J Korean Med Sci.  2010 Nov;25(11):1574-1581. 10.3346/jkms.2010.25.11.1574.

Disruption of Microtubules Sensitizes the DNA Damage-induced Apoptosis Through Inhibiting Nuclear Factor kappaB (NF-kappaB) DNA-binding Activity

• Lee H
• Jeon J
• Ryu YS
• Jeong JE
• Shin S
• Zhang T
• Kang SW
• Hong JH
• Hur GM

• KMID: 1779226
• DOI: http://doi.org/10.3346/jkms.2010.25.11.1574

Abstract

The massive reorganization of microtubule network involves in transcriptional regulation of several genes by controlling transcriptional factor, nuclear factor-kappa B (NF-kappaB) activity. The exact molecular mechanism by which microtubule rearrangement leads to NF-kappaB 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-kappaB binding activity, even though these could enhance NF-kappaB signaling in the absence of other stimuli. Moreover this suppressed NF-kappaB 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-kappaB 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.

Keyword

NF-kappa B; Apoptosis; DNA Damage; Microtubules; Signaling; DNA Damage

MeSH Terms

Animals
Antibiotics, Antineoplastic/therapeutic use
*Apoptosis
Caspases/metabolism
Cell Line
Colchicine/pharmacology
DNA/metabolism
*DNA Damage
Doxorubicin/therapeutic use
Humans
Mice
Microtubules/chemistry/*drug effects/metabolism
NF-kappa B/antagonists & inhibitors/*metabolism
Neoplasms/drug therapy
Nocodazole/pharmacology
Protein Binding
Signal Transduction
Tubulin Modulators/*pharmacology
Vinblastine/pharmacology

Figure

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  Fig. 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.

• 

  Fig. 2 DNA damaging agents induces NF-κB activation. (A) HeLa cells were treated with adriamycin (Adr, 10 µg/mL) or comptothecin (Cpt, 100 µM) for various times as indicated. Cell extracts were applied to SDS-PAGE for immunoblotting with anti-IκB-α and anti-IKK-β antibodies. (B) HeLa cells were treated as described in A for various times as indicated. Nuclear extracts were prepared and 5 µg of nuclear extracts from each sample was used to analyze NF-κB binding activity by EMSA with a NF-κB probe.

• 

  Fig. 3 Microtubule disrupting agents suppresses the DNA damage-induced NF-κB binding activity. HeLa cells were treated with Adr (10 µg/mL) or Cpt (100 µM) for various times as indicated in the presence or absence of Col (A, 10 µM), Vin (B, 1 µM) and Noc (C, 0.5 µM). Nuclear extracts were prepared and 5 µg of nuclear extracts from each sample was used to analyze NF-κB binding activity by EMSA as described in Fig. 2B. As a control, 50 µg of the nuclear extracts were applied to SDS-PAGE for immunoblotting with anti-SP1 antibody.

• 

  Fig. 4 Microtubule disrupting agents does not affect the DNA damage-induced IκB-α degradation and nuclear translocation of NF-κB subunit p65. HeLa cells were treated with Adr (10 µg/mL) or Cpt (100 µM) for various times as indicated in the presence or absence of Col (10 µM), Vin (1 µM) and Noc (0.5 µM). (A, B) Total cell extracts were prepared and applied to SDS-PAGE for immunoblotting with anti-IκB-α and anti-IKK-β antibodies. (C, D) Nuclear extracts were obtained as described Fig.1B, and the nuclear translocation of NF-κB subunit p65 was analyzed by immunoblotting with anti-p65 and anti-p84N5 antibodies from nuclear extracts (50 µg) from each sample.

• 

  Fig. 5 Microtubule disrupting agents sensitizes DNA damage-induced apoptotic cell death. (A) HeLa cells were treated with Adr (10 µg/mL) for various times as indicated in the presence or absence of pan-caspase inhibitor z-VAD-FMK (20 µM) and cell extracts were analyzed by immunoblotting with antibodies against casapse-9, -3 and PARP. As a protein loading control, the same amounts of extracts were analyzes by immunoblotting with anti-actin antibody. The asterisks indicate cleaved products of casapse-3 and PARP upon Adr treatment. (B) Wild-type and p65-/- MEF cells were treated with various concentrations of Adr as indicated. 12 hr after treatment, the percentage of cell death was determined by trypan blue exclusion assay as described under Materials and Methods. The results represent the mean values of at least three independent experiments. *, P<0.05, compared with Adr-treated wild-type MEF cells. (C) The expression levels of p65 and IKK-β in wild-type and p65-/- MEF cells. The equal amount of cell extract from each cells was analyzed by immunoblotting with antibodies against p65, IKK-β and actin. (D) HeLa cells were pretreated with the NF-κB specific inhibitors, 5 µM Bay-11 or 1 µM TPCA-1 for 30 min and then treated with 10 µg/mL Adr for 12 hr. Cell death was quantified as described in (B), and each column shows mean±S.E. of at least three independent experiments. *, P<0.05, compared with Adr-treated group. (E) HeLa cells were treated with Adr (10 µg/mL) for 15 hr in the presence or absence of Col (10 µM), Vin (1 µM) and Noc (0.5 µM), or z-VAD-FMK (20 µM). Then, cells were visualized with a normal light microscope with an inverted microscope. (F) HeLa cells were treated with Adr (10 µg/mL) for various times as indicated in the presence or absence of Col (10 µM), Vin (1 µM) and Noc (0.5 µM). Cell death was quantified as described in (B), and each column shows mean±S.E. of at least three independent experiments. (G) HeLa cells were treated as described in (C). Cell extracts form each sample were analyzed by SDS-PAGE followed by immunoblotting with antibodies against casapse-9, -3, PARP and actin. The asterisks indicate cleaved products of casapse-3 and PARP.

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