bims-medica 2024-02-25 papers

bims-medica

Biomed News

on Metabolism and diet in cancer

Issue of 2024–02–25
33 papers selected by
Brett Chrest, East Carolina University

Metabolic Responses of Lung Adenocarcinoma Cells to Survive under Stressful Conditions Associated with Tumor Microenvironment.
Modulation of Pyruvate Export and Extracellular Pyruvate Concentration in Primary Astrocyte Cultures.
Mitochondrial and cytosolic one-carbon metabolism is a targetable metabolic vulnerability in cisplatin-resistant ovarian cancer.
Arginase-induced cell death pathways and metabolic changes in cancer cells are not altered by insulin.
Dietary methionine restriction in cancer development and antitumor immunity.
Calorie Restriction and Time-Restricted Feeding: Effective Interventions in Overweight or Obese Patients Undergoing Radiotherapy Treatment with Curative Intent for Cancer.
An engineered biosensor enables dynamic aspartate measurements in living cells.
CD24 negativity reprograms mitochondrial metabolism to PPARα and NF-κB-driven fatty acid β-oxidation in triple-negative breast cancer.
Enhanced Expression of Glycolytic Enzymes and Succinate Dehydrogenase Complex Flavoprotein Subunit A by Mesothelin Promotes Glycolysis and Mitochondrial Respiration in Myeloblasts of Acute Myeloid Leukemia.
The Structure of the Cardiac Mitochondria Respirasome Is Adapted for the β-Oxidation of Fatty Acids.
A Hepatocellular Cancer Patient-derived Organoid Xenograft Model to Investigate Impact of Liver Regeneration on Tumor Growth.
Modeling Acute Myeloid Leukemia Using StarPEG-Heparin Hydrogels.
Development and validation of a multiplexed LC-MS/MS ketone body assay for clinical diagnostics.
Panobinostat sensitizes AraC-resistant AML cells to the combination of azacitidine and venetoclax.
HSK3486 Inhibits Colorectal Cancer Growth by Promoting Oxidative Stress and ATPase Inhibitory Factor 1 Activation.
Calcium oscillations optimize the energetic efficiency of mitochondrial metabolism.
PRMT1 sustains de novo fatty acid synthesis by methylating PHGDH to drive chemoresistance in triple-negative breast cancer.
Enteric and systemic postprandial lactate shuttle phases and dietary carbohydrate carbon flow in humans.
Intermittent energy restriction inhibits tumor growth and enhances paclitaxel response in a transgenic mouse model of endometrial cancer.
Cancer stem cell hypothesis 2.0 in glioblastoma: Where are we now and where are we going?
Low-dose hypomethylating agents cooperate with ferroptosis inducers to enhance ferroptosis by regulating the DNA methylation-mediated MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway in acute myeloid leukemia.
Addressing a Pre-Clinical Pipeline Gap: Development of the Pediatric Acute Myeloid Leukemia Patient-Derived Xenograft Program at Texas Children's Hospital at Baylor College of Medicine.
Dysregulated inter-mitochondrial crosstalk in glioblastoma cells revealed by in situ cryo-electron tomography.
Combating Acute Myeloid Leukemia via Sphingosine Kinase 1 Inhibitor-Nanomedicine Combination Therapy with Cytarabine or Venetoclax.
Targeting glutamine metabolism exhibits anti-tumor effects in thyroid cancer.
Plasma metabolomic differences in early-onset compared to average-onset colorectal cancer.
Opinion: The Key Steps in the Origin of Life to the Formation of the Eukaryotic Cell.
A Simple, Fast, Sensitive LC-MS/MS Method to Quantify NAD(H) in Biological Samples: Plasma NAD(H) Measurement to Monitor Brain Pathophysiology.
Characterization of Single-Spheroid Oxygen Consumption Using a Microfluidic Platform and Fluorescence Lifetime Imaging Microscopy.
Stress hyperglycaemia following trauma - a survival benefit or an outcome detriment?
Relationship Between Serum β-Hydroxybutyrate and Hepatic Fatty Acid Oxidation in Individuals with Obesity and NAFLD.
Effect of a 6-Week Carbohydrate-Reduced High-Protein Diet on Levels of FGF21 and GDF15 in People With Type 2 Diabetes.
Rapid time-lapse 3D oxygen tension measurements within hydrogels using widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) and image segmentation.

Metabolites. 2024 Feb 02. pii: 103. [Epub ahead of print]14(2):

Metabolic Responses of Lung Adenocarcinoma Cells to Survive under Stressful Conditions Associated with Tumor Microenvironment.

Angeles Carlos-Reyes, Susana Romero-Garcia, Heriberto Prado-Garcia.

  Solid tumors frequently present a heterogeneous tumor microenvironment. Because tumors have the potential to proliferate quickly, the consequence is a reduction in the nutrients, a reduction in the pH (<6.8), and a hypoxic environment. Although it is often assumed that tumor clones show a similar growth rate with little variations in nutrient consumption, the present study shows how growth-specific rate (µ), the specific rates of glucose, lactate, and glutamine consumption (qS), and the specific rates of lactate and glutamate production (qP) of 2D-cultured lung tumor cells are affected by changes in their environment. We determined in lung tumor cells (A427, A549, Calu-1, and SKMES-1) the above mentioned kinetic parameters during the exponential phase under different culture conditions, varying the predominant carbon source, pH, and oxygen tension. MCF-7 cells, a breast tumor cell line that can consume lactate, and non-transformed fibroblast cells (MRC-5) were included as controls. We also analyzed how cell-cycle progression and the amino acid transporter CD98 expression were affected. Our results show that: (1) In glucose presence, μ increased, but qS Glucose and qP Lactate decreased when tumor cells were cultured under acidosis as opposed to neutral conditions; (2) most lung cancer cell lines consumed lactate under normoxia or hypoxia; (3) although qS Glutamine diminished under hypoxia or acidosis, it slightly increased in lactate presence, a finding that was associated with CD98 upregulation; and (4) under acidosis, G0/G1 arrest was induced in A427 cancer cells, although this phenomenon was significantly increased when glucose was changed by lactate as the predominant carbon-source. Hence, our results provide an understanding of metabolic responses that tumor cells develop to survive under stressful conditions, providing clues for developing promising opportunities to improve traditional cancer therapies.

Keywords:  glucose deprivation; hypoxia; lactic acidosis; specific growth rate; tumor metabolism; tumor microenvironment

DOI:  https://doi.org/10.3390/metabo14020103
Neurochem Res. 2024 Feb 20.

Modulation of Pyruvate Export and Extracellular Pyruvate Concentration in Primary Astrocyte Cultures.

Nadine Denker, Ralf Dringen.

  Astrocyte-derived pyruvate is considered to have neuroprotective functions. In order to investigate the processes that are involved in astrocytic pyruvate release, we used primary rat astrocyte cultures as model system. Depending on the incubation conditions and medium composition, astrocyte cultures established extracellular steady state pyruvate concentrations in the range between 150 µM and 300 µM. During incubations for up to 2 weeks in DMEM culture medium, the extracellular pyruvate concentration remained almost constant for days, while the extracellular lactate concentration increased continuously during the incubation into the millimolar concentration range as long as glucose was present. In an amino acid-free incubation buffer, glucose-fed astrocytes released pyruvate with an initial rate of around 60 nmol/(h × mg) and after around 5 h an almost constant extracellular pyruvate concentration was established that was maintained for several hours. Extracellular pyruvate accumulation was also observed, if glucose had been replaced by mannose, fructose, lactate or alanine. Glucose-fed astrocyte cultures established similar extracellular steady state concentrations of pyruvate by releasing pyruvate into pyruvate-free media or by consuming excess of extracellular pyruvate. Inhibition of the monocarboxylate transporter MCT1 by AR-C155858 lowered extracellular pyruvate accumulation, while inhibition of mitochondrial pyruvate uptake by UK5099 increased the extracellular pyruvate concentration. Finally, the presence of the uncoupler BAM15 or of the respiratory chain inhibitor antimycin A almost completely abolished extracellular pyruvate accumulation. The data presented demonstrate that cultured astrocytes establish a transient extracellular steady state concentration of pyruvate which is strongly affected by modulation of the mitochondrial pyruvate metabolism.

Keywords:  Astrocytes; MCT1; Metabolism; Mitochondria; Pyruvate; Transport

DOI:  https://doi.org/10.1007/s11064-024-04120-0
Mol Cancer Ther. 2024 Feb 20.

Mitochondrial and cytosolic one-carbon metabolism is a targetable metabolic vulnerability in cisplatin-resistant ovarian cancer.

