Joseph A. Bauer

Suppression of NF-κB Survival Signaling by Nitrosylcobalamin Sensitizes Neoplasms to the Anti-tumor Effects of Apo2L/TRAIL

We have previously demonstrated the anti-tumor activity of nitrosylcobalamin (NO-Cbl), an analog of vitamin B12 that delivers nitric oxide (NO) and increases the expression of tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) and its receptors in human tumors. The specific aim of this study was to examine whether NO-Cbl could sensitize drug-resistant melanomas to Apo2L/TRAIL. Antiproliferative effects of NO-Cbl and Apo2L/TRAIL were assessed in malignant melanomas and non-tumorigenic melanocyte and fibroblast cell lines. Athymic nude mice bearing human melanoma A375 xenografts were treated with NO-Cbl and Apo2L/TRAIL. Apoptosis was measured by TUNEL and confirmed by examining levels and activity of key mediators of apoptosis. The activation status of NF-κB was established by assaying DNA binding, luciferase reporter activity, the phosphorylation status of IκBα, and in vitro IKK activity. NO-Cbl sensitized Apo2L/TRAIL-resistant melanoma cell lines to growth inhibition by Apo2L/TRAIL but had minimal effect on normal cell lines. NO-Cbl and Apo2L/TRAIL exerted synergistic anti-tumor activity against A375 xenografts. Treatment with NO-Cbl followed by Apo2L/TRAIL induced apoptosis in Apo2L/TRAIL-resistant tumor cells, characterized by cleavage of caspase-3, caspase-8, and PARP. NO-Cbl inhibited IKK activation, characterized by decreased phosphorylation of IκBα and inhibition of NF-κB DNA binding activity. NO-Cbl suppressed Apo2L/TRAIL- and TNF-α-mediated activation of a transfected NF-κB-driven luciferase reporter. XIAP, an inhibitor of apoptosis, was inactivated by NO-Cbl. NO-Cbl treatment rendered Apo2L/TRAIL-resistant malignancies sensitive to the anti-tumor effects of Apo2L/TRAIL in vitro and in vivo. The use of NO-Cbl and Apo2L/TRAIL capitalizes on the tumor-specific properties of both agents and represents a promising anti-cancer combination.

Nitrosylcobalamin Promotes Cell Death via S Nitrosylation of Apo2L/TRAIL Receptor DR4

ABSTRACT We have previously demonstrated that nitrosylcobalamin (NO-Cbl), an analogue of vitamin B12 that delivers nitric oxide (NO), had potent antiproliferative activity against several human cancer cell lines. NO-Cbl induced apoptosis via a death receptor/caspase-8 pathway. In this study, we demonstrate that a functional Apo2L/TRAIL receptor was necessary for the induction of cell death by NO-Cbl. Furthermore, the Apo2L/TRAIL death receptor DR4 (TRAIL R1) was S nitrosylated following NO-Cbl treatment. Human melanoma (A375), renal carcinoma (ACHN), and ovarian carcinoma (NIH-OVCAR-3) cells were treated with NO-Cbl and subjected to the biotin switch assay; S-nitrosylated DR4 was detected in all three cell lines. NO-Cbl treatment did not cause S nitrosylation of DR5. The seven cysteine residues located in the cytoplasmic domain of DR4 were individually point mutated to alanines. NIH-OVCAR-3 cells expressing the DR4 C336A mutation lacked S nitrosylation following NO-Cbl treatment. Overexpression of wild-type DR4 sensitized cells to growth inhibition by NO-Cbl. Cells expressing the DR4 C336A mutant were more resistant to NO-Cbl and Apo2L/TRAIL than were the other six C-A mutations or wild-type cells. The C336A mutant also displayed blunted caspase-8 enzymatic activity following NO-Cbl treatment compared to the other mutants. Thus, DR4 residue C336 becomes S nitrosylated and promotes apoptosis following NO-Cbl treatment.

