Functional inhibition of Hsp70 by Pifithrin-µ switches Gambogic acid induced caspase dependent cell death to caspase independent cell death in human bladder cancer cells.

Heat shock protein-70kDa (Hsp70) is a member of molecular chaperone family, involved in the proper folding of various proteins. Hsp70 is important for tumor cell survival and is also reported to be involved in enhancing the drug resistance of various cancer types. Hsp70 controls apoptosis both upstream and downstream of the mitochondria by regulating the mitochondrial membrane permeabilization (MMP) and apoptosome formation respectively. In the present study, we have elucidated the role of Hsp70 in Gambogic acid (GA) induced apoptosis in bladder cancer cells. We observed that functional inhibition of Hsp70 by Pifithrin-µ switches GA induced caspase dependent (apoptotic) cell death to caspase independent cell death. However, this cell death was not essentially necrotic in nature, as shown by the observations like intact plasma membranes, cytochrome-c release and no significant effect on nuclear condensation/fragmentation. Inhibition of Hsp70 by Pifithrin-µ shows differential effect on MMP. GA induced MMP and cytochrome-c release was inhibited by Pifithrin-µ at 12h but enhanced at 24h. Pifithrin-µ also reverted back GA inhibited autophagy which resulted in the degradation of accumulated ubiquitinated proteins. Our results demonstrate that Hsp70 plays an important role in GA induced apoptosis by regulating caspase activation. Therefore, inhibition of Hsp70 may hamper with the caspase dependent apoptotic pathways induced by most anti-cancer drugs and reduce their efficacy. However, the combination therapy with Pifithrin-µ may be particularly useful in targeting apoptotic resistant cancer cells as Pifithrin-µ may initiate alternative cell death program in these resistant cells.

Gambogic acid induced oxidative stress dependent caspase activation regulates both apoptosis and autophagy by targeting various key molecules (NF-κB, Beclin-1, p62 and NBR1) in human bladder cancer cells.

BACKGROUND: Gambogic acid is a potent anticancer agent and has been found effective against various types of cancer cells. The present study was addressed to explore the cytotoxic potential of Gambogic acid and the modulation of autophagy and apoptosis in bladder cancer cells T24 and UMUC3. METHODS: Bladder cancer cell lines T24 and UMUC3 were treated with Gambogic acid, apoptosis was checked by flow-cytometry and expression of various autophagy and apoptosis related proteins was monitored by Western blotting. Confocal microscope was used for colocalization of p62 and Beclin-1. RESULTS: Gambogic acid induces reactive oxygen species, and elicits a strong autophagic response by activating JNK at earlier time points, which is inhibited at later time points with the activation of caspases. Reactive oxygen species mediated caspase activation causes degradation of autophagic proteins, cleavage of molecular chaperones (Hsp90 and GRP-78) and adaptor proteins (p62 and NBR1). Gambogic acid treatment results in mitochondrial hyperpolarization and cytochrome c release and activates caspases involved in both extrinsic and intrinsic apoptotic pathways. Gambogic acid abrogates NF-κB activation by ROS mediated inhibition of IκB-α phosphorylation. Functionally Gambogic acid induced autophagy acts as a strong cell survival response and delays caspase activation. CONCLUSION: Our study provides the new insights about the mechanism of Gambogic acid induced modulation of autophagy and apoptosis in bladder cancer cells. All the molecular events responsible for Gambogic acid induced autophagy and apoptosis are mediated by reactive oxygen species. GENERAL SIGNIFICANCE: Since Gambogic acid targets various cell survival molecules therefore, it may be considered as a potential anticancer agent against bladder cancer.

Inhibition of bfl-1/A1 by siRNA inhibits mycobacterial growth in THP-1 cells by enhancing phagosomal acidification.

