Mitochondrial Membrane Hyperpolarization Research Articles

Cytotoxic activity of cholesterol oxidase produced by Streptomyces sp. AKHSS against cancerous cell lines: mechanism of action in HeLa cells.

Re-occurrence of cancer is the major drawback for the currently available anticancer therapies. Therefore, study of an efficient enzyme, cholesterol oxidase produced by various kinds of microbes especially obtained from unexplored marine actinobacterial species against human cancer cell lines and understanding its mechanism of action helps to identify an irreversible and potent anticancer agent. The cytotoxic potential of cholesterol oxidase produced by a marine Streptomyces sp. AKHSS against four different human cancer cell lines was demonstrated through MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Fluorescent confocal microscopy and flow cytometry based experiments were performed to understand the efficiency of the enzymatic action on HeLa cells. Further, the apoptotic related proteins were detected through western blotting. Interestingly, the enzyme exhibited potent cytotoxicity at very low concentrations (0.093-0.327µM) against all the cells tested. Fluorescent confocal microscopy revealed the morphological variations induced by the enzyme on cancer cell lines such as the formation of lipid droplets and condensation of nuclei. The enzyme treated cell-free extracts of HeLa cells analyzed through gas chromatography mass spectrometry showed the depletion of membrane cholesterol and the presence of substituted enzyme oxidized product, cholest-4-ene-3-one. The enzyme had induced significant inhibitory effects on the cell viability such as cell cycle arrest (G1 phase), apoptosis and rise of reactive oxygen species as evident through flow cytometry. Besides, hyperpolarization of mitochondrial membrane, reduced rates of phosphorylation of pAkt and the expression of apoptotic death markers like Fas, Fas L, caspases (8 and 3) and PARP-1 were recorded in the enzyme treated HeLa cells. Thus, cholesterol oxidase purified from a marine Streptomyces sp. AKHSS exhibits potent cytotoxicity at very low concentrations against human cancer cell lines. All the ex vivo experiments portrayed the substantial inhibitory effect of the enzyme on HeLa cells suggesting that cholesterol oxidase of Streptomyces sp. AKHSS could be a prominent cancer chemotherapeutic agent.

Mitochondrial clearance of calcium facilitated by MICU2 controls insulin secretion

ObjectiveTransport of Ca2+ into pancreatic β cell mitochondria facilitates nutrient-mediated insulin secretion. However, the underlying mechanism is unclear. Recent establishment of the molecular identity of the mitochondrial Ca2+ uniporter (MCU) and associated proteins allows modification of mitochondrial Ca2+ transport in intact cells. We examined the consequences of deficiency of the accessory protein MICU2 in rat and human insulin-secreting cells and mouse islets. MethodssiRNA silencing of Micu2 in the INS-1 832/13 and EndoC-βH1 cell lines was performed; Micu2−/− mice were also studied. Insulin secretion and mechanistic analyses utilizing live confocal imaging to assess mitochondrial function and intracellular Ca2+ dynamics were performed. ResultsSilencing of Micu2 abrogated GSIS in the INS-1 832/13 and EndoC-βH1 cells. The Micu2−/− mice also displayed attenuated GSIS. Mitochondrial Ca2+ uptake declined in MICU2-deficient INS-1 832/13 and EndoC-βH1 cells in response to high glucose and high K+. MICU2 silencing in INS-1 832/13 cells, presumably through its effects on mitochondrial Ca2+ uptake, perturbed mitochondrial function illustrated by absent mitochondrial membrane hyperpolarization and lowering of the ATP/ADP ratio in response to elevated glucose. Despite the loss of mitochondrial Ca2+ uptake, cytosolic Ca2+ was lower in siMICU2-treated INS-1 832/13 cells in response to high K+. It was hypothesized that Ca2+ accumulated in the submembrane compartment in MICU2-deficient cells, resulting in desensitization of voltage-dependent Ca2+ channels, lowering total cytosolic Ca2+. Upon high K+ stimulation, MICU2-silenced cells showed higher and prolonged increases in submembrane Ca2+ levels. ConclusionsMICU2 plays a critical role in β cell mitochondrial Ca2+ uptake. β cell mitochondria sequestered Ca2+ from the submembrane compartment, preventing desensitization of voltage-dependent Ca2+ channels and facilitating GSIS.

