Kwang Seok Ahn

Targeting Signal-Transducer-and-Activator-of-Transcription-3 for Prevention and Therapy of Cancer

Abstract:  Recent evidence indicates a convergence of molecular targets for both prevention and therapy of cancer. Signal‐transducer‐and‐activator‐of‐transcription‐3 (STAT3), a member of a family of six different transcription factors, is closely linked with tumorigenesis. Its role in cancer is indicated by numerous avenues of evidence, including the following: STAT3 is constitutively active in tumor cells; STAT3 is activated by growth factors (e.g., EGF, TGF‐α, IL‐6, hepatocyte growth factor) and oncogenic kinases (e.g., Src); STAT3 regulates the expression of genes that mediate proliferation (e.g., c‐myc and cyclin D1), suppress apoptosis (e.g., Bcl‐xL and survivin), or promote angiogenesis (e.g, VEGF); STAT3 activation has been linked with chemoresistance and radioresistance; and chemopreventive agents have been shown to suppress STAT3 activation. Thus inhibitors of STAT3 activation have potential for both prevention and therapy of cancer. Besides small peptides and oligonucleotides, numerous small molecules have been identified as blockers of STAT3 activation, including synthetic molecules (e.g., AG 490, decoy peptides, and oligonucleotides) and plant polyphenols (e.g., curcumin, resveratrol, flavopiridol, indirubin, magnolol, piceatannol, parthenolide, EGCG, and cucurbitacin). This article discusses these aspects of STAT3 in more detail.

Transcription Factor NF-κB: A Sensor for Smoke and Stress Signals

Nuclear factor‐kappa B (NF‐κB) is a transcription factor that resides in the cytoplasm of every cell and translocates to the nucleus when activated. Its activation is induced by a wide variety of agents including stress, cigarette smoke, viruses, bacteria, inflammatory stimuli, cytokines, free radicals, carcinogens, tumor promoters, and endotoxins. On activation, NF‐κB regulates the expression of almost 400 different genes, which include enzymes (e.g., COX‐2, 5‐LOX, and iNOS), cytokines (such as TNF, IL‐1, IL‐6, IL‐8, and chemokines), adhesion molecules, cell cycle regulatory molecules, viral proteins, and angiogenic factors. The constitutive activation of NF‐κB has been linked with a wide variety of human diseases, including asthma, atherosclerosis, AIDS, rheumatoid arthritis, diabetes, osteoporosis, Alzheimers disease, and cancer. Several agents are known to suppress NF‐κB activation, including Th2 cytokines (IL‐4, IL‐13, and IL‐10), interferons, endocrine hormones (LH, HCG, MSH, and GH), phytochemicals, corticosteroids, and immunosuppressive agents. Because of the strong link of NF‐κB with different stress signals, it has been called a “smoke‐sensor” of the body.

Plumbagin (5-Hydroxy-2-methyl-1,4-naphthoquinone) Suppresses NF-κB Activation and NF-κB-regulated Gene Products Through Modulation of p65 and IκBα Kinase Activation, Leading to Potentiation of Apoptosis Induced by Cytokine and Chemotherapeutic Agents

Plumbagin, derived from the medicinal plant Plumbago zeylanica, modulates cellular proliferation, carcinogenesis, and radioresistance, all known to be regulated by the activation of the transcription factor NF-κB, suggesting plumbagin might affect the NF-κB activation pathway. We found that plumbagin inhibited NF-κB activation induced by TNF, and other carcinogens and inflammatory stimuli (e.g. phorbol 12-myristate 13-acetate, H2O2, cigarette smoke condensate, interleukin-1β, lipopolysaccharide, and okadaic acid). Plumbagin also suppressed the constitutive NF-κB activation in certain tumor cells. The suppression of NF-κB activation correlated with sequential inhibition of the tumor necrosis factor (TNF)-induced activation of IκBα kinase, IκBα phosphorylation, IκBα degradation, p65 phosphorylation, p65 nuclear translocation, and the NF-κB-dependent reporter gene expression activated by TNF, TNFR1, TRAF2, NIK, IKK-β, and the p65 subunit of NF-κB. Plumbagin also suppressed the direct binding of nuclear p65 and recombinant p65 to the DNA, and this binding was reversed by dithiothreitol both in vitro and in vivo. However, plumbagin did not inhibit p65 binding to DNA when cells were transfected with the p65 plasmid containing cysteine 38 mutated to serine. Plumbagin down-regulated the expression of NF-κB-regulated anti-apoptotic (IAP1, IAP2, Bcl-2, Bcl-xL, cFLIP, Bfl-1/A1, and survivin), proliferative (cyclin D1 and COX-2), and angiogenic (matrix metalloproteinase-9 and vascular endothelial growth factor) gene products. This led to potentiation of apoptosis induced by TNF and paclitaxel and inhibited cell invasion. Overall, our results indicate that plumbagin is a potent inhibitor of the NF-κB activation pathway that leads to suppression of NF-κB-regulated gene products. This may explain its cell growth modulatory, anticarcinogenic, and radiosensitizing effects previously described.

