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

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-kappaB, 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.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-kappaB activation.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-kappaB DNA binding activity, inhibition of IkappaB kinase (IKK) enzymatic activity, decreased AKT activation, increased caspase-8 and PARP cleavage, and decreased cellular XIAP protein levels.The use of NO-Cbl to inhibit survival signaling may enhance drug efficacy by preventing concomitant activation of NF-kappaB or AKT

IκB kinase (IKK) activity. IKK activity was assessed using recombinant GST-IκBα(1-54) and γ³²P-ATP as substrates.

<p>The phosphorylated GST fusion protein was detected by autoradiography. IKK activity was determined in cells that were pre-treated with NO-Cbl (300 µM, 16 h) followed by doxorubicin (20 µM, 4 h) or cisplatin (20 µM, 1 h) or 5 flurouracil (5-FU, 100 µM, 5 h) or etoposide (20 µM, 4 h) or paclitaxel (20 µΜ, 5 h). Anti-β-actin antibody was used as an irrelevant antibody control for immunoprecipitation and yielded no signal. After exposure to film, the gel was stained with Coomassie blue to visualize total protein and demonstrated equal loading of the GST-IκBα(1-54) substrate. The same cell extracts were probed for total IKK by immunoblot analysis and demonstrated equal loading of IKK.</p

Western blot analysis of phospho-AKT.

<p>Cells were pre-treated with NO-Cbl (300 µM, 16 h) followed by doxorubicin (20 µM, 4 h) or cisplatin (20 µM, 1 h) or 5 flurouracil (5-FU, 100 µM, 5 h) or etoposide (20 µM, 4 h) or paclitaxel (20 µΜ, 5 h). Whole cell lysates were probed with anti-phospho-AKT and then re-probed with anti-AKT (unphosphorylated) which served as a loading control.</p

Effects of nitrosylcobalamin (NO-Cbl) and chemotherapeutic agents on the proliferation of A375 (melanoma), A549 (lung), ACHN (renal), HeLa (cervical) HEY (ovarian), HT29 (colon), MCF-7 (breast), OC-2 and OC-3 (platinum and paclitaxel refractory ovarian), OVCAR-3 (ovarian), WM9 and WM3211 (melanoma), and P388 (murine leukemia).

<p>Cells were pre-treated with NO-Cbl for 16 h followed by addition of chemotherapeutic agents for five days, and growth was measured by the colorimetric sulforhodamine B assay(37, 75). Data points represent the combination index comparing low, medium, and high combinations of NO-Cbl and each chemotherapeutic agent (mean of eight replicates) to assess synergy. Synergy between NO-Cbl and various chemotherapeutic agents was determined by median effect analysis(38), (combination index >1 indicates antagonism,  = 1 indicates additivity, and <1 indicates synergy). The sequential treatment of NO-Cbl and chemotherapeutic drugs induced synergistic antiproliferative activity in 77.7% of the combinations examined.</p

Western blot analysis of XIAP.

<p>Cells were pre-treated with NO-Cbl (300 µM, 16 h) followed by doxorubicin (20 µM, 4 h) or cisplatin (20 µM, 1 h) or 5 flurouracil (5-FU, 100 µM, 5 h) or etoposide (20 µM, 4 h) or paclitaxel (20 µΜ, 5 h). XIAP protein levels were determined in whole cell lysates. GAPDH was used as a loading control.</p

Western blot analysis of mediators of apoptosis.

<p>A, Time course analysis of A375 cells pre-treated with NO-Cbl (300 µM, 16 h) followed by doxorubicin (20 µM, 4 h). Phospho-AKT, XIAP, caspase-8 and PARP immunoblots were performed on whole cell lysates. Note that caspase-8 and PARP cleavage were maximal with combination treatment at all time points. Degradation of XIAP was increased following combination treatment at all time points. B, OVCAR-3 cells were pre-treated with NO-Cbl (300 µM, 16 h) followed by etoposide (20 µM, 4 h). XIAP protein levels were determined. GAPDH was used as a loading control.</p

Effects of NO-Cbl and chemotherapeutic agents <i>in vivo.</i>

<p>A, NCR male athymic nude (nu/nu) mice (<i>n</i> = 10 per group) were injected subcutaneously (s.c.) with 2×10⁶ NIH-OVCAR-3 cells. Daily drug treatments of control (PBS), NO-Cbl (150 mg/kg, s.c.), etoposide (2 mg/kg, s.c.), and the combination began on day 2 following inoculation. Tumor volume was measured every other day. Points represent the mean tumor volume±95% CI. B, Kaplan-Meir survival curve. DBA/2 male mice (n = 5) were inoculated intraperitoneally (i.p.) with 10⁵ P388 murine leukemia cells. NO-Cbl was given twice daily (165 mg/kg, i.p.) and doxorubicin (4 mg/kg, i.p.) was administered once weekly, starting on day 2. Treatment in the combination group ceased on day 40 and the animals continued to be monitored for ninety days. Significance comparing the survival of groups was calculated using the logrank test.</p