ABSTRACT
Wide spread use of Di-(2-ethylhexyl) phthalate (DEHP) has made it a ubiquitous contaminant in today’s environment, responsible for possible carcinogenic and endocrine disrupting effects. In the present investigation an integrative toxico-proteomic approach was made to study the estrogenic potential of DEHP. In vitro experiments carried out with DEHP (0.1-100 μM) induced proliferations (E-screen assay) in human estrogen receptors-α (ERα) positive MCF-7 and ERα negative MDA-MB-231 breast cancer cells irrespective of their ERα status. Further, DEHP suppressed tamoxifen (a potent anti-breast cancer drug) induced apoptosis in both cell types as shown by flowcytometric cell cycle analysis. Label-free quantitative proteomics analysis of the cell secretome of both the cell lines indicated a wide array of stress related, structural and receptor binding proteins that were affected due to DEHP exposure. The secretome of DEHP treated MCF-7 cells revealed the down regulation of lactotransferrin, an ERα responsive iron transport protein. The results indicated that toxicological effects of DEHP did not follow an ERα signaling pathway. However, the differential effects in MCF-7 and MDA-MB-231 cell lines indicate that ERα might have an indirect modulating effect on DEHP induced toxicity.
Subject(s)
Apoptosis/drug effects , Breast Neoplasms/pathology , Cell Cycle/drug effects , Cell Division/drug effects , Cell Line, Tumor/drug effects , Cell Line, Tumor/metabolism , Diethylhexyl Phthalate/toxicity , Environmental Pollutants/toxicity , Estrogen Receptor alpha/drug effects , Estrogen Receptor alpha/physiology , Estrogens , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lactoferrin/biosynthesis , Lactoferrin/genetics , Lactoferrin/metabolism , MCF-7 Cells/drug effects , MCF-7 Cells/metabolism , Mass Spectrometry/instrumentation , Microchemistry/instrumentation , Neoplasm Proteins/drug effects , Neoplasm Proteins/physiology , Neoplasm Proteins/metabolism , Neoplasms, Hormone-Dependent/pathology , Proteomics , Tamoxifen/antagonists & inhibitors , Tamoxifen/pharmacologyABSTRACT
A correlation between cancer and prothrombotic states has long been described. More recently, a number of studies have focused on the procoagulant mechanisms exhibited by tumor cells. In the present study, we dissected the molecular mechanisms responsible for the procoagulant activity of MV3, a highly aggressive human melanoma cell line. It was observed that tumor cells strongly accelerate plasma coagulation as a result of: i) expression of the blood clotting initiator protein, a tissue factor, as shown by flow cytometry and functional assays (factor Xa formation in the presence of cells and factor VIIa), and ii) direct activation of prothrombin to thrombin by cells, as evidenced by hydrolysis of the synthetic substrate, S-2238, and the natural substrate, fibrinogen. This ability was highly potentiated by the addition of exogenous factor Va, which functions as a co-factor for the enzyme factor Xa. In contrast, prothrombin activation was not observed when cells were previously incubated with DEGR-factor Xa, an inactive derivative of the enzyme. Moreover, a monoclonal antibody against bovine factor Xa reduced the prothrombin-converting activity of tumor cells. In conclusion, the data strongly suggest that MV3 cells recruit factor Xa from the culture medium, triggering an uncommon procoagulant mechanism.
Subject(s)
Humans , Cysteine Endopeptidases/physiology , Melanoma/metabolism , Neoplasm Proteins/physiology , Prothrombin/metabolism , Thrombin/metabolism , Thromboplastin/metabolism , Cell Line, Tumor/chemistry , Cysteine Endopeptidases/drug effects , Flow Cytometry , Factor V/pharmacology , Factor VIIa/pharmacology , Factor Xa/pharmacology , Melanoma/chemistry , Neoplasm Proteins/drug effectsABSTRACT
Retinoic acids (RA) play a key role in myeloid differentiation through their agonistic nuclear receptors (RAR alpha/RXR) to modulate the expression of target genes. In acute promyelocytic leukemia (APL) cells with rearrangement of retinoic acid receptor a (RAR alpha) (including: PML-RAR alpha, PLZF-RAR alpha, NPM-RAR alpha, NuMA- RAR alpha or STAT5b-RAR alpha) as a result of chromosomal translocations, the RA signal pathway is disrupted and myeloid differentiation is arrested at the promyelocytic stage. Pharmacologic dosage of all-trans retinoic acid (ATRA) directly modulates PML-RAR alpha and its interaction with the nuclear receptor co-repressor complex, which restores the wild-type RAR alpha/RXR regulatory pathway and induces the transcriptional expression of downstream genes. Analysing gene expression profiles in APL cells before and after ATRA treatment represents a useful approach to identify genes whose functions are involved in this new cancer treatment. A chronologically well coordinated modulation of ATRA-regulated genes has thus been revealed which seems to constitute a balanced functional network underlying decreased cellular proliferation, initiation and progression of maturation, and maintenance of cell survival before terminal differentiation.