Changes In Membrane Potential Research Articles

Moringa oleifera L. leaf extract induces cell cycle arrest and mitochondrial apoptosis in Dalton's Lymphoma: An in vitro and in vivo study

Ethnopharmacological relevanceThe present work is based on a wide spectrum of evidences available from scientific literature which reflects nutritional and medicinal values of natural products such as plants and their extracts. Moringa oleifera is one such popular plant species amidst indigenous tribal communities which is frequently used to treat ailments such as piles, sore throat, eye and ear infections and even poisonous bites of tropical fauna such as insects or snakes. Furthermore decoction of leaf and bark was used to cure fever and cough. Evidences further reveal that Moringa oleifera L. (Family Moringaceae), is widely distributed not only over the Indian sub-continent, but also over Philippines, Central America, Saudi Arabia and the Caribbean Islands and have been traditionally used to treat cancers since ancient times. However, therapeutic effects of Moringa oleifera on Non-Hodgkin Lymphoma (NHL) are yet to be established. Aim of the studyThe study aims to investigate the anti-cancer effects of Moringa oleifera leaf extract against murine NHL Non-Hodgkin cells in vitro and in vivo. Material and methodsThe pharmacologically active compounds of Moringa oleifera leaf extract were identified by GC-HRMS analysis. Tests of Moringa oleifera leaf extract's cytotoxicity against DL cells were carried out using the MTT assay. Chromatin condensation along with other morphological alterations were visualized through Fluorescence microscopy. Changes in the mitochondrial membrane potential (ΔΨm), the cell cycle, and apoptosis were analysed through flow cytometer. We tried to identify proteins involved in apoptosis and cell cycle through Western blotting using BALB/c mice as a model organism. ResultsGC-HRMS study revealed that a methanol based leaf extract of Moringa oleifera (MOML) comprises of a variety of bioactive chemicals. Our results indicate that MOML successfully reduced the proliferation of DL cells by lowering ΔΨm, changing overall cell morphology. DL cells treated with MOML showed arrested cell cycle at the G2/M phase and substantially up-regulated the expression of p53 and p21. Elevated levels of Bax, Cyt-c, and Caspase-3 and lowered expression levels of Bcl-2 protein suggested induction of apoptosis. Mechanistically, the anticancer efficacy of MOML is attributed to MEK/ERK-mediated pathway inactivation in DL cells. It is also interesting to note that MOML-mediated inhibition of DL growth was accompanied by apoptosis induction and improvement in hematological parameters in DL-bearing mice. ConclusionOur finding suggested that MOML induces apoptosis and abrogates the growth of Dalton's lymphoma both in vitro and in vivo.

Mitochondria-targeted photosensitizer based nanoplatform loading glutathione inhibitor for enhanced breast cancer photodynamic therapy

Photodynamic therapy (PDT) is an approved photoactivated technique that has been widely used for anti-tumor therapy through the generation of excessive ROS. However, the poor water solubility and targeting ability of photosensitizer and ROS depleting by high concentrations of glutathione (GSH) in the tumor microenvironment will limit the efficiency of PDT and synergistic therapy. Herein, we report a mitochondria targeted heterodimeric photosensitizer (TPP-TK-PPa) that can form co-delivery nanoplatform together with DSPE-PEG2000 and GSH-depleting agent DEM through the self-assembly. The TPP-TK-PPa/DEM NPs reveals excellent mitochondrial targeting ability and can efficiently deliver the therapeutic agents to tumor cells and release them into the mitochondria in an ROS-responsive manner. Additionally, The TPP-TK-PPa/DEM NPs can induce in situ production of ROS during PDT and reduce ROS consumption by decreasing intracellular GSH levels, which is attributed to the progressive release of DEM after cellular internalization. In addition, generation of ROS during PDT leads to distinct changes in mitochondrial membrane potential and morphology, which ultimately leads to apoptosis. More importantly, the in vitro antitumor activity results demonstrate that TPP-TK-PPa/DEM NPs showed remarkable phototoxicity and the IC50 values of TPP-TK-PPa/DEM NPs in MDA-MB-231 breast cancer cells was as low as 250 nM after PDT. This work provides a strategy for the development of photosensitizers with specific organelle targeting ability, which is of great importance for precise and effective PDT.

Cytotoxic Effects of Cannabidiol on Neonatal Rat Cortical Neurons and Astrocytes: Potential Danger to Brain Development.

