BRAF Mutant Melanoma Adjusts to BRAF/MEK Inhibitors via Dependence on Increased Antioxidant SOD2 and Increased Reactive Oxygen Species Levels
Abstract
:1. Introduction
2. Results
2.1. ROS Levels Are Increased in BRAF Mutant Melanoma in Response to Treatment with BRAF and MEK Inhibitors
2.2. ROS Level Is Upregulated upon Drug Resistance in BRAF Mutant Melanoma Cell Lines
2.3. BRAF Mutant Melanoma Cells Resistant to BRAF and MEK Inhibition Show Increases in DNA Damage
2.4. SOD2 and NF-κB Levels Are Increased in BRAF Pathway Inhibitor-Resistant Melanoma Cells
2.5. Knockdown of SOD2 Suppresses the Growth of BRAF Pathway Inhibitor Resistant Melanoma Cells
2.6. NAC, a ROS Scavenger, Can Re-Sensitize the BRAF Inhibitor in Melanoma Cells
3. Discussion
4. Material and Methods
4.1. Cell Culture and Inhibitors
4.2. Generation of Trametinib- and Dabrafenib-Resistant (TDR) Cell Lines
4.3. Immunoblotting
4.4. Transient Transfection
4.5. MTT Cell Proliferation Assay
4.6. Crystal Violet Cell Proliferation Assay
4.7. Measurement of Intracellular ROS
4.8. qPCR Analysis of Antioxidant mRNA
4.9. Immunofluorescence
4.10. Generation of Stable Reporter Cell Lines
4.11. In Vitro Luciferase Assays
4.12. In Vivo Xenograft Experiments
4.13. In Vivo Luciferase Experiments
4.14. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Yuan, L.; Mishra, R.; Patel, H.; Alanazi, S.; Wei, X.; Ma, Z.; Garrett, J.T. BRAF Mutant Melanoma Adjusts to BRAF/MEK Inhibitors via Dependence on Increased Antioxidant SOD2 and Increased Reactive Oxygen Species Levels. Cancers 2020, 12, 1661. https://doi.org/10.3390/cancers12061661
Yuan L, Mishra R, Patel H, Alanazi S, Wei X, Ma Z, Garrett JT. BRAF Mutant Melanoma Adjusts to BRAF/MEK Inhibitors via Dependence on Increased Antioxidant SOD2 and Increased Reactive Oxygen Species Levels. Cancers. 2020; 12(6):1661. https://doi.org/10.3390/cancers12061661
Chicago/Turabian StyleYuan, Long, Rosalin Mishra, Hima Patel, Samar Alanazi, Xin Wei, Zhijun Ma, and Joan T. Garrett. 2020. "BRAF Mutant Melanoma Adjusts to BRAF/MEK Inhibitors via Dependence on Increased Antioxidant SOD2 and Increased Reactive Oxygen Species Levels" Cancers 12, no. 6: 1661. https://doi.org/10.3390/cancers12061661