This (ReadMe) file was created on 2024-10-03 by Matic Legiša

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GENERAL INFORMATION
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Name of the dataset: Small-moleculs inhibitors of 6-phosphofructo-1-kinase simultaneously suppress Lactate and Superoxide generation in cancer cells – data used in the article

Author/leading researcher details
Name and surname: Matic Legiša
ORCID: 0000-0001-7355-4504
Institution: National Institute of Chemistry
Address: Hajdrihova 19, Ljubljana, Slovenia
Email: matic.legisa@ki.si

Information about the author(s)

Matic Legiša
Samo Lešnik
Janez Konc
Tina Vodopivec
Katja Čarmernik
Urška Potokar

Date of data collection: from 2024-09-12 to 2024-10-04

Geographical location of data collection: Ljubljana, Slovenia

Information on the funders/programmes/projects that made the data collection possible:Research Agency of Republic of Slovenia N0. P4-0176 and N0. N1-0142

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SHARING/ACCESSING INFORMATION
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Data licences/restrictions: CC BY 4.0

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VIEWING DATA AND FILES
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Abstract

Deregulated energy metabolism is a hallmark of cancer, characterized by increased glycolytic flux. Cancer-specific modification of 6-phosphofructo-1-kinase (PFK) impairs its ability to regulate the enzyme’s activity and increase glycolytic flux. Consequently, excessive cytosolic NADH formation triggers a harmful redox imbalance in the cancer cells, which is rapidly neutralized by the formation of lactic acid and superoxide (SOX). In the present study, inhibition of modified PFK1 activity was shown to suppress redundant NADH formation in cancer cells while simultaneously preventing lactate and SOX formation.
First, the atomic model of the crystal structure of human PFK1 was used to dock with the catalytic binding sites of the enzyme by virtually screening the ZING Drug NOW database using a supercomputer. Preliminary screening of selected compounds for suppression of lactate and SOX were performed on Jurkat cells (acute T-cell leukemia) and three other tumorigenic cell lines. Two compounds showing no cytostatic or cytotoxic effects were further tested for their inhibitory abilities. Sequential re-administration of compounds at low concentrations (10µM) was found to be effective in reducing lactate and SOX formation in all tumorigenic cell lines tested. In other experiment, Jurkat cells treated with compounds were observed to successfully reduce respiration and glycolytic rate compared to untreated cells. Lactate is known to cause acidosis in tumor matrix, which impairs T cell efficiency. Reduced PFK1 activity could therefore prevent immune escape in treated Jurkat cells. Indeed, increased apoptosis was observed by the combined growth of compound-treated Jurkat cells and activated T cells compared to the co-culture with untreated cells.                                                                                                                                                      
Inhibition of cancer-specific PFK1 activity reduces glycolytic flux, avoids abundant NADH formation and restores redox balance in the cytosol thus preventing deleterious effects of lactate such as immune escape, angiogenesis and metastasis, as well as SOX-induced endogenic mutagenesis in the cancer cells.                                                                                                                                                       

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METHODOLOGICAL INFORMATION
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Description of the methods used to collect/obtain the data: 

MATERIALS AND METHODS   

PFK1 molecular modeling and small-molecule inhibitors screening

To find the drugs inhibiting highly active, cancer-specific PFK1s, the atomic model of the human PFK-P iso-enzyme was designed based on the crystal structure of the human PFK-P tetramer (UniProt Q01813) in combination with ATP–Mg2+ at a resolution of 3.1 Å (Protein Data Bank accession number 4XYJ) [34]. In collaboration with the Laboratory for Molecular Modelling at the National Institute of Chemistry, Ljubljana, the ZINC Drug NOW database was first filtered to exclude expected aggregators and poorly soluble compounds. By using a supercomputer (CROW 16, National Institute of Chemistry, Ljubljana, Slovenia) with approximately 3,000 processor cores, large-scale virtual screening was performed by docking with ProBiS-Dock algorithm [35], [36] to the catalytic ATP binding site of PFK-P/PFK-M and PFK-L isoenzymes. 

