Induced ATP Depletion Research Articles

Arenicolide Family Macrolides Provide a New Therapeutic Lead Combating Multidrug‐resistant Tuberculosis

The emergence of multidrug‐resistant (MDR) and extensively drug‐resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) poses a significant threat to health globally. During searching for new chemical entities regulating MDR‐ and XDR‐Mtb, chemical investigation of the black oil beetle gut bacterium Micromonospora sp. GR10 led to the discovery of eight new members of arenicolides along with the identification of arenicolide A (Ar‐A, 1), which was a previously reported macrolide with incomplete configuration. Genomic analysis of the bacterial strain GR10 revealed their putative biosynthetic pathway. Quantum mechanics‐based computation, chemical derivatizations, and bioinformatic analysis established the absolute stereochemistry of Ar‐A and arenicolides D‐K (Ar‐D‐K, 2‐9) completely for the first time. Biological studies of 1‐9 revealed their antimicrobial activity against MDR and XDR strains of Mtb. Ar‐A had the most potent in vitro antimicrobial efficacy against MDR‐ and XDR‐Mtb. Mechanistically, Ar‐A induced ATP depletion and destabilized Mtb cell wall, thereby inhibiting growth. Notably, Ar‐A exerted a significant antimicrobial effect against Mtb in macrophages, was effective in the treatment of Mtb infections, and showed a synergistic effect with amikacin (AMK) in a mouse model of MDR‐Mtb lung infection. Collectively, our findings indicate Ar‐A to be a promising drug lead for drug‐resistant tuberculosis.

Arenicolide Family Macrolides Provide a New Therapeutic Lead Combating Multidrug-resistant Tuberculosis.

The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis (Mtb) poses a significant threat to health globally. During searching for new chemical entities regulating MDR- and XDR-Mtb, chemical investigation of the black oil beetle gut bacterium Micromonospora sp. GR10 led to the discovery of eight new members of arenicolides along with the identification of arenicolide A (Ar-A, 1), which was a previously reported macrolide with incomplete configuration. Genomic analysis of the bacterial strain GR10 revealed their putative biosynthetic pathway. Quantum mechanics-based computation, chemical derivatizations, and bioinformatic analysis established the absolute stereochemistry of Ar-A and arenicolides D-K (Ar-D-K, 2-9) completely for the first time. Biological studies of 1-9 revealed their antimicrobial activity against MDR and XDR strains of Mtb. Ar-A had the most potent in vitro antimicrobial efficacy against MDR- and XDR-Mtb. Mechanistically, Ar-A induced ATP depletion and destabilized Mtb cell wall, thereby inhibiting growth. Notably, Ar-A exerted a significant antimicrobial effect against Mtb in macrophages, was effective in the treatment of Mtb infections, and showed a synergistic effect with amikacin (AMK) in a mouse model of MDR-Mtb lung infection. Collectively, our findings indicate Ar-A to be a promising drug lead for drug-resistant tuberculosis.

In Vitro Methylene Blue and Carboplatin Combination Triggers Ovarian Cancer Cells Death.

Ovarian cancer presents a dire prognosis and high mortality rates, necessitating the exploration of alternative therapeutic avenues, particularly in the face of platinum-based chemotherapy resistance. Conventional treatments often overlook the metabolic implications of cancer, but recent research has highlighted the pivotal role of mitochondria in cancer pathogenesis and drug resistance. This study delves into the metabolic landscape of ovarian cancer treatment, focusing on modulating mitochondrial activity using methylene blue (MB). Investigating two epithelial ovarian cancer (EOC) cell lines, OV1369-R2 and OV1946, exhibiting disparate responses to carboplatin, we sought to identify metabolic nodes, especially those linked to mitochondrial dysfunction, contributing to chemo-resistance. Utilizing ARPE-19, a normal retinal epithelial cell line, as a control model, our study reveals MB's distinct cellular uptake, with ARPE-19 absorbing 5 to 7 times more MB than OV1946 and OV1369-R2. Treatment with 50 µM MB (MB-50) effectively curtailed the proliferation of both ovarian cancer cell lines. Furthermore, MB-50 exhibited the ability to quell glutaminolysis and the Warburg effect in cancer cell cultures. Regarding mitochondrial energetics, MB-50 spurred oxygen consumption, disrupted glycolytic pathways, and induced ATP depletion in the chemo-sensitive OV1946 cell line. These findings highlight the potential of long-term MB exposure as a strategy to improve the chemotherapeutic response in ovarian cancer cells. The ability of MB to stimulate oxygen consumption and enhance mitochondrial activity positions it as a promising candidate for ovarian cancer therapy, shedding light on the metabolic pressures exerted on mitochondria and their modulation by MB, thus contributing to a deeper understanding of mitochondrial dysregulation and the metabolic underpinnings of cancer cell proliferation.