Virulent tubercle bacilli inhibit apoptosis to establish a safe environment within the host cells. Here, we report that NF-kappaB dependent antiapoptotic protein bfl-1/A1 plays an important role in this process. Both virulent and avirulent mycobacteria bearing THP-1 cells expressed considerable amount of bfl-1/A1 after 4 h of infection. However, after 48 h of infection, bfl-1/A1 expression was evident only in Mycobacterium tuberculosis H37Rv but not in M. tuberculosis H37Ra infected cells. When parallel experiments were performed with Human monocyte-derived macrophages (MDMs), differential expression of bfl-1/A1 mRNA was observed in case of M. tuberculosis H37Rv and M. tuberculosis H37Ra infection. siRNA mediated inhibition of bfl-1/A1 induced apoptosis in M. tuberculosis H37Rv infected THP-1 and MDMs. Reduction in intracellular mycobacterial growth was observed in bfl-1/A1 siRNA transfected, M. tuberculosis H37Rv infected THP-1 cells. Enhancement of phagosome-lysosome fusion was observed in bfl-1/A1 siRNA treated and M. tuberculosis H37Rv infected THP-1 cells. These results clearly indicated that differential expression of bfl-1/A1 in M. tuberculosis H37Rv and M. tuberculosis H37Ra infected THP-1 cells probably account for the difference in infection outcome.

Differential expression of NF-kappaB in mycobacteria infected THP-1 affects apoptosis.

The present study was conducted to see the role of NF-kappaB in virulent (Mycobacterium tuberculosis H37Rv) and avirulent (M. tuberculosis H37Ra) mycobacterial infection in THP-1 cells. To inactivate NF-kappaB, pCMV-IkappaBalphaM dn containing THP-1 cell line was generated which showed marked increase in apoptosis with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Infected THP-1-IkappaBalphaM dn cells showed decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and enhanced TNF-alpha production. Increase in apoptosis of infected THP-1-IkappaBalphaM dn cells resulted in inhibition of intracellular mycobacterial growth. Differential NF-kappaB activation potential was observed with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Both the strains activated NF-kappaB after 4 h in THP-1 cells however after 48 h only M. tuberculosis H37Rv activated NF-kappaB which lead to up-regulation of bcl-2 family anti-apoptotic member, bfl-1/A1. Our results indicated that NF-kappaB activation may be a determinant factor for the success of virulent mycobacteria within macrophages.

Nitric oxide induces apoptosis in cutaneous T cell lymphoma (HuT-78) by downregulating constitutive NF-kappaB.

Constitutive active NF-kappaB have been shown to protect cutaneous T cell lymphoma (CTCL) cells from apoptosis. In the present study, we have studied the cytotoxic potential of nitric oxide generating compound, sodium nitroprusside (SNP) on CTCL cell line, HuT-78. Treatment of cells with SNP resulted in decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and poly (ADP ribose) polymerase cleavage. SNP treatment inhibited activation of NF-kappaB in a concentration dependent manner. SNP increased the expression of IkappaBalpha without affecting the phosphorylation of IkappaBalpha. Downregulation of NF-kappaB by SNP decreased p65 nuclear translocation as evident by confocal fluorescence microscopy. Further it was found that SNP treatment caused downregulation of Bcl-2 family member (Bcl-xl) in HuT-78 cells. Thus, we have provided evidence that SNP induces apoptosis in CTCL cell line, HuT-78 by downregulating constitutive NF-kappaB and thereby Bcl-xl expression.

Ursolic acid inhibits nuclear factor-kappaB activation induced by carcinogenic agents through suppression of IkappaBalpha kinase and p65 phosphorylation: correlation with down-regulation of cyclooxygenase 2, matrix metalloproteinase 9, and cyclin D1.

The process of tumorigenesis requires cellular transformation, hyperproliferation, invasion, angiogenesis, and metastasis. Several genes that mediate these processes are regulated by the transcription factor nuclear factor-kappaB (NF-kappaB). The latter is activated by various carcinogens, inflammatory agents, and tumor promoters. Thus, agents that can suppress NF-kappaB activation have the potential to suppress carcinogenesis. Ursolic acid, a pentacyclic triterpene acid, has been shown to suppress the expression of several genes associated with tumorigenesis, but whether ursolic acid mediates its effects through suppression of NF-kappaB is not understood. In the study described in the present report, we found that ursolic acid suppressed NF-kappaB activation induced by various carcinogens including tumor necrosis factor (TNF), phorbol ester, okadaic acid, H(2)O(2), and cigarette smoke. These effects were not cell type specific. Ursolic acid inhibited DNA binding of NF-kappaB consisting of p50 and p65. Ursolic acid inhibited IkappaBalpha degradation, IkappaBalpha phosphorylation, IkappaBalpha kinase activation, p65 phosphorylation, p65 nuclear translocation, and NF-kappaB-dependent reporter gene expression. Ursolic acid also inhibited NF-kappaB-dependent reporter gene expression activated by TNF receptor, TNF receptor-associated death domain, TNF receptor-associated factor, NF-kappaB-inducing kinase, IkappaBalpha kinase, and p65. The inhibition of NF-kappaB activation correlated with suppression of NF-kappaB-dependent cyclin D1, cyclooxygenase 2, and matrix metalloproteinase 9 expression. Thus, overall, our results indicate that ursolic acid inhibits IkappaBalpha kinase and p65 phosphorylation, leading to the suppression of NF-kappaB activation induced by various carcinogens. These actions of ursolic acid may mediate its antitumorigenic and chemosensitizing effects.

