Doxorubicin-Induced Autophagolysosome Formation Is Partly Prevented by Mitochondrial ROS Elimination in DOX-Resistant Breast Cancer Cells

Since its discovery, mitophagy has been viewed as a protective mechanism used by cancer cells to prevent the induction of mitochondrial apoptosis. Most cancer treatments directly or indirectly cause mitochondrial dysfunction in order to trigger signals for cell death. Elimination of these dysfunctional mitochondria by mitophagy could thus prevent the initiation of the apoptotic cascade. In breast cancer patients, resistance to doxorubicin (DOX), one of the most widely used cancer drugs, is an important cause of poor clinical outcomes. However, the role played by mitophagy in the context of DOX resistance in breast cancer cells is not well understood. We therefore tried to determine whether an increase in mitophagic flux was associated with the resistance of breast cancer cells to DOX. Our first objective was to explore whether DOX-resistant breast cancer cells were characterized by conditions that favor mitophagy induction. We next tried to determine whether mitophagic flux was increased in DOX-resistant cells in response to DOX treatment. For this purpose, the parental (MCF-7) and DOX-resistant (MCF-7dox) breast cancer cell lines were used. Our results show that mitochondrial reactive oxygen species (ROS) production and hypoxia-inducible factor-1 alpha (HIF-1 alpha) expression are higher in MCF-7dox in a basal condition compared to MCF-7, suggesting DOX-resistant breast cancer cells are prone to stimuli to induce a mitophagy-related event. Our results also showed that, in response to DOX, autophagolysosome formation is induced in DOX-resistant breast cancer cells. This mitophagic step following DOX treatment seems to be partly due to mitochondrial ROS production as autophagolysosome formation is moderately decreased by the mitochondrial antioxidant mitoTEMPO.     

The Effects of Cadmium at Low Environmental Concentrations on THP-1 Macrophage Apoptosis

Cadmium at environmental concentrations is a risk factor for many diseases, including cardiovascular and neurodegenerative diseases, in which macrophages play an important role. The aim of this study was to evaluate the effects of cadmium at low environmental (nanomolar) concentrations on apoptotic processes in THP-1(acute monocytic leukemia cells line)-derived macrophages, with special focus on mitochondrial events involved. Macrophages were incubated with various cadmium chloride (CdCl₂) solutions for 48 h at final concentrations of 5 nM, 20 nM, 200 nM and 2 µM CdCl₂. Cell viability was measured using flow cytometry. Flow cytometric measurement (annexin V/FITC (annexin V/fluorescein isothiocyanate) and PI (propidium iodide) double staining) was used to quantify the extent of apoptosis. Fluorescence and confocal microscopy were used for imaging of apoptosis process. Changes in mitochondrial membrane potential were monitored using cytofluorimetry after cell staining with JC-1(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyane iodide) probe. Mitochondrial ROS (reactive oxygen species) levels were measured cytofluorimetrically after incubation of cells with mitochondrial superoxide indicator (MitoSOX) red fluorescent marker. The mRNA expression of Bcl-2 and Bax was analysed with qRT-PCR. Our study demonstrates that cadmium, even at low environmental concentrations, exerts mitochondrial toxicity in THP-1 macrophages. Forty-eight-hour exposure to very low concentrations reduces cell viability and results in cell death by apoptosis and necrosis. The decrease in mitochondrial membrane potential, increased ROS production, increased Bax and decreased Bcl-2 mRNA expression are mitochondrial events involved in cadmium-induced apoptosis.     

DHA Protects Hepatocytes from Oxidative Injury through GPR120/ERK-Mediated Mitophagy

Oxidative stress occurs in a variety of clinical liver diseases and causes cellular damage and mitochondrial dysfunction. The clearance of damaged mitochondria by mitophagy may facilitate mitochondrial biogenesis and enhance cell survival. Although the supplementation of docosahexaenoic acid (DHA) has been recognized to relieve the symptoms of various liver diseases, the antioxidant effect of DHA in liver disease is still unclear. The purpose of our research was to investigate the antioxidant effect of DHA in the liver and the possible role of mitophagy in this. In vitro, H₂O₂-induced injury was caused in AML12 cells. The results showed that DHA repressed the level of reactive oxygen species (ROS) induced by H₂O₂ and stimulated the cellular antioxidation response. Most notably, DHA restored oxidative stress-impaired autophagic flux and promoted protective autophagy. In addition, PINK/Parkin-mediated mitophagy was activated by DHA in AML12 cells and alleviated mitochondrial dysfunction. The ERK1/2 signaling pathway was inhibited during oxidative stress but reactivated by DHA treatment. It was proven that the expression of ERK1/2 was involved in the regulation of mitophagy by the ERK1/2 inhibitor. We further proved these results in vivo. DHA effectively alleviated the liver oxidative damage caused by CCl₄ and enhanced antioxidation capacity; intriguingly, autophagy was also activated. In summary, our data demonstrated that DHA protected hepatocytes from oxidative damage through GPR120/ERK-mediated mitophagy.     

