Madan M. Chaturvedi

Oxidative stress, inflammation, and cancer: How are they linked?

Extensive research during the past 2 decades has revealed the mechanism by which continued oxidative stress can lead to chronic inflammation, which in turn could mediate most chronic diseases including cancer, diabetes, and cardiovascular, neurological, and pulmonary diseases. Oxidative stress can activate a variety of transcription factors including NF-κB, AP-1, p53, HIF-1α, PPAR-γ, β-catenin/Wnt, and Nrf2. Activation of these transcription factors can lead to the expression of over 500 different genes, including those for growth factors, inflammatory cytokines, chemokines, cell cycle regulatory molecules, and anti-inflammatory molecules. How oxidative stress activates inflammatory pathways leading to transformation of a normal cell to tumor cell, tumor cell survival, proliferation, chemoresistance, radioresistance, invasion, angiogenesis, and stem cell survival is the focus of this review. Overall, observations to date suggest that oxidative stress, chronic inflammation, and cancer are closely linked.

NF-κB addiction and its role in cancer: ‘one size does not fit all’

Activation of nuclear factor (NF)-κB, one of the most investigated transcription factors, has been found to control multiple cellular processes in cancer including inflammation, transformation, proliferation, angiogenesis, invasion, metastasis, chemoresistance and radioresistance. NF-κB is constitutively active in most tumor cells, and its suppression inhibits the growth of tumor cells, leading to the concept of ‘NF-κB addiction’ in cancer cells. Why NF-κB is constitutively and persistently active in cancer cells is not fully understood, but multiple mechanisms have been delineated including agents that activate NF-κB (such as viruses, viral proteins, bacteria and cytokines), signaling intermediates (such as mutant receptors, overexpression of kinases, mutant oncoproteins, degradation of IκBα, histone deacetylase, overexpression of transglutaminase and iNOS) and cross talk between NF-κB and other transcription factors (such as STAT3, HIF-1α, AP1, SP, p53, PPARγ, β-catenin, AR, GR and ER). As NF-κB is ‘pre-active’ in cancer cells through unrelated mechanisms, classic inhibitors of NF-κB (for example, bortezomib) are unlikely to mediate their anticancer effects through suppression of NF-κB. This review discusses multiple mechanisms of NF-κB activation and their regulation by multitargeted agents in contrast to monotargeted agents, thus ‘one size does not fit all’ cancers.

Sanguinarine (Pseudochelerythrine) Is a Potent Inhibitor of NF-κB Activation, IκBα Phosphorylation, and Degradation

The nuclear factor NF-κB is a pleiotropic transcription factor whose activation results in inflammation, viral replication, and growth modulation. Due to its role in pathogenesis, NF-κB is considered a key target for drug development. In the present report we show that sanguinarine (a benzophenanthridine alkaloid), a known anti-inflammatory agent, is a potent inhibitor of NF-κB activation. Treatment of human myeloid ML-1a cells with tumor necrosis factor rapidly activated NF-κB, this activation was completely suppressed by sanguinarine in a dose- and time-dependent manner. Sanguinarine did not inhibit the binding of NF-κB protein to the DNA but rather inhibited the pathway leading to NF-κB activation. The reversal of inhibitory effects of sanguinarine by reducing agents suggests a critical sulfhydryl group is involved in NF-κB activation. Sanguinarine blocked the tumor necrosis factor-induced phosphorylation and degradation of IκBα, an inhibitory subunit of NF-κB, and inhibited translocation of p65 subunit to the nucleus. As sanguinarine also inhibited NF-κB activation induced by interleukin-1, phorbol ester, and okadaic acid but not that activated by hydrogen peroxide or ceramide, the pathway leading to NF-κB activation is likely different for different inducers. Overall, our results demonstrate that sanguinarine is a potent suppressor of NF-κB activation and it acts at a step prior to IκBα phosphorylation.