A systematic review and BMD modeling approach to develop an AOP for humidifier disinfectant-induced pulmonary fibrosis and cell death

Dosimetry modeling and point of departure (POD) estimation using in vitro data are essential for mechanism-based hazard identification and risk assessment. This study aimed to develop a putative adverse outcome pathway (AOP) for humidifier disinfectant (HD) substances used in South Korea through a systematic review and benchmark dose (BMD) modeling. We collected in vitro toxicological studies on HD substances, including polyhexamethylene guanidine hydrochloride (PHMG-HCl), PHMG phosphate (PHMG-p), a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (CMIT/MIT), CMIT, and MIT from scientific databases. A total of 193 sets of dose-response data were extracted from 34 articles reporting in vitro experimental results of HD toxicity. The risk of bias (RoB) in each study was assessed following the office of health assessment and translation (OHAT) guideline. The BMD of each HD substance at different toxicity endpoints was estimated using the US Environmental Protection Agency (EPA) BMD software (BMDS). Interspecies- or interorgan differences or most critical effects in the toxicity of the HD substances were analyzed using a 95% lower confidence limit of the BMD (BMDL). We found a critical molecular event and cells susceptible to each HD substance and constructed an AOP of PHMG-p- or CMIT/MIT-induced damage. Notably, PHMG-p induced ATP depletion at the lowest in vitro concentration, endoplasmic reticulum (ER) stress, epithelial-to-mesenchymal transition (EMT), inflammation, leading to fibrosis. CMIT/MIT enhanced mitochondrial reactive oxygen species (ROS) production, oxidative stress, mitochondrial dysfunction, resulting in cell death. Our approach will increase the current understanding of the effects of HD substances on human health and contribute to evidence-based risk assessment of these compounds.

Diesel exhaust particulate matter induces GC-1 spg cells oxidative stress by KEAP1-NRF2 pathway and inhibition of ATP5α1 S-sulfhydration

Diesel exhaust particle (DEP) exposure induces a variety of toxicological effects through oxidative stress and inflammation responses. This research investigated the mechanisms underlying DEP-induced GC-1spg cells oxidative stress by examining ROS accumulation, antioxidant defense systems activation, mitochondrial dysfunction, and the Nrf2/Keap1/HO-1 pathway response. Subsequently, we further evaluated the ATP levels, ATP5α synthase activity and ATP5α synthase S-sulfhydrated modification in DEP-exposed GC-1 spg cells. The results showed that DEP exposure significantly inhibited cell proliferation and viability, increased intracellular ROS production, decreased MMP, down-regulated antioxidant capacity, activated the Nrf2/Keap1/HO-1 pathway. However, DEP-induced oxidative stress was partially alleviated by GSH and exogenous H2S. In addition, DEP exposure induced ATP depletion and ATP5α synthase inactivity in GC-1 spg cells, accompanied by ATP5α synthase S-sulfhydrated modification. In conclusion, our research showed that DEP may incapacitate mitochondria through oxidative stress injury, leading to GC-1 spg cells oxidative stress. This process may be associated with the reduction of ATP5α1 S-sulfhydrated modification. It provides a new perspective for the research of the mechanism related to male reproductive toxicity due to air pollution.

