Depolarization Of Mitochondrial Membrane Potential Research Articles

Cuproptosis associated cytoskeletal destruction contributes to podocyte injury in chronic kidney disease.

The podocyte cytoskeleton determines the stability of podocyte structure and function, and their imbalance plays a pathogenic role in podocyte diseases. However, the underlying mechanism of podocyte cytoskeleton damage is not fully understood. Here, we investigate the specific role of cuproptosis in inducing podocyte cytoskeleton injury. In vitro and in vivo studies, exposure to high levels of copper and adriamycin (ADR) caused significant increases in copper concentration in intracellular and renal tissue. Moreover, excessive accumulation of copper induced cuproptosis, resulting in the destruction of the podocyte cytoskeleton. However, inhibition of copper accumulation to reduce cuproptosis also significantly alleviated the damage of podocyte cytoskeleton. In addition, inhibition of cuproptosis mitigated ADR-induced mitochondrial damage as well as the production of reactive oxygen species and depolarization of mitochondrial membrane potential, and restored ATP synthesis. Among the transcriptome sequencing data, the difference of CXCL5 was the most significant. Both high copper and ADR exposure can cause up-regulation of CXCL5, and CXCL5 deletion inhibits the occurrence of cuproptosis, thereby alleviating the podocyte cytoskeleton damage. This suggests that CXCL5 may act upstream of cuproptosis that mediates podocyte cytoskeleton damage. In conclusion, cuproptosis induced by excessive copper accumulation may induce podocyte cytoskeleton damage by promoting mitochondrial dysfunction, thereby causing podocyte injury. This indicates that cuproptosis plays an important role in the pathogenesis of podocyte injury and provides a basis for seeking potential targets for the treatment of chronic kidney disease.

Neferine mitigates angiotensin II-induced atrial fibrillation and fibrosis via upregulation of Nrf2/HO-1 and inhibition of TGF-β/p-Smad2/3 pathways.

Atrial fibrillation (AF) is often associated with atrial fibrosis and oxidative stress. Neferine, a bisbenzylisoquinoline alkaloid, has been reported to exert an antiarrhythmic effect. However, its impact on Angiotensin II (Ang II) infusion-induced AF and the underlying mechanism remains unclear. This study aimed to investigate whether neferine alleviates Ang II-induced AF and explore the underlying mechanisms. Mice subjected to Ang II infusion to induce AF were concurrently treated with neferine or saline. AF incidence, myocardial cell size, fibrosis, and oxidative stress were then examined. Neferine treatment inhibited Ang II-induced AF, atrial size augmentation, and atrial fibrosis. Additionally, we observed that Ang II increased reactive oxygen species (ROS) generation, induced mitochondrial membrane potential depolarization, and reduced glutathione (GSH) and superoxide dismutase (SOD) levels, which were reversed to some extent by neferine. Mechanistically, neferine activated the Nrf2/HO-1 signaling pathway and inhibited TGF-β/p-Smad2/3 in Ang II-infused atria. Zinc Protoporphyrin (ZnPP), an HO-1 inhibitor, reduced the anti-oxidative effect of neferine to some extent and subsequently abolished the beneficial effect of neferine on Ang II-induced AF. These findings provide hitherto undocumented evidence that the protective role of neferine in Ang II-induced AF is dependent on HO-1.

Caffeic acid phenethyl ester restores mitochondrial homeostasis against peritoneal fibrosis induced by peritoneal dialysis through the AMPK/SIRT1 pathway