[53] Assay for redox-sensitive transcription factor

Publisher Summary To delineate the mechanism of response to oxidative stress, an assay is required for transcription activation. This chapter describes an assay, the electrophoretic mobility shift assay (EMSA), used commonly to assay for redox-sensitive transcription factors. The method described is very suitable for measuring the activation of transcription factors such as nuclear factor-ĸB (NF-ĸB) in response to a variety of prooxidant stimuli, including tumor necrosis factor (TNF), interleukin-1, hydrogen peroxide (H 2 O 2 ), ceramide, okadaic acid, lipopolysaccharides, and phorbol myristate acetate (PMA). The method is extremely sensitive, as the activation of NF-ĸB can be detected within minutes after treatment of cells with as little as 1 p M TNF. It also uses the protocol to detect the activation of activator protein-1 (AP-1) by a variety of stimuli. The involvement of ROS in NF-ĸB activation is demonstrated by its inhibition by pyrrolidine dithiocarbamate. The nuclear extract prepared by the method described here has also been used to assay other constitutively active transcription factors such as Oct-l, SP-1, and TF-IID.

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

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.

Targeting Inflammatory Pathways by Flavonoids for Prevention and Treatment of Cancer

Observational studies have suggested that lifestyle risk factors such as tobacco, alcohol, high-fat diet, radiation, and infections can cause cancer and that a diet consisting of fruits and vegetables can prevent cancer. Evidence from our laboratory and others suggests that agents either causing or preventing cancer are linked through the regulation of inflammatory pathways. Genes regulated by the transcription factor NF- kappaB have been shown to mediate inflammation, cellular transformation, tumor cell survival, proliferation, invasion, angiogenesis, and metastasis. Whereas various lifestyle risk factors have been found to activate NF- kappaB and NF- kappaB-regulated gene products, flavonoids derived from fruits and vegetables have been found to suppress this pathway. The present review describes various flavones, flavanones, flavonols, isoflavones, anthocyanins, and chalcones derived from fruits, vegetables, legumes, spices, and nuts that can suppress the proinflammatory cell signaling pathways and thus can prevent and even treat the cancer.

MODULATION OF TRANSCRIPTION FACTORS BY CURCUMIN

Curcumin is the active ingredient of turmeric that has been consumed as a dietary spice for ages. Turmeric is widely used in traditional Indian medicine to cure biliary disorders, anorexia, cough, diabetic wounds, hepatic disorders, rheumatism, and sinusitis. Extensive investigation over the last five decades has indicated that curcumin reduces blood cholesterol, prevents low-density lipoprotein oxidation, inhibits platelet aggregation, suppresses thrombosis and myocardial infarction, suppresses symptoms associated with type II diabetes, rheumatoid arthritis, multiple sclerosis, and Alzheimers disease, inhibits HIV replication, enhances wound healing, protects from liver injury, increases bile secretion, protects from cataract formation, and protects from pulmonary toxicity and fibrosis. Evidence indicates that the divergent effects of curcumin are dependent on its pleiotropic molecular effects. These include the regulation of signal transduction pathways and direct modulation of several enzymatic activities. Most of these signaling cascades lead to the activation of transcription factors. Curcumin has been found to modulate the activity of several key transcription factors and, in turn, the cellular expression profiles. Curcumin has been shown to elicit vital cellular responses such as cell cycle arrest, apoptosis, and differentiation by activating a cascade of molecular events. In this chapter, we briefly review the effects of curcumin on transcription factors NF-KB, AP-1, Egr-1, STATs, PPAR-gamma, beta-catenin, nrf2, EpRE, p53, CBP, and androgen receptor (AR) and AR-related cofactors giving major emphasis to the molecular mechanisms of its action.

Anethole blocks both early and late cellular responses transduced by tumor necrosis factor : effect on NF-κB, AP-1, JNK, MAPKK and apoptosis

Anethole, a chief constituent of anise, camphor, and fennel, has been shown to block both inflammation and carcinogenesis, but just how these effects are mediated is not known. One possibility is TNF-mediated signaling, which has also been associated with both inflammation and carcinogenesis. In the present report we show that anethole is a potent inhibitor of TNF-induced NF-κB activation (an early response) as monitored by electrophoretic mobility shift assay, IκBα phosphorylation and degradation, and NF-κB reporter gene expression. Suppression of IκBα phosphorylation and NF-κB reporter gene expression induced by TRAF2 and NIK, suggests that anethole acts on IκBα kinase. Anethole also blocked the NF-κB activation induced by a variety of other inflammatory agents. Besides NF-κB, anethole also suppressed TNF-induced activation of the transcription factor AP-1, c-jun N-terminal kinase and MAPK-kinase. In addition, anethole abrogated TNF-induced apoptosis as measured by both caspase activation and cell viability. The anethole analogues eugenol and isoeugenol also blocked TNF signaling. Anethole suppressed TNF-induced both lipid peroxidation and ROI generation. Overall, our results demonstrate that anethole inhibits TNF-induced cellular responses, which may explain its role in suppression of inflammation and carcinogenesis.

