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. 2016 Feb 23;4(2):e00212.
doi: 10.1002/prp2.212. eCollection 2016 Apr.

Deguelin-induced blockade of PI3K/protein kinase B/MAP kinase signaling in zebrafish and breast cancer cell lines is mediated by down-regulation of fibroblast growth factor receptor 4 activity

Wei Wu  1 , Yang Hai  1 , Lu Chen  2 , Rui-Jin Liu  1 , Yu-Xiang Han  2 , Wen-Hao Li  1 , Song Li  2 , Shuo Lin  3 , Xin-Rong Wu  1
Affiliations

Deguelin-induced blockade of PI3K/protein kinase B/MAP kinase signaling in zebrafish and breast cancer cell lines is mediated by down-regulation of fibroblast growth factor receptor 4 activity

Wei Wu et al. Pharmacol Res Perspect. .

Abstract

Deguelin, a natural component derived from leguminous plants, has been used as pesticide in some regions. Accumulating evidence show that deguelin has promising chemopreventive and therapeutic activities against cancer cells. This study shows that low concentrations of deguelin can lead to significant delay in zebrafish embryonic development through growth inhibition and induction of apoptosis. Furthermore, we identified fibroblast growth factor receptor 4 (FGFR4) as the putative target of deguelin. The candidate was initially identified by a microarray approach and then validated through in vitro experiments using hormone-responsive (MCF-7) and nonresponsive (MDA-MB-231) human breast cancer cell lines. The results show that deguelin suppressed cell proliferation and induced apoptosis in both cancer cell lines, but not in Hs 578Bst cells, by blocking PI3K/AKT and mitogen-activated protein kinases (MAPK) signaling. The FGFR4 mRNA and protein level also diminished in a dose-dependent manner. Interestingly, we found that forced FGFR4 overexpression attenuated deguelin-induced proliferative suppression and apoptotic cell death in both zebrafish and MCF-7 cell lines, p-AKT and p-ERK levels were restored upon FGFR4 overexpression. Taken together, our results strongly suggest that deguelin inhibition of PI3K/AKT and MAPK signaling in zebrafish and breast cancer cell lines is partially mediated through down-regulation of FGFR4 activity.

Keywords: Breast cancer; FGFR4; deguelin; zebrafish.