Mitochondria as a Novel Target for Cancer Chemoprevention: Emergence of Mitochondrial-targeting Agents.

Cancer chemoprevention is the most effective approach to control cancer in the population. Despite significant progress, chemoprevention has not been widely adopted because agents that are safe tend to be less effective and those that are highly effective tend to be toxic. Thus, there is an urgent need to develop novel and effective chemopreventive agents, such as mitochondria-targeted agents, that can prevent cancer and prolong survival. Mitochondria, the central site for cellular energy production, have important functions in cell survival and death. Several studies have revealed a significant role for mitochondrial metabolism in promoting cancer development and progression, making mitochondria a promising new target for cancer prevention. Conjugating delocalized lipophilic cations, such as triphenylphosphonium cation (TPP+), to compounds of interest is an effective approach for mitochondrial targeting. The hyperpolarized tumor cell membrane and mitochondrial membrane potential allow for selective accumulation of TPP+ conjugates in tumor cell mitochondria versus those in normal cells. This could enhance direct killing of precancerous, dysplastic, and tumor cells while minimizing potential toxicities to normal cells.

Anti-Biofilm Activity of Cannabidiol against Candida albicans

Candida albicans is a common fungal pathogen in humans. Biofilm formation is an important virulence factor of C. albicans infections. We investigated the ability of the plant-derived cannabidiol (CBD) to inhibit the formation and removal of fungal biofilms. Further, we evaluated its mode of action. Our findings demonstrate that CBD exerts pronounced time-dependent inhibitory effects on biofilm formation as well as disruption of mature biofilm at a concentration range below minimal inhibitory and fungicidal concentrations. CBD acts at several levels. It modifies the architecture of fungal biofilm by reducing its thickness and exopolysaccharide (EPS) production accompanied by downregulation of genes involved in EPS synthesis. It alters the fungal morphology that correlated with upregulation of yeast-associated genes and downregulation of hyphae-specific genes. Importantly, it represses the expression of C. albicans virulence-associated genes. In addition, CBD increases ROS production, reduces the intracellular ATP levels, induces mitochondrial membrane hyperpolarization, modifies the cell wall, and increases the plasma membrane permeability. In conclusion, we propose that CBD exerts its activity towards C. albicans biofilm through a multi-target mode of action, which differs from common antimycotic agents, and thus can be explored for further development as an alternative treatment against fungal infections.

Anti-Tumor Activity of Hypericum perforatum L. and Hyperforin through Modulation of Inflammatory Signaling, ROS Generation and Proton Dynamics.

In this paper we review the mechanisms of the antitumor effects of Hypericum perforatum L. (St. John’s wort, SJW) and its main active component hyperforin (HPF). SJW extract is commonly employed as antidepressant due to its ability to inhibit monoamine neurotransmitters re-uptake. Moreover, further biological properties make this vegetal extract very suitable for both prevention and treatment of several diseases, including cancer. Regular use of SJW reduces colorectal cancer risk in humans and prevents genotoxic effects of carcinogens in animal models. In established cancer, SJW and HPF can still exert therapeutic effects by their ability to downregulate inflammatory mediators and inhibit pro-survival kinases, angiogenic factors and extracellular matrix proteases, thereby counteracting tumor growth and spread. Remarkably, the mechanisms of action of SJW and HPF include their ability to decrease ROS production and restore pH imbalance in tumor cells. The SJW component HPF, due to its high lipophilicity and mild acidity, accumulates in membranes and acts as a protonophore that hinders inner mitochondrial membrane hyperpolarization, inhibiting mitochondrial ROS generation and consequently tumor cell proliferation. At the plasma membrane level, HPF prevents cytosol alkalization and extracellular acidification by allowing protons to re-enter the cells. These effects can revert or at least attenuate cancer cell phenotype, contributing to hamper proliferation, neo-angiogenesis and metastatic dissemination. Furthermore, several studies report that in tumor cells SJW and HPF, mainly at high concentrations, induce the mitochondrial apoptosis pathway, likely by collapsing the mitochondrial membrane potential. Based on these mechanisms, we highlight the SJW/HPF remarkable potentiality in cancer prevention and treatment.

