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Y-box binding protein 1 enhances DNA topoisomerase 1 activity and sensitivity to camptothecin via direct interaction.

Wu Y, Wang KY, Li Z, Liu YP, Izumi H, Uramoto H, Nakayama Y, Ito K, Kohno K - J. Exp. Clin. Cancer Res. (2014)

Bottom Line: We found that YB-1 interacts directly with TOPO1 (but not with TOPO2) and promotes TOPO1 catalytic activity.Furthermore, we found that the interaction is prevented by pretreatment with the antioxidant agent, N-acetyl cysteine, and that YB-1 downregulation renders cells resistant to CPT.Our findings suggest that nuclear YB-1 serves as an intracellular promoter of TOPO1 catalytic activity that enhances CPT sensitivity through its direct interaction with TOPO1.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, the First Hospital, China Medical University, Shenyang, China. cmuwuying@hotmail.co.

ABSTRACT

Background: The Y-box binding protein 1 (YB-1) possesses pleiotropic functions through its interactions with various cellular proteins, and its high expression levels make it a potential useful prognostic biomarker for cancer cells. Eukaryotic DNA topoisomerases, such as DNA topoisomerase 1 (TOPO1) and DNA topoisomerase 2 (TOPO2), are the essential DNA metabolism regulators that usually overexpressed in cancer cells, and multiple proteins have been reported to regulate the enzyme activity and the clinical efficacy of their inhibitors. The present study unraveled the interaction of YB-1 with TOPO1, and further investigated the related function and potential mechanisms during the interaction.

Methods: The direct association of TOPO1 with specific domain of YB-1 was explored by co-immunoprecipitation and GST pull-down assays. The interaction function was further clarified by DNA relaxation assays, co-immunoprecipitation and WST-8 assays with in vitro gain- and loss- of function models.

Results: We found that YB-1 interacts directly with TOPO1 (but not with TOPO2) and promotes TOPO1 catalytic activity. Interactions between YB-1 and TOPO1 increased when cancer cells were treated with the TOPO1 inhibitor, camptothecin (CPT), but not with the TOPO2 inhibitor, adriamycin (ADM). Furthermore, we found that the interaction is prevented by pretreatment with the antioxidant agent, N-acetyl cysteine, and that YB-1 downregulation renders cells resistant to CPT.

Conclusions: Our findings suggest that nuclear YB-1 serves as an intracellular promoter of TOPO1 catalytic activity that enhances CPT sensitivity through its direct interaction with TOPO1.

