ABSTRACT
OBJECTIVE: The aim of this study was to test the safety of diazoxide and to search for signs of efficacy in patients with relapsing-remitting multiple sclerosis (RRMS). METHODS: In this multicenter, randomized, placebo-controlled, double-blind trial (treatment allocation was concealed), 102 patients with RRMS were randomized to receive a daily oral dose of diazoxide (0.3 and 4 mg/d) or placebo for 24 weeks (NCT01428726). The primary endpoint was the cumulative number of new T1 gadolinium-enhancing lesions per patient, recorded every 4 weeks from week 4 to week 24. Secondary endpoints included brain MRI variables such as the number of new/enlarging T2 lesions and the percentage brain volume change (PBVC); clinical variables such as the percentage of relapse-free patients, relapse rate, and change in the Expanded Disability Status Scale score; and safety and tolerability. RESULTS: Diazoxide was well-tolerated and it produced no serious adverse events other than 1 case of Hashimoto disease. At the 2 doses tested, diazoxide did not improve the primary endpoint or the MRI and clinical variables related to the presence of new lesions or relapses. Patients treated with diazoxide showed reduced PBVC compared with the placebo group, although such changes could be confounded by the higher disease activity of the treated group and the vascular effects of diazoxide. CONCLUSION: At the doses tested, oral diazoxide did not decrease the appearance of new lesions evident by MRI. The effects in slowing the progression of brain atrophy require further validation. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that for patients with RRMS, diazoxide (0.3 and 4 mg/d) does not significantly change the number of new MRI T1 gadolinium-enhancing lesions.
ABSTRACT
Acadesine is a nucleoside analogue with known activity against B-cell malignancies. Herein, we showed that in mantle cell lymphoma (MCL) cells acadesine induced caspase-dependent apoptosis through turning on the mitochondrial apoptotic machinery. At the molecular level, the compound triggered the activation of the AMPK pathway, consequently modulating known downstream targets, such as mTOR and the cell motility-related vasodilator-stimulated phosphoprotein (VASP). VASP phosphorylation by acadesine was concomitant with a blockade of CXCL12-induced migration. The inhibition of the mTOR cascade by acadesine, committed MCL cells to enter in apoptosis by a translational downregulation of the antiapoptotic Mcl-1 protein. In contrast, Bcl-2 protein levels were unaffected by acadesine and MCL samples expressing high levels of Bcl-2 tended to have a reduced response to the drug. Targeting Bcl-2 with the selective BH3-mimetic agent ABT-199 sensitized Bcl-2high MCL cells to acadesine. This effect was validated in vivo, where the combination of both agents displayed a more marked inhibition of tumor outgrowth than each drug alone. These findings support the notions that antiapoptotic proteins of the Bcl-2 family regulate MCL cell sensitivity to acadesine and that the combination of this agent with Bcl-2 inhibitors might be an interesting therapeutic option to treat MCL patients.
Subject(s)
Aminoimidazole Carboxamide/analogs & derivatives , Bridged Bicyclo Compounds, Heterocyclic/administration & dosage , Cell Adhesion Molecules/metabolism , Lymphoma, Mantle-Cell/metabolism , Microfilament Proteins/metabolism , Phosphoproteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Ribonucleosides/administration & dosage , Sulfonamides/administration & dosage , Actins/chemistry , Aminoimidazole Carboxamide/administration & dosage , Animals , Apoptosis , Cell Line, Tumor/drug effects , Cell Movement , Chemokine CXCL12/metabolism , Chemotaxis , Down-Regulation , Drug Resistance, Neoplasm , Drug Synergism , Female , Flow Cytometry , Gene Expression Regulation, Neoplastic , Humans , Mice , Mice, SCID , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Neoplasm TransplantationABSTRACT
Mantle cell lymphoma (MCL) is considered one of the most challenging lymphoma, with limited responses to current therapies. Acadesine, a nucleoside analogue has shown antitumoral effects in different preclinical cancer models as well as in a recent phase I/II clinical trial conducted in patients with chronic lymphocytic leukemia. Here we observed that acadesine exerted a selective antitumoral activity in the majority of MCL cell lines and primary MCL samples, independently of adverse cytogenetic factors. Moreover, acadesine was highly synergistic, both in vitro and in vivo, with the anti-CD20 monoclonal antibody rituximab, commonly used in combination therapy for MCL. Gene expression profiling analysis in harvested tumors suggested that acadesine modulates immune response, actin cytoskeleton organization and metal binding, pointing out a substantial impact on metabolic processes by the nucleoside analog. Rituximab also induced changes on metal binding and immune responses.The combination of both drugs enhanced the gene signature corresponding to each single agent, showing an enrichment of genes involved in inflammation, metabolic stress, apoptosis and proliferation. These effects could be important as aberrant apoptotic and proinflammatory pathways play a significant role in the pathogenesis of MCL. In summary, our results suggest that acadesine exerts a cytotoxic effect in MCL in combination with rituximab, by decreasing the proliferative and survival signatures of the disease, thus supporting the clinical examination of this strategy in MCL patients.