γ-Tocotrienol Inhibits Nuclear Factor-κB Signaling Pathway through Inhibition of Receptor-interacting Protein and TAK1 Leading to Suppression of Antiapoptotic Gene Products and Potentiation of Apoptosis

Unlike the tocopherols, the tocotrienols, also members of the vitamin E family, have an unsaturated isoprenoid side chain. In contrast to extensive studies on tocopherol, very little is known about tocotrienol. Because the nuclear factor-κB (NF-κB) pathway has a central role in tumorigenesis, we investigated the effect of γ-tocotrienol on the NF-κB pathway. Althoughγ-tocotrienol completely abolished tumor necrosis factor α (TNF)-induced NF-κB activation, a similar dose of γ-tocopherol had no effect. Besides TNF, γ-tocotrienol also abolished NF-κB activation induced by phorbol myristate acetate, okadaic acid, lipopolysaccharide, cigarette smoke, interleukin-1β, and epidermal growth factor. Constitutive NF-κB activation expressed by certain tumor cells was also abrogated by γ-tocotrienol. Reducing agent had no effect on the γ-tocotrienol-induced down-regulation of NF-κB. Mevalonate reversed the NF-κB inhibitory effect of γ-tocotrienol, indicating the role of hydroxymethylglutaryl-CoA reductase. γ-Tocotrienol blocked TNF-induced phosphorylation and degradation of IκBα through the inhibition of IκBα kinase activation, thus leading to the suppression of the phosphorylation and nuclear translocation of p65. γ-Tocotrienol also suppressed NF-κB-dependent reporter gene transcription induced by TNF, TNFR1, TRADD, TRAF2, TAK1, receptor-interacting protein, NIK, and IκBα kinase but not that activated by p65. Additionally, the expressions of NF-κB-regulated gene products associated with antiapoptosis (IAP1, IAP2, Bcl-xL, Bcl-2, cFLIP, XIAP, Bfl-1/A1, TRAF1, and Survivin), proliferation (cyclin D1, COX2, and c-Myc), invasion (MMP-9 and ICAM-1), and angiogenesis (vascular endothelial growth factor) were down-regulated byγ-tocotrienol. This correlated with potentiation of apoptosis induced by TNF, paclitaxel, and doxorubicin. Overall, our results demonstrate that γ-tocotrienol inhibited the NF-κB activation pathway, leading to down-regulation of various gene products and potentiation of apoptosis.

Targeting Nuclear Factor-κB Activation Pathway by Thymoquinone: Role in Suppression of Antiapoptotic Gene Products and Enhancement of Apoptosis

