Anacardic acid (6-nonadecyl salicylic acid), an inhibitor of histone acetyltransferase, suppresses expression of nuclear factor-kappaB-regulated gene products involved in cell survival, proliferation, invasion, and inflammation through inhibition of the inhibitory subunit of nuclear factor-kappaBalpha kinase, leading to potentiation of apoptosis

Abstract

Anacardic acid (6-pentadecylsalicylic acid) is derived from traditional medicinal plants, such as cashew nuts, and has been linked to anticancer, anti-inflammatory, and radiosensitization activities through a mechanism that is not yet fully understood. Because of the role of nuclear factor-kappaB (NF-kappaB) activation in these cellular responses, we postulated that anacardic acid might interfere with this pathway. We found that this salicylic acid potentiated the apoptosis induced by cytokine and chemotherapeutic agents, which correlated with the down-regulation of various gene products that mediate proliferation (cyclin D1 and cyclooxygenase-2), survival (Bcl-2, Bcl-xL, cFLIP, cIAP-1, and survivin), invasion (matrix metalloproteinase-9 and intercellular adhesion molecule-1), and angiogenesis (vascular endothelial growth factor), all known to be regulated by the NF-kappaB. We found that anacardic acid inhibited both inducible and constitutive NF-kappaB activation; suppressed the activation of IkappaBalpha kinase that led to abrogation of phosphorylation and degradation of IkappaBalpha; inhibited acetylation and nuclear translocation of p65; and suppressed NF-kappaB-dependent reporter gene expression. Down-regulation of the p300 histone acetyltransferase gene by RNA interference abrogated the effect of anacardic acid on NF-kappaB suppression, suggesting the critical role of this enzyme. Overall, our results demonstrate a novel role for anacardic acid in potentially preventing or treating cancer through modulation of NF-kappaB signaling pathway.

Figure 1

Anacardic acid potentiates apoptosis induced by TNF and chemotherapeutic agents. (A) Structure of anacardic acid (AA). (B) Anacardic acid potentiates TNF-induced apoptosis. KBM-5 cells were pretreated with 25 μmol/L anacardic acid for 4 hours and then incubated with 1 nmol/L TNF for 16 hours. The cells were stained with the Live/Dead assay reagent for 30 minutes and then analyzed under a fluorescence microscope. The results shown are representative of 3 independent experiments. (C) Cells were pretreated with 25 μmol/L anacardic acid for 4 hours and then incubated with 1 nmol/L TNF for 16 hours. Cells were incubated with an anti–annexin V antibody conjugated with FITC and then analyzed by flow cytometry for early apoptotic effects. (D) Cells were pretreated with 25 μmol/L anacardic acid for 4 hours and then incubated with 1 nmol/L TNF for 16 hours. Cells were fixed, stained with TUNEL assay reagent, and then analyzed by flow cytometry for apoptotic effects. (E) KBM-5 cells were incubated with 25 μmol/L anacardic acid for 4 hours and then treated with 1 nmol/L TNF for 24 hours. Whole-cell extracts were prepared and analyzed by Western blotting using the indicated antibodies. (F) Cells were pretreated with 25 μmol/L anacardic acid for 4 hours and then incubated with 1 nmol/L TNF for the indicated times. Whole-cell extracts were prepared and subjected to Western blot analysis using an anti-PARP antibody.

Figure 2

Anacardic acid represses TNF-induced NF-κB–dependent expression of antiapoptosis-, proliferation-, and metastasis-related gene products. (A) Antiapoptotic proteins. (B) Proliferative and metastatic proteins. KBM-5 cells were incubated with 25 μmol/L anacardic acid for 4 hours and then treated with 1 nmol/L TNF for the indicated times. Whole-cell extracts were prepared and subjected to Western blot analysis using the relevant antibodies.

