A. Ferraretto

A. Fiorilli

B.M. Donida

E. Donetti

G. Lombardi

G. Tettamanti

M. Bedoni

S. Cosentino

B. M. Donida

AIR Universita degli studi di Milano

SCUOLA DI DOTTORATO DI RICERCA IN SCIENZE BIOCHIMICHE, NUTRIZIONALI E METABOLICHE DOTTORATO DI RICERCA IN BIOCHIMICA XXII CICLORole of the Caseinophosphopeptides and Vitamin D on calcium uptake and cell functions in human cancer intestinal cell lines differentiated in culture: a possible correlation between nutrients and colon cancerDocente Guida: Chiar.ma Prof.ssa Vanna CHIGORNODott. Giovanni LombardiMatr.: R0724445 63-Gly-Ser-Glu-Ser-Thr…Glu-Ser-Ile-Ser-Ser-Ser-Glu-Glu-P           P                        P P     P    P55-Gly-Ser-Ser-Ser-Glu-Glu-Ser-Ala-Glu-Val-Ala-Thr-Glu-Glu-Val-Lys-P     P     P Pβ-Casein13                                                              34-Val-Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu…Gln-Ser-Glu-P      P    P     P Pαs1 -Caseinαs2 -CaseinCASEINPHOSPHOPEPTIDES To study the responses, to CPP administration, of differentiated and undifferentiated intestinal cellsÎ Necessity of intestinal epithelium in vitro modelsAIM OF THE STUDY (1th)THE INTESTINAL MUCOSA AND INTESTINAL CELL LINESÎ HT-29 human adenocarcinoma cell lineÎ Caco-2 human adenocarcinoma cell lineEVALUATION OF HT-29 CELL DIFFERENTIATIONSHT-29 DMEM10000xHT-29 RPMI10000xHT-29 NaBu10000xHT-29 Gala14000xCaco-2 CELL DIFFERENTIATION: A NEW METHODFerraretto et al. (2007) Anticancer Research IN PRESSMORPHOLOGICAL EVALUATION OF Caco-2 CELL DIFFERENTIATIONFerraretto et al. (2007) Anticancer Research IN PRESS11th Passage 14000x18th Passage 4800x 40th Passage 20000xLow Passages(6thP)IntermediatePassages (24thP)High Passages(41thP)FUNCTIONAL EVALUATION OF Caco-2 CELL DIFFERENTIATIONFerraretto et al. (2007) Anticancer Research IN PRESSSpecificActivity(mU/mg Proteins)SpecificActivity(mU/mg Proteins)OUR NEW METHOD OF DIFFERENTIATION FOR Caco-2 CELLS IS EFFECTIVE IN REPRODUCING THE MORPHOLOGY AND THE FUNCTIONALITY OF THE INTESTINAL EPITHELIUMCaco-2 CELL DIFFERENTIATION: A NEW METHODEFFECT OF CPP ADMINISTRATION ON [Ca2+]i0.00         75.0           150            225            300 [Ca2+]i (nM)340/380 50s                150s              250s[Ca2+]i 100nMHT-29 DMEM340/380 50s                162s              220s[Ca2+]i 100nMHT-29 RPMI340/380 50s                150s              220s[Ca2+]i 100nMTime (s)Caco-218thPCPP ADMINISTRATION INDUCES TRANSIENT [Ca2+]i INCREMENTS ONLY IN DIFFERENTIATED INTESTINAL CELLSÎHT-29 induced to differentiate (growth in RPMI-1640 medium, treatment with NaBu, gradual adaptation toGalactose)ÎCaco-2 grown for above 18 passages before the post-confluence stageEFFECT OF CPP ADMINISTRATION ON [Ca2+]i To study the responses, to CPPs administration, of differentiated and undifferentiated intestinal cells To study the effects of 1,25-(OH)2D3 on cell differentiation and responsiveness to CPP administrationÎ Pretreatment