Patrizia Marini

Irradiation specifically sensitises solid tumour cell lines to TRAIL mediated apoptosis

BackgroundTRAIL (tumor necrosis factor related apoptosis inducing ligand) is an apoptosis inducing ligand with high specificity for malignant cell systems. Combined treatment modalities using TRAIL and cytotoxic drugs revealed highly additive effects in different tumour cell lines. Little is known about the efficacy and underlying mechanistic effects of a combined therapy using TRAIL and ionising radiation in solid tumour cell systems. Additionally, little is known about the effect of TRAIL combined with radiation on normal tissues.MethodsTumour cell systems derived from breast- (MDA MB231), lung- (NCI H460) colorectal- (Colo 205, HCT-15) and head and neck cancer (FaDu, SCC-4) were treated with a combination of TRAIL and irradiation using two different time schedules. Normal tissue cultures from breast, prostate, renal and bronchial epithelia, small muscle cells, endothelial cells, hepatocytes and fibroblasts were tested accordingly. Apoptosis was determined by fluorescence microscopy and western blot determination of PARP processing. Upregulation of death receptors was quantified by flow cytometry.ResultsThe combined treatment of TRAIL with irradiation strongly increased apoptosis induction in all treated tumour cell lines compared to treatment with TRAIL or irradiation alone. The synergistic effect was most prominent after sequential application of TRAIL after irradiation. Upregulation of TRAIL receptor DR5 after irradiation was observed in four of six tumour cell lines but did not correlate to tumour cell sensitisation to TRAIL. TRAIL did not show toxicity in normal tissue cell systems. In addition, pre-irradiation did not sensitise all nine tested human normal tissue cell cultures to TRAIL.ConclusionsBased on the in vitro data, TRAIL represents a very promising candidate for combination with radiotherapy. Sequential application of ionising radiation followed by TRAIL is associated with an synergistic induction of cell death in a large panel of solid tumour cell lines. However, TRAIL receptor upregulation may not be the sole mechanism by which sensitation to TRAIL after irradiation is induced.

Aurora kinase inhibitor ZM447439 induces apoptosis via mitochondrial pathways

ZM447439 (ZM) is a potent and selective inhibitor of aurora-A and -B kinase with putative anti-tumoral activity. Inhibitors of aurora kinases were shown to induce apoptosis in vitro and in vivo. To investigate the underlying mechanisms, cell death pathways triggered by ZM was analysed in HCT-116 colorectal cancer cells. Through correlation of polyploidization and apoptosis in different knockout cells, the interrelation of these cellular responses to ZM was investigated. ZM induced apoptosis in a concentration- and time-dependent manner. ZM-induced apoptosis was associated with an upregulation of p53, breakdown of the mitochondrial membrane potential (DeltaPsim) and activation of caspase-3. To precisely define key components for ZM-induced apoptosis, knockout cells lacking p53, Bak, Bax or both Bak and Bax were used. Lack of p53 reduced ZM-induced apoptosis and breakdown of DeltaPsim, while lack of Bak, Bax or both almost completely inhibited apoptosis and breakdown of DeltaPsim. Since no difference in apoptosis induction was detectable between HCT-116 cells lacking Bak, Bax or both, apoptosis induction depended non-redundantly on both Bak and Bax. Phenomenally, ZM induced notable polyploidization in all examined cells, especially in p53-/- cells. A correlation between polyploidization and apoptosis was observed in wild-type, and also in p53-/- cells, albeit with a modest extent of apoptosis. Moreover, in Bak-/-, Bax-/- and Bak/Bax-/- cells apoptosis was totally inhibited in spite of the strongest polyploidization, suggesting apoptosis may be a secondary event following polyploidization in HCT-116 cells. Thus ZM-induced apoptosis depends not only on polyploidization, but also on the intracellular apoptotic signaling.

Molecular requirements for the combined effects of TRAIL and ionising radiation.

BACKGROUND AND PURPOSE Previously it was shown that combination of death ligand TRAIL and irradiation strongly increases cell kill in several human tumour cell lines. Since Bcl-2 overexpression did not strongly interfere with the efficacy, components of the mitochondrial death pathway are not required for an effective combined treatment. In the present study the minimal molecular prerequisites for the efficacy of a combined treatment were determined. MATERIALS AND METHODS Apoptosis induction in control, caspase-8 and FADD negative Jurkat cells, BJAB control and FADD-DN cells was analysed by FACS. Activation of caspase-8, -10 and -3 and cleavage of PARP was determined by immunoblotting. TRAIL receptors were activated using recombinant human TRAIL. Surface expression of TRAIL receptors DR4 and DR5 was analysed by FACS. RESULTS Jurkat T-cells express the agonistic DR5 receptor but not DR4. Presence of FADD was found to be essential for TRAIL induced apoptosis. Caspase-8 negative cells show very low rates of apoptosis after prolonged stimulation with TRAIL. No combined effects of TRAIL with irradiation could be found in FADD-DN overexpressing and FADD deficient cells. However, the combination of TRAIL and irradiation clearly lead to a combined effect in caspase-8 negative Jurkat cells, albeit with reduced death rates. In these cells activation of the alternative initiator caspase-10 could be detected after combined treatment. CONCLUSION Our data show that a combined therapy with TRAIL and irradiation will only be effective in cells expressing at least one agonistic TRAIL receptor, FADD and caspase-8 or caspase-10.

