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Endothelin-1 promotes vascular endothelial growth factor-dependent angiogenesis in human chondrosarcoma cells

Abstract

Chondrosarcoma is the second most common sarcoma in bone malignancy and is characterized by a high metastatic potential. Angiogenesis is essential for the cancer metastasis. Endothelin-1 (ET-1) has been implicated in tumor angiogenesis and metastasis. However, the relationship of ET-1 with vascular endothelial growth factor (VEGF) expression and angiogenesis in human chondrosarcoma cells is mostly unknown. Here, we found that the expression of ET-1 and VEGF were correlated with tumor stage and were significantly higher than that in the normal cartilage. Exogenous ET-1 with chondrosarcoma cells promoted VEGF expression and subsequently increased migration and tube formation in endothelial progenitor cells. ET-1 increased VEGF expression and angiogenesis through ETAR, integrin-linked kinase (ILK), Akt and hypoxia-inducible factor-1α (HIF-1α) signaling cascades. Knockdown of ET-1 decreased VEGF expression and also abolished chondrosarcoma conditional medium-mediated angiogenesis in vitro as well as angiogenesis effects in the chick chorioallantoic membrane and Matrigel plug nude mice model in vivo. In addition, in the xenograft tumor angiogenesis model, knockdown of ET-1 significantly reduced tumor growth and tumor-associated angiogenesis. Taken together, these results indicate that ET-1 occurs through ETAR, ILK and Akt, which in turn activates HIF-1α, resulting in the activation of VEGF expression and contributing to the angiogenesis and tumor growth of human chondrosarcoma cells.

Introduction

Chondrosarcoma is the second most common sarcoma arising in bone malignancy after myeloma and osteosarcoma. It is characterized by its rapid-progressing, pathologically diverse and highly malignant form.1 Currently, surgical resection remains the primary mode of remedy for chondrosarcoma treatment. The distant metastatic potential of chondrosarcoma also has been reported previously.1 Therefore, better strategies of treatment will ultimately require understanding of the molecular mechanisms of the metastasis step of human chondrosarcoma and identifying and specifically targeting the critical signaling effectors.

Tumor growth and metastasis depend on the ability of cells to induce their own blood supply. Angiogenesis is essential for the development, growth and progression of human cancers.2, 3 The angiogenic process is balanced by various positive (such as vascular endothelial growth factor (VEGF)) and negative (such as thrombospondin-1) regulatory molecules of endothelial proliferation and migration.4 VEGF-A has been shown to play an important role in wound healing, embryonic development, growth of certain solid tumors and angiogenesis.5 Treatment with a VEGF antagonist significantly attenuated angiogenesis and tumor growth.4 Therefore, VEGF is a critical target for cancer therapy and metastasis.

Endothelin-1 (ET-1) is a potent vasoconstrictor and the most abundantly and widely expressed member of the endothelin family of proteins (ET-1, ET-2 and ET-3). Aberrant ET-1 is implicated in the pathobiology of a wide range of human tumors.6 ET-1 acts as an autocrine or paracrine growth, survival and angiogenic factor selectively through the specific receptor ETAR7 or ETBR8 in several tumor cell types. Although ET-1 is associated with many secreted factors and matrix proteins, it plays an important role in tumor progression and angiogenesis.9 However, a newly identified mechanism of tumor growth and angiogenesis has been observed, which associates with VEGF secreted in tumor cells, such as melanoma,10 meningiomas,11 and prostate cancer.12 Therefore, ET-1-induced, VEGF-dependent angiogenesis may be a novel target for tumor angiogenesis and metastasis.

ET-1 has been demonstrated to induce migration in human chondrosarcoma through matrix metalloproteinase-13 upregulation.13 On the other hand, VEGF is a potent angiogenic factor that is pivotal in angiogenesis and tumor growth. Although a role for ET-1 in VEGF production has been implicated in some cancer cells, the signaling pathway for ET-1 in VEGF expression in human chondrosarcoma has not been studied extensively. In this study, we explored the intracellular signaling pathways involved in ET-1-induced VEGF production in human chondrosarcoma. The results show that ET-1 and ETAR interaction activates integrin-linked kinase (ILK), Akt and the hypoxia-inducible factor-1α (HIF-1α) pathway, leading to the upregulation of VEGF expression, resulting in angiogenesis and tumor growth.

