Tumour Necrosis Factor alpha (TNF alpha), is an inflammatory cytokine produced by macrophages/monocytes during acute inflammation and is responsible for a diverse range of signalling events within cells, leading to necrosis or apoptosis. The protein is also important for resistance to infection and cancers. Tumor necrosis factor (TNF) is a multifunctional cytokine that plays important roles in diverse cellular events such as cell survival, proliferation, differentiation, and death. As a pro-inflammatory cytokine, TNF is secreted by inflammatory cells, which may be involved in inflammation-associated carcinogenesis. TNF exerts its biological functions through activating distinct signaling pathways such as nuclear factor κB (NF-κB) and c-Jun N-terminal kinase (JNK). NF-κB is a major cell survival signal that is anti-apoptotic while sustained JNK activation contributes to cell death. The crosstalk between the NF-κB and JNK is involved in determining cellular outcomes in response to TNF. In regard to cancer, TNF is a double-dealer. On one hand, TNF could be an endogenous tumor promoter, because TNF stimulates cancer cells’ growth, proliferation, invasion and metastasis, and tumor angiogenesis. On the other hand, TNF could be a cancer killer. The property of TNF in inducing cancer cell death renders it a potential cancer therapeutic, although much work is needed to reduce its toxicity for systematic TNF administration. Recent studies have focused on sensitizing cancer cells to TNF-induced apoptosis through inhibiting survival signals such as NF-κB, by combined therapy.
There are two receptors for TNF, namely TNF receptor 1 (TNFR-1, p55 receptor) and TNFR-2 (p75 receptor). TNFR-1 is ubiquitously expressed while TNFR-2 is mainly expressed in immune cells . Although both the receptors bind TNF, the main receptor mediating TNF’s cellular effects in most cell types is TNFR-1. TNFR-1 is a death domain (DD)-containing receptor with an extracellular domain (ECD), a transmembrane domain (TMD), and an intracellular domain (ICD). TNFR-1 is an important member of the death receptor family that shares the capability of inducing apoptotic cell death. TNFR-2 does not possess a DD, although it can mediate a cell death signal, which may be indirect through TNFR-1 .
The pathways mediated by TNFR-1 have been extensively studied. Upon binding by TNF, which is a natural homotrimer, TNFR-1 forms a homotrimer to recruit TNFR-associated death domain (TRADD) through the homologous binding of the DDs of both proteins. TRADD serves as a platform to recruit downstream adaptor proteins to generate signals for distinct signaling pathways. These adaptor proteins include receptor interacting protein (RIP), TNFR-associated factor 2 (TRAF-2), and Fas-associated death domain (FADD) that further recruit key molecules that are responsible for intracellular signaling to activate NF-κB, mitogen-activated protein kinases (MAPKs), and cell death, respectively (Fig. 1).
TNF-induced NF-κB activation is initiated by activation of inhibitor of κB (IκB) kinase (IKK). During TNFR-1 signaling IKK is recruited to the TNFR-1 signaling complex (Complex I), which consists of TRADD, TRAF2, and RIP. IKK is activated by a RIP-dependent mechanism that involves MEKK3, TAK1, and TAB2 [5, 6]. The activated IKK phosphorylates IκB, which retains NF-κB in the cytoplasm, to trigger its rapid polyubiquitination followed by degradation in the 26S proteasome. This process causes the NF-κB nuclear localization signal to be exposed, allowing its nuclear translocation to promote transcription of its target genes. Among NF-κB’s target genes, A20, cIAP-1, cIAP-2, Bcl-xL, XIAP, and IEX-1L are found to have anti-apoptotic properties . Induction of the antioxidant manganese superoxide dismutase (MnSOD) by NF-κB is also implicated in anti-apoptotic and necrotic cell death . The transcriptional activity of NF-κB is further regulated by phosphorylation and acetylation, which modulate the DNA binding by NF-κB and interaction with transcriptional co-activators and/or co-repressors .
TNF is able to activate MAPKs (extracellular signal-regulated protein kinases [ERK], JNK and p38). In most cell types, JNK is the main MAPK induced by TNF [4, 9]. TNFR-1-mediated JNK activation is transduced by TRAF2 and RIP through sequential phosphorylation of the MAP kinase module, MAP3K-MAP2-MAPK[9, 10]. MAP3Ks for JNK include members of the MAP/ERK kinase kinase (MEKK) family such as apoptosis signal-regulating kinase 1 (ASK1), mixed lineage kinase (MLK), and transforming growth factor activated kinase 1 (TAK1). Genetic disruption of MEKK1 in mice abrogates TNF-activated JNK. There are two MAP2Ks (JNKK1/MKK4/SEK1 and JNKK2/MKK7) that activate JNK. The two JNK kinases (JNKKs) phosphorylate JNK at Thr183 and Tyr185, leading to its activation . The role of JNK activation in cell death regulation is controversial, but recent studies suggested that sustained JNK activation, which is suppressed by NF-κB, is pro-apoptotic [12, 13]. As is similar to JNK, the activation of ERK and p38 by TNF involves TRAF2 and RIP .
