Dottorato in Medicina Molecolare

Belisario DC, Rocafull MA, del Castillo JR. Dimas. Purification and characterization of the ouabain-sensitive H+/K+-ATPase from guinea-pig distal colon. Arch Biochem Biophys 2010; 496(1):21-32.

Riganti C, Pinto H, Bolli E, Belisario DC, Calogero RA, Bosia A, Cavallo F. Atorvastatin modulates anti-proliferative and pro-proliferative signals in Her2/neu-positive mammary cancer. Biochem Pharmacol 2011 ;82(9):1079-89.

Doublier S, Belisario DC, Polimeni M, Annaratone L, Riganti C, Allia E, Ghigo D, Bosia A, Sapino A. HIF-1 activation induces doxorubicin resistance in MCF7 3-D spheroids via P-glycoprotein expression: a potential model of the chemo-resistance of invasive micropapillary carcinoma of the breast. BMC Cancer. 2012 Jan 4;12:4. The two first authors contributed equally.

 

Arch Biochem Biophys. 2010 Apr 1;496(1):21-32. Epub 2010 Feb 1.

 Purification and characterization of the ouabain-sensitive H+/K+-ATPase from guinea-pig distal colon.

Belisario DC, Rocafull MA, del Castillo JR.

Source

Laboratorio de Fisiología Molecular, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas (IVIC), Apartado 21827, Caracas 1020-A, Venezuela.

Abstract

Distal colon absorbs K+ through a Na+-independent, ouabain-sensitive H+/K+-exchange, associated to an apical ouabain-sensitive H+/K+-ATPase. Expression of HKalpha2, gene associated with this ATPase, induces K+-transport mechanisms, whose ouabain susceptibility is inconsistent. Both ouabain-sensitive and ouabain-insensitive K+-ATPase activities have been described in colonocytes. However, native H+/K+-ATPases have not been identified as unique biochemical entities. Herein, a procedure to purify ouabain-sensitive H+/K+-ATPase from guinea-pig distal colon is described. H+/K+-ATPase is Mg2+-dependent and activated by K+, Cs+ and NH4+ but not by Na+ or Li+, independently of K+-accompanying anion. H+/K+-ATPase was inhibited by ouabain and vanadate but insensitive to SCH-28080 and bafilomycin-A. Enzyme was phosphorylated from [32P]-gamma-ATP, forming an acyl-phosphate bond, in an Mg2+-dependent, vanadate-sensitive process. K+ inhibited phosphorylation, effect blocked by ouabain. H+/K+-ATPase is an alpha/beta-heterodimer, whose subunits, identified by Tandem-mass spectrometry, seems to correspond to HKalpha2 and Na+/K+-ATPase beta1-subunit, respectively. Thus, colonic ouabain-sensitive H+/K+-ATPase is a distinctive P-type ATPase.

2010 Elsevier Inc. All rights reserved.

Biochem Pharmacol. 2011 Nov 1;82(9):1079-89. Epub 2011 Jul 23.

Atorvastatin modulates anti-proliferative and pro-proliferative signals in Her2/neu-positive mammary cancer.

Riganti C, Pinto H, Bolli E, Belisario DC, Calogero RA, Bosia A, Cavallo F.

Source

Department of Genetics, Biology and Biochemistry,UniversityofTorino, via Santena 5/bis, 10126,Torino,Italy. chiara.riganti@unito.it

Abstract

The widely used anticholesterolemic drugs statins decrease the synthesis of cholesterol and the isoprenylation and activity of small G-proteins such as Ras andRho, the effectors of which are often critical in cell proliferation. Thanks to this property, it has been hypothesized that statins may have anti-tumor activities. We investigated this issue in BALB-neuT mice, which developed Her2/neu-positive mammary cancers with 100% penetrance, and in TUBO cells, a cell line established from these tumors. Contrary to the mammary glands of BALB/c mice, the tumor tissue from BALB-neuT animals had constitutively activated Ras and ERK1/2. These were reduced by the oral administration of atorvastatin, but the statin did not prevent tumor growth in mice nor reduce the proliferation of TUBO cells, although it lowered the activity of mevalonate pathway and Ras/ERK1/2 signaling. By decreasing the mevalonate pathway-derived metabolite geranylgeranyl pyrophosphate and the RhoA/RhoA kinase signaling, atorvastatin activated NF-κB, that sustained cell proliferation. Unexpectedly Her2-positive cells were much more sensitive to the inhibition of RhoA-dependent pathways than to the suppression of Ras-dependent pathways elicited by atorvastatin. Only the simultaneous inhibition of RhoA/RhoA-kinase/NF-κB and Ras/ERK1/2 signaling allowed the statin to decrease tumor cell proliferation. Our study demonstrates that Her2-positive mammary cancers have redundant signals to sustain their proliferation and shows that statins simultaneously reduce the pro-proliferative Ras/ERK1/2 axis and activate the pro-proliferative RhoA/RhoA-kinase/NF-κB axis. The latter event dissipates the antitumor efficacy that may arise from the former one. Only the association of statins and NF-κB-targeted therapies efficiently decreased proliferation of tumor cells.