Results

ET-1 induces VEGF expression and promotes angiogenesis in vitro

ET-1 has been reported to promote VEGF-dependent angiogenesis in human cancer cells.14, 15 However, little is known about the correlation between ET-1 and VEGF in human chondrosarcoma cells. We first examined tissues obtained from human chondrosarcoma patients for the expression of the ET-1 and VEGF using immunohistochemistry (IHC). The expression of ET-1 and VEGF in chondrosarcoma patients was significantly higher than that in the normal cartilage (Figures 1a and b). In addition, the high level of ET-1 expression correlated strongly with VEGF expression and tumor stage (Figures 1a and b). The quantitative data also show that the expression of ET-1 is correlated with the expression of VEGF in human chondrosarcoma patients (Figure 1c). Next, we directly applied ET-1 to human chondrosarcoma cell line and examined the expression of VEGF (an important angiogentic factor). The results showed that ET-1 increased VEGF mRNA expression in a concentration-dependent manner (Figure 2a). On the other hand, stimulation of cells with ET-1 also led to increase in the protein expression of VEGF in a concentration-dependent manner, as shown by enzyme-linked immunosorbent assay (ELISA) and western blotting (Figures 2b and c). In angiogenic processes, endothelial cells must undergo migration, proliferation and tube formation to form new blood vessels.16 We then examined whether ET-1-dependent VEGF expression induced angiogenesis by using an endothelial progenitor cell (EPC) model in vitro. We found that the conditioned medium (CM) from ET-1-treated chondrosarcoma cells promoted migration and tube formation in EPCs concentration-dependently (VEGF was used as positive control) (Figures 2d and e). To elucidate whether ET-1-dependent VEGF plays an important role in angiogenesis, the VEGF antibody was used. As shown in Figures 2d and e, pre-treatment of JJ012 cells with VEGF antibody reduced ET-1-induced migration and tube formation in EPCs. These data indicated that ET-1-dependent VEGF expression promotes angiogenesis in human chondrosarcoma cells.

Figure 1
figure1

The correlation of ET-1, VEGF and tumor grades in human chondrosarcoma tissues. IHC of ET-1 (a) and VEGF (b) expressions in normal cartilage and chondrosarcoma tissue. The correlation and quantitative data are shown in (c).

Figure 2
figure2

ET-1 promotes VEGF expression and subsequently increases angiogenesis in human osteosarcoma cells. (ac) JJ012 cells were incubated with ET-1 (30–300 ng/ml) for 24 h, and VEGF expression was examined by qPCR, ELISA, and western blotting. (d and e) JJ012 cells were pre-treated for 30 min with VEGF antibody (5 μg/ml), followed by stimulation with ET-1 (100 ng/ml) or incubated with ET-1 (10–100 ng/ml) for 24 h. The medium was collected as CM and then applied to EPCs for 24 h. The cell migration and capillary-like structure formation in EPCs was examined by Transwell and tube formation assay. Results are expressed as mean±s.e. *P<0.05 compared with control; #P<0.05 compared with ET-1-treated group.

The ETAR/ILK/Akt signaling pathway is involved in ET-1-mediated VEGF expression and angiogenesis

The exertion of ET-1 affects VEGF and angiogenesis by binding to the cell surface of two distinct receptors (ETAR and ETBR).17, 18 Therefore, we hypothesized that the ET receptors may be involved in ET-1-induced VEGF expression and angiogenesis in human chondrosarcoma cells. Pre-treatment of chondrosarcoma cells with ETAR antagonist BQ123 but not ETBR antagonist BQ788 abolished ET-1-increased VEGF expression (Figures 3a and b). On the other hand, CM from chondrosarcoma also demonstrated that BQ123 but not BQ788 reduced ET-1-mediated migration and tube formation in EPCs. These data suggest that ET-1-dependent VEGF promoted angiogenesis through ETAR.

Figure 3
figure3

ET-1 increases VEGF expression and angiogenesis via ERAR receptor. (a and b) JJ012 cells were pre-treated with the BQ123 (5 μM) or BQ788 (10 μM) for 30 min, followed by stimulation with ET-1 (100 nM) for 24 h, and VEGF expression was examined by qPCR and ELISA. (c and d) JJ012 cells were pre-treated with BQ123 (5 μM) or BQ788 (10 μM) for 30 min, followed by stimulation with ET-1 (100 nM) for 24 h. The medium was collected as CM and then applied to EPCs for 24 h. The cell migration and capillary-like structure formation in EPCs was examined by Transwell and tube formation assay. Results are expressed as mean±s.e. *P<0.05 compared with control; #P<0.05 compared with ET-1-treated group.

