Mitochondrial Membrane Permeabilization Research Articles

EFHD1 promotes osteosarcoma proliferation and drug resistance by inhibiting the opening of the mitochondrial membrane permeability transition pore (mPTP) by binding to ANT3

Chemoresistance is the main obstacle in the clinical treatment of osteosarcoma (OS). In this study, we investigated the role of EF-hand domain-containing protein 1 (EFHD1) in OS chemotherapy resistance. We found that the expression of EFHD1 was highly correlated with the clinical outcome after chemotherapy. We overexpressed EFHD1 in 143B cells and found that it increased their resistance to cell death after drug treatment. Conversely, knockdown of EFHD1 in 143BR cells (a cisplatin-less-sensitive OS cell line derived from 143B cells) increased their sensitivity to treatment. Mechanistically, EFHD1 bound to adenine nucleotide translocase-3 (ANT3) and inhibited its conformational change, thereby inhibiting the opening of the mitochondrial membrane permeability transition pore (mPTP). This effect could maintain mitochondrial function, thereby favoring OS cell survival. The ANT3 conformational inhibitor carboxyatractyloside (CATR), which can promote mPTP opening, enhanced the chemosensitivity of EFHD1-overexpressing cells when combined with cisplatin. The ANT3 conformational inhibitor bongkrekic acid (BKA), which can inhibit mPTP opening, restored the resistance of EFHD1 knockdown cells. In conclusion, our results suggest that EFHD1-ANT3-mPTP might be a promising target for OS therapy in the future.

Silver ciprofloxacin (CIPAG): a multitargeted metallodrug in the development of breast cancer therapy.

The anti-proliferative activity of the known metalloantibiotic {[Ag(CIPH)2]NO3∙0.75MeOH∙1.2H2O} (CIPAG) (CIPH = ciprofloxacin) against the human breast adenocarcinoma cancer cells MCF-7 (hormone dependent (HD)) and MDA-MB-231 (hormone independent (HI)) is evaluated. The in vitro toxicity and genotoxicity of the metalloantibiotic were estimated toward fetal lung fibroblast (MRC-5) cells. The molecular mechanism of the CIPAG activity against MCF-7 cells was clarified by the (i) cell morphology, (ii) cell cycle arrest, (iii) mitochondrial membrane permeabilization, and (iv) by the assessment of the possible differential effect of CIPAG on estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) transcriptional activation, applying luciferase reporter gene assay. Moreover, the ex vivo mechanism of CIPAG was clarified by its binding affinity toward calf thymus (CT-DNA).

Oxidized guanosines induce mitochondrial dysfunction and loss of viability in β-cells

The production of reactive oxygen species and oxidative stress promote β-cell dysfunction and impair insulin secretion, thereby contributing to the pathogenesis of type 2 diabetes mellitus (T2DM). The nucleobase guanine is highly sensitive to oxidation, which results in the formation of 8-oxoguanosine (8oxoG) and 8-oxodeoxyguanosine (8oxodG). The urinary excretion of 8oxoG is associated with the risk of mortality in people with T2DM, including from diabetic complications such as cardiovascular disease. However, the cellular mechanisms responsible for this association are poorly defined. Therefore, in this study, we examined the effect of 8oxoG, 8oxodG and other oxidized guanosine derivatives, on the INS-1E β-cell line. Exposure of INS-1E cells to 8oxoG and 8oxodG decreased metabolic activity and promoted cell death by apoptosis. The change in cell viability was similar to that induced by treatment of INS-1E cells with the inflammatory cytokines interleukin 1β (Il-1β) and tumour necrosis factor α (TNFα). Changes in mitochondrial membrane permeability and superoxide radical formation were also observed with 8oxoG, but there was no significant change in the oxidation state of mitochondrial cytochromes or hydrogen peroxide levels in the INS-1E cells. Interestingly, exposure to 8oxoG and 8-oxodG also increased the mRNA expression of stress response genes, including NADPH dehydrogenase quinone 1 (NQO1), and thioredoxin-interacting protein (TXNIP). Together, these results support a potential role of oxidized guanosine derivatives in the induction of β-cell dysfunction, which could be relevant to the pathogenesis of T2DM.

