Curcumin (diferuloylmethane), an anti-inflammatory agent used in traditional medicine, has been shown to suppress cellular transformation, proliferation, invasion, angiogenesis, and metastasis through a mechanism not fully understood. Because several genes that mediate these processes are regulated by nuclear factor-B (NF-B), we have postulated that curcumin mediates its activity by modulating NF-B activation. Indeed, our laboratory has shown previously that curcumin can suppress NF-B activation induced by a variety of agents (J Biol Chem 270:24995-50000, 1995) . In the present study, we investigated the mechanism by which curcumin manifests its effect on NF-B and NF-B-regulated gene expression. Screening of 20 different analogs of curcumin showed that curcumin was the most potent analog in suppressing the tumor necrosis factor (TNF)-induced NF-B activation. Curcumin inhibited TNF-induced NF-B-dependent reporter gene expression in a dose-dependent manner. Curcumin also suppressed NF-B reporter activity induced by tumor necrosis factor receptor (TNFR)1, TNFR2, NF-B-inducing kinase, IB kinase complex (IKK), and the p65 subunit of NF-B. Such TNFinduced NF-B-regulated gene products involved in cellular proliferation [cyclooxygenase-2 (COX-2), cyclin D1, and c-myc], antiapoptosis [inhibitor of apoptosis protein (IAP)1, IAP2, X-chromosome-linked IAP, Bcl-2, Bcl-x L , Bfl-1/A1, TNF receptor-associated factor 1, and cellular Fas-associated death domain protein-like interleukin-1␤-converting enzyme inhibitory protein-like inhibitory protein], and metastasis (vascular endothelial growth factor, matrix metalloproteinase-9, and intercellular adhesion molecule-1) were also down-regulated by curcumin. COX-2 promoter activity induced by TNF was abrogated by curcumin. We found that curcumin suppressed TNFinduced nuclear translocation of p65, which corresponded with the sequential suppression of IB␣ kinase activity, IB␣ phosphorylation, IB␣ degradation, p65 phosphorylation, p65 nuclear translocation, and p65 acetylation. Curcumin also inhibited TNF-induced Akt activation and its association with IKK. Glutathione and dithiothreitol reversed the effect of curcumin on TNF-induced NF-B activation. Overall, our results indicated that curcumin inhibits NF-B activation and NF-B-regulated gene expression through inhibition of IKK and Akt activation.It is generally assumed that traditional medicines are safe and efficacious, given that they have been used for centuries. Despite the widespread use of these medicines, however, research to definitively establish safety and efficacy of many of them is lacking. Between 1981 and 2002, almost 74% (48 of 65) of all drugs approved for cancer were either natural products or based on natural products (typically, analogs or mimics) (Newman et al., 2003). The delineation of their Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.doi:10.1124/mol.105.017400. ABBREVIATIONS:COX-2, cyclooxygenase-2; MMP, matrix metalloproteinase; TNF, tumor necro...

Key words: HNSCC; NF-B; IB␣; IKK; apoptosis; MMP-9; cyclin D1; COX-2Head and neck squamous cell carcinoma (HNSCC) includes epithelial malignancies that arise from the mucosal lining of the oral cavity, oropharynx, nose and pernasal sinuses, hypopharynx and larynx. These cancers occur more than twice as often in men as in women. 1 HNSCC are among the most morbid of human cancers and affect annually nearly 500,000 people world-wide and approximately 40,000 cases in the United States, making it the sixth most common cancer type.Cigarette-smoking, tobacco-chewing and betel or areca nut chewing are environmental factors associated with increased risk of developing HNSCC. [1][2][3] Despite standard treatment strategies that involve surgery, radiotherapy or chemotherapy, the survival rate of patients with this cancer has remained poor. A total of 30 -50% of patients develop local or regional recurrence, and an increasing number of patients are developing distant metastases. 4 Another 10 -40% patients develop second primary tumors of the aerodigestive tract as a result of field cancerization. 5 Preventing cancer by inhibiting carcinoma before invasive tumors develop is a promising strategy, but current strategies have limited efficacy and documented toxicity. 6,7 Modulating the expression of proinflammatory and proangiogenic factors associated with aggressive tumor growth and decreased survival of patients with HNSCC is one avenue that holds potential. 8 -12 Several of the cytokines expressed by HNSCC are regulated by the nuclear transcription factor NF-B. NF-B consists of a group of 5 proteins: c-Rel, RelA (p65), Rel B, NF-B1 (p50 and p105) and NF-B2 (p52). 13 In resting state, NF-B is sequestered in the cytoplasm through its tight association with a specific inhibitory proteins. These proteins are inhibitors of NF-B (IB) and belong to a gene family consisting of IB␣, IB␤, IB⑀, IB␥, Bcl-3, p100 and p105. 13 On activation by agents such as TNF, the IB␣ is phosphorylated at serine residue 32 and 36, ubiquitinated at lysine residue 21 and 22 and degraded through the proteasomal pathway, exposing the nuclear localization signals on the p50 -p65 heterodimer. p65 then undergoes phosphorylation, leading to nuclear translocation and binding to a specific sequence in DNA. This results in gene transcription. The phosphorylation of IB␣ is catalyzed by the IB␣ kinase (IKK) consisting of IKK-␣, IKK-␤ and IKK-␥ (also called NF-B essential modulator (NEMO). 13 Gene deletion studies have indicated that IKK-␤ is essential for NF-B activation by TNF. 14 -16 IKK-␣ deletion, however, has no effect on NF-B activation by most agents. Determining which kinase induces the phosphorylation of p65 is controversial but the role of PKA, casein kinase II, glycogen synthase kinase-3␤, IKK-␣ and IKK-␤ have been implicated. [17][18][19][20][21][22][23] It has been shown that the phosphorylation of p65 at serine 529 is required for TNF-induced transcriptional activity of NF-B. 24 NF-B is activated by a wide variety of agents including various carcinog...