Subject(s)
Aminoimidazole Carboxamide/analogs & derivatives , Antibodies, Monoclonal, Murine-Derived/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Lymphoma, Mantle-Cell/drug therapy , Ribonucleosides/pharmacology , Aminoimidazole Carboxamide/administration & dosage , Aminoimidazole Carboxamide/pharmacology , Animals , Antibodies, Monoclonal, Murine-Derived/administration & dosage , Antibodies, Monoclonal, Murine-Derived/immunology , Antigens, CD20/immunology , Apoptosis/drug effects , Cell Line, Tumor , Cohort Studies , Disease Models, Animal , Drug Synergism , Female , Humans , Mice , Mice, SCID , Random Allocation , Ribonucleosides/administration & dosage , Rituximab , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Acadesine has shown in vitro to selectively induce apoptosis in B cells from chronic lymphocytic leukemia (CLL) patients. We conducted a phase I/II open-label clinical study, to determine the safety and tolerability of acadesine given intravenously as a 4-h infusion to CLL patients. METHODS: Patient population included CLL patients with relapsed/refractory disease who had received one or more prior lines of treatment including either a fludarabine or an alkylator-based regimen. Twenty-four patients were included: eighteen in Part I treated at single doses of 50-315 mg/kg, and six in Part II, three with two doses at 210 mg/kg and three with five doses at 210 mg/kg. RESULTS: A manageable and predictable safety profile was demonstrated for acadesine at single doses between 50 and 210 mg/kg; 210 mg/kg was the maximum tolerated dose (MTD) and optimal biological dose (OBD). Grade ≥2 hyperuricemia occurred commonly but was not clinically significant and resolved with the administration of prophylactic allopurinol. Other adverse events included transient anemia and/or thrombocytopenia (not clinically significant), renal impairment, and transient infusion-related hypotension (clinically significant). Trends of efficacy such as a reduction of peripheral CLL cells and reduction in lymphadenopathy were observed; however, the results were variable due to the small population and the range of doses tested. CONCLUSIONS: A MTD of 210 mg/kg was established with single acadesine dose. Multiple dose administrations at the OBD were tested with an acceptable safety profile, showing that acadesine might be a valuable agent for the treatment of relapsed/refractory CLL patients.
