Gynecologic Oncology 131 (2013) 734–743
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The synthetic flavonoid WYC02-9 inhibits cervical cancer cell migration/invasion and angiogenesis via MAPK14 signaling Yun-Ju Chen a,b,c,d, Yu-Jen Cheng e,f,1, Amos C. Hung a,b,c,d, Yang-Chang Wu g,h,i, Ming-Feng Hou j,k, Yu-Chang Tyan l,m,n,⁎, Shyng-Shiou F. Yuan a,b,c,d,⁎⁎ a
Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan Translational Research Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan d School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan e Division of Thoracic Surgery, Department of Surgery, and Cancer Center, E-DA Hospital, Kaohsiung, Taiwan f Department of Health Management, I-Shou University, Kaohsiung, Taiwan g School of Pharmacy, College of Pharmacy, China Medical University, Taichung, Taiwan h Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung, Taiwan i Center for Molecular Medicine, China Medical University Hospital, Taichung, Taiwan j Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan k Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan l Department of Medical Imaging and Radiological Sciences, Kaohsiung Medical University, Kaohsiung, Taiwan m National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, Kaohsiung, Taiwan n Biomedical Engineering and System Bioinformatics Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan b c
H I G H L I G H T S • WYC02-9 inhibits cervical cancer cell proliferation, migration, invasion and angiogenesis in vitro and in vivo. • WYC02-9 inhibits cervical cancer cell migration, invasion and angiogenesis through suppression of MAPK14 activity. • WYC02-9 may be a promising drug candidate for cervical cancer chemotherapy.
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Article history: Received 26 August 2013 Accepted 11 October 2013 Available online 18 October 2013 Keywords: Cervical cancer WYC02-9 Viability Migration Invasion Angiogenesis
a b s t r a c t Objective. Development of flavonoids as potential chemotherapeutic agents for cervical cancer may open new avenues in anticancer drug design. In this study, the cytotoxic activity and anti-migration/invasion/angiogenesis efficiency of the synthetic flavonoid WYC02-9 on cervical cancer and the underlying mechanisms are explored. Methods. XTT cell viability assay, apoptosis assays, cell cycle analysis, and immunoblotting analysis were applied to study the biologic activity of WYC02-9. Anchorage independent soft agar assay and xenograft nude mouse model were applied to study the anti-tumor effect of WYC02-9 in vivo. Wound healing assay, transwell invasion assay, and gelatin zymography analysis were applied to study the effect of WYC02-9 on cancer cell migration and invasion. Tube formation analysis, zebrafish angiogenesis model, and nude mice Matrigel plug angiogenesis assay were applied to study the effect of WYC02-9 on angiogenesis. Results. WYC02-9 induced cytotoxicity on cervical cancer cells by promoting apoptosis and G2/M cell cycle arrest. WYC02-9 inhibited cervical cancer cell migration/invasion and angiogenesis in vitro and in vivo via MAPK14 pathway. Conclusion. WYC02-9 significantly inhibited cervical cancer cell proliferation/migration/invasion and angiogenesis in vitro and in vivo. WYC02-9 may be a promising drug candidate for cervical cancer chemotherapy. © 2013 Elsevier Inc. All rights reserved.
Introduction ⁎ Correspondence to: Y.-C. Tyan, Department of Medical Imaging and Radiological Sciences, Kaohsiung Medical University, Kaohsiung, Taiwan. ⁎⁎ Correspondence to: S.-S. F. Yuan, Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung 80756, Taiwan. E-mail address: [email protected] (S.-S.F. Yuan). 1 Co-first author. 0090-8258/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygyno.2013.10.012
Cervical cancer is the most common cancer among women in developing countries and the second most common cancer in women worldwide [1]. Platinum-based chemotherapy, either neoadjuvant chemotherapy before surgery or concurrent use during radiotherapy,
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significantly improves the survival of cervical cancer patients [2–4]. However, resistance to platinum-based chemotherapy is a common problem [5,6] and therefore development of new chemotherapeutic agents is required. Neoplastic metastasis is a major cause of cancer-mediated death [7]. Angiogenesis promotes both primary tumor growth and dissemination of tumor cells to distant organs [8] via several mechanisms. High levels of vascular endothelial growth factor-A (VEGF-A) in tumor microenvironment and the consequent activation of its signaling pathway stimulate the growth and migration of vessel endothelial cells [9]. In addition, matrix metalloproteinase-2 (MMP-2) and MMP-9, two major MMPs secreted from HeLa cells, are involved in the proteolytic events required for tumor migration, metastasis and angiogenesis [10,11]. Mitogen-activated protein kinases (MAPKs) are a family of protein kinases include extracellular signal-regulated kinase (ERK1/2, also referred to as MAPK1/3), c-Jun NH2-terminal kinase (JNK, also referred to as MAPK8), and MAPK14 (also referred to as p38 MAPK) [12]. MAPK1/3 regulates cell growth, survival, and differentiation [13], while MAPK8 and MAPK14 are involved in cytotoxicity via mitochondrial dysfunction and caspase activation [14,15]. Notably, MAPK14 is a critical mediator of several antitumor agents, including cisplatin, camptothecin, doxorubicin, oxaliplatin and tamoxifen [16,17]. The polyphenolic secondary metabolites “flavonoids” possess varieties of anticancer potentials including antioxidant, antiproliferation, cell cycle arrest, induction of apoptosis, as well as inhibition of cancer cell migration, invasion and angiogenesis [18]. Therefore, by exploring the potential anti-tumor effect of various flavonoids may open new avenues in anticancer drug design. Apigenin, anthocyanin and quercetin decrease cervical cancer cell viability and inhibited cancer cell–matrix adhesion, migration, invasion and angiogenesis [19–21]. WYC02, a synthetic flavonoid, suppresses cervical cancer cell proliferation through inhibition of PIK3 signaling pathway [22]. WYC02-9 (WYC02 analog with a C-7 modification) exhibits significant antitumor activity on prostate cancer cells by inducing DNA damage and apoptosis through accumulation of reactive oxygen species [23]. Furthermore, WYC02-9 inhibits colorectal cancer cell growth through a ROS- and MAPK14-mediated apoptotic pathway [24]. In this study, the antitumor effect of WYC02-9 on cervical cancer and the underlying mechanisms are investigated using both in vitro and in vivo approaches.
Materials and methods Materials The plant-derived natural flavonoid protoapigenone was isolated from Thelypteris torresiana (Gaud.), followed by total synthesis and renamed WYC02 [25,26]. WYC02-9 is a derivative of WYC02 with a C-7 modification [23,26]. DMEM medium was purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum, penicillin, streptomycin, and amphotericin B were purchased from Biological Industries (Haemek, Israel). Other reagents employed in this study were indicated separately wherever possible.
Cell cultures Human cervical cancer cell lines HeLa, SiHa, and C33A and mouse endothelial cell line MS-1 were obtained from the American Type Culture Collection. The genotypes and phenotypes of the cell lines were authenticated by the Bioresource Collection and Research Center, Taiwan (www.bcrc.firdi.org.tw). Cells were cultured in DMEM medium supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin and 2.5 μg/mL amphotericin B at 37 °C in a 5% CO2 incubator.
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XTT cell viability assay Cells were seeded at 5 × 103 cells/well in 96-well plates and allowed to grow for 24 h before treatment with WYC02-9. After treatment for 48 h, the cytotoxicity was determined by XTT cell viability assay kit from Sigma-Aldrich (St Louis, MO, USA). The procedures were followed according to a previous report [27]. Three independent experiments were performed. Cell cycle analysis Procedures for fluorescence-activated cell sorting (FACS) analysis were described in a previous report [27]. Three independent experiments were performed. Immunoblotting analysis Procedures for immunoblotting analysis were described in a previous report [27]. Antibodies against phospho-CDC2 (Thr161), cleaved CASP3, cleaved CASP8, cleaved CASP9, cleaved PARP1, phospho-MAPK14 (Thr180/Tyr182), MAPK14 and phospho-PIK3 (Tyr467) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against RB1, ACTB, phospho-AKT1 (Thr308), phospho-AKT1 (Ser473) and AKT1 were purchased from GeneTex (Irvine, CA, USA). Antibodies against phospho-RB1 (Thr356), phosphoMAPK1/3 (Thr202/Tyr204), MAPK1/3, phospho-MAPK8 (Thr183/ Tyr185) and MAPK8 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Annexin V apoptosis assay Cells (1 × 104 cells/well) were plated in an 8-well chamber slide from Nunc (Naperville, IL, USA). After treatment with the indicated concentrations of WYC02-9 for 18 h, cells were washed twice with cold 1X PBS and stained with the binding buffer containing annexin V-FITC (1 μg/mL) (Strong Biotech, Taipei, Taiwan). Detection of annexin V was carried out by fluorescent microscopy according to a previous article [28]. Three independent experiments were performed and 1000 cells were counted in each experiment. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay Cells were treated with the indicated concentrations of WYC02-9 for 24h and then stained for apoptotic cells using the DeadEnd Colorimetric TUNEL system from Promega (Madison, WI, USA). The procedures were followed according to a previous article [28]. Three independent experiments were performed and 1000 cells were counted in each experiment. Wound healing assay Cells were seeded in 12-well plates and allowed to reach 100% confluence. The cell monolayer was scratched with a 200 μL pipette tip to yield constant widths. Cells were then incubated with the indicated treatments and wound healing assay was carried out according to a previous article [27]. Three independent experiments were performed. Transwell invasion assay Cells were seeded on 8 μm-pore ECM-coated insert chambers from Corning (Corning, NY, USA) and allowed to reach 100% confluence, followed by incubation of the cells with the indicated treatments. The invasion assay was then carried out according to a previous article [27]. Three independent experiments were performed.
