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Inhibition of metabotropic glutamate receptor 1 suppresses tumor growth and angiogenesis in experimental non-small cell lung cancer
European Journal of Pharmacology 783 (2016) 103–111
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Inhibition of metabotropic glutamate receptor 1 suppresses tumor growth and angiogenesis in experimental non-small cell lung cancer Hui Xia a,1, Ying-Nan Zhao a,1, Chang-Hai Yu a, Yun-Long Zhao a, Yang Liu b,n a b
Department of Thoracic-Cardio Surgery, The First Affiliated Hospital of the General Hospital of PLA, Beijing 100048, China Department of Thoracic Surgery, The General Hospital of PLA, 28 Fuxing Road, Beijing 100853, China
art ic l e i nf o
a b s t r a c t
Article history: Received 8 January 2016 Received in revised form 27 April 2016 Accepted 28 April 2016 Available online 29 April 2016
Metabotropic glutamate receptor 1 (mGlu1 receptor) is expressed in many cancer cell types as compared to normal counterparts underscoring its potential role in tumor behavior. The aim of present study was to test the role of mGlu1 receptor in experimental non-small cell lung cancer (NSCLC). First, protein expression of mGlu1 receptor was higher in human NSCLC cell lines, including both adenocarcinoma and squamous carcinoma subtypes, when compared to normal bronchial epithelial cells. Inhibition of mGlu1 receptor by BAY36-7620 (an mGlu1 receptor-specific inhibitor) inhibited tumor growth and prolonged survival of mice with tumors of A549 or H1299. Treatment with BAY36-7620 suppressed AKT phosphorylation in A549 tumors and pre-treatment with BAY36-7620 blocked the L-quisqualate (a potent mGlu1 receptor agonist)-induced AKT phosphorylation in A549 cells. Treatment with BAY36-7620 reduced cellular proliferation of A549 cells. Treatment with BAY36-7620 enhanced cleaved PARP levels and reduced protein expression of bcl-2, HIF-1α, and VEGF. In contrast, treatment with L-quisqualate reduced cleaved PARP levels and enhanced protein expression of bcl-2, HIF-1α, VEGF, and IL-8, which was reversed by co-incubation with MK2206 (an AKT inhibitor). Pre-treatment with BAY36-7620 blocked the VEGF-induced AKT phosphorylation in HUVECs. Treatment of HUVECs with L-quisqualate resulted in enhancement of capillary tube formation, which was reversed by co-incubation with MK2206. Furthermore, mGlu1 receptor knockdown suppressed tumor growth and prolonged survival of mice with tumors of A549 or H1299. Collectively, inhibition of mGlu1 receptor suppressed tumor growth and angiogenesis in experimental NSCLC. & 2016 Elsevier B.V. All rights reserved.
Keywords: Metabotropic glutamate receptor 1 Non-small cell lung cancer Tumor growth Angiogenesis
1. Introduction Lung cancer is the leading cause of cancer death worldwide, with non-small cell lung cancers (NSCLCs) accounting for more than 85% of all lung cancer cases (Jemal et al., 2007). Despite the enormous improvements made in chemotherapy and radiotherapy over the past few decades, the outlook for patients with NSCLC was dismal, with only slightly more than 15% of patients alive 5 years following diagnosis. Thus, molecular targeted therapy based on a better understanding of the molecular mechanisms of NSCLC is urgently required. Metabotropic glutamate receptor 1 (mGlu1 receptor), a member of the G-protein coupled receptor family, is most notably known for its role in nervous system development, function, and pathology (Monaghan et al., 1989; Hollmann and Heinemann, n
Corresponding author. E-mail address: [email protected] (Y. Liu). 1 The first two authors contributed equally to this study.
http://dx.doi.org/10.1016/j.ejphar.2016.04.053 0014-2999/& 2016 Elsevier B.V. All rights reserved.
1994; Hamilton et al., 2015), which mediates responses to a diverse array of signaling molecules that include hormones, neurotransmitters, and chemokines and acts in an autocrine or paracrine fashion. Recently, several reports have indicated an important role of mGlu1 receptor in tumorigenesis. The mGlu1 receptor protein was highly expressed in the nervous system, so initial discoveries of mGlu1 receptor in human tumors were reported in neuro-glial derived tumors such as gliomas, neuroblastoma, and medulloblastoma (Takano et al., 2001; Haas et al., 2013). Chen et al. provided the first proof of the involvement of mGlu1 receptor in nonneuronal tumorigenesis of melanoma (Chen et al., 1996; Zhu et al., 1998). More recent work has demonstrated that mGlu1 receptor was involved in the tumorigenesis of renal cell carcinoma (Martino et al., 2013), breast cancer (Mehta et al., 2013; Speyer et al., 2012), prostate cancer (Koochekpour et al., 2012), and oral squamous cell carcinoma (Park et al., 2007). In this work, we found that mGlu1 receptor protein was high expressed in all seven NSCLC cell lines including A549, H1650, H1299, SK-MES-1, SPCA-1, 95-D, and H23. The aim of this work
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Fig. 1. mGlu1 receptor protein expression. Analysis of mGlu1 receptor expression levels in NSCLC cell lines (A549, H1650, H1299, SK-MES-1, SPCA-1, 95-D, and H23) and the normal bronchial epithelial cell line (16HBE) by Western blotting analysis. mGlu1 receptor, metabotropic glutamate receptor 1; *P o 0.05 versus 16HBE group.
was to test the role of mGlu1 receptor in experimental NSCLC.