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Related in: MedlinePlus

Chemical treatment promotes binding of TOPO1 and YB-1 through oxidative stress. A. Identification of the YB-1- TOPO1 interaction in Flag-YB-1 stably-transfected PC-3 cells. Whole-cell lysates (500 μg) prepared from two clones (cl35 and cl37) of PC-3 cells transfected with the pcDNA3-Flag-YB-1 expression plasmid were subjected to SDS–PAGE and western blotting. Transferred proteins were probed with anti-Flag (upper panel) or anti-YB-1 (lower panel) antibodies. Both Flag-YB-1 and endogenous YB-1 are shown (left panel). Nuclear extracts (500 μg) of control cells or cl37 PC-3 cells were immunoprecipitated with the anti-Flag (M2) antibody. The resulting immunocomplexes were separated by SDS-PAGE. And then two parallel gels were subjected to western blot and Coomassie blue staining, respectively. Transferred proteins were probed with the anti-TOPO1 antibody. The band labeled with an asterisk was identified as TOPO1. The Flag-YB-1 protein band is indicated by an arrow. B. Chemical treatment increases YB-1-TOPO1 complex. Cl37 Flag-YB-1 stably-transfected PC-3 cells were treated for 4 h with the concentrations of CPT or ADM indicated. Nuclear extracts were immunoprecipitated with the anti- Flag (M2) antibody, and the resulting immunocomplexes were subjected to western blot analysis with anti-Flag and anti-TOPO1 antibodies. C. Longer CPT treatment increased YB-1-TOPO1 interaction and YB-1 expression. Cl37 Flag-YB-1 stably-transfected PC-3 cells were treated with the indicated concentrations of CPT for 4 h and 24 h respectively. Nuclear extracts were immunoprecipitated with the anti-Flag (M2) antibody, and the resulting immunocomplexes were subjected to western blot analysis with anti-Flag and anti-TOPO1 antibodies. D. Pretreatment with NAC prevented the chemically augmented YB-1-TOPO1 interaction. Cl37 of Flag-YB-1 stably-transfected PC-3 cells were untreated or preincubated with NAC (30 mM, 1 h) before addition of CPT. Nuclear extracts were immunoprecipitated with the anti-Flag (M2) antibody, and the resulting immunocomplexes were subjected to western blot analysis with anti-Flag and anti-TOPO1 antibodies.
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Fig4: Chemical treatment promotes binding of TOPO1 and YB-1 through oxidative stress. A. Identification of the YB-1- TOPO1 interaction in Flag-YB-1 stably-transfected PC-3 cells. Whole-cell lysates (500 μg) prepared from two clones (cl35 and cl37) of PC-3 cells transfected with the pcDNA3-Flag-YB-1 expression plasmid were subjected to SDS–PAGE and western blotting. Transferred proteins were probed with anti-Flag (upper panel) or anti-YB-1 (lower panel) antibodies. Both Flag-YB-1 and endogenous YB-1 are shown (left panel). Nuclear extracts (500 μg) of control cells or cl37 PC-3 cells were immunoprecipitated with the anti-Flag (M2) antibody. The resulting immunocomplexes were separated by SDS-PAGE. And then two parallel gels were subjected to western blot and Coomassie blue staining, respectively. Transferred proteins were probed with the anti-TOPO1 antibody. The band labeled with an asterisk was identified as TOPO1. The Flag-YB-1 protein band is indicated by an arrow. B. Chemical treatment increases YB-1-TOPO1 complex. Cl37 Flag-YB-1 stably-transfected PC-3 cells were treated for 4 h with the concentrations of CPT or ADM indicated. Nuclear extracts were immunoprecipitated with the anti- Flag (M2) antibody, and the resulting immunocomplexes were subjected to western blot analysis with anti-Flag and anti-TOPO1 antibodies. C. Longer CPT treatment increased YB-1-TOPO1 interaction and YB-1 expression. Cl37 Flag-YB-1 stably-transfected PC-3 cells were treated with the indicated concentrations of CPT for 4 h and 24 h respectively. Nuclear extracts were immunoprecipitated with the anti-Flag (M2) antibody, and the resulting immunocomplexes were subjected to western blot analysis with anti-Flag and anti-TOPO1 antibodies. D. Pretreatment with NAC prevented the chemically augmented YB-1-TOPO1 interaction. Cl37 of Flag-YB-1 stably-transfected PC-3 cells were untreated or preincubated with NAC (30 mM, 1 h) before addition of CPT. Nuclear extracts were immunoprecipitated with the anti-Flag (M2) antibody, and the resulting immunocomplexes were subjected to western blot analysis with anti-Flag and anti-TOPO1 antibodies.

Mentions: To determine the physiologically relevance of YB-1-TOPO1 association in cells, further co-immunoprecipitation was performed with a stable PC-3 cell line expressing a Flag-YB-1 construct. Of the two clones generated, clone 37 (cl37) with slightly higher expression of Flag-YB-1 (left panel, Figure 4A) was used for further experiments, and the immunoprecipitation results showed that the Flag-YB-1 precipitate contained TOPO1 protein (right panel). As seen in Figure 4B, YB-1-TOPO1 complex formation increased respectively by 334% and 221% after 4 h treatment with a 0.05 and 0.1 μM concentration CPT, while no significant increase was observed in YB-1 expression. There was no significant increase in the quantity of the YB-1-TOPO1 complex after ADM treatment compared with untreated PC-3 cells. Higher concentrations of these drugs were toxic to the cells (data not shown). Comparing with 4 h, 24 h incubation of cells with CPT resulted in more YB-1-TOPO1 complex formation and an increase in YB-1 expression. At 24 h treatment, CPT increased YB-1-TOPO1 complex formation and YB-1 expression by 520% and 152%, and by 469% and 170% at the concentration of 0.05 and 0.1 μM, respectively. However, the relative ratio of TOPO1 over YB-1 signals demonstrated no significant difference between 4 h and 24 h incubation when CPT was applied at the concentration not more than 0.05 μM (Figure 4C). Furthermore, pretreatment of PC-3 cells with N-acetyl-cysteine (NAC), which can inhibit reactive oxygen species (ROS) generation [27,28], prevented the CPT-induced increase in YB-1-TOPO1 complexes (Figure 4D).Figure 4


Y-box binding protein 1 enhances DNA topoisomerase 1 activity and sensitivity to camptothecin via direct interaction.

