Tumor chemical suffocation therapy by dual respiratory inhibitions

The extraordinarily rapid growth of malignant tumors depends heavily on the glucose metabolism by the pathways of glycolysis and mitochondrial oxidative phosphorylation to generate adenosine 5′-triphosphate (ATP) for maintaining cell proliferation and tumor growth. This study reports a tumor chemical suffocation therapeutic strategy by concurrently suppressing both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) via the co-deliveries of EDTA and rotenone into a glutathione (GSH)-overexpressed tumor microenvironment. EDTA is to block the glycolytic pathway through inhibiting the activity of glycolytic enzymes via the chelation of magnesium ion, a co-worker of glycolytic enzymes, despite the presence of Ca²⁺. Meanwhile rotenone is to inhibit the mitochondrial OXPHOS. This work provides a novel tumor suffocation strategy by the co-deliveries of glucose metabolism inhibitors, especially by de-functioning glycolytic enzymes via eliminating their co-worker magnesium.

The EDTA- and Rotenone-loaded MPER nanoparticles have been synthesized to suffocate tumor cells to death through inhibiting glycolytic process and mitochondrial oxidative phosphorylation simultaneously in vitro and in vivo.     

ENERGETICS OF DNA REPAIR IN UV-IRRADIATED PERIPHERAL BLOOD LEUKOCYTES FROM CHRONIC MYELOID LEUKAEMIA PATIENTS

Abstract— The relationship between energy metabolism and UV-induced (251 nm) DNA repair has been investigated in leukocytes obtained from peripheral blood of chronic myeloid leukaemia patients. Energy supply was modulated by using inhibitors of respiratory (antimvcin-A) and glycolytic (2-deoxy-o-glucose and other glucose analogues) pathways for adenosine-triphosphatc (ATP) production. DNA repair was measured by the unscheduled DNA synthesis technique. Parameters of energy metabolism like glucose utilization. lactate production and ATP content of cells. were measured under similar experimental conditions. The observations made indicate the following results: (1) ATP for DNA repair can be supplied by the respiratory and/or by the glycolytic pathway. (2) In the absence of respiration, the rate of glycolysis bears a linear correlation with DNA repair. (3) A minimum threshold rate of ATP-production is necessary for DNA repair.     

The disubstituted adamantyl derivative LW1564 inhibits the growth of cancer cells by targeting mitochondrial respiration and reducing hypoxia-inducible factor (HIF)-1α accumulation

Targeting cancer metabolism has emerged as an important cancer therapeutic strategy. Here, we describe the synthesis and biological evaluation of a novel class of hypoxia-inducible factor (HIF)-1α inhibitors, disubstituted adamantyl derivatives. One such compound, LW1564, significantly suppressed HIF-1α accumulation and inhibited the growth of various cancer cell lines, including HepG2, A549, and HCT116. Measurements of the oxygen consumption rate (OCR) and ATP production rate revealed that LW1564 suppressed mitochondrial respiration, thereby increasing the intracellular oxygen concentration to stimulate HIF-1α degradation. LW1564 also significantly decreased overall ATP levels by inhibiting mitochondrial electron transport chain (ETC) complex I and downregulated mammalian target of rapamycin (mTOR) signaling by increasing the AMP/ATP ratio, which increased AMP-activated protein kinase (AMPK) phosphorylation. Consequently, LW1564 promoted the phosphorylation of acetyl-CoA carboxylase, which inhibited lipid synthesis. In addition, LW1564 significantly inhibited tumor growth in a HepG2 mouse xenograft model. Taken together, the results indicate that LW1564 inhibits the growth of cancer cells by targeting mitochondrial ETC complex I and impairing cancer cell metabolism. We, therefore, suggest that LW1564 may be a potent therapeutic agent for a subset of cancers that rely on oxidative phosphorylation for ATP generation.Subject terms: Cancer metabolism, Cancer metabolism, Drug development     

