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.

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.

Anti-CD33-antibodies labelled with the alpha-emitter Bismuth-213 kill CD33-positive acute myeloid leukaemia cells specifically by activation of caspases and break radio- and chemoresistance by inhibition of the anti-apoptotic proteins X-linked inhibitor of apoptosis protein and B-cell lymphoma-extra large.

AIM: The emerging interest in radioimmunotherapies employing alpha-emitters for cancer treatment like high risk-leukaemia leads to the question of how these radionuclides exhibit their cytotoxicity. To clarify the molecular mechanisms of cell death induction, we investigated the molecular effects of the alpha-emitter Bismuth-213 (Bi-213) bound to a monoclonal anti-CD33-antibody ([Bi-213]anti-CD33) on the cell cycle and on apoptosis induction in sensitive as well as in beta- and gamma-radiation-resistant CD33-positive acute myeloid leukaemia (AML) cells. METHODS: The cytotoxic potential of the radioimmunoconjugate [Bi-213]anti-CD33 was analysed in the CD33-expressing human AML cell line HL-60 and in radiation- and chemoresistant HL-60-derived cell lines. Cell cycle and apoptosis induction analyses were performed via flow cytometry. Activation of apoptosis pathways was determined by immunodetection. RESULTS: [Bi-213]anti-CD33 induced apoptotic cell death in CD33-positive AML cells specifically. Molecular analyses revealed that the intrinsic mitochondrial pathway of apoptosis was activated resulting in caspase-9 activation. In the apoptotic executioner cascade caspase-3 was activated and its substrate poly (ADP-ribose) polymerase (PARP) was cleaved. Notably, [Bi-213]anti-CD33 overcame radio- and chemoresistance by reversing deficient activation of apoptosis pathways in resistant CD33-positive AML cells and by the downregulation of inhibitors of apoptosis B-cell lymphoma-extra large (Bcl-xL) and X-linked inhibitor of apoptosis protein (XIAP) involved in leukaemia resistance. CONCLUSION: [Bi-213]anti-CD33 exhibits its cytotoxic effects specifically in CD33-expressing AML cells via induction of the intrinsic, mitochondrial pathway of apoptosis. The abrogation of chemo- and radioresistances and the reactivation of apoptotic pathways seem to be promising for the treatment of patients with so far untreatable resistant AML and underline the importance of this emerging therapeutic approach of targeted alpha-therapies.

Targeted alpha-therapy using [Bi-213]anti-CD20 as novel treatment option for radio- and chemoresistant non-Hodgkin lymphoma cells.

Radioimmunotherapy (RIT) is an emerging treatment option for non-Hodgkin lymphoma (NHL) producing higher overall response and complete remission rates compared with unlabelled antibodies. However, the majority of patients treated with conventional or myeloablative doses of radiolabelled antibodies relapse. The development of RIT with alpha-emitters is attractive for a variety of cancers because of the high linear energy transfer (LET) and short path length of alpha-radiation in human tissue, allowing higher tumour cell kill and lower toxicity to healthy tissues. In this study, we investigated the molecular effects of the alpha-emitter Bi-213 labelled to anti-CD20 antibodies ([Bi-213]anti-CD20) on cell cycle and cell death in sensitive and radio-/chemoresistant NHL cells. [Bi-213]anti-CD20 induced apoptosis, activated caspase-3, caspase-2 and caspase-9 and cleaved PARP specifically in CD20-expressing sensitive as well as in chemoresistant, beta-radiation resistant and gamma-radiation resistant NHL cells. CD20 negative cells were not affected by [Bi-213]anti-CD20 and unspecific antibodies labelled with Bi-213 could not kill NHL cells. Breaking radio-/ chemoresistance in NHL cells using [Bi-213]anti-CD20 depends on caspase activation as demonstrated by complete inhibition of [Bi-213]anti-CD20-induced apoptosis with zVAD.fmk, a specific inhibitor of caspases activation. This suggests that deficient activation of caspases was reversed in radioresistant NHL cells using [Bi-213]anti- CD20. Activation of mitochondria, resulting in caspase-9 activation was restored and downregulation of Bcl-x(L) and XIAP, death-inhibiting proteins, was found after [Bi- 213]anti-CD20 treatment in radio-/chemosensitive and radio-/chemoresistant NHL cells. [Bi-213]anti-CD20 seems to be a promising radioimmunoconjugate to improve therapeutic success by breaking radio- and chemoresistance selectively in CD20- expressing NHL cells via re-activating apoptotic pathways through reversing deficient activation of caspases and the mitochondrial pathway and downregulation of XIAP and Bcl-x(L).