Evidence that TNF-TNFR1-TRADD-TRAF2-RIP-TAK1-IKK pathway mediates constitutive NF-κB activation and proliferation in human head and neck squamous cell carcinoma

Constitutively activated nuclear factor-κB (NF-κB) has been associated with a variety of aggressive tumor types, including head and neck squamous cell carcinoma (HNSCC); however, the mechanism of its activation is not fully understood. Therefore, we investigated the molecular pathway that mediates constitutive activation of NF-κB in a series of HNSCC cell lines. We confirmed that NF-κB was constitutively active in all HNSCC cell lines (FaDu, LICR-LON-HN5 and SCC4) examined as indicated by DNA binding, immunocytochemical localization of p65, by NF-κB-dependent reporter gene expression and its inhibition by dominant-negative (DN)-inhibitory subunit of NF-κB (IκBα), the natural inhibitor of NF-κB. Constitutive NF-κB activation in HNSCC was found to be due to constitutive activation of IκBα kinase (IKK); and this correlated with constitutive expression of phosphorylated forms of IκBα and p65 proteins. All HNSCC showed the expression of p50, p52, p100 and receptor-interacting protein; all linked with NF-κB activation. The expression of constitutively active NF-κB in HNSCC is mediated through the tumor necrosis factor (TNF) signaling pathway, as NF-κB reporter activity was inhibited by DN-TNF receptor-associated death domain (TRADD), DN-TNF receptor-associated factor (TRAF)2, DN-receptor-interacting protein (RIP), DN-transforming growth factor-β-activated kinase 1 (TAK1), DN-κ-Ras, DN-AKT and DN-IKK but not by DN-TRAF5 or DN-TRAF6. Constitutive NF-κB activation was also associated with the autocrine expression of TNF, TNF receptors and receptor-activator of NF-κB and its ligand in HNSCC cells but not interleukin (IL)-1β. All HNSCC cell lines expressed IL-6, a NF-κB-regulated gene product. Furthermore, treatment of HNSCC cells with anti-TNF antibody downregulated constitutively active NF-κB, and this was associated with inhibition of IL-6 expression and cell proliferation. Our results clearly demonstrate that constitutive activation of NF-κB is mediated through the TRADD-TRAF2-RIP-TAK1-IKK pathway, making TNF a novel target in the treatment of head and neck cancer.

Berberine Modifies Cysteine 179 of IκBα Kinase, Suppresses Nuclear Factor-κB–Regulated Antiapoptotic Gene Products, and Potentiates Apoptosis

Berberine, an isoquinoline alkaloid derived from a plant used traditionally in Chinese and Ayurvedic medicine, has been reported to exhibit chemopreventive and anti-inflammatory activities through unknown mechanism. Because of the critical role of the transcription factor nuclear factor-kappaB (NF-kappaB) in these processes, we investigated the effect of berberine on this pathway. We found that berberine suppressed NF-kappaB activation induced by various inflammatory agents and carcinogens. This alkaloid also suppressed constitutive NF-kappaB activation found in certain tumor cells. Suppression of NF-kappaB activation occurred through the inhibition of phosphorylation and degradation of IkappaBalpha by the inhibition of IkappaB kinase (IKK) activation, leading to suppression of phosphorylation and nuclear translocation of p65, and finally to inhibition of NF-kappaB reporter activity. Inhibition of IKK by berbeine was direct and could be reversed by reducing agents. Site-specific mutagenesis suggested the involvement of cysteine residue 179 in IKK. Berberine also suppressed the expression of NF-kappaB-regulated gene products involved in antiapoptosis (Bcl-xL, Survivin, IAP1, IAP2, and cFLIP), proliferation (cyclin D1), inflammation (cyclooxygenase-2), and invasion (matrix metalloproteinase-9). Suppression of antiapoptotic gene products correlated with enhancement of apoptosis induced by tumor necrosis factor (TNF)-alpha and chemotherapeutic agents and with inhibition of TNF-induced cellular invasion. Overall, our results indicate that chemopreventive, apoptotic, and anti-inflammatory activities displayed by berberine may be mediated in part through the suppression of the NF-kappaB activation pathway. This may provide the molecular basis for the ability of berberine to act as an anticancer and anti-inflammatory agent.