Maria Belmonte

Università della Calabria, Rende, Calabria, Italy

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Publications (5)18.69 Total impact

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    ABSTRACT: Peroxisome proliferator-activated receptor gamma (PPARgamma) has been demonstrated to be anti-neoplastic against various human tumors. The aim of this study was to delineate the molecular mechanism underlying PPARgamma ligand rosiglitazone (BRL) antiproliferative effects in follicular WRO and anaplastic FRO human thyroid carcinoma cells. BRL upregulated the p21Cip1/WAF1 levels in the two thyroid cancer cells, while did not modify the p53 protein content. Different evidences indicate that the p21Cip1/WAF1 upregulation by BRL requires a functional PPARgamma, since it was reversed by silencing PPARgamma and pretreatment with GW9662, an irreversible PPARgamma antagonist. Transient transfection assays showed that BRL triggered the transcriptional activity of p21Cip1/WAF1 promoter gene in a p53-independent way, being a p21Cip1/WAF1 promoter construct deleted in the p53 sites still activated by BRL. The Sp1 inhibitor mithramycin silenced the p21Cip1/WAF1 promoter activity suggesting an important role of Sp1 in mediating BRL activation. The electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) assays evidenced a functional interaction between PPARgamma and Sp1 in regulating p21Cip1/WAF1. Intriguingly, ChIP analysis revealed in the p21Cip1/WAF1 gene promoter an increased recruitment of the RNA Pol II associated with an increased histone H3 acetylation and a reduced H3 methylation. The biological event, consistent with PPARgamma-induced WRO and FRO cell growth inhibition, was reversed by p21Cip1/WAF1 antisense oligonucleotides and was confirmed by increasing the PPARgamma expression, suggesting a crucial role exerted by p21Cip1/WAF1 in PPARgamma action. Our results further candidate BRL as a potential agent able to inhibit tumor progression of follicular and anaplastic thyroid carcinoma.
    Endocrine Related Cancer 07/2008; 15(2):545-57. · 5.26 Impact Factor
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    ABSTRACT: In just over a decade, apart from established metabolic actions, peroxisome proliferator-activated receptor gamma (PPARgamma) has evolved as key therapeutic target in cancer disease. Fas ligand (FasL), a trans-membrane protein, induces apoptosis by crosslinking with the Fas receptor. Despite the FasL relevance, little is available on the regulation of its expression. In the current study, we explored for the first time the potential role of PPARgamma in triggering apoptotic events through the Fas/FasL pathway in breast cancer cells. In MCF7 cells, by reverse transcription-polymerase chain reaction and Western blotting, we showed that the synthetic PPARgamma ligand rosiglitazone (BRL) enhanced FasL expression, that was abrogated by a specific PPARgamma antagonist GW9662. Transient transfection assays demonstrated that BRL transactivated human FasL promoter gene in a PPARgamma-dependent manner. Progressive 5' deletion analysis has identified a minimal promoter fragment spanning nucleotides from -318 to -237 bp, which is still sensitive to BRL treatment. FasL promoter activity was abrogated by mithramycin, suggesting an involvement of Sp1 transcription factor in PPARgamma action. Electrophoretic mobility shift and chromatin immuno-precipitation assays demonstrated that BRL increased the binding of PPARgamma and Sp1 to the Sp1 sequence located within the FasL gene promoter. The role of PPARgamma and Fas/FasL pathways in BRL-induced apoptotic events was assessed by caspase 8 cleavage in the presence of GW as well as PPARgamma and FasL RNA interferences. Our results indicate that PPARgamma positively regulates the FasL gene expression also in MDA-MB231 and in BT20, revealing a new molecular mechanism by which BRL induces apoptosis in breast cancer cells.
    Breast Cancer Research and Treatment 03/2008; 113(3):423-34. · 4.47 Impact Factor