The influence of cannabidiol (CBD) on brain development is inadequately understood. Since CBD is considered a non-intoxicating drug, it has attracted great interest concerning its potential medical applicability, including in pregnant women and children. Here, we elucidated the response of perinatal rat cortical neurons and astrocytes to CBD at submicromolar (0.1, 0.5, 1, 5 µM) concentrations attainable in humans. The effect of CBD was concentration- and time-dependent and cell-specific. In neurons, 0.1 µM CBD induced an early and transient change in mitochondrial membrane potential (ΔΨm), ATP depletion, and caspase-8 activation, followed by rapid ATP recovery and progressive activation of caspase-9 and caspase-3/7, resulting in early apoptotic cell death with reduction and shortening of dendrites, cell shrinkage, and chromatin condensation. The decrease in neuronal viability, ATP depletion, and caspase activation due to CBD exposure was prevented by transient receptor potential vanilloid 1 (TRPV1) antagonist. In astrocytes, 0.5 µM CBD caused an immediate short-term dysregulation of ΔΨm, followed by ATP depletion with transient activation of caspase-8 and progressive activation of caspase-9 and caspase-3/7, leading to early apoptosis and subsequent necroptosis. In astrocytes, both TRPV1 and cannabinoid receptor 1 (CB1) antagonists protected viability and prevented apoptosis. Given that CBD is a non-intoxicating drug, our results clearly show that this is not the case during critical periods of brain development when it can significantly interfere with the endogenous cannabinoid system.

Modulation of Neutrophil Activity by Soluble Complement Cleavage Products—An In-Depth Analysis

The cellular and fluid phase-innate immune responses of many diseases predominantly involve activated neutrophil granulocytes and complement factors. However, a comparative systematic analysis of the early impact of key soluble complement cleavage products, including anaphylatoxins, on neutrophil granulocyte function is lacking. Neutrophil activity was monitored by flow cytometry regarding cellular (electro-)physiology, cellular activity, and changes in the surface expression of activation markers. The study revealed no major effects induced by C3a or C4a on neutrophil functions. By contrast, exposure to C5a or C5a des-Arg stimulated neutrophil activity as reflected in changes in membrane potential, intracellular pH, glucose uptake, and cellular size. Similarly, C5a and C5a des-Arg but no other monitored complement cleavage product enhanced phagocytosis and reactive oxygen species generation. C5a and C5a des-Arg also altered the neutrophil surface expression of several complement receptors and neutrophil activation markers, including C5aR1, CD62L, CD10, and CD11b, among others. In addition, a detailed characterization of the C5a-induced effects was performed with a time resolution of seconds. The multiparametric response of neutrophils was further analyzed by a principal component analysis, revealing CD11b, CD10, and CD16 to be key surrogates of the C5a-induced effects. Overall, we provide a comprehensive insight into the very early interactions of neutrophil granulocytes with activated complement split products and the resulting neutrophil activity. The results provide a basis for a better and, importantly, time-resolved and multiparametric understanding of neutrophil-related (patho-)physiologies.

The role of Phe150 in human voltage-gated proton channel

The voltage-gated proton channel Hv1 is a member of voltage-gated ion channels containing voltage-sensing domains (VSDs). The VSDs are made of four membrane-spanning segments (S1 through S4), and their function is to detect changes in membrane potential in the cells. A highly conserved phenylalanine 150 (F150) is located in the S2 segment of human voltage-gated proton channels. We previously discovered that the F150 is a binding site for the open channel blocker 2GBI. Here, we show that the Hv1 VSD voltage-dependent activation requires a hydrophobic group at position F150. We perform double-mutant cycle analysis to probe interactions between F150 and positively charged arginines in the S4 segment of the channel. Our results indicate that F150 interacts with two arginines (R2 and R3) in the S4 segment and catalyzes the transfer of the S4 arginines in the process of voltage-dependent activation.

Antitumor activity of the protein kinase inhibitor 1-(β-D-2′-deoxyribofuranosyl)-4,5,6,7-tetrabromo- 1H-benzimidazole in breast cancer cell lines

BackgroundThe protein kinases CK2 and PIM-1 are involved in cell proliferation and survival, the cell cycle, and drug resistance, and they are found overexpressed in virtually all types of human cancer, including breast cancer. In this study, we investigated the antitumor activity of a deoxynucleoside derivative, the protein kinase inhibitor compound 1-(β-D-2′-deoxyribofuranosyl)-4,5,6,7-tetrabromo-1H-benzimidazole (K164, also termed TDB), inter alia CK2 and PIM-1, on breast cancer cell lines (MDA-MB-231, MCF-7, and SK-BR-3).MethodsAn evaluation of the cytotoxic and proapoptotic effects, mitochondrial membrane potential (ΔΨm), and cell cycle progression was performed using an MTT assay, flow cytometry, and microscopic analysis. The Western blotting method was used to analyze the level of proteins important for the survival of breast cancer cells and proteins phosphorylated by the CK2 and PIM-1 kinases.ResultsThe examined compound demonstrated the inhibition of cell viability in all the tested cell lines and apoptotic activity, especially in the MCF-7 and SK-BR-3 cells. Changes in the mitochondrial membrane potential (ΔΨm), cell cycle progression, and the level of the proteins studied were also observed.ConclusionsThe investigated CK2 and PIM-1 kinase inhibitor K164 is a promising compound that can be considered a potential agent in targeted therapy in selected types of breast cancer; therefore, further research is necessary.