Subject(s)
Aminoimidazole Carboxamide/analogs & derivatives , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Ribonucleosides/therapeutic use , Aged , Aminoimidazole Carboxamide/adverse effects , Aminoimidazole Carboxamide/pharmacokinetics , Aminoimidazole Carboxamide/therapeutic use , B-Lymphocytes/drug effects , Cohort Studies , Drug Resistance, Neoplasm , Female , Humans , Karnofsky Performance Status , Lymph/cytology , Lymph/drug effects , Lymphocyte Count , Male , Maximum Tolerated Dose , Middle Aged , Recurrence , Ribonucleosides/adverse effects , Ribonucleosides/pharmacokinetics , T-Lymphocytes/drug effectsABSTRACT
5-Aminoimidazole-4-carboxamide riboside or acadesine (AICAR) induces apoptosis in chronic lymphocytic leukemia (CLL) cells. A clinical study of AICAR is currently being performed in patients with this disease. Here, we have analyzed the mechanisms involved in AICAR-induced apoptosis in CLL cells in which it activates its only well-known molecular target, adenosine monophosphate-activated protein kinase (AMPK). However, AMPK activation with phenformin or A-769662 failed to induce apoptosis in CLL cells and AICAR also potently induced apoptosis in B lymphocytes from Ampkα1(-/-) mice, demonstrating an AMPK-independent mechanism of cell death. Importantly, AICAR induced apoptosis irrespective of the tumor suppressor TP53 or ataxia telangiectasia mutated (ATM) status via induction of the mitochondrial pathway. Apoptosis was preceded by an increase in mRNA and protein levels of proapoptotic BCL-2 family proteins of the BH3-only subgroup, including BIM, NOXA, and PUMA in CLL cells. Strikingly, B lymphocytes from Noxa(-/-) or Bim(-/-) mice were partially protected from the cytotoxic effects of AICAR. Consistently, B cells from Noxa(-/-)/Bim(-/-) mice resisted induction of apoptosis by AICAR as potently as B lymphocytes overexpressing transgenic BCL-2. These findings support the notion that AICAR is an interesting alternative therapeutic option for CLL patients with impaired p53 function and resistance to conventional chemotherapy.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Aminoimidazole Carboxamide/analogs & derivatives , Antineoplastic Agents/pharmacology , Apoptosis Regulatory Proteins/genetics , Apoptosis/drug effects , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Membrane Proteins/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins/genetics , Ribonucleosides/pharmacology , Tumor Suppressor Protein p53/metabolism , Adult , Aged , Aged, 80 and over , Aminoimidazole Carboxamide/pharmacology , Animals , Bcl-2-Like Protein 11 , Cell Line, Tumor , Cells, Cultured , Female , Gene Expression Regulation, Leukemic/drug effects , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Male , Mice , Middle Aged , Up-Regulation/drug effectsABSTRACT
PURPOSE: To assess the pharmacodynamic effects of nimotuzumab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody with intermediate affinity for the receptor, in skin and tumor tissues from head and neck cancer patients. EXPERIMENTAL DESIGN: Pharmacodynamic study in patients with advanced squamous cell carcinoma of the head and neck, unsuitable for chemoradiotherapy, enrolled in a single-center trial. Patients received 8 weekly infusions of nimotuzumab. The first nimotuzumab infusion was administered 1 week before starting radiation, whereas the remaining doses were administered concomitantly with irradiation. Paired biopsies were taken from skin and primary tumors, before (pretherapy) and 1 week (on single-agent therapy) after first infusion. Immunohistochemistry was conducted to assay the effects of nimotuzumab on total and phosphorylated EGFR, phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), p-AKT, and proliferation (Ki-67). RESULTS: Nimotuzumab was well tolerated and there was no evidence of skin rash. Objective response was achieved in 9 of 10 patients. The pharmacodynamic assays showed inhibition of p-EGFR in both skin and tumor (P = 0.042 in skin and P = 0.034 in tumor). No significant changes in p-ERK1/2, p-AKT, or Ki-67 were detected in skin. In addition, lymphocytic infiltrates, folliculitis, or perifolliculitis were not observed. In tumor samples, there was an upregulation of p-AKT (P = 0.043), a reduction in proliferation index (P = 0.012), and a nonsignificant trend toward a decrease of p-ERK1/2 (P = 0.091). CONCLUSIONS: The pharmacodynamic data confirmed the ability of nimotuzumab to decrease EGFR phosphorylation. Downstream effects were observed in tumor cells but not in skin, a finding that may help to explain the lack of skin rash in patients treated with nimotuzumab.