The ILK/Akt signaling pathway has been reported to regulate VEGF expression and angiogenesis.19 We next examined whether the ILK/Akt pathway is involved in the ET-1-mediated VEGF expression and angiogenesis. Pre-treatment of JJ012 cells with ILK inhibitor KP392 or Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate)) reduced ET-1-induced expression of VEGF (Figures 4a and b). In addition, ET-1-mediated EPC migration and tube formation was also diminished by pre-treatment with ILK inhibitor KP392 or Akt inhibitor. Moreover, incubation of JJ012 cells with ET-1 increased ILK kinase activity and Akt phosphorylation in a time-dependent manner (Figure 4e). On the basis of these results, it appears that the ET-1 acted through the ILK/Akt pathway to enhance VEGF expression and angiogenesis in human chondrosarcoma cells.

Figure 4
figure4

ILK/Akt pathway is involved ET-1-induced VEGF expression and angiogenesis in human chondrosarcoma cells. (a and b) JJ012 cells were pre-treated with the KP392 (10 μM) or Akt inhibitor (10 μM) for 30 min, followed by stimulation with ET-1 (100 nM) for 24 h, and VEGF expression was examined by qPCR and ELISA. (c and d) JJ012 cells were pre-treated with the KP392 (10 μM) or Akt inhibitor (10 μM) for 30 min, followed by stimulation with ET-1 (100 nM) for 24 h. The medium was collected as CM and then applied to EPCs for 24 h. The cell migration and capillary-like structure formation in EPCs was examined by Transwell and tube formation assay. (e) JJ012 cells were incubated with ET-1 (100 ng/ml) for the indicated times, and ILK activation and Akt phosphorylation were determined by ILK kinase assay and western blotting. Results are expressed as mean±s.e. *P<0.05 compared with control; #P<0.05 compared with ET-1-treated group.

Involvement of HIF-1α in ET-1 induced angiogenesis and VEGF expression

Recent studies have documented that ET-1 promoted VEGF expression by HIF-1α transactivation.20, 21 Pre-treatment of JJ012 cells with HIF-1α inhibitor antagonized ET-1-increased VEGF expression (Figures 5a and b). On the other hand, HIF-1α inhibitor treatment also reduced ET-1-mediated EPC migration and tube formation. To confirm the results from the chemical inhibitor, small interfering RNA (siRNA) transfection was used. Transfection of cells with ETAR, ILK, Akt and HIF-1α siRNA reduced ET-1-induced VEGF expression. In addition, ET-1-mediated EPC migration and tube formation was also diminished by transfection with ETAR, ILK, Akt and HIF-1α siRNA (Supplementary Figures S1B–D). It has been reported that HIF-1α nuclear translocation is necessary for the transcriptional activation of HIF-1-regulated VEGF expression.22 We therefore used western blotting to examine the nuclear translocation of HIF-1α protein after ET-1 treatment. We found that ET-1 increased the accumulation of HIF-1α in the nucleus in a time-dependent manner (Figure 5e). We also further examined whether ET-1 could upregulate HIF-1 activation through the increase of mRNA level. Stimulation of JJ012 cells with ET-1 induced HIF-1α mRNA expression (Figure 5f). Therefore, HIF-1α transactivation plays a critical role in ET-1-induced VEGF expression and angiogenesis.

Figure 5
figure5

HIF-1αtransactivation is involved in ET-1-induced VEGF expression and angiogenesis. (a and b) JJ012 cells were pre-treated with the HIF-1αinhibitor (10 μM) for 30 min, followed by stimulation with ET-1 (100 nM) for 24 h, and VEGF expression was examined by qPCR and ELISA. (c and d) JJ012 cells were pre-treated with the HIF-1αinhibitor (10 μM) for 30 min, followed by stimulation with ET-1 (100 nM) for 24 h. The medium was collected as CM and then applied to EPCs for 24 h. The cell migration and capillary-like structure formation in EPCs was examined by Transwell and tube formation assay. (e) JJ012 cells were incubated with ET-1 (100 ng/ml) for the indicated times and HIF-1αaccumulation in the nucleus was determined by western blotting. (f) JJ012 cells were incubated with ET-1 (30–300 ng/ml) for 24 h, and HIF-1α mRNA expression was examined by qPCR. Results are expressed as mean±s.e. *P<0.05 compared with control; #P<0.05 compared with ET-1-treated group.

