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  • As GSH cellular redox status

    2022-07-01

    As GSH cellular redox status is critical for various biological phenomena including apoptosis and inflammation [34], [35] we hypothesized that inflammation could modulate GSTP1-1 gene expression. Indeed, reactive oxygen species (ROS) are released at inflammation sites and can be eliminated from Boc-D-FMK after conjugation to GSH by GSTP1-1. TNFα also known as cachectin or differentiation-inducing factor (DIF) [36] causes inflammation, tumorigenesis, cellular differentiation and proliferation, necrotic and apoptotic cell death, by binding to TNF receptors (TNFR) 1 and 2 [37], [38], [39]. However, it is widely admitted that binding of TNFα to TNFR1 initiates the majority of the biological activities of TNFα [40]. This interaction leads to the dissociation of the silencer of the death domain from the intracellular death domain (DD) of TNFR1. The TNF receptor-associated death domain (TRADD) then interacts with TNFR1 through its DD sequence and recruits other adaptor proteins among which the TNF-R-associated receptor 2 (TRAF2). TRAF2 interacts with the NF-κB inducing kinase (NIK) which phosphorylates the enzyme inhibitor of κB kinase (IKK), itself implicated in the activation of the NF-κB transcription factors family by phosphorylation of inhibitor of κB (IκB) leading to its degradation by the proteasome. The mammalian NF-κB transcription factors are involved in immune and inflammatory responses as well as cell growth. They reside in the cytoplasm in an inactive homo- or heterodimer form bound to inhibitory proteins of the IκB family [41], [42]. NF-κB activation results in its translocation to the nucleus where it will interact with its target genes, although NF-κB may shuttle between nucleus and cytoplasm in unstimulated cells [43], [44]. Redox-sensitive transcription factors such as NF-κB and AP-1 are known to play a key role in proinflammatory processes such as transcription of cytokine genes, and in induction of protective antioxidant genes [45], [46], [47]. These transcription factors are modulated by TNFα [48], [49] and anticancer drugs which may induce oxidative stress stimulating various signaling pathways including NF-κB [50], [51]. Considering the role of the GSTP1-1 enzyme in the cellular response to oxidative stress, we investigated the involvement of both TNFα and NF-κB in the regulation of GSTP1-1 gene expression in K562 cells. We demonstrated that the GSTP1-1 promoter is activated by TNFα and NF-κBp65 overexpression in K562 cells and we identified a new TNFα-inducible NF-κB binding site located at −323 within the GSTP1-1 promoter. The deletion of this site led to a drastic decrease of the TNFα-induced activation of the GSTP1-1 promoter gene. Altogether, we report here for the first time that the GSTP1-1 gene expression is inducible by the TNFα signaling cascade leading to NF-κB-activated GSTP1-1 promoter.
    Materials and methods
    Results
    Discussion TNFα regulates various cellular mechanisms, particularly immune responses, by induction of specific early responsive genes among which transcription factors such as c-jun and NF-κB [55], [56]. TNFα production is increased in a number of stressful and pathological states and as a proinflammatory cytokine it promotes cell damage through several mechanisms including the overproduction of ROS which is responsible for TNFα toxicity especially in cancer cells [23], [57], [58]. Besides, it has been reported that TNFα induces several protective genes among which enzymes of the GSH metabolism such as γ-glutamylcysteine synthetase in HepG2 cells [59] and the murine GSTA4 in regenerating liver [60]. Our present study investigates for the first time the potential role of TNFα as well as the NF-κB family of transcription factors in GSTP1-1 gene expression in human leukemia cells. First of all, an increase in mRNA expression was observed in TNFα-treated K562 cells and transient transfection assays clearly showed an activation of the GSTP1-1 promoter gene. In addition, inhibition of TNFα-induced mRNA expression using the specific IκBα phosphorylation inhibitor BAY11-7082 [61] supported our hypothesis that NF-κB is involved in the GSTP1-1 gene activation by TNFα. Results obtained by Mori et al.[62] recently demonstrated that BAY11-7082 could be used as a suitable therapeutic agent to treat T cell leukemia since NF-κB pathway is crucial in the development of this pathology and apoptotic resistance. Other detoxifying enzymes including the multidrug resistance P-glycoprotein (MDR1) are inhibited by BAY11-7082 in kidney proximal tubule cells when stimulated with cadmium [63]. Studies by Hideshima et al.[64] demonstrate that proteasome inhibitors such as PS1145 and PS341 inhibit TNFα-induced NF-κB activation in a dose and time dependant fashion in multiple myeloma cells. Therefore, proteasome inhibitors are also of interest as therapeutic tools for inhibition of GSTP1-1-related drug resistance mechanisms.