ATP Reduction Research Articles

Five‐to‐Seven Carbon Glycols Severely Impair Bioenergetics and Metabolism of Aggressive Lung Cancer Cells

Lung cancer is the leading cause of cancer deaths. Nonsmall cell lung cancer (NSCLC) accounts for the vast majority of the histological subtypes and at advanced stage exhibits extremely low survival rate. Certain compounds, such as glycols, inhibit migration and decreased viability of the highly metastatic NSCLC cells A549_3R has been recently shown. The glycols-induced effects are associated with a parallel reduction of ATP production, detected in A549_3R spheroids embedded in 3D desmoplastic-like extracellular matrix, following overnight treatment. Herein, selected glycols are tested for their ability to acutely compromise bioenergetics, metabolism, and viability of A549_3R cells. Therefore, 1,5-pentanediol (1,5-PD), 1,6-hexanediol (1,6-HD), and 1,7-heptanediol (1,7-Hept) turn out to be the most effective. They interfere with key enzymes in glycolysis and elicit most or all of the following responses: acute collapse of the critical mitochondrial membrane potential, substantial reduction of ATP, pyruvate and L-lactate levels, and massive increase in reactive oxygen species production. Moreover, computational docking analysis reveals that the glycols bind to the substrate-binding residues of lactate dehydrogenase A, which catalyzes the interconversion of pyruvate and lactate and is upregulated in aggressive tumors. Hence, the effective glycols may be promising anticancer compounds, or lead substances, for localized and intratumoral applications.

Mitochondrial Dysfunction and Cardiovascular Disease: Pathophysiology and Emerging Therapies.

Mitochondria ensure the supply of cellular energy through the production of ATP via oxidative phosphorylation. The alteration of this process, called mitochondrial dysfunction, leads to a reduction in ATP and an increase in the production of reactive oxygen species (ROS). Mitochondrial dysfunction can be caused by mitochondrial/nuclear DNA mutations, or it can be secondary to pathological conditions such as cardiovascular disease, aging, and environmental stress. The use of therapies aimed at the prevention/correction of mitochondrial dysfunction, in the context of the specific treatment of cardiovascular diseases, is a topic of growing interest. In this context, the data are conflicting since preclinical studies are numerous, but there are no large randomized studies.

α-Lipoic acid up-regulates gene expression but reduces protein levels of fibroblast growth factor 21 in HepG2 cells.

Fibroblast growth factor 21 (FGF21) is a metabolism-regulating hepatokine, and its expression is finely controlled by the nutrients and cellular stressors. α-Lipoic acid (ALA) regulates fuel metabolism as a nutrient, but it also arouses mitochondrial and endoplasmic reticulum (ER) stress as well as oxidative stress in hepatocytes. However, the role of cellular stress in ALA-regulated FGF21 expression has not been demonstrated as yet. The present study found that ALA up-regulated FGF21 gene expression while it reduced FGF21 protein levels in HepG2 cells, which was accompanied by mitochondrial damage that was shown by ATP reduction and ROS elevation. ALA led to mitochondrial stress and ER stress as shown by the increased expression of HSP60, ATF6 and ATF4. Inhibition of ER stress by 4-PBA significantly attenuated ALA-stimulated FGF21 gene expression while it did not influence the reduction of FGF21 protein levels. H2 O2 -induced oxidative stress reduced FGF21 protein levels in HepG2 cells, and anti-oxidation by Tempol blocked ALA-induced reduction of FGF21 proteins. In conclusion, ALA up-regulates FGF21 gene expression through the stimulation of mitochondrial and ER stress while it reduces FGF21 protein levels through the induction of oxidative stress in HepG2 cells. Further studies are needed to demonstrate the in vivo effect of ALA on hepatic FGF21 expression.

Endogenous ROS-Mediated Covalent Immobilization of Gold Nanoparticles in Mitochondria: A "Sharp Sword" in Tumor Radiotherapy.