Thymoquinone (TQ), derived from the medicinal plant Nigella sativa, exhibits antiinflammatory and anticancer activities through mechanism(s) that is not fully understood. Because numerous effects modulated by TQ can be linked to interference with the nuclear factor-κB (NF-κB) signaling, we investigated in detail the effect of this quinone on NF-κB pathway. As examined by DNA binding, we found that TQ suppressed tumor necrosis factor–induced NF-κB activation in a dose- and time-dependent manner and inhibited NF-κB activation induced by various carcinogens and inflammatory stimuli. The suppression of NF-κB activation correlated with sequential inhibition of the activation of IκBα kinase, IκBα phosphorylation, IκBα degradation, p65 phosphorylation, p65 nuclear translocation, and the NF-κB–dependent reporter gene expression. TQ specifically suppressed the direct binding of nuclear p65 and recombinant p65 to the DNA, and this binding was reversed by DTT. However, TQ did not inhibit p65 binding to DNA when cells were transfected with the p65 plasmid containing cysteine residue 38 mutated to serine. TQ also down-regulated the expression of NF-κB–regulated antiapoptotic (IAP1, IAP2, XIAP Bcl-2, Bcl-xL, and survivin), proliferative (cyclin D1, cyclooxygenase-2, and c-Myc), and angiogenic (matrix metalloproteinase-9 and vascular endothelial growth factor) gene products. This led to potentiation of apoptosis induced by tumor necrosis factor and chemotherapeutic agents. Overall, our results indicate that the anticancer and antiinflammatory activities previously assigned to TQ may be mediated in part through the suppression of the NF-κB activation pathway, as shown here, and thus may have potential in treatment of myeloid leukemia and other cancers. (Mol Cancer Res 2008;6(6):1059–70)

Ursolic Acid Inhibits STAT3 Activation Pathway Leading to Suppression of Proliferation and Chemosensitization of Human Multiple Myeloma Cells

The activation of signal transducers and activators of transcription 3 (STAT3) has been linked with the proliferation of a variety of human cancer cells, including multiple myeloma. Agents that can suppress STAT3 activation have potential for prevention and treatment of cancer. In the present report, we tested an agent, ursolic acid, found in basil, apples, prunes, and cranberries, for its ability to suppress STAT3 activation. We found that ursolic acid, a pentacyclic triterpenoid, inhibited both constitutive and interleukin-6–inducible STAT3 activation in a dose- and time-dependent manner in multiple myeloma cells. The suppression was mediated through the inhibition of activation of upstream kinases c-Src, Janus-activated kinase 1, Janus-activated kinase 2, and extracellular signal–regulated kinase 1/2. Vanadate treatment reversed the ursolic acid–induced down-regulation of STAT3, suggesting the involvement of a tyrosine phosphatase. Indeed, we found that ursolic acid induced the expression of tyrosine phosphatase SHP-1 protein and mRNA. Moreover, knockdown of SHP-1 by small interfering RNA suppressed the induction of SHP-1 and reversed the inhibition of STAT3 activation, thereby indicating the critical role of SHP-1 in the action of this triterpene. Ursolic acid down-regulated the expression of STAT3-regulated gene products such as cyclin D1, Bcl-2, Bcl-xL, survivin, Mcl-1, and vascular endothelial growth factor. Finally, ursolic acid inhibited proliferation and induced apoptosis and the accumulation of cells in G1-G0 phase of cell cycle. This triterpenoid also significantly potentiated the apoptotic effects of thalidomide and bortezomib in multiple myeloma cells. Overall, these results suggest that ursolic acid is a novel blocker of STAT3 activation that may have a potential in prevention and treatment of multiple myeloma and other cancers. (Mol Cancer Res 2007;5(9):943–55)

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.

Potential role of signal transducer and activator of transcription (STAT)3 signaling pathway in inflammation, survival, proliferation and invasion of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies, and is also the fourth most common cancer worldwide with around 700,000 new cases each year. Currently, first line chemotherapeutic drugs used for HCC include fluorouracil, cisplatin, doxorubicin, paclitaxel and mitomycin, but most of these are non-selective cytotoxic molecules with significant side effects. Sorafenib is the only approved targeted therapy by the U.S. Food and Drug Administration for HCC treatment, but patients suffer from various kinds of adverse effects, including hypertension. The signal-transducer-and-activator-of-transcription 3 (STAT3) protein, one of the members of STATs transcription factor family, has been implicated in signal transduction by different cytokines, growth factors and oncogenes. In normal cells, STAT3 activation is tightly controlled to prevent dysregulated gene transcription, whereas constitutively activated STAT3 plays an important role in tumorigenesis through the upregulation of genes involved in anti-apoptosis, proliferation and angiogenesis. Thus, pharmacologically safe and effective agents that can block STAT3 activation have the potential both for the prevention and treatment of HCC. In the present review, we discuss the possible role of STAT3 signaling cascade and its interacting partners in the initiation of HCC and also analyze the role of various STAT3 regulated genes in HCC progression, inflammation, survival, invasion and angiogenesis.

