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Disruption of microtubules sensitizes the DNA damage-induced apoptosis through inhibiting nuclear factor κB (NF-κB) DNA-binding activity.

Lee H, Jeon J, Ryu YS, Jeong JE, Shin S, Zhang T, Kang SW, Hong JH, Hur GM - J. Korean Med. Sci. (2010)

Bottom Line: The exact molecular mechanism by which microtubule rearrangement leads to NF-κB activation largely remains to be identified.Interestingly pretreatment of microtubule disrupting agents (colchicine, vinblastine and nocodazole) was observed to lead to paradoxical suppression of DNA damage-induced NF-κB binding activity, even though these could enhance NF-κB signaling in the absence of other stimuli.Moreover this suppressed NF-κB binding activity subsequently resulted in synergic apoptotic response, as evident by the combination with Adr and low doses of microtubule disrupting agents was able to potentiate the cytotoxic action through caspase-dependent pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Research Institute for Medical Science, Daejeon Regional Cancer Center, Daejeon, Korea.

ABSTRACT
The massive reorganization of microtubule network involves in transcriptional regulation of several genes by controlling transcriptional factor, nuclear factor-kappa B (NF-κB) activity. The exact molecular mechanism by which microtubule rearrangement leads to NF-κB activation largely remains to be identified. However microtubule disrupting agents may possibly act in synergy or antagonism against apoptotic cell death in response to conventional chemotherapy targeting DNA damage such as adriamycin or comptothecin in cancer cells. Interestingly pretreatment of microtubule disrupting agents (colchicine, vinblastine and nocodazole) was observed to lead to paradoxical suppression of DNA damage-induced NF-κB binding activity, even though these could enhance NF-κB signaling in the absence of other stimuli. Moreover this suppressed NF-κB binding activity subsequently resulted in synergic apoptotic response, as evident by the combination with Adr and low doses of microtubule disrupting agents was able to potentiate the cytotoxic action through caspase-dependent pathway. Taken together, these results suggested that inhibition of microtubule network chemosensitizes the cancer cells to die by apoptosis through suppressing NF-κB DNA binding activity. Therefore, our study provided a possible anti-cancer mechanism of microtubule disrupting agent to overcome resistance against to chemotherapy such as DNA damaging agent.

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Related in: MedlinePlus

Microtubule disrupting agents induces NF-κB activation through nuclear translocation of NF-κB subunit p65. (A) HeLa cells were treated with microtubule disrupting agents (upper panel) including colchicines (Col, 10 µM), vinblastine (Vin, 1 µM) and nocodazole (Noc, 0.5 µM) or TNF (lower panel) for various times as indicated. Cell extracts were applied to SDS-PAGE for immunoblotting with anti-IκB-α and anti-p65 antibodies. As a protein loading control, the same amounts of extracts were analyzes by immunoblotting with anti-IKK-β antibody. (B, C) After HeLa cells were treated with 10 µM of Col, 1 µM of Vin or 0.5 µM of Noc for various times as indicated, nuclear extracts were obtained as described under Materials and Methods. (B) The nuclear translocation of NF-κB subunit p65 was analyzed by immunoblotting with anti-p65 and anti-p84N5 antibodies from nuclear extracts from each sample. (C) EMSAs were performed with labeled NF-κB oligomers and 5 µg of nuclear extracts to analyze NF-κB binding activity. As a control, 50 µg of the nuclear extracts were applied to SDS-PAGE for immunoblotting with anti-SP1 antibody.
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Figure 1: Microtubule disrupting agents induces NF-κB activation through nuclear translocation of NF-κB subunit p65. (A) HeLa cells were treated with microtubule disrupting agents (upper panel) including colchicines (Col, 10 µM), vinblastine (Vin, 1 µM) and nocodazole (Noc, 0.5 µM) or TNF (lower panel) for various times as indicated. Cell extracts were applied to SDS-PAGE for immunoblotting with anti-IκB-α and anti-p65 antibodies. As a protein loading control, the same amounts of extracts were analyzes by immunoblotting with anti-IKK-β antibody. (B, C) After HeLa cells were treated with 10 µM of Col, 1 µM of Vin or 0.5 µM of Noc for various times as indicated, nuclear extracts were obtained as described under Materials and Methods. (B) The nuclear translocation of NF-κB subunit p65 was analyzed by immunoblotting with anti-p65 and anti-p84N5 antibodies from nuclear extracts from each sample. (C) EMSAs were performed with labeled NF-κB oligomers and 5 µg of nuclear extracts to analyze NF-κB binding activity. As a control, 50 µg of the nuclear extracts were applied to SDS-PAGE for immunoblotting with anti-SP1 antibody.

