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Mitochondrial energy metabolism and signalling in human glioblastoma cell lines with different PTEN gene status

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Abstract

Glioblastomas epidemiology and aggressiveness demand for a well characterization of biochemical mechanisms of the cells. The discovery of oxidative tumours related to chemoresistance is changing the prevalent view of dysfunctional mitochondria in cancer cells. Thus, glioblastomas metabolism is now an area of intense research, wherein was documented a high heterogeneity in energy metabolism and in particular in mitochondrial OxPhos. We report results gained by investigating mitochondrial OxPhos and bioenergetics, in a model of three human glioblastoma cell lines characterized by a different PTEN gene status. Functional data are analysed in relation to the expression levels of some main transcription factors and signalling proteins, which can be involved in the regulation of mitochondrial biogenesis and activity. Collectively, our observations indicate for the three cell lines a similar bioenergetic phenotype maintaining a certain degree of mitochondrial oxidative activity, with some difference for PTEN-wild type SF767 cells respect to PTEN-deleted A172 and U87MG characterized by a loss-of-function point mutation of PTEN. SF767 has lower ATP content and higher ADP/ATP ratio, higher AMPK activating-phosphorylation evoking energy impairment, higher OxPhos complexes and PGC1α-Sirt3-p53 protein abundance, in line with a higher respiration. Finally, SF767 shows a similar mitochondrial energy supply, but higher non-phosphorylating respiration linked to dissipation of protonmotive force. Intriguingly, it is now widely accepted that a regulated mitochondrial proton leak attenuate ROS generation and in tumours may be at the base of pro-survival advantage and chemoresistance.

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Abbreviations

ADP:

Adenosine 5′-diphosphate

AMPK:

AMP-activated protein kinase

ATP:

Adenosine 5′-trphosphate

CS:

Citrate synthase

2-DG:

2-deoxyglucose

DMEM:

Dulbecco’s modified Eagle’s medium

ECAR:

Extracellular acidification rate

EDTA:

Ethylenediaminetetraacetic acid

ETS:

Electron transport system

FCCP:

Carbonyl cyanide p-trifluoro-methoxyphenyl hydrazine

HSP60:

Heat shock protein 60

LKB1:

Liver kinase B1

MDM2:

Mouse double minute 2

mTOR:

Mammalian target of rapamycin

mTORC1:

Mammalian target of rapamycin complex 1

OCR:

Oxygen consumption rate

OxPhos:

Oxidative phosphorylation

PBS:

Phosphate–buffered saline

PGC1α:

Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha

PS:

Phosphorylating system

PTEN:

Phosphatase and tensin homolog

ROS:

Reactive oxygen species

SDS-PAGE:

Sodium dodecyl sulphate - polyacrylamide gel electrophoresis

Sirt3:

Silent mating-type information regulation 2 homolog sirtuin

SCO2:

Cytochrome c oxidase assembly protein

TCA:

Tricarboxylic acid

TFAM:

Mitochondrial transcription factor A

TIGAR:

TP53-induced glycolysis and apoptosis regulator

TMPD:

Tetramethyl-p-phenylenediamine dihydrochloride

TOM:

Translocase of outer mitochondrial membrane

WHO:

World health organization

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Acknowledgements

The authors gratefully acknowledge doctor Pier Giorgio Mastroberardino, Department of Genetics Erasmus Medical Center Rotterdam, for the useful discussion of data, as well as prof. Claudio Brancolini and prof. Gianluca Tell, Department of Medicine, University of Udine for glioblastoma cell lines, and prof. Giovanna Lippe, Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine, for TOM20 antibody. Additionally, Silvia Lolini and Luca Bazzichetto, Department of Medicine, University of Udine, for technical support.

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Authors and Affiliations

  1. Department of Medicine and MATI Centre of Excellence, University of Udine, p.le Kolbe 4, 33100, Udine, Italy

    Marina Comelli, Ivan Pretis, Alessia Buso & Irene Mavelli

  2. INBB Istituto Nazionale Biostrutture e Biosistemi, Viale Medaglie d’Oro, 00136, Rome, Italy

    Irene Mavelli

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  1. Marina Comelli
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  2. Ivan Pretis
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Contributions

I.M. and M.C. designed research and interpreted results of experiments; M.C. and I.P. maintained cell lines and performed experiments of mitochondria imaging, cell respiration and bioenergetic phenotype; M.C. analysed data, prepared figures and drafted manuscript; I.P. and A.B. performed quantitative WB analyses; A.B. organised “Supplementary Content”; I.M. edited and revised manuscript.

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Correspondence to Marina Comelli.

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Comelli, M., Pretis, I., Buso, A. et al. Mitochondrial energy metabolism and signalling in human glioblastoma cell lines with different PTEN gene status. J Bioenerg Biomembr 50, 33–52 (2018). https://doi.org/10.1007/s10863-017-9737-5

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  • DOI: https://doi.org/10.1007/s10863-017-9737-5

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