Preclinical report 379
The therapeutic potential of a novel non-ATP-competitive fibroblast growth factor receptor 1 inhibitor on gastric cancer Chaochao Xua,c,*, Wulan Lia,b,*, Peihong Qiua, Yiqun Xiac, Xiaojing Duc, Fen Wange, Lailai Shena, Qiuxiang Chenc, Yunjie Zhaoa, Rong Jinc,d, Jianzhang Wua, Guang Lianga and Xiaokun Lia Previous studies showed that fibroblast growth factor receptor 1 (FGFR1) is an attractive target in gastric cancer therapy. In the current study, we aimed to investigate whether the compound L6123, a novel non-ATP-competitive FGFR1 inhibitor, could show better antitumor activity than the leading compound, nordihydroguaiaretic acid (NDGA), in FGFR1-overexpressing gastric cancer cells. Using an MTT assay, we investigated the inhibitory effect of L6123 on the viability of three gastric cancer cells (MGC-803, SGC-7901, and BGC-823) overexpressing FGFR1, wild-type mouse embryonic fibroblast (MEF-WT), and MEF expressing FGFR1, FGFR2, and FRS2α gene knockout (MEFFGFR1-FGFR2-FRS2α-ko ). We studied the antitumor mechanism of L6123 against the gastric cancer cell line SGC-7901 by western blot analysis. The antitumor effects of L6123 on the gastric cancer cell line SGC-7901 were detected by flow cytometry, Hoechst staining, western blot analysis, and Transwell invasion assay. L6123 had lower IC50 in all three gastric cancer cells than NDGA and showed better inhibitory activity against MEF-WT cells than against MEFFGFR1-FGFR2-FRS2α-ko cells. In the SGC-7901 gastric cell, L6123 inhibited the FGF2-induced phosphorylation of FGFR1/FRS2α/ERK1/2 in a dose-dependent manner, induced the activation of the apoptosis-related proteins, cleaved-PARP and cleaved-caspase-3, decreased the expression of pro-caspase-3 and Bcl-2, and induced tumor cell apoptosis. L6123 also dose-dependently reduced cell
Introduction The incidence of gastric cancer (GC) has decreased markedly worldwide. However, 989 600 new cases of stomach cancer (8% of the total new cancer cases) and 738 000 deaths (10% of the total deaths) have occurred in the past 7 years [1]. Surgical techniques and postoperative adjuvant chemotherapies have improved, but the total survival rate of GC remains unsatisfactory. Therefore, understanding the molecular mechanisms of GC and developing new targeted therapies to improve the survival rate of GC patients are important. Investigation of targeted inhibitors of receptor tyrosine kinases (RTKs) is currently one of the prevalent topics of cancer treatment research; many inhibitor drugs, including EGFR and VEGFR inhibitors, are used clinically [2,3]. For GC, some studies report that there are gene amplification, mutations, and rearrangements of 0959-4973 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
invasion ability, and showed better activity than NDGA at the same concentration. A novel non-ATP-competitive inhibitor L6123 showed excellent antigastric cancer activity by inhibiting the FGFR1 signaling pathway. Thus, we discovered a potential agent for the treatment of FGFR1-overexpressing gastric cancer. Anti-Cancer Drugs 26:379–387 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Anti-Cancer Drugs 2015, 26:379–387 Keywords: fibroblast growth factor receptor 1, gastric cancer, molecular targeted therapy a
Chemical Biology Research Center, College of Pharmaceutical Sciences, College of Information Science and Computer Engineering, Wenzhou Medical University, Departments of cDigestive Diseases, dEpidemiology, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China and eThe Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A&M University, Houston, Texas, USA b
Correspondence to Rong Jin, MD, Department of Digestive Diseases, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 323000, China Tel/fax: + 86 577 555 79999; e-mail: [email protected] Correspondence to Jianzhang Wu, PhD, Chemical Biology Research Center, College of Pharmaceutical Sciences, Wenzhou Medical Universtiy, Wenzhou, Zhejiang 325035, China Tel/fax: + 86 577 866 99396; e-mail: [email protected] *Chaochao Xu and Wulan Li contributed equally to the writing of this article. Received 14 May 2014 Revised form accepted 14 November 2014
RTKs; RTK inhibition might be a new therapeutic strategy for GC. For example, foretinib (GSK1363089), imatinib, regorafenib, and other RTK inhibitors have shown great therapeutic potential against GC [4–6]. FGFR, which is an RTK, can activate downstream signaling pathways, including FRS2α, PI3K/AKT, and ERK1/2 pathways, after ligand [fibroblast growth factor 2 (FGF2)] binding [7]. Overexpression of fibroblast growth factor receptors (FGFRs) extensively involves the regulation of physiological and pathological functions, including cell proliferation, apoptosis, metastasis, and angiogenesis [8]. In GC, FGFRs are overactivated by several mechanisms, including gene amplification, chromosomal translocation, and mutation [9]. Compared with other FGFRs, FGFR1 expression is amplified in GC cell lines and tissues [10]. The development of targeted inhibitors of FGFR1 may be a promising therapeutic strategy for GC. DOI: 10.1097/CAD.0000000000000195
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Fig. 1
CH3 HO
O
OH HO
OH
OH CH3
HO
NDGA FGFR1 inhibition: IC50=24.5μmol/l
HO
S
OH
L6123 FGFR1 inhibition: IC50=0.6μmol/l
Structures of L6123 and NDGA. The IC50 of FGFR1 inhibition: 0.6 μmol/l for L6123; 24.5 μmol/l for NDGA. FGFR1, fibroblast growth factor receptor 1; NDGA, nordihydroguaiaretic acid.
Nordihydroguaiaretic acid (NDGA) is a novel non-ATPcompetitive FGFR inhibitor [11]. In our previous study, we designed and obtained a novel non-ATP-competitive FGFR1 inhibitor L6123. L67123 showed better inhibitory activity on the level of FGFR1 kinases than NDGA, which was used as a positive control (Fig. 1). L6123 was identified as a selective FGFR1 inhibitor with therapeutic potential in nonsmall cell lung cancer in vivo and in vitro (data not shown). Therefore, we hypothesized that L6123 has therapeutic potential in FGFR1-overexpressing GC cells. The results obtained were in agreement with our hypothesis that L6123 showed excellent anti-GC activity by inhibiting the FGFR signaling pathway.
Methods Cell lines, compounds, and reagents
Human GC cells MGC-803, SGC-7901, and BGC-823 were purchased from the Shanghai Institute of Biosciences and Cell Resources Center (Chinese Academy of Sciences, Shanghai, China), Wuhan Boster Biological Engineering Co. Ltd (Wuhan, People’s Republic of China), and the Xiangya Cell Center of Central South University (Changsha, China), respectively. SGC-7901, BGC-823, and MGC-803 cells were maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin. The wild-type mouse embryonic fibroblast (MEF-WT) and MEF-FGFR1-FGFR2-FRS2α-KO (FGFR1, FGFR2, and FRS2α gene knockout) cell lines were kindly provided by Fen Wang (Professor), Texas A&M University, and were cultured in Dulbecco’s modified Eagle’s medium with 1% penicillin and 10% FBS (Gibco, Eggenstein, Germany). All cells were incubated at 37°C in an atmosphere of 5% CO2. L6123 was synthesized and purified in our laboratory, and the purity was 98.0%. NDGA was purchased from Sigma (St Louis, Missouri, USA). Recombinant human FGF2 was purchased from the Institute of Biological and Natural Product, Wenzhou Medical University, Wenzhou, China. Cell proliferation assay
An MTT assay was performed to evaluate the antiproliferative activity of the compound L6123 against the GC cell lines SGC-7901, BGC-823, and MGC-803, and the MEF-WT and MEF-FGFR1-FGFR2-FRS2α-KO cells. All cells (4000 cells/well) were seeded in a 96-well plate.
After 24 h, the culture medium was removed and all cells were treated with increasing concentrations (20/27, 20/9, 20/3, 20, and 60 μmol/l) of compound L6123 for 72 h. NDGA was used as a positive control. After treatment, the viability of the cells was tested using an MTT assay.
Antibodies and western blotting assay
All antibodies, including the goat anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase (HRP) and mouse anti-goat IgG-HRP antibodies, were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). Anti-pFRS2α and anticleaved-caspase-3 were purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). The western blot was operated by standard means. After washing with PBS, 60 μl/well cell lysates were added to a six-well plate at 4°C for 3 min. Then, cell lysates were collected and centrifuged to remove the insoluble components. The supernatant was run on 10% SDS-PAGE and then transferred to a PVDF membrane. After blocking with 5% nonfat dry milk in TBST, the membrane was incubated with the primary antibodies (anti-pFGFR1, anti-FGFR1, anti-pFRS2α, anti-FRS2α, anti-pERK1/2, anti-ERK1/2, anticleaved-caspase-3, anti-procaspase-3, anti-Bcl-2, anticleaved-PARP, and anti-GAPDH) overnight and subsequently incubated with goat anti-rabbit IgG or mouse anti-goat IgG HRP-linked antibody. The blots were detected using an ECL detection kit (Bio-Rad, Hercules, California, USA) according to the manufacturer’s procedure. The results were analyzed using Quantity One software (BioRad) to determine the relative band density ratio.
Cell apoptosis assay
The percentage of apoptotic cells was detected by flow cytometry using the annexin V and propidium iodide (PI) staining. SGC-7901 cells were exposed to different compound concentrations (5, 10, and 20 μmol/l) for 48 h and were subsequently harvested. NDGA was used as a positive control. Finally, cells were incubated with 1 × binding buffer, annexin V (5 μl), and PI (1 μl) in a dark room for 15 min. Cells were analyzed by FACScalibur Flow Cytometry (BD Biosciences, San Jose, California, USA).
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FGFR1 inhibitor on gastric cancer Xu et al. 381
Hoechst assay
SGC-7901 cells (4 × 10 cells/well) were exposed to different L6123 concentrations (5, 10, and 20 μmol/l) for 48 h before staining with Hoechst 33 342 dye (Beyotime Biotech, Nantong, China). NDGA (20 μmol/l) was used as a positive control. The stained cells were observed under a fluorescence microscope (Nikon, Tokyo, Japan). Images were viewed under a × 200 objective. 5
Transwell invasion assay
SGC-7901 cell migration was performed in Corning Transwell insert chambers (8.0 μm pore size) according to the manufacturer’s instructions. SGC-7901 cells (1 × 105) were paved in 200 μl of serum-free RPMI 1640 medium in the upper well (precoated with diluted Matrigel) before incubation with various concentrations (5, 10, and 20 μmol/l) of L6123 for 24 h. RPMI 1640 medium (500 μl) with 10% FBS was added to the lower chambers. After 24 h at 37°C, cells were removed from the upper surface of the membrane by a cotton swab. The membranes were cut from the chamber and fixed with 4% paraformaldehyde for 15–20 min. The membranes were stained with crystal violet for 25–30 min. Finally, cells that adhered to the lower surface of the membranes were counted and imaged by phase microscopy. Images were observed under a × 200 objective. Statistical analysis
All data were assayed in triplicate (n = 3) and were represented as mean ± SEM. Statistical analyses were calculated using Student’s t-test and one-way analysis of variance using GraphPad Prism 5.0 (GraphPad, San Diego, California, USA). A P value of less than 0.05 was considered statistically significant.