In vivo chronic exposure to inorganic mercury worsens hypercholesterolemia, oxidative stress and atherosclerosis in the LDL receptor knockout mice

Heavy metal exposure leads to multiple system dysfunctions. The mechanisms are likely multifactorial and involve inflammation and oxidative stress. The aim of this study was to evaluate markers and risk factors for atherosclerosis in the LDL receptor knockout mouse model chronically exposed to inorganic mercury (Hg) in the drinking water. Results revealed that Hg exposed mice present increased plasma levels of cholesterol, without alterations in glucose. As a major source and target of oxidants, we evaluated mitochondrial function. We found that liver mitochondria from Hg treated mice show worse respiratory control, lower oxidative phosphorylation efficiency and increased H2O2 release. In addition, Hg induced mitochondrial membrane permeability transition. Erythrocytes from Hg treated mice showed a 50% reduction in their ability to take up oxygen, lower levels of reduced glutathione (GSH) and of antioxidant enzymes (SOD, catalase and GPx). The Hg treatment disturbed immune system cells counting and function. While lymphocytes were reduced, monocytes, eosinophils and neutrophils were increased. Peritoneal macrophages from Hg treated mice showed increased phagocytic activity. Hg exposed mice tissues present metal impregnation and parenchymal architecture alterations. In agreement, increased systemic markers of liver and kidney dysfunction were observed. Plasma, liver and kidney oxidative damage indicators (MDA and carbonyl) were increased while GSH and thiol groups were diminished by Hg exposure. Importantly, atherosclerotic lesion size in the aorta root of Hg exposed mice were larger than in controls. In conclusion, in vivo chronic exposure to Hg worsens the hypercholesterolemia, impairs mitochondrial bioenergetics and redox function, alters immune cells profile and function, causes several tissues oxidative damage and accelerates atherosclerosis development.

Isoquercitrin alleviates pirarubicin-induced cardiotoxicity in vivo and in vitro by inhibiting apoptosis through Phlpp1/AKT/Bcl-2 signaling pathway.

Introduction: Due to the cardiotoxicity of pirarubicin (THP), it is necessary to investigate new compounds for the treatment of THP-induced cardiotoxicity. Isoquercitrin (IQC) is a natural flavonoid with anti-oxidant and anti-apoptosis properties. Thus, the present study aimed to investigate the influence of IQC on preventing the THP-induced cardiotoxicity in vivo and in vitro. Methods: The optimal concentration and time required for IQC to prevent THP-induced cardiomyocyte damage were determined by an MTT assay. The protective effect was further verified in H9c2 and HCM cells using dichlorodihydrofluorescein diacetate fluorescent probes, MitoTracker Red probe, enzyme-linked immunosorbent assay, JC-1 probe, and real time-quantitative polymerase chain reaction (RT-qPCR). Rats were administered THP to establish cardiotoxicity. An electrocardiogram (ECG) was performed, and cardiac hemodynamics, myocardial enzymes, oxidative stress indicators, and hematoxylin-eosin staining were studied. Voltage-dependent anion channel 1 (VDAC1), adenine nucleotide translocase 1 (ANT1), and cyclophilin D (CYPD) were detected by qRT-PCR, and the Phlpp1/AKT/Bcl-2 axis proteins were detected by western blot, confirming that IQC markedly increased cell viability and superoxide dismutase (SOD) levels, diminished the levels of ROS and MDA, and elevated mitochondrial function and apoptosis in vivo and in vitro. Results: Results showed that IQC reduced THP-induced myocardial histopathological injury, electrocardiogram (ECG) abnormalities, and cardiac dysfunction in vivo. IQC also decreased serum levels of MDA, BNP, CK-MB, c-TnT, and LDH, while increasing levels of SOD and GSH. We also found that IQC significantly reduced VDAC1, ANT1, and CYPD mRNA expression. In addition, IQC controlled apoptosis by modulating Phlpp1/AKT/Bcl-2 signaling pathways. IQC markedly increased H9c2 and HCM cell viability and SOD levels, diminished the levels of ROS and MDA, and elevated mitochondrial function in H9c2 and HCM cells to defend against THP-induced cardiomyocyte apoptosis in vitro. The AKT inhibitor IMQ demonstrated that IQC lacked antioxidant and anti-apoptotic properties. Moreover, our data showed that IQC regulates Phlpp1 expression, thereby influencing the expression levels of p-AKT, cytochrome c, caspase-3, caspase-9, Bcl-2, and Bax. Discussion: In conclusion, our results indicate that IQC protects the changes in mitochondrial membrane permeability in cardiomyocytes by regulating the Phlpp1/AKT/Bcl-2 signaling pathway, inhibits the release of cytc from the mitochondrial inner membrane to the cytoplasm, forms apoptotic bodies, induces cell apoptosis, and reduces THP induced cardiotoxicity.

