Chalcone-Acridine Hybrid Suppresses Melanoma Cell Progression via G2/M Cell Cycle Arrest, DNA Damage, Apoptosis, and Modulation of MAP Kinases Activity
Abstract
:1. Introduction
2. Results
2.1. MTT Screening Assay
2.2. BrdU Cell Proliferation Assay
2.3. AO/PI Apoptosis Analysis
2.4. Cell Cycle Analysis
2.5. Apoptosis Detection
2.6. Effect of Chalcone 1C on Cell Cycle-Related Protein Expression
2.6.1. p53 Protein
2.6.2. p21 Protein
2.6.3. Chk1 Kinase
2.6.4. Cyclin B1
2.6.5. Effect on Tubulins
2.7. Effect of Chalcone 1C on Mitochondrial Apoptosis Pathway Proteins
2.7.1. Effect on Bcl-2 Family Proteins
2.7.2. Cytochrome c Release
2.7.3. Mitochondrial Membrane Potential (MMP)
2.7.4. Caspase 3/7 Activity
2.7.5. Cleavage of PARP
2.8. DNA Damage Analysis
2.8.1. ATM Kinase
2.8.2. SMC1 Protein
2.8.3. Histone HA2.X
2.9. Chalcone 1C Modulates Signalling Pathways/Changes in Expression and Phosphorylation of MAPK Proteins
3. Discussion
4. Materials and Methods
4.1. Tested Compounds
4.2. Cell Culture
4.3. MTT Viability Assay
4.4. BrdU (5-Bromo-2′-deoxyuridine) Cell Proliferation Assay
4.5. AO/PI Viability Assay
4.6. Cell Cycle Analysis
4.7. Apoptosis Detection
4.8. Flow Cytometric Analyses
4.9. Western Blot Analyses
4.10. Statistical Analyses
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Compound | Cell Lines | ||
---|---|---|---|
A2058 | BLM | MCF-10A | |
1C | 7.96 ± 0.38 | 17.93 ± 0.87 | 36.54 ± 0.87 |
ZKCH-11A | 31.90 ± 0.95 | 35.27 ± 6.10 | 85.01 ± 2.19 |
ZKCH-11C | 41.64 ± 0.73 | 38.55 ± 0.06 | >100 |
ZKCH-11E | 37.34 ± 2.28 | 33.75 ± 2.14 | 44.71 ± 4.96 |
ZKCH-11F | 45.82 ± 2.54 | 47.69 ± 1.49 | >100 |
ZKCH-11G | 39.96 ± 1.37 | 39.45 ± 3.90 | 51.27 ± 3.94 |
ZKCH-11H | 42.43 ± 2.98 | 40.85 ± 2.62 | 60.68 ± 3.09 |
Compound | Assay | Cell Lines | ||
---|---|---|---|---|
A2058 | BLM | MCF-10A | ||
1C | MTT | 7.96 ± 0.38 | 17.93 ± 0.87 | 36.54 ± 0.87 |
BrdU | 8.40 ± 0.05 | 16.51 ± 0.01 | 32.86 ± 1.56 | |
Selectivity index | 4.6/3.9 | 2.0/1.99 |
A2058 | |||||
---|---|---|---|---|---|
Time | Treatment | subG0 | G1 | S | G2/M |
24 h | DMSO | 0.55 ± 0.05 | 50.75 ± 0.04 | 16.85 ± 0.61 | 31.85 ± 0.53 |
1C | 1.30 ± 1.11 | 34.67 ± 4.92 ** | 11.79 ± 1.51 * | 52.23 ± 7.03 ** | |
48 h | DMSO | 0.54 ± 0.15 | 51.40 ± 0.33 | 17.15 ± 1.43 | 30.90 ± 1.63 |
1C | 7.54 ± 3.73 * | 24.60 ± 11.45 ** | 8.12 ± 1.15 * | 59.73 ± 12.23 ** | |
72 h | DMSO | 0.98 ± 0.02 | 54.20 ± 0.73 | 13.00 ± 0.65 | 31.80 ± 0.08 |
