thapsigargin and efavirenz

Topics: Adipose Tissue; Alkynes; Anti-Retroviral Agents; Benzoxazines; Cyclopropanes; Diabetes Mellitus, Type 2; Drug Combinations; Endoplasmic Reticulum Stress; Fibroblast Growth Factors; Hep G2 Cells; HIV Infections; HIV Integrase Inhibitors; Humans; Klotho Proteins; Liver; Lopinavir; Maraviroc; Membrane Proteins; Muscle, Skeletal; Obesity; Oxidative Stress; Protease Inhibitors; Quinolones; Reverse Transcriptase Inhibitors; Ritonavir; Thapsigargin; Tunicamycin

Mitochondria-associated membranes (MAMs) are specific endoplasmic reticulum (ER) domains that enable it to interact directly with mitochondria and mediate metabolic flow and Ca. This work compares mitochondrial/ER contact during combined ER stress/mitochondrial dysfunction using a model of human hepatoma cells (Hep3B cell line) treated for 24 h with classic pharmacological inducers of ER stress (thapsigargin), mitochondrial dysfunction (carbonyl cyanide m-chlorophenyl hydrazone or rotenone) or both (the antiretroviral drug efavirenz used at clinically relevant concentrations).. Markers of mitochondrial dynamics (dynamin-related protein 1, optic atrophy 1 and mitofusin 2) were expressed differently with these stimuli, pointing to a specificity of combined ER/mitochondrial stress. Lon, a matrix protease involved in protein and mtDNA quality control, was up-regulated at mRNA and protein levels under all conditions. However, only efavirenz decreased the mitochondrial content of Lon while increasing its extramitochondrial presence and its localization to MAMs. This latter effect resulted in an enhanced mitochondria/ER interaction, as shown by co-immunoprecipitation experiments of MAMs protein partners and confocal microscopy imaging.. A specific dual drug-induced mitochondria-ER effect enhances the MAMs content of Lon and its extramitochondrial expression. This is the first report of this phenomenon and suggests a novel MAMs-linked function of Lon protease.

Topics: Alkynes; Benzoxazines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclopropanes; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Humans; Liver Neoplasms; Microscopy, Confocal; Mitochondria; Mitochondrial Membranes; Protease La; Rotenone; Thapsigargin

ER stress is associated with a growing number of liver diseases, including drug-induced hepatotoxicity. The non-nucleoside analogue reverse transcriptase inhibitor Efavirenz, a cornerstone of the multidrug strategy employed to treat HIV1 infection, has been related to the development of various adverse events, including metabolic disturbances and hepatic toxicity, the mechanisms of which remain elusive. Recent evidence has pinpointed a specific mitochondrial effect of Efavirenz in human hepatic cells. This study assesses the induction of ER stress by Efavirenz in the same model and the implication of mitochondria in this process.. Primary human hepatocytes and Hep3B were treated with clinically relevant concentrations of Efavirenz and parameters of ER stress were studied using standard cell biology techniques.. ER stress markers, including CHOP and GRP78 expression (both protein and mRNA), phosphorylation of eIF2α, and presence of the spliced form of XBP1 were upregulated. Efavirenz also enhanced cytosolic Ca(2+) content and induced morphological changes in the ER suggestive of ER stress. This response was greatly attenuated in cells with altered mitochondrial function (Rho°). The effects of Efavirenz on the ER, and particularly in regard to the mitochondrial involvement, differed from those elicited by a standard pharmacological ER stressor.. This newly discovered mechanism of cellular insult involving ER stress and UPR response may help comprehend the hepatic toxicity that has been associated with the widespread and life-long use of Efavirenz. In addition, the specificity of the actions of Efavirenz observed expands our knowledge of the mechanisms that trigger ER stress and shed some light on the mitochondria/ER interplay in drug-induced hepatic challenge.

Topics: Alkynes; Anti-HIV Agents; Benzoxazines; Biomarkers; Calcium; Cell Line; Cells, Cultured; Cyclopropanes; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Hepatocytes; HIV Reverse Transcriptase; Humans; Microscopy, Electron, Transmission; Mitochondria, Liver; Models, Biological; Reverse Transcriptase Inhibitors; Thapsigargin