Colorectal cancer (CRC) is a malignant epithelial tumor that develops in the colon, rectum, or vermiform appendix and may spread to the liver. It is the third most common type of cancer in Poland, and the most widespread gastrointestinal tract cancer in the world [1]. Due to the aggressiveness of CRC and the lack of targeted therapies, it seems reasonable to investigate the key metabolic pathways involved in cancer development and progression.
Metabolic adaptation to rapid environmental changes is essential for cancer cell survival and proliferation [2]. The tumor milieu is characterized by a high variability of oxygen and nutrient supply, resulting from rapid proliferation and dynamic processes such as necrosis and angiogenesis [3]. Hypoxia leads to an increase of hypoxia-inducible factor 1 (HIF-1) transcription factor activity, which stimulates the transcription of glucose transporters and glycolytic enzyme genes, resulting in elevated lactate synthesis with simultaneous inhibition of mitochondrial pyruvate metabolism [4, 5, 6, 7]. Under normoxic conditions, however, lactate is produced not only by glycolysis but also by a glutaminolysis process. It is known that both processes are stimulated by the c-Myc transcription factor [8, 9, 10]. Most cancer cells produce large amounts of lactate even under sufficient availability of oxygen. This phenomenon is called the Warburg effect [11, 12].There may be differences in mitochondrial metabolism between the cells of primary and metastatic cancer, as metastases exhibit better environmental adaptation and more dynamic growth. In order to increase the rate of proliferation at hypoxia, cancer cell metabolism is adapted to facilitate the uptake of mainly glucose and glutamine for energy production and macromolecule synthesis needed for cell growth. It is known that cancer and normal rapidly proliferating cells exhibit a high glycolytic rate, however, positron emission tomography (PET) using fluorine-labeled deoxyglucose (18FDG) as a tumor marker showed low effects of targeting glycolysis in cancer treatment [13, 14, 15, 16]. The latest studies indicated that rapidly proliferating cells (neoplastic and normal) depend also on glutaminolysis, which is another metabolic pathway providing energy and substrates for nucleic acids, proteins, and lipids synthesis [17, 18].
It is known that under routine culture cell conditions, i.e. at 21% atmospheric normoxia, glutaminolysis is conducted by both mitochondrial enzymes such as - glutaminase (GLS; the first enzyme in glutaminolysis), aspartate aminotransferase 2 (AST2) and several Krebs cycle enzymes and cytosolic enzymes such as ATP-citrate lyase (ACL) and aspartate aminotransferase 1 (AST1).
Under these conditions, mitochondrial glutaminase converts L-glutamine into L- glutamate, that is transaminated by AST2 to α-ketoglutarate (α-KG) - one of the Krebs cycle intermediates. Next, α-KG is converted to citrate that can be metabolized by the Krebs cycle enzymes or transported into cytosol, where it is splitted by ACL into acetyl-CoA and oxaloacetate, that can be transaminated by AST1 to L-aspartate (L-Asp) (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.
Recently, two alternative pathways of glutaminolysis (cytoplasmic and mitochondrial) have been proposed in cancer cells with reduced oxygen availability [20, 21].During alternative cytoplasmic glutaminolysis, L-glutamate formed by mitochondrial GLS1 can be transported to cytosol by GC (glutamate transporter) and is transaminated by aspartate aminotransferase 1 to α-ketoglutarate that in reductive carboxylation is converted to citrate by cytoplasmic isoforms of isocitrate dehydrogenase and cis-aconitase. In the alternative mitochondrial glutaminolysis the beginning of the pathway is the same as under normal oxygen availability (L-Gln→ L-Glu→ α-KG), however because of hypoxia, α-ketoglutarate is not effectively involved in the Krebs cycle, but it is rather reduced and carboxylated to citrate by mitochondrial isoforms of isocitrate dehydrogenase and cisaconitase (figure 2) [20, 21, 22].
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.
Thus, the reductive carboxylation of α-ketoglutarate to citrate, is a common feature of the two alternative pathways.
The above mentioned alternative glutaminolysis pathways were based only on the study of labeled metabolites [23]. In literature there is no data about the expression or activity of enzymes in volved in alternative glutaminolysis and expression of the mitochondrial L-glutamate transporter. Besides, there is no experimental study concerning its role in the L-glutamate cytoplasmic and mitochondrial balance.
The most
In the current work, for the first time, we study the gene expression of enzymatic and transporter proteins involved in the alternative glutaminolysis pathways at tissue normoxia (10% oxygen) in comparison to tissue hypoxia and atmospheric normoxia.