Mitochondria as one of the key subcellular organelles have been well recognized as a promising druggable target and are closely associated with energy supply and various cellular functions. Realizing high accumulation and prolonged retention of radiosensitizers in the cellular mitochondria of tumors is an effective way to improve radiotherapeutic efficacy. Herein, we develop mitochondria-targeting and protein sulfenic acid (PSA)-reactive gold nanoparticles (dAuNP-TPP) that are fabricated by incorporating triphenylphosphine and 1,3-cyclohexanedione onto the surface of AuNPs (∼20 nm) to improve CT imaging and radiotherapeutic efficacy of tumors. Taking advantage of the specific mitochondrial targeting and PSA-mediated on-site covalent immobilization, this nanosystem shows significantly enhanced accumulation and retention in mitochondria with approximately 5.22-fold higher enrichment than nonimmobilizable AuNP-TPP. More notably, the covalent immobilization of dAuNP-TPP in cellular mitochondria could induce a dramatic reduction of ATP, leading to serious mitochondrial disruption, combined with the radiosensitization effect, in consequence achieving efficient radiotherapy of breast tumors in vivo. This subcellular organelle-targeted and immobilizable strategy may offer a valuable and universal tool for efficient tumor treatment.

Mechanisms of regenerative preconditioning in pigs with subtotal hepatectomies.

Portal vein occlusion shortly before extended hepatic resections has hepatoprotective properties, but its molecular effects have not been elucidated. We characterized the impact of regenerative preconditioning by portal vein embolization (PVE) on hepatic energy metabolism and cytokine expression. About 90% hepatectomies were performed in normal pigs (Control) and in pigs that underwent a PVE 24 h before the surgery (n = 10/group). Blood biochemistry and coagulation, liver damage, liver function (ICG), hepatic content of adenine nucleotides, and hepatic expression of inflammatory mediators (RT-PCR and WB) were determined before the hepatectomy, 15 min, and 24 h later. All PVE and hepatectomies were successfully accomplished. The 90% hepatectomy resulted in: Immediate reduction of ATP, leading to persistent decreases of energy load and ATP/ADP ratio up to the 24-h time-point; and pro-inflammatory expression profile of cytokines in the remnant liver. Prior performance of PVE attenuated the bioenergetic alterations and prevented many of the changes in hepatic cytokine expression. Regenerative preconditioning by PVE improved hepatic energy metabolism and modulated inflammatory mediators in the remnant liver in pigs undergoing major hepatectomies, potentially contributing to its hepatoprotective effects.

PKC-Mediated Orai1 Channel Phosphorylation Modulates Ca2+ Signaling in HeLa Cells.

The overexpression of the Orai1 channel inhibits SOCE when using the Ca2+ readdition protocol. However, we found that HeLa cells overexpressing the Orai1 channel displayed enhanced Ca2+ entry and a limited ER depletion in response to the combination of ATP and thapsigargin (TG) in the presence of external Ca2+. As these effects require the combination of an agonist and TG, we decided to study whether the phosphorylation of Orai1 S27/S30 residues had any role using two different mutants: Orai1-S27/30A (O1-AA, phosphorylation-resistant) and Orai1-S27/30D (O1-DD, phosphomimetic). Both O1-wt and O1-AA supported enhanced Ca2+ entry, but this was not the case with O1-E106A (dead-pore mutant), O1-DD, and O1-AA-E106A, while O1-wt, O1-E106A, and O1-DD inhibited the ATP and TG-induced reduction of ER [Ca2+], suggesting that the phosphorylation of O1 S27/30 interferes with the IP3R activity. O1-wt and O1-DD displayed an increased interaction with IP3R in response to ATP and TG; however, the O1-AA channel decreased this interaction. The expression of mCherry-O1-AA increased the frequency of ATP-induced sinusoidal [Ca2+]i oscillations, while mCherry-O1-wt and mCherry-O1-DD decreased this frequency. These data suggest that the combination of ATP and TG stimulates Ca2+ entry, and the phosphorylation of Orai1 S27/30 residues by PKC reduces IP3R-mediated Ca2+ release.

In Vitro Antithrombotic, Hematological Toxicity, and Inhibitor Studies of Protocatechuic, Isovanillic, and p-Hydroxybenzoic Acids from Maclura tricuspidata (Carr.) Bur.