Efficacy of a Triple Treatment with Irradiation, Agonistic TRAIL Receptor Antibodies and EGFR Blockade

Background and Purpose:Since the efficacy of a single targeted agent in combination with ionizing radiation is limited by putative treatment resistances, a rationally designed triple treatment consisting of an agonistic antibody targeting either TRAIL-R1 (mapatumumab) or TRAIL-R2 (lexatumumab), radiation and an epidermal growth factor receptor-(EGFR-)inhibiting antibody (cetuximab) was tested.Material and Methods:Induction of apoptosis after triple treatment was determined in Colo205, HCT116 and FaDu cells by Hoechst 33342 stain. The degree of interaction was determined by isobologram analysis. A knockout variant of HCT116 was used to examine Bax dependence of the triple treatment. The role of Akt/PKB signaling was analyzed using the phosphatidylinositol 3-kinase inhibitor LY294002. Clonogenic assays were performed to examine the effect on clonogenic survival of tumor cells.Results:A synergistic effect of radiation, cetuximab and agonistic TRAIL-R antibodies was demonstrated in cell lines derived from colorectal tumors or head-and-neck cancers. The efficacy of this multimodal approach was dependent on Bax and inhibition of Akt/PKB in the cell systems used. The results also show a positive impact on clonogenic cell death in several cell lines.Conclusion:These data suggest that rationally designed multimodal therapy approaches integrating radiation with more than one targeted agent will open new perspectives in radiation oncology.Hintergrund und Ziel:Die Wirkung einer Kombination von singulären molekular gezielten Substanzen mit ionisierender Strahlung kann durch Behandlungsresistenzen limitiert sein. Daher wurde geprüft, ob eine molekular gezielte Dreifachkombination, bestehend aus einem agonistischen Antikörper gegen entweder TRAIL-R1 (Mapatumumab) oder TRAIL-R2 (Lexatumumab) sowie einem Antikörper (Cetuximab) gegen den epidermalen Wachstumsfaktor-Rezeptor (EGFR), zur optimierten Tumorzellabtötung in vitro beitragen kann.Material und Methodik:Die Apoptoseinduktion nach Dreifachtherapie wurde in den Zelllinien Colo205, HCT116 und FaDu durch Hoechst-33342-Färbung bestimmt. Der Interaktionsgrad wurde mittels Isobologrammanalyse ermittelt. Eine Knock-out-Variante der Zelllinie HCT116 wurde verwendet, um die Bax-Abhängigkeit der Dreifachtherapie zu untersuchen. Die Rolle des Akt/PKB-Signalwegs wurde mit dem Phosphatidylinositol-3-Kinase-Inhibitor LY294002 analysiert. Koloniebildungstests wurden durchgeführt, um den Effekt auf das klonogene Überleben der Tumorzellen zu untersuchen.Ergebnisse:Ein synergistischer Effekt von Bestrahlung, Cetuximab und agonistischen TRAIL-R-Antikörpern ließ sich in Adeno- und Plattenepithelkarzinomzellsystemen nachweisen. Die Effektivität dieses multimodalen Ansatzes war abhängig von Bax und der Inhibierung von Akt/PKB. Parallel mit einer erhöhten Apoptoserate fand sich ein optimierte Eradikation klonogener Tumorzellen.Schlussfolgerung:Diese Daten deuten darauf hin, dass zielgerichtete multimodale Therapieansätze unter Einbeziehung mehrerer molekularer Zielstrukturen neue Perspektiven in der Radioonkologie eröffnen könnten.

