O 1
FENRETINIDE (4HPR) IS ABLE TO TARGET PARENTAL AND CANCER-INITIATING MEDULLOBLASTOMA CELLS
Barbara Bassani (1) - Antonino Bruno (1) - Katiuscia Dallaglio (2) - Arianna
Pagani (3) - Elisa Principi (1) - Douglas Noonan (3) - Adriana Albini (2)
Fondazione MultiMedica Onlus, Fondazione MultiMedica Onlus, Milan, Italy (1) - IRCCS “Tecnologie Avanzate e Modelli Assistenziali in Oncolo-
gia”, Arcispedale S. Maria Nuova, Reggio Emilia, Italy (2) - Department of
Biotechnology and Life Sciences, University of Insubria, Varese, Italy (3)
Introduction: Medulloblastoma (MB) is a neuroectodermal
tumor arising in the cerebellum that represents the most fre- quent malignant childhood brain tumor. Current treatments for MB include surgery and combination radio- and chemo-therapy, leading to severe side effects, thus novel therapeutic strategies are urgently needed. N-(4-Hydroxyphenyl)retinamide (4HPR, fenretinide), a synthetic analog of all-trans retinoic acid, has emerged as a promising and well-tolerated cancer chemopre- vention and chemotherapy agent.
Materials and methods: Primary and metastatic medulloblas-
toma cell lines (MB-DAOY and ONS-76, respectively) were used to assess the ability of 4-HPR to affect cell proliferation and survival. Flow cytometry analysis was performed to assess 4-HPR induction of apoptosis and oxygen reactive species (ROS) production, as well as cell cycle effects, on MB cell lines. Moreover, functional analysis to determine whether 4- HPR is able to decrease MB chemotaxis and invasion were per- formed. The main pathways affected by 4-HPR treatment, in- cluding b-catenin/wint3a, were determined by western blot analysis. The ability of 4-HPR to target MB- cancer-stem-like cells was evaluated by flow cytometry and quantitative real- time PCR and to inhibit MB tumor growth in vivo in nude mice.
Results: We show that 4HPR induces caspase-dependent cell
death in (DAOY and ONS-76) medulloblastoma cell lines asso- ciated with increased ROS generation, suggesting that free radi- cal intermediates might be directly involved. 4HPR induces cell cycle arrest in G1/S phase, inactivated β-catenin and inhibited migration and invasion of MB cells. We observed inhibition of
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transcription factor activation in both cell lines, affecting path- ways frequently over-expressed in MB. 4HPR inhibited sphe- roid formation by both DAOY and ONS-76, known to be en- riched in the tumor stem/initiating cell component, in terms of size, invasive/migratory properties as well as Oct-4, Sox-2 and Nanog expression levels. This inhibition was associated with decreased levels of CD133+ and ABCG2+cells, markers of can-
cer initiating cells. In vivo tumor growth of MB cell lines was inhibited by 4HPR.
Conclusions: Our data suggest that 4HPR also targets the tumor
initiating cell population. Since 4HPR exerts low toxicity, it could represent a valid molecule in the management of human MB.
This work was supported by the AIRC “Caterina Forni” fellow- ship to Dr. A.P.
O 2
ANTI ANGIOGENIC AND ANTI TUMOUR ACTIVITIES OF OLIVE MILL WASTEWATERS
Teresa Rossi (1) - Antonino Bruno (2) - Annarita Cantelmo (2) - Barbara
Bassani (2) - Sara Canali (2) - Adriana Albini (1)
IRCCS “Tecnologie Avanzate e Modelli Assistenziali in Oncologia”, Ar- cispedale S. Maria Nuova, Reggio Emilia, Italy (1) - Fondazione Multi-
Medica Onlus, Fondazione MultiMedica Onlus, Milan, Italy (2)
Introduction: Angiogenesis is a crucial event for cancer pro-
gression, since a cancer-associated bloodstream provides nutri- ents and oxygen to cancer cells and represent the “roadways” through transformed cells can invade distant organs and tissues. Several diet derived compounds have been reported to exert an- ti-oxidant, anti-proliferative, anti-angiogenic and pro-apoptotic effects in a variety of cancers, including leukemia, prostate, breast, colon, brain, melanoma, and pancreatic tumors. Further, they also affect cellular metabolism, targeting cancer cells though their metabolic derangements as well.