In blood coagulation, circulating platelets and coagulation factors are crucial for the primary process because thrombi are generated by fibrin clotting with fibrinogen, thrombin, FXIIIa, and platelet activation. Therefore, strategies to reduce the activity of key coagulation factors, or interfere with their functions and delay the activation of platelets can be used as important tools to suppress excessive blood clot formation and platelet hyperactivation. This study examined the antithrombotic activity and hematological toxicity of PA, IVA, and 4-HA isolated from M. tricuspidata (Carr.) Bur in several in vitro experiments and inhibitor assays. We found that PA, IVA, and 4-HA attenuated the formation of fibrin polymers/clots and degraded the blood clots. These compounds inhibited the activities of procoagulant proteases and fibrinoligase, and prolonged the coagulation time. There was a significant reduction in platelet function and ATP or serotonin levels in thrombin-activated platelets. An inhibitor study showed that PA exhibited a mixed inhibition type for thrombin, an uncompetitive inhibition type for FXa, and a non-competitive inhibition type for FXIIIa and IVA, while 4-HA exhibited an uncompetitive inhibition type for thrombin and non-competitive inhibition type for FXa and FXIIIa. These three compounds (up to 50 μg/mL) were not toxic to blood cells.

Ablation of PRAK exacerbates the mitochondria dysfunction in C2C12 myoblasts exposed to palmitate stress

Backgroundp38 regulated/activated protein kinase (PRAK) plays a crucial role in modulating survival and metabolism. However, the role of PRAK in the regulation of palmitate‐induced metabolic stress remains unknown. We examined whether deletion of PRAK would suppress cell survival and mitochondrial function and whether PRAK‐induced detrimental effect was mitigated by irisin in C2C12 myoblasts in response to palmitate (PA) stress.MethodsPRAK of C2C12 myoblasts was knocked out by using CRISPR/Cas‐9 genome editing technology. Both wild type and PRAK‐/‐ C2C12 cells were exposed to palmitate at the concentration of 200 mmol/L for 48 h to induce metabolic stress. The effect of irisin, an adipomyokine, on both wild type and PRAK‐/‐cells was determined to explore its relationship with RPAK in response to palmitate stress. Cell viability, ATP product, glucose uptake, mitochondrial membrane potential and mitochondrial swelling were assessed.ResultsPRAK knockout decreased C2C12 viability in response to PA stress as evident by MTT assay in association with the reduction of ATP and glucose uptake. PRAK knockout enhanced apoptosis of C2C12 myoblasts in response to PA treatment, consistent with an impairment in mitochondrial function, by decreasing mitochondrial membrane potential and increasing mitochondrial swelling. However, PRAK knockout induced an impairment of mitochondrial and cell damage in C2C12 cells exposed to PA stress were rescued by treatment of irisin (10ng/ml).ConclusionOur finding indicates a pivotal role of PRAK in preserving cellular survival, mitochondrial function and glucose uptake in C2C12 cells exposed to palmitate induced metabolic stress.

Abstract 518: Erk5 S496 Phosphorylation, But Not Erk5 Kinase Or Transcriptional Activity, Is Responsible For Promoting Macrophage Inflammation And Mitochondrial Dysfunction Via Upregulating Novel Site Of Nrf2 K518 Sumoylation