Mentions: To characterize the NF-κB signaling pathway in HeLa cells affected by the disruption of the microtubule network, we compared the kinetics of the degradation of IκB-α at various time points after tumor necrosis factor (TNF) or tubulin polymerization inhibitors (colchicine, vinblastine and nocodazole). As shown in the bottom panel of Fig. 1A, when cells were treated with TNF, the expression of IκB-α started to be degraded at 15 min after treatment, and was recovered by 60 min. In contrast to the cells treated with TNF, the IκB-α degradation and its recovery were slower and weaker upon treatment with the microtubule disrupting agents (top three panels of Fig. 1A), which suggest that disruption of the microtubule network affects NF-κB activity in a different manner with a classical pathway. Since the nuclear translocation of NF-κB subunit, relA (p65) is essential for the NF-κB signaling pathway, we next examined the effect of the microtubule disrupting agents on the nuclear translocation of p65. As expected, the treatment of microtubule disrupting agents resulted in enhanced translocation of p65 into the nucleus, even though the extent of accumulated p65 into nucleus was lower than that of TNF treatment (Fig. 1B). As previously reported (19), the expression of nuclear protein encoded by N5 gene (p84N5) occurred principally in the nuclear fraction (Fig. 1B, bottom panels). However, the expression levels of p65 on the total cell extract were unaffected by treatment with microtubule disrupting agents (Fig. 1A). To confirm these observations, nuclear extracts were examined for NF-κB binding activity by EMSA using a palindromic NF-κB binding site. Consistent with above results, the binding activity of NF-κB subunit complex was gradually increased in response to microtubule agents, indicating that microtubule disruption directly induce NF-κB binding activity through nuclear translocation of NF-κB subunit, p65 without the expressional change of NF-κB. As a control of nuclear protein contents, the SP-1 protein level was examined by immunoblotting with an anti-SP-1 antibody and no difference was observed (Fig. 2B, bottom panels).


Disruption of microtubules sensitizes the DNA damage-induced apoptosis through inhibiting nuclear factor κB (NF-κB) DNA-binding activity.

Lee H, Jeon J, Ryu YS, Jeong JE, Shin S, Zhang T, Kang SW, Hong JH, Hur GM - J. Korean Med. Sci. (2010)

Microtubule disrupting agents induces NF-κB activation through nuclear translocation of NF-κB subunit p65. (A) HeLa cells were treated with microtubule disrupting agents (upper panel) including colchicines (Col, 10 µM), vinblastine (Vin, 1 µM) and nocodazole (Noc, 0.5 µM) or TNF (lower panel) for various times as indicated. Cell extracts were applied to SDS-PAGE for immunoblotting with anti-IκB-α and anti-p65 antibodies. As a protein loading control, the same amounts of extracts were analyzes by immunoblotting with anti-IKK-β antibody. (B, C) After HeLa cells were treated with 10 µM of Col, 1 µM of Vin or 0.5 µM of Noc for various times as indicated, nuclear extracts were obtained as described under Materials and Methods. (B) The nuclear translocation of NF-κB subunit p65 was analyzed by immunoblotting with anti-p65 and anti-p84N5 antibodies from nuclear extracts from each sample. (C) EMSAs were performed with labeled NF-κB oligomers and 5 µg of nuclear extracts to analyze NF-κB binding activity. As a control, 50 µg of the nuclear extracts were applied to SDS-PAGE for immunoblotting with anti-SP1 antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2966993&req=5

