DOTTORATO DI RICERCA IN BIOCHIMICA XXII CICLO
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1 SCUOLA DI DOTTORATO DI RICERCA IN SCIENZE BIOCHIMICHE, NUTRIZIONALI E METABOLICHE DOTTORATO DI RICERCA IN BIOCHIMICA XXII CICLO 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 Docente Guida: Chiar.ma Prof.ssa Vanna CHIGORNO Dott. Giovanni Lombardi Matr.: R07244
2 CASEINPHOSPHOPEPTIDES α s1 -Casein Gly-Ser-Glu-Ser-Thr Glu-Ser-Ile-Ser-Ser-Ser-Glu-Glu- P P P P P P α s2 -Casein 55 -Gly-Ser-Ser-Ser-Glu-Glu-Ser-Ala-Glu-Val-Ala-Thr-Glu-Glu-Val-Lys- P P P P β-casein Val-Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu Gln-Ser-Glu- P P P P P
3 AIM OF THE STUDY (1 th ) To study the responses, to CPP administration, of differentiated and undifferentiated intestinal cells Necessity of intestinal epithelium in vitro models
4 THE INTESTINAL MUCOSA AND INTESTINAL CELL LINES HT-29 human adenocarcinoma cell line Caco-2 human adenocarcinoma cell line
5 EVALUATION OF HT-29 CELL DIFFERENTIATIONS HT-29 DMEM HT-29 RPMI 10000x 10000x HT-29 NaBu 10000x HT-29 Gala 14000x
6 Caco-2 CELL DIFFERENTIATION: A NEW METHOD Ferraretto et al. (2007) Anticancer Research IN PRESS
7 MORPHOLOGICAL EVALUATION OF Caco-2 CELL DIFFERENTIATION Low Passages (6 th P) Intermediate Passages (24 th P) 11 th Passage 14000x High Passages (41 th P) 18 th Passage 4800x 40 th Passage 20000x Ferraretto et al. (2007) Anticancer Research IN PRESS
8 FUNCTIONAL EVALUATION OF Caco-2 CELL DIFFERENTIATION Ferraretto et al. (2007) Anticancer Research IN PRESS Specific Activity (mu/mg Proteins) Specific Activity (mu/mg Proteins)
9 Caco-2 CELL DIFFERENTIATION: A NEW METHOD OUR NEW METHOD OF DIFFERENTIATION FOR Caco-2 CELLS IS EFFECTIVE IN REPRODUCING THE MORPHOLOGY AND THE FUNCTIONALITY OF THE INTESTINAL EPITHELIUM
![EFFECT OF CPP ADMINISTRATION ON [Ca 2+ ] i 0.00 75.](/docs-images/75/71572197/images/10-2.jpg "0 150 225 300 [Ca 2+ ] i (nm) HT-29 DMEM 340/380 50s 150s 250s [Ca 2+ ] i 100nM HT-29 RPMI 340/380 50s 162s 220s [Ca 2+ ]")
10 EFFECT OF CPP ADMINISTRATION ON [Ca 2+ ] i [Ca 2+ ] i (nm) HT-29 DMEM 340/380 50s 150s 250s [Ca 2+ ] i 100nM HT-29 RPMI 340/380 50s 162s 220s [Ca 2+ ] i 100nM Caco-2 18 th P [Ca 2+ ] i 100nM 340/380 50s 150s 220s Time (s)
11 EFFECT OF CPP ADMINISTRATION ON [Ca 2+ ] i CPP ADMINISTRATION INDUCES TRANSIENT [Ca 2+ ] i INCREMENTS ONLY IN DIFFERENTIATED INTESTINAL CELLS HT-29 induced to differentiate (growth in RPMI-1640 medium, treatment with NaBu, gradual adaptation to Galactose) Caco-2 grown for above 18 passages before the postconfluence stage
12 AIM OF THE STUDY (2 nd ) To study the responses, to CPPs administration, of differentiated and undifferentiated intestinal cells To study the effects of 1,25-(OH) 2 D 3 on cell differentiation and responsiveness to CPP administration Pretreatment with 1,25(OH) 2 D 3 100nM for 48h
13 1,25-(OH) 2 D 3 It is a differentiating agent for intestinal cells Well defined antineoplastic activity It is the main regulator of intestinal calcium absorption: Regulation of the transcellular calcium transport Regulation of the epithelial paracellular permeability It is an inductor of the extracellular calcium signalling (CaSR)
14 EFFECT OF 1,25-(OH) 2 D 3 ON CELL DIFFERENTIATION HT-29 Caco-2 SUCRASE-ISOMALTASE Specific Activity (% of Control) ALP Specific Activity (% of Control)
15 EFFECT OF CPP ADMINISTRATION ON [Ca 2+ ] i AFTER 1,25-(OH) 2 D 3 PRETREATMENT HT-29 DMEM + 1,25-(OH) 2 D 3 HT-29 RPMI + 1,25-(OH) 2 D 3 Caco-2 12 th P + 1,25-(OH) 2 D 3
16 THE 1,25-(OH) 2 D 3 IS ABLE TO INDUCE A DIFFERENTIATED PHENOTYPE IN INTESTINAL TUMOR CELLS EFFECT OF 1,25-(OH) 2 D 3 PRETREATMENT INCREASED SUCRASE-ISOMALTASE SPECIFIC ACTIVITY IN PRETREATED HT-29 DMEM INCREASED ALP SPECIFIC ACTIVITY IN LOW PASSAGES PRETREATED Caco-2 1,25-(OH) 2 D 3 TREATMENT IS NOT EFFECTIVE ON THE FUNCTIONAL DIFFERENTIATION IN DIFFERENTIATED HT-29 AND Caco-2 CELLS UNDIFFERENTIATED CELLS PRETREATED WITH 1,25-(OH) 2 D 3 RESPOND TO CPP ADMINISTRATION
17 EFFECT OF 1,25-(OH) 2 D 3 PRETREATMENT CELLULAR RESPONSES TO CPP ADMINISTRATION IN PRETREATED CELLS DIFFER WITH RESPECT TO THEIR CONTROL FOR: PRESENCE OF [Ca 2+ ] i OBSCILLATIONS PERSISTING ALL OVER THE EXPERIMENT IN HT-29 CELL LINE BIPHASIC SPIKES IN Caco-2 CELL LINE
18 CONCLUSIONS To enhance the dairy calcium fraction ready to be taken up by intestinal cells CPPs Usefull tools for the study of the interaction between nutrients and the phenomenon underlying the intestinal calcium absorption processes USE AS FUNCTIONAL FOODS OR NUTRACEUTICS
19 CONCLUSIONS DAIRY FOOD COMPONENTS THAT MAY BE PROTECTIVE AGAINST COLON CANCER Calcium Vitamin D Conjugated linoleic acid Sphingolipidis Butyric acid Bacterial cultures From: Studies of calcium in food supplements in humans; P.R. Holt Ann. NY Acad. Sci. 1999, 889:
20 FUTURE 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: PROLIFERATION DIFFERENTIATION APOPTOSIS EVALUATION OF THE INTERPLAY OF CPPs AND 1,25-(OH) 2 D 3 IN THESE PHYSIOLOGIC PROCESSES
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