- Email: [email protected]
p73 Regulated Gene That Modulates Survival in Tumour Prone Mouse Model
Poster Sessions
european journal of cancer 48, suppl. 5 (2012) S25–S288
Methods: The lead compound 3-b-D-galactopyranosyl-oxymethyl-4-sulfatomethyl-furan (GSF) was modified by adding a second sulphate to the 6 position of galactose (GSSF). Galactose was substituted with glucose (GluSF) and the sulphate in furan with fluorine (GFF) (Frank M. et al. Europ Patent Appl 11000103.9, 2011). The human melanoma WM-266−4 and HBMEC-60 cells were used. Cytotoxicity was determined with the sulforhodamin-B assay. Twodimensional migration of the melanoma cells was determined with the ‘woundhealing assay’, as well as the OrisTM assay. Adhesion of cells to the ECM proteins was determined by labelling adherent cells with methylene blue. Results and Discussion: Cytotoxicity of GluSF and GSF were comparable with 5 mM being cytostatic after more than 48h. GSSF was more toxic, here 2.5 mM was cytostatic and 5 mM cytotoxic. After 24 h none of the compounds damaged cells up to 5 mM. At this concentration, GSF effectively inhibited wound healing of WM-266−4 cells. GFF was active at 1 mM and GSSF at 0.1 mM. The quantitative effects determined with the OrisTM assay, were similar if less pronounced. Cell adhesion of melanoma cells to fibronectin was more effectively inhibited by GluSF and GFF than by GSF. Adhesion of HBMEC60 cells to ECM proteins was not inhibited to a greater extent by GSSF than by GSF. Earlier studies had led us to postulate that furan-based saccharide mimetics bind to integrins rather than lectins and blind docking/molecular dynamics studies showed GSF (Marano G. et al submitted) and its derivatives to bind to the RGD-binding site on integrin avb3. Conclusions: The optimisation of our lead compound GSF was successful and first structure-activity relationships can be deduced: melanoma and endothelial cells react differently to the new compounds, although they had shown similar sensitivity to GSF. Optimisation was mainly achieved for melanoma cells’ migration and adhesion. Both properties are important for the metastatic spread of solid tumours. Chelating properties in a molecule, achieved with a second sulphate improved anti-migratory property 50-fold concomitant with only a mild increase in cytotoxicity. Replacement of galactose by glucose improved the anti-adhesive effect, similar to the substitution of sulphate by fluorine. Molecular modelling studies had predicted the improved activities seen in the cell experiments. 922 DRAGO, a P53/p73 Regulated Gene That Modulates Survival in Tumour Prone Mouse Model P. Rusconi1 , F. Polato1 , A. Musi1 , M. Broggini1 . 1 Mario Negri Institute for Pharmacological Research, Oncology, Milan, Italy Introduction: DRAGO (DRug Activated Gene Overexpressed) was cloned in our Laboratory and in vitro experiments demonstrated that it is a p53/p73 responsive gene, whose expression is up-regulated in response to chemotherapeutic compounds with different mechanism of action. DRAGO sequence matches KIAA0247, an uncharacterized, highly evolutionaryconserved gene and both transiently and stably transfected cell lines expressing the gene showed toxic phenotype. These evidences are in accordance with a potential oncosuppressive role for DRAGO. Nevertheless in vivo experiments showed that mice knockout for the gene do not exhibit any evident phenotype and their lifespan is comparable to wild type mice. Our hypothesis is that DRAGO may cooperate with p53 in counteracting tumor onset. To verify this hypothesis we crossed p53 and DRAGO knockout mice. We assessed genotype distribution in the offspring and compared survival of double transgenic mice with single p53 knockout mice, which develop spontaneous tumors. We also analyzed the distribution of such tumors in the different genotype subpopulations. Material and Methods: DRAGO knockout mice were generated by Cre recombinase-mediated-cassette-exchange approach. p53 knockout mice were purchased from Jackson Lab. Mice were bred and PCR genotyped. Survival analysis were performed with GraphPad Prism 5 ® . Formalin fixed and paraffin embedded histological samples were hematoxylin and eosin stained and assessed by pathologists for diagnosis. Results and