ΔΨm Disruption Research Articles

Dehydrocorydaline attenuates myocardial ischemia-reperfusion injury via the FoXO signalling pathway: A multimodal study based on network pharmacology, molecular docking, and experimental study

Ethnopharmacological relevanceDehydrocorydaline (DHC), an active component of Corydalis yanhusuo (Y.H. Chou & Chun C. Hsu) W.T. Wang ex Z.Y. Su & C.Y. Wu (Papaveraceae), exhibits protective and pain-relieving effects on coronary heart disease, but the underlying mechanism still remains unknown. Aim of the studyNetwork pharmacology and experimental validation both in vivo and in vitro were applied to assess whether DHC can treat myocardial ischemia-reperfusion injury (MIRI) by regulating the forkhead box O (FoxO) signalling pathway to inhibit apoptosis. Materials and methodsDHC and MIRI targets were retrieved from various databases. Molecular docking and microscale thermophoresis (MST) determined potential binding affinity. An in vivo mouse model of MIRI was established by ligating the left anterior descending coronary artery. C57BL/6N mice were divided into sham, MIRI, and DHC (intraperitoneal injection of 5 mg/kg DHC) groups. Haematoxylin and eosin, Masson, and immunohistochemical stainings verified DHC treatment effects and the involved signalling pathways. In vitro, H9c2 cells were incubated with DHC and underwent hypoxia/reoxygenation. TUNEL, JC-1, and reactive oxygen species stainings and western blots were used to explore the protective effects of DHC and the underlying mechanisms. ResultsVenny analysis identified 120 common targets from 121 DHC and 23,354 MIRI targets. DHC exhibited high affinity for CCND1, CDK2, and MDM2 (<−7 kcal/mol). In vivo, DHC attenuated decreases in left ventricular ejection fraction and fractional shortening, reduced infarct sizes, and decreased cTnI and lactate dehydrogenase levels. In vitro, DHC alleviated apoptosis and oxidative stress in the hypoxia/reoxygenation model by attenuating ΔΨm disruption; reducing the production of reactive oxygen species; upregulating Bax and CCND1 via the FoxO signalling pathway, as well as cleaved-caspase 8; downregulating the apoptosis-associated proteins Bcl-2, Bid, cleaved-caspase 3, and cleaved-caspase 9; and promoting the phosphorylation of FOXO1A and MDM2. ConclusionBy upregulating the FoxO signaling pathway to inhibit apoptosis, DHC exerts a cardioprotective effect, which could serve as a potential therapeutic option for MIRI.

Silver nanoparticles exert toxic effects in human monocytes and macrophages associated with the disruption of Δψm and release of pro-inflammatory cytokines.

Silver nanoparticles (AgNP) are the most widely produced type of nanoparticles due to their antimicrobial and preservative properties. However, their systemic bioavailability may be considered a potential hazard. When AgNP reach the bloodstream, they interact with the immune cells, contributing to the onset and development of an inflammatory response. Monocytes and macrophages play a pivotal role in our defense system, but the interaction of AgNP with these cells is still not clear. Therefore, the main objective of this work was to assess the cytotoxic and pro-inflammatory effects induced by 5, 10, and 50nm AgNP coated with polyvinylpyrrolidone (PVP) and citrate, in concentrations that could be attained in vivo (0-25μg/mL), in human monocytes isolated from human blood and human macrophages derived from a monocytic cell line (THP-1). The effects of PVP and citrate-coated AgNP on cell viability, mitochondrial membrane potential, and cytokines release were evaluated. The results evidenced that AgNP exert strong harmful effects in both monocytes and macrophages, through the establishment of a strong pro-inflammatory response that culminates in cell death. The observed effects were dependent on the AgNP concentration, size and coating, being observed more pronounced cytotoxic effects with smaller PVP coated AgNP. The results showed that human monocytes seem to be more sensitive to AgNP exposure than human macrophages. Considering the increased daily use of AgNP, it is imperative to further explore the adverse outcomes and mechanistic pathways leading to AgNP-induced pro-inflammatory effects to deep insight into the molecular mechanism involved in this effect.

