Cellular ATP Levels Research Articles

Leaching components and initial biocompatibility of novel bioactive restorative materials.

Bioactive restorative materials were developed on the premise that direct restorations should not only serve the purpose of reconstructing dental hard tissue defects but also exhibit biological features that prevent secondary caries development, without having adverse effects on the host cells. This study focuses on assessing the in vitro biocompatibility of two novel bioactive restorative materials. Specimens of the bioactive restorative materials, Cention Forte (CF) and ACTIVA BioACTIVE RESTORATIVE (AB), a glass ionomer cement/glass hybrid (EQUIA Forte HT, EF) and an established nanohybrid composite (Venus Diamond, VD) were produced and finished. The specimens were eluted in water and methanol and the resulting eluates were analyzed via gas chromatography-mass spectrometry. hGF-1 cells were exposed to eluates prepared in cell culture medium. Cellular ATP levels, oxidized glutathione concentration, caspase-3/7 activity and the inflammatory response (IL-6 and PGE2 levels) were determined. Microscopic images were taken to examine the cell morphology. Methyl methacrylate and 2-Hydroxyethyl methacrylate were the main monomers detected in CF and AB eluates. All materials inhibited cell proliferation and led to significantly reduced ATP-levels. The cells exhibited a healthy morphology in the presence of CF and AB. Cells exposed to VD showed increased oxidized glutathione levels. Only EF led to enhanced caspase-3/7 activity. CF and AB caused IL-6 levels to increase, while EF and AB led to enhanced PGE2 levels. CF and AB are promising materials from a biological point of view and seem to have improved bioactive properties compared to glass ionomer cements.

Genome-Wide Investigation of Apyrase (APY) Genes in Peanut (Arachis hypogaea L.) and Functional Characterization of a Pod-Abundant Expression Promoter AhAPY2-1p

Peanut (Arachis hypogaea L.) is an important food and feed crop worldwide and is affected by various biotic and abiotic stresses. The cellular ATP levels decrease significantly during stress as ATP molecules move to extracellular spaces, resulting in increased ROS production and cell apoptosis. Apyrases (APYs) are the nucleoside phosphatase (NPTs) superfamily members and play an important role in regulating cellular ATP levels under stress. We identified 17 APY homologs in A. hypogaea (AhAPYs), and their phylogenetic relationships, conserved motifs, putative miRNAs targeting different AhAPYs, cis-regulatory elements, etc., were studied in detail. The transcriptome expression data were used to observe the expression patterns in different tissues and under stress conditions. We found that the AhAPY2-1 gene showed abundant expression in the pericarp. As the pericarp is a key defense organ against environmental stress and promoters are the key elements regulating gene expression, we functionally characterized the AhAPY2-1 promoter for its possible use in future breeding programs. The functional characterization of AhAPY2-1P in transgenic Arabidopsis plants showed that it effectively regulated GUS gene expression in the pericarp. GUS expression was also detected in flowers of transgenic Arabidopsis plants. Overall, these results strongly suggest that APYs are an important future research subject for peanut and other crops, and AhPAY2-1P can be used to drive the resistance-related genes in a pericarp-specific manner to enhance the defensive abilities of the pericarp.

The alphavirus nonstructural protein 2 NTPase induces a host translational shut-off through phosphorylation of eEF2 via cAMP-PKA-eEF2K signaling.

