1. Biobanking e Colture cellulari: Interazione degli estratti di vinaccioli sull’attività dell’oxaliplatino in colture cellulari di carcinoma del colon Istituto Tumori G Paolo II IRCCS Bari Amalia Azzariti Laboratorio di Farmacologia Clinica e Preclinica

2. Grape seed extract (GSE) National Cancer Institute Bari - ITALY  Grape seed extract is widely consumed as a dietary supplement on the basis of its potent anti- oxidant, antiinflammatory and purported, anti- neoplastic properties.  Grape seed extract may represent a new therapeutic option to decrease the symptoms of intestinal mucositis, a serious disorder of the gastrointestinal tract that results from cancer chemotherapy (Cheah et al. 2014)  Italia grape (I-GSE)  Palieri grape (P-GSE)

3.  To evaluate the efficacy of GSE (from Italia grape and Palieri grape) in colon cancer cell lines  To evaluate the capability of GSE to affect oxaliplatin effectiveness National Cancer Institute Bari - ITALY Aim of the study  The GSE was able to reduce intestinal damage both in rat models of intestinal mucositis and ulcerative colitis (Cheah, 2014)  The combination of GSE and 5-FU further enhanced toxicity in colon cancer cells (Cheah, 2014) Rationale  The GSE showed a protective role against skin, breast, prostate, head and necks, and lung cancers (Katiyar, 2013). (Katiyar, 2013)

4. National Cancer Institute Bari - ITALY Colon cancer in vitro models Cell line IC 50 oxaliplatin (  M) HCT11632 ± 4 HT-292,5 ± 0,7 Colo2052,6 ± 0,6 LoVo0,6 ± 0,1  GSE reduced cell proliferation Cell Growth (%) GSE (  g/ml) Colo205 HCT116 HT-29 LoVo  GSE reduced oxaliplatin effectiveness only in HT-29 and LoVo cells and did not in HCT116 and Colo205… HCT116 Colo205 HT-29 LoVo Cell Growth (%) Oxaliplatin (  M) Oxaliplatin 50ug/ml GSE + oxa 100ug/ml GSE + oxa ? GSE activity alone and in combination with oxaliplatin

5. National Cancer Institute Bari - ITALY Analysis of platinum amount Inductively coupled plasma mass spectrometry (ICP-MS) was performed with a Varian 820-MS ICP mass spectrometer. Platinum (ppm) 0 9 4 3 2 1 HCT116 COLO205 HT-29 LoVo  Platinum increased in function of concentration HCT116COLO205LoVoHT-29 Platinum (ppm)  GSE strongly increased platinum intracellular concentration

6. National Cancer Institute Bari - ITALY Plasencia et al. 2006 Transport systems of platinum derivatives Tadini-Buoninsegni et al. 2014 McLaren et al. 2012

7. National Cancer Institute Bari - ITALY Transport systems of platinum derivatives hCTR1 GSE didn’t affect the transporter expression oxaliplatin increased hCTR1 expression GSE + oxaliplatin partially reduced hCTR1 Ctrl oxaliplatin GSE oxaliplatin GSE + oxaliplatin IMPORT ATP7A GSE reduced the transporter expression oxaliplatin didn’ t increase ATP7A expression GSE + oxaliplatin reduced ATP7A Ctrl oxaliplatin GSE oxaliplatin GSE + oxaliplatin EFFLUX oxaliplatin stimulated its import BUT down-regulated it, when in combination with GSE GSE reduced the efflux of this platimun derivative

8. National Cancer Institute Bari - ITALY Mechanism of action of platinum derivatives p-ERK1/2 CELL DEATH REDUCTION OF CELL PROLIFERATION

9. National Cancer Institute Bari - ITALY Mechanism of action of oxaliplatin in HT-29 and LoVo Apoptosis modulation GSE plus oxaliplatin:  the pulse to activate the repair of DNA increased Cell Growth (%) Oxaliplatin (  M) Oxaliplatin 50ug/ml GSE + oxa 100ug/ml GSE + oxa LoVo LoVo -  H2AX p-Erk1/2 Erk1/2 Ctrloxa GSE (50) GSE (100) GSE +oxa oxaliplatin GSE + oxa CTRL GSE  the impairment of the proliferation pathway is reducted

10. National Cancer Institute Bari - ITALY Mechanism of action of oxaliplatin in HCT116 and Colo205 Cell Growth (%) Oxaliplatin (  M) Oxaliplatin 50ug/ml GSE + oxa 100ug/ml GSE + oxa COLO205 p-Erk1/2 Erk1/2 Ctrloxa GSE (100) GSE +oxa GSE (50) Colo205  H2AX GSE + oxa oxaliplatin Ctrl/GSE GSE plus oxaliplatin:  the pulse to activate the DNA repair decreased  Erk1/2 activation is persistent

11. CONCLUSIONS National Cancer Institute Bari - ITALY  GSE, from both Palieri and Italia grape, reduced colon cancer cell proliferation  GSE didn’t affect or reduced the effectiveness of oxaliplatin  GSE strongly increased the intracellular concentration of oxaliplatin  GSE affected both the transport and the activity of oxaliplatin  Which is the interaction between GSE and oxaliplatin? Is it possible the chelation of oxaliplatin by GSE compounds?  Which components of GSE are responsable for the modulation of oxaliplatin effectiveness? The fractioning of metabolites in GSE will be performed in order to allocate the different biological activities to single or groups of interacting molecules  Which molecular or cellular target is responsable for the different effects on p- Erk1/2? Transcriptome profiling of GSE treated cells (Agilent microarray platform) will be performed. OPEN QUESTIONS and PERSPECTIVES

12. National Cancer Institute Bari - ITALY GRAZIE! Istituto Tumori Giovanni Paolo II Consiglio per la Ricerca e la Sperimentazione in Agricoltura Dott. G. Simone Dott.ssa L. Porcelli Dott.ssa AE. Quatrale Dott.ssa RM. Iacobazzi Dott.ssa DA. Stolfa Dott.ssa A. Mangia Dott. D. Antonacci Dott. C. Bergamini Dott. P. Crupi Dott.ssa R. Milella National Cancer Institute Bari - ITALY