Results L6123 suppressed the growth of GC cell lines
On the level of FGFR1 kinases, L6123 showed better inhibitory activity than NDGA (FGFR1 inhibition IC50 of 0.6 μmol/l for L6123 and 24.5 μmol/l for NDGA). FGFR1 was highly expressed in the GC cell lines SGC-7901, BGC-823, and MGC-803. The proliferation and colony formation of GC cells were inhibited by silencing FGFR1 [10]. The antitumor effect of L6123 and NDGA on GC cells was tested using an MTT assay. All GC cell lines were treated with various concentrations of L6123 and NDGA for 72 h. L6123 had lower IC50 than NDGA in SGC-7901, BGC-823, and MGC-803 (Fig. 2a). Therefore, L6123 showed better antitumor effect than NDGA. To detect the target selectivity of L6123, the antiproliferative activity of L6123 on MEF-WT and MEF-FGFR1-FGFR2-FRS2α-KO cells was studied using an MTT assay. MEF-WT and MEF-FGFR1-FGFR2-FRS2α-KO cells were treated with various concentrations of L6123 or NDGA for 72 h. L6123 showed better inhibitory activity against MEF-WT than MEF-FGFR1-FGFR2-FRS2α -KO cells (Fig. 2b). FGFR1 knockout inhibited the antiproliferative activity of L6123. Moreover, L6123 showed higher IC50 than NDGA
in MEF-FGFR1-FGFR2-FRS2α -KO cells (IC50: 68.1 ± 2.9 μmol/l for L6123 and 53.7 ± 6.5 μmol/l for NDGA). L6123 downregulated the phosphorylation of FGFR1 and downstream signaling pathways
In GC cell lines, L6123 had better antitumor activity than NDGA, but the mechanism remains unclear. The expression of FGFR1 protein in SGC-790 was the highest [10] among the GC cell lines (SGC-7901, BGC-823, and MGC-803), which was consistent with our findings (data not shown). Meanwhile, L6123 and NDGA showed the best inhibitory activity against SGC-790. Hence, SGC-7901 was used to investigate (by western blot analysis) whether L6123 could downregulate FGFR1 and its downstream signaling pathways. L6123 induced an evident and dose-dependent decrease in FGF2-induced phosphorylation of FGFR1, FRS2α, and ERK1/2 (Fig. 3). Therefore, L6123 showed significant antitumor activity by downregulating the FGFR1/ FRS2α/ERK1/2 pathway in GC cells. NDGA could exert an antitumor effect by inhibiting RTK activity [11–13], which is in agreement with our finding that NDGA (20 μmol/l) could arrest the FGFR1/FRS2α/ERK1/2 pathway (Fig. 3). Compound L6123 enhanced apoptosis in the GC cells
The effects of L6123 on cell apoptosis were analyzed by flow cytometry, Hoechst staining, and western blot. We investigated whether the mitochondria-mediated apoptotic pathway was involved in L6123 induced apoptosis. The apoptosis-related molecules, namely, Bcl-2, cleavedcaspase-3, pro-caspase-3, and cleaved-PARP, were used to evaluate the apoptotic ability of SGC-7901 cells treated with varying concentrations of L6123. The expression of cleaved-caspase-3, pro-caspase-3, and Bcl-2 decreased, and cleaved-PARP expression increased in SGC-7901 cells after pretreatment with varying concentrations of L6123. However, the effect of NDGA on cell apoptosis was not obvious (Fig. 4). The result of flow cytometry using the annexin V-PI dual-labeling technique showed that L6123 increased the number of apoptotic SGC-7901 cells to 0.7, 2.9, and 14.1% at concentrations of 5, 10, and 20 μmol/l, respectively. However, the total apoptotic cell percentage was only 1.8% after pretreatment with NDGA (20 μmol/l) (Fig. 5a). The same result was found in Hoechst staining. SGC-7901 cell number increased after pretreatment with different concentrations of L6123 for 48 h, and nuclear schizolysis and condensations occurred in a dose-dependent manner (Fig. 5b). Thus, L6123 could induce GC cell apoptosis. Compound L6123 inhibited cell invasion
We detected the invasion ability of SGC-7901 cells treated with varying concentrations of L6123. The cell invasion capacity was measured using a Transwell invasion assay with diluted Matrigel chamber precoating. SGC-7901 cells (1 × 105) were paved in 200 μl of serum-free RPMI 1640 medium in the upper well, and were treated or not treated with various concentrations of L6123. Cells that passed
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382 Anti-Cancer Drugs 2015, Vol 26 No 4
Fig. 2
(a) IC50 (μmol/l) Compound BGC-823
MGC-803
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Compounds L6123 and NDGA inhibited cell proliferation. (a) The IC50 of L6123 and NDGA inhibiting GC cells (SGC-7901, BGC-823, and MGC-803) proliferation. (b) L6123 and NDGA all showed better inhibitory effect on MEF-WT cells than that of MEF-FGFR1-FGFR2-FRS2α-KO cells. NDGA, nordihydroguaiaretic acid.
through the Matrigel and Transwell membrane were stained. The results of the Transwell cell invasion are presented in Fig. 6. The addition of L6123 (at 5, 10, and 20 μmol/l) in the upper well downregulated the invading cell numbers in a dose-dependent manner. In addition, NDGA (20 μmol/l) failed to reduce the number of invading cells.
Discussion In recent years, genetic amplifications of FGFR family members have been discovered in GC. Many researchers found FGFR2 amplification in ∼ 4–10% of GCs; FGFR2 amplification was associated with depth of invasion, lymph node metastasis, distant metastasis, and TMN staging, but was not associated with age, sex, and degree of differentiation of GC patients [14–16]. Targeted FGFR2 inhibitors, such as GP369, Ki23057, and FGFR2 monoclonal antibodies, could effectively inhibit GC growth and enhance the chemosensitivity of drugresistant GC cells [17–20]. By detecting FGFR1 in GC tissue specimens, Oki et al. [21] found that FGFR1 expression was amplified, and the overexpression was related closely to EphA4 expression; both FGFR1 and EphA4 expressions play an important role in GC. Wen et al. [10] also found that FGFR1 was amplified not only in gastric tissue samples but also in a variety of GC cell lines; when the FGFR1 was silenced by miR-133b, the growth of GC cell lines was inhibited. FGFR1 might be
a promising target in the therapy of GCs. Targeting FGFR1 inhibitors with minimal side effects and low cell toxicity is important in GC therapy. Because of multiple targets and the limited selectivity of the ATP-competitive inhibitor, we attempted to discover a novel kinase inhibitor. Non-ATP-competitive inhibitors may show targeted selectivity; the research and development of non-ATP-competitive inhibitors has become the focus of research [22,23]. However, except for ARQ 069 [24], information on other non-ATPcompetitive FGFR1 inhibitors is limited. NDGA is a non-ATP-competitive FGFR3 kinase inhibitor that inhibits FGFR3 autophosphorylation and downstream signaling in multiple myeloma in vitro and in vivo [11]. We found that NDGA showed better selective inhibitory effect against FGFR1 than against FGFR3. Hence, using NDGA as a positive control, we successfully designed a series of novel non-ATP-competitive FGFR1 kinase inhibitors, which showed better inhibition activity against FGFR1 than NDGA [25]. Among the active FGFR1 inhibitors, the compound L6123 showed significant inhibitory activity against FGFR1 (IC50 : 0.6 μmol/l). Thus, the antitumor activity of L6123 on GC was explored. L6123 showed a good antiproliferative effect on GC cell lines, including SGC-7901, BGC-823, and MGC-803, in which FGFR1 was overexpressed. The activity of L6123 against three GC cell lines was better than that of NDGA, and this finding was in agreement
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FGFR1 inhibitor on gastric cancer Xu et al. 383
Fig. 3
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Compound L6123 suppressed FGF2-induced phosphorylation of FGFR1, FES2α, and ERK1/2 in a dose-dependent manner. SGC-7901 cell was treated with compound L6123 (5, 10 and 20 μmol/l) for 1 h, starved for 24 h, and then stimulated with FGF2 (20 ng/ml) for 10 min; cell lysates was collected and the phosphorylation levels of FGFR1, FRS2α, and ERK1/2 were tested using a western blotting assay. NDGA (20 μmol/l) was used as a positive control. The column figure was the normalized optical density as a percentage of the relevant total protein. Data are presented as the mean ± SD of three independent experiments conducted in triplicate.**P < 0.01; ***P < 0.001. Com., compound; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor 2; NDGA, nordihydroguaiaretic acid; NS, not significant.
