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. 2017 Apr;39(2):161-173.
doi: 10.1007/s11357-017-9970-1. Epub 2017 Apr 4.

Apigenin Suppresses the Senescence-Associated Secretory Phenotype and Paracrine Effects on Breast Cancer Cells

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Free PMC article

Apigenin Suppresses the Senescence-Associated Secretory Phenotype and Paracrine Effects on Breast Cancer Cells

Kevin M Perrott et al. Geroscience. .
Free PMC article

Abstract

Apigenin (4',5,7,-trihydroxyflavone) is a flavonoid found in certain herbs, fruits, and vegetables. Apigenin can attenuate inflammation, which is associated with many chronic diseases of aging. Senescent cells-stressed cells that accumulate with age in mammals-display a pro-inflammatory senescence-associated secretory phenotype (SASP) that can drive or exacerbate several age-related pathologies, including cancer. Flavonoids, including apigenin, were recently shown to reduce the SASP of a human fibroblast strain induced to senesce by bleomycin. Here, we confirm that apigenin suppresses the SASP in three human fibroblast strains induced to senesce by ionizing radiation, constitutive MAPK (mitogen-activated protein kinase) signaling, oncogenic RAS, or replicative exhaustion. Apigenin suppressed the SASP in part by suppressing IL-1α signaling through IRAK1 and IRAK4, p38-MAPK, and NF-κB. Apigenin was particularly potent at suppressing the expression and secretion of CXCL10 (IP10), a newly identified SASP factor. Further, apigenin-mediated suppression of the SASP substantially reduced the aggressive phenotype of human breast cancer cells, as determined by cell proliferation, extracellular matrix invasion, and epithelial-mesenchymal transition. Our results support the idea that apigenin is a promising natural product for reducing the impact of senescent cells on age-related diseases such as cancer.

Keywords: Flavonoids; Human fibroblasts; IL-1A; IL-6; IRAK1/4; Invasion; NF-κB; Proliferation.