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Table 1 Cytotoxicitya of WYC02-9 and cisplatin on various cervical cancer cell lines.
A
IC50 (μM) Cell line
WYC02-9
Cisplatin
HeLa SiHa C33A
3.17 ± 0.14b 6.62 ± 0.21 4.53 ± 0.18
11.8 ± 0.96 8.92 ± 1.23 7.69 ± 0.72
a The cells were treated with WYC02-9 or cisplatin at different concentrations for 48 h, before being evaluated by the XTT assay. b Mean ± SEM of six independent experiments.
Gelatin zymography analysis Cells were plated in 24-well plates and allowed to reach 100% confluence. Cells were then incubated with the indicated treatments and gelatin zymography analysis was carried out according to a previous article [29]. Three independent experiments were performed.
B
Soft agar anchorage-independent assay Cells were treated with the indicated concentrations of WYC02-9 for 3 h, followed by soft agar analysis with the procedures described in a previous study [27]. Three independent experiments were performed.
C
Ex vivo tumor xenograft study Six-week-old female immunodeficient (Foxnlnu/Foxnlnu) mice were injected subcutaneously with 5 × 106 HeLa cells at the right flank. When tumors became visible (approximately an average diameter of 3 mm), mice were treated with intraperitoneal injection without or with WYC02-9 at 0.88 μg/g body weight in PBS (a dose equals to the IC50) every two days. Tumor volumes were calculated according to a standard formula of width2 × length/2 [28]. Immunohistochemistry Immunohistochemistry protocol was followed according to a previous report [30]. Xenograft tumor sections were incubated overnight at 4 °C with 100 × diluted CD31 antibody (GeneTex). After development with the Universal LSAB + Kit/HRP system (DAKO, Denmark), the images were photographed using Nikon Eclipse 80i light microscope (Nikon, Tokyo, Japan) and the intratumoral microvascular density (MVD) was measured based on the appearance of positive staining of endothelial cell lumens or clusters that were considered as microvessel formation [31]. Tube formation analysis Tube formation of mouse MS-1 endothelial cells on Matrigel was carried out as described before [32]. After WYC02-9 treatment for 3 h, 1 × 103 MS-1 cells were plated onto Matrigel-coated 12 mm cover glass for 24 h and then the cells were imaged using Nikon Eclipse 80i light microscope and analyzed by the Wimasis program (www. wimasis.com). The percentage of tube formation was estimated by the loop number of triplicate samples in each group. Three independent experiments were performed. Fig. 1. WYC02-9 induced G2/M arrest and apoptosis in HeLa cervical cancer cells. HeLa cells were treated with vehicle control or WYC02-9 for 24 h and analyzed for (A) cell cycle distribution by FACS analysis, (B) the expression of cell cycle regulatory proteins by immunoblotting analysis, (C) apoptosis by annexin V and TUNEL assays, and (D) the expression of apoptosis-related proteins by immunoblotting analysis. The data were presented as mean ± SEM. * Indicates a significant difference (P b 0.05) when compared with the vehicle control.
D
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In vivo Matrigel plug angiogenesis assay Female immunodeficient (Foxnlnu/Foxnlnu) mice were injected subcutaneously with 500 μL of Matrigel containing 50 ng of VEGF and 10U of heparin, in combination with 1μM of WYC02-9 or vehicle control (PBS). After 14 days, mice were sacrificed and the Matrigel plugs were dissected out for quantitation of hemoglobin levels using the Darkin's reagent kit (Sigma-Aldrich) according to a previous article [33].
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absence of cleaved CASP3 and PARP1 (Supplemental Fig. 1) as well as the [IC50] = 10.71 μM (data not shown). WYC02-9 also decreased the activities of MMP-2 and MMP-9 in HeLa cells (Fig. 2A). In this study, WYC02-9 treatment significantly increased the phosphorylation of MAPK14 in HeLa cells (Fig. 2B), while pretreatment with MAPK14 specific inhibitor SB203580 for 2 h reversed the biological effects of WYC02-9 on cell migration/invasion and MMPs activities (Fig. 2D), but had no effect on the WYC02-9-suppressed cell viability (Fig. 2C).
In vivo angiogenesis in zebrafish model WYC02-9 inhibited HeLa tumor growth and microvascular formation Blood vessel patterning is highly characteristic in the developing zebrafish embryo and the sub-intestinal vessel (SIV) can be stained and visualized microscopically. Eggs were generated by natural pairwise mating [34] and immediately incubated with WYC02-9 or vehicle control, both containing 0.003% phenyl-2-thiourea, at 28 °C for 72 h. At 72 h post-fertilization (hpf), zebrafish embryos were anesthetized with 0.5 g/L ethyl 3-aminobenzoate methanesulfonic acid salt for 30 min and fixed in 4% paraformaldehyde for 2 h, and then stained for endogenous alkaline phosphatase activity according to a previous article [35]. The SIVs were imaged and counted under the Nikon Eclipse 80i microscope. VEGF-A enzyme-linked immunosorbent assay (ELISA) The conditioned media obtained from MS-1 cells were centrifuged at 2000 g for 10 min and the supernatants were subjected for determination of VEGF-A concentration using RayBio human VEGFA ELISA Kit (RayBiotech, GA, USA). Each sample was performed in duplicate and the procedures were followed according to the manufacturer's instruction. Statistical analysis Quantitative data were presented as mean ± SEM. Two-sided Student's t-test was used to determine the significance between different treatment groups. P b 0.05 was considered statistically significant. Results WYC02-9 inhibited cervical cancer cell viability and induced apoptotic cell death The cytotoxicity of WYC02-9 on three human cervical cancer cell lines HeLa, SiHa and C33A was analyzed (Table 1). Notably, WYC02-9 had higher cytotoxic activity than cisplatin in all the cervical cancer cell lines examined (Table 1). Furthermore, WYC02-9 treatment caused accumulation of cells at G2/M phase in a dose-dependent manner (Fig. 1A). In contrast to the decreased level of G2/M regulator P-CDC2 (Thr161), an increased phosphorylation of RB1, a G1 to S regulator, was observed in HeLa cells after WYC02-9 treatment (Fig. 1B). To evaluate apoptotic cell death upon WYC02-9 treatment, annexin V (marker of early apoptosis) and TUNEL (marker of late apoptosis) assays were applied. As shown in Fig. 1C, the numbers of annexin V- and TUNEL-positive cells significantly increased in HeLa cells after WYC02-9 treatment. Furthermore, WYC02-9-induced apoptosis was mediated by activation of CASP8, CASP9, CASP3, and poly-ADP ribose polymerase-1 (PARP1) (Fig. 1D). WYC02-9 inhibited cervical cancer cell migration, invasion, and MMP-2/-9 activities via MAPK14 activation WYC02-9 decreased cell migration and invasion in HeLa cells (Fig. 2A). Notably, no significant apoptosis was observed in 100% confluent HeLa cells after 2 μM WYC02-9 treatment, judged by the
Cancer cells that proliferate without extracellular matrix (ECM) are considered conducive to enhance tumor progression. Our data revealed that treatment of HeLa cells with WYC02-9 significantly decreased the ECM-independent cell growth in soft-agar assay (Fig. 3A). In a mouse xenograft model, WYC02-9 significantly suppressed the xenografted tumor growth (Fig. 3B), but did not impair body weight, hematopoiesis, liver or renal function (data not shown), or cervical epithelium (Supplemental Fig. 3). We also analyzed the expression of endothelial marker CD31 in the xenografted tumor by immunohistochemistry and the result showed that WYC02-9 treatment decreased CD31 staining and tumor microvascular density (MVD) in the xenografted tumors (Fig. 3C). WYC02-9 inhibited angiogenesis in vivo and in vitro The in vivo significance of WYC02-9 on VEGF-induced angiogenesis was evaluated in mice by measuring hemoglobin levels in the Matrigel plugs at 14 days after implantation. When compared to VEGF treatment alone, the presence of WYC02-9 significantly reduced the VEGF-induced angiogenesis (Fig. 4A). Further zebrafish model also showed that WYC02-9 significantly decreased the number of sub-intestinal vessel (SIV) (Fig. 4B). WYC02-9 inhibited the migration, invasion, and MMP-2/-9 activities of MS-1 endothelial cells (Supplemental Fig. 4). MAPK14 is a negative regulator during angiogenesis [36,37]. In MS-1 cells, WYC02-9 treatment significantly increased the phosphorylation of MAPK14 (Fig. 5A), but had no effects on the phosphorylation of MAPK1/3 and MAPK8 (data not shown). Pretreatment of MS-1 cells with SB203580 partially but significantly reversed the anti-migration/invasion (Fig. 5B), antitube formation (Fig. 5C) and anti-VEGF-A secretion (Fig. 5D) activities of WYC02-9. Notably, no significant apoptosis was observed in the 100% confluent MS-1 cells after 2 μM WYC02-9 treatment for 24 h ([IC50] = 9.61 μM; data not shown). Discussion The synthetic flavonoid WYC02-9 inhibits cervical cancer cell growth, migration, invasion and angiogenesis Flavonoids have been shown to possess multiple anti-tumor activities in a variety of cancer types including cervical carcinomas [18]. The current study showed that the synthetic flavonoid WYC02-9 exhibited a number of inhibitory effects on cervical cancer cells. First, WYC02-9 induced cytotoxicity in cervical cancer cell lines (Table 1). Second, WYC02-9 arrested cervical cancer cells at G2/M phases (Fig. 1A and B) and increased their apoptosis (Fig. 1C and D), which were in agreement with the reduced cervical tumor growth in WYC02-9-treated mice (Fig. 3B). Furthermore, the ECM-independent cell growth was decreased upon WYC02-9 treatment in HeLa cells (Fig. 3A). WYC02-9 also inhibited cervical cancer migration, invasion and angiogenesis. The in vitro wound healing and invasion as well as the metastasis-related proteinases MMP-2/-9 secretion were suppressed in WYC02-9-treated HeLa cells (Fig. 2A). The inhibitory effect of
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Fig. 2. WYC02-9 decreased HeLa cell migration, invasion, and MMP-2/-9 activities through MAPK14 signaling. (A) HeLa cells were treated with WYC02-9 for 24 h and the migration and invasion efficiencies were determined by wound healing assay and ECM-coated transwell system respectively. MMP-2/-9 activities were measured using gelatin zymography analysis. (B) HeLa cells were treated with WYC02-9 for 4 h and cell lysates were analyzed for the phosphorylated and total forms of signal proteins, including MAPK (MAPK14, MAPK1/3 and MAPK8) pathway by immunoblotting analysis. (C) Cells were pretreated with 5 μM of SB203580 for 2 h, followed by WYC02-9 treatment for 48 h. The cell viability was measured by XTT assay. (D) Cells were pretreated with 5 μM of SB203580 for 2 h, followed by WYC02-9 treatment for 24 h. The migration/invasion efficiencies as well as MMP-2/-9 activities were measured. The data were presented as mean ± SEM. * indicates a significant difference (P b 0.05) when compared with the vehicle control. # indicates a significant difference (P b 0.05) when compared with the corresponding WYC02-9 treatment without SB203580.
