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STAR Protocols Mar 2022Mitochondrial electron transport chain (ETC) dysfunction elevates the NADH/NAD ratio to cause metabolic derangements. Here we describe a protocol to measure the NADH/NAD...
Mitochondrial electron transport chain (ETC) dysfunction elevates the NADH/NAD ratio to cause metabolic derangements. Here we describe a protocol to measure the NADH/NAD ratio and analyze the rewiring of glucose metabolism using [4-H]-glucose, [3-H]-glucose, and [U-C]-glucose in ETC-inhibited human cancer cells. We also describe a protocol to analyze the NADH/NAD ratio-sensitive metabolites in mouse plasma and mouse liver following phenformin treatment. These protocols comprehensively analyze the metabolic derangements resulting from increased NADH/NAD ratio in and models. For complete details on the use and execution of this profile, please refer to Liu et al. (2021).
Topics: Animals; Cell Line; Glucose; Metabolic Diseases; Mice; NAD; Oxidation-Reduction
PubMed: 35106502
DOI: 10.1016/j.xpro.2021.101120 -
Investigational New Drugs Jun 2022Myeloproliferative neoplasms (MPN) are disorders characterized by an alteration at the hematopoietic stem cell (HSC) level, where the JAK2 mutation is the most common...
BACKGROUND
Myeloproliferative neoplasms (MPN) are disorders characterized by an alteration at the hematopoietic stem cell (HSC) level, where the JAK2 mutation is the most common genetic alteration found in classic MPN (polycythemia vera, essential thrombocythemia, and primary myelofibrosis). We and others previously demonstrated that metformin reduced splenomegaly and platelets counts in peripheral blood in JAK2 pre-clinical MPN models, which highlighted the antineoplastic potential of biguanides for MPN treatment. Phenformin is a biguanide that has been used to treat diabetes, but was withdrawn due to its potential to cause lactic acidosis in patients.
AIMS
We herein aimed to investigate the effects of phenformin in MPN disease burden and stem cell function in Jak2-knockin MPN mice.
RESULTS
In vitro phenformin treatment reduced cell viability and increased apoptosis in SET2 JAK2 cells. Long-term treatment with 40 mg/kg phenformin in Jak2 knockin mice increased the frequency of LSK, myeloid progenitors (MP), and multipotent progenitors (MPP) in the bone marrow. Phenformin treatment did not affect peripheral blood counts, spleen weight, megakaryocyte count, erythroid precursors frequency, or ex vivo clonogenic capacity. Ex vivo treatment of bone marrow cells from Jak2 knockin mice with phenformin did not affect hematologic parameters or engraftment in recipient mice.
CONCLUSIONS
Phenformin increased the percentages of LSK, MP, and MPP populations, but did not reduce disease burden in Jak2-knockin mice. Additional studies are necessary to further understand the effects of phenformin on early hematopoietic progenitors.
Topics: Animals; Bone Marrow; Disease Models, Animal; Humans; Janus Kinase 2; Mice; Mutation; Myeloproliferative Disorders; Phenformin; Polycythemia Vera
PubMed: 35015172
DOI: 10.1007/s10637-022-01212-y -
The Journal of Toxicological Sciences 2022The liver microphysiological system (MPS) model is an in-vitro culture method that mimics physiological blood flow, which enhances basal cellular functions. However, the...
The liver microphysiological system (MPS) model is an in-vitro culture method that mimics physiological blood flow, which enhances basal cellular functions. However, the liver MPS model has not been tested in the preclinical stage because of its obscure utility. It can overcome the major problem of conventional systems-rapid loss of mitochondrial activity in cultured hepatocytes due to limited oxygen supply-by supplying oxygen to cultured hepatocytes using a perfusion device. In this study, we developed a new perfusion culture system that can detect mitochondrial toxicity. Primary mouse hepatocytes were cultured under perfusion condition for 48 hr. The hepatocytes showed increased oxygen consumption and reduced lactate release. These results indicated that the ATP-production pathway was switched from glycolysis to mitochondrial oxidative phosphorylation in the perfusion culture system. Furthermore, ATP levels were considerably reduced in the perfusion culture system after exposure to phenformin, a mitochondrial complex I inhibitor. To summarize, the perfusion culture system could improve the mitochondrial activity in primary mouse hepatocytes, and thus, has potential implications in the detection of mitochondrial toxicity.