with the activities of L6123 and NDGA against FGFR1 kinase. Thus, compound L6123 showed better activity than NDGA not only at the level of FGFR1 kinases but also at the cellular level. Target selectivity is very important for a kinase inhibitor. The antiproliferative activities of L6123 against MEF-FGFR1-FGFR2-FRS2α-KO (FGFR1 knockout) and MEF-WT were detected using an MTT assay, and the results were compared. L6123 showed higher inhibitory effect against MEF-WT than against MEFFGFR1-FGFR2-FRS2α-KO . FGFR1 knockout downregulated the antiproliferative activity of L6123 and L6123 showed an antitumor effect by targeting FGFR1, at least partly. Similar to ATP-competitive FGFR1 inhibitors AZD4547
[26] and BGJ398 [27] and non-ATP-competitive FGFR1 inhibitor ARQ 069 [24], L6123 showed a satisfactory inhibitory effect on FGFR2 and FGFR3 (data not shown). Thus, the target selectivity of L6123 needs improvement. We are designing novel analogs with L6123 as the leading compound. ERKs are members of the MAPK superfamily that play a major role in cell proliferation. In many cancers, ERK1/2 functions as a downstream signaling molecule of FGFRs [22]. FRS2α, which is a substrate of FGFR1, can induce the activation of ERK1/2 and FRS2α and is vital for the FGF/FGFR pathway [28]. FGFR1/FRS2α/ERK1/2 signaling has not been investigated previously in GC cell lines. We confirmed that FGF2 can activate the FGFR1/
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384 Anti-Cancer Drugs 2015, Vol 26 No 4
Fig. 4
L6123 −
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Compound L6123 activated caspase-3 and PARP and suppressed Bcl-2 in a dose-dependent manner in SCG-7901 cells. SCG-7901 cells were exposed to L6123 or NDGA at indicated concentrations for 48 h. The levels of Bcl-2, cleaved-caspase-3, pro-caspase-3, and cleaved-PARP were detected by western blot analysis. GAPDH was used as a criterion. Data are presented as the mean ± SD of three independent experiments conducted in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001. Com., compound; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NDGA, nordihydroguaiaretic acid.
FRS2α/ERK1/2 pathway. L6123 showed inhibitory effects on the growth of GC cells by inhibiting this signal pathway. Recently, Zhao et al. [29] confirmed that FGF signaling regulates cell survival and migration by the regulation of NFκB and CXCL4-SDF1 pathways. Further studies are required to determine whether L6123 suppresses cell survival and migration by the NFκB and CXCL4-SDF1 pathways. Bcl-2, caspase-3, and PARP are apoptosis-related molecules that participate in the mitochondria-mediated apoptotic pathway. siRNA silencing of FGFR expression in the human GC cell line MGC-803 induced the activity of the apoptosis pathway by the upregulation of Bax and caspase-3 [30]. Moreover, FGFR inhibitor PD173074 caused apoptotic death by decreasing the expression of Bcl-2, survivin, and Mcl-1 and by increasing the expression of Bax [31]. L6123 enhanced apoptosis
of the GC cells in response to FGFR1 downregulation, as confirmed by the change in apoptosis-related molecules Bcl-2, cleaved-caspase-3, pro-caspase-3, and cleavedPARP. The epithelial–mesenchymal transition (EMT) is a core process in tumor progression and its deregulation is associated with the cancer cell’s ability to invade stromal tissues and migrate to other regions [32]. The FGFR1 inhibitor PD173074 induces EMT transition through the transcription factor AP-1 in head and neck squamous cell carcinoma [33]. L6123 could prohibit gastric cell invasion. Further studies are required to determine whether L6123 could induce EMT and to elucidate the mechanism underlying this activity. FGFR1-targeted therapy is a promising strategy for the treatment and diagnosis of GC. In this study, we proved that L6123 inhibits the phosphorylation of FGFR1 and its downstream signaling molecules at the cellular level. L6123
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FGFR1 inhibitor on gastric cancer Xu et al. 385
Fig. 5
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Compound L6123 induced SCG-7901 cells apoptosis. (a) SGC-7901 cells were incubated with indicated concentrations for 48 h and then stained with annexin V and PI. The number of apoptotic cells was detected by flow cytometry. The figures were representative of three separate experiments. (b) Hoechst staining and morphological changes was observed in SCG-7901 cells cultured with L6123 (5, 10, and 20 μmol/l) or NDGA (20 μmol/l) for 48 h. Figures (×200) were representative of more than three separate experiments.**P < 0.01. Com., compound; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; NDGA, nordihydroguaiaretic acid; PI, propidium iodide.
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386 Anti-Cancer Drugs 2015, Vol 26 No 4
Fig. 6
L6123 (5 μmol/l)
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significantly repressed proliferation, inhibited invasion, and induced apoptosis in FGFR1-overexpressing GC cells.