Antitumoral synergism between a copper(II) complex and cisplatin improves in vitro and in vivo anticancer activity against melanoma, lung and breast cancer cells

BackgroundIntrinsic resistance of cancer cells is a major concern for the success of chemotherapy, and this undesirable feature stimulates further research into the design of new compounds and/or alternative multiple drug chemotherapy protocols. MethodsIn this study, we investigated the antitumoral potential of the coordination compounds [Cu(HPClNOL)Cl]Cl (1), [Fe(HPClNOL)Cl2]NO3(2) and [Mn(HPClNOL)Cl2] (3). Using the human, MCF-7 and A549, and the murine melanoma, B16-F10, cell lines, we determined the cytotoxicity, DCFH oxidation, disruption of mitochondrial membrane potential (ΔΨm), Sub-G1 and TUNEL positive cells, and caspase 8 and 9 activities. Fractional inhibitory concentration (FIC) and xenograft models were also assessed to evaluate the efficacy of antitumoral potential. ResultsWe observed that only complex 1 was cytotoxic. The treatment of cancer cells with complex 1 triggered ROS generation and promoted the disruption of ΔΨm. Complex 1 increased the number of Sub-G1 and TUNEL positive cells, and the measurement of caspase 8 and 9 activity confirmed that apoptosis was triggered by the intrinsic pathway. FIC demonstrated that the combination of complex 1 with cisplatin was additive for the A549 cells whilst it was synergic for MCF-7 and B16-F10. Treatment with complex 1, either alone or combined with cisplatin, reduced tumor growth on xenograft models. ConclusionsThe present study brings new clues regarding the mechanism of action of [Cu(HPClNOL)Cl]Cl, either alone or in combination with cisplatin. General significanceThese results indicate that complex 1, administered either singly or in combination with current drugs, has real potential for use in cancer therapy.

Swertia chirayita suppresses the growth of non-small cell lung cancer A549 cells and concomitantly induces apoptosis via downregulation of JAK1/STAT3 pathway.

Lung carcinoma is the leading cause of cancer-related mortalities worldwide, and present therapeutical interventions are not successful enough to treat this disease in many cases. Recent years have witnessed a surge in exploring natural compounds for their antiproliferative efficacy to expedite the characterization of novel anticancer chemotherapeutics. Swertia chirayita is a valued medicinal herb and possess intrinsic pharmaceutical potential. However, elucidation of its anticancer effects at molecular levels remains unclear and needs to be investigated. We assessed the anticancer and apoptotic efficacy of S. chirayita ethanolic extract (Sw-EtOH) on non-small cell lung cancer (NSCLC) A549 cells during this exploratory study. The results elucidated that S. chirayita extract induced toxic effects within lung cancer cells by ~1 fold during cytotoxicity and LDH release assay at a 400 μg/ml concentration. Sw-EtOH extract elevates the level of ROS, resulting in the disruption of Δψm and release of cytosolic cytochrome c by 3.15 fold. Activation of caspases-3, -8 & -9 also escalated by ~1 fold, which further catalyze the augmentation of PARP cleavage (~3 folds), resulting in a four-fold increase in Sw-EtOH induced apoptosis. The gene expression analysis further demonstrated that Sw-EtOH extracts inhibited JAK1/STAT3 signaling pathway by down-regulating the levels of JAK1 and STAT3 to nearly half a fold. Treatment of Sw-EtOH modulates the expression level of various STAT3 associated proteins, including Bcl-XL, Bcl-2, Mcl-1, Bax, p53, Fas, Fas-L, cyclinD1, c-myc, IL-6, p21 and p27 in NSCLC cells. Thus, our study provided a strong impetus that Sw-EtOH holds the translational potential of being further evaluated as efficient cancer therapeutics and a preventive agent for the management of NSCLC.

The antimammary tumor effects of ethanolic extract of propolis from Adamawa region (Cameroon) are by apoptosis via reactive oxygen species-mediated mitochondrial pathway.

Identification of novel natural treatment to combat cancer is a current need. This study was aimed at assessing the anticancer effects of ethanol-extracted Cameroonian propolis (EEP). The antitumor effect of EPP was evaluated in vitro by measuring; cell viability, cell cycle, cell death mechanism, cell migration/invasion, reactive oxygen species (ROS), mitochondrial potential (ΔΨm), caspase activity, and apoptosis-regulating proteins (Bcl-2 and Bcl-XL) in cell lines. In vivo, the effect of EEP against 7,12 dimethylbenz(a)anthracene (DMBA)-induced breast tumorigenesis in rats was assessed. EEP was found to induce cytotoxicity against ER negative MDA-MB-231 breast cancer cells by activating apoptosis through ROS-mediated mitochondrial pathway. The extract equally triggered caspase-3 and caspase-9, increment of ROS level, disruption of ΔΨm and down-regulation of Bcl-XL and Bcl-2 proteins. Besides, EPP prevented migration and invasion activities by inhibiting MMP-2 activity. At all doses it prevented breast tumor incidence (20% in EEP 150 mg/kg vs 70% in DMBA) as well as tumor burden. Tumor sections from EEP-treated rats showed middle proliferation of mammary ducts with weak inflammatory responses. In summary, Cameroonian propolis exhibited antimammary tumor effects via the intrinsic pathway of apoptosis.