ERK5 is a dual kinase-transcription factor, which contains two transcriptional transactivation domains in the C-terminus and a kinase domain in the N-terminus. Many ERK5 kinase inhibitors have been developed, and are being tested in clinical studies for cancer and inflammatory diseases. Recent data has raised questions regarding the functional role of these ERK5 kinase inhibitors. Specifically, the possible link between blockade of pro-inflammatory ERK5 S496 phosphorylation and the anti-inflammatory effects of ERK5-specific kinase inhibitors has largely been neglected. In this study, we aimed to study the role and regulatory mechanisms of ERK5 S496 phosphorylation on macrophage inflammation and the impact of ERK5-specific kinase inhibitors. ATP binding site deletion mutant of ERK5b (a kinase-dead mutant) inhibited KLF2 induction but not oxidized LDL (oxLDL)-induced ERK5 S496 phosphorylation and TNFα expression. In contrast, both specific ERK5 kinase inhibitors (AX15836 and XMD8-92) and a dual phosphorylation site mutant of ERK5 (AEF) inhibited not only KLF2 but also oxLDL-induced ERK5 S496 phosphorylation and TNFα induction. These data suggested that ERK5 S496 phosphorylation, but not ERK5 kinase activity, plays a crucial role in ERK5-mediated pro-inflammatory effects. We also discovered a key effect of ERK5 S496 phosphorylation on SUMOylation at a novel site of NRF2 (i.e., K518), which inhibited NRF2 transcriptional activity without affecting ERK5 kinase activity, and antagonized oxLDL-induced macrophage inflammation. The role of NRF2 activation on the efficiency of oxidative phosphorylation (OXPHOS) and ATP synthesis had previously been reported, and we found that both ERK5 S496A and NRF2 K518R mutants abolished oxLDL-induced reduction of OXPHOS, ATP, and NAD + levels. In summary, we discovered a novel mechanism in which ERK5 S496 phosphorylation directly inhibited NRF2 activity via SUMOylation of NRF2 at K518 and thereby induced macrophage inflammation and mitochondrial dysfunction. The often-neglected role of ERK S496 signaling should be carefully considered in the interpretation of prior reports of ERK5 knockdown and pharmacological kinase inhibition relative to cellular inflammation and mitochondrial dysfunction.

Characterizing Metabolic Changes in Cancer Cells after Treatment with an Extract Created from Walnuts

One of the main hallmarks of cancer cells is the reprogramming of energy metabolism to support the high energy and metabolite demands of constant proliferation. Although previously observed as mainly a shift from oxidative phosphorylation to glycolysis, also known as the Warburg Effect, recent research has shown that cancer cells can utilize both pathways to make energy, which has been referred to as metabolic flux. This suggests that cancer cells can adapt to their environment by reactivating or continuing to use the oxidative phosphorylation pathway to make energy. Thus, the energy metabolism of cancer cells is of therapeutic interest. The triple‐negative breast cancer cell line MDA‐MB‐231 can undergo metabolic flux, making it an ideal cell line to study the effects of metabolism‐altering drugs. Previous work from our lab showed that treatment with a Walnut Extract (WE) induced cell death in MDA‐MB‐231 cells, as well as HeLa cells. We also observed that the WE treatment targets the mitochondria by destabilizing the mitochondrial outer membrane potential, suggesting that the extract may be able to impact oxidative phosphorylation. To examine the effects on energy production, we have taken advantage of the Agilent Seahorse XFe96 Cell Metabolic Analyzer to monitor, in real time, metabolic changes in MDA‐MB‐231 and HeLa cells treated with WE. WE treatment had impacts on both glycolytic and oxidative phosphorylation processes in both cell lines. Both time‐ and dose‐dependent responses were observed, with WE treated MDA‐MB‐231 cells exhibiting a 70% reduction in basal oxygen consumption rate compared to vehicle treated cells. Additionally, basal glycolytic rate was reduced by up to 41%, and compensatory glycolysis was reduced 70%. In HeLa cells, basal oxygen consumption rate was reduced by 74%, with a 56% reduction in basal glycolytic rate and a 76% reduction in compensatory glycolysis. Quantification of ATP production rate showed an overall reduction of 73%, with a 91% reduction in mitochondrially contributed ATP production, as well as a 47% reduction in glycolytic ATP production in MDA‐MB‐231 cells. Quantitation of total ATP within the cell using a separate assay shows an 62% decrease in ATP in treated HeLa cells. We have also looked at the status of mTOR and AMPK in the cells after treatment with the WE, because of their role in energy sensing and energy production. Interestingly, our preliminary data suggests that phosphorylated mTOR levels do not change and we also do not detect any phosphorylation of AMPK, suggesting that the WE is not effecting its changes on these proteins despite the reduction in overall ATP in the cell. Taken together, our results suggest that the WE treatment can alter and reduce the rates of glycolysis and oxidative phosphorylation, and by extension, the metabolic flux of these cancer cells.