WYC02-9 on angiogenesis was evidenced from both in vivo and in vitro studies (Figs. 3C, 4, 5). Taken together, the data support a view that WYC02-9 may have the potential to be developed as a therapeutic agent for cervical cancer treatment. Activation of PIK3/AKT1 is considered oncogenic in cervical cancer and other cancer types [38,39]. In contrast, activation of MAPK14 leads to cancer cell death [24]. We previously reported that WYC02, the precursor compound from which WYC02-9 was derived, inhibited cervical cancer cell proliferation and tumorigenesis via suppression of PIK3 signaling pathway [22]. Our current data showed that PIK3/AKT1 activities were inhibited while MAPK14 activity was enhanced by WCY02-9 (Fig. 2B and Supplemental Fig. 2). These observations were in accordance with the cytotoxic effect of WCY02-9 reported in our present and previous studies [23,24]. We noticed that the presence of MAPK14 inhibitor SB203580 could not reverse the WCY02-9-induced cytotoxicity (Fig. 2C). On the other hand, PIK3 inhibitor wortmannin alone showed cytotoxicity on HeLa cells and no synergistic effect between wortmannin and WYC02-9 was observed (Supplemental Fig. 2), suggesting that WYC02-9 may exert its cytotoxic effect through PIK3 pathway but not MAPK14
pathway. More experiments may be required to elucidate the differential demands on PIK3 and MAPK14 pathways in WYC02-9induced cervical cancer cell death. WYC02-9 decreases cervical cancer cell migration and invasion via activation of MAPK14 pathway The anti-migratory and anti-invasive effects of flavonoids were mediated through various signaling molecules. For example, (−)-epigallocatechin-3-gallate (EGCG) downregulates the hepatocyte growth factor-activated MMP-9 and urokinase-type plasminogen activator (uPA) in hypopharyngeal carcinoma [40]. Combined treatment of EGCG and TRAIL synergistically decreased the expression of MMP-2/3/-9, VEGF, uPA, and angiopoietin 1/2 in prostate cancer cells [41]. In addition, the inhibitory effects of flavonoids on cancer cell migration and invasion are associated with MAPKs, c-jun, NF-kappaB, and PIK3/AKT pathways [22,24,40,42]. In the current study, WYC02-9 treatment increased the expression of phospho-MAPK14 in HeLa cells (Fig. 2B). Notably, the WYC02-9-inhibited HeLa cell migration, invasion, and MMP-2/-9 activation were reversed in the presence
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A
B
C
Fig. 3. WYC02-9 suppressed cervical tumor growth and the formation of intratumoral vessels. (A) HeLa cells were treated with the indicated concentrations of WYC02-9 for 3 h, and then grown in soft-agar for 14 days before counting of anchorage-independent colonies. (B) Female nude mice bearing cervical tumors were treated with vehicle control (PBS) or WYC02-9. Tumor volumes were measured weekly for each group. (C) Tumor xenografts were analyzed for the expression of endothelial marker CD31 by immunohistochemistry analysis, from which the intratumoral microvascular density (MVD) was measured. The data were presented as mean ± SEM. * indicates a significant difference (P b 0.05) when compared with the vehicle control.
of the MAPK14 inhibitor SB203580 (Fig. 2D). These results suggest that MAPK14 may act as a crucial mediator of the anti-migration/ invasion effect of WYC02-9. WYC02-9 inhibits angiogenesis via activation of MAPK14 pathway Angiogenesis is required for tumor growth and metastasis. It is for this reason that anti-angiogenic therapy constitutes a profound
strategy for the control of tumor progression [43]. In such instances, VEGF signalings are the most relevant regulators of angiogenesis via the recruitment and activation of both cancer cells and endothelial cells, and therefore the VEGF-related pathways are often targeted for cancer treatment [44]. To verify the anti-angiogenic activity of WYC02-9, both in vivo and in vitro models were employed in the present study. WYC02-9 decreased the expression of endothelial cell marker CD31 in xenografted
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A
B
Fig. 4. WYC02-9 decreased in vivo angiogenesis in Matrigel plug angiogenesis assay and zebrafish SIV model. (A) Matrigels containing 50 ng of VEGF and 10 U of heparin, in combination with vehicle control (PBS) or 1 μM of WYC02-9, were injected subcutaneously to the female nude mice. After 14 days of implantation, Matrigel plugs (N = 6 in each group) were dissected out and the hemoglobin levels were determined by the Darkin's reagent kit. (B) Treatment with 20 μM of WYC02-9 for 72 h caused a reduction of the SIV number in 72 hpf zebrafish embryos (N = 120 in each group). Arrow showed the characteristic pattern of SIV. White arrowhead indicates the pronephric duct which served as a positive control for AP staining [35]. The data were presented as mean ± SEM. * Indicates a significant difference (P b 0.05) when compared with the vehicle control.
cervical tumor (Fig. 3C). In addition, WYC02-9 inhibited VEGF-induced blood vessel formation in Matrigel plug angiogenesis mouse model (Fig. 4A) as well as in zebrafish model (Fig. 4B). Moreover, the migratory properties, tube formation, and VEGF-A secretion of endothelial cells were reduced by WYC02-9, partially through an MAPK14-dependent pathway (Fig. 5). Since anti-angiogenic therapy in combination with irradiation or chemotherapy enhance the treatment efficacy [45,46], it will be valuable to investigate whether combination of WYC02-9 with radiation or chemotherapy will further inhibit cancer progression.
Conclusion Our current data showed the inhibitory effect of WYC02-9 on cervical tumorigenesis and angiogenesis both in vitro and in vivo. In addition, the previously unreported functions of WYC02-9 on cervical cancer migration, invasion and angiogenesis through MAPK14 activation were identified. All of the results suggest that WYC02-9 may be a promising drug candidate to be developed for cervical cancer chemotherapy.
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A
B
0
C
D
Fig. 5. WYC02-9 decreased migration, invasion, MMP-2/-9 secretion, and in vitro angiogenesis through MAPK14 activation in MS-1 endothelial cells. (A) MS-1 cells were treated with WYC02-9 for 4 h, followed by immunoblotting analysis for the phosphorylated and total forms of MAPK14 proteins. (B–D) MS-1 cells were pretreated with 5 μM of SB203580 for 2 h, followed by WYC02-9 treatment for 24 h. (B) The migration and invasion efficiencies were determined by wound healing assay and ECM-coated transwell system respectively. MMP2/-9 activities were measured using gelatin zymography analysis. (C) Tube formation in MS-1 cells upon WYC02-9 treatment was determined by counting the loop numbers. A typical photo for tube formation upon WYC02-9 treatment was shown. (D) VEGF-A secretion were measured by ELISA. The data were presented as mean ± SEM. * Indicates a significant difference (P b 0.05) when compared with the vehicle control. # Indicates a significant difference (P b 0.05) when compared with the corresponding WYC02-9 treatment without SB203580.
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ygyno.2013.10.012.
Conflict of interest statement No potential conflicts of interest were disclosed.