Hyperpolarized mitochondria accumulate in Drosophila Hipk-overexpressing cells to drive tumor-like growth.

ABSTRACTBoth functional and dysfunctional mitochondria are known to underlie tumor progression. Here, we establish use of the proto-oncogene Drosophila Homeodomain-interacting protein kinase (Hipk) as a new tool to address this paradox. We find that, in Hipk-overexpressing tumor-like cells, mitochondria accumulate and switch from fragmented to highly fused interconnected morphologies. Moreover, elevated Hipk promotes mitochondrial membrane hyperpolarization. These mitochondrial changes are at least in part driven by the upregulation of Myc. Furthermore, we show that the altered mitochondrial energetics, but not morphology, is required for Hipk-induced tumor-like growth, because knockdown of pdsw (also known as nd-pdsw; NDUFB10 in mammals; a Complex I subunit) abrogates the growth. Knockdown of ATPsynβ (a Complex V subunit), which produces higher levels of reactive oxygen species (ROS) than pdsw knockdown, instead synergizes with Hipk to potentiate JNK activation and the downstream induction of matrix metalloproteinases. Accordingly, ATPsynβ knockdown suppresses Hipk-induced tumor-like growth only when ROS scavengers are co-expressed. Together, our work presents an in vivo tumor model featuring the accumulation of hyperfused and hyperpolarized mitochondria, and reveals respiratory complex subunit-dependent opposing effects on tumorigenic outcomes.This article has an associated First Person interview with the first author of the paper.

Cell-based and multi-omics profiling reveals dynamic metabolic repurposing of mitochondria to drive developmental progression of Trypanosoma brucei.

Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite's metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro-induced differentiation. Our results provide insights into the mechanisms of the parasite's mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.

Determination of critical quality attributes of mesenchymal stem cell by defining critical process parameters and efficacy of the product

Background & Aim Today, despite thousands of MSC clinical trials conducted, we have not reached any conclusion on what are the Critical Quality Attribute's (CQA's) for MSC. MSC is generated from various mesodermal tissues and can differentiate to mesoderm lineage cells or secret immuno-modulatory cytokines after transplant, which makes the definition of CQA vague. To resolve this, we propose to specify the origin of the cell, the mechanism and scope of indication, and then consider to name the tissue specific-stem cell instead of ‘MSC’. Methods, Results & Conclusion We select immuno-suppression as an indication of tissue specific-stem cells to define the CQA easily. In this case, it will be defined mainly by two major events: having proliferative potential as a process parameter and expecting anti-inflammatory effect as an efficacy. Tissue stem cells cease to proliferate after certain number of mitosis, which relates to the “aging” process. “Aged” cells can be described by increased production of ROS, hyperpolarization of mitochondrial membrane, decreased Rho activity related to spindle shape of cell and low mobility, decrease serine uptake and increase kynurenine secretion that could relate to the loss of intracellular anti-oxidization mechanism. The above parameters can explain the status of exponential cell growth, non-senescence stage in a real time manner via in process monitoring, and can be used as critical process parameters (CPPs) to verify the CQA of the product. As process parameters may vary depending on the culture condition, it is essential to define an optimized culture condition first to address CPPs and CQA. CPPs are used as monitoring points during manufacturing (quality by design) and are well matched to automated mass culturing systems with sensors, not manual manufacturing that verifies the quality of final product only at the release stage (quality by testing) due to lack of information on material and process variability. Efficacy of the final product also constitutes a CQA of the product. We propose to set up a simple immuno-modulation assay such as measuring INF-g or IL-4 secreted from PBMC in Th1 or Th2 induction conditions in the presence of tissue stem cells to address immunosuppressant effect of the product, such an assay needs further refinement in a future study. We hope our approach to define CQA can contribute to find the answer of a decades-long question “what is MSC” and will pave the way to commercial manufacturing of cell products.