Norcantharidin Sensitizes Colorectal Cancer Cells to Radiotherapy via Reactive Oxygen Species-DRP1-Mediated Mitochondrial Damage.

Norcantharidin (NCTD), a cantharidin derivative, induces ROS generation and is widely used to treat CRC. In this study, we clarified the role and mechanism of action of norcantharidin in increasing CRC sensitivity to radiotherapy. We treated the CRC cell lines LoVo and DLD-1 with NCTD (10 or 50 μmol/L), ionizing radiation (IR, 6 Gy), and a combination of the two and found that NCTD significantly inhibited the proliferation of CRC cells and enhanced their sensitivity to radiotherapy. NCTD induced ROS generation by decreasing the mitochondrial membrane potential, increasing mitochondrial membrane permeability, and promoting cytochrome C release from mitochondria into the cytoplasm. IR combined with NCTD induced ROS production, which activated the mitochondrial fission protein DRP1, leading to increased mitochondrial fission and CRC sensitivity to radiotherapy. NCTD also reduced CRC cell resistance to radiotherapy by blocking the cell cycle at the G2/M phase and decreasing p-CHK2, cyclin B1, and p-CDC2 expression. NCTD and IR also inhibited radiation resistance by causing DNA damage. Our findings provide evidence for the potential therapeutic use of NCTD and IR against CRC. Moreover, this study elucidates whether NCTD can overcome CRC radiation tolerance and provides insights into the underlying mechanisms.

Induction of IFIT1/IFIT3 and inhibition of Bcl-2 orchestrate the treatment of myeloma and leukemia via pyroptosis

Induction of pyroptosis is proposed as a promising strategy for the treatment of hematological malignancies, but little is known. In the present study, we find clioquinol (CLQ), an anti-parasitic drug, induces striking myeloma and leukemia cell pyroptosis on a drug screen. RNA sequencing reveals that the interferon-inducible genes IFIT1 and IFIT3 are markedly upregulated and are essential for CLQ-induced GSDME activation and cell pyroptosis. Specifically, IFIT1 and IFIT3 form a complex with BAX and N-GSDME therefore directing N-GSDME translocalization to mitochondria and increasing mitochondrial membrane permeabilization and triggering pyroptosis. Furthermore, venetoclax, an activator of BAX and an inhibitor of Bcl-2, displays strikingly synergistic effects with CLQ against leukemia and myeloma via pyroptosis. This study thus reveals a novel mechanism for mitochondrial GSDME in pyroptosis and it also illustrates that induction of IFIT1/T3 and inhibition of Bcl-2 orchestrate the treatment of leukemia and myeloma via pyroptosis.

Long-term preservation of established fibroblast lines from six-banded armadillos (Euphractus sexcintus, Linnaeus, 1758) by extended passage and cryopreservation.

Establishing new somatic cell cultures has raised significant attention as an effective and convenient way to preserve genetic samples for different applications. Although many lines have been established in model animals, none derived from six-banded armadillo species is currently available. We report the successful isolation and characterization of fibroblasts from six-banded armadillos, evaluating the cell quality after extended culture and cryopreservation. Initially, we collected ear skin from five captive adult individuals and identified fibroblast lines by morphology, karyotyping, and immunophenotyping assays. The isolated fibroblasts were evaluated after several passages (fourth, seventh, and tenth passages) and cryopreservation by slow freezing. Cell morphology, viability, metabolism, proliferative activity, mitochondrial membrane potential, and apoptosis levels were analyzed. The skin explants had great adhesion, and cell outgrowth could be seen after 3-6 d. The cells were verified as fibroblasts at the fourth passage by vimentin expression and normal karyotype (2n = 58). The viability remained high (> 87%) and constant from the fourth to the tenth passage (p > 0.05). The passages did not change the cell morphology and metabolic and growth rates. Moreover, cryopreservation did not affect most evaluated parameters; post-thawed cells maintained their viability, growth, metabolism, and apoptosis levels. Nevertheless, cryopreservation increased mitochondrial membrane permeability and cell population doubling time compared to non-cryopreserved cells (p < 0.05). In summary, viable fibroblasts can be obtained from six-banded armadillo skin while conserving their quality as the number of passages increases and featuring few changes after cryopreservation.