Increasing evidence suggests that peritoneal fibrosis induced by peritoneal dialysis (PD) is linked to oxidative stress. However, there are currently no effective interventions for peritoneal fibrosis. In the present study, we explored whether adding caffeic acid phenethyl ester (CAPE) to peritoneal dialysis fluid (PDF) improved peritoneal fibrosis caused by PD and explored the molecular mechanism. We established a peritoneal fibrosis model in Sprague–Dawley rats through intraperitoneal injection of PDF and lipopolysaccharide (LPS). Rats in the PD group showed increased peritoneal thickness, submesothelial collagen deposition, and the expression of TGFβ1 and α-SMA. Adding CAPE to PDF significantly inhibited PD-induced submesothelial thickening, reduced TGFβ1 and α-SMA expression, alleviated peritoneal fibrosis, and improved the peritoneal ultrafiltration function. In vitro, peritoneal mesothelial cells (PMCs) treated with PDF showed inhibition of the AMPK/SIRT1 pathway, mitochondrial membrane potential depolarization, overproduction of mitochondrial reactive oxygen species (ROS), decreased ATP synthesis, and induction of mesothelial-mesenchymal transition (MMT). CAPE activated the AMPK/SIRT1 pathway, thereby inhibiting mitochondrial membrane potential depolarization, reducing mitochondrial ROS generation, and maintaining ATP synthesis. However, the beneficial effects of CAPE were counteracted by an AMPK inhibitor and siSIRT1. Our results suggest that CAPE maintains mitochondrial homeostasis by upregulating the AMPK/SIRT1 pathway, which alleviates oxidative stress and MMT, thereby mitigating the damage to the peritoneal structure and function caused by PD. These findings suggest that adding CAPE to PDF may prevent and treat peritoneal fibrosis.

The toxic effects induced by benzethonium chloride on Daphnia carinata over two generations: Resistance and higher sublethal toxicity

With the widespread utilization of the sanitizing product benzethonium chloride (BEC) throughout the coronavirus pandemic, concerns have emerged regarding its potential hazards. Nevertheless, the long-term and multigenerational toxic effects of BEC on aquatic organisms remains unexplored. This study investigates acute and chronic toxicity, oxidative stress, mitochondrial membrane potential, ATP concentrations, and gene expression using Daphnia carinata as the model organism. Meanwhile, hierarchical clustering analysis was utilized to investigate phenotypic effects among different treatment groups. The integrated biomarker response index version 2 (IBRv2) was employed to estimate the deviation in toxic effects over two generations. These results indicated that D. carinata in the second generation exhibited higher survival rate and lower levels of oxidative stress than the first generation. However, the higher sublethal effects were found in the second generation as follows, the weakened growth performance, mitochondrial membrane potential depolarization, reduced ATP concentrations, and down-regulated gene expression. The mitochondrial toxicity induced by BEC may account for the distinct toxic effects exhibited in two generations. The findings here can assist with the evaluation of potential risk for BEC on aquatic organisms, and provide new insight into the cross-generational toxicity mechanisms of pollutants in aquatic ecosystems.

Female Sex Protects Cerebral Arteries from Mitochondrial Membrane Potential Depolarization and Cell Death Induced by Reactive Oxygen Species

Stroke and traumatic brain injury augment the production of reactive oxygen species (ROS) leading to apoptosis in cerebral arteries, and females are more resilient to vascular damage than males. Depolarization of mitochondrial membrane potential (ΔΨm) is a key event in apoptosis, however, when ΔΨm is suffciently depolarized, ATP synthase can reverse directions and act as a proton pump to limit ΔΨm depolarization. We hypothesized that ATP synthase attenuates depolarization of ΔΨm in cerebral arteries during acute oxidative stress thus limiting cell death. Posterior cerebral arteries (PCA; ~80 μm diameter) from male and female mice (age, 4-6 mo) were isolated, cannulated, and pressurized to 90 cm H2O at 36°C. Cell death was quantified with Hoechst 33342 (1 μM, labels all nuclei) and propidium iodide (2 μM, labels dead nuclei), and ΔΨm was evaluated with tetramethylrhodamine methyl ester (TMRM, 10 nM). Vessels were exposed to H2O2 (200 μM) in the presence/absence of ATP synthase inhibitor oligomycin (2 μM). H2O2 exposure (50 min) led to significantly (p<0.05) greater smooth muscle cell death in males compared to females (30±7.4% vs. 7±3%; n=8); there was a similar trend for endothelial cell death. Oligomycin greatly augmented apoptosis in PCAs from both males and females to ~80% and eliminated differences between sexes. Consistently, H2O2 evoked a more robust depolarization of ΔΨm in males vs. females and oligomycin enhanced ΔΨm depolarization to H2O2 leading to a similar response in both sexes. To directly depolarize ΔΨm, PCAs were treated with the protonophore FCCP (10 μM). Surprisingly, depolarization of ΔΨm to FCCP was greater in males vs. females, and oligomycin augmented depolarization and eliminated differences between sexes. FCCP initiated minimal (<5%) cell death at 50-min or 3-h exposure; oligomycin did not alter cell death to FCCP. We conclude that: 1) cerebral vessels from female mice possess greater resilience to H2O2 -induced apoptosis than males by limiting depolarization of ΔΨm; and 2) oxidative stress triggers cell death through pathways in addition to ΔΨm depolarization. Support: NIH R01NS134690. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Versatile Fluorescence Lifetime-Based Copper Probe to Quantify Mitochondrial Membrane Potential and Reveal Its Interaction with Protein Aggregation.

