Cell death sensitization of leukemia cells by opioid receptor activation

Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies

Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma

Glioblastoma are the most frequent and malignant human brain tumors, having a very poor prognosis. The enhanced radio- and chemoresistance of glioblastoma and the glioblastoma stem cells might be the main reason why conventional therapies fail. The second messenger cyclic AMP (cAMP) controls cell proliferation, differentiation, and apoptosis. Downregulation of cAMP sensitizes tumor cells for anti-cancer treatment. Opioid receptor agonists triggering opioid receptors can activate inhibitory Gi proteins, which, in turn, block adenylyl cyclase activity reducing cAMP. In this study, we show that downregulation of cAMP by opioid receptor activation improves the effectiveness of anti-cancer drugs in treatment of glioblastoma. The µ-opioid receptor agonist D,L-methadone sensitizes glioblastoma as well as the untreatable glioblastoma stem cells for doxorubicin-induced apoptosis and activation of apoptosis pathways by reversing deficient caspase activation and deficient downregulation of XIAP and Bcl-xL, playing critical roles in glioblastomas' resistance. Blocking opioid receptors using the opioid receptor antagonist naloxone or increasing intracellular cAMP by 3-isobutyl-1-methylxanthine (IBMX) strongly reduced opioid receptor agonist-induced sensitization for doxorubicin. In addition, the opioid receptor agonist D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux, whereas doxorubicin increased opioid receptor expression in glioblastomas. Furthermore, opioid receptor activation using D,L-methadone inhibited tumor growth significantly in vivo. Our findings suggest that opioid receptor activation triggering downregulation of cAMP is a promising strategy to inhibit tumor growth and to improve the effectiveness of anti-cancer drugs in treatment of glioblastoma and in killing glioblastoma stem cells

Suicide gene therapy for pediatric tumors

Tumor gene therapy is potentially very specific and efficacious. Suicide genes are promising tools in the arsenal of tumor gene therapy. However, problems of tumor targeting, low in vivo efficacy of nucleic acid transfer, and recent reports of adverse effects hinder the translation of this approach into clinical practice. Therefore vector design, tumor targeting, mechanisms of cell kill and killing of untransfected tumor cells must be improved. Once these problems are solved in vitro and in animal models, gene therapy holds great promise for pediatric oncology given the abundance of specific targets in pediatric tumors. This review describes the current state of preclinical research in tumor suicide gene therapy, provides an outline of pediatric suicide gene therapy protocols, and identifies potential targets in pediatric malignancies

Small molecule XIAP inhibitors sensitize childhood acute leukemia cells for CD95-induced apoptosis.

Escape of apoptosis may contribute to treatment failure in childhood acute lymphoblastic leukemia (ALL) calling for new approaches to overcome apoptosis resistance. Here, we provide for the first time evidence that small molecule inhibitors that target the anti-apoptotic protein X-linked inhibitor of apoptosis (XIAP) sensitize ALL cells for CD95-induced apoptosis. XIAP inhibitors at subtoxic concentrations, but not a structurally related control compound, act synergistically with agonistic anti-CD95 antibodies or MegaFasL, a hexameric form of CD95 ligand, to induce apoptosis in ALL cells. Further, XIAP inhibitors co-operate with MegaFasL to reduce clonogenic survival of ALL cells demonstrating their effect also on long-term survival. In contrast, XIAP inhibitors show little effect on MegaFasL-mediated apoptosis in normal peripheral blood lymphocytes (PBLs), pointing to some tumor selectivity. Molecular studies reveal that XIAP inhibitors enhance CD95-induced activation of caspases, loss of mitochondrial membrane potential and cytochrome c release in a caspase-dependent manner. Importantly, XIAP inhibitors sensitize primary leukemic blasts from children with ALL for MegaFasL-induced apoptosis. Thus, small molecule XIAP inhibitors present a promising novel approach to enhance CD95-induced apoptosis in childhood acute leukemia

Mitochondrial amplification of death signals determines thymidine kinase/ ganciclovir-triggered activation of apoptosis

Previous clinical experience shows that the efficacy of suicide gene transfer in tumor therapy is limited, resulting from inefficient gene transfer or alternatively, from intrinsic resistance of the tumor in vivo. Herpes simplex virus thymidine kinase/ganciclovir (TK/GCV), a paradigmatic suicide gene therapy system, has been described to exert its cytotoxic effect, at least in part, by inducing apoptosis in target cells. Here, we report that mitochondria amplify TK/GCV-induced apoptosis by regulating p53 accumulation and the effector phase of apoptosis. Treatment with TK/GCV led to mitochondrial perturbations including loss of the mitochondrial membrane potential and release of cytochrome c from mitochondria into the cytosol, inducing caspase activation and nuclear fragmentation. Inhibition of TK/GCV- induced mitochondrial perturbations by Bcl-2 overexpression or by the mitochondrion-specific inhibitor bongkrekic acid also strongly inhibited TK/GCV-induced activation of caspases and apoptosis. TK/GCV-induced mitochondrial perturbations depended on caspase activity possibly initiated by death receptor signaling. Perturbation of mitochondrial function mediated accumulation of wild-type p53 protein, since Bcl-2 overexpression, bongkrekic acid, or inhibition of mitochondrial protein synthesis with chloramphenicol strongly reduced TK/GCV-induced accumulation of wild-type p53 protein. These findings suggest that TK/GCV therapy may be less efficient in tumors in which the mitochondrial amplification of TK/GCV-induced apoptosis is blocked, e.g., by Bcl-2 overexpression. Given the low efficacy of currently used gene therapy systems, our data on molecular mechanisms that regulate sensitivity or resistance toward TK/GCV-induced cytotoxicity might have important implications to improve the clinical application of suicide gene therapy

Teilprojekt G: T Zelldepletion durch Apoptose bei AIDS Schlussbericht

Human peripheral blood lymphocytes of infected and non-infected individuals were tested for sensitivity towards CD4 and CXCR4-mediated apoptosis. We showed that peripheral blood lymphocytes of infected individuals are significantly more senstive. These results will influence strategies of vaccination. The hypothesis that CD4"+ T helper cells in HIV-positive individuals are directly eliminated by virus loses ground. Therefore, the damage done by regulatory HIV proteins starts to play an eminent role in the discussion of AIDS pathogenesis. In this context we could generate a monoclonal antibody against HIV1 Tat which can now be used to determine Tat in serum by a sandwich ELISA. This will enable us to determine Tat even in small concentration and to correlate its level with stages of the disease. Despite a longstanding successful collaboration and despite the fact that several important discoveries have been made in the context of the present AIDS program, the financing of the Heidelberg AIDS Verbund was discontinued. This procedure puts at great danger to the continuation of important research in the basic and the preclinical arena. (orig.)SIGLEAvailable from TIB Hannover: DtF QN1(79,38) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung und Forschung (BMBF), Bonn (Germany)DEGerman

Klinisch-biomedizinische Forschung an ausseruniversitaeren Einrichtungen der biomedizinischen Grundlagenforschung in Kooperation mit Hochschulkliniken. APO-1 (Fas/CD95) vermittelte Apoptose und ihre Bedeutung fuer die Therapie von Tumoren Wissenschaftlicher Abschlussbericht

SIGLEAvailable from TIB Hannover: DtF QN1(63,4) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman