Depletion Of Cellular ATP Research Articles

Pyrazolopyrimidinones, a novel class of copper-dependent bactericidal antibiotics against multi-drug resistant S. aureus.

The treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections poses a therapeutic challenge as even last resort drugs become increasingly ineffective. As the demand for antibiotics with novel modes of action is growing, new approaches are needed to probe a greater spectrum of antimicrobial activities for their potential efficacy against drug-resistant pathogens. The use of copper (Cu) by the innate immune system to mount an antimicrobial response against bacterial invaders has created an opportunity to explore a role for Cu in antimicrobial therapy. Here we describe pyrazolopyrimidinones (PZP) as novel copper-dependent inhibitors (CDI) of S. aureus. 5-Benzyl-3-(4-chlorophenyl)-2-methyl-4H,7H-pyrazolo[1,5-a]pyrimidin-7-one (PZP-915) showed potent bactericidal properties at sub-micromolar concentrations and activity against clinical MRSA isolates and biofilms cultures. This cupricidal activity is founded on the molecule's ability to coordinate Cu and induce accumulation of Cu ions inside S. aureus cells. We demonstrate that exposure to 915 + Cu led to an almost instantaneous collapse of the membrane potential which was accompanied by a complete depletion of cellular ATP, loss of cell-associated K+, a substantial gain of cell associated Na+, and an inability to control the influx of protons in slightly acidic medium, while the integrity of the cell membrane remained intact. These findings highlight PZP-915 as a novel membrane-directed metalloantibiotic against S. aureus that is likely to target a multiplicity of membrane associated protein functions rather than imposing physical damage to the membrane structure.

The combination of ribose and adenine promotes adenosine release and attenuates the intensity and frequency of epileptiform activity in hippocampal slices: Evidence for the rapid depletion of cellular ATP during electrographic seizures.

In addition to being the universal cellular energy source, ATP is the primary reservoir for the neuromodulator adenosine. Consequently, adenosine is produced during ATP‐depleting conditions, such as epileptic seizures, during which adenosine acts as an anticonvulsant to terminate seizure activity and raise the threshold for subsequent seizures. These actions protect neurones from excessive ionic fluxes and hence preserve the remaining cellular content of ATP. We have investigated the consequences of manipulation of intracellular ATP levels on adenosine release and epileptiform activity in hippocampal slices by pre‐incubating slices (3 h) with creatine (1 mM) and the combination of ribose (1 mM) and adenine (50 μM; RibAde). Creatine buffers and protects the concentration of cellular ATP, whereas RibAde restores the reduced cellular ATP in brain slices to near physiological levels. Using electrophysiological recordings and microelectrode biosensors for adenosine, we find that, while having no effect on basal synaptic transmission or paired‐pulse facilitation, pre‐incubation with creatine reduced adenosine release during Mg2+−free/4‐aminopyridine‐induced electrographic seizure activity, whereas RibAde increased adenosine release. This increased release of adenosine was associated with an attenuation of both the intensity and frequency of seizure activity. Given the depletion of ATP after injury to the brain, the propensity for seizures after trauma and the risk of epileptogenesis, therapeutic strategies elevating the cellular reservoir of adenosine may have value in the traumatized brain. Ribose and adenine are both in use in man and thus their combination merits consideration as a potential therapeutic for the acutely injured central nervous system.

Synthesis and biological evaluation of Complex I inhibitor R419 and its derivatives as anticancer agents in HepG2 cells

In this study, Complex I inhibitor R419 was firstly revealed to have significant anticancer activity against HepG2 cells (IC50 = 5.2 ± 0.9 μM). Based on this finding, a series of R419 derivatives were synthesized and biologically evaluated. As results, 9 derivatives were found to have obvious anticancer activity. Among them, H20 exhibited the most potent activity (IC50 = 2.8 ± 0.4 μM). Mechanism study revealed that H20 caused severe depletion of cellular ATP, dose-dependently activated AMPK, decreased Bcl-2/Bax ratio and induced necrotic cell death. Most importantly, H20 displayed definite inhibitory activity against Complex I.

Cytosolic increased labile Zn2+ contributes to arrhythmogenic action potentials in left ventricular cardiomyocytes through protein thiol oxidation and cellular ATP depletion

Intracellular labile (free) Zn2+-level ([Zn2+]i) is low and increases markedly under pathophysiological conditions in cardiomyocytes. High [Zn2+]i is associated with alterations in excitability and ionic-conductances while exact mechanisms are not clarified yet. Therefore, we examined the elevated-[Zn2+]i on some sarcolemmal ionic-mechanisms, which can mediate cardiomyocyte dysfunction. High-[Zn2+]i induced significant changes in action potential (AP) parameters, including depolarization in resting membrane-potential and prolongations in AP-repolarizing phases. We detected also the time-dependent effects such as induction of spontaneous APs at the time of ≥ 3 min following [Zn2+]i increases, a manner of cellular ATP dependent and reversible with disulfide-reducing agent dithiothreitol, DTT. High-[Zn2+]i induced inhibitions in voltage-dependent K+-channel currents, such as transient outward K+-currents, Ito, steady-state currents, Iss and inward-rectifier K+-currents, IK1, reversible with DTT seemed to be responsible from the prolongations in APs. We, for the first time, demonstrated that lowering cellular ATP level induced significant decreaeses in both Iss and IK1, while no effect on Ito. However, the increased-[Zn2+]i could induce marked activation in ATP-sensitive K+-channel currents, IKATP, depending on low cellular ATP and thiol-oxidation levels of these channels. The mRNA levels of Kv4.3, Kv1.4 and Kv2.1 were depressed markedly with increased-[Zn2+]i with no change in mRNA level of Kv4.2, while the mRNA level of IKATP subunit, SUR2A was increased significantly with increased-[Zn2+]i, being reversible with DTT. Overall we demonstrated that high-[Zn2+]i, even if nanomolar levels, alters cardiac function via prolonged APs of cardiomyocytes, at most, due to inhibitions in voltage-dependent K+-currents, although activation of IKATP is playing cardioprotective role, through some biochemical changes in cellular ATP- and thiol-oxidation levels. It seems, a well-controlled [Zn2+]i can be novel therapeutic target for cardiac complications under pathological conditions including oxidative stress.

Elimination of stem-like cancer cell side-population by auranofin through modulation of ROS and glycolysis

Cancer side-population (SP) represents a sub-population of stem-like cancer cells that have an important role in drug resistance due to their high expression of the ATP-binding cassette transporter ABCG2 involved in drug export. Auranofin (AF), a clinical drug of gold complex that is used in treatment of rheumatoid arthritis, has been reported inducing tumor antiproliferation. However, whether AF can impact SP cells remains unclear. Our study showed that AF caused a depletion of SP cells and a downregulation of stem cell markers, and impaired their ability to form tumor colonies in vitro and incidence to develop tumors in vivo of lung cancer cells. Reactive oxygen species (ROS) had an important role in mediating AF-induced depletion of SP cells, which could be reversed by antioxidant NAC. Further study revealed that AF could also cause ATP depletion by inhibition of glycolysis. The depletion of cellular ATP might impair the function of ABCG2 pump, leading to increased drug accumulation within the cells and thus enhancing anticancer activity of chemotherapeutic agents such as adriamycin. Synergistic effect of AF and adriamycin was demonstrated both in vitro and in vivo. Simultaneous increase of ROS and inhibition of glycolysis is a novel strategy to eliminate stem-like cancer cells. Combination of AF with adriamycin seems to be promising to enhance therapeutic effectiveness.

EEF-2 Kinase-targeted miR-449b confers radiation sensitivity to cancer cells

The roles of microRNA in regulation of various biological processes and in modulation of therapeutic effects have been widely appreciated. In this study, we found a positive correlation between miR-449 b expression and radiation sensitivity in cancer cells and in tumor specimens from patients. We showed that eEF-2 kinase, a negative regulator of global protein synthesis, is a target of miR-449 b. Introducing a miR-449 b mimic into cancer cells led to suppression of eEF-2 kinase expression, leading to increases of protein synthesis and depletion of cellular ATP. Further, we demonstrated that the miR-449 b mimic rendered the cancer cells more sensitive to ionizing radiation both in vitro (cell culture) and in vivo (animal xenograft model). Moreover, the radiation sensitivity conferred by miR-449 b could be blunted by cycloheximide, an inhibitor of protein synthesis, or by direct delivery of ATP liposome, supporting eEF-2 kinase as a mediator of the radio-sensitizing effects of miR-449 b. These results indicate that miR-449 b, which is frequently down-regulated in radio-resistant cancers, may represent a new critical determinant of radio-sensitivity.

Abstract B023: ONC201 kills breast cancer cells by targeting mitochondria

Abstract ONC201 is a small molecule originally identified as a TRAIL (TNF-related apoptosis-inducing ligand) inducing compound currently being tested in phase1/2 clinical trials in multiple cancer types. Two recent studies reported that ONC201 also induces an atypical stress response mediated in part by ATF4 and CHOP. In this study, we tested ONC201 toxicity in breast cancer cell lines. ONC201 was obtained from Oncoceutics, Inc. Cell viability was tested with MTS assay and CytoTox-Glo assay. ATP level was measured with CellTiter-Glo 2.0 assay. RNA-seq and Western blotting were performed to investigate change of gene expression. Mitochondrial respiration was monitored by Seahorse XF analyzer. Live cell imaging was performed to examine the mode of cell death. Confocal and electron microscopy analysis were performed to study mitochondrial morphology. Mitochondrial DNA (mtDNA) copy number was analyzed by Quantitative PCR (qPCR). Mitochondrial defective (rho0) cell lines were generated by ethidium bromide treatment. We tested the effects of ONC201 on 18 human breast cancer cell lines that represent ER+, HER2-amplified, basal A triple-negative (TNBC), and basal B TNBC breast cancer. ONC201 reduced cell viability in breast cancer cell lines in all subtypes tested with IC50s ranging from 0.8-5 μM, similar to what has been reported for other cancer cell types. Unexpectedly, ONC201 toxicity was not dependent on TRAIL receptors or caspases. Live cell imaging revealed ONC201 induces cell membrane ballooning followed by rupture. By contrast, GST-TRAIL induced TRAIL-receptor dependent caspase mediated death and classic apoptosis morphology. These results suggested that ONC201 kills breast cancer cells via a caspase-independent, TRAIL-receptor-independent mechanism. Western blots confimed that ONC201 induces ATF4 and CHOP, consistent with reported observations. ONC201 also induced phosphorylation of AMP-dependent kinase (AMPK) and depletion of cellular ATP in multiple breast cancer cell lines. Cytotoxicity and ATP depletion induced by ONC201 were significantly enhanced in nonglucose (galactose) medium compared with glucose-containing medium, suggesting that ONC201 targets mitochondrial respiration. Supplementing glucose to cells grown in galactose medium rescued ONC201-dependent ATP depletion, cytotoxicity, and induction of p-AMPK, ATF4, and CHOP. Seahorse XF analyzer indicated that ONC201 inhibits mitochondrial respiration via an indirect mechanism. Confocal imaging revealed that ONC201 induces mitochondrial fission and reduces membrane potential. Electron microscopic analysis revealed that ONC201 induces mitochondrial structural damages, such as mitochondrial swelling, matrix lysis, and fragmentation. Confocal imaging and qPCR revealed that ONC201 decreases mtDNA. Importantly, ONC201 induced mitochondrial damage and mtDNA depletion even in ONC201-resistant cells, implying that cells that are not dependent on mitochondrial respiration are ONC201 resistant. Indeed, multiple rho0 cell lines were ONC201-resistant. RNAseq analysis confirmed that ONC201 inhibits expression of multiple mtDNA-encoded genes and nuclear encoded mitochondrial genes involved in oxidative phosphorylation and other mitochondrial functions, and Western blot reinforced those findings. In summary, our data demonstrate that ONC201 kills breast cancer cells by targeting mitochondria. Citation Format: Yoshimi Endo Greer, Samuel Gilbert, Celia Islam, Ashley Ubaldini, Christina Stuelten, Natalie Porat-Shliom, Roberto Weigert, Yun Ji, Luca Gattinoni, Ferri Soheilian, Kunio Nagashima, Xiantao Wang, Markus Hafner, Jyoti Shetty, Bao Tran, Vishal Koparde, Parthav Jailwala, Maggie Cam, Dan Crooks, W. Marston Linehan, Donna Voeller, William Reinhold, Vinodh Rajapakse, Yves Pommier, Stanley Lipkowitz. ONC201 kills breast cancer cells by targeting mitochondria [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B023.

The Purine Salvage Pathway and the Restoration of Cerebral ATP: Implications for Brain Slice Physiology and Brain Injury

Brain slices have been the workhorse for many neuroscience labs since the pioneering work of Henry McIlwain in the 1950s. Their utility is undisputed and their acceptance as appropriate models for the central nervous system is widespread, if not universal. However, the skeleton in the closet is that ATP levels in brain slices are lower than those found in vivo, which may have important implications for cellular physiology and plasticity. Far from this being a disadvantage, the ATP-impoverished slice can serve as a useful and experimentally-tractable surrogate for the injured brain, which experiences similar depletion of cellular ATP. We have shown that the restoration of cellular ATP in brain slices to in vivo values is possible with a simple combination of d-ribose and adenine (RibAde), two substrates for ATP synthesis. Restoration of ATP in slices to physiological levels has implications for synaptic transmission and plasticity, whilst in the injured brain in vivo RibAde shows encouraging positive results. Given that ribose, adenine, and a third compound, allopurinol, are all separately in use in man, their combined application after acute brain injury, in accelerating ATP synthesis and increasing the reservoir of the neuroprotective metabolite, adenosine, may help reduce the morbidity associated with stroke and traumatic brain injury.

Antioxidant defense in quiescent cells determines selectivity of electron transport chain inhibition-induced cell death

Mitochondrial electron transport chain (ETC) targeting shows a great promise in cancer therapy. It is particularly effective in tumors with high ETC activity where ETC-derived reactive oxygen species (ROS) are efficiently induced. Why modern ETC-targeted compounds are tolerated on the organismal level remains unclear. As most somatic cells are in non-proliferative state, the features associated with the ETC in quiescence could account for some of the specificity observed. Here we report that quiescent cells, despite increased utilization of the ETC and enhanced supercomplex assembly, are less susceptible to cell death induced by ETC disruption when glucose is not limiting. Mechanistically, this is mediated by the increased detoxification of ETC-derived ROS by mitochondrial antioxidant defense, principally by the superoxide dismutase 2 – thioredoxin axis. In contrast, under conditions of glucose limitation, cell death is induced preferentially in quiescent cells and is correlated with intracellular ATP depletion but not with ROS. This is related to the inability of quiescent cells to compensate for the lost mitochondrial ATP production by the upregulation of glucose uptake. Hence, elevated ROS, not the loss of mitochondrially-generated ATP, are responsible for cell death induction by ETC disruption in ample nutrients condition, e.g. in well perfused healthy tissues, where antioxidant defense imparts specificity. However, in conditions of limited glucose, e.g. in poorly perfused tumors, ETC disruption causes rapid depletion of cellular ATP, optimizing impact towards tumor-associated dormant cells. In summary, we propose that antioxidant defense in quiescent cells is aided by local glucose limitations to ensure selectivity of ETC inhibition-induced cell death.

Abstract 1487: ONC201 kills breast cancer cells by inhibiting mitochondrial respiration

Abstract Background: ONC201 is a small molecule originally identified as a TRAIL inducing compound currently being tested in phase1/2 clinical trials in multiple cancer types. Two recent studies reported that ONC201 also induces an atypical stress response mediated in part by ATF4 and CHOP. Methods: ONC201 was obtained from Oncoceutics, Inc. Recombinant GST-TRAIL was prepared in the laboratory. Cell viability was tested with MTS assay and CellTiter-Glo luminescent cell viability assay. ATP level was measured with CellTiter-Glo 2.0 assay. RNAseq and western blotting were performed to investigate change of gene expression. Mitochondrial respiration was monitored by Seahorse XF analyzer. Live cell imaging was performed to examine the mode of cell death. Confocal microscopy and electron microscopy analysis were performed to study mitochondrial morphology. Results: We tested the effects of ONC201 on 18 human breast cancer cell lines that represent ER+, HER2 amplified, TNBC basal A and TNBC basal B breast cancer. ONC201 reduced cell viability in breast cancer cell lines in all subtypes tested with IC50s ranging from 0.8-5 μM, similar to what has been reported for other cancer cell types. Unexpectedly, ONC201 toxicity was not dependent on TRAIL receptors or caspases and live cell imaging revealed ONC201 induces cell membrane ballooning followed by rupture. By contrast, GST-TRAIL induced TRAIL-receptor dependent caspase mediated death and classic apoptosis morphology. These results suggest that ONC201 kills breast cancer cells via a caspase-independent, TRAIL-receptor-independent mechanism distinct from TRAIL-induced apoptosis. Western blots revealed that ONC201 induces the stress pathway proteins ATF4 and CHOP, consistent with the recently published observations. ONC201 also induced phosphorylation of AMP-dependent kinase (AMPK) and depletion of cellular ATP in multiple breast cancer cell lines. Seahorse XF analysis found that ONC201 inhibited mitochondrial oxygen consumption rate but did not inhibit glycolysis as measured by the extracellular acidification rate. Both ONC201-induced toxicity and ATP depletion were enhanced when cells were cultured in non-glucose (galactose) medium. Supplementing glucose to cells grown in galactose medium partially prevented ONC201-dependent ATP depletion, induction of phospho-AMPK, ATF4 and CHOP, and cell death. These data are consistent with an inhibition of oxidative phosphorylation (OxPhos) by ONC201. RNAseq revealed ONC201 inhibits expression of multiple mitochondrial genes involved in OxPhos and other mitochondrial functions, and western blot confirmed those findings. Confocal and electron microscopic evaluation revealed abnormal mitochondrial morphology. Conclusion: Our data demonstrate that ONC201 can kill breast cancer cells by a novel mechanism involving disruption of mitochondrial morphology and inhibition of mitochondrial respiration. Citation Format: Yoshimi Greer, Samuel Gilbert, Celia Islam, Yun Ji, Luca Gattinoni, Christina Stuelten, Natalie Porat-Shliom, Roberto Weigert, Xiantao Wang, Markus Hafner, Kunio Nagashima, Donna Voeller, Stanley Lipkowitz. ONC201 kills breast cancer cells by inhibiting mitochondrial respiration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1487. doi:10.1158/1538-7445.AM2017-1487

A study of the dynamics of PTEN proteins in living cells using in vivo fluorescence correlation spectroscopy

PTEN (phosphatase and tensin homolog on chromosome 10) is one of the most important tumor-suppressor proteins, which plays a key role in negative regulation of the PI3K/AKT pathway, and governs many cellular processes including growth, proliferation, survival and migration. The dynamics of PTEN proteins in single living cells is as yet unclear owing to a shortage of suitable in vivo approaches. Here, we report a single-molecule method for in vivo study of the dynamics of PTEN proteins in living cells using fluorescence correlation spectroscopy (FCS). First, we established a monoclonal H1299 stable cell line expressing enhanced green fluorescent protein (EGFP) and PTEN (EGFP-PTEN) fusion proteins; we then developed an in vivo FCS method to study the dynamics of EGFP-PTEN both in the nucleus and the cytoplasm. We investigated the diffusion behaviors of EGFP and EGFP-PTEN in solution, nucleus and cytosol, and observed that the motion of PTEN in living cells was restricted compared with EGFP. Finally, we investigated the protein dynamics in living cells under oxidative stress stimulation and a cellular ATP depletion treatment. Under oxidative stress stimulation, the EGFP-PTEN concentration increased in the nucleus, but slightly decreased in the cytoplasm. The diffusion coefficient and alpha value of EGFP-PTEN reduced significantly both in the nucleus and cytoplasm; the significantly decreased alpha parameter indicates a more restricted Brownian diffusion behavior. Under the cellular ATP depletion treatment, the concentration of EGFP-PTEN remained unchanged in the nucleus and decreased significantly in cytosol. The diffusion coefficient of EGFP-PTEN decreased significantly in cytosol, but showed no significant change in the nucleus; the alpha value decreased significantly in both the nucleus and cytoplasm. These results suggest that the concentration and mobility of PTEN in the nucleus and cytoplasm can be regulated by stimulation methods. Our approach provides a unique method for real-time monitoring of protein dynamics in different subcellular compartments under different stimulation treatments.

Simultaneously Inducing and Tracking Cancer Cell Metabolism Repression by Mitochondria-Immobilized Rhenium(I) Complex.

Mitochondrial metabolism is essential for tumorigenesis, and the development of cancer is usually accompanied by alternations of mitochondrial function. Emerging studies have demonstrated that targeting mitochondria and mitochondrial metabolism is an effective strategy for cancer therapy. In this work, eight phosphorescent organometallic rhenium(I) complexes have been synthesized and explored as mitochondria-targeted theranostic agents, capable of inducing and tracking the therapeutic effect simultaneously. Complexes 1b-4b can quickly and efficiently penetrate into A549 cells, specifically localizing within mitochondria, and their cytotoxicity is superior to cisplatin against the cancer cells screened. Notably, complex 3b [Re(CO)3(DIP) (py-3-CH2Cl)]+ containing thiol-reactive chloromethylpyridyl moiety for mitochondria immobilization shows higher cytotoxicity and selectivity against cancer cells than other Re(I) complexes without mitochondria-immobilization properties. Mechanistic studies show that complexes 1b-4b induce a cascade of mitochondria-dependent events including mitochondrial damage, mitochondrial respiration inhibition, cellular ATP depletion, reactive oxygen species (ROS) elevation, and caspase-dependent apoptosis. By comparison, mitochondria-immobilized 3b causes more effective repression of mitochondrial metabolism than mitochondrial-nonimmobilized complexes. The excellent phosphorescence and O2-sensitive lifetimes of mitochondria-immobilized 3b can be utilized for real-time tracking of the morphological changes of mitochondria and mitochondrial respiration repression during therapy process, accordingly providing reliable information for understanding anticancer mechanisms.

Light-Up Mitophagy in Live Cells with Dual-Functional Theranostic Phosphorescent Iridium(III) Complexes.

Phosphorescent Ir(III) complexes are expected to be new multifunctional theranostic platforms that enable the integration of imaging capabilities and anticancer properties. Mitophagy is an important selective autophagic process that degrades dysfunctional mitochondria. Until now, the regulation of mitophagy is still poorly understood. Herein, we present two phosphorescent cyclometalated iridium(III) complexes (Ir1 and Ir2) that can accumulate in mitochondria and induce mitophagy. Because of their intrinsic phosphorescence, they can specially image mitochondria and track mitochondrial morphological alterations. Mechanism studies show that Ir1 and Ir2 induce mitophagy by depolarization of mitochondrial membrane potential, depletion of cellular ATP, perturbation in mitochondrial metabolic status, and induction of oxidative stress. Moreover, no sign of apoptosis is observed in Ir1- and Ir2-treated cells under the same conditions that an obvious mitophagic response is initiated. We demonstrate that Ir1 is a promising theranostic agent that can induce mitophagy and visualize changes in mitochondrial morphology simultaneously.

Physiological responses to acid stress by Saccharomyces cerevisiae when applying high initial cell density.

High initial cell density is used to increase volumetric productivity and shorten production time in lignocellulosic hydrolysate fermentation. Comparison of physiological parameters in high initial cell density cultivation of Saccharomyces cerevisiae in the presence of acetic, formic, levulinic and cinnamic acids demonstrated general and acid-specific responses of cells. All the acids studied impaired growth and inhibited glycolytic flux, and caused oxidative stress and accumulation of trehalose. However, trehalose may play a role other than protecting yeast cells from acid-induced oxidative stress. Unlike the other acids, cinnamic acid did not cause depletion of cellular ATP, but abolished the growth of yeast on ethanol. Compared with low initial cell density, increasing initial cell density reduced the lag phase and improved the bioconversion yield of cinnamic acid during acid adaptation. In addition, yeast cells were able to grow at elevated concentrations of acid, probable due to the increase in phenotypic cell-to-cell heterogeneity in large inoculum size. Furthermore, the specific growth rate and the specific rates of glucose consumption and metabolite production were significantly lower than at low initial cell density, which was a result of the accumulation of a large fraction of cells that persisted in a viable but non-proliferating state.

Grifolin inhibits tumor cells adhesion and migration via suppressing interplay between PGC1α and Fra-1 / LSF- MMP2 / CD44 axes.

Grifolin, a farnesyl phenolic compound isolated from the fresh fruiting bodies of the mushroom Albatrellus confluens, exhibits effective antitumor bioactivity in previous study of our group and other lab. In this study, we observed that grifolin inhibited tumor cells adhesion and migration. Moreover, grifolin reduced reactive oxygen species (ROS) production and caused cellular ATP depletion in high-metastatic tumor cells. PGC1α (Peroxisome proliferator-activated receptor γ, coactivator 1α) encodes a transcriptional co-activator involved in mitochondrial biogenesis and respiration and play a critical role in the maintenance of energy homeostasis. Interestingly, grifolin suppressed the mRNA as well as protein level of PGC1α. We further identified that MMP2 and CD44 expressions were PGC1α inducible. PGC1α can bind with metastatic-associated transcription factors: Fra-1 and LSF and the protein-protein interaction was attenuated by grifolin treatment. Overall, these findings suggest that grifolin decreased ROS generation and intracellular ATP to suppress tumor cell adhesion/migration via impeding the interplay between PGC1α and Fra-1 /LSF-MMP2/CD44 axes. Grifolin may develop as a promising lead compound for antitumor therapies by targeting energy metabolism regulator PGC1α signaling.

Fatty Acid Oxidation Mediated by Acyl-CoA Synthetase Long Chain 3 Is Required for Mutant KRAS Lung Tumorigenesis

KRAS is one of the most commonly mutated oncogenes in human cancer. Mutant KRAS aberrantly regulates metabolic networks. However, the contribution of cellular metabolism to mutant KRAS tumorigenesis is not completely understood. We report that mutant KRAS regulates intracellular fatty acid metabolism through Acyl-coenzyme A (CoA) synthetase long-chain family member 3 (ACSL3), which converts fatty acids into fatty Acyl-CoA esters, the substrates for lipid synthesis and β-oxidation. ACSL3 suppression is associated with depletion of cellular ATP and causes the death of lung cancer cells. Furthermore, mutant KRAS promotes the cellular uptake, retention, accumulation, and β-oxidation of fatty acids in lung cancer cells in an ACSL3-dependent manner. Finally, ACSL3 is essential for mutant KRAS lung cancer tumorigenesis invivo and is highly expressed in human lung cancer. Our data demonstrate that mutant KRAS reprograms lipid homeostasis, establishing a metabolic requirement that could be exploited for therapeutic gain.

Addition of lysophospholipids with large head groups to cells inhibits Shiga toxin binding.

Shiga toxin (Stx), an AB5 toxin, binds specifically to the neutral glycosphingolipid Gb3 at the cell surface before being transported into cells. We here demonstrate that addition of conical lysophospholipids (LPLs) with large head groups inhibit Stx binding to cells whereas LPLs with small head groups do not. Lysophosphatidylinositol (LPI 18:0), the most efficient LPL with the largest head group, was selected for in-depth investigations to study how the binding of Stx is regulated. We show that the inhibition of Stx binding by LPI is reversible and possibly regulated by cholesterol since addition of methyl-β-cyclodextrin (mβCD) reversed the ability of LPI to inhibit binding. LPI-induced inhibition of Stx binding is independent of signalling and membrane turnover as it occurs in fixed cells as well as after depletion of cellular ATP. Furthermore, data obtained with fluorescent membrane dyes suggest that LPI treatment has a direct effect on plasma membrane lipid packing with shift towards a liquid disordered phase in the outer leaflet, while lysophosphoethanolamine (LPE), which has a small head group, does not. In conclusion, our data show that cellular treatment with conical LPLs with large head groups changes intrinsic properties of the plasma membrane and modulates Stx binding to Gb3.

Evaluation of nitric oxide donors impact on cisplatin resistance in various ovarian cancer cell lines

Ovarian cancer chemoresistance, both intrinsic and acquired, is the main obstacle in improving the outcome of anticancer therapies. Therefore the development of new treatment strategies, including the use of new compounds that can support the standard therapeutics is required. Among many candidates, nitric oxide (NO) donors, agents with multivalent targeted activities in cancer cells, are worth considering. The aim of this study was evaluation of SPER/NO and DETA/NO ability to enhance cisplatin cytotoxicity against different ovarian cancer cell lines. Obtained data indicate that NO donors action varies between different cancer cell lines and is strongest in low aggressive and cisplatin sensitive cells. While statistically significant, the enhancement of cisplatin cytotoxicity by NO donors is of low magnitude. The rise in the percentage of late apoptotic/necrotic ovarian cancer cells may suggest that NO donors enhancement action might be based on the cellular ATP depletion. Nevertheless, no significant impact of the NO donors, cisplatin or their combination on the expressions of ABCB1, BIRC5 and PTEN genes has been found. Although our data puts the therapeutical potential of NO donors to aid cisplatin action in question it may also point out at the further approach to utilize these compounds in therapies.