Topics: Animals; Cells, Cultured; Glycolysis; Hepatocytes; Liver; Mice; Oxidative Phosphorylation; Oxygen Consumption; Perfusion
PubMed: 34987137
DOI: 10.2131/jts.47.13 -
ChemMedChem Mar 2022Metformin and other biguanides represent a new class of inhibitors of mitochondrial complex I that show promising antitumor effects. However, stronger inhibition of...
Metformin and other biguanides represent a new class of inhibitors of mitochondrial complex I that show promising antitumor effects. However, stronger inhibition of mitochondrial complex I is generally associated with upregulation of glycolysis and higher risk of lactic acidosis. Herein we report a novel biguanide derivative, N-cystaminylbiguanide (MC001), which was found to inhibit mitochondrial complex I with higher potency while inducing lactate production to a similar degree as metformin.Furthermore, MC001 was found to efficiently inhibit a panel of colorectal cancer (CRC) cells in vitro and to suppress tumor growth in a HCT116 xenograft nude mouse model, while not enhancing lactate production relative to metformin, exhibiting a superior safety profile to other potent biguanides such as phenformin. Mechanistically, MC001 efficiently inhibits mitochondrial complex I, activates AMPK, and represses mTOR, leading to cell-cycle arrest and apoptosis. Notably, MC001 inhibits both oxidative phosphorylation (OXPHOS) and glycolysis. We therefore propose that MC001 warrants further investigation in cancer treatment.
Topics: Animals; Humans; Mice; Cell Line, Tumor; Electron Transport Complex I; Glycolysis; Lactates; Metformin; Oxidative Phosphorylation
PubMed: 34984842
DOI: 10.1002/cmdc.202100674 -
Journal of Nanobiotechnology Nov 2021Mild-temperature photothermal therapy (mild-PTT) has emerged as a highly promising antitumor strategy by triggering immunogenic cell death (ICD) to elicit both innate...
Selectively down-regulated PD-L1 by albumin-phenformin nanoparticles mediated mitochondrial dysfunction to stimulate tumor-specific immunological response for enhanced mild-temperature photothermal efficacy.
BACKGROUND
Mild-temperature photothermal therapy (mild-PTT) has emerged as a highly promising antitumor strategy by triggering immunogenic cell death (ICD) to elicit both innate and adaptive immune responses for tumor control. However, mild-PTT still leads to the risk of tumor recurrence or metastasis because it could hardly completely eradicate tumors due to its impaired immunological efficacy owing to the enhanced PD-L1 expression in tumor cells after treatment.
RESULTS
In this study, we described a hydrogen peroxide (HO) responsive manganese dioxide mineralized albumin nanocomposite loading with mitochondria function inhibitor phenformin (PM) and near-infrared photothermal dye indocyanine green (ICG) by modified two-step biomineralization method. In combination with ICG induced mild-PTT and PM mediated mitochondria dysfunction, PD-L1 expression was obviously down-regulated and the generated immunological responses was able to effectively attack the remaining tumor cells. Meanwhile, the risk of tumor metastasis was effectively inhibited by reducing the expression of tumor invasion-related signal molecules (TGF-β and vimentin) after combining treatment.
CONCLUSION
Such a strategy offers novel insight into the development of nanomedicine for mild-PTT as well as cancer immunotherapy, which can provide protection against tumor relapse post elimination of their initial and metastatic tumors.