The consumption of extra virgin olive oil represents an im- portant constituent of the Mediterranean diet. Compared to oth- er vegetable oils, the presence of several phenolic antioxidants, including hydroxytyrosol, in olive oil is believed to prevent the occurrence of a variety of pathological processes, including cancer. While the strong antioxidant potential of these mole- cules is well characterized, their anti-angiogenic activities re- main unknown. Here we assessed the anti-angiogenic and anti- tumor properties exerted by extracts from olive mill wastewaters (OMWs) in vitro and in vivo.
Material and methods: OMWs ability to affect cell prolifera-
tion and survival were evaluated on human umbilical vein endo- thelial cells (HUVECs) and murine C26 colorectal carcinoma cells with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while the induction of apoptosis and re- active oxygen species were assessed by flow cytometry. Fur- ther, functional studies evaluated the capacity of OMWs to in- terfere with endothelial cell tube formation, migration and inva- sive activities were performed by morphogenesis and Boyden chamber assays. Finally, the inhibition of angiogenesis and tu- mor cell growth was evaluated in vivo, by the matrigel sponge assay and on tumor xenograft growth.
Results: OMWs were able to inhibit both HUVEC and C26 cell
growth in a dose dependent manner, exerting a stronger inhibi- tory effect as compared with purified hydroxytyrosol alone. This effect was directly associated with the induction of apopto- sis and oxygen reactive species (ROS) on HUVECs. Moreover, OMWs were able to inhibit HUVEC migration and invasive abilities in a dose dependent manner. Finally, OMWs inhibited tumor angiogenesis and C26 tumor cell growth in vivo.
Conclusions: Taken together, OMWs, which represent a waste
product from olive oil industry, could represent a valid source of polyphenols to be used for and angiopreventive approaches with natural compounds.
O 3
DIET DERIVED FLAVONOIDS: ANALYSIS OF ANTI-ANGIOGENIC ACTIVITY AND POTENTIAL ROLE IN TUMOR PREVENTION OF A BEER HOP DERIVATIVE
Cristina Gallo (1) - Teresa Rossi (1) - Sally Maramotti (1) - Candida Bonelli (1)
- Adriana Albini (2)
Laboratory of Translational Research, IRCCS-Arcispedale Santa Maria Nuova, Reggio Emilia, Italy (1) - Department Of Research And Statistics
Infrastructure And Laboratory Of Translational Research, Irccs- Arcispedale Santa Maria Nuova, Reggio Emilia, Italy (2)
Introduction: The process of carcinogenesis is sustained by
new vessel formation and chemoprevention of angiogenesis (angioprevention) represents a possible strategy to block/reverse the progression of tumors. Chemopreventive drugs are mole- cules preventing or retarding cancer progression and develop- ment. Recently, diet flavonoids have been recognized as chemo- and angiopreventive agents. Among these, Xanthohumol (XN), is the principal prenylated chalcone of the female inflorescence of the hop plant (Humulus lupulus L.) with chemo/angio- preventive properties. The biological effects of the chalcones mostly depend on their chemical structure, whose variations in- fluence their anti-tumor effects. In order to identify novel poten- tial chemo/angio-preventive agents, we analysed the effects of eight different synthetic derivatives of XN on tumor cells and normal primary endothelial cells (ECs) proliferation. AMP- activated protein kinase (AMPK), an energy and metabolic sen- sor, is a key target in chemoprevention, also involved in the regulation of angiogenesis. We therefore investigated the in- volvement of AMPK in XN effects on ECs.
Materials and methods: Tumor cell lines (A375 (Malignant
Melanoma), MCF7 (Breast Carcinoma), PC3-DU145 (Prostate cancer), H29 (Colorectal cancer)), were used as in vitro models of highly vascularized and hormone-responsive tumors. Human Umbilical Vein Endothelial Cells (HUVEC), were used as the gold standard for ECs analysis.
We tested the anti-proliferative properties of XN synthetic de- rivatives by MTT assay. The effects of XN derivatives were compared to those of XN “master molecule”, by calculating IC50 values (half maximal inhibitory concentration) at 96h. To assess the molecular mechanism underlying XN effects on ECs proliferation, HUVEC were stimulated with 10uM XN and
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lysed after 5-10-30 min and 1 h. AMPK phosphorylation at Thr172 was evaluated by Western blotting.