Impact of Complex Apoptotic Signaling Pathways on Cancer Cell Sensitivity to Therapy.

Anticancer drugs induce apoptotic and non-apoptotic cell death in various cancer types. The signaling pathways for anticancer drug-induced apoptotic cell death have been shown to differ between drug-sensitive and drug-resistant cells. In atypical multidrug-resistant leukemia cells, the c-Jun/activator protein 1 (AP-1)/p53 signaling pathway leading to apoptotic death is altered. Cancer cells treated with anticancer drugs undergo c-Jun/AP-1-mediated apoptotic death and are involved in c-Jun N-terminal kinase activation and growth arrest- and DNA damage-inducible gene 153 (Gadd153)/CCAAT/enhancer-binding protein homologous protein pathway induction, regardless of the p53 genotype. Gadd153 induction is associated with mitochondrial membrane permeabilization after anticancer drug treatment and involves a coupled endoplasmic reticulum stress response. The induction of apoptosis by anticancer drugs is mediated by the intrinsic pathway (cytochrome c, Cyt c) and subsequent activation of the caspase cascade via proapoptotic genes (e.g., Bax and Bcl-xS) and their interactions. Anticancer drug-induced apoptosis involves caspase-dependent and caspase-independent pathways and occurs via intrinsic and extrinsic pathways. The targeting of antiapoptotic genes such as Bcl-2 enhances anticancer drug efficacy. The modulation of apoptotic signaling by Bcl-xS transduction increases the sensitivity of multidrug resistance-related protein-overexpressing epidermoid carcinoma cells to anticancer drugs. The significance of autophagy in cancer therapy remains to be elucidated. In this review, we summarize current knowledge of cancer cell death-related signaling pathways and their alterations during anticancer drug treatment and discuss potential strategies to enhance treatment efficacy.

Extracellular freezing induces a permeability transition in the inner membrane of muscle mitochondria of freeze-sensitive but not freeze-tolerant Chymomyza costata larvae.

Background: Many insect species have evolved the ability to survive extracellular freezing. The search for the underlying principles of their natural freeze tolerance remains hampered by our poor understanding of the mechanistic nature of freezing damage itself. Objectives: Here, in search of potential primary cellular targets of freezing damage, we compared mitochondrial responses (changes in morphology and physical integrity, respiratory chain protein functionality, and mitochondrial inner membrane (IMM) permeability) in freeze-sensitive vs. freeze-tolerant phenotypes of the larvae of the drosophilid fly, Chymomyza costata. Methods: Larvae were exposed to freezing stress at -30°C for 1h, which is invariably lethal for the freeze-sensitive phenotype but readily survived by the freeze-tolerant phenotype. Immediately after melting, the metabolic activity of muscle cells was assessed by the Alamar Blue assay, the morphology of muscle mitochondria was examined by transmission electron microscopy, and the functionality of the oxidative phosphorylation system was measured by Oxygraph-2K microrespirometry. Results: The muscle mitochondria of freeze-tolerant phenotype larvae remained morphologically and functionally intact after freezing stress. In contrast, most mitochondria of the freeze-sensitive phenotype were swollen, their matrix was diluted and enlarged in volume, and the structure of the IMM cristae was lost. Despite this morphological damage, the electron transfer chain proteins remained partially functional in lethally frozen larvae, still exhibiting strong responses to specific respiratory substrates and transferring electrons to oxygen. However, the coupling of electron transfer to ATP synthesis was severely impaired. Based on these results, we formulated a hypothesis linking the observed mitochondrial swelling to a sudden loss of barrier function of the IMM.