Chikungunya virus (CHIKV) is a reemerging alphavirus. Since 2005, it has infected millions of people during outbreaks in Africa, Asia, and South/Central America. CHIKV replication depends on host cell factors at many levels and is expected to have a profound effect on cellular physiology. To obtain more insight into host responses to infection, stable isotope labeling with amino acids in cell culture and liquid chromatography-tandem mass spectrometry were used to assess temporal changes in the cellular phosphoproteome during CHIKV infection. Among the ~3,000 unique phosphorylation sites analyzed, the largest change in phosphorylation status was measured on residue T56 of eukaryotic elongation factor 2 (eEF2), which showed a >50-fold increase at 8 and 12 h p.i. Infection with other alphaviruses (Semliki Forest, Sindbis and Venezuelan equine encephalitis virus (VEEV)) triggered a similarly strong eEF2 phosphorylation. Expression of a truncated form of CHIKV or VEEV nsP2, containing only the N-terminal and NTPase/helicase domains (nsP2-NTD-Hel), sufficed to induce eEF2 phosphorylation, which could be prevented by mutating key residues in the Walker A and B motifs of the NTPase domain. Alphavirus infection or expression of nsP2-NTD-Hel resulted in decreased cellular ATP levels and increased cAMP levels. This did not occur when catalytically inactive NTPase mutants were expressed. The wild-type nsP2-NTD-Hel inhibited cellular translation independent of the C-terminal nsP2 domain, which was previously implicated in directing the virus-induced host shut-off for Old World alphaviruses. We hypothesize that the alphavirus NTPase activates a cellular adenylyl cyclase resulting in increased cAMP levels, thus activating PKA and subsequently eukaryotic elongation factor 2 kinase. This in turn triggers eEF2 phosphorylation and translational inhibition. We conclude that the nsP2-driven increase of cAMP levels contributes to the alphavirus-induced shut-off of cellular protein synthesis that is shared between Old and New World alphaviruses. MS Data are available via ProteomeXchange with identifier PXD009381.

Investigating the adverse outcome pathways (AOP) of neurotoxicity induced by DBDPE with a combination of in vitro and in silico approaches

Current studies have shown an association between DBDPE and neurotoxicity. In this study, the adverse outcome pathway (AOP) and mechanistic analysis of DBDPE-induced neurotoxicity were explored by a combination of in vitro and in silico approaches in SK-N-SH cells. DBDPE-induced oxidative stress caused DNA strand breaks, resulting in the activation of poly (ADP-ribose) (PAR) polymerase-1 (PARP-1). Activation of PARP1 could cause toxic damage in various organ systems, especially in the nervous system. DBDPE-induced apoptosis via the caspase-dependent intrinsic mitochondrial pathway and the PARP1-dependent pathway. Activation of PARP1 by DBDPE was deemed the initiating event, thereby affecting the key downstream biochemical events (e.g., ROS production, DNA damage, membrane potential changes, and ATP reduction), which induced apoptosis. Furthermore, excessive activation of PARP1 was accompanied by the translocation of the apoptosis-inducing factor (AIF), which was associated with PARP1-dependent cell death. The inhibition of PARP1 by PJ34 reduced DBDPE-induced apoptosis and maintained cellular ATP levels. PJ34 also prevented the translocation of AIF from the mitochondria to the nucleus. These findings improve the understanding of the mechanism of DBDPE-induced neurotoxic effects and provide a theoretical basis for the ecological risk of DBDPE.

ACTA1 H40Y mutant iPSC-derived skeletal myocytes display mitochondrial defects in an in vitro model of nemaline myopathy

Nemaline myopathies (NM) are a group of congenital myopathies that lead to muscle weakness and dysfunction. While 13 genes have been identified to cause NM, over 50% of these genetic defects are due to mutations in nebulin (NEB) and skeletal muscle actin (ACTA1), which are genes required for normal assembly and function of the thin filament. NM can be distinguished on muscle biopsies due to the presence of nemaline rods, which are thought to be aggregates of the dysfunctional protein. Mutations in ACTA1 have been associated with more severe clinical disease and muscle weakness. However, the cellular pathogenesis linking ACTA1 gene mutations to muscle weakness are unclear To evaluate cellular disease phenotypes, iPSC-derived skeletal myocytes (iSkM) harboring an ACTA1 H40Y point mutation were used to model NM in skeletal muscle. These were generated by Crispr-Cas9, and include one non-affected healthy control (C) and 2 NM iPSC clone lines, therefore representing isogenic controls. Fully differentiated iSkM were characterized to confirm myogenic status and subject to assays to evaluate nemaline rod formation, mitochondrial membrane potential, mitochondrial permeability transition pore (mPTP) formation, superoxide production, ATP/ADP/phosphate levels and lactate dehydrogenase release. C- and NM-iSkM demonstrated myogenic commitment as evidenced by mRNA expression of Pax3, Pax7, MyoD, Myf5 and Myogenin; and protein expression of Pax4, Pax7, MyoD and MF20. No nemaline rods were observed with immunofluorescent staining of NM-iSkM for ACTA1 or ACTN2, and these mRNA transcript and protein levels were comparable to C-iSkM. Mitochondrial function was altered in NM, as evidenced by decreased cellular ATP levels and altered mitochondrial membrane potential. Oxidative stress induction revealed the mitochondrial phenotype, as evidenced by collapsed mitochondrial membrane potential, early formation of the mPTP and increased superoxide production. Early mPTP formation was rescued with the addition of ATP to media. Together, these findings suggest that mitochondrial dysfunction and oxidative stress are disease phenotypes in the in vitro model of ACTA1 nemaline myopathy, and that modulation of ATP levels was sufficient to protect NM-iSkM mitochondria from stress-induced injury. Importantly, the nemaline rod phenotype was absent in our in vitro model of NM. We conclude that this in vitro model has the potential to recapitulate human NM disease phenotypes, and warrants further study.