2. Materials and methods 2.1. Animals and chemicals Female 8 week-old athymic nude mice (Vital River Laboratory Animal Technology Co. Ltd., China) were maintained in a pathogen-free environment and given ad libitum access to food and water, under controlled temperature (23 7 2 °C) and 12-h light and 12-h dark cycles. All the mice used in this work received humane care in compliance with institutional animal care guidelines, and were approved by the Local Institutional Committee. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010). Unless otherwise specified, drugs and regents were purchased from Sigma-Aldrich (MO, USA). The mGlu1 receptor shRNA lentiviral particles (sc-61027-V) and negative lentiviral particles (sc-108080) were purchased from Santa Cruz (CA, USA). 2.2. Cell culture Human umbilical vascular endothelial cells (HUVECs), seven NSCLC cell lines (A549, H1650, H1299, SK-MES-1, SPCA-1, 95-D, and H23), and a normal human bronchial epithelial cell line (16HBE) were obtained from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). HUVECs were cultured on 0.1% gelatin-coated flasks in M199/20% FBS supplemented with penicillin/streptomycin, and endothelial cell growth supplement (BD Biosciences, San Jose, CA, USA). A549, H1650, H1299, SK-MES-1, SPCA-1, 95-D, H23, and 16HBE were cultured in Kaighn's F-12 media (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen) and 1% penicillin-streptomycin at 37 °C in a humidified 5% CO2 atmosphere, unless otherwise specified. The culture medium was renewed every 48 h. 2.3. Subcutaneous implantation Nude mice were injected with 5 105 cells into the right dorsal flank, and treated as indicated above. Tumor volumes (V¼L W2 π/6) were measured every six days, where ‘L’ is the length and ‘W’ is the width of the tumor. Following 24 days of
implantation, all mice were terminated by CO2 asphyxiation, the tumors were harvested. After washing with PBS and weighing, tumors were frozen and stored in liquid nitrogen until it was used for assays. Survival studies: For long-term survival studies, the treatment continued up to the end of the survival studies, and animals were killed when tumor volumes reached 1500 mm3. 2.4. Cell proliferation assay A549 or H1299 of cells were seeded in 12-well plates at a density of 7500 cells/ml using RPMI Medium 1640 supplemented with 10% FBS under 5% CO2 at 37 °C. After treatment, cell proliferation was determined on day three by measuring the conversion of tetrazolium salt into a formazan product using the CellTiter Non-Radioactive Cell Proliferation Assay (MTT Assay) according to manufacturer's protocol (Promega, Wisconsin, USA). 2.5. Hypoxic conditions A549 cells were incubated under hypoxic conditions to investigate the effect of mGlu1 receptor/AKT on HIF-1α/VEGF. Hypoxic conditions were achieved in an incubator containing a gaseous mixture of 94% N2, 5% CO2, and 1% O2 (v/v). 2.6. Western blotting analysis Cultured cells were lysed in RIPA buffer (50 mM Tris (pH 7.5), 300 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM PMSF and 2 μM leupeptin). The tumors were homogenized in ice cold buffer (0.15 M NaCl, 5 mM EDTA, 10 mM Tris-Cl, 1% Triton X-100 and protease inhibitors cocktail). The homogenates were centrifuged (12,000g for 15 min at 4 °C) and the supernatants were collected. Total protein levels were quantified by BCA assay (Thermo Scientific, Rockford, IL, USA). The proteins (15 μg) were separated on 10% polyacrylamideSDS gel and electro-blotted onto polyvinylidene difluoride membrane. After blocking with TBS/5% skim milk, the membrane was incubated overnight at 4 °C with primary antibodies (Santa Cruz, CA, USA), followed by incubation with HRP-conjugated secondary antibodies. As a loading control, the blots were stripped and reprobed with anti-GAPDH antibody.
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Fig. 2. BAY36-7620 inhibited tumor growth and prolonged survival of athymic nude mice with A549 and H1299 lung tumors. Athymic nude mice were injected with 5 105 cells of A549 or H1299 into the flank, and treated with BAY36-7620 (5, 10 mg/kg/day, i.p.). Tumor volumes (A and B) were measured every six days. Overall survival was calculated from the day of inoculation to end of survival. Survival (C and D) was estimated using the Kaplan-Meier product limit method. n ¼ 10 in each group; *P o 0.05 versus control group.
Fig. 3. BAY36-7620 inhibited AKT phosphorylation in A549 tumors in vivo and A549 cells in vitro. Athymic nude mice were injected with 5 105 cells into the flank, and treated with BAY36-7620 (5, 10 mg/kg/day, i.p.). 24 days after implantation, the tumors were removed for determination of protein expression of p-AKT (Ser-473) and AKT (A) by Western blotting analysis. *P o 0.05 versus control group. A549 cells were stimulated with the mGlu1 receptor agonist, L-quisqualate (10 μM) in the presence or absence of BAY36-7620 (25 μM). Cells were collected at indicated time for measuring AKT phosphorylation (B). mGlu1 receptor, metabotropic glutamate receptor 1. *Po 0.05 versus non-treated group.
2.7. In vitro angiogenesis assay Following 48 h of treatment, HUVECs tube formation assay was performed using an ECMatrix assay kit (Millipore, Temecula, CA, USA) according to the manufacturer’s instruction. Briefly, chilled ECMatrix (10 μl) was transferred to each well of a pre-cooled 1 μSlide Angiogenesis ibiTreat chamber (ibidi GmbH, Germany) and allowed to solidify for 30 min (37 °C). HUVECs were seeded onto the surface of polymerized ECMatrix and incubated for 8 h (37 °C,
5% CO2). Tube networks were observed and photographed. Tube formation was quantified by measuring area of these capillary-like structures in five fields of view in each well. The experiment was repeated in triplicate. 2.8. Elisa assay The VEGF and IL-8 protein levels were determined with Quantikine ELISA kits (R&D Systems, Minneapolis, MN), according
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Fig. 4. Inhibition of cell growth by mGlu1 receptor antagonist. A549 cells were exposed to vehicle (0.05% DMSO, control) or BAY36-7620 (10, 25 μM) and cell concentrations (A) were determined by MTT assay. Significant inhibition of cell proliferation was observed in the BAY36-7620-treated groups after 4 days of incubation. A549 cells were treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. Following 4 days incubation, cells were collected for determination of cleaved-PARP, PARP, and bcl-2 protein expression (B). Under condition of hypoxia, A549 cells were treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. Following 4 days incubation, cells were collected for determination of protein levels of HIF-1α (C) by Western blotting analysis and HIF activity (D) by using the dual luciferase reporter assay system. Supernatants were collected for determination of protein levels of VEGF (E) and IL-8 (F) by ELISA method. HIF-1α, hypoxia-inducible factor 1 alpha; VEGF, vascular endothelial growth factor; mGlu1 receptor, metabotropic glutamate receptor 1; PARP, poly(ADPribose) polymerase; bcl-2, B-cell lymphoma-2; *P o 0.05 versus non-treated (control) group; # P o 0.05 versus L-quisqualate-treated alone group.