Ginsenoside Re regulates mitochondrial biogenesis through Nrf2/HO-1/PGC-1α pathway to reduce hypoxia/reoxygenation injury in H9c2 cells

This article explored the mechanism by which ginsenoside Re reduces hypoxia/reoxygenation(H/R) injury in H9c2 cells by regulating mitochondrial biogenesis through nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/peroxisome prolife-rator-activated receptor gamma coactivator-1α(PGC-1α) pathway. In this study, H9c2 cells were cultured in hypoxia for 4 hours and then reoxygenated for 2 hours to construct a cardiomyocyte H/R injury model. After ginsenoside Re pre-administration intervention, cell activity, superoxide dismutase(SOD) activity, malondialdehyde(MDA) content, intracellular reactive oxygen species(Cyto-ROS), and intramitochondrial reactive oxygen species(Mito-ROS) levels were detected to evaluate the protective effect of ginsenoside Re on H/R injury of H9c2 cells by resisting oxidative stress. Secondly, fluorescent probes were used to detect changes in mitochondrial membrane potential(ΔΨ_m) and mitochondrial membrane permeability open pore(mPTP), and immunofluorescence was used to detect the expression level of TOM20 to study the protective effect of ginsenoside Re on mitochondria. Western blot was further used to detect the protein expression levels of caspase-3, cleaved caspase-3, Cyto C, Nrf2, HO-1, and PGC-1α to explore the specific mechanism by which ginsenoside Re protected mitochondria against oxidative stress and reduced H/R injury. Compared with the model group, ginse-noside Re effectively reduced the H/R injury oxidative stress response of H9c2 cells, increased SOD activity, reduced MDA content, and decreased Cyto-ROS and Mito-ROS levels in cells. Ginsenoside Re showed a good protective effect on mitochondria by increasing ΔΨ_m, reducing mPTP, and increasing TOM20 expression. Further studies showed that ginsenoside Re promoted the expression of Nrf2, HO-1, and PGC-1α proteins, and reduced the activation of the apoptosis-related regulatory factor caspase-3 to cleaved caspase-3 and the expression of Cyto C protein. In summary, ginsenoside Re can significantly reduce I/R injury in H9c2 cells. The specific mechanism is related to the promotion of mitochondrial biogenesis through the Nrf2/HO-1/PGC-1α pathway, thereby increasing the number of mitochondria, improving mitochondrial function, enhancing the ability of cells to resist oxidative stress, and alleviating cell apoptosis.

Ethanolic extract from leaves of tithonia diversifolia induces apoptosis in HCT-116 cells through oxidative stress

ABSTRACT Tithonia diversifolia is a perennial bushy plant found in South America with significant ethnopharmacological importance as an antimalarial, antidiabetic, antibacterial, and anticancer agent. The aim of the present study was to determine the cytotoxicity of the ethanolic extract from leaves of T. diversifolia (TdE) on human cancer cell lines (HCT-116, SNB-19, NCIH-460 and MCF-7), as well as the mechanism of action involved in cell death and cellular modulation of oxidative stress. The TdE exhibited significant activity with IC50 values ranging from 7.12 to 38.41 μg/ml, with HCT-116 being the most sensitive cell line. Subsequent experiments were conducted with HCT-116 cell line. TdE decreased the number of viable cells, followed by induction of apoptotic events, increase in mitochondrial membrane permeabilization, and enhanced G2/M phase of the cell cycle. Pro-oxidative effects including elevated acidic vesicular organelle formation, lipid peroxidation, and nitric oxide by-products, as well as reduced levels of intracellular glutathione and reactive oxygen species production were also observed following incubation with TdE, which may lead to DNA damage followed by apoptotic cell death. These results demonstrate the potential of TdE ethanolic leaf extraction for biological activity and enhance the importance of continuing to study natural sources of plants for the development of anticancer agents.

Antifungal Activity of Cedrol from Cunninghamia lanceolate var. konishii against Phellinus noxius and Its Mechanism.