Adrianne Wallace-Povirk, Carrie O'Connor, Aamod S Dekhne, Xun Bao, Md Junayed Nayeen, Mathew Schneider, Jade M Katinas, Jennifer Wong-Roushar, Seongho Kim, Lisa Polin, Jing Li, Jessica B Back, Charles E Dann, Aleem Gangjee, Zhanjun Hou, Larry H Matherly.

One-carbon (C1) metabolism is compartmentalized between the cytosol and mitochondria with the mitochondrial C1 pathway as the major source of glycine and C1 units for cellular biosynthesis. Expression of mitochondrial C1 genes including SLC25A32, serine hydroxymethyl transferase (SHMT) 2, 5,10-methylene tetrahydrofolate dehydrogenase 2, and 5,10-methylene tetrahydrofolate dehydrogenase 1-like was significantly elevated in primary epithelial ovarian cancer (EOC) specimens compared to normal ovaries. 5-Substituted pyrrolo[3,2-d]pyrimidine antifolates (AGF347, AGF359, AGF362) inhibited proliferation of cisplatin sensitive (A2780, CaOV3, IGROV1) and resistant (A2780-E80, SKOV3) EOC cells. In SKOV3 and A2780-E80 cells, colony formation was inhibited. AGF347 induced apoptosis in SKOV3 cells. In IGROV1 cells, AGF347 was transported by folate receptor (FR) α. AGF347 was also transported into IGROV1 and SKOV3 cells by the proton-coupled folate transporter (SLC46A1) and the reduced folate carrier (SLC19A1). AGF347 accumulated to high levels in the cytosol and mitochondria of SKOV3 cells. By targeted metabolomics with [2,3,3-2H]L-serine, AGF347, AGF359 and AGF362 inhibited SHMT2 in the mitochondria. In the cytosol, SHMT1 and de novo purine biosynthesis (i.e., glycinamide ribonucleotide formyltransferase, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase) were targeted; AGF359 also inhibited thymidylate synthase. Antifolate treatments of SKOV3 cells depleted cellular glycine, mitochondrial NADH and glutathione, and showed synergistic in vitro inhibition toward SKOV3 and A2780-E80 cells when combined with cisplatin. In vivo studies with subcutaneous SKOV3 EOC xenografts in SCID mice confirmed significant antitumor efficacy of AGF347. Collectively, our studies demonstrate a unique metabolic vulnerability in EOC involving mitochondrial and cytosolic C1 metabolism that offers a promising new platform for therapy.

DOI: https://doi.org/10.1158/1535-7163.MCT-23-0550
Sci Rep. 2024 02 19. 14(1): 4112

Arginase-induced cell death pathways and metabolic changes in cancer cells are not altered by insulin.

Hui Yi Chew, Goran Cvetkovic, Slobodan Tepic, James W Wells.

Arginine, a semi-essential amino acid, is critical for cell growth. Typically, de novo synthesis of arginine is sufficient to support cellular processes, however, it becomes vital for cancer cells that are unable to synthesise arginine due to enzyme deficiencies. Targeting this need, arginine depletion with enzymes such as arginase (ARG) has emerged as a potential cancer therapeutic strategy. Studies have proposed using high dose insulin to induce a state of hypoaminoacidaemia in the body, thereby further reducing circulating arginine levels. However, the mitogenic and metabolic properties of insulin could potentially counteract the therapeutic effects of ARG. Our study examined the combined impact of insulin and ARG on breast, lung, and ovarian cell lines, focusing on cell proliferation, metabolism, apoptosis, and autophagy. Our results showed that the influence of insulin on ARG uptake varied between cell lines but failed to promote the proliferation of ARG-treated cells or aid recovery post-ARG treatment. Moreover, insulin was largely ineffective in altering ARG-induced metabolic changes and did not prevent apoptosis. In vitro, at least, these findings imply that insulin does not offer a growth or survival benefit to cancer cells being treated with ARG.

DOI: https://doi.org/10.1038/s41598-024-54520-z
Trends Endocrinol Metab. 2024 Feb 20. pii: S1043-2760(24)00023-7. [Epub ahead of print]

Dietary methionine restriction in cancer development and antitumor immunity.

Ming Ji, Qing Xu, Xiaoling Li.

  Methionine restriction (MR) has been shown to suppress tumor growth and improve the responses to various anticancer therapies. However, methionine itself is required for the proliferation, activation, and differentiation of T cells that are crucial for antitumor immunity. The dual impact of methionine, that influences both tumor and immune cells, has generated concerns regarding the potential consequences of MR on T cell immunity and its possible role in promoting cancer. In this review we systemically examine current literature on the interactions between dietary methionine, cancer cells, and immune cells. Based on recent findings on MR in immunocompetent animals, we further discuss how tumor stage-specific methionine dependence of immune cells and cancer cells in the tumor microenvironment could ultimately dictate the response of tumors to MR.

Keywords:  antitumor immunity; gut microbiota; methylation; redox homeostasis; sulfur metabolism

DOI:  https://doi.org/10.1016/j.tem.2024.01.009
Nutrients. 2024 Feb 07. pii: 477. [Epub ahead of print]16(4):

Calorie Restriction and Time-Restricted Feeding: Effective Interventions in Overweight or Obese Patients Undergoing Radiotherapy Treatment with Curative Intent for Cancer.

Carmen Vega, Esteban Barnafi, César Sánchez, Francisco Acevedo, Benjamin Walbaum, Alejandra Parada, Nicolás Rivas, Tomás Merino.

  This study assesses the feasibility of calorie restriction (CR) and time-restricted feeding (TRF) in overweight and obese cancer patients who realized little to no physical activity undergoing curative radiotherapy, structured as a prospective, interventional, non-randomized open-label clinical trial. Of the 27 participants initially enrolled, 21 patients with breast cancer were selected for analysis. The participants self-selected into two dietary interventions: TRF, comprising a sugar and saturated fat-free diet calibrated to individual energy needs consumed within an 8 h eating window followed by a 16 h fast, or CR, involving a 25% reduction in total caloric intake from energy expenditure distributed across 4 meals and 1 snack with 55% carbohydrates, 15% protein, and 30% fats, excluding sugars and saturated fats. The primary goal was to evaluate the feasibility of these diets in the specific patient group. The results indicate that both interventions are effective and statistically significant for weight loss and reducing one's waist circumference, with TRF showing a potentially stronger impact and better adherence. Changes in the LDL, HDL, total cholesterol, triglycerides, glucose and insulin were not statistically significant.

Keywords:  calorie restriction; cancer; curative; diet; intermittent fasting; nutrition; obesity; overweight; radiotherapy; time-restricted feeding

DOI:  https://doi.org/10.3390/nu16040477
Elife. 2024 Feb 23. pii: RP90024. [Epub ahead of print]12

An engineered biosensor enables dynamic aspartate measurements in living cells.

Kristian Davidsen, Jonathan S Marvin, Abhi Aggarwal, Timothy A Brown, Lucas B Sullivan.

  Intracellular levels of the amino acid aspartate are responsive to changes in metabolism in mammalian cells and can correspondingly alter cell function, highlighting the need for robust tools to measure aspartate abundance. However, comprehensive understanding of aspartate metabolism has been limited by the throughput, cost, and static nature of the mass spectrometry (MS)-based measurements that are typically employed to measure aspartate levels. To address these issues, we have developed a green fluorescent protein (GFP)-based sensor of aspartate (jAspSnFR3), where the fluorescence intensity corresponds to aspartate concentration. As a purified protein, the sensor has a 20-fold increase in fluorescence upon aspartate saturation, with dose-dependent fluorescence changes covering a physiologically relevant aspartate concentration range and no significant off target binding. Expressed in mammalian cell lines, sensor intensity correlated with aspartate levels measured by MS and could resolve temporal changes in intracellular aspartate from genetic, pharmacological, and nutritional manipulations. These data demonstrate the utility of jAspSnFR3 and highlight the opportunities it provides for temporally resolved and high-throughput applications of variables that affect aspartate levels.

Keywords:  aspartate; biochemistry; biosensor; cell biology; chemical biology; glutamine; human; metabolism; mitochondria; protein engineering

DOI:  https://doi.org/10.7554/eLife.90024
Cancer Lett. 2024 Feb 17. pii: S0304-3835(24)00117-4. [Epub ahead of print] 216724

CD24 negativity reprograms mitochondrial metabolism to PPARα and NF-κB-driven fatty acid β-oxidation in triple-negative breast cancer.

Divya Murthy, Debasmita Dutta, Kuldeep S Attri, Tagari Samanta, Sukjin Yang, Kwang Hwa Jung, Sarah G Latario, Vasanta Putluri, Shixia Huang, Nagireddy Putluri, Jun Hyoung Park, Benny Abraham Kaipparettu.