Discussion: Mice heterozygous for both genes (p53+/−; DRAGO+/−) were crossed in order to obtain all possible genotype combinations. Genotype distribution followed the expected frequencies. DRAGO knockout negatively influenced survival determining different effects depending on the p53 background: on a p53−/− background, mice carrying one or both DRAGO alleles showed prolonged survival (median survival: 166 and 190 days respectively) compared with DRAGO−/− mice (139 days). On a p53+/− background only DRAGO+/+ mice displayed prolonged survival (570 days) compared with DRAGO−/− and DRAGO+/− mice (446 and 431 days respectively). DRAGO genotype did not influence tumor spectrum as all p53−/− subpopulations showed high incidence of lymphomas (70%) followed by sarcomas (30%), while p53+/− subpopulations developed mostly sarcomas (60%) and secondly lymphomas (20%) and carcinomas (20%). Conclusions: Crossing p53 and DRAGO knockout mice allowed us to demonstrate the oncosuppressive role of DRAGO in vivo and its cooperation with p53. Deletion of one or both DRAGO alleles determined a progressive effect in survival decrease for p53−/− mice, while on a p53+/− background deletion of one or both DRAGO alleles reduced survival to the same extent. Furthermore DRAGO genotype did not affect tumor incidence since p53−/−
S223
and p53+/− subpopulations displayed tumor distributions in accordance with the literature. 923 Characterization of the Activity of Human Immunodeficiency Virus Protease Inhibitors Against Acute Myeloid Leukemia in Vitro 2 2 M. Kraus1 , J. Bader1 , H. Muller-Ide ¨ , T. Ruckrich ¨ , H. Overkleeft3 , C. Driessen1 . 1 Kantonsspital St. Gallen, MRC Lab Exp. Oncology, St. Gallen, ¨ Department of Medicine II, Tubingen, ¨ Switzerland, 2 University of Tubingen, Germany, 3 University of Leiden, Leiden Institute of Chemistry, Leiden, The Netherlands
Introduction: HIV protease inhibitors (HIV-PI) are oral drugs for HIV treatment with putative antitumor activity via the induction of ER stress and the inhibition of p-AKT and proteasome activity. Several clinical trials explore HIV-PI in solid tumors. We here characterize the activity of HIV-PI on AML cells to provide a basis for future clinical testing in AML. Material and Method: We compared the effects of HIV-PI AML cells regarding cytotoxicity, proteasome activity, ER-stress induction and AKTphosphorylation, and further evaluated synergistic activity with proteasome inhibitors, lenalidomide and sorafenib in vitro (AML cell lines, AML patient blasts). Results and Discussion: Lopinavir, Nelfinavir, Ritonavir and Saquinavir showed biological and molecular activity at concentrations within or near therapeutic drug levels (10−20 mM). In this dose range, they triggered ER stress-induced apoptosis, inhibited AKT-phosphorylation and showed synergistic cytotoxicity with bortezomib, carfilzomib, lenalidomide and sorafenib. Cell lines and normal control cells were significantly less sensitive towards HIV-PI induced cytotoxicity than primary cells. Nelfinavir was the only HIV-PI with proteasome-inhibiting activity in the tested dose range in intact cells, inhibiting also the bortezomib- and carfilzomib-insensitive b2 proteasome subunit. Conclusion: Nelfinavir, ritonavir, lopinavir and saquinavir in the 10−20 mM dose range are cytotoxic against primary AML cells in vitro, in contrast to control cells or cell lines, and act synergistically with bortezomib, carfilzomib, lenalidomide and sorafenib. Only nelfinavir inhibits proteasome activity in intact cells in vitro and in vivo. We consider nelfinavir as the most promising candidate HIV-PI that warrants clinical tested in AML. 924 The Ascidian Natural Product Eusynstyelamide B Induces G2/M Phase Arrest in Human Breast Cancer Cells M.S. Liberio1 , A.F.G. Quest2 , M. Sadowski3 , R.J. Quinn1 , R.A. Davis1 , C. Nelson3 . 