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. 2013 Aug 1;8(8):e71333.
doi: 10.1371/journal.pone.0071333. Print 2013.

Trichothecin induces cell death in NF-κB constitutively activated human cancer cells via inhibition of IKKβ phosphorylation

Jia Su  1 , Peiji Zhao, Lingmei Kong, Xingyao Li, Juming Yan, Ying Zeng, Yan Li
Affiliations

Trichothecin induces cell death in NF-κB constitutively activated human cancer cells via inhibition of IKKβ phosphorylation

Jia Su et al. PLoS One. .

Abstract

Constitutive activation of the transcription factor nuclear factor-κB (NF-κB) is involved in tumorigenesis and chemo-resistance. As the key regulator of NF-κB, IKKβ is a major therapeutic target for various cancers. Trichothecin (TCN) is a metabolite isolated from an endophytic fungus of the herbal plant Maytenus hookeri Loes. In this study, we evaluated the anti-tumor activity of TCN and found that TCN markedly inhibits the growth of cancer cells with constitutively activated NF-κB. TCN induces G0/G1 cell cycle arrest and apoptosis in cancer cells, activating pro-apoptotic proteins, including caspase-3, -8 and PARP-1, and decreasing the expression of anti-apoptotic proteins Bcl-2, Bcl-xL, and survivin. Reporter activity assay and target genes expression analysis illustrated that TCN works as a potent inhibitor of the NF-κB signaling pathway. TCN inhibits the phosphorylation and degradation of IκBα and blocks the nuclear translocation of p65, and thus inhibits the expression of NF-κB target genes XIAP, cyclin D1, and Bcl-xL. Though TCN does not directly interfere with IKKβ kinase, it suppresses the phosphorylation of IKKβ. Overexpression of constitutively activated IKKβ aborted TCN induced cancer cell apoptosis, whereas knockdown of endogenous IKKβ with siRNA sensitized cancer cells toward apoptosis induced by TCN. Moreover, TCN showed a markedly weaker effect on normal cells. These findings suggest that TCN may be a potential therapeutic candidate for cancer treatment, targeting NF-κB signaling.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. TCN induces apoptosis of NF-κB constitutively activated cancer cells.
(A) Chemical structure of trichothecin. (B) Cell cytotoxic effects of trichothecin at successive concentrations. After 48 h treatment, cell viability was determined using MTT assays. (C) Annexin V-FITC/PI analysis of apoptosis in cells treated with TCN for 24 h. (D) HL-60, HepG2, A549 and PANC-1 cells were treated with TCN for 24 h and cell lysates were subjected to western blot analysis with antibodies indicated. β-actin were used as loading controls. Each column represents the mean ± SD of triplicates in three independent experiments.
Figure 2
Figure 2. TCN inhibits NF-κB signaling and induces cell cycle arrest.
(A) HEK 293T cells were transiently transfected with pNF-κB-Luc plasmids followed by treatment with TCN for 1 h before being stimulated with 25 ng/mL TNF-α for 18 h. (B) Lysates fromcells treated with TCN for 24 h were subjected to western blot analysis with p65, XIAP, cyclin D1 and Bcl-xL antibodies. (C) HepG2 cells were treated with 2.5 µM TCN for 8, 16 and 24 h. Cells were harvested and subjected to cell cycle analysis. The percentage of cells of different phases of cell cycle was analyzed by FlowJo. Experiments were done independently in triplicate, results are reported as means and standard deviations. Statistical significance was analyzed by One-way ANOVA, **p<0.01.
Figure 3
Figure 3. TCN blocks TNF-α induced p65 nuclear translocation and the phosphorylation of IκBα.
(A) HepG2 cells pretreated with 2.5 µM TCN were stimulated with 25 ng/mL TNF-α for 20 min and processed for immunostaining with anti-p65 antibody. Nuclei of cells were stained with DAPI (blue) and p65 was visualized by green fluorescence. (B) HEK293T cells were transiently transfected with pNF-κB-Luc and p65 expression plasmids followed by pretreatment of 0.3 µM TCN and stimulation with 25 ng/mL TNF-α. Reporter activity was then measured. (C) HepG2 cells pretreated with TCN were collected after stimulation with 25 ng/mL TNF-α for 10 min. Cell lysates were then analyzed by western blot using antibodies against phospho-IκBα, phospho-p65 and IκBα. Experiments were done independently in triplicate and the results are reported as means and standard deviations. Statistical analysis was perform with Student’s t-test, *p<0.05.
Figure 4
Figure 4. TCN inhibits the phosphorylation of IKKβ.
(A) IKKβ phosphorylation was detected by phospho-IKKβ antibody in HepG2 cells stimulated with 25 ng/mL TNF-α for 10 min. Cells were fixed, permeabilized, and examined by fluorescence microscope. (B) Western blot analysis showing the inhibition of IKKβ phosphorylation in cells treated with 2.5 µM TCN. Cells were collected after stimulated with 25 ng/mL TNF-α for 10 min. (C) IKKβ kinase activity was analyzed with recombinant IKKβ using the Z’-LYTE™ kinase assay kit. Fluorescence was detected at 400 nm for the excitation wavelengths and 445 nm and 520 nm for the emission wavelengths. Experiments were done independently in triplicate and the results are reported as means and standard deviations.
Figure 5
Figure 5. TCN induced cancer cell apoptosis is mediated by inhibition of IKKβ phosphorylation.
(A) HEK 293T cells were transiently transfected with IKKβ CA or empty vector for 12 h, then pretreated with 2.5 µM TCN and stimulated with 25 ng/mL TNF-α for 18 h. Cells subjected to analysis of luciferase activity. (B) HepG2 cells were transiently transfected with IKKβ CA or empty vector for 12 h, and then treated with 2.5 µM TCN for 24 h. Cells subjected to apoptosis analysis. (C) HepG2 cells were transfected with IKKβ CA or empty vector for 12 h, and then treated with 2.5 µM TCN for 24 h. Cells subjected to western blot analysis for the expression of indicated proteins. (D) HepG2 cells were transiently transfected with a scrambled siRNA or IKKβ-siRNA for 48 h, treated with 2.5 µM TCN for 24 h and subjected to apoptosis analysis. (E) HepG2 cells transiently transfected with scrambled siRNA or IKKβ-siRNA were treated with 2.5 µM TCN for 24 h. Cell lysates were collected and subjected to western blot analysis with the specified antibodies. Experiments were done independently in triplicate and the results are reported as means and standard deviations. Statistical analysis was perform with Student’s t-test, *p<0.05.
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Grants and funding

This work was supported by the 100 Talents Program of the Chinese Academy of Sciences (Y. Li), the Major State Basic Research Development Program of China (No. 2009CB522300), the Natural Science Foundation of China (No.81173076), and the Recruited Top Talent of Sciences and Technology of Yunnan Province (2009CI120). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.