Changes in the mitochondrial membrane potential in endothelial cells can be detected by Raman microscopy

The role of mitochondria goes beyond their capacity to create molecular fuel and includes e.g. the production of reactive oxygen species and the regulation of cell death. In endothelial cells, mitochondria have a significant impact on cellular function under both healthy and pathological conditions. Endothelial dysfunction contributes to the development of various lifestyle diseases and the key players in their pathogenesis are among others vascular inflammation and oxidative stress. The latter is very closely related to mitochondrial dysfunction; however, it is not straightforward. First, because mitochondria are small cellular structures, and second, it requires a sensitive method to follow the subtle biochemical changes. For this purpose, Raman microscopy (RM) was used here, which is considered a high-resolution method and can be applied in situ, usually as a non-labeled technique. In this work, we show that RM can not only locate mitochondria in the cell but also track their functional changes. Moreover, we test if labeling cells with Raman probes (Rp) can improve the specificity and sensitivity of RM (compared to conventional labeled techniques such as fluorescence, and the non-labeled Raman technique). MitoBADY Rp was used to detect changes in mitochondrial membrane potential as an indicator of mitochondrial activity, e.g. hyperpolarization or distortion of the proton gradient in the intermembrane space (depolarization). Thus, we show and compare RM, in the form of a label and non-labeled, to such a subtle cellular analysis.

Mechanisms of Action of the Antimicrobial Peptide Cecropin in the Killing of Candida albicans.

The development of drug resistance has caused fungal infections to become a global health concern. Antimicrobial peptides (AMPs) offer a viable solution to these pathogens due to their resistance to drug resistance and their diverse mechanisms of actions, which include direct killing and immunomodulatory properties. The peptide Cecropin, which is expressed by genetically engineered bacteria, has antifungal effects on Candida albicans. The minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) of Candida albicans were 0.9 μg/mL and 1.8 μg/mL, respectively, detected by the micro-broth dilution method. According to the killing kinetics, the MFC of Cecropin could kill Candida albicans in 40 min. The electron microscope indicated that Cecropin could cause the cell wall to become rough and nicked, eventually killing Candida albicans. The effects of Cecropin on the cell membrane of treated C. albicans, using the 1,6-diphenyl-1,3,5-hexatriene and propidium iodide protocol, showed that they could change the permeability and fluidity, destroy it, and lead to cell necrosis. In addition, Cecropin can also induce cells to produce excessive reactive oxygen species, causing changes in the mitochondrial membrane potential. Therefore, this study provides a certain theoretical basis for the antifungal infection of new antifungal agents.

Mechanisms of 15-Epi-LXA4-Mediated HO-1 in Cytoprotection Following Inflammatory Injury