to the manufacturer's instructions. 2.9. Microvessel density analysis Tumor sections were incubated overnight at 4 °C with antiCD31 antibody (1:500, BD Biosciences, San Jose, CA, USA) and then incubated for 1 h at room temperature with FITC-conjugated goat anti-rat antibody (1:100). To assess mean vessel density, the number of vessels, determined by positive CD31 staining, were counted in 6–8 fields and averaged to get a mean value for each tumor. 2.10. Measurement of HIF activity Reverse transfection into A549 cells was made with a mixture of a transcription factorresponsive firefly luciferase construct ‘HIF Reporter’ (Cignal Finder Pathway Reporter Assays, SABiosciences, Frederick, MD, USA) and constitutively expressing Renilla luciferase construct. A mixture of non-inducible firefly luciferase construct and constitutively expressing Renilla luciferase construct used as negative control and a mixture of constitutively expressing firefly and Renilla luciferase constructs used as positive control were also transfected. At 24 h after transfection, A549 cells were treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or
MK2206 (5 μM), either alone or in combination under condition of hypoxia, and 4 days later, the activities of HIF was measured with the Dual-Glo luciferase assay (Promega). 2.11. Study design Experiment 1: The Western blotting analysis was performed to examine the expression of mGlu1 receptor in seven human NSCLC cell lines (A549, H1650, H1299, SK-MES-1, SPCA-1, 95-D, and H23), as well as in normal bronchial epithelial cell line (16HBE). Experiment 2: Athymic nude mice were injected with 5 105 cells of A549 or H1299 into the flank, and treated with BAY367620 (5, 10 mg/kg/day, i.p.). Tumor volumes were measured every six days. Overall survival was calculated from the day of inoculation to end of survival. Experiment 3: Athymic nude mice were injected with 5 105 cells of A549 into the flank, and treated with BAY36-7620 (5, 10 mg/kg/day, i.p.). 24 days after implantation, the tumors were removed for determination of AKT phosphorylation (Ser-473) and microvessel density. Experiment 4: A549 cells were pre-treated for 30 min with BAY36-7620 (25 μM) or not, then they were stimulated with L-quisqualate (10 μM). At various time points after treatment, cells were collected for determination of AKT phosphorylation (Ser-
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Fig. 5. HUVECs were stimulated with VEGF (100 ng/ml) in the presence or absence of BAY36-7620 (25 μM). Cells were collected at indicated time for measuring AKT phosphorylation (Ser-473, A). HUVECs were plated onto Matrigel coated 24 well plates at 2 105 cells per well and incubated in the presence of VEGF (100 ng/ml) and treated with BAY36–7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM). About 24 h later, capillary tubes formed (B) were evaluated. *P o0.05 versus untreated group; #Po 0.05 versus L-quisqualate-treated alone group. Athymic nude mice were injected with 5 105 cells into the flank, and treated with BAY36-7620 (5, 10 mg/kg/day, i.p.). 24 days after implantation, the tumors were removed for determination of microvessel density by CD31 immunohistochemisty (C, n ¼8 in each group). *P o 0.05 versus control group. VEGF, vascular endothelial growth factor; HUVECs, human umbilical vein endothelial cells.
473). Experiment 5: A549 cells were treated with BAY36-7620 (10 and 25 μM) or vehicle (0.05% DMSO, control). At various time points after treatment, cell proliferation was determined. Experiment 6: A549 cells were treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. Following 4 days of incubation, cells were collected for determination of cleaved-PARP, PARP, and bcl-2 protein expression. Experiment 7: Under condition of hypoxia, A549 cells were treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. Following 4 days incubation, cells were collected for determination of HIF-1α protein expression and HIF activity and supernatants were collected for determination of VEGF and IL-8 protein levels. Experiment 8: HUVECs were pre-treated for 30 min with BAY36-7620 (25 μM) or not, then they were stimulated with VEGF (100 ng/ml). At various time points after treatment, cells were collected for determination of AKT phosphorylation (Ser-473). Experiment 9: HUVECs were plated onto Matrigel coated 24 well plates at 2 105 cells per well and incubated in the presence of VEGF (100 ng/ml) and treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. About 48 h later, capillary tubes formed were evaluated.
Experiment 10: A549 and H1299 cells were transfected with mGlu1 receptor shRNA lentiviral particles or negative lentiviral particles according to manufacturer's instructions for 12 h. After transfection, cells were washed out with PBS and changed with fresh medium and cultured for 48 h. 48 h later, cells were collected for determination of mGlu1 receptor protein expression by Western blotting. Athymic nude mice were injected with 5 105 cells transfected with mGlu1 receptor shRNA into the flank. Tumor volumes were measured every six days. Overall survival was calculated from the day of inoculation to end of survival. 24 days after implantation, the tumors were removed for determination of protein expression of p-AKT, AKT, cleaved PARP, PARP, Bcl-2, and HIF-1α by Western blotting, and protein levels of VEGF by ELISA.
2.12. Ethics All the mice used in this work received humane care in compliance with institutional animal care guidelines, and were approved by the Local Institutional Committee. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010).
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Inhibition of mGlu1 receptor by BAY36-7620 treatment markedly prolonged the survival (Fig. 2(C) and (D)) of inoculated mice when compared to control group. 3.3. mGlu1 receptor/AKT signaling Athymic nude mice were injected with 5 105 cells of A549 into the flank, and treated with BAY36-7620 (an mGlu1 receptorspecific inhibitor, 5, 10 mg/kg/day, i.p.). It was found that treatment with BAY36-7620 lowered the phosphorylated AKT (Ser-473) levels in A549 tumors (Fig. 3(A)), but had no significant effect on phosphorylation of JNK, ERK, and p38 (Fig. S1, shown in the supplementary data). In SK-MES-1 tumors, treatment with BAY367620 also suppressed phosphorylation of AKT (Fig. S2, shown in the supplementary data). To determine whether AKT is a downstream mediator of mGlu1 receptor signaling in NSCLC cells, A549 cells were stimulated with L-quisqualate (a potent mGlu1 receptor agonist), either in the presence or absence of BAY36-7620. L-quisqualate stimulation led to increased AKT phosphorylation levels. However, pre-treatment with BAY36-7620 prior to L-quisqualate stimulation blocked this increase in AKT phosphorylation levels (Fig. 3(B)), indicating the role of AKT in mGlu1 receptor signaling in NSCLC cells. Fig. 6. Protein expression of mGlu1 receptor. The A549 or H1299 cells was cultured and transfected with mGlu1 receptor shRNA lentiviral particles or negative lentiviral particles according to manufacturer's instructions. 60 h later, cells were collected for determination of mGlu1 receptor levels by Western blotting (Western blotting results and corresponding quantification). mGlu1 receptor, metabotropic glutamate receptor 1; *Po 0.05 versus control group.
2.13. Statistical analysis All data except overall survival are expressed as mean 7 S.D. Statistical analysis was performed by one-way ANOVA followed by a multiple comparison procedure with the Student-Newman Keuls method. Overall survival was calculated from the day of inoculation to end of survival. Survival was estimated using the KaplanMeier product limit method, and differences among the groups were assessed with the generalized Wilcoxon test. A value of o0.05 was considered significant.
3. Results 3.1. mGlu1 receptor expression To determine whether mGlu1 receptor protein is active in NSCLC cells, we first assessed several NSCLC cell lines for mGlu1 receptor expression. The mGlu1 receptor protein was detected in all seven NSCLC cell lines (A549, H1650, H1299, SK-MES-1, SPCA-1, 95-D, and H23) as well as in normal bronchial epithelial cells (16HBE, Fig. 1), and it was higher expressed in NSCLC cell lines than that in normal bronchial epithelial cells. 3.2. The role of mGlu1 receptor in NSCLC Athymic nude mice were injected with 5 105 cells of A549 or H1299 into the flank, and treated with BAY36-7620 (an mGlu1 receptor-specific inhibitor, 5, 10 mg/kg/day, i.p.). 24 days after implantation, volumes of both A549 (Fig. 2(A)) and H1299 tumors (Fig. 2(B)) were lower in BAY36-7620-treated group than that in control group, indicating that mGlu1 receptor inhibition by BAY367620 suppressed tumor growth in athymic mice with lung tumors. According to Kaplan–Meier analysis, the survival curves for the control and BAY36-7620-treated mice differed significantly.