We next explored whether the ETAR/ILK/Akt pathway is an upstream molecule in ET-1-mediated HIF-1α transactivation. Our data found that ETAR, ILK or Akt inhibitor but not ETBR inhibitor reduced ET-1-increased mRNA expression of HIF-1α (Figures 6a and b). Furthermore, the in vivo binding of HIF-1α to the HRE element of the VEGF promoter occurred after ET-1 stimulation (Figure 6c). The binding of HIF-1α to the HRE element by ET-1 was attenuated by KP392 and Akt inhibitor (Figure 6c). HIF-1α activation was also evaluated by analyzing the HRE luciferase activity. As shown in Figure 6d, ET-1 treatment for 24 h caused increases in HRE luciferase activity concentration-dependently. ET-1-mediated HRE luciferase activity was reduced by pre-treatment with BQ123, KP392 and Akt inhibitor or by co-transfection with ILK siRNA and Akt mutant (Figures 6e and f). Therefore, ETAR, ILK and Akt signaling pathways are involved in ET-1-mediated HIF-1α activation.

Figure 6
figure6

ETAR/ILK/Akt pathway is involved in ET-1-mediated HIF-1α activation. (a) JJ012 cells were pre-treated with the BQ123, BQ788, KP392 and Akt inhibitor for 30 min, followed by stimulation with ET-1 (100 nM) for 24 h, and HIF-1α mRNA expression was examined by qPCR. (b) JJ012 cells were pre-treated with KP392 and Akt inhibitor for 30 min, followed by stimulation with ET-1 for 2 h, and then chromatin immunoprecipitation assay was then performed. Chromatin was immunoprecipitated with anti-HIF-1α. The percentage of precipitated chromatin was assayed to verify equal loading (input). (ce) JJ012 cells were incubated with ET-1 (10–100 ng/ml) for 24 h or pre-treated with BQ123, BQ788, KP392 and Akt inhibitor for 30 min or co-transfected with ILK siRNA and Akt mutant for 24 h before exposure to ET-1. The HRE-driven luciferase activity was measured, and the results were normalized to the β-galactosidase activity and expressed as mean±s.e. for three independent experiments performed in triplicate. Results are expressed as mean±s.e. *P<0.05 compared with control; #P<0.05 compared with ET-1-treated group.

Knockdown of ET-1 decreases angiogenesis and tumor growth in vivo

To confirm if ET-1 mediated VEGF-dependent angiogenesis in human chondrosarcoma cells, the ET-1-short hairpin (shRNA) expression in cells was established. Knockdown of ET-1 did not affect the cell viability in human chondrosarcoma cells (Supplementary Figure S1A). The expression of ET-1 and VEGF was reduced by ET-1-shRNA in JJ012/ET-1-shRNA cells (Figure 7a). CM from JJ012/control-shRNA cells promoted tube formation and cell migration in EPCs (Figures 7b and c). However, knockdown of ET-1 reduced CM-mediated tube formation and cell migration (Figures 7b and c). In addition, the effect of ET-1 on angiogenesis in vivo was evaluated by using the in vivo model of chick embryo chorioallantoic membrane (CAM) assay. CM from JJ012/control-shRNA increased angiogenesis in CAM was observed clearly (Figure 7d). In contrast, ET-1 shRNA completely reduced angiogenesis in CAM (Figure 7d). We also analyzed Matrigel plug formation following subcutaneous implantation in mice. Matrigel mixed with CM from JJ012/control-shRNA cells increased blood vessel growth (Figure 7e). On the other hand, CM from JJ012/ET-1-shRNA significantly inhibited neovascularization (Figure 7e). Furthermore, knockdown of ET-1 also reduced vascular formation in the Matrigel by analyzing the CD31 and hemoglobin content (Figures 7f and g). Overall, these results indicated that ET-1 from chondrosarcoma promotes angiogenesis in vivo.