IntroductionHeme oxygenase-1 (HO-1) is a protective protein in oxidative stress response. LXA4 is an “inflammatory braking signal” that is widely studied at present. The purpose of this study was to elucidate that LXA4 can protect cells by inducing HO-1 in human pulmonary microvascular endothelial cells (HPMECs) as in vitro model to explain acute lung injury after severe acute pancreatitis. MethodsThis study was performed in two parts: (1) To investigate the mechanisms of lipoxin A4-induced HO-1 expression in vitro, the study subjects were divided into four groups: a control group, LXA4 group (50 ng/mL LXA4), inhibitor group (50 ng/mL LXA4 + 20 μM LY294002 or 50 ng/mL LXA4 + 2 nmol/mL Bis II), and agonist group (50 ng/mL insulin-like growth factor 1, PMA). Western blotting was used to detect the expression of p-Akt, Akt, protein kinase C (PKC), p-Nrf2, Nrf2, and Keap1, and the location of Nrf2 was detected using immunofluorescence. The activation of antioxidant responsive element induced by Nrf2 was detected using Electrophoretic Mobility Shift Assay and (2) to investigate the cytoprotection of HO-1 induced by LXA4 in vitro, the subjects were divided into four groups: a control group, tumor necrosis factor α (TNF-α) group (50 ng/mL), LXA4 group (50 ng/mL TNF-α + 50 ng/mL LXA4), and Zinc protoporphyrin IX group (pretreated with 0.5 μM Zinc protoporphyrin IXfor 12 h, followed by 50 ng/mL TNF-α + 50 ng/mL LXA4). BCECF/AM-labeled THP-1 cells were used to analyze the adhesion of HPMECs, and a mitochondrial membrane potential assay kit with JC-1 was used to analyze the apoptosis of HPMECs. ResultsIn part one, (1) LXA4 upregulated the expression of HO-1 in a dose-dependent manner and (2) LXA4 activated the PI3K/Akt and PKC pathways and modulated the phosphorylation and subsequent depolymerization of Nrf2 from Keap1, promoting the translocation of Nrf2 to the nucleus. In part two, (1) LXA4 reversed the changes in mitochondrial membrane potential to alleviate apoptosis in HPMECs and (2) LXA4 attenuated the adhesion of HPMECs induced by TNF-α. ConclusionsLXA4 can activate the PI3K/Akt and PKC pathways and induce the phosphorylation of Nrf2, resulting in the upregulation of HO-1. In addition, LXA4 alleviates adhesion and protects mitochondrial function by upregulating the expression of HO-1, which exerts cytoprotection in severe acute pancreatitis–induced lung injury.

Hyperpolarization induces cytosolic alkalization of mouse sperm flagellum probably through sperm Na+/H+ exchanger.

Hyperpolarization of the membrane potential is a crucial step for mammalian sperm maturation. This work demonstrates that this membrane potential change likely activates a sperm-specific sodium/proton exchanger to induce alkalization in mouse sperm flagellum. The sperm-specific sodium/proton exchanger (sNHE) is an indispensable protein for male fertility in mammals. Nevertheless, it is still unknown how mammalian sNHE is regulated. Evidence obtained from sea urchin sNHE indicates that hyperpolarization of plasma membrane potential (Vm), which is a hallmark of mammalian capacitation, positively regulates the sNHE. Therefore, we explored the activity of sNHE in mouse and human sperm by fluorescence imaging of intracellular pH (pHi) with a ratiometric dye, SNARF-5F. A valinomycin-induced Vm hyperpolarization elevated sperm flagellar pHi of WT mouse but not in sNHE-KO mouse. Moreover, this pHi increase was inhibited in a high K+ (40 mM) medium. These results support the idea that mouse sNHE is activated by Vm hyperpolarization. Interestingly, we observed different types of kinetics derived from valinomycin-induced alkalization, including some (30%) without any pHi changes. Our quantitative pHi determinations revealed that unresponsive cells had a high resting pHi (>7.5), suggesting that the activity of mouse sNHE is regulated by the resting pHi. On the other hand, valinomycin did not increase the pHi of human sperm in the head or the flagellum, regardless of their resting pHi values. Our findings suggest that the regulatory mechanisms of mammalian sNHEs are probably distinct depending on the species.

Albumins as Extracellular Protein Nanoparticles Collaborate with Plasma Ions to Control Biological Osmotic Pressure.

IntroductionPlasma albumins as protein nanoparticles (PNs) exert essential functions in the control of biological osmotic pressure (OP), being involved in regulating water metabolism, cell morphology and cell tension. Understanding how plasma albumins and different electrolytes co-determine biological OP effects is crucial for correct interpretation of hemodynamic disorders, and practical treatment of hypo/hyper-proteinemia.MethodsOptical measurement based on intermediate filament (IF) tension probe was used for real-time evaluation of transmembrane osmotic effects in live cells. Ion fluorescent probes were employed to evaluate intracellular ion levels, and a current clamp was used to measure membrane potential, thus exploring association of electrochemical and osmotic effects.ResultsAlbumins are involved in regulation of intracellular osmolarity by a quantitative relationship. Extracellular PNs can alter membrane potentials by adsorbing counterions, induce production of intracellular PNs and further control the opening of ion channels and ion flow, contributing to electrochemical and osmotic re-equilibrium. Furthermore, various ions interplay with extracellular PNs, showing different osmotic effects: increased levels of calcium ions result in a hypotonic effect, whereas potassium ions induce hyper-osmolarity.ConclusionExtracellular PNs and Ca2+/K+ display antagonistic or synergetic effects in regulating biological OP. Live cells can spontaneously regulate osmotic effects through changing membrane potential and controlling intracellular ion content. Various plasma components need to be comprehensively analyzed, further developing a diagnostic index that considers the biological OP effects of various blood components and improves the evaluation of symptoms and diseases, such as calcium/potassium-hemodynamic disorders and edema.

