Indole-3-carbinol suppresses NF-kappaB and IkappaBalpha kinase activation, causing inhibition of expression of NF-kappaB-regulated antiapoptotic and metastatic gene products and enhancement of apoptosis in myeloid and leukemia cells - PubMed (original) (raw)

Indole-3-carbinol suppresses NF-kappaB and IkappaBalpha kinase activation, causing inhibition of expression of NF-kappaB-regulated antiapoptotic and metastatic gene products and enhancement of apoptosis in myeloid and leukemia cells

Yasunari Takada et al. Blood. 2005.

Abstract

Indole-3-carbinol, found in Brassica species vegetables (such as cabbage, cauliflower, and brussels spouts), exhibits antitumor effects through poorly defined mechanisms. Because several genes that regulate apoptosis, proliferation, and metastasis are regulated by nuclear factor-kappaB (NF-kappaB), we postulated that indole-3-carbinol must mediate its activity through NF-kappaB modulation. We demonstrated that indole-3-carbinol suppressed constitutive NF-kappaB activation and activation induced by tumor necrosis factor (TNF), interleukin-1beta (IL-1beta), phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), and cigarette smoke; the suppression was not cell type specific, because activation was inhibited in myeloid, leukemia, and epithelial cells. This activation correlated with the sequential suppression of the IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha ubiquitination, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, p65 acetylation, and NF-kappaB-dependent reporter gene expression. The NF-kappaB-regulated gene products cyclin D1, cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), survivin, inhibitor-of-apoptosis protein-1 (IAP1), IAP2, X chromosome-linked IAP (XIAP), Bcl-2, Bfl-1/A1, TNF receptor-associated factor-1 (TRAF1), and Fas-associated death domain protein-like interleukin-1beta-converting enzyme inhibitory protein (FLIP) were all down-regulated by indole-3-carbinol. This down-regulation led to the potentiation of apoptosis induced by cytokines and chemotherapeutic agents. Indole-3-carbinol suppressed constitutive NF-kappaB activation in mononuclear cells derived from bone marrow of acute myelogenous leukemia patients, and this correlated with inhibition of cell growth. Overall, our results indicated that indole-3-carbinol inhibits NF-kappaB and NF-kappaB-regulated gene expression and that this mechanism may provide the molecular basis for its ability to suppress tumorigenesis.

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Figures

Figure 1.

Figure 1.

Effect of I3C on TNF-induced NF-κB activation. (A) I3C blocks NF-κB activation induced by TNF, PMA, LPS, and CSC. Jurkat cells were preincubated with 50 μM I3C for 24 hours; treated with 0.1 nM TNF and 10 μg/mL LPS for 30 minutes; treated with 100 ng/mL IL-1β, 15 ng/mL PMA, and 1 μg/mL CSC for 1 hour; and then analyzed for NF-κB activation as described in “Materials and methods.” (B) Jurkat cells were preincubated at 37°C with 50 μM I3C for the indicated times and then treated with 0.1 nM TNF at 37°C for 30 minutes. Nuclear extracts were then prepared and assayed for NF-κB activation by EMSA. Cell viability was determined by the trypan blue dye exclusion method. (C) I3C suppresses TNF-induced NF-κB in a dose-dependent manner in Jurkat, KBM-5, and H1299 cells. Cells were incubated with different concentrations of I3C for 24 hours, followed by an incubation with 0.1 nM TNF for 30 minutes. Nuclear extracts were then prepared and assayed for NF-κB activation by EMSA. (D) I3C inhibits constitutive NF-κB activation. MM.1, U266, and SCC-4 cells were incubated with different concentrations of I3C for 24 hours. Nuclear extracts were then prepared and assayed for NF-κB activation by EMSA.

Figure 2.

Figure 2.