  CD24 is a well-characterized breast cancer (BC) stem cell (BCSC) marker. Primary breast tumor cells having CD24-negativity together with CD44-positivity is known to maintain high metastatic potential. However, the functional role of CD24 gene in triple-negative BC (TNBC), an aggressive subtype of BC, is not well understood. While the significance of CD24 in regulating immune pathways is well recognized in previous studies, the significance of CD24 low expression in onco-signaling and metabolic rewiring is largely unknown. Using CD24 knock-down and over-expression TNBC models, our in vitro and in vivo analysis suggest that CD24 is a tumor suppressor in metastatic TNBC. Comprehensive in silico gene expression analysis of breast tumors followed by lipidomic and metabolomic analyses of CD24-modulated cells revealed that CD24 negativity induces mitochondrial oxidative phosphorylation and reprograms TNBC metabolism toward the fatty acid beta-oxidation (FAO) pathway. CD24 silencing activates PPARα-mediated regulation of FAO in TNBC cells. Further analysis using reverse-phase protein array and its validation using CD24-modulated TNBC cells and xenograft models nominated CD24-NF-κB-CPT1A signaling pathway as the central regulatory mechanism of CD24-mediated FAO activity. Overall, our study proposes a novel role of CD24 in metabolic reprogramming that can open new avenues for the treatment strategies for patients with metastatic TNBC.

Keywords:  CD24; Fatty acid β-oxidation; Metabolic reprogramming; NF-κB; Triple-negative breast cancer

DOI:  https://doi.org/10.1016/j.canlet.2024.216724
Int J Mol Sci. 2024 Feb 10. pii: 2140. [Epub ahead of print]25(4):

Enhanced Expression of Glycolytic Enzymes and Succinate Dehydrogenase Complex Flavoprotein Subunit A by Mesothelin Promotes Glycolysis and Mitochondrial Respiration in Myeloblasts of Acute Myeloid Leukemia.

Yunseon Jang, Jeong Suk Koh, Jung-Hyun Park, Suyoung Choi, Pham Thi Thuy Duong, Bu Yeon Heo, Sang Woo Lee, Jung Yeon Kim, Myung-Won Lee, Seok-Hwan Kim, Ik-Chan Song.

  Acute myeloid leukemia (AML) is an aggressive malignancy characterized by rapid growth and uncontrolled proliferation of undifferentiated myeloid cells. Metabolic reprogramming is commonly observed in the bone marrow of AML patients, as leukemia cells require increased ATP supply to support disease progression. In this study, we examined the potential role of mesothelin as a metabolic modulator in myeloid cells in AML. Mesothelin is a well-known marker of solid tumors that promotes cancer cell proliferation and survival. We initially analyzed alterations in mesothelin expression in the myeloblast subpopulations, defined as SSC-Alow/CD45dim, obtained from the bone marrow of AML patients using flow cytometry. Our results showed overexpression of mesothelin in 34.8% of AML patients. Subsequently, metabolic changes in leukemia cells were evaluated by comparing the oxygen consumption rates (OCR) of bone marrow samples derived from adult AML patients. Notably, a higher OCR was observed in the mesothelin-positive compared to the mesothelin-low and non-expressing groups. Treatment with recombinant human mesothelin protein enhanced OCR and increased the mRNA expression of glycolytic enzymes and mitochondrial complex II in KG1α AML cells. Notably, siRNA targeting mesothelin in KG1α cells led to the reduction of glycolysis-related gene expression but had no effect on the mitochondrial complex gene. The collective results demonstrate that mesothelin induces metabolic changes in leukemia cells, facilitating the acquisition of a rapid supply of ATP for proliferation in AML. Therefore, the targeting of mesothelin presents a potentially promising approach to mitigating the progression of AML through the inhibition of glycolysis and mitochondrial respiration in myeloid cells.

Keywords:  acute myeloid leukemia; glycolysis; mesothelin; oxygen consumption rate

DOI:  https://doi.org/10.3390/ijms25042140
Int J Mol Sci. 2024 Feb 18. pii: 2410. [Epub ahead of print]25(4):

The Structure of the Cardiac Mitochondria Respirasome Is Adapted for the β-Oxidation of Fatty Acids.

Alexander V Panov.

  It is well known that in the heart and kidney mitochondria, more than 95% of ATP production is supported by the β-oxidation of long-chain fatty acids. However, the β-oxidation of fatty acids by mitochondria has been studied much less than the substrates formed during the catabolism of carbohydrates and amino acids. In the last few decades, several discoveries have been made that are directly related to fatty acid oxidation. In this review, we made an attempt to re-evaluate the β-oxidation of long-chain fatty acids from the perspectives of new discoveries. The single set of electron transporters of the cardiac mitochondrial respiratory chain is organized into three supercomplexes. Two of them contain complex I, a dimer of complex III, and two dimers of complex IV. The third, smaller supercomplex contains a dimer of complex III and two dimers of complex IV. We also considered other important discoveries. First, the enzymes of the β-oxidation of fatty acids are physically associated with the respirasome. Second, the β-oxidation of fatty acids creates the highest level of QH2 and reverses the flow of electrons from QH2 through complex II, reducing fumarate to succinate. Third, β-oxidation is greatly stimulated in the presence of succinate. We argue that the respirasome is uniquely adapted for the β-oxidation of fatty acids. The acyl-CoA dehydrogenase complex reduces the membrane's pool of ubiquinone to QH2, which is instantly oxidized by the smaller supercomplex, generating a high energization of mitochondria and reversing the electron flow through complex II, which reverses the electron flow through complex I, increasing the NADH/NAD+ ratio in the matrix. The mitochondrial nicotinamide nucleotide transhydrogenase catalyzes a hydride (H-, a proton plus two electrons) transfer across the inner mitochondrial membrane, reducing the cytosolic pool of NADP(H), thus providing the heart with ATP for muscle contraction and energy and reducing equivalents for the housekeeping processes.

Keywords:  heart mitochondria; oxidative phosphorylation; respirasome; respiratory chain; tricarboxylic acid cycle; ubiquinol; ubiquinone; β-oxidation of fatty acids

DOI:  https://doi.org/10.3390/ijms25042410
J Vis Exp. 2024 Feb 02.

A Hepatocellular Cancer Patient-derived Organoid Xenograft Model to Investigate Impact of Liver Regeneration on Tumor Growth.

Fabian Haak, Gabriel Fridolin Hess, Philipp Sedlaczek, Savas Deniz Soysal, Jürg Vosbeck, Salvatore Piscuoglio, Mairene Coto-Llerena, Otto Kollmar.

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. Recurrence stems from undetected micro-metastasis or de novo cancer, potentially triggered by postsurgical liver regeneration. Prior research employed HCC cell lines in orthotopic models to study the impact of liver regeneration, but their limited validity prompted the need for a more representative model. Here, we introduce a novel approach utilizing patient-derived HCC organoids to investigate the influence of liver regeneration on HCC. Patient tumor tissues are processed to create tumor organoids, embedded in a three-dimensional basement membrane matrix, and cultured in a liver-specific medium. One million organoids are injected into the right superior lobe (RSL) of immunodeficient mice, confirming macroscopic tumor growth through sonography. The intervention group undergoes resection of the left lateral lobe (LLL) (30% of total liver volume) or additionally, the middle lobe (ML) (65% of total liver volume) to induce liver regeneration within the tumor site. The control group experiences re-laparotomy without liver tissue resection. After 2 weeks, both groups undergo tumor and normal tissue explantation. In conclusion, this patient-derived HCC organoid model offers a robust platform to investigate the impact of liver regeneration post-cancer resection. Its multi-cellular composition, genetic diversity, and prolonged culture capabilities make it an invaluable tool for studying HCC recurrence mechanisms and potential interventions.

DOI: https://doi.org/10.3791/66245
Methods Mol Biol. 2024 ;2764 61-73

Modeling Acute Myeloid Leukemia Using StarPEG-Heparin Hydrogels.

P Lewen Holloway, Akhilandeshwari Ravichandran, Julien Clegg, Claudia Bruedigam, Laura J Bray.

  Biomimetic semi-synthetic hydrogels formed from a combination of star-shaped poly(ethylene glycol) (starPEG) and the glycosaminoglycan, heparin, allows for the three-dimensional (3D) culture of various cells and tissues. In this chapter, we describe methods for the use of starPEG-heparin hydrogels to cultivate primary and immortalized human acute myeloid leukemia (AML) cells. The resulting 3D culture models allow for the study of AML development and response to chemotherapeutic agents.

Keywords:  3D model; Acute myeloid leukemia; Hydrogel

DOI:  https://doi.org/10.1007/978-1-0716-3674-9_6
J Mass Spectrom Adv Clin Lab. 2024 Jan;31 49-58

Development and validation of a multiplexed LC-MS/MS ketone body assay for clinical diagnostics.