Increasing evidence from diverse sources during the past several years has indicated that long-term, low level, chronic inflammation mediates several chronic diseases including cancer, arthritis, obesity, diabetes, cardiovascular diseases, and neurological diseases. The inflammatory molecules and transcription factors, adhesion molecules, AP-1, chemokines, C-reactive protein (CRP), cyclooxygenase (COX)-2, interleukins (ILs), 5-lipooxygenase (5-LOX), matrix metalloproteinases (MMPs), nuclear factor (NF)-kB, signal transducer and activator of transcription 3 (STAT3), tumor necrosis factor (TNF), and vascular endothelial growth factor (VEGF) are molecular links between inflammation and chronic diseases. Thus, suppression of inflammatory molecules could be potential strategy for the prevention and therapy of chronic diseases. The currently available drugs against chronic diseases are highly expensive, minimally effective and produce several side effects when taken for long period of time. The focus of this review is to discuss the potential of nutraceuticals derived from “Mother Nature” such as apigenin, catechins, curcumin, ellagic acid, emodin, epigallocatechin gallate, escin, fisetin, flavopiridol, genistein, isoliquiritigenin, kaempferol, mangostin, morin, myricetin, naringenin, resveratrol, silymarin, vitexin, and xanthohumol in suppression of these inflammatory pathways. Thus, these nutraceuticals offer potential in preventing or delaying the onset of chronic diseases. We provide evidence for the potential of these nutraceuticals from pre-clinical and clinical studies.

Vitamin A (retinol) is essential for normal regulation of cell growth and differentiation. We have shown that the retinol metabolite retinoic acid (RA) induces mucous cell differentiation of normal human tracheobronchial epithelial (NHTBE) cells. However, early biological effects of RA in the differentiation of bronchial epithelia are largely unknown. Here, we showed that RA rapidly activated cAMP response element-binding protein (CREB). However, RA did not use the conventional retinoic acid receptor (RAR)/retinoid X receptor (RXR) to activate CREB. RA activated CREB in NHTBE and H1734 cells in which RARs/RXR were silenced with small interfering RNA (siRNA) targeting RAR/RXR expression or deactivated by antagonist. Inhibition of protein kinase C (PKC) or extracellular regulated kinase (ERK1/2) blocked the RA-mediated activation of CREB. In addition, depletion of p90 ribosomal S6 kinase (RSK) via siRSK1/2 completely abolished the activation, suggesting that PKC, ERK, and RSK are required for the activation. Altogether, this study provides the first evidence that RA rapidly activates CREB transcription factor via PKC, ERK, and RSK in a retinoid receptor-independent manner in normal bronchial epithelial cells. This noncanonical RA signaling pathway may play an important role in mediating early biological effects in the mucociliary differentiation of bronchial epithelia. INTRODUCTIONVitamin A (retinol) and its related analogs, collectively called retinoids, play a pivotal role in cell growth and differentiation (for review, see Gudas et al., 1994). In the respiratory system, retinoic acid (RA), the natural metabolite of vitamin A, controls the development and maintenance of differentiated mucociliary epithelial cells. It has been well documented that RA is essential for induction and maintenance of normal mucociliary airway epithelium (McDowell et al., 1984a(McDowell et al., , 1984b. RA deficiency causes squamous metaplasia, and supplementation with RA restores the normal mucous cell phenotype in cultured primary bronchial epithelial cells (Koo et al., 1999). However, the early effect of RA on mucous cell differentiation of bronchial epithelia is largely unknown.It has been well documented that the effects of RA at the genomic level are mediated mainly through heterodimerized forms of retinoid receptors, RARs and RXRs, which recognize expression of their target genes through the RA response element (A/G)G(G/T)TCA upon binding of RA and activate gene expression (Giguere, 1994;Mangelsdorf and Evans, 1995;Chambon, 1996). Recent data showed, however, that RA action could be diversified to other cellular signaling pathways, the so-called nongenomic action of RA. For example, RA has been shown to modulate PKC activity by binding directly to PKC. RA binds directly to the C2-domain of PKC␣ (Ochoa et al., 2002), as determined by structural analysis of the cocrystalized C2-domain of PKC with RA. In addition, RA binding to the phosphatidyl serine binding site of PKC␣ was determined by photoaffinity labeling assay (Radominska-P...