Metformin Restores Parkin-Mediated Mitophagy,Suppressed by Cytosolic p53

Metformin is known to alleviate hepatosteatosis by inducing 5’ adenosine monophosphate (AMP)-kinase-independent, sirtuin 1 (SIRT1)-mediated autophagy. Dysfunctional mitophagy in response to glucolipotoxicities might play an important role in hepatosteatosis. Here, we investigated the mechanism by which metformin induces mitophagy through restoration of the suppressed Parkin-mediated mitophagy. To this end, our ob/ob mice were divided into three groups: (1) ad libitum feeding of a standard chow diet; (2) intraperitoneal injections of metformin 300 mg/kg; and (3) 3 g/day caloric restriction (CR). HepG2 cells were treated with palmitate (PA) plus high glucose in the absence or presence of metformin. We detected enhanced mitophagy in ob/ob mice treated with metformin or CR, whereas mitochondrial spheroids were observed in mice fed ad libitum. Metabolically stressed ob/ob mice and PA-treated HepG2 cells showed an increase in expression of endoplasmic reticulum (ER) stress markers and cytosolic p53. Cytosolic p53 inhibited mitophagy by disturbing the mitochondrial translocation of Parkin, as demonstrated by immunoprecipitation. However, metformin decreased ER stress and p53 expression, resulting in induction of Parkin-mediated mitophagy. Furthermore, pifithrin-α, a specific inhibitor of p53, increased mitochondrial incorporation into autophagosomes. Taken together, these results indicate that metformin treatment facilitates Parkin-mediated mitophagy rather than mitochondrial spheroid formation by decreasing the inhibitory interaction with cytosolic p53 and increasing degradation of mitofusins.     

Rapamycin Ameliorates Defects in Mitochondrial Fission and Mitophagy in Glioblastoma Cells

Glioblastoma (GBM) cells feature mitochondrial alterations, which are documented and quantified in the present study, by using ultrastructural morphometry. Mitochondrial impairment, which roughly occurs in half of the organelles, is shown to be related to mTOR overexpression and autophagy suppression. The novelty of the present study consists of detailing an mTOR-dependent mitophagy occlusion, along with suppression of mitochondrial fission. These phenomena contribute to explain the increase in altered mitochondria reported here. Administration of the mTOR inhibitor rapamycin rescues mitochondrial alterations. In detail, rapamycin induces the expression of genes promoting mitophagy (PINK1, PARKIN, ULK1, AMBRA1) and mitochondrial fission (FIS1, DRP1). This occurs along with over-expression of VPS34, an early gene placed upstream in the autophagy pathway. The topographic stoichiometry of proteins coded by these genes within mitochondria indicates that, a remarkable polarization of proteins involved in fission and mitophagy within mitochondria including LC3 takes place. Co-localization of these proteins within mitochondria, persists for weeks following rapamycin, which produces long-lasting mitochondrial plasticity. Thus, rapamycin restores mitochondrial status in GBM cells. These findings add novel evidence about mitochondria and GBM, while fostering a novel therapeutic approach to restore healthy mitochondria through mTOR inhibition.     

Xanthohumol-Induced Rat Glioma C6 Cells Death by Triggering Mitochondrial Stress

AIM: To investigate the underlying mechanisms of xanthohumol (XN) on the proliferation inhibition and death of C6 glioma cells. METHODS: To determine the effects of XN on C6 cells, cell proliferation and mortality after XN treatment were assessed by SRB assay and trypan blue assay respectively. Apoptotic rates were evaluated by flowcytometry after Annexin V-FITC/PI double staining. The influence of XN on the activity of caspase-3 was determined by Western blot (WB); and nuclear transposition of apoptosis-inducing factor (AIF) was tested by immunocytochemistry and WB. By MitoSOX^TM staining, the mitochondrial ROS were detected. Mitochondrial function was also tested by MTT assay (content of succinic dehydrogenase), flow cytometry (mitochondrial membrane potential (MMP)—JC-1 staining; mitochondrial abundance—mito-Tracker green), immunofluorescence (MMP—JC-1 staining; mitochondrial morphology—mito-Tracker green), WB (mitochondrial fusion-fission protein—OPA1, mfn2, and DRP1; mitophagy-related proteins—Pink1, Parkin, LC3B, and P62), and high-performance liquid chromatography (HPLC) (energy charge). Finally, mitochondrial protein homeostasis of C6 cells after XN treatment with and without LONP1 inhibitor bortezomib was investigated by trypan blue assay (proliferative activity and mortality) and WB (mitochondrial protease LONP1). All cell morphology images were taken by a Leica Microsystems microscope. RESULTS: XN could lead to proliferation inhibition and death of C6 cells in a time- and dose-dependent manner and induce apoptosis of C6 cells through the AIF pathway. After long incubation of XN, mitochondria of C6 cells were seriously impaired, and mitochondria had a diffuse morphology and mitochondrial ROS were increased. The content of succinic dehydrogenase per cell was significantly decreased after XN insults of 24, 48, and 72 h. The energy charge was weakened after XN insult of 24 h. Furthermore, the MMP and mitochondrial abundance were significantly decreased; the protein expression levels of OPA1, mfn2, and DRP1 were down-regulated; and the protein expression levels of Pink1, Parkin, LC3B-II/LC3B-I, and p62 were up-regulated in long XN incubation times (24, 48, and 72 h). XN incubation with bortezomib for 48 h resulted in lower proliferative activity and higher mortality of C6 cells and caused the cell to have visible vacuoles. Moreover, the protein expression levels of LONP1 was up-regulated gradually as XN treatment time increased. CONCLUSION: These data supported that XN could induce AIF pathway apoptosis of the rat glioma C6 cells by affecting the mitochondria.     

