Barbosa, Ana Margarida

Casal, Margarida

Miranda, Joana

Moreira, Roxana

Queirós, Odília

Vieira, Joana Margarida Pereira

English

Universidade do Minho: RepositoriUM

IntroductionThe majority of cancer cells present an altered energeticmetabolism resorting to aerobic glycolysis, even under aerobicconditions (“Warburg” effect) and this can be the basis for thedevelopment of new and more effective antitumor agents. 3-bromopyruvate (3-BP) is an alkylating agent putativelytransported by the monocarboxylate transporters (MCTs), whichtargets cancer cell metabolism, and it has been demonstrated tobe a powerful and specific antitumor agent either in in vitro or invivo models. 3-BP inhibits tumor energetic metabolism, causingdepletion of intracellular ATP, thus acting as a cytotoxic agent.Our published results and preliminary transport assays show thatMCT1/4 and CD147 might play a key role in 3-BP uptake.We investigated the effect of 3-BP in glioblastomas, a veryaggressive cancer, and in colorectal cancer cell lines, whichappears in top three ranking of the most frequent cancer types[1]. 3-BP cytotoxic effect was assessed at different extracellularpH (pHe) values and correlated with MCTs expression [2, 3]. 3-BPeffect on migratory capacity and cell metabolism in the differentcell lines was also determined.References:[1] Ferlay, J. et al. (2014). Cancer incidence and mortality worldwide: sources, methods and major patterns inGLOBOCAN 2012. International Journal of Cancer. 1-76.[2] Azevedo-Silva, J. et al. (2015) The cytotoxicity of 3-Bromopyruvate in breast cancer cells depends onextracellular pH. Biochem J. 467: 247–258.[3] Queiros, O. et al. (2012) Butyrate activates the monocarboxylate transporter MCT4 expression in breast cancercells and enhances the antitumor activity of 3-bromopyruvate. J Bioenerg Biomembr. 44: 141-53.MethodsSulforhodamine B: The cytotoxic effect of 3-BP in glioblastomaand colorectal cancer cell lines was evaluated at basal conditionsand at different extracellular pHs. The different cell lines wereexposed in the assayed conditions to 3-BP for 16 hour and cellviability was determined after this period of time.Wound healing assay: Cells were plated in 6-well plates andgrown until total confluence. Two wounds were then created inthe confluent cells by manual scratching. Cells were treated with½ IC50 and IC50 values of the 3-BP, being untreated cells used ascontrol. Photograph records were obtained cells at 0, 12 and 24hour. Percentage of cell migration relative to time zero of thecontrol was evaluated with the GraphPad Prism 5 software.Glucose and lactate quantification: The different cell lines wereexposed at ½ IC50 and IC50 values of 3-BP and lactate and glucosewere measured in extracellular medium. As control, untreatedcells were used. The obtained results were normalized to theprotein biomass using SRB assay.Western Blot: Protein extracts of different cancer cell lines wereused to evaluate the MCT1 and MCT4 expression by Western-blotting. α-tubulin was used as loading control and the density ofeach band was determined with the software Image J (version1.48, NIH).Final Remarks•3-BP presented cytotoxic effect to both glioblastoma andcolorectal cancer cell lines, but with different sensitivities.•3-BP was more cytotoxic at lower pHe in most cell lines assayed,according to its probable mechanism of transport by a proton-symport.•The migratory capacity as well as energetic metabolism wereinhibited by 3-BP, being this effect more evident when higherconcentrations of the compound were used.• In glioblastomas, MCT4 was more expressed in the mostsensitive cell line, which also present a considerable expressionof MCT1. As 3-BP is transported by MCTs, its cytotoxic effectprobably be associated to the MCTs expression. However, incolorectal cancer cell lines no direct association was found.This work was supported by the CESPU project BioCat-CESPU-2016Metabolic profile in glioblastoma and colorectal cancer cell linesCell migration capacity in glioblastoma and colorectal cancer cell linesMCTs expression in glioblastoma and colorectal cancer cell linesTable 1. IC50 (µM) values for different cancer cell lines exposed to 3-BP.Figure 1. Quantification of the extracellular glucose and lactate in different cancer cell lines. Significantly different between groups: *p < 0.05; **p <0.01; ***p < 0.001; ****p < 0.0001; ns: no statistically significant compared to control.