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Proliferative Glioblastoma Cancer Cells Exhibit Persisting Temporal Control of Metabolism and Display Differential Temporal Drug Susceptibility in Chemotherapy

Molecular Neurobiology volume 56, pages 1276–1292 (2019)Cite this article

Abstract

Even in immortalized cell lines, circadian clocks regulate physiological processes in a time-dependent manner, driving transcriptional and metabolic rhythms, the latter being able to persist without transcription. Circadian rhythm disruptions in modern life (shiftwork, jetlag, etc.) may lead to higher cancer risk. Here, we investigated whether the human glioblastoma T98G cells maintained quiescent or under proliferation keep a functional clock and whether cells display differential time responses to bortezomib chemotherapy. In arrested cultures, mRNAs for clock (Per1, Rev-erbα) and glycerophospholipid (GPL)-synthesizing enzyme genes, 32P-GPL labeling, and enzyme activities exhibited circadian rhythmicity; oscillations were also found in the redox state/peroxiredoxin oxidation. In proliferating cells, rhythms of gene expression were lost or their periodicity shortened whereas the redox and GPL metabolisms continued to fluctuate with a similar periodicity as under arrest. Cell viability significantly changed over time after bortezomib treatment; however, this rhythmicity and the redox cycles were altered after Bmal1 knock-down, indicating cross-talk between the transcriptional and the metabolic oscillators. An intrinsic metabolic clock continues to function in proliferating cells, controlling diverse metabolisms and highlighting differential states of tumor suitability for more efficient, time-dependent chemotherapy when the redox state is high and GPL metabolism low.

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Abbreviations

Chokα:

Choline kinase α

CTP:

Phosphoethanolamine cytidylyltransferase 2 (Pcyt-2)

CCG:

Clock-control gene

GPL:

Glycerophospholipid

DAG:

Diacylglycerol

DEX:

Dexamethasone

LPLAT:

Lysophospholipid acyl transferase

PC:

Phosphatidylcholine and

PE:

Phosphatidylethanolamine

PAP:

Phosphatidate phosphohydrolase

ROS:

Reactive oxygen species

ICC:

Immunocytochemistry

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Acknowledgments

The authors are grateful to Mrs. Susana Deza and Gabriela Schanner for their excellent technical support and to Dr. Laura Allende for the gift of the PX459-plasmid.

Funding

This work has been supported by Agencia Nacional de Promoción Científica y Técnica (FONCyT, PICT 2010 No. 647 and PICT 2013 No. 021), Consejo Nacional de Investigaciones Científicas y Tecnológicas de la República Argentina (CONICET) (PIP 2014), Secretaría de Ciencia y Tecnología de la Universidad Nacional de Córdoba (SeCyT-UNC), and John Simon Guggenheim Memorial Foundation for Latinoamérica and Caribe (2009) in Natural Sciences.

Author information

Author notes

  1. Lucas G. Sosa Alderete

    Present address: Department of Molecular Biology, UNRC, CONICET, Río Cuarto, Argentina

  2. Paula M. Wagner and Lucas G. Sosa Alderete contributed equally to this work.

Authors and Affiliations

  1. CIQUIBIC-CONICET, Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre s/n, Ciudad Universitaria, 5000, Córdoba, Argentina

    Paula M. Wagner, Lucas G. Sosa Alderete & Mario E. Guido

  2. Departamento de Química Biológica “Ranwel Caputto”, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, 5000, Córdoba, Argentina

    Paula M. Wagner, Lucas G. Sosa Alderete & Mario E. Guido

  3. Facultad de Ciencias Exactas Físicas y Naturales, Universidad Nacional de Córdoba, Córdoba, Argentina

    Lucas D. Gorné

  4. Consejo Nacional de Investigaciones Científicas y Técnicas, CONICET, Instituto Multidisciplinario de Biología Vegetal (IMBiV), Córdoba, Argentina

    Lucas D. Gorné

  5. Departamento de Biología, Bioquímica y Farmacia, Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB, UNS-CONICET), Universidad Nacional del Sur, 8000, Bahía Blanca, Argentina

    Virginia Gaveglio, Gabriela Salvador & Susana Pasquaré

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Contributions

LSA, PMW, MEG designed research; LSA, PMW, VG, GAS, SJP performed research; MEG, SJP, GAS contribute new reagents/analytic tools; LSA, PMW, LDG, GAS, SJP, MEG analyzed data; MEG wrote; All authors read and approved the final manuscript.

