- Email: [email protected]
338 ONCOLYTIC VIROTHERAPY AND THE PROTEASOME INHIBITOR BORTEZOMIB ACT SYNERGISTICALLY IN THE TREATMENT OF HEPATOCELLULAR CARCINOMA
03A. LIVER TUMORS – A) EXPERIMENTAL 337 THE ONCOLYTIC ADENOVIRUS ADP53-SENSOR FOR SELECTIVE REPLICATION IN P53-ALTERED TUMORS AND POTENTIAL TARGET SPECTRUM IS NOT LIMITED BY THE P73 STATUS F. Kuehnel, E. Guerlevik, T.C. Wirth, L. Zender, M.P. Manns, S. Kubicka. Gastroenterology, Hepatology and Endocrinology, Medical School Hannover, Hannover, Germany E-mail: kuehnel.fl[email protected] Background and Aims: Oncolytic virotherapy provides a promising mean for the treatment of solid tumors. A central goal of virotherapeutic vectors are tight restriction of viral replication to tumor cells and a broad range of potential target tumors. Aim of our study was the establishment of a conditionally replicating adenovirus able to recognize the transcriptional p53-status of target cells and able to consequently respond by the selective onset of viral replication in cells lacking functional p53. Methods: We constructed Adp53-sensor by combining E1A-expression controlled by a GAL4-binding CMV-promoter to the expression of the GAL4KRAB-repressor under control of the p53-sensitive promoter prMinRGC. Vector selectivity and therapeutic efficacy were characterized by luciferase assays, western blots, oncolysis assays, replication kinetics, and tumor size measurements in vivo. Results: First, the artificial promoter prMinRGC included in Adp53sensor showed enormous induction by endogenous or overexpressed p53, but was silent in p53-aberrant cells. Though the promoter was sensitive to overexpressed p73-b, but no further members of the p53 family, prMinRGC was silent in p53-altered cells including p73-positive cells. As endogenous p73 seems to be insufficient to activate prMinRGC, the p73-status should not limit the potential tumor spectrum of therapeutic Adp53sensor application. We could further show selectivity and oncolytic efficacy of Adp53sensor by comparing a large panel of cell lines with different p53/p73 status including HCC-cells. Furthermore, p53-selectivity was confirmed in human hepatocytes where Adp53-sensor replication was inhibited. Western Blots demonstrated correct function of the p53dependent, GAL4-KRAB regulated E1A expression in cells dependent on the p53-status of target cells. Doxorubicin pretreatment significantly enhanced selectivity of Adp53-sensor. In oncolysis assays Adp53-sensor showed slightly reduced oncolytic properties compared to Ad-wt but was both more p53-selective and efficient compared to ONYX-015. The vector was also more efficient in the treatment of s.c. grown Hep3B-xenografts and treatment resulted in partial remissions. Conclusions: Our data suggest that Adp53-sensor represents an effective tool for p53-selective virotherapy of cancer. p53-selectivity and oncolytic properties of Adp53-sensor are improved compared to ONYX-015. The vector can be applied to a broad tumor spectrum as the selective function is not limited by the p73 status of the target tumors.
338 ONCOLYTIC VIROTHERAPY AND THE PROTEASOME INHIBITOR BORTEZOMIB ACT SYNERGISTICALLY IN THE TREATMENT OF HEPATOCELLULAR CARCINOMA B. Boozari, T.C. Wirth, N. Woller, M.P. Manns, S. Kubicka, F. Kuehnel. Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany E-mail: kuehnel.fl[email protected] Background and Aims: Oncolytic Therapy using conditionally replicating adenoviral vectors provides a challenging tool for the treatment of solid tumors. Recently, the proteasome inhibitor Bortezomib has been established as novel anti-cancer agent. However, potential interference with virotherapy if applied as combination treatment is unkown. Aim of this work was to investigate adenoviral replication under Bortezomib treatment in Huh-7 cells.