• 3-BP treatment induced a decrease of glucose consumption and lactate production in all cell lines.• In glioblastoma cell lines, MCT4 is more expressed in the most sensitive cell line, U373MG. MCT1 is more expressed in U251MG and U373MG cell lines. • In colorectal, there was no relevant differences between cell lines in MCTs expression.Table 3. Expression of MCT1 and MCT4 in different cancer cell lines.Cytotoxic effect of the 3-BP in glioblastoma and colorectal cancer cell linesResults3-Bromopyruvate induces cytotoxicity, inhibits glycolysis and decreases migratory capacity in glioblastomas and colorectal cancer cell lines Ana Margarida Barbosa1, Joana Vieira1,2, Joana Miranda1, Roxana Moreira1,2, Margarida Casal2 and Odília Queirós1,21CESPU, Institute of Research and Advanced Training in Health Sciences and Technologies, Rua Central de Gandra, 1317, 4585-116 Gandra PRD, Portugal; 2CBMA - Center of Molecular and Environmental Biology, University of Minho, Campus de Gualtar, Braga, 4710-057, PortugalGlioblastoma Cell Lines Colorectal Cancer Cell linesU373MG U87MG U251MG HCT-15 Caco-2 HT-29Medium with NaHCO364.71 ±7.0165.92 ±6.9595.72 ±7.4331.76 ±6.28234.15 ±17.38263,67 ± 74.89Medium without NaHCO3pHe 6.626.18 ±1.5359.16 ±5.0043.55 ±4.7015.95 ±6.07ND NDpHe 7.425.35 ±3.6875.86 ±5.4457.41 ±5.4719.55 ±3.98ND ND• The cell lines more resistant to 3-BP were U251MG and HT-29, in glioblastoma and colorectal cancer cell lines, respectively.• 3-BP was more cytotoxic at pHe 6.6 than pHe 7.4. It was not possible determinate IC50 value at different pHe for Caco-2 and HT-29 in colorectal cancer cell lines.• This effect was more evident whenhigher concentrations of the compoundwere used, namely after 24 hours oftreatment.• 3-BP inhibits migratory capacity of bothglioblastoma and colorectal cancer celllines.Glioblastoma cell linesControl50IC1/2 50IC0.00.51.01.52.0****U373MG***[glucose] mmol/LControl50IC1/2 50IC0.000.010.020.030.040.05*******[lactate] mmol/LControl50IC1/2 50IC0.00.51.01.52.0**** U87MG*[glucose] mmol/LControl50IC1/2 50IC0.000.010.020.030.040.05**ns[lactate] mmol/LControl50IC1/2 50IC0.00.51.01.52.0****U251MG***[glucose]mmol/LControl50 IC1/250IC0.000.010.020.030.040.05*******[lactate] mmol/LColorectal cell linesControl50 IC1/250IC0510152025HCT-15** **[glucose] mmol/LControl50 IC1/250IC0246810nsns[lactate]  mmol/LControl50 IC1/250IC0510152025Caco-2ns ns[glucose] mmol/LControl50 IC1/250IC0246810ns ns[lactate] mmol/LControl50 IC1/250IC0510152025HT-29ns ns[glucose]mmol/LControl50 IC1/250IC0246810* *[lactate] mmol/LFigure 4. Effect of 3-BP in migration capacity in different cancer cell lines. Significantlydifferent between groups: *P < 0.1;**P < 0.01; ***P < 0.001; ****P < 0.0001; ns: nostatistically significant compared to control.Colorectal cell linesGlioblastoma cell linesU373MG02040608012h 24h***********% Cell migrationU251MG020406080Control1/2 IC50IC5012h 24h**********% Cell migrationCaco-2010203012h 24hnsns****% Cell migrationHT-290102030 Control1/2 IC50IC5012h 24hns*******% Cell migrationFigure 5. Expression profile of MCT1 and MCT4 in different cancer cell lines.Glioblastoma cell lines Colorectal cell lines020406080100120MCT1MCT4U373MG U87MG U251MG% MCTs expression020406080100120MCT1MCT4HCT-15 Caco-2 HT-29%MCTs expressionU373MGU87MGU251MGHCT-15HT-29Caco-2Tubulin MCT1 MCT4Glioblastoma Cell LinesTubulin MCT1 MCT4Colorectal Cancer Cell Lines

3-Bromopyruvate induces cytotoxicity, inhibits glycolysis and decreases migratory capacity in glioblastomas and colorectal cancer cell lines

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3-Bromopyruvate induces cytotoxicity, inhibits glycolysis and decreases migratory capacity in glioblastomas and colorectal cancer cell lines

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