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Nitric Oxide Down-Regulates Topoisomerase I and Induces Camptothecin Resistance in Human Breast MCF-7 Tumor Cells.

Sharma NK, Kumar A, Kumari A, Tokar EJ, Waalkes MP, Bortner CD, Williams J, Ehrenshaft M, Mason RP, Sinha BK - PLoS ONE (2015)

Bottom Line: Here we show that ·NO/·NO-derived species induces a significant down-regulation of topoisomerase I protein via the ubiquitin/26S proteasome pathway in human colon (HT-29) and breast (MCF-7) cancer cell lines.Importantly, ·NO treatment induced a significant resistance to CPT only in MCF-7 cells.This up-regulation of bcl2 protein in MCF-7 cells was wtp53 dependent as pifithrine-α, a small molecule inhibitor of wtp53 function, completely reversed CPT resistance, suggesting that wtp53 and bcl2 proteins played important roles in CPT resistance.

View Article: PubMed Central - PubMed

Affiliation: Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle, Park, Durham, North Carolina, United States of America.

ABSTRACT
Camptothecin (CPT), a topoisomerase I poison, is an important drug for the treatment of solid tumors in the clinic. Nitric oxide (·NO), a physiological signaling molecule, is involved in many cellular functions, including cell proliferation, survival and death. We have previously shown that ·NO plays a significant role in the detoxification of etoposide (VP-16), a topoisomerase II poison in vitro and in human melanoma cells. ·NO/·NO-derived species are reported to modulate activity of several important cellular proteins. As topoisomerases contain a number of free sulfhydryl groups which may be targets of ·NO/·NO-derived species, we have investigated the roles of ·NO/·NO-derived species in the stability and activity of topo I. Here we show that ·NO/·NO-derived species induces a significant down-regulation of topoisomerase I protein via the ubiquitin/26S proteasome pathway in human colon (HT-29) and breast (MCF-7) cancer cell lines. Importantly, ·NO treatment induced a significant resistance to CPT only in MCF-7 cells. This resistance to CPT did not result from loss of topoisomerase I activity as there were no differences in topoisomerase I-induced DNA cleavage in vitro or in tumor cells, but resulted from the stabilization/induction of bcl2 protein. This up-regulation of bcl2 protein in MCF-7 cells was wtp53 dependent as pifithrine-α, a small molecule inhibitor of wtp53 function, completely reversed CPT resistance, suggesting that wtp53 and bcl2 proteins played important roles in CPT resistance. Because tumors in vivo are heterogeneous and contaminated by infiltrating macrophages, ·NO-induced down-regulation of topoisomerase I protein combined with bcl2 protein stabilization could render certain tumors highly resistant to CPT and drugs derived from it in the clinic.

No MeSH data available.


Related in: MedlinePlus

·NO-induced down-regulation of topo I protein levels in MCF-7 cells.(A) Cells were treated for various times with PPNO and cells were collected, lysed and analyzed by Western blots for topo I, (B) Quantification of the resulting topo I protein levels, and (C) Cytotoxicity of CPT (■-■) alone and in the presence of PPNO (100 μM; ●-●, 50 μM; ▲-▲) in MCF-7 cells. Values are mean SEM from at least 3 separate experiments carried out in triplicate (n = 3). *P<0.05, **P<0.01, # # and ***P<0.001, # # # and with respect to concentration-matched samples.
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pone.0141897.g004: ·NO-induced down-regulation of topo I protein levels in MCF-7 cells.(A) Cells were treated for various times with PPNO and cells were collected, lysed and analyzed by Western blots for topo I, (B) Quantification of the resulting topo I protein levels, and (C) Cytotoxicity of CPT (■-■) alone and in the presence of PPNO (100 μM; ●-●, 50 μM; ▲-▲) in MCF-7 cells. Values are mean SEM from at least 3 separate experiments carried out in triplicate (n = 3). *P<0.05, **P<0.01, # # and ***P<0.001, # # # and with respect to concentration-matched samples.

Mentions: Treatment of MCF-7 cells with ·NO (via PPNO) resulted in a significant down-regulation of topo I protein levels in a dose- and time-dependent manner (Fig 4). These results are similar to those described for HT-29 cells; however, in contrast to HT-29 cells, the level of topo I protein was further down-regulated up to 18 h following ·NO treatment (up to 70% at 100 μM PPNO). ·NO did not affect this down-regulation at the topo I gene (Fig 1C). Rather, the down-regulation of the protein was due to degradation of the topo I protein via 26 S proteasome-mediated pathways, as the inclusion of MG 132 completely inhibited this degradation (Fig 4A).