AMP-activated protein kinase (AMPK) suppresses Ibaraki virus propagation

The Ibaraki virus (IBAV) causes Ibaraki disease in cattle. Our previous studies have shown that IBAV uses macropinocytosis to enter the host cell and exit from the endosome to the cytosol in response to endosomal acidification. To further explore the mechanism of IBAV infection and replication, we examined the effect of inhibitors of mitochondrial oxidative phosphorylation, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and antimycin A, on IBAV propagation. These inhibitors significantly suppressed IBAV propagation, with reduced cellular ATP levels resulting from suppression of ATP synthesis. Furthermore, we identified AMP-activated protein kinase (AMPK), which is activated by CCCP or antimycin A, as a key signaling molecule in IBAV suppression. We also observed that IBAV infection induces ATP depletion and increases AMPK activity. Our findings suggest that AMPK is a potential target in Ibaraki disease.

The potential for citrate to reinforce epigenetic therapy by promoting apoptosis

Epigenetic drugs induce ATP depletion, promoting a glycolysis-to-oxidative phosphorylation (OXPHOS) shift which sometimes favors tumor growth by promoting necroptosis over apoptosis. To restore effective apoptosis in tumors, we propose that the administration of citrate could inhibit ATP production, activate caspase-8 (a key necroptosis inhibitor), and downregulate key anti-apoptotic proteins (Bcl-xL and MCL1).

PDHA1 hyperacetylation-mediated lactate overproduction promotes sepsis-induced acute kidney injury via Fis1 lactylation

The increase of lactate is an independent risk factor for patients with sepsis-induced acute kidney injury (SAKI). However, whether elevated lactate directly promotes SAKI and its mechanism remain unclear. Here we revealed that downregulation of the deacetylase Sirtuin 3 (SIRT3) mediated the hyperacetylation and inactivation of pyruvate dehydrogenase E1 component subunit alpha (PDHA1), resulting in lactate overproduction in renal tubular epithelial cells. We then found that the incidence of SAKI and renal replacement therapy (RRT) in septic patients with blood lactate ≥ 4 mmol/L was increased significantly, compared with those in septic patients with blood lactate < 2 mmol/L. Further in vitro and in vivo experiments showed that additional lactate administration could directly promote SAKI. Mechanistically, lactate mediated the lactylation of mitochondrial fission 1 protein (Fis1) lysine 20 (Fis1 K20la). The increase in Fis1 K20la promoted excessive mitochondrial fission and subsequently induced ATP depletion, mitochondrial reactive oxygen species (mtROS) overproduction, and mitochondrial apoptosis. In contrast, PDHA1 activation with sodium dichloroacetate (DCA) or SIRT3 overexpression decreased lactate levels and Fis1 K20la, thereby alleviating SAKI. In conclusion, our results show that PDHA1 hyperacetylation and inactivation enhance lactate overproduction, which mediates Fis1 lactylation and exacerbates SAKI. Reducing lactate levels and Fis1 lactylation attenuate SAKI.

Nephrotoxicity of iopamidol is associated with mitochondrial impairment in human cell and teleost models

Iopamidol is a nonionic, low-osmolar iodinated contrast agent used for angiography. Its clinical use is associated with renal dysfunction. Patients suffering from preexisting kidney disease have an increased risk of renal failure upon iopamidol administration. Studies in animals confirmed renal toxicity, but the involved mechanisms remain unclear.Therefore, the aim of the present study was to use human embryonic kidney cells (HEK293T) as a general cell model of mitochondrial damage, as well as, zebrafish larvae, and isolated proximal tubules of killifish to investigate factors promoting renal tubular toxicity of iopamidol with a focus on mitochondrial damage.Results from in vitro HEK293T cell-based assays indicate that iopamidol affects mitochondrial function Treatment with iopamidol induces ATP depletion, reduces the mitochondrial membrane potential, and elevates mitochondrial superoxide and reactive oxygen species accumulation. Similar results were obtained with gentamicin sulfate and cadmium chloride, two well-known model compounds associated with renal tubular toxicity. Confocal microscopy confirms changes in mitochondrial morphology, such as mitochondrial fission. Importantly, these results were confirmed in proximal renal tubular epithelial cells using ex vivo and in vivo teleost models.In conclusion, this study provides evidence for iopamidol-induced mitochondrial damage in proximal renal epithelial cells. Teleost models allow studying proximal tubular toxicity with translational relevance for humans.