SiMa, a new neuroblastoma cell line combining poor prognostic cytogenetic markers with high adrenergic differentiation

We describe the establishment and characterization of a new neuroblastoma (Nb) cell line, SiMa, carrying the major recurrent chromosome changes associated with poor prognosis Nb, including amplification of N-MYC by formation of double minutes (dmin), der(1)t(1;17)(p35;q12) and der(22)t(17;22)(q22;p13), and loss of chromosome 11, documented at both initiation and late passage. In contrast to these cytogenetic stigmata of poor prognosis, analysis of catecholamine synthesis by high pressure liquid chromatography (HPLC) measurement revealed an advanced degree of adrenergic differentiation with high rates of 3,4-Dihydroxyphenylalanine (DOPA), noradrenaline, homovanillic acid (HVA), and vanillylmandelic acid (VMA) production. Contrastingly advanced differentiation and poor prognostic genetic markers combine to render SiMa a unique instrument for investigating the pathology and therapy of Nb.

Combined treatment with lexatumumab and irradiation leads to strongly increased long term tumour control under normoxic and hypoxic conditions

PurposeThe combination of ionizing radiation with the pro-apoptotic TRAIL receptor antibody lexatumumab has been shown to exert considerable synergistic apoptotic effects in vitro and in short term growth delay assays. To clarify the relevance of these effects on local tumour control long-term experiments using a colorectal xenograft model were conducted.Materials and methodsColo205-xenograft bearing NMRI (nu/nu) nude mice were treated with fractionated irradiation (5× 3 Gy, d1-5) and lexatumumab (0.75 mg/kg, d1, 4 and 8). The tumour bearing hind limbs were irradiated with graded single top up doses at d8 under normoxic (ambient) and acute hypoxic (clamped) conditions. Experimental animals were observed for 270 days. Growth delay and local tumour control were end points of the study. Statistical analysis of the experiments included evaluation of tumour regrowth and local tumour control.ResultsCombined treatment with irradiation and lexatumumab led to a pronounced tumour regrowth-delay when compared to irradiation alone. The here presented long-term experiments revealed a highly significant rise of local tumour control for normoxic (ambient) (p = 0. 000006) and hypoxic treatment (p = 0. 000030).ConclusionOur data show that a combination of the pro-apoptotic antibody lexatumumab with irradiation reduces tumour regrowth and leads to a highly increased local tumour control in a nude mouse model. This substantial effect was observed under ambient and more pronounced under hypoxic conditions.

Death receptor ligands: new strategies for combined treatment with ionizing radiation.

The major goal of modern radiation oncology is the achievement of a maximal tumor control with minimal normal tissue damage. However, normal tissue tolerance may preclude the application of tumoricidal radiation doses. In order to overcome this limitation, strategies either to increase normal tissue tolerance or to reduce the radiation dose required may prove beneficial. In this regard, attempts to minimize the required radiation dose by reducing the number of malignant clonogenic cells are promising. Therefore, therapies which induce programmed cell death (apoptosis) in tumor cells, may prove to be suitable approaches. TRAIL (TNFalpha-related apoptosis inducing ligand)/Apo2L is a very promising member of the family of death ligands. The ligand preferentially induces apoptotic cell death in a wide range of tumor cells but not in normal cells. TRAIL/Apo2L triggers apoptosis even in cells not undergoing apoptosis in response to radiation, since ionizing radiation induce apoptosis by a different pathway as death ligands although an overlapping set of molecules is involved. Combination of both modalities has been shown to induce additive or synergistic apoptotic effects and eradication of clonogenic tumor cells thereby increasing the therapeutic efficacy. The present article reviews this novel biological strategy for optimized radiotherapy based on the combination of ionizing irradiation and death receptor triggered cell death.

Combination of the Pro-Apoptotic TRAIL-Receptor Antibody Mapatumumab With Ionizing Radiation Strongly Increases Long-Term Tumor Control Under Ambient and Hypoxic Conditions

PURPOSE Mapatumumab, an agonistic tumor necrosis factor-related apoptosis inducing ligand-receptor antibody, exerts highly synergistic apoptotic effects in vitro and in short-term growth delay assays when combined with irradiation. Because it remained unclear in how far these effects influence local tumor control, long-term experiments using a colorectal xenograft model were undertaken. MATERIAL AND METHODS Experiments were performed with irradiation (5 x 3 Gy, d1-5) and mapatumumab (10 mg/kg) in Colo205-xenograft-bearing NMRI (nu/nu) nude mice. Graded top up doses were delivered on the tumor-bearing hind leg under ambient and hypoxic conditions; follow-up was 270 days. Growth delay and local tumor control were end points of the study. Statistical analysis of the experiments included calculation of tumor regrowth and local tumor control. RESULTS After combined treatment, a pronounced tumor regrowth-delay was observed when compared with irradiation alone. Long-term experiments revealed a highly significant increase in local tumor control for ambient (p = 0.00076) and hypoxic treatment (p = 0.000069). CONCLUSIONS The present data demonstrate for the first time that combination of a pro-apoptotic antibody with irradiation results in evidently reduced tumor regrowth times and subsequently highly increased local tumor control under normoxic and hypoxic conditions in a xenograft mouse model.