Diclofenac Sodium Triggers p53-Dependent Apoptosis in Human Corneal Epithelial Cells via ROS-Mediated Crosstalk.

Diclofenac sodium (DFS), a nonsteroidal anti-inflammatory drug, is frequently used in ophthalmology, but it causes negative effects on corneas. The mechanisms underlying the toxicities to corneas remains unclear. The present study was designed to assess the cytotoxicity of DFS to human corneal epithelial (HCEP) cells in vitro and further investigate its related mechanisms. The HCEP cells were treated with DFS at different concentrations ranging from 0.003 125% to 0.1%. DFS showed a dose- and time-dependent cytotoxicity to HCEP cells including abnormal morphology and declined viability. The 0.05% DFS-treated HCEP cells presented cell cycle arrest at S phase, reactive oxygen species (ROS) overproduction, and positive staining of phosphorylated H2AX, suggesting that DFS caused ROS-mediated DNA damage. The upregulation of p53 expression, formation of apoptotic body, phosphatidylserine externalization, and DNA ladder demonstrated that the p53-dependent apoptosis pathway was involved in the cytotoxicity of DFS. Furthermore, DFS activated caspase-8, caspase-9, and caspase-3 altered the expression levels of Bcl-2 family proteins including tBid, Bax, and Bcl-2, as well as increased poly(ADP-ribose) polymerase (PARP) cleavage. DFS also induced ΔΨm disruption, resulting in the release of cytochrome c and apoptosis-inducing factor into the cytoplasm. Additionally, the DFS-induced apoptosis was alleviated by p53 inhibitor. Taken together, DFS triggered p53-dependent apoptosis in HCEP cells via ROS-mediated crosstalk between the extrinsic and intrinsic pathways.

Chrysophyllum cainito stem bark extract induces apoptosis in Human hepatocarcinoma HepG2 cells through ROS-mediated mitochondrial pathway.

Hepatocellular carcinoma is the most common type of primary liver cancer in humans. This study aimed to demonstrate anticancer properties of an aqueous extract from Chrysophyllum cainito stem bark (CE) and its underlying mechanisms. Our MTT assay results showed that CE significantly reduced human hepatocellular carcinoma (HepG2) cell viability with the IC50of 100 µg/mL, while human dermal primary fibroblast (HDFa) cells showed less susceptibility in every concentration tested. Determined by Annexin V staining, the proportion of apoptotic HepG2 cells increased in a dose-dependent fashion after 24 hour-exposure of CE. The results from Western blot analysis confirmed that CE reduced procaspase-3, suggesting apoptosis by activating caspase-3 cleavage. Using the DCFH-DA and DiOC6 fluorescent probes, it was found that CE significantly stimulated the generation of reactive oxygen species (ROS) and reduced mitochondrial membrane potential (Δψ m), respectively. According to cell cycle analysis, CE (100 µg/mL) profoundly increased the percentage of cells in the sub-G1 phase, indicating cell apoptosis. These data suggest that CE induces apoptosis and cell death in human hepatocellular carcinoma via generation of intracellular ROS and disruption of Δψm. This is the first demonstration of the anticancer activity with proposed underlying mechanism of CE in liver cancer cells.

Dose- and Time-Dependent Cytotoxicity of Carteolol in Corneal Endothelial Cells and the Underlying Mechanisms.

