Glutamine Starvation Affects Cell Cycle, Oxidative Homeostasis and Metabolism in Colorectal Cancer Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Growth Curves
2.3. Colony Forming Assay
2.4. Fluorescence-Activated Cell Sorting (FACS) Analysis
2.5. Sample Preparation for Metabolomics Analysis
2.6. Gas Chromatography-Mass Spectrometry Analysis
2.7. Glucose Uptake Assay
2.8. GLUT1 Protein Expression by Immunofluorescence
2.9. 2-Deoxyglucose Treatment and MTT Viability Test
2.10. Determination of Intracellular Aminothyol Levels
2.11. Statistical Analysis
3. Results
3.1. Glutamine Starvation Alters Growth Rate in Colorectal Cancer Cells
3.2. Glutamine Starvation Reduces Proliferative Capacity in Colorectal Cancer Cells
3.3. Glutamine Starvation Triggers Cell Death and G0/G1 Cell Cycle Arrest
3.4. Metabolomic Alterations under Glutamine Deprivation
3.5. Glutamine Deprivation Altered Glucose Uptake and GLUT1 Expression
3.6. The Combined Treatment with Glutamine Deprivation and 2-Deoxy-D-Glucose Affected CRC Survival more than Glutamine Deprivation
3.7. Glutamine-Deprivation-Induced Reduction in Antioxidant Defenses in CRC Cells
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Cell line | Caco-2 | HCT116 | HT29 | SW480 | References |
---|---|---|---|---|---|
APC | c.4099C>T | Wild type | c.2557G>T | c.4012C>T | [27,28] |
BRAF | Wild type | Wild type | c.1799T>A | Wild type | ATCC [27,29] |
CTNNB1 | c.734G>C | c.133_ 135delTCT | Wild type | Wild type | [27,30] |
KRAS | Wild type | c.38G>A | Wild type | c.35G>T | [27,28] |
PIK3CA | Wild type | c.3140A>G | c.1345C>A | Wild type | [27] |
SMAD4 | c.1051G>C | Wild type | c.931C>T | Wild type | [27,28] |
TP53 | c.610G>T | Wild type | c.818G>A | c.818G>A | [27,31] |
Histotype | Colorectal carcinoma | Colorectal carcinoma | Colorectal adenocarcinoma | Colorectal adenocarcinoma | [29] |
Duke’s stage | / | D | C | B | [29] |
Cell Lines | Cell Viability in -Gln Condition (% of Controls) | ±SD |
---|---|---|
Caco-2 | 55.02 | 5.04 |
HCT116 | 62.01 | 3.25 |
HT29 | 48.83 | 7.07 |
SW480 | 49.34 | 0.57 |
Cell Lines | Live | Apoptotic | Necrotic | ||||
---|---|---|---|---|---|---|---|
% of Cells ±SD | p-Value (vs. Cont) | % of Cells ±SD | p-Value (vs. Cont) | % of Cells ±SD | p-Value (vs. Cont) | ||
Caco-2 | Cont | 70.93 ± 2.39 | 7.05 ± 2.48 | 22.02 ± 0.86 | |||
-Gln | 41.36 ± 1.27 | <0.0001 | 2.20 ± 0.86 | 0.0025 | 56.44 ± 1.67 | <0.0001 | |
HCT116 | Cont | 80.24 ± 1.15 | 1.84 ± 0.33 | 17.93 ± 1.28 | |||
-Gln | 81.73 ± 1.46 | ns | 1.24 ± 0.21 | ns | 17.02 ± 1.48 | ns | |
HT29 | Cont | 88.92 ± 0.49 | 2.32 ± 0.90 | 8.77 ± 0.40 | |||
-Gln | 82.36 ± 0.56 | <0.0001 | 4.16 ± 0.88 | 0.0395 | 16.24 ± 0.63 | <0.0001 | |