Interactions of cubilin with megalin and the product of the amnionless gene (AMN): effect on its stability

Cubilin, a 456 kDa multipurpose receptor lacking in both transmembrane and cytoplasmic domains is expressed in the apical BBMs (brush border membranes) of polarized epithelia. Cubilin interacts with two transmembrane proteins, AMN, a 45-50 kDa protein product of the amnionless gene, and megalin, a 600 kDa giant endocytic receptor. In vitro, three fragments of cubilin, the 113-residue N-terminus and CUB domains 12-17 and 22-27, demonstrated Ca2+-dependent binding to megalin. Immunoprecipitation and immunoblotting studies using detergent extracts of rat kidney BBMs revealed that cubilin interacts with both megalin and AMN. Ligand (intrinsic factor-cobalamin)-affinity chromatography showed that in renal BBMs, functional cubilin exists as a complex with both AMN and megalin. Cubilin and AMN levels were reduced by 80% and 55-60% respectively in total membranes and BBMs obtained from kidney of megalin antibody-producing rabbits. Immunohistochemical analysis and turnover studies for cubilin in megalin or AMN gene-silenced opossum kidney cells showed a significant reduction (85-90%) in cubilin staining and a 2-fold decrease in its half-life. Taken together, these results indicate that three distinct regions of cubilin bind to megalin and its interactions with both megalin and AMN are essential for its intracellular stability.

Apo2L/TRAIL induction and nuclear translocation of inositol hexakisphosphate kinase 2 during IFN-β-induced apoptosis in ovarian carcinoma

Previously, we have reported that overexpression of IHPK2 (inositol hexakisphosphate kinase 2) sensitized NIH-OVCAR-3 ovarian carcinoma cell lines to the growth-suppressive and apoptotic effects of IFN-beta (interferon-beta) treatment and gamma-irradiation. In the present study, we demonstrate that Apo2L/TRAIL (Apo2L/tumour-necrosis-factor-related apoptosis-inducing ligand) is a critical mediator of IFN-induced apoptosis in these cells. Compared with IFN-alpha2, IFN-beta is a more potent inducer of Apo2L/TRAIL and IHPK2 activity. Overexpression of IHPK2 converts IFN-alpha2-resistant cells into cells that readily undergo apoptosis in response to IFN-alpha2. In untreated cells transfected with IHPK2-eGFP (where eGFP stands for enhanced green fluorescent protein), the fusion protein is localized to the cytoplasm and perinuclear region. After treatment with IFN-beta, IHPK2-eGFP translocated to the nucleus. In cells transfected with mutant IHPK2-NLS-eGFP (where NLS stands for nuclear localization sequence), containing point mutations in the NLS, the fusion protein remained trapped in the cytoplasm, even after IFN-beta treatment. Cells expressing mutant NLS mutation were more resistant to IFN-beta. The IC50 value of IHPK2-expressing cells was 2-3-fold lower than vector control. The IC50 value of NLS-mutant-expressing cells was 3-fold higher than vector control. Blocking antibodies to Apo2L/TRAIL or transfection with a dominant negative Apo2L/TRAIL receptor (DR5Delta) inhibited the antiproliferative effects of IFN-beta. Thus overexpression of IHPK2 enhanced apoptotic effects of IFN-beta, and expression of the NLS mutant conferred resistance to IFN-beta. Apo2L/TRAIL expression and nuclear localization of IHPK2 are both required for the induction of apoptosis by IFN-beta in ovarian carcinoma.

