ROS is produced in non-phagocytic cells as a result of various signaling pathways such as receptor tyrosine kinases (RTKs) which become activated by growth factors – epidermal growth factor, platelet derived growth factor, fibroblast growth factor as well as cytokines (tumor necrosis factor, γ-interferon and interleukins) leading to an intracellular tyrosine phosphorylation cascade [64
]. The ROS activated signal transduction pathways are regulated by two distinct protein families – the Mitogen Activated Protein Kinase (MAPK) and the redox sensitive kinases. The MAPKs transduce signals from the cell membrane to the nucleus in response to a wide range of stimuli. MAPKs are serine/threonine kinases that, upon stimulation, phosphorylate their specific substrates at serine and/or threonine residues. Such phosphorylation events can either positively or negatively regulate substrate, and thus entire signaling cascade activity. Thus, the MAPK signaling pathways modulate gene expression, mitosis, proliferation, motility, metabolism, and programmed cell death. Conventional MAPKs consist of three family members: the extracellular signal-regulated kinase (ERK, subdivided into ERK1 and 2); the c-Jun NH2-terminal kinase (JNK, subdivided into JNK1, 2 and 3); and the p38 MAPK (subdivided into α, β, γ, and δ p38-MAPK [92
MAPKs regulate processes important in carcinogenesis including proliferation, differentiation, and apoptosis. MAPK modulate gene expression through phosphorylation of a wide array of transcription factors. Of the three subfamilies, the ERK pathway has most commonly been associated with the regulation of cell proliferation. Activation of the ERK, JNK, and p38 subfamilies has been observed in response to changes in the cellular redox balance. The balance between ERK and JNK activation is a key determinant for cell survival as both a decrease in ERK and an increase in JNK is required for the induction of apoptosis. Activation of MAPKs directly leads to increased AP-1 activity resulting in increased cell proliferation. One of the genes regulated by AP-1 is cyclin D1. AP-1 binding sites have been identified in the cyclin D1 promoter and AP-1 activates this promoter, resulting in activation of cyclin-dependent kinase (cdks), which promotes entry into the cell division cycle. c-Jun also stimulates the progression into the cell cycle both by induction of cyclin D1 and suppression of p21waf, a protein that inhibits cell cycle progression. JunB, considered a negative regulator of c-jun-induced cell proliferation, represses c-jun-induced cyclin D1 activation by the transcription of p16INK4a, a protein that inhibits the G1 to S phase transition.
NF-κB activation has been linked to the carcinogenesis process because of its roles in inflammation, differentiation and cell growth. NF-κB regulates several genes involved in cell transformation, proliferation, and angiogenesis. Carcinogens and tumor promoters including UV radiation, phorbol esters, asbestos, alcohol, and benzo(a)pyrene are among the external stimuli that activate NF-κB. The expression of several genes regulated by NF-κB (bcl-2, bcl-xL
, TRAF1, TRAF2, SOD, and A20) promotes cell survival at least in part through inhibition of apoptotic pathways. Expression of NF-κB has been shown to promote cell proliferation, whereas inhibition of NF-κB activation blocks cell proliferation. Additionally, tumor cells from blood neoplasms, and colon, breast, pancreas, and squamous cell carcinoma cell lines have all been reported to constitutively express activated NF-κB [93
The second family consists of signaling factors that use cysteine motifs as redox-sensitive sulphydryl switches to modulate specific signal transduction cascades regulating downstream proteins. The redox-sensitive signaling cascade involves the cytoplasmic factors (thioredoxins), nuclear signaling factors such as Ref-1 (Redox factor-1) and transcription factors (AP-1, NF-κB, Nfr-1, Egr-1). The cytoplasmic sulphydryl containing proteins such as thioredoxins are critical upstream signaling proteins that regulate multiple intracellular processes such as DNA synthesis, cell growth, etc. The signaling cascades elicited by ROS culminates in the activation of c-Jun and c-Fos subunits of the active nuclear transcription factor, AP-1 (activator protein-1), that activate genes involved in cellular proliferation. Redox-sensitive signaling factors regulate multiple processes including proliferation, cell cycle and anti-apoptotic signaling pathways. Inhibition of thioredoxins inhibits several pro-survival transcription factors such as Egr-1, AP-1 and NF-κB resulting in a G1 phase arrest [94
] (see figure ).
The various signaling cascades of ROS involved in the regulation and activation of redox sensitive genes.
The role of reactive oxygen species in cell growth regulation is complex, being cell specific and dependent upon the form of the oxidant as well as the concentration of the particular reactive oxygen species. The modification of gene expression by reactive oxygen species has direct effects on cell proliferation and apoptosis through the activation of transcription factors including MAPK, AP-1, and NF-κB pathways. Oxidant-mediated AP-1 activation results in enhanced expression of cyclin D1 and cdks, which in turn promotes entry into mitosis and cell division. Likewise, reactive oxygen species function as second messengers involved in activation of NF-κB by tumor necrosis factor and cytokines. DNA damage, mutation, and altered gene expression are all required participants in the process of carcinogenesis. Although these events may be derived by different mechanisms, a common theme is the involvement of reactive oxygen species and oxidative stress in neoplastic transformation.