Butyrate prevents the migration and invasion, and aerobic glycolysis in gastric cancer via inhibiting Wnt/β-catenin/c-Myc signaling.

Gastric cancer (GC) remains a common cause of cancer death worldwide. Evidence has found that butyrate exhibited antitumor effects on GC cells. However, the mechanism by which butyrate regulate GC cell proliferation, migration, invasion, and aerobic glycolysis remains largely unknown. The proliferation, migration, and invasion of GC cells were tested by EdU staining, transwell assays. Additionally, protein expressions were determined by western blot assay. Next, glucose uptake, lactate production, and cellular ATP levels in GC cells were detected. Furthermore, the antitumor effects of butyrate in tumor-bearing nude mice were evaluated. We found, butyrate significantly prevented GC cell proliferation, migration, and invasion (p < .01). Additionally, butyrate markedly inhibited GC cell aerobic glycolysis, as shown by the reduced expressions of GLUT1, HK2, and LDHA (p < .01). Moreover, butyrate notably decreased nuclear β-catenin and c-Myc levels in GC cells (p < .01). Remarkably, through activating Wnt/β-catenin signaling with LiCl, the inhibitory effects of butyrate on the growth and aerobic glycolysis of GC cells were diminished (p < .01). Moreover, butyrate notably suppressed tumor volume and weight in GC cell xenograft nude mice in vivo (p < .01). Meanwhile, butyrate obviously reduced nuclear β-catenin, c-Myc, GLUT1, HK2 and LDHA levels in tumor tissues in GC cell xenograft mice (p < .01). Collectively, butyrate could suppress the growth and aerobic glycolysis of GC cells in vitro and in vivo via downregulating wnt/β-catenin/c-Myc signaling. These findings are likely to prove useful in better understanding the role of butyrate in GC.

Oncolytic Avian Reovirus σA-Modulated Upregulation of the HIF-1α/C-myc/glut1 Pathway to Produce More Energy in Different Cancer Cell Lines Benefiting Virus Replication.

Our previous reports proved that the structural protein σA of avian reovirus (ARV) is an energy activator which can regulate cellular metabolism that is essential for virus replication. This study has further demonstrated that the ARV protein σA is able to upregulate the HIF-1α/myc/glut1 pathway in three cancer cell lines (A549, B16-F10, and HeLa) to alter the metabolic pathway of host cells. Quantitative real-time RT-PCR and Western blotting results have revealed that σA protein could enhance both mRNA and the protein levels of HIF-1α, c-myc, and glut1 in these cancer cell lines. In this work, ATeam immunofluorescence staining was used to reveal that knockdown of HIF-1α, c-myc, and glut1 by shRNAs decreased cellular ATP levels. Our data reveal that the ARV σA protein can downregulate lactate fermentation and upregulate glutaminolysis. The σA protein upregulates glutaminase, which converts glutamate into the TCA cycle intermediate α-ketoglutarate, activating the TCA cycle. In the lactate fermentation pathway, ARV σA protein suppresses lactate dehydrogenase A (LDHA), implying the Warburg effect does not occur in these cancer cell lines. This study provides a novel finding revealing that ARV σA protein upregulates glycolysis and glutaminolysis to produce energy using the HIF-1α/c-myc/glut1 pathway to benefit virus replication in these cancer cell lines.