292 (PB072) - Development of selective MCL-1 heterobifunctional degraders

Background: MCL1 is a member of the Bcl-2 family that plays a key role in cellular homeostasis through the regulation of apoptosis as well as other less characterized functions. The growing recognition of MCL-1 role in cancer cell survival and its association with the development of anticancer drug resistance makes it an attractive target for cancer therapy. Several inhibitors have been developed during the past decade and some of them have entered clinical trials, but no drugs have been approved for clinical use so far. Targeted protein degradation using heterobifunctional degraders has emerged as a novel therapeutic modality in drug discovery. This technology has many potential advantages over traditional inhibitors, e.g. reduced side effects and drug resistance, extended duration of action, elevated selectivity, and the opportunity to target “undruggable” proteins. In this study, we report the development of the first-in-class MCL-1 bifunctional degraders with in vivo PD effect. Material and Methods: Compounds’ affinity to CRBN (Cereblon), an E3 ligase, and to MCL-1 was determined by FP and SPR, respectively. AlphaLISA and HTRF were utilized to confirm ternary complex formation. Cell viability was assessed by CellTiter-Glo Assay. Apoptosis induction and changes in mitochondrial membrane potential were assessed by flow cytometry using Annexin/PI and TMRE staining, respectively. Targeted protein degradation in cells treated with compounds in the absence or presence of apoptosis inhibitor was assessed by WB. The mechanism of action of the compounds was confirmed in cancer cells pre-treated with proteasome or NEDD8-Activating Enzyme inhibitors, or CRBN-targeting siRNA, and then treated with the compounds. MCL-1 abundance and expression of apoptosis markers were confirmed by WB. PK studies were conducted in CD1 mice and Sprague Dawley rats, and PD studies in CB-17 SCID mice xenografted with MV4–11 cells. Results: Synthesized compounds bind CRBN and MCL-1 with high affinity, form a ternary complex in vitro and induce the degradation of MCL-1 in cells, which is caspase- and proteasome-dependent and leads to apoptosis induction. These series of compounds showed a nM range cytotoxic activity in haematological cancer cell lines with the best compounds having an IC50 <100 nM. Moreover, MCL-1 degradation in vivo can be achieved by IP administration leading to the initiation of the programmed cell death. Conclusions: Our compounds have a broad cytotoxic activity toward cultured human cancer cells and a promising in vivo pharmacokinetic profile. Our data indicate that targeting MCL-1 by employing bifunctional degraders represents a potentially new and effective strategy for cancer treatment. No conflict of interest.

322 (PB102) - Cationic amphiphilic drugs as potential anticancer therapy for PDAC

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the sixth leading cause of death worldwide. PDAC carries a 5-y survival of less than 10%, as it is often diagnosed at a late stage and is widely refractory to available therapies. PDAC tumors are hypoperfused, resulting in poor nutrient delivery. To exist in this hostile microenvironment, PDAC cells rely on intracellular and extracellular scavenging pathways to acquire metabolic substrates for growth. Autophagy and other lysosome-dependent recycling pathways are aberrantly regulated in PDAC. Although autophagy modulation is an emerging therapeutic strategy for PDAC, the co-dependences induced by lysosomal inhibition have not been systematically explored. Cationic amphiphilic drugs (CADs) accumulate in lysosomes, destabilize their membranes, and can have anti-tumorigenic effects. CADs include hundreds of pharmacologic agents used to treat a broad spectrum of common diseases. The aim of this study was to investigate anti-PDAC activity of several clinically-approved and newly synthesized CADs. Material and methods: Imidazoline, quinoline, and chrysene derivatives were examined. Anti-tumor activity of CADs was evaluated in human PDAC cell lines in vitro and in Tg(fli1:EGFP) zebrafish model in vivo. Effects on mitochondria, lysosomes, and autophagy flux were examined by immunofluorescent microscopy. Levels of reactive oxygen species (ROS), changes in the mitochondrial membrane potential, and induction of apoptosis by the selected CADs were evaluated by flow cytometry. Results: The newly synthesized derivatives of quinoline and chrysene induced apoptosis in PDAC cells in vitro in the 2–10μM range, while the only FDA-approved imidazoline with apoptotic activity was rilmenidine, at doses higher than 100μM. Most of the tested compounds induced the expansion of the acidic compartment, as measured by acridine orange and LAMP1 staining, and modulated autophagy, as seen by LC3 staining intensity and distribution. Mitochondrial oxidative stress was induced by the tested CADs as well as the dissipation of the mitochondrial membrane potential (ΔΨm). Ultimately, both newly synthesized CADs and rilmenidine limited the growth and spread of PANC1 cancer cells in the Tg(fli1:EGFP) zebrafish model. Conclusion: Quinoline and chrysene CADs had the potential for a dual lysosome/mitochondrion targeting in PDAC cells and were inducing apoptosis almost in a nM range. While rilmenidine was less potent than newly synthetized CADs in inducing cell death in vitro, all the tested compounds had significant anti-tumor effects in the zebrafish model. These results imply that CADs have promising anti-PDAC effects and that effects on the other cells in the tumor microenvironment other than the cancer cells themselves have to be examined. No conflict of interest.