5
Acknowledgements The work was supported by the ZheJiang Province Natural Science Funding of China (LY12H30004, LY14H160044), the National Natural Science Foundation of China (81102310, 81402839, 81272462), and the Technology Foundation for Medical Science of Zhejiang Province (2012KYA129, 2012 KYB127). Conflicts of interest
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There are no conflicts of interest. 11
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The therapeutic potential of a novel non-ATP-competitive fibroblast growth factor receptor 1 inhibitor on gastric cancer Chaochao Xua,c,*, Wulan Lia,b,*, Peihong Qiua, Yiqun Xiac, Xiaojing Duc, Fen Wange, Lailai Shena, Qiuxiang Chenc, Yunjie Zhaoa, Rong Jinc,d, Jianzhang Wua, Guang Lianga and Xiaokun Lia Previous studies showed that fibroblast growth factor receptor 1 (FGFR1) is an attractive target in gastric cancer therapy. In the current study, we aimed to investigate whether the compound L6123, a novel non-ATP-competitive FGFR1 inhibitor, could show better antitumor activity than the leading compound, nordihydroguaiaretic acid (NDGA), in FGFR1-overexpressing gastric cancer cells. Using an MTT assay, we investigated the inhibitory effect of L6123 on the viability of three gastric cancer cells (MGC-803, SGC-7901, and BGC-823) overexpressing FGFR1, wild-type mouse embryonic fibroblast (MEF-WT), and MEF expressing FGFR1, FGFR2, and FRS2α gene knockout (MEFFGFR1-FGFR2-FRS2α-ko ). We studied the antitumor mechanism of L6123 against the gastric cancer cell line SGC-7901 by western blot analysis. The antitumor effects of L6123 on the gastric cancer cell line SGC-7901 were detected by flow cytometry, Hoechst staining, western blot analysis, and Transwell invasion assay. L6123 had lower IC50 in all three gastric cancer cells than NDGA and showed better inhibitory activity against MEF-WT cells than against MEFFGFR1-FGFR2-FRS2α-ko cells. In the SGC-7901 gastric cell, L6123 inhibited the FGF2-induced phosphorylation of FGFR1/FRS2α/ERK1/2 in a dose-dependent manner, induced the activation of the apoptosis-related proteins, cleaved-PARP and cleaved-caspase-3, decreased the expression of pro-caspase-3 and Bcl-2, and induced tumor cell apoptosis. L6123 also dose-dependently reduced cell
Introduction The incidence of gastric cancer (GC) has decreased markedly worldwide. However, 989 600 new cases of stomach cancer (8% of the total new cancer cases) and 738 000 deaths (10% of the total deaths) have occurred in the past 7 years [1]. Surgical techniques and postoperative adjuvant chemotherapies have improved, but the total survival rate of GC remains unsatisfactory. Therefore, understanding the molecular mechanisms of GC and developing new targeted therapies to improve the survival rate of GC patients are important. Investigation of targeted inhibitors of receptor tyrosine kinases (RTKs) is currently one of the prevalent topics of cancer treatment research; many inhibitor drugs, including EGFR and VEGFR inhibitors, are used clinically [2,3]. For GC, some studies report that there are gene amplification, mutations, and rearrangements of 0959-4973 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
invasion ability, and showed better activity than NDGA at the same concentration. A novel non-ATP-competitive inhibitor L6123 showed excellent antigastric cancer activity by inhibiting the FGFR1 signaling pathway. Thus, we discovered a potential agent for the treatment of FGFR1-overexpressing gastric cancer. Anti-Cancer Drugs 26:379–387 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Anti-Cancer Drugs 2015, 26:379–387 Keywords: fibroblast growth factor receptor 1, gastric cancer, molecular targeted therapy a
Chemical Biology Research Center, College of Pharmaceutical Sciences, College of Information Science and Computer Engineering, Wenzhou Medical University, Departments of cDigestive Diseases, dEpidemiology, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China and eThe Center for Cancer and Stem Cell Biology, Institute of Biosciences and Technology, Texas A&M University, Houston, Texas, USA b
Correspondence to Rong Jin, MD, Department of Digestive Diseases, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 323000, China Tel/fax: + 86 577 555 79999; e-mail: [email protected] Correspondence to Jianzhang Wu, PhD, Chemical Biology Research Center, College of Pharmaceutical Sciences, Wenzhou Medical Universtiy, Wenzhou, Zhejiang 325035, China Tel/fax: + 86 577 866 99396; e-mail: [email protected] *Chaochao Xu and Wulan Li contributed equally to the writing of this article. Received 14 May 2014 Revised form accepted 14 November 2014
RTKs; RTK inhibition might be a new therapeutic strategy for GC. For example, foretinib (GSK1363089), imatinib, regorafenib, and other RTK inhibitors have shown great therapeutic potential against GC [4–6]. FGFR, which is an RTK, can activate downstream signaling pathways, including FRS2α, PI3K/AKT, and ERK1/2 pathways, after ligand [fibroblast growth factor 2 (FGF2)] binding [7]. Overexpression of fibroblast growth factor receptors (FGFRs) extensively involves the regulation of physiological and pathological functions, including cell proliferation, apoptosis, metastasis, and angiogenesis [8]. In GC, FGFRs are overactivated by several mechanisms, including gene amplification, chromosomal translocation, and mutation [9]. Compared with other FGFRs, FGFR1 expression is amplified in GC cell lines and tissues [10]. The development of targeted inhibitors of FGFR1 may be a promising therapeutic strategy for GC. DOI: 10.1097/CAD.0000000000000195
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Fig. 1
CH3 HO
O
OH HO
OH
OH CH3
HO
NDGA FGFR1 inhibition: IC50=24.5μmol/l
HO
S
OH
L6123 FGFR1 inhibition: IC50=0.6μmol/l
Structures of L6123 and NDGA. The IC50 of FGFR1 inhibition: 0.6 μmol/l for L6123; 24.5 μmol/l for NDGA. FGFR1, fibroblast growth factor receptor 1; NDGA, nordihydroguaiaretic acid.
Nordihydroguaiaretic acid (NDGA) is a novel non-ATPcompetitive FGFR inhibitor [11]. In our previous study, we designed and obtained a novel non-ATP-competitive FGFR1 inhibitor L6123. L67123 showed better inhibitory activity on the level of FGFR1 kinases than NDGA, which was used as a positive control (Fig. 1). L6123 was identified as a selective FGFR1 inhibitor with therapeutic potential in nonsmall cell lung cancer in vivo and in vitro (data not shown). Therefore, we hypothesized that L6123 has therapeutic potential in FGFR1-overexpressing GC cells. The results obtained were in agreement with our hypothesis that L6123 showed excellent anti-GC activity by inhibiting the FGFR signaling pathway.
