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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.

Mileo AM, Di Venere D, Abbruzzese C, Miccadei S - Oxid Med Cell Longev (2015)

Bottom Line: 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.Inhibition of ROS generation by N-acetylcysteine (NAC) attenuates the antiproliferative effect.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.

View Article: PubMed Central - PubMed

Affiliation: Regina Elena National Cancer Institute, Via Elio Chianesi 53, 00144 Rome, Italy.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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fig1: 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.

Mentions: We have previously demonstrated [25] that high concentrations of AEs (from 200 to 800 μM for 24 h) are able to activate an apoptotic program in MDA-MB231 to halt tumour progression. Since bioactive compounds concentrations required to induce apoptosis in tumour cell lines might not be reachable in target tissues, we asked whether AEs chronically administered at low and sublethal doses can affect the growth of tumour cells as well. To this purpose, we focused on MDA-MB231, which provide the scientific rationale for testing AEs as an antitumour agent against invasive and hormone resistant breast cancer phenotype. After 10-day treatment (from 2.5 μM to 60 μM) direct cell count assay indicated that the cellular growth was significantly inhibited by chronic exposure to low doses of AEs (Figure 1(a)). Treatments up to 30 μM resulted in a relevant inhibition of cell proliferation in viable cells (about 90%); the highest concentration (60 μM) induced a dramatic growth arrest and was slightly cytotoxic (about 70% viability). Based on these data, we focused on two noncytotoxic concentrations of AEs, namely 10 and 30 μM, that modulate cell growth. Furthermore, we investigated whether these treatments caused a reduction in the number of living cells through the activation of caspases pathways. As shown in Figure 1(b), in these experimental conditions protein expression analysis revealed that caspase-9 was not activated. Conversely, high concentration of AEs (400 μM), used as positive control [25], triggers a significant activation/cleavage of caspase-9 after 24 h treatment. In addition, long term exposure to low concentrations of AEs did not activate caspase-8 (unpublished results). Altogether our results strongly suggested that low doses of AEs inhibit breast cancer cell growth via a caspases-independent mechanism.


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.

Mileo AM, Di Venere D, Abbruzzese C, Miccadei S - Oxid Med Cell Longev (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4477242&req=5

fig1: 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.
Mentions: We have previously demonstrated [25] that high concentrations of AEs (from 200 to 800 μM for 24 h) are able to activate an apoptotic program in MDA-MB231 to halt tumour progression. Since bioactive compounds concentrations required to induce apoptosis in tumour cell lines might not be reachable in target tissues, we asked whether AEs chronically administered at low and sublethal doses can affect the growth of tumour cells as well. To this purpose, we focused on MDA-MB231, which provide the scientific rationale for testing AEs as an antitumour agent against invasive and hormone resistant breast cancer phenotype. After 10-day treatment (from 2.5 μM to 60 μM) direct cell count assay indicated that the cellular growth was significantly inhibited by chronic exposure to low doses of AEs (Figure 1(a)). Treatments up to 30 μM resulted in a relevant inhibition of cell proliferation in viable cells (about 90%); the highest concentration (60 μM) induced a dramatic growth arrest and was slightly cytotoxic (about 70% viability). Based on these data, we focused on two noncytotoxic concentrations of AEs, namely 10 and 30 μM, that modulate cell growth. Furthermore, we investigated whether these treatments caused a reduction in the number of living cells through the activation of caspases pathways. As shown in Figure 1(b), in these experimental conditions protein expression analysis revealed that caspase-9 was not activated. Conversely, high concentration of AEs (400 μM), used as positive control [25], triggers a significant activation/cleavage of caspase-9 after 24 h treatment. In addition, long term exposure to low concentrations of AEs did not activate caspase-8 (unpublished results). Altogether our results strongly suggested that low doses of AEs inhibit breast cancer cell growth via a caspases-independent mechanism.

Bottom Line: 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.Inhibition of ROS generation by N-acetylcysteine (NAC) attenuates the antiproliferative effect.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.

View Article: PubMed Central - PubMed

Affiliation: Regina Elena National Cancer Institute, Via Elio Chianesi 53, 00144 Rome, Italy.

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.

No MeSH data available.


Related in: MedlinePlus