Figure 3

Anacardic acid inhibits TNF-dependent NF-κB activation. (A) Effect of anacardic acid per dose. KBM-5 cells were preincubated with indicated concentrations of anacardic acid for 4 hours, treated with 0.1 nmol/L TNF for 30 minutes, and then subjected to EMSA to test for NF-κB activation. (B) Effect of anacardic acid according to exposure duration. Cells were preincubated with 25 μmol/L anacardic acid for the indicated times, treated with 0.1 nmol/L TNF for 30 minutes, and then subjected to EMSA to test for NF-κB activation. (C) NF-κB induced by TNF is composed of p65 and p50 subunits. Nuclear extracts from untreated or TNF-treated cells were incubated with the indicated antibody, preimmune serum, unlabeled NF-κB oligoprobe, or mutant oligoprobe and then assayed for NF-κB activation by EMSA. (D) The direct effect of anacardic acid on NF-κB complex was investigated. Nuclear extracts were prepared from untreated cells or cells treated with 0.1 nmol/L TNF and incubated for 30 minutes with the indicated concentrations of anacardic acid. They were then assayed for NF-κB activation by EMSA. (E) Anacardic acid blocks NF-κB activation induced by TNF, IL-1β, LPS, PMA, OA, and EGF. KBM-5 cells were preincubated with 25 μmol/L anacardic acid for 4 hours and then treated with 0.1 nmol/L TNF, 100 ng/mL IL-1β, or 10 μg/mL LPS for 30 minutes; 500 nmol/L OA for 4 hours or 25 μg/mL PMA or 100 ng/mL EGF for 2 hours. The cells were then analyzed for NF-κB activation by EMSA. (F-I) Inhibition of NF-κB activation by anacardic acid is not cell type–specific. H1299, Jurkat, Du145, and SCC4 cells were incubated with 25 μmol/L anacardic acid for 4 hours and then incubated with 0.1 nmol/L TNF for 30 minutes. Nuclear extracts were then prepared and assayed for NF-κB activation by EMSA.

Figure 4

Anacardic acid inhibits TNF-dependent IκBα phosphorylation, IκBα degradation, p65 phosphorylation, and p65 nuclear translocation. (A) Anacardic acid inhibits TNF-induced activation of NF-κB. KBM-5 cells were incubated with 25 μmol/L anacardic acid for 4 hours, treated with 0.1 nmol/L TNF for the indicated times, and then analyzed for NF-κB activation by EMSA. (B) Effect of anacardic acid on TNF-induced IκBα degradation, p65 phosphorylation, and p65 nuclear translocation. Cells were incubated with 25 μmol/L anacardic acid for 4 hours and treated with 0.1 nmol/L TNF for the indicated times. Cytoplasmic extracts (CE) and nuclear extracts (NE) were prepared, fractionated on SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed using the indicated antibody. An anti–β-actin antibody was the loading control. (C) Effect of anacardic acid on the phosphorylation of IκBα by TNF. Cells were preincubated with 25 μmol/L anacardic acid for 4 hours, incubated with 50 μg/mL ALLN for 30 minutes, and then treated with 0.1 nmol/L TNF for 10 minutes. Cytoplasmic extracts were fractionated and then subjected to Western blot analysis using a phospho-specific anti-IκBα antibody. The same membrane was reblotted with anti-IκBα antibody. (D) Anacardic acid inhibits TNF-induced IκBα kinase activity. Whole-cell extracts were immunoprecipitated with antibody against IKKα and analyzed by an immune complex kinase assay. To examine the effect of anacardic acid on the level of expression of IKK proteins, whole-cell extracts were fractionated on SDS-PAGE and examined by Western blot analysis using anti-IKKα and anti-IKKβ antibodies. (E) Direct effect of anacardic acid on IKK activation induced by TNF. Whole-cell extracts were prepared from KBM-5 cells treated with 1 nmol/L TNF and immunoprecipitated with anti-IKKα antibody. The immunocomplex kinase assay was performed in the absence or presence of the indicated concentration of anacardic acid. (F) Effect of anacardic acid on TNF-induced acetylation of p65. Cells were treated with 25 μmol/L anacardic acid for 4 hours and then exposed to 1 nmol/L TNF. Whole-cell extracts were prepared, immunoprecipitated with an anti-p65 antibody, and subjected to Western blot analysis using an anti–acetyl-lysine antibody. The same blots were reprobed with anti-p65 antibody. (G) Effect of anacardic acid on TNF-induced protein acetylation. Cells were treated with 25 μmol/L anacardic acid for 4 hours and then exposed to 1 nmol/L TNF for 20 minutes. Whole cell extracts were prepared and subjected to Western blot analysis using an anti–acetyl-lysine antibody. (H) Immunocytochemical analysis of p65 localization. Cells were incubated with 25 μmol/L anacardic acid for 4 hours and then treated with 1 nmol/L TNF for 15 minutes. Cells were subjected to immunocytochemical analysis.