Methods Cell lines, compounds, and reagents
Human GC cells MGC-803, SGC-7901, and BGC-823 were purchased from the Shanghai Institute of Biosciences and Cell Resources Center (Chinese Academy of Sciences, Shanghai, China), Wuhan Boster Biological Engineering Co. Ltd (Wuhan, People’s Republic of China), and the Xiangya Cell Center of Central South University (Changsha, China), respectively. SGC-7901, BGC-823, and MGC-803 cells were maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin. The wild-type mouse embryonic fibroblast (MEF-WT) and MEF-FGFR1-FGFR2-FRS2α-KO (FGFR1, FGFR2, and FRS2α gene knockout) cell lines were kindly provided by Fen Wang (Professor), Texas A&M University, and were cultured in Dulbecco’s modified Eagle’s medium with 1% penicillin and 10% FBS (Gibco, Eggenstein, Germany). All cells were incubated at 37°C in an atmosphere of 5% CO2. L6123 was synthesized and purified in our laboratory, and the purity was 98.0%. NDGA was purchased from Sigma (St Louis, Missouri, USA). Recombinant human FGF2 was purchased from the Institute of Biological and Natural Product, Wenzhou Medical University, Wenzhou, China. Cell proliferation assay
An MTT assay was performed to evaluate the antiproliferative activity of the compound L6123 against the GC cell lines SGC-7901, BGC-823, and MGC-803, and the MEF-WT and MEF-FGFR1-FGFR2-FRS2α-KO cells. All cells (4000 cells/well) were seeded in a 96-well plate.
After 24 h, the culture medium was removed and all cells were treated with increasing concentrations (20/27, 20/9, 20/3, 20, and 60 μmol/l) of compound L6123 for 72 h. NDGA was used as a positive control. After treatment, the viability of the cells was tested using an MTT assay.
Antibodies and western blotting assay
All antibodies, including the goat anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase (HRP) and mouse anti-goat IgG-HRP antibodies, were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). Anti-pFRS2α and anticleaved-caspase-3 were purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). The western blot was operated by standard means. After washing with PBS, 60 μl/well cell lysates were added to a six-well plate at 4°C for 3 min. Then, cell lysates were collected and centrifuged to remove the insoluble components. The supernatant was run on 10% SDS-PAGE and then transferred to a PVDF membrane. After blocking with 5% nonfat dry milk in TBST, the membrane was incubated with the primary antibodies (anti-pFGFR1, anti-FGFR1, anti-pFRS2α, anti-FRS2α, anti-pERK1/2, anti-ERK1/2, anticleaved-caspase-3, anti-procaspase-3, anti-Bcl-2, anticleaved-PARP, and anti-GAPDH) overnight and subsequently incubated with goat anti-rabbit IgG or mouse anti-goat IgG HRP-linked antibody. The blots were detected using an ECL detection kit (Bio-Rad, Hercules, California, USA) according to the manufacturer’s procedure. The results were analyzed using Quantity One software (BioRad) to determine the relative band density ratio.
Cell apoptosis assay
The percentage of apoptotic cells was detected by flow cytometry using the annexin V and propidium iodide (PI) staining. SGC-7901 cells were exposed to different compound concentrations (5, 10, and 20 μmol/l) for 48 h and were subsequently harvested. NDGA was used as a positive control. Finally, cells were incubated with 1 × binding buffer, annexin V (5 μl), and PI (1 μl) in a dark room for 15 min. Cells were analyzed by FACScalibur Flow Cytometry (BD Biosciences, San Jose, California, USA).
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FGFR1 inhibitor on gastric cancer Xu et al. 381
Hoechst assay
SGC-7901 cells (4 × 10 cells/well) were exposed to different L6123 concentrations (5, 10, and 20 μmol/l) for 48 h before staining with Hoechst 33 342 dye (Beyotime Biotech, Nantong, China). NDGA (20 μmol/l) was used as a positive control. The stained cells were observed under a fluorescence microscope (Nikon, Tokyo, Japan). Images were viewed under a × 200 objective. 5
Transwell invasion assay
SGC-7901 cell migration was performed in Corning Transwell insert chambers (8.0 μm pore size) according to the manufacturer’s instructions. SGC-7901 cells (1 × 105) were paved in 200 μl of serum-free RPMI 1640 medium in the upper well (precoated with diluted Matrigel) before incubation with various concentrations (5, 10, and 20 μmol/l) of L6123 for 24 h. RPMI 1640 medium (500 μl) with 10% FBS was added to the lower chambers. After 24 h at 37°C, cells were removed from the upper surface of the membrane by a cotton swab. The membranes were cut from the chamber and fixed with 4% paraformaldehyde for 15–20 min. The membranes were stained with crystal violet for 25–30 min. Finally, cells that adhered to the lower surface of the membranes were counted and imaged by phase microscopy. Images were observed under a × 200 objective. Statistical analysis
All data were assayed in triplicate (n = 3) and were represented as mean ± SEM. Statistical analyses were calculated using Student’s t-test and one-way analysis of variance using GraphPad Prism 5.0 (GraphPad, San Diego, California, USA). A P value of less than 0.05 was considered statistically significant.