3.4. Role of mGlu1 receptor/AKT in A549 cells A549 cells were treated with BAY36-7620 (10 and 25 μM) or vehicle (0.05% DMSO, control). Treatment with BAY36-7620 reduced cellular proliferation of A549 cells when compared to control group (Fig. 4(A)). Similar result was also found in SK-MES-1 cells (Fig. S3, shown in the supplementary data). In addition, incubation with BAY36-7620 had no significant effect on cell viability of 16HBE (Fig. S4, shown in the supplementary data). A549 cells were treated with BAY36-7620 (an mGlu1 receptorspecific inhibitor, 25 μM), L-quisqualate (a potent mGlu1 receptor agonist, 10 μM) or MK2206 (an AKT inhibitor, 5 μM), either alone or in combination. Following 4 days of incubation, treatment of A549 cells with BAY36-7620 enhanced cleaved PARP levels and reduced bcl-2 protein expression. Treatment of A549 cells with L-quisqualate reduced cleaved PARP levels and enhanced bcl-2 protein expression; Co-incubation with MK2206 blocked the effect of L-quisqualate on bcl-2, but did not affect its effect on cleaved PARP (Fig. 4(B)). Under condition of hypoxia, A549 cells were treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. Following 4 days incubation, treatment of A549 cells with BAY36-7620 reduced HIF-1α (Fig. 4 (C)) protein expression and HIF activity (Fig. 4(D)) and secretion of VEGF (Fig. 4(E)) and IL-8 (Fig. 4(F)) into supernatants. Treatment of A549 cells with L-quisqualate enhanced HIF-1α protein expression and HIF activity and secretion of VEGF and IL-8 in supernatants, which was abolished by co-incubation with MK2206. 3.5. Role of mGlu1 receptor/AKT in HUVECs HUVECs were pre-treated for 30 min with BAY36-7620 (25 μM) or not, then they were stimulated with VEGF (100 ng/ml). VEGF stimulation led to increased AKT phosphorylation levels. However, pre-treatment with BAY36-7620 prior to VEGF stimulation blocked this increase in AKT phosphorylation levels (Fig. 5(A)), indicating that BAY36-7620 treatment blocked VEGF/AKT signaling in HUVECs. HUVECs were plated onto Matrigel coated 24 well plates at 2 105 cells per well and incubated in the presence of VEGF (100 ng/ml) and treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. About
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Fig. 7. Effects of mGlu1 receptor knockdown on cell proliferation of A549 and H1299, and tumor growth and survival of athymic mice with A549 and H1299 lung tumors. Cell numbers (A and B) were determined after 1, 3, 5, 7 days of incubation. 5 105 cells were injected s.c. into the flank of athymic mice. Tumor volumes (C and D) were measured every six days (n¼ 8 in each group). Overall survival was calculated from the day of inoculation to end of survival. Survival (E and F) was estimated using the Kaplan-Meier product limit method. n ¼8 in each group with A549 lung tumors; n¼ 9 in each group with H1299 lung tumors; Microvessel density was determined by CD31 immunohistochemisty (G and H). mGlu1 receptor, metabotropic glutamate receptor 1; *P o0.05 versus control group.
48 h later, it was found that treatment with BAY36-7620 led to a reduction in capillary tube formation. Treatment of HUVECs with L-quisqualate resulted in enhancement of capillary tube formation, which was blocked by co-incubation with MK2206 (Fig. 5(B)). When compared to tumors of control group, microvessel density marked by positive CD31 staining was significantly less in tumors treated with BAY36-7620 (Fig. 5(C)), indicating that inhibition of mGlu1 receptor by BAY36-7620 suppressed
angiogenesis in A549 lung tumors. 3.6. Effect of mGlu1 receptor knockdown on NSCLC A549 and H1299 cells were transfected with mGlu1 receptor shRNA lentiviral particles or negative lentiviral particles according to manufacturer's instructions for 12 h. After transfection, cells were washed out with PBS and changed with fresh medium and cultured
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Fig. 8. Effect of mGlu1 receptor knockdown on expression of several proteins in A549 tumors. 5 105 A549 cells were injected s.c. into the flank of athymic mice. 24 days after implantation, tumors were removed for determination of p-AKT, AKT, cleaved PARP, PARP, Bcl-2, and HIF-1α by Western blotting (A, B), and VEGF by ELISA (C). HIF-1α, hypoxia-inducible factor 1 alpha; VEGF, vascular endothelial growth factor; mGlu1 receptor, metabotropic glutamate receptor1; PARP, poly(ADP-ribose) polymerase; bcl-2, B-cell lymphoma-2; mGlu1 receptor, metabotropic glutamate receptor1; n ¼ 8 in each group; * P o 0.05 versus control group.
for 48 h. Transfection of A549 cells and H1299 cells with mGlu1 receptor shRNA lentiviral particles reduced protein expression (Fig. 6) of mGlu1 receptor. When compared to the Blank group (cells with no transfection), transfection of cells with control lentiviral particles had no significant effect on mGlu1 receptor expression. A549 and H1299 cells transfected with mGlu1 receptor shRNA lentiviral particles exhibited a significant decrease in cellular proliferation after about 5 days of incubation (Fig. 7(A) and (B)). Athymic nude mice were injected in the right dorsal flank with 5 105 control or mGlu1 receptor-RNAi cells. 24 days after implantation, volumes of both A549 (Fig. 7(C)) and H1299 tumors (Fig. 7(D)) were lower in mGlu1 receptor-RNAi group than that in control group, indicating that mGlu1 receptor knockdown inhibited NSCLC tumor growth in vivo. According to Kaplan–Meier analysis, the survival curves for the control and mGlu1 receptor-RNAi mice differed significantly. The mGlu1 receptor knockdown markedly prolonged the survival (Fig. 7(E) and (F)) of inoculated mice when compared to control group. Microvessel density (Fig. 7(G) and (H)) marked by positive CD31 staining was significantly less in tumors of mGlu1 receptorRNAi group than that in control group. In A549 tumors, ratio of p-AKT/AKT (Fig. 8(A) and (B)) and protein expression Bcl-2 (Fig. 8(A) and (B)), HIF-1α (Fig. 8(A) and (B)), and VEGF (Fig. 8(C)) were lower, and ratio of cleaved PARP/ PARP (Fig. 8(A) and (B)) was higher in mGlu1 receptor-RNAi group than that in control group.
