Table 1.
Primer sequences for qRT-PCR.
Figure 1.
FOXO1-GFP translocation in stably transfected U-2 OS (human osteosarcoma cells).
Intracellular localization of the forkhead box transcription factor O1 labelled with green fluorescent protein FOXO1-GFP visualized by fluorescence microscopy after nuclear staining with DAPI (colour coded red). Merged images in left panel: Cytoplasm green from FOXO1-GFP, nuclei red→orange→yellow→green depending on GFP overlay from FOXO1-GFP accumulated in nuclei (A–C). Right panel GFP images (A′–C′). (A, A′) Transfected U-2 OS cells with pEGFP-FOXO1 (after 1 h starvation in DMEM without FBS) treated with DMSO 0.15% (control) 2 h with cytoplasmic and perinuclear localization of FOXO1-GFP. (B, B′) Apigenin 30 µM in 0.15% DMSO induced nuclear accumulation of FOXO1-GFP. (C, C′) Luteolin 30 µM in DMSO 0.15% induced translocation of FOXO1-GFP from cytoplasm into nuclei in nearly all transfected U-2 OS cells with stable expression of GFP tagged FOXO1.
Figure 2.
Dose-dependent induction of FOXO-GFP translocation by apigenin and luteolin, competition by insulin.
Stably transfected human osteosarcoma cells with FOXO1-GFP (U2OS-FOXO1-GFP) treated with apigenin (A), luteolin (B) 1–100 µM for 2 h −/+ addition of insulin 100 nM after 30 minutes. Cells were fixed and stained with DAPI. Fluorescence microscopic detection of nuclei, segmentation of cells, quantification of GFP intensities measured in nuclear and cytoplasmic areas and calculation of the GFP-ratio nucleus/cytoplasm were performed for all FOXO-GFP expressing cells by BD Image Data Explorer. Nonlinear regression was performed with Graph Pad Prism. Results are presented as mean ± SEM of quadruplets with *(p<0.05) significant differences vs. control (ANOVA + Post Hoc Tests). (A) Dose-dependent accumulation of FOXO1 in nuclei induced by apigenin 1–100 µM shown as mean ratio of FOXO1 nucleus/cytoplasm + SEM (n = 4) *p<0.05 (Dunnett T3) and EC50 = 13 µM calculated by nonlinear regression from sigmoidal dose response. Reversion by insulin 100 nM induced FOXO1 translocation from nuclei into the cytoplasm. (B) Luteolin induced FOXO1 nuclear accumulation shown as mean ratio of FOXO1 nucleus/cytoplasm + SEM (n = 4) *p<0.05 (Bonferroni) with EC50 = 12 µM and competing insulin effect.
Figure 3.
Time-dependent induction of FOXO-GFP translocation by apigenin A) and luteolin B) obtained by fluorescence-microscopic life cell imaging.
Stably transfected human osteosarcoma cells with FOXO1-GFP (U2OS-FOXO1-GFP) were incubated with apigenin 10 µM (A) and transiently transfected human hepatic cells with FOXO1-GFP (HepG2-FOXO1-GFP) were incubated with luteolin 10 µM (B), respectively, for 1 h at 37°C in an incubation chamber connected to the inverted fluorescence microscope Axio Observer.Z1 from Zeiss. Images were taken every minute with the filter for GFP up to 60 minutes. FOXO1-GFP translocation with nuclear accumulation of FOXO1 is shown in a time-dependent course.
Figure 4.
Time-dependent FOXO-GFP translocation induced by apigenin and reversed by insulin.
Stably transfected human osteosarcoma cells with FOXO1-GFP (U2OS-FOXO1-GFP) were treated with apigenin 10 µM up to 1 h −/+ addition of insulin 100 nM after 30 minutes. Cells were fixed at indicated time points. GFP-ratio nucleus/cytoplasm was normalized to control at 0 minutes. Apigenin induced a significant FOXO1 nuclear translocation within 5–60 minutes of stimulation with maximal nuclear accumulation after 30 minutes. This accumulation was completely reversed by insulin during an incubation period from 30–60 minutes by competing export from nuclei into cytoplasm induced via the insulin signaling cascade. Experiments were performed in quadruplicates for each time interval and treatment condition. Cells were fixed and stained with DAPI for defining nuclear areas. Fluorescence microscopic analyses were performed in BD Pathway 435 system with BD Attovision using segmentation of cells by Cyto-Nuc Ring Band, quantification of GFP intensities measured in nuclear and cytoplasmic areas. The calculation of the GFP-ratios nucleus/cytoplasm were performed by BD Image Data Explorer. Results are presented as means of ratios normalized to control (0 minutes) ± SEM of 3 independent treatments. Significances versus control are shown for apigenin and insulin induced time dependent translocation of FOXO1 analyzed by Oneway ANOVA with Post Hoc Dunnett T3 and Bonferroni, respectively *(p<0.05).