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Acknowledgment This work was supported by grants from E-DA Hospital (EDAHP101014, EDAHP-101023 and EDAHP-102025), National Health Research Institutes, Taiwan (NHRI-EX98, 99,100-9829BI, NHRI-EX102-10212BI), and Department of Health, Taiwan (DOH101-TD-C-111-002). References [1] Wright Jr TC, Kuhn L. Alternative approaches to cervical cancer screening for developing countries. Best Pract Res Clin Obstet Gynaecol 2012;26:197–208. [2] Devita VT, Hellman S, Rosenberg SA, editors. Cancer: principles and practice of oncology. 8th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2007. [3] González-Martín A, González-Cortijo L, Carballo N, Garcia JF, Lapuente F, Rojo A, et al. The current role of neoadjuvant chemotherapy in the management of cervical carcinoma. Gynecol Oncol 2008;110:S36–40. [4] Peters III WA, Liu PY, Barrett II RJ, Stock RJ, Monk BJ, Berek JS, et al. Concurrent chemotherapy and pelvic radiation therapy compared with pelvic radiation therapy alone as adjuvant therapy after radical surgery in high-risk early stage cancer of the cervix. J Clin Oncol 2000;18:1606–13. [5] Cadron I, Van Gorp T, Amant F, Leunen K, Neven P, Vergote I. Chemotherapy for recurrent cervical cancer. Gynecol Oncol 2007;107:S113–8. [6] Siddik ZH. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 2003;22:7265–79. [7] Sporn MB, Suh N. Chemoprevention of cancer. Carcinogenesis 2000;21:525–30. [8] Albini A, Tosetti F, Li VW, Noonan DM, Li WW. Cancer prevention by targeting angiogenesis. Nat Rev Clin Oncol 2012;9:498–509. [9] Appelmann I, Liersch R, Kessler T, Mesters RM, Berdel WE. Angiogenesis inhibition in cancer therapy: platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) and their receptors: biological functions and role in malignancy. Recent Results Cancer Res 2010;180:51–81. [10] Giannelli G, Antonaci S. MMP and TIMP assay in cancer. Biological and clinical significance. Int J Cancer 2005;116:1002–3. [11] Roomi MW, Monterrey JC, Kalinovsky T, Rath M, Niedzwiecki A. In vitro modulation of MMP-2 and MMP-9 in human cervical and ovarian cancer cell lines by cytokines, inducers and inhibitors. Oncol Rep 2010;23:605–14. [12] Tibbles LA, Woodgett JR. The stress-activated protein kinase pathways. Cell Mol Life Sci 1999;55:1230–54. [13] Husain SS, Szabo IL, Pai R, Soreghan B, Jones MK, Tarnawski AS. MAPK (ERK2) kinase —a key target for NSAIDs-induced inhibition of gastric cancer cell proliferation and growth. Life Sci 2001;69:3045–54. [14] El Mchichi B, Hadji A, Vazquez A, Leca G. p38 MAPK and MSK1 mediate caspase-8 activation in manganese-induced mitochondria-dependent cell death. Cell Death Differ 2007;14:1826–36. [15] Taylor CA, Zheng Q, Liu Z, Thompson JE. Role of p38 and JNK MAPK signaling pathways and tumor suppressor p53 on induction of apoptosis in response to Ad-eIF5A1 in A549 lung cancer cells. Mol Cancer 2013;12:35. [16] Mandlekar S, Kong AN. Mechanisms of tamoxifen-induced apoptosis. Apoptosis 2001;6:469–77. [17] Olson JM, Hallahan AR. p38 MAP kinase: a convergence point in cancer therapy. Trends Mol Med 2004;10:125–9. [18] Weng CJ, Yen GC. Flavonoids, a ubiquitous dietary phenolic subclass, exert extensive in vitro anti-invasive and in vivo anti-metastatic activities. Cancer Metastasis Rev 2012;31:323–51. [19] Chen PN, Kuo WH, Chiang CL, Chiou HL, Hsieh YS, Chu SC. Black rice anthocyanins inhibit cancer cells invasion via repressions of MMPs and u-PA expression. Chem Biol Interact 2006;163:218–29. [20] Czyz J, Madeja Z, Irmer U, Korohoda W, Hülser DF. Flavonoid apigenin inhibits motility and invasiveness of carcinoma cells in vitro. Int J Cancer 2005;114:12–8. [21] Zhang FL, Zhang W, Chen XM, Luo RY. Effects of quercetin and quercetin in combination with cisplatin on adhesion, migration and invasion of HeLa cells. Zhonghua Fu Chan Ke Za Zhi 2008;43:619–21. [22] Chen YJ, Kay N, Yang JM, Lin CT, Chang HL, Wu YC, et al. Total synthetic protoapigenone WYC02 inhibits cervical cancer cell proliferation and tumour growth through PIK3 signalling pathway. Basic Clin Pharmacol Toxicol 2013;113:8–18.
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[23] Chen HM, Chang FR, Hsieh YC, Cheng YJ, Hsieh KC, Tsai LM, et al. A novel synthetic protoapigenone analogue, WYC02-9, induces DNA damage and apoptosis in DU145 prostate cancer cells through generation of reactive oxygen species. Free Radic Biol Med 2011;50:1151–62. [24] Chen YJ, Chen HP, Cheng YJ, Lin YH, Liu KW, Chen YJ, et al. The synthetic flavonoid WYC02-9 inhibits colorectal cancer cell growth through ROS-mediated activation of MAPK14 pathway. Life Sci 2013;92:1081–92. [25] Lin AS, Chang FR, Wu CC, Liaw CC, Wu YC. New cytotoxic flavonoids from Thelypteris torresiana. Planta Med 2005;71:867–70. [26] Lin AS, Nakagawa-Goto K, Chang FR, Yu D, Morris-Natschke SL, Wu CC, et al. First total synthesis of protoapigenone and its analogues as potent cytotoxic agents. J Med Chem 2007;50:3921–7. [27] Yuan SS, Hou MF, Hsieh YC, Huang CY, Lee YC, Chen YJ, et al. Role of MRE11 in cell proliferation, tumor invasion, and DNA repair in breast cancer. J Natl Cancer Inst 2012;104:1485–502. [28] Chang HL, Wu YC, Su JH, Yeh YT, Yuan SS. Protoapigenone, a novel flavonoid, induces apoptosis in human prostate cancer cells through activation of p38 mitogenactivated protein kinase and c-Jun NH2-terminal kinase 1/2. J Pharmacol Exp Ther 2008;325:841–9. [29] Ke FC, Chuang LC, Lee MT, Chen YJ, Lin SW, Wang PS, et al. The modulatory role of transforming growth factor beta1 and androstenedione on follicle-stimulating hormone-induced gelatinase secretion and steroidogenesis in rat granulosa cells. Biol Reprod 2004;70:1292–8. [30] Yeh YT, Ou-Yang F, Chen IF, Yang SF, Wang YY, Chuang HY, et al. STAT3 ser727 phosphorylation and its association with negative estrogen receptor status in breast infiltrating ductal carcinoma. Int J Cancer 2006;118:2943–7. [31] Weidner N. Current pathologic methods for measuring intratumoral microvessel density within breast carcinoma and other solid tumors. Breast Cancer Res Treat 1995;36:169–80. [32] Brown AC, Shah C, Liu J, Pham JT, Zhang JG, Jadus MR. Ginger's (Zingiber officinale Roscoe) inhibition of rat colonic adenocarcinoma cells proliferation and angiogenesis in vitro. Phytother Res 2009;23:640–5. [33] Park CC, Morel JC, Amin MA, Connors MA, Harlow LA, Koch AE. Evidence of IL-18 as a novel angiogenic mediator. J Immunol 2001;167:1644–53. [34] Westerfield M. The zebrafish book: a guide for the laboratory use of zebrafish. Eugene, Oregon: The University of Oregon Press; 1993. [35] Serbedzija GN, Flynn E, Willett CE. Zebrafish angiogenesis: a new model for drug screening. Angiogenesis 1999;3:353–9. [36] Issbrücker K, Marti HH, Hippenstiel S, Springmann G, Voswinckel R, Gaumann A, et al. p38 MAP kinase—a molecular switch between VEGF-induced angiogenesis and vascular hyperpermeability. FASEB J 2003;17:262–4. [37] Yin H, Liu Z, Li F, Ni M, Wang B, Qiao Y, et al. Ginsenoside-Rg1 enhances angiogenesis and ameliorates ventricular remodeling in a rat model of myocardial infarction. J Mol Med (Berl) 2011;89:363–75. [38] Denley A, Gymnopoulos M, Kang S, Mitchell C, Vogt PK. Requirement of phosphatidylinositol(3,4,5)trisphosphate in phosphatidylinositol 3-kinase-induced oncogenic transformation. Mol Cancer Res 2009;7:1132–8. [39] Schwarz JK, Payton JE, Rashmi R, Xiang T, Jia Y, Huettner P, et al. Pathway-specific analysis of gene expression data identifies the PI3K/Akt pathway as a novel therapeutic target in cervical cancer. Clin Cancer Res 2012;18:1464–71. [40] Lim YC, Park HY, Hwang HS, Kang SU, Pyun JH, Lee MH, et al. (−)-Epigallocatechin3-gallate (EGCG) inhibits HGF-induced invasion and metastasis in hypopharyngeal carcinoma cells. Cancer Lett 2008;271:140–52. [41] Siddiqui IA, Malik A, Adhami VM, Asim M, Hafeez BB, Sarfaraz S, et al. Green tea polyphenol EGCG sensitizes human prostate carcinoma LNCaP cells to TRAILmediated apoptosis and synergistically inhibits biomarkers associated with angiogenesis and metastasis. Oncogene 2008;27:2055–63. [42] Vayalil PK, Katiyar SK. Treatment of epigallocatechin-3-gallate inhibits matrix metalloproteinases-2 and −9 via inhibition of activation of mitogen-activated protein kinases, c-Jun and NF-kappaB in human prostate carcinoma DU-145 cells. Prostate 2004;59:33–42. [43] Sato Y. Aminopeptidases and angiogenesis. Endothelium 2003;10:287–90. [44] Pircher A, Hilbe W, Heidegger I, Drevs J, Tichelli A, Medinger M. Biomarkers in tumor angiogenesis and anti-angiogenic therapy. Int J Mol Sci 2011;12:7077–99. [45] Gorski DH, Beckett MA, Jaskowiak NT, Calvin DP, Mauceri HJ, Salloum RM, et al. Blockage of the vascular endothelial growth factor stress response increases the antitumor effects of ionizing radiation. Cancer Res 1999;59:3374–8. [46] Scappaticci FA. Mechanisms and future directions for angiogenesis based cancer therapies. J Clin Oncol 2002;18:3906–27.