Depletion of SOD2 enhances nasopharyngeal carcinoma cell radiosensitivity via ferroptosis induction modulated by DHODH inhibition

BackgroundRecurrence due to the development of radioresistance remains a major challenge in the clinical management of nasopharyngeal carcinoma. The objective of this study was to increase the sensitivity of nasopharyngeal carcinoma cells to ionizing radiation by enhancing oxidative stress and ferroptosis caused by disrupting the mitochondrial anti-oxidant enzyme system.MethodsOxidative stress cell model was constructed by SOD2 knockdown using shRNA. The expression and activity of DHODH was suppressed by siRNA and brequinar in SOD2 depleted cells. Protein levels were determined by western blotting and ferroptosis was assessed by C11 BODIPY and malondialdehyde assay. Cell viability was evaluated using CCK-8 assay while radiotoxicity was assessed by colony formation assay. Cellular ATP level was determined by ATP assay kits, ROS was determined by DCFD and DHE, while mitochondrial oxygen consumption was determined by seahorse assay. Data were analyzed by two-tailed independent t-test.ResultsRadiation upregulated SOD2 expression and SOD2 depletion increased cellular O2.−, malondialdehyde, and the fluorescence intensity of oxidized C11 BODIPY. It also resulted in mitochondrial damage. Its depletion decreased colony formation both under ionizing and non-ionizing radiation conditions. The ferroptosis inhibitor, deferoxamine, rescued cell viability and colony formation in SOD2 depleted cells. Cellular level of malondialdehyde, fluorescence intensity of oxidized C11 BODIPY, O2.− level, ATP, and mitochondrial oxygen consumption decreased following DHODH inhibition in SOD2 depleted cells. Cell viability and colony formation was rescued by DHODH inhibition in SOD2 depleted cells.ConclusionInducing oxidative stress by SOD2 inhibition sensitized nasopharyngeal carcinoma cells to ionizing radiation via ferroptosis induction. This was found to be dependent on DHODH activity. This suggests that DHODH inhibitors should be used with caution during radiotherapy in nasopharyngeal carcinoma patients.

Interaction between Butyrate and Tumor Necrosis Factor α in Primary Rat Colonocytes.

Butyrate, a short-chain fatty acid, is utilized by the gut epithelium as energy and it improves the gut epithelial barrier. More recently, it has been associated with beneficial effects on immune and cardiovascular homeostasis. Conversely, tumor necrosis factor alpha (TNFα) is a pro-inflammatory and pro-hypertensive cytokine. While butyrate and TNFα are both linked with hypertension, studies have not yet addressed their interaction in the colon. Here, we investigated the capacity of butyrate to modulate a host of effects of TNFα in primary rodent colonic cells in vitro. We measured ATP levels, cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), mitochondrial oxidative phosphorylation, and glycolytic activity in colonocytes following exposure to either butyrate or TNFα, or both. To address the potential mechanisms, transcripts related to oxidative stress, cell fate, and cell metabolism (Pdk1, Pdk2, Pdk4, Spr, Slc16a1, Slc16a3, Ppargc1a, Cs, Lgr5, Casp3, Tnfr2, Bax, Bcl2, Sod1, Sod2, and Cat) were measured, and untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to profile the metabolic responses of colonocytes following exposure to butyrate and TNFα. We found that both butyrate and TNFα lowered cellular ATP levels towards a quiescent cell energy phenotype, characterized by decreased oxygen consumption and extracellular acidification. Co-treatment with butyrate ameliorated TNFα-induced cytotoxicity and the reduction in cell viability. Butyrate also opposed the TNFα-mediated decrease in MMP and mitochondrial-to-intracellular calcium ratios, suggesting that butyrate may protect colonocytes against TNFα-induced cytotoxicity by decreasing mitochondrial calcium flux. The relative expression levels of pyruvate dehydrogenase kinase 4 (Pdk4) were increased via co-treatment of butyrate and TNFα, suggesting the synergistic inhibition of glycolysis. TNFα alone reduced the expression of monocarboxylate transporters slc16a1 and slc16a3, suggesting effects of TNFα on butyrate uptake into colonocytes. Of the 185 metabolites that were detected with LC-MS, the TNFα-induced increase in biopterin produced the only significant change, suggesting an alteration in mitochondrial biogenesis in colonocytes. Considering the reports of elevated colonic TNFα and reduced butyrate metabolism in many conditions, including in hypertension, the present work sheds light on cellular interactions between TNFα and butyrate in colonocytes that may be important in understanding conditions of the colon.