BACKGROUND: Despite earlier studies demonstrating in vitro propagation of solid tumour cancer stem cells (CSCs) as non-adherent tumour spheres, it remains controversial as to whether CSCs can be maintained in vitro. Additional validation of the CSC properties of tumour spheres would support their use as CSC models and provide an opportunity to discover additional CSC cell surface markers to aid in CSC detection and potential elimination. METHODS: Primary tumour cells isolated from 13 surgically resected colon tumour specimens were propagated using serum-free CSCselective conditions. The CSC properties of long-term cultured tumour spheres were established and mass spectrometry-based proteomics performed. RESULTS: Freshly isolated CD133 þ colorectal cancer cells gave rise to long-term tumour sphere (or spheroids) cultures maintaining CD133 expression. These spheroid cells were able to self-renew and differentiate into adherent epithelial lineages and recapitulate the phenotype of the original tumour. Relative to their differentiated progeny, tumour spheroid cells were more resistant to the chemotherapeutic irinotecan. Finally, CD44, CD166, CD29, CEACAM5, cadherin 17, and biglycan were identified by mass spectrometry to be enriched in CD133 þ tumour spheroid cells. CONCLUSION: Our data suggest that ex vivo-expanded colon CSCs isolated from clinical specimens can be maintained in culture enabling the identification of CSC cell surface-associated proteins.

Chemotherapeutic agents with low toxicity to normal tissues are a major goal in cancer research. In this regard, the therapeutic activities of cationic dyes, such as rhodamine 6G, toward cancer cells have been studied for decades with observed toxicities toward normal and cancer cells. Herein, we report rhodamine 6G-based organic salts with varying counter-anions that are stable under physiological conditions, display excellent fluorescence photostability, and more importantly have tunable chemotherapeutic properties. Our in-vitro studies indicate that the hydrophobic compounds of this series allow production of nanoparticles which are non-toxic to normal cells and toxic to cancer cells. Furthermore, the anions, in combination with cations such as sodium, were observed to be non-toxic to both normal and cancer cells. To the best of our knowledge, this is the first demonstration that both the cation and anion play an extremely important and cooperative role in the antitumor properties of these compounds.

Genes regulated by cyclic AMP response element-binding protein (CREB) have been reported to suppress apoptosis, induce cell proliferation, and mediate inflammation and tumor metastasis. However, it is not clear whether CREB is critically involved in the carcinogenesis of lung cancer. We found that non-small cell lung cancer (NSCLC) cell lines exhibited elevated constitutive activity in CREB; in its immediate upstream kinases, ribosomal s6 kinase and extracellular signal kinase; and in the CREB-regulated cell survival proteins, Bcl-2 and Bcl-xL. We hypothesized that constitutively active CREB is important to lung cancer cell growth and survival and therefore could be a potential therapeutic target for NSCLC. Ectopic expression of dominant-repressor CREB and transfection with small interfering RNA against CREB suppressed the growth and survival of NSCLC cells and induced apoptotic cell death. Furthermore, treating H1734 NSCLC cells with an inhibitor of the CREB signaling pathway, Ro-31-8220, inhibited CREB activation by blocking the activity of extracellular signal kinase and ribosomal s6 kinase, arrested the cell cycle at the G2/M phase, and subsequently induced apoptosis with the suppression of Bcl-2 and Bcl-xL expression. Ro-31-8220 suppressed both the anchorage-dependent and the independent growth of NSCLC cells, but its cytotoxic effect was much less prominent in normal bronchial epithelial cells. Our results indicate that active CREB plays an important role in NSCLC cell growth and survival. Thus, agents that suppress CREB activation could have potential therapeutic value for NSCLC treatment.

Inactivation of the dead-end (Dnd1) gene in the Ter mouse strain results in depletion of primordial germ cells (PGCs) so that mice become sterile. However, on the 129 mouse strain background, loss of Dnd1 also increases testicular germ cell tumor incidence in parallel to PGC depletion. We report that inactivation of Dnd1 also affects embryonic viability in the 129 strain. Mouse Dnd1 encodes two protein isoforms, DND1-isoform α (DND1-α) and DND1-isoform β (DND1-β). Using isoform specific antibodies, we determined DND1-α is expressed in embryos and embryonic gonads whereas DND1-β expression is restricted to germ cells of the adult testis. Our data implicates DND1-α isoform to be necessary for germ cell viability and therefore its loss in Ter mice results in PGC depletion, germ cell tumor development and partial embryonic lethality in the 129 strain.