[Death inducing ligands in combination with ionizing radiation: objective and current knowledge].

Hintergrund: Zukünftige Forschungsstrategien in der Strahlentherapie werden neben der weiteren Verbesserung der Bestrahlungstechnologie Wege zur biologischen Optimierung umfassen. Hier können mehrere Bereiche abgegrenzt werden: Prädiktion, Entwicklung neuer zytotoxischer Wirkprinzipien, Erhöhung derNormalgewebstoleranz und Optimierung der Radiochemotherapie. Essenziell für die wissenschaftliche Bearbeitung der genannten Ansätze ist die Aufklärung der grundlegenden Mechanismen des strahleninduzierten Zelltods. Material und Methode: In der vorliegenden Arbeit soll eine neuartige biologische Strategie zur Optimierung der Strahlenwirkung vorgestellt werden, die auf Basis molekularer Zelltodabläufe entwickelt wurde. Ergebnisse: Die durch Todesliganden induzierte Apoptose weist einen distinkten Signaltransduktionsweg im Vergleich zur strahleninduzierten Apoptose auf. TRAIL (TNFα-related apoptosis inducing ligand), ein Mitglied der TNF-Todesligandfamilie, induziert Apoptose in Tumorzelllinien, ohne nach heutigem Kenntnisstand in Normalgeweben toxisch zu sein. Aufgrund der getrennten Signalkaskaden ist eine Kombination von TRAIL mit ionisierender Strahlung sinnvoll. In der vorliegenden Arbeit werden die Rationale, eigene Daten sowie Ergebnisse anderer Gruppen vorgestellt und der Stand der wissenschaftlichen Erkenntnisse über TRAIL dargelegt. Schlussfolgerungen: Eine Apoptoseresistenz gegenüber ionisierender Strahlung ist nicht gleichbedeutend mit einer globalen Apoptoseresistenz. Zelltodliganden wie TRAIL induzieren Apoptose auch in Zellen mit gestörter Apoptoseinduktion nach Bestrahlung. Eine Kombination beider Modalitäten führt zu additiven und teilweise synergistischen Effekten hinsichtlich der Apoptoseinduktion und der Eradikation klonaler Tumorzellen. Anhand der vorgestellten Kombination von TRAIL mit Bestrahlung wird deutlich, dass es möglich ist, auf Basis molekular- oder zellbiologischer Untersuchungen völlig neue Strategien zur Optimierung der Strahlenwirkung zu entwickeln.Background: Apart from optimization of the radiation technology, future new strategies in radiation oncology will focus on the biological optimisation leading to improved adaptation of the tumor therapy on each tumor entity. In this regard, different areas of biological research may be distinguished: prediction, development of new cytotoxic agents, improvement of the tolerance of normal tissue and the optimisation of radiochemotherapy. Material and Method: For the development of new strategies the knowledge of molecular mechanisms of radiation induced cell death is essential. In the present article a novel biological strategy for optimisation of radiotherapy is introduced. Results: Distinct pathways mediate apoptosis in response to death receptor stimulation and ionizing radiation although an overlapping set of molecules is involved. TRAIL (TNFα-related apoptosis inducing ligand) is a type-II membrane protein belonging to the TNF family, which preferentially induces apoptotic cell death in a wide variety of tumor cells but not in normal cells. Based on the assumption of distinct signalling pathways, combination of TRAIL and ionizing radiation seems ideal to increase the therapeutic efficacy. Our study presents the rationale, own data, data of other groups as well as the current status of the latest findings regarding the function and physiological role of the TRAIL death ligand. Conclusions: Apoptosis resistance towards ionising radiation is not associated with a general apoptosis resistance. Death ligands, for example TRAIL trigger apoptosis even in cells not undergoing apoptosis in response to radiation. A combination of both modalities induces additive or synergistic effects in regard to apoptosis induction and eradication of clonogenic tumor cells. Thus, the combination of TRAIL with radiation proofs that novel treatment strategies may be developed on the basis of molecular or cell biological research.

Inhibition of the Membrane Localization of p21 ras Proteins by Lovastatin in Tumor Cells Possessing a Mutated N-ras Gene

Mutated ras genes are found in a variety of human tumors. For biological activity the gene product p21 ras needs to be bound to the cell membrane by a farnesyl residue. Treatment of tumor cells with lovastatin reduces the availability of farnesyl pyrophosphate for the modification of the ras proteins. The membrane localization of p21 ras has been reduced by 30-36% after the tumor cells have grown in the presence of 10 microM lovastatin for 7 days. The extent of the inhibition depends on the growth kinetics of the cell lines.