How Many Sirtuin Genes Are Out There? Evolution of Sirtuin Genes in Vertebrates With a Description of a New Family Member.

Studying the evolutionary history of gene families is a challenging and exciting task with a wide range of implications. In addition to exploring fundamental questions about the origin and evolution of genes, disentangling their evolution is also critical to those who do functional/structural studies to allow a deeper and more precise interpretation of their results in an evolutionary context. The sirtuin gene family is a group of genes that are involved in a variety of biological functions mostly related to aging. Their duplicative history is an open question, as well as the definition of the repertoire of sirtuin genes among vertebrates. Our results show a well-resolved phylogeny that represents an improvement in our understanding of the duplicative history of the sirtuin gene family. We identified a new sirtuin gene family member (SIRT3.2) that was apparently lost in the last common ancestor of amniotes but retained in all other groups of jawed vertebrates. According to our experimental analyses, elephant shark SIRT3.2 protein is located in mitochondria, the overexpression of which leads to an increase in cellular levels of ATP. Moreover, in vitro analysis demonstrated that it has deacetylase activity being modulated in a similar way to mammalian SIRT3. Our results indicate that there are at least eight sirtuin paralogs among vertebrates and that all of them can be traced back to the last common ancestor of the group that existed between 676 and 615 millions of years ago.

Effect of Sirolimus/Metformin Co-Treatment on Hyperglycemia and Cellular Respiration in BALB/c Mice.

Sirolimus (SRL) is widely used as an immunosuppressant to prevent graft rejection, despite the risk of impairing glucose metabolism. Metformin (MET) can reduce the detrimental effects of SRL in many patients, including diabetes and renal transplant recipients. Limited in vivo studies have reported on SRL and MET therapy, particularly in relation to cellular bioenergetics, glucose metabolism, and insulin resistance. Herein, we investigated the efficacy of SRL and MET co-treatment in BALB/c mice over 4 weeks. Balb/c mice (4-6 weeks old) were divided into four groups and injected intraperitoneally (i.p.) with water (control, CTRL), MET (200 µg/g), SRL (5 µg/g), or MET (200 µg/g) +SRL (5 µg/g) over a period of one month. We evaluated the body weight, food consumption rate, random blood glucose (BG), insulin levels, serum biochemistry parameters (ALT, Albumin, BUN, Creatinine), and histomorphology in all groups using standardized techniques and assays. All drug-treated groups showed a statistically significant decrease in weight gain compared to the CTRL group, despite normal food intake. Treatment with SRL caused elevated BG and insulin levels, which were restored with SRL + MET combination. Serum biochemical parameters were within the normal range in all the studied groups. SRL+ MET co-treatment decreased liver cellular respiration and increased cellular ATP levels in the liver. In the pancreas, co-treatment resulted in increased cellular respiration and decreased cellular ATP levels. Liver and pancreatic histology were unchanged in all groups. This study showed that co-treatment of SRL with MET alleviates hyperglycemia induced by SRL without any deleterious effects. These results provide initial insights into the potential use of SRL + MET therapy in various settings.

Restoration of mitochondrial structure and function within Helicobacter pylori VacA intoxicated cells.

The Helicobacter pylori vacuolating cytotoxin (VacA) is an intracellular, mitochondrial-targeting exotoxin that rapidly causes mitochondrial dysfunction and fragmentation. Although VacA targeting of mitochondria has been reported to alter overall cellular metabolism, there is little known about the consequences of extended exposure to the toxin. Here, we describe studies to address this gap in knowledge, which have revealed that mitochondrial dysfunction and fragmentation are followed by a time-dependent recovery of mitochondrial structure, mitochondrial transmembrane potential, and cellular ATP levels. Cells exposed to VacA also initially demonstrated a reduction in oxidative phosphorylation, as well as increase in compensatory aerobic glycolysis. These metabolic alterations were reversed in cells with limited toxin exposure, congruent with the recovery of mitochondrial transmembrane potential and the absence of cytochrome c release from the mitochondria. Taken together, these results are consistent with a model that mitochondrial structure and function are restored in VacA-intoxicated cells.