Blockade of ß-Adrenergic Receptors by Nebivolol Enables Tumor Control Potential for Uveal Melanoma in 3D Tumor Spheroids and 2D Cultures.

Uveal melanoma (UM) is the most common primary cancer of the eye in adults. A new systemic therapy is needed to reduce the high metastasis and mortality rate. As β-blockers are known to have anti-tumor effects on various cancer entities, this study focuses on investigating the effect of β1-selective blockers atenolol, celiprolol, bisoprolol, metoprolol, esmolol, betaxolol, and in particular, nebivolol on UM. The study was performed on 3D tumor spheroids as well as 2D cell cultures, testing tumor viability, morphological changes, long-term survival, and apoptosis. Flow cytometry revealed the presence of all three β-adrenoceptors with a dominance of β2-receptors on cell surfaces. Among the blockers tested, solely nebivolol concentration-dependently decreased viability and altered 3D tumor spheroid structure. Nebivolol blocked the repopulation of cells spreading from 3D tumor spheroids, indicating a tumor control potential at a concentration of ≥20 µM. Mechanistically, nebivolol induced ATP depletion and caspase-3/7 activity, indicating that mitochondria-dependent signaling is involved. D-nebivolol or nebivolol combined with the β2-antagonist ICI 118.551 displayed the highest anti-tumor effects, suggesting a contribution of both β1- and β2-receptors. Thus, the present study reveals the tumor control potential of nebivolol in UM, which may offer a perspective for co-adjuvant therapy to reduce recurrence or metastasis.

Pomegranate Extract (POMx) Induces Mitochondrial Dysfunction and Apoptosis of Oral Cancer Cells.

The anticancer effect of pomegranate polyphenolic extract POMx in oral cancer cells has rarely been explored, especially where its impact on mitochondrial functioning is concerned. Here, we attempt to evaluate the proliferation modulating function and mechanism of POMx against human oral cancer (Ca9-22, HSC-3, and OC-2) cells. POMx induced ATP depletion, subG1 accumulation, and annexin V/Western blotting-detected apoptosis in these three oral cancer cell lines but showed no toxicity to normal oral cell lines (HGF-1). POMx triggered mitochondrial membrane potential (MitoMP) disruption and mitochondrial superoxide (MitoSOX) generation associated with the differential downregulation of several antioxidant gene mRNA/protein expressions in oral cancer cells. POMx downregulated mitochondrial mass, mitochondrial DNA copy number, and mitochondrial biogenesis gene mRNA/protein expression in oral cancer cells. Moreover, POMx induced both PCR-based mitochondrial DNA damage and γH2AX-detected nuclear DNA damage in oral cancer cells. In conclusion, POMx provides antiproliferation and apoptosis of oral cancer cells through mechanisms of mitochondrial impairment.

P1600KIDNEY PERFUSION WITH MESENCHYMAL STROMAL CELLS OR EXTRACELLULAR VESICLES PREVENTS ISCHAEMIC DAMAGE THROUGH CD73/ADO SYSTEM IN A RAT MODEL OF DCD DONATION