Major Depressive Disorder is Associated with Impaired Mitochondrial Function in Skin Fibroblasts.

Mitochondrial malfunction is supposed to be involved in the etiology and pathology of major depressive disorder (MDD). Here, we aimed to identify and characterize the molecular pathomechanisms related to mitochondrial dysfunction in adult human skin fibroblasts, which were derived from MDD patients or non-depressive control subjects. We found that MDD fibroblasts showed significantly impaired mitochondrial functioning: basal and maximal respiration, spare respiratory capacity, non-mitochondrial respiration and adenosine triphosphate (ATP)-related oxygen consumption was lower. Moreover, MDD fibroblasts harbor lower ATP levels and showed hyperpolarized mitochondrial membrane potential. To investigate cellular resilience, we challenged both groups of fibroblasts with hormonal (dexamethasone) or metabolic (galactose) stress for one week, and found that both stressors increased oxygen consumption but lowered ATP content in MDD as well as in non-depressive control fibroblasts. Interestingly, the bioenergetic differences between fibroblasts from MDD or non-depressed subjects, which were observed under non-treated conditions, could not be detected after stress. Our findings support the hypothesis that altered mitochondrial function causes a bioenergetic imbalance, which is associated with the molecular pathophysiology of MDD. The observed alterations in the oxidative phosphorylation system (OXPHOS) and other mitochondria-related properties represent a basis for further investigations of pathophysiological mechanisms and might open new ways to gain insight into antidepressant signaling pathways.

Mitochondrial Dysfunction Combined with High Calcium Load Leads to Impaired Antioxidant Defense Underlying the Selective Loss of Nigral Dopaminergic Neurons.

Mitochondrial dysfunction is critically involved in Parkinson's disease, characterized by loss of dopaminergic neurons (DaNs) in the substantia nigra (SNc), whereas DaNs in the neighboring ventral tegmental area (VTA) are much less affected. In contrast to VTA, SNc DaNs engage calcium channels to generate action potentials, which lead to oxidant stress by yet unknown pathways. To determine the molecular mechanisms linking calcium load with selective cell death in the presence of mitochondrial deficiency, we analyzed the mitochondrial redox state and the mitochondrial membrane potential in mice of both sexes with genetically induced, severe mitochondrial dysfunction in DaNs (MitoPark mice), at the same time expressing a redox-sensitive GFP targeted to the mitochondrial matrix. Despite mitochondrial insufficiency in all DaNs, exclusively SNc neurons showed an oxidized redox-system, i.e., a low reduced/oxidized glutathione (GSH-GSSG) ratio. This was mimicked by cyanide, but not by rotenone or antimycin A, making the involvement of reactive oxygen species rather unlikely. Surprisingly, a high mitochondrial inner membrane potential was maintained in MitoPark SNc DaNs. Antagonizing calcium influx into the cell and into mitochondria, respectively, rescued the disturbed redox ratio and induced further hyperpolarization of the inner mitochondrial membrane. Our data therefore show that the constant calcium load in SNc DaNs is counterbalanced by a high mitochondrial inner membrane potential, even under conditions of severe mitochondrial dysfunction, but triggers a detrimental imbalance in the mitochondrial redox system, which will lead to neuron death. Our findings thus reveal a new mechanism, redox imbalance, which underlies the differential vulnerability of DaNs to mitochondrial defects.SIGNIFICANCE STATEMENT Parkinson's disease is characterized by the preferential degeneration of dopaminergic neurons (DaNs) of the substantia nigra pars compacta (SNc), resulting in the characteristic hypokinesia in patients. Ubiquitous pathological triggers cannot be responsible for the selective neuron loss. Here we show that mitochondrial impairment together with elevated calcium burden destabilize the mitochondrial antioxidant defense only in SNc DaNs, and thus promote the increased vulnerability of this neuron population.