1 Griffith University, Eskitis Institute, Brisbane, Australia, 2 Universidad de Chile, Laboratory of Cellular Communication Center for Molecular Studies of the Cell (CEMC), Santiago, Chile, 3 Queensland University of Technology, Australian Prostate Cancer Research Centre, Brisbane, Australia Researchers have shown that marine ascidians are an excellent source of new anticancer compounds that can exhibit new mechanisms of action. The Great Barrier Reef is a rich environment harbouring prolific ascidian biodiversity, and many of the species have never been explored for bioactive natural products. We selected a total of 143 ascidian specimens belonging to the family Didemnidae from the Eskitis Biota Library for organic solvent extraction. We tested the 143 extracts for anti-proliferative effects in the breast cancer cell line MDA-MB-231 using the real-time cell analyser xCELLigence System. Twentyone hit extracts were identified and investigated in MDA-MB-231 cells for their effects on cell proliferation, cell cycle, morphology and the level of the antiapoptotic protein Birc5/survivin. The most interesting extracts were selected for bioassay-guided fractionation in order to purify the bioactive compound(s). From these studies we identified a previously reported modified tryptophanarginine dipeptide dimer, named eusynstyelamide B (EB). We found that EB inhibited proliferation of MDA-MB-231 cells with an IC50 of 5 mM and arrested the cell cycle in G2/M phase, which was accompanied by cell enlargement. Apoptosis investigation was performed by FACS analysis measuring subG1 peak and annexin V staining. The slightly increase of apoptotic cells was consistent with the elevated level of the anti-apoptotic protein surviving in EBtreated MDA-MB-231 cells. Studies are ongoing to further characterize the potential anti-cancer properties of this compound and validate the findings in additional cancer cell lines. 925 F14512, a Polyamine Vectorized Anti-cancer Drug Exhibits a Marked Antileukemic Activity, Alone and in Combination With AraC 1 A. Kruczynski1 , A. Pillon1 , L. Creancier ´ , I. Vandenberghe1 , B. Gomes1 , V. Brel1 , V. Cartron1 , N. Chansard1 , J. Verdier1 , N. Guilbaud1 , J.-P. Annereau1 . 1 Institut De Recherche Pierre Fabre, Centre de Recherche en Oncologie ´ Experimentale, Toulouse, France
Background: Although cytotoxic chemotherapy induced complete remissions in AML patients, relapse rates are still very high, resulting in a poor outcome in most cases. Therefore the development of more effective drugs for AML represents a high level of priority. F14512 combines an epipodophyllotoxin
european journal of cancer 48, suppl. 5 (2012) S25–S288
Methods: The lead compound 3-b-D-galactopyranosyl-oxymethyl-4-sulfatomethyl-furan (GSF) was modified by adding a second sulphate to the 6 position of galactose (GSSF). Galactose was substituted with glucose (GluSF) and the sulphate in furan with fluorine (GFF) (Frank M. et al. Europ Patent Appl 11000103.9, 2011). The human melanoma WM-266−4 and HBMEC-60 cells were used. Cytotoxicity was determined with the sulforhodamin-B assay. Twodimensional migration of the melanoma cells was determined with the ‘woundhealing assay’, as well as the OrisTM assay. Adhesion of cells to the ECM proteins was determined by labelling adherent cells with methylene blue. Results and Discussion: Cytotoxicity of GluSF and GSF were comparable with 5 mM being cytostatic after more than 48h. GSSF was more toxic, here 2.5 mM was cytostatic and 5 mM cytotoxic. After 24 h none of the compounds damaged cells up to 5 mM. At this concentration, GSF effectively inhibited wound healing of WM-266−4 cells. GFF was active at 1 mM and GSSF at 0.1 mM. The quantitative effects determined with the OrisTM assay, were similar if less pronounced. Cell adhesion of melanoma cells to fibronectin was more effectively inhibited by GluSF and GFF than by GSF. Adhesion of HBMEC60 cells to ECM proteins was not inhibited to a greater extent by GSSF than by GSF. Earlier studies had led us to postulate that furan-based saccharide mimetics bind to integrins rather than lectins and blind docking/molecular dynamics studies showed GSF (Marano G. et al submitted) and its derivatives to bind to the RGD-binding site on integrin avb3. Conclusions: The optimisation of our lead compound GSF was successful and first structure-activity relationships can be deduced: melanoma and endothelial cells react differently to the new compounds, although they had shown similar sensitivity to GSF. Optimisation was mainly achieved for melanoma cells’ migration and adhesion. Both properties are important for the metastatic spread of solid tumours. Chelating properties in a molecule, achieved with a second sulphate improved anti-migratory property 50-fold concomitant with only a mild increase in cytotoxicity. Replacement of galactose by glucose improved the anti-adhesive effect, similar to the substitution of sulphate by fluorine. Molecular modelling studies had predicted the improved activities seen in the cell experiments. 