Pentoxifylline triggers autophagy via ER stress response that interferes with Pentoxifylline induced apoptosis in human melanoma cells.

Pentoxifylline (PTX), a non-specific phosphodiesterase inhibitor is known to inhibit the growth of various cancer cells including melanoma. Here in this study, we have found that PTX induces autophagy in human melanoma cell lines (A375 and MeWo). Induction of autophagy is associated with the increase in Atg5 expression as knockdown of Atg5 effectively inhibited PTX mediated autophagy. A decrease in mTOR activation was also observed after PTX treatment. We observed that autophagy was activated as a downstream effector mechanism of ER stress induced by PTX. ER stress response was confirmed by upregulation of IRE-1α, GRP78 and CHOP expression. PTX treatment also resulted in an increase in intracellular calcium (Ca(2+)) level. Ca(2+) is the central player as blocking Ca(2+) by intracellular calcium chelator (BAPTA-AM) effectively inhibited the PTX induced ER stress response as well as autophagy. Moreover, silencing of CHOP also resulted in autophagy inhibition with a decrease in Atg5 expression. Collectively, PTX triggers ER stress response followed by induction of autophagy via involvement of Ca(2+)→CHOP→Atg5 signalling cascade. Interestingly, inhibition of intracellular calcium level by BAPTA-AM significantly increased PTX mediated cell death by augmenting intrinsic apoptotic pathway. Inhibition of autophagy by the ATG5 siRNA and pharmacological inhibitor, chloroquine also enhances PTX induced cell death. Taken together, our results clearly indicate that activation of ER stress response and autophagy provides resistance to PTX mediated apoptosis, and thus, interferes with the anticancer activity of PTX in human melanoma cells.

Adenosine suppresses activation of nuclear factor-kappaB selectively induced by tumor necrosis factor in different cell types.

Adenosine is an endogenous immunomodulator that has been shown to exhibit anti-inflammatory and immunosuppressive properties through a mechanism that is not fully established. Owing to the pivotal role of nuclear factor (NF)-kappaB in these responses, we tested the hypothesis that adenosine mediates its effects through suppression of NF-kappaB activation. We investigated the effects of adenosine on NF-kappaB activation induced by various inflammatory agents in human myeloid KBM-5 cells. The treatment of these cells with adenosine suppressed TNF-induced NF-kappaB activation, but had no effect on activation of another redox-sensitive transcription factor, AP-1. These effects were not restricted to myeloid cells, as NF-kappaB activation in other lymphocytic and epithelial cell types was also inhibited. The effect on TNF-induced NF-kappaB activation was selective as adenosine had minimal effect on NF-kappaB activation induced by H(2)O(2), PMA, LPS, okadaic acid, or ceramide, suggesting differences in the pathway leading to NF-kappaB activation by different agents. Adenosine also suppressed NF-kappaB-dependent reporter gene expression activated by TNF or by overexpression of TNFR1, TRAF 2, NIK, and p65 subunit of NF-kappaB. The suppression of TNF-induced NF-kappaB activation by adenosine was found not to be because of inhibition of TNF-induced IkappaBalpha phosphorylation and degradation or IkappaBalpha kinase activation. The suppression of TNF-induced NF-kappaB activation was unique to adenosine, as neither its metabolites (inosine, AMP, and ATP) nor pyrimidines (thymidine and uridine) had any effect. Overall, our results clearly demonstrate that adenosine selectively suppresses TNF-induced NF-kappaB activation, which may contribute to its role in suppression of inflammation and of the immune system.

IL-27 inhibits IFN-γ induced autophagy by concomitant induction of JAK/PI3 K/Akt/mTOR cascade and up-regulation of Mcl-1 in Mycobacterium tuberculosis H37Rv infected macrophages.

