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A short peptide reverses the aggressive phenotype of prostate cancer cells
Author’s Accepted Manuscript A short peptide reverses the aggressive phenotype of prostate cancer cells Hongjiao Liu, Xiaomian Lin, Tao Huang, Li Song, Cairong Zhu, Hongmin Ma, Tianzhu Long, Huixuan Zeng, Rongzhen Li, Heng Wang, Yishan Huang, Liankuai Chen, Xiaoping Wu www.elsevier.com/locate/ejphar
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S0014-2999(18)30530-2 https://doi.org/10.1016/j.ejphar.2018.09.013 EJP71982
To appear in: European Journal of Pharmacology Received date: 22 May 2018 Revised date: 5 September 2018 Accepted date: 5 September 2018 Cite this article as: Hongjiao Liu, Xiaomian Lin, Tao Huang, Li Song, Cairong Zhu, Hongmin Ma, Tianzhu Long, Huixuan Zeng, Rongzhen Li, Heng Wang, Yishan Huang, Liankuai Chen and Xiaoping Wu, A short peptide reverses the aggressive phenotype of prostate cancer cells, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.09.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A short peptide reverses the aggressive phenotype of prostate cancer cells
Hongjiao Liua1 •Xiaomian Linb•Tao Huangb •Li Songb • Cairong Zhua1 •Hongmin Maa1 •Tianzhu Longa•Huixuan Zengb •Rongzhen Lib • Heng Wangb •Yishan Huang •Liankuai Chenb •Xiaoping Wub2
a
Guangzhou Women and Children’s Medical Center, Guangzhou Medical University,
Guangzhou 510623, China b
Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou
510632, China [email protected] (C. Zhu) [email protected] (X. Wu)
ABSTRACT Previous studies have demonstrated that fibroblast growth factor 8b (FGF8b) is up-regulated in a large proportion of prostate cancer patients, and plays a key role in the aggressive progress of prostate cancer. Herein, we investigated the effects of a short peptide derived from the gN helix domain of FGF8b on the metastatic behaviors of prostate cancer cells. The results demonstrated that the synthetic peptide might 1 2
Hongjiao Liu, Xiaomian Lin, and Tao Huang have contributed equally to this work. Tel: 8620-85220732 1
reverse the effects of FGF8b on cell proliferation, migration and invasion by suppressing the activation of MAPK and Akt signaling cascades, and reducing the expression of the metastasis-related proteins, resulting in the improvement of the aggressive phenotype of the prostate cancer cells. Collectively, these results underline the therapeutic potential of the FGF8b mimic peptide in advanced prostate cancer.
Keywords: fibroblast growth factor 8; aggressive phenotype; prostate cancer
1. Introduction Prostate cancer is the second most common type of cancer in men, with more frequent occurrence in the developed countries and increasing rates in the developing world (Baade, et al. 2009). Hormonal therapy may be effective. However, metastatic recurrence is often hormone-refractory, resulting in shorter median survival (Koo, et al. 2015). Indeed, metastasis-relevant events like vessel invasion, positive metastatic lymph nodes, and bone metastasis have been observed in advanced prostate cancer patients (Gdowski, et al. 2017; Quayle, et al. 2015). Preventing metastasis of the advanced prostate cancer is still a great challenge in clinic (Lassi and Dawson 2011). It has been proposed that cell proliferation, motility and invasion of tumor cells are involved in the metastatic process. But the pivotal factors that modulate the metastasis in prostate cancer and the related targeting agents remain to be explored. Compelling evidence indicated that fibroblast growth factor 8b (FGF8b), the major isoform of FGF8 up-regulated in prostate cancer (Gnanapragasam, et al. 2003; 2
Valta, et al. 2008), plays a key role in conferring an aggressive phenotype to prostate cancer cells via binding and activating the tyrosine kinase FGF receptors (FGFRs) (Dorkin, et al. 1999; Mattila and Harkonen 2007; Song, et al. 2000). Therefore, FGF8b/FGFRs signal axis has been considered as the potential target for reversing metastatic behaviors of prostate cancer. We have demonstrated previously that a short peptide (named 8b-13) derived from the gN helix domain of FGF8b responsible for the high affinity and specificity of FGF8b to its receptors has potentials of antagonizing the bioactivity of FGF8b (Li, et al. 2013). Herein, we further investigated the effects of 8b-13 peptides on FGF8b-mediated aggressive behaviors of prostate cancer cells.
2. Materials and Methods 2.1 Materials Human FGF8b was purchased from PeproTechInc (Rocky Hill, NJ, USA). The 8b-13 peptide (PNFTQHVREQSLV) and its scrambled version H13 (QRVSQFENHPVTL) with the purity greater than 98% were synthesized at SBS Genetech (Beijing, China). The cell proliferation assay reagent MTT was obtained from Sigma (CA, USA). Anti-FGF8b antibody was purchased from Millipore (Billerica, MA, USA).Anti-phospho-Erk1/2, anti-Erk1/2, anti-phospho-P38, anti-P38, anti-phospho-Akt, anti-Akt, anti-GAPDH antibodies and horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody were obtained from Cell Signaling Technology (Danvers, MA, USA).Erk1/2 inhibitor (U0126), Akt inhibitor 3
(LY294002) and P38 inhibitor (SB202190) were the products of Selleck (Houston, Texas, United States). The polyvinylidenedifluoride (PVDF) membrane was from Millipore (Billerica, MA, USA). The enhanced chemiluminescence (ECL) detection kit was the product of Pierce (Rockford, IL, USA). TRIZOL was purchased from Life technologies (New York, USA). RevertAid First strand cDNA synthesis kit was from Thermo Scientific (Waltham, MA, USA),and SYBR green Q-PCR kit was purchased from Bio-Rad (Hercules, CA, USA). Matrigel was purchased from BD Biosciences (San Jose, CA, USA).
2.2 Methods 2.2.1 Cell culture The prostate cancer cell line PC-3M was kept by the Institute of Tissue Transplantation and Immunology of Jinan University, and TRAMP-C2 cell line was kindly provided by Dr. Fen Wang in the Institute of Biosciences and Technology, Texas A&M Health Science Center. The chronicmyelogenous leukemia cell line K562 was a gift from Professor Yifei Wang in Department of Cellular Biology of Jinan University. All cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) and maintained in a humidified atmosphere containing 5% CO2 at 37 °C.