1C | 12.12 ± 5.84 * | 18.67 ± 8.56 ** | 8.85 ± 2.70 * | 60.37 ± 4.42 ** | |
BLM | |||||
Time | Treatment | subG0 | G1 | S | G2/M |
24 h | DMSO | 2.43 ± 0.24 | 56.70 ± 0.90 | 15.90 ± 0.33 | 25.00 ± 0.98 |
1C | 8.22 ± 1.62 * | 19.35 ± 1.43 ** | 9.19 ± 0.28 * | 63.25 ± 0.45 *** | |
48 h | DMSO | 2.10 ± 0.08 | 61.60 ± 3.76 | 14.75 ± 1.18 | 21.55 ± 2.65 |
1C | 17.05 ± 0.45 * | 11.65 ± 0.69 ** | 16.35 ± 0.20 | 54.95 ± 0.45 ** | |
72 h | DMSO | 1.87 ± 0.02 | 63.90 ± 2.45 | 16.40 ± 0.33 | 17.85 ± 2.08 |
1C | 24.30 ± 3.84 ** | 13.85 ± 1.18 *** | 32.60 ± 2.69 * | 29.25 ± 0.04 * |
A2058 | |||||
---|---|---|---|---|---|
Time | Treatment | Live An−/PI− | Early Apo An+/PI− | Late Apo An+/PI+ | Dead An−/PI+ |
24 h | DMSO | 88.60 ± 3.67 | 5.83 ± 2.04 | 3.93 ± 1.25 | 1.65 ± 0.35 |
1C | 84.80 ± 0.24 | 7.53 ± 1.17 | 4.22 ± 0.47 | 3.41 ± 0.46 | |
48 h | DMSO | 88.75 ± 0.94 | 4.69 ± 1.56 | 4.63 ± 0.61 | 1.96 ± 0.02 |
1C | 68.70 ± 1.63 ** | 11.52 ± 0.42 * | 13.85 ± 2.57 * | 5.94 ± 0.52 | |
72 h | DMSO | 84.20 ± 0.65 | 7.76 ± 0.00 | 6.88 ± 1.42 | 1.19 ± 0.06 |
1C | 52.00 ± 5.23 ** | 13.30 ± 1.71 * | 23.05 ± 4.94 ** | 11.65 ± 1.43 * | |
BLM | |||||
Time | Treatment | Live An−/PI− | Early Apo An+/PI− | Late Apo An+/PI+ | Dead An−/PI+ |
24 h | DMSO | 85.00 ± 1.39 | 8.60 ± 0.14 | 3.95 ± 0.60 | 2.42 ± 0.66 |
1C | 73.90 ± 0.49 * | 12.96 ± 0.77 * | 6.90 ± 0.09 | 6.25 ± 0.20 | |
48 h | DMSO | 85.95 ± 4.04 | 7.37 ± 0.96 | 4.29 ± 1.88 | 2.41 ± 1.18 |
1C | 61.50 ± 2.61 ** | 20.25 ± 1.27 * | 12.40 ± 0.49 * | 5.81 ± 0.86 | |
72 h | DMSO | 83.70 ± 1.71 | 7.95 ± 0.21 | 6.17 ± 1.29 | 2.18 ± 0.24 |
1C | 39.60 ± 0.90 ** | 25.95 ± 3.80 ** | 28.55 ± 3.23 ** | 5.90 ± 1.47 * |
Analysis | Staining | Manufacturer |
---|---|---|
Caspase activation | CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit | Thermo Scientific, Rockford, IL, USA |
Cytochrome c release | Cytochrome c Antibody (6H2) FITC Conjugate | Invitrogen, Carlsbad, CA, USA |
Mitochondrial membrane potential | TMRE (Tetramethylrhodamine ethyl ester perchlorate) final concentration 0.1 μmol/L | Sigma-Aldrich, St. Louis, MO, USA |
DNA damage | FlowCellect™ Multi-Color DNA Damage Response Kit | Millipore Corporation, Temecula, CA, USA |
Primary Antibodies | Mr (kDa) | Origin | Dilution | Manufacturer |
---|---|---|---|---|
Bad | 22 | Rabbit | 1:1000 | Abcam, Cambridge, UK |