922 DRAGO, a P53/p73 Regulated Gene That Modulates Survival in Tumour Prone Mouse Model P. Rusconi1 , F. Polato1 , A. Musi1 , M. Broggini1 . 1 Mario Negri Institute for Pharmacological Research, Oncology, Milan, Italy Introduction: DRAGO (DRug Activated Gene Overexpressed) was cloned in our Laboratory and in vitro experiments demonstrated that it is a p53/p73 responsive gene, whose expression is up-regulated in response to chemotherapeutic compounds with different mechanism of action. DRAGO sequence matches KIAA0247, an uncharacterized, highly evolutionaryconserved gene and both transiently and stably transfected cell lines expressing the gene showed toxic phenotype. These evidences are in accordance with a potential oncosuppressive role for DRAGO. Nevertheless in vivo experiments showed that mice knockout for the gene do not exhibit any evident phenotype and their lifespan is comparable to wild type mice. Our hypothesis is that DRAGO may cooperate with p53 in counteracting tumor onset. To verify this hypothesis we crossed p53 and DRAGO knockout mice. We assessed genotype distribution in the offspring and compared survival of double transgenic mice with single p53 knockout mice, which develop spontaneous tumors. We also analyzed the distribution of such tumors in the different genotype subpopulations. Material and Methods: DRAGO knockout mice were generated by Cre recombinase-mediated-cassette-exchange approach. p53 knockout mice were purchased from Jackson Lab. Mice were bred and PCR genotyped. Survival analysis were performed with GraphPad Prism 5 ® . Formalin fixed and paraffin embedded histological samples were hematoxylin and eosin stained and assessed by pathologists for diagnosis. Results and Discussion: Mice heterozygous for both genes (p53+/−; DRAGO+/−) were crossed in order to obtain all possible genotype combinations. Genotype distribution followed the expected frequencies. DRAGO knockout negatively influenced survival determining different effects depending on the p53 background: on a p53−/− background, mice carrying one or both DRAGO alleles showed prolonged survival (median survival: 166 and 190 days respectively) compared with DRAGO−/− mice (139 days). On a p53+/− background only DRAGO+/+ mice displayed prolonged survival (570 days) compared with DRAGO−/− and DRAGO+/− mice (446 and 431 days respectively). DRAGO genotype did not influence tumor spectrum as all p53−/− subpopulations showed high incidence of lymphomas (70%) followed by sarcomas (30%), while p53+/− subpopulations developed mostly sarcomas (60%) and secondly lymphomas (20%) and carcinomas (20%). Conclusions: Crossing p53 and DRAGO knockout mice allowed us to demonstrate the oncosuppressive role of DRAGO in vivo and its cooperation with p53. Deletion of one or both DRAGO alleles determined a progressive effect in survival decrease for p53−/− mice, while on a p53+/− background deletion of one or both DRAGO alleles reduced survival to the same extent. Furthermore DRAGO genotype did not affect tumor incidence since p53−/−
S223
and p53+/− subpopulations displayed tumor distributions in accordance with the literature. 923 Characterization of the Activity of Human Immunodeficiency Virus Protease Inhibitors Against Acute Myeloid Leukemia in Vitro 2 2 M. Kraus1 , J. Bader1 , H. Muller-Ide ¨ , T. Ruckrich ¨ , H. Overkleeft3 , C. Driessen1 . 1 Kantonsspital St. Gallen, MRC Lab Exp. Oncology, St. Gallen, ¨ Department of Medicine II, Tubingen, ¨ Switzerland, 2 University of Tubingen, Germany, 3 University of Leiden, Leiden Institute of Chemistry, Leiden, The Netherlands