Abstract Background and Aims Adenosine (Ado), the main substrate of ATP, is a potent endogenous anti-inflammatory nucleoside. Hypoxia induces ATP depletion, AMP extracellular increase that is phosphohydrolyzed to ADO by the enzyme ecto-5′-nucleotidase (CD73). Constitutive CD73 expression is higher in deep cortex outer medulla that is the most vulnerable region to ischemic injury. Notably CD73 is also expressed on Mesenchymal Stromal Cell (MSC) and is essential phenotypic marker. Previously in a rat model of Donation after Circulatory Death (DCD), we demonstrated that the perfusion of ischemic kidney with MSC or MSC derived Extracellular Vesicles (EV) protects tissue up regulating mitochondrial energetic metabolism genes. The aim of this study was to investigate the role of CD73/Ado system in MSC/EV protection from ischemia. Method Fisher rats were used as kidney donors and Lewis rats as MSC donors. MSC missing CD73 were produced by electroporation in the presence of specific siRNA to block CD73 expression (siMSC). EV were isolated from supernatants of MSC and siMSC (siEV) medium through differential ultracentrifugations. Size distribution and enumeration analysis were performed using NanoSight NS300. EV phenotypic characterization was performed in flow cytometer using CD45, CD49e, CD63, CD9, CD81 and CD73 monoclonal antibodies. DCD model was obtained by renal artery clamping for 20 minutes. This warm ischemia time represents the “no touch period” imposed by the Italian law to death declaration. After nephrectomy, kidneys were perfused in hypothermia (4°C) with Belzer Solution (BS), or BS supplemented with 3x106 MSC (MSC) or BS supplemented with 28,5x109 EV/siEV (EV/siEV). The effluent fluid (EF) was collected at the beginning (T0), every hours (T1, T2, T3) and at the end of the perfusion (T4). Ado and ATP determinations in EF and tissues were performed by HPLC and ELISA, respectively. Results EF Ado was significantly higher in MSC vs BS from T1 and in EV vs BS from T0 to T4 (p&amp;lt;0,05). Only in EV, EF Ado concentration increased over time from T2 to T4 ( p&amp;lt;0,05), while in BS, MSC and siEV there was a steady trend over time. There was a negative correlation between EF and tissue levels of Ado (r=0,67 p=0,01). Tissue ATP was higher in EV and significantly in MSC vs BS (p&amp;lt;0,01). There was a negative correlation between tissue ATP and EF ADO levels (r= 0,79 p&amp;lt; 0,0001). Tissue ATP/Ado ratio was significantly higher in EV vs BS (p&amp;lt;0,01). There was a negative correlation between tissue ATP/Ado ratio and EF Ado (r= 0,84 p &amp;lt; 0.0001). siEV cancelled EV effects on ATP and Ado levels in EF and renal tissue. Conclusion CD73 expressed on MSC /EV impacts on cell energy metabolism pathway and ATP generation. This is the first evidence that MSC/EV act through CD73/Ado system to prevent ischaemic damage.

2,3-Dimethoxy-5-methyl-p-benzoquinone (Coenzyme Q0) Disrupts Carbohydrate Metabolism of HeLa Cells by Adduct Formation with Intracellular Free Sulfhydryl-Groups, and Induces ATP Depletion and Necrosis.

2,3-Dimethoxy-5-methyl-p-benzoquinone is a common chemical structure of coenzyme Q (CoQ) that conjugates different lengths of an isoprenoid side chain at the 6-position of the p-benzoquinone ring. In a series of studies to explore the cytotoxic mechanism of CoQ homologues with a short isoprenoid side chain, we found that a CoQ analogue without an isoprenoid side chain, CoQ0, showed marked toxicity against HeLa cells in comparison with cytotoxic homologues. Therefore, we examined the cytotoxic mechanism of CoQ0. Different from the cytotoxic CoQ homologues that induced apoptosis, 100 µM CoQ0 induced necrosis of HeLa cells. The CoQ0-induced cell death was accompanied by a decrease in endogenous non-protein and protein-associated sulfhydryl (SH)-groups, but this improved with the concomitant addition of compounds with SH-groups but not antioxidants without SH-groups. In addition, UV-spectrum analysis suggested that CoQ0 could rapidly form S-conjugated adducts with compounds with SH-groups by Michael addition. On the other hand, enzyme activities of both glyceraldehyde-3-phosphate dehydrogenase, which has a Cys residue in the active site, and α-ketoglutarate dehydrogenase complex, which requires cofactors with SH-groups, CoA and protein-bound α-lipoic acid, and CoA and ATP contents in the cells were significantly decreased by the addition of CoQ0 but not CoQ1. Furthermore, the decrease of an endogenous antioxidant, glutathione (GSH), by CoQ0 treatment was much greater than the predicted increase of endogenous GSH disulfide. These results suggest that CoQ0 rapidly forms S-conjugate adducts with these endogenous non-protein and protein-associated SH-groups of HeLa cells, which disrupts carbohydrate metabolism followed by intracellular ATP depletion and necrotic cell death.