Phospho Bad | 23 | Rabbit | 1:1000 | Cell Signalling Technology®, Danvers, MA, USA |
Bcl-2 | 26 | Mouse | 1:1000 | Abcam, Cambridge, UK |
Phospho Bcl-2 | 28 | Rabbit | 1:1000 | Cell Signalling Technology®, Danvers, MA, USA |
Bax | 23 | Mouse | 1:1000 | Santa Cruz Biotechnology, Inc. Dallas, TX, USA |
Bcl-xL | 30 | Rabbit | 1:1000 | |
α Tubulin | 55 | Rabbit | 1:1000 | |
α1C Tubulin | 50 | Mouse | 1:1000 | |
β Tubulin | 55 | Rabbit | 1:1000 | |
p21 | 21 | Rabbit | 1:1000 | Cell Signalling Technology®, Danvers, MA, USA |
Phospho-Cyclin B1 | 55 | Rabbit | 1:1000 | |
Phosho-Chk1 | 56 | Rabbit | 1:1000 | |
p38 MAPK | 43 | Rabbit | 1:1000 | |
Phospho p38 MAPK | 43 | Rabbit | 1:1000 | |
p44/42 MAPK (Erk1/2) | 42/44 | Rabbit | 1:1000 | |
Phospho p44/42 MAPK (Erk1/2) | 42/44 | Mouse | 1:1000 | |
JNK | 48 | Mouse | 1:1000 | Thermo Scientific, Rockford, IL, USA |
Phospho SAPK/JNK | 46/54 | Mouse | 1:1000 | Cell Signalling Technology®, Danvers, MA, USA |
PARP | 116/89 | Rabbit | 1:1000 | |
p53 | 53 | Rabbit | 1:1000 | |
Phospho p53 | 53 | Rabbit | 1:1000 | |
β-actin | 45 | Mouse | 1:2500 | |
Secondary Antibodies | ||||
Anti-mouse IgG HRP | - | Goat | 1:1000 | Cell Signalling Technology®, Danvers, MA, USA |
Anti-rabbit IgG HRP | - | Goat | 1:1000 |
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Gazdova, M.; Michalkova, R.; Kello, M.; Vilkova, M.; Kudlickova, Z.; Baloghova, J.; Mirossay, L.; Mojzis, J. Chalcone-Acridine Hybrid Suppresses Melanoma Cell Progression via G2/M Cell Cycle Arrest, DNA Damage, Apoptosis, and Modulation of MAP Kinases Activity. Int. J. Mol. Sci. 2022, 23, 12266. https://doi.org/10.3390/ijms232012266
Gazdova M, Michalkova R, Kello M, Vilkova M, Kudlickova Z, Baloghova J, Mirossay L, Mojzis J. Chalcone-Acridine Hybrid Suppresses Melanoma Cell Progression via G2/M Cell Cycle Arrest, DNA Damage, Apoptosis, and Modulation of MAP Kinases Activity. International Journal of Molecular Sciences. 2022; 23(20):12266. https://doi.org/10.3390/ijms232012266
Chicago/Turabian StyleGazdova, Maria, Radka Michalkova, Martin Kello, Maria Vilkova, Zuzana Kudlickova, Janette Baloghova, Ladislav Mirossay, and Jan Mojzis. 2022. "Chalcone-Acridine Hybrid Suppresses Melanoma Cell Progression via G2/M Cell Cycle Arrest, DNA Damage, Apoptosis, and Modulation of MAP Kinases Activity" International Journal of Molecular Sciences 23, no. 20: 12266. https://doi.org/10.3390/ijms232012266