Combination of metformin/efavirenz/fluoxetine exhibits profound anticancer activity via a cancer cell-specific ROS amplification

ABSTRACT The possible anticancer activity of combination (M + E + F) of metformin (M), efavirenz (E), and fluoxetine (F) was investigated in normal HDF cells and HCT116 human colon cancer cells. Metformin increased cellular FOXO3a, p-FOXO3a, AMPK, p-AMPK, and MnSOD levels in HDFs but not in HCT116 cells. Cellular ATP level was decreased only in HDFs by metformin. Metformin increased ROS level only in HCT116 cells. Transfection of si-FOXO3a into HCT116 reversed the metformin-induced cellular ROS induction, indicating that FOXO3a/MnSOD is the key regulator for cellular ROS level. Viability readout with M, E, and F alone decreased slightly, but the combination of three drugs dramatically decreased cell survival in HCT116, A549, and SK-Hep-1 cancer cells but not in HDF cells. ROS levels in HCT116 cells were massively increased by M + E + F combination, but not in HDF cells. Cell cycle analysis showed that of M + E + F combination caused cell death only in HCT116 cells. The combination of M + E + F reduced synergistically mitochondrial membrane potential and mitochondrial electron transport chain complex I and III activities in HCT116 cells when compared with individual treatments. Western blot analysis indicated that DNA damage, apoptosis, autophagy, and necroptosis-realated factors increased in M + E + F-treated HCT116 cells. Oral administration with M + E + F combination for 3 weeks caused dramatic reductions in tumor volume and weight in HCT116 xenograft model of nude mice when compared with untreated ones. Our results suggest that M + E + F have profound anticancer activity both in vitro and in vivo via a cancer cell-specific ROS amplification (CASRA) through ROS-induced DNA damage, apoptosis, autophagy, and necroptosis.

Restoration of CPT1A-mediated fatty acid oxidation in mesothelial cells protects against peritoneal fibrosis.

Background: Peritoneal dialysis (PD) is limited by gradual fibrotic remodeling in the peritoneum, a process involving profibrotic response of mesothelial cells. However, the role of fatty acid oxidation (FAO) and carnitine palmitoyltransferase 1A (CPT1A) in this process remains unexplored. Methods: FAO and CPT1A expression were characterized in mesothelial cells from patients on long-term PD and from a mouse model of PD using multiple experimental methods, including single-cell sequencing, seahorse assay, real-time quantitative PCR, Western blot, and immunofluorescence staining. Overexpression of CPT1A was achieved in a human mesothelial cell line and in primary mouse mesothelial cells. Finally, genetic and pharmacological manipulations of CPT1A were performed in a mouse model of PD. Results: Herein, FAO and CPT1A expression were reduced in mesothelial cells from patients on long-term PD, which negatively correlated with expression of fibrogenic markers in these cells. This was corroborated in PD mice, as well as in mouse and human mesothelial cells incubated with transforming growth factor (TGF) β1. CPT1A overexpression in mesothelial cells, which prevented TGFβ1-induced suppression of mitochondrial respiration, restored cellular ATP levels and downregulated the expression of fibrogenic markers. Furthermore, restoration of FAO by overexpressing CPT1A in PD mice reversed profibrotic phenotype in mesothelial cells and reduced fibrotic lesions in the peritoneum. Treatment with the CPT1A activator C75 induced similar therapeutic benefit in PD mice. In contrast, inhibition of FAO with a CPT1 inhibitor caused more severe fibrosis in PD mice. Conclusions: A defective FAO is responsible for the profibrotic response of mesothelial cells and thus the peritoneal fibrogenesis. This aberrant metabolic state could be improved by modulating CPT1A in mesothelial cells, suggesting FAO enhancement in mesothelial cells is a potential treatment of peritoneal fibrosis.

Macrophage Resistance to Ionizing Radiation Exposure Is Accompanied by Decreased Cathepsin D and Increased Transferrin Receptor 1 Expression.

