207: A tea polyphenol epigallocatechin gallate (EGCG) displays a superior effect on enzyme inhibition of human ornithine decarboxylase

EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 Results: We provide the first evidence that NSCLC patients exhibit various pattern of VEGF165 b tumor expression and that high levels of VEGF165 b correlate with lymph node metastasis. At the molecular level, we identify a VEGFR1/VEGFR2-activated autocrine loop by which VEGF165 b triggers a more invasive phenotype in NSCLC cells. Consistently, VEGF165 b and P-VEGFR1 (Tyr1213) expression levels are significantly associated in NSCLC (p = 0.0061). Furthermore, we show that VEGF165 inhibits this VEGFRdependent autocrine loop and triggers apoptosis of NSCLC cells, unraveling an unexpected anti-invasive function of this specific isoform in NSCLC. Finally, we demonstrate that bevacizumab treatment combined or not with chemotherapy increases VEGF165 b expression, leading to activation of the VEGFR-dependent autocrine pathway. Conclusions: It is known since one decade that VEGF-A pre-mRNA alternative splicing generates splice isoforms with antagonist activities on endothelial cells. Here, we demonstrate that VEGF-A pre-mRNA alternative splicing also controls VEGF-A autocrine functions on tumor cells in an opposite manner and more importantly is regulated by anti-angiogenic therapies. No conflict of interest. 206 GDF15 knockdown induces resistance to temozolomide treatment in glioblastoma cell line M. Baroni1 , P.F. Fedatto2 , A.F. Andrade3 , V.K. Suazo2 , R.G.P. Queiroz2 , ˜ Preto-USP, Clinical L.G. Tone2 , C.A. Scrideli2 . 1 Medical School of Ribeirao ˜ Preto − SP, Brazil, 2 Medical Oncology Stem Cells and Cell Therapy, Ribeirao ˜ Preto-USP, Pediatrics, Ribeirao ˜ Preto − SP, Brazil, School of Ribeirao 3 ˜ Preto-USP, Genetics, Ribeirao ˜ Preto − SP, Medical School of Ribeirao Brazil Background: Glioblastoma (GBM) is one of the most frequent and malignant human tumors. GBM displays high heterogeneity with complex genetic alterations, characterized by resistance to traditional treatments such as radiotherapy and treatment with the drug Temozolomide (TMZ). Among the genetic changes, our group observed a high GDF15 expression in GBM primary tumor samples and cell lines. The GDF15 is a growth factor that in normal physiological conditions is poorly expressed, but in cases of inflammation and malignancies your expression is increased. This study aims to correlate GDF15 knockdown with sensitivity to TMZ treatment in child and adult cell lines of GBM. Material and Methods: To examine these correlations, first was analyzed the GDF15 expression of nine GBM cells lines: U87, T98G, U251, U138, LN319, MO59K, U343, KNS42 and SF188, by qRT-PCR. For this analysis, it was used Taqman gene probes for GDF15 and endogenous controls, TBP and HPRT . For silencing assays, lentiviral shRNA construct for GDF15 and lentiviral control shRNA in pLKO vector were used. The expression reduction of GDF15 gene and protein was confirmed respectively by qRT-PCR and Western Blotting. For cellular proliferation analysis, we realized Resazurin assay in 96 wells plate, in quadruplicate samples in three independent experiments. Cells were treated with different doses of TMZ (250, 500, 1000 and 1500mM) and times (24−96 h). One-ANOVA and Bonferoni post-hoc was performed for statistical analysis with the Package SPSS Statistics 20.0. Results: Eight from nine GBM cell lines showed GDF15 expression 2.5 times higher than white matter human controls. The cell lines with highest expression, when compared to control, was the adult cell line U343 and the pediatric cell line KNS42, showing 22 and 194 times more expression of GDF15, respectively. In these two cell lines, the GDF15 was silenced by shRNA. The efficiency of gene silencing was confirmed by protein levels, decreasing 56% for KNS42 and 73% for U343. Cell proliferation assay was performed with GDF15 knockdown cells treated with TMZ. The KNS42 GDF15 silenced cells did not show difference when compared to control cells at all doses of TMZ and times. It was observed that U343 GDF15 knockdown cells were more resistant to TMZ treatment. The strongest effect was after 72 hours at the doses of 250 and 500mM of TMZ (p < 0.05). Conclusion: This study showed that GDF15 knockdown does not sensitize pediatric GBM cell line to TMZ while for adult cell line the gene silencing induces resistance to treatment. To better understand GDF15 correlation with cell sensitization, further studies will be conducted. No conflict of interest. 207 A tea polyphenol epigallocatechin gallate (EGCG) displays a superior effect on enzyme inhibition of human ornithine decarboxylase H.C. Hung1 , L. Pan1 . 1 National Chung Hsing University, Department of Life Sciences, Taichung city, Taiwan Introduction: Ornithine decarboxylase (ODC) catalyzes the decarboxylation of ornithine to putrescine and is the rate-limiting enzyme inthe polyamine biosynthesis pathway. Because ODC activity and the cellular levels of polyamine are crucial for cell proliferation and are critical for the beginning and progression of neoplastic diseases, ODChas been recognized as an oncogenic