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Biomarkers, Tumor/metabolism , Carcinoma, Squamous Cell/drug therapy , Head and Neck Neoplasms/drug therapy , Aged , Antibodies, Monoclonal, Humanized , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cohort Studies , ErbB Receptors/immunology , ErbB Receptors/metabolism , Female , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/pathology , Humans , Male , Middle Aged , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Skin/metabolism , Survival Rate , Treatment OutcomeABSTRACT
PURPOSE: Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) dephosphorylates mitogen-activated protein kinase [extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38], mediates breast cancer chemoresistance, and is repressible by doxorubicin in breast cancer cells. We aimed to characterize doxorubicin effects on MKP-1 and phospho-MAPKs in human breast cancers and to further study the clinical relevance of MKP-1 expression in this disease. EXPERIMENTAL DESIGN: Doxorubicin effects on MKP-1, phospho-ERK1/2 (p-ERK1/2), phospho-JNK (p-JNK), and phospho-p38 were assayed in a panel of human breast cancer cells by Western blot and in human breast cancer were assayed ex vivo by immunohistochemistry (n = 50). MKP-1 expression was also assayed in a range of normal to malignant breast lesions (n = 30) and in a series of patients (n = 96) with breast cancer and clinical follow-up. RESULTS: MKP-1 was expressed at low levels in normal breast and in usual ductal hyperplasia and at high levels in in situ carcinoma. MKP-1 was overexpressed in approximately 50% of infiltrating breast carcinomas. Similar to what was observed in breast cancer cell lines, ex vivo exposure of breast tumors to doxorubicin down-regulated MKP-1, and up-regulated p-ERK1/2 and p-JNK, in the majority of cases. However, in a proportion of tumors overexpressing MKP-1, doxorubicin did not significantly affect MKP-1 or phospho-MAPKs. With regard to patient outcome, MKP-1 overexpression was an adverse prognostic factor for relapse both by univariate (P < 0.001) and multivariate analysis (P = 0.002). CONCLUSIONS: MKP-1 is overexpressed during the malignant transformation of the breast and independently predicts poor prognosis. Furthermore, MKP-1 is repressed by doxorubicin in many human breast cancers.
Subject(s)
Breast Neoplasms/pathology , Doxorubicin/pharmacology , Dual Specificity Phosphatase 1/metabolism , Antibiotics, Antineoplastic/pharmacology , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Cluster Analysis , Dual Specificity Phosphatase 1/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immunohistochemistry , In Vitro Techniques , JNK Mitogen-Activated Protein Kinases/metabolism , Kaplan-Meier Estimate , Neoplasm Recurrence, Local , Phosphorylation/drug effects , Predictive Value of Tests , Prognosis , Reverse Transcriptase Polymerase Chain Reaction , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The Third Annual Drug Repositioning Summit 2008 was held at the Hyatt Harborside Hotel in Boston, Massachusetts, October 6-7, and focused on new strategies for drug repositioning. The meeting, organized by The Cambridge Healthtech Institute, brought together a panel of speakers from the industry and the academia, who discussed and proposed new routes for success in drug repositioning based on their own experience in the field. This meeting report summarizes the most relevant presentations and issues discussed.
Subject(s)
Drug Discovery/economics , Drug Discovery/methods , Drug Industry/trends , Clinical Trials as Topic , HumansABSTRACT
BACKGROUND AND OBJECTIVES: The potential anticancer agent 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a translocator protein (18KDa) (TSPO) ligand, facilitates the induction of cell death by a variety of cytotoxic and chemotherapeutic agents. Primary chronic lymphocytic leukemia (CLL) cells overexpress TSPO. The aim of this study was to examine the effects of PK11195 on CLL cells. Table 1. Characteristics of the patients with chronic lymphocytic leukemia. DESIGN AND METHODS: Using cytometric analysis, we studied the cytotoxic effects of PK11195 on peripheral B and T lymphocytes from patients with CLL and from healthy donors. Western blot and cytometric analyses were used to study the