Isoliquiritigenin inhibits migration and invasion of prostate cancer cells: possible mediation by decreased JNK/AP-1 signaling

Structure of ISL.

EGF, but not IGF-1 or HRG-β, induces migration of DU145 cells. The migration of DU145 cells through a Type IV collagen-coated Transwell filter was assessed with 10 μg/L EGF, 10 nmol/L IGF-I or 20 μg/L HRG-β. Cells were incubated for 4 h. (A) Photographs of migrated H&E-stained cells are shown (×100). (B) Quantitative analysis of the migrated cells. Each bar represents the mean±S.E.M. (n=3). Means without a common letter differ (P<.05).

ISL decreases migration of DU145 cells — Wound migration assay. Ninety percent confluent DU145 cells were treated with 1 mg/L mitomycin C, and the injury line was made with a tip. The cells were then incubated with or without 20 μmol/L ISL in the absence or presence of 10 μg/L EGF in DMEM/F12 containing 1% charcoal-stripped FBS for 0, 6, 12 or 24 h. (A) Cell migration was observed by microscopy at the indicated time points (×40). (B) The measured width of injury lines was plotted as a percentage of the width at 0 h. Each bar represents the mean±S.E.M. (n=3). Means at a time without a common letter differ (P<.05).

ISL decreases migration and invasion of prostate cancer cells — Transwell filter migration and invasion assays. The migration of DU145 (A) and LNCaP (B) cells through a Type IV collagen coated filter was performed as described in Fig. 2. (C) DU145 cell invasion was measured with the same procedure described in Fig. 2 except that a matrigel-coated Transwell filter was used. The migrated (A, B) or invaded cells (C) were quantified by counting of H&E-stained cells. Each bar represents the mean±S.E.M. (n=3). Means without a common letter differ (P<.05).

ISL decreases MMP-9 secretion and increases TIMP-2 secretion in DU145 cells. Serum-starved DU145 cells were incubated with 0–20 μmol/L ISL in DMEM/F12 containing 0 or 10 μg/L EGF for 48 h. Forty-eight-hour conditioned media were collected and concentrated for gelatin zymography (A) and Western blotting (B). The volumes of media loaded onto the gel were adjusted for equivalent protein levels. (C) Serum-starved cells were incubated with ISL and/or EGF for 2 h. Total RNA was isolated for RT-PCR analysis. Photographs of the Commassie blue-stained gel (A), chemiluminescent detection of the blots (B) and ethidium bromide-stained gels (C), which are representative of three independent experiments, are shown. In the first lane of (A), serum-free HT 1080 cell-conditioned medium was loaded. The relative abundance of each band (B) or that to its own β-actin (C) was quantified, and the control levels were set at 100%. The adjusted mean±S.E.M. (n=3) of each band is shown above each blot. Means without a common letter differ (P<.05).

ISL decreases VEGF secretion. Serum-starved DU145 cells were incubated with 0–20 μmol/L ISL in DMEM/F12 containing 0 or 10 μg/L EGF. Twenty-four-hour conditioned media were collected for VEGF ELISA (A), and 48-h conditioned media were collected for Western blotting (B). Serum-starved cells were incubated with ISL and/or EGF for 2 h. Total RNA was isolated for RT-PCR analysis (C). Each bar represents the mean±S.E.M. (n=4) (A). Photographs of the chemiluminescent detection of the blots (B) and ethidium bromide-stained gels (C), which are representative of three independent experiments, are shown. The relative abundance of each protein band (B) and mRNA band to its own β-actin (C) was quantified, and the control levels were set at 100%. The adjusted mean±S.E.M. (n=3) of each band is shown above each blot. Means without a common letter differ (P<.05).

ISL decreases cell adhesion of DU145 cells. (A) DU145 cells were plated in human collagen Type I-coated CytoMatrix Cell Adhesion Strips and incubated in DMEM/F12 containing 1% charcoal-stripped FBS with 0–20 μmol/L ISL in the absence or presence of 10 μg/L EGF for 45 min. Cells were stained with crystal violet, and the cell-bound stains quantified. Each bar represents the mean±S.E.M. (n=3). (B) Serum-starved DU145 cells were incubated with 0–20 μmol/L ISL in serum-free media containing 0 or 10 μg/L EGF for 48 h. Total cell lysates were prepared for immunoblotting. (C) Serum-starved cells were incubated with ISL and/or EGF for 2 h. Total RNA was isolated for RT-PCR analysis. Photographs of chemiluminescent detection of the blots (B) and ethidium bromide-stained gels (C), which are representative of three independent experiments, are shown. The relative abundance of each band to its own β-actin was quantified, and the control levels were set at 100%. The adjusted mean±S.E.M. (n=3) of each band is shown above each blot. Means without a common letter differ (P<.05).

ISL inhibits EGF-induced Akt and JNK signaling in DU145 cells. Serum-starved cells were incubated with 0 or 20 μmol/L ISL for 2 h and lysed without stimulation (0) or after 15 min of stimulation with EGF. Total cell lysates were subjected to immunoblotting. Photographs of chemiluminescent detection of the blots, which are representative of three independent experiments, are shown. The relative abundance of each phosphorylated band to its own control band (ratios of pAkt/Akt, p-p38/p38, p-ERK1/2/ERK1/2, p-JNK/JNK or p-c-Jun/c-Jun) was quantified, and the control levels were set at 100%. The adjusted mean±S.E.M. (n=3) of each band is shown above each blot. Means without a common letter differ (P<.05).

The JNK inhibitor SP600125 decreases cell migration and secretion of proteins related to metastasis in DU145 cells. (A) Serum-starved cells were incubated with 0 or 10 μmol/L SP600125 for 2 h and lysed without stimulation (−) or after 15 min of stimulation (+) with EGF. Total cell lysates were prepared for immunoblotting. Photographs of chemiluminescent detection of the blots, which are representative of three independent experiments, are shown. The relative abundance of each phosphorylated band to its own control band (ratios of p-JNK/JNK or p-c-Jun/c-Jun) was quantified, and the control levels were set at 100%. The adjusted mean±S.E.M. (n=3) of each band is shown above each blot. (B) Serum-starved cells were incubated with 0 or 10 μmol/L SP600125 in serum free media with or without 10 μg/L EGF. Forty-eight-hour conditioned media were collected and concentrated for Western blotting. The volumes of media loaded onto the gel were adjusted for equivalent proteins. Photographs of chemiluminescent detection of the blots, which are representative of three independent experiments, are shown. The relative abundance of each band was quantified, and the control levels were set at 100%. The adjusted mean±S.E.M. (n=3) of each band is shown above each blot. (C) The effect of EGF and/or SP600125 on the migration of DU145 cells through a Transwell filter was assessed. Each bar represents the mean±S.E.M. (n=3). (D) Cells were treated with ISL or SP600125 for 2 h and stimulated with EGF as described above. Nuclear extracts were prepared for EMSA. An autoradiography of the dried gels, which were representative of three independent experiments, is shown. The relative abundance of each band was quantified, and the control levels were set at 100%. The adjusted mean±S.E.M. (n=3) of each band is shown above each blot. Means without a common letter differ (P<.05).