Figure 7
figure7

Knockdown of ET-1 reduces angiogenetic effects in vitro and in vivo. (a) The protein expression of ET-1 and VEGF in JJ012/control-shRNA and JJ012/ET-1-shRNA cells was examined by western blotting. (b and c) EPCs were incubated with CM from JJ012/control-shRNA or JJ012/ET-1-shRNA for 24 h, and tube formation or cell migration was photographed under the microscope or examined by Transwell assay. (d) Chick embryos were incubated with PBS, JJ012/control-shRNA CM or JJ012/ET-1-shRNA CM for 4 days, and then resected, fixed and photographed with a stereomicroscope. (eg) Mice were injected subcutaneously with Matrigel mixed with PBS, JJ012/control-shRNA CM or JJ012/ET-1-shRNA CM for 7 days. The plugs were excised from mice and photographed, stained with CD31 and quantified the hemoglobin content (n=10 in each group). Results are expressed as mean±s.e. *P<0.05 compared with control; #P<0.05 compared with ET-1-treated group.

To further investigate whether knockdown of ET-1 is able to inhibit tumor-induced angiogenesis, a xenograft tumor-induced angiogenesis model was used. Chondrosarcoma cells were mixed with Matrigel and injected into the flanks of mice. We found that JJ012/control-shRNA cells profoundly induced tumor mass formation. However, knockdown of ET-1 reduced tumor growth in mice (Figures 8a–c). On the other hand, we also quantified the level of angiogenesis by determining the hemoglobin content of the plugs and found that reducing ET-1 expression diminished chondrosarcoma-induced angiogenesis in vivo (Figure 8d). The hemoglobin content also correlated with tumor volume on these tumors (Figure 8e). Overall these results suggest that ET-1 promotes angiogenesis and tumor growth in vivo.

Figure 8
figure8

Knockdown of ET-1 decreases tumor-associated angiogenesis in mice. (ad) JJ012/control-shRNA or JJ012/ET-1-shRNA cells were mixed with Matrigel and injected into flank sites of mice for 10 days, and then resected. The tumors were photographed with a microscope, measured weight and volume, and quantified the hemoglobin levels. The correlation between tumor volume and hemoglobin levels is shown in (e) (n=10 in each group). Results are expressed as mean±s.e. *P<0.05 compared with control.

Discussion

Unlike other mesenchymal malignancies, such as osteosarcoma and Ewing’s sarcoma, which cause dramatic increases in long-term survival with the advent of systemic chemotherapy, chondrosarcoma continues to have a poor prognosis because of the absence of an effective adjuvant therapy. Chondrosarcoma shows a predilection for metastasis to the lungs. Furthermore, angiogenesis is essential for the cancer metastasis. Therefore, it is important to explore the potential target for preventing chondrosarcoma angiogenesis and metastasis. Using IHC analysis, we found that the expression of ET-1 and VEGF in chondrosarcoma patients correlated with tumor stage and higher-than-normal cartilages. Chondrosarcoma CM-mediated tube formation and angiogenesis was abolished by ET-1 shRNA. In addition, ET-1 knockdown reduced angiogenesis and tumor growth in vivo. Our study suggests that ET-1 increased VEGF expression and subsequently promoted angiogenesis in human chondrosarcoma cells. One of the mechanisms underlying ET-1 increased VEGF production and angiogenesis through the activation of ETAR, ILK, Akt and HIF-1αpathways. In this study, we provided the data from clinical patients, cell experiment and animal model. Our results suggest that ET-1 promotes VEGF-dependent angiogenesis in human chondrosarcoma cells. Therefore, ET-1 may be a novel target for chondrosarcoma angiogenesis and metastasis.

ET-1 axis represents one of the key regulators of tumorigenesis and tumor progression in several tumor cells.23 Previous studies have shown that ET-1’s contribution to VEGF production through ETAR or ETBR has a critical role in the progression of tumor malignancy.24, 25 While, in particular, ET-1 acting through the ETAR has been implicated in progression of tumor angiogenesis.7, 26 It has also been demonstrated that ETBR blockade by the antagonist BQ788 inhibited tumor growth and death of melanoma cells in vivo and in vitro.27 We previously reported that ET-1 increased cell migration and matrix metalloproteinase-13 expression in human chondrosarcoma through ETAR and ETBR receptor.13 In contrast, this study shows that ETAR-specific inhibitor BQ123 but not ETBR-specific inhibitor BQ788 abolished ET-1-induced VEGF expression and angiogenesis. These results indicate that ET-1 mediates angiogenesis and migration in human chondrosarcoma cells through a different ETR receptor.