Results: Overall all synthetic derivatives of XN display a lower
IC50 value at 96h as compared to XN master molecule.
Further, XN activated AMPK in a time-dependent manner with a peak of activation after 5 minutes of exposure, up to 1h. Acti- vation of AMPK signalling pathway by XN was confirmed ACC phosphorylation at Ser-79 at the same timepoints.
Conclusions: Since XN and some derivatives are able to inhibit
tumor cell proliferation, without compromising ECs functions, they might be good candidates for both tumor chemo- prevention and angio-protection. Moreover, preliminary results shown that AMPK seems to be a new target of XN in angio- prevention.
O 4
EFFECTS OF METFORMIN ON ENDOTHELIAL AND CANCER CELLS
Tiziana Petrachi (1) - Katiuscia Dallaglio (1) - Antonino Bruno (2) - Cristina
Masini (1) - Ulrich Pfeffer (3) - Francesco Bertolini (4) - Douglas M. Noonan (5) - Adriana Albini (6)
Translational Research Laboratory, IRCCS "Tecnologie Avanzate e Mod- elli Assistenziali in Oncologia", Arcispedale S. Maria Nuova, Reggio Emi- lia, Italy (1) - Scientific and Technologic Park, IRCCS MultiMedica,
MILANO, Italy (2) - Integrated Molecular Pathology, IRCCS AOU San Mar-
tino, IST Istituto Nazionale per la Ricerca sul Cancro, GENOVA, Italy (3) -
Laboratory of Hematology-Oncology, European Institute of Oncology (IEO), Milano, Italy (4) - Department of Biotechnology and Life Sciences,
University of Insubria, Varese, Italy (5) - Department of Research and Sta-
tistics Infrastructure, IRCCS "Tecnologie Avanzate e Modelli Assistenziali in Oncologia", Arcispedale S. Maria Nuova, Reggio Emilia, Italy (6)
Background: Epidemiological evidence has suggested that
metformin, an anti-hyperglycemic agent commonly used in the treatment of type 2 diabetes, is a potential cancer preventive agent. Anti-angiogenesis represents a key mechanism in cancer prevention, a concept termed angioprevention. Since conflicting data concerning the anti-angiogenic action of metformin are emerging, we elucidate the effects of metformin, on endothelial and tumor cells as well as on angiogenesis. Further, since met- formin activates the energy and stress sensor AMP activated kinase (AMPK), we evaluated the involvement of AMPK in these mechanisms.
Materials and methods: We used Human Umbelical Vein En-
dothelial cells (HUVEC) to test the effects of metformin in vitro. We used in vitro assays to analyze HUVEC viability, pro- liferation, expression and functional analysis as well as in vivo assays and transcriptomic approaches. In order to test AMPK involvement in this mechanism, we transfected HUVE cells with an AMPK specific siRNA.
Results: We show that metformin inhibits endothelial cell abil-
ity to organize into capillary-like networks; this effect is partial- ly dependent on the energy sensor AMPK. Gene expression and proteins profiling revealed paradoxic effects on several angio- genesis associated factors. We found induction of VEGF, COX2 and CXCR4 at the mRNA level and down-regulation of ADAMTS1. Interestingly, antibody array analysis showed es- sentially opposite regulation of numerous angiogenesis-
associated proteins in endothelial and breast cancer cells. We also show that endothelial production of cytochrome p450 fami- ly member CYP1B1 was up-regulated by tumor cell superna- tants (breast and prostate cancer), while metformin blocked this effect by acting on AMPK. Metformin anti-angiogenic activity was exerted through inhibition of ERK1/2 activation, even in the presence of VEGF, while blocking AMPK activity abrogat- ed this effect. Metformin inhibited angiogenesis induced by VEGF in matrigel pellets in vivo and contrasted the increase in microvessel density in obese mice on a high fat diet. Further, it down-regulated the number of endothelial precursor cells from white adipose tissue in obese mice.
Conclusions: Our data show that metformin has an anti-
angiogenic activity in vitro and in vivo, which is associated with a contradictory enhancement of chemokines and other inflam- matory pro-angiogenic mediators, as well as a different regula- tion in endothelial and tumor cells.