- Email: [email protected]
mTOR signaling
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 of Rana palustris and the net charge and hydrophobicity is +5 and 48%, respectively. Material and Methods: Estrogen receptor (ER) positive cell line, MCF-7 and ER(−) cell line, MDA-MB 231 were used in this study. Antitumor activity was determined with cell proliferation assay and cell cytotoxicity assay after treatment with 0–200 mg/ml Esculentin-2PLa for 24 hours. The cell death mechanism was investigated by measuring activity of caspase-3 and counting stained the apoptotic and necrotic cells with acridine orange-ethidium bromide. The cell morphology after the peptide treatment was observed by scanning electron microscopy. In addition, possible electrostatic interactions between cell membranes and the peptide were researched by solid-phase heparan sulfate and chondroitin sulfate binding assay and by measuring the affinity of peptide to cancer cell membranes. Results: Esculentin-2PLa reduced cell viability in a dose dependent manner in the two cell lines and MDA-MB 231 cell line was more sensitive. Beside this, the peptide caused the release of lactate dehydrogenase (LDH) from the cells. In addition, necrotic cell death was detected in both cell lines by observing membrane pores with scanning electrone microscope and this finding was consistent with the increase in the number of cell nuclei stained with etidium bromide. Moreover, Esculentin-2PLa attached to heparan sulfate and chondroitin sulfate proteoglycans which are abundant on cancer cell membranes in a dose dependent manner. Furthermore, this peptide has a high affinity to cancer cell membranes via electrostatic interaction. Conclusions: Overall, these results suggest that Esculentin-2PLa induced cell death through membranolytic effects and its anticancer activity may arise from its cationic and hydrophobic characters which facilitate the interaction with cancer cell membranes. Our findings indicate that Esculentin-2PLa has the potential for development as anticancer agent. No conflict of interest. 601 Bioactivities of novel boehmeriasin derivatives in liver cancer cells E. Akhan Guzelcan1 , M. Baumann2 , I. Baxendale2 , R. Cetin Atalay1 . 1 Middle East Technical University, Bioinformatics Department, Ankara, Turkey, 2 University of Durham, Department of Chemistry, Durham, United Kingdom Introduction: Hepatocellular carcinoma (HCC) is the second most deadly cancer type and the fifth most common cancer in the world which occurs in patients with chronic liver disease/infection or cirrhosis. The incidence of HCC has increased over the past decades due to obesity but still an effective therapy has not been developed. Sorafenib, which is the only FDA approved agent, can improve the patient survival just for a few months, therefore liver transplantation is the most efficient way of treatment up to date. Hence, more efficient therapeutic agents must be developed. Boehmeriasin A, an alkaloid isolated from extracts of Boehmeria siamensis Craib has been shown to possess promising anticancer activities in various cancer types such as lung, breast, kidney, colon, prostate and leukemia. However, the bioactivities of boehmeriasin A and several of its novel synthetic derivatives remain unclear in liver cancer. In this study, we demonstrated that boehmeriasin A and other boehmeriasin derivatives boehmeriasin-OH, -ketone and -K-salt have cytotoxic bioactivities in HCC cells. Material and Methods: The initial cytotoxicity of boehmeriasin A and -OH, -ketone -K-salt were assessed with Sulforhodamine B (SRB) assay on epithelial cancer cells: Huh7, HCT116 and MCF7. Then the compounds were tested in a panel of HCC cell lines: Huh7, HepG2, Hep3B, Mahlavu, FOCUS and SNU475. The cytotoxicity of the boehmeriasin A and its hydroxyl-derivative were further confirmed by Real-time cell electronic sensing assay (RT-CES) over 96 hours. The cell cycle analysis of treated HCC cells was assessed with flow cytometry assays. Finally, the cell signaling mechanisms leading to cells death with boehmeriasin derivatives were analyzed by immunofluorescence and western blot assays. Results: In this study, we showed highly potent cytotoxic activities of boehmeriasin A and boehmeriasin-OH, and -ketone on HCC cells. The IC50 values of both boehmeriasin A and its -OH derivative were in the nanomolar range in all HCC cells tested. The real time cell growth analysis also supported this significant anti-proliferative effect of the compounds on HCC cells in a time dependent manner. The compounds were also shown to induce apoptosis as demonstrated by cytochrome-c release and induction of other apoptotic proteins. Apoptotic arrest was further confirmed by cell cycle analysis. In parallel ROS and SIRT pathways, which was previously described in boehmeriasin action were analyzed in detail. Conclusion: Novel boehmeriasin derivatives were identified as powerful therapeutics due to their strong cytotoxicity and apoptotic effect on HCC cells. These compounds can be considered as potential therapeutic agents in primary liver cancer treatment. No conflict of interest.
