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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 phospho-AKT.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.
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pone-0001313-g004: Western blot analysis of phospho-AKT.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.

Mentions: Next we examined the role of the pro-survival protein AKT and the effects of NO-Cbl on its activation (Figure 4), using the same lysates utilized in the previous two experiments. In A375 cells, phosphorylation of AKT was enhanced following treatment by cisplatin, 5-FU and etoposide. NO-Cbl inhibited AKT activation following cisplatin treatment by 51.24% (95% CI = 50.73% to 51.73%), 5-FU by 42.82% (95% CI = 39.84% to 47.26%), and with etoposide by 46.85% (95% CI = 44.00% to 49.32%).


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 phospho-AKT.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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2117345&req=5

pone-0001313-g004: Western blot analysis of phospho-AKT.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.
Mentions: Next we examined the role of the pro-survival protein AKT and the effects of NO-Cbl on its activation (Figure 4), using the same lysates utilized in the previous two experiments. In A375 cells, phosphorylation of AKT was enhanced following treatment by cisplatin, 5-FU and etoposide. NO-Cbl inhibited AKT activation following cisplatin treatment by 51.24% (95% CI = 50.73% to 51.73%), 5-FU by 42.82% (95% CI = 39.84% to 47.26%), and with etoposide by 46.85% (95% CI = 44.00% to 49.32%).

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