Contents lists available at ScienceDirect
Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno
The synthetic flavonoid WYC02-9 inhibits cervical cancer cell migration/invasion and angiogenesis via MAPK14 signaling Yun-Ju Chen a,b,c,d, Yu-Jen Cheng e,f,1, Amos C. Hung a,b,c,d, Yang-Chang Wu g,h,i, Ming-Feng Hou j,k, Yu-Chang Tyan l,m,n,⁎, Shyng-Shiou F. Yuan a,b,c,d,⁎⁎ a
Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan Translational Research Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan d School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan e Division of Thoracic Surgery, Department of Surgery, and Cancer Center, E-DA Hospital, Kaohsiung, Taiwan f Department of Health Management, I-Shou University, Kaohsiung, Taiwan g School of Pharmacy, College of Pharmacy, China Medical University, Taichung, Taiwan h Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung, Taiwan i Center for Molecular Medicine, China Medical University Hospital, Taichung, Taiwan j Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan k Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan l Department of Medical Imaging and Radiological Sciences, Kaohsiung Medical University, Kaohsiung, Taiwan m National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, Kaohsiung, Taiwan n Biomedical Engineering and System Bioinformatics Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan b c
H I G H L I G H T S • WYC02-9 inhibits cervical cancer cell proliferation, migration, invasion and angiogenesis in vitro and in vivo. • WYC02-9 inhibits cervical cancer cell migration, invasion and angiogenesis through suppression of MAPK14 activity. • WYC02-9 may be a promising drug candidate for cervical cancer chemotherapy.
a r t i c l e
i n f o
Article history: Received 26 August 2013 Accepted 11 October 2013 Available online 18 October 2013 Keywords: Cervical cancer WYC02-9 Viability Migration Invasion Angiogenesis
a b s t r a c t Objective. Development of flavonoids as potential chemotherapeutic agents for cervical cancer may open new avenues in anticancer drug design. In this study, the cytotoxic activity and anti-migration/invasion/angiogenesis efficiency of the synthetic flavonoid WYC02-9 on cervical cancer and the underlying mechanisms are explored. Methods. XTT cell viability assay, apoptosis assays, cell cycle analysis, and immunoblotting analysis were applied to study the biologic activity of WYC02-9. Anchorage independent soft agar assay and xenograft nude mouse model were applied to study the anti-tumor effect of WYC02-9 in vivo. Wound healing assay, transwell invasion assay, and gelatin zymography analysis were applied to study the effect of WYC02-9 on cancer cell migration and invasion. Tube formation analysis, zebrafish angiogenesis model, and nude mice Matrigel plug angiogenesis assay were applied to study the effect of WYC02-9 on angiogenesis. Results. WYC02-9 induced cytotoxicity on cervical cancer cells by promoting apoptosis and G2/M cell cycle arrest. WYC02-9 inhibited cervical cancer cell migration/invasion and angiogenesis in vitro and in vivo via MAPK14 pathway. Conclusion. WYC02-9 significantly inhibited cervical cancer cell proliferation/migration/invasion and angiogenesis in vitro and in vivo. WYC02-9 may be a promising drug candidate for cervical cancer chemotherapy. © 2013 Elsevier Inc. All rights reserved.
Introduction ⁎ Correspondence to: Y.-C. Tyan, Department of Medical Imaging and Radiological Sciences, Kaohsiung Medical University, Kaohsiung, Taiwan. ⁎⁎ Correspondence to: S.-S. F. Yuan, Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung 80756, Taiwan. E-mail address: [email protected] (S.-S.F. Yuan). 1 Co-first author. 0090-8258/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygyno.2013.10.012
Cervical cancer is the most common cancer among women in developing countries and the second most common cancer in women worldwide [1]. Platinum-based chemotherapy, either neoadjuvant chemotherapy before surgery or concurrent use during radiotherapy,
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significantly improves the survival of cervical cancer patients [2–4]. However, resistance to platinum-based chemotherapy is a common problem [5,6] and therefore development of new chemotherapeutic agents is required. Neoplastic metastasis is a major cause of cancer-mediated death [7]. Angiogenesis promotes both primary tumor growth and dissemination of tumor cells to distant organs [8] via several mechanisms. High levels of vascular endothelial growth factor-A (VEGF-A) in tumor microenvironment and the consequent activation of its signaling pathway stimulate the growth and migration of vessel endothelial cells [9]. In addition, matrix metalloproteinase-2 (MMP-2) and MMP-9, two major MMPs secreted from HeLa cells, are involved in the proteolytic events required for tumor migration, metastasis and angiogenesis [10,11]. Mitogen-activated protein kinases (MAPKs) are a family of protein kinases include extracellular signal-regulated kinase (ERK1/2, also referred to as MAPK1/3), c-Jun NH2-terminal kinase (JNK, also referred to as MAPK8), and MAPK14 (also referred to as p38 MAPK) [12]. MAPK1/3 regulates cell growth, survival, and differentiation [13], while MAPK8 and MAPK14 are involved in cytotoxicity via mitochondrial dysfunction and caspase activation [14,15]. Notably, MAPK14 is a critical mediator of several antitumor agents, including cisplatin, camptothecin, doxorubicin, oxaliplatin and tamoxifen [16,17]. The polyphenolic secondary metabolites “flavonoids” possess varieties of anticancer potentials including antioxidant, antiproliferation, cell cycle arrest, induction of apoptosis, as well as inhibition of cancer cell migration, invasion and angiogenesis [18]. Therefore, by exploring the potential anti-tumor effect of various flavonoids may open new avenues in anticancer drug design. Apigenin, anthocyanin and quercetin decrease cervical cancer cell viability and inhibited cancer cell–matrix adhesion, migration, invasion and angiogenesis [19–21]. WYC02, a synthetic flavonoid, suppresses cervical cancer cell proliferation through inhibition of PIK3 signaling pathway [22]. WYC02-9 (WYC02 analog with a C-7 modification) exhibits significant antitumor activity on prostate cancer cells by inducing DNA damage and apoptosis through accumulation of reactive oxygen species [23]. Furthermore, WYC02-9 inhibits colorectal cancer cell growth through a ROS- and MAPK14-mediated apoptotic pathway [24]. In this study, the antitumor effect of WYC02-9 on cervical cancer and the underlying mechanisms are investigated using both in vitro and in vivo approaches.
Materials and methods Materials The plant-derived natural flavonoid protoapigenone was isolated from Thelypteris torresiana (Gaud.), followed by total synthesis and renamed WYC02 [25,26]. WYC02-9 is a derivative of WYC02 with a C-7 modification [23,26]. DMEM medium was purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum, penicillin, streptomycin, and amphotericin B were purchased from Biological Industries (Haemek, Israel). Other reagents employed in this study were indicated separately wherever possible.
Cell cultures Human cervical cancer cell lines HeLa, SiHa, and C33A and mouse endothelial cell line MS-1 were obtained from the American Type Culture Collection. The genotypes and phenotypes of the cell lines were authenticated by the Bioresource Collection and Research Center, Taiwan (www.bcrc.firdi.org.tw). Cells were cultured in DMEM medium supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin and 2.5 μg/mL amphotericin B at 37 °C in a 5% CO2 incubator.
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XTT cell viability assay Cells were seeded at 5 × 103 cells/well in 96-well plates and allowed to grow for 24 h before treatment with WYC02-9. After treatment for 48 h, the cytotoxicity was determined by XTT cell viability assay kit from Sigma-Aldrich (St Louis, MO, USA). The procedures were followed according to a previous report [27]. Three independent experiments were performed. Cell cycle analysis Procedures for fluorescence-activated cell sorting (FACS) analysis were described in a previous report [27]. Three independent experiments were performed. Immunoblotting analysis Procedures for immunoblotting analysis were described in a previous report [27]. Antibodies against phospho-CDC2 (Thr161), cleaved CASP3, cleaved CASP8, cleaved CASP9, cleaved PARP1, phospho-MAPK14 (Thr180/Tyr182), MAPK14 and phospho-PIK3 (Tyr467) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against RB1, ACTB, phospho-AKT1 (Thr308), phospho-AKT1 (Ser473) and AKT1 were purchased from GeneTex (Irvine, CA, USA). Antibodies against phospho-RB1 (Thr356), phosphoMAPK1/3 (Thr202/Tyr204), MAPK1/3, phospho-MAPK8 (Thr183/ Tyr185) and MAPK8 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Annexin V apoptosis assay Cells (1 × 104 cells/well) were plated in an 8-well chamber slide from Nunc (Naperville, IL, USA). After treatment with the indicated concentrations of WYC02-9 for 18 h, cells were washed twice with cold 1X PBS and stained with the binding buffer containing annexin V-FITC (1 μg/mL) (Strong Biotech, Taipei, Taiwan). Detection of annexin V was carried out by fluorescent microscopy according to a previous article [28]. Three independent experiments were performed and 1000 cells were counted in each experiment. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay Cells were treated with the indicated concentrations of WYC02-9 for 24h and then stained for apoptotic cells using the DeadEnd Colorimetric TUNEL system from Promega (Madison, WI, USA). The procedures were followed according to a previous article [28]. Three independent experiments were performed and 1000 cells were counted in each experiment. Wound healing assay Cells were seeded in 12-well plates and allowed to reach 100% confluence. The cell monolayer was scratched with a 200 μL pipette tip to yield constant widths. Cells were then incubated with the indicated treatments and wound healing assay was carried out according to a previous article [27]. Three independent experiments were performed. Transwell invasion assay Cells were seeded on 8 μm-pore ECM-coated insert chambers from Corning (Corning, NY, USA) and allowed to reach 100% confluence, followed by incubation of the cells with the indicated treatments. The invasion assay was then carried out according to a previous article [27]. Three independent experiments were performed.