Portoamides A and B are mitochondrial toxins and induce cytotoxicity on the proliferative cell layer of in vitro microtumours

Cyanobacteria are known to produce many toxins and other secondary metabolites. The study of their specific mode of action may reveal the biotechnological potential of such compounds. Portoamides A and B (PAB) are cyclic peptides isolated from the cyanobacteria Phormidium sp. due to their growth repression effect on microalgae and were shown to be cytotoxic against certain cancer cell lines. In the present work, viability was assessed on HCT116 colon cancer cells grown as monolayer culture and as multicellular spheroids (MTS), non-carcinogenic cells and on zebrafish larvae. HCT116 cells and epithelial RPE-1hTERT cells showed very similar degrees of sensitivities to PAB. PAB were able to penetrate the MTS, showing a four-fold high IC50 compared to monolayer cultures. The toxicity of PAB was similar at 4 °C and 37 °C suggesting energy-independent uptake. PAB exposure decreased ATP production, mitochondrial maximal respiration rates and induced mitochondrial membrane hyperpolarization. PAB induced general organelle stress response, indicated by an increase of the mitochondrial damage sensor PINK-1, and of phosphorylation of eIF2α, characteristic for endoplasmic reticulum stress. In summary, these findings show general toxicity of PAB on immortalized cells, cancer cells and zebrafish embryos, likely due to mitochondrial toxicity.

Circulating levels of mitochondrial uncoupling protein 2, but not prohibitin, are lower in humans with type 2 diabetes and correlate with brachial artery flow-mediated dilation

BackgroundExcessive reactive oxygen species from endothelial mitochondria in type 2 diabetes individuals (T2DM) may occur through multiple related mechanisms, including production of mitochondrial reactive oxygen species (mtROS), inner mitochondrial membrane (Δψm) hyperpolarization, changes in mitochondrial mass and membrane composition, and fission of the mitochondrial networks. Inner mitochondrial membrane proteins uncoupling protein-2 (UCP2) and prohibitin (PHB) can favorably impact mtROS and mitochondrial membrane potential (Δψm). Circulating levels of UCP2 and PHB could potentially serve as biomarker surrogates for vascular health in patients with and without T2DM.MethodsPlasma samples and data from a total of 107 individuals with (N = 52) and without T2DM (N = 55) were included in this study. Brachial artery flow mediated dilation (FMD) was measured by ultrasound. ELISA was performed to measure serum concentrations of PHB1 and UCP2. Mitochondrial membrane potential was measured from isolated leukocytes using JC-1 dye.ResultsSerum UCP2 levels were significantly lower in T2DM subjects compared to control subjects (3.01 ± 0.34 vs. 4.11 ± 0.41 ng/mL, P = 0.04). There were no significant differences in levels of serum PHB. UCP2 levels significantly and positively correlated with FMDmm (r = 0.30, P = 0.03) in T2DM subjects only and remained significant after multivariable adjustment. Within T2DM subjects, serum PHB levels were significantly and negatively correlated with UCP2 levels (ρ = − 0.35, P = 0.03).ConclusionCirculating UCP2 levels are lower in T2DM patients and correlate with endothelium-dependent vasodilation in conduit vessels. UCP2 could be biomarker surrogate for overall vascular health in patients with T2DM and merits additional investigation.

Time-resolved characterization of the mechanisms of toxicity induced by silica and amino-modified polystyrene on alveolar-like macrophages

Macrophages play a major role in the removal of foreign materials, including nano-sized materials, such as nanomedicines and other nanoparticles, which they accumulate very efficiently. Because of this, it is recognized that for a safe development of nanotechnologies and nanomedicine, it is essential to investigate potential effects induced by nano-sized materials on macrophages. To this aim, in this work, a recently established model of primary murine alveolar-like macrophages was used to investigate macrophage responses to two well-known nanoparticle models: 50 nm amino-modified polystyrene, known to induce cell death via lysosomal damage and apoptosis in different cell types, and 50 nm silica nanoparticles, which are generally considered non-toxic. Then, a time-resolved study was performed to characterize in detail the response of the macrophages following exposure to the two nanoparticles. As expected, exposure to the amino-modified polystyrene led to cell death, but surprisingly no lysosomal swelling or apoptosis were detected. On the contrary, a peculiar mitochondrial membrane hyperpolarization was observed, accompanied by endoplasmic reticulum stress (ER stress), increased cellular reactive oxygen species (ROS) and changes of metabolic activity, ultimately leading to cell death. Strong toxic responses were observed also after exposure to silica, which included mitochondrial ROS production, mitochondrial depolarization and cell death by apoptosis. Overall, these results showed that exposure to the two nanoparticles led to a very different series of intracellular events, suggesting that the macrophages responded differently to the two nanoparticle models. Similar time-resolved studies are required to characterize the response of macrophages to nanoparticles, as a key parameter in nanosafety assessment.