Topics: Albumins; Animals; Antineoplastic Agents; B7-H1 Antigen; Biomineralization; Cell Line, Tumor; Down-Regulation; Hydrogen Peroxide; Indocyanine Green; Manganese Compounds; Mice; Mitochondria; Nanoparticles; Oxides; Phenformin; Photothermal Therapy
PubMed: 34794446
DOI: 10.1186/s12951-021-01124-8 -
Molecules (Basel, Switzerland) Oct 2021The results presented in this paper confirm the beneficial role of an easy-to-use and low-cost thin-layer chromatography (TLC) technique for describing the retention... (Comparative Study)
Comparative Study
The results presented in this paper confirm the beneficial role of an easy-to-use and low-cost thin-layer chromatography (TLC) technique for describing the retention behavior and the experimental lipophilicity parameter of two biguanide derivatives, metformin and phenformin, in both normal-phase (NP) and reversed-phase (RP) TLC systems. The retention parameters (R, R) obtained under different chromatographic conditions, i.e., various stationary and mobile phases in the NP-TLC and RP-TLC systems, were used to determine the lipophilicity parameter (R) of metformin and phenformin. This study confirms the poor lipophilicity of both metformin and phenformin. It can be stated that the optimization of chromatographic conditions, i.e., the kind of stationary phase and the composition of mobile phase, was needed to obtain the reliable value of the chromatographic lipophilicity parameter (R) in this study. The fewer differences in the R values of both biguanide derivatives were ensured by the RP-TLC system composed of RP2, RP18, and RP18W plates and the mixture composed of methanol, propan-1-ol, and acetonitrile as an organic modifier compared to the NP-TLC analysis. The new calculation procedures for logP of drugs based on topological indices χ, χ, χ, M, and M may be a certain alternative to other algorithms as well as the TLC procedure performed under optimized chromatographic conditions. The knowledge of different lipophilicity parameters of the studied biguanides can be useful in the future design of novel and more therapeutically effective metformin and phenformin formulations for antidiabetic and possible anticancer treatment. Moreover, the topological indices presented in this work may be further used in the QSAR study of the examined biguanides.
Topics: Chromatography, Reverse-Phase; Chromatography, Thin Layer; Hydrophobic and Hydrophilic Interactions; Metformin; Molecular Structure; Phenformin
PubMed: 34771022
DOI: 10.3390/molecules26216613 -
Cancer Biology & Therapy Dec 2021Biguanide drugs (metformin and phenformin) have drawn interest for potential cancer treatments, and laboratory studies show that some cancer cells are selectively...
Biguanide drugs (metformin and phenformin) have drawn interest for potential cancer treatments, and laboratory studies show that some cancer cells are selectively sensitive to growth-inhibitory effects of biguanides. Examining metabolic pathways affected by biguanide treatments in cancer cells that are highly sensitive to biguanides, we found that biguanide treatment depletes cellular levels of both aspartate and NAD+. Experiments to replenish these metabolites or block steps of the aspartate-malate shuttle suggest that depletion of both metabolites, rather than either aspartate of NAD+ individually, is critical for growth-inhibitory effects of biguanide exposure. Even in sensitive cancer cells, though, biguanide treatment alone over a broad range of doses only inhibits cell replication without significantly affecting cell viability. Noting that clinical observations of biguanide efficacy have used combinations of agents that typically include cisplatin, we found that biguanide treatment at a cytostatic level substantially decreases survival of lung cancer and breast cancer cells when co-treated with cisplatin at doses that alone are also non-cytotoxic. This striking enhancement of cisplatin toxicity by biguanides depends on reductions of levels of NAD+ and aspartate, since addition of either of these metabolites prevented this potentiation of cisplatin cytotoxicity. Thus, biguanide drugs can have cytotoxic effects when used in combination with other cancer drugs, such as cisplatin, and depleting cellular levels of NAD+ and aspartate is critical for enhancing the cytotoxicity of cisplatin by biguanide drugs in sensitive cancer cells.
Topics: Antineoplastic Agents; Aspartic Acid; Cisplatin; Metformin; NAD; Neoplasms; Pharmaceutical Preparations
PubMed: 34720054
DOI: 10.1080/15384047.2021.1982599 -
The Journal of Clinical Investigation Sep 2021Mitochondrial electron transport chain complex I (ETCC1) is the essential core of cancer metabolism, yet potent ETCC1 inhibitors capable of safely suppressing tumor...
