Abstract: Concomitant inhibition of ErbB1/2- and VEGF receptor-signaling synergizes when used in combination with DNA-damaging agents. Here, we investigated for the first time the combined treatment modality of the novel dual specific receptor tyrosine kinase inhibitor AEE788 with ionizing radiation and analyzed treatment-induced end points in situ as indicators for a potential sensitizing mechanism. Furthermore, we assessed tumor hypoxia in response to different antiangiogenic and antiproliferative treatment modalities. The combined treatment effect was investigated in a spontaneously growing mammary carcinoma model and against Her-2/neu-overexpressing mammary carcinoma allografts. In tumor allografts derived from murine mammary carcinoma cells of mouse mammary tumor virus/c-neu transgenic mice, a minimal treatment regimen with AEE788 and fractionated irradiation resulted in an at least additive tumor response. Treatment response in the corresponding spontaneous tumor model strongly exceeded the response induced in the isogenic allografts. Treatment-induced changes of tumor proliferation, apoptosis, and microvessel density were similar in the two tumor models. Treatment with AEE788 alone or in combination with IR strongly improved tumor oxygenation in both tumor models as determined by the detection of endogenous and exogenous markers of tumor hypoxia. Specific inhibition of the VEGF-receptor tyrosine kinase versus Erb1/2-receptor tyrosine kinase indicated that it is the antiproliferative and not the antiangiogenic potency of AEE788 that mediates the hypoxia-reducing effect of this dual kinase-specific inhibitor. Overall, we show that concomitant inhibition of ErbB- and VEGF-receptor signaling by AEE788, in combination with ionizing radiation, is a promising treatment approach, especially in hypoxic, oncogenic ErbB-driven tumors.
Abstract: In current applied radiobiology, there exists a tremendous effort in basic and translational research to identify novel treatment modalities combining ionizing radiation with anticancer agents. This is mainly due to the highly improved molecular understanding of intrinsic radioresistance and the profiling of cellular stress responses to irradiation during recent years. Ionizing radiation not only damages DNA but also affects multiple cellular components that induce a multi-layered stress response. The treatment responses can be restricted to the individual cell level but might also be part of an intercellular stress communication network. Both DNA damage-induced signaling (which results in cell cycle arrest and induction of the DNA-repair machinery) and also ionizing radiation-induced signal transduction cascades, which are generated at cellular sites distant from and independent of DNA-damage, represent interesting targets for anticancer treatment modalities to sensitize for ionizing radiation. Due to the lack of molecular knowledge classic radiobiology assembled the cellular and tissue responses into four groups (4 R's of radiotherapy) which describe biological factors influencing the treatment response to fractionated radiotherapy. These classic 4 R's are Repair, Reassortment, Repopulation and Reoxygenation. With the tremendous progress in molecular oncology we now begin to understand theses factors on the molecular level. At the same time this classification may guide modern molecular radiobiologists to identify novel pharmaceuticals and antisignaling agents which can modulate the treatment response to irradiation. In this review we describe current approaches to sensitize tumor cells with novel anticancer agents along the lines of these 4 R's.
Abstract: Interference with microtubule function is a promising antitumoral concept. Paclitaxel is a clinically validated tubulin-targeting agent; however, treatment with paclitaxel is often limited by taxane-related toxicities and is ineffective in tumors with multidrug-resistant cells. Patupilone (EPO906, epothilone B) is a novel non-taxane-related microtubule-stabilizing natural compound that retains full activity in multidrug-resistant tumors and is clinically less toxic than paclitaxel. Here we have investigated the effect of combined treatment with ionizing radiation and patupilone or paclitaxel in the P-glycoprotein-overexpressing, p53-mutated human colon adenocarcinoma cell line SW480 and in murine, genetically defined E1A/ras-transformed paclitaxel-sensitive embryo fibroblasts. Patupilone and paclitaxel alone and in combination with ionizing radiation reduced the proliferative activity of the E1A/ras-transformed cell line with similar potency in the sub and low nanomolar range. SW480 cells were only sensitive to patupilone, and combined treatment with low-dose patupilone (0.1 nmol/L) followed by clinically relevant doses of ionizing radiation (2 and 5 Gy) resulted in a supra-additive cytotoxic effect. Inhibition of the drug efflux protein P-glycoprotein with verapamil resensitized SW480 cells to treatment with low doses of paclitaxel alone and in combination with IR. In tumor xenografts derived from SW480 cells a minimal treatment regimen with patupilone and fractionated irradiation (1 x 2 mg/kg plus 4 x 3 Gy) resulted in an at least additive tumor response with extended tumor growth arrest. Analysis by flow cytometry in vitro revealed an apoptosis- and G(2)-M-independent mode of radiosensitization by patupilone. Interestingly though, a transient accumulation of cells in S phase was observed on combined treatment.Overall, patupilone might be a promising alternative in paclitaxel-resistant, P-glycoprotein-overexpressing tumors for a combined treatment regimen using ionizing radiation and a microtubule inhibitor.