2.2.2 Detection of the endogenous FGF8b and FGFRs The PC-3M, TRAMP-C2, and K562 cells were plated in 6-well plates. After 4
cultured for 48 h, cells were lysed with 1×S.D.S-PAGE loading buffer. The lysate was syringed, boiled for 5 min and subjected to SDS–PAGE followed by transferal to PVDF membranes at a current 350 mA for 70 min. The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h, probed with rabbit monoclonal FGF8b antibody at room temperature for 1 h followed by a HRP conjugated goat anti-rabbit secondary antibody before visualization with ECL reagents. The results were analyzed by Quantity One software to determine the relative ratio and presented in graphs as expression relative to that of GAPDH. Real-Time quantitative PCR was further applied to determine the expressions of FGFRs. Briefly, One microgram of total RNA extracted from PC-3M and TRAMP-C2 cells by TRIZOL was reverse-transcribed using the First-Strand cDNA Synthesis Kit to produce cDNA, which was used as a template for quantitative PCR amplification with the SYBR green qPCR Kit. The quantitative PCR was performed using the CFX96 Touch Deep Well real-time PCR Detection System as follows: 1 cycle of 95℃ for 3 min; 40 cycles of (95℃ for 15 s, 58℃ for 5 s and 72℃ for 10 s). The mRNA levels of FGFRs were expressed as the relative ratio to the GAPDH mRNA level.
2.2.3 Cell viability assay PC-3M and TRAMP-C2 cells were seeded in 96-well plates at a density of 5×104 cells per well, and allowed to attach overnight. After starved for 24 h, cells were treated with serial dilutions of the 8b-13 or H13 peptides, 40 ng/ml FGF8b alone, or 5
40 ng/ml FGF8b plus with serial dilutions of the 8b-13 or H13 peptides. Starved cells were pretreated with the 8b-13 for 10 min before addition of FGF8b. Cell viability was measured 48 h later using the methylthiazoletetrazolium (MTT) assay.
2.2.4 Analysis of Akt and MAP kinase activation PC-3M and TRAMP-C2 cells were seeded in 6-well plates at a density of 5×104 cells per well for western blot analysis. Briefly, after starved cultivation in DMEM with 0.4% FBS for 24 h, cells were pretreated with the 8b-13 for 10 min before the addition of FGF8b (40 ng/ml) for 15 min. Cells were collected and lysed in 1×SDS-PAGE loading buffer. The lysate was subjected to run on SDS–PAGE gels followed by transferal to PVDF membranes. The membrane was blocked with 5% non-fat dry milk at room temperature for 1 h, probed with the primary antibodies at 4℃ overnight followed by incubation with HRP-linked goat anti-rabbit IgG for 1 h at room temperature. An ECL detection kit was applied to detect the blots according to the manufacturer’s procedure. The results were analyzed by Quantity One software to determine the relative ratio as presented in graphs.
2.2.5 In vitro wound healing assay PC-3M and TRAMP-C2 cells were seeded into 6-well plates at a density of 1×105 cells per well and allowed to adhere completely. Cells were starved for 24 h in DMEM with 0.4% FBS. A sterilized 200-μl disposable pipette tip was used to scrape the cell monolayer in a straight line to create a scratch wound in each well. After 6
washing three times with PBS to remove debris, cells were pretreated with 125 nM 8b-13, 10 μM U0126, 10 μM LY294002, or 10 μM SB202190 for 15 min before addition of 40 ng/ml FGF8b. The images were acquired using a phase-contrast microscope at defined time frames, and the scratch distance was quantitatively analyzed using the Image Pro-Plus 6.0 software. The migration rate was calculated following the formula: Migrationrate= [𝑠𝑐𝑟𝑎𝑡𝑐ℎ 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 (0 ℎ)－𝑠𝑐𝑟𝑎𝑡𝑐ℎ 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 (𝑛 ℎ)] / 𝑠𝑐𝑟𝑎𝑡𝑐ℎ 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 (0 ℎ)
2.2.6 Cell invasion assay Transwell chambers (24-well) were applied for cell invasion assay. After Matrigel (1:20) was coated on the filter of the transwell chambers and incubated at 37 °C for 30 min, starved PC-3M and TRAMP-C2 cells suspended in DMEM medium with 0.4 % FBS containing 40 ng/ml FGF8b alone, 40 ng/ml FGF8b plus 125 nM 8b-13, 40 ng/ml FGF8b plus 10 μM U0126, 40 ng/ml FGF8b plus 10 μM LY294002, or 40 ng/ml FGF8b plus 10 μMSB202190, were seeded onto the upper chamber. Medium containing 10 % FBS was added in the lower chamber as a chemo-attractant. After incubation for 24 h, cells on the upper side of the filter were removed with a cotton swab, and cells on the lower side of the filter were stained with crystal violet and counted using an inverted microscope.
2.2.7 Real-Time quantitative PCR analysis of uPA and VEGF expression PC-3M and TRAMP-C2 cells were seeded in 6-well plates at a density of 5×104 7
cells per well, starved for 24 h and treated with 40 ng/ml FGF8b alone or 40 ng/ml FGF8b plus 125 nM 8b-13 for 48 h. Total RNA extraction and reverse transcription were carried out according to the manufacturer’s instructions. The quantitative PCR was performed as follows: 1 cycle of 95℃ for 3 min; 40 cycles of (95℃ for 15 s, 59℃ for 5 s and 72℃ for 10 s). The sequences of the PCR primers were as follow: uPA (h): 5'-CGCAGTCACACCAAGGAAGAGAATG-3' (F), 5'-TCTGTGCAGAGCCTATCTTCCCAGT-3' (R); VEGF (h): 5'-GGCAGAATCATCACGAAG-3' (F), 5'-TGTGCTGTAGGAAGCTCA-3' (R); GADPH (h): 5'-CCCACT CCTCCACCTTTGAC-3' (F), 5'-TGTGCTGTAGGAAGCTCA -3' (R); uPA (m): 5'-AGGTGGAGAACCAGCCCT-3' (F), 5'-AGGGCTGGTTCTCCACCT-3' (R); VEGF (m): 5'-AATTGAGACCCTGGTGGA-3' (F), 5'-CTCTGACGTGGGCACGCA -3' (R); GADPH (m): 5'-TGGTGAAGGTCGGTGTGA -3' (F), 5'-TGAGTGGAGTCATACTGG-3' (R). The mRNA level of the detected protein was expressed as the relative ratio to the GAPDH mRNA level.
2.2.8 Statistical analysis Statistical analysis was performed using the Graph Pad Prism software 5.0. The student’s t-test was used to compare data between two groups, and an oneway ANOVA followed by Tukey’s multiple comparison test was used for multiple comparison data. Significance was set at p< 0.05.