Robin H J Kemperman, Rebecca D Ganetzky, Stephen R Master.

   Objectives: Ketone bodies (KBs) serve as important energy sources that spare glucose, providing the primary energy for cardiac muscle, skeletal muscle during aerobic exercise, and the brain during periods of catabolism. The levels and relationships between the KBs are critical indicators of metabolic health and disease. However, challenges in separating isomeric KBs and concerns about sample stability have previously limited their clinical measurement.
Methods: A novel 6.5-minute liquid chromatography-mass spectrometry-based assay was developed, enabling the precise measurement of alpha-, beta- and gamma-hydroxybutyrate, beta-hydroxyisobutyrate, and acetoacetate. This method was fully validated for human serum and plasma samples by investigating extraction efficiency, matrix effects, accuracy, recovery, intra- and inter-precision, linearity, lower limit of quantitation (LLOQ), carryover, specificity, stability, and more. From 107 normal samples, reference ranges were established for all analytes and the beta-hydroxybutyrate/acetoacetate ratio.
Results: All five analytes were adequately separated chromatographically. An extraction efficiency between 80 and 120 % was observed for all KBs. Accuracy was evaluated through spike and recovery using 10 random patient samples, with an average recovery of 85-115 % for all KBs and a coefficient of variation of ≤ 3 %. Coefficients of variation for intra- and inter-day imprecision were < 5 %, and the total imprecision was < 10 %. No significant interferences were observed. Specimens remained stable for up to 6 h on ice or 2 h at room temperature.
Conclusions: The developed method is highly sensitive and robust. It has been validated for use with human serum and plasma, overcoming stability concerns and providing a reliable and efficient quantitative estimation of ketone bodies.

Keywords:  Acetoacetate; Beta-hydroxybutyrate; Clinical diagnostics; Ketone bodies; LC-MS/MS; Mitochondrial disease testing

DOI:  https://doi.org/10.1016/j.jmsacl.2024.01.004
Biochem Pharmacol. 2024 Feb 17. pii: S0006-2952(24)00048-0. [Epub ahead of print] 116065

Panobinostat sensitizes AraC-resistant AML cells to the combination of azacitidine and venetoclax.

Jianlei Zhao, Shuangshuang Wu, Deying Wang, Holly Edwards, Jenna Thibodeau, Seongho Kim, Paul Stemmer, Guan Wang, Jingji Jin, Süreyya Savasan, Jeffrey W Taub, Yubin Ge.

  The majority of acute myeloid leukemia (AML) patients respond to intensive induction therapy, consisting of cytarabine (AraC) and an anthracycline, though more than half experience relapse. Relapsed/refractory (R/R) AML patients are difficult to treat, and their clinical outcomes remain dismal. Venetoclax (VEN) in combination with azacitidine (AZA) has provided a promising treatment option for R/R AML, though the overall survival (OS) could be improved (OS ranges from 4.3 to 9.1 months). Overexpression of c-Myc is associated with chemoresistance in AML. Histone deacetylase (HDAC) inhibitors have been shown to suppress c-Myc and enhance the antileukemic activity of VEN, as well as AZA, though combination of all three has not been fully explored. In this study, we investigated the HDAC inhibitor, panobinostat, in combination with VEN + AZA against AraC-resistant AML cells. Panobinostat treatment downregulated c-Myc and Bcl-xL and upregulated Bim, which enhanced the antileukemic activity of VEN + AZA against AraC-resistant AML cells. In addition, panobinostat alone and in combination with VEN + AZA suppressed oxidative phosphorylation and/or glycolysis in AraC-resistant AML cells. These findings support further development of panobinostat in combination with VEN + AZA for the treatment of AraC-resistant AML.

Keywords:  Acute myeloid leukemia; Azacitidine; Panobinostat; Venetoclax

DOI:  https://doi.org/10.1016/j.bcp.2024.116065
Dig Dis Sci. 2024 Feb 20.

HSK3486 Inhibits Colorectal Cancer Growth by Promoting Oxidative Stress and ATPase Inhibitory Factor 1 Activation.

Ke Nan, Ziwen Zhong, Ying Yue, Wenchang Zhou, Xingfeng Sun, Yang Shen, Mengdi Qu, Zhaoyuan Chen, Jiahui Gu, Caihong Sun, Xun Sun, Lihong Lu, Jie Zhang, Changhong Miao, Minli Sun.

   BACKGROUND: HSK3486 (ciprofol), a new candidate drug similar to propofol, exerts sedative and hypnotic effects through gamma-aminobutyric acid type A receptors; however, its potential role in colorectal cancer is currently unknown.
AIMS: This study aimed to evaluate the effects of HSK3486 on colorectal cancer cell proliferation.
METHODS: Imaging was performed to detect reactive oxygen species and mitochondrial membrane potential. Western blotting was used to determine the expression of target signals. The HSK3486 molecular mechanism was investigated through ATPase inhibitory factor 1 knockdown and xenograft model experiments to assess mitochondrial function in colorectal cancer cells.
RESULTS: Cell Counting Kit-8 and Annexin V/propidium iodide double staining assays showed that HSK3486 inhibited colorectal cancer cell proliferation in a concentration-dependent manner. In addition, HSK3486 treatment increased the expression of B-cell lymphoma-2-associated X, cleaved caspase 3, and cleaved poly (ADP-ribose) polymerase, whereas myeloid cell leukemia-1 and B-cell lymphoma 2 expression decreased. HSK3486 promoted mitochondrial dysfunction by inducing ATPase inhibitor factor 1 expression. Furthermore, HSK3486 promoted oxidative stress, as shown by the increase in reactive oxygen species and lactate dehydrogenase levels, along with a decrease in mitochondrial membrane potential and ATP levels. ATPase inhibitor factor 1 small interfering RNA pretreatment dramatically increased the mitochondrial membrane potential and tumor size in a xenograft model following exposure to HSK3486.
CONCLUSION: Collectively, our findings revealed that HSK3486 induces oxidative stress, resulting in colorectal cancer cell apoptosis, making it a potential candidate therapeutic strategy for colorectal cancer.

Keywords:  ATPIF1; Anesthesia; Apoptosis; Colorectal cancer; GABA receptor agonist; Oxidative stress

DOI:  https://doi.org/10.1007/s10620-023-08213-8
iScience. 2024 Mar 15. 27(3): 109078

Calcium oscillations optimize the energetic efficiency of mitochondrial metabolism.

Valérie Voorsluijs, Francesco Avanzini, Gianmaria Falasco, Massimiliano Esposito, Alexander Skupin.

  Energy transduction is central to living organisms, but the impact of enzyme regulation and signaling on its thermodynamic efficiency is generally overlooked. Here, we analyze the efficiency of ATP production by the tricarboxylic acid cycle and oxidative phosphorylation, which generate most of the chemical energy in eukaryotes. Calcium signaling regulates this pathway and can affect its energetic output, but the concrete energetic impact of this cross-talk remains elusive. Calcium enhances ATP production by activating key enzymes of the tricarboxylic acid cycle while calcium homeostasis is ATP-dependent. We propose a detailed kinetic model describing the calcium-mitochondria cross-talk and analyze it using nonequilibrium thermodynamics: after identifying the effective reactions driving mitochondrial metabolism out of equilibrium, we quantify the mitochondrial thermodynamic efficiency for different conditions. Calcium oscillations, triggered by extracellular stimulation or energy deficiency, boost the thermodynamic efficiency of mitochondrial metabolism, suggesting a compensatory role of calcium signaling in mitochondrial bioenergetics.

Keywords:  Biological sciences; Human metabolism; Molecular biology; Natural sciences

DOI:  https://doi.org/10.1016/j.isci.2024.109078
Cancer Res. 2024 Feb 22.

PRMT1 sustains de novo fatty acid synthesis by methylating PHGDH to drive chemoresistance in triple-negative breast cancer.

Takehiro Yamamoto, Tetsu Hayashida, Yohei Masugi, Kiyotaka Oshikawa, Noriyo Hayakawa, Mai Itoh, Chiyoko Nishime, Masami Suzuki, Aiko Nagayama, Yuko Kawai, Takako Hishiki, Tomomi Matsuura, Yoshiko Naito, Akiko Kubo, Arisa Yamamoto, Yujiro Yoshioka, Tomokazu Kurahori, Misa Nagasaka, Minako Takizawa, Naoharu Takano, Koji Kawakami, Michiie Sakamoto, Masatoshi Wakui, Takushi Yamamoto, Yuko Kitagawa, Yasuaki Kabe, Kenichi Horisawa, Atsushi Suzuki, Masaki Matsumoto, Makoto Suematsu.