Abstract: In locally advanced epithelial malignancies, local control can be achieved with high doses of radiotherapy (RT). Concurrent chemoradiotherapy can improve tumor control in selected solid epithelial adult tumors; however, treatment-related toxicity is of major concern and the therapeutic window often small. Therefore, novel pharmacologic radiosensitizers with a tumor-specific molecular target and a broad therapeutic window are attractive. Because of clonal heterogeneity and the high mutation rate of these tumors, combined treatment with single molecular target radiosensitizers and RT are unlikely to improve sustained local tumor control substantially. Therefore, radiosensitizers modulating entire tumor cell survival pathways in epithelial tumors are of potential clinical use. We discuss the preclinical efficacy and the mechanism of three different, potential radiosensitizers targeting the PTEN/PI3K/Akt survival pathway. These compounds were initially thought to act as single-target agents against growth factor receptors (PKI 166 and PTK 787) or protein kinase C isoforms (PKC 412). We describe an additional target for these compounds. PKI 166 (an epidermal growth factor [EGF] receptor inhibitor) and PKC 412, target the PTEN/PI3K/Akt pathway mainly in tumor cells, and PTK 787 (a vascular endothelial growth factor [VEGF] receptor inhibitor) in endothelial cells. Even for these broader range molecular radiosensitizers, the benefit could be restricted to human epithelial tumor cell clones with a distinct molecular profile. Therefore, these potential radiosensitizers have to be carefully tested in specific model systems before introduction in early clinical trials.
Abstract: Proteolytic activation of zymogens or controlled degradation of inhibitory factors is part of a major regulatory system on the post-translational level to regulate treatment induced cellular stress responses. The identification of differential activity based substrates is thus of high interest to prioritize and validate candidate targets for drug discovery. Here we present a novel subtractive substrate phage display screening method for the selection of treatment induced post-translational peptide modifications in complex proteomes. We investigated this approach with tumor cells in response to a protease activating anticancer treatment modality using subtractive and iterative screening of cellular extracts derived from control and treated cells. Specific phage were identified that served as substrates for proteolytic activities in response to treatment related activity changes and could be distinguished from substrates for unspecific proteolytic background activities. Novel, selected peptide substrates were investigated in vitro and in vivo and showed high substrate specificity and functional biological significance.
Abstract: Cell cycle checkpoints are activated in response to DNA-damage to ensure that accurate copies of the cellular genome are passed on to the next generation and to avoid replication and segregation of damaged DNA. These cellular control systems can be overcome by combining conventional DNA-damaging agents with compounds that target the cell cycle regulatory pathways, to enhance cytotoxicity. Tumor cells often comprise a corrupted G(1) cell cycle checkpoint while the G(2) cell cycle checkpoint is still intact. This review describes the concept of G(2) checkpoint abrogation with recognized (methylxanthines, UCN-01) and novel G(2) checkpoint abrogators to potentiate the cytotoxicity of DNA-damaging drugs and ionizing radiation. It illustrates the potential of G(2) checkpoint abrogators to preferentially sensitize p53-mutated, treatment resistant tumor cells for genotoxic treatment. Identification of the targets of caffeine and UCN-01 to be key-players of the G(2) checkpoint (ATM/ATR and Chk1, respectively) promoted the search for novel inhibitors of this checkpoint. Even though a direct causal link between G(2) checkpoint abrogation and chemo-/radiosensitization is difficult to prove the multitude of these novel compounds validate that inhibition of critical elements of the G(2) checkpoint (ATM/ATR-Chk1/Chk2-CDC25C-cascade) potentiates the cytotoxicity of DNA-damaging agents.