Results L6123 suppressed the growth of GC cell lines
On the level of FGFR1 kinases, L6123 showed better inhibitory activity than NDGA (FGFR1 inhibition IC50 of 0.6 μmol/l for L6123 and 24.5 μmol/l for NDGA). FGFR1 was highly expressed in the GC cell lines SGC-7901, BGC-823, and MGC-803. The proliferation and colony formation of GC cells were inhibited by silencing FGFR1 [10]. The antitumor effect of L6123 and NDGA on GC cells was tested using an MTT assay. All GC cell lines were treated with various concentrations of L6123 and NDGA for 72 h. L6123 had lower IC50 than NDGA in SGC-7901, BGC-823, and MGC-803 (Fig. 2a). Therefore, L6123 showed better antitumor effect than NDGA. To detect the target selectivity of L6123, the antiproliferative activity of L6123 on MEF-WT and MEF-FGFR1-FGFR2-FRS2α-KO cells was studied using an MTT assay. MEF-WT and MEF-FGFR1-FGFR2-FRS2α-KO cells were treated with various concentrations of L6123 or NDGA for 72 h. L6123 showed better inhibitory activity against MEF-WT than MEF-FGFR1-FGFR2-FRS2α -KO cells (Fig. 2b). FGFR1 knockout inhibited the antiproliferative activity of L6123. Moreover, L6123 showed higher IC50 than NDGA
in MEF-FGFR1-FGFR2-FRS2α -KO cells (IC50: 68.1 ± 2.9 μmol/l for L6123 and 53.7 ± 6.5 μmol/l for NDGA). L6123 downregulated the phosphorylation of FGFR1 and downstream signaling pathways
In GC cell lines, L6123 had better antitumor activity than NDGA, but the mechanism remains unclear. The expression of FGFR1 protein in SGC-790 was the highest [10] among the GC cell lines (SGC-7901, BGC-823, and MGC-803), which was consistent with our findings (data not shown). Meanwhile, L6123 and NDGA showed the best inhibitory activity against SGC-790. Hence, SGC-7901 was used to investigate (by western blot analysis) whether L6123 could downregulate FGFR1 and its downstream signaling pathways. L6123 induced an evident and dose-dependent decrease in FGF2-induced phosphorylation of FGFR1, FRS2α, and ERK1/2 (Fig. 3). Therefore, L6123 showed significant antitumor activity by downregulating the FGFR1/ FRS2α/ERK1/2 pathway in GC cells. NDGA could exert an antitumor effect by inhibiting RTK activity [11–13], which is in agreement with our finding that NDGA (20 μmol/l) could arrest the FGFR1/FRS2α/ERK1/2 pathway (Fig. 3). Compound L6123 enhanced apoptosis in the GC cells
The effects of L6123 on cell apoptosis were analyzed by flow cytometry, Hoechst staining, and western blot. We investigated whether the mitochondria-mediated apoptotic pathway was involved in L6123 induced apoptosis. The apoptosis-related molecules, namely, Bcl-2, cleavedcaspase-3, pro-caspase-3, and cleaved-PARP, were used to evaluate the apoptotic ability of SGC-7901 cells treated with varying concentrations of L6123. The expression of cleaved-caspase-3, pro-caspase-3, and Bcl-2 decreased, and cleaved-PARP expression increased in SGC-7901 cells after pretreatment with varying concentrations of L6123. However, the effect of NDGA on cell apoptosis was not obvious (Fig. 4). The result of flow cytometry using the annexin V-PI dual-labeling technique showed that L6123 increased the number of apoptotic SGC-7901 cells to 0.7, 2.9, and 14.1% at concentrations of 5, 10, and 20 μmol/l, respectively. However, the total apoptotic cell percentage was only 1.8% after pretreatment with NDGA (20 μmol/l) (Fig. 5a). The same result was found in Hoechst staining. SGC-7901 cell number increased after pretreatment with different concentrations of L6123 for 48 h, and nuclear schizolysis and condensations occurred in a dose-dependent manner (Fig. 5b). Thus, L6123 could induce GC cell apoptosis. Compound L6123 inhibited cell invasion
We detected the invasion ability of SGC-7901 cells treated with varying concentrations of L6123. The cell invasion capacity was measured using a Transwell invasion assay with diluted Matrigel chamber precoating. SGC-7901 cells (1 × 105) were paved in 200 μl of serum-free RPMI 1640 medium in the upper well, and were treated or not treated with various concentrations of L6123. Cells that passed
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Fig. 2
(a) IC50 (μmol/l) Compound BGC-823
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Compounds L6123 and NDGA inhibited cell proliferation. (a) The IC50 of L6123 and NDGA inhibiting GC cells (SGC-7901, BGC-823, and MGC-803) proliferation. (b) L6123 and NDGA all showed better inhibitory effect on MEF-WT cells than that of MEF-FGFR1-FGFR2-FRS2α-KO cells. NDGA, nordihydroguaiaretic acid.
through the Matrigel and Transwell membrane were stained. The results of the Transwell cell invasion are presented in Fig. 6. The addition of L6123 (at 5, 10, and 20 μmol/l) in the upper well downregulated the invading cell numbers in a dose-dependent manner. In addition, NDGA (20 μmol/l) failed to reduce the number of invading cells.
Discussion In recent years, genetic amplifications of FGFR family members have been discovered in GC. Many researchers found FGFR2 amplification in ∼ 4–10% of GCs; FGFR2 amplification was associated with depth of invasion, lymph node metastasis, distant metastasis, and TMN staging, but was not associated with age, sex, and degree of differentiation of GC patients [14–16]. Targeted FGFR2 inhibitors, such as GP369, Ki23057, and FGFR2 monoclonal antibodies, could effectively inhibit GC growth and enhance the chemosensitivity of drugresistant GC cells [17–20]. By detecting FGFR1 in GC tissue specimens, Oki et al. [21] found that FGFR1 expression was amplified, and the overexpression was related closely to EphA4 expression; both FGFR1 and EphA4 expressions play an important role in GC. Wen et al. [10] also found that FGFR1 was amplified not only in gastric tissue samples but also in a variety of GC cell lines; when the FGFR1 was silenced by miR-133b, the growth of GC cell lines was inhibited. FGFR1 might be
a promising target in the therapy of GCs. Targeting FGFR1 inhibitors with minimal side effects and low cell toxicity is important in GC therapy. Because of multiple targets and the limited selectivity of the ATP-competitive inhibitor, we attempted to discover a novel kinase inhibitor. Non-ATP-competitive inhibitors may show targeted selectivity; the research and development of non-ATP-competitive inhibitors has become the focus of research [22,23]. However, except for ARQ 069 [24], information on other non-ATPcompetitive FGFR1 inhibitors is limited. NDGA is a non-ATP-competitive FGFR3 kinase inhibitor that inhibits FGFR3 autophosphorylation and downstream signaling in multiple myeloma in vitro and in vivo [11]. We found that NDGA showed better selective inhibitory effect against FGFR1 than against FGFR3. Hence, using NDGA as a positive control, we successfully designed a series of novel non-ATP-competitive FGFR1 kinase inhibitors, which showed better inhibition activity against FGFR1 than NDGA [25]. Among the active FGFR1 inhibitors, the compound L6123 showed significant inhibitory activity against FGFR1 (IC50 : 0.6 μmol/l). Thus, the antitumor activity of L6123 on GC was explored. L6123 showed a good antiproliferative effect on GC cell lines, including SGC-7901, BGC-823, and MGC-803, in which FGFR1 was overexpressed. The activity of L6123 against three GC cell lines was better than that of NDGA, and this finding was in agreement
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FGFR1 inhibitor on gastric cancer Xu et al. 383
Fig. 3
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Compound L6123 suppressed FGF2-induced phosphorylation of FGFR1, FES2α, and ERK1/2 in a dose-dependent manner. SGC-7901 cell was treated with compound L6123 (5, 10 and 20 μmol/l) for 1 h, starved for 24 h, and then stimulated with FGF2 (20 ng/ml) for 10 min; cell lysates was collected and the phosphorylation levels of FGFR1, FRS2α, and ERK1/2 were tested using a western blotting assay. NDGA (20 μmol/l) was used as a positive control. The column figure was the normalized optical density as a percentage of the relevant total protein. Data are presented as the mean ± SD of three independent experiments conducted in triplicate.**P < 0.01; ***P < 0.001. Com., compound; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor 2; NDGA, nordihydroguaiaretic acid; NS, not significant.