Interleukin-27 (IL-27), a key immunoregulatory cytokine plays an important role in host response to mycobacterial infection as neutralization of IL-27 augments intracellular killing of mycobacteria. Autophagy has a pivotal role in host immunity and is regulated by various cytokines. Here, we report that IL-27 inhibits IFN-γ and starvation induced autophagy and as a result blocks phagosome maturation and promotes intracellular survival of Mycobacterium tuberculosis H37Rv. Addition of exogenous IL-27 induces the activation of mTOR through JAK/PI3 K pathway and inhibits IFN-γ stimulated autophagy. Furthermore, blockade of JAKs obstructs the inhibitory effect of IL-27 on IFN-γ induced autophagy. Besides this, IL-27 also up-regulates Mcl-1 through PI3 K pathway. We further show that in mTOR or Mcl-1 silenced THP-1 cells, IL-27 could no longer inhibit IFN-γ mediated autophagy in M. tuberculosis H37Rv infected cells. Altogether, our study demonstrates that IL-27 by concurrent activation of JAK/PI3 K/Akt/mTOR cascade as well as up-regulation of Mcl-1 inhibits IFN-γ induced autophagy and elimination of intracellular mycobacteria in macrophages.

Oxidative stress induced by curcumin promotes the death of cutaneous T-cell lymphoma (HuT-78) by disrupting the function of several molecular targets.

Curcumin is known to exert its anticancer effect either by scavenging or by generating reactive oxygen species (ROS). In this study, we report that curcumin-mediated rapid generation of ROS induces apoptosis by modulating different cell survival and cell death pathways in HuT-78 cells. Curcumin induces the activation of caspase-8, -2, and -9, alteration of mitochondrial membrane potential, release of cytochrome c, and activation of caspase-3 and concomitant PARP cleavage, but the addition of caspase inhibitors only partially blocked the curcumin-mediated apoptosis. Curcumin also downregulates the expression of antiapoptotic proteins c-FLIP, Bcl-xL, cellular inhibitor of apoptosis protein, and X-linked IAP in a ROS-dependent manner. Curcumin disrupts the integrity of IKK and beclin-1 by degrading Hsp90. Degradation of IKK leads to the inhibition of constitutive NF-κB. Degradation of beclin-1 by curcumin leads to the accumulation of autophagy-specific marker, microtubule-associated protein-I light chain 3 (LC3), LC3-I. Our findings indicate that HuT-78 cells are vulnerable to oxidative stress induced by curcumin and as a result eventually undergo cell death.

IL-6 inhibits IFN-γ induced autophagy in Mycobacterium tuberculosis H37Rv infected macrophages.

The significance of IL-6 production in tuberculosis is yet to be fully elucidated, although it is known for quite some time that IL-6 interferes with IFN-γ induced signal. In order to know which cellular process induced by IFN-γ is actually counteracted by IL-6, we studied the role of IL-6 on IFN-γ induced autophagy formation in virulent Mycobacterium tuberculosis infection in THP-1 cells, since it is well characterized that induction of autophagy by IFN-γ eliminates intracellular mycobacterium by overcoming the phagosome maturation block imposed by bacilli. We report here that IL-6 inhibits both IFN-γ and starvation induced autophagy in M. tuberculosis H37Rv infected cells. M. tuberculosis H37Rv infection results in time dependent production of IL-6 in THP-1 cells and neutralization of this endogenous IL-6 by anti-IL-6 antibody significantly enhances the IFN-γ mediated killing of the intracellular bacteria. IL-6 time dependently lowers Atg12-Atg5 complex and therefore inhibits autophagosome biogenesis rather than autophagolysosome formation. IL-6 also affects IFN-γ mediated stimulation of mTOR, p-38 and JNK pathways. These results clearly indicate that virulent mycobacteria strategically upregulate IL-6 production to combat innate immunity.

Bfl-1/A1 acts as a negative regulator of autophagy in mycobacteria infected macrophages.