Mitochondria play a crucial role in maintaining cellular homeostasis, and the depolarization of mitochondrial membrane potential (MMP) is an important signal of apoptosis. Additionally, protein misfolding and aggregation are closely related to diseases including neurodegenerative diseases, diabetes, and cancers. However, the interaction between MMP changes and disease-related protein aggregation was rarely studied. Herein, we report a novel "turn-on" fluorescent probe MitoRhB that specifically targets to mitochondria for Cu2+ detection in situ. The fluorescence lifetime (τ) of MitoRhB exhibits a positive correlation with MMP changes, allowing us to quantitatively determine the relative MMP during SOD1 (A4 V) protein aggregation. Finally, we found that (1) the increasing concentrations of copper will accelerate the depolarization of mitochondria and reduce MMP; (2) the depolarization of mitochondria can intensify the degree of protein aggregation, suggesting a new routine of copper-induced cell death mediated through abnormal MMP depolarization and protein aggregation.

Isobavachalcone induces hepatotoxicity in zebrafish embryos and HepG2 cells via the System Xc--GSH-GPX4 signaling pathway in ferroptosis response.

Isobavachalcone (IBC) is a flavonoid component of the traditional Chinese medicine Psoraleae Fructus, with a range of pharmacological properties. However, IBC causes some hepatotoxicity, and the mechanism of toxicity is unclear. The purpose of this paper was to investigate the possible mechanism of toxicity of IBC on HepG2 cells and zebrafish embryos. The results showed that exposure to IBC increased zebrafish embryo mortality and decreased hatchability. Meanwhile, IBC induced liver injury and increased expression of ALT and AST activity. Further studies showed that IBC caused the increase of ROS and MDA the decrease of CAT, GSH, and GSH-Px; the increase of Fe2+ content; and the changes of ferroptosis related genes (acsl4, gpx4, and xct) and iron storage related genes (tf, fth, and fpn) in zebrafish embryos. Through in vitro verification, it was found that IBC also caused oxidative stress and increased Fe2+ content in HepG2 cells. IBC caused depolarization of mitochondrial membrane potential (MMP) and reduction of mitochondrial ATP, as well as altered expression of ACSl4, SLC7A11, GPX4, and FTH1 proteins. Treatment of HepG2 cells with ferrostatin-1 could reverse the effect of IBC. Targeting the System Xc--GSH-GPX4 pathway of ferroptosis and preventing oxidative stress damage might offer a theoretical foundation for practical therapy and prevention of IBC-induced hepatotoxicity.

Unveil the mechanism for EHMT -- A novel triterpenoid inhibits proliferation and induces apoptosis in colon cancer through ROS-mediated JNK signaling pathway

Colon cancer ranks among the most prevalent malignancies worldwide, trailing only lung and breast cancer in incidence. Despite the availability of numerous therapeutic strategies, the burden of new cases and fatalities remains high in countries undergoing socioeconomic transitions. Natural products offer promising avenues for developing more effective and less toxic anticancer agents, expanding the clinical arsenal. In this investigation, we isolated a triterpenoid, (21 S,23 R,24 R)-21,23-epoxy-24-hydroxy-21-methoxytirucalla-7,25-dien-3-one (EHMT), from the fruits of Melia azedarach L., which exhibited significant inhibitory activity against colon cancer cells while sparing normal cells. EHMT effectively curtailed colony formation and induced apoptosis and cell cycle arrest in the HCT116 cell line. Furthermore, EHMT prompted the generation of reactive oxygen species (ROS) and the depolarization of mitochondrial membrane potential. Notably, EHMT treatment triggered ROS-mediated cell apoptosis via activation of the JNK signaling pathway in HCT116 cells. Additionally, our findings extended to Caenorhabditis elegans, where EHMT induced ROS accumulation and apoptosis. Collectively, these findings position EHMT as a promising candidate for the development of anticancer agents in the treatment of colon cancer, offering new hope in the battle against this formidable disease.