Phellinus noxius is a highly destructive fungus that causes brown root disease in trees, leading to decay and death. In Taiwan, five prized woods-Taiwania cryptomerioides, Calocedrus macrolepis var. formosana, Cunninghamia lanceolata var. konishii, Chamaecyparis formosensis, and Chamaecyparis obtusa var. formosana-are known for their fragrance and durability. This study aims to explore the anti-brown-root-rot-fungus activity of Cunninghamia lanceolata var. konishii (CL) essential oil (CLOL) and its primary components, while also delving into their mechanisms of action and inhibition pathways. The essential oil (CLOL) from CL wood demonstrated significant efficacy against P. noxius, with an inhibitory concentration (IC50) of 37.5 µg/mL. Cedrol, the major component (78.48%) in CLOL, emerged as a potent antifungal agent, surpassing the reference drug triflumizole. Further assays with cedrol revealed a stronger anti-brown-root-disease activity (IC50 = 15.7 µg/mL) than triflumizole (IC50 = 32.1 µg/mL). Scanning electron microscopy showed deformation and rupture of fungal hyphae treated with CLOL and cedrol, indicating damage to the fungal cell membrane. Cedrol-induced oxidative stress in P. noxius was evidenced by increased reactive oxygen species (ROS) levels, leading to DNA fragmentation, mitochondrial membrane potential reduction, and fungal apoptosis through the mitochondrial pathway. Gel electrophoresis confirmed cedrol-induced DNA fragmentation, whereas TUNEL staining demonstrated increased apoptosis with rising cedrol concentrations. Moreover, protein expression analysis revealed cedrol-triggered release of cytochrome c, activation of caspase-9, and subsequent caspase-3 activation, initiating a caspase cascade reaction. This groundbreaking study establishes cedrol as the first compound to induce apoptosis in P. noxius while inhibiting its growth through oxidative stress, an increase in mitochondrial membrane permeability, and activation of the mitochondrial pathway. The findings offer compelling evidence for cedrol's potential as an effective antifungal agent against the destructive brown root disease caused by P. noxius.

Augmented mitochondrial apoptotic signaling impairs C2C12 myoblast differentiation following cellular aging through sequential passaging.

Aging is associated with the steady decline of several cellular processes. The loss of skeletal muscle mass, termed sarcopenia, is one of the major hallmarks of aging. Aged skeletal muscle exhibits a robust reduction in its regenerative capacity due to dysfunction (i.e., senescence, lack of self-renewal, and impaired differentiation) of resident muscle stem cells, called satellite cells. To replicate aging in vitro, immortalized skeletal muscle cells (myoblasts) can be treated with various agents to mimic age-related dysfunction; however, these come with their own set of limitations. In the present study, we used sequential passaging of mouse myoblasts to mimic impaired differentiation that is observed in aged skeletal muscle. Further, we investigated mitochondrial apoptotic mechanisms to better understand the impaired differentiation in these "aged" cells. Our data shows that sequential passaging (>20 passages) of myoblasts is accompanied with significant reductions in differentiation and elevated cell death. Furthermore, high-passage (HP) myoblasts exhibit greater mitochondrial-mediated apoptotic signaling through mitochondrial BAX translocation, CYCS and AIFM1 release, and caspase-9 activation. Finally, we show that inhibition of mitochondrial outer membrane permeability partly recovered differentiation in HP myoblasts. Together, our findings suggests that mitochondrial apoptotic signaling is a contributing factor to the diminished differentiation that is observed in aged myoblasts.

Effects of Concomitant Use of N-acetylcysteine and Cyclosporine A on Acetaminophen-induced Acute Kidney Injury in Mice

Background: Acetaminophen (APAP), a commonly used analgesic, causes acute kidney injury (AKI) in overdose although it is rare. Mitochondrial dysfunction plays a major role in the pathophysiology of renal damage, although the exact molecular mechanism is unknown. This study aimed to evaluate the potential therapeutic effect of cyclosporin A (CsA), a mitochondrial membrane permeability transition pore (MPTP) inhibitor, with N-acetylcysteine (NAC) in APAP-induced AKI. Methods: Male BALB/c mice were divided into control, APAP, APAP+NAC, APAP+CsA and APAP+NAC+CsA groups (n=6). A single dose of APAP (400 mg/kg) was administered intraperitoneally. All other treatments (1200 mg/kg NAC, 50 mg/kg CsA) were performed intraperitoneally 3 h after APAP administration. All animals were decapitated and blood samples and kidney tissue samples were collected for evaluation. Serum creatinine (Cr) and blood urea nitrogen (BUN) levels were measured. The kidney tissue 8-hydroxy-deoxyguanosine (8-OHdG), cytochrome c (Cytc) and 3-nitrotyrosine (3-NT) levels and cytochrome c (Cytc) expressions were determined. Result: Increased Cr and BUN levels, histopathological examinations and expressions of 8-OHdG, 3-NT and Cytc were detected in the APAP group. Combined NAC+CsA treatment sufficiently reversed oxidative stress, serum Cr and BUN levels and histopathological alterations induced by APAP. Moreover, cytc levels and renal tubular injury were remarkably reduced by combined drug treatment compared to the APAP+NAC group. These data suggest that the therapeutic effect of combined NAC+CsA treatment in mice with APAP-induced nephrotoxicity can be related to the combination of the antioxidant effect of NAC and the mitochondrial MPTP inhibitor effect of CsA.