Novel Potassium Channels in Kidney Mitochondria: The Hyperpolarization-Activated and Cyclic Nucleotide-Gated HCN Channels.

Hyperpolarization-activated cationic HCN channels comprise four members (HCN1–4) that control dendritic integration, synaptic transmission and action potential firing. In the kidney, HCN1, HCN2 and HCN3 are differentially expressed and contribute to the transport of sodium, potassium (K+) and ammonium into the nephrons. HCN3 is regulated by K+ diets in the kidney. In this work we performed a proteomic analysis of HCN3 expressed in human embryonic kidney cells (HEK293 cells). More than 50% of the interacting proteins belonged to mitochondria. Therefore, we explored the presence of HCN channels in kidney mitochondria. By immunoblotting and immunogold electron microscopy HCN3 protein expression was found in rat kidney mitochondria; it was also confirmed in human kidney. Patch-clamp recordings of renal mitochondria and mitochondria from HEK293 cells overexpressing HCN1, HCN2 and HCN3 channels, stained with MitoTracker Green FM, indicated that only HCN3 could produce inwardly K+ currents that were inhibited by ZD7288, a specific blocker of HCN channels. Furthermore, ZD7288 caused inhibition of the oxygen consumption coupled to ATP synthesis and hyperpolarization of the inner mitochondrial membrane. In conclusion, we show for the first time that pacemaker HCN channels contribute to K+ transport in mitochondria facilitating the activity of the respiratory chain and ATP synthesis by controlling the inner mitochondrial membrane potential.

Estrogen-dependent disruption of intracellular iron metabolism augments the cytotoxic effects of doxorubicin in select breast and ovarian cancer cells.

Introduction: Increased iron content in cancer cells is associated with resistance to chemotherapy. Recent studies have demonstrated that estrogen (E2) suppresses hepcidin synthesis and enhances intracellular iron efflux. Herein, we investigated whether E2-driven intracellular iron efflux renders cancer cells more susceptible to doxorubicin (Dox)-induced cytotoxicity.Methods: Breast, ovarian, and liver cancer cell lines treated with E2, Dox, or a combination of both were assessed for intracellular iron status, mitochondrial function, cell cycle, and apoptosis.Results: E2+Dox treatment in MCF7, SKOV3 and MDA-MB231 cells resulted in enhanced apoptosis compared with Dox-treated cells. Expression of γH2AX was significantly higher and that of survivin significantly lower in E2+Dox-treated cells than Dox-treated cells. At 48 hours, E2+Dox had induced a significant increase in the percentage of sub-G1 apoptotic cells, increased CHK1 expression, and decreased cyclin D1, CDK4, and CDK6 expression. Ferroportin and ferritin expression was significantly higher and that of TfR1 significantly lower in E2+Dox-treated cells than Dox-treated cells. Intracellular iron content was significantly reduced in E2+Dox-treated cells at 48 hours posttreatment. Lastly, E2+Dox-treated cells showed higher levels of mitochondrial membrane hyperpolarization than Dox-treated cells.Conclusion: These findings suggest that E2 disrupts intracellular iron metabolism in such a way that increases cell susceptibility to Dox-induced cytotoxicity.