Abstract 2407: Extracellular acidosis inhibits glucose starvation-induced lung cancer cell death by suppressing overall metabolic rate

Abstract Bioenergetic tumor metabolism is essential to meet ATP requirements for energy homeostasis. Cancer cells utilize aerobic glycolysis and proliferate continuously beyond the capacity of blood supply. These phenotypic alterations lead to various microenvironmental stresses, such as hypoxia, nutrient (glucose) deprivation, and extracellular acidosis, to which cancer cells must metabolically adapt in order to survive and proliferate. However, compared to hypoxia, relatively little is known about the metabolic adaptations in response to glucose deprivation and acidosis. In the present study, we explored the effects of extracellular acidosis on cancer metabolism under glucose deprivation stress, as extracellular acidosis and insufficient glucose supply usually co-exist in the tumor microenvironment. To this aim, A549 lung cancer cells were treated with acidosis, glucose deprivation, or a combination. Then, cell survival, total cellular ATP turnover, relative glycolytic and mitochondrial dependency and capacity, and de novo rRNA and protein synthesis were determined. We found that acidosis (pH 6.8) inhibited ATP depletion and cell death induced by glucose deprivation. The cytoprotective effect of acidosis against glucose deprivation-induced cell death was mediated by two distinct mechanisms. First, acidosis shifted cellular ATP production from glycolysis to OXPHOS that became independent of glucose uptake but dependent on glutaminolysis and fatty acid β-oxidation. Second, acidosis reduced ATP consumption by inhibiting de novo rRNA and protein synthesis that utilizes ATP. In contrast, the protective effect of acidosis on glucose starvation-induced cell death was independent of AMPK, PI3K, ERK, and p38 MAPK. Based on this mechanistic knowledge of acidosis-dependent metabolic alterations, we tested the effect of the mitochondrial complex I inhibitor metformin on death of lung cancer cells. Metformin and a combination of rotenone and AICAR mimicking an action of metformin induced ATP depletion and cell death in glucose-free medium, but under acidosis the cytotoxic effect of metformin was significantly attenuated because overall ATP turnover was reduced under low pH. These results suggest that during acidosis, cells are forced to a reduced glycolytic energy production, but the decrease in protein synthesis and ATP requirement serves as an adaptive response to glucose deprivation stress. This acidosis-dependent, glucose starvation-resistant phenotype may provide an adaptive strategy in an energy-restricted tumor microenvironment. Our findings highlight the importance of considering the two factors, acidosis and nutrient starvation in combination to understand their contributions to tumor metabolism. Citation Format: Yuki Iwai, Ryota Kikuchi, Yasutaka Watanabe, Nobuyuki Koyama, Koichi Hagiwara, Hiroyuki Nakamura, Kazutetsu Aoshiba. Extracellular acidosis inhibits glucose starvation-induced lung cancer cell death by suppressing overall metabolic rate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2407.

Identification of a nucleoside analog active against adenosine kinase-expressing plasma cell malignancies.

Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase-inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI-sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers.