Introduction: HIV protease inhibitors (HIV-PI) are oral drugs for HIV treatment with putative antitumor activity via the induction of ER stress and the inhibition of p-AKT and proteasome activity. Several clinical trials explore HIV-PI in solid tumors. We here characterize the activity of HIV-PI on AML cells to provide a basis for future clinical testing in AML. Material and Method: We compared the effects of HIV-PI AML cells regarding cytotoxicity, proteasome activity, ER-stress induction and AKTphosphorylation, and further evaluated synergistic activity with proteasome inhibitors, lenalidomide and sorafenib in vitro (AML cell lines, AML patient blasts). Results and Discussion: Lopinavir, Nelfinavir, Ritonavir and Saquinavir showed biological and molecular activity at concentrations within or near therapeutic drug levels (10−20 mM). In this dose range, they triggered ER stress-induced apoptosis, inhibited AKT-phosphorylation and showed synergistic cytotoxicity with bortezomib, carfilzomib, lenalidomide and sorafenib. Cell lines and normal control cells were significantly less sensitive towards HIV-PI induced cytotoxicity than primary cells. Nelfinavir was the only HIV-PI with proteasome-inhibiting activity in the tested dose range in intact cells, inhibiting also the bortezomib- and carfilzomib-insensitive b2 proteasome subunit. Conclusion: Nelfinavir, ritonavir, lopinavir and saquinavir in the 10−20 mM dose range are cytotoxic against primary AML cells in vitro, in contrast to control cells or cell lines, and act synergistically with bortezomib, carfilzomib, lenalidomide and sorafenib. Only nelfinavir inhibits proteasome activity in intact cells in vitro and in vivo. We consider nelfinavir as the most promising candidate HIV-PI that warrants clinical tested in AML. 924 The Ascidian Natural Product Eusynstyelamide B Induces G2/M Phase Arrest in Human Breast Cancer Cells M.S. Liberio1 , A.F.G. Quest2 , M. Sadowski3 , R.J. Quinn1 , R.A. Davis1 , C. Nelson3 . 1 Griffith University, Eskitis Institute, Brisbane, Australia, 2 Universidad de Chile, Laboratory of Cellular Communication Center for Molecular Studies of the Cell (CEMC), Santiago, Chile, 3 Queensland University of Technology, Australian Prostate Cancer Research Centre, Brisbane, Australia Researchers have shown that marine ascidians are an excellent source of new anticancer compounds that can exhibit new mechanisms of action. The Great Barrier Reef is a rich environment harbouring prolific ascidian biodiversity, and many of the species have never been explored for bioactive natural products. We selected a total of 143 ascidian specimens belonging to the family Didemnidae from the Eskitis Biota Library for organic solvent extraction. We tested the 143 extracts for anti-proliferative effects in the breast cancer cell line MDA-MB-231 using the real-time cell analyser xCELLigence System. Twentyone hit extracts were identified and investigated in MDA-MB-231 cells for their effects on cell proliferation, cell cycle, morphology and the level of the antiapoptotic protein Birc5/survivin. The most interesting extracts were selected for bioassay-guided fractionation in order to purify the bioactive compound(s). From these studies we identified a previously reported modified tryptophanarginine dipeptide dimer, named eusynstyelamide B (EB). We found that EB inhibited proliferation of MDA-MB-231 cells with an IC50 of 5 mM and arrested the cell cycle in G2/M phase, which was accompanied by cell enlargement. Apoptosis investigation was performed by FACS analysis measuring subG1 peak and annexin V staining. The slightly increase of apoptotic cells was consistent with the elevated level of the anti-apoptotic protein surviving in EBtreated MDA-MB-231 cells. Studies are ongoing to further characterize the potential anti-cancer properties of this compound and validate the findings in additional cancer cell lines. 925 F14512, a Polyamine Vectorized Anti-cancer Drug Exhibits a Marked Antileukemic Activity, Alone and in Combination With AraC 1 A. Kruczynski1 , A. Pillon1 , L. Creancier ´ , I. Vandenberghe1 , B. Gomes1 , V. Brel1 , V. Cartron1 , N. Chansard1 , J. Verdier1 , N. Guilbaud1 , J.-P. Annereau1 . 1 Institut De Recherche Pierre Fabre, Centre de Recherche en Oncologie ´ Experimentale, Toulouse, France
Background: Although cytotoxic chemotherapy induced complete remissions in AML patients, relapse rates are still very high, resulting in a poor outcome in most cases. Therefore the development of more effective drugs for AML represents a high level of priority. F14512 combines an epipodophyllotoxin