Figure 5.
FOXO1-GFP translocation induced by apigenin and luteolin in the presence of N-acetyl-L-cysteine, reduced reversion by insulin.
Stably transfected human osteosarcoma cells with FOXO1-GFP (U2OS-FOXO1-GFP) were incubated with the antioxidant N-acetyl-L-cysteine (NAC) 5 mM and 25 mM for 30 minutes before treatment with apigenin 30 µM and luteolin 30 µM −/+ insulin 100 nM for 2 h and 24 h respectively. Cells were fixed and stained with DAPI. Experiments were performed in quadruplets and fluorescence microscopic analyses performed with the BD Pathway 435 system, BD Attovision and BD Image Data Explorer. GFP-ratios nucleus/cytoplasm were normalized to untreated control cells. Results are presented as relative FOXO1 translocations resulting from means of ratios GFP Nuc/Cyt normalized to control ± SEM. Oneway ANOVA and Dunnett T3 significances are shown *(p<0.05), **(p<0.01), and ***(p<0.001) in blue for flavone induced FOXO1 nuclear accumulation vs control DMEM, in red for insulin induced FOXO1 export into cytoplasm, and in yellow for NAC effect on FOXO1 translocation.
Table 2.
Cell proliferation assay.
Figure 6.
Time- and dose-dependent modulation of gene expression in HepG2 cells induced by apigenin and luteolin.
A–D: Human hepatoma cells (HepG2) were cultivated in EMEM + 10% FBS and starved without FBS 16 h before stimulation. Apigenin and luteolin were applied in the range of 1–100 µM diluted in EMEM. Incubation of HepG2 was performed for 2 h and 24 h respectively. Total RNA was extracted with Nucleospin RNA II isolation kit and reverse transcribed with the High capacity cDNA reverse transcription kit for quantitative realtime PCR (qRT-PCR) in triplicates using the Power SYBR green PCR master mix with primers pairs described in Table 1. qRT-PCR was run in triplicates using cDNA from control cells treated with DMSO 0.5% for standard dilutions. Levels of mRNA were normalized to the houskeeping gene ribosomal protein (RPL32). Three independent experiments were performed with different passages of HepG2. Results are presented as fold mRNA expression normalized to control expression as means ± SEM and significances versus control *(p<0.05). Gluconeogenic (A) phosphoenolpyruvate carboxykinase (PEPCK) and (B) glucose-6-phosphatase (G6Pc), lipogenic (C) fatty-acid synthase (FASN) and (D) acetyl-CoA-carboxylase (ACC).
Figure 7.
FOXO targetgene-expression in HepG2 (human hepatoma) cells modulated by polyphenolic resveratrol in a time-dependent course.
A–B: HepG2 cell cultures grown in EMEM + FBS 10% and starved for 16 h without FBS were stimulated with resveratrol 50 µM in 0.125% DMSO in EMEM for 1–24 h. RNA was extracted with Nucleospin RNA II isolation kit and reverse transcribed with the High capacity cDNA reverse transcription kit for quantitative realtime PCR (qRT-PCR) in triplicates using the Power SYBR green PCR master mix with primers pairs described in Table 1. Modulated mRNA levels normalized to ribosomal protein (RPL32) housekeeping gene are shown as fold mRNA of basal expression in mock stimulated HepG2 means ± SEM (n = 3) of 3 independent experiments with different passages of HepG2 with significances (t-test) versus DMSO-control *p<0.05. (A) gluconeogenic phosphoenolpyruvate carboxykinase (PEPCK) up-regulated by resveratrol in a biphasic manner (1–4 h and 16–24 h) and glucose-6-phosphatase (G6Pc) with continuous increase (4–24 h), (B) lipogenic fatty-acid synthase (FASN) and acetyl-CoA-carboxylase (ACC) slight induction 8–24 h and 16–24 h respectively.