SW480 | Cont | 78.43 ± 4.00 | 2.66 ± 1.80 | 18.91 ± 2.22 | |||
-Gln | 82.48 ± 2.60 | ns | 8.33 ± 2.83 | 0.0311 | 9.19 ± 0.23 | 0.0030 |
Cell Line | G0/G1 Phase | S Phase | G2/M Phase | PI | |||||
---|---|---|---|---|---|---|---|---|---|
% of Cells ±SD | p-Value (vs. Cont) | % of Cells ±SD | p-Value (vs. Cont) | % of Cells ±SD | p-Value (vs. Cont) | % of Cells ±SD | p-Value (vs. Cont) | ||
Caco-2 | Cont | 64.71 ± 1.57 | 15.85 ± 0.84 | 14,76 ± 0.20 | 30.61 ± 1.04 | ||||
-Gln | 76.12 ± 2.18 | <0.0001 | 13.06 ± 0.45 | ns | 8.41 ± 1.25 | 0.0017 | 21.46 ± 1.59 | 0.0059 | |
HCT116 | Cont | 73.77 ± 2.71 | 5.79 ± 1.03 | 13.98 ± 1.01 | 19.77 ± 2.03 | ||||
-Gln | 83.28 ± 1.75 | <0.0001 | 4.28 ± 0.79 | ns | 7.81 ± 0.79 | 0.0009 | 12.09 ± 0.31 | 0.0029 | |
HT29 | Cont | 67.10 ± 3.02 | 12.16 ± 1.35 | 6.51 ± 0.62 | 18.66 ± 1.95 | ||||
-Gln | 75.45 ± 1.64 | 0.0005 | 7.22 ± 2.42 | 0.0243 | 6.31 ± 1.40 | ns | 13.54 ± 1.67 | 0.0258 | |
SW480 | Cont | 66.21 ± 4.11 | 15.49 ± 1.87 | 10.12 ± 2.08 | 25.61 ± 3.62 | ||||
-Gln | 78.61 ± 0.86 | <0.0001 | 13.50 ± 0.14 | ns | 4.26 ± 0.65 | 0.0143 | 17.77 ± 0.79 | 0.0215 |
Metabolites | Caco-2 | HCT116 | HT29 | SW480 | ||||
---|---|---|---|---|---|---|---|---|
Fold Change -Gln/Cont | p-Value | Fold Change -Gln/Cont | p-Value | Fold Change -Gln/Cont | p-Value | Fold Change -Gln/Cont | p-Value | |
Adenosine monophosphate | 0.60 | 0.0135 | 0.40 | ns | 0.48 | 0.0017 | 0.82 | ns |
Aminomalonc acid | 1.62 | 0.0003 | 4.04 | 0.0012 | - | - | 3.96 | 0.0154 |
Beta-Alanine | 0.36 | 0.0007 | 0.51 | 0.0054 | 0.43 | <0.0001 | 0.51 | 0.0016 |
Beta-Glycerophosphoric acid | 1.02 | ns | 1.23 | ns | - | - | - | - |
Cholesterol | 1.56 | 0.0169 | 0.98 | ns | 0.56 | ns | 0.87 | ns |
Citric acid | 0.36 | <0.0001 | 0.85 | ns | 0.69 | 0.0029 | 0.96 | ns |
Creatinine | 1.67 | 0.0059 | 1.40 | 0.0342 | - | - | - | - |
D-Fructose | 0.80 | 0.0010 | 0.93 | ns | 1.75 | 0.0062 | - | - |
D-Galactose | 1.53 | 0.0004 | 2.57 | 0.0021 | 1.50 | 0.0065 | 3.82 | 0.0042 |
D-Glucose | 1.66 | 0.0011 | 2.47 | 0.0039 | 1.51 | 0.0049 | 4.11 | 0.0030 |
D-Glucose 6-phosphate | 1.42 | ns | 1.31 | ns | 0.92 | ns | 1.42 | 0.0036 |
D-Ribose 5-phosphate | 0.29 | 0.0010 | 0.77 | ns | 0.49 | ns | 1.56 | ns |
Fumaric acid | 0.05 | ns | 0.69 | 0.0022 | 0.14 | <0.0001 | 0.53 | 0.0381 |
Gamma-Aminobutyric acid | 0.19 | 0.0025 | 0.36 | 0.0210 | 1.08 | ns | 0.28 | 0.0199 |
Glycerol 3-phosphate | 1.37 | 0.0256 | 1.01 | ns | 1.58 | <0.0001 | 1.95 | 0.0008 |
Glycine | 3.56 | <0.0001 | 3.87 | <0.0001 | 8.44 | <0.0001 | 3.68 | 0.0002 |
L-Alanine | 0.13 | 0.0070 | 0.46 | ns | 0.20 | 0.0431 | 0.71 | ns |
L-Aspartic acid | 0.27 | 0.0039 | 0.29 | 0.0050 | - | - | 0.16 | 0.0091 |