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    ABSTRACT: The peroxisome proliferation-activated receptor gamma (PPARgamma) is mainly expressed in the adipose tissue and integrates the control of energy, lipid, and glucose homeostasis. The present study, by means of RT-PCR, Western blot, and immunofluorescence techniques, demonstrates that human sperm express the PPARgamma. The functionality of the receptor was evidenced by 15-deoxy-12,14-prostaglandin J(2) (PGJ2) and rosiglitazone (BRL) PPARgamma-agonists that were tested on capacitation, acrosome reaction, and motility. Both treatments also increase AKT phosphorylations and influence glucose and lipid metabolism in sperm. The specificity of PGJ2 and BRL effects through PPARgamma on human sperm was confirmed by an irreversible PPARgamma antagonist, GW9662. Our findings provide evidence that human sperm express a functional PPARgamma whose activation influences sperm physiology. In conclusion, the presence of PPARgamma in male gamete broadens the field of action of this nuclear receptor, bringing us to look towards sperm as an endocrine mobile unit independent of the systemic regulation.
    Journal of Cellular Physiology 01/2007; 209(3):977-86. · 4.22 Impact Factor
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    ABSTRACT: The aim of the present study was to provide new mechanistic insight into the growth arrest and apoptosis elicited by peroxisome proliferator-activated receptor (PPAR)gamma in breast cancer cells. We ascertained that PPARgamma mediates the inhibition of cycle progression in MCF7 cells exerted by the specific PPARgamma agonist rosiglitazone [BRL4653 (BRL)], because this response was no longer notable in the presence of the receptor antagonist GW9662. We also provided evidence that BRL is able to up-regulate mRNA and protein levels of the tumor suppressor gene p53 and its effector p21(WAF1/Cip1) in a time- and dose-dependent manner. Moreover, in transfection experiments with deletion mutants of the p53 gene promoter, we documented that the nuclear factor-kappaB sequence is required for the transcriptional response to BRL. Interestingly, EMSA showed that PPARgamma binds directly to the nuclear factor-kappaB site located in the promoter region of p53, and chromatin immunoprecipitation experiments demonstrated that BRL increases the recruitment of PPARgamma on the p53 promoter sequence. Next, both PPARgamma and p53 were involved in the cleavage of caspases-9 and DNA fragmentation induced by BRL, given that GW9662 and an expression vector for p53 antisense blunted these effects. Our findings provide evidence that the PPARgamma agonist BRL promotes the growth arrest and apoptosis in MCF7 cells, at least in part, through a cross talk between p53 and PPARgamma, which may be considered an additional target for novel therapeutic interventions in breast cancer patients.
    Molecular Endocrinology 01/2007; 20(12):3083-92. · 4.75 Impact Factor
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    ABSTRACT: Peroxisome proliferator-activated receptor g (PPARg) has been demonstrated to be anti- neoplastic against various human tumors. The aim of this study was to delineate the molecular mechanism underlying PPARg ligand rosiglitazone (BRL) antiproliferative effects in follicular WRO and anaplastic FRO human thyroid carcinoma cells. BRL upregulated the p21Cip1/WAF1 levels in the two thyroid cancer cells, while did not modify the p53 protein content. Different evidences indicate that the p21Cip1/WAF1 upregulation by BRL requires a functional PPARg, since it was reversed by silencing PPARg and pretreatment with GW9662, an irreversible PPARg antagonist. Transient transfection assays showed that BRL triggered the transcriptional activity of p21Cip1/WAF1 promoter gene in a p53-independent way, being a p21Cip1/WAF1 promoter construct deleted in the p53 sites still activated by BRL. The Sp1 inhibitor mithramycin silenced the p21Cip1/WAF1 promoter activity suggesting an important role of Sp1 in mediating BRL activation. The electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) assays evidenced a functional interaction between PPARg and Sp1 in regulating p21Cip1/WAF1. Intriguingly, ChIP analysis revealed in the p21Cip1/WAF1 gene promoter an increased recruitment of the RNA Pol II associated with an increased histone H3 acetylation and a reduced H3 methylation. The biological event, consistent with PPARg-induced WRO and FRO cell growth inhibition, was reversed by p21Cip1/WAF1 antisense oligonucleotides and was confirmed by increasing the PPARg expression, suggesting a crucial role exerted by p21Cip1/WAF1 in PPARg action. Our results further candidate BRL as a potential agent able to inhibit tumor progression of follicular and anaplastic thyroid carcinoma. Endocrine-Related Cancer (2008) 15 545-557