BMC Cancer. 2012 Jan 4;12:4.

 HIF-1 activation induces doxorubicin resistance in MCF7 3-D spheroids via P-glycoprotein expression: a potential model of the chemo-resistance of invasive micropapillary carcinoma of the breast.

Doublier S, Belisario DC, Polimeni M, Annaratone L, Riganti C, Allia E, Ghigo D, Bosia A, Sapino A.

Source

Department of Genetics, Biology and Biochemistry,UniversityofTurin, Via Santena, 5/bis, 10126Turin,Italy. sophie.doublier@unito.it

Abstract

BACKGROUND:

Invasive micropapillary carcinoma (IMPC) of the breast is a distinct and aggressive variant of luminal type B breast cancer that does not respond to neoadjuvant chemotherapy. It is characterized by small pseudopapillary clusters of cancer cells with inverted cell polarity. To investigate whether hypoxia-inducible factor-1 (HIF-1) activation may be related to the drug resistance described in this tumor, we used MCF7 cancer cells cultured as 3-D spheroids, which morphologically simulate IMPC cell clusters.

METHODS:

HIF-1 activation was measured by EMSA and ELISA in MCF7 3-D spheroids and MCF7 monolayers. Binding of HIF-1α to MDR-1 gene promoter and modulation of P-glycoprotein (Pgp) expression was evaluated by ChIP assay and FACS analysis, respectively. Intracellular doxorubicin retention was measured by spectrofluorimetric assay and drug cytotoxicity by annexin V-FITC measurement and caspase activity assay.

RESULTS:

In MCF7 3-D spheroids HIF-1 was activated and recruited to participate to the transcriptional activity of MDR-1 gene, coding for Pgp. In addition, Pgp expression on the surface of cells obtained from 3-D spheroids was increased. MCF7 3-D spheroids accumulate less doxorubicin and are less sensitive to its cytotoxic effects than MCF7 cells cultured as monolayer. Finally, HIF-1α inhibition either by incubating cells with 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (a widely used HIF-1α inhibitor) or by transfecting cells with specific siRNA for HIF-1α significantly decreased the expression of Pgp on the surface of cells and increased the intracellular doxorubicin accumulation in MCF7 3-D spheroids.

CONCLUSIONS:

MCF7 breast cancer cells cultured as 3-D spheroids are resistant to doxorubicin and this resistance is associated with an increased Pgp expression in the plasma membrane via activation of HIF-1. The same mechanism may be suggested for IMPC drug resistance.

 

22a RIUNIONE NAZIONALE “A. Castellani” DEI DOTTORANDI DI RICERCA IN DISCIPLINE BIOCHIMICHE Brallo di Pregola (PV) 9 – 12 Giugno 2009.

23a RIUNIONE NAZIONALE “A. Castellani” DEI DOTTORANDI DI RICERCA IN DISCIPLINE BIOCHIMICHE Brallo di Pregola (PV) 8 – 11 Giugno 2010

The 3rd FEBS Special Meeting: “ ATP-Binding Cassette (ABC) Proteins: From Genetic Disease to Multidrug Resistance”, February 27-March  5, 2010,Innsbruck,Austria.

“Convegno annuale SIB/LLP” , sezione ligure-lombardo-piemontese della Società Italiana di Biochimica e Biologia Molecolare (SIB), Novara, 20 Maggio 2011           

“The Biology of Cancer Oncogene and Microenvironment”, Fondazione Piemontese per la Ricerca sul Cancro-ONLUS, Torino 29 Maggio 2011

 - “Novel nanoparticles capturing low abundance biological peptides: application for cancer biomarker discovery”, Dott.ssa Claudia Fredolini, 24 Gennaio 2011

-“BLIMP2 is a tumor suppressor gene frequently  in activate B cell-like diffuse large B cell lymphoma”, Prof.  Riccardo dalla Favera,  7 Febbraio 2011

-“The escape of  cancer from immune surveillance: MHC expression and tumor rejection”, Prof. Federico Garrido, 21 Marzo 2011

-"Senescence and cancer", Prof. Daniel S. Peeper, 15 Aprile 2011

 -"Manipulation of senescence pathways for  cancer therapy: from experimental model to clinic", Prof. Andrea Alimonti, 19 Aprile 2011

-"D-Day" Scuola di Dottorato in Scienze della Vita e della Salute, 19 settembre 2011