S133
Methods: Huh-7 cells were treated with single or combined treaments of Bortezomib (10 nM) and hTERT-Ad virotherapy and were investigated by different analytical methods. Results: First, LC3 I-LC3 II Western Blots showed no alteration of autophagic status compared to control. As Caspase-3 was highly active at the same time this suggests that Bortezomib kills cells solely by apoptosis. This was confirmed by a Bortezomib-dependent downregulation of XIAP, a potent antiapoptotic factor. Adenoviral infection usually leads to EIF2aphosphorylation, repression of host cell mRNA translation and preferred translation of viral transcripts. We found, that Bortezomib inhibits EIF2a phosphorylation in adenoviral infected cells. As this observation suggests a negative interference between adenoviral infection and Bortezomib we investigated viral replication and viral protein expression. Interestingly, late viral proteins were expressed much earlier suggesting that proteasome inhibition leads to early accumulation of viral proteins that could compensate for the inhibition of EIF2-a phosphorylation. Concomitantly, 10 nM Bortezomib did not influence viral DNA replication and only slightly reduced the particle numbers. In contrast, we could show that adenoviral infection significantly increased Bortezomib-induced caspase-3 activation. Caspase-3 was activated in a synergistic manner as adenovirus alone did not result in caspase-3 activation. The synergism was confirmed by oncolysis assays showing an increased cell killing by combination of both treatments and could also be confirmed in vivo in s.c. xenografted Huh7 tumors on nude mice. Conclusion: We could show that low dose Bortezomib treatment does not significantly interfere with adenoviral replication but acts synergistically in inducing apoptosis in virotherapy of HCC cells. Coapplication of proteasome inhibition by Bortezomib together with virotherapy could therefore provide a promising approach for the treatment of HCC 339 TARGETING THE HISTONE DEACETYLASE-SIRT1 FOR ANTI-TUMOR THERAPY: INHIBITION OF SIRT1 DOWN-REGULATES HIF-1 A. Laemmle, S. Vorburger, A. Keogh, V. Roh, D. Candinas, D. Stroka. Dept. of Visceral & Transplantation Surgery, Dept. Clinical Research, Inselspital, Bern University Hospital, and University of Bern, Switzerland E-mail: [email protected] Background and Aims: SIRT1 is a broadly conserved NAD-dependent deacetylase (class III histone deacetylase), that downregulates stressinduced p53 and FoxO pathways of apoptosis, thus, favoring survival under stress. Inhibition of SIRT1 promotes cell cycle arrest and apoptosis in human cancer cells and therefore is a potential target for anti-tumoral therapies. Activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) plays a key role in adaptation of tumor cells to a hypoxic microenvironment by stimulating proangiogenic factors and inducing enzymes necessary for anaerobic metabolism. Here, we investigated whether the inhibition of SIRT1 has a growth inhibitory effect in hepatocellular carcinoma cells (HCC) and impairs the expression and function of HIF-1. Methods: Three HCC cell lines (Hep3B, HepG2 and Huh-7) were pretreated with 100mM Sirtinol (a specific small molecule inhibitor of SIRT1) or with shRNA against SIRT1 in a lentiviral vector. For hypoxic conditions cells were incubated in a hypoxic chamber with 1.5% O2. Results: The inhibition of SIRT1 led to growth inhibition and apoptosis in all three HCC cell lines. In cells treated with Sirtinol, cell viability was decreased to 65% after 24 hours and selective targeting of SIRT1 by using shRNA killed over 80% of cells within 7 days. Both methods of SIRT1 inhibition strongly down regulated HIF-1a protein levels, what was shown by Western blot. In HCC cells transiently transfected with a luciferase construct driven by a HIF-1-dependent promoter, HIF-1 transactivation ability was decreased over 50% upon Sirtinol treatment. HIF-1 inhibition was confirmed by real-time RT-PCR of VEGF and EPO (known HIF-1 target genes). Sirtinol inhibited the hypoxic induction of EPO mRNA from a 20-fold increase down to normoxic levels in Huh-7 cells and from a 80fold down to a 5-fold increase in Hep3B cells.
338 ONCOLYTIC VIROTHERAPY AND THE PROTEASOME INHIBITOR BORTEZOMIB ACT SYNERGISTICALLY IN THE TREATMENT OF HEPATOCELLULAR CARCINOMA B. Boozari, T.C. Wirth, N. Woller, M.P. Manns, S. Kubicka, F. Kuehnel. Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany E-mail: kuehnel.fl[email protected] Background and Aims: Oncolytic Therapy using conditionally replicating adenoviral vectors provides a challenging tool for the treatment of solid tumors. Recently, the proteasome inhibitor Bortezomib has been established as novel anti-cancer agent. However, potential interference with virotherapy if applied as combination treatment is unkown. Aim of this work was to investigate adenoviral replication under Bortezomib treatment in Huh-7 cells.