Carteolol is a non-selective β-adrenoceptor antagonist used for the treatment of glaucoma, and its abuse might be cytotoxic to the cornea. However, its cytotoxicity and underlying mechanisms need to be elucidated. Herein, we used an in vivo model of feline corneas and an in vitro model of human corneal endothelial cells (HCECs), respectively. In vivo results displayed that 2% carteolol (clinical dosage) could induce monolayer density decline and breaking away of feline corneal endothelial (FCE) cells. An in vitro model of HCECs that were treated dose-dependently (0.015625–2%) with carteolol for 2–28 h, resulted in morphological abnormalities, declining in cell viability and elevating plasma membrane (PM) permeability in a dose- and time- dependent manner. High-dose (0.5–2%) carteolol treatment induced necrotic characteristics with uneven distribution of chromatin, marginalization and dispersed DNA degradation, inactivated caspase-2/-8, and increased RIPK1, RIPK3, MLKL, and pMLKL expression. The results suggested that high-dose carteolol could induce necroptosis via the RIPK/MLKL pathway. While low-dose (0.015625–0.25%) carteolol induced apoptotic characteristics with chromatin condensation, typical intranucleosomal DNA laddering patterns, G1 cell-cycle arrest, phosphatidylserine (PS) externalization, and apoptotic body formation in HCECs. Meanwhile, 0.25% carteolol treatment resulted in activated caspase-2, -3, -8, and -9, downregulation of Bcl-2 and Bcl-xL, upregulation of Bax and Bad, ΔΨm disruption, and release of cytoplasmic cytochrome c (Cyt.c) and AIF into the cytoplasm. These observations suggested that low-dose carteolol could induce apoptosis via a caspase activated and mitochondrial-dependent pathway. These results suggested that carteolol should be used carefully, as low as 0.015625% cartelol caused apoptotic cell death in HCECs in vitro.

Phenylephrine induces necroptosis and apoptosis in corneal epithelial cells dose- and time-dependently

In the present study, the toxicity of phenylephrine, a selective α1-adrenergic receptor agonist, in corneal epithelial cells and its underlying mechanisms were investigated using an in vitro model of human corneal epithelial cells (HCEPCs) and an in vivo model of New Zealand white rabbit corneas. The HCEPCs treated with phenylephrine at concentrations from 10% to 0.078125% displayed abnormal morphology, decline of cell viability and elevation of plasma membrane permeability time- and dose-dependently. Moreover, 10%–1.25% phenylephrine induce necrosis characteristics of marginalization and uneven distribution of chromatin through up-regulation of RIPK1, RIPK3 and MLKL along with inactivation of caspase-8 and caspase-2, whereas 0.625% phenylephrine induced condensed chromatin, S phase arrest, phosphatidylserine externalization, DNA fragmentation and apoptotic body formation in the HCECs through activation of caspase-2, -8, -9 and -3 as well as down-regulation of Bcl-2, up-regulation of Bad, ΔΨm disruption and release of cytochrome c and AIF into cytosol. At last, 10% phenylephrine induced destruction of the corneal epithelia and apoptosis of corneal epithelial cells in rabbit corneas. In conclusion, 10% to 1.25% phenylephrine cause necroptosis via RIPK1-RIPK3-MLKL axis and 0.625% phenylephrine induce apoptosis via a mitochondrion-dependent and death receptor-mediated signal pathway in HCEPCs.

Mitochondrial protection by simvastatin against angiotensin II-mediated heart failure.

Background and PurposeMitochondrial dysfunction plays a role in the progression of cardiovascular diseases including heart failure. 3‐Hydroxy‐3‐methylglutaryl‐CoA reductase inhibitors (statins), which inhibit ROS synthesis, show cardioprotective effects in chronic heart failure. However, the beneficial role of statins in mitochondrial protection in heart failure remains unclear.Experimental ApproachRats were treated with angiotensin II (1.5 mg·kg−1·day−1) or co‐administered simvastatin (oral, 10 mg·kg−1) for 14 days; and then administration was stopped for the following 14 days. Cardiac structure/function was examined by wheat germ agglutinin staining and echocardiography. Mitochondrial morphology and the numbers of lipid droplets, lysosomes, autophagosomes, and mitophagosomes were determined by transmission electron microscopy. Human cardiomyocytes were stimulated, and intracellular ROS and mitochondrial membrane potential (ΔΨ m) changes were measured by flow cytometry and JC‐1 staining, respectively. Autophagy and mitophagy‐related and mitochondria‐regulated apoptotic proteins were identified by immunohistochemistry and western blotting.Key ResultsSimvastatin significantly reduced ROS production and attenuated the disruption of ΔΨ m. Simvastatin induced the accumulation of lipid droplets to provide energy for maintaining mitochondrial function, promoted autophagy and mitophagy, and inhibited mitochondria‐mediated apoptosis. These findings suggest that mitochondrial protection mediated by simvastatin plays a therapeutic role in heart failure prevention by modulating antioxidant status and promoting energy supplies for autophagy and mitophagy to inhibit mitochondrial damage and cardiomyocyte apoptosis.Conclusion and ImplicationsMitochondria play a key role in mediating heart failure progression. Simvastatin attenuated heart failure, induced by angiotensin II, via mitochondrial protection and might provide a new therapy to prevent heart failure.