Sumac Induces Mitochondria‐Dependent Cellular Toxicity in Human Pancreatic Cancer Cell Lines

Pancreatic cancer is a devastating disease that kills about 57,000 Americans and 250,000 people worldwide each year. Chemoresistance, lack of symptoms, and ineffective therapy have resulted in gaps in our understanding of the molecular events underlying its etiology. We aim to identify natural small molecule compounds that are synthetic lethal in cellular models of mutant KRAS‐dependent pancreatic adenocarcinoma. Our preliminary experiments demonstrate that aqueous extracts of the Middle Eastern spice‐plant sumac induce cytotoxicity in two cultured human pancreatic cancer cell lines with KRAS gene mutations; MIA PaCa‐2 (G12C) and PANC‐1 (G12V and G12D) but not in normal fibroblasts. Pancreatic cancer patients, 90% of whom have mutations in the KRAS oncogene, do not respond well to traditional chemotherapy. We hypothesized that the phytochemical sumac added as an adjunct to the therapeutic regimen would skew the otherwise chemoresistant pancreatic cancer cells into an apoptotic phenotype. We hypothesized that sumac’s cytotoxicity was a consequence of mitochondrial targeting, specifically a derangement of mitochondrial membrane potential, leading to mitochondrial compromise, and eventually apoptosis. To test our hypothesis, we treated MIA PaCa‐2 and PANC‐1 cells in vitro with increasing concentrations of aqueous extracts of sumac (0, 0.03, 0.3, and 3.0 mg/mL) for varying time points and measured 1) mitochondrial membrane potential using TMRE assays, 2) ROS production using DHT assays, 3) ATP levels using fluorimetry, and 4) activated caspases 3 and 7. Our data show that sumac treatment 1) ameliorated ROS production, 2) abrogated mitochondrial membrane potential, 3) activated caspases 3 and 7, and 4) reduced cellular ATP levels in a dose‐dependent manner. These changes were accompanied by a dose‐dependent change in the protein expression of the energy‐sensing mitochondrial PGC‐1α, which increased in both cell lines, the anti‐apoptotic protein Bcl‐2, which decreased in the PANC‐1 cell line but not in the MIA PaCa‐2 cell line, the pro‐apoptotic Bax, and mitofusin‐1, which is responsible for the fusion of the mitochondrial outer membranes. These data suggest that sumac mediates “synthetic lethality” in pancreatic cancer cells based on the type of KRAS gene mutation. We also hypothesized that the observed ATP reduction would alter levels of SIRT3, which is major mitochondrial physiology regulating deacetylase enzyme. Immunoblot data show a paradoxical increase in SIRT3 in a dose‐dependent manner, suggesting other roles for SIRT‐3 in this pathway. Furthermore, using HPLC‐based NMR spectroscopy, we identified the active compound in the aqueous sumac extract to be [(2R, 3R, 4S, 5R)‐3, 4, 5, 6‐tetrakis [(2‐deuterio‐3, 4, 5‐trihydroxybenzoyl)oxy]oxan‐2‐yl] methyl‐2‐deuterio‐3, 4, 5‐trihydroxybenzoate. In conclusion, sumac targets hitherto unknown mitochondrial signal transduction pathways and is synthetic lethal for the KRAS mutant G12V and G12D.

Changes in Astroglial K+ upon Brief Periods of Energy Deprivation in the Mouse Neocortex.

Malfunction of astrocytic K+ regulation contributes to the breakdown of extracellular K+ homeostasis during ischemia and spreading depolarization events. Studying astroglial K+ changes is, however, hampered by a lack of suitable techniques. Here, we combined results from fluorescence imaging, ion-selective microelectrodes, and patch-clamp recordings in murine neocortical slices with the calculation of astrocytic [K+]. Brief chemical ischemia caused a reversible ATP reduction and a transient depolarization of astrocytes. Moreover, astrocytic [Na+] increased by 24 mM and extracellular [Na+] decreased. Extracellular [K+] increased, followed by an undershoot during recovery. Feeding these data into the Goldman–Hodgkin–Katz equation revealed a baseline astroglial [K+] of 146 mM, an initial K+ loss by 43 mM upon chemical ischemia, and a transient K+ overshoot of 16 mM during recovery. It also disclosed a biphasic mismatch in astrocytic Na+/K+ balance, which was initially ameliorated, but later aggravated by accompanying changes in pH and bicarbonate, respectively. Altogether, our study predicts a loss of K+ from astrocytes upon chemical ischemia followed by a net gain. The overshooting K+ uptake will promote low extracellular K+ during recovery, likely exerting a neuroprotective effect. The resulting late cation/anion imbalance requires additional efflux of cations and/or influx of anions, the latter eventually driving delayed astrocyte swelling.