4. Discussion Prior studies demonstrated that cancer cells released glutamate (Sharma et al., 2010), and increased glutamate efflux occurred in hepatocellular carcinoma cells in response to hypoxic conditions (Hu et al., 2014), indicating the important role of glutamate signaling in cancers. Several groups reported that glutamate receptor antagonists inhibited proliferation of cancer cells in vitro and limited tumor growth in vivo (Luksch et al., 2011; Wu et al., 2010). Stepulak et al. (2009) reported that mGlu1 receptor was expressed in A549 cells and it was shown that A549 cells depolarized in response to application of glutamate agonists and that this effect was reversed by glutamate receptor antagonists, which was similar with TE671 cells (a human rhabdomyosarcoma/medulloblastoma cell line). In this work, we found that mGlu1 receptor protein was detected in all four NSCLC cell lines, and first demonstrated that inhibition of mGlu1 receptor by BAY36-7620 suppressed tumor growth and prolonged the survival of inoculated mice with A549 and H1299 cells. AKT, as a pathway central in regulating cell proliferation,
survival, death, migration, and angiogenesis in NSCLC (Lee et al., 2011), was reported to be the primary downstream mediator of mGlu1 receptor signaling in melanoma (Shin et al., 2010). In this work, we demonstrated that AKT was also one of the downstream targets of mGlu1 in A549 cells and HUVECs. Members of the Bcl-2 protein family are important regulators of apoptosis, contributing to prolonged cell survival through their ability to block apoptosis (Kelly and Strasser, 2011). Down-regulation of Bcl-2 expression induces sensitivity to anti-cancer drugs (Oltersdorf et al., 2005; Li et al., 2009) and increases survival (Letai et al., 2004). In this work, we found that inhibition of mGlu1 receptor by BAY36-7620 treatment reduced bcl-2 protein expression; AKT signaling mediated the effect of mGlu1 receptor on bcl-2 in A549 cells, which was similar the report by Shin et al. (2010). In addition, we found that treatment of BAY36-7620 led to a rise in levels of another apoptotic marker, the cleaved form of PARP; however, it was not dependent on AKT signaling. Pathological angiogenesis is a relatively early event in carcinogenesis, and increased tumor angiogenesis is correlated with invasive tumor growth and metastasis and a poor prognosis (Vamesu, 2008; Vermeulen et al., 2010). Our results revealed that inhibition of mGlu1 receptor by BAY36-7620 reduced angiogenesis in A549 tumors. HIF-1α/VEGF pathway plays a critical role in tumor angiogenesis. VEGF, which is the most extensively characterized endothelial cell-specific angiogenic factor, leads to increased vascular permeability and plays a significant role in physiological and pathological angiogenesis (Tammela et al., 2005; Gabhann and Popel, 2008). It was reported that the expression of HIF-1α and VEGF was up-regulated in NSCLC and was related to a poor prognosis and worse overall survival (Rovina et al., 2011; Park et al., 2011). It was reported that mGlu1 receptor signal transduction promoted angiogenesis in melanoma through activation of the AKT/mTOR/HIF-1α signaling pathway (Wen et al., 2014). In A549 cells, inhibition of mGlu1 receptor reduced protein expression of HIF-1α and secretion of VEGF via inactivation of AKT. It was shown that glutamate caused a loss in human cerebral endothelial barrier integrity through activation of NMDA receptor (Sharp et al., 2003). Recently, it was found that Group I metabotropic glutamate receptors (including mGlu1 receptor and mGlu5 receptor) was also expressed in vascular endothelial cells and they could prevent endothelial programmed cell death independent from MAP kinase p38 activation (Lin et al., 2001). Speyer et al. (2014) detected expression of mGlu1 receptor by human dermal microvascular endothelial cells, HUVECs, and human pulmonary microvascular endothelial cells, and first demonstrated a novel role for mGlu1 receptor in mediating various steps of the angiogenic process including EC proliferation, Matrigel tube formation and vessel formation in vitro. AKT signaling is crucial for the signaling of angiogenic growth factors
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that promote endothelial cell migration, proliferation, microtubule formation, and survival (Shiojima and Walsh, 2002). In this work, we found that inhibition of mGlu1 receptor blocked VEGF-induced activation of AKT in HUVECs and reduced capillary tube formation. Therefore, inhibition of mGlu1 receptor suppressed tumor angiogenesis via suppressing AKT/HIF-1α/VEGF pathway in tumor cells and blocking VEGF/AKT pathway in endothelial cells. In addition, it was reported that non-small cell lung carcinoma cells synthesized and released norepinephrine and epinephrine (Al-Wadei et al., 2012), and exogenous norepinephrine treatment upregulated the expression of VEGF and IL-8 and stimulated tumor growth in vivo (Deng et al., 2014). The specific agonist of Group I mGluRs, DHPG, was able to stimulate the release of norepinephrine and epinephrine in several kinds of cells (Arce et al., 2004; Luccini et al., 2007). Therefore, norepinephrine and epinephrine might be involved in the function of mGlu1 receptor in NSCLC, which required further investigation. In conclusion, inhibition of metabotropic glutamate receptor 1 suppressed tumor growth and angiogenesis in experimental NSCLC, partly via inactivation of AKT signaling.
Conflicts of interest disclosure The authors declare that we have no conflict of interest.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2016.04.053.
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Full length article
Inhibition of metabotropic glutamate receptor 1 suppresses tumor growth and angiogenesis in experimental non-small cell lung cancer Hui Xia a,1, Ying-Nan Zhao a,1, Chang-Hai Yu a, Yun-Long Zhao a, Yang Liu b,n a b
Department of Thoracic-Cardio Surgery, The First Affiliated Hospital of the General Hospital of PLA, Beijing 100048, China Department of Thoracic Surgery, The General Hospital of PLA, 28 Fuxing Road, Beijing 100853, China
art ic l e i nf o
a b s t r a c t
Article history: Received 8 January 2016 Received in revised form 27 April 2016 Accepted 28 April 2016 Available online 29 April 2016
Metabotropic glutamate receptor 1 (mGlu1 receptor) is expressed in many cancer cell types as compared to normal counterparts underscoring its potential role in tumor behavior. The aim of present study was to test the role of mGlu1 receptor in experimental non-small cell lung cancer (NSCLC). First, protein expression of mGlu1 receptor was higher in human NSCLC cell lines, including both adenocarcinoma and squamous carcinoma subtypes, when compared to normal bronchial epithelial cells. Inhibition of mGlu1 receptor by BAY36-7620 (an mGlu1 receptor-specific inhibitor) inhibited tumor growth and prolonged survival of mice with tumors of A549 or H1299. Treatment with BAY36-7620 suppressed AKT phosphorylation in A549 tumors and pre-treatment with BAY36-7620 blocked the L-quisqualate (a potent mGlu1 receptor agonist)-induced AKT phosphorylation in A549 cells. Treatment with BAY36-7620 reduced cellular proliferation of A549 cells. Treatment with BAY36-7620 enhanced cleaved PARP levels and reduced protein expression of bcl-2, HIF-1α, and VEGF. In contrast, treatment with L-quisqualate reduced cleaved PARP levels and enhanced protein expression of bcl-2, HIF-1α, VEGF, and IL-8, which was reversed by co-incubation with MK2206 (an AKT inhibitor). Pre-treatment with BAY36-7620 blocked the VEGF-induced AKT phosphorylation in HUVECs. Treatment of HUVECs with L-quisqualate resulted in enhancement of capillary tube formation, which was reversed by co-incubation with MK2206. Furthermore, mGlu1 receptor knockdown suppressed tumor growth and prolonged survival of mice with tumors of A549 or H1299. Collectively, inhibition of mGlu1 receptor suppressed tumor growth and angiogenesis in experimental NSCLC. & 2016 Elsevier B.V. All rights reserved.
