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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

Effects of NO-Cbl and chemotherapeutic agents in vivo.A, NCR male athymic nude (nu/nu) mice (n = 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.
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pone-0001313-g007: Effects of NO-Cbl and chemotherapeutic agents in vivo.A, NCR male athymic nude (nu/nu) mice (n = 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.

Mentions: In vitro combination therapy with NO-Cbl on tumor cells in culture exhibited positive effects in inhibiting cell proliferation and decreasing cell survival signaling (Figs. 1–6). To test drug combinations in vivo, subcutaneous NIH-OVCAR-3 xenografts were established in athymic nude mice. Daily drug treatments began on day 2 after cell inoculation, at which time tumors were both visible and palpable (Figure 7a). After 25 days, the tumors from mice treated with NO-Cbl were reduced in volume by 66.94% (95% CI = 60.90% to 69.81%; P = 0.00068) compared to controls. Tumors from mice treated with etoposide were smaller by 27.30% (95% CI = 19.70% to 30.90%; P = 0.14099) compared to control tumors. Mice receiving the combination of NO-Cbl and etoposide displayed tumors that were inhibited by 99.01% (95% CI = 98.48% to 100.12%; P = .000012) compared to controls. In two mice, tumors disappeared completely and did not recur 60 days after treatment cessation.


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)

Effects of NO-Cbl and chemotherapeutic agents in vivo.A, NCR male athymic nude (nu/nu) mice (n = 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2117345&req=5

pone-0001313-g007: Effects of NO-Cbl and chemotherapeutic agents in vivo.A, NCR male athymic nude (nu/nu) mice (n = 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.
Mentions: In vitro combination therapy with NO-Cbl on tumor cells in culture exhibited positive effects in inhibiting cell proliferation and decreasing cell survival signaling (Figs. 1–6). To test drug combinations in vivo, subcutaneous NIH-OVCAR-3 xenografts were established in athymic nude mice. Daily drug treatments began on day 2 after cell inoculation, at which time tumors were both visible and palpable (Figure 7a). After 25 days, the tumors from mice treated with NO-Cbl were reduced in volume by 66.94% (95% CI = 60.90% to 69.81%; P = 0.00068) compared to controls. Tumors from mice treated with etoposide were smaller by 27.30% (95% CI = 19.70% to 30.90%; P = 0.14099) compared to control tumors. Mice receiving the combination of NO-Cbl and etoposide displayed tumors that were inhibited by 99.01% (95% CI = 98.48% to 100.12%; P = .000012) compared to controls. In two mice, tumors disappeared completely and did not recur 60 days after treatment cessation.

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