S133
Methods: Huh-7 cells were treated with single or combined treaments of Bortezomib (10 nM) and hTERT-Ad virotherapy and were investigated by different analytical methods. Results: First, LC3 I-LC3 II Western Blots showed no alteration of autophagic status compared to control. As Caspase-3 was highly active at the same time this suggests that Bortezomib kills cells solely by apoptosis. This was confirmed by a Bortezomib-dependent downregulation of XIAP, a potent antiapoptotic factor. Adenoviral infection usually leads to EIF2aphosphorylation, repression of host cell mRNA translation and preferred translation of viral transcripts. We found, that Bortezomib inhibits EIF2a phosphorylation in adenoviral infected cells. As this observation suggests a negative interference between adenoviral infection and Bortezomib we investigated viral replication and viral protein expression. Interestingly, late viral proteins were expressed much earlier suggesting that proteasome inhibition leads to early accumulation of viral proteins that could compensate for the inhibition of EIF2-a phosphorylation. Concomitantly, 10 nM Bortezomib did not influence viral DNA replication and only slightly reduced the particle numbers. In contrast, we could show that adenoviral infection significantly increased Bortezomib-induced caspase-3 activation. Caspase-3 was activated in a synergistic manner as adenovirus alone did not result in caspase-3 activation. The synergism was confirmed by oncolysis assays showing an increased cell killing by combination of both treatments and could also be confirmed in vivo in s.c. xenografted Huh7 tumors on nude mice. Conclusion: We could show that low dose Bortezomib treatment does not significantly interfere with adenoviral replication but acts synergistically in inducing apoptosis in virotherapy of HCC cells. Coapplication of proteasome inhibition by Bortezomib together with virotherapy could therefore provide a promising approach for the treatment of HCC 339 TARGETING THE HISTONE DEACETYLASE-SIRT1 FOR ANTI-TUMOR THERAPY: INHIBITION OF SIRT1 DOWN-REGULATES HIF-1 A. Laemmle, S. Vorburger, A. Keogh, V. Roh, D. Candinas, D. Stroka. Dept. of Visceral & Transplantation Surgery, Dept. Clinical Research, Inselspital, Bern University Hospital, and University of Bern, Switzerland E-mail: [email protected] Background and Aims: SIRT1 is a broadly conserved NAD-dependent deacetylase (class III histone deacetylase), that downregulates stressinduced p53 and FoxO pathways of apoptosis, thus, favoring survival under stress. Inhibition of SIRT1 promotes cell cycle arrest and apoptosis in human cancer cells and therefore is a potential target for anti-tumoral therapies. Activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) plays a key role in adaptation of tumor cells to a hypoxic microenvironment by stimulating proangiogenic factors and inducing enzymes necessary for anaerobic metabolism. Here, we investigated whether the inhibition of SIRT1 has a growth inhibitory effect in hepatocellular carcinoma cells (HCC) and impairs the expression and function of HIF-1. Methods: Three HCC cell lines (Hep3B, HepG2 and Huh-7) were pretreated with 100mM Sirtinol (a specific small molecule inhibitor of SIRT1) or with shRNA against SIRT1 in a lentiviral vector. For hypoxic conditions cells were incubated in a hypoxic chamber with 1.5% O2. Results: The inhibition of SIRT1 led to growth inhibition and apoptosis in all three HCC cell lines. In cells treated with Sirtinol, cell viability was decreased to 65% after 24 hours and selective targeting of SIRT1 by using shRNA killed over 80% of cells within 7 days. Both methods of SIRT1 inhibition strongly down regulated HIF-1a protein levels, what was shown by Western blot. In HCC cells transiently transfected with a luciferase construct driven by a HIF-1-dependent promoter, HIF-1 transactivation ability was decreased over 50% upon Sirtinol treatment. HIF-1 inhibition was confirmed by real-time RT-PCR of VEGF and EPO (known HIF-1 target genes). Sirtinol inhibited the hypoxic induction of EPO mRNA from a 20-fold increase down to normoxic levels in Huh-7 cells and from a 80fold down to a 5-fold increase in Hep3B cells.