Cytotoxic effects, inflammatory response and apoptosis induction of cyclophosphamide in the peripheral blood leukocyte of blunt snout bream (Megalobrama amblycephala)

The present study was aimed to evaluate the effects of the cyclophosphamide (CY) exposure (Control, 0.032, 0.32, 1.0, 1.6 and 3.2 mg/mL) on the damage in the peripheral blood leukocytes of blunt snout bream for 24 h, which including cell viability, apoptosis, lactate dehydrogenase (LDH) release, mitochondrial membrane potential (Δѱm), ROS, antioxidant enzyme activity and the relative mRNA levels of apoptosis. Results showed that cell viability and Δѱm effects of CY were greatly reduced, and occurred in a dose-dependent manner. CY exposure (0.32–3.2 mg/mL) significantly increased the LDH release and induced apoptosis accompanied by ΔΨm disruption and ROS generation compared to the control. The cellular ROS was significantly increased with increase of CY level from 0.032 mg/mL to 1 mg/mL and the plateau occurred at 0.32 mg/mL. Additionally CY exposure led to oxidative stress as evidenced by significantly the decrease of SOD and CAT and increase of MDA concentration after treating cells with 3.2 mg/mL of CY. Besides, the relative mRNA levels of caspase-3 in the dose of 0.032, 0.32 mg/mL CY, caspase-9 and interleukins-1β (IL-1β) in the dose of 0.32 mg/mL CY, tumor necrosis factor-alpha (TNF-α) in the dose of 0.032 mg/mL CY significantly higher than that of the control. In conclusion, 0.32–3.2 mg/mL CY could lead to cytotoxic effect, inflammatory response and induce the apoptosis of the peripheral blood leukocyte of Megalobrama amblycephala.

Novel peptide myristoly-CM4 induces selective cytotoxicity in leukemia K562/MDR and Jurkat cells by necrosis and/or apoptosis pathway.

Purpose: There is an urgent need for the development of novel, effective, and less toxic drugs to treat leukemia. Antimicrobial peptides (AMPs) have received much more attention as alternative chemotherapeutic agents. This study aimed to examined the cytotoxicity of a novel AMP myristoly-CM4 against chronic myeloid leukemia cells (K562/MDR) and acute lymphocytic leukemia cells (Jurkat), and further investigated its selectivity to clarify the cytotoxic mechanism.Materials and methods: In this study, the cytotoxicity and selectivity of myristoly-CM4 against K562/MDR and Jurkat cells were assessed in vitro, and the anticancer mechanism responsible for its cytotoxicity and selectivity was further investigated.Results: Myristoly-CM4 was cytotoxic to these leukemia cell lines (IC50 2–4 μM) and was less cytotoxic to normal cells (HEK-293, L02 cells, peripheral blood mononuclear cells, and erythrocytes). Myristoyl-CM4 had stronger affinity to K562/MDR and Jurkat cells than to normal cells, while the contents of phosphatidylserine and sialic acids on the cell surfaces of K562/MDR and Jurkat cells were significantly higher than that of HEK293 cells. The myristoyl group effectively mediated the internalization of myristoyl-CM4 to leukemia cells. After internalization, myristoyl-CM4 could target mitochondria and affected mitochondrial function, including disruption of Δψm, increasing the accumulation of ROS, increasing the Bax/Bcl-2 ratio, activating caspase 9 and 3, and PARP to induce mitochondria-dependent apoptosis in both K562/MDR and Jurkat cells. Myristoyl-CM4 also induced K562/MDR cell necrosis by directive membrane disruption, and significantly decreased the level of P-glycoprotein in K562/MDR cells.Conclusion: These results suggested that myristoyl-CM4 showed selective cytotoxicity to leukemia K562/MDR and Jurkat cells by apoptosis and/or necrosis pathway. Myristoyl-CM4, thus, appears to be a promising candidate for leukemia treatment, including multidrug-resistant leukemia.

Synthesis, characterization, and photodynamic therapy activity of 5,10,15,20-Tetrakis(carboxyl)porphyrin.