A cytotoxic Petiveria alliacea dry extract induces ATP depletion and decreases β-F1-ATPase expression in breast cancer cells and promotes survival in tumor-bearing mice

Metabolic plasticity in cancer cells assures cell survival and cell proliferation under variable levels of oxygen and nutrients. Therefore, new anticancer treatments endeavor to target such plasticity by modifying main metabolic pathways as glycolysis or oxidative phosphorylation. In American traditional medicine Petiveria alliacea L., Phytolaccacea, leaf extracts have been used for leukemia and breast cancer treatments. Herein, we study cytotoxicity and antitumoral effects of P. alliacea extract in tumor/non-tumorigenic cell lines and murine breast cancer model. Breast cancer cells treated with P. alliacea dry extract showed reduction in β-F1-ATPase expression, glycolytic flux triggering diminished intracellular ATP levels, mitochondrial basal respiration and oxygen consumption. Consequently, a decline in cell proliferation was observed in conventional and three-dimension spheres breast cancer cells culture. Additionally, in vivo treatment of BALB/c mice transplanted with the murine breast cancer TS/A tumor showed that P. alliacea extract via i.p. decreases the primary tumor growth and increases survival in the TS/A model.

Synergistic Anticancer Action of Lysosomal Membrane Permeabilization and Glycolysis Inhibition

We investigated the in vitro and in vivo anticancer effect of combining lysosomal membrane permeabilization (LMP)-inducing agent N-dodecylimidazole (NDI) with glycolytic inhibitor 2-deoxy-d-glucose (2DG). NDI-triggered LMP and 2DG-mediated glycolysis block synergized in inducing rapid ATP depletion, mitochondrial damage, and reactive oxygen species production, eventually leading to necrotic death of U251 glioma cells but not primary astrocytes. NDI/2DG-induced death of glioma cells was partly prevented by lysosomal cathepsin inhibitor E64 and antioxidant α-tocopherol, suggesting the involvement of LMP and oxidative stress in the observed cytotoxicity. LMP-inducing agent chloroquine also displayed a synergistic anticancer effect with 2DG, whereas glucose deprivation or glycolytic inhibitors iodoacetate and sodium fluoride synergistically cooperated with NDI, thus further indicating that the anticancer effect of NDI/2DG combination was indeed due to LMP and glycolysis block. The two agents synergistically induced ATP depletion, mitochondrial depolarization, oxidative stress, and necrotic death also in B16 mouse melanoma cells. Moreover, the combined oral administration of NDI and 2DG reduced in vivo melanoma growth in C57BL/6 mice by inducing necrotic death of tumor cells, without causing liver, spleen, or kidney toxicity. Based on these results, we propose that NDI-triggered LMP causes initial mitochondrial damage that is further increased by 2DG due to the lack of glycolytic ATP required to maintain mitochondrial health. This leads to a positive feedback cycle of mitochondrial dysfunction, ATP loss, and reactive oxygen species production, culminating in necrotic cell death. Therefore, the combination of LMP-inducing agents and glycolysis inhibitors seems worthy of further exploration as an anticancer strategy.

TIGAR regulates glycolysis in ischemic kidney proximal tubules.

Tp53-induced glycolysis and apoptosis regulator (TIGAR) activation blocks glycolytic ATP synthesis by inhibiting phosphofructokinase-1 activity. Our data indicate that TIGAR is selectively induced and activated in renal outermedullary proximal straight tubules (PSTs) after ischemia-reperfusion injury in a p53-dependent manner. Under severe ischemic conditions, TIGAR expression persisted through 48 h postinjury and induced loss of renal function and histological damage. Furthermore, TIGAR upregulation inhibited phosphofructokinase-1 activity, glucose 6-phosphate dehydrogenase (G6PD) activity, and induced ATP depletion, oxidative stress, autophagy, and apoptosis. Small interfering RNA-mediated TIGAR inhibition prevented the aforementioned malevolent effects and protected the kidneys from functional and histological damage. After mild ischemia, but not severe ischemia, G6PD activity and NADPH levels were restored, suggesting that TIGAR activation may redirect the glycolytic pathway into gluconeogenesis or the pentose phosphate pathway to produce NADPH. The increased level of NADPH maintained the level of GSH to scavenge ROS, resulting in a lower sensitivity of PST cells to injury. Under severe ischemia, G6PD activity and NADPH levels were reduced during reperfusion; however, blockade of TIGAR enhanced their levels and reduced oxidative stress and apoptosis. Collectively, these results demonstrate that inhibition of TIGAR may protect PST cells from energy depletion and apoptotic cell death in the setting of severe ischemia-reperfusion injury. However, under low ischemic burden, TIGAR activation induces the pentose phosphate pathway and autophagy as a protective mechanism.