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3. Results 3.1 The synthetic peptide suppresses FGF8b-stimulated cell proliferation To determine the potential role of 8b-13 in prostate cancer progression, we first detected the effects of the synthetic peptides on the proliferation of both the androgen dependent prostate cancer cell line TRAMP-C2 and the androgen independent prostate cancer cell line PC-3M, both of which expressed abundant endogenous FGF8b and its high affinity receptors (FGFR1-3) (Fig. 1A and B). The results indicated that 8b-13 peptide mediated strong proliferation inhibition in both cell lines stimulated by the exogenous FGF8b, whereas little inhibitory effect was observed in the groups treated with the scrambled peptide H13 (Fig. 1C and D). In addition, the administration of 8b-13 alone also suppressed the growth of two cell lines expressing abundant endogenous FGF8b and its receptors (Fig. 1E and F), suggesting 8b-13 might inhibit basal cell proliferation via targeting the endogenous FGF8b stimulation.
3.2 The synthetic peptide blocks FGF8b-induced activation of signal pathways FGF8b binds to its receptors and activates multiple signal cascades to trigger diverse biological responses. To determine whether 8b-13 influences FGF8b signal pathways, we first evaluate the capacity of 8b-13 to suppress the activation of Akt and MAP kinases stimulated with the exogenous FGF8b by western blotting. As shown in Fig. 2, FGF8b significantly triggered the intense phosphorylation of Erk1/2 and Akt rather than P38 in PC-3M cells (Fig. 2A), and activated Erk1/2 and P38, but not Akt signal molecule in TRAMP-C2 cells (Fig. 2B). Pretreatment of the cells with different 9
concentrations of 8b-13 (1, 5, 25, 125 nM) for 10 min led to significant blockage of the exogenous FGF8b-induced phosphorylation of the corresponding signal molecules in a dose-dependent manner in both cell lines (Fig.2A and 2B). As PC-3M and TRAMP-C2 cell lines expresses abundant endogenous FGF8b (Fig. 1E), we further detected the effects of 8b-13 on the basal activation levels of the signal cascades. The results demonstrated that 8b-13 also down-regulated the basal activation levels of Erk1/2 and Akt in PC-3M cells (Fig. 2C), and Erk1/2 and P38 in TRAMP-C2 cells (Fig. 2D).
3.3 The synthetic peptide reduces cell migration induced by FGF8b Cell migration of cancer cells is an essential step for the metastatic process in the malignant tumors. Therefore, we examined the role of the synthetic 8b-13 peptides in cell migration using the wound healing assay. A “wound” was created by scraping the cell monolayer in a straight line. Images taken post-scratch showed almost complete wound closure was observed at 24 h in PC-3M and TRAMP-C2 cells treated with FGF8b, while only a partial closure was found at the corresponding time in both cell lines with 8b-13 pretreatment (Fig. 3A and 3B). Analysis of the migration distance indicated that higher migration rate induced by FGF8b was significantly reduced by 8b-13 addition, suggesting that 8b-13 has the potential of reversing the ability of FGF8b-triggered cell migration. As the above results shown in Fig. 2 demonstrated that FGF8b activated Erk1/2 and Akt cascades in PC-3M cells, and Erk1/2 and P38 MAPK signal axis in TRAMP-C2 10
cells, we speculated MAPK and Akt cascades may be involved in FGF8b-induced cell migration. As expected, inhibition of the Erk1/2 pathway with U0126 or the Akt pathway with LY294002 in PC-3M cells, and suppression of the Erk1/2 pathway with U0126 or the P38 pathway with SB202190 in TRAMP-C2 cells significantly attenuated the FGF8b-stimulated cell migration (Fig. 3C and 3D), suggesting that FGF8b promoted cell migration via activation of MAPK and Akt cascades.
3.4 The synthetic peptide reverses FGF8b-stimulated cell invasion As enhanced cell invasion is also involved in the metastatic cascade, we further detected whether 8b-13 influences the ability of cell invasion using Matrigel-coated transwell culture chambers. As showed in Fig. 4, FGF8b treatment enhanced the invasive ability of both PC-3M and TRAMP-C2 cells, whereas 8b-13 significantly reduced cell invasion stimulated by FGF8b. Similar to the effects of the individual signal pathway inhibitor on cell migration, treatment of PC-3M cells with U0126 or LY294002, and TRAMP-C2 cells with U0126 or SB202190 led to inhibition of FGF8b-induced cell invasion.
3.5 The synthetic peptide decreases the expression of uPA and VEGF enhanced by FGF8b Accumulating findings indicate the importance of the urokinase type plasminogen activator (uPA) and the vascular endothelial growth factor (VEGF) in tumor metastasis (Jiang, et al. 2015; Roberts, et al. 2013; Sheng 2001). To determine 11
whether uPA and VEGF were involved in the mechanisms of 8b-13 inhibiting cell migration and invasion, real time quantitative PCR was used to detect the influence of 8b-13 on the expression of uPA and VEGF. As shown in Fig. 5, 8b-13 significantly reversed the enhanced effects of FGF8b on the expression of both proteins, implying that down-regulation of the metastasis-related factors may contribute to the anti-metastasis potential of 8b-13 targeting FGF8b stimulation.