In Situ Raman Spectroscopy Reveals Cytochrome c Redox-Controlled Modulation of Mitochondrial Membrane Permeabilization That Triggers Apoptosis.

The selective interaction of cytochrome c (Cyt c) with cardiolipin (CL) is involved in mitochondrial membrane permeabilization, an essential step for the release of apoptosis activators. The structural basis and modulatory mechanism are, however, poorly understood. Here, we report that Cyt c can induce CL peroxidation independent of reactive oxygen species, which is controlled by its redox states. The structural basis of the Cyt c-CL binding was unveiled by comprehensive spectroscopic investigation and mass spectrometry. The Cyt c-induced permeabilization and its effect on membrane collapse, pore formation, and budding are observed by confocal microscopy. Moreover, cytochrome c oxidase dysfunction is found to be associated with the initiation of Cyt c redox-controlled membrane permeabilization. These results verify the significance of a redox-dependent modulation mechanism at the early stage of apoptosis, which can be exploited for the design of cytochrome c oxidase-targeted apoptotic inducers in cancer therapy.

New cyclophilin D inhibitor rescues mitochondrial and cognitive function in Alzheimer's disease.

Mitochondrial dysfunction is an early pathological feature of Alzheimer disease and plays a crucial role in the development and progression of Alzheimer's disease. Strategies to rescue mitochondrial function and cognition remain to be explored. Cyclophilin D (CypD), the peptidylprolyl isomerase F (PPIase), is a key component in opening the mitochondrial membrane permeability transition pore, leading to mitochondrial dysfunction and cell death. Blocking membrane permeability transition pore opening by inhibiting CypD activity is a promising therapeutic approach for Alzheimer's disease. However, there is currently no effective CypD inhibitor for Alzheimer's disease, with previous candidates demonstrating high toxicity, poor ability to cross the blood-brain barrier, compromised biocompatibility and low selectivity. Here, we report a new class of non-toxic and biocompatible CypD inhibitor, ebselen, using a conventional PPIase assay to screen a library of ∼2000 FDA-approved drugs with crystallographic analysis of the CypD-ebselen crystal structure (PDB code: 8EJX). More importantly, we assessed the effects of genetic and pharmacological blockade of CypD on Alzheimer's disease mitochondrial and glycolytic bioenergetics in Alzheimer's disease-derived mitochondrial cybrid cells, an ex vivo human sporadic Alzheimer's disease mitochondrial model, and on synaptic function, inflammatory response and learning and memory in Alzheimer's disease mouse models. Inhibition of CypD by ebselen protects against sporadic Alzheimer's disease- and amyloid-β-induced mitochondrial and glycolytic perturbation, synaptic and cognitive dysfunction, together with suppressing neuroinflammation in the brain of Alzheimer's disease mouse models, which is linked to CypD-related membrane permeability transition pore formation. Thus, CypD inhibitors have the potential to slow the progression of neurodegenerative diseases, including Alzheimer's disease, by boosting mitochondrial bioenergetics and improving synaptic and cognitive function.

Bioactive compound D-Pinitol-loaded graphene oxide-chitosan-folic acid nanocomposite induced apoptosis in human hepatoma HepG-2 cells