Table 3.
siRNA-knockdown.
Figure 8.
Gene expression profiling upon siRNA knockdowns and analysis of modulations by apigenin and luteolin.
A–D: Subconfluent human hepatoma cells (HepG2) were transfected with silencing RNA (siRNA) for forkhead box transcription factor O1 (FOXO1), forkhead box transcription factor O3a (FOXO3a), sirtuin1 (SIRT1), protein kinase B (PKB/AKT), nuclear factor (erythroid-derived2)-like2 (NRF2) and non targeting (NT)-siRNA with DharmaFECT4 in EMEM + 10% FBS for 48 h including a starvation period without FBS of 16 h preceeding stimulation with apigenin and luteolin each 20 µM for 24 h. RNA was extracted, reverse transcribed and cDNA from control cells after treatment with DMSO 0.1% were used for standard dilutions. For 14 targets qRT-PCR was run with SYBR green in triplicates. Levels of mRNA were normalized to the expression of houskeeping ribosomal protein (RPL32) mRNA. At least four experiments (n = 4–8) transfecting each siRNA or combined siRNAs for single and double knockdowns and control transfections with NT-siRNA followed by apigenin, luteolin or mock treatment with DMSO 0.1% were performed with different passages of HepG2. Ratios of mRNA levels vs basal expression in NT-siRNA transfected cells were calculated for knockdown induced fold mRNA of basal levels (grey columns). T-tests were performed for independent samples and significances versus control are shown #p<0.05, ## p<0.01 and ###p<0.001 according to Levene statistics for equality of variances with corrections for equal variances. Expression profiles are shown for DMSO mock stimulation (grey) apigenin 20 µM (blue) and luteolin 20 µM (red) + SEM and significant differences upon each knockdown condition obtained by Oneway ANOVA and posthoc tests with multiple comparisons Dunnet T3 for Levene unequal or Bonferroni for Levene equal variances indicated as *p<0.05, **p<0.01 and ***p<0.001 (blue apigenin vs DMSO, red luteolin vs DMSO, purple apigenin vs luteolin). (A) phosphoenolpyruvate carboxykinase (PEPCK)-, (B) glucose-6-phosphatase (G6Pc)-, (C) fatty-acid synthase (FASN)-, (D) acetyl-CoA carboxylase (ACC).
Figure 9.
Posttranslational phosphorylation in the AKT-signaling cascade.
A–E: Modifications of molecules at nodal points of AKT intracellular signaling were analysed in lysates of human hepatoma (HepG2) cells treated with apigenin 20 µM, luteolin 20 µM or DMSO 0.1% for 30′ ± pretreatment with insulin 100 nM for 15′. Cell lysis was performed in presence of phosphatase inhibitors and lysates analyzed for protein phosphorylation or cleavage using the PathScan Intracellular Signaling Array Kit (fluorescent readout) from Cell Signaling technology. Data are shown as means of integrated intensities of three independent experiments (n = 3) + SEM normalized to untreated control cells and significances shown as # p<0.05 and ## p<0.01 vs mock stimulated HepG2 with DMSO 0.1% (T-Test) or * p<0.05, ** p<0.01 and ***p<0.001 as indicated (Oneway ANOVA and posthoc Bonferroni or Dunnett T3 multiple comparisons). (A) protein kinase B PKB/AKT (Thr308), (B) AKT (Ser473), (C) proline-rich AKT/PKB substrate 40 kDa PRAS40 (Thr246), (D) mammalian target of rapamycin mTor (Ser2448), (E) p70S6 kinase p70S6K (Thr389), (F) ribosomal protein S6 (Ser235/236).
Figure 10.
Effect of apigenin on the auto-phosphorylation of the IGF-1 receptor.
Human embryonic kidney (HEK) cells overexpressing the insulin-like growth factor receptor (IGF-1R) were incubated with different concentrations of IGF-1 in the presence or absence of apigenin 20 µM for 16 minutes. Stimulated cells were lysed and transferred to a normal ELISA-sandwich assay using a mouse monoclonal IGF-1R antibody as a capture antibody and anti-mouse horseradish peroxidase-conjugated anti-phosphotyrosine monoclonal antibody as a detection antibody to quantify the phosphorylation of IGF-1 receptor. Bars show means ± SEM of two experiments performed in duplicate.