[solved]-Causes of drug failure
One of the most common causes of drug failure is drug-induced liver injuries (DILIs). The majority of these failures are idiosyncratic reactions, which occur in small patient populations (between 1 in 1.000-10.000) in an unpredictable manner.1 The underlying mechanism of this type of DILI is very complex and still not completely understood.2 However, recent data have suggested that the crosstalk between cytokine-mediated pro-apoptotic signalling and drug reactive metabolite-mediated intracellular stress responses is essential in the comprehension of DILI.3
Various xenobiotics (e.g. diclofenac) can induce liver damage via the tumor necrosis factor ?? (TNF??) pathway. Excretion of this major cytokine will initiate through liver macrophages (Kuppfer cells) after exposure to bacterial endotoxins (e.g. Lipopolysaccharide).4 After binding of TNF?? to its receptor (TNFR1), the transcription factor nuclear factor kappa-B (NF-??B) is activated.5 In general, NF-??B is detained in the cytoplasm by binding to an inhibitor of ??B (I??B) complex. The initiated NF-??B leads to activation of I??B kinase (IKK), which eventually leads to the ubiquitination and phosphorylation of the I??B complex.6 Subsequently, this complex is targeted for proteosomal degradation. Hereafter, NF-??B translocates to the nucleus in an oscillatory way and activates the transcription of several genes which primarily encode survival proteins, such as cellular FLICE-like inhibitory protein (c-FLIP), inhibitor of apoptosis proteins (IAPs) and negative regulators proteins (e.g. A20, I??B??).7 After protein synthesis, A20 and I??B?? will inhibit the function of NF-??B in a negative feedback manner (Figure 1). Modified TNF??-induced NF??B translocation by various compounds is believed to shift the balance between cell survival and cell death.
Furthermore, reactive compound metabolites are capable of altering cellular molecules, which could lead to intracellular disturbances and eventually to the induction of various stress response or toxicity pathways.8 These pathways, combined with a decreased response for cell damage recovery and protection, will enhance the susceptibility to cell death of various cells. Up to now, insufficient studies have been performed to investigate the contribution of various pathways to DILI. It still remains uncertain which drug-induced toxicity pathways modulate the pro-apoptotic activity of TNF?? signaling in DILI reactions. However, there are different stress responses which are most likely involved in the formation of DILI. The Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2) antioxidant response pathway and the endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR) have been studied in drug-induced toxicity of hepatocytes [2]. The Keap1/Nrf2 pathway is essential in recognizing ROS and/or cellular oxidative stress [6]. Keap1 maintains Nrf2 in the cytoplasm and guides it toward proteasomal degradation under normal circumstances. Nrf2 signaling is important in the cytoprotective response against ROS, but its role in the TNF??/drug interaction in idiosyncratic DILI remains unclear.
Furthermore, the ER stress-mediated UPR is a stress response due to enhanced translation and/or disturbed protein folding. Should the modification fail, a pro-apoptotic system will be initiated to eliminate the injured cell. The exact mechanism and role of the ER stress signalling response in managing DILI in relation to TNF??-induced apoptosis still remains unclear.
In this research, we hypothesize that stress response mechanisms (e.g. ER stress responses, oxidative stress responses) are involved in the delay of NF-??B nuclear translocation upon exposure to various NF-??B nuclear translocation compounds.
In this project, a human HepG2 cell line will be used to study the interaction between five different compounds (amiodarone, carbamazepine, diclofenac, nefazodone, ximelagatran) and cytokine TNF alpha. To investigate the overall percentage of cell death, a lactate dehydrogenase (LDH) assay will be performed. Furthermore, in order to quantify the amount of apoptotic cells, an Annexin V affinity assay will be executed. It is expected that the concentration-dependant toxicity of the compounds is enhanced with the presence of TNF??. Live cell imaging with HepG2 GFPp65 cells will be used to follow the NF-??B translocation after exposure to the five various compounds. Subsequently, an automated image quantification of the p65 signal intensity ratio of nucleus/cytoplasm is measured to show the exact onset of the second nuclear entry of NF-??B. It is estimated that the data of the NF-??B translocation will show a compound-induced delayed onset of NF-??B.
The activation of NF-??B target genes cIAP and c-FLIP will be measured using a Western Blot analysis. Moreover, the negative regulators of NF-??B, A20 and I??B??, will be studied to investigate the negative feedback loop of NF-??B. We anticipate that the data of the Western Blot analysis will show a decrease in production of the investigated target genes, because of the reduced TNF??-induced NF-??B transcriptional activity.
Ultimately, a data analysis will be applied on the results using t-test or two-way analysis of variance (ANOVA) in case of multiple comparisons.
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