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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 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.

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

Cytotoxicity of CPT in HT-29 cells.(A) Cell count-based cytotoxicity of CPT was carried out as described in the methods sections. HT-29 cells were seeded in a 6-well plate in triplicates and allowed to attach for 18 h. PPNO (100 μM) treatment was carried out in medium containing 0.5–1% FBS without antibiotics for 6 h. Medium was changed to 10% FBS before adding various concentrations of CPT, and incubated for 48 h, and surviving cells were counted. Data represent at least three separate experiments. CPT alone (■-■); CPT in the presence of PPNO (○-○) and cytokine-induced HT-29 cells (▲-▲). (B) DNA cleavage induced in pHOT1 DNA by CPT (10 μM) in the presence of topo I (5 units) and various concentrations of PPNO. The DNA cleavage assay was carried out as described in the Methods section. Lane 1, control DNA; lane 2, in the presence of topo I; lane 3, in the presence of 10 μM CPT and topo I; lanes 4–7, in the presence of 25, 50, 100 and 200 μM PPNO, 10 μM CPT and topo I, respectively.
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pone.0141897.g003: Cytotoxicity of CPT in HT-29 cells.(A) Cell count-based cytotoxicity of CPT was carried out as described in the methods sections. HT-29 cells were seeded in a 6-well plate in triplicates and allowed to attach for 18 h. PPNO (100 μM) treatment was carried out in medium containing 0.5–1% FBS without antibiotics for 6 h. Medium was changed to 10% FBS before adding various concentrations of CPT, and incubated for 48 h, and surviving cells were counted. Data represent at least three separate experiments. CPT alone (■-■); CPT in the presence of PPNO (○-○) and cytokine-induced HT-29 cells (▲-▲). (B) DNA cleavage induced in pHOT1 DNA by CPT (10 μM) in the presence of topo I (5 units) and various concentrations of PPNO. The DNA cleavage assay was carried out as described in the Methods section. Lane 1, control DNA; lane 2, in the presence of topo I; lane 3, in the presence of 10 μM CPT and topo I; lanes 4–7, in the presence of 25, 50, 100 and 200 μM PPNO, 10 μM CPT and topo I, respectively.

Mentions: We evaluated whether an ·NO-mediated degradation of topo I protein results in altered sensitivity to CPT. PPNO had no significant effects on CPT cytotoxicity in HT-29 cells (Fig 3A), suggesting that down-regulation of the topo I protein level in HT-29 cells does not contribute significantly to CPT toxicity. Similar results were observed in HT-29 cells induced by a cytokine mixture (Fig 3;Table 1).


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)

Cytotoxicity of CPT in HT-29 cells.(A) Cell count-based cytotoxicity of CPT was carried out as described in the methods sections. HT-29 cells were seeded in a 6-well plate in triplicates and allowed to attach for 18 h. PPNO (100 μM) treatment was carried out in medium containing 0.5–1% FBS without antibiotics for 6 h. Medium was changed to 10% FBS before adding various concentrations of CPT, and incubated for 48 h, and surviving cells were counted. Data represent at least three separate experiments. CPT alone (■-■); CPT in the presence of PPNO (○-○) and cytokine-induced HT-29 cells (▲-▲). (B) DNA cleavage induced in pHOT1 DNA by CPT (10 μM) in the presence of topo I (5 units) and various concentrations of PPNO. The DNA cleavage assay was carried out as described in the Methods section. Lane 1, control DNA; lane 2, in the presence of topo I; lane 3, in the presence of 10 μM CPT and topo I; lanes 4–7, in the presence of 25, 50, 100 and 200 μM PPNO, 10 μM CPT and topo I, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141897.g003: Cytotoxicity of CPT in HT-29 cells.(A) Cell count-based cytotoxicity of CPT was carried out as described in the methods sections. HT-29 cells were seeded in a 6-well plate in triplicates and allowed to attach for 18 h. PPNO (100 μM) treatment was carried out in medium containing 0.5–1% FBS without antibiotics for 6 h. Medium was changed to 10% FBS before adding various concentrations of CPT, and incubated for 48 h, and surviving cells were counted. Data represent at least three separate experiments. CPT alone (■-■); CPT in the presence of PPNO (○-○) and cytokine-induced HT-29 cells (▲-▲). (B) DNA cleavage induced in pHOT1 DNA by CPT (10 μM) in the presence of topo I (5 units) and various concentrations of PPNO. The DNA cleavage assay was carried out as described in the Methods section. Lane 1, control DNA; lane 2, in the presence of topo I; lane 3, in the presence of 10 μM CPT and topo I; lanes 4–7, in the presence of 25, 50, 100 and 200 μM PPNO, 10 μM CPT and topo I, respectively.
Mentions: We evaluated whether an ·NO-mediated degradation of topo I protein results in altered sensitivity to CPT. PPNO had no significant effects on CPT cytotoxicity in HT-29 cells (Fig 3A), suggesting that down-regulation of the topo I protein level in HT-29 cells does not contribute significantly to CPT toxicity. Similar results were observed in HT-29 cells induced by a cytokine mixture (Fig 3;Table 1).

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 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.

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