Tumorigenic cell lines
The tumorigenic cell lines Jurkat (acute T cell leukemia; TIB-152), Caco-2 (colorectal ade-nocarcinoma; HTB-37), COLO 829 (melanoma; CRL-1974), were purchased from American Type Culture Collection (Manassas, Virginia, USA) while MDA-MB-231 cells (breast gland adenocarcinoma; CRM-HTB-26) were obtained through the courtesy of investigators at Josef Stefan Institute (Ljubljana, Slovenia). Mycoplasma testing was performed on all cell lines before being used in the experiments. 
If not specified differently the cells were cultivated in the RPMI 1640 GlutaMAX medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% of fetal bovine se-rum (FBS). The cells were incubated in 1mL volume (24 wells plate) at 5% CO2, 37oC. 
Cellular and metabolic analyses
If not specified differently, the tumorigenic cells were plated in the RPMI 1640 GlutaMax medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS at the concentration of 5x105 cells per mL. Different concentrations of specific inhibitors were added to the medium at the beginning of inoculation. As a vehicle, adequate amounts of DMSO as used in the solution with the specific concentrations of inhibitors were supplemented into the medium. The cells were incubated at 37oC and 5% CO2. 
Lactic acid Measurements
For measuring lactate concentrations in the medium, 20 µL samples were taken at specified time intervals, centrifuged at 1200 rpm for 5 minutes at 4oC and 15 µL supernatants were preserved at 4oC until analyzed. 
Lactate levels were determined enzymatically using an L-Lactic acid assay kit (K-LATE, Megazyme, Bray, Ireland) according to the manufacturer’s instructions. 
SOX/ROS measurements

For measuring intracellular levels of reactive oxygen species, tumorigenic cell lines were initially grown in the RPMI 1640 GlutaMax medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% of FBS and incubated at 5% CO2, 37oC. The cells were incubated in a 24-well plate with 1 mL of medium per well. The individual inhibitor was first added to the medium at a specified concentration at the start of incubation, followed by sequential re-insertion of the inhibitor at 24-hour intervals. After 78 hours of growth, the cells were washed by centrifugation and an aliquot of 1x105 cells was placed into 1 mL of phenol red-free IMDM medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% of FBS. As reported phenol red might interfere with fluorescent dyes present in ROS/SOX assays [37]. Simultaneously specific inhibitor of pre-defined concentration was added to each well. The cells were incubated for an additional 3 hours. As a vehicle, the equivalent amount of DMSO, as used for a specific concentration of inhibitor was added to the medium. For the detection of ROS and SOX levels in the cells, the cells in 100 µL of medium were washed with PBS by standard centrifugation protocol and then placed into a well on the 96-well black wall/clear bottom plate. To each well 100 µL ROS detection reagent (Green) and SOX detection reagent (Orange) were added as specified in the Cellular ROS/Superoxide Detection Assay Kit (ab139476 Abcam, Cambridge, UK). For measuring superoxide Cellular Superoxide Detection Assay Kit (ab139477 Abcam, Cambridge, UK) was used that contains “Orange” but not the “Green” reagent.  Relative fluorescence was measured by a microplate reader (Biotek, Vermont, USA), using Ex/Em 525 nm for the “Green” and Ex/Em 550/620 for the “Orange” reagent. For the positive control (K+) ROS inducer Pyocyanin was used and for the negative control (K-) ROS inhibitor N-acetyl-L-cysteine was used according to the manufacturer’s instructions. 
Cytostatic and cytotoxic assays