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Table 1 Cytotoxicitya of WYC02-9 and cisplatin on various cervical cancer cell lines.
A
IC50 (μM) Cell line
WYC02-9
Cisplatin
HeLa SiHa C33A
3.17 ± 0.14b 6.62 ± 0.21 4.53 ± 0.18
11.8 ± 0.96 8.92 ± 1.23 7.69 ± 0.72
a The cells were treated with WYC02-9 or cisplatin at different concentrations for 48 h, before being evaluated by the XTT assay. b Mean ± SEM of six independent experiments.
Gelatin zymography analysis Cells were plated in 24-well plates and allowed to reach 100% confluence. Cells were then incubated with the indicated treatments and gelatin zymography analysis was carried out according to a previous article [29]. Three independent experiments were performed.
B
Soft agar anchorage-independent assay Cells were treated with the indicated concentrations of WYC02-9 for 3 h, followed by soft agar analysis with the procedures described in a previous study [27]. Three independent experiments were performed.
C
Ex vivo tumor xenograft study Six-week-old female immunodeficient (Foxnlnu/Foxnlnu) mice were injected subcutaneously with 5 × 106 HeLa cells at the right flank. When tumors became visible (approximately an average diameter of 3 mm), mice were treated with intraperitoneal injection without or with WYC02-9 at 0.88 μg/g body weight in PBS (a dose equals to the IC50) every two days. Tumor volumes were calculated according to a standard formula of width2 × length/2 [28]. Immunohistochemistry Immunohistochemistry protocol was followed according to a previous report [30]. Xenograft tumor sections were incubated overnight at 4 °C with 100 × diluted CD31 antibody (GeneTex). After development with the Universal LSAB + Kit/HRP system (DAKO, Denmark), the images were photographed using Nikon Eclipse 80i light microscope (Nikon, Tokyo, Japan) and the intratumoral microvascular density (MVD) was measured based on the appearance of positive staining of endothelial cell lumens or clusters that were considered as microvessel formation [31]. Tube formation analysis Tube formation of mouse MS-1 endothelial cells on Matrigel was carried out as described before [32]. After WYC02-9 treatment for 3 h, 1 × 103 MS-1 cells were plated onto Matrigel-coated 12 mm cover glass for 24 h and then the cells were imaged using Nikon Eclipse 80i light microscope and analyzed by the Wimasis program (www. wimasis.com). The percentage of tube formation was estimated by the loop number of triplicate samples in each group. Three independent experiments were performed. Fig. 1. WYC02-9 induced G2/M arrest and apoptosis in HeLa cervical cancer cells. HeLa cells were treated with vehicle control or WYC02-9 for 24 h and analyzed for (A) cell cycle distribution by FACS analysis, (B) the expression of cell cycle regulatory proteins by immunoblotting analysis, (C) apoptosis by annexin V and TUNEL assays, and (D) the expression of apoptosis-related proteins by immunoblotting analysis. The data were presented as mean ± SEM. * Indicates a significant difference (P b 0.05) when compared with the vehicle control.
D
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In vivo Matrigel plug angiogenesis assay Female immunodeficient (Foxnlnu/Foxnlnu) mice were injected subcutaneously with 500 μL of Matrigel containing 50 ng of VEGF and 10U of heparin, in combination with 1μM of WYC02-9 or vehicle control (PBS). After 14 days, mice were sacrificed and the Matrigel plugs were dissected out for quantitation of hemoglobin levels using the Darkin's reagent kit (Sigma-Aldrich) according to a previous article [33].
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absence of cleaved CASP3 and PARP1 (Supplemental Fig. 1) as well as the [IC50] = 10.71 μM (data not shown). WYC02-9 also decreased the activities of MMP-2 and MMP-9 in HeLa cells (Fig. 2A). In this study, WYC02-9 treatment significantly increased the phosphorylation of MAPK14 in HeLa cells (Fig. 2B), while pretreatment with MAPK14 specific inhibitor SB203580 for 2 h reversed the biological effects of WYC02-9 on cell migration/invasion and MMPs activities (Fig. 2D), but had no effect on the WYC02-9-suppressed cell viability (Fig. 2C).
In vivo angiogenesis in zebrafish model WYC02-9 inhibited HeLa tumor growth and microvascular formation Blood vessel patterning is highly characteristic in the developing zebrafish embryo and the sub-intestinal vessel (SIV) can be stained and visualized microscopically. Eggs were generated by natural pairwise mating [34] and immediately incubated with WYC02-9 or vehicle control, both containing 0.003% phenyl-2-thiourea, at 28 °C for 72 h. At 72 h post-fertilization (hpf), zebrafish embryos were anesthetized with 0.5 g/L ethyl 3-aminobenzoate methanesulfonic acid salt for 30 min and fixed in 4% paraformaldehyde for 2 h, and then stained for endogenous alkaline phosphatase activity according to a previous article [35]. The SIVs were imaged and counted under the Nikon Eclipse 80i microscope. VEGF-A enzyme-linked immunosorbent assay (ELISA) The conditioned media obtained from MS-1 cells were centrifuged at 2000 g for 10 min and the supernatants were subjected for determination of VEGF-A concentration using RayBio human VEGFA ELISA Kit (RayBiotech, GA, USA). Each sample was performed in duplicate and the procedures were followed according to the manufacturer's instruction. Statistical analysis Quantitative data were presented as mean ± SEM. Two-sided Student's t-test was used to determine the significance between different treatment groups. P b 0.05 was considered statistically significant. Results WYC02-9 inhibited cervical cancer cell viability and induced apoptotic cell death The cytotoxicity of WYC02-9 on three human cervical cancer cell lines HeLa, SiHa and C33A was analyzed (Table 1). Notably, WYC02-9 had higher cytotoxic activity than cisplatin in all the cervical cancer cell lines examined (Table 1). Furthermore, WYC02-9 treatment caused accumulation of cells at G2/M phase in a dose-dependent manner (Fig. 1A). In contrast to the decreased level of G2/M regulator P-CDC2 (Thr161), an increased phosphorylation of RB1, a G1 to S regulator, was observed in HeLa cells after WYC02-9 treatment (Fig. 1B). To evaluate apoptotic cell death upon WYC02-9 treatment, annexin V (marker of early apoptosis) and TUNEL (marker of late apoptosis) assays were applied. As shown in Fig. 1C, the numbers of annexin V- and TUNEL-positive cells significantly increased in HeLa cells after WYC02-9 treatment. Furthermore, WYC02-9-induced apoptosis was mediated by activation of CASP8, CASP9, CASP3, and poly-ADP ribose polymerase-1 (PARP1) (Fig. 1D). WYC02-9 inhibited cervical cancer cell migration, invasion, and MMP-2/-9 activities via MAPK14 activation WYC02-9 decreased cell migration and invasion in HeLa cells (Fig. 2A). Notably, no significant apoptosis was observed in 100% confluent HeLa cells after 2 μM WYC02-9 treatment, judged by the
Cancer cells that proliferate without extracellular matrix (ECM) are considered conducive to enhance tumor progression. Our data revealed that treatment of HeLa cells with WYC02-9 significantly decreased the ECM-independent cell growth in soft-agar assay (Fig. 3A). In a mouse xenograft model, WYC02-9 significantly suppressed the xenografted tumor growth (Fig. 3B), but did not impair body weight, hematopoiesis, liver or renal function (data not shown), or cervical epithelium (Supplemental Fig. 3). We also analyzed the expression of endothelial marker CD31 in the xenografted tumor by immunohistochemistry and the result showed that WYC02-9 treatment decreased CD31 staining and tumor microvascular density (MVD) in the xenografted tumors (Fig. 3C). WYC02-9 inhibited angiogenesis in vivo and in vitro The in vivo significance of WYC02-9 on VEGF-induced angiogenesis was evaluated in mice by measuring hemoglobin levels in the Matrigel plugs at 14 days after implantation. When compared to VEGF treatment alone, the presence of WYC02-9 significantly reduced the VEGF-induced angiogenesis (Fig. 4A). Further zebrafish model also showed that WYC02-9 significantly decreased the number of sub-intestinal vessel (SIV) (Fig. 4B). WYC02-9 inhibited the migration, invasion, and MMP-2/-9 activities of MS-1 endothelial cells (Supplemental Fig. 4). MAPK14 is a negative regulator during angiogenesis [36,37]. In MS-1 cells, WYC02-9 treatment significantly increased the phosphorylation of MAPK14 (Fig. 5A), but had no effects on the phosphorylation of MAPK1/3 and MAPK8 (data not shown). Pretreatment of MS-1 cells with SB203580 partially but significantly reversed the anti-migration/invasion (Fig. 5B), antitube formation (Fig. 5C) and anti-VEGF-A secretion (Fig. 5D) activities of WYC02-9. Notably, no significant apoptosis was observed in the 100% confluent MS-1 cells after 2 μM WYC02-9 treatment for 24 h ([IC50] = 9.61 μM; data not shown). Discussion The synthetic flavonoid WYC02-9 inhibits cervical cancer cell growth, migration, invasion and angiogenesis Flavonoids have been shown to possess multiple anti-tumor activities in a variety of cancer types including cervical carcinomas [18]. The current study showed that the synthetic flavonoid WYC02-9 exhibited a number of inhibitory effects on cervical cancer cells. First, WYC02-9 induced cytotoxicity in cervical cancer cell lines (Table 1). Second, WYC02-9 arrested cervical cancer cells at G2/M phases (Fig. 1A and B) and increased their apoptosis (Fig. 1C and D), which were in agreement with the reduced cervical tumor growth in WYC02-9-treated mice (Fig. 3B). Furthermore, the ECM-independent cell growth was decreased upon WYC02-9 treatment in HeLa cells (Fig. 3A). WYC02-9 also inhibited cervical cancer migration, invasion and angiogenesis. The in vitro wound healing and invasion as well as the metastasis-related proteinases MMP-2/-9 secretion were suppressed in WYC02-9-treated HeLa cells (Fig. 2A). The inhibitory effect of
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Fig. 2. WYC02-9 decreased HeLa cell migration, invasion, and MMP-2/-9 activities through MAPK14 signaling. (A) HeLa cells were treated with WYC02-9 for 24 h and the migration and invasion efficiencies were determined by wound healing assay and ECM-coated transwell system respectively. MMP-2/-9 activities were measured using gelatin zymography analysis. (B) HeLa cells were treated with WYC02-9 for 4 h and cell lysates were analyzed for the phosphorylated and total forms of signal proteins, including MAPK (MAPK14, MAPK1/3 and MAPK8) pathway by immunoblotting analysis. (C) Cells were pretreated with 5 μM of SB203580 for 2 h, followed by WYC02-9 treatment for 48 h. The cell viability was measured by XTT assay. (D) Cells were pretreated with 5 μM of SB203580 for 2 h, followed by WYC02-9 treatment for 24 h. The migration/invasion efficiencies as well as MMP-2/-9 activities were measured. The data were presented as mean ± SEM. * indicates a significant difference (P b 0.05) when compared with the vehicle control. # indicates a significant difference (P b 0.05) when compared with the corresponding WYC02-9 treatment without SB203580.