Triple-negative breast cancer (TNBC) chemoresistance hampers the ability to effectively treat patients. Identification of mechanisms driving chemoresistance can lead to strategies to improve treatment. Here, we revealed that protein arginine methyltransferase-1 (PRMT1) simultaneously methylates D-3-phosphoglycerate dehydrogenase (PHGDH), a critical enzyme in serine synthesis, and the glycolytic enzymes PFKFB3 and PKM2 in TNBC cells. 13C metabolic flux analyses showed that PRMT1 methylation of these three enzymes diverts glucose toward intermediates in the serine-synthesizing and serine/glycine cleavage pathways, thereby accelerating the production of methyl donors in TNBC cells. Mechanistically, PRMT1-dependent methylation of PHGDH at R54 or R20 activated its enzymatic activity by stabilizing 3-phosphoglycerate binding and suppressing polyubiquitination. PRMT1-mediated PHGDH methylation drove chemoresistance independently of glutathione synthesis. Rather, activation of the serine synthesis pathway supplied α-ketoglutarate and citrate to increase palmitate levels through activation of fatty acid synthase (FASN). Increased palmitate induced protein S-palmitoylation of PHGDH and FASN to further enhance fatty acid synthesis in a PRMT1-dependent manner. Loss of PRMT1 or pharmacological inhibition of FASN or protein S-palmitoyltransferase reversed chemoresistance in TNBC. Furthermore, immunohistochemistry coupled with imaging mass spectrometry in clinical TNBC specimens substantiated that PRMT1-mediated methylation of PHGDH, PFKFB3, and PKM2 correlates with chemoresistance and that metabolites required for methylation and fatty acid synthesis are enriched in TNBC. Together, these results suggest that enhanced de novo fatty acid synthesis mediated by coordinated protein arginine methylation and protein S-palmitoylation is a therapeutic target for overcoming chemoresistance in TNBC.

DOI: https://doi.org/10.1158/0008-5472.CAN-23-2266
Nat Metab. 2024 Feb 22.

Enteric and systemic postprandial lactate shuttle phases and dietary carbohydrate carbon flow in humans.

Robert G Leija, Casey C Curl, Jose A Arevalo, Adam D Osmond, Justin J Duong, Melvin J Huie, Umesh Masharani, George A Brooks.

Dietary glucose in excess is stored in the liver in the form of glycogen. As opposed to direct conversion of glucose into glycogen, the hypothesis of the postprandial lactate shuttle (PLS) proposes that dietary glucose uptake is metabolized to lactate in the gut, thereby being transferred to the liver for glycogen storage. In the present study, we provide evidence of a PLS in young healthy men and women. Overnight fasted participants underwent an oral glucose tolerance test, and arterialized lactate concentration and rate of appearance were determined. The concentration of lactate in the blood rose before the concentration of glucose, thus providing evidence of an enteric PLS. Secondary increments in the concentration of lactate in the blood and its rate of appearance coincided with those of glucose, which indicates the presence of a larger, secondary, systemic PLS phase driven by hepatic glucose release. The present study challenges the notion that lactate production is the result of hypoxia in skeletal muscles, because our work indicates that glycolysis proceeds to lactate in fully aerobic tissues and dietary carbohydrate is processed via lactate shuttling. Our study proposes that, in humans, lactate is a major vehicle for carbohydrate carbon distribution and metabolism.

DOI: https://doi.org/10.1038/s42255-024-00993-1
bioRxiv. 2024 Feb 07. pii: 2024.02.02.578679. [Epub ahead of print]

Intermittent energy restriction inhibits tumor growth and enhances paclitaxel response in a transgenic mouse model of endometrial cancer.

Ziyi Zhao, Jiandong Wang, Weimin Kong, Ziwei Fang, Michael Coleman, Ginger Milne, Wesley C Burkett, Meredith A Newton, Douglas Lee, Beor Deng, Xiaochang Shen, Hongyan Suo, Wenchuan Sun, Stephen Hursting, Chunxiao Zhou, Victoria L Bae-Jump.

Objective: Overweight/obesity is the strongest risk factor for endometrial cancer (EC), and weight management can reduce that risk and improve survival. We aimed to establish the differential abilities of intermittent energy restriction (IER) and low-fat diet (LFD), alone and in combination with paclitaxel, to reverse the procancer effects of high-fat diet (HFD)-induced obesity in a mouse model of EC.
Methods: Lkb1fl/flp53fl/fl mice were fed high-fat diet (HFD) or LFD to generate obese and lean phenotypes, respectively. Obese mice were maintained on HFD or switched to LFD (HFD-LFD) or IER (HFD-IER). Ten weeks after induction of endometrial tumor, mice in each group received paclitaxel or placebo for 4 weeks. Body and tumor weights; tumoral transcriptomic, metabolomic and oxylipin profiles; and serum metabolic hormones and chemocytokines were assessed.
Results: HFD-IER and HFD-LFD, relative to HFD, reduced body weight; reversed obesity-induced alterations in serum insulin, leptin and inflammatory factors; and decreased tumor incidence and mass, often to levels emulating those associated with continuous LFD. Concurrent paclitaxel, versus placebo, enhanced tumor suppression in each group, with greatest benefit in HFD-IER. The diets produced distinct tumoral gene expression and metabolic profiles, with HFD-IER associated with a more favorable (antitumor) metabolic and inflammatory environment.
Conclusion: In Lkb1 fl/fl p53 fl/fl mice, IER is generally more effective than LFD in promoting weight loss, inhibiting obesity-related endometrial tumor growth (particularly in combination with paclitaxel), and reversing detrimental obesity-related metabolic effects. These findings lay the foundation for further investigations of IER as a EC prevention strategy in women with overweight/obesity.

DOI: https://doi.org/10.1101/2024.02.02.578679
Neuro Oncol. 2024 Feb 23. pii: noae011. [Epub ahead of print]

Cancer stem cell hypothesis 2.0 in glioblastoma: Where are we now and where are we going?

Anthony R Sloan, Daniel J Silver, Sam Kint, Marco Gallo, Justin D Lathia.

  Over the past 2 decades, the cancer stem cell (CSC) hypothesis has provided insight into many malignant tumors, including glioblastoma (GBM). Cancer stem cells have been identified in patient-derived tumors and in some mouse models, allowing for a deeper understanding of cellular and molecular mechanisms underlying GBM growth and therapeutic resistance. The CSC hypothesis has been the cornerstone of cellular heterogeneity, providing a conceptual and technical framework to explain this longstanding phenotype in GBM. This hypothesis has evolved to fit recent insights into how cellular plasticity drives tumor growth to suggest that CSCs do not represent a distinct population but rather a cellular state with substantial plasticity that can be achieved by non-CSCs under specific conditions. This has further been reinforced by advances in genomics, including single-cell approaches, that have used the CSC hypothesis to identify multiple putative CSC states with unique properties, including specific developmental and metabolic programs. In this review, we provide a historical perspective on the CSC hypothesis and its recent evolution, with a focus on key functional phenotypes, and provide an update on the definition for its use in future genomic studies.

Keywords:  cancer stem cell; glioblastoma; heterogeneity; proliferation

DOI:  https://doi.org/10.1093/neuonc/noae011
Exp Hematol Oncol. 2024 Feb 20. 13(1): 19

Low-dose hypomethylating agents cooperate with ferroptosis inducers to enhance ferroptosis by regulating the DNA methylation-mediated MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway in acute myeloid leukemia.

Shuya Feng, Yigang Yuan, Zihan Lin, Min Li, Daijiao Ye, Liuzhi Shi, Danyang Li, Min Zhao, Chen Meng, Xiaofei He, Shanshan Wu, Fang Xiong, Siyu Ye, Junjun Yang, Haifeng Zhuang, Lili Hong, Shenmeng Gao.