Figure 1: Microtubule disrupting agents induces NF-κB activation through nuclear translocation of NF-κB subunit p65. (A) HeLa cells were treated with microtubule disrupting agents (upper panel) including colchicines (Col, 10 µM), vinblastine (Vin, 1 µM) and nocodazole (Noc, 0.5 µM) or TNF (lower panel) for various times as indicated. Cell extracts were applied to SDS-PAGE for immunoblotting with anti-IκB-α and anti-p65 antibodies. As a protein loading control, the same amounts of extracts were analyzes by immunoblotting with anti-IKK-β antibody. (B, C) After HeLa cells were treated with 10 µM of Col, 1 µM of Vin or 0.5 µM of Noc for various times as indicated, nuclear extracts were obtained as described under Materials and Methods. (B) The nuclear translocation of NF-κB subunit p65 was analyzed by immunoblotting with anti-p65 and anti-p84N5 antibodies from nuclear extracts from each sample. (C) EMSAs were performed with labeled NF-κB oligomers and 5 µg of nuclear extracts to analyze NF-κB binding activity. As a control, 50 µg of the nuclear extracts were applied to SDS-PAGE for immunoblotting with anti-SP1 antibody.
Mentions: To characterize the NF-κB signaling pathway in HeLa cells affected by the disruption of the microtubule network, we compared the kinetics of the degradation of IκB-α at various time points after tumor necrosis factor (TNF) or tubulin polymerization inhibitors (colchicine, vinblastine and nocodazole). As shown in the bottom panel of Fig. 1A, when cells were treated with TNF, the expression of IκB-α started to be degraded at 15 min after treatment, and was recovered by 60 min. In contrast to the cells treated with TNF, the IκB-α degradation and its recovery were slower and weaker upon treatment with the microtubule disrupting agents (top three panels of Fig. 1A), which suggest that disruption of the microtubule network affects NF-κB activity in a different manner with a classical pathway. Since the nuclear translocation of NF-κB subunit, relA (p65) is essential for the NF-κB signaling pathway, we next examined the effect of the microtubule disrupting agents on the nuclear translocation of p65. As expected, the treatment of microtubule disrupting agents resulted in enhanced translocation of p65 into the nucleus, even though the extent of accumulated p65 into nucleus was lower than that of TNF treatment (Fig. 1B). As previously reported (19), the expression of nuclear protein encoded by N5 gene (p84N5) occurred principally in the nuclear fraction (Fig. 1B, bottom panels). However, the expression levels of p65 on the total cell extract were unaffected by treatment with microtubule disrupting agents (Fig. 1A). To confirm these observations, nuclear extracts were examined for NF-κB binding activity by EMSA using a palindromic NF-κB binding site. Consistent with above results, the binding activity of NF-κB subunit complex was gradually increased in response to microtubule agents, indicating that microtubule disruption directly induce NF-κB binding activity through nuclear translocation of NF-κB subunit, p65 without the expressional change of NF-κB. As a control of nuclear protein contents, the SP-1 protein level was examined by immunoblotting with an anti-SP-1 antibody and no difference was observed (Fig. 2B, bottom panels).

Bottom Line: The exact molecular mechanism by which microtubule rearrangement leads to NF-κB activation largely remains to be identified.Interestingly pretreatment of microtubule disrupting agents (colchicine, vinblastine and nocodazole) was observed to lead to paradoxical suppression of DNA damage-induced NF-κB binding activity, even though these could enhance NF-κB signaling in the absence of other stimuli.Moreover this suppressed NF-κB binding activity subsequently resulted in synergic apoptotic response, as evident by the combination with Adr and low doses of microtubule disrupting agents was able to potentiate the cytotoxic action through caspase-dependent pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Research Institute for Medical Science, Daejeon Regional Cancer Center, Daejeon, Korea.

ABSTRACT
The massive reorganization of microtubule network involves in transcriptional regulation of several genes by controlling transcriptional factor, nuclear factor-kappa B (NF-κB) activity. The exact molecular mechanism by which microtubule rearrangement leads to NF-κB activation largely remains to be identified. However microtubule disrupting agents may possibly act in synergy or antagonism against apoptotic cell death in response to conventional chemotherapy targeting DNA damage such as adriamycin or comptothecin in cancer cells. Interestingly pretreatment of microtubule disrupting agents (colchicine, vinblastine and nocodazole) was observed to lead to paradoxical suppression of DNA damage-induced NF-κB binding activity, even though these could enhance NF-κB signaling in the absence of other stimuli. Moreover this suppressed NF-κB binding activity subsequently resulted in synergic apoptotic response, as evident by the combination with Adr and low doses of microtubule disrupting agents was able to potentiate the cytotoxic action through caspase-dependent pathway. Taken together, these results suggested that inhibition of microtubule network chemosensitizes the cancer cells to die by apoptosis through suppressing NF-κB DNA binding activity. Therefore, our study provided a possible anti-cancer mechanism of microtubule disrupting agent to overcome resistance against to chemotherapy such as DNA damaging agent.

Show MeSH
Related in: MedlinePlus