Inhibition of mTOR by apigenin in UVB-irradiated keratinocytes: A new implication of skin cancer prevention

Abstract

Ultraviolet B (UVB) radiation is the major environmental risk factor for developing skin cancer, the most common cancer worldwide, which is characterized by aberrant activation of Akt/mTOR (mammalian target of rapamycin). Importantly, the link between UV irradiation and mTOR signaling has not been fully established. Apigenin is a naturally occurring flavonoid that has been shown to inhibit UV-induced skin cancer. Previously, we have demonstrated that apigenin activates AMP-activated protein kinase (AMPK), which leads to suppression of basal mTOR activity in cultured keratinocytes. Here, we demonstrated that apigenin inhibited UVB-induced mTOR activation, cell proliferation and cell cycle progression in mouse skin and in mouse epidermal keratinocytes. Interestingly, UVB induced mTOR signaling via PI3K/Akt pathway, however, the inhibition of UVB-induced mTOR signaling by apigenin was not Akt-dependent. Instead, it was driven by AMPK activation. In addition, mTOR inhibition by apigenin in keratinocytes enhanced autophagy, which was responsible, at least in part, for the decreased proliferation in keratinocytes. In contrast, apigenin did not alter UVB-induced apoptosis. Taken together, our results indicate the important role of mTOR inhibition in UVB protection by apigenin, and provide a new target and strategy for better prevention of UV-induced skin cancer.

Keywords: AMPK; Akt; Apigenin; Autophagy; UVB; mTOR.

Fig. 1. Apigenin inhibits UVB-induced proliferation and cell cycle progression in mouse keratinocytes

(A-D) SKH-1 mice were subjected to UVB radiation (1000 J/m² daily, 5 days), or topical apigenin (Api, 5 μmol, in 200 μl vehicle, DMSO: acetone 1:9) was applied 1 h prior to each UVB exposure. Mice were also sham-irradiated and treated with apigenin, and the control group of mice was subjected to sham irradiation and vehicle. 24 h after the final UVB exposure, the mice were euthanized, dorsal skin was harvested, fixed in formalin and paraffin-embedded. (A) Representative Hematoxylin and Eosin (H&E) staining of skin sections (scale bar, 100 μm). (B) Quantitation of epidermal thickness (mean ± SD), *, P < 0.001; **, P< 0.0001. Three sections per mouse and 3 mice per group were evaluated. (C) Immunohistochemistry (IHC) for proliferation marker Ki-67 (scale bar, 100 μm). (D) Quantification of Ki-67 staining (mean ± SD), *, P < 0.01; **, P < 0.0001. Three fields from 3 independent sections/group were examined, Ki-67 positive nuclei were calculated per linear 100 μm of epidermis and hair follicles were not included in analysis. (E) Cell cycle analysis of mouse 308 keratinocytes: determination of cells in S-phase (mean ± SD), *, P < 0.01; **, P < 0.001. 308 cells at 80-90% confluence were synchronized by nutrient withdrawal overnight, pre-treated for 1 hr with apigenin (25 μM), and irradiated with UVB (500 J/m²), as indicated. Cells were harvested at 24 h post irradiation and analyzed by flow cytometry. Data are from 3 independent experiments. (F) 308 cells were treated as above and CDK2 expression was analyzed by Western blot. (G) IHC for CDK2. The mice were treated as in (A-D).

Fig. 2. Apigenin suppresses mTOR activationby UVB

(A) Western blot analysis of mTOR downstream targets p-p70S6K and p4E-BP1 in 308 cells exposed to UVB (500 J/m²) and/or pre-treated with apigenin (25 μM) for 1 h where indicated, and harvested at 24 h post irradiation. (B) Dose-dependent effect of apigenin on baseline mTOR activation. 308 cells were treated with apigenin or vehicle for 24 h and harvested for immunoblotting. (C) Inhibition of mTOR activation in vivo: Control and UVB-irradiated SKH-1 mice (see Fig.1 for details) were treated topically with apigenin or vehicle 1 hr prior to irradiation. IHC was performed on dorsal epidermis sections using antibodies for active phosphorylated mTOR (p-mTOR, Ser 2448).