WYC02-9 on angiogenesis was evidenced from both in vivo and in vitro studies (Figs. 3C, 4, 5). Taken together, the data support a view that WYC02-9 may have the potential to be developed as a therapeutic agent for cervical cancer treatment. Activation of PIK3/AKT1 is considered oncogenic in cervical cancer and other cancer types [38,39]. In contrast, activation of MAPK14 leads to cancer cell death [24]. We previously reported that WYC02, the precursor compound from which WYC02-9 was derived, inhibited cervical cancer cell proliferation and tumorigenesis via suppression of PIK3 signaling pathway [22]. Our current data showed that PIK3/AKT1 activities were inhibited while MAPK14 activity was enhanced by WCY02-9 (Fig. 2B and Supplemental Fig. 2). These observations were in accordance with the cytotoxic effect of WCY02-9 reported in our present and previous studies [23,24]. We noticed that the presence of MAPK14 inhibitor SB203580 could not reverse the WCY02-9-induced cytotoxicity (Fig. 2C). On the other hand, PIK3 inhibitor wortmannin alone showed cytotoxicity on HeLa cells and no synergistic effect between wortmannin and WYC02-9 was observed (Supplemental Fig. 2), suggesting that WYC02-9 may exert its cytotoxic effect through PIK3 pathway but not MAPK14
pathway. More experiments may be required to elucidate the differential demands on PIK3 and MAPK14 pathways in WYC02-9induced cervical cancer cell death. WYC02-9 decreases cervical cancer cell migration and invasion via activation of MAPK14 pathway The anti-migratory and anti-invasive effects of flavonoids were mediated through various signaling molecules. For example, (−)-epigallocatechin-3-gallate (EGCG) downregulates the hepatocyte growth factor-activated MMP-9 and urokinase-type plasminogen activator (uPA) in hypopharyngeal carcinoma [40]. Combined treatment of EGCG and TRAIL synergistically decreased the expression of MMP-2/3/-9, VEGF, uPA, and angiopoietin 1/2 in prostate cancer cells [41]. In addition, the inhibitory effects of flavonoids on cancer cell migration and invasion are associated with MAPKs, c-jun, NF-kappaB, and PIK3/AKT pathways [22,24,40,42]. In the current study, WYC02-9 treatment increased the expression of phospho-MAPK14 in HeLa cells (Fig. 2B). Notably, the WYC02-9-inhibited HeLa cell migration, invasion, and MMP-2/-9 activation were reversed in the presence
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Fig. 3. WYC02-9 suppressed cervical tumor growth and the formation of intratumoral vessels. (A) HeLa cells were treated with the indicated concentrations of WYC02-9 for 3 h, and then grown in soft-agar for 14 days before counting of anchorage-independent colonies. (B) Female nude mice bearing cervical tumors were treated with vehicle control (PBS) or WYC02-9. Tumor volumes were measured weekly for each group. (C) Tumor xenografts were analyzed for the expression of endothelial marker CD31 by immunohistochemistry analysis, from which the intratumoral microvascular density (MVD) was measured. The data were presented as mean ± SEM. * indicates a significant difference (P b 0.05) when compared with the vehicle control.
of the MAPK14 inhibitor SB203580 (Fig. 2D). These results suggest that MAPK14 may act as a crucial mediator of the anti-migration/ invasion effect of WYC02-9. WYC02-9 inhibits angiogenesis via activation of MAPK14 pathway Angiogenesis is required for tumor growth and metastasis. It is for this reason that anti-angiogenic therapy constitutes a profound
strategy for the control of tumor progression [43]. In such instances, VEGF signalings are the most relevant regulators of angiogenesis via the recruitment and activation of both cancer cells and endothelial cells, and therefore the VEGF-related pathways are often targeted for cancer treatment [44]. To verify the anti-angiogenic activity of WYC02-9, both in vivo and in vitro models were employed in the present study. WYC02-9 decreased the expression of endothelial cell marker CD31 in xenografted
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Fig. 4. WYC02-9 decreased in vivo angiogenesis in Matrigel plug angiogenesis assay and zebrafish SIV model. (A) Matrigels containing 50 ng of VEGF and 10 U of heparin, in combination with vehicle control (PBS) or 1 μM of WYC02-9, were injected subcutaneously to the female nude mice. After 14 days of implantation, Matrigel plugs (N = 6 in each group) were dissected out and the hemoglobin levels were determined by the Darkin's reagent kit. (B) Treatment with 20 μM of WYC02-9 for 72 h caused a reduction of the SIV number in 72 hpf zebrafish embryos (N = 120 in each group). Arrow showed the characteristic pattern of SIV. White arrowhead indicates the pronephric duct which served as a positive control for AP staining [35]. The data were presented as mean ± SEM. * Indicates a significant difference (P b 0.05) when compared with the vehicle control.
cervical tumor (Fig. 3C). In addition, WYC02-9 inhibited VEGF-induced blood vessel formation in Matrigel plug angiogenesis mouse model (Fig. 4A) as well as in zebrafish model (Fig. 4B). Moreover, the migratory properties, tube formation, and VEGF-A secretion of endothelial cells were reduced by WYC02-9, partially through an MAPK14-dependent pathway (Fig. 5). Since anti-angiogenic therapy in combination with irradiation or chemotherapy enhance the treatment efficacy [45,46], it will be valuable to investigate whether combination of WYC02-9 with radiation or chemotherapy will further inhibit cancer progression.
Conclusion Our current data showed the inhibitory effect of WYC02-9 on cervical tumorigenesis and angiogenesis both in vitro and in vivo. In addition, the previously unreported functions of WYC02-9 on cervical cancer migration, invasion and angiogenesis through MAPK14 activation were identified. All of the results suggest that WYC02-9 may be a promising drug candidate to be developed for cervical cancer chemotherapy.
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Fig. 5. WYC02-9 decreased migration, invasion, MMP-2/-9 secretion, and in vitro angiogenesis through MAPK14 activation in MS-1 endothelial cells. (A) MS-1 cells were treated with WYC02-9 for 4 h, followed by immunoblotting analysis for the phosphorylated and total forms of MAPK14 proteins. (B–D) MS-1 cells were pretreated with 5 μM of SB203580 for 2 h, followed by WYC02-9 treatment for 24 h. (B) The migration and invasion efficiencies were determined by wound healing assay and ECM-coated transwell system respectively. MMP2/-9 activities were measured using gelatin zymography analysis. (C) Tube formation in MS-1 cells upon WYC02-9 treatment was determined by counting the loop numbers. A typical photo for tube formation upon WYC02-9 treatment was shown. (D) VEGF-A secretion were measured by ELISA. The data were presented as mean ± SEM. * Indicates a significant difference (P b 0.05) when compared with the vehicle control. # Indicates a significant difference (P b 0.05) when compared with the corresponding WYC02-9 treatment without SB203580.
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ygyno.2013.10.012.
Conflict of interest statement No potential conflicts of interest were disclosed.