Effect of I3C on IκBα phosphorylation and degradation induced by TNF. (A) I3C inhibits TNF-induced activation of NF-κB. Jurkat cells were incubated with 50 μM I3C for 24 hours, treated with 0.1 nM TNF for indicated times, and then analyzed for NF-κB activation by EMSA. (B) Effect of I3C on TNF-induced degradation of IκBα. Jurkat cells were incubated with 50 μM I3C for 24 hours and treated with 0.1 nM TNF for the indicated times. Cytoplasmic extracts were prepared, fractionated on 10% SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed with anti-IκBα antibody. Anti-β-actin antibody was the loading control. (C) Effect of I3C on the phosphorylation of IκBα by TNF. Jurkat cells were incubated with of 50 μM I3C for 24 hours and treated with 0.1 nM TNF for the indicated times. Cytoplasmic extracts were fractionated and then blotted using phosphospecific anti-IκBα antibody. (D) Effect of I3C on TNF-induced ubiquitination of IκBα. Jurkat cells were pretreated with 50 μM I3C for 24 hours, then with 100 μg/mL N-acetyl-leucyl-leucyl-norleucinal (ALLN) for 1 hour, and finally with 0.1 nM TNF for 15 minutes. Whole-cell extracts were prepared, fractionated, and examined by Western blot analysis using anti-IκBα antibody. (E) Effect of I3C on the activation of IKK by TNF. Jurkat cells were incubated with 50 μM I3C for 24 hours and then activated with 1 nM TNF for different times. Whole-cell extracts were immunoprecipitated with antibody against IKK-α and analyzed by immunocomplex kinase assay. To examine the effect of I3C on the level of expression of IKK proteins, whole-cell extracts were fractionated on SDS-PAGE and examined by Western blot analysis using anti-IKK-α and anti-IKK-β antibodies.

Figure 3.

Figure 3.

Effect of I3C on the TNF-induced translocation of p65 into the nucleus. (A) Western blot analysis of phospho-p65 and p65 using cytoplasmic extracts (CE). Jurkat cells were incubated with 50 μM I3C for 24 hours and treated with 0.1 nM TNF for the indicated times in minutes. Cytoplasmic extracts were prepared and subjected to Western blot analysis using anti-p65 antibody and phosphospecific anti-p65 antibody. (B) Western blot analysis of phospho-p65 and p65 using nuclear extracts (NE). Jurkat cells were incubated with 50 μM I3C for 24 hours and treated with 0.1 nM TNF for the indicated times. Nuclear extracts were prepared and subjected to Western blot analysis using anti-p65 and phosphospecific anti-p65 antibodies. For loading control of nuclear protein, the membrane was blotted with anti-PARP antibody. (C) Effect of I3C on TNF-induced acetylation of p65. Jurkat cells were incubated with 50 μM I3C for 24 hours and then treated with 1 nM TNF for the indicated times. Whole-cell extracts were prepared, immunoprecipitated (IP) with anti-p65 antibody, and then examined by Western blot analysis (WB) using anti-acetyl-lysine antibody. Whole-cell extracts were subjected to Western blot analysis using anti-p65 antibody. (D) Immunocytochemical analysis of p65 localization after treatment with 1 nM TNF in the absence or presence of 50 μM I3C. Jurkat cells were incubated with I3C for 24 hours and then treated with 1 nM TNF for 30 minutes. Cells were subjected to immunocytochemistry as described in “Materials and methods.” Stained slides were mounted with mounting medium (Sigma-Aldrich) and analyzed under an epifluorescence microscope (Labophot-2; Nikon, Tokyo, Japan). Pictures were captured using a Photometrics Coolsnap CF color camera (Nikon, Lewisville, TX) and MetaMorph Version 4.6.5 software (Universal Imaging, Downingtown, PA). Original magnification, ×200.

Figure 4.

Figure 4.