ILK activation has been reported to regulate VEGF gene expression.28 Recent studies have also indicated that growth factors and chemokines increased ILK activation. In this study, we examined whether ILK activation is involved in ET-1-mediated VEGF production. Stimulation of chondrosarcoma with ET-1 increased the activity of ILK, and ET-1-mediated VEGF expression and tube formation in EPCs were both inhibited by the specific ILK inhibitor KP-392. These data suggest that ILK activation is an important signaling molecule in ET-1-induced VEGF expression and angiogenesis. Akt is the most common downstream molecule in ILK-regulated cellular function pathway.29 Here we found that ET-1 increased the phosphorylation of Akt. Akt inhibitor and mutant also reduced ET-1-mediated VEGF expression and angiogenesis. These results suggested that ILK-dependent Akt activation is mediated by ET-1-enhanced VEGF expression and angiogenesis. In addition to gene expression, a similar signal pathway has also been reported in the ET-1-induced cell migration of ovarian carcinoma, which involved ILK-dependent Akt activation.30 ILK-dependent Akt pathway was also involved in ET-1-inhibited prolyl hydroxylase domain 2 expression,10 and in cancer metastasis response to ET-1, which activated the ILK/Akt pathway.31 Taken together, our results provided evidence that ET-1 promotes VEGF expression and angiogenesis via ILK-dependent Akt activation.

Tumor metastasis is the spread of tumor cells from a primary tumor to colonize other sites of the body. Invasion, intravasation and extravasation from the circulatory system, colonization and finally angiogenesis at a distant site are the most common features of tumor metastasis. Because of the prognosis of patients with chondrosarcoma, distant metastasis is generally considered as very poor. Thus, development of an antiangiogenic and antimetastatic therapy could conceivably be useful in these patients. Here we found that ET-1 induced VEGF expression and subsequently promoted angiogenesis and tumor growth in human chondrosarcoma cells through the activation of ETAR, ILK, Akt and HIF-1α signaling pathways. These findings may provide a better understanding of the mechanisms of metastasis and may lead to the development of effective therapies of chondrosarcoma.

Materials and methods

Materials

Rabbit polyclonal antibodies specific for mammalian target of glycogen synthase kinase 3β (GSK3β) and phospho-GSK3β were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for ILK, p-Akt, Akt, β-actin, CD31, ILK siRNA, control siRNA, control-shRNA plasmid and ET-1 shRNA plasmid were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). ET-1, HIF-1α and VEGF antibodies were purchased from Abcam (Cambridge, MA, USA). Recombinant human VEGF was purchased from R&D Systems (Minneapolis, MN, USA). Akt inhibitor was purchased from Calbiochem (San Diego, CA, USA). KP-392 was purchased from Kinetek Pharmaceuticals (Vancouver, BC, Canada). Dulbecco’s modified Eagle’s medium, α-minimum essential medium (MEM), fetal bovine serum and all the other cell culture reagents were purchased from Gibco-BRL Life technologies (Grand Island, NY, USA). Akt (Akt K179A) mutant and pHRE-luciferase construct were provided by Dr WM Fu (National Taiwan University, Taipei City, Taiwan). pSV-β-galactosidase vector and luciferase assay kit were purchased from Promega (Madison, WI, USA). All other chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA). The migration assay, ELISA assay, transfection of siRNAs and hemoglobin assay are provided in Supplementary Data.

Cell culture

The human chondrosarcoma cell line (JJ012) was kindly provided by the laboratory of Dr Sean P Scully (University of Miami School of Medicine, Miami, FL, USA). The cells were cultured in complete medium containing Dulbecco’s modified Eagle’s medium /α-MEM supplemented with 10% fetal bovine serum. All experiments with cells were carried out at the permissive temperature (37 °C) and maintained in a humidified atmosphere of 5% CO2.

Isolation and cultivation of circulating EPCs

The study protocol was approved by the Institutional Review Board of Mackay Medical College (New Taipei City, Taiwan) (reference number: P1000002), and all subjects gave informed written consent before enrollment in this study. After collecting peripheral blood (80 ml) from healthy donors, the peripheral blood mononuclear cells were fractionated from other blood components by centrifugation on Ficoll-Paque plus (Amersham Biosciences, Uppala, Sweden) according to the manufacturer’s instructions. CD34-positive progenitor cells were obtained from the isolated peripheral blood mononuclear cells using CD34 MicroBead kit and MACS Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany). The maintenance and characterization of CD34-positive EPCs were performed as described previously.32 Briefly, human CD34-positive EPCs were maintained and propagated in MV2 complete medium, consisting of MV2 basal medium and growth supplement (PromoCell, Heidelberg, Germany), and supplied with 20% defined fetal bovine serum (HyClone, Logan, UT, USA). The cultures were seeded onto 1% gelatin-coated plasticware and maintained at 37 °C in a humidified atmosphere of 5% CO2.