mitochondrial effects of PK11195 on CLL cells. Moreover, we analyzed the cytotoxic effect of PK11195 in patients' cells with mutated p53 or ATM. RESULTS: PK11195 induces apoptosis and had additive effects with chemotherapeutic drugs in primary CLL cells. Other TSPO ligands such as RO 5-4864 and FGIN-1-27 also induce apoptosis in CLL cells. PK11195 induces mitochondrial depolarization and cytochrome c release upstream of caspase activation, and dithiocyana-tostilbene-2,2- disulfonic acid (DIDS), a voltage-dependent anion channel (VDAC) inhibitor, inhibits PK11195-induced apoptosis, demonstrating a direct involvement of mitochondria. CLL cells and normal B cells are more sensitive than T cells to PK11195-induced apoptosis. Interestingly, PK11195 induced apoptosis in CLL cells irrespective of their p53 or ATM status. INTERPRETATION AND CONCLUSIONS: These results suggest that PK11195 alone or in combination with chemotherapeutic drugs might be a new therapeutic option for the treatment of CLL.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Cycle Proteins/genetics , DNA-Binding Proteins/genetics , Isoquinolines/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Protein Serine-Threonine Kinases/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Proteins/genetics , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Adult , Aged , Aged, 80 and over , Antineoplastic Agents/therapeutic use , Ataxia Telangiectasia Mutated Proteins , B-Lymphocytes/metabolism , Benzodiazepinones/pharmacology , Caspase Inhibitors , Caspases/metabolism , Cell Cycle Proteins/metabolism , Cytochromes c/metabolism , DNA-Binding Proteins/metabolism , Drug Screening Assays, Antitumor , Enzyme Activation/drug effects , Female , Humans , Hypolipidemic Agents/pharmacology , Indoleacetic Acids/pharmacology , Isoquinolines/therapeutic use , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Male , Membrane Potential, Mitochondrial/drug effects , Middle Aged , Mitochondria/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptors, GABA/genetics , Receptors, GABA/metabolism , T-Lymphocytes/metabolism , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/metabolism , Voltage-Dependent Anion Channels/antagonists & inhibitors , Voltage-Dependent Anion Channels/metabolismABSTRACT
OBJECTIVE: Antiapoptotic Bcl-2 is overexpressed in most cases of chronic lymphocytic leukemia (CLL). The inhibition of the antiapoptotic Bcl-2 proteins is an attractive strategy for either restoring normal apoptotic process in cancer cells or making these cells more susceptible to conventional chemotherapy. We studied the effect of Bcl-2 inhibitors on the viability of cells from CLL and other mature B-cell neoplasms. MATERIALS AND METHODS: We studied the cytotoxic effects of four nonpeptidic cell-permeable Bcl-2 inhibitors (HA14-1, antimycin A, GX15-003, and GX15-070) on B cells from patients with CLL, mantle cell lymphoma (MCL), and splenic marginal zone lymphoma (SMZL). Moreover, we analyzed the effect of these inhibitors in combination with fludarabine or chlorambucil. RESULTS: HA14-1 induced apoptosis with an EC50 lower than 50 microM in 26 of the 36 CLL samples analyzed. The mean EC50 for these sensitive patients was 23 +/- 2 microM. Antimycin A induced apoptosis in 13 of the 18 CLL samples analyzed. Both HA14-1 and antimycin A induced cytochrome c release from mitochondria and caspase-3 activation. Moreover, HA14-1 induced apoptosis in peripheral cells from MCL and SMZL. HA14-1 also induced apoptosis in CLL samples with alterations in p53 or ATM. Finally, GX compounds induced apoptosis in B cells from 9 of the 11 CLL samples tested. The combination of either HA14-1, antimycin A, or GX compounds with fludarabine or chlorambucil had additive cytotoxic effects on CLL cells. CONCLUSION: Bcl-2 inhibitors induce apoptosis in CLL cells ex vivo and could be used in CLL as monotherapy or given in combination with current chemotherapy.
Subject(s)
Antimycin A/pharmacology , Apoptosis/drug effects , Benzopyrans/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Lymphoma, Mantle-Cell/metabolism , Nitriles/pharmacology , Pyrroles/pharmacology , Splenic Neoplasms/metabolism , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Drug Synergism , Humans , Indoles , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Lymphoma, Mantle-Cell/drug therapy , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Splenic Neoplasms/drug therapyABSTRACT
