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.
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.
Table 1
The types of primers used
| Gene symbol | Gene name | Assay ID |
|---|---|---|
| Housekeeping gene | ||
| 18S rRNA | 18S ribosomal RNA | Hs99999901_s1 |
| MT-ATP6 | Mitochondrially encoded ATP synthase 6 | Hs02596862_g1 |
| Studied genes | ||
| AST1 | Aspartate aminotransferase 1 | Hs00157798_m1 |
| AST2 | Aspartate aminotransferase 2 | Hs00751057_s1 |
| GLS1 | Glutaminase 1 | Hs00248163_m1 |
| PC | Pyruvate carboxylase | Hs00559398_m1 |
| ACL | ATP- citrate lyase | Hs00982738_m1 |
| GC1 | Glutamate transporter 1 | Hs01017349_m1 |
| GC2 | Glutamate transporter 2 | Hs00368705_m1 |
| HIF-1α | Hypoxia inducible factor 1 | Hs00153153_m1 |
| GLUT 1 | Glucose transporter 1 | Hs00892681_m1 |
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.
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.
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.
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
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.