Effect of TP53 deficiency and KRAS signaling on the bioenergetics of colon cancer cells in response to different substrates: A single cell study.

Metabolic reprogramming is a hallmark of cancer. Somatic mutations in genes involved in oncogenic signaling pathways, including KRAS and TP53, rewire the metabolic machinery in cancer cells. We here set out to determine, at the single cell level, metabolic signatures in human colon cancer cells engineered to express combinations of activating KRAS gene mutations and TP53 gene deletions. Specifically, we explored how somatic mutations in these genes and substrate availability (lactate, glucose, substrate deprivation) from the extracellular microenvironment affect bioenergetic parameters, including cellular ATP, NADH and mitochondrial membrane potential dynamics. Employing cytosolic and mitochondrial FRET-based ATP probes, fluorescent NADH sensors, and the membrane-permeant cationic fluorescent probe TMRM in HCT-116 cells as a model system, we observed that TP53 deletion and KRAS mutations drive a shift in metabolic signatures enabling lactate to become an efficient metabolite to replenish both ATP and NADH following nutrient deprivation. Intriguingly, cytosolic, mitochondrial and overall cellular ATP measurements revealed that, in WT KRAS cells, TP53 deficiency leads to an enhanced ATP production in the presence of extracellular lactate and glucose, and to the greatest increase in ATP following a starvation period. On the other hand, oncogenic KRAS in TP53-deficient cells reversed the alterations in cellular ATP levels. Moreover, cell population measurements of mitochondrial and glycolytic metabolism using a Seahorse analyzer demonstrated that WT KRAS TP53-silenced cells display an increase of the basal respiration and tightly-coupled mitochondria, in the presence of glucose as substrate, compared to TP53 competent cells. Furthermore, cells possessing oncogenic KRAS, independently of TP53 status, showed less pronounced mitochondrial membrane potential changes in response to metabolic nutrients. Furthermore, analysis of cytosolic and mitochondrial NADH levels revealed that the simultaneous presence of TP53 deletion and oncogenic KRAS showed the most pronounced alteration in cytosolic and mitochondrial NADH during metabolic stress. In conclusion, our findings demonstrate how activating KRAS mutation and loss of TP53 remodel cancer metabolism and lead to alterations in bioenergetics under metabolic stress conditions by modulating cellular ATP production, NADH oxidation, mitochondrial respiration and function.

ASIC1a induces mitochondrial apoptotic responses in acute lung injury

AimThis study aimed to investigate the promoting effect of acid-sensing ion channel 1a (ASIC1a) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and its mechanisms. Methods: In this experiment, the ALI rat model was induced by intratracheal injection of LPS, and the ASIC1a specific blocker psalmotoxin-1 (PcTx-1) was injected into the tail vein before LPS administration once. Western blot, immunofluorescence, immunohistochemistry and real-time PCR methods were used to detect ASIC1a and apoptosis-related proteins expressions in lung tissue and RLE-6TN rat type II alveolar epithelial cells. Confocal Laser Scanning Microscopy was used to detect Ca2+ fluorescence intensity in RLE-6TN cells. Results: PcTx-1 pretreatment not only inhibited the pathological changes of LPS-induced ALI in lung tissue, but also inhibited lung dysfunction. PcTx-1 also reduced the increased levels of the apoptosis-related proteins B-cell lymphoma-2-associated X (Bax) and cleaved cysteinyl aspartate specific proteinase 3 (Cleaved caspase-3) and increased the decreased level of B-cell lymphoma-2 (Bcl-2) in the lung tissue of the model group. LPS-induced changes in mitochondrial membrane potential and calcium influx in alveolar epithelial cells were also reversed by PcTx-1. Conclusion: ASIC1a induces an apoptotic response in ALI through mitochondrial apoptosis.

P53 Promotes Ferroptosis in Macrophages Treated with Fe3O4 Nanoparticles.