Synthesis and biological evaluation of folic acid-rotenol conjugate as a potent targeted anticancer prodrug

Rotenone, a plant-based agricultural insecticide, has been shown to have anti-tumor activity through targeting mitochondrial complex I in cancer cells. However, off-target toxic side effect on nervous systems have greatly restricted the application of rotenone as anticancer drugs. Here, a folic acid-rotenol (FA-rotenol) conjugate was prepared by covalent coupling of the tumor-targeting ligand folic acid with rotenone derivative—rotenol to enhance its accumulation at tumor site. FA-rotenol conjugates present high in vitro cytotoxicties against several cell lines by inducing mitochondrial membrane potential depolarization and increasing the level of intracellular reactive oxygen species (ROS) to activate the mitochondrial pathway of apoptosis and enhance the G2/M cell cycle arrest. Because of the high affinity with over-expressed folate receptors, FA-rotenol conjugate demonstrated more effective in vivo therapeutic outcomes in 4T1 tumor-bearing mice than rotenone and rotenol. In addition, FA-rotenol conjugate can markedly inhibit the cell migration and invasion of HepG-2 cells. These studies confirm the feasibility of tumor-targeted ligand conjugated rotenone derivatives for targeted antitumor therapy; likewise, they lay the foundations for the development of other rotenol-conjugates with antitumor potential.

Mitochondrial flashes are interlinked with adaptive thermogenesis in brown adipocytes

Mitochondrial flash (mitoflash) is the electrochemical excitation of a single mitochondrion and plays diverse signaling roles in a variety of cells. From the viewpoint of bioenergetics, it represents the transient uncoupling of mitochondrial respiration from ATP synthesis. Brown adipose tissue (BAT) produces heat, primarily via mitochondrial uncoupled respiration. Here, we aim to illustrate the mitoflash activity in BAT thermogenesis. We show that BAT mitochondria undergo stochastic and intermittent mitoflashes at the levels of isolated mitochondria, cultured brown adipocytes, and even intact BAT. A BAT mitoflash contains multiple signals, including transient depolarization of mitochondrial membrane potential, transient matrix alkalization, and bursting production of reactive oxygen species. Importantly, thermogenic activation by β3-adrenergic stimulation dramatically augments mitoflash incidence, with mild effects on its unitary characteristics. Ablation of the thermogenic uncoupling protein 1 (UCP1) showed little effect on mitoflash biogenesis during heat production. Collectively, our results suggest that mitoflash might constitute an elemental signaling activity of mitochondria to regulate BAT thermogenesis.

Apoptotic Potential and Molecular Docking of 3,4-Dihydro-lactucin, a Compound With Anticancer Properties Derived from Microbispora rosea AL22.

Tetrazolium-based cell proliferation assays using MDA-MB-231 and HeLa cells revealed that 3,4-dihydro-lactucin (3,4-DHL), a compound isolated from Microbispora rosea AL22, possesses anticancer properties. Apoptotic cell death was observed in 3,4-DHL-treated cells. Lactucopicrin, a related compound, reportedly exerts anticancer activity against different cancer types. However, data on the anticancer mechanism of lactucins are limited. This study aimed to investigate apoptosis induction in MDA-MB-231 cells treated with 3,4-DHL. Morphological changes, changes in mitochondrial membrane potential, and apoptosis induction in MDA-MB-231 cells treated with 3,4-DHL were investigated. Furthermore, molecular docking and absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis of anti-apoptotic proteins were performed to determine the effector mechanism of 3,4-DHL. 3,4-DHL induced cytotoxicity at a half-maximal inhibitory concentration of 37.62 μg/ml, along with various morphological alterations in apoptotic and viable cells. Furthermore, 3,4-DHL-treated cells showed mitochondrial membrane potential depolarization, intense annexin V-fluorescein isothiocyanate staining, and increased caspase 3 and 8 activities. Molecular-docking studies demonstrated that 3,4-DHL should bind to the active site of various anti-apoptotic proteins, forming stable complexes. Our findings revealed that 3,4-DHL has great potential to be used as an apoptosis-inducing agent in cancer therapy. However, further in-vivo confirmation is required in evaluation of 3,4-DHL as an anticancer agent in cancer chemotherapy.

