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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

IκB kinase (IKK) activity. IKK activity was assessed using recombinant GST-IκBα(1-54) and γ32P-ATP as substrates.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.
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pone-0001313-g003: IκB kinase (IKK) activity. IKK activity was assessed using recombinant GST-IκBα(1-54) and γ32P-ATP as substrates.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.

Mentions: IκB kinase (IKK) mediates phosphorylation of IκBα, marking it for eventual polyubiquitination and proteolysis thereby resulting in NF-κB activation[39]. Hence, we examined the effect of NO-Cbl upon IKK activity (Figure 3) using the same lysates used to assess NF-κB DNA binding activity. NO-Cbl treatment reduced basal IKK activity in all treatments, although IKK activity was not noticeably increased in A375 cells following treatment with any of the chemotherapeutic agents.


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)

IκB kinase (IKK) activity. IKK activity was assessed using recombinant GST-IκBα(1-54) and γ32P-ATP as substrates.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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001313-g003: IκB kinase (IKK) activity. IKK activity was assessed using recombinant GST-IκBα(1-54) and γ32P-ATP as substrates.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.
Mentions: IκB kinase (IKK) mediates phosphorylation of IκBα, marking it for eventual polyubiquitination and proteolysis thereby resulting in NF-κB activation[39]. Hence, we examined the effect of NO-Cbl upon IKK activity (Figure 3) using the same lysates used to assess NF-κB DNA binding activity. NO-Cbl treatment reduced basal IKK activity in all treatments, although IKK activity was not noticeably increased in A375 cells following treatment with any of the chemotherapeutic agents.

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