Mitochondrial electron transport chain complex I (ETCC1) is the essential core of cancer metabolism, yet potent ETCC1 inhibitors capable of safely suppressing tumor growth and metastasis in vivo are limited. From a plant extract screening, we identified petasin (PT) as a highly potent ETCC1 inhibitor with a chemical structure distinct from conventional inhibitors. PT had at least 1700 times higher activity than that of metformin or phenformin and induced cytotoxicity against a broad spectrum of tumor types. PT administration also induced prominent growth inhibition in multiple syngeneic and xenograft mouse models in vivo. Despite its higher potency, it showed no apparent toxicity toward nontumor cells and normal organs. Also, treatment with PT attenuated cellular motility and focal adhesion in vitro as well as lung metastasis in vivo. Metabolome and proteome analyses revealed that PT severely depleted the level of aspartate, disrupted tumor-associated metabolism of nucleotide synthesis and glycosylation, and downregulated major oncoproteins associated with proliferation and metastasis. These findings indicate the promising potential of PT as a potent ETCC1 inhibitor to target the metabolic vulnerability of tumor cells.
Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Electron Transport Complex I; Endoplasmic Reticulum Stress; Female; Humans; Lung Neoplasms; Metabolome; Metformin; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Neoplasm Metastasis; Neoplasms, Experimental; Petasites; Phenformin; Sesquiterpenes; Unfolded Protein Response; Xenograft Model Antitumor Assays
PubMed: 34623325
DOI: 10.1172/JCI139933 -
ACS Omega Sep 2021Graphene-based drug carriers provide a promising addition to current cancer drug delivery options. Increased accessibility of high-quality graphene made by...
Graphene-based drug carriers provide a promising addition to current cancer drug delivery options. Increased accessibility of high-quality graphene made by plasma-enhanced chemical vapor deposition (PE-CVD) makes it an attractive material to revisit in comparison to the widely studied graphene oxide (GO) in drug delivery. Here, we show the potential of repurposing the metabolic drug phenformin for cancer treatment in terms of stability, binding, and pH-responsive release. Using covalent attachment of poly(ethylene glycol) (PEG) onto pristine (PE-CVD) graphene, we show that PEG stabilized graphene nanosheets (PGNS) are stable in aqueous solutions and exhibit higher binding affinity toward phenformin than GO. Moreover, we experimentally demonstrate an improved drug release from PGNS than GO at pH levels lower than physiological conditions, yet comparable to that found in tumor microenvironments.
PubMed: 34604644
DOI: 10.1021/acsomega.1c03283 -
International Journal of Molecular... Aug 2021Microglial functioning depends on Ca signaling. By using Ca sensitive fluorescence dye, we studied how inhibition of mitochondrial respiration changed spontaneous Ca...
Microglial functioning depends on Ca signaling. By using Ca sensitive fluorescence dye, we studied how inhibition of mitochondrial respiration changed spontaneous Ca signals in soma of microglial cells from 5-7-day-old rats grown under normoxic and mild-hypoxic conditions. In microglia under normoxic conditions, metformin or rotenone elevated the rate and the amplitude of Ca signals 10-15 min after drug application. Addition of cyclosporin A, a blocker of mitochondrial permeability transition pore (mPTP), antioxidant trolox, or inositol 1,4,5-trisphosphate receptor (IP3R) blocker caffeine in the presence of rotenone reduced the elevated rate and the amplitude of the signals implying sensitivity to reactive oxygen species (ROS), and involvement of mitochondrial mPTP together with IP3R. Microglial cells exposed to mild hypoxic conditions for 24 h showed elevated rate and increased amplitude of Ca signals. Application of metformin or rotenone but not phenformin before mild hypoxia reduced this elevated rate. Thus, metformin and rotenone had the opposing fast action in normoxia after 10-15 min and the slow action during 24 h mild-hypoxia implying activation of different signaling pathways. The slow action of metformin through inhibition of complex I could stabilize Ca homeostasis after mild hypoxia and could be important for reduction of ischemia-induced microglial activation.
Topics: Animals; Caffeine; Calcium Signaling; Chromans; Cyclosporine; Electron Transport Complex I; Female; Hypoxia; Male; Metformin; Microglia; Mitochondria; Mitochondrial Membrane Transport Proteins; Neurons; Primary Cell Culture; Rats; Reactive Oxygen Species; Rotenone
PubMed: 34502402
DOI: 10.3390/ijms22179493