Nitric Oxide Down-Regulates Topoisomerase I and Induces Camptothecin Resistance in Human Breast MCF-7 Tumor Cells.

Sharma NK, Kumar A, Kumari A, Tokar EJ, Waalkes MP, Bortner CD, Williams J, Ehrenshaft M, Mason RP, Sinha BK - PLoS ONE (2015)

·NO-induced down-regulation of topo I protein levels in MCF-7 cells.(A) Cells were treated for various times with PPNO and cells were collected, lysed and analyzed by Western blots for topo I, (B) Quantification of the resulting topo I protein levels, and (C) Cytotoxicity of CPT (■-■) alone and in the presence of PPNO (100 μM; ●-●, 50 μM; ▲-▲) in MCF-7 cells. Values are mean SEM from at least 3 separate experiments carried out in triplicate (n = 3). *P<0.05, **P<0.01, # # and ***P<0.001, # # # and with respect to concentration-matched samples.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4635000&req=5

pone.0141897.g004: ·NO-induced down-regulation of topo I protein levels in MCF-7 cells.(A) Cells were treated for various times with PPNO and cells were collected, lysed and analyzed by Western blots for topo I, (B) Quantification of the resulting topo I protein levels, and (C) Cytotoxicity of CPT (■-■) alone and in the presence of PPNO (100 μM; ●-●, 50 μM; ▲-▲) in MCF-7 cells. Values are mean SEM from at least 3 separate experiments carried out in triplicate (n = 3). *P<0.05, **P<0.01, # # and ***P<0.001, # # # and with respect to concentration-matched samples.
Mentions: Treatment of MCF-7 cells with ·NO (via PPNO) resulted in a significant down-regulation of topo I protein levels in a dose- and time-dependent manner (Fig 4). These results are similar to those described for HT-29 cells; however, in contrast to HT-29 cells, the level of topo I protein was further down-regulated up to 18 h following ·NO treatment (up to 70% at 100 μM PPNO). ·NO did not affect this down-regulation at the topo I gene (Fig 1C). Rather, the down-regulation of the protein was due to degradation of the topo I protein via 26 S proteasome-mediated pathways, as the inclusion of MG 132 completely inhibited this degradation (Fig 4A).

Bottom Line: Here we show that ·NO/·NO-derived species induces a significant down-regulation of topoisomerase I protein via the ubiquitin/26S proteasome pathway in human colon (HT-29) and breast (MCF-7) cancer cell lines.Importantly, ·NO treatment induced a significant resistance to CPT only in MCF-7 cells.This up-regulation of bcl2 protein in MCF-7 cells was wtp53 dependent as pifithrine-α, a small molecule inhibitor of wtp53 function, completely reversed CPT resistance, suggesting that wtp53 and bcl2 proteins played important roles in CPT resistance.

View Article: PubMed Central - PubMed

Affiliation: Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle, Park, Durham, North Carolina, United States of America.

ABSTRACT
Camptothecin (CPT), a topoisomerase I poison, is an important drug for the treatment of solid tumors in the clinic. Nitric oxide (·NO), a physiological signaling molecule, is involved in many cellular functions, including cell proliferation, survival and death. We have previously shown that ·NO plays a significant role in the detoxification of etoposide (VP-16), a topoisomerase II poison in vitro and in human melanoma cells. ·NO/·NO-derived species are reported to modulate activity of several important cellular proteins. As topoisomerases contain a number of free sulfhydryl groups which may be targets of ·NO/·NO-derived species, we have investigated the roles of ·NO/·NO-derived species in the stability and activity of topo I. Here we show that ·NO/·NO-derived species induces a significant down-regulation of topoisomerase I protein via the ubiquitin/26S proteasome pathway in human colon (HT-29) and breast (MCF-7) cancer cell lines. Importantly, ·NO treatment induced a significant resistance to CPT only in MCF-7 cells. This resistance to CPT did not result from loss of topoisomerase I activity as there were no differences in topoisomerase I-induced DNA cleavage in vitro or in tumor cells, but resulted from the stabilization/induction of bcl2 protein. This up-regulation of bcl2 protein in MCF-7 cells was wtp53 dependent as pifithrine-α, a small molecule inhibitor of wtp53 function, completely reversed CPT resistance, suggesting that wtp53 and bcl2 proteins played important roles in CPT resistance. Because tumors in vivo are heterogeneous and contaminated by infiltrating macrophages, ·NO-induced down-regulation of topoisomerase I protein combined with bcl2 protein stabilization could render certain tumors highly resistant to CPT and drugs derived from it in the clinic.

No MeSH data available.


Related in: MedlinePlus