PURPOSE: To investigate whether nuclear factor kappaB (NF-kappaB)/interleukin 6 (IL-6) was linked to docetaxel response in human prostate cancer cell lines, and whether inhibition of NF-kappaB sensitized tumor cells to docetaxel. We also aimed to correlate IL-6 (as a surrogate marker of NF-kappaB) and docetaxel response in hormone-independent prostate cancer (HIPC) patients. EXPERIMENTAL DESIGN: Hormone-dependent (LNCaP) and hormone-independent (PC-3 and DU-145) prostate cancer cell lines were exposed to docetaxel alone or combined with the NF-kappaB inhibitor PS-1145 (an inhibitor of IkappaB kinase-2). Effects of dose, exposure time, and schedule dependence were assessed. Activation of NF-kappaB was assayed by electrophoresis mobility shift assay and luciferase reporter assay, IL-6 levels by ELISA, and cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell cycle and apoptosis were assessed by fluorescence-activated cell sorting analysis. Apoptosis was also measured by detection of cleavage of poly(ADP-ribose) polymerase. In patients with metastatic HIPC receiving docetaxel-based chemotherapy, IL-6 serum levels were assayed before chemotherapy and every 3 to 4 weeks thereafter. RESULTS: PC-3 and DU-145 cells had higher NF-kappaB activity, secreted more IL-6, and were more resistant to docetaxel than LNCaP cells. NF-kappaB activity was induced by docetaxel. Cotreatment with docetaxel and PS-1145 prevented docetaxel-induced NF-kappaB activation, reduced IL-6 production, and increased docetaxel effects on cell viability in PC-3 and DU-145 cells but not in LNCaP. Synergism with docetaxel and PS-1145, as assayed by median-effect principle, was observed in DU-145 and PC-3. In HIPC patients, pretreatment IL-6 serum levels correlated to prostate-specific antigen (PSA) response: median IL-6 level was 10.8+/-9.5 pg/mL in PSA responders versus 36.7+/-20.8 pg/mL (P=0.006) in nonresponders. A PSA response was also linked to a decline in IL-6 levels during treatment. Median overall survival was 6.8 months in patients with high IL-6 versus 16.6 months in those with low IL-6 (P=0.0007). On multivariate analysis, pretreatment IL-6 (P=0.05) was an independent prognostic factor for time to disease progression and survival. CONCLUSIONS: Inhibition of NF-kappaB emerges as an attractive strategy to enhance docetaxel response in prostate cancer. The interest of this view is further supported by a significant association between high IL-6 in sera of HIPC patients and decreased response to docetaxel.
Subject(s)
Drug Resistance, Neoplasm/physiology , Heterocyclic Compounds, 3-Ring/therapeutic use , Interleukin-6/physiology , NF-kappa B/antagonists & inhibitors , Prostatic Neoplasms/drug therapy , Pyridines/therapeutic use , Taxoids/therapeutic use , Aged , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Bone Neoplasms/drug therapy , Cell Survival , Disease Progression , Docetaxel , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Drug Synergism , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , Interleukin-6/biosynthesis , Interleukin-6/blood , Lymph Nodes , Male , Middle Aged , NF-kappa B/metabolism , Neoplasm Metastasis/drug therapy , Prognosis , Prostate-Specific Antigen/analysis , Prostatic Neoplasms/mortality , Prostatic Neoplasms/secondary , Pyridines/pharmacology , Statistics as Topic , Survival Analysis , Tumor Cells, CulturedABSTRACT
Apoptosis of B cell chronic lymphocytic leukemia (B-CLL) cells is regulated by the PI-3K-Akt pathway. In the present work, we have analyzed the mechanisms of Akt phosphorylation in B-CLL cells. Freshly isolated cells present basal Akt phosphorylation, which is PI-3K-dependent, as incubation with the PI-3K inhibitor LY294002 decreased Ser-473 and Thr-308 phosphorylation in most samples analyzed (seven out of 10). In three out of 10 cases, inhibition of protein kinase C (PKC) inhibited basal Akt phosphorylation. Stromal cell-derived factor-1alpha, IL-4, and B cell receptor activation induced PI-3K-dependent Akt phosphorylation. PMA induced the phosphorylation of Akt at Ser-473 and Thr-308 and the phosphorylation of Akt substrates, independently of PI-3K in B-CLL cells. In contrast, PKC-mediated phosphorylation of Akt was PI-3K-dependent in normal B cells. Finally, a specific inhibitor of PKCbeta blocked the phosphorylation and activation of Akt by PMA in B-CLL cells. Taken together, these results suggest a model in which Akt could be activated by two different pathways (PI-3K and PKCbeta) in B-CLL cells.