To identify a molecular signature of macrophages exposed to clinically relevant ionizing radiation (IR) doses, mirroring radiotherapy sessions. Human monocyte-derived macrophages were exposed to 2 Gy/ fraction/ day for 5 days, mimicking one week of cancer patient's radiotherapy. Protein expression profile by proteomics was performed. A gene ontology analysis revealed that radiation-induced protein changes are associated with metabolic alterations, which were further supported by a reduction of both cellular ATP levels and glucose uptake. Most of the radiation-induced deregulated targets exhibited a decreased expression, as was the case of cathepsin D, a lysosomal protease associated with cell death, which was validated by Western blot. We also found that irradiated macrophages exhibited an increased expression of the transferrin receptor 1 (TfR1), which is responsible for the uptake of transferrin-bound iron. TfR1 upregulation was also found in tumor-associated mouse macrophages upon tumor irradiation. In vitro irradiated macrophages also presented a trend for increased divalent metal transporter 1 (DMT1), which transports iron from the endosome to the cytosol, and a significant increase in iron release. Irradiated macrophages present lower ATP levels and glucose uptake, and exhibit decreased cathepsin D expression, while increasing TfR1 expression and altering iron metabolism.

The ADP-ribose hydrolase NUDT5 is important for DNA repair.

DNA damage leads to rapid synthesis of poly(ADP-ribose) (pADPr), which is important for damage signaling and repair. pADPr chains are removed by poly(ADP-ribose) glycohydrolase (PARG), releasing free mono(ADP-ribose) (mADPr). Here, we show that the NUDIX hydrolase NUDT5, which can hydrolyze mADPr to ribose-5-phosphate and either AMP or ATP, is recruited to damage sites through interaction with PARG. NUDT5 does not regulate PARP or PARG activity. Instead, loss of NUDT5 reduces basal cellular ATP levels and exacerbates the decrease in cellular ATP that occurs during DNA repair. Further, loss of NUDT5 activity impairs RAD51 recruitment, attenuates the phosphorylation of key DNA-repair proteins, and reduces both H2A.Z exchange at damage sites and repair by homologous recombination. The ability of NUDT5 to hydrolyze mADPr, and/or regulate cellular ATP, may therefore be important for efficient DNA repair. Targeting NUDT5 to disrupt PAR/mADPr and energy metabolism may be an effective anti-cancer strategy.

ATP hydrolysis kinetics and thermodynamics as determinants of calcium oscillation in pancreatic β cells

Cellular ATP plays an important role in the calcium oscillation signal transduction pathway of pancreatic $\ensuremath{\beta}$ cells. It triggers oscillation by binding the ATP-sensitive ${\text{K}}^{+}$ channels $({\text{K}}_{\mathrm{ATP}})$, and maintains the oscillation by providing ATP hydrolysis free energy for the normal function of ion pumps on the plasma membrane. To reveal how cellular ATP level and ATP hydrolysis free energy affect calcium oscillation, we first constructed a simplified kinetic model of ${\text{K}}_{\mathrm{ATP}}$ and calcium pumps, then analyzed their thermodynamic characteristics. Bifurcation of calcium oscillation is determined by both cellular ATP concentration and ATP hydrolysis free energy such that an insufficient ATP energy supply would result in dysfunctional calcium oscillation. Second, to investigate the glucose sensing in $\ensuremath{\beta}$ cells, we developed a glycolysis-calcium model that considers the dynamics of ATP and free energy levels. The model simulated three calcium patterns in wild type cells and impaired calcium response of ${\text{K}}_{\mathrm{ATP}}$ mutant cells, allowing the use of the ATP-free energy phase plane to explore the underlying mechanism. Our results reveal the thermodynamics of calcium oscillation and provide a framework for understanding the thermodynamics of other ion transport systems.

A new inhibitor of human peptide deformylase suppresses cell proliferation and induces cell apoptosis and autophagy in cancers.

