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Influence of oxygen availability on expression of glutaminolysis genes in human colon cancer cells

Postępy Higieny i Medycyny Doświadczalnej
Volume 75 (2021): Issue 1 (January 2021)
By:
Open Access
|Dec 2021

Figures & Tables

Figure 1

The course of classical glutaminolysis in cancer cells under atmospheric normoxia acc. Dimitros A. [19], independent; GLS; glutaminase, AST2; aspartate aminotransferase 2, ACL; ATP-citrate lyase, AST1; aspartate aminotransferase 1. Black arrows - classic glutaminolysis.
The course of classical glutaminolysis in cancer cells under atmospheric normoxia acc. Dimitros A. [19], independent; GLS; glutaminase, AST2; aspartate aminotransferase 2, ACL; ATP-citrate lyase, AST1; aspartate aminotransferase 1. Black arrows - classic glutaminolysis.

Figure 2

The course of alternative glutaminolysis pathways under hypoxic conditions in cancer cells acc. to Grabon W. et al. [22] GLS; glutaminase, AST2; aspartate aminotransferase 2, ACL; ATP-citrate lyase, AST1; aspartate aminotransferase 1, GC1; glutamate transporter 1, GC2; glutamate transporter 2 Black arrows - classic glutaminolysis; light grey arrows - alternative cytosolic glutaminolysis, black double arrows - alternative mitochondrial glutaminolysis, black arrows - common reactions of all glutaminolysis pathways.
The course of alternative glutaminolysis pathways under hypoxic conditions in cancer cells acc. to Grabon W. et al. [22] GLS; glutaminase, AST2; aspartate aminotransferase 2, ACL; ATP-citrate lyase, AST1; aspartate aminotransferase 1, GC1; glutamate transporter 1, GC2; glutamate transporter 2 Black arrows - classic glutaminolysis; light grey arrows - alternative cytosolic glutaminolysis, black double arrows - alternative mitochondrial glutaminolysis, black arrows - common reactions of all glutaminolysis pathways.

Figure 3

Effect of oxygen on the number of viable colon cancer cells (TB method) and on the viable cells with active mitochondria (MTT method). Trypan blue exclusion dye method (A), MTT test (B). Cells were cultured as indicated in the Material and Methods. Results were calculated from 6 separate experiments and expressed as means ± SD. *P < 0.001 relative to 10% oxygen concentration.
Effect of oxygen on the number of viable colon cancer cells (TB method) and on the viable cells with active mitochondria (MTT method). Trypan blue exclusion dye method (A), MTT test (B). Cells were cultured as indicated in the Material and Methods. Results were calculated from 6 separate experiments and expressed as means ± SD. *P < 0.001 relative to 10% oxygen concentration.

Figure 4

HIF1α and GLUT1 mRNA expression in CRC cell lines under hypoxia and normoxia conditions Expression was carried out by real-time quantitative polymerase chain reaction, as described in the Material and Methods. Results were shown as the ratio of expression of studied genes GLUT1 and HIF1 to the expression of housekeeping genes (ΔCt Method) and expressed as means (± SD). Each assay was performed in duplicates and repeated twice. Results are expressed as the mean value from 3 experiments. Black Bars, GLUT1; Gray Bars, HIF1α.*P<0.001.
HIF1α and GLUT1 mRNA expression in CRC cell lines under hypoxia and normoxia conditions Expression was carried out by real-time quantitative polymerase chain reaction, as described in the Material and Methods. Results were shown as the ratio of expression of studied genes GLUT1 and HIF1 to the expression of housekeeping genes (ΔCt Method) and expressed as means (± SD). Each assay was performed in duplicates and repeated twice. Results are expressed as the mean value from 3 experiments. Black Bars, GLUT1; Gray Bars, HIF1α.*P<0.001.