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Acknowledgment This work was supported by grants from E-DA Hospital (EDAHP101014, EDAHP-101023 and EDAHP-102025), National Health Research Institutes, Taiwan (NHRI-EX98, 99,100-9829BI, NHRI-EX102-10212BI), and Department of Health, Taiwan (DOH101-TD-C-111-002). References [1] Wright Jr TC, Kuhn L. Alternative approaches to cervical cancer screening for developing countries. Best Pract Res Clin Obstet Gynaecol 2012;26:197–208. [2] Devita VT, Hellman S, Rosenberg SA, editors. Cancer: principles and practice of oncology. 8th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2007. [3] González-Martín A, González-Cortijo L, Carballo N, Garcia JF, Lapuente F, Rojo A, et al. The current role of neoadjuvant chemotherapy in the management of cervical carcinoma. Gynecol Oncol 2008;110:S36–40. [4] Peters III WA, Liu PY, Barrett II RJ, Stock RJ, Monk BJ, Berek JS, et al. Concurrent chemotherapy and pelvic radiation therapy compared with pelvic radiation therapy alone as adjuvant therapy after radical surgery in high-risk early stage cancer of the cervix. J Clin Oncol 2000;18:1606–13. [5] Cadron I, Van Gorp T, Amant F, Leunen K, Neven P, Vergote I. Chemotherapy for recurrent cervical cancer. Gynecol Oncol 2007;107:S113–8. [6] Siddik ZH. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 2003;22:7265–79. [7] Sporn MB, Suh N. Chemoprevention of cancer. Carcinogenesis 2000;21:525–30. [8] Albini A, Tosetti F, Li VW, Noonan DM, Li WW. Cancer prevention by targeting angiogenesis. Nat Rev Clin Oncol 2012;9:498–509. [9] Appelmann I, Liersch R, Kessler T, Mesters RM, Berdel WE. Angiogenesis inhibition in cancer therapy: platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) and their receptors: biological functions and role in malignancy. Recent Results Cancer Res 2010;180:51–81. [10] Giannelli G, Antonaci S. MMP and TIMP assay in cancer. Biological and clinical significance. Int J Cancer 2005;116:1002–3. [11] Roomi MW, Monterrey JC, Kalinovsky T, Rath M, Niedzwiecki A. In vitro modulation of MMP-2 and MMP-9 in human cervical and ovarian cancer cell lines by cytokines, inducers and inhibitors. Oncol Rep 2010;23:605–14. [12] Tibbles LA, Woodgett JR. The stress-activated protein kinase pathways. Cell Mol Life Sci 1999;55:1230–54. [13] Husain SS, Szabo IL, Pai R, Soreghan B, Jones MK, Tarnawski AS. MAPK (ERK2) kinase —a key target for NSAIDs-induced inhibition of gastric cancer cell proliferation and growth. Life Sci 2001;69:3045–54. [14] El Mchichi B, Hadji A, Vazquez A, Leca G. p38 MAPK and MSK1 mediate caspase-8 activation in manganese-induced mitochondria-dependent cell death. Cell Death Differ 2007;14:1826–36. [15] Taylor CA, Zheng Q, Liu Z, Thompson JE. Role of p38 and JNK MAPK signaling pathways and tumor suppressor p53 on induction of apoptosis in response to Ad-eIF5A1 in A549 lung cancer cells. Mol Cancer 2013;12:35. [16] Mandlekar S, Kong AN. Mechanisms of tamoxifen-induced apoptosis. Apoptosis 2001;6:469–77. [17] Olson JM, Hallahan AR. p38 MAP kinase: a convergence point in cancer therapy. Trends Mol Med 2004;10:125–9. [18] Weng CJ, Yen GC. Flavonoids, a ubiquitous dietary phenolic subclass, exert extensive in vitro anti-invasive and in vivo anti-metastatic activities. Cancer Metastasis Rev 2012;31:323–51. [19] Chen PN, Kuo WH, Chiang CL, Chiou HL, Hsieh YS, Chu SC. Black rice anthocyanins inhibit cancer cells invasion via repressions of MMPs and u-PA expression. Chem Biol Interact 2006;163:218–29. [20] Czyz J, Madeja Z, Irmer U, Korohoda W, Hülser DF. Flavonoid apigenin inhibits motility and invasiveness of carcinoma cells in vitro. Int J Cancer 2005;114:12–8. [21] Zhang FL, Zhang W, Chen XM, Luo RY. Effects of quercetin and quercetin in combination with cisplatin on adhesion, migration and invasion of HeLa cells. Zhonghua Fu Chan Ke Za Zhi 2008;43:619–21. [22] Chen YJ, Kay N, Yang JM, Lin CT, Chang HL, Wu YC, et al. Total synthetic protoapigenone WYC02 inhibits cervical cancer cell proliferation and tumour growth through PIK3 signalling pathway. Basic Clin Pharmacol Toxicol 2013;113:8–18.
743
[23] Chen HM, Chang FR, Hsieh YC, Cheng YJ, Hsieh KC, Tsai LM, et al. A novel synthetic protoapigenone analogue, WYC02-9, induces DNA damage and apoptosis in DU145 prostate cancer cells through generation of reactive oxygen species. Free Radic Biol Med 2011;50:1151–62. [24] Chen YJ, Chen HP, Cheng YJ, Lin YH, Liu KW, Chen YJ, et al. The synthetic flavonoid WYC02-9 inhibits colorectal cancer cell growth through ROS-mediated activation of MAPK14 pathway. Life Sci 2013;92:1081–92. [25] Lin AS, Chang FR, Wu CC, Liaw CC, Wu YC. New cytotoxic flavonoids from Thelypteris torresiana. Planta Med 2005;71:867–70. [26] Lin AS, Nakagawa-Goto K, Chang FR, Yu D, Morris-Natschke SL, Wu CC, et al. First total synthesis of protoapigenone and its analogues as potent cytotoxic agents. J Med Chem 2007;50:3921–7. [27] Yuan SS, Hou MF, Hsieh YC, Huang CY, Lee YC, Chen YJ, et al. Role of MRE11 in cell proliferation, tumor invasion, and DNA repair in breast cancer. J Natl Cancer Inst 2012;104:1485–502. [28] Chang HL, Wu YC, Su JH, Yeh YT, Yuan SS. Protoapigenone, a novel flavonoid, induces apoptosis in human prostate cancer cells through activation of p38 mitogenactivated protein kinase and c-Jun NH2-terminal kinase 1/2. J Pharmacol Exp Ther 2008;325:841–9. [29] Ke FC, Chuang LC, Lee MT, Chen YJ, Lin SW, Wang PS, et al. The modulatory role of transforming growth factor beta1 and androstenedione on follicle-stimulating hormone-induced gelatinase secretion and steroidogenesis in rat granulosa cells. Biol Reprod 2004;70:1292–8. [30] Yeh YT, Ou-Yang F, Chen IF, Yang SF, Wang YY, Chuang HY, et al. STAT3 ser727 phosphorylation and its association with negative estrogen receptor status in breast infiltrating ductal carcinoma. Int J Cancer 2006;118:2943–7. [31] Weidner N. Current pathologic methods for measuring intratumoral microvessel density within breast carcinoma and other solid tumors. Breast Cancer Res Treat 1995;36:169–80. [32] Brown AC, Shah C, Liu J, Pham JT, Zhang JG, Jadus MR. Ginger's (Zingiber officinale Roscoe) inhibition of rat colonic adenocarcinoma cells proliferation and angiogenesis in vitro. Phytother Res 2009;23:640–5. [33] Park CC, Morel JC, Amin MA, Connors MA, Harlow LA, Koch AE. Evidence of IL-18 as a novel angiogenic mediator. J Immunol 2001;167:1644–53. [34] Westerfield M. The zebrafish book: a guide for the laboratory use of zebrafish. Eugene, Oregon: The University of Oregon Press; 1993. [35] Serbedzija GN, Flynn E, Willett CE. Zebrafish angiogenesis: a new model for drug screening. Angiogenesis 1999;3:353–9. [36] Issbrücker K, Marti HH, Hippenstiel S, Springmann G, Voswinckel R, Gaumann A, et al. p38 MAP kinase—a molecular switch between VEGF-induced angiogenesis and vascular hyperpermeability. FASEB J 2003;17:262–4. [37] Yin H, Liu Z, Li F, Ni M, Wang B, Qiao Y, et al. Ginsenoside-Rg1 enhances angiogenesis and ameliorates ventricular remodeling in a rat model of myocardial infarction. J Mol Med (Berl) 2011;89:363–75. [38] Denley A, Gymnopoulos M, Kang S, Mitchell C, Vogt PK. Requirement of phosphatidylinositol(3,4,5)trisphosphate in phosphatidylinositol 3-kinase-induced oncogenic transformation. Mol Cancer Res 2009;7:1132–8. [39] Schwarz JK, Payton JE, Rashmi R, Xiang T, Jia Y, Huettner P, et al. Pathway-specific analysis of gene expression data identifies the PI3K/Akt pathway as a novel therapeutic target in cervical cancer. Clin Cancer Res 2012;18:1464–71. [40] Lim YC, Park HY, Hwang HS, Kang SU, Pyun JH, Lee MH, et al. (−)-Epigallocatechin3-gallate (EGCG) inhibits HGF-induced invasion and metastasis in hypopharyngeal carcinoma cells. Cancer Lett 2008;271:140–52. [41] Siddiqui IA, Malik A, Adhami VM, Asim M, Hafeez BB, Sarfaraz S, et al. Green tea polyphenol EGCG sensitizes human prostate carcinoma LNCaP cells to TRAILmediated apoptosis and synergistically inhibits biomarkers associated with angiogenesis and metastasis. Oncogene 2008;27:2055–63. [42] Vayalil PK, Katiyar SK. Treatment of epigallocatechin-3-gallate inhibits matrix metalloproteinases-2 and −9 via inhibition of activation of mitogen-activated protein kinases, c-Jun and NF-kappaB in human prostate carcinoma DU-145 cells. Prostate 2004;59:33–42. [43] Sato Y. Aminopeptidases and angiogenesis. Endothelium 2003;10:287–90. [44] Pircher A, Hilbe W, Heidegger I, Drevs J, Tichelli A, Medinger M. Biomarkers in tumor angiogenesis and anti-angiogenic therapy. Int J Mol Sci 2011;12:7077–99. [45] Gorski DH, Beckett MA, Jaskowiak NT, Calvin DP, Mauceri HJ, Salloum RM, et al. Blockage of the vascular endothelial growth factor stress response increases the antitumor effects of ionizing radiation. Cancer Res 1999;59:3374–8. [46] Scappaticci FA. Mechanisms and future directions for angiogenesis based cancer therapies. J Clin Oncol 2002;18:3906–27.