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enzyme. Therefore, ODC inhibitors and negativeregulators of the polyamine pathway could be beneficial in the treatment of many cancers. Material and Methods: In the present study, the effects of green tea polyphenols (catechin, gallic acid EGCG, GCG, ECG and EGC), on ODC enzyme inhibition were examined. In addition, the size-distribution analysis measured by analytical ultracentrifugation was performed to examine the possible change in quaternary structure of ODC in the presence of these tea catechins. Results and Discussion: The inhibition studies indicated that EGCG is most effective to inhibit ODC enzyme activity, GCG and EGC showed secondly and thirdly, respectively. The IC50 values for these three catechins were 10.2, 30.1 and 66.3 mM, respectively. The other catechins, catechin, gallic acid and ECG, show little or no enzyme inhibition, indicating this inhibitory effect is structure-specific. The size distribution analysis showed that EGCG induced ODC polymerization, especially to form a double dimer. Because ODC is a dimeric enzyme, and the dimeric structure is essential for ODC enzyme activity. The inhibitory effect of EGCG may due to its binding toward the dimerinterface of ODC and then to induce the formation of the double-dimers. Conclusion: This study provides information to better understand the molecular mechanisms underlying the biological activities of catechins and to design new polyphenol-based ODC-specific inhibitors. No conflict of interest. 208 GALNT1 knockdown suppresses hepatocellular carcinoma cell malignant behaviours and decreases EGFR activation and recycling M. Huang1 , Y.M. Wu2 , M.C. Huang1 . 1 National Taiwan University College of Medicine, Graduate Institute of Anatomy and Cell Biology, Taipei City, Taiwan, 2 National Taiwan University Hospital, Department of Surgery, Taipei City, Taiwan Introduction: N-acetylgalactosaminyltransferase (GALNT) 1 is one of the 20 GALNT isoenzymes that initiate the biosynthesis of mucin-type O-glycosylation producing GalNAca1-O-serine/threonine, also known as cancer-associated Tn-antigens, that is fundamental for further complex O-glycan formation. GALNT1 is the major GALNT enzyme expressed in liver cancer. Very few studies have reported the function of GALNT1 in cancer, in particular, hepatocellular carcinoma (HCC), a highly lethal cancer that is ranked the third leading cancer-caused death worldwide. This study aims to investigate the role of GALNT1 mediated O-glycosylation in HCC pathogenesis as the basis for potential therapeutic drug development. Material and Method: Data retrieved from public microarray database and real-time RT-PCR quantification of GALNT1 of 140 HCC tumours collected from the National Taiwan University Hospital (NTUH) were statistically analysed. This study was approved by the NTUH Ethics Committee and patient informed consents were obtained prior to collection. Western blotting was used for protein expression and signal transduction analysis. siRNA knockdown and GALNT1/pcDNA3.1A overexpression of GALNT1 in PLC5 and HA22T cells were subjected to transwell migration and matrigel invasion assays using growth factors, including, EGF, FGF, HGF, IGF, PDGF, TGFb, and VEGF as chemoattractants. Immunoprecipitation and Vicia villosa (VVA) lectin pull down assays were used to determine the Tn-antigen expression on EGFR. EGFR recycling was analysed by immunofluorescence microscopy. Results and Discussion: Public microarray data indicate that GALNT1 is frequently up-regulated in HCC compared with normal liver tissues. Our data show that GALNT1 mRNA and protein expression is predominantly up-regulated in paired HCC tumours and higher GALNT1 expression is associated with poorer overall survival. GALNT1 knockdown suppressed while overexpression enhanced PLC5 and HA22T cell migration and invasion. GALNT1 knockdown most significantly suppressed EGF-induced cell migration and invasion. GALNT1 knockdown reduced EGFR O-glycan expression and EGF-induced EGFR activation. GALNT1 knockdown facilitated EGF-induced EGFR degradation and hindered EGFR recycling. Conclusion: GALNT1 is frequently up-regulated in HCC and in vitro studies show that knockdown of GALNT1 suppresses HCC cell malignant behaviours by decreasing EGFR O-glycosylation, which in turn reduces EGF-induced EGFR activation and recycling. In conclusion, GALNT1 plays an important role in HCC pathogenesis rendering it a potential as a target for therapeutic drug development. No conflict of interest. 209 K06, a novel CD43 epitope on human thymocytes, is involved in caspase-independent cell death T.J. Kim1 , Y. Bae2 . 1 Sungkyunkwan University School of Medicine, Immunology, Suwon, Korea, 2 Seoul National University College of Medicine, Parasitology and Tropical Medicine, Seoul, Korea Background: The K06 antigen is a 120 kDa molecule which is highly expressed on the double positive thymocytes. We present immunological and biological evidence demonstrating that K06 is a variant of CD43. The functional