Fe3O4 nanoparticles are the most widely used magnetic nanoparticles in the biomedicine field. The biodistribution of most nanoparticles in vivo is determined by the capture of macrophages; however, the effects of nanoparticles on macrophages remain poorly understood. Here, we demonstrated that Fe3O4 nanoparticles could reduce macrophage viability after 48 h of treatment and induce a shift in macrophage polarization toward the M1 phenotype; RNA sequencing revealed the activation of the ferroptosis pathway and p53 upregulation compared to the control group. The expression in p53, xCT, glutathione peroxidase 4 (GPX4), and transferrin receptor (TFR) in macrophages was similar to that in erastin-induced ferroptosis in macrophages, and the ultrastructural morphology of mitochondria was consistent with that of erastin-treated cells. We used DCFH-DA to estimate the intracellular reactive oxygen species content in Fe3O4 nanoparticles treated with Ana-1 and JC-1 fluorescent probes to detect the mitochondrial membrane potential change; both showed to be time-dependent. Fer-1 inhibited the reduction of the glutathione/oxidized glutathione (GSH/GSSG) ratio and inhibited intracellular oxidative stress states; therefore, Fe3O4 nanoparticles induced ferroptosis in macrophages. Finally, we used pifithrin-α hydrobromide (PFT) as a p53 inhibitor to verify whether the high expression of p53 is involved in mediating this process. After PFT treatment, the live/dead cell rate, TFR, p53 expression, and GPX4 consumption were inhibited and mitigated the GSH/GSSG ratio reduction as well. This indicates that p53 may contribute to Fe3O4 nanoparticle-induced ferroptosis of macrophages. We provide a theoretical basis for the molecular mechanisms of ferroptosis in macrophages and the biotoxicity in vivo induced by Fe3O4 nanoparticles.

H2S regulation of ferroptosis attenuates sepsis‑induced cardiomyopathy.

Ferroptosis is a non-apoptotic form of cell death mediated by reactive oxygen species (ROS). Iron metabolism disorders play a key role in sepsis-induced cardiomyopathy (SIC). While hydrogen sulfide (H2S) inhibits SIC, it is unknown if it does so by controlling ferroptosis. The present study evaluated whether sodium hydrosulfide (NaHS), an H2S donor, alleviates SIC by decreasing ferroptosis. Lipopolysaccharide (LPS) was employed to induce an in vitro model of septic myocardial injury in rat H9c2 cardiomyocytes. The myocardial injury model of septic rats was established by cecal ligation and puncture (CLP). Cardiomyocyte injury was evaluated using Cell Counting Kit-8 and myocardial enzyme assay and hematoxylin and eosin (H&E) staining. The cardiac function of rats was assessed using echocardiography and changes in myocardial fibers and mitochondria were evaluated using H&E staining and transmission electron microscopy, respectively. Fe2+, glutathione and malondialdehyde levels in cardiomyocytes were detected using assay kits, ROS and mitochondrial membrane potential changes were detected using fluorescent probes and ferroptosis and Beclin 1 (BECN1) signaling pathway-associated protein expression levels were semi-quantified using western blotting. NaHS decreased ferroptosis of H9c2 cells induced by LPS and decreased injury of myocardial cells by improving iron metabolism disorder and oxidative stress levels. Furthermore, in vivo results demonstrated that NaHS attenuated CLP-induced septic myocardial ferroptosis and significantly improved cardiac dysfunction in septic rats compared with the CLP group. NaHS was demonstrated to attenuate sepsis-induced myocardial cell and tissue injury by significantly inhibiting the phosphorylation of BECN1 and significantly increasing expression levels of the ferroptosis regulatory proteins solute carrier family 7 member 11 and glutathione peroxidase 4. The results of the present study suggested that by regulating the BECN1 signaling pathway, NaHS may decrease the incidence of myocardial ferroptosis, thereby improving SIC.

SHARPIN promotes cell proliferation of cholangiocarcinoma and inhibits ferroptosis via p53/SLC7A11/GPX4 signaling.