S133
602 Improving nelarabine efficacy in refractory/relapsed T-cell acute lymphoblastic leukemia (T-ALL) by targeting aberrant PI3K/mTOR signaling F. Chiarini1 , A. Cappellini2 , A. Lonetti3 , A. Bertaina4 , F. Locatelli4 , F. Melchionda5 , A. Pession5 , A.M. Martelli3 . 1 National Research Council, Institute of Molecular Genetics, Bologna, Italy, 2 University of Cassino, Department of Human Social and Health Sciences, Cassino, Italy, 3 University of Bologna, Department of Biomedical and Neuromotor Sciences, Bologna, Italy, 4 IRCCS Bambino Gesu` Children’s Hospital, Department of Pediatric Hematology and Oncology, Rome, Italy, 5 S. Orsola-Malpighi HospitalUniversity of Bologna, Pediatric Oncology and Hematology Unit “Lalla Seragnoli”, Bologna, Italy Background: The introduction of novel chemotherapy protocols has improved the outcome of T-ALL patients, but refractory and/or relapsing disease remains a major concern. In this context, a major contribution was provided by the introduction of the nelarabine, approved for salvage treatment of refractory/relapsed T-ALL patients. Aims: Nelarabine could induce a dose-dependent neurotoxicity. To improve its efficacy, it is essential to study its molecular targets, testing selective inhibitors of such targets, to be administered in combination with nelarabine, allowing for a lower dosage of the drug. Methods: Human T-ALL cell lines and primary T-ALL refractory/relapsed lymphoblasts from patients were incubated with increasing concentrations of nelarabine alone or combinated with PI3K/mTOR inhibitors for cell viability assays. Apoptosis and phenotyping analyses were performed by flow cytometry. Protein expression was evaluated by Western Blot. ENT1/2 gene expression was measured by quantitative real time PCR in T-ALL settings. Results: Cell viability assays indicated the presence of T-ALL cell lines sensitive to nelarabine (IC50 <5 mM) and others which displayed higher IC50 (>15 mM). Nelarabine sensitive cells showed a significant increase of apoptotic cells after 48 h treatment with 2−5 mM nelarabine, as demonstrated by caspases and PARP cleavage. In contrast, resistant T-ALL cells were not perturbated. Levels of expression of ENT1/2 nucleoside transporters could be related to in vitro nelarabine sensitivity of T-ALL cells, and could be also dependent on interactions between leukemic cells and tumor microenvironment. No significant differences in ENT1/2 mRNA levels between samples sensitive or resistant to nelarabine were seen. Modulation of ENT1/2 gene expression was not related to nelarabine treatment, even if in some cases the co-culture with human stromal HS-5 cells supported cell survival. Upregulated PI3K/mTOR signaling is a common feature of T-ALL, where it portends a poorer prognosis by influencing leukemic cell proliferation/survival/drug-resistance. Sensitive T-ALL cells treated with nelarabine showed a strong decrease in the phosphorylation of Ser473 p-Akt and Ser235/236 p-S6 ribosomal protein. In contrast, resistant cells, showed a hyperactivation of PI3K/mTOR signaling pathway. The combination of nelarabine with the pan PI3K inhibitors ZSTK474 or BKM120 was synergistic in reducing proliferation and in inducing strong apoptosis in all the resistant cell lines and in relapsed T-ALL patient samples with upregulated PI3K/mTOR. Conclusions: Nelarabine combined with PI3K inhibitors efficiently reduced cell viability and induced apoptosis in T-ALL settings, allowing for a lower dosage of nelarabine and therefore, synergizing with conventional therapies in relapsed/refractory T-ALL patients with upregulation of PI3K signaling. No conflict of interest. 603 S-phase arrest independent activation of the DNA-damage response under hypoxic conditions M. Likhatcheva1 , R. Gieling1 , C. Demonacos1 , K. Williams1 . 1 The University of Manchester, Pharmacy, Manchester, United Kingdom Introduction: Hypoxia is a common feature of all solid tumours that arises due to a high proliferation rate and aberrant angiogenesis. Hypoxia is associated with poor prognosis, a more aggressive tumour phenotype as well as radio and chemoresistance. Recently, it has been proposed that tumour hypoxia leads to the activation of the DNA damage response (DDR) during tumour growth and development. Evidence suggests that the activation of DDR in hypoxia occurs as a direct consequence of S-phase cell cycle arrest. This arrest leads to the activation of ATM via ATR. Furthermore, changes in chromatin structure have been associated with the activation of ATM. It has been shown that ATM activation is dependent on the chromatin context, under normoxia as well as under hypoxia. Specifically this is related to the induction of H3K9me3. In response to the DNA damage in normoxia, Tip60 is the link between the DNA damage induced-changes in the chromatin structure and the activation of ATM. Whether the same mechanism operates under hypoxic conditions in not known and is under evaluation in this study. Materials and Methods: The activation of ATR and ATM was assessed in U87 and FTC133 cell lines by Western blot under hypoxic (0.1%) and anoxic (0%) conditions. Cell cycle distribution was analysed at different time points by FACS. The induction of H3K9me3 marker under hypoxic and anoxic conditions was established by Western blot and immunofluorescence. The activity of
