Single Cause Single Cure Foundation. Fundação para a Ciência e Tecnologia (FCT)  through the PhD grant (SFR/BD/146065/2019)NewG Lab Pharm

Baltazar, Fátima

Casal, Margarida

Celeiro, Sónia Pires

Granja, Sara Costa

Ko, Young

Matos, Ana Catarina Barbosa

Preto, Ana

Queirós, Odília

Vieira, Joana Margarida Pereira

English

Universidade do Minho: RepositoriUM

3-bromopyruvate boosts the effect of chemotherapy in acute myeloid leukaemia by reducing cell antioxidant defenceJoana Pereira-Vieira1,2, Sónia Pires Celeiro1,2, Sara Granja1,2, Catarina Matos-Barbosa1,2, Ana Preto3, O Queirós4, Young H Ko5, Margarida Casal3, Fátima Baltazar1,21Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho,Campus of Gualtar, Braga, Portugal;2ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal;3Center of molecular and Environmental Biology (CBMA), University of Minho, Portugal;4CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias daSaúde, Rua Central de Gandra, 1317, 4585-116, Gandra, PRD, Portugal;5KoDiscovery, LLC, University of Maryland Bio Park, Baltimore, Maryland, USA.AML is most common type of leukemia in adults1 in 4 adults survive longerthan 5 yearMetabolism involved in chemoresistanceLeukemic Stem CellsSelf-renewalLeukemic Myeloid ProgenitorsMyeloid BlastsAcute Myeloid Leukemia (AML) AIM: Explore the metabolic inhibition as a therapeutic approach in AML in combination with classical chemotherapy regimens2. MATERIALS AND METHODS 3. RESULTS/CONCLUSIONS1. INTRODUCTION 4. PROPOSED MECHANISM AND ACKNOWLEDGMENTSGlutamineGlutamateGSH GSSGNADP+Glucose Glucose-6-P Ribose-5-PFructose bisPNADPHGlycolysisPPPGlutaminolysis2. MATERIALS AND METHODS 3. RESULTS/CONCLUSIONS1. INTRODUCTION 4. PROPOSED MECHANISM AND ACKNOWLEDGMENTSPre-treatment (PT)PTCTR16 hours glycolytic inhibitor: 3-Bromopyruvate (3-BP)Treatment 48 hours chemotherapeutic agentsMOLM13KG-1EffectCell viabilityMetabolic profileMitochondrial activityGlutathione levelsDaunorubicin (DNR)Cytarabine (Ara-C)AML cell lines20.0001 0.001 0.01 0.1 1050100150 KG-1Daunorubicin (µM)0.0001 0.001 0.01 0.1 1050100150 MOLM13Daunorubicin (µM)0.01 0.1 1 10 100050100150*MOLM13Cytarabine (M)0.0001 0.001 0.01 0.1 1050100150 KG-1Daunorubicin (µM)0.01 0.1 1 10 100050100150 KG-1Cytarabine (µM)0.0001 0.001 0.01 0.1 1050100150Daunorubicin (µM)IC50 = 0.008 ±0.005 µMIC50 = 0.021 ±0.009 µM IC50 = 0.368 ±0.175 µMIC50 = 0.139 ±0.064 µMIC50 = 3.627 ±2.131 µMIC50 = 1.767 ±1.698 µMIC50 = 0.017 ±0.008 µMIC50 = 0.020 ±0.009 µM0.0001 0.001 0.01 0.1 1050100150 MOLM13Daunorubicin (µM)0.0001 0.001 0.01 0.1 1050100150 KG-1Daunorubicin (µM)5µM 3-BP sensitize AML cells to DNR and/or Ara-C3.1Figure 1. Effect of 3-BP pre-treatment on daunorubicin and cytarabine cytotoxicity in KG-1 andMOLM13 cells by trypan blue assay.5µM 3-BP do not induce cell death or loss in viability of AML cells3.2Untreated 5µM 3-BP Untreated 5µM 3-BPKG-1 MOLM13Propidium iodide (PI)Annexin V (AnneV)050100AnneV-/PI- (Viable cells)AnneV+/PI- (Early apoptotic cells)AnneV+/PI+ (Late apoptotic/necrotic cells)AnneV-/PI+ (Necrotic cells)PBS 3-BPKG-1Cells %050100AnneV-/PI- (Viable cells)AnneV+/PI- (Early apoptotic cells)AnneV+/PI+ (Late apoptotic/necrotic cells)AnneV-/PI+ (Necrotic cells)PBS 3-BPMOLM13Cells %%CellsUntreated%CellsUntreated5µM 3-BP 5µM 3-BPOLM13-1050100AnneV-/PI- (Viable cells)AnneV+/PI- (Early apoptotic cells)AnneV+/PI+ (Late apoptotic/necrotic cells)AnneV-/PI+ (Necrotic cells)PBS 3-BPMOLM13Cells %050100AnneV-/PI- (Viable cells)AnneV+/PI- (Early apoptotic cells)AnneV /PI+ (Late apoptoti /necrotic cells)AnneV-/PI+ (Necrotic cells)PBS 3-BPL 13Cells %050100AnneV-/PI- (Viable cells)AnneV+/PI- (Early apoptotic