Bovine serum albumin (BSA), trypan blue (TB) and Thiazolyl Blue Tetrazolium Bromide (MTT) were purchased from Sigma-Aldrich (St Louis, MO, USA). PBS, fetal bovine serum (FBS), 0.25% trypsin-0.02% EDTA, penicillin/streptomycin were supplied by Gibco BRL (San Francisco, CA, USA). MEM with Earle’s balanced salt solution (EBSS) (with 5.55 mM glucose and 2.0 mM L-Gln), TaqMan gene expression assays and 1 M HEPES were obtained from ThermoSci (Waltham, MA, USA) and qPCR/real time PCR Master MIX DLP4 by GeneON (Germany). Trizol Reagent, RNaseOut, 5x concentrated buffer (first-stand buffer), reverse transcriptase (M-MLV), dNTP (deoxynucleotide triphosphates) were supplied from Invitrogen (USA). All cell culture plastics were purchased from BD Falcon Biosciences.
Experiments were performed on human cell lines SW480 (primary colon cancer) and SW620 (metastatic lymph node of the same patient) purchased from the American Type Culture Collection (ATCC). Both cells lines were cultured until 8090% confluence in MEM (Minimum Essential Medium) with L-Gln, and without L-Ser, Gly, L-Asp, L-Glu, L-Ala, pyruvate; (ThermoSci, USA) supplemented with 10% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 μg/mL), and HEPES (20 mM), in 37°C/5% CO2 humidified incubator. Next, cells were harvested by treatment with 0.25% trypsin–0.02% EDTA in phosphate buffered saline solution and used for experiments.
To determine the influence of oxygen, cells were seeded in 6-well plates (2x105 cells) for TB assay and qPCR or in 96-well plates (1x104) for the MTT method. The cells were cultured at various oxygen concentrations (1% - chronic hypoxia, 10% - tissue normoxia, 21% - atmospheric normoxia) in a Hypoxia Chamber with oxygen controller (Coy Laboratory Products INC, USA).
Trypan Blue exclusion (TB) assay
After a 120h incubation, the cells were washed twice with PBS, trypsinized and harvested. The total cell count and viability were determined by trypan blue exclusion dye assay using automated cell counter (Countess Invitrogen, Waltham, MA). Each experiment was performed six times.
To estimate the number of viable cells, MTT assay was performed with eight replicates for each cell lines. After a 72h incubation, MTT solution (5 mg/mL) was added to each well, and incubated for 4 h in 37°C/5% CO2. Next, medium was aspirated and a mixture of DMSO with isopropanol (1:1) was added. The number of cells was evaluated by measuring the absorbance in a UVM 340 reader (ASYS Hitech GmbH, Austria) at a wavelength of 570 nm.
Total RNA was extracted from SW480 and SW620 cells using Trizol Reagent (Invitrogen, USA) according to the manufacturer’s instructions (Molecular Research Center, Inc). Cells were harvested in 1 mL of TRIzol reagent. To each sample tube 0.2 mL of chloroform was added. RNA left in water phase was precipitated by is opropanol and freezing in 70°C by 24 h, then washed with 75% ethanol, and dissolved in H2O (DEPC). Next, RNA concentration was determined at 260 nm, and the purity assessed from the absorbance ratio 260/280 nm using Nanodrop spectrophotometer (Nanodrop Technologies).
2 μg of total RNA from each sample was reverse transcribed to a single-stranded cDNA according to the manufacturer’s instructions (Invitrogen, USA) in a total volume of 20 μL. The synthesized cDNA was immediately used in real time PCR or stored at -20°C for later experiments.
Expression of genes (
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 |
Aspartate aminotransferase 2 | Hs00751057_s1 | |
GLS1 | Glutaminase 1 | Hs00248163_m1 |
Pyruvate carboxylase | Hs00559398_m1 | |
ATP- citrate lyase | Hs00982738_m1 | |
Glutamate transporter 1 | Hs01017349_m1 | |
Glutamate transporter 2 | Hs00368705_m1 | |
Hypoxia inducible factor 1 | Hs00153153_m1 | |
Glucose transporter 1 | Hs00892681_m1 |
The statistical analysis was performed using Statistica 13.0 (StatSoft, Inc, Oklahoma, USA). Statistically significant differences in the expression of the examined genes were performed using Student’s t-test. The analysis of ANOVA variance was used to evaluate the dependence of the mRNA level of the tested genes and the oxygen concentration. Correlation between the expression of individual genes and SW480, SW620 cells proliferation was determined using the Pearson test. Results calculated from 6 separate experiments, were expressed as means ± SD, and considered statistically significant at P<0.05.