Abstract: DNA double strand breaks are the pivotal cellular damage induced by ionizing radiation. A plethora of molecular and cellular processes are activated as part of the cellular stress response that result in cell cycle arrest and induction of the DNA-repair machinery to restore the damage of DNA or to activate a cell death program. However ionizing radiation also initiates signal transduction cascades that are generated at cellular sites distant from and independent of DNA-damage. These signaling processes are similar to hormone activated growth factor receptor controlled signal transduction cascades and represent interesting targets for anticancer treatment modalities combining ionizing radiation with molecular defined pharmacological compounds. Activation of these signal transduction cascades upon irradiation or upregulation of growth factor mediated pathways due to oncogene-transformation often contribute to an acquired or inherent treatment resistance in malignant cells. Therefore pharmacological compounds inhibiting specific key-entities of these signal transduction cascades potentially sensitize for radiation induced cell death. Here we describe current preclinical concepts of combined treatment strategies with locoregional-applied ionizing radiation and molecular defined signal transduction inhibitors to overcome a high treatment threshold in tumor cells.
Abstract: In many human hematologic and solid malignancies, intrinsic or acquired treatment resistance remains a major obstacle for successful cancer therapy. The molecular understanding of how tumor cells respond to chemotherapy and ionizing radiation is rapidly evolving. Induction of programmed cell death, apoptosis, is one important strategy for successful cancer therapy. This has been shown convincingly for oncogene-transformed normal cells as well as tumor cells of lymphoid origin. However, the relevance of apoptosis in solid human malignancies is less clear. Loss of apoptosis might be linked to specific mutations in the often tissue-specific apoptotic pathways due to aberrations in the stress-related signal transduction cascades. Restoration of a dysfunctional apoptotic program in cancer tissue where apoptosis has been identified as an important mechanism for tissue homeostasis is one rational approach for innovative cancer therapy. In this review, we focus on the relevance of the tumor suppressor p53 for apoptosis-induction and successful cancer therapy outlining the importance of an intact caspase machinery for apoptosis execution. Strategies are discussed to overcome treatment resistance and a high apoptotic threshold in human malignancies where apoptosis is the dominant mode of cell death and the status of p53 is an important determinant for apoptosis induction.
Abstract: Activation of the phosphatidylinositol 3'-kinase (PI3K)/Akt survival pathway protects against apoptotic stress stimuli. Therefore, compounds that down-regulate this pathway are of clinical interest for single and combined anticancer treatment modalities. Here we demonstrate that the cytotoxic effect of the protein kinase C (PKC)-inhibitor N-benzoylated staurosporine (PKC412) is mediated via the PI3K/Akt pathway. Dose-dependent down-regulation of the proliferative activity, activation of the apoptotic machinery, and cell killing by PKC412 (0-1 microM) in Rat1a-fibroblasts and H-ras-oncogene-transformed fibroblasts correlated with a decrease of Akt phosphorylation and a reduced phosphorylation of the endogenous Akt-substrate GSK3-alpha. Expression of the dominant-active myristoylated form of Akt abrogated this cytotoxic effect of PKC412. Experiments with Apaf-1-deficient cells revealed that PKC412-induced cytotoxicity depends on an intact apoptosome but that the decrease of Akt phosphorylation is not attributable to apoptosis execution. Comparative experiments indicate that PKC412 and the parent-compound staurosporine down-regulate this survival pathway upstream or at the level of Akt but by a different mechanism than the PI3K-inhibitor LY294002. Furthermore, inhibition of this pathway by PKC412 is relevant for sensitization to ionizing radiation. These results demonstrate the specific role of this signaling pathway for the PKC412-mediated down-regulation of an apoptotic threshold and its cytotoxicity.