Plant-derived mitochondria-targeting cysteine-rich peptide modulates cellular bioenergetics

Mitochondria are attractive therapeutic targets for developing agents to delay age-related frailty and diseases. However, few promising leads have been identified from natural products. Previously, we identified roseltide rT1, a hyperstable 27-residue cysteine-rich peptide from Hibiscus sabdariffa, as a knottin-type neutrophil elastase inhibitor. Here, we show that roseltide rT1 is also a cell-penetrating, mitochondria-targeting peptide that increases ATP production. Results from flow cytometry, live-cell imaging, pulldown assays, and genetically-modified cell lines supported that roseltide rT1 enters cells via glycosaminoglycan-dependent endocytosis, and enters the mitochondria through TOM20, a mitochondrial protein import receptor. We further showed that roseltide rT1 increases cellular ATP production via mitochondrial membrane hyperpolarization. Using biotinylated roseltide rT1 for target identification and proteomic analysis, we showed that human mitochondrial membrane ATP synthase subunit O is an intramitochondrial target. Collectively, these data support our discovery that roseltide rT1 is a first-in-class mitochondria-targeting, cysteine-rich peptide with potentials to be developed into tools to further our understanding of mitochrondria-related diseases.

Synthetic Cannabinoids JWH-122 and THJ-2201 Disrupt Endocannabinoid-Regulated Mitochondrial Function and Activate Apoptotic Pathways as a Primary Mechanism of In Vitro Nephrotoxicity at In Vivo Relevant Concentrations.

The widespread recreational use of synthetic cannabinoids (SCBs) represents a major public health issue, as reports of intoxications and deaths following SCB use rapidly mount up. Specifically, a direct link between SCB use and acute kidney injury (AKI) has been established, although the pathophysiologic mechanisms remain undefined. Here we assessed the in vitro nephrotoxicity of 3 commonly detected and structurally distinct SCBs-AB-FUBINACA, JWH-122, and THJ-2201-in human proximal tubule cells (HK-2), to ascertain potential similarities and/or differences regarding their nephrotoxicity signatures. We showed that 2 of the 3 SCBs tested, namely JWH-122 and THJ-2201, at in vivo relevant concentrations (1 nM-1 μM), triggered apoptotic cell death pathways, mainly through a shared mechanism involving the deregulation of mitochondrial function (ie, with mitochondrial membrane hyperpolarization and increased intracellular ATP levels), as the primary molecular signature of nephrotoxicity mechanism. Noteworthy, no SCB affected cell viability (MTT reduction, lactate dehydrogenase release, Neutral Red inclusion). Use of the cannabinoid receptor (CBR) antagonists SR141716A and SR144528, as well as HEK293T cells, which do not express CBRs, confirmed the involvement of these receptors in SCB-mediated mitochondrial membrane hyperpolarization but not on other events, suggesting an off-target action regulating SCB-induced kidney cell death. Our results further strengthen the relevance of the endocannabinoid system in maintaining mitochondrial function in kidney cells, as we demonstrate that HK-2 incubation with CBR antagonists or inhibitors of endocannabinoid biosynthesis (ie, methyl arachydonyl fluorophosphonate, tetrahydrolipstatin) alone produced deleterious effects similar to those now reported for SCBs. Overall, SCB-induced nephrotoxicity seems to be mainly regulated at the mitochondrial level, but the specific mechanisms involved require further clarification.

Impact of erythromycin on a non-target organism: Cellular effects on the freshwater microalga Pseudokirchneriella subcapitata

The increasing and indiscriminate use of antibiotics is the origin of their introduction in aquatic systems through domestic and livestock effluents. The occurrence of erythromycin (ERY), a macrolide antibiotic, in water bodies raises serious concerns about its potential toxic effect in aquatic biota (non-target organisms), particularly in microalgae, the first organisms in contact with aquatic contaminants. This study aimed to evaluate the possible toxic effects of ERY on relevant cell targets of the freshwater microalga Pseudokirchneriella subcapitata. Algal cells incubated with significant environmental ERY concentrations presented disturbance of the photosynthetic apparatus (increased algal autofluorescence and reduction of chlorophyll a content) and mitochondrial function (hyperpolarization of mitochondrial membrane). These perturbations can apparently be attributed to the similarity of the translational machinery of these organelles (chloroplasts and mitochondria) with the prokaryotic cells. P. subcapitata cells treated with ERY showed a modification of metabolic activity (increased esterase activity) and redox state (alteration of intracellular levels of reactive oxygen species and reduced glutathione content) and an increased biovolume. ERY induced an algistatic effect: reduction of growth rate without loss of cell viability (plasma membrane integrity). The present study shows that chronic exposure (72 h), at low (μg L−1) ERY concentrations (within the range of concentrations detected in surface and ground waters), induce disturbances in the physiological state of the alga P. subcapitata. Additionally, this work alerts to the possible negative impact of the uncontrolled use of ERY on the aquatic systems.