Expression of Bcl-2 family protein, Bfl-1/A1 has been found to differ considerably amongst macrophages infected with virulent Mycobacterium tuberculosis H37Rv or with avirulent M. tuberculosis H37Ra. Present work was undertaken to deduce the significance of differential expression of Bfl-1/A1 in the outcome of mycobacterial infection. We have studied the role of Bfl-1/A1 particularly in autophagy formation in tubercle bacilli infected cells since autophagy has been recognized as a component of innate immunity against pathogenic mycobacteria. First, we have confirmed that upon infection virulent strain H37Rv retain Bfl-1/A1 for longer period and impose autophagosome maturation block within infected cells as evident from confocal microscopy. Moreover, down regulation of Bfl-1/A1 by siRNA induced autophagy formation and reduced bacterial growth. Furthermore, even the avirulent strain H37Ra resist autophagosome maturation and survive if the cellular level of Bfl-1 is maintained in THP-1 cells by stable transfection (Bfl-1 overexpressing cells). No noteworthy difference in mTOR expression was observed between normal THP-1 and Bfl-1 overexpressing THP-1 cells infected with either strain of mycobacteria. Interestingly, we found that not only mTOR but also Bfl-1/A1 is involved in rapamycin induced autophagy in mycobacteria infected macrophages. We have found that Bfl-1 physically interacts with Beclin 1 in Bfl-1 overexpressing THP-1 as well as in H37Rv infected THP-1 cells as they co-precipitated. Taken together, our results clearly demonstrated that Bfl-1/A1 negatively regulates autophagy and expression of Bfl-1/A1 in H37Rv infected macrophages provides the bacteria a survival strategy to overcome host defense.

Pentoxifylline augments TRAIL/Apo2L mediated apoptosis in cutaneous T cell lymphoma (HuT-78 and MyLa) by modulating the expression of antiapoptotic proteins and death receptors.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a promising anticancer agent but cutaneous T lymphoma cells (CTCL) are less sensitive to TRAIL-induced apoptosis. Here, we report that pentoxifylline (PTX), a phosphodiesterase inhibitor, augments TRAIL-mediated apoptosis in HuT-78 and MyLa cells through modulating extrinsic death receptors and intrinsic mitochondria dependent pathways. Our results clearly show that PTX augments TRAIL-mediated activation of caspase-8 and induces cleavage of Bid, although PTX alone cannot activate caspase-8. This is followed by cytochrome c release and subsequent, activation of caspase-9 and caspase-3 and cleavage of poly (ADP ribose) polymerase (PARP). Combined treatment downregulates the expression of various antiapoptotic proteins including c-FLIP, Bcl-xl, cIAP-1, cIAP-2 and XIAP. PTX induces the expression of death receptors DR4 and DR5 on cell surface of both the cell types where c-Jun NH2-terminal kinase (JNK) pathway plays an important role. Moreover, combined silencing of DR4 and DR5 by small interfering RNA abrogates the ability of PTX to induce TRAIL-mediated apoptosis. Thus, this is the first demonstration that PTX can potentiate TRAIL-mediated apoptosis through downregulation of cell survival gene products and upregulation of death receptors.

Pentoxifylline induces apoptosis in vitro in cutaneous T cell lymphoma (HuT-78) and enhances FasL mediated killing by upregulating Fas expression.

Constitutive nuclear factor-kappaB (NF-kappaB) is known to play an important role in the survival of HuT-78 cells, a cutaneous T cell lymphoma (CTCL) cell line. Here, we have demonstrated that pentoxifylline (PTX), a phosphodiesterase inhibitor, can trigger a series of events leading to apoptosis in HuT-78 cells without affecting NF-kappaB. Apoptosis was ascertained by sub-G1 peak analysis and TUNEL assay. Apoptosis induced by PTX in HuT-78 cells involved mitochondrial hyperpolarization, cytochrome c release, caspase-3 activation and PARP cleavage. Further, it was found that PTX treatment downregulated Bcl-xl and c-FLIP expression without affecting constitutive NF-kappaB but upregulated activator protein-1 (AP-1). Low concentration of PTX upregulated Fas and TRAIL expression in HuT-78 cells. In addition, PTX can act as a scavenger of reactive oxygen intermediate and it could enhance FasL mediated killing in HuT-78 cells. Our results taken together indicated that PTX may be a potential agent for killing CTCL cells.

Charged nylon membrane substrate for convenient and versatile high resolution microscopic analysis of Escherichia coli & mammalian cells in suspension culture.