Cytostatic assay

Total Cell numbers at the end of incubation (72 hours) were determined by the XTT cell proliferation assay Kit (Cat. No. 30-1011K, ATCC, Manassas, Virginia) according to the manufacturer’s instructions. Untreated cells and cells treated with different concentrations of inhibitors were incubated for 72 hours before alive cellular biomass was determined according to the manufacturer’s instructions. As a vehicle, untreated cells grown in the medium with added adequate amounts of DMSO were assessed.
Cytotoxicity
Cell viability was determined by the Cytotoxicity Detection Kit (LDH) (Cat. No. 11 644 793 001, Sigma-Aldrich, Steinheim, Germany) according to the manufacturer’s instructions. As a negative control, 100 µL Triton X-100 was added to wells containing 1mL medium with cells. For the survival evaluation in the dose-dependent tests, the measurements were conducted at the end of incubation at 72 hours of growth in the medium with a single addition of a specific inhibitor (80 µM) or with an adequate amount of DMSO in the vehicle.  
Measurements of respiratory and glycolytic rates and capacities 
The efficiency of PFK1 inhibitors was evaluated by measuring respiratory and glycolytic rates and capacities in treated and untreated Jurkat cells. For this experiment, the Seahorse XFp instrument (Agilent Technology, Santa Clara, CA, USA) was used under standard conditions before and after the addition of 1µM of oligomycin A and 0.25 µM of Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP). The cells were incubated in RPMI 1650 GlutaMAX medium and inoculated with inhibitors No. 9 and No. 30 at the final concentration of 10 µM. An adequate amount of DMSO was added to the vehicle. After 24 hours of growth, the cell numbers were normalized to (3.33x106 per mL).  For the experiment 400 µl culture was washed by centrifugation and placed into XF Base medium (Agilent) with added glucose (10 mM), glutamine (2 mM), and Na-pyruvate (1 mM). A day before the experiment the Kit boxes were poured with poly-l-lysine (50 µL) (cell sticker) which was removed before the medium with Jurkat cells was added. The measurements were conducted essentially as described in the Seahorse XFp protocol. After 20 minutes of incubation respiration and glycolytic basal rates were measured, followed by adding 1 µM of oligomycin A (ATP synthase inhibitor) and 0.25 µM of FCCP (mitochondrial oxidative phosphorylation uncoupler). After 6.5 minutes OCR and ECAR values were determined again to compute maximal respiration and glycolytic capacity. 
Co-culture of tumorigenic Jurkat cells and activated T-cells.  
Isolating immune cells 
Peripheral Blood Monocyte Cells (PBMCs) were donated by Dr. V. Forstnerič, a colleague in the lab. Samples were obtained with informed consent and according to the study protocol approved by the National Medical Ethics Committee (0120-21/2020/4). Depletion of non-T cells from the PBMCs was conducted by Ficoll® Paque gradient centrifugation followed by Pan T Cell Isolation kit according to the manufacturer’s instructions (Miltenyi Biotec GmbH, Auburn, CA). Consequently, unharmed T-cells were isolated containing levels of enriched CD4 helper T-cells and CD8 cytotoxic T-cells. Isolated T-cells were grown in 12 well plates containing 1 mL medium. Growth stimulating medium contained in a total volume of 50 mL, X-Vivo 15 medium (Lonza, Basel, Switzerland) plus 5% FBS; 6.25 µL of 80 IU/mL IL-2; 70 µL/mL ImmunoCultTM human CD3/CD28 T-cell activator (Stemcell Technologies, Vancouver, Canada); and 0.17 µL 5 µM 2-mercaptoethanol. The T-cells were seeded in the medium at a concentration of 5.105 cells per mL. After 3 days of incubation when the number of stimulated cells reached 2-3.106 cells per mL, DMSO was added to the final concentration of 10 %, and the activated T-cells were stored at -80oC until use.
The combined growth of Jurkat cells and activated T-cells.

Before the start of the co-culture, the Jurkar cells were pre-grown in RPMI 1640 GlutaMax medium without sodium bicarbonate to enable pH alternation (Product number R6504, Sigma-Aldrich, Steinheim, Germany) with added FBS to the final concentration of 10% and incubated as specified above. The specific inhibitors were added to 10 µM concentration and sequentially reintroduced at 24-hour intervals. As a control, an equivalent amount of DMSO as used in inhibitors was supplemented to the medium. The tests started with 1x105 cells per mL. After 72 hours of Jurkat cell growth, immediately after the sequential reintroduction of inhibitors, the cells were collected by centrifuging, and the supernatant was saved for further experiments. An aliquot of precipitated Jurkat cells was added to 1mL of pre-used supernatant to reach a final cell number of 1.104 cells per mL. The pre-used medium has been taken to maintain the pH value of the medium during the co-culture experiment. Finally, to each well with Jurkat cells an aliquot of activated T-cells was added (5.104 per mL). As recommended previously [38] the co-culture was incubated under standard conditions for 18 hours.

Measuring apoptosis in a co-culture of Jurkat cells and activated T-cells.

Early and late-stage apoptosis of Jurkat cells in a co-culture were detected after washing cells with PBS and adding fluorescently labeled apoptotic dye Annexin V and viability dye 7-AAD. eBioscience Annexin V Apoptosis Detection Kit eFluor 450 (Cat. No 88-8006) (Thermo Fisher Scientific, Waltham, MA, USA) was used for all experiments according to the manufacturer’s instructions. 
Fluorescence was measured using a Spectral Flow Cytometer (Aurora Cytometer, Cytek Biosciences, Amsterdam, The Netherlands) and analyzed using FloJo software (Tree Star Inc., San Carlos, CA, USA). Annexin V+/7-AAD+ (late apoptotic) and annexin V+/7-AAD- (early apoptotic) cells were quantified by the frequency of fluorescently labeled cells and statistical significance was assessed by the two sample T-test (independent variable).  

Ref.: 34. doi:10.1038/nature14405
Ref.: 35. doi.org/10.1021/acs.jcim.1c01176
Ref.: 36.: DOI: 10.1093/nar/gkab294
Ref.: 37.: doi:10.3791/3357
Ref.: 38. doi.org/10.1080/2162402x.2019.1599635

Data processing methods: Biochemical laboratories, super-computers

Software information: "Ubuntu 14.04.6LT"

Standards and calibration data: Standart deviation, Student's T-tests (Graph Pad Prism version 3.0) (Graph Pad Software).