UPP regulates the stability of multiple proteins, affecting the expression and function of regulatory proteins (e.g., JNK), tumor suppressors (e.g., p53), transcription factors (e.g., NFkB), and proto-oncogenes (e.g., c-Jun). Inhibition of proteasome action can lead to apoptosis by inhibiting NFkB activity, increasing p53 and Bax protein expressions, and accumulating cyclin-dependent kinase inhibitors p27 and p21.29
Inhibition of NFkB activity by proteasome inhibitors takes place by blocking the degradation of IkB.30 This inhibition leads to decreased levels in the proapoptotic proteins Bcl-2 and A1/Bfl-1, triggering cytochrome c release, caspase-9 activation, and apoptosis.31
Among UPP inhibitors, bortezomib (Velcade) was the first to enter clinical studies. On the basis of data resulting from clinical trials, the US Food and Drug Administration (FDA) granted accelerated approval for this drug for the treatment of patients with multiple myeloma on May 13, 2003. The promising preclinical and clinical activity exhibited by bortezomib on multiple myeloma and other malignancies has confirmed the proteasome as a relevant and important target in the treatment of cancers (for a review, see Rajkumar et al.32). NFkB inhibition is probably one of the main mechanisms by which bortezomib induces apoptosis and overcomes drug resistance in multiple myeloma.30 In addition, JNK activation seems to be an important pathway for bortezomib-induced multiple myeloma cell apoptosis. Bortezomib activates JNK, leading to Fas upregulation and caspase-8 and caspase-3 activation. This caspase-8-mediated apoptotic pathway is independent of the caspase-9-mediated pathway. On the other hand, induction of caspase-3 leads to mdm2 degradation and p53 phosphorylation, thereby increasing p53 activity and apoptosis. Bortezomib also induces FasL expression, probably because of increased c-myc expression that occurs as a result of proteasome inhibition.31
Furthermore, the inhibition of growth in multiple myeloma cell lines and primary multiple myeloma cells is a consequence of the inhibiting effect of bortezomib on IL6-induced Ras/Raf/MAP kinase pathway activation.30 Bortezomib has no effect on IL6-induced signaling through the JAK/STAT3 pathway. However, the specific effects of proteasome inhibition in malignancy and the precise mechanism of action of bortezomib require further investigation to be fully understood in the future.
NFkB transcription factor is a dimeric complex of subunits belonging to the Rel family (p105/50, p100/52, p65 (Rel A), Rel B, and c-Rel). NFkB proteins share the Rel homology domain (RHD), allowing DNA binding, dimerization, and nuclear localization. NFkB proteins are sequestered in the cytoplasm by a family of IkB proteins through interactions between inhibitor ankyrin repeats and RHD. Upon stimulation by various activators such as cytokines, LPS, growth factors, stress inducers, or chemotherapeutic agents, IkB is phosphorylated on two serine residues, which triggers its ubiquitinylation and degradation by the 26S proteasome (see Section 18.104.22.168.1). NFkB is then free to enter the nucleus and to activate the transcription of target genes by binding to its cognate decameric DNA sequence 5'-GGGRNNYYCC-3', where R indicates A or G, Y indicates C or T, and N can be any base. NFkB is involved in the transcription of many proinflammatory as well as antiapoptotic genes, and is thus a key player in the progression of carcinogenesis and inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, and asthma.33 Therapies aiming to suppress NFkB-induced survival genes are thus an interesting approach to fight and cure these diseases.
As previously stated, activation of NFkB through IkB phosphorylation and degradation depends on the activation of IkB kinases (IKKs) (Figure 4). The IKK complex is composed of three subunits, the catalytic subunits IKKa and IKKb and the regulatory subunit IKKg (or NEMO, NFkB essential modulator).21 The different components of this complex were identified and characterized34-38 and knockout experiments have determined their role in NFkB activation. From these studies, it appears that IKKg is required for IKK activity and classical NFkB activation through IkB phosphorylation and degradation pathway,39,40 but not for the alternative pathway leading to p52/RelB dimer translocation.41 IKKb gene disruption in mice leads to death between days 12 and 15 post gestation.