Preparation of isolated cells and microorganisms for ultrastructural examination always provides a challenge in terms of adequate immobilization of the cells and prevention of subsequent sample loss and damage during various steps of sample processing. Using a positively charged nylon membrane substrate we demonstrate that it is possible to easily immobilize and retain a sample of isolated cells in culture for a wide variety of microscopy-based techniques. Radiolabelled E. coli cells when immobilized on the charged membrane were seen to be highly resistant to detachment when subjected to the normal sample processing procedures associated with microscopy. In contrast cells on regular millipore membranes were rapidly lost during sample preparation. We demonstrate the utility of charged nylon membranes for a wide variety of microscopy based analysis including scanning and transmission electron microscopy (SEM and TEM), atomic force microscopy (AFM), TEM based immunogold labelling, laser confocal microscopy and SEM based elemental analysis.

Thalidomide suppresses NF-kappa B activation induced by TNF and H2O2, but not that activated by ceramide, lipopolysaccharides, or phorbol ester.

Thalidomide ([+]-alpha-phthalimidoglutarimide), a psychoactive drug that readily crosses the blood-brain barrier, has been shown to exhibit anti-inflammatory, antiangiogenic, and immunosuppressive properties through a mechanism that is not fully established. Due to the central role of NF-kappaB in these responses, we postulated that thalidomide mediates its effects through suppression of NF-kappaB activation. We investigated the effects of thalidomide on NF-kappaB activation induced by various inflammatory agents in Jurkat cells. The treatment of these cells with thalidomide suppressed TNF-induced NF-kappaB activation, with optimum effect occurring at 50 microg/ml thalidomide. These effects were not restricted to T cells, as other hematopoietic and epithelial cell types were also inhibited. Thalidomide suppressed H(2)O(2)-induced NF-kappaB activation but had no effect on NF-kappaB activation induced by PMA, LPS, okadaic acid, or ceramide, suggesting selectivity in suppression of NF-kappaB. The suppression of TNF-induced NF-kappaB activation by thalidomide correlated with partial inhibition of TNF-induced degradation of an inhibitory subunit of NF-kappaB (IkappaBalpha), abrogation of IkappaBalpha kinase activation, and inhibition of NF-kappaB-dependent reporter gene expression. Thalidomide abolished the NF-kappaB-dependent reporter gene expression activated by overexpression of TNFR1, TNFR-associated factor-2, and NF-kappaB-inducing kinase, but not that activated by the p65 subunit of NF-kappaB. Overall, our results clearly demonstrate that thalidomide suppresses NF-kappaB activation specifically induced by TNF and H(2)O(2) and that this may contribute to its role in suppression of proliferation, inflammation, angiogenesis, and the immune system.

Piceatannol inhibits TNF-induced NF-kappaB activation and NF-kappaB-mediated gene expression through suppression of IkappaBalpha kinase and p65 phosphorylation.

Piceatannol is an anti-inflammatory, immunomodulatory, and anti-proliferative stilbene that has been shown to interfere with the cytokine signaling pathway. Previously, we have shown that resveratrol suppresses the activation of the nuclear transcription factor NF-kappaB. Piceatannol, previously reported as a selective inhibitor of protein tyrosine kinase Syk, is structurally homologous to resveratrol. Whether piceatannol can also suppress NF-kappaB activation was investigated. The treatment of human myeloid cells with piceatannol suppressed TNF-induced DNA binding activity of NF-kappaB. In contrast, stilbene or rhaponticin (another analog of piceatannol) had no effect, suggesting the critical role of hydroxyl groups. The effect of piceatannol was not restricted to myeloid cells, as TNF-induced NF-kappaB activation was also suppressed in lymphocyte and epithelial cells. Piceatannol also inhibited NF-kappaB activated by H(2)O(2), PMA, LPS, okadaic acid, and ceramide. Piceatannol abrogated the expression of TNF-induced NF-kappaB-dependent reporter gene and of matrix metalloprotease-9, cyclooxygenase-2, and cyclin D1. When examined for the mechanism, we found that piceatannol inhibited TNF-induced IkappaBalpha phosphorylation, p65 phosphorylation, p65 nuclear translocation, and IkappaBalpha kinase activation, but had no significant effect on IkappaBalpha degradation. Piceatannol inhibited NF-kappaB in cells with deleted Syk, indicating the lack of involvement of this kinase. Overall, our results clearly demonstrate that hydroxyl groups of stilbenes are critical and that piceatannol, a tetrahydroxystilbene, suppresses NF-kappaB activation induced by various inflammatory agents through inhibition of IkappaBalpha kinase and p65 phosphorylation.