L-Glutamic acid | 0.13 | 0.0021 | 0.72 | 0.0109 | 0.31 | 0.0012 | 0.26 | 0.0270 |
L-Isoluecine | 1.08 | ns | 2.27 | 0.0068 | - | - | 2.20 | 0.0193 |
L-Lactic acid | 0.67 | 0.0039 | 0.30 | <0.0001 | 0.54 | 0.0001 | 0.92 | ns |
L-Leucine | 0.29 | ns | 2.71 | 0.0011 | - | - | 2.50 | 0.0024 |
L-Phenylalanine | 2.00 | 0.0064 | 4.08 | <0.0001 | - | - | 3.53 | 0.0016 |
L-Proline | 0.97 | ns | 0.41 | 0.0068 | - | - | 0.86 | ns |
L-Sorbose | 0.83 | 0.0114 | 1.06 | ns | 1.93 | 0.0004 | - | - |
L-Threonine | 2.31 | <0.0001 | 5.07 | <0.0001 | 2.63 | 0.0121 | 2.99 | <0.0001 |
L-Tryptophan | 1.34 | ns | 2.14 | <0.0001 | - | - | 1.99 | 0.0030 |
L-Tyrosine | 2.69 | 0.0007 | 3.15 | 0.0006 | 2.42 | ns | 3.16 | 0.0014 |
L-Valine | 1.17 | 0.0242 | 2.92 | 0.0093 | 1.63 | ns | 1.74 | 0.0244 |
Malic acid | 0.13 | <0.0001 | 0.79 | ns | 0.14 | <0.0001 | 0.51 | ns |
Mannitol | 1.99 | 0.0022 | 0.54 | 0.0006 | - | - | 1.60 | 0.0095 |
Mannose 6-phosphate | 0.48 | 0.001 | 1.38 | ns | 0.81 | ns | 1.36 | 0.0180 |
Myo-Inositol | 0.78 | 0.0093 | 0.65 | 0.0010 | 0.63 | 0.0001 | 0.38 | <0.0001 |
Myo-Inositol 1-phosphate | 1.03 | ns | 0.86 | ns | 1.07 | ns | 0.92 | ns |
Niacinamide | - | - | 0.86 | ns | - | - | 0.98 | ns |
Pantothenic acid | 0.92 | ns | 0.90 | ns | 0.92 | ns | 1.42 | 0.0112 |
Pyroglutamic acid | 0.10 | 0.0022 | 0.12 | 0.0333 | 0.13 | <0.0001 | 0.16 | 0.0011 |
Serine | 7.14 | <0.0001 | 7.94 | <0.0001 | 13.71 | 0.03052 | 2.46 | 0.0003 |
Taurine | 2.21 | ns | 0.90 | ns | - | - | 1.10 | ns |
Uridine 5-monophosphate | 1.09 | ns | 0.42 | 0.0001 | 0.40 | ns | - | - |
5′Methylthioadenosine | 0.32 | 0.0498 | 1.07 | ns | 0.94 | ns | 0.88 | ns |
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Spada, M.; Piras, C.; Diana, G.; Leoni, V.P.; Frau, D.V.; Serreli, G.; Simbula, G.; Loi, R.; Noto, A.; Murgia, F.; et al. Glutamine Starvation Affects Cell Cycle, Oxidative Homeostasis and Metabolism in Colorectal Cancer Cells. Antioxidants 2023, 12, 683. https://doi.org/10.3390/antiox12030683
Spada M, Piras C, Diana G, Leoni VP, Frau DV, Serreli G, Simbula G, Loi R, Noto A, Murgia F, et al. Glutamine Starvation Affects Cell Cycle, Oxidative Homeostasis and Metabolism in Colorectal Cancer Cells. Antioxidants. 2023; 12(3):683. https://doi.org/10.3390/antiox12030683
Chicago/Turabian StyleSpada, Martina, Cristina Piras, Giulia Diana, Vera Piera Leoni, Daniela Virginia Frau, Gabriele Serreli, Gabriella Simbula, Roberto Loi, Antonio Noto, Federica Murgia, and et al. 2023. "Glutamine Starvation Affects Cell Cycle, Oxidative Homeostasis and Metabolism in Colorectal Cancer Cells" Antioxidants 12, no. 3: 683. https://doi.org/10.3390/antiox12030683