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Figures

Figure 1
Figure 1
Growth repression and apoptosis induction caused by deguelin. (A) Morphological change in zebrafish with or without deguelin treatment. Significant growth retardation can be found in 200 and 500 nmol/L deguelin‐treated group. (B) Whole‐mount embryos labeled with anti‐pH3 antibody to examine proliferating cells in zebrafish larvae. The numbers of pH3‐positive cells decreased dramatically and rarely expressed with 200 nmol/L deguelin treatment (magnification 50×). (C) Phenotypic assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. There was a dose‐dependent increase of apoptotic cells in TUNEL assay. (magnification 50×).
Figure 2
Figure 2
Microarray analysis. Fibroblast growth factor receptor 4 (FGFR4) is substantially down‐regulated after deguelin treatment.
Figure 3
Figure 3
Reduced levels of FGFR4 and related downstream genes induced by deguelin. (A) Real‐time reverse transcription‐PCR for FGFR4 was conducted to examine FGFR4 mRNA expression. Deguelin dose‐dependently suppressed FGFR4 release, which was validated by positive control group. Three individual experiments were conducted. Each bar indicates the mean ± SD. *P < 0.05; **P < 0.01;***P < 0.001 (t‐test). Each drug‐treated group was compared to respective control group with DMSO treatment to decide its significance. FGFR inhibitor SU5402 was used as a positive control. (B) Immunoblot was performed with specialized antibodies for FGFR4, p‐AKT, p‐ERK, and ERK to detect the protein levels. The results showed that deguelin inhibited FGFR4 expression and constitutive phosphorylation of AKT and ERK in zebrafish. FGFR inhibitor SU5402 was used as a positive control. Bar graph, Density analysis results from each concentration in western Blot. a.u. represent arbitrary units. *P < 0.05; **P < 0.01;***P < 0.001 (t‐test). Each drug‐treated group was compared to control group with DMSO treatment to decide its significance. FGFR4, fibroblast growth factor receptor 4; PCR, polymerase chain reaction; p‐AKT, phospho‐protein kinase B; p‐ERK, phospho‐extracellular regulated protein kinases; DMSO, dimethyl sulfoxide.
Figure 4
Figure 4
Growth inhibition of MCF‐7 and MDA‐MB‐231 induced by deguelin. (A) MTT assay. MCF‐7, MDA‐MB‐231 and Hs 578Bst were exposed to deguelin at indicated concentrations (0–500 nmol/L) for 24, 48, 72 h. Cell viability was measured by a spectrophotometer. Each spot indicates the mean ± SD of 6 samples. *P < 0.05; **P < 0.01;***P < 0.001 (t‐test). Each drug‐treated group was compared to respective control group with DMSO treatment to decide its significance. (B) colony formation assay. The breast cancer cells were treated in the presence or absence of deguelin for 14 days, cell colonies were labeled by crystal violet and counted in Image J. Each bar indicates the mean ± SD of 3 samples. *P < 0.05; **P < 0.01;***P < 0.001 (t‐test). Each drug‐treated group was compared to respective control group with DMSO treatment to decide its significance. MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide; DMSO, dimethyl sulfoxide.
Figure 5
Figure 5
Induction of apoptosis by deguelin in MCF‐7 and MDA‐MB‐231. (A) Deguelin induced a dose‐dependent apoptosis in breast cancer cells determined by in situ cell death detection and Hoechst 33258 staining. In upper pictures of each group, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)‐positive cells increased with rising concentrations of deguelin treatment (magnification 100×). Pictures in the bottom right‐hand corner of each TUNEL image were amplified to show the morphology more clearly (magnification 400×). Likewise, all kinds of cells with the same treatment of deguelin were labeled with Hoechst 33258, condensed chromatin and apoptotic nucleus fragmentations were observed in breast cancer cells. (B) Deguelin exerted marginal toxic influence on normal cells Hs 578Bst. TUNEL and Hoechst 33258 assays were applied to Hs 578Bst as indicated, the morphology of Hs 578Bst cells were as normal as the control group after deguelin treatment. Three independent experiments were made to validate the result.
Figure 6
Figure 6
The reduced expression of FGFR4 in breast cancer cells after the treatment of deguelin. (A) Real‐time reverse transcription–PCR for FGFR4 expression in two breast cancer cells. After deguelin treatment for 48 h, the RNA was extracted and detected by real‐time RT‐PCR. FGFR4 expression gradually decreased in a dose‐dependent manner. Three individual experiments were conducted. Each bar indicates the mean ± SD. *P < 0.05; **P < 0.01;***P < 0.001 (t‐test). Each drug‐treated group was compared to respective control group with DMSO treatment to decide its significance. (B) Western blot analysis on FGFR4 and related gene protein expression in MCF‐7 and MDA‐MB‐231. The cells cultured with 0–500 nmol/L deguelin for indicated time witnessed a declined expression of FGFR4, p‐AKT, and p‐ERK without effects on total ERK expression, which was validated by the positive‐control group (SU5402). Bar graph, Density analysis results from each concentration in western Blot. a.u. represent arbitrary units. *P < 0.05; **P < 0.01;***P < 0.001 (t‐test). Each drug‐treated group was compared to control group with DMSO treatment to decide its significance. FGFR4, fibroblast growth factor receptor 4; PCR, polymerase chain reaction; DMSO, dimethyl sulfoxide.
Figure 7
Figure 7
The counteractant effect of overexpressing FGFR4 in zebrafish after deguelin treatment. (A) Zebrafish embryos were labeled with anti‐pH3 antibody after injection of pEGFP‐C3 containing FGFR4 (Z‐FGFR4 and H‐FGFR4 stand for zebrafish and human FGFR4, respectively) to detect proliferating cells. DyLight 594 secondary antibody was used to avoid green fluorescence emitted by pEGFP‐C3 vector. Up‐regulation of FGFR4 has partly restored proliferating cells compared with the control group. PH3‐positive cells are counted in Image J. **P < 0.01 (t‐test) (B). TUNEL assay was conducted to analyze the apoptosis after the injection of FGFR4. Apoptotic cells were reduced in the trunk areas in injected groups. FGFR4, fibroblast growth factor receptor 4; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.
Figure 8
Figure 8
The counteractant effect of overexpressing FGFR4 in MCF‐7 cells after deguelin treatment. (A) MTT assay. MCF‐7 cancer cells transfected of pEGFP‐C3 with or without FGFR4 were treated with deguelin for 2 days. Transfected group showed better cell viability. Each bar indicates the mean ± SD of 6 samples. *P < 0.05, t‐test. (B) In situ cell death detection and Hoechst 33258 staining were performed to detect apoptotic cells. The apoptosis were dramatically decreased in the transfected group. FGFR4, fibroblast growth factor receptor 4; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide.
Figure 9
Figure 9
The effect of FGFR4 overexpression on PI3K/AKT/MAPK pathway. The injection in zebrafish embryos or transfected in MCF‐7 cells with human FGFR4 could regain the expression levels of p‐AKT and p‐ERK. Bar graph, Density analysis results from each concentration in western Blot. a.u. represent arbitrary units. ***P < 0.001 (t‐test). Each drug‐treated group was compared to control group with DMSO treatment to decide its significance. FGFR4, fibroblast growth factor receptor 4; PI3K, phosphoinositide 3‐kinase; AKT, protein kinase B; MAPK, mitogen‐activated protein kinases; DMSO, dimethyl sulfoxide.
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