Excessive Fluoride Consumption Leads to Accelerated Death of Erythrocytes and Anemia in Rats

The present study was performed to evaluate an overall effect of long-term consumption of excessive fluoride (F) amounts by rats on their erythrocytes. The animals were administered regular drinking water (0.4ppm F) or the same water supplemented with 2, 10, and 20ppm F (as NaF) for 12months. Chronic exposure of the rats to increasing F doses induced a progressive rise of the plasma F concentration accompanied by a dose-dependent fall of hematocrit and decrease in the mean erythrocyte volume. Consumption of 10 and 20ppm F resulted in appearance of morphologically abnormal cells (stomatocytes and echinocytes) in the peripheral blood. Rise of the water F concentration to 20ppm F led to significant increase in the number of phosphatidylserine-exposing erythrocytes, although suppression of cell viability was revealed in all three groups of F-poisoned rats. A compensatory enhanced release of reticulocytes was not sufficient to compensate for erythrocyte loss. Dose-dependent accumulation of free cytosolic Ca(2+) appears to be a major pathophysiological process underlying the development of F-induced death processes in rat erythrocytes. In addition, 10 and 20ppm F induced ATP depletion and generation of peroxides in erythrocytes, whereas superoxide and glutathione levels were not altered. Thus, long-term intoxication of the rats with F triggers premature death of their erythrocytes due to intrinsic death-associated biochemical defects and development of anemia.

Hexavalent chromium induces energy metabolism disturbance and p53-dependent cell cycle arrest via reactive oxygen species in L-02 hepatocytes

Hexavalent chromium [Cr(VI)] has become a non-negligible pollutant in the world. Cr(VI) exposure leads to severe damage to the liver, but the mechanisms involved in Cr(VI)-mediated toxicity in the liver are unclear. The present study aimed to explore whether Cr(VI) induces energy metabolism disturbance and cell cycle arrest in human L-02 hepatocytes. We showed that Cr(VI) inhibited state 3 respiration, respiratory control rate (RCR), and subsequently induced energy metabolism disturbance with decreased ATP production. Interestingly, cell cycle analysis by flow cytometry and protein expression analysis by western blotting revealed that low dose of Cr(VI) (4 uM) exposure induced S phase cell cycle arrest with decreased mediator of replication checkpoint 1 (Mrc1) and cyclin-dependent kinase 2 (CDK2), while higher doses of Cr(VI) (16, 32 uM) exposure resulted in G2/M phase arrest with decreased budding uninhibited by benzimidazoles-related 1 (BubR1) and cell division cycle 25 (CDC25). Mechanism study revealed that Cr(VI) decreased the activities of mitochondrial respiratory chain complex (MRCC) I and II, thus leading to ROS accumulation. Moreover, inhibiting ROS production by antioxidant N-acetyl-L-cysteine (NAC) rescued Cr(VI)-induced ATP depletion and cell cycle arrest. ROS-mediated p53 activation was found to involve in Cr(VI)-induced cell cycle arrest, and p53 inhibitor Pifithrin-α (PFT-α) rescued Cr(VI)-induced reduction of check point proteins Mrc1 and BubR1, thus inhibiting cell cycle arrest. In summary, the present study provides experimental evidence that Cr(VI) leads to energy metabolism disturbance and p53-dependent cell cycle arrest via ROS in L-02 hepatocytes.