cells)AnneV+/PI+ (Late apoptotic/necrotic cells)AnneV-/PI+ (Necrotic cells)PBS 3-BPMOLM13Cells %- I- ( i l  ll )I- ( rl  t ti  ll )I ( t  t ti r ti  ll )- I ( r ti  ll )Cells %Figure 2. Characterization of apoptotic effect of 5 µM of 3-BP in AML cell lines by flow cytometry.Cell line KG-1 MOLM133-BP (µM) 0 5 20 0 5 20Cell viability (%) 100.00 109.86 38.96 100.00 146.26 70.70± SD 4.94 8.43 22.06 7.20 23.93 14.38Table 1. Effect of 5 µM 3-BP on AML cell viability. MOLM13 and KG-1 cell viabilitywas evaluated by MTS assay.0.0001 0.001 0.01 0.1 1050100150 KG-1 DMSOPTDaunorubicin (µM)Pre-treated with 5 µM 3-BP0.0001 0.001 0.01 0.1 1050100150 KG-1 DMSOPTDaunorubicin (µM)UntreatedCell viability (%)Cell viability (%)0110- 0 7210- 0 7310- 0 7MOLM 13Glucose consumptionµg/nº cells* ns0110 - 0 7210 - 0 7310 - 0 7MOLM 13Control 5 µM 3-BP 10 µM 3-BPExtracellular lactate***GlucoseGlucose-6-P PyruvateLactate3-BPGlucoseLactateFigure 3. Characterization of 3-BP effect on AML glycolytic profile using commercial enzymatic colorimetric kits.*p < 0.05; **p < 0.01; ns: not statistically significantµg / n°cells5µM 3-BP decrease glucose consumption in AML cells3.30110 - 0 7210 - 0 7310 - 0 7410 - 0 7510 - 0 7KG-1Glucose consumptionµg/nº cells**0110 - 0 7210 - 0 7310 - 0 7410 - 0 7510 - 0 7KG-1Control 5 µM 3BP 10 µM 3BPExtracellular lactatensnsµg / n°cells5µM 3-BP do not affect AML extracellular lactate levels, suggesting that non-toxic concentration of 3-BP may affect other metabolic pathway(s)Untreated2. MATERIALS AND METHODS 3. RESULTS/CONCLUSIONS1. INTRODUCTION 4. PROPOSED MECHANISM AND ACKNOWLEDGMENTS0.00.51.01.52.010 µM 3BPControl 5 µM 3BP*ns nsnsKG-1MOLM 13030060030006000900012000Mitotrackers(MFI)Green RedKG-1ns nsns ns03006009003000600090001200015000Mitotrackers(MFI)Green RedMOLM13ns nsnsnsDHE(MFI)Mitochondrial activityROS levelsGlutathione levelsNADP+GPoxNADPH2GSH GSSGH2O2 H2OGRROS5µM 3-BP induce oxidative stress, increasing ROS levels in AML cells3.4Mitotrackers(MFI)Mitotrackers(MFI)Figure 4. Characterization of mitochondrial activity and ROS levels of AMLcells in the presence of 3-BP for 16 h. *p < 0.05; ns: no statistically significant.Figure 5. Characterization of 5µM of 3-BP effect on glutathione levels of MOLM13 and KG-1 cell lines. **p < 0.01.5µM 3-BP decrease the redox potential of AML cells, increasing oxidized glutathione (GSSG)3.5GlucoseGlucose-6-PPyruvateGlucoseROS3-BPUntreated2. MATERIALS AND METHODS 3. RESULTS/CONCLUSIONS1. INTRODUCTION 4. PROPOSED MECHANISM AND ACKNOWLEDGMENTS012345MOLM13KG-1Control 5 µM 3BP****Ratio(GSH / GSSG)UntreatedDNRFree RadicalsDNA damage2. MATERIALS AND METHODS 3. RESULTS/CONCLUSIONS1. INTRODUCTION 4. PROPOSED MECHANISM AND ACKNOWLEDGMENTSThis project has financial support through a grant provided by the Single Cause Single Cure Foundation, NewG Lab Pharma (UMINHO/BI/211/2019) and by a PhD grant (SFRH/BD/146065/2019) of “Fundação para a Ciência e Tecnologia”.Pre-treatment with a non-toxic concentration of 3-BP boosts effect of chemotherapy in AML cell lines as result of combined mechanismsEffect of PT with 5µM 3-BP Boost of the chemotherapy effectGSHResistance to oxidative stressAntioxidant capacityEl Sayed, et al. (2017)Niedźwieckal, et al. (2016)Calviño, et al. (2014)ROS3-BPGlucoseGlucoseGlucose-6-PLactateHK IIPyruvateLactateGAPDH

3-bromopyruvate boosts the effect of chemotherapy in acute myeloid leukemia by reducing cell antioxidant defense

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3-bromopyruvate boosts the effect of chemotherapy in acute myeloid leukemia by reducing cell antioxidant defense

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