Quercetin protects cardiomyoblasts against hypertonic cytotoxicity by abolishing intracellular Ca2+ elevations and mitochondrial depolarisation

Background and AimOsmotic changes represent a burden for the body and their limitation would be beneficial. We hypothesized that ubiquitous natural compounds could guard against cytotoxic effects of osmotic stress. We evaluated the anti-hypertonic mechanism of quercetin and 2,3-dehydrosilybin in H9c2 cells in vitro. Experimental procedureProtective effect of both compounds was determined by neutral red assay, cell apoptosis was estimated by measuring caspase-3 activity and verified by western blot and annexin V assay. Phosphorylation level of selected proteins was also detected. Mitochondrial membrane potential was evaluated using dye JC-1. Ca2+ signals were evaluated using genetically encoded fluorescent Ca2+ biosensor GCaMP7f. Formation of reactive oxygen species was measured using an oxidant-sensing probe dihydrofluorescein diacetate. Key resultsQuercetin protected H9c2 cells against hypertonic stress-induced cell death. We observed a significant increase in intracellular Ca2+ levels ([Ca2+]cyto) when cells originally placed in a hypertonic solution were returned to a normotonic environment. Quercetin was found to prevent this increase in [Ca2+]cyto and also the depolarization of mitochondrial membrane potential. Conclusions and implicationsQuercetin, but not 2,3-dehydrosilybin, reduced adverse effects of osmotic stress mainly by dampening the elevation of [Ca2+]cyto and mitochondrial Ca2+ overload. This may consequently prevent MPTP pore opening and activation of apoptosis.

Hesperetin Inhibits Bladder Cancer Cell Proliferation and Promotes Apoptosis and Cycle Arrest by PI3K/AKT/FoxO3a and ER Stress-mitochondria Pathways.

Hesperetin (HSE) is a natural flavonoid derived from the hydrolysis of Hesperidin, which is mainly found in traditional natural Chinese herbs, such as Chenpi and Hovenia caryophyllus. HSE displays anti-inflammatory and antioxidant activities. However, its potential mechanism of action on bladder cancer (BLCA) remains unknown. The aim of this study was to investigate the potential mechanism of action of HSE on BLCA cells. Network pharmacology analysis was used to construct a composite target network, combined with Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to identify HSE-induced cell death patterns and signaling pathway alterations. Cytotoxicity evaluation was determined by CCK-8 assay. A clone formation assay was performed to assess cell proliferative capacity. Scratch and Transwell assays were performed to evaluate cell migration and invasion ability. Hoechst 33342 staining was visualized to observe morphological features of apoptosis. Apoptosis, cycle distribution, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP) changes were examined by flow cytometry. Western blot analysis was performed to analyze the expression of key proteins associated with cell proliferation, apoptosis, cycle block, PI3K/AKT/FoxO3a and endoplasmic reticulum (ER) stress-mitochondrial pathways. Network pharmacology analysis was performed to identify 155 potential candidate targets of HSE-BLCA, and further topological analysis was performed to obtain 34 hub-gene. Enrichment analysis yielded patterns of death and key pathways, revealing that the anti-BLCA effect of HSE may be related to the positive regulation of PI3K/AKT/FoxO3a and ER stress-mitochondrial pathways. in vitro results showed that HSE blocked cell proliferation, migration, and invasion in a concentration-dependent manner and triggered apoptosis, G0/G1 phase blockade, ROS production, and MMP depolarization. In addition, Western blot results showed that HSE downregulated phosphorylated (p)-3-phosphoinositide-dependent kinase-1 (PI3K), phosphorylated (p)-AKT serine/threonine kinase 1 (AKT), phosphorylated (p)-Forkhead box O 3a (FoxO3a), anti-apoptotic proteins, proliferation-associated proteins, and cell cycle promoters, whereas the levels of proteins related to the expression of cell cycle regulators, pro-apoptotic proteins, and ER stress-mitochondrial pathway were up-regulated in BLCA cells by the intervention of HSE. PI3K agonist (YS-49) and ER stress inhibitor (4-PBA) partially or completely reversed HSE-mediated proliferation, apoptosis, and cycle blockade in BLCA cells. The anticancer effects of HSE in BLCA may be attributed to its coordination of actions, inhibiting cell proliferation, migration, and invasion, inducing apoptosis, G0/G1 phase arrest, generating reactive oxygen species, causing MMP loss, and engaging the caspase protein family. These actions are likely mediated through the PI3K/AKT/FoxO3a and ER stress-mitochondrial pathways. Thus, our findings suggest that HSE is a promising novel therapeutic candidate for the prevention and treatment of BLCA.