The cell viability for both studied cell lines in all oxygen concentrations was similar and counted from 92%
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.
The number of viable SW480 and SW620 cells did not correlate with increasing oxygen concentrations and was higher in hypoxia and atmospheric normoxia in comparison to tissue normoxia.
According to the TB method, the number of viable SW480 and SW620 cells was comparable at 1% and 10%, whereas at 21% oxygen the number of SW620 cells was significantly higher than SW480 cells (P<0.001).
At hypoxia, the number of viable cells for both studied cell lines was over 3-fold higher than at tissue normoxia, whereas at 21% oxygen was 2.5-fold higher for SW480 cells and 5-fold higher for SW620 cells in comparison to tissue normoxia (P<0.001) (fig. 3 A).
The results of quantitative RT-PCR analysis showed that mRNA expression of
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
The level of
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
In SW480 cells the level of
The level of
Effect of oxygen on pyruvate carboxylase (
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
The
At all studied oxygen concentrations the profile of
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
The
ANOVA analysis showed that in SW480 cells the expression of
Variable oxygen availability resulting from the disproportions in the dynamics of cancer cell proliferation and endothelial cells responsible for angiogenesis may result in local hypoxia in the insufficiently vascularized area of the tissue. Therefore, cancer cells have developed mechanisms that allow them to adapt to differing oxygen and nutrient availability [24, 25].
Among such adaptations to hypoxia is the activation of HIF-1α factor, that leads to the expression of genes involved in things such as energy metabolism, glucose transport and cell proliferation (GLUT1, vascular endothelial growth factor (VEGF), and others) [26, 27]. It is known that in various tumors
In this study, we found that the number of both SW480 and SW620 cells was higher in 1% hypoxia than in 10% oxygen (physiological conditions) and it indicates that oxygen supply is a limiting factor in the growth of colon cancer cells
The cancerous process is considered to be a metabolic disease. Changes in cellular metabolism are induced by various levels of oxygen availability, which influence genes’ expression of proteins involved in glycolysis and glutaminolysis pathways [34].
According to the literature, the activity and expression of glutaminase (the first glutaminolysis enzyme) is increased in liver, prostate, or breast cancer [35, 36, 37, 38]. We have shown previously that
It is known that
The next step of glutaminolysis is conversion of L-glutamate to α-ketoglutarate, catalyzed by mitochondrial aspartate aminotransferase (AST2; transamination) or glutamate dehydrogenase (GLUD1; oxidative deamination) [46, 47].
We found that
According to literature data, α-KG produced with the participation of AST2, depending on the prevailing aerobic conditions, is converted to citrate in the classical or alternative glutaminolysis pathway [50].
In the classical glutaminolysis under normoxia conditions, α-KG is included in the Krebs cycle, where it is converted to oxaloacetate, which after condensation with acetyl-CoA forms citrate. The citrate is transported to the cytoplasm, where it is split into acetyl-CoA and oxaloacetate in an ACL-catalyzed reaction.
In the current paper,
According to the literature, the expression of the glutamate transporter (GC) has never been studied before under hypoxia and tissue normoxia conditions.
The presence of two mitochondrial isoforms of the GC transporter (GC1, GC2) in normal tissues was confirmed in many studies, but there is no data about their existence in colon cancer cells [36, 56, 57, 58]. We showed for the first time the presence of both GC isoforms in human colorectal cancer cells and the effect of oxygen on their expression. The expression of the
We also confirmed our previous findings that studies routinely conducted under non-physiological atmospheric normoxia may not reflect the real situation
In summary, the above-mentioned findings suggest a difference in metabolic adaptation to oxygen availability
Higher expression of glutaminolytic genes at 1% oxygen in SW480 cells indicates adaptation to a deficiency of nutrients in the tumor microenvironment at chronic hypoxia, whereas higher expression in SW620 cells at 10% may result from increased oxygen-dependent mitochondrial biogenesis. It is known that metastatic SW620 cancer cells exhibit higher mitochondrial metabolism (c-Myc, α-PGC 1) and show features similar to cancer stem cells (CSC) [59].
To the best of our knowledge, the present study comparing the influence of physiological oxygen levels on glutaminolytic gene expression in cancer cells is pioneering research. Since the oxygen level in the tumor milieu never exceeds 10%, we suggest carrying out future studies at tissue normoxia rather than at atmospheric normoxia. In the present work we focused on expression of glutaminolysis genes under differing physiological oxygen levels. Our results give support for further comprehensive studies involving enzymatic protein expression and factorsi.e HIF-1α, c-Myc affecting their activity.