SHARPIN is a tumor-associated gene involved in the growth and proliferation of many tumor types. A function of SHARPIN in cholangiocarcinoma (CCA) is so far unclear. Here, we studied the role and function of SHARPIN in CCA and revealed its relevant molecular mechanism. The expression of SHARPIN was analyzed in cholangiocarcinoma tissues from patients using immunohistochemistry, quantitative PCR, and western blot analysis. Expression of SHARPIN was suppressed/overexpressed by siRNA silencing or lentiviral overexpression vector, and the effect on cell proliferation was determined by the CCK-8 assay and flow cytometry. Accumulation of reactive oxygen species was measured with MitoTracker, and JC-1 staining showed mitochondrial fission/fusion and mitochondrial membrane potential changes as a result of the silencing or overexpression. The ferroptosis marker solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), and the antioxidant enzymes superoxide dismutase 1 (SOD-1) and SOD-2 were analyzed by western blot. The results showed that SHARPIN expression was increased in CCA tissue, and this was involved in cell proliferation. SHARPIN silencing resulted in accumulated reactive oxygen species, reduced mitochondrial fission, and a reduced mitochondrial membrane potential. Silencing of SHARPIN inhibited the ubiquitination and degradation of p53, and downregulated levels of SLC7A11, GPX4, SOD-1, and SOD-2, all of which contributed to excessive oxidative stress that leads to ferroptosis. Overexpression of SHARPIN would reverse the above process. The collected data suggest that in CCA, SHARPIN-mediated cell ferroptosis via the p53/SLC7A11/GPX4 signaling pathway is inhibited. Targeting SHARPIN might be a promising approach for the treatment of CCA.

Neuroprotective investigation of tanshinone in the cerebral infarction model in the Keap1-Nrf2/ARE pathway

ABSTRACT It was to investigate the neuroprotective mechanism of tanshinone after cerebral infarction via the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant reaction element (ARE) signaling pathway. Forty specific pathogen-free (SPF) Sprague Dawley (SD) rats were selected, all of which were male, approximately seven weeks old, weighing 250 ± 25 g. They were randomly divided into a model group, a non-model operation group, a positive control group, and an experimental group with ten SD rats in each group. The model of cerebral infarction in rats was established by the wire occlusion method. The model group and non-model operation group (control group) were injected with normal saline daily, the negative control group was injected with Keap1 gene inhibitor daily, and the experimental group was injected with tanshinone IIA (10 mg·kg−1·d−1) daily. Animal behavior analysis was performed on the 7th day after the operation, and pathology and the neuroprotective effects of tanshinone IIA on cells were assessed, including cell proliferation, autophagy, oxidative damage, and mitochondrial membrane permeability. The neuroprotective mechanism based on the Keap1-Nrf2/ARE pathway was explored and analyzed. Compared with the model group, the number of Keap1 proteins in the experimental group and the control group was substantially reduced (P < 0.05), and the experimental group was substantially different from the model group (P < 0.01). The protein expression of Nrf2, HO-1, and NQO1 increased substantially (P < 0.05), and the experimental group was substantially different from the model group (P < 0.01). In summary, tanshinone IIA promoted the proliferation of nerve cells, inhibited the production of cellular reactive oxygen species, inhibited the change in mitochondrial membrane potential, and activated the Keap1-Nrf2/ARE signaling pathway. It also induced and regulated the upregulation of downstream NQO1, HO-1, etc. and protected cells from cerebral infarction.

Efficacy of malachite green mediated photodynamic therapy on treatment of Cutaneous Leishmaniasis: In vitro study

BackgroundLeishmaniasis is a common zoonotic disease that is transmitted by phlebotomus and causes several clinical conditions, from self healing lesion to deadly internal organ involvement. Photodynamic therapy (PDT) is a treatment method that leads to the generation of cytotoxic species and consequently to cell death and tissue destruction by visible light in the presence of a photosensitizer and oxygen. The aim of this study was to investigate effect of malachite green (MG)-mediated PDT in Leishmania tropica (L. tropica) promastigotes. Material and methodsParasites were incubated with 0.19, 0.39, 1.56, 3.25 and 6.25 μM of MG for one hour and subjected to 46.4 J/cm2 light irradiation. Trypan blue assay was used to evaluate the viability of the cells and mitochondirial activity alteration was determined by MTT. Morphological changes were analyzed by Giemsa staining and Scanning electron microscopy (SEM) analyses. Flow cytometry was used to quantify the fluorescence emitted by cell volume, JC-1, Cell Cycle and Annexin V/PI staining reagents. ResultsMalachite green mediated photodynamic therapy at 1.56 and 3.125 μM decreased the viability of the L. tropica promastigotes and induced changes in the mitochondrial membrane potential. L.tropica promastigotes was bloked in G0/G1 phase. The morphology of the parasite was affected at the 1.56 and 3.125 μM MG+PDT, resulting in rounded cells with loss of flagellum and irregular shape. ConclusionsThis study demonstrated that antileishmanial effects through mitochondrial dysfunction, cell cycle arrest, and apoptosis-like cell death to parasites. This work showed PDT with MG effectedparasites. Therefore, MG-mediated PDT may provide a promising approach for L. tropica promastigotes.