D-Amino acids differentially trigger an inflammatory environment in vitro

Studies in vivo have demonstrated that the accumulation of d-amino acids (d-AAs) is associated with age-related diseases and increased immune activation. However, the underlying mechanism(s) of these observations are not well defined. The metabolism of d-AAs by d-amino oxidase (DAO) produces hydrogen peroxide (H2O2), a reactive oxygen species involved in several physiological processes including immune response, cell differentiation, and proliferation. Excessive levels of H2O2 contribute to oxidative stress and eventual cell death, a characteristic of age-related pathology. Here, we explored the molecular mechanisms of d-serine (d-Ser) and d-alanine (d-Ala) in human liver cancer cells, HepG2, with a focus on the production of H2O2 the downstream secretion of pro-inflammatory cytokine and chemokine, and subsequent cell death. In HepG2 cells, we demonstrated that d-Ser decreased H2O2 production and induced concentration-dependent depolarization of mitochondrial membrane potential (MMP). This was associated with the upregulation of activated NF-кB, pro-inflammatory cytokine, TNF-α, and chemokine, IL-8 secretion, and subsequent apoptosis. Conversely, d-Ala-treated cells induced H2O2 production, and were also accompanied by the upregulation of activated NF-кB, TNF-α, and IL-8, but did not cause significant apoptosis. The present study confirms the role of both d-Ser and d-Ala in inducing inflammatory responses, but each via unique activation pathways. This response was associated with apoptotic cell death only with d-Ser. Further research is required to gain a better understanding of the mechanisms underlying d-AA-induced inflammation and its downstream consequences, especially in the context of aging given the wide detection of these entities in systemic circulation.

Screening of low-toxic zinc oxide nanomaterials and study the apoptosis mechanism of NSC-34 cells.

With the increasing application of ZnO nanomaterials (ZnO-NMts) in the biomedical field, it is crucial to assess their potential risks to humans and the environment. Therefore, this study aimed to screen for ZnO-NMts with low toxicity and establish safe exposure limits, and investigate their mechanisms of action. The study synthesized 0D ZnO nanoparticles (ZnO NPs) and 3D ZnO nanoflowers (ZnO Nfs) with different morphologies using a hydrothermal approach for comparative research. The ZnO-NMts were characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Mouse brain neuronal cells (NSC-34) were incubated with ZnO NMts for 6, 12, and 24h, and the cell morphology was observed using TEM. The toxic effects of ZnO Nfs on NSC-34 cells were studied using CCK-8 cell viability detection, reactive oxygen species (ROS) measurement, caspase-3 activity detection, Annexin V-FITC/PI apoptosis assay, and mitochondrial membrane potential (Δφm) measurement. The results of the research showed that ZnO-NMts caused cytoplasmic vacuolization and nuclear pyknosis. After incubating cells with 12.5µgmL-1 ZnO-NMts for 12h, ZnO NRfs exhibited the least toxicity and ROS levels. Additionally, there was a significant increase in caspase-3 activity, depolarization of mitochondrial membrane potential (Δφm), and the highest rate of early apoptosis.This study successfully identified ZnO NRfs with the lowest toxicity and determined the safe exposure limit to be<12.5µgmL-1 (12h). These findings will contribute to the clinical use of ZnO NRfs with low toxicity and provide a foundation for further research on their potential applications in brain disease treatment.