Muscle Glycogen in Elite Soccer - A Perspective on the Implication for Performance, Fatigue, and Recovery.

Based on extrapolation of current trends in modern soccer, physiological loading has increased markedly, and the game will continue to become even more demanding in the future, which will exacerbate fatigue at the end of a game and between games. Soccer is a glycogen consuming activity due to its high-intensity intermittent nature, and muscle glycogen is a key factor associated with fatigue late in a game, as well as in determining recovery after a game or an intense training session. Low glycogen in individual muscle fibers and subcellular compartments in the muscle cell is likely to negatively affect several essential steps in the excitation-contraction coupling such as action potential propagation, calcium handling and cross-bridge cycling through reductions in muscle ATP which are suggested sites of muscle function impairment inducing muscle fatigue. Recovery of physical performance and muscle glycogen after a soccer game is a slow process, which challenges the reality in modern elite soccer with increased game and training frequency and physiological loading. We suggest a markedly higher prioritization of fitness training modalities, nutritional approaches and general recovery strategies that optimizes muscle glycogen storage prior to games and training sessions. Also, the soccer community including the governing bodies of the sport must acknowledge and plan according to the high and increasing demands of the modern game, as well as the consequences this has on fatigue and recovery. These aspects are paramount to consider in the planning of training and games, as well as in the process of structuring soccer tournaments and developing competitive regulations in the future to optimize performance and player health.

Chi@HMPB@CBD nanocomplexes for laser-assisted therapy of MRSA-infected cutaneous wounds in normal and MKR diabetic mice

The emergence and spreading of methicillin-resistant Staphylococcus aureus (MRSA) have become one of the main reasons for the surgical failure even for the death. Thus, there is an urgent need to develop efficient antimicrobial alternatives for eradicating it so as to improve the therapy efficiency of related diseases. Although the synthesized metal nanoparticles showed the MRSA-killing function in vitro, their biosafety in vivo application is still in controversy due to the potential toxicity and induced multidrug resistance. Recently, plant natural compounds with high antibacterial activity and special mechanisms different from antibiotics have attracted wide interest for developing new anti-MRSA regents. In this work, new nanocomplexes of Chi@HMPB@CBD based on natural compounds of cannabidiol (CBD) and chitosan (Chi) were designed for eradicating MRSA. In vitro results demonstrate that Chi@HMPB@CBD NPs plus laser irradiation can efficiently kill MRSA by inducing bacterial surface perforation, content leakage, ROS production, and ATP reduction. In vivo results show that the combination of Chi@HMPB@CBD NPs plus laser can effectively remove bacteria and accelerate wound healing of MRSA-infected normal and diabetes mellitus mice by up-regulating VEGF and CD31. Moreover, Chi@HMPB@CBD NPs possessing excellent biocompatibility and ultra-low toxicity in vitro significantly delayed the time for multidrug-resistance induction, comparing with silver nanoparticles. In our point, the “green therapeutics” against MRSA show great potential in clinical application.

Methylglyoxal Induces Inflammation, Metabolic Modulation and Oxidative Stress in Myoblast Cells.

