ROS-Dependent Antiproliferative Effect of Brassinin Derivative Homobrassinin in Human Colorectal Cancer Caco2 Cells
Abstract
:1. Introduction
2. Results and Discussion
2.1. Effect of Brassinin and Its Derivatives on Cell Proliferation
Compound | Cancer Cell Lines | |||||||
---|---|---|---|---|---|---|---|---|
Jurkat | Caco2 | HepG2 | HCT-116 | A549 | HeLa | MCF-7 | MDA-MB-231 | |
1 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 |
K1 | 28.2 ± 1.2 | 8.0 ± 0.6 | 21.3 ± 2.3 | 27.3 ± 1.8 | 33.4 ± 0.8 | 26.1 ± 2.4 | 35.0 ± 2.1 | 22.8 ± 1.4 |
K10 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 |
K124 | 30.0 ± 0.5 | >100 | >100 | 30.7 ± 2.9 | >100 | 34.0 ± 0.7 | 92.0 ± 2.7 | >100 |
K49 | 39.6 ± 1.8 | 89.4 ± 1.3 | 37.4 ± 3.7 | 76.0 ± 2.5 | 22.8 ± 0.5 | 45.6 ± 1.4 | 62.0 ± 3.4 | >100 |
K170 | 44.3 ± 3.2 | >100 | >100 | 53.7 ± 2.1 | 96.5 ± 4.1 | >100 | >100 | >100 |
47 | >100 | >100 | >100 | >100 | >100 | >100 | >100 | >100 |
2.2. K1 Blocks Cell Cycle at G2/M Transition
Treatment | Time (h) | Sub-G1 | G0/G1 | S | G2/M |
---|---|---|---|---|---|
Control | 0.29 ± 0.15 | 40.81 ± 3.29 | 18.25 ± 3.90 | 36.64 ± 2.29 | |
K1 | 24 | 1.49 ± 0.37 | 21.34 ± 3.13 ** | 6.69 ± 1.56 * | 70.48 ± 2.28 *** |
K1 | 48 | 3.36 ± 1.67 * | 30.89 ± 2.81 * | 9.58 ± 2.43 | 56.20 ± 2.58 ** |
K1 | 72 | 5.58 ± 1.16 * | 30.17 ± 1.65 * | 11.47 ± 1.65 | 52.79 ± 2.77 ** |
2.3. K1 Induces Apoptotic Cell Death
Treatment | Time (h) | An−/PI− | An+/PI− | An+/PI+ |
---|---|---|---|---|
Control | 94.07 ± 1.32 | 3.17 ± 0.60 | 2.05 ± 0.50 | |
K1 | 24 | 67.81 ± 1.18 ** | 18.54 ± 1.63 ** | 13.75 ± 2.43 * |
K1 | 48 | 49.92 ± 3.20 *** | 38.55 ± 2.47 *** | 11.81 ± 2.24 * |
K1 | 72 | 42.36 ± 3.65 *** | 37.07 ± 2.66 *** | 20.85 ± 3.69 ** |
2.4. Effect of K1 on ROS Formation
2.5. K1-Induced Mitochondrial Dysfunction
2.6. Activation of Caspase-3 by K1
2.7. Effect of K1 on Gene Expression of Tubulins
Genes | Normalized Ratio | ||
---|---|---|---|
24 h | 48 h | 72 h | |
α-tubulin | 5.59 | 1.68 | 3.35 |
α1-tubulin | 1.89 | 0.65 | 1.47 |
β5-tubulin | 0.29 | 0.99 | 0.48 |
2.8. Effect of K1 on Cytoskeletal Tubulins Assembly
2.9. Discussion
3. Experimental Section
3.1. Test Compounds
3.2. Cell Culture
3.3. Growth Inhibition Assay
3.4. 5-Bromo-2'-Deoxyuridine (BrdU) Cell Proliferation Assay
3.5. xCELLigence Cell Analysis System
3.6. Experimental Design for Flow Cytometry Analysis
3.7. Analysis of Cell Cycle
3.8. Annexin V-FITC Labelling
3.9. Measurement of ROS
3.10. Detection of MMP
3.11. Detection of Active Caspase 3
3.12. DNA Fragmentation Assay
3.13. DAPI Staining
3.14. RNA Isolation and cDNA Synthesis
3.15. Quantitative Real-Time PCR
3.16. Western Blot Analysis
3.17. Statistical Analysis
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Kello, M.; Drutovic, D.; Chripkova, M.; Pilatova, M.; Budovska, M.; Kulikova, L.; Urdzik, P.; Mojzis, J. ROS-Dependent Antiproliferative Effect of Brassinin Derivative Homobrassinin in Human Colorectal Cancer Caco2 Cells. Molecules 2014, 19, 10877-10897. https://doi.org/10.3390/molecules190810877
Kello M, Drutovic D, Chripkova M, Pilatova M, Budovska M, Kulikova L, Urdzik P, Mojzis J. ROS-Dependent Antiproliferative Effect of Brassinin Derivative Homobrassinin in Human Colorectal Cancer Caco2 Cells. Molecules. 2014; 19(8):10877-10897. https://doi.org/10.3390/molecules190810877
Chicago/Turabian StyleKello, Martin, David Drutovic, Martina Chripkova, Martina Pilatova, Mariana Budovska, Lucia Kulikova, Peter Urdzik, and Jan Mojzis. 2014. "ROS-Dependent Antiproliferative Effect of Brassinin Derivative Homobrassinin in Human Colorectal Cancer Caco2 Cells" Molecules 19, no. 8: 10877-10897. https://doi.org/10.3390/molecules190810877