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, 2015, 363827

Long Term Exposure to Polyphenols of Artichoke (Cynara Scolymus L.) Exerts Induction of Senescence Driven Growth Arrest in the MDA-MB231 Human Breast Cancer Cell Line

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Long Term Exposure to Polyphenols of Artichoke (Cynara Scolymus L.) Exerts Induction of Senescence Driven Growth Arrest in the MDA-MB231 Human Breast Cancer Cell Line

Anna Maria Mileo et al. Oxid Med Cell Longev.

Abstract

Polyphenolic extracts from the edible part of artichoke (Cynara scolymus L.) have been shown to be potential chemopreventive and anticancer dietary compounds. High doses of polyphenolic extracts (AEs) induce apoptosis and decrease the invasive potential of the human breast cancer cell line, MDA-MB231. However, the molecular mechanism underlying AEs antiproliferative effects is not completely understood. We demonstrate that chronic and low doses of AEs treatment at sublethal concentrations suppress human breast cancer cell growth via a caspases-independent mechanism. Furthermore, AEs exposure induces a significant increase of senescence-associated β-galactosidase (SA-β-gal) staining and upregulation of tumour suppressor genes, p16(INK4a) and p21(Cip1/Waf1) in MDA-MB231 cells. AEs treatment leads to epigenetic alterations in cancer cells, modulating DNA hypomethylation and lysine acetylation levels in total proteins. Cell growth arrest correlates with increased reactive oxygen species (ROS) production in AEs treated breast cancer cells. Inhibition of ROS generation by N-acetylcysteine (NAC) attenuates the antiproliferative effect. These findings demonstrate that chronic AEs treatment inhibits breast cancer cell growth via the induction of premature senescence through epigenetic and ROS-mediated mechanisms. Our results suggest that artichoke polyphenols could be a promising dietary tool either in cancer chemoprevention or/and in cancer treatment as a nonconventional, adjuvant therapy.

Figures

Figure 1
Figure 1
Low doses of AEs suppress breast cancer cell growth via a caspases-independent mechanism. (a) Cell growth inhibition by AEs. MDA-MB231 cells were treated with increasing concentrations of AEs (from 2.5 to 60 μM) for 10 days. The results are the mean ± SD of at least three independent experiments. Significant statistical differences are indicated by asterisks: ∗∗∗ p < 0.0001. (b) Effects of AEs on caspases pathway. The cells were treated with low doses of AEs (10 and 30 μM) for 10 days or with a high concentration of AEs (400 μM) for 24 h, as a positive control. Whole cell lysates (25 μg/lane) were tested for activation and cleavage of caspase-9. β-Actin was used as a protein loading control.
Figure 2
Figure 2
Chronic treatment of AEs induces cell senescence. MDA-MB231 cells were treated with low doses of AEs (10 and 30 μM) for 10 days and then analyzed for the SA-β-gal, senescence-associated β-galactosidase activity. The image shown is representative of at least three independent experiments. Scale bar: 50 μm. The senescent cells versus total cells were counted in random fields under an inverted microscope (20x) and the following data are the mean ± SD: C = 7.7 ± 0.5, 10 μM AEs = 31 ± 5.6, p = 0.0044∗∗, 30 μM AEs = 51 ± 6.6, p = 0.0005∗∗∗. Significant statistical differences are indicated by asterisks. Boxed area, regarding blue cells with a typical senescent flattened and enlarged morphology, is magnified (2x).
Figure 3
Figure 3
Chronic treatment of AEs induces increased p16INK4a and p21Cip1/Waf1 expression. MDA-MB231 cells were treated with low doses of AEs (10 and 30 μM) for 10 days. Whole cell lysates were tested for (a) p16INK4a and (b) p21Cip1/Waf1 protein expression. Immunoblots are representative of at least three independent experiments. Intensities of electrophoretic bands relative to the immunoblot shown were quantified by densitometry and the values are reported.
Figure 4
Figure 4
Effect of AEs on global DNA methylation levels in MDA-MB231 cells. Low doses of AEs (2.5–30 μM) for 10 days treatment decreased levels of 5-mC, 5-methylcytosine, in a dose-dependent manner. 5-mC specific antibody was used for (a) and (b) cytostaining and (c) for dot blot analysis on genomic DNA. (a) The image shown is representative of at least three independent experiments. Scale bar: 100 μm. (b) The 5-methylcytosine positive cells versus total cells were counted by using an inverted microscope (20x) and the results are reported as mean ± SD. (c) The intensity of individual DNA dots shown was quantified by densitometry.
Figure 5
Figure 5
Effect of AEs on lysine acetylation of total proteins in MDA-MB231 cells. Cells were treated with low doses of AEs for 10 days. Cell lysates were analyzed for lysine acetylation of total proteins. Immunoblot is representative of at least three experiments. The intensities of electrophoretic bands relative to the immunoblot shown were quantified by densitometry.
Figure 6
Figure 6
ROS production in MDA-MB231 cells treated with AEs. (a) The presence of ROS was detected by DHE fluorescent staining after 10 days treatment. Scale bar: 50 μm, original magnification 20x. (b) Red fluorescent-stained cells versus total cells were counted using an inverted fluorescence microscope. The results are the mean ± SD of at least three independent experiments. Significant statistical differences are indicated by asterisks: ∗∗ p = 0.0012. (c) NAC reduced the antiproliferative effect of AEs (30 μM for 10 days). The results are the mean ± SD of at least three independent experiments. Significant statistical differences are indicated by asterisks: ∗∗ p = 0.0012.

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