Uremic sarcopenia is a serious clinical problem associated with physical disability and increased morbidity and mortality. Methylglyoxal (MG) is a highly reactive, dicarbonyl uremic toxin that accumulates in the circulatory system in patients with chronic kidney disease (CKD) and is related to the pathology of uremic sarcopenia. The pathophysiology of uremic sarcopenia is multifactorial; however, the details remain unknown. We investigated the mechanisms of MG-induced muscle atrophy using mouse myoblast C2C12 cells, focusing on intracellular metabolism and mitochondrial injury. We found that one of the causative pathological mechanisms of uremic sarcopenia is metabolic flow change to fatty acid synthesis with MG-induced ATP shortage in myoblasts. Evaluation of cell viability revealed that MG showed toxic effects only in myoblast cells, but not in myotube cells. Expression of mRNA or protein analysis revealed that MG induces muscle atrophy, inflammation, fibrosis, and oxidative stress in myoblast cells. Target metabolomics revealed that MG induces metabolic alterations, such as a reduction in tricarboxylic acid cycle metabolites. In addition, MG induces mitochondrial morphological abnormalities in myoblasts. These changes resulted in the reduction of ATP derived from the mitochondria of myoblast cells. Our results indicate that MG is a pathogenic factor in sarcopenia in CKD.

A hydrogen sulphide-responsive and depleting nanoplatform for cancer photodynamic therapy

Hydrogen sulfide (H2S) as an important biological gasotransmitter plays a pivotal role in many physiological and pathological processes. The sensitive and quantitative detection of H2S level is therefore crucial for precise diagnosis and prognosis evaluation of various diseases but remains a huge challenge due to the lack of accurate and reliable analytical methods in vivo. In this work, we report a smart, H2S-responsive and depleting nanoplatform (ZNNPs) for quantitative and real-time imaging of endogenous H2S for early diagnosis and treatment of H2S-associated diseases. We show that ZNNPs exhibit unexpected NIR conversion (F1070 → F720) and ratiometric photoacoustic (PA680/PA900) signal responsiveness towards H2S, allowing for sensitive and quantitative visualization of H2S in acute hepatotoxicity, cerebral hemorrhage model as well as colorectal tumors in living mice. ZNNPs@FA simultaneously scavenges the mitochondrial H2S in tumors leading to significant ATP reduction and severe mitochondrial damage, together with the activated photodynamic effect, resulting in efficient suppression of colorectal tumor growth in mice. We believe that this platform may provide a powerful tool for studying the vital impacts of H2S in related diseases.

Effects of lysophosphatidic acid (LPA) signaling via LPA receptors on cellular functions associated with ATP reduction in osteosarcoma cells treated with ethidium bromide.

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) exhibits a variety of malignant properties in cancer cells. Intracellular ATP depletion leads to the development of necrosis and apoptosis. The present study aimed to evaluate the effects of LPA receptor-mediated signaling on the regulation of cancer cell functions associated with ATP reduction. Long-term ethidium bromide (EtBr) treated (MG63-EtBr) cells were established from osteosarcoma MG-63 cells. The intracellular ATP levels of MG63-EtBr cells were significantly lower than that of MG-63 cells. LPAR2, LPAR3, LPAR4 and LPAR6 gene expressions were elevated in MG63-EtBr cells. The cell motile and invasive activities of MG63-EtBr cells were markedly higher than those of MG-63 cells. The cell motile activity of MG-63 cells was increased by LPA4 and LPA6 knockdowns. In cell survival assay, cells were treated with cisplatin (CDDP) every 24h for 3 days. The cell survival to CDDP of MG63-EtBr cells was lower than that of MG-63 cells. LPA2 knockdown decreased the cell survival to CDDP of MG-63 cells. The cell survival to CDDP of MG-63 cells was inhibited by (2S)-OMPT (LPA3 agonist). Moreover, the cell survival to CDDP of MG-63 cells was enhanced by LPA4 and LPA6 knockdowns. These results indicate that LPA signaling via LPA receptors is involved in the regulation of cellular functions associated with ATP reduction in MG-63 cells treated with EtBr.

Evaluation of Platinum Anticancer Drug-Induced Kidney Injury in Primary Culture of Rat Kidney Tissue Slices by Using Gas-Permeable Plates.