   BACKGROUND: Ferroptosis is a new form of nonapoptotic and iron-dependent type of cell death. Glutathione peroxidase-4 (GPX4) plays an essential role in anti-ferroptosis by reducing lipid peroxidation. Although acute myeloid leukemia (AML) cells, especially relapsed and refractory (R/R)-AML, present high GPX4 levels and enzyme activities, pharmacological inhibition of GPX4 alone has limited application in AML. Thus, whether inhibition of GPX4 combined with other therapeutic reagents has effective application in AML is largely unknown.
METHODS: Lipid reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) assays were used to assess ferroptosis in AML cells treated with the hypomethylating agent (HMA) decitabine (DAC), ferroptosis-inducer (FIN) RAS-selective lethal 3 (RSL3), or their combination. Combination index (CI) analysis was used to assess the synergistic activity of DAC + RSL3 against AML cells. Finally, we evaluated the synergistic activity of DAC + RSL3 in murine AML and a human R/R-AML-xenografted NSG model in vivo.
RESULTS: We first assessed GPX4 expression and found that GPX4 levels were higher in AML cells, especially those with MLL rearrangements, than in NCs. Knockdown of GPX4 by shRNA and indirect inhibition of GPX4 enzyme activity by RSL3 robustly induced ferroptosis in AML cells. To reduce the dose of RSL3 and avoid side effects, low doses of DAC (0.5 µM) and RSL3 (0.05 µM) synergistically facilitate ferroptosis by inhibiting the AMP-activated protein kinase (AMPK)-SLC7A11-GPX4 axis. Knockdown of AMPK by shRNA enhanced ferroptosis, and overexpression of SLC7A11 and GPX4 rescued DAC + RSL3-induced anti-leukemogenesis. Mechanistically, DAC increased the expression of MAGEA6 by reducing MAGEA6 promoter hypermethylation. Overexpression of MAGEA6 induced the degradation of AMPK, suggesting that DAC inhibits the AMPK-SLC7A11-GPX4 axis by increasing MAGEA6 expression. In addition, DAC + RSL3 synergistically reduced leukemic burden and extended overall survival compared with either DAC or RSL3 treatment in the MLL-AF9-transformed murine model. Finally, DAC + RSL3 synergistically reduced viability in untreated and R/R-AML cells and extended overall survival in two R/R-AML-xenografted NSG mouse models.
CONCLUSIONS: Our study first identify vulnerability to ferroptosis by regulating MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway. Combined treatment with HMAs and FINs provides a potential therapeutic choice for AML patients, especially for R/R-AML.

Keywords:  AMPK; Acute myeloid leukemia; Ferroptosis; Glutathione peroxidase-4; Hypomethylating agent

DOI:  https://doi.org/10.1186/s40164-024-00489-4
Biomedicines. 2024 Feb 08. pii: 394. [Epub ahead of print]12(2):

Addressing a Pre-Clinical Pipeline Gap: Development of the Pediatric Acute Myeloid Leukemia Patient-Derived Xenograft Program at Texas Children's Hospital at Baylor College of Medicine.

Alexandra M Stevens, Maci Terrell, Raushan Rashid, Kevin E Fisher, Andrea N Marcogliese, Amos Gaikwad, Pulivarthi Rao, Chelsea Vrana, Michael Krueger, Michael Loken, Andrew J Menssen, Jacqueline A Cook, Noah Keogh, Michelle Alozie, Hailey Oviedo, Alan K Gonzalez, Tamilini Ilangovan, Julia Kim, Sohani Sandhu, Michele S Redell.

  The survival rate of pediatric acute myeloid leukemia (pAML) is currently around 60%. While survival has slowly increased over the past few decades, the development of novel agents likely to further improve survival for this heterogeneous patient population has been limited by gaps in the pAML pre-clinical pipeline. One of the major hurdles in evaluating new agents for pAML is the lack of pAML patient-derived xenograft (PDX) models. Unlike solid tumors and other types of leukemias, AML is notoriously hard to establish in mouse models, likely due in part to the need for specific human microenvironment elements. Our laboratory at TCH/BCM addressed this gap by establishing a systematic PDX workflow, leveraging advanced immunodeficient hosts and capitalizing on our high volume of pAML patients and close coordination between labs and clinical sections. Patients treated at TCH are offered the chance to participate in specimen banking protocols that allow blood and bone marrow collection as well as the collection of relevant clinical data. All patients who consent and have samples available are trialed for PDX development. In addition, samples from the Children's Oncology Group (COG) are also trialed for PDX generation. Serially transplanting PDX models are validated using short tandem repeat (STR) and characterized using both targeted DNA/RNA next generation sequencing and RNAseq. As of March 2023, this systematic approach has resulted in 26 serially transplanting models. Models have been shared with requesting labs to facilitate external pAML pre-clinical studies. Available PDX models can be located through the BCM PDX Portal. We expect our growing PDX resource to make a significant contribution to expediting the testing of promising novel therapeutics for pAML.

Keywords:  BCM PDX portal; Children’s Oncology Group (COG); MISTRG; MISTRG6; NSGS; PDX development; future development; novel therapeutics; serial transplanting

DOI:  https://doi.org/10.3390/biomedicines12020394
Proc Natl Acad Sci U S A. 2024 Feb 27. 121(9): e2311160121

Dysregulated inter-mitochondrial crosstalk in glioblastoma cells revealed by in situ cryo-electron tomography.

Rui Wang, Huan Lei, Hongxiang Wang, Lei Qi, Yu'e Liu, Yunhui Liu, Yufeng Shi, Juxiang Chen, Qing-Tao Shen.

  Glioblastomas (GBMs) are the most lethal primary brain tumors with limited survival, even under aggressive treatments. The current therapeutics for GBMs are flawed due to the failure to accurately discriminate between normal proliferating cells and distinctive tumor cells. Mitochondria are essential to GBMs and serve as potential therapeutical targets. Here, we utilize cryo-electron tomography to quantitatively investigate nanoscale details of randomly sampled mitochondria in their native cellular context of GBM cells. Our results show that compared with cancer-free brain cells, GBM cells own more inter-mitochondrial junctions of several types for communications. Furthermore, our tomograms unveil microtubule-dependent mitochondrial nanotunnel-like bridges in the GBM cells as another inter-mitochondrial structure. These quantified inter-mitochondrial features, together with other mitochondria-organelle and intra-mitochondrial ones, are sufficient to distinguish GBM cells from cancer-free brain cells under scrutiny with predictive modeling. Our findings decipher high-resolution inter-mitochondrial structural signatures and provide clues for diagnosis and therapeutic interventions for GBM and other mitochondria-related diseases.

Keywords:  cryo-electron tomography; glioblastoma; mitochondria; organelle crosstalk

DOI:  https://doi.org/10.1073/pnas.2311160121
Pharmaceutics. 2024 Jan 31. pii: 209. [Epub ahead of print]16(2):

Combating Acute Myeloid Leukemia via Sphingosine Kinase 1 Inhibitor-Nanomedicine Combination Therapy with Cytarabine or Venetoclax.

Thao M Nguyen, Paul Joyce, David M Ross, Kristen Bremmell, Manasi Jambhrunkar, Sook S Wong, Clive A Prestidge.

  MP-A08 is a novel sphingosine kinase 1 (SPHK1) inhibitor with activity against acute myeloid leukemia (AML). A rationally designed liposome-based encapsulation and delivery system has been shown to overcome the physicochemical challenges of MP-A08 and enable its effective delivery for improved efficacy and survival of mice engrafted with human AML in preclinical models. To establish therapies that overcome AML's heterogeneous nature, here we explored the combination of MP-A08-loaded liposomes with both the standard chemotherapy, cytarabine, and the targeted therapy, venetoclax, against human AML cell lines. Cytarabine (over the dose range of 0.1-0.5 µM) in combination with MP-A08 liposomes showed significant synergistic effects (as confirmed by the Chou-Talalay Combination Index) against the chemosensitised human AML cell lines MV4-11 and OCI-AML3. Venetoclax (over the dose range of 0.5-250 nM) in combination with MP-A08 liposomes showed significant synergistic effects against the chemosensitised human AML cell lines, particularly in venetoclax-resistant human AML cells. This strong synergistic effect is due to multiple mechanisms of action, i.e., inhibiting MCL-1 through SPHK1 inhibition, leading to ceramide accumulation, activation of protein kinase R, ATF4 upregulation, and NOXA activation, ultimately resulting in MCL-1 degradation. These combination therapies warrant further consideration and investigation in the search for a more comprehensive treatment strategy for AML.

Keywords:  acute myeloid leukemia; anti-cancer activity; combination therapy; cytarabine; drug resistance; liposomal drug encapsulation; nanocarrier drug delivery; sphingosine kinase 1 inhibitors; targeted cancer therapy; venetoclax

DOI:  https://doi.org/10.3390/pharmaceutics16020209
J Endocrinol Invest. 2024 Feb 22.

Targeting glutamine metabolism exhibits anti-tumor effects in thyroid cancer.

G-Q Zhang, C Xi, N-T Ju, C-T Shen, Z-L Qiu, H-J Song, Q-Y Luo.