As a death receptor TNFR-1 signals cells to die. It is well established that TNF induces apoptosis in a variety of cell types. This pathway is initiated by TNFR1 internally signaling Complex I to form Complex II that consists of TRADD, RIP, FADD, and caspase-8. Caspase-8 is auto-activated to trigger activation of the executor capsases-3, and -7, and the endonucleases, resulting in destruction of cell component proteins, fragmentation of DNA, and, eventually, apoptotic cell death. This death receptor-mediated apoptosis pathway is also called the extrinsic apoptosis pathway. TNF-induced apoptosis also uses the mitochondria-mediated (intrinsic) apoptosis pathway. This is achieved by caspase-8 activating BCL-2 interacting domain (Bid), a BH3-only Bcl2 family member. Cleavage of Bid by caspase-8 generates tBid, which migrates to the mitochondria and causes lose of mitochondrial membrane potential, and release of cytochrome c and second mitochondria-derived activator of caspase (Smac)/ direct IAP binding protein with low pI (DIABLO) from mitochondria to the cytosol. Cytochrome c binds to apoptotic protease activating factor 1(Apaf-1) and pro-caspase-9 to form apoptosome, resulting in caspase-9-mediated activation of the executor caspases . Smac binds to and inhibits the inhibitor of apoptosis proteins (IAP, including c-IAP1, c-IAP2, X-linked Inhibitor of Apoptosis Protein [XIAP], and survivin), releasing the brake to accelerate apoptosis. In some cancer cells this apoptosis pathway is suppressed partly through the suppression of caspase-8 by cellular FLICE inhibitory protein (c-FLIP), a caspase-8 homolog that competes with caspase 8-for binding to FADD, and suppression of the mitochondrial pathway by the anti-apoptotic Bcl2 family members .
Despite the well-known Complex II-mediated apoptosis pathway, an additional TNF-induced apoptosis pathway has recently been discovered. Although the exact mechanism has not yet been determined, it is clearly distinct from the well-known pathway that is mediated by FADD, RIP dispensable, and can be suppressed by c-FLIP. The new pathway is mediated by RIP and FADD, independent on TRADD, and is suppressed by c-IAP[16, 17] . Both the pathways use caspase-8 as the initiator caspase to activate the apoptotic execution enzymes. Interestingly, in some cancer cells this new apoptosis pathway is partially or completely suppressed .
In addition to apoptosis, TNF can also induce necrotic cell death. Reactive oxygen species (ROS) play a critical role in mediating necrotic cell death because ROS scavenger BHA can effectively block this pathway . This TNF-induced necrosis requires RIP kinase activity[19, 20]. Furthermore, this pathway involves RIP-dependent activation of the NADPH oxidase Nox1 . However, because this pathway is damaged in some cancer cells, these cells are capable of evading all of the TNF-induced cell death pathways, resulting in their malignant proliferation. Understanding the mechanism behind this capability would improve TNF’s anticancer value.
Aforementioned crosstalk among the TNF-induced pathways plays a key role in TNF’s biological effects on cancer. For example, NF-κB suppresses the apoptotic JNK activation and mediates expression of anti-apoptotic and antioxidant genes, blocking cell death and facilitating cancer cell proliferation [4, 8, 12]. Activation of caspase-8 causes cleavage of the key NF-κB mediator RIP to shut off the cell survival signal while it concurrently promotes the apoptosis pathway . The mitochondria-released Smac suppresses IAPs, releasing the apoptosis brake. Therefore, the balance of TNF-induced survival- and death-signaling is pivotal in determining the fate of TNF-responding cells. Modulating this balance could help to prevent cancer development and facilitate using TNF for cancer therapy.
Natural compounds as a source of TNF-a Inhibitors
Plants produce a huge diversity of compounds belonging to different classes from which drugs can be developed. Many compounds are known to reduce TNF-a levels or disrupt the various pro-inﬂammatory mediators that are actively involved in TNF-a expression (Kuhnau 1976). These compounds may providean alternative for treatment of inﬂammatory diseases.Here we will review these compounds according totheir biosynthetic-chemical classiﬁcation. For reasonsof comparison in Tables 1,2and 3we have summarized some activity data of well known anti-inﬂam-matory medicines that effect TNF-a.
Flavonoids are among the most wide-spread secondary metabolites in the plant kingdom.The capacity of ﬂavonoids to act as anti-inﬂammatory agents has long been utilized in Chinese medicine and in the cosmetic industry in the form of crude plant extracts (Ratty and Das 1988). The Western diet is richin ﬂavonoids; the daily intake is about 1 g of various dietary ﬂavonoids including luteolin, apigenin, kaempferol, quercetin, myricetin ,naringenin , catechin, phloretin, butein ,pelargonidin and cyanidin were potent inhibitors of TNF-a with IC50 values ranging from 3 to37 lM.