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. 2016 Sep;37(9):918-928.
doi: 10.1093/carcin/bgw071. Epub 2016 Jun 22.

Dietary Flavonoid Fisetin Increases Abundance of High-Molecular-Mass Hyaluronan Conferring Resistance to Prostate Oncogenesis

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Dietary Flavonoid Fisetin Increases Abundance of High-Molecular-Mass Hyaluronan Conferring Resistance to Prostate Oncogenesis

Rahul K Lall et al. Carcinogenesis. .
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Abstract

We and others have shown previously that fisetin, a plant flavonoid, has therapeutic potential against many cancer types. Here, we examined the probable mechanism of its action in prostate cancer (PCa) using a global metabolomics approach. HPLC-ESI-MS analysis of tumor xenografts from fisetin-treated animals identified several metabolic targets with hyaluronan (HA) as the most affected. Efficacy of fisetin on HA was then evaluated in vitro and also in vivo in the transgenic TRAMP mouse model of PCa. Size exclusion chromatography-multiangle laser light scattering (SEC-MALS) was performed to analyze the molar mass (Mw) distribution of HA. Fisetin treatment downregulated intracellular and secreted HA levels both in vitro and in vivo Fisetin inhibited HA synthesis and degradation enzymes, which led to cessation of HA synthesis and also repressed the degradation of the available high-molecular-mass (HMM)-HA. SEC-MALS analysis of intact HA fragment size revealed that cells and animals have more abundance of HMM-HA and less of low-molecular-mass (LMM)-HA upon fisetin treatment. Elevated HA levels have been shown to be associated with disease progression in certain cancer types. Biological responses triggered by HA mainly depend on the HA polymer length where HMM-HA represses mitogenic signaling and has anti-inflammatory properties whereas LMM-HA promotes proliferation and inflammation. Similarly, Mw analysis of secreted HA fragment size revealed less HMM-HA is secreted that allowed more HMM-HA to be retained within the cells and tissues. Our findings establish that fisetin is an effective, non-toxic, potent HA synthesis inhibitor, which increases abundance of antiangiogenic HMM-HA and could be used for the management of PCa.