Wu Y, Wang KY, Li Z, Liu YP, Izumi H, Uramoto H, Nakayama Y, Ito K, Kohno K - J. Exp. Clin. Cancer Res. (2014)

Chemical treatment promotes binding of TOPO1 and YB-1 through oxidative stress. A. Identification of the YB-1- TOPO1 interaction in Flag-YB-1 stably-transfected PC-3 cells. Whole-cell lysates (500 μg) prepared from two clones (cl35 and cl37) of PC-3 cells transfected with the pcDNA3-Flag-YB-1 expression plasmid were subjected to SDS–PAGE and western blotting. Transferred proteins were probed with anti-Flag (upper panel) or anti-YB-1 (lower panel) antibodies. Both Flag-YB-1 and endogenous YB-1 are shown (left panel). Nuclear extracts (500 μg) of control cells or cl37 PC-3 cells were immunoprecipitated with the anti-Flag (M2) antibody. The resulting immunocomplexes were separated by SDS-PAGE. And then two parallel gels were subjected to western blot and Coomassie blue staining, respectively. Transferred proteins were probed with the anti-TOPO1 antibody. The band labeled with an asterisk was identified as TOPO1. The Flag-YB-1 protein band is indicated by an arrow. B. Chemical treatment increases YB-1-TOPO1 complex. Cl37 Flag-YB-1 stably-transfected PC-3 cells were treated for 4 h with the concentrations of CPT or ADM indicated. Nuclear extracts were immunoprecipitated with the anti- Flag (M2) antibody, and the resulting immunocomplexes were subjected to western blot analysis with anti-Flag and anti-TOPO1 antibodies. C. Longer CPT treatment increased YB-1-TOPO1 interaction and YB-1 expression. Cl37 Flag-YB-1 stably-transfected PC-3 cells were treated with the indicated concentrations of CPT for 4 h and 24 h respectively. Nuclear extracts were immunoprecipitated with the anti-Flag (M2) antibody, and the resulting immunocomplexes were subjected to western blot analysis with anti-Flag and anti-TOPO1 antibodies. D. Pretreatment with NAC prevented the chemically augmented YB-1-TOPO1 interaction. Cl37 of Flag-YB-1 stably-transfected PC-3 cells were untreated or preincubated with NAC (30 mM, 1 h) before addition of CPT. Nuclear extracts were immunoprecipitated with the anti-Flag (M2) antibody, and the resulting immunocomplexes were subjected to western blot analysis with anti-Flag and anti-TOPO1 antibodies.
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Fig4: Chemical treatment promotes binding of TOPO1 and YB-1 through oxidative stress. A. Identification of the YB-1- TOPO1 interaction in Flag-YB-1 stably-transfected PC-3 cells. Whole-cell lysates (500 μg) prepared from two clones (cl35 and cl37) of PC-3 cells transfected with the pcDNA3-Flag-YB-1 expression plasmid were subjected to SDS–PAGE and western blotting. Transferred proteins were probed with anti-Flag (upper panel) or anti-YB-1 (lower panel) antibodies. Both Flag-YB-1 and endogenous YB-1 are shown (left panel). Nuclear extracts (500 μg) of control cells or cl37 PC-3 cells were immunoprecipitated with the anti-Flag (M2) antibody. The resulting immunocomplexes were separated by SDS-PAGE. And then two parallel gels were subjected to western blot and Coomassie blue staining, respectively. Transferred proteins were probed with the anti-TOPO1 antibody. The band labeled with an asterisk was identified as TOPO1. The Flag-YB-1 protein band is indicated by an arrow. B. Chemical treatment increases YB-1-TOPO1 complex. Cl37 Flag-YB-1 stably-transfected PC-3 cells were treated for 4 h with the concentrations of CPT or ADM indicated. Nuclear extracts were immunoprecipitated with the anti- Flag (M2) antibody, and the resulting immunocomplexes were subjected to western blot analysis with anti-Flag and anti-TOPO1 antibodies. C. Longer CPT treatment increased YB-1-TOPO1 interaction and YB-1 expression. Cl37 Flag-YB-1 stably-transfected PC-3 cells were treated with the indicated concentrations of CPT for 4 h and 24 h respectively. Nuclear extracts were immunoprecipitated with the anti-Flag (M2) antibody, and the resulting immunocomplexes were subjected to western blot analysis with anti-Flag and anti-TOPO1 antibodies. D. Pretreatment with NAC prevented the chemically augmented YB-1-TOPO1 interaction. Cl37 of Flag-YB-1 stably-transfected PC-3 cells were untreated or preincubated with NAC (30 mM, 1 h) before addition of CPT. Nuclear extracts were immunoprecipitated with the anti-Flag (M2) antibody, and the resulting immunocomplexes were subjected to western blot analysis with anti-Flag and anti-TOPO1 antibodies.
Mentions: To determine the physiologically relevance of YB-1-TOPO1 association in cells, further co-immunoprecipitation was performed with a stable PC-3 cell line expressing a Flag-YB-1 construct. Of the two clones generated, clone 37 (cl37) with slightly higher expression of Flag-YB-1 (left panel, Figure 4A) was used for further experiments, and the immunoprecipitation results showed that the Flag-YB-1 precipitate contained TOPO1 protein (right panel). As seen in Figure 4B, YB-1-TOPO1 complex formation increased respectively by 334% and 221% after 4 h treatment with a 0.05 and 0.1 μM concentration CPT, while no significant increase was observed in YB-1 expression. There was no significant increase in the quantity of the YB-1-TOPO1 complex after ADM treatment compared with untreated PC-3 cells. Higher concentrations of these drugs were toxic to the cells (data not shown). Comparing with 4 h, 24 h incubation of cells with CPT resulted in more YB-1-TOPO1 complex formation and an increase in YB-1 expression. At 24 h treatment, CPT increased YB-1-TOPO1 complex formation and YB-1 expression by 520% and 152%, and by 469% and 170% at the concentration of 0.05 and 0.1 μM, respectively. However, the relative ratio of TOPO1 over YB-1 signals demonstrated no significant difference between 4 h and 24 h incubation when CPT was applied at the concentration not more than 0.05 μM (Figure 4C). Furthermore, pretreatment of PC-3 cells with N-acetyl-cysteine (NAC), which can inhibit reactive oxygen species (ROS) generation [27,28], prevented the CPT-induced increase in YB-1-TOPO1 complexes (Figure 4D).Figure 4