Anticancer effect of organotin complexes derived from 2,6-naphthalenedicarboxylic acid by enhancing the ROS generation

Enhancing ROS generation is crucial for anti-metastasis and anti-proliferation effects in anticancer chemotherapeutics. Herein, five organotin complexes [(Ph3Sn)2L] (1) [(R3Sn)2L]n (R = Et 2, R = n-Bu 3), [(n-Bu3Sn)2L(4,4′-bpe)]n (4), [(n-Bu3Sn)2L(4,4′-bipy)]n (5) were synthesized based on appropriate organotin precursors, 2,6-naphthalenedicarboxylic acid (H2L) and auxiliary ligand (4,4′-bipy = 4,4′-bipyridine, 4,4′-bpe = 1,2-di(4-pyridyl)ethylene). It was found that complex 1 exhibits a tetra-coordinated monomeric structure, complexes 2 and 3 show the 2D coordination polymer, and complexes 4 and 5 display the 1D chain with the central tin atoms as all penta-coordinated. These complexes showed high anti-tumor activity in vitro against four cancer cells (A549, HCT116, HepG-2 and HeLa), with IC50 values changed from 0.34±0.01 μM to 25.54 ± 1.55 μM. Additionally, complex 1 could be effective in inhibiting HepG-2 cell migration. Simultaneously, anticancer mechanism studies show that complex 1 induces excessive ROS production in HepG-2 cells, which in turn leads to the occurrence of ROS-induced downstream events, followed by the disintegration of the redox balance, depolarization of mitochondrial membrane potential, nucleus damage and ultimately apoptosis. This work suggests that enhancing the ability of organotin complexes to generate reactive oxygen species and disrupt intracellular redox balance may be a promising anticancer strategy.

Weak base drug-induced endolysosome iron dyshomeostasis controls the generation of reactive oxygen species, mitochondrial depolarization, and cytotoxicity.

Approximately 75 % of marketed drugs have the physicochemical property of being weak bases. Weak-base drugs with relatively high pKa values enter acidic organelles including endosomes and lysosomes (endolysosomes), reside in and de-acidify endolysosomes, and induce cytotoxicity. Divalent cations within endolysosomes, including iron, are released upon endolysosome de-acidification. Endolysosomes are "master regulators of iron homeostasis", and neurodegeneration is linked to ferrous iron (Fe2+)-induced reactive oxygen species (ROS) generation via Fenton chemistry. Because endolysosome de-acidification-induced lysosome-stress responses release endolysosome Fe2+, it was crucial to determine the mechanisms by which a functionally and structurally diverse group of weak base drugs including atropine, azithromycin, fluoxetine, metoprolol, and tamoxifen influence endolysosomes and cause cell death. Using U87MG astrocytoma and SH-SY5Y neuroblastoma cells, we conducted concentration-response relationships for 5 weak-base drugs to determine EC50 values. From these curves, we chose pharmacologically and therapeutically relevant concentrations to determine if weak-base drugs induced lysosome-stress responses by de-acidifying endolysosomes, releasing endolysosome Fe2+ in sufficient levels to increase cytosolic and mitochondria Fe2+ and ROS levels and cell death. Atropine (anticholinergic), azithromycin (antibiotic), fluoxetine (antidepressant), metoprolol (beta-adrenergic), and tamoxifen (anti-estrogen) at pharmacologically and therapeutically relevant concentrations (1) de-acidified endolysosomes, (2) decreased Fe2+ levels in endolysosomes, (3) increased Fe2+ and ROS levels in cytosol and mitochondria, (4) induced mitochondrial membrane potential depolarization, and (5) increased cell death; effects prevented by the endocytosed iron-chelator deferoxamine. Weak-base pharmaceuticals induce lysosome-stress responses that may affect their safety profiles; a better understanding of weak-base drugs on Fe2+ interorganellar signaling may improve pharmacotherapeutics.

Lysosome-targeted ruthenium(II) complex encapsulated with pluronic® F-127 induces oncosis in A549 cells.