I3C inhibits the TNF-induced expression of NF-κB-dependent genes. (A) I3C inhibits the NF-κB-dependent reporter gene expression induced by TNF and TNFR1. A293 cells were transiently transfected with an NF-κB-containing plasmid alone or with TNFR1-expressing plasmids for 24 hours. After transfection, cells were washed and treated with the indicated concentrations of I3C for 24 hours. For TNF-treated cells, cells were washed and treated with 1 nM TNF for an additional 24 hours. The supernatants of the culture medium were assayed for SEAP activity as described in “Materials and methods.” Values represent the mean ± SD of triplicate cultures. (B) I3C inhibits TNF-induced cyclin D1, COX-2, and MMP-9 expression. Jurkat cells were incubated with 25 μM I3C and then treated with 1 nM TNF for the indicated times. Whole-cell extracts were prepared and analyzed by Western blot analysis using antibodies against cyclin D1, COX-2, and MMP-9. (C) I3C inhibits the expression of TNF-induced antiapoptotic proteins. Jurkat cells were incubated with 25 μM I3C and then treated with 1 nM TNF for the indicated times. Whole-cell extracts were prepared and analyzed by Western blot analysis using antibodies against survivin, IAP1, IAP2, XIAP, Bcl-2, Bfl-1/A1, TRAF1, FLIP, and β-actin.

Figure 5.

Figure 5.

I3C enhances TNF-induced cytotoxicity. (A) I3C enhances TNF- and chemotherapy-induced cytotoxicity. A total of 5000 Jurkat cells were seeded in triplicate in 96-well plates. Cells were pretreated with 1 μM I3C and then incubated with indicated concentrations of TNF (i), cisplatin (ii), or doxorubicin (iii) for 72 hours. Thereafter, cell viability was analyzed by the MTT method as described in “Materials and methods.” Values represent the mean ± SD of triplicate cultures. (B) Jurkat cells were pretreated with 25 μM I3C and then incubated with 1 nM TNF for the indicated times. Whole-cell extracts were prepared, subjected to SDS-PAGE, and blotted with anti-PARP antibody. (C) Jurkat cells were pretreated with 25 μM I3C and then incubated with 1 nM TNF for 16 hours. Cells were stained with Live and Dead assay reagent for 30 minutes and then analyzed under a fluorescence microscope as described in “Materials and methods.” (D) Jurkat cells were pretreated with 25 μM I3C and then incubated with 1 nM TNF for 16 hours. Cells were fixed, stained with TUNEL assay reagent, and then analyzed under a fluorescence microscope as described in “Materials and methods.” Stained slides were mounted with mounting medium (Sigma-Aldrich) and analyzed under an epifluorescence microscope (Labophot-2; Nikon). Pictures were captured using a Photometrics Coolsnap CF color camera (Nikon, Lewisville, TX) and Meta Morph version 4.6.5 software (Universal Imaging). Original magnification, ×200.

Figure 6.

Figure 6.

I3C inhibits constitutively active NF-κB activity and proliferation of AML cells. A total of 1 × 107 cells were resuspended in RPMI 1640 medium and treated with 50 μM I3C for 24 hours, and nuclear extracts were prepared and then analyzed for NF-κB activity by EMSA.

Figure 7.

Figure 7.

A schematic diagram of the effect of I3C on TNF-induced NF-κB activation and apoptosis.

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References

    1. Loub WD, Wattenberg LW, Davis DW. Aryl hydrocarbon hydroxylase induction in rat tissues by naturally occurring indoles of cruciferous plants. J Natl Cancer Inst. 1975;54: 985-988. - PubMed
    1. Wattenberg LW, Loub WD. Inhibition of polycyclic aromatic hydrocarbon-induced neoplasia by naturally occurring indoles. Cancer Res. 1978;38: 1410-1413. - PubMed
    1. Brandi G, Paiardini M, Cervasi B, et al. A new indole-3-carbinol tetrameric derivative inhibits cyclin-dependent kinase 6 expression, and induces G1 cell cycle arrest in both estrogen-dependent and estrogen-independent breast cancer cell lines. Cancer Res. 2003;63: 4028-4036. - PubMed
    1. Chinni SR, Li Y, Upadhyay S, Koppolu PK, Sarkar FH. Indole-3-carbinol (I3C) induced cell growth inhibition, G1 cell cycle arrest and apoptosis in prostate cancer cells. Oncogene. 2001;20: 2927-2936. - PubMed
    1. Bonnesen C, Eggleston IM, Hayes JD. Dietary indoles and isothiocyanates that are generated from cruciferous vegetables can both stimulate apoptosis and confer protection against DNA damage in human colon cell lines. Cancer Res. 2001;61: 6120-6130. - PubMed

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