with the activities of L6123 and NDGA against FGFR1 kinase. Thus, compound L6123 showed better activity than NDGA not only at the level of FGFR1 kinases but also at the cellular level. Target selectivity is very important for a kinase inhibitor. The antiproliferative activities of L6123 against MEF-FGFR1-FGFR2-FRS2α-KO (FGFR1 knockout) and MEF-WT were detected using an MTT assay, and the results were compared. L6123 showed higher inhibitory effect against MEF-WT than against MEFFGFR1-FGFR2-FRS2α-KO . FGFR1 knockout downregulated the antiproliferative activity of L6123 and L6123 showed an antitumor effect by targeting FGFR1, at least partly. Similar to ATP-competitive FGFR1 inhibitors AZD4547
[26] and BGJ398 [27] and non-ATP-competitive FGFR1 inhibitor ARQ 069 [24], L6123 showed a satisfactory inhibitory effect on FGFR2 and FGFR3 (data not shown). Thus, the target selectivity of L6123 needs improvement. We are designing novel analogs with L6123 as the leading compound. ERKs are members of the MAPK superfamily that play a major role in cell proliferation. In many cancers, ERK1/2 functions as a downstream signaling molecule of FGFRs [22]. FRS2α, which is a substrate of FGFR1, can induce the activation of ERK1/2 and FRS2α and is vital for the FGF/FGFR pathway [28]. FGFR1/FRS2α/ERK1/2 signaling has not been investigated previously in GC cell lines. We confirmed that FGF2 can activate the FGFR1/
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Fig. 4
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DMSO
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L6123 NDGA Bcl-2/GAPDH
Compound L6123 activated caspase-3 and PARP and suppressed Bcl-2 in a dose-dependent manner in SCG-7901 cells. SCG-7901 cells were exposed to L6123 or NDGA at indicated concentrations for 48 h. The levels of Bcl-2, cleaved-caspase-3, pro-caspase-3, and cleaved-PARP were detected by western blot analysis. GAPDH was used as a criterion. Data are presented as the mean ± SD of three independent experiments conducted in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001. Com., compound; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NDGA, nordihydroguaiaretic acid.
FRS2α/ERK1/2 pathway. L6123 showed inhibitory effects on the growth of GC cells by inhibiting this signal pathway. Recently, Zhao et al. [29] confirmed that FGF signaling regulates cell survival and migration by the regulation of NFκB and CXCL4-SDF1 pathways. Further studies are required to determine whether L6123 suppresses cell survival and migration by the NFκB and CXCL4-SDF1 pathways. Bcl-2, caspase-3, and PARP are apoptosis-related molecules that participate in the mitochondria-mediated apoptotic pathway. siRNA silencing of FGFR expression in the human GC cell line MGC-803 induced the activity of the apoptosis pathway by the upregulation of Bax and caspase-3 [30]. Moreover, FGFR inhibitor PD173074 caused apoptotic death by decreasing the expression of Bcl-2, survivin, and Mcl-1 and by increasing the expression of Bax [31]. L6123 enhanced apoptosis
of the GC cells in response to FGFR1 downregulation, as confirmed by the change in apoptosis-related molecules Bcl-2, cleaved-caspase-3, pro-caspase-3, and cleavedPARP. The epithelial–mesenchymal transition (EMT) is a core process in tumor progression and its deregulation is associated with the cancer cell’s ability to invade stromal tissues and migrate to other regions [32]. The FGFR1 inhibitor PD173074 induces EMT transition through the transcription factor AP-1 in head and neck squamous cell carcinoma [33]. L6123 could prohibit gastric cell invasion. Further studies are required to determine whether L6123 could induce EMT and to elucidate the mechanism underlying this activity. FGFR1-targeted therapy is a promising strategy for the treatment and diagnosis of GC. In this study, we proved that L6123 inhibits the phosphorylation of FGFR1 and its downstream signaling molecules at the cellular level. L6123
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FGFR1 inhibitor on gastric cancer Xu et al. 385
Fig. 5
(a)
L6123 (5 μmol/l)
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Compound L6123 induced SCG-7901 cells apoptosis. (a) SGC-7901 cells were incubated with indicated concentrations for 48 h and then stained with annexin V and PI. The number of apoptotic cells was detected by flow cytometry. The figures were representative of three separate experiments. (b) Hoechst staining and morphological changes was observed in SCG-7901 cells cultured with L6123 (5, 10, and 20 μmol/l) or NDGA (20 μmol/l) for 48 h. Figures (×200) were representative of more than three separate experiments.**P < 0.01. Com., compound; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; NDGA, nordihydroguaiaretic acid; PI, propidium iodide.
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386 Anti-Cancer Drugs 2015, Vol 26 No 4
Fig. 6
L6123 (5 μmol/l)
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Compound L6123 suppressed the invasion ability of SGC-7901 cells in a dose-dependent manner. After incubation with compound L6123 (5, 10, and 20 μmol/l) for 24 h, the invasion ability was test using a Transwell invasion assay. NDGA (20 μmol/l) was used as a positive control. Figures (×200) were representative of three separate experiments. Com., compound; DMSO, dimethyl sulfoxide; NDGA, nordihydroguaiaretic acid.
significantly repressed proliferation, inhibited invasion, and induced apoptosis in FGFR1-overexpressing GC cells.
5
Acknowledgements The work was supported by the ZheJiang Province Natural Science Funding of China (LY12H30004, LY14H160044), the National Natural Science Foundation of China (81102310, 81402839, 81272462), and the Technology Foundation for Medical Science of Zhejiang Province (2012KYA129, 2012 KYB127). Conflicts of interest
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There are no conflicts of interest. 11
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