Transition metal complexes with characteristics of unique packaging in nanoparticles and remarkable cancer cell cytotoxicity have emerged as potential alternatives to platinum-based antitumor drugs. Here we report the synthesis, characterization, and antitumor activities of three new Ruthenium complexes that introduce 5-fluorouracil-derived ligands. Notably, encapsulation of one such metal complex, Ru3, within pluronic® F-127 micelles (Ru3-M) significantly enhanced Ru3 cytotoxicity toward A549 cells by a factor of four. To determine the mechanisms underlying Ru3-M cytotoxicity, additional in vitro experiments were conducted that revealed A549 cell treatment with lysosome-targeting Ru3-M triggered oxidative stress, induced mitochondrial membrane potential depolarization, and drastically reduced intracellular ATP levels. Taken together, these results demonstrated that Ru3-M killed cells mainly via a non-apoptotic pathway known as oncosis, as evidenced by observed Ru3-M-induced cellular morphological changes including cytosolic flushing, cell swelling, and cytoplasmic vacuolation. In turn, these changes together caused cytoskeletal collapse and activation of porimin and calpain1 proteins with known oncotic functions that distinguished this oncotic process from other cell death processes. In summary, Ru3-M is a potential anticancer agent that kills A549 cells via a novel mechanism involving Ru(II) complex triggering of cell death via oncosis.

Protective effects of amphetamine and methylphenidate against dopaminergic neurotoxicants in SH-SY5Y cells

Full treatment of the second most common neurodegenerative disorder, Parkinson’s disease (PD), is still considered an unmet need. As the psychostimulants, amphetamine (AMPH) and methylphenidate (MPH), were shown to be neuroprotective against stroke and other neuronal injury diseases, this study aimed to evaluate their neuroprotective potential against two dopaminergic neurotoxicants, 6-hydroxydopamine (6-OHDA) and paraquat (PQ), in differentiated human dopaminergic SH-SY5Y cells.Neither cytotoxicity nor mitochondrial membrane potential changes were seen following a 24-hour exposure to either therapeutic concentration of AMPH or MPH (0.001–10 μM). On the other hand, a 24-hour exposure to 6-OHDA (31.25–500 μM) or PQ (100–5000 μM) induced concentration-dependent mitochondrial dysfunction, assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and lysosomal damage, evaluated by the neutral red uptake assay. The lethal concentrations 25 and 50 retrieved from the concentration-toxicity curves in the MTT assay were 99.9 µM and 133.6 µM for 6-OHDA, or 422 µM and 585.8 µM for PQ. Both toxicants caused mitochondrial membrane potential depolarization, but only 6-OHDA increased reactive oxygen species (ROS). Most importantly, PQ-induced toxicity was partially prevented by 1 μM of AMPH or MPH. Nonetheless, neither AMPH nor MPH could prevent 6-OHDA toxicity in this experimental model.According to these findings, AMPH and MPH may provide some neuroprotection against PQ-induced neurotoxicity, but further investigation is required to determine the exact mechanism underlying this protection.

Mitochondria-targeting biocompatible fluorescent BODIPY probes.

An increase in the mitochondrial membrane potential (MMP) is a characteristic feature of cancer and cardiovascular disease. Therefore, it remains of crucial importance to develop new and improved fluorescent probes that are sensitive to the MMP, to report on mitochondrial health and function. Reported here are the design, synthesis, photophysical properties and biological characterisation of a series of BODIPY dyes, BODIPY-Mito-n, for mitochondria-targeted fluorescence imaging applications. Six BODIPY-Mito-n analogues were synthesised under mild conditions, and displayed excellent fluorescence quantum yields of between 0.59 and 0.72 in aqueous environments at physiological pH (pH = 7.4). The incorporation of poly(ethylene glycol) (PEG) chains to the triarylphosphonium cation moiety significantly improved the biocompatibility of the probes (BODIPY-Mito-6, IC50 > 50 μM). All BODIPY-Mito-n compounds demonstrated a high MMP-sensitive localisation in the mitochondria, with Pearson's correlation coefficients (PCC) of between 0.76 and 0.96. Compounds BODIPY-Mito-2 and BODIPY-Mito-6 revealed the highest sensitivity to the MMP, with a decrease in the emission intensity of 62% and 75%, respectively following MMP depolarisation. It is anticipated that the highest MMP sensitivity and enhanced biocompatibility of BODIPY-Mito-6 could lead to the development of new probes for mitochondrial imaging in the future.