Subject(s)
Apoptosis , Leukemia, Lymphocytic, Chronic, B-Cell/enzymology , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase C/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Apoptosis/drug effects , Carcinogens/pharmacology , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Humans , Models, Biological , Morpholines/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Protein Kinase C/antagonists & inhibitors , Protein Kinase C beta , Protein Processing, Post-Translational/drug effects , Receptors, Cytokine/metabolism , Signal Transduction/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Tumor Cells, CulturedABSTRACT
B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5(+) B lymphocytes. Several drugs currently used in the therapy of B-CLL act, at least partially, through activation of the p53 pathway. Recently, nongenotoxic small-molecule activators of p53, the nutlins, have been developed that inhibit p53-MDM2 binding. We have investigated the antitumor potential of nutlin-3 in B-CLL and find that it can activate the p53 pathway and effectively induce apoptosis in cells with wild-type p53, including cells with dysfunctional ataxia telangiectasia mutated, but not mutant p53. Nutlin-3 stabilized p53 and induced p53 target genes, including MDM2, p21(CIP1), PUMA, BAX, PIG3, and WIG1. Nutlin-3 synergized with the genotoxic drugs doxorubicin, chlorambucil, and fludarabine, but not with acadesine, which induces p53-independent apoptosis. Normal human T cells showed lower sensitivity to nutlin-3 than B-CLL cells and no synergism with the genotoxic drugs. These results suggest that MDM2 antagonists alone or in combination with chemotherapeutic drugs may offer a new treatment option for B-CLL.
Subject(s)
Antineoplastic Agents/pharmacology , Lymphoma, B-Cell/drug therapy , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Tumor Suppressor Protein p53/metabolism , Antigens, CD , Apoptosis , CD5 Antigens , Cell Line, Tumor , Flow Cytometry , Humans , Imidazoles/pharmacology , Piperazines/pharmacologyABSTRACT
Acadesine, 5-aminoimidazole-4-carboxamide (AICA) riboside, induced apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells in all samples tested (n = 70). The half-maximal effective concentration (EC(50)) for B-CLL cells was 380 +/- 60 microM (n = 5). The caspase inhibitor Z-VAD.fmk completely blocked acadesine-induced apoptosis, which involved the activation of caspase-3, -8, and -9 and cytochrome c release. Incubation of B-CLL cells with acadesine induced the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), indicating that it is activated by acadesine. Nitrobenzylthioinosine (NBTI), a nucleoside transport inhibitor, 5-iodotubercidin, an inhibitor of adenosine kinase, and adenosine completely inhibited acadesine-induced apoptosis and AMPK phosphorylation, demonstrating that incorporation of acadesine into the cell and its subsequent phosphorylation to AICA ribotide (ZMP) are necessary to induce apoptosis. Inhibitors of protein kinase A and mitogen-activated protein kinases did not protect from acadesine-induced apoptosis in B-CLL cells. Moreover, acadesine had no effect on p53 levels or phosphorylation, suggesting a p53-independent mechanism in apoptosis triggering. Normal B lymphocytes were as sensitive as B-CLL cells to acadesine-induced apoptosis. However, T cells from patients with B-CLL were only slightly affected by acadesine at doses up to 4 mM. AMPK phosphorylation did not occur in T cells treated with acadesine. Intracellular levels of ZMP were higher in B-CLL cells than in T cells when both were treated with 0.5 mM acadesine, suggesting that ZMP accumulation is necessary to activate AMPK and induce apoptosis. These results suggest a new pathway involving AMPK in the control of apoptosis in B-CLL cells and raise the possibility of using acadesine in B-CLL treatment.