IFN-α2b and thalidomide synergistically inhibit tumor-induced angiogenesis

Angiogenesis is an absolute requirement for tumor growth and metastasis. The purpose of this study was to evaluate the antiangiogenic activity of interferon-alpha2b (IFN-alpha2b) and thalidomide, as single agents and in combination. The murine dermis model was used to assess tumor-induced angiogenesis in nude mice. Human ACHN (renal), NIH-OVCAR-3 (ovarian), LNCaP (prostate), and SK-Mel-1 (melanoma) tumor cells were inoculated intradermally into the flanks of nude mice. IFN-alpha2b and thalidomide, administered daily, were effective inhibitors of angiogenesis induced by all four tumor types. The combination of IFN-alpha2b and thalidomide caused a synergistic decrease in mean vessel count in tumors that were resistant to the antiproliferative effects of IFN-alpha2b and thalidomide in vitro. This enhanced suppression of angiogenesis translated into synergistic antitumor activity in a xenograft model. Pegylated IFN-alpha (PEG-IFN-alpha2b) (10(6) U) administered once in 10 days was as effective as daily IFN-alpha2b treatment (10(6) U x 10 days). IFN-alpha2b and thalidomide have potentiated antiangiogenic activity when used in combination. A single dose of PEG-IFN-alpha2b (10(6) U) was as effective at suppressing vessel growth as an equivalent dose of IFN-alpha2b given daily for 10 days.

Nitrosylcobalamin Potentiates the Anti-Neoplastic Effects of Chemotherapeutic Agents via Suppression of Survival Signaling

Background Nitrosylcobalamin (NO-Cbl) is a chemotherapeutic pro-drug derived from vitamin B12 that preferentially delivers nitric oxide (NO) to tumor cells, based upon increased receptor expression. NO-Cbl induces Apo2L/TRAIL-mediated apoptosis and inhibits survival signaling in a variety of malignant cell lines. Chemotherapeutic agents often simultaneously induce an apoptotic signal and activation of NF-κB, which has the undesired effect of promoting cell survival. The specific aims of this study were to 1) measure the anti-tumor effects of NO-Cbl alone and in combination with conventional chemotherapeutic agents, and to 2) examine the mechanism of action of NO-Cbl as a single agent and in combination therapy. Methodology Using anti-proliferative assays, electrophoretic mobility shift assay (EMSA), immunoblot analysis and kinase assays, we demonstrate an increase in the effectiveness of chemotherapeutic agents in combination with NO-Cbl as a result of suppressed NF-κB activation. Results Eighteen chemotherapeutic agents were tested in combination with NO-Cbl, in thirteen malignant cell lines, resulting in a synergistic anti-proliferative effect in 78% of the combinations tested. NO-Cbl pre-treatment resulted in decreased NF-κB DNA binding activity, inhibition of IκB kinase (IKK) enzymatic activity, decreased AKT activation, increased caspase-8 and PARP cleavage, and decreased cellular XIAP protein levels. Conclusion The use of NO-Cbl to inhibit survival signaling may enhance drug efficacy by preventing concomitant activation of NF-κB or AKT.

Effect of Inositol Hexakisphosphate Kinase 2 on Transforming Growth Factor β-activated Kinase 1 and NF-κB Activation

We previously showed that inositol hexakisphosphate kinase 2 (IHPK2) functions as a growth-suppressive and apoptosis-enhancing kinase during cell stress. Overexpression of IHPK2 sensitized ovarian carcinoma cell lines to the growth-suppressive and apoptotic effects of interferon β (IFN-β), IFN-α2, and γ-irradiation. Expression of a kinase-dead mutant abrogated 50% of the apoptosis induced by IFN-β. Because the kinase-dead mutant retained significant response to cell stressors, we hypothesized that a portion of the death-promoting function of IHPK2 was independent of its kinase activity. We now demonstrate that IHPK2 binds to tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2 and interferes with phosphorylation of transforming growth factor β-activated kinase 1 (TAK1), thereby inhibiting NF-κB signaling. IHPK2 contains two sites required for TRAF2 binding, Ser-347 and Ser-359. Compared with wild type IHPK2-transfected cells, cells expressing S347A and S359A mutations displayed 3.5-fold greater TAK1 activation following TNF-α. This mutant demonstrated a 6–10-fold increase in NF-κB DNA binding following TNF-α compared with wild type IHPK2-expressing cells in which NF-κB DNA binding was inhibited. Cells transfected with wild type IHPK2 or IHPK2 mutants that lacked S347A and S359A mutations displayed enhanced terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining following TNF-α. We believe that IHPK2-TRAF2 binding leads to attenuation of TAK1- and NF-κB-mediated signaling and is partially responsible for the apoptotic activity of IHPK2.