Keywords: Metabotropic glutamate receptor 1 Non-small cell lung cancer Tumor growth Angiogenesis
1. Introduction Lung cancer is the leading cause of cancer death worldwide, with non-small cell lung cancers (NSCLCs) accounting for more than 85% of all lung cancer cases (Jemal et al., 2007). Despite the enormous improvements made in chemotherapy and radiotherapy over the past few decades, the outlook for patients with NSCLC was dismal, with only slightly more than 15% of patients alive 5 years following diagnosis. Thus, molecular targeted therapy based on a better understanding of the molecular mechanisms of NSCLC is urgently required. Metabotropic glutamate receptor 1 (mGlu1 receptor), a member of the G-protein coupled receptor family, is most notably known for its role in nervous system development, function, and pathology (Monaghan et al., 1989; Hollmann and Heinemann, n
Corresponding author. E-mail address: [email protected] (Y. Liu). 1 The first two authors contributed equally to this study.
http://dx.doi.org/10.1016/j.ejphar.2016.04.053 0014-2999/& 2016 Elsevier B.V. All rights reserved.
1994; Hamilton et al., 2015), which mediates responses to a diverse array of signaling molecules that include hormones, neurotransmitters, and chemokines and acts in an autocrine or paracrine fashion. Recently, several reports have indicated an important role of mGlu1 receptor in tumorigenesis. The mGlu1 receptor protein was highly expressed in the nervous system, so initial discoveries of mGlu1 receptor in human tumors were reported in neuro-glial derived tumors such as gliomas, neuroblastoma, and medulloblastoma (Takano et al., 2001; Haas et al., 2013). Chen et al. provided the first proof of the involvement of mGlu1 receptor in nonneuronal tumorigenesis of melanoma (Chen et al., 1996; Zhu et al., 1998). More recent work has demonstrated that mGlu1 receptor was involved in the tumorigenesis of renal cell carcinoma (Martino et al., 2013), breast cancer (Mehta et al., 2013; Speyer et al., 2012), prostate cancer (Koochekpour et al., 2012), and oral squamous cell carcinoma (Park et al., 2007). In this work, we found that mGlu1 receptor protein was high expressed in all seven NSCLC cell lines including A549, H1650, H1299, SK-MES-1, SPCA-1, 95-D, and H23. The aim of this work
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Fig. 1. mGlu1 receptor protein expression. Analysis of mGlu1 receptor expression levels in NSCLC cell lines (A549, H1650, H1299, SK-MES-1, SPCA-1, 95-D, and H23) and the normal bronchial epithelial cell line (16HBE) by Western blotting analysis. mGlu1 receptor, metabotropic glutamate receptor 1; *P o 0.05 versus 16HBE group.
was to test the role of mGlu1 receptor in experimental NSCLC.
2. Materials and methods 2.1. Animals and chemicals Female 8 week-old athymic nude mice (Vital River Laboratory Animal Technology Co. Ltd., China) were maintained in a pathogen-free environment and given ad libitum access to food and water, under controlled temperature (23 7 2 °C) and 12-h light and 12-h dark cycles. All the mice used in this work received humane care in compliance with institutional animal care guidelines, and were approved by the Local Institutional Committee. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010). Unless otherwise specified, drugs and regents were purchased from Sigma-Aldrich (MO, USA). The mGlu1 receptor shRNA lentiviral particles (sc-61027-V) and negative lentiviral particles (sc-108080) were purchased from Santa Cruz (CA, USA). 2.2. Cell culture Human umbilical vascular endothelial cells (HUVECs), seven NSCLC cell lines (A549, H1650, H1299, SK-MES-1, SPCA-1, 95-D, and H23), and a normal human bronchial epithelial cell line (16HBE) were obtained from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). HUVECs were cultured on 0.1% gelatin-coated flasks in M199/20% FBS supplemented with penicillin/streptomycin, and endothelial cell growth supplement (BD Biosciences, San Jose, CA, USA). A549, H1650, H1299, SK-MES-1, SPCA-1, 95-D, H23, and 16HBE were cultured in Kaighn's F-12 media (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen) and 1% penicillin-streptomycin at 37 °C in a humidified 5% CO2 atmosphere, unless otherwise specified. The culture medium was renewed every 48 h. 2.3. Subcutaneous implantation Nude mice were injected with 5 105 cells into the right dorsal flank, and treated as indicated above. Tumor volumes (V¼L W2 π/6) were measured every six days, where ‘L’ is the length and ‘W’ is the width of the tumor. Following 24 days of
implantation, all mice were terminated by CO2 asphyxiation, the tumors were harvested. After washing with PBS and weighing, tumors were frozen and stored in liquid nitrogen until it was used for assays. Survival studies: For long-term survival studies, the treatment continued up to the end of the survival studies, and animals were killed when tumor volumes reached 1500 mm3. 2.4. Cell proliferation assay A549 or H1299 of cells were seeded in 12-well plates at a density of 7500 cells/ml using RPMI Medium 1640 supplemented with 10% FBS under 5% CO2 at 37 °C. After treatment, cell proliferation was determined on day three by measuring the conversion of tetrazolium salt into a formazan product using the CellTiter Non-Radioactive Cell Proliferation Assay (MTT Assay) according to manufacturer's protocol (Promega, Wisconsin, USA). 2.5. Hypoxic conditions A549 cells were incubated under hypoxic conditions to investigate the effect of mGlu1 receptor/AKT on HIF-1α/VEGF. Hypoxic conditions were achieved in an incubator containing a gaseous mixture of 94% N2, 5% CO2, and 1% O2 (v/v). 2.6. Western blotting analysis Cultured cells were lysed in RIPA buffer (50 mM Tris (pH 7.5), 300 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1 mM PMSF and 2 μM leupeptin). The tumors were homogenized in ice cold buffer (0.15 M NaCl, 5 mM EDTA, 10 mM Tris-Cl, 1% Triton X-100 and protease inhibitors cocktail). The homogenates were centrifuged (12,000g for 15 min at 4 °C) and the supernatants were collected. Total protein levels were quantified by BCA assay (Thermo Scientific, Rockford, IL, USA). The proteins (15 μg) were separated on 10% polyacrylamideSDS gel and electro-blotted onto polyvinylidene difluoride membrane. After blocking with TBS/5% skim milk, the membrane was incubated overnight at 4 °C with primary antibodies (Santa Cruz, CA, USA), followed by incubation with HRP-conjugated secondary antibodies. As a loading control, the blots were stripped and reprobed with anti-GAPDH antibody.