Cancer cell metabolism: implications for therapeutic targets

Cancer cell metabolism is characterized by an enhanced uptake and utilization of glucose, a phenomenon known as the Warburg effect. The persistent activation of aerobic glycolysis in cancer cells can be linked to activation of oncogenes or loss of tumor suppressors, thereby fundamentally advancing cancer progression. In this respect, inhibition of glycolytic capacity may contribute to an anticancer effect on malignant cells. Understanding the mechanisms of aerobic glycolysis may present a new basis for cancer treatment. Accordingly, interrupting lactate fermentation and/or other cancer-promoting metabolic sites may provide a promising strategy to halt tumor development. In this review, we will discuss dysregulated and reprogrammed cancer metabolism followed by clinical relevance of the metabolic enzymes, such as hexokinase, phosphofructokinase, pyruvate kinase M2, lactate dehydrogenase, pyruvate dehydrogenase kinase and glutaminase. The proper intervention of these metabolic sites may provide a therapeutic advantage that can help overcome resistance to chemotherapy or radiotherapy.     

Light-induced tetracycline accumulation by Rhodopseudomonas sphaeroides.

Light has been used as a primary energy source in studies of tetracycline transport by Rhodopseudomonas sphaeroides. Accumulation of the antibiotic occurs in light, while efflux occurs in dark. Both fluorescence enhancement and radioisotopic tracing have been used to monitor transport. Km's obtained from both techniques are similar. Light-induced accumulation of tetracyclines is inhibited by a variety of inhibitors, including antimycin A, N-ethylmaleimide, carbonylcyanide m-chlorophenylhydrazone, and 2,4-dinitrophenol. A rapid efflux is observed after loading when cells are placed in the dark or treated with inhibitors.     

2-Deoxy-d-glucose Promotes Buforin IIb-Induced Cytotoxicity in Prostate Cancer DU145 Cells and Xenograft Tumors

Inhibition of the glycolytic pathway is a critical strategy in anticancer therapy because of the role of aerobic glycolysis in cancer cells. The glycolytic inhibitor 2-Deoxy-d-glucose (2-DG) has shown potential in combination with other anticancer agents. Buforin IIb is an effective antimicrobial peptide (AMP) with broad-spectrum anticancer activity and selectivity. The efficacy of combination treatment with 2-DG and buforin IIb in prostate cancer remains unknown. Here, we tested the efficacy of buforin IIb as a mitochondria-targeting AMP in the androgen-independent human prostate cancer cell line DU145. Combining 2-DG with buforin IIb had a synergistic toxic effect on DU145 cells and mouse xenograft tumors. Combination treatment with 2-DG and buforin IIb caused stronger proliferation inhibition, greater G1 cell cycle arrest, and higher apoptosis than either treatment alone. Combination treatment dramatically decreased L-lactate production and intracellular ATP levels, indicating severe inhibition of glycolysis and ATP production. Flow cytometry and confocal laser scanning microscopy results indicate that 2-DG may increase buforin IIb uptake by DU145 cells, thereby increasing the mitochondria-targeting capacity of buforin IIb. This may partly explain the effect of combination treatment on enhancing buforin IIb-induced apoptosis. Consistently, 2-DG increased mitochondrial dysfunction and upregulated Bax/Bcl-2, promoting cytochrome c release to initiate procaspase 3 cleavage induced by buforin IIb. These results suggest that 2-DG sensitizes prostate cancer DU145 cells to buforin IIb. Moreover, combination treatment caused minimal hemolysis and cytotoxicity to normal WPMY-1 cells. Collectively, the current study demonstrates that dual targeting of glycolysis and mitochondria by 2-DG and buforin IIb may be an effective anticancer strategy for the treatment of some advanced prostate cancer.     

EFFECTS OF HEMATOPORPHYRIN DERIVATIVE ON THE CELLULAR ENERGY METABOLISM IN THE ABSENCE AND PRESENCE OF LIGHT

Effects of Photofrin II on energy metabolism and metabolic viability were studied in a mammalian transformed cell line (BHK-21) in dark and after photo-irradiation with visible light. Cells were allowed to accumulate Photofrin by incubating for 4 h in buffer containing Photofrin (5-60 micrograms/ml). The results show that Photofrin significantly affects the cellular energy metabolism even in the absence of light; activity of cytochrome c oxidase is decreased and glucose utilization and lactate production (glycolysis) are increased. Irradiation with light resulted in a significant decrease in the activity of cytochrome c oxidase, glycolysis, ATP content, energy charge, ratios of adenine nucleotides like ATP/ADP, ATP/AMP and cell viability (dye exclusion test). Presence of inhibitors of energy metabolism, potassium cyanide (respiration) and 2-deoxyglucose (glycolysis), further enhanced the cytotoxic effects induced by hematoporphyrin derivative and light.     