S133
602 Improving nelarabine efficacy in refractory/relapsed T-cell acute lymphoblastic leukemia (T-ALL) by targeting aberrant PI3K/mTOR signaling F. Chiarini1 , A. Cappellini2 , A. Lonetti3 , A. Bertaina4 , F. Locatelli4 , F. Melchionda5 , A. Pession5 , A.M. Martelli3 . 1 National Research Council, Institute of Molecular Genetics, Bologna, Italy, 2 University of Cassino, Department of Human Social and Health Sciences, Cassino, Italy, 3 University of Bologna, Department of Biomedical and Neuromotor Sciences, Bologna, Italy, 4 IRCCS Bambino Gesu` Children’s Hospital, Department of Pediatric Hematology and Oncology, Rome, Italy, 5 S. Orsola-Malpighi HospitalUniversity of Bologna, Pediatric Oncology and Hematology Unit “Lalla Seragnoli”, Bologna, Italy Background: The introduction of novel chemotherapy protocols has improved the outcome of T-ALL patients, but refractory and/or relapsing disease remains a major concern. In this context, a major contribution was provided by the introduction of the nelarabine, approved for salvage treatment of refractory/relapsed T-ALL patients. Aims: Nelarabine could induce a dose-dependent neurotoxicity. To improve its efficacy, it is essential to study its molecular targets, testing selective inhibitors of such targets, to be administered in combination with nelarabine, allowing for a lower dosage of the drug. Methods: Human T-ALL cell lines and primary T-ALL refractory/relapsed lymphoblasts from patients were incubated with increasing concentrations of nelarabine alone or combinated with PI3K/mTOR inhibitors for cell viability assays. Apoptosis and phenotyping analyses were performed by flow cytometry. Protein expression was evaluated by Western Blot. ENT1/2 gene expression was measured by quantitative real time PCR in T-ALL settings. Results: Cell viability assays indicated the presence of T-ALL cell lines sensitive to nelarabine (IC50 <5 mM) and others which displayed higher IC50 (>15 mM). Nelarabine sensitive cells showed a significant increase of apoptotic cells after 48 h treatment with 2−5 mM nelarabine, as demonstrated by caspases and PARP cleavage. In contrast, resistant T-ALL cells were not perturbated. Levels of expression of ENT1/2 nucleoside transporters could be related to in vitro nelarabine sensitivity of T-ALL cells, and could be also dependent on interactions between leukemic cells and tumor microenvironment. No significant differences in ENT1/2 mRNA levels between samples sensitive or resistant to nelarabine were seen. Modulation of ENT1/2 gene expression was not related to nelarabine treatment, even if in some cases the co-culture with human stromal HS-5 cells supported cell survival. Upregulated PI3K/mTOR signaling is a common feature of T-ALL, where it portends a poorer prognosis by influencing leukemic cell proliferation/survival/drug-resistance. Sensitive T-ALL cells treated with nelarabine showed a strong decrease in the phosphorylation of Ser473 p-Akt and Ser235/236 p-S6 ribosomal protein. In contrast, resistant cells, showed a hyperactivation of PI3K/mTOR signaling pathway. The combination of nelarabine with the pan PI3K inhibitors ZSTK474 or BKM120 was synergistic in reducing proliferation and in inducing strong apoptosis in all the resistant cell lines and in relapsed T-ALL patient samples with upregulated PI3K/mTOR. Conclusions: Nelarabine combined with PI3K inhibitors efficiently reduced cell viability and induced apoptosis in T-ALL settings, allowing for a lower dosage of nelarabine and therefore, synergizing with conventional therapies in relapsed/refractory T-ALL patients with upregulation of PI3K signaling. No conflict of interest. 603 S-phase arrest independent activation of the DNA-damage response under hypoxic conditions M. Likhatcheva1 , R. Gieling1 , C. Demonacos1 , K. Williams1 . 1 The University of Manchester, Pharmacy, Manchester, United Kingdom Introduction: Hypoxia is a common feature of all solid tumours that arises due to a high proliferation rate and aberrant angiogenesis. Hypoxia is associated with poor prognosis, a more aggressive tumour phenotype as well as radio and chemoresistance. Recently, it has been proposed that tumour hypoxia leads to the activation of the DNA damage response (DDR) during tumour growth and development. Evidence suggests that the activation of DDR in hypoxia occurs as a direct consequence of S-phase cell cycle arrest. This arrest leads to the activation of ATM via ATR. Furthermore, changes in chromatin structure have been associated with the activation of ATM. It has been shown that ATM activation is dependent on the chromatin context, under normoxia as well as under hypoxia. Specifically this is related to the induction of H3K9me3. In response to the DNA damage in normoxia, Tip60 is the link between the DNA damage induced-changes in the chromatin structure and the activation of ATM. Whether the same mechanism operates under hypoxic conditions in not known and is under evaluation in this study. Materials and Methods: The activation of ATR and ATM was assessed in U87 and FTC133 cell lines by Western blot under hypoxic (0.1%) and anoxic (0%) conditions. Cell cycle distribution was analysed at different time points by FACS. The induction of H3K9me3 marker under hypoxic and anoxic conditions was established by Western blot and immunofluorescence. The activity of