Water-soluble porphyrins are considered promising drug candidates for photodynamic therapy (PDT). This study investigated the PDT activity of a new water-soluble, anionic porphyrin (1-Zn), which possesses four negative charges. The photodynamic anticancer activity of 1-Zn was investigated by the MTT assay, with mTHPC as a positive control. The cellular distribution was determined by fluorescence microscopy. Holographic and phase contrast images were recorded after 1-Zn treatment with a HoloMonitor™ M3 instrument. The inhibition of A549 cell growth achieved by inducing apoptosis was investigated by flow cytometry and fluorescence microscopy. DNA damage was investigated by the comet assay. The expression of apoptosis-related proteins was also measured by western blot assays. 1-Zn had better phototoxicity against A549 cells than HeLa and HepG2 cancer cells. Interestingly, 1-Zn was clearly located almost entirely in the cell cytoplasmic region/organelles. The late apoptotic population was less than 1.0% at baseline in the untreated and only light-treated cells and increased to 40.5% after 1-Zn treatment and irradiation (P < 0.05). 1-Zn triggered significant ROS generation after irradiation, causing ΔΨm disruption (P < 0.01) and DNA damage. 1-Zn induced A549 cell apoptosis via the mitochondrial apoptosis pathway. In addition, 1-Zn bound in the groove of DNA via an outside binding mode by pi-pi stacking and hydrogen bonding. 1-Zn exhibits good photonuclease activity and might serve as a potential photosensitizer (PS) for lung cancer cells.

Mitochondrial permeability transition pore in sea urchin female gametes

Mitochondrial permeability transition pore (MPTP) has been associated to calcium homeostasis and reactive oxygen species (ROS) generation in several cell types. While extensively investigated in somatic cells, there are few data regarding MPTP phenomenon in gametes. The aim of the present work was to investigate MPTP occurrence in sea urchin female gametes. The protonophores CCCP and FCCP, and the Ca2+ ionophore ionomycin, were used as pore inductors. Pore opening was monitored by mitochondrial potential sensitive probes and cobalt-quenched calcein assay. The pore desensitizer cyclosporin A (CsA) prevented the loss of mitochondrial inner membrane potential (ΔΨm) and pore opening induced by MPTP activators. The disruption of ΔΨm led to an increase in ROS generation, which was completely prevented by CsA. Our data also demonstrated that the increase in ROS production induced by MPTP opening requires extracellular Ca2+. In summary, the current study provides evidence about the occurrence of MPTP in sea urchin eggs in a similar manner as described in vertebrate somatic cells - CsA-sensitive, voltage- and Ca2+-triggered - and shows MPTP as a highly conserved physiological event through the evolution.

Suppressed de novo lipogenesis by plasma membrane citrate transporter inhibitor promotes apoptosis in HepG2 cells.

Suppression of the expression or activities of enzymes that are involved in the synthesis of de novo lipogenesis (DNL) in cancer cells triggers cell death via apoptosis. The plasma membrane citrate transporter (PMCT) is the initial step that translocates citrate from blood circulation into the cytoplasm for de novo long‐chain fatty acids synthesis. This study investigated the antitumor effect of the PMCT inhibitor (PMCTi) in inducing apoptosis by inhibiting the DNL pathway in HepG2 cells. The present findings showed that PMCTi reduced cell viability and enhanced apoptosis through decreased intracellular citrate levels, which consequently caused inhibition of fatty acid and triacylglycerol productions. Thus, as a result of the reduction in fatty acid synthesis, the activity of carnitine palmitoyl transferase‐1 (CPT‐1) was suppressed. Decreased CPT‐1 activity also facilitated the disruption of mitochondrial membrane potential (ΔΨm) leading to stimulation of apoptosis. Surprisingly, primary human hepatocytes were not affected by PMCTi. Increased caspase‐8 activity as a consequence of reduction in fatty acid synthesis was also found to cause disruption of ΔΨm. In addition, apoptosis induction by PMCTi was associated with an enhanced reactive oxygen species generation. Taken together, we suggest that inhibition of the DNL pathway following reduction in citrate levels is an important regulator of apoptosis in HepG2 cells via suppression of CPT‐1 activity. Thus, targeting the DNL pathway mediating CPT‐1 activity by PMCTi may be a selective potential anticancer therapy.

Dronedarone-Induced Cardiac Mitochondrial Dysfunction and Its Mitigation by Epoxyeicosatrienoic Acids.