4. Discussion Prostate cancer is the most common malignant tumor occurred in men worldwide. Metastatic recurrence causes shorter median survival in patients suffering from advanced prostate cancer (Krishna and Bergan 2014). Several innovative approaches, including cytotoxic chemotherapy, targeted therapy, and Immunotherapy have recently been approved for clinical trials or application in metastatic prostate cancer (Lauer, et al. 2015; Wadosky and Koochekpour 2016). Two small-molecule multikinase inhibitors-dovitinib and nintedanib-targeting FGFRs simultaneously with other tyrosine kinases are in clinical development for advanced prostate cancer (Corn, et al. 2013). It has been demonstrated that high level of FGF8b was positively correlated with metastasis and poor survival in prostate, breast and colorectal cancer (Dorkin, et al. 1999; Liu, et al. 2015; Marsh, et al. 1999; Valta, et al. 2008). FGF8b confers an aggressive phenotype to cancer cells via binding and activating its receptors (FGFRs). Interruption of the interaction between FGF8b and its receptors is a promising strategy for reversing the metastatic behaviors induced by 12
FGF8b. A short peptide 8b-13 designed on the basis of the structure of FGF8b-FGFR complex displayed potentials of antagonizing the bioactivity of FGF8b by impeding FGF8b/FGFRs triggered RAS/MAPK and PI3K/Akt cascades, implying that 8b-13 may serve as an anti-metastasis peptide targeting FGF8b/FGFRs. Our results demonstrated that the synthetic 8b-13 peptide has the potentials of reversing the aggressive phenotype including proliferation, migration, and invasion of the prostate cancer cells caused by FGF8b. Compelling evidence indicates that activation of RAS/MAPK and PI3K/Akt signal cascades plays an essential role in the aggressive nature of prostate cancer (Drake, et al. 2013; Yin, et al. 2007). Augmentation of p-MAPK and p-Akt levels was commonly found in metastastic prostate cancer (Suzuki, et al. 2008; Toren and Zoubeidi 2014). Moreover, RAS/MAPK and PI3K/Akt pathways collaborate to promote metastasis initiated from prostate cancer stem/progenitor cells (Mulholland, et al. 2012). In order to explore the molecular mechanisms underlying the inhibition effects of 8b-13 peptide on the aggressive phenotype of prostate cancer cells, we evaluated the capacity of 8b-13 to suppress FGF8b-triggered signal cascades governing the aggressive behaviors. The results indicated that 8b-13 inhibited the activation effects of the exogenous and endogenous FGF8b on FGFR-mediated Erk1/2 and Akt cascades in PC-3M cells, and Erk1/2 and P38 MAPK signal axis in TRAMP-C2 cells. The corresponding kinase inhibitors respectively attenuated the FGF8b-stimulated cell migration and invasion in PC-3M and TRAMP-C2 cells. Combined with the results that 8b-13 attenuated FGF8b-induced activation of MAPK 13
and Akt signal pathways in both cell lines, we suggested that MAPK and Akt cascades might mediate the inhibitory effects of 8b-13 on FGF8b-induced cell migration and invasion. It has been proposed that uPA and VEGF play essential roles in the metastatic process of tumor via fibrin degradation and vascular formation effects (Bekes, et al. 2011; Conn, et al. 2009; Jamison, et al. 2015; Li and Cozzi 2007). They form a positive feedback loop to degrade the extracellular matrix and basement membrane, providing an advantageous microenvironment for tumorous aggressiveness (Jiang, et al. 2012). In addition, previous report also found a significant correlation between VEGF and FGF-8 expression in human prostate cancer (West, et al. 2001). Combined with our results showing that 8b-13 significantly reversed the enhanced effects of FGF8b on the expression of uPA and VEGF, it is reasonable to speculate that 8b-13 might down-regulate the expression of uPA and VEGF, and interfere with the FGF8b-induced microenvironment suitable for tumorous metastasis, leading to suppression of the aggressive phenotype of prostate cancer cells. In summary, the results of the present study demonstrated that the synthetic 8b-13 peptides have the potentials of antagonizing the metastatic actions of FGF8b in prostate cancer cells. Combined with our previous study showing that 8b-13 peptide exhibit potent inhibitory effects on angiogenesis (Lin, et al. 2017), which greatly contributes to tumor metastasis, we proposed that 8b-13 might serve as a potent FGF8b antagonist for treatment of advanced prostate cancer with abnormal FGF8b upregulation. 14
Acknowledgments The present study was supported by the National Science Foundation of China (81573334), the Science and Technology Planning Project of Guangdong Province of China (2017A020211029, 2015A020211017, 2011B061300065), the Opening Project of Zhejiang Provincial Top Key Discipline of Pharmaceutical Sciences, the Natural Science Foundation of Zhejiang Province of China (LY14H310013), the Undergraduate Innovation and Entrepreneurship Training Project (201610559036), and the Guangdong Provincial ‘Thousand-Hundred-Ten Talent Project”.
Conflicts of interest None of the authors have any relevant conflicts of interest or disclosures.
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Figure legends Fig. 1. The synthetic peptide 8b-13 suppresses cell proliferation. (A) Analysis of the endogenous FGF8b in PC-3M, TRAMP-C2, and K562 cells by western blotting. No detectable FGF8b expression was observed in K562 cells. (B) RT-qPCR was applied to determine the expression levels of FGFRs in PC-3M and TRAMP-C2 cells. (C-D) Starved PC-3M and TRAMP-C2 cells were treated with 40 19
ng/ml FGF8b plus 8b-13 or the scrambled peptide H13 at the indicated concentrations, and cell viability was measured 48 h later using MTT assay. (E-F) Starved PC-3M and TRAMP-C2 cells were treated with 8b-13 or the scrambled peptide H13 for 48 h prior to assessment of cell viability by MTT assay. Data are presented as the mean (± S.D.) of three independent experiments performed in triplicate. #p< 0.05, ##p< 0.01 versus control; *P< 0.05, **P< 0.01, ***P< 0.001 versus FGF8b group.
Fig. 2. The synthetic peptide 8b-13 blocks activation of Akt and MAP kinases. (A-B) Starved PC-3M and TRAMP-C2 cells were pretreated with serially diluted 8b-13 peptides for 10 min before stimulation with 40 ng/ml FGF8b for 15 min. The phosphorylated and total levels of Erk1/2, P38 and Akt were determined by western blot analysis. (C-D) Starved PC-3M and TRAMP-C2 cells were treated with serially diluted 8b-13 peptides for 20 min. The phosphorylated and total levels of Erk1/2, P38 and Akt were determined by western blot analysis. Data are presented as the mean ± S.D. of three independent experiments. *P< 0.05, **P< 0.01, ***P< 0.001.
Fig. 3. The synthetic peptide 8b-13 reduces FGF8b-stimulated cell migration. (A-B) Starved cells were treated with FGF8b and FGF8b plus 125 nM 8b-13 for 24 h. Cell migration ability was examined by scratch wound healing assay under a microscope (100×). (C-D) Starved cells were treated with 125 nM 8b-13, U0126, LY294002, or SB202190 for 15 min before addition of 40 ng/ml FGF8b. Cell images were examined using ZENinverted microscope (100×). The migration rate was 20
evaluated by measuring the remaining scratch distance and expressed as percentage of the initial scratch distance. Data are shown as mean ± S.D. (n = 3). *P< 0.05, **P< 0.01, ***P
< 0.001.
Fig. 4. The synthetic peptide 8b-13 reverses FGF8b-stimulated cell invasion. Starved cells were seeded onto the upper chamber with FGF8b alone, or plus 8b-13, U0126, LY294002, or SB202190 at the indicated concentrations. After incubation for 24 h, cells on the lower side of the filter were stained with crystal violet and counted using an inverted microscope. Data are shown as mean ± S.D. (n = 3). *P< 0.05, **P< 0.01, ***P< 0.001.
Fig. 5. The synthetic peptide 8b-13 decreases the expression of uPA and VEGF enhanced by FGF8b. (A-B) Starved cells were treated with 40 ng/ml FGF8b alone or 40 ng/ml FGF8b plus 125 nM 8b-13 for 48 h. Total RNA was extracted by TRIZOL for RT-qPCR to determine the relative mRNA levels of uPA and VEGF. Data are presented as the mean ± S.D. of four independent experiments.* P< 0.05, **P< 0.01, ***P< 0.001.