   BACKGROUND: Effective treatment for patients with advanced thyroid cancer is lacking. Metabolism reprogramming is required for cancer to undergo oncogenic transformation and rapid tumorigenic growth. Glutamine is frequently used by cancer cells for active bioenergetic and biosynthetic needs. This study aims to investigate whether targeting glutamine metabolism is a promising therapeutic strategy for thyroid cancer.
METHODS: The expression of glutaminase (GLS) and glutamate dehydrogenase (GDH) in thyroid cancer tissues was evaluated by immunohistochemistry, and glutamine metabolism-related genes were assessed using real time-qPCR and western blotting. The effects of glutamine metabolism inhibitor 6-diazo-5-oxo-l-norleucine (DON) on thyroid cancer cells were determined by CCK-8, clone formation assay, Edu incorporation assay, flow cytometry, and Transwell assay. The mechanistic study was performed by real time-qPCR, western blotting, Seahorse assay, and gas chromatography-mass spectrometer assay. The effect of DON prodrug (JHU-083) on thyroid cancer in vivo was assessed using xenograft tumor models in BALB/c nude mice.
RESULTS: GLS and GDH were over-expressed in thyroid cancer tissues, and GLS expression was positively associated with lymph-node metastasis and TNM stage. The growth of thyroid cancer cells was significantly inhibited when cultured in glutamine-free medium. Targeting glutamine metabolism with DON inhibited the proliferation of thyroid cancer cells. DON treatment did not promote apoptosis, but increased the proportion of cells in the S phase, accompanied by the decreased expression of cyclin-dependent kinase 2 and cyclin A. DON treatment also significantly inhibited the migration and invasion of thyroid cancer cells by reducing the expression of N-cadherin, Vimentin, matrix metalloproteinase-2, and matrix metalloproteinase-9. Non-essential amino acids, including proline, alanine, aspartate, asparagine, and glycine, were reduced in thyroid cancer cells treated with DON, which could explain the decrease of proteins involved in migration, invasion, and cell cycle. The efficacy and safety of DON prodrug (JHU-083) for thyroid cancer treatment were verified in a mouse model. In addition to suppressing the proliferation and metastasis potential of thyroid cancer in vivo, enhanced innate immune response was also observed in JHU-083-treated xenograft tumors as a result of decreased expression of cluster of differentiation 47 and programmed cell death ligand 1.
CONCLUSIONS: Thyroid cancer exhibited enhanced glutamine metabolism, as evidenced by the glutamine dependence of thyroid cancer cells and high expression of multiple glutamine metabolism-related genes. Targeting glutamine metabolism with DON prodrug could be a promising therapeutic option for advanced thyroid cancer.

Keywords:  DON; Glutamine; Metabolism reprogramming; Thyroid cancer; Treatment

DOI:  https://doi.org/10.1007/s40618-023-02294-y
Sci Rep. 2024 02 21. 14(1): 4294

Plasma metabolomic differences in early-onset compared to average-onset colorectal cancer.

Thejus Jayakrishnan, Arshiya Mariam, Nicole Farha, Daniel M Rotroff, Federico Aucejo, Shimoli V Barot, Madison Conces, Kanika G Nair, Smitha S Krishnamurthi, Stephanie L Schmit, David Liska, Alok A Khorana, Suneel D Kamath.

  Deleterious effects of environmental exposures may contribute to the rising incidence of early-onset colorectal cancer (eoCRC). We assessed the metabolomic differences between patients with eoCRC, average-onset CRC (aoCRC), and non-CRC controls, to understand pathogenic mechanisms. Patients with stage I-IV CRC and non-CRC controls were categorized based on age ≤ 50 years (eoCRC or young non-CRC controls) or ≥ 60 years (aoCRC or older non-CRC controls). Differential metabolite abundance and metabolic pathway analyses were performed on plasma samples. Multivariate Cox proportional hazards modeling was used for survival analyses. All P values were adjusted for multiple testing (false discovery rate, FDR P < 0.15 considered significant). The study population comprised 170 patients with CRC (66 eoCRC and 104 aoCRC) and 49 non-CRC controls (34 young and 15 older). Citrate was differentially abundant in aoCRC vs. eoCRC in adjusted analysis (Odds Ratio = 21.8, FDR P = 0.04). Metabolic pathways altered in patients with aoCRC versus eoCRC included arginine biosynthesis, FDR P = 0.02; glyoxylate and dicarboxylate metabolism, FDR P = 0.005; citrate cycle, FDR P = 0.04; alanine, aspartate, and glutamate metabolism, FDR P = 0.01; glycine, serine, and threonine metabolism, FDR P = 0.14; and amino-acid t-RNA biosynthesis, FDR P = 0.01. 4-hydroxyhippuric acid was significantly associated with overall survival in all patients with CRC (Hazards ratio, HR = 0.4, 95% CI 0.3-0.7, FDR P = 0.05). We identified several unique metabolic alterations, particularly the significant differential abundance of citrate in aoCRC versus eoCRC. Arginine biosynthesis was the most enriched by the differentially altered metabolites. The findings hold promise in developing strategies for early detection and novel therapies.

Keywords:  Arginine biosynthesis pathway; Citric acid cycle; Early onset colorectal cancer; Metabolomics; Pathway analysis; Synthetic lethality; Translational research

DOI:  https://doi.org/10.1038/s41598-024-54560-5
Life (Basel). 2024 Feb 05. pii: 226. [Epub ahead of print]14(2):

Opinion: The Key Steps in the Origin of Life to the Formation of the Eukaryotic Cell.

Clifford F Brunk, Charles R Marshall.

  The path from life's origin to the emergence of the eukaryotic cell was long and complex, and as such it is rarely treated in one publication. Here, we offer a sketch of this path, recognizing that there are points of disagreement and that many transitions are still shrouded in mystery. We assume life developed within microchambers of an alkaline hydrothermal vent system. Initial simple reactions were built into more sophisticated reflexively autocatalytic food-generated networks (RAFs), laying the foundation for life's anastomosing metabolism, and eventually for the origin of RNA, which functioned as a genetic repository and as a catalyst (ribozymes). Eventually, protein synthesis developed, leading to life's biology becoming dominated by enzymes and not ribozymes. Subsequent enzymatic innovation included ATP synthase, which generates ATP, fueled by the proton gradient between the alkaline vent flux and the acidic sea. This gradient was later internalized via the evolution of the electron transport chain, a preadaptation for the subsequent emergence of the vent creatures from their microchamber cradles. Differences between bacteria and archaea suggests cellularization evolved at least twice. Later, the bacterial development of oxidative phosphorylation and the archaeal development of proteins to stabilize its DNA laid the foundation for the merger that led to the formation of eukaryotic cells.

Keywords:  ATP synthase; LUCA; RNA world; acetyl-CoA pathway; alkaline hydrothermal vent microchambers; archaea; bacteria; chemiosmosis; origin of life; prebiotic chemistry

DOI:  https://doi.org/10.3390/life14020226
Int J Mol Sci. 2024 Feb 15. pii: 2325. [Epub ahead of print]25(4):

A Simple, Fast, Sensitive LC-MS/MS Method to Quantify NAD(H) in Biological Samples: Plasma NAD(H) Measurement to Monitor Brain Pathophysiology.

Tamaki Ishima, Natsuka Kimura, Mizuki Kobayashi, Ryozo Nagai, Hitoshi Osaka, Kenichi Aizawa.

  Nicotinamide adenine dinucleotide (NAD) is a cofactor in redox reactions and an essential mediator of energy metabolism. The redox balance between NAD+ and NADH affects various diseases, cell differentiation, and aging, and in recent years there has been a growing need for measurement techniques with improved accuracy. However, NAD(H) measurements, representing both NAD+ and NADH, have been limited by the compound's properties. We achieved highly sensitive simultaneous measurement of NAD+ and NADH under non-ion pairing, mobile phase conditions of water, or methanol containing 5 mM ammonium acetate. These were achieved using a simple pre-treatment and 7-min analysis time. Use of the stable isotope 13C5-NAD+ as an internal standard enabled validation close to BMV criteria and demonstrated the robustness of NAD(H) determination. Measurements using this method showed that brain NAD(H) levels correlate strongly with plasma NAD(H) levels in the same mouse, indicating that NAD(H) concentrations in brain tissue are reflected in plasma. As NAD(H) is involved in various neurodegenerative diseases and cerebral ischemia, as well as brain diseases such as mitochondrial myopathies, monitoring changes in NADH levels in plasma after drug administration will be useful for development of future diagnostics and therapeutics.

Keywords:  LC-MS/MS; NAD+; NADH; brain; endogenous; plasma; protein precipitation; validation

DOI:  https://doi.org/10.3390/ijms25042325
Biosensors (Basel). 2024 Feb 11. pii: 96. [Epub ahead of print]14(2):

Characterization of Single-Spheroid Oxygen Consumption Using a Microfluidic Platform and Fluorescence Lifetime Imaging Microscopy.

Santhosh Kannan, Chien-Chung Peng, Hsiao-Mei Wu, Yi-Chung Tung.

