02 cells than in the controls. PFOA stimulated the biosynthesis of arginine and the metabolism of vitamin B6 in A549 cells. Arginine and vitamin B6 supplemented into cell culture effectively decreased the levels of IL-6 and IL-8. The inhibition of purine metabolism by PFOA resulted in the arrestation of DLD-1 cells at the G0/G1-phase. Our results suggest that the differential toxicity of PFOA related to exposure pathways could be elucidated by metabolic profiles specific to various human cells.

Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) in Pancreatic Ductal Adenocarcinoma (PDA): An integrative analysis of a novel therapeutic target.

We investigated biomarker CEACAM6, a highly abundant cell surface adhesion receptor that modulates the extracellular matrix (ECM) in pancreatic ductal adenocarcinoma (PDA). The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) RNA-Seq data from PDA patients were analyzed for CEACAM6 expression and evaluated for overall survival, association, enrichment and correlations. A CRISPR/Cas9 Knockout (KO) of CEACAM6 in PDA cell line for quantitative proteomics, mitochondrial bioenergetics and tumor growth in mice were conducted. We found CEACAM6 is over-expressed in primary and metastatic basal and classical PDA subtypes. Highest levels are in classical activated stroma subtype. CEACAM6 over-expression is universally a poor prognostic marker in KRAS mutant and wild type PDA. High CEACAM6 expression is associated with low cytolytic T-cell activity in both basal and classical PDA subtypes and correlates with low levels of T-REG markers. In HPAF-II cells knockout of CEACAM6 alters ECM-cell adhesion, catabolism, immune environment, transmembrane transport and autophagy. CEACAM6 loss increases mitochondrial basal and maximal respiratory capacity. HPAF-II CEACAM6-/- cells are growth suppressed by >65% vs. wild type in mice bearing tumors. CEACAM6, a key regulator affects several hallmarks of PDA including the fibrotic reaction, immune regulation, energy metabolism and is a novel therapeutic target in PDA.

Degradation pathways of 1-methylphenanthrene in bacterial Sphingobium sp. MP9-4 isolated from petroleum-contaminated soil.

Alkylated polycyclic aromatic hydrocarbons (PAHs) are abundant in petroleum, and alkylated phenanthrenes are considered as the primary PAHs during some oil spill events. Bacterial strain of Sphingobium sp. MP9-4, isolated from petroleum-contaminated soil, was efficient to degrade 1-methylphenanthrene (1-MP). A detailed metabolism map of 1-MP in this strain was delineated based on analysis of metabolites with gas chromatograph-mass spectrometer (GC-MS). 1-MP was initially oxidized via two different biochemical strategies, including benzene ring and methyl-group attacks. Benzene ring attack was initiated with dioxygenation of the non-methylated aromatic ring via similar degradation pathways of phenanthrene (PHE) by bacteria. For methyl-group attack, mono oxygenase system was involved and more diverse enzymes were needed than that of PHE degradation. This study enhances the understanding of the metabolic pathways of alkylated PAHs and shows the significant potential of Sphingobium sp. MP9-4 for the bioremediation of alkylated PAHs contaminated environments.

Effects of an epidermal growth factor receptor inhibitor on arsenic associated toxicity in the rat bladder epithelium.

Arsenite (As(III)), an inorganic arsenical, is a known human carcinogen, inducing tumors of the skin, urinary bladder and lung. It is known to be metabolized to organic methylated arsenicals in vivo. As(III) has been reported to have the ability to up-regulate the epidermal growth factor receptor (EGFR)-associated pathway in epithelial cells, including human urothelial cells in vitro. EGFR is a cell-surface receptor belonging to the ErbB family of receptor tyrosine kinases, and the EGFR-associated signaling pathway has been reported to play an important role in carcinogenesis and cancer progression, including in bladder cancer. In this study, we investigated the growth effects of As(III) and an organic trivalent arsenical, dimethylarsinous acid (DMA(III)), and the effects of co-exposure of gefitinib, an EGFR inhibitor, with As(III) to a rat urothelial cell line (MYP3). We also investigated the effects of co-administration of dietary As(III) and gefitinib in vivo. In vitro, concentrations of 1.0microM As(III) or 0.5microM DMA(III) induced cytotoxicity. However, lower concentrations of As(III) treatment had a slight mitogenic growth effect whereas lower concentrations of DMA(III) did not. Gefitinib blocked As(III)-induced cell growth in vitro. In vivo, a high dose of gefitinib alone induced slight urothelial cytotoxicity, and did not reduce cytotoxicity and regenerative cell proliferation when co-administered with As(III). The majority of arsenic metabolites present in the urine of As(III)-treated rats were organic arsenicals, mainly dimethylarsinic acid (DMA(V)). As(III) was also present, and its concentration was higher than the concentration required to produce cytotoxicity in vitro. These data suggest that an EGFR inhibitor has the ability to block As(III)-induced cell proliferation in vitro but not in vivo in a short-term study.

Increased catalytic efficiency following gene fusion of bifunctional methionine sulfoxide reductase enzymes from Shewanella oneidensis.

Methionine sulfoxide reductase enzymes MsrA and MsrB have complementary stereospecificities that reduce the S and R stereoisomers of methionine sulfoxide (MetSO), respectively, and together function as critical antioxidant enzymes. In some pathogenic and metal-reducing bacteria, these genes are fused to form a bifunctional methionine sulfoxide reductase (i.e., MsrBA) enzyme. To investigate how gene fusion affects the substrate specificity and catalytic activities of Msr, we have cloned and expressed the MsrBA enzyme from Shewanella oneidensis, a metal-reducing bacterium and fish pathogen. For comparison, we also cloned and expressed the wild-type MsrA enzyme from S. oneidensis and a genetically engineered MsrB protein. MsrBA is able to completely reduce (i.e., repair) MetSO in the calcium regulatory protein calmodulin (CaM), while only partial repair is observed using both MsrA and MsrB enzymes together at 25 degrees C. A restoration of the normal protein fold is observed co-incident with the repair of MetSO in oxidized CaM (CaMox by MsrBA, as monitored by time-dependent increases in the anisotropy associated with the rigidly bound multiuse affinity probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH). Underlying the efficient repair of MetSO in CaMox is the coordinate activity of the two catalytic domains in the MsrBA fusion protein, which results in a 1 order of magnitude rate enhancement in comparison to those of the individual MsrA or MsrB enzyme alone. The coordinate binding of both domains of MsrBA permits the full repair of all MetSO in CaMox. The common expression of Msr fusion proteins in bacterial pathogens is consistent with an important role for this enzyme activity in the maintenance of protein function necessary for bacterial survival under highly oxidizing conditions associated with pathogenesis or bioremediation.