Cadmium Modifies the Cell Cycle and Apoptotic Profiles of Human Breast Cancer Cells Treated with 5-Fluorouracil
Abstract
:1. Introduction
2. Results
2.1. Effect of Cd and 5-FU on Cell Cycle Analysis and Apoptosis
2.2. Gene Expression
2.3. Protein Expression
3. Discussion
4. Experimental Section
4.1. Cell Culture
4.2. Drugs
4.3. Cell Cycle Distribution Analysis
4.4. Apoptosis Detection by Staining with Annexin V-FITC and Propidium Iodide
4.5. Gene Expression
4.6. Protein Expression
4.7. Statistical Analysis
5. Conclusions
Acknowledgments
Conflict of Interest
References
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M | Cd | 5FU | 5FU + Cd | Cd + 5FU½ | 5FU + Cd½ | |
---|---|---|---|---|---|---|
6 h | ||||||
G0/G1 | 56.7 ± 1.2 | 61.1 ± 1.64 | 59.0 ± 0.85 | 61.5 ± 1.23 | 62.9 ± 0.86 | 61.1 ± 2.34 |
S | 28.0 ± 0.95 | 23.0 ± 1.06 | 28.6 ± 0.19 | 24.2 ± 1.27 | 24.9 ± 0.56 | 26.8 ± 1.21 |
G2/M | 14.0 ± 0.35 | 15.8 ± 0.57 | 12.2 ± 0.78 | 14.2 ± 0.19 | 11.3 ± 0.75 | 15.7 ± 0.1 |
12 h | ||||||
G0/G1 | 61.6 ± 2.07 * | 49.0 ± 1.19 ** | 62.1 ± 1.27 * | 58.9 ± 1.21 | 61.8 ± 0.93 | 60.0 ± 1.54 |
S | 21.7 ± 1.54 * | 26.1 ± 0.56 * | 24.6 ± 0.29 *** | 26.2 ± 0.31 | 22.2 ± 1.07 * | 26.9 ± 0.6 |
G2/M | 16.2 ± 0.77 * | 6.9 ± 0.74 * | 12.9 ± 0.35 | 14.4 ± 0.43 | 14.6 ± 0.65 * | 12.6 ± 0.22 *** |
24 h | ||||||
G0/G1 | 81.7 ± 2.88 ** | 66.9 ± 1.2 * | 75.2 ± 2.33 ** | 63.0 ± 1.54 | 74.9 ± 1.76 ** | 72.2 ± 2.81 |
S | 9.5 ± 0.32 *** | 18.1 ± 1.35 * | 15.3 ± 0.88 *** | 15.7 ± 1.02 ** | 14.0 ± 0.58 *** | 15.2 ± 0.76 ** |
G2/M | 8.5 ± 0.08 *** | 5.5 ± 0.1 ** | 8.8 ± 0.55 * | 2.3 ± 0.12 *** | 3.1 ± 0.05 *** | 9.5 ± 0.56 *** |
48 h | ||||||
G0/G1 | 85.9 ± 3.21 ** | 60.5 ± 2.03 | 82.9 ± 3.08 ** | 72.3 ± 2.44 * | 75.5 ± 1.56 ** | 78.2 ± 1.8 ** |
S | 5.8 ± 0.88 *** | 23.5 ± 1.1 | 9.2 ± 0.77 *** | 11.5 ± 1.1 ** | 12.3 ± 0.9 *** | 9.0 ± 1.3 ** |
G2/M | 6.7 ± 0.5 *** | 12.3 ± 0.21 ** | 4.9 ± 0.4 ** | 0.3 ± 0.02 *** | 0.2 ± 0.01 *** | 0.9 ± 0.03 *** |
Time points | ||
---|---|---|
M | Control non treated cells | 6 h, 24 h, 48 h |
Cd | Cells treated with Cd | 6 h, 24 h, 48 h |
5FU | Cells treated with 5FU | 6 h, 24 h, 48 h |
Cd + 5FU | Cells treated with both drugs | 6 h, 24 h, 48 h |
Cd + 5FU½ | Cells treated with Cd plus 5-FU added after the half of time from the experiment started | 6 h, 24 h, 48 h |
5FU + Cd½ | Cells treated with 5-FU plus Cd added after the half time from the experiment started | 6 h, 24 h, 48 h |
Primer | Annealing temperature (°C) | |
---|---|---|
bcl-2 | Forward: 5′-TGGTGGTTTGACCTTTAGAGA-3′ Reverse: 5′-AGGTCTGATCATTCTGTTC-3′ | 55 |
p 53 | Forward: 5′-GGCATTCTGGGAGCTTCATCT-3′ Reverse: 5′-CCCAAGCAATGGATGATTTGA-3′ | 58.5 |
bax | Forward: 5′-TGCTTCAGGGTTTCATCCAG-3′ Reverse: 5′-GGCGGCAATCATCCTCTG-3′ | 55 |
caspase 8 | Forward: 5′-AGGAGGAGATGGAAAGGGAACTT-3′ Reverse: 5′-ACCTCAATTCTGATCTGCTCACTTCT-3′ | 55 |
caspase 9 | Forward: 5′-CCTCAAACTCTCAAGAGCAC-3′ Reverse: 5′-GAGTCAGGCTCTTCCTTTG-3′ | 58.5 |
c-myc | Forward: 5′-GGACGACGAGACCTTCATCAA-3′ Reverse: 5′-CCAGCTTCTCTGAGACGAGCTT-3′ | 55 |
cyclin D1 | Forward: 5′-CCGTCCATGCGGAAGATC-3′ Reverse: 5′-ATGGCCAGCGGGAAGAC-3′ | 55 |
cyclin A1: | Forward: 5′-GCACCCTGCTCGTCACTTG-3′ Reverse: 5′-AGCCCCCAATAAAAGATCCAG-3′ | 55 |
GAPDH | Forward: 5′-CAAGGAGTAAGACCCCTGGAC-3′ Reverse: 5′-TCTACATGGCAACTGTGAGGAG-3′ | 58.5 |
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Asara, Y.; Marchal, J.A.; Carrasco, E.; Boulaiz, H.; Solinas, G.; Bandiera, P.; Garcia, M.A.; Farace, C.; Montella, A.; Madeddu, R. Cadmium Modifies the Cell Cycle and Apoptotic Profiles of Human Breast Cancer Cells Treated with 5-Fluorouracil. Int. J. Mol. Sci. 2013, 14, 16600-16616. https://doi.org/10.3390/ijms140816600
Asara Y, Marchal JA, Carrasco E, Boulaiz H, Solinas G, Bandiera P, Garcia MA, Farace C, Montella A, Madeddu R. Cadmium Modifies the Cell Cycle and Apoptotic Profiles of Human Breast Cancer Cells Treated with 5-Fluorouracil. International Journal of Molecular Sciences. 2013; 14(8):16600-16616. https://doi.org/10.3390/ijms140816600
Chicago/Turabian StyleAsara, Yolande, Juan A. Marchal, Esther Carrasco, Houria Boulaiz, Giuliana Solinas, Pasquale Bandiera, Maria A. Garcia, Cristiano Farace, Andrea Montella, and Roberto Madeddu. 2013. "Cadmium Modifies the Cell Cycle and Apoptotic Profiles of Human Breast Cancer Cells Treated with 5-Fluorouracil" International Journal of Molecular Sciences 14, no. 8: 16600-16616. https://doi.org/10.3390/ijms140816600