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S0014-2999(18)30530-2 https://doi.org/10.1016/j.ejphar.2018.09.013 EJP71982
To appear in: European Journal of Pharmacology Received date: 22 May 2018 Revised date: 5 September 2018 Accepted date: 5 September 2018 Cite this article as: Hongjiao Liu, Xiaomian Lin, Tao Huang, Li Song, Cairong Zhu, Hongmin Ma, Tianzhu Long, Huixuan Zeng, Rongzhen Li, Heng Wang, Yishan Huang, Liankuai Chen and Xiaoping Wu, A short peptide reverses the aggressive phenotype of prostate cancer cells, European Journal of Pharmacology, https://doi.org/10.1016/j.ejphar.2018.09.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A short peptide reverses the aggressive phenotype of prostate cancer cells
Hongjiao Liua1 •Xiaomian Linb•Tao Huangb •Li Songb • Cairong Zhua1 •Hongmin Maa1 •Tianzhu Longa•Huixuan Zengb •Rongzhen Lib • Heng Wangb •Yishan Huang •Liankuai Chenb •Xiaoping Wub2
a
Guangzhou Women and Children’s Medical Center, Guangzhou Medical University,
Guangzhou 510623, China b
Institute of Tissue Transplantation and Immunology, Jinan University, Guangzhou
510632, China [email protected] (C. Zhu) [email protected] (X. Wu)
ABSTRACT Previous studies have demonstrated that fibroblast growth factor 8b (FGF8b) is up-regulated in a large proportion of prostate cancer patients, and plays a key role in the aggressive progress of prostate cancer. Herein, we investigated the effects of a short peptide derived from the gN helix domain of FGF8b on the metastatic behaviors of prostate cancer cells. The results demonstrated that the synthetic peptide might 1 2
Hongjiao Liu, Xiaomian Lin, and Tao Huang have contributed equally to this work. Tel: 8620-85220732 1
reverse the effects of FGF8b on cell proliferation, migration and invasion by suppressing the activation of MAPK and Akt signaling cascades, and reducing the expression of the metastasis-related proteins, resulting in the improvement of the aggressive phenotype of the prostate cancer cells. Collectively, these results underline the therapeutic potential of the FGF8b mimic peptide in advanced prostate cancer.
Keywords: fibroblast growth factor 8; aggressive phenotype; prostate cancer
1. Introduction Prostate cancer is the second most common type of cancer in men, with more frequent occurrence in the developed countries and increasing rates in the developing world (Baade, et al. 2009). Hormonal therapy may be effective. However, metastatic recurrence is often hormone-refractory, resulting in shorter median survival (Koo, et al. 2015). Indeed, metastasis-relevant events like vessel invasion, positive metastatic lymph nodes, and bone metastasis have been observed in advanced prostate cancer patients (Gdowski, et al. 2017; Quayle, et al. 2015). Preventing metastasis of the advanced prostate cancer is still a great challenge in clinic (Lassi and Dawson 2011). It has been proposed that cell proliferation, motility and invasion of tumor cells are involved in the metastatic process. But the pivotal factors that modulate the metastasis in prostate cancer and the related targeting agents remain to be explored. Compelling evidence indicated that fibroblast growth factor 8b (FGF8b), the major isoform of FGF8 up-regulated in prostate cancer (Gnanapragasam, et al. 2003; 2
Valta, et al. 2008), plays a key role in conferring an aggressive phenotype to prostate cancer cells via binding and activating the tyrosine kinase FGF receptors (FGFRs) (Dorkin, et al. 1999; Mattila and Harkonen 2007; Song, et al. 2000). Therefore, FGF8b/FGFRs signal axis has been considered as the potential target for reversing metastatic behaviors of prostate cancer. We have demonstrated previously that a short peptide (named 8b-13) derived from the gN helix domain of FGF8b responsible for the high affinity and specificity of FGF8b to its receptors has potentials of antagonizing the bioactivity of FGF8b (Li, et al. 2013). Herein, we further investigated the effects of 8b-13 peptides on FGF8b-mediated aggressive behaviors of prostate cancer cells.
2. Materials and Methods 2.1 Materials Human FGF8b was purchased from PeproTechInc (Rocky Hill, NJ, USA). The 8b-13 peptide (PNFTQHVREQSLV) and its scrambled version H13 (QRVSQFENHPVTL) with the purity greater than 98% were synthesized at SBS Genetech (Beijing, China). The cell proliferation assay reagent MTT was obtained from Sigma (CA, USA). Anti-FGF8b antibody was purchased from Millipore (Billerica, MA, USA).Anti-phospho-Erk1/2, anti-Erk1/2, anti-phospho-P38, anti-P38, anti-phospho-Akt, anti-Akt, anti-GAPDH antibodies and horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody were obtained from Cell Signaling Technology (Danvers, MA, USA).Erk1/2 inhibitor (U0126), Akt inhibitor 3
(LY294002) and P38 inhibitor (SB202190) were the products of Selleck (Houston, Texas, United States). The polyvinylidenedifluoride (PVDF) membrane was from Millipore (Billerica, MA, USA). The enhanced chemiluminescence (ECL) detection kit was the product of Pierce (Rockford, IL, USA). TRIZOL was purchased from Life technologies (New York, USA). RevertAid First strand cDNA synthesis kit was from Thermo Scientific (Waltham, MA, USA),and SYBR green Q-PCR kit was purchased from Bio-Rad (Hercules, CA, USA). Matrigel was purchased from BD Biosciences (San Jose, CA, USA).
2.2 Methods 2.2.1 Cell culture The prostate cancer cell line PC-3M was kept by the Institute of Tissue Transplantation and Immunology of Jinan University, and TRAMP-C2 cell line was kindly provided by Dr. Fen Wang in the Institute of Biosciences and Technology, Texas A&M Health Science Center. The chronicmyelogenous leukemia cell line K562 was a gift from Professor Yifei Wang in Department of Cellular Biology of Jinan University. All cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) and maintained in a humidified atmosphere containing 5% CO2 at 37 °C.