Subject(s)
Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/pharmacology , Antimetabolites, Antineoplastic/pharmacology , Apoptosis/drug effects , B-Lymphocytes/drug effects , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Multienzyme Complexes/metabolism , Neoplasm Proteins/metabolism , Neoplastic Stem Cells/drug effects , Protein Serine-Threonine Kinases/metabolism , Ribonucleosides/pharmacology , Tubercidin/analogs & derivatives , AMP-Activated Protein Kinases , Adenosine/pharmacology , Amino Acid Chloromethyl Ketones/pharmacology , Aminoimidazole Carboxamide/antagonists & inhibitors , Aminoimidazole Carboxamide/metabolism , B-Lymphocytes/enzymology , B-Lymphocytes/pathology , Caspases/metabolism , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/enzymology , MAP Kinase Signaling System/drug effects , Mitochondria/drug effects , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Neoplastic Stem Cells/enzymology , Neoplastic Stem Cells/pathology , Phosphorylation/drug effects , Protein Processing, Post-Translational/drug effects , Ribonucleosides/antagonists & inhibitors , Ribonucleotides/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/enzymology , Tubercidin/pharmacology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/enzymology , Tumor Cells, Cultured/pathologyABSTRACT
5-Aminoimidazole-4-carboxamide (AICA) riboside, a precursor of purine nucleotide biosynthesis, induces apoptosis in Jurkat cells. Incorporation of AICAriboside into the cells is necessary for this effect since addition of nitrobenzylthioinosine, a nucleoside-transport inhibitor, completely protects Jurkat cells from apoptosis. Adenosine, but not other nucleosides, also protects Jurkat cells from AICAriboside-induced apoptosis. The apoptotic effect is caspase-dependent since caspases 9 and 3 are activated and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) blocks apoptosis. Furthermore, AICAriboside induces mitochondrial cytochrome c release. AICAriboside, when phosphorylated to AICAribotide (ZMP), is a specific activator of the AMP-activated protein kinase (AMPK) in certain cell types. However, AICAriboside does not activate AMPK in Jurkat cells. Moreover, 5-iodotubercidin, an inhibitor of AICAriboside phosphorylation, does not inhibit apoptosis in Jurkat cells. These results indicate that AICAriboside induces apoptosis independently of ZMP synthesis and AMPK activation in Jurkat cells.
Subject(s)
Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/pharmacology , Apoptosis/drug effects , Cyclic AMP-Dependent Protein Kinases/metabolism , Ribonucleosides/pharmacology , Thioinosine/analogs & derivatives , Adenosine/pharmacology , Affinity Labels/pharmacology , Amino Acid Chloromethyl Ketones/metabolism , Apoptosis/physiology , Caspases/metabolism , Cytochrome c Group/metabolism , Enzyme Inhibitors/metabolism , Humans , Jurkat Cells , Phosphorylation , Thioinosine/pharmacologyABSTRACT
The involvement of several protein kinase pathways in the regulation of apoptosis and cell survival has been analyzed in a wide range of models. This article reviews current understanding of the protein kinases involved in the control of apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells. Protein kinase C (PKC), phosphatidylinositol 3-kinase (P13K) and nuclear factor-kappa B (NF-kappaB) play important roles in the survival of these leukemic cells. These survival pathways affect proteins involved in the control of apoptosis by altering their expression or function. The elucidation of the signal transduction network involved in the survival of B-CLL cells could provide novel pharmacological targets for the therapy of B-CLL.
Subject(s)
Apoptosis , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , NF-kappa B/physiology , Phosphatidylinositol 3-Kinases/physiology , Protein Kinase C/physiology , Animals , Enzyme Inhibitors/pharmacology , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/enzymology , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , NF-kappa B/antagonists & inhibitors , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase C/antagonists & inhibitors , Signal TransductionABSTRACT
B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5(+) B lymphocytes. TPA (12-O-tetradecanoylphorbol 13- acetate) and interleukin-4 (IL-4) inhibit apoptosis of B-CLL lymphocytes ex vivo. We used specific inhibitors of protein kinase C (PKC), extracellular-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3-kinase) to study their involvement in TPA- and IL-4-induced survival of B-CLL lymphocytes. BisI, a specific inhibitor of PKC, induced apoptosis and inhibited the antiapoptotic activity of TPA and IL-4. B-CLL cells have a basal PKC activity that was increased by TPA but not by IL-4. TPA, but not IL-4, induced ERK activation. However, the inhibition of ERK activation did not affect the viability of B-CLL lymphocytes, demonstrating that this pathway is not involved in their survival. Inhibition of PI3-kinase by LY294002 induced apoptosis of B-CLL cells and inhibited the survival effect of IL-4 and TPA. In addition, Akt, a downstream effector of PI3-kinase activity, was phosphorylated by TPA and IL-4 in B-CLL cells, though PI3-kinase had no effect on PKC-dependent phosphorylation of Akt. Furthermore, the inhibition of PKC or PI3-kinase increased dexamethasone- and fludarabine-induced apoptosis ex vivo in the presence of survival factors. These results demonstrate that PKC and PI3-kinase are involved in the survival of B-CLL cells and suggest that inhibitors of these pathways could be combined with the drugs used in the treatment of B-CLL.