Identification of small molecule inhibitors that distinguish between non-transferrin bound iron uptake and transferrin-mediated iron transport

Chemical genetics is an emerging field that takes advantage of combinatorial chemical and small molecule libraries to dissect complex biological processes. Here we establish a fluorescence-based assay to screen for inhibitors of iron uptake by mammalian cells. Using this approach, we screened the National Cancer Institute's Diversity Set library for inhibitors of non-transferrin bound iron uptake. This screen identified 10 novel small molecule inhibitors of iron transport with IC(50) values that ranged from 5 to 30 microM. Of these ten compounds, only two blocked uptake of iron mediated by transferrin. Thus, this study characterizes the first small molecule inhibitors that distinguish between different pathways of iron transport.     

Zelkovamycin is an OXPHOS Inhibitory Member of the Argyrin Natural Product Family

Natural products (NPs) are an important inspirational source for developing drugs and chemical probes. In 1999, the group of Ōmura reported the constitutional elucidation of zelkovamycin. Although largely unrecognized so far, this NP displays structural similarities as well as differences to the argyrin NP family, a class of peptidic NPs with promising anticancer activities and diverse mode-of-action at the molecular level. By a combination of structure elucidation experiments, the first total synthesis of zelkovamycin and bioassays, the zelkovamycin configuration was determined and its previously proposed molecular structure was revised. The full structure assignment proves zelkovamycin as an additional member of the argyrins with however unique OXPHOS inhibitory properties. Zelkovamycin may therefore not only serve as a new starting point for chemical inhibitors of the OXPHOS system, but also guide customized argyrin NP isolation and biosynthesis studies.     

Surface Immobilization of Redox‐Labile Fluorescent Probes: Enabling Single‐Cell Co‐Profiling of Aerobic Glycolysis and Oncogenic Protein Signaling Activities

An analytical method is described for profiling lactate production in single cells via the use of coupled enzyme reactions on surface‐grafted resazurin molecules. The immobilization of the redox‐labile probes was achieved through chemical modifications on resazurin, followed by bio‐orthogonal click reactions. The lactate detection was demonstrated to be sensitive and specific. The method was incorporated into a single‐cell barcode chip for simultaneous quantification of aerobic glycolysis activities and oncogenic signaling phosphoproteins in cancer. The interplay between glycolysis and oncogenic signaling activities was interrogated on a glioblastoma cell line. Results revealed a drug‐induced oncogenic signaling reliance accompanying shifted metabolic paradigms. A drug combination that exploits this induced reliance exhibited synergistic effects in growth inhibition.     

Shuttles for translocation of NADH in isolated liver cells from fed rats during oxidation of xylitol.

Translocation of xylitol-derived NADH via malate-aspartate and alpha-glycerophosphate shuttles was studied in liver cells isolated from fed rats. In bicarbonate medium amino-oxyacetate, rotenone and antimycin A, were equally efficient in depressing the xylitol removal. Incubation of cells in nonbicarbonate medium did not affect the rate of xylitol removal. In this medium amino-oxyacetate and antimycin A, but not rotenone, inhibited xylitol removal. Xylitol inhibited the lactate accumulation found when the cells were incubated without any exogenous substrates. Glucose was the main end product of xylitol oxidation. In nonbicarbonate medium ketogenesis was high, whereas in bicarbonate medium a low rate of ketone body formation was found. Xylitol had no effect on the rate of ketone body formation in either medium tested. Xylitol markedly decreased the ATP and Pi contents of the cells, but no change in the ATP/ADP x Pi ratio or the rate of oxygen consumption was found. The results suggest that NADH formed during xylitol oxidation is translocated to the mitochondria mainly through the malate-aspartate shuttle and only when this shuttle is inhibited does the alpha-glycerophosphate shuttle transfer NADH. Intramitochondrial reactions which form NADH and FADH2 are also suggested to be important regulators of the activity of the alpha-glycerophosphate shuttle.     