2.2.2 Detection of the endogenous FGF8b and FGFRs The PC-3M, TRAMP-C2, and K562 cells were plated in 6-well plates. After 4
cultured for 48 h, cells were lysed with 1×S.D.S-PAGE loading buffer. The lysate was syringed, boiled for 5 min and subjected to SDS–PAGE followed by transferal to PVDF membranes at a current 350 mA for 70 min. The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h, probed with rabbit monoclonal FGF8b antibody at room temperature for 1 h followed by a HRP conjugated goat anti-rabbit secondary antibody before visualization with ECL reagents. The results were analyzed by Quantity One software to determine the relative ratio and presented in graphs as expression relative to that of GAPDH. Real-Time quantitative PCR was further applied to determine the expressions of FGFRs. Briefly, One microgram of total RNA extracted from PC-3M and TRAMP-C2 cells by TRIZOL was reverse-transcribed using the First-Strand cDNA Synthesis Kit to produce cDNA, which was used as a template for quantitative PCR amplification with the SYBR green qPCR Kit. The quantitative PCR was performed using the CFX96 Touch Deep Well real-time PCR Detection System as follows: 1 cycle of 95℃ for 3 min; 40 cycles of (95℃ for 15 s, 58℃ for 5 s and 72℃ for 10 s). The mRNA levels of FGFRs were expressed as the relative ratio to the GAPDH mRNA level.
2.2.3 Cell viability assay PC-3M and TRAMP-C2 cells were seeded in 96-well plates at a density of 5×104 cells per well, and allowed to attach overnight. After starved for 24 h, cells were treated with serial dilutions of the 8b-13 or H13 peptides, 40 ng/ml FGF8b alone, or 5
40 ng/ml FGF8b plus with serial dilutions of the 8b-13 or H13 peptides. Starved cells were pretreated with the 8b-13 for 10 min before addition of FGF8b. Cell viability was measured 48 h later using the methylthiazoletetrazolium (MTT) assay.
2.2.4 Analysis of Akt and MAP kinase activation PC-3M and TRAMP-C2 cells were seeded in 6-well plates at a density of 5×104 cells per well for western blot analysis. Briefly, after starved cultivation in DMEM with 0.4% FBS for 24 h, cells were pretreated with the 8b-13 for 10 min before the addition of FGF8b (40 ng/ml) for 15 min. Cells were collected and lysed in 1×SDS-PAGE loading buffer. The lysate was subjected to run on SDS–PAGE gels followed by transferal to PVDF membranes. The membrane was blocked with 5% non-fat dry milk at room temperature for 1 h, probed with the primary antibodies at 4℃ overnight followed by incubation with HRP-linked goat anti-rabbit IgG for 1 h at room temperature. An ECL detection kit was applied to detect the blots according to the manufacturer’s procedure. The results were analyzed by Quantity One software to determine the relative ratio as presented in graphs.
2.2.5 In vitro wound healing assay PC-3M and TRAMP-C2 cells were seeded into 6-well plates at a density of 1×105 cells per well and allowed to adhere completely. Cells were starved for 24 h in DMEM with 0.4% FBS. A sterilized 200-μl disposable pipette tip was used to scrape the cell monolayer in a straight line to create a scratch wound in each well. After 6
washing three times with PBS to remove debris, cells were pretreated with 125 nM 8b-13, 10 μM U0126, 10 μM LY294002, or 10 μM SB202190 for 15 min before addition of 40 ng/ml FGF8b. The images were acquired using a phase-contrast microscope at defined time frames, and the scratch distance was quantitatively analyzed using the Image Pro-Plus 6.0 software. The migration rate was calculated following the formula: Migrationrate= [𝑠𝑐𝑟𝑎𝑡𝑐ℎ 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 (0 ℎ)－𝑠𝑐𝑟𝑎𝑡𝑐ℎ 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 (𝑛 ℎ)] / 𝑠𝑐𝑟𝑎𝑡𝑐ℎ 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 (0 ℎ)
2.2.6 Cell invasion assay Transwell chambers (24-well) were applied for cell invasion assay. After Matrigel (1:20) was coated on the filter of the transwell chambers and incubated at 37 °C for 30 min, starved PC-3M and TRAMP-C2 cells suspended in DMEM medium with 0.4 % FBS containing 40 ng/ml FGF8b alone, 40 ng/ml FGF8b plus 125 nM 8b-13, 40 ng/ml FGF8b plus 10 μM U0126, 40 ng/ml FGF8b plus 10 μM LY294002, or 40 ng/ml FGF8b plus 10 μMSB202190, were seeded onto the upper chamber. Medium containing 10 % FBS was added in the lower chamber as a chemo-attractant. After incubation for 24 h, cells on the upper side of the filter were removed with a cotton swab, and cells on the lower side of the filter were stained with crystal violet and counted using an inverted microscope.
2.2.7 Real-Time quantitative PCR analysis of uPA and VEGF expression PC-3M and TRAMP-C2 cells were seeded in 6-well plates at a density of 5×104 7
cells per well, starved for 24 h and treated with 40 ng/ml FGF8b alone or 40 ng/ml FGF8b plus 125 nM 8b-13 for 48 h. Total RNA extraction and reverse transcription were carried out according to the manufacturer’s instructions. The quantitative PCR was performed as follows: 1 cycle of 95℃ for 3 min; 40 cycles of (95℃ for 15 s, 59℃ for 5 s and 72℃ for 10 s). The sequences of the PCR primers were as follow: uPA (h): 5'-CGCAGTCACACCAAGGAAGAGAATG-3' (F), 5'-TCTGTGCAGAGCCTATCTTCCCAGT-3' (R); VEGF (h): 5'-GGCAGAATCATCACGAAG-3' (F), 5'-TGTGCTGTAGGAAGCTCA-3' (R); GADPH (h): 5'-CCCACT CCTCCACCTTTGAC-3' (F), 5'-TGTGCTGTAGGAAGCTCA -3' (R); uPA (m): 5'-AGGTGGAGAACCAGCCCT-3' (F), 5'-AGGGCTGGTTCTCCACCT-3' (R); VEGF (m): 5'-AATTGAGACCCTGGTGGA-3' (F), 5'-CTCTGACGTGGGCACGCA -3' (R); GADPH (m): 5'-TGGTGAAGGTCGGTGTGA -3' (F), 5'-TGAGTGGAGTCATACTGG-3' (R). The mRNA level of the detected protein was expressed as the relative ratio to the GAPDH mRNA level.
2.2.8 Statistical analysis Statistical analysis was performed using the Graph Pad Prism software 5.0. The student’s t-test was used to compare data between two groups, and an oneway ANOVA followed by Tukey’s multiple comparison test was used for multiple comparison data. Significance was set at p< 0.05.