Dronedarone and amiodarone are structurally similar antiarrhythmic drugs. Dronedarone worsens cardiac adverse effects with unknown causes while amiodarone has no cardiac adversity. Dronedarone induces preclinical mitochondrial toxicity in rat liver and exhibits clinical hepatotoxicity. Here, we further investigated the relative potential of the antiarrhythmic drugs in causing mitochondrial injury in cardiomyocytes. Differentiated rat H9c2 cardiomyocytes were treated with dronedarone, amiodarone, and their respective metabolites namely N-desbutyldronedarone (NDBD) and N-desethylamiodarone (NDEA). Intracellular ATP content, mitochondrial membrane potential (Δψm), and inhibition of carnitine palmitoyltransferase I (CPT1) activity and arachidonic acid (AA) metabolism were measured in H9c2 cells. Inhibition of electron transport chain (ETC) activities and uncoupling of ETC were further studied in isolated rat heart mitochondria. Dronedarone, amiodarone, NDBD and NDEA decreased intracellular ATP content significantly (IC50 = 0.49, 1.84, 1.07, and 0.63 µM, respectively) and dissipated Δψm potently (IC50 = 0.5, 2.94, 12.8, and 7.38 µM, respectively). Dronedarone, NDBD, and NDEA weakly inhibited CPT1 activity while amiodarone (IC50 > 100 µM) yielded negligible inhibition. Only dronedarone inhibited AA metabolism to its regioisomeric epoxyeicosatrienoic acids (EETs) consistently and potently. NADH-supplemented ETC activity was inhibited by dronedarone, amiodarone, NDBD and NDEA (IC50 = 3.07, 5.24, 11.94, and 16.16 µM, respectively). Cytotoxicity, ATP decrease and Δψm disruption were ameliorated via exogenous pre-treatment of H9c2 cells with 11, 12-EET and 14, 15-EET. Our study confirmed that dronedarone causes mitochondrial injury in cardiomyocytes by perturbing Δψm, inhibiting mitochondrial complex I, uncoupling ETC and dysregulating AA-EET metabolism. We postulate that cardiac mitochondrial injury is one potential contributing factor to dronedarone-induced cardiac failure exacerbation.

β-adrenergic Receptor-stimulated Cardiac Myocyte Apoptosis: Role of Cytochrome P450 ω-hydroxylase.

Prolonged or excessive β-adrenergic activation leads to cardiac myocyte loss and heart dysfunction; however, the underlying cellular mechanisms are still unclear. Therefore, we first confirmed the effect of isoproterenol (ISO), a β-adrenergic receptor agonist, on cardiac toxicity using TUNEL and caspase activity assays in cultured rat cardiomyocytes. ISO treatment significantly increased cardiomyocyte apoptosis. Persistent ISO stimulation of cardiomyocytes also increased the expression of CYP4A3, a major CYP450 ω-hydroxylase that produces 20-hydroxyeicosatetraenoic acid (20-HETE) in a time-dependent manner. Next, we examined the effect of ISO and 20-HETE on cardiomyocyte apoptosis using annexin V and propidium iodide staining. Treatment with either 20-HETE or ISO significantly increased cardiomyocyte apoptosis, and inhibition of 20-HETE production using 17-ODYA, a CYP450 ω-hydroxylase inhibitor, dramatically attenuated ISO-induced cardiomyocyte apoptosis. To determine the apoptotic pathway involved, the mitochondrial membrane potential (ΔΨm) was measured by detecting the ratio of JC-1 green/red emission intensity. The results demonstrated that 17-ODYA significantly abolished ISO-induced disruption of ΔΨm and that 20-HETE alone induced a marked disruptive effect on ΔΨm in cardiomyocytes. In addition, 20-HETE-induced disruption of ΔΨm and apoptosis was significantly attenuated by KN93, a CaMKII inhibitor. Taken together, these results demonstrate that 20-HETE treatment induces significant apoptosis via mitochondrial-dependent pathways, and that inhibition of 20-HETE production using 17-ODYA attenuates ISO-induced cardiomyocyte apoptosis.

Adrenergic Receptor‐Stimulated Cardiac Myocyte Apoptosis: Role of Cytochrome P450 ω‐Hydroxylase Cytochrome P450 ω‐Hydroxylase

