N-Acetylcysteine Promotes Metastatic Spread of Melanoma in Mice
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Culture of Melanoma Cells and Keratinocytes
2.2. Mice and Orthotopic Xenografts
2.3. NAC Administration to Tumor-Bearing Mice
2.4. Determination of NAC, Cys and Cystine Levels in Plasma
2.5. Isolation of Melanoma Cells Using Enzymatic Digestion and a Double Ficoll Gradient
2.6. Experimental Metastases
2.7. Enzyme Assays
2.8. RT-PCR and Detection of mRNA
2.9. Western Blots
2.10. GSH and GSSG Determination
2.11. Knockdown of GCLC, SLC7A10 and SLC7A11genes
2.12. Analysis of Amino Acids
2.13. Rate of Cyst(e)ine Uptake
2.14. Rates of Glucose and Glutamine Utilization
2.15. H2O2 and O2·− Generation Assays
2.16. Rate of Oxygen Utilization
2.17. Statistics
3. Results
3.1. NAC Supplementation Does Not Increase the Growth of Melanoma Xenografts
3.2. NAC Supplementation Promotes Metastatic Spread despite Surgical Removal of Primary Growing Melanoma Xenografts
3.3. shRNA-Induced Downregulation of Cystine Uptake or γ-GCS Activity Decreases Metastatic Melanoma Growth
3.4. Melanoma Metastases Show Higher ROS Generation in Mice Treated with NAC
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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A | ||||||
Amino Acid Concentration (μM) in Plasma | ||||||
Non-Tumor-Bearing Mice | B16-F10-Bearing Mice | |||||
- | +NAC | - | +NAC | |||
Gln | 543 ± 31 | 526 ± 39 | 416 ± 51 * | 385 ± 32 * | ||
Glu | 132 ± 26 | 125 ± 17 | 87 ± 23 * | 106 ± 21 | ||
Gly | 327 ± 43 | 318 ± 40 | 306 ± 38 | 312± 36 | ||
Ser | 169 ± 25 | 159 ± 37 | 152 ± 26 | 149 ± 24 | ||
Met | 64 ± 14 | 59 ± 15 | 37 ± 10 * | 30 ± 6 * | ||
Cyst(e)ine | 16 ± 3 | 184 ± 35 + | 21 ± 5 | 227 ± 42 + | ||
B | ||||||
GGT Activity and Cyst(e)ine Uptake in B16-F10 Cells | ||||||
- | +NAC | |||||
GGT (mU/106 cells) | 35.5 ± 6.4 | 36.0 ± 5.3 | ||||
Cys uptake (nmol/mg protein × min) | 4.3 ± 1.2 | 8.5 ± 2.3 + | ||||
Cystine uptake (nmol/mg protein × min) | 27.2 ± 4.6 | 60.7 ± 9.7 + |
Cys Uptake (nmol/mg prot. × min) | Cystine Uptake (nmol/mg prot. × min) | GSH (nmol/106 cells) | Metastatic Activity (% of the Liver Volume Occupied by Metastases) | |||||
---|---|---|---|---|---|---|---|---|
Treatment | - | +NAC | - | +NAC | - | +NAC | - | +NAC |
Control | 4.0 ± 0.9 | 8.3 ± 1.7 + | 25.4 ± 3.8 | 62.3 ± 8.2 + | 26.2 ± 4.1 | 40.4 ± 5.0 + | 17.4 ± 3.6 | 30.2 ± 4.5 + |
Anti-ASC1-shRNA | 0.7 ± 0.2 * | 0.9 ± 0.3 * | 26.2 ± 3.5 | 60.4 ± 7.7 + | 23.5 ± 3.3 | 38.3 ± 5.7 + | 15.9 ± 2.8 | 27.4 ± 3.6 + |
Anti-xCT-shRNA | 3.9 ± 0.7 | 8.5 ± 2.0 + | 3.5 ± 0.6 * | 6.4 ± 1.2 *,+ | 7.9 ± 1.3 * | 9.6 ± 3.4 * | 9.2 ± 1.5 * | 16.7 ± 2.8 *,+ |
Anti-GCLC-shRNA | 4.2 ± 1.0 | 8.5 ± 1.8 + | 27.9 ± 4.2 | 61.7 ± 6.9 + | 2.0 ± 0.5 * | 3.5 ± 0.7 *,+ | 4.1 ± 0.7 * | 5.8 ± 1.2 * |
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Obrador, E.; Salvador-Palmer, R.; López-Blanch, R.; Oriol-Caballo, M.; Moreno-Murciano, P.; Estrela, J.M. N-Acetylcysteine Promotes Metastatic Spread of Melanoma in Mice. Cancers 2022, 14, 3614. https://doi.org/10.3390/cancers14153614
Obrador E, Salvador-Palmer R, López-Blanch R, Oriol-Caballo M, Moreno-Murciano P, Estrela JM. N-Acetylcysteine Promotes Metastatic Spread of Melanoma in Mice. Cancers. 2022; 14(15):3614. https://doi.org/10.3390/cancers14153614
Chicago/Turabian StyleObrador, Elena, Rosario Salvador-Palmer, Rafael López-Blanch, María Oriol-Caballo, Paz Moreno-Murciano, and José M. Estrela. 2022. "N-Acetylcysteine Promotes Metastatic Spread of Melanoma in Mice" Cancers 14, no. 15: 3614. https://doi.org/10.3390/cancers14153614
APA StyleObrador, E., Salvador-Palmer, R., López-Blanch, R., Oriol-Caballo, M., Moreno-Murciano, P., & Estrela, J. M. (2022). N-Acetylcysteine Promotes Metastatic Spread of Melanoma in Mice. Cancers, 14(15), 3614. https://doi.org/10.3390/cancers14153614