A Potent Autophagy Inhibitor (Lys05) Enhances the Impact of Ionizing Radiation on Human Lung Cancer Cells H1299
Abstract
:1. Introduction
2. Results
2.1. Treatment of H1299 Cells by Autophagy Inhibitors and IR Causes a Decrease in Proliferation in a Concentration- and Dose-Dependent Manner
2.2. Lys05 Renders H1299 Cells More Sensitive to IR
2.3. The Combination of Inhibitors with Irradiation Shows an Additive Effect
2.4. Lys05 Does Not Affect the Apical Parts of mTOR Signaling but Results in Increased LC3-II and p62/SQSTM1
2.5. Lys05 Induces the Early-Stage Autophagy but Subsequently Leads to Its Inhibition Resulting in Lysosome Accumulation
2.6. Lys05 Potentiates Autophagy Inhibition in H1299 Cells via Accumulation of Autophagosomes
3. Discussion
4. Materials and Methods
4.1. Cell Cultures and Cultivation
4.2. Cell Treatment
4.3. Cell Growth Real-Time Monitoring
4.4. Clonogenic Assay
4.5. The Combination Index and a Time-Dependent Combined Toxicity Level Calculations
4.6. Western Blot
4.7. Real-Time Quantitative Reverse Transcription PCR
4.8. Transmission Electron Microscopy
4.9. Fluorescence Microscopy
4.10. Flow Cytometry
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
Baf | Bafilomycin A1 |
BNIP3 | Bcl2 interacting protein 3 |
CI | Combination Index |
CQ | Chloroquine |
H1299 | Non-small cell lung carcinoma cell line |
HCQ | Hydroxychloroquine |
IR | Ionizing radiation |
LC3 | Microtubule-associated protein 1A/1B light chain 3 |
LSG | Lysosensor Green DND-189 |
Lys05 | Coding name for an autophagy inhibitor |
mTOR | Mammalian target of rapamycin |
NSCLC | Non-small cell lung carcinoma |
p53 | Cellular tumor antigen 53 |
p62 | Ubiquitin-binding protein p62 |
PRAS40 | Proline-rich Akt substrate of 40 kDa |
RT | Radiation therapy |
SQSTM1 | Sequestosome-1 |
TEM | Transmission electron microscopy |
ULK-1 | Unc-51 like autophagy activating kinase 1 |
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IR Dose [Gy] | Dose of Lys05 [µM] | Fa | CI | IR Dose [Gy] | Dose of Spautin-1 [µM] | Fa | CI |
---|---|---|---|---|---|---|---|
2 | 2 | 0.39 | 1.02 | 2 | 2 | 0.43 | 1.04 |
2 | 5 | 0.47 | 0.94 | 2 | 5 | 0.39 | 1.91 |
2 | 10 | 0.52 | 0.97 | 2 | 10 | 0.65 | 0.83 |
4 | 2 | 0.52 | 1.23 | 4 | 2 | 0.65 | 0.92 |
4 | 5 | 0.65 | 0.93 | 4 | 5 | 0.63 | 1.14 |
4 | 10 | 0.58 | 1.27 | 4 | 10 | 0.74 | 0.89 |
8 | 2 | 0.77 | 1.23 | 8 | 2 | 0.85 | 0.93 |
8 | 5 | 0.82 | 1.02 | 8 | 5 | 0.82 | 1.07 |
8 | 10 | 0.78 | 1.20 | 8 | 10 | 0.85 | 0.96 |
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Cechakova, L.; Ondrej, M.; Pavlik, V.; Jost, P.; Cizkova, D.; Bezrouk, A.; Pejchal, J.; Amaravadi, R.K.; Winkler, J.D.; Tichy, A. A Potent Autophagy Inhibitor (Lys05) Enhances the Impact of Ionizing Radiation on Human Lung Cancer Cells H1299. Int. J. Mol. Sci. 2019, 20, 5881. https://doi.org/10.3390/ijms20235881
Cechakova L, Ondrej M, Pavlik V, Jost P, Cizkova D, Bezrouk A, Pejchal J, Amaravadi RK, Winkler JD, Tichy A. A Potent Autophagy Inhibitor (Lys05) Enhances the Impact of Ionizing Radiation on Human Lung Cancer Cells H1299. International Journal of Molecular Sciences. 2019; 20(23):5881. https://doi.org/10.3390/ijms20235881
Chicago/Turabian StyleCechakova, Lucie, Martin Ondrej, Vojtech Pavlik, Petr Jost, Dana Cizkova, Ales Bezrouk, Jaroslav Pejchal, Ravi K. Amaravadi, Jeffrey D. Winkler, and Ales Tichy. 2019. "A Potent Autophagy Inhibitor (Lys05) Enhances the Impact of Ionizing Radiation on Human Lung Cancer Cells H1299" International Journal of Molecular Sciences 20, no. 23: 5881. https://doi.org/10.3390/ijms20235881