Figure 5

Anacardic acid represses NF-κB–dependent reporter gene expression induced by TNF and various plasmids. (A) Anacardic acid inhibits the NF-κB–dependent reporter gene expression induced by TNF. A293 cells were transiently transfected with a NF-κB–containing plasmid for 24 hours. After transfection, the cells were incubated with the indicated concentrations of anacardic acid for 4 hours and then treated with 1 nmol/L TNF for an additional 24 hours. The supernatants of the culture media were assayed for SEAP activity. Data are presented as mean (± SD). (B) Anacardic acid inhibits the NF-κB–dependent reporter gene expression induced by TNF, TNFR1, TRADD, TRAF2, NIK, IKK, p65, and TAK1/TAB1. Cells were transiently transfected with a NF-κB–containing plasmid alone or with the indicated plasmids. After transfection, cells were incubated with 25 μmol/L anacardic acid for 4 hours and then incubated with the relevant plasmid for an additional 24 hours. TNF-treated cells were incubated with 25 μmol/L anacardic acid for 4 hours and then treated with 1 nmol/L TNF for an additional 24 hours. The supernatants of the culture media were assayed for SEAP activity. Data are presented as mean (± SD). (C) Anacardic acid inhibits the COX-2 promoter activity induced by TNF. Cells were transiently transfected with a COX-2 promoter linked to the luciferase reporter gene plasmid for 24 hours and treated with the indicated concentrations of anacardic acid for 4 hours. Cells were then treated with 1 nmol/L TNF for an additional 24 hours, lysed, and subjected to a luciferase assay. Data are presented as mean (± SD).

Figure 6

Down-regulation of p300 HAT abrogates the effect of anacardic acid. (A) TNF regulates antiapoptotic gene expression. The wild-type and p65^−/− MEF cells were pretreated with 25 μmol/L anacardic acid for 4 hours and then incubated with 1 nmol/L TNF for the indicated times. Whole-cell extracts were prepared and subjected to Western blot analysis using the relevant antibodies. (B) Anacardic acid inhibits binding of NF-κB to the COX-2 and MMP-9 promoter. KBM-5 cells were pretreated with 25 μmol/L anacardic acid for 4 hours and treated with 1 nmol/L TNF for the indicated times, and the proteins were cross-linked with DNA by formaldehyde and then subjected to chromatin immunoprecipitation (ChIP) assay using an anti-p65 antibody with the COX-2 and MMP-9 primers. Reaction products were resolved by electrophoresis. (C) Down-regulation of p300 by RNA interference reverses the effect of anacardic acid. A293 cells were transfected with indicated concentration of p300 siRNA or scrambled (SC) control. After 48 hours, cells were harvested, and whole-cell extracts were prepared and analyzed by Western blotting with an anti-p300 antibody. (D) Transfected cells were preincubated with 25 μmol/L anacardic acid for 4 hours and then treated with 0.1 nmol/L TNF for 30 minutes. The nuclear extracts were prepared and assayed for NF-κB activation by EMSA.