Human peptide deformylase (hsPDF) has been found overexpressed in many cancer cells and its inhibitors exhibit antitumor activity. Studies were performed to validate that hsPDF is a good antitumor target. The inhibitory effect of PDF64 on hsPDF enzymatic activity was measured and confirmed by computation analysis. Antiproliferation activity was determined and in-vivo antitumor activity were analyzed in HCT116 and HL60 nude mice xenografts. Mitochondrial membrane potential (MMP), cell apoptosis, and autophagic cell death were analyzed by flow cytometry. ATP level was quantified using an ATP assay kit. Protein expression and kinase phosphorylation were determined by western blotting. A new hsPDF inhibitor PDF64 was identified. It showed evident antiproliferation activity in 10 cancer cells and significantly suppressed tumor growth in HCT116 and HL60 xenografts. It induced an obvious decrease in MMP and caused apparent cell apoptosis and autophagy in HCT116 and Jurkat cells. PDF64 treatment also led to an evident decrease in cellular ATP levels in these cells. Moreover, PDF64 downregulated c-Myc expression and had some effects on extracellular regulated protein kinases (ERK) and protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) pathways. PDF64 exhibited good antitumor effects both in vivo and in vitro . It caused cell apoptosis and autophagic death in HCT116 and Jurkat cells. The effects may be mediated by inhibiting c-Myc expression and ERK or PI3K-Akt-mTOR pathway. Therefore, PDF64 may be a promising reagent for antitumor drug development, which further supports that hsPDF is a good antitumor drug target.

Characterization of zinc(II) complex of 1,4,7,10-tetrazacyclododecane and deprotonated 5-fluorouracil (FU) in crystalline/solution states and evaluation of anticancer activity: Approach for improving the anticancer activity of FU

Structural and biological properties of 5-fluorouracil (FU) bound zinc(II)–cyclen complex (FU––ZnL2+, L = cyclen; 1,4,7,10-tetrazacyclododecane) were studied in to develop an anticancer drug candidate. The complex formation was analyzed by X-ray crystallography and pH-titration study, and the anticancer activity was investigated by cytotoxicity examination against HL-60 cells. The crystal structure revealed that FU– and ZnL+ form the complex with 1:1 stoichiometry. The pH titration study proved that FU– has a strong affinity to ZnL+ at the physiological pH range (6.5–9.0). The IC50 values of FU––ZnL2+ against HL-60 cells growth was 132 μM in a quantitating cellular ATP levels. These results provide the basis for designing novel anticancer drug complexes and controlling FU release.

Nanofibrillar peptide hydrogels for self-delivery of lonidamine and synergistic photodynamic therapy

The use of lonidamine (LND) in photodynamic therapy (PDT) provides a viable approach to develop low-dose PDT modules with high efficacy, for LND potentiates cytotoxicity of photosensitizers through dysregulation of mitochondrial function. Yet, the efficacy of LND is restricted by its low accumulation in cancer cells, especially in the mitochondrial compartments. To address the problem, we design an LND-derived self-assembling peptide molecule (LND-K) that dually targets integrin receptors and mitochondria of cancer cells. The targeted cellular delivery of LND-K gives higher efficacy in ablation of mitochondrial function in melanoma cells A375, as compared to free LND or the control molecule that lacks mitochondria-targeting moieties. To integrate LND-K in a typical PDT module, we develop a nanofibrillar hydrogel system through co-assembly of LND-K and TPPS4, an anionic photosensitizer that forms tight electrostatic interactions with cationic residues of LND-K. Notably, hydrogel formulation of LND-K/TPPS4 facilitates slow release of TPPS4 over 14 days in vitro, and displays a longer retention time than aqueous solution of TPPS4in vivo. By integrating a mitochondria-targeted molecule (LND-K) in a typical PDT module, we achieve synergistic killing of A375 cells with dual drugs, where LND-K not only serves as a chemotherapeutic drug, but also potentiates the cytotoxicities of TPPS4 toward A375 cells in vitro and in vivo. The peptide-based drug self-delivery system promises the development of efficacious combination treatments against cancer, that integrate cell sensitization with existing anticancer modules (e.g., chemotherapy and PDT) for enhanced therapeutic efficacy. Statement of significanceThis study reports the design and synthesis of a lonidamine (LND)-derived self-assembling peptide (LND-K) that dually targets integrin receptors and mitochondria of cancer cells. Under the precision guidance of a mitochondria-targeting sequence, LND-K-containing nanofibers target mitochondria and ablate mitochondrial functions. On one hand, the targeted delivery of LND-K reduces cell viabilities through a chemotherapy route; on the other hand, LND-K sensitizes cancer cells for subsequent PDT treatment with enhanced efficacy, which is mediated by induction of ROS, loss of mitochondrial membrane potential, and decrease of cellular ATP level. We believe that the design of mitochondria-targeted drug delivery systems with a self-assembling molecule provides a new approach to potentiate cytotoxicity of photosensitizers in a low-dose PDT module.