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3. Results 3.1 The synthetic peptide suppresses FGF8b-stimulated cell proliferation To determine the potential role of 8b-13 in prostate cancer progression, we first detected the effects of the synthetic peptides on the proliferation of both the androgen dependent prostate cancer cell line TRAMP-C2 and the androgen independent prostate cancer cell line PC-3M, both of which expressed abundant endogenous FGF8b and its high affinity receptors (FGFR1-3) (Fig. 1A and B). The results indicated that 8b-13 peptide mediated strong proliferation inhibition in both cell lines stimulated by the exogenous FGF8b, whereas little inhibitory effect was observed in the groups treated with the scrambled peptide H13 (Fig. 1C and D). In addition, the administration of 8b-13 alone also suppressed the growth of two cell lines expressing abundant endogenous FGF8b and its receptors (Fig. 1E and F), suggesting 8b-13 might inhibit basal cell proliferation via targeting the endogenous FGF8b stimulation.
3.2 The synthetic peptide blocks FGF8b-induced activation of signal pathways FGF8b binds to its receptors and activates multiple signal cascades to trigger diverse biological responses. To determine whether 8b-13 influences FGF8b signal pathways, we first evaluate the capacity of 8b-13 to suppress the activation of Akt and MAP kinases stimulated with the exogenous FGF8b by western blotting. As shown in Fig. 2, FGF8b significantly triggered the intense phosphorylation of Erk1/2 and Akt rather than P38 in PC-3M cells (Fig. 2A), and activated Erk1/2 and P38, but not Akt signal molecule in TRAMP-C2 cells (Fig. 2B). Pretreatment of the cells with different 9
concentrations of 8b-13 (1, 5, 25, 125 nM) for 10 min led to significant blockage of the exogenous FGF8b-induced phosphorylation of the corresponding signal molecules in a dose-dependent manner in both cell lines (Fig.2A and 2B). As PC-3M and TRAMP-C2 cell lines expresses abundant endogenous FGF8b (Fig. 1E), we further detected the effects of 8b-13 on the basal activation levels of the signal cascades. The results demonstrated that 8b-13 also down-regulated the basal activation levels of Erk1/2 and Akt in PC-3M cells (Fig. 2C), and Erk1/2 and P38 in TRAMP-C2 cells (Fig. 2D).
3.3 The synthetic peptide reduces cell migration induced by FGF8b Cell migration of cancer cells is an essential step for the metastatic process in the malignant tumors. Therefore, we examined the role of the synthetic 8b-13 peptides in cell migration using the wound healing assay. A “wound” was created by scraping the cell monolayer in a straight line. Images taken post-scratch showed almost complete wound closure was observed at 24 h in PC-3M and TRAMP-C2 cells treated with FGF8b, while only a partial closure was found at the corresponding time in both cell lines with 8b-13 pretreatment (Fig. 3A and 3B). Analysis of the migration distance indicated that higher migration rate induced by FGF8b was significantly reduced by 8b-13 addition, suggesting that 8b-13 has the potential of reversing the ability of FGF8b-triggered cell migration. As the above results shown in Fig. 2 demonstrated that FGF8b activated Erk1/2 and Akt cascades in PC-3M cells, and Erk1/2 and P38 MAPK signal axis in TRAMP-C2 10
cells, we speculated MAPK and Akt cascades may be involved in FGF8b-induced cell migration. As expected, inhibition of the Erk1/2 pathway with U0126 or the Akt pathway with LY294002 in PC-3M cells, and suppression of the Erk1/2 pathway with U0126 or the P38 pathway with SB202190 in TRAMP-C2 cells significantly attenuated the FGF8b-stimulated cell migration (Fig. 3C and 3D), suggesting that FGF8b promoted cell migration via activation of MAPK and Akt cascades.
3.4 The synthetic peptide reverses FGF8b-stimulated cell invasion As enhanced cell invasion is also involved in the metastatic cascade, we further detected whether 8b-13 influences the ability of cell invasion using Matrigel-coated transwell culture chambers. As showed in Fig. 4, FGF8b treatment enhanced the invasive ability of both PC-3M and TRAMP-C2 cells, whereas 8b-13 significantly reduced cell invasion stimulated by FGF8b. Similar to the effects of the individual signal pathway inhibitor on cell migration, treatment of PC-3M cells with U0126 or LY294002, and TRAMP-C2 cells with U0126 or SB202190 led to inhibition of FGF8b-induced cell invasion.
3.5 The synthetic peptide decreases the expression of uPA and VEGF enhanced by FGF8b Accumulating findings indicate the importance of the urokinase type plasminogen activator (uPA) and the vascular endothelial growth factor (VEGF) in tumor metastasis (Jiang, et al. 2015; Roberts, et al. 2013; Sheng 2001). To determine 11
whether uPA and VEGF were involved in the mechanisms of 8b-13 inhibiting cell migration and invasion, real time quantitative PCR was used to detect the influence of 8b-13 on the expression of uPA and VEGF. As shown in Fig. 5, 8b-13 significantly reversed the enhanced effects of FGF8b on the expression of both proteins, implying that down-regulation of the metastasis-related factors may contribute to the anti-metastasis potential of 8b-13 targeting FGF8b stimulation.