Immunohistochemistry

The human chondrosarcoma tissue array was purchased from Biomax (Rockville, MD, USA; 11 cases for normal cartilage, 4 cases for type Ib chondrosarcoma and 9 cases for type IIb chondrosarcoma). The tissues were placed on glass slides, rehydrated and incubated in 3% hydrogen peroxide to block the endogenous peroxidase activity. After trypsinization, sections were blocked by incubation in 3% bovine serum albumin in phosphate-buffered saline (PBS). The primary antibody monoclonal mouse anti-human ET-1 or VEGF were applied to the slides at a dilution of 1:50 and incubated at 4 °C overnight. After being washed three times in PBS, the samples were treated with goat anti-mouse IgG biotin-labeled secondary antibodies at a dilution of 1:50. Bound antibodies were detected with an ABC kit (Vector Laboratories, Burlingame, CA, USA). The slides were stained with chromogen diaminobenzidine, washed, counterstained with Delafield’s hematoxylin, dehydrated, treated with xylene and mounted. The sum of the intensity and percentage score was used as the final staining scores (0–5).

Quantitative real-time PCR

Total RNA was extracted from cancer cells using a TRIzol kit (MDBio Inc., Taipei, Taiwan). The reverse transcription reaction was performed using 2 μg of total RNA that was reverse transcribed into cDNA using oligo (dT) primer.33, 34 The quantitative real-time polymerase chain reaction (qPCR) analysis was carried out using Taqman one-step PCR Master Mix (Applied Biosystems, Foster City, CA, USA). A volume of 100 ng total cDNA was added per 25-μl reaction with sequence-specific primers and Taqman probes. Sequences for all target gene primers and probes were purchased commercially (β-actin was used as internal control) (Applied Biosystems, Foster City, CA, USA). qPCR assays were carried out in triplicate on a StepOnePlus Sequence Detection System. The cycling conditions were 10 min polymerase activation at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 60 s. The threshold was set above the non-template control background and within the linear phase of target gene amplification to calculate the cycle number at which the transcript was detected (denoted as CT).

Western blot analysis

The cellular lysates were prepared as described previously.35, 36 Proteins were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to Immobilon polyvinyldifluoride membranes. The blots were blocked with 4% bovine serum albumin for 1 h at room temperature and then probed with rabbit anti-human antibodies against ILK, GSK3β, and p-GSK3β, Akt, p-Akt, HIF-1α, PCNA or VEGF (1:1000) for 1 h at room temperature. After three washes, the blots were subsequently incubated with a donkey anti-rabbit peroxidase-conjugated secondary antibody (1:1000) for 1 h at room temperature. The blots were visualized by enhanced chemiluminescence using Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY, USA).

ILK kinase assay

ILK enzymatic activity was assayed in cells lysed in Nonidet P-40 buffer (0.5% sodiumdeoxycholate, 1% Nonidet P-40, 50 mM HEPES (pH 7.4), 150 mM NaCl) as reported previously.37 Briefly, ILK was immunoprecipitated with ILK antibody overnight at 4 °C from 250 μg of lysate. After immunoprecipitation, beads were resuspended in 30 μl of kinase buffer containing 1 μg of recombinant substrate (GSK3β fusion protein) and 200 μM cold ATP, and the reaction was carried out for 30 min at 30 °C. The phosphorylated substrate was visualized by western blot with phospho-GSK3β antibody. Total GSK3β was detected with the appropriate antibody.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation analysis was performed as described previously.38 DNA immunoprecipitated by anti-HIF-1α antibody was purified. The DNA was then extracted with phenol–chloroform. The purified DNA pellet was subjected to PCR. PCR products were then resolved by 1.5% agarose gel electrophoresis and visualized by UV light. The primers 5′-IndexTermCCTTTGGGTTTTGCCAGA-3′ and 5′-IndexTermCCAAGTTTGTGGAGCTGA-3′ were utilized to amplify across the VEGF promoter region.39