Figures

Figure 1.
Figure 1.
Identification of HA as a unique target of fisetin. ( A ) Metabolites of NB11 and NB26 xenograft tissues with or without fisetin treatment ( n = 6 animals/group) analyzed using untargeted metabolomics (HPLC–ESI–MS) in positive-ion mode. A representative list of 20 metabolites identified from the METLIN database is shown; m/z = 776.25 was identified as HA (Student’s t -test; * P ≤ 0.1). For the complete list of metabolites, please see Supplementary Table 8 , available at Carcinogenesis Online (MS excel file). ( B ) HPLC separation (retention time = 2546.73) and relative intensity of HA metabolite peak identified in NB26 xenografts between control versus fisetin-treated animals. All six biological replicates showed similar analytical separation between the control and treated groups and a representative picture is shown. ( C ) Comparative plot of relative abundance of HA between control and fisetin-treated animal groups in NB11 and NB26 xenografts. Error bars represent mean ± SEM among six biological replicates (fisetin dose was 1mg/animal thrice weekly; * P ≤ 0.1). ( D ) Representative images showing immunofluorescence for HA between control and fisetin-treated NB11 and NB26 xenografts. Tumor tissues were harvested, and each of the six biological replicates was performed in triplicates for HA staining. Images were captured by a confocal microscope as described in Materials and methods. Scale bar, 30 µm. Magnification for NB11 xenograft images are at ×20 and for NB26 xenograft images at ×40. 4′,6-Diamidino-2-phenylindole (DAPI) was used as a nuclear staining control. Fluorescence intensity was measured and plotted (see Supplementary Figure 2 , available at Carcinogenesis Online).
Figure 2.
Figure 2.
Fisetin decreases abundance of HA in PCa cells. ( A ) Representative images showing immunofluorescence for HA in NB11, NB26, PC3 and DU145 cells with or without fisetin (40 µM) treatment for 48h. Three biological replicates for each were subjected to staining and performed in triplicate. Images were captured by a confocal microscope as described in Materials and methods. Scale bar, 30 µm. Magnification for NB11 images are at ×20 and for NB26 images at ×40. 4′,6-Diamidino-2-phenylindole (DAPI) was used as a nuclear staining control. Fluorescence intensity was measured and plotted (see Supplementary Figure 3 , available at Carcinogenesis Online). ( B ) Histogram showing intracellular HA levels in NB11, NB26, PC3 and DU145 cells measured with or without fisetin (40 µM) treatment for 48h. Error bars represent mean ± SEM among three independent experiments/group and each of the three biological replicate was performed in triplicate to measure HA levels using ELISA ( **P ≤ 0.01). All samples were normalized using 100 µg protein of starting whole cell lysates. ( C ) Histogram showing extracellular HA levels secreted in NB11, NB26, PC3 and DU145 cell culture media with or without fisetin treatment (40 µM) for 48h. Error bars represent mean ± SEM among three independent experiments/group, and each of the three biological replicate was performed in triplicate to measure HA levels using ELISA (** P ≤ 0.01). All samples were normalized using 1ml of collected cultured media.
Figure 3.
Figure 3.
Fisetin decreases HA levels in vivo . ( A ) Representative images showing immunofluorescence for HA between TRAMP and TRAMP + fisetin groups with increasing age and PCa progression. Prostatic tumor tissues were harvested from three biological replicates and performed in triplicate for HA staining. Images were captured by a confocal microscope as described in Materials and methods. Scale bar, 30 µm; magnification: ×20. 4′,6-Diamidino-2-phenylindole (DAPI) was used as a nuclear staining control. ( B ) HA expression in Figure 3A graphed as relative fluorescence intensity (arbitrary unit) between control and fisetin-treated TRAMP animals with increasing age and PCa progression. Error bars represent mean ± SEM of three biological replicates/group, and each replicate was performed in triplicate for HA staining. Statistical differences were seen in fisetin treatment groups when compared with the respective control group (** P ≤ 0.01). ( C ) Histogram showing HA levels (% control) secreted in TRAMP mice serum between vehicle- and fisetin-treated groups (1mg/animal; 3 times/week) with increasing stages of PCa progression (PIN, prostatic intraepithelial neoplasia; WDA, well-differentiated carcinoma; MDA, moderately differentiated carcinoma; PDA, poorly differentiated carcinoma). Error bars represent mean ± SEM among three biological replicates/group, and each biological replicate was performed in triplicate for HA levels using ELISA (** P ≤ 0.01).
Figure 4.
Figure 4.
Fisetin reduces HA synthesis and degradation enzymes both in vitro / in vivo . ( A ) Histograms represent relative HAS (1, 2 and 3) and HYAL (1, 2, 3 and 4) mRNA expression in NB11 and NB26 cells treated with or without fisetin (40 µM). Gene expression was measured at 6h by qPCR and normalized to housekeeping control, GapDH. Error bars represent mean ± SEM among three independent experiments, and each experiment was performed in triplicate. Statistical difference was seen in gene expression on fisetin treatment when compared with the respective control (* P ≤ 0.05, ** P ≤ 0.01). ( B ) Immunoblot images of NB11 and NB26 cells treated with or without increasing doses of fisetin (0–10–20–40 µM) for 48h and HAS (2 and 3) and HYAL (1, 2 and 3) protein expression were measured. Three independent experiments were performed, and each experiment was analyzed in triplicate. β-Actin was used as a loading control. Quantitative densitometry was analyzed and plotted (see Supplementary Figure 4 , available at Carcinogenesis Online). ( C ) Histograms represents relative HAS2 and HAS3 mRNA expression measured by qPCR in prostatic tissues of control and fisetin (1mg/animal thrice weekly) treated TRAMP animals with increasing age and PCa progression. 18sRNA was used as a housekeeping gene for normalization. Error bars represent mean ± SEM among three independent experiments, and each experiment was performed in triplicates (* P ≤ 0.05, ** P ≤ 0.01). ( D ) Histogram showing intracellular ROS generation levels (% control) with or without fisetin treatment (40 µM) in a time-dependent manner (0–0.5–2–4–6–12–24–48h). H 2 O 2 (40 µM) was used as a positive control. Error bars represent mean ± SEM among three independent experiments, and each experiment was performed in triplicate (* P ≤ 0.05).
Figure 5.
Figure 5.
Fisetin increases the abundance of HMM-HA in PCa cells. ( A ) SEC-MALS chromatogram showing molar mass distribution profile of HA fragments between normal RWPE1 and cancerous PC3 cells. Refractive index traces (solid line) and the molar mass values calculated from the light scattering data (dotted clusters) are shown, indicating the relative amount and size of HA present in RWPE1 and PC3 cells. The size of HA fragments is represented as HA-1 (HMM-HA clusters), HA-2 (medium-molar-mass HA clusters) and HA-3 (LMM-HA clusters). BSA was used as an internal standard. HA was isolated from three independent experiments and analyzed using SEC-MALS. ( B ) Intracellular and extracellular HA was isolated from cells and culture media with or without fisetin treatment (40 µM; 48h) and analyzed using SEC-MALS to generate a molar mass distribution profile of HA fragments. Refractive index traces (solid line) and the molar mass values calculated from the light-scattering data (dotted clusters) are shown, indicating the relative amount and size of HA present in PC3 cells (top left) and PC3 cells + fisetin (40 µM, 48h; top right). Similarly, secreted HA sizes are shown in the cell culture media of PC3 cells (bottom left) and PC3 cells + fisetin (40 µM, 48h; bottom right). The size of HA fragments is represented as HA-1 (HMM-HA clusters), HA-2 (medium molar mass HA clusters) and HA-3 (LMM-HA clusters). BSA was used as an internal standard. HA was isolated from three independent experiments and analyzed using SEC-MALS.
Figure 6.
Figure 6.
Fisetin increases abundance of HMM-HA in TRAMP mouse model. ( A ) Intracellular HA was isolated from tumors (top) of 24 week TRAMP and TRAMP + fisetin animals ( n = 3 biological replicates/group) and analyzed using SEC-MALS. Refractive index traces (solid line) and the molar mass values calculated from the light-scattering data (dotted clusters) are shown, indicating the relative amount and size of intact HA present in both animal groups. The size of HA fragments is represented as HA-1 (HMM-HA clusters), HA-2 (medium-molar-mass HA clusters) and HA-3 (LMM-HA clusters). BSA was used as an internal standard. ( B ) Extracellular HA (bottom) was isolated from serum of 24 week TRAMP and TRAMP + fisetin animals ( n = 3 biological replicates/group) and analyzed using SEC-MALS. Refractive index traces (solid line) and the molar mass values calculated from the light-scattering data (dotted clusters) are shown, indicating the relative amount and size of secreted HA in serum of both animal groups. The size of HA fragments is represented as HA-1 (HMM-HA clusters), HA-2 (medium-molar-mass HA clusters) and HA-3 (LMM-HA clusters). BSA was used as an internal standard.

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