Fig. 3. mTOR activation by UVB involves PI3K/Akt signaling

(A) Time course of Akt activation in UVB irradiated 308 cells. Cells were irradiated (500 J/m²) for indicated time periods, and phospho-Akt assessed by immunoblotting. Total Akt and Actin were used to as certain equal loading. (B) PI3K inhibitor Wortmannin attenuates UVB-induced mTOR activation. 308 keratinocytes were pre-treated with 5 μM wortmannin for 1 h, where indicated, irradiated and harvested 12 h (for Akt analysis) or 24 h (for p70S6K and 4E-BP1) following UVB irradiation. Representative Western blots are shown.

Fig. 4. Inhibition of UVB-induced mTOR by apigenin is independent of its suppression of Akt

(A) 308 cells were transiently transfected with an expression vector encoding constitutively active Akt (CA-Akt) or a control vector, incubated overnight. Apigenin (25 μM) was added 1 h prior to UVB irradiation (500 J/m²) and the cells were harvested 24 after UVB irradiation. mTOR activation (phosphorylation of its target p70S6K) and Akt activation/phosphorylation were assessed by Western blot. (B) 308 cells were pre-treated with Akt inhibitor MK-2206 (1 μM) or in combination with different doses of apigenin for 1 h before subjecting to UVB irradiation (500 J/m²), or (C) 308 cells were transfected with siRNA against Akt or non-targeting control siRNA, and 72 h later cells were exposed to apigenin and UVB irradiation as described above. All samples were collected 24 post irradiation. Western blotting was performed to measure phosphorylated p70S6K/Akt and total p70S6K/Akt.

Fig. 5. AMPK is essential for the blockade of UVB-induced mTOR pathway by apigenin

(A) Apigenin activates AMPK at baseline and in UVB-irradiated cells. 308 cells were treated as indicated. Apigenin (25 μM) was added 1 hr prior to irradiation. The cells were harvested at 24 h after irradiation. (B) Apigenin activates AMPK in mouse skin. SKH-1 mice were irradiated as described in Fig.1. Apigenin was given topically 1 hr prior to irradiation at 5 and 10 μmol. Whole-skin protein extracts were immunoblotted for phosphorylated and total AMPK and p44/42 MAPK was used as loading control. (C) 308 cells were pre-treated with 5 μM compound C or vehicle (DMSO) for 1 h, then 25 μM apigenin was added to the media for another 1 h and followed by UVB exposure (500 J/m²). (D) 308 cells were transfected with AMPK siRNA or non-targeting control siRNA, and 48 h later cells were exposed to apigenin and UVB irradiation as described above. (E) 308 cells were pre-treated with different doses of AMPK activator A-769662 for 1 h before subjecting to UVB irradiation. All samples in (C-D) were harvested 24 h after irradiation and phosphorylated and total p70S6K/AMPK were assessed by Western blot.

Fig. 6. The effect of apigenin on UVB-induced autophagy and apoptosis

(A) Immunoblot of endogenous LC3 (LC3-I and LC3-II) in 308 cells exposed to UVB (500 J/m²) and/or pre-treated with apigenin (25 μM) for 1 h where indicated, and harvested at 24 h post irradiation. (B) 308 cell were treated as above, and the LC3 puncta (autophagosomes, white arrows) were monitored by immunofluorescence. (C) The percentage of cells with >5 LC-3 positive puncta was calculated using least 200 cells per treatment. (D) 308 cells were treated as above, or pre-treated with autophagy inhibitor chloroquine (50 μM) for 1 h before apigenin treatment and UVB irradiation, and apoptosis detected by annexin-V staining followed by flow cytometry. Mean ± SD are shown, *, P < 0.01. (E) Determination of cells in S-phase was by carried out by cell cycle analysis as described in Fig.1, except that cells were pre-treated with chloroquine as above.

Fig. 7. Signaling pathways involved in inhibition of mTOR activation by apigenin and subsequent cellular reaction in keratinocytes

UVB activates mTOR pathway through PI3K/Akt signaling, while apigenin inhibits UVB-induced mTOR activation mainly by AMPK, further induces autophagy and suppresses cell proliferation.