Dual inhibition of glucose uptake and energy supply synergistically restrains the growth and metastasis of breast cancer

Metastatic breast cancer (MBC) is one of the most common and knotty diseases in female population which could place them in a life-threatening condition. For malignant proliferation and migration, cancer cells require a large amount of glucose and energy to meet the demand of rapid metabolism. Hence, efficiently diminishing the utilization of energy substances by cancer cells is emerging as validated therapeutic strategies for cancer therapy. Herein, a nanoplatform with dual-inhibition of glucose uptake and oxidative phosphorylation (OXPHOS) was designed, which consisted of albendazole (ABZ) and atovaquone (ATO) by simple carrier-free self-assembling. The introduction of ABZ could evidently decrease glucose uptake to reduce the main “energy fuel” of cancer cells. Meanwhile, as a blocker of OXPHOS, ATO would reduce adenosine triphosphate (ATP) production and ameliorate hypoxia microenvironment by suppressing mitochondrial respiratory chain. Under such dual inhibition of energy metabolism, AA NPs exerted synergistic energy exhaustion effect and outstanding hypoxia improvement function, efficiently inhibiting tumor growth and metastasis. This research not only illustrates the feasibility of energy metabolism therapy by co-inhibiting glucose uptake and OXPHOS, but also provides an ingenious tactic to diminish metastasis during MBC treatment     

Discovery of bioactive small-molecule inhibitor of poly adp-ribose polymerase: implications for energy-deficient cells

Poly (ADP-ribose) polymerase (PARP1) is a nuclear protein that, when overactivated by oxidative stress-induced DNA damage, ADP ribosylates target proteins leading to dramatic cellular ATP depletion. We have discovered a biologically active small-molecule inhibitor of PARP1. The discovered compound inhibited PARP1 enzymatic activity in vitro and prevented ATP loss and cell death in a surrogate model of oxidative stress in vivo. We also investigated a new use for PARP1 inhibitors in energy-deficient cells by using Huntington's disease as a model. Our results showed that insult with the oxidant hydrogen peroxide depleted cellular ATP in mutant cells below the threshold of viability. The protective role of PARP1 inhibitors against oxidative stress has been shown in this model system.     

同位素示踪技术定量分析肿瘤细胞中的代谢重编

肿瘤代谢在最近几年重新获得重视,讨论肿瘤代谢我们无法避开瓦伯格效应,尽管瓦伯格效应在20世纪20年代就提出来了。瓦伯格效应也称需氧糖酵解,是指在有氧的条件下癌细胞过量摄取葡萄糖,产生更多的乳酸。此外,癌细胞也可摄取其他物质,如谷氨酰胺、丝氨酸和甘氨酸等作为能量需求与营养来源。为了描述癌细胞中改变了的代谢通路,同位素标记示踪技术提供了一种新的全局观点,让我们更好地理解在癌基因或抑癌基因调控下的代谢切换。色谱与质谱联用是一种重要的分析工具,可定量检测带有同位素标记的代谢物,从而剖析肿瘤细胞中的生物化学反应。在本文中,我们总结了基于质谱的肿瘤代谢分析的研究概况并提出未来的研究方向。重点讨论碳-13标记与氘代标记的示踪技术分析中间代谢产物,包括还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)。目前生物化学教科书中认为NADPH主要来源于磷酸戊糖途径。然而,氘代示踪技术分析表明,包括一碳代谢在内的其他代谢通路对产生NADPH也很重要。以上内容都在本文中进行了讨论。     

Pharmaceutical Drug Metformin and MCL1 Inhibitor S63845 Exhibit Anticancer Activity in Myeloid Leukemia Cells via Redox Remodeling