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Fig. 2. BAY36-7620 inhibited tumor growth and prolonged survival of athymic nude mice with A549 and H1299 lung tumors. Athymic nude mice were injected with 5 105 cells of A549 or H1299 into the flank, and treated with BAY36-7620 (5, 10 mg/kg/day, i.p.). Tumor volumes (A and B) were measured every six days. Overall survival was calculated from the day of inoculation to end of survival. Survival (C and D) was estimated using the Kaplan-Meier product limit method. n ¼ 10 in each group; *P o 0.05 versus control group.
Fig. 3. BAY36-7620 inhibited AKT phosphorylation in A549 tumors in vivo and A549 cells in vitro. Athymic nude mice were injected with 5 105 cells into the flank, and treated with BAY36-7620 (5, 10 mg/kg/day, i.p.). 24 days after implantation, the tumors were removed for determination of protein expression of p-AKT (Ser-473) and AKT (A) by Western blotting analysis. *P o 0.05 versus control group. A549 cells were stimulated with the mGlu1 receptor agonist, L-quisqualate (10 μM) in the presence or absence of BAY36-7620 (25 μM). Cells were collected at indicated time for measuring AKT phosphorylation (B). mGlu1 receptor, metabotropic glutamate receptor 1. *Po 0.05 versus non-treated group.
2.7. In vitro angiogenesis assay Following 48 h of treatment, HUVECs tube formation assay was performed using an ECMatrix assay kit (Millipore, Temecula, CA, USA) according to the manufacturer’s instruction. Briefly, chilled ECMatrix (10 μl) was transferred to each well of a pre-cooled 1 μSlide Angiogenesis ibiTreat chamber (ibidi GmbH, Germany) and allowed to solidify for 30 min (37 °C). HUVECs were seeded onto the surface of polymerized ECMatrix and incubated for 8 h (37 °C,
5% CO2). Tube networks were observed and photographed. Tube formation was quantified by measuring area of these capillary-like structures in five fields of view in each well. The experiment was repeated in triplicate. 2.8. Elisa assay The VEGF and IL-8 protein levels were determined with Quantikine ELISA kits (R&D Systems, Minneapolis, MN), according
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Fig. 4. Inhibition of cell growth by mGlu1 receptor antagonist. A549 cells were exposed to vehicle (0.05% DMSO, control) or BAY36-7620 (10, 25 μM) and cell concentrations (A) were determined by MTT assay. Significant inhibition of cell proliferation was observed in the BAY36-7620-treated groups after 4 days of incubation. A549 cells were treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. Following 4 days incubation, cells were collected for determination of cleaved-PARP, PARP, and bcl-2 protein expression (B). Under condition of hypoxia, A549 cells were treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. Following 4 days incubation, cells were collected for determination of protein levels of HIF-1α (C) by Western blotting analysis and HIF activity (D) by using the dual luciferase reporter assay system. Supernatants were collected for determination of protein levels of VEGF (E) and IL-8 (F) by ELISA method. HIF-1α, hypoxia-inducible factor 1 alpha; VEGF, vascular endothelial growth factor; mGlu1 receptor, metabotropic glutamate receptor 1; PARP, poly(ADPribose) polymerase; bcl-2, B-cell lymphoma-2; *P o 0.05 versus non-treated (control) group; # P o 0.05 versus L-quisqualate-treated alone group.
to the manufacturer's instructions. 2.9. Microvessel density analysis Tumor sections were incubated overnight at 4 °C with antiCD31 antibody (1:500, BD Biosciences, San Jose, CA, USA) and then incubated for 1 h at room temperature with FITC-conjugated goat anti-rat antibody (1:100). To assess mean vessel density, the number of vessels, determined by positive CD31 staining, were counted in 6–8 fields and averaged to get a mean value for each tumor. 2.10. Measurement of HIF activity Reverse transfection into A549 cells was made with a mixture of a transcription factorresponsive firefly luciferase construct ‘HIF Reporter’ (Cignal Finder Pathway Reporter Assays, SABiosciences, Frederick, MD, USA) and constitutively expressing Renilla luciferase construct. A mixture of non-inducible firefly luciferase construct and constitutively expressing Renilla luciferase construct used as negative control and a mixture of constitutively expressing firefly and Renilla luciferase constructs used as positive control were also transfected. At 24 h after transfection, A549 cells were treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or
MK2206 (5 μM), either alone or in combination under condition of hypoxia, and 4 days later, the activities of HIF was measured with the Dual-Glo luciferase assay (Promega). 2.11. Study design Experiment 1: The Western blotting analysis was performed to examine the expression of mGlu1 receptor in seven human NSCLC cell lines (A549, H1650, H1299, SK-MES-1, SPCA-1, 95-D, and H23), as well as in normal bronchial epithelial cell line (16HBE). Experiment 2: Athymic nude mice were injected with 5 105 cells of A549 or H1299 into the flank, and treated with BAY367620 (5, 10 mg/kg/day, i.p.). Tumor volumes were measured every six days. Overall survival was calculated from the day of inoculation to end of survival. Experiment 3: Athymic nude mice were injected with 5 105 cells of A549 into the flank, and treated with BAY36-7620 (5, 10 mg/kg/day, i.p.). 24 days after implantation, the tumors were removed for determination of AKT phosphorylation (Ser-473) and microvessel density. Experiment 4: A549 cells were pre-treated for 30 min with BAY36-7620 (25 μM) or not, then they were stimulated with L-quisqualate (10 μM). At various time points after treatment, cells were collected for determination of AKT phosphorylation (Ser-
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Fig. 5. HUVECs were stimulated with VEGF (100 ng/ml) in the presence or absence of BAY36-7620 (25 μM). Cells were collected at indicated time for measuring AKT phosphorylation (Ser-473, A). HUVECs were plated onto Matrigel coated 24 well plates at 2 105 cells per well and incubated in the presence of VEGF (100 ng/ml) and treated with BAY36–7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM). About 24 h later, capillary tubes formed (B) were evaluated. *P o0.05 versus untreated group; #Po 0.05 versus L-quisqualate-treated alone group. Athymic nude mice were injected with 5 105 cells into the flank, and treated with BAY36-7620 (5, 10 mg/kg/day, i.p.). 24 days after implantation, the tumors were removed for determination of microvessel density by CD31 immunohistochemisty (C, n ¼8 in each group). *P o 0.05 versus control group. VEGF, vascular endothelial growth factor; HUVECs, human umbilical vein endothelial cells.