Transfection and reporter assay

Human chondrosarcoma cells were transfected with reporter plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. At 24 h after transfection, the cells were pre-treated with inhibitors for 30 min, and then ET-1 or vehicle was added for 24 h. Cell extracts were then prepared, and luciferase and β-galactosidase activities were measured.13

Preparation of CM

The human chondrosarcoma cell line (JJ012 cells) was grown to confluence. On reaching confluence, culture media were changed with Dulbecco’s modified Eagle’s medium/α-MEM without fetal bovine serum. CM was collected 2 days after the change of medium and stored at −70 °C until use. In the series of experiments, JJ012 cells were pre-treated for 30 min with inhibitors, including BQ123, BQ788, KP392, Akt inhibitor, HIF-1α inhibitor or vehicle control (0.1% dimethylsulfoxide), and followed by treatment with ET-1 for 24 h to prevent signaling via the ET-1.

Tube formation assay

Matrigel (BD Biosciences, Bedford, MA, USA) was dissolved at 4 °C overnight and 48-well plates were prepared with 150 μl Matrigel in each well after coating and incubating at 37 °C for 30 min. EPCs (5 × 104) were cultured in 100 μl cultured media, which also included 50% EGM-MV2 media and 50% CM. After 16 h of incubation at 37 °C, EPC tube formation was assessed with a photomicroscope, and each well was photographed at × 200 magnification under a light microscope. Tube branches and total tube lengths were calculated using MacBiophotonics Image J software (Bethesda, MD, USA).

CAM assay

Angiogenic activity was determined using a CAM assay as described previously.40 Fertilized chicken eggs were incubated at 38 °C in an 80% humidified atmosphere. On day 8, CM from JJ012/control-shRNA or JJ012/ET-1-shRNA cells (2 × 104 cells) were deposited in the center of the chorioallantoic. CAM results were analyzed on the fourth day. Chorioallantoid membranes were collected for microscopy and photographic documentation. Angiogenesis was quantified by counting the number of blood vessel branch; at least 10 viable embryos were tested for each treatment. All animal works were carried out in accordance with a protocol approved by the China Medical University (Taichung, Taiwan) institutional animal care and use committees.

In vivo tumor xenograft study

Four-week-old male nude mice were used and randomized into two groups. For experimental cells growing exponentially, each implanted into 10 nude mice by subcutaneous injection, 1 × 106 cells (JJ012/control-shRNA or JJ012/ET-1-shRNA) were resuspended in 0.1 ml of serum-free DMEM/α-MEM and injected into the right flank. After 4 weeks, the mice were killed by overdose with anesthetic. The tumor were removed and fixed in 10% formalin. The tumor volume, weight and hemoglobin concentration of tumor was measured.

Matrigel plug assay

Matrigel plug angiogenesis assay was adapted from previously described assay.41 Thirty 4-week male nude mice were randomized into three groups: PBS (control), JJ012/control-shRNA or JJ012/ET-1-shRNA resuspended with Matrigel. Mice were subcutaneously injected with 0.4 ml Matrigel containing 2 × 105 cells. On day 7, Matrigel pellets were harvested, were fixed partly with 4% formalin, embedded in paraffin and subsequently processed for IHC staining for CD31. They were also partly estimated by the Drabkin’s method with Drabkin’s reagent kit (Sigma) to quantify blood vessel formation.

Statistics

Data are presented as mean±s.e.m.. Statistical comparison of two groups was performed using the Student’s t-test. Statistical comparisons of more than two groups were performed using one-way analysis of variance with Bonferroni’s post hoc test. In all cases, P<0.05 was considered significant.

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Acknowledgements

We thank Dr WM Fu for providing Akt mutant and HRE promoter construct. This work was supported by grants from the National Science Council of Taiwan (NSC99-2320-B-039-003-MY3 and NSC100-2320-B-039-028-MY3).

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Correspondence to C-H Tang.

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Wu, MH., Huang, CY., Lin, JA. et al. Endothelin-1 promotes vascular endothelial growth factor-dependent angiogenesis in human chondrosarcoma cells. Oncogene 33, 1725–1735 (2014). https://doi.org/10.1038/onc.2013.109

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Keywords

  • ET-1
  • VEGF
  • angiogenesis
  • chondrosarcoma

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