Acute Glucose Shift Induces the Activation of the NLRP3 Inflammasome in THP-1 Cells

We aimed to investigate the effect of acute glucose shift on the activation of the NLRP3 inflammasome, IL-1β secretion, and underlying signaling pathways in THP-1 cells. THP-1 cells were divided into four groups and exposed to the following glucose concentrations for 24 h: constant normal glucose (NG, 5.5 mM), constant high glucose (HG, 25 mM), normal to high glucose shift (NG-to-HG, 5.5 to 25 mM), and high to normal glucose shift (HG-to-NG, 25 to 5.5 mM). Cell viability, oxidative stress, and the levels of NLRP3 inflammasome components were assessed. Both directions of the acute glucose shift increased the activation of the NLRP3 inflammasome, generation of reactive oxygen species (ROS), and expression of phosphorylated p38 MAPK, JNK, and NF-κB compared with either constant NG or HG. Treatment with N-acetylcysteine, a pharmacological antioxidant, inhibited the acute glucose shift-induced generation of ROS, activation of NLRP3 inflammasome, and upregulation of MAPK-NF-κB. Further analysis using inhibitors of p38 MAPK, JNK, and NF-κB indicated that acute glucose shifts promoted IL-1β secretion by activating the signaling pathway in a ROS-MAPK-NF-κB-NLRP3 inflammasome in THP-1 cells. These findings suggested that acute changes in glucose concentration might cause monocyte inflammation, which is associated with diabetic complications.     

Targeted siRNA Screens Identify ER-to-Mitochondrial Calcium Exchange in Autophagy and Mitophagy Responses in RPE1 Cells

Autophagy is an important stress response pathway responsible for the removal and recycling of damaged or redundant cytosolic constituents. Mitochondrial damage triggers selective mitochondrial autophagy (mitophagy), mediated by a variety of response factors including the Pink1/Parkin system. Using human retinal pigment epithelial cells stably expressing autophagy and mitophagy reporters, we have conducted parallel screens of regulators of endoplasmic reticulum (ER) and mitochondrial morphology and function contributing to starvation-induced autophagy and damage-induced mitophagy. These screens identified the ER chaperone and Ca²⁺ flux modulator, sigma non-opioid intracellular receptor 1 (SIGMAR1), as a regulator of autophagosome expansion during starvation. Screens also identified phosphatidyl ethanolamine methyl transferase (PEMT) and the IP3-receptors (IP3Rs) as mediators of Parkin-induced mitophagy. Further experiments suggested that IP3R-mediated transfer of Ca²⁺ from the ER lumen to the mitochondrial matrix via the mitochondrial Ca²⁺ uniporter (MCU) primes mitochondria for mitophagy. Importantly, recruitment of Parkin to damaged mitochondria did not require IP3R-mediated ER-to-mitochondrial Ca²⁺ transfer, but mitochondrial clustering downstream of Parkin recruitment was impaired, suggesting involvement of regulators of mitochondrial dynamics and/or transport. Our data suggest that Ca²⁺ flux between ER and mitochondria at presumed ER/mitochondrial contact sites is needed both for starvation-induced autophagy and for Parkin-mediated mitophagy, further highlighting the importance of inter-organellar communication for effective cellular homeostasis.     