Exposure to a Nonionic Surfactant Induces a Response Akin to Heat-Shock Apoptosis in Intestinal Epithelial Cells: Implications for Excipients Safety.

Amphipathic, nonionic, surfactants are widely used in pharmaceutical, food, and agricultural industry to enhance product features; as pharmaceutical excipients, they are also aimed at increasing cell membrane permeability and consequently improving oral drugs absorption. Here, we report on the concentration- and time-dependent succession of events occurring throughout and subsequent exposure of Caco-2 epithelium to a "typical" nonionic surfactant (Kolliphor HS15) to provide a molecular explanation for nonionic surfactant cytotoxicity. The study shows that the conditions of surfactant exposure, which increase plasma membrane fluidity and permeability, produced rapid (within 5 min) redox and mitochondrial effects. Apoptosis was triggered early during exposure (within 10 min) and relied upon an initial mitochondrial membrane hyperpolarization (5-10 min) as a crucial step, leading to its subsequent depolarization and caspase-3/7 activation (60 min). The apoptotic pathway appears to be triggered prior to substantial surfactant-induced membrane damage (observed ≥60 min). We hence propose that the cellular response to the model nonionic surfactant is triggered via surfactant-induced increase in plasma membrane fluidity, a phenomenon akin to the stress response to membrane fluidization induced by heat shock, and consequent apoptosis. Therefore, the fluidization effect that confers surfactants the ability to enhance drug permeability may also be intrinsically linked to the propagation of their cytotoxicity. The reported observations have important implications for the safety of a multitude of nonionic surfactants used in drug delivery formulations and to other permeability enhancing compounds with similar plasma membrane fluidizing mechanisms.

Hexane extract from Spondias tuberosa (Anacardiaceae) leaves has antioxidant activity and is an anti-Candida agent by causing mitochondrial and lysosomal damages

BackgroundSpondias tuberosa is a plant that produces a fruit crop with high economic relevance at Brazilian Caatinga. Its roots and leaves are used in folk medicine.MethodsChemical composition of a hexane extract from S. tuberosa leaves was evaluated by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and 1H nuclear magnetic resonance (NMR). Antioxidant potential was investigated by DPPH and ABTS assays. Antifungal action on Candida species was evaluated determining the minimal inhibitory concentration (MIC50) and putative mechanisms were determined by flow cytometry analysis. In addition, hemolytic activity on human erythrocytes was assessed and the concentration required to promote 50% hemolysis (EC50) was determined.ResultsPhytochemical analysis by TLC showed the presence of flavonoids, hydrolysable tannins, saponins and terpenes. The HPLC profile of the extract suggested the presence of gallic acid (0.28 ± 0.01 g%) and hyperoside (1.27 ± 0.01 g%). The representative 1H NMR spectrum showed saturated and unsaturated fatty acids among the main components. The extract showed weak and moderate antioxidant activity in DPPH (IC50: 234.00 μg/mL) and ABTS (IC50: 123.33 μg/mL) assays, respectively. It was able to inhibit the growth of C. albicans and C. glabrata with MIC50 of 2.0 and 0.078 mg/mL, respectively. The treatment of C. glabrata cells with the extract increased levels of mitochondrial superoxide anion, caused hyperpolarization of mitochondrial membrane, and compromised the lysosomal membrane. Weak hemolytic activity (EC50: 740.8 μg/mL) was detected.ConclusionThe results demonstrate the pharmacological potential of the extract as antioxidant and antifungal agent, aggregating biotechnological value to this plant and stimulating its conservation.