4. Discussion Prostate cancer is the most common malignant tumor occurred in men worldwide. Metastatic recurrence causes shorter median survival in patients suffering from advanced prostate cancer (Krishna and Bergan 2014). Several innovative approaches, including cytotoxic chemotherapy, targeted therapy, and Immunotherapy have recently been approved for clinical trials or application in metastatic prostate cancer (Lauer, et al. 2015; Wadosky and Koochekpour 2016). Two small-molecule multikinase inhibitors-dovitinib and nintedanib-targeting FGFRs simultaneously with other tyrosine kinases are in clinical development for advanced prostate cancer (Corn, et al. 2013). It has been demonstrated that high level of FGF8b was positively correlated with metastasis and poor survival in prostate, breast and colorectal cancer (Dorkin, et al. 1999; Liu, et al. 2015; Marsh, et al. 1999; Valta, et al. 2008). FGF8b confers an aggressive phenotype to cancer cells via binding and activating its receptors (FGFRs). Interruption of the interaction between FGF8b and its receptors is a promising strategy for reversing the metastatic behaviors induced by 12
FGF8b. A short peptide 8b-13 designed on the basis of the structure of FGF8b-FGFR complex displayed potentials of antagonizing the bioactivity of FGF8b by impeding FGF8b/FGFRs triggered RAS/MAPK and PI3K/Akt cascades, implying that 8b-13 may serve as an anti-metastasis peptide targeting FGF8b/FGFRs. Our results demonstrated that the synthetic 8b-13 peptide has the potentials of reversing the aggressive phenotype including proliferation, migration, and invasion of the prostate cancer cells caused by FGF8b. Compelling evidence indicates that activation of RAS/MAPK and PI3K/Akt signal cascades plays an essential role in the aggressive nature of prostate cancer (Drake, et al. 2013; Yin, et al. 2007). Augmentation of p-MAPK and p-Akt levels was commonly found in metastastic prostate cancer (Suzuki, et al. 2008; Toren and Zoubeidi 2014). Moreover, RAS/MAPK and PI3K/Akt pathways collaborate to promote metastasis initiated from prostate cancer stem/progenitor cells (Mulholland, et al. 2012). In order to explore the molecular mechanisms underlying the inhibition effects of 8b-13 peptide on the aggressive phenotype of prostate cancer cells, we evaluated the capacity of 8b-13 to suppress FGF8b-triggered signal cascades governing the aggressive behaviors. The results indicated that 8b-13 inhibited the activation effects of the exogenous and endogenous FGF8b on FGFR-mediated Erk1/2 and Akt cascades in PC-3M cells, and Erk1/2 and P38 MAPK signal axis in TRAMP-C2 cells. The corresponding kinase inhibitors respectively attenuated the FGF8b-stimulated cell migration and invasion in PC-3M and TRAMP-C2 cells. Combined with the results that 8b-13 attenuated FGF8b-induced activation of MAPK 13
and Akt signal pathways in both cell lines, we suggested that MAPK and Akt cascades might mediate the inhibitory effects of 8b-13 on FGF8b-induced cell migration and invasion. It has been proposed that uPA and VEGF play essential roles in the metastatic process of tumor via fibrin degradation and vascular formation effects (Bekes, et al. 2011; Conn, et al. 2009; Jamison, et al. 2015; Li and Cozzi 2007). They form a positive feedback loop to degrade the extracellular matrix and basement membrane, providing an advantageous microenvironment for tumorous aggressiveness (Jiang, et al. 2012). In addition, previous report also found a significant correlation between VEGF and FGF-8 expression in human prostate cancer (West, et al. 2001). Combined with our results showing that 8b-13 significantly reversed the enhanced effects of FGF8b on the expression of uPA and VEGF, it is reasonable to speculate that 8b-13 might down-regulate the expression of uPA and VEGF, and interfere with the FGF8b-induced microenvironment suitable for tumorous metastasis, leading to suppression of the aggressive phenotype of prostate cancer cells. In summary, the results of the present study demonstrated that the synthetic 8b-13 peptides have the potentials of antagonizing the metastatic actions of FGF8b in prostate cancer cells. Combined with our previous study showing that 8b-13 peptide exhibit potent inhibitory effects on angiogenesis (Lin, et al. 2017), which greatly contributes to tumor metastasis, we proposed that 8b-13 might serve as a potent FGF8b antagonist for treatment of advanced prostate cancer with abnormal FGF8b upregulation. 14
Acknowledgments The present study was supported by the National Science Foundation of China (81573334), the Science and Technology Planning Project of Guangdong Province of China (2017A020211029, 2015A020211017, 2011B061300065), the Opening Project of Zhejiang Provincial Top Key Discipline of Pharmaceutical Sciences, the Natural Science Foundation of Zhejiang Province of China (LY14H310013), the Undergraduate Innovation and Entrepreneurship Training Project (201610559036), and the Guangdong Provincial ‘Thousand-Hundred-Ten Talent Project”.
Conflicts of interest None of the authors have any relevant conflicts of interest or disclosures.
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Figure legends Fig. 1. The synthetic peptide 8b-13 suppresses cell proliferation. (A) Analysis of the endogenous FGF8b in PC-3M, TRAMP-C2, and K562 cells by western blotting. No detectable FGF8b expression was observed in K562 cells. (B) RT-qPCR was applied to determine the expression levels of FGFRs in PC-3M and TRAMP-C2 cells. (C-D) Starved PC-3M and TRAMP-C2 cells were treated with 40 19
ng/ml FGF8b plus 8b-13 or the scrambled peptide H13 at the indicated concentrations, and cell viability was measured 48 h later using MTT assay. (E-F) Starved PC-3M and TRAMP-C2 cells were treated with 8b-13 or the scrambled peptide H13 for 48 h prior to assessment of cell viability by MTT assay. Data are presented as the mean (± S.D.) of three independent experiments performed in triplicate. #p< 0.05, ##p< 0.01 versus control; *P< 0.05, **P< 0.01, ***P< 0.001 versus FGF8b group.
Fig. 2. The synthetic peptide 8b-13 blocks activation of Akt and MAP kinases. (A-B) Starved PC-3M and TRAMP-C2 cells were pretreated with serially diluted 8b-13 peptides for 10 min before stimulation with 40 ng/ml FGF8b for 15 min. The phosphorylated and total levels of Erk1/2, P38 and Akt were determined by western blot analysis. (C-D) Starved PC-3M and TRAMP-C2 cells were treated with serially diluted 8b-13 peptides for 20 min. The phosphorylated and total levels of Erk1/2, P38 and Akt were determined by western blot analysis. Data are presented as the mean ± S.D. of three independent experiments. *P< 0.05, **P< 0.01, ***P< 0.001.
Fig. 3. The synthetic peptide 8b-13 reduces FGF8b-stimulated cell migration. (A-B) Starved cells were treated with FGF8b and FGF8b plus 125 nM 8b-13 for 24 h. Cell migration ability was examined by scratch wound healing assay under a microscope (100×). (C-D) Starved cells were treated with 125 nM 8b-13, U0126, LY294002, or SB202190 for 15 min before addition of 40 ng/ml FGF8b. Cell images were examined using ZENinverted microscope (100×). The migration rate was 20
evaluated by measuring the remaining scratch distance and expressed as percentage of the initial scratch distance. Data are shown as mean ± S.D. (n = 3). *P< 0.05, **P< 0.01, ***P
< 0.001.
Fig. 4. The synthetic peptide 8b-13 reverses FGF8b-stimulated cell invasion. Starved cells were seeded onto the upper chamber with FGF8b alone, or plus 8b-13, U0126, LY294002, or SB202190 at the indicated concentrations. After incubation for 24 h, cells on the lower side of the filter were stained with crystal violet and counted using an inverted microscope. Data are shown as mean ± S.D. (n = 3). *P< 0.05, **P< 0.01, ***P< 0.001.
Fig. 5. The synthetic peptide 8b-13 decreases the expression of uPA and VEGF enhanced by FGF8b. (A-B) Starved cells were treated with 40 ng/ml FGF8b alone or 40 ng/ml FGF8b plus 125 nM 8b-13 for 48 h. Total RNA was extracted by TRIZOL for RT-qPCR to determine the relative mRNA levels of uPA and VEGF. Data are presented as the mean ± S.D. of four independent experiments.* P< 0.05, **P< 0.01, ***P< 0.001.
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