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Fig. 1
Fig. 1
Apigenin downregulates IL-6 secretion and moderately reduces fibroblast proliferation but does not induce apoptosis. a The indicated compounds from the Prestwick Library were used at 2 μM to treat primary human HCA2 fibroblasts immediately following X-irradiation at 10 Gy. Cells were treated for 10 days, after which conditioned media (CM) were collected and analyzed using the Perkin-Elmer IL-6 AlphaLISA assay. CM from non-senescent (NS) cells treated with vehicle (DMSO) provided a baseline for IL-6 secretion. Senescent cells were also treated with DMSO (negative control) and corticosterone (100 nM) (positive control). b HCA2 fibroblasts were induced to senesce (10 Gy X-irradiation) and immediately treated with increasing concentrations of apigenin for 10 days. CM were collected and analyzed for IL-6 secretion. IL-6 levels secreted by senescent cells were set at 100%. c HCA2 cells were induced to senesce as above and 7 days later were reseeded in 96-well plates (10,000 cells/well) and allowed to recover for 24 h. In parallel, non-senescent cells were seeded at 2500 cells/well. After 24 h, both senescent and non-senescent cells were treated with DMSO, 1 μM staurosporin or 10 μM apigenin, incubated for 48 h, and then caspase-3 activity was measured by luminescence using the Promega ApoTox Glo kit and arbitrary units. d HCA2 fibroblasts were seeded at 5000 cells/well in five 12-well plates, one for each day of a 5-day treatment with DMSO, 5, 10, or 20 μM apigenin. Three samples corresponding to each concentration were counted from one plate daily to determine cell number
Fig. 2
Fig. 2
Effects of apigenin treatment on senescence-associated phenotypes. a Human BJ, IMR90, and HCA2 fibroblasts were non-senescent (NS) or induced to senesce as described in the legend to Fig. 1 (IR) and treated with DMSO or apigenin for 10 days. Thirty-six hours before collection of senescent samples, NS cells were plated at 10,000/cm2 and allowed to recover overnight, and media on NS and IR cultures were replaced with serum-free media containing DMSO or apigenin. The following day, cells were stained for SA-β-gal activity and 100–150 cells were counted. b Cells were prepared as in (a) except 10,000/cm2 of senescent or non-senescent cells were seeded into each well of a 8-well slide, and EdU was added to the serum-free media and 100–150 cells were counted. c HCA2 fibroblasts were infected with L3P (insertless vector), MKK6EE, or H-RASV12 containing lentiviruses. Infected cells were selected with 1 μg/ml puromycin for 24 h, allowed to recover for 4 days, and reseeded. Five days later, all media were replaced with serum-free media containing apigenin or DMSO for 24 h. Then, CM was collected and analyzed for IL-6 secretion. d Replicatively senescent (PD 60.2) HCA2 fibroblasts were seeded at 10,000/cm2 onto 12-well plate and allowed to recover for 48 h. Cells were then treated with DMSO or apigenin (media refreshed every 48 h). On day 8, non-senescent cells were seeded onto 6-well plates at 10,000/cm2 and allowed to recover overnight. On day 9, all samples were given serum-free media supplemented with DMSO or apigenin, and collected 24 h later (day 10) and analyzed for IL-6 secretion
Fig. 3
Fig. 3
Effect of timing of apigenin treatment on IL-6 secretion. a, b HCA2 fibroblasts were seeded at 10,000/cm2 into two 24-well plates and induced to senesce by IR. 10 days later, we varied the length of continuous treatment with apigenin starting immediately after IR in one plate (a), and in the other plate, we varied the day of initiation of treatment with apigenin after IR (b). Duplicate samples were treated with media containing apigenin or DMSO and refreshed every 48 h. On day 9, media were replaced with serum-free media containing apigenin or DMSO, and 24 h later, cells were counted and conditioned media analyzed for IL-6 secretion. c HCA2 fibroblasts were seeded at 10,000/cm2 into a 24-well plate and induced to senesce by IR. Immediately following IR, media were refreshed with DMSO or apigenin and incubated for 10 days (media refreshed every 48 h with DMSO or apigenin). On day 10, cells were washed and incubated with serum-containing media except for the first sample (day 11) that was replaced with serum-free media. Samples for subsequent time points were similarly washed and media replaced with serum-free media 24 h before collection. After the final time point on day 20, CM for all time points were analyzed for IL-6
Fig. 4
Fig. 4
Effects of apigenin on the SASP and its regulation. a We compared the levels of selected cytokines (from the Luminex screening) secreted by treated (apigenin) and untreated (DMSO), NS, or IR senescent IMR90 fibroblasts. Levels of IP10, IL-8, GROA, and IL-6 in the CM from senescent cells are shown relative to NS. b Using qPCR, we determined the mRNA level of IP10 in DMSO- and apigenin-treated non-senescent (NS) and senescent (IR) BJ fibroblasts. c The kinetics of phosphorylation of IRAK4, IRAK1, and p38MAPK in non-senescent BJ fibroblasts stimulated with IL-1A and treated or not with apigenin, was examined using stabilization of phosphorylation by calyculin A (Cal). d IRAK4 phosphorylation in response to apigenin treatment was compared between senescent and non-senescent BJ fibroblasts
Fig. 5
Fig. 5
Apigenin suppresses the ability of the SASP to induce cancer cell aggressiveness. a MDA-MB231 (left panel) and ZR75.1 (right panel) breast cancer cells were cultured in presence of conditioned media (CM) from DMSO- or apigenin (Api)-treated fibroblasts and monitored for proliferation over 3 days. b CM were prepared from non-senescent (NS) cells or senescent (IR) cells, treated or not with apigenin (Api). CM were assayed for ability to stimulate MDA-MB231 human breast cancer cells to invade a basement membrane, as described in the “Materials and methods” section. Invasion stimulated by NS CM was given a value of one, and other conditions were normalized to this value. Error bars indicate the standard deviation around the mean. c ZR75.1 cells were incubated with the indicated CM for 3 days and immunostained for the tight junction protein ZO-1. d Using western blotting, we analyzed the expression of ZO-1, the epithelial marker cytokeratin 18 (K-18), and the mesenchymal marker vimentin (Vim) in ZR75-1 cells. The effect of CM from apigenin-treated NS and IR fibroblasts on the expression of these three markers is presented on lanes 2 and 4, whereas lanes 1 and 3 show the expression in cells cultured in CM from DMSO-treated NS and IR fibroblasts, respectively

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