In the present study, we formulated a nanocomposite—comprising graphene oxide, chitosan, and folic acid—as a nanocarrier loaded with the phytochemical d-Pinitol and then subjected to spectroscopic and microscopic analyses to assess the shape, size, and structure of the nanocomposite. The nanocomposite was further subjected to cytotoxic analysis against hepatoma HepG-2 and non-malignant Vero cells using the MTT assay. The generation of ROS, disruption of mitochondrial membrane permeability, and induction of cell death were assessed with corresponding fluorescent staining. Finally, the apoptotic proteins Bax, Bcl2, and caspases were quantified using commercial kits. The characterization analysis confirmed the synthesis of the agglomerated spherical GO Chitosan D-Pinitol FA nanocomposites with an average particle size of 161.50 nm. The nanocomposite remarkably inhibited the HepG-2 cell viability with an IC50 concentration of 7.5 μg/mL but did not disturb the growth of non-malignant Vero cells. It effectively promoted the generation of ROS, disrupted MMP, and induced apoptosis in hepatoma HepG-2 cells, whereas it rendered minimal cytotoxicity even at high doses in mammalian Vero cells. It also promoted the caspase-3, -9, and Bax expressions but inhibited the Bcl-2 expression in the HepG-2 cells. Our findings suggest that the GO Chitosan D-Pinitol FA nanocomposite is a potent drug-loaded nanocarrier that can effectively induce an anticancer effect in hepatoma HepG-2 cells.

Protective role of melatonin in ultraviolet radiation- induced oxidative stress in human skin photoaging

The ultraviolet radiation of the sun that reaches the earth is made up of ultraviolet A (95%) and ultraviolet B (5%). Exposure to ultraviolet radiation (UVR) is the main factor in photoaging. Chronic exposure to sunlight acts as an environmental stressor, leading to oxidative damage or stress. Oxidative damage stimulates the accumulation of free radicals, such as reactive oxygen species (ROS) or reactive nitrogen species (RNS) that are responsible for premature skin aging. Photoaged skin is characterized externally by irregular pigmentation, wrinkles, and reduced skin elasticity, and internally by the breakdown of dermal collagen and elastin. Free radicals can be scavenged and the skin can be protected from further oxidative damage by antioxidants. Melatonin is a hormone produced mainly by the pineal gland, as well as many other organs, including the skin. One of the functions of melatonin is exerted by the antioxidative melatoninergic system to prevent ultraviolet (UV)-induced skin photoaging. The aim of this review was to study the protective effect of melatonin on skin photoaging resulting from UVR exposure. The references were tracked using various databases, such as Google Scholar and PubMed with regard to publications in English for the last 5 years (2019-2023). Melatonin inhibits UVR-induced aging in multiple ways, such as protecting skin cells, binding free radicals, accelerating the activity of antioxidant enzymes, preventing increased mitochondrial membrane permeability, and assisting electron transport efficiency in the mitochondrial respiratory chain. Exogenous application of melatonin is usually by the oral route, but for localized effects on the skin, topical administration is recommended, with consideration of preparations with a better half-life and bioavailability. Understanding the protective antioxidant function of melatonin in UV-induced skin photoaging helps to optimize its application. The protective properties of melatonin against UVR-oxidative stress-induced photoaging will be further explored in this review.

VDAC1, as a downstream molecule of MLKL, participates in OGD/R-induced necroptosis by inducing mitochondrial damage

Ischemia-reperfusion (I/R) injury constitutes a significant risk factor for a range of diseases, including ischemic stroke, myocardial infarction, and trauma. Following the restoration of blood flow post-tissue ischemia, oxidative stress can lead to various forms of cell death, including necrosis, apoptosis, autophagy, and necroptosis. Recent evidence has highlighted the crucial role of mitochondrial dysfunction in I/R injury. Nevertheless, there remains much to be explored regarding the molecular signaling network governing cell death under conditions of oxidative stress. Voltage-dependent anion channel 1 (VDAC1), a major component in the outer mitochondrial membrane, is closely involved in the regulation of cell death. In a cellular model of oxygen-glucose deprivation and reoxygenation (OGD/R), which effectively simulates I/R injury in vitro, our study reveals that OGD/R induces VDAC1 oligomerization, consequently exacerbating cell death. Furthermore, we have revealed the translocation of mixed lineage kinase domain-like protein (MLKL) to the mitochondria, where it interacts with VDAC1 following OGD/R injury, leading to an increased mitochondrial membrane permeability. Notably, the inhibition of MLKL by necrosulfonamide hinders the binding of MLKL to VDAC1, primarily by affecting the membrane translocation of MLKL, and reduces OGD/R-induced VDAC1 oligomerization. Collectively, our findings provide preliminary evidence of the functional association between MLKL and VDAC1 in the regulation of necroptosis.