The type of method adopted for the evaluation of drug-induced kidney injury (DIKI) plays an important role during the drug discovery process. In the present study, the usefulness of cultured rat kidney tissue slices maintained on gas-permeable poly(dimethylsiloxane) (PDMS) plates for DIKI was assessed by monitoring the ATP content as a marker of cell viability. The amount of ATP in the kidney slices cultured on the PDMS plates was higher than that in the slices cultured on gas-impermeable polystyrene plates. The protein expression of organic cation transporter-2 (Oct2) was maintained for 3 d. Cisplatin showed a time- and concentration-dependent reduction in ATP in the slices with a half-effective concentration value of 24 µM, which was alleviated by cimetidine, an Oct2 inhibitor, suggesting that cisplatin-induced kidney injury in the cultured slices was regulated by the basolateral uptake transporter Oct2. Furthermore, the intensity of platinum anticancer drug-induced nephrotoxicity in the cultured slices was consistent with that of the in vivo study. In conclusion, the primary culture of rat kidney tissue slices on gas-permeable plates is expected to aid in the prediction of the extent of nephrotoxicity of drugs, even when transporters are responsible for the accumulation of drugs in kidney tissues.

Identification of the mitochondrial protein ADCK2 as a therapeutic oncotarget of NSCLC.

The aarF domain containing kinase 2 (ADCK2) is a mitochondria-locating protein, important for fatty acid metabolism and coenzyme Q biosynthesis. The bioinformatics results show that elevated ADCK2 transcripts in NSCLC correlate with poor overall survival and poor anti-PD-1/PD-L1 therapy response. ADCK2 is overexpressed in local human NSCLC tissues and various primary and established NSCLC cells. In NSCLC cells, ADCK2 shRNA or CRISPR/Cas9 knockout remarkably suppressed cell viability, proliferation, cell cycle progression, cell mobility, and provoked cell apoptosis. Moreover, ADCK2 depletion disrupted mitochondrial functions in NSCLC cells, causing cytochrome C release, mitochondrial depolarization, DNA damage and ATP reduction. Contrarily, ectopic ADCK2 overexpression promoted NSCLC cell growth. Further studies revealed that ADCK2 depletion inactivated Akt-mTOR signaling in primary NSCLC cells. NSCLC xenograft growth in nude mice was significantly hindered after ADCK2 silencing or knockout. ADCK2 depletion, apoptosis induction and oxidative injury as well as ATP reduction and Akt-mTOR inactivation were detected in ADCK2-silenced or ADCK2-knockout NSCLC xenograft tissues. Together overexpressed ADCK2 is important for the growth of NSCLC cells, representing an important therapeutic molecular oncotarget.

Screening for Mitochondrial tRNA Mutations in 318 Patients with Dilated Cardiomyopathy

Objectives: Dilated cardiomyopathy (DCM) is a complex cardiovascular disease with unknown etiology. Although nuclear genes play active roles in DCM, mitochondrial dysfunction was believed to be involved in the pathogenesis of DCM. The objective of this study was to analyze the association between mitochondrial tRNA (mt-tRNA) mutations and DCM. Materials and Methods: We performed a mutational analysis of mt-tRNA genes in a cohort of 318 patients with DCM and 200 age- and gender-matched control subjects. To further assess their pathogenicity, phylogenetic analysis and mitochondrial functions including mtDNA copy number, ATP, and ROS were analyzed. Results: Seven possible pathogenic mutations, i.e., MT-TL1 3302A>G, MT-TI 4295A>G, MT-TM 4435A>G, MT-TA 5655T>C, MT-TH 12201T>C, MT-TE 14692A>G, and MT-TT 15927G>A, were identified in the DCM group but were absent in controls. These mutations occurred at extremely conserved nucleotides of corresponding tRNAs and led to the failure in tRNAs metabolism. Moreover, a significant reduction in ATP and mtDNA copy number and a marked increase in ROS level were observed in polymononuclear leukocytes (PMNs) derived from the DCM patients carrying these mt-tRNA mutations, suggesting that these mutations may cause mitochondrial dysfunction that was responsible for DCM. Conclusions: Our data indicated that mt-tRNA mutations may be the molecular basis for DCM, which provides novel insights into the pathophysiology of DCM that manifested as mitochondrial dysfunction.