Reference Gene Selection for Quantitative PCR Studies in Sheep Neutrophils

Reference genes are essential for studying mRNA expression with quantitative PCR (qPCR). We investigated 11 potential neutrophil reference genes (RPL19, GAPDH, ACTB, B2M, HPRT, G6PD, TFRC, PGK1, YWHAZ, SDHA and GYPC) for sheep under disease conditions of foot rot (FR) and with or without Se supplementation. Initial screening was based on gene expression level (<28 Cq cycles) and variability (SD < 1.5 Cq cycles) and excluded TFRC, GYPC and HPRT from further analysis. Expression stability of the remaining genes was evaluated using four software programs: geNorm, NormFinder, BestKeeper and the comparative delta Cq method. The neutrophil reference genes, G6PD, YWHAZ, GAPDH, RPL19 and SDHA, consistently ranked among the top five most stable genes under these experimental conditions. The SDHA gene expression was not stable in FR-diseased sheep receiving Se treatment and, thus, cannot be recommended as a reference gene. The commonly used genes, PGK1, ACTB and B2M, were not reliable reference genes, underscoring the need to validate neutrophil reference genes under different experimental conditions. Multiple references genes rather than a single gene may provide more robust and reliable results. The best pair of reference genes was SDHA/G6PD in healthy sheep and GADPH/YWHAZ in FR-diseased sheep.     

ROS/TNF-α Crosstalk Triggers the Expression of IL-8 and MCP-1 in Human Monocytic THP-1 Cells via the NF-κB and ERK1/2 Mediated Signaling

IL-8/MCP-1 act as neutrophil/monocyte chemoattractants, respectively. Oxidative stress emerges as a key player in the pathophysiology of obesity. However, it remains unclear whether the TNF-α/oxidative stress interplay can trigger IL-8/MCP-1 expression and, if so, by which mechanism(s). IL-8/MCP-1 adipose expression was detected in lean, overweight, and obese individuals, 15 each, using immunohistochemistry. To detect the role of reactive oxygen species (ROS)/TNF-α synergy as a chemokine driver, THP-1 cells were stimulated with TNF-α, with/without H₂O₂ or hypoxia. Target gene expression was measured by qRT-PCR, proteins by flow cytometry/confocal microscopy, ROS by DCFH-DA assay, and signaling pathways by immunoblotting. IL-8/MCP-1 adipose expression was significantly higher in obese/overweight. Furthermore, IL-8/MCP-1 mRNA/protein was amplified in monocytic cells following stimulation with TNF-α in the presence of H₂O₂ or hypoxia (p ˂ 0.0001). Synergistic chemokine upregulation was related to the ROS levels, while pre-treatments with NAC suppressed this chemokine elevation (p ≤ 0.01). The ROS/TNF-α crosstalk involved upregulation of CHOP, ERN1, HIF1A, and NF-κB/ERK-1,2 mediated signaling. In conclusion, IL-8/MCP-1 adipose expression is elevated in obesity. Mechanistically, ROS/TNF-α crosstalk may drive expression of these chemokines in monocytic cells by inducing ER stress, HIF1A stabilization, and signaling via NF-κB/ERK-1,2. NAC had inhibitory effect on oxidative stress-driven IL-8/MCP-1 expression, which may have therapeutic significance regarding meta-inflammation.     

Proteomic Analyses Reveal that Sky1 Modulates Apoptosis and Mitophagy in Saccharomyces cerevisiae Cells Exposed to Cisplatin

Sky1 is the only member of the SR (Serine–Arginine) protein kinase family in Saccharomyces cerevisiae. When yeast cells are treated with the anti-cancer drug cisplatin, Sky1 kinase activity is necessary to produce the cytotoxic effect. In this study, proteome changes in response to this drug and/or SKY1 deletion have been evaluated in order to understand the role of Sky1 in the response of yeast cells to cisplatin. Results reveal differential expression of proteins previously related to the oxidative stress response, DNA damage, apoptosis and mitophagy. With these precedents, the role of Sky1 in apoptosis, necrosis and mitophagy has been evaluated by flow-cytometry, fluorescence microscopy, biosensors and fluorescence techniques. After cisplatin treatment, an apoptotic-like process diminishes in the ∆sky1 strain in comparison to the wild-type. The treatment does not affect mitophagy in the wild-type strain, while an increase is observed in the ∆sky1 strain. The increased resistance to cisplatin observed in the ∆sky1 strain may be attributable to a decrease of apoptosis and an increase of mitophagy.     

Effects of Acrylamide on the Activity and Structure of Human Brain Creatine Kinase

Acrylamide is widely used worldwide in industry and it can also be produced by the cooking and processing of foods. It is harmful to human beings, and human brain CK (HBCK) has been proposed to be one of the important targets of acrylamide. In this research, we studied the effects of acrylamide on HBCK activity, structure and the potential binding sites. Compared to CKs from rabbit, HBCK was fully inactivated at several-fold lower concentrations of acrylamide, and exhibited distinct properties upon acrylamide-induced inactivation and structural changes. The binding sites of acrylamide were located at the cleft between the N- and C-terminal domains of CK, and Glu232 was one of the key binding residues. The effects of acrylamide on CK were proposed to be isoenzyme- and species-specific, and the underlying molecular mechanisms were discussed.