Bottom Line: We found that YB-1 interacts directly with TOPO1 (but not with TOPO2) and promotes TOPO1 catalytic activity.Furthermore, we found that the interaction is prevented by pretreatment with the antioxidant agent, N-acetyl cysteine, and that YB-1 downregulation renders cells resistant to CPT.Our findings suggest that nuclear YB-1 serves as an intracellular promoter of TOPO1 catalytic activity that enhances CPT sensitivity through its direct interaction with TOPO1.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, the First Hospital, China Medical University, Shenyang, China. cmuwuying@hotmail.co.

ABSTRACT

Background: The Y-box binding protein 1 (YB-1) possesses pleiotropic functions through its interactions with various cellular proteins, and its high expression levels make it a potential useful prognostic biomarker for cancer cells. Eukaryotic DNA topoisomerases, such as DNA topoisomerase 1 (TOPO1) and DNA topoisomerase 2 (TOPO2), are the essential DNA metabolism regulators that usually overexpressed in cancer cells, and multiple proteins have been reported to regulate the enzyme activity and the clinical efficacy of their inhibitors. The present study unraveled the interaction of YB-1 with TOPO1, and further investigated the related function and potential mechanisms during the interaction.

Methods: The direct association of TOPO1 with specific domain of YB-1 was explored by co-immunoprecipitation and GST pull-down assays. The interaction function was further clarified by DNA relaxation assays, co-immunoprecipitation and WST-8 assays with in vitro gain- and loss- of function models.

Results: We found that YB-1 interacts directly with TOPO1 (but not with TOPO2) and promotes TOPO1 catalytic activity. Interactions between YB-1 and TOPO1 increased when cancer cells were treated with the TOPO1 inhibitor, camptothecin (CPT), but not with the TOPO2 inhibitor, adriamycin (ADM). Furthermore, we found that the interaction is prevented by pretreatment with the antioxidant agent, N-acetyl cysteine, and that YB-1 downregulation renders cells resistant to CPT.

Conclusions: Our findings suggest that nuclear YB-1 serves as an intracellular promoter of TOPO1 catalytic activity that enhances CPT sensitivity through its direct interaction with TOPO1.

Show MeSH
Related in: MedlinePlus