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Nitrosylcobalamin potentiates the anti-neoplastic effects of chemotherapeutic agents via suppression of survival signaling.

Bauer JA, Lupica JA, Schmidt H, Morrison BH, Haney RM, Masci RK, Lee RM, Didonato JA, Lindner DJ - PLoS ONE (2007)

Bottom Line: Chemotherapeutic agents often simultaneously induce an apoptotic signal and activation of NF-kappaB, which has the undesired effect of promoting cell survival.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.

View Article: PubMed Central - PubMed

Affiliation: Taussig Cancer Center, Center for Hematology and Oncology Molecular Therapeutics, The Cleveland Clinic Foundation, Cleveland, Ohio, United States of America. bauerj@ccf.org <bauerj@ccf.org>

ABSTRACT

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-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.

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-kappaB 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-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.

Conclusion: The use of NO-Cbl to inhibit survival signaling may enhance drug efficacy by preventing concomitant activation of NF-kappaB or AKT.

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Related in: MedlinePlus

Western blot analysis of mediators of apoptosis.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.
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pone-0001313-g006: Western blot analysis of mediators of apoptosis.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.

Mentions: A375 cells were stimulated with doxorubicin, followed by assessment of AKT phosphorylation, XIAP expression, and caspase-8 and PARP cleavage. (Figure 6a). NO-Cbl inhibited doxorubicin-mediated AKT activation at all time points, resulting in reduction of 85.73% (95% CI = 85.26% to 86.24%) at 8 h, 94.05% (95% CI = 92.44% to 96.02%) at 12 h, 60.94% (95% CI = 58.93% to 63.73%) at 16 h, and completely abrogated the signal at 24 h. In addition, degradation of XIAP was enhanced when NO-Cbl was combined with doxorubicin; this inhibition was 82.94% (95% CI = 81.27% to 84.14%) at 8 h, 49.36% (95% CI = 41.51% to 58.74%) at 12 h, 64.67% (95% CI = 60.68% to 67.69%) at 16 h, and 35.81% (95% CI = 33.93% to 36.54%) at 24 h.


Nitrosylcobalamin potentiates the anti-neoplastic effects of chemotherapeutic agents via suppression of survival signaling.

Bauer JA, Lupica JA, Schmidt H, Morrison BH, Haney RM, Masci RK, Lee RM, Didonato JA, Lindner DJ - PLoS ONE (2007)

Western blot analysis of mediators of apoptosis.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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2117345&req=5

pone-0001313-g006: Western blot analysis of mediators of apoptosis.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.
Mentions: A375 cells were stimulated with doxorubicin, followed by assessment of AKT phosphorylation, XIAP expression, and caspase-8 and PARP cleavage. (Figure 6a). NO-Cbl inhibited doxorubicin-mediated AKT activation at all time points, resulting in reduction of 85.73% (95% CI = 85.26% to 86.24%) at 8 h, 94.05% (95% CI = 92.44% to 96.02%) at 12 h, 60.94% (95% CI = 58.93% to 63.73%) at 16 h, and completely abrogated the signal at 24 h. In addition, degradation of XIAP was enhanced when NO-Cbl was combined with doxorubicin; this inhibition was 82.94% (95% CI = 81.27% to 84.14%) at 8 h, 49.36% (95% CI = 41.51% to 58.74%) at 12 h, 64.67% (95% CI = 60.68% to 67.69%) at 16 h, and 35.81% (95% CI = 33.93% to 36.54%) at 24 h.

Bottom Line: Chemotherapeutic agents often simultaneously induce an apoptotic signal and activation of NF-kappaB, which has the undesired effect of promoting cell survival.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.

View Article: PubMed Central - PubMed

Affiliation: Taussig Cancer Center, Center for Hematology and Oncology Molecular Therapeutics, The Cleveland Clinic Foundation, Cleveland, Ohio, United States of America. bauerj@ccf.org <bauerj@ccf.org>

ABSTRACT

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-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.

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-kappaB 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-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.

Conclusion: The use of NO-Cbl to inhibit survival signaling may enhance drug efficacy by preventing concomitant activation of NF-kappaB or AKT.

Show MeSH
Related in: MedlinePlus