IKKb and IKKg are required for the canonical pathway of IkB phosphorylation and degradation in response to proinflammatory stimuli, while IKKa is dispensable for IkB phosphorylation, but is involved and necessary for inducible p100 processing. RelB-p100 forms an inactive dimer and the IKKa-dependent degradation of the C-terminal part of p100 allows nuclear translocation of the so-formed RelB-p52 complex. Activation of this dimer is important for lymphoid organ development and adaptive immune response but not for other NFkB-dependent functions such as apoptosis inhibition and innate immunity.42,43
Apoptotic cell death is characterized by morphological changes and involves the activation of a family of cysteine aspartate proteases, called caspases. These proteases exist in unstimulated cells as inactive zymogens and are activated by proteolytic cleavage to form a heterotetramer consisting of two large and two small subunits. Two major pathways of
Cytokine (TNF-a )
Cytokine (TNF-a )
caspase activation are well characterized to date: (1) the death receptors (DRs) or extrinsic pathway; and (2) the mitochondrial or intrinsic pathway.44
Apoptotic signaling through the extrinsic pathway is triggered by the engagement of members of the TNF-receptor family (TNFR1, Fas/CD95, DR3, DR4, DR5, and DR6) by their ligands (TNF-a, lymphotoxin a, FasL, Apo3L, and TNF-related apoptosis-inducing ligand (TRAIL)), resulting in receptor trimerization and activation of the procaspase-8 and -10. DNA damage or cell stress leading to activation of p53 or Bcl-2 family proapoptotic proteins such as Bax and Bak initiate the intrinsic pathway and induce the mitochondrial release of apoptogenic molecules such as cytochrome c, apoptosis-inducing factor (AIF) or Smac/DIABLO (second mitochondria-derivated activator of caspases/direct inhibitors of apoptosis (IAP)-binding protein with low pI). Cytochrome c binds to the apoptotic protease-activating factor (Apaf)-1 and this dimer forms the apoptosome complex with procaspase-9.45 Extrinsic and intrinsic pathways are interconnected: caspase-8 proteolysis through the extrinsic pathway leads to cleavage and activation of Bid, a proapoptotic member of the Bcl-2 family. Truncated Bid incorporates into the mitochondrial membrane and induces cytochrome c release, which in turn leads to apoptosome formation and thus intrinsic apoptotic pathway.46
Transcription factors of the NFkB family play a role in the regulation of the apoptotic program in different cell lines.47 The first evidence of the NFkB cytoprotective role came from the analysis of p65-/- mice that died on embryonic day 15 from massive liver apoptosis, which could be reestablished by p65 reexpression.48'49 NFkB transcriptional activity leads to the expression of many antiapoptotic genes. Among those gene products, some are directly implicated in inhibiting the apoptotic cascade, such as IAPs, c-FLIP (caspase-8/FADD-like-IL1^-converting-enzyme (caspase-8/FLICE) inhibitory protein), or Bcl-2 family proteins. IAPs are a family of antiapoptotic proteins highly conserved throughout evolution that have the ability to inhibit the activation of various caspases by direct binding and interaction through their baculoviral-inhibitory repeat (BIR).50 NFkB inhibits TNF-a-induced apoptosis through transcriptional activation of IAP1, IAP2, TNF-receptor-associated factor (TRAF) 1 and TRAF2, thus inhibiting caspase-8 activity.51 c-FLIP is a proteolytically inactive analog of caspase-8 and competes with procaspases-8 and -10 for binding to FADD, thus preventing their activation and subsequent apoptosis.52'53 Bcl-2 family proteins such as Bfl-1/A1, Bcl-XL, or Bcl-2 prevent apoptosis by inhibiting cytochrome c leakage from the mitochondria, thus avoiding apoptosome formation and apoptotic cascade. NFkB activation also leads to repression of several proapoptotic genes. For example, two genes coding for the transcription factors Forkhead and growth arrest and DNA-damage-inducible gene (GADD) 153/C/EBP-homologous protein (CHOP),54'55 as well as the proapoptotic gene bax,56'57 are repressed by the activity of NFkB. NFkB activation also interferes with p53 proapoptotic function, since both transcription factors can inhibit each other's transcriptional activity by competing for a limiting pool of CBP/p300 complexes.57
NFkB may have a proapoptotic activity in some cell types under certain conditions. It regulates the expression of genes whose products can induce apoptosis, such as TNF-receptor superfamily members DR4, DR5, DR6, Fas, and the FasL ligand.58-61 Moreover, NFkB activity can sometimes be correlated to apoptosis. NFkB is activated during serum starvation-induced human embryonic kidney cell apoptosis, and its inhibition partially protects cells from this death.62
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