with 1,25(OH)2D3 100nM for 48hAIM OF THE STUDY (2nd)1,25-(OH)2 D3• It is a differentiating agent for intestinal cellsÎ Well defined antineoplastic activity• It is the main regulator of intestinal calcium absorption:Î Regulation of the transcellular calcium transportÎ Regulation of the epithelial paracellularpermeability• It is an inductor of the extracellular calcium signalling (CaSR)SUCRASE-ISOMALTASESpecificActivity(% of Control)ALP SpecificActivity (% of Control)HT-29EFFECT OF 1,25-(OH)2 D3 ON CELL DIFFERENTIATIONCaco-2EFFECT OF CPP ADMINISTRATION ON [Ca2+]i AFTER 1,25-(OH)2 D3 PRETREATMENTHT-29 DMEM+ 1,25-(OH)2 D3HT-29 RPMI+ 1,25-(OH)2 D3Caco-2 12thP+ 1,25-(OH)2 D3• THE 1,25-(OH)2 D3 IS ABLE TO INDUCE A DIFFERENTIATED PHENOTYPE IN INTESTINAL TUMOR CELLSÎ INCREASED SUCRASE-ISOMALTASE SPECIFIC ACTIVITY IN PRETREATED HT-29 DMEM  Î INCREASED ALP SPECIFIC ACTIVITY IN LOW PASSAGES PRETREATED Caco-2 • 1,25-(OH)2 D3 TREATMENT IS NOT EFFECTIVE ON THE FUNCTIONAL DIFFERENTIATION IN DIFFERENTIATED HT-29 AND Caco-2 CELLS  • UNDIFFERENTIATED CELLS PRETREATED WITH 1,25-(OH)2 D3 RESPOND TO CPP ADMINISTRATIONEFFECT OF 1,25-(OH)2 D3 PRETREATMENT• CELLULAR RESPONSES TO CPP ADMINISTRATION IN PRETREATED CELLS DIFFER WITH RESPECT TO THEIR CONTROL FOR:Î PRESENCE OF [Ca2+]i OBSCILLATIONS PERSISTING ALL OVER THE EXPERIMENT IN HT-29 CELL LINEÎ BIPHASIC SPIKES IN Caco-2 CELL LINEEFFECT OF 1,25-(OH)2 D3 PRETREATMENTCONCLUSIONSÎ USE AS FUNCTIONAL FOODS OR NUTRACEUTICSCPPsUsefull tools for the study of the interaction between nutrients and the phenomenon underlying the intestinal calcium absorption processesTo enhance the dairy calcium fraction ready to be taken up by intestinal cellsDAIRY FOOD COMPONENTS THAT MAY BE PROTECTIVE AGAINST COLON CANCERCalciumVitamin DConjugated linoleic acidSphingolipidisButyric acidBacterial culturesFrom: Studies of calcium in food supplements in humans; P.R. HoltAnn. NY Acad. Sci. 1999, 889: 128-137.CONCLUSIONSFUTURE PERSPECTIVES• EVALUATION OF THE EFFECTS OF A LONG TERM TREATMENT WITH CPPs ON INTRACELLULAR CALCIUM SIGNALLING• TO TEST THE EFFECTS OF A LONG TERM TREATMENT WITH CPPs ON:Î PROLIFERATIONÎ DIFFERENTIATIONÎ APOPTOSIS• EVALUATION OF THE INTERPLAY OF CPPs AND 1,25-(OH)2 D3 IN THESE PHYSIOLOGIC PROCESSES

Role of the Caseinophosphopeptides and Vitamin D on calcium uptake and cell functions in human cancer intestinal cell lines differentiated in culture : a possible correlation between nutrients and colon cancer. A.A. 2006-2007

https://air.unimi.it/retrieve/handle/2434/63627/106372/Role%20of%20the%20Caseinophosphopeptides%20and%20Vitamin%20D%20on%20calcium%20uptake.%20AA%202006-2007.pdf

Role of the Caseinophosphopeptides and Vitamin D on calcium uptake and cell functions in human cancer intestinal cell lines differentiated in culture : a possible correlation between nutrients and colon cancer. A.A. 2006-2007

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