β-Caryophyllene oxide inhibits growth and induces apoptosis through the suppression of PI3K/AKT/mTOR/S6K1 pathways and ROS-mediated MAPKs activation.

Both PI3K/AKT/mTOR/S6K1 and mitogen activated protein kinase (MAPK) signaling cascades play an important role in cell proliferation, survival, angiogenesis, and metastasis of tumor cells. In the present report, we investigated the effects of β-caryophyllene oxide (CPO), a sesquiterpene isolated from essential oils of medicinal plants such as guava (Psidium guajava), oregano (Origanum vulgare L.), cinnamon (Cinnamomum spp.) clove (Eugenia caryophyllata), and black pepper (Piper nigrum L.) on the PI3K/AKT/mTOR/S6K1 and MAPK activation pathways in human prostate and breast cancer cells. We found that CPO not only inhibited the constitutive activation of PI3K/AKT/mTOR/S6K1 signaling cascade; but also caused the activation of ERK, JNK, and p38 MAPK in tumor cells. CPO induced increased reactive oxygen species (ROS) generation from mitochondria, which is associated with the induction of apoptosis as characterized by positive Annexin V binding and TUNEL staining, loss of mitochondrial membrane potential, release of cytochrome c, activation of caspase-3, and cleavage of PARP. Inhibition of ROS generation by N-acetylcysteine (NAC) significantly prevented CPO-induced apoptosis. Subsequently, CPO also down-regulated the expression of various downstream gene products that mediate cell proliferation (cyclin D1), survival (bcl-2, bcl-xL, survivin, IAP-1, and IAP-2), metastasis (COX-2), angiogenesis (VEGF), and increased the expression of p53 and p21. Interestingly, we also observed that CPO can significantly potentiate the apoptotic effects of various pharmacological PI3K/AKT inhibitors when employed in combination in tumor cells. Overall, these findings suggest that CPO can interfere with multiple signaling cascades involved in tumorigenesis and used as a potential therapeutic candidate for both the prevention and treatment of cancer.

Embelin, an inhibitor of X chromosome-linked inhibitor-of-apoptosis protein, blocks nuclear factor-κB (NF-κB) signaling pathway leading to suppression of NF-κB-regulated antiapoptotic and metastatic gene products

Identifying the active chemical ingredients of ancient medicines and the molecular targets of those ingredients is an attractive therapeutic objective. Embelin, identified primarily from the Embelia ribes plant, is one such compound shown to exhibit chemopreventive, anti-inflammatory, and apoptotic activities through an unknown mechanism. Because nuclear factor-κB (NF-κB) regulates several genes associated with inflammation, proliferation, carcinogenesis, and apoptosis, we postulated that embelin might mediate its activity through modulation of NF-κB activation. We found that embelin inhibited tumor necrosis factor (TNF) α-induced NF-κB activation. Both inducible and constitutive NF-κB activation were abrogated by embelin. In addition, NF-κB activated by diverse stimuli such as interleukin-1β, lipopolysaccharide, phorbol myristate acetate, okadaic acid, hydrogen peroxide, and cigarette smoke condensate also was suppressed. We found that embelin inhibited sequentially the TNFα-induced activation of the inhibitory subunit of NF-κBα (IκBα) kinase, IκBα phosphorylation, IκBα degradation, and p65 phosphorylation and nuclear translocation. Embelin also suppressed NF-κB-dependent reporter gene transcription induced by TNFα, TNF receptor-1 (TNFR1), TNFR1-associated death domain protein, TNFR-associated factor-2, NF-κB-inducing kinase, and IκBα kinase but not by p65. Furthermore, we found that embelin down-regulated gene products involved in cell survival, proliferation, invasion, and metastasis of the tumor. This down-regulation was associated with enhanced apoptosis by cytokine and chemotherapeutic agents. Together, our results indicate that embelin is a novel NF-κB blocker and potential suppressor of tumorigenesis.