prostaglandin-a2 and Breast-Neoplasms

Here, we report Prostaglandin A2 (PGA2) induced binding of HSP70 to a novel site on phi1 SMAR1 5' UTR which stabilizes the wild type transcript and leads to subsequent increase in SMAR1 protein levels. SMAR1 mediated cell cycle arrest is perturbed in PGA2-treated cells when HSP70 is knocked-down. Contrarily HSP70, unlike SMAR1, is overexpressed in breast cancers. We demonstrate that this is because of the inability of HSP70 to bind to the phi17 SMAR1 UTR variant which is the predominant form in breast cancers.

Topics: 5' Untranslated Regions; Base Sequence; Breast Neoplasms; Cell Cycle Proteins; DNA-Binding Proteins; Gene Expression Regulation; HSP70 Heat-Shock Proteins; Humans; Macromolecular Substances; Models, Biological; Nuclear Proteins; Nucleic Acid Conformation; Nucleoproteins; Prostaglandins A; Protein Binding; RNA Stability; RNA, Messenger; Tumor Cells, Cultured; Tumor Suppressor Proteins

Cyclopentenone prostaglandins (cyPGs) exert diverse cellular functions, such as anti-inflammatory and cytoprotective effects, via multiple mechanisms. CyPGs, especially those of the A and J series, are characterized by the presence of a chemically reactive alpha,beta-unsaturated carbonyl group. 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a representative cyPG of the J series, has been reported to directly inhibit the activity of redox-sensitive transcription factors, such as activator protein-1 and nuclear factor-kappaB. In the present study, we examined the effects of 15d-PGJ(2) on activation of p53 tumor suppressor in human breast cancer (MCF-7) cells. MCF-7 cells treated with 15d-PGJ(2) exhibited elevated p53 protein expression in time- and concentration-related manners, whereas prostaglandin A(2) (PGA(2)) and the nonprostaglandin derivative 2-cyclopenten-1-one exerted an effect to a lesser extent than did 15d-PGJ(2). In addition, MCF-7 cells exposed to 15d-PGJ(2) significantly accumulated p53 in both cytosolic and nuclear fractions. Despite the elevated levels of p53, its DNA-binding activity was reduced in 15d-PGJ(2)-treated MCF-7 cells. Moreover, isolated MCF-7 nuclear extracts directly treated with 15d-PGJ(2) exhibite diminished DNA-binding ability of p53, while the same concentration of PGA(2) or 2-cyclopenten-1-one was much less inhibitory. Thus, the electrophilic carbon center located in the alpha,beta-unsaturated carbonyl moiety of the cyclopentenone ring might be critical for the control of DNA-binding activity as well as cellular levels of p53 by 15d-PGJ(2).

Topics: Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cell-Free System; Cyclopentanes; Cytosol; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Female; Humans; Immunohistochemistry; Microscopy, Confocal; Molecular Structure; Prostaglandin D2; Prostaglandins A; Protein Binding; Time Factors; Tumor Suppressor Protein p53

Prostaglandins are anticancer agents known to inhibit tumor cell proliferation both in vitro and in vivo by affecting the mRNA stability. Here we report that a MAR-binding protein SMAR1 is a target of Prostaglandin A2 (PGA2) induced growth arrest. We identify a regulatory mechanism leading to stabilization of SMAR1 transcript. Our results show that a minor stem and loop structure present in the 5' UTR of SMAR1 (1-UTR) is critical for nucleoprotein complex formation that leads to SMAR1 stabilization in response to PGA2. This results in an increased SMAR1 transcript and altered protein levels, that in turn causes downregulation of Cyclin D1 gene, essential for G1/S phase transition. We also provide evidence for the presence of a variant 5' UTR SMAR1 (17-UTR) in breast cancer-derived cell lines. This form lacks the minor stem and loop structure required for mRNA stabilization in response to PGA2. As a consequence of this, there is a low level of endogenous tumor suppressor protein SMAR1 in breast cancer-derived cell lines. Our studies provide a mechanistic insight into the regulation of tumor suppressor protein SMAR1 by a cancer therapeutic PGA2, that leads to repression of Cyclin D1 gene.

Topics: 5' Untranslated Regions; Antineoplastic Agents; Base Sequence; Breast Neoplasms; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D; Cyclins; DNA-Binding Proteins; Humans; Molecular Sequence Data; Nuclear Proteins; Nucleic Acid Conformation; Prostaglandins A; RNA Stability; RNA, Messenger

The effects of 20 microg/ml exogenous prostaglandin A(2) (PGA(2)) were evaluated on cell numbers in HeLa (human epithelial cervix carcinoma) and MCF-7 (human breast carcinoma) cells. In HeLa cells, PGA(2) reduced cell numbers significantly to 75% after 24 h (P < 0.05) and exposure of 48 h decreased cell numbers to 61% (P < 0.05) of the control. In MCF-7 cells, PGA(2) significantly reduced cell numbers to 48% after 24 h and to 20% after 48 h, compared to vehicle-treated control cells (P < 0.05). The anti-mitogenic effects were confirmed by morphological studies conducted after 48 h of exposure to PGA(2), when optimal effects were observed. HeLa and MCF-7 cells exposed to PGA(2), showed chromatin aggregation, cell membrane blebbing and uneven distribution of chromosomes. Cell cycle progression analysis of HeLa and MCF-7 cells, showed an increase in DNA content preceding the G(0)/G(1) peak after 48 h of exposure, which is indicative of apoptotic body formation.

Topics: Apoptosis; Breast Neoplasms; Cell Count; Cell Cycle; Cell Division; Female; HeLa Cells; Humans; Prostaglandins A

Prostaglandin A2 (PGA2) potently inhibits cell proliferation and suppresses tumor growth in vivo, but little is known regarding the molecular mechanisms mediating these effects. Here we demonstrate that treatment of breast carcinoma MCF-7 cells with PGA2 leads to G1 arrest associated with a dramatic decrease in the levels of cyclin D1 and cyclin-dependent kinase 4 (cdk4) and accompanied by an increase in the expression of p21. We further show that these effects occur independent of cellular p53 status. The decline in cyclin D and cdk4 protein levels is correlated with loss in cdk4 kinase activity, cdk2 activity is also significantly inhibited in PGA2-treated cells, an effect closely associated with the upregulation of p21. Immunoprecipitation experiments verified that p21 was indeed complexed with cdk2 in PGA2-treated cells. Additional experiments with synchronized MCF-7 cultures stimulated with serum revealed that treatment with PGA2 prevents the progression of cells from G1 to S. Accordingly, the kinase activity associated with cdk4, cyclin E, and cdk2 immunocomplexes, which normally increases following serum addition, was unchanged in PGA2-treated cells. Furthermore, the retinoblastoma protein (Rb), a substrate of cdk4 and cdk2 whose phosphorylation is necessary for cell cycle progression, remains underphosphorylated in PGA2-treated serum-stimulated cells. These findings indicate that PGA2 exerts its growth-inhibitory effects through modulation of the expression and/or activity of several key G1 regulatory proteins. Our results highlight the chemotherapeutic potential of PGA2, particularly for suppressing growth of tumors lacking p53 function.

Topics: Breast Neoplasms; Carcinoma; Cell Division; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Oncogene Proteins; Prostaglandins A; Proto-Oncogene Proteins; Tumor Cells, Cultured; Tumor Suppressor Protein p53