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Correspondence to Mario E. Guido.

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Supplementary Fig.1

Flow cytometry of cultures of T98G cells. Cells kept in serum-free DMEM (arrest, a, left panel) or in the presence of serum (proliferation, b, right panel) were synchronized with a shock of 100 nM DEX for 20 min and collected at different times from 0 to 48 h. Percentages of T98G cells in G0/G1 (black) and S + G2/M (gray) cell cycle phases are plotted. Cells were collected at 4–8-h intervals after DEX synchronization and analyzed by flow cytometry with propidium iodide staining as described in “Materials and Methods.” Cells kept in the presence of serum (proliferation, c, bottom panel) were transfected with PX459-Bmal1 plasmid, synchronized with a shock of 100 nM DEX for 20 min and collected at 24 h. Transfected cells exhibited a higher percentage in G0/G1 phases (77%) than in controls (61%). See text for further details. b Temporal variations in clock genes Per1and Rev-erbα mRNAs in T98G cell cultures. Cells were kept in serum-free DMEM (arrest, a, left panels) or in the presence of serum (proliferation, b, right panels) and synchronized with DEX (100 nM) for 20 min and collected at different times. Per1 (a, b) and Rev-erbα (c, d) mRNA levels were assessed by RT-qPCR with RNA extracted from cells collected at different times during 48 h after DEX treatment at time 0; values were normalized according to the expression of the housekeeping gene TBP. A significant adjustment to a periodic function was obtained, illustrated by means of dashed lines (p < 0.05). The results are mean ± SEM (triplicate samples from three independent experiments, n = 3–5/time). See text and Table 1 for further details on statistical analysis and period determination. (PNG 302 kb)

High-resolution image (TIF 13083 kb)

Supplemenatry Fig. 2

Relative levels of GLP endogenous content in proliferative T98G cells. Endogenous levels of PC (top) and PE content (middle) and the PC/PE ratio (bottom panel) in DEX synchronized T98G cells kept under proliferation in the presence of serum. Cells were synchronized with dexamethasone (DEX, 100 nM) for 20 min and collected at different times from 0 to 36 h. Lipids extracted and separated by TLC as described in “Materials and Methods.” The COSINOR revealed a significant time effect of for PE content and the PC/PE ratio with a period of 24 h for both parameters (p ≤ 0.03). The results are mean ± SEM (triplicate samples from three independent experiments, n = 3–6/time). See text and Table 1 for further details on statistical analysis and period determination. (PNG 240 kb)

High-resolution image (TIF 16501 kb)

Supplementary Fig. 3

Rhythms of LPLAT enzymatic activity in T98G cell cultures. Cells kept in serum-free DMEM (arrest, a, left panels) or in the presence of serum (proliferation, b, right panels) and synchronized with DEX (100 nM) for 20 min and collected at different times. LPLAT activity was determined in cell homogenates and measured as the incorporation of [3H]-oleate into lysophosphatidate (LPA) (a, d), lysophosphatidylcholine (LPC) (b, e), and lysophosphatidylethanolamine (LPE) (c, f). LPLAT activity exhibited significant temporal changes only for LPE (p ≤ 0.02 by COSINOR). Results are the mean ± SEM of two independent experiments (n = 4/group). See text for further details and Table 1 for the statistical analysis. (PNG 436 kb)

High-resolution image (TIF 6983 kb)

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Wagner, P.M., Sosa Alderete, L.G., Gorné, L.D. et al. Proliferative Glioblastoma Cancer Cells Exhibit Persisting Temporal Control of Metabolism and Display Differential Temporal Drug Susceptibility in Chemotherapy. Mol Neurobiol 56, 1276–1292 (2019). https://doi.org/10.1007/s12035-018-1152-3

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Keywords

  • Circadian rhythm
  • Tumor cell
  • Glioblastoma
  • Clock gene
  • Glycerophospholipid metabolism
  • Redox state