  Oxygen consumption has been used to evaluate various cellular activities. In addition, three-dimensional (3D) spheroids have been broadly exploited as advanced in vitro cell models for various biomedical studies due to their capability of mimicking 3D in vivo microenvironments and cell arrangements. However, monitoring the oxygen consumption of live 3D spheroids poses challenges because existing invasive methods cause structural and cell damage. In contrast, optical methods using fluorescence labeling and microscopy are non-invasive, but they suffer from technical limitations like high cost, tedious procedures, and poor signal-to-noise ratios. To address these challenges, we developed a microfluidic platform for uniform-sized spheroid formation, handling, and culture. The platform is further integrated with widefield frequency domain fluorescence lifetime imaging microscopy (FD-FLIM) to efficiently characterize the lifetime of an oxygen-sensitive dye filling the platform for oxygen consumption characterization. In the experiments, osteosarcoma (MG-63) cells are exploited as the spheroid model and for the oxygen consumption analysis. The results demonstrate the functionality of the developed approach and show the accurate characterization of the oxygen consumption of the spheroids in response to drug treatments. The developed approach possesses great potential to advance spheroid metabolism studies with single-spheroid resolution and high sensitivity.

Keywords:  frequency domain fluorescence lifetime imaging microscopy (FD-FLIM); microfluidics; oxygen consumption; spheroid

DOI:  https://doi.org/10.3390/bios14020096
Curr Opin Anaesthesiol. 2024 Jan 22.

Stress hyperglycaemia following trauma - a survival benefit or an outcome detriment?

Christopher Rugg, Stefan Schmid, Johannes Zipperle, Janett Kreutziger.

PURPOSE OF REVIEW: Stress hyperglycaemia occur often in critically injured patients. To gain new consideration about it, this review compile current as well as known immunological and biochemical findings about causes and emergence.
RECENT FINDINGS: Glucose is the preferred energy substrate for fending immune cells, reparative tissue and the cardiovascular system following trauma. To fulfil these energy needs, the liver is metabolically reprogrammed to rebuild glucose from lactate and glucogenic amino acids (hepatic insulin resistance) at the expenses of muscles mass and - to a less extent - fat tissue (proteolysis, lipolysis, peripheral insulin resistance). This inevitably leads to stress hyperglycaemia, which is evolutionary preserved and seems to be an essential and beneficial survival response. It is initiated by damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), intensified by immune cells itself and mainly ruled by tumour necrosis factor (TNF)α and catecholamines with lactate and hypoxia inducible factor (HIF)-1α as intracellular signals and lactate as an energy shuttle. Important biochemical mechanisms involved in this response are the Warburg effect as an efficient metabolic shortcut and the extended Cori cycle.
SUMMARY: Stress hyperglycaemia is beneficial in an acute life-threatening situation, but further research is necessary, to prevent trauma patients from the detrimental effects of persisting hyperglycaemia.

DOI: https://doi.org/10.1097/ACO.0000000000001350
Am J Physiol Endocrinol Metab. 2024 Feb 21.

Relationship Between Serum β-Hydroxybutyrate and Hepatic Fatty Acid Oxidation in Individuals with Obesity and NAFLD.

Mary P Moore, Grace Shryack, Isabella Alessi, Nicole Wieschhaus, Grace M Meers, Sarah A Johnson, Andrew A Wheeler, Jamal A Ibdah, Elizabeth J Parks, R Scott Rector.

  NAFLD is characterized by excess lipid accumulation which can progress to inflammation (NASH), and fibrosis. Serum β-hydroxybutyrate (β-HB), a product of the ketogenic pathway, is commonly employed as a surrogate marker for hepatic fatty acid oxidation (FAO). However, it remains uncertain whether this relationship holds true in the context of NAFLD in humans. We compared fasting serum (β-HB) levels with direct measurement of liver mitochondrial palmitate oxidation in humans stratified based on NAFLD severity (n = 142). Patients were stratified based on NAFLD activity Score (NAS): NAS=0 (no disease), NAS=1-2 (mild), NAS=3-4 (moderate), and NAS³5 (advanced). Moderate and advanced NAFLD associated with reductions in liver HMGCS2, serum β-HB, but not HMGCL mRNA, relative to no disease. Worsening liver mitochondrial complete palmitate oxidation corresponded with lower HMGCS2 mRNA but not total (complete + incomplete) palmitate oxidation. Interestingly, we found that liver HMGCS2 mRNA and serum β-HB correlated with liver mitochondrial β-HAD activity and CPT1A mRNA. Also, lower mitochondrial mass and markers of mitochondrial turnover positively correlated with lower HMGCS2 in the liver. These data suggest that liver ketogenesis and FAO occur at comparable rates in individuals with NAFLD. Our findings support the utility of serum β-HB to serve as a marker of liver injury and hepatic FAO in the context of NAFLD.

Keywords:  HMGCS2; MASH; MASLD; ketones; mitochondria

DOI:  https://doi.org/10.1152/ajpendo.00336.2023
J Endocr Soc. 2024 Feb 19. 8(4): bvae008

Effect of a 6-Week Carbohydrate-Reduced High-Protein Diet on Levels of FGF21 and GDF15 in People With Type 2 Diabetes.

Michael M Richter, Mads N Thomsen, Mads J Skytte, Sasha A S Kjeldsen, Amirsalar Samkani, Jan Frystyk, Faidon Magkos, Jens J Holst, Sten Madsbad, Thure Krarup, Steen B Haugaard, Nicolai J Wewer Albrechtsen.

   Context: Fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15) are increased in type 2 diabetes and are potential regulators of metabolism. The effect of changes in caloric intake and macronutrient composition on their circulating levels in patients with type 2 diabetes are unknown.
Objective: To explore the effects of a carbohydrate-reduced high-protein diet with and without a clinically significant weight loss on circulating levels of FGF21 and GDF15 in patients with type 2 diabetes.
Methods: We measured circulating FGF21 and GDF15 in patients with type 2 diabetes who completed 2 previously published diet interventions. Study 1 randomized 28 subjects to an isocaloric diet in a 6 + 6-week crossover trial consisting of, in random order, a carbohydrate-reduced high-protein (CRHP) or a conventional diabetes (CD) diet. Study 2 randomized 72 subjects to a 6-week hypocaloric diet aiming at a ∼6% weight loss induced by either a CRHP or a CD diet. Fasting plasma FGF21 and GDF15 were measured before and after the interventions in a subset of samples (n = 24 in study 1, n = 66 in study 2).
Results: Plasma levels of FGF21 were reduced by 54% in the isocaloric study (P < .05) and 18% in the hypocaloric study (P < .05) in CRHP-treated individuals only. Circulating GDF15 levels increased by 18% (P < .05) following weight loss in combination with a CRHP diet but only in those treated with metformin.
Conclusion: The CRHP diet significantly reduced FGF21 in people with type 2 diabetes independent of weight loss, supporting the role of FGF21 as a "nutrient sensor." Combining metformin treatment with carbohydrate restriction and weight loss may provide additional metabolic improvements due to the rise in circulating GDF15.

Keywords:  hepatokines; metformin; nutrients; weight loss; weight maintenance

DOI:  https://doi.org/10.1210/jendso/bvae008
Analyst. 2024 Feb 20.

Rapid time-lapse 3D oxygen tension measurements within hydrogels using widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) and image segmentation.

Dao-Ming Chang, Heng-Hua Hsu, Ping-Liang Ko, Wei-Jen Chang, Tung-Han Hsieh, Hsiao-Mei Wu, Yi-Chung Tung.

Understanding the influence of oxygen tension on cellular functions and behaviors is crucial for investigating various physiological and pathological conditions. In vitro cell culture models, particularly those based on hydrogel extracellular matrices, have been developed to study cellular responses in specific oxygen microenvironments. However, accurately characterizing oxygen tension variations with great spatiotemporal resolutions, especially in three dimensions, remains challenging. This paper presents an approach for rapid time-lapse 3D oxygen tension measurements in hydrogels using a widely available inverted fluorescence microscope. Oxygen-sensitive fluorescent microbeads and widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) are utilized for oxygen tension estimation. To incorporate the third dimension, a motorized sample stage is implanted that enables automated image acquisition in the vertical direction. A machine learning algorithm based on K-means clustering is employed for microbead position identification. Using an upside-down microfluidic device, 3D oxygen gradients are generated within a hydrogel sample, and z-stack images are acquired using the FD-FLIM system. Analyses of the acquired images, involving microbead position identification, lifetime calculation, and oxygen tension conversion, are then performed offline. The results demonstrate the functionality of the developed approach for rapid time-lapse 3D oxygen tension measurements in hydrogels. Furthermore, the 3D oxygen tension adjacent to a tumor spheroid within a hydrogel during media exchange is characterized. The results further confirm that the 3D spatiotemporal oxygen tension profiles can be successfully measured quantitatively using the established setup and analysis process and that the approach may have great potential for investigating cellular activities within oxygen microenvironments.

DOI: https://doi.org/10.1039/d3an01625k