Figure 5

Effect of oxygen on glutaminolysis gene expression in colon cancer cells Expression was carried out by real-time quantitative polymerase chain reaction, as described in the Material and Methods. Results were shown as the ratio of expression of studied genes GLS1, AST2, AST1, ACL in comparison to the expression of housekeeping genes (ΔCt Method) and expressed as means (± SD) in SW480 (A) and SW620 (B). Each assay was performed in duplicates and repeated twice. Results are expressed as the mean (± SD) from 3 experiments. *P<0.01; **P<0.001; ***P<0.0001.
Effect of oxygen on glutaminolysis gene expression in colon cancer cells Expression was carried out by real-time quantitative polymerase chain reaction, as described in the Material and Methods. Results were shown as the ratio of expression of studied genes GLS1, AST2, AST1, ACL in comparison to the expression of housekeeping genes (ΔCt Method) and expressed as means (± SD) in SW480 (A) and SW620 (B). Each assay was performed in duplicates and repeated twice. Results are expressed as the mean (± SD) from 3 experiments. *P<0.01; **P<0.001; ***P<0.0001.

Figure 6

Effect of oxygen on pyruvate carboxylase (PC) mRNA expression in SW480, SW620 cells. Expression was carried out by real-time quantitative polymerase chain reaction, as described in the Material and Methods. Results were shown as the ratio of expression of studied gene PC to the expression of housekeeping genes (ΔCt Method) and expressed as means (± SD). Each assay was performed in duplicates and repeated twice. Each value is the mean from 3 experiments. * P<0.001; **P<0,0001
Effect of oxygen on pyruvate carboxylase (PC) mRNA expression in SW480, SW620 cells. Expression was carried out by real-time quantitative polymerase chain reaction, as described in the Material and Methods. Results were shown as the ratio of expression of studied gene PC to the expression of housekeeping genes (ΔCt Method) and expressed as means (± SD). Each assay was performed in duplicates and repeated twice. Each value is the mean from 3 experiments. * P<0.001; **P<0,0001

Figure 7

Effect of oxygen on two glutamate transporter isoforms of mRNA expression in colon cancer cells Expression was carried out by real-time quantitative polymerase chain reaction, as described in the Material and Methods. Results were shown as the ratio of expression of studied genes GC1 and GC2 to the expression of housekeeping genes (ΔCt Method) and expressed as means (± SD). Each assay was performed in duplicates and repeated twice. Each value is the mean from 3 experiments.*P<0.002; **P<0.01; ***P<0.05.
Effect of oxygen on two glutamate transporter isoforms of mRNA expression in colon cancer cells Expression was carried out by real-time quantitative polymerase chain reaction, as described in the Material and Methods. Results were shown as the ratio of expression of studied genes GC1 and GC2 to the expression of housekeeping genes (ΔCt Method) and expressed as means (± SD). Each assay was performed in duplicates and repeated twice. Each value is the mean from 3 experiments.*P<0.002; **P<0.01; ***P<0.05.

The types of primers used

Gene symbolGene nameAssay ID
Housekeeping gene
18S rRNA18S ribosomal RNAHs99999901_s1
MT-ATP6Mitochondrially encoded ATP synthase 6Hs02596862_g1
Studied genes
AST1Aspartate aminotransferase 1Hs00157798_m1
AST2Aspartate aminotransferase 2Hs00751057_s1
GLS1Glutaminase 1Hs00248163_m1
PCPyruvate carboxylaseHs00559398_m1
ACLATP- citrate lyaseHs00982738_m1
GC1Glutamate transporter 1Hs01017349_m1
GC2Glutamate transporter 2Hs00368705_m1
HIF-1αHypoxia inducible factor 1Hs00153153_m1
GLUT 1Glucose transporter 1Hs00892681_m1
DOI: https://doi.org/10.2478/ahem-2021-0032 | Journal eISSN: 1732-2693
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Language: English
Page range: 923 - 932
Submitted on: Nov 9, 2020
Accepted on: Jul 29, 2021
Published on: Dec 20, 2021
Published by: Hirszfeld Institute of Immunology and Experimental Therapy
In partnership with: Paradigm Publishing Services
Publication frequency: 1 issue per year
Keywords:
Colorectal cancer,
glutaminolysis,
hypoxia,
tissue normoxia
Related subjects:
Life sciences,
Molecular biology,
Microbiology and virology,
Medicine,
Basic medical science,
Immunology

© 2021 Dagmara Otto-Ślusarczyk, Wojciech Graboń, Magdalena Mielczarek-Puta, Alicja Chrzanowska, Anna Barańczyk-Kuźma, published by Hirszfeld Institute of Immunology and Experimental Therapy
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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