Metabolic landscape and sensitivity to apoptosis induction play a crucial role in acute myeloid leukemia (AML) resistance. Therefore, we investigated the effect of metformin, a medication that also acts as an inhibitor of oxidative phosphorylation (OXPHOS), and MCL-1 inhibitor {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540","term_text":"S63845"}}S63845 in AML cell lines NB4, KG1 and chemoresistant KG1A cells. The impact of compounds was evaluated using fluorescence-based metabolic flux analysis, assessment of mitochondrial Δψ and cellular ROS, trypan blue exclusion, Annexin V-PI and XTT tests for cell death and cytotoxicity estimations, also RT-qPCR and Western blot for gene and protein expression. Treatment with metformin resulted in significant downregulation of OXPHOS; however, increase in glycolysis was observed in NB4 and KG1A cells. In contrast, treatment with {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540","term_text":"S63845"}}S63845 slightly increased the rate of OXPHOS in KG1 and KG1A cells, although it profoundly diminished the rate of glycolysis. Generally, combined treatment had stronger inhibitory effects on cellular metabolism and ATP levels. Furthermore, results revealed that treatment with metformin, {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540","term_text":"S63845"}}S63845 and their combinations induced apoptosis in AML cells. In addition, level of apoptotic cell death correlated with cellular ROS induction, as well as with downregulation of tumor suppressor protein MYC. In summary, we show that modulation of redox-stress could have a potential anticancer activity in AML cells.     

Sequencing,modeling, and selective inhibition of Trypanosoma brucei hexokinase

For Trypanosoma brucei, a parasite responsible for African sleeping sickness, carbohydrate metabolism is the only source of ATP, and glycolytic enzymes are localized within membrane-bound organelles called glycosomes. Hexokinase, the first enzyme of the glycolytic pathway, was chosen as a target for selective drug design. We have cloned and sequenced the hexokinase gene of T. brucei. In parallel, we have synthesized several inhibitors. Kinetic analysis revealed differences in the binding mode of these compounds toward yeast and T. brucei hexokinases, while the m-bromophenyl glucosamide was found to be selective for T. brucei. The modeled structure of T. brucei hexokinase-inhibitor complex (using the crystal structure of the Schistosoma mansoni hexokinase as a template) allows us to propose a mode of action of this inhibitor for the trypanosome hexokinase and to account for the observed selectivity.     

Discovery of novel Pim-1 kinase inhibitors by a hierarchical multistage virtual screening approach based on SVM model, pharmacophore, and molecular docking

In this investigation, we describe the discovery of novel potent Pim-1 inhibitors by employing a proposed hierarchical multistage virtual screening (VS) approach, which is based on support vector machine-based (SVM-based VS or SB-VS), pharmacophore-based VS (PB-VS), and docking-based VS (DB-VS) methods. In this approach, the three VS methods are applied in an increasing order of complexity so that the first filter (SB-VS) is fast and simple, while successive ones (PB-VS and DB-VS) are more time-consuming but are applied only to a small subset of the entire database. Evaluation of this approach indicates that it can be used to screen a large chemical library rapidly with a high hit rate and a high enrichment factor. This approach was then applied to screen several large chemical libraries, including PubChem, Specs, and Enamine as well as an in-house database. From the final hits, 47 compounds were selected for further in vitro Pim-1 inhibitory assay, and 15 compounds show nanomolar level or low micromolar inhibition potency against Pim-1. In particular, four of them were found to have new scaffolds which have potential for the chemical development of Pim-1 inhibitors.     

具有全新机理的DNA旋转酶抑制剂的筛选及抑菌活性

利用具有新机制的抗耐药菌DNA旋转酶抑制剂GSK299423与DNA旋转酶的晶体复合物(PDB code:2XCS)构建基于配体-受体复合物的药效团模型, 诱骗集(Decoy set)验证结果表明该药效团模型具有较强的活性识别能力. 将药效团模型与分子对接相结合用于筛选化合物库, 通过抑菌活性测定, 获得了具有抗多药耐药菌活性的DNA旋转酶抑制剂LTH02.     

Caminoside A,an antimicrobial glycolipid isolated from the marine sponge Caminus sphaeroconia

Extracts of the marine sponge Caminus sphaeroconia showed potent activity in a screen for bacterial type III secretion inhibitors. Bioassay guided fractionation of the extract led to the isolation of the novel antimicrobial glycolipid caminoside A (1). The structure of caminoside A was elucidated by analysis of spectroscopic data and chemical degradation.[structure: see text]