Cytotoxic effects of psychotropic benzofuran derivatives, N-methyl-5-(2-aminopropyl)benzofuran and its N-demethylated derivative, on isolated rat hepatocytes.

The novel psychoactive compounds derived from amphetamine have been illegally abused as recreational drugs, some of which are known to be hepatotoxic in humans and experimental animals. The cytotoxic effects and mechanisms of 5-(2-aminopropyl)benzofuran (5-APB) and N-methyl-5-(2-aminopropyl)benzofuran (5-MAPB), both of which are benzofuran analogues of amphetamine, and 3,4-methylenedioxy-N-methamphetamine (MDMA) were studied in freshly isolated rat hepatocytes. 5-MAPB caused not only concentration-dependent (0-4.0 mm) and time-dependent (0-3 h) cell death accompanied by the depletion of cellular ATP and reduced glutathione and protein thiol levels, but also accumulation of oxidized glutathione. Of the other analogues examined at a concentration of 4 mm, 5-MAPB/5-APB-induced cytotoxicity with the production of reactive oxygen species and loss of mitochondrial membrane potential was greater than that induced by MDMA. In isolated rat liver mitochondria, the benzofurans resulted in a greater increase in the rate of state 4 oxygen consumption than did MDMA, with a decrease in the rate of state 3 oxygen consumption. Furthermore, the benzofurans caused more of a rapid mitochondrial swelling dependent on the mitochondrial permeability transition than MDMA. 5-MAPB at a weakly toxic level (1 mm) was metabolized slowly: levels of 5-MAPB and 5-APB were approximately 0.9 mm and 50 μm, respectively, after 3 h incubation. Taken collectively, these results indicate that mitochondria are target organelles for the benzofuran analogues and MDMA, which elicit cytotoxicity through mitochondrial failure, and the onset of cytotoxicity may depend on the initial and/or residual concentrations of 5-MAPB rather than on those of its metabolite 5-APB. Copyright © 2016 John Wiley & Sons, Ltd.

Altered expression of Mg2 + transport proteins during Parkinson's disease-like dopaminergic cell degeneration in PC12 cells

Mg2+ is an essential cation to maintain cellular functions, and intracellular Mg2+ concentration ([Mg2+]i) is regulated by Mg2+ channels and transporters. In our previous study, we demonstrated that MPP+ elicits Mg2+ influx across the cell membrane and Mg2+ mobilization from mitochondria, and the resulting [Mg2+]i is an important determinants of the cell viability in MPP+ model of Parkinson's disease (PD). It indicates that cellular Mg2+ transport is one of the important factors to determine the progress of PD. However, whether the expression levels of Mg2+ transport proteins change in the progress of PD has still been obscure. In this study, we estimated the mRNA expression levels of Mg2+ transport proteins upon the exposure to MPP+. In thirteen Mg2+ transport proteins examined, mRNA expression level of SLC41A2 was increased and that of ACDP2, NIPA1 and MMgT2 were decreased. Knockdown of SLC41A2, ACDP2 or NIPA1 accelerated the MPP+-induced cell degeneration, and overexpression attenuated it. The decrease in the mRNA expression levels of NIPA1 and MMgT2 were also elicited by rotenone, H2O2 and FCCP, indicating that mitochondrial dysfunction related to this down-regulation. The increase in that of SLC41A2 was induced by an uncoupler, FCCP, as well as MPP+, suggesting that it is an intrinsic protection mechanism against depolarized mitochondrial membrane potential and/or cellular ATP depletion. Our results shown here indicate that alteration of Mg2+ transport proteins is implicated in the MPP+ model of PD, and it affects cell degeneration.