Prolonged or excessive β‐adrenergic activation leads to cardiomyocyte loss and heart dysfunction. However, the underlying cellular mechanisms are still unclear. Therefore, we first confirmed the effect of isoproterenol (ISO), a β‐adrenergic receptor agonist, on cardiac toxicity using TUNEL and Caspase activity assays in cultured rat cardiomyocytes. ISO treatment significantly increased cardiomyocyte apoptosis. Persistent ISO stimulation of cardiomyocytes also increased the expression of CYP4A3, a major CYP450 ω‐hydroxylase that produces 20‐Hydroxyeicosatetraenoic acid (20‐HETE), in a time‐dependent manner. Next, we examined the effect of ISO and 20‐HETE on cardiomyocyte apoptosis using annexin V and propidium iodide staining. Treatment with either 20‐HETE or ISO significantly increased cardiomyocyte apoptosis, and inhibition of 20‐HETE production using 17‐ODYA, a CYP450 ω‐hydroxylase inhibitor, dramatically attenuated ISO‐induced cardiomyocyte apoptosis. To determine the apoptotic pathway involved, the mitochondrial membrane potential (ΔΨm) was measured by detecting the ratio of JC‐1 green/red emission intensity. The results demonstrated that 17‐ODYA significantly abolished ISO‐induced disruption in ΔΨm and that 20‐HETE alone induced a marked disruptive effect on ΔΨm in cardiomyocytes. 20‐HETE‐induced ΔΨm disruption and apoptosis were attenuated by KN93, a CaMKII blocker, suggesting that this action of 20‐HETE is mediated by a CaMKII‐dependent mechanism. Taken together, these results demonstrate that 20‐HETE treatment induces significant apoptosis via mitochondrial‐dependent pathways, and that inhibition of 20‐HETE production using 17‐ODYA attenuates ISO‐induced cardiomyocyte apoptosis. Thus, CYP450 ω‐hydroxylase could be a novel target to protect against cardiac damage induced by excessive β‐adrenergic activation or hypersympathoexcitability.Support or Funding InformationThis work was supported by Grants from the National Institute of Neurological Disorders and Stroke, National Institutes of Health (NIH, NS55008), American Heart Association (10GRNT3170012), Norman Bethune Program of Jilin University (2015221), and Education Department of Jilin Province (2015498).

MitoTEMPO Prevents Oxalate Induced Injury in NRK-52E Cells via Inhibiting Mitochondrial Dysfunction and Modulating Oxidative Stress.

As one of the major risks for urolithiasis, hyperoxaluria can be caused by genetic defect or dietary intake. And high oxalate induced renal epithelial cells injury is related to oxidative stress and mitochondrial dysfunction. Here, we investigated whether MitoTEMPO, a mitochondria-targeted antioxidant, could protect against oxalate mediated injury in NRK-52E cells via inhibiting mitochondrial dysfunction and modulating oxidative stress. MitoSOX Red was used to determine mitochondrial ROS (mtROS) production. Mitochondrial membrane potential (Δψm) and quantification of ATP synthesis were measured to evaluate mitochondrial function. The protein expression of Nox4, Nox2, and p22 was also detected to explore the effect of oxalate and MitoTEMPO on NADPH oxidase. Our results revealed that pretreatment with MitoTEMPO significantly inhibited oxalate induced lactate dehydrogenase (LDH) and malondialdehyde (MDA) release and decreased oxalate induced mtROS generation. Further, MitoTEMPO pretreatment restored disruption of Δψm and decreased ATP synthesis mediated by oxalate. In addition, MitoTEMPO altered the protein expression of Nox4 and p22 and decreased the protein expression of IL-6 and osteopontin (OPN) induced by oxalate. We concluded that MitoTEMPO may be a new candidate to protect against oxalate induced kidney injury as well as urolithiasis.

Anticancer effect of thalidomide invitro on human osteosarcoma cells.

Osteosarcoma is a high‑grade malignant tumor frequently found in children and adolescents. Thalidomide has been reported for treatment of various malignancies. Thalidomide was added to osteosarcoma cells and studied by cytotoxicity assay, evaluating apoptosis, cell cycle arrest, mitochondrial membrane potential(ΔΨm), and reactive oxygen species(ROS) levels and the expression of Bcl‑2, Bax, caspase‑3 and NF‑κB. The results showed that thalidomide could inhibit the proliferation of MG‑63 and U2OS cells in a concentration‑and time‑dependent manner. Morphological changes of apoptosis were also observed. Thalidomide increased the apoptosis rate of MG‑63 cells and induced cell cycle arrest by increasing the number of cells in the G0/G1 phase and decreasing the percentage of S phase in MG‑63 cells. Further investigation showed that a disruption of ΔΨm and upregulation of ROS were induced by thalidomide in high concentration. By western blot analysis, thalidomide resulted in the decreasing expression of Bcl‑2 and NF‑κB, and the increasing expression of Bcl‑2/Bax and caspase‑3. Here, we provide evidence that thalidomide could cause apoptosis in osteosarcoma cells. Taken together, these results indicate that thalidomide could be an antitumor drug in the therapy of osteosarcoma.