473). Experiment 5: A549 cells were treated with BAY36-7620 (10 and 25 μM) or vehicle (0.05% DMSO, control). At various time points after treatment, cell proliferation was determined. Experiment 6: A549 cells were treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. Following 4 days of incubation, cells were collected for determination of cleaved-PARP, PARP, and bcl-2 protein expression. Experiment 7: Under condition of hypoxia, A549 cells were treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. Following 4 days incubation, cells were collected for determination of HIF-1α protein expression and HIF activity and supernatants were collected for determination of VEGF and IL-8 protein levels. Experiment 8: HUVECs were pre-treated for 30 min with BAY36-7620 (25 μM) or not, then they were stimulated with VEGF (100 ng/ml). At various time points after treatment, cells were collected for determination of AKT phosphorylation (Ser-473). Experiment 9: HUVECs were plated onto Matrigel coated 24 well plates at 2 105 cells per well and incubated in the presence of VEGF (100 ng/ml) and treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. About 48 h later, capillary tubes formed were evaluated.
Experiment 10: A549 and H1299 cells were transfected with mGlu1 receptor shRNA lentiviral particles or negative lentiviral particles according to manufacturer's instructions for 12 h. After transfection, cells were washed out with PBS and changed with fresh medium and cultured for 48 h. 48 h later, cells were collected for determination of mGlu1 receptor protein expression by Western blotting. Athymic nude mice were injected with 5 105 cells transfected with mGlu1 receptor shRNA into the flank. Tumor volumes were measured every six days. Overall survival was calculated from the day of inoculation to end of survival. 24 days after implantation, the tumors were removed for determination of protein expression of p-AKT, AKT, cleaved PARP, PARP, Bcl-2, and HIF-1α by Western blotting, and protein levels of VEGF by ELISA.
2.12. Ethics All the mice used in this work received humane care in compliance with institutional animal care guidelines, and were approved by the Local Institutional Committee. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010).
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Inhibition of mGlu1 receptor by BAY36-7620 treatment markedly prolonged the survival (Fig. 2(C) and (D)) of inoculated mice when compared to control group. 3.3. mGlu1 receptor/AKT signaling Athymic nude mice were injected with 5 105 cells of A549 into the flank, and treated with BAY36-7620 (an mGlu1 receptorspecific inhibitor, 5, 10 mg/kg/day, i.p.). It was found that treatment with BAY36-7620 lowered the phosphorylated AKT (Ser-473) levels in A549 tumors (Fig. 3(A)), but had no significant effect on phosphorylation of JNK, ERK, and p38 (Fig. S1, shown in the supplementary data). In SK-MES-1 tumors, treatment with BAY367620 also suppressed phosphorylation of AKT (Fig. S2, shown in the supplementary data). To determine whether AKT is a downstream mediator of mGlu1 receptor signaling in NSCLC cells, A549 cells were stimulated with L-quisqualate (a potent mGlu1 receptor agonist), either in the presence or absence of BAY36-7620. L-quisqualate stimulation led to increased AKT phosphorylation levels. However, pre-treatment with BAY36-7620 prior to L-quisqualate stimulation blocked this increase in AKT phosphorylation levels (Fig. 3(B)), indicating the role of AKT in mGlu1 receptor signaling in NSCLC cells. Fig. 6. Protein expression of mGlu1 receptor. The A549 or H1299 cells was cultured and transfected with mGlu1 receptor shRNA lentiviral particles or negative lentiviral particles according to manufacturer's instructions. 60 h later, cells were collected for determination of mGlu1 receptor levels by Western blotting (Western blotting results and corresponding quantification). mGlu1 receptor, metabotropic glutamate receptor 1; *Po 0.05 versus control group.
2.13. Statistical analysis All data except overall survival are expressed as mean 7 S.D. Statistical analysis was performed by one-way ANOVA followed by a multiple comparison procedure with the Student-Newman Keuls method. Overall survival was calculated from the day of inoculation to end of survival. Survival was estimated using the KaplanMeier product limit method, and differences among the groups were assessed with the generalized Wilcoxon test. A value of o0.05 was considered significant.
3. Results 3.1. mGlu1 receptor expression To determine whether mGlu1 receptor protein is active in NSCLC cells, we first assessed several NSCLC cell lines for mGlu1 receptor expression. The mGlu1 receptor protein was detected in all seven NSCLC cell lines (A549, H1650, H1299, SK-MES-1, SPCA-1, 95-D, and H23) as well as in normal bronchial epithelial cells (16HBE, Fig. 1), and it was higher expressed in NSCLC cell lines than that in normal bronchial epithelial cells. 3.2. The role of mGlu1 receptor in NSCLC Athymic nude mice were injected with 5 105 cells of A549 or H1299 into the flank, and treated with BAY36-7620 (an mGlu1 receptor-specific inhibitor, 5, 10 mg/kg/day, i.p.). 24 days after implantation, volumes of both A549 (Fig. 2(A)) and H1299 tumors (Fig. 2(B)) were lower in BAY36-7620-treated group than that in control group, indicating that mGlu1 receptor inhibition by BAY367620 suppressed tumor growth in athymic mice with lung tumors. According to Kaplan–Meier analysis, the survival curves for the control and BAY36-7620-treated mice differed significantly.
3.4. Role of mGlu1 receptor/AKT in A549 cells A549 cells were treated with BAY36-7620 (10 and 25 μM) or vehicle (0.05% DMSO, control). Treatment with BAY36-7620 reduced cellular proliferation of A549 cells when compared to control group (Fig. 4(A)). Similar result was also found in SK-MES-1 cells (Fig. S3, shown in the supplementary data). In addition, incubation with BAY36-7620 had no significant effect on cell viability of 16HBE (Fig. S4, shown in the supplementary data). A549 cells were treated with BAY36-7620 (an mGlu1 receptorspecific inhibitor, 25 μM), L-quisqualate (a potent mGlu1 receptor agonist, 10 μM) or MK2206 (an AKT inhibitor, 5 μM), either alone or in combination. Following 4 days of incubation, treatment of A549 cells with BAY36-7620 enhanced cleaved PARP levels and reduced bcl-2 protein expression. Treatment of A549 cells with L-quisqualate reduced cleaved PARP levels and enhanced bcl-2 protein expression; Co-incubation with MK2206 blocked the effect of L-quisqualate on bcl-2, but did not affect its effect on cleaved PARP (Fig. 4(B)). Under condition of hypoxia, A549 cells were treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. Following 4 days incubation, treatment of A549 cells with BAY36-7620 reduced HIF-1α (Fig. 4 (C)) protein expression and HIF activity (Fig. 4(D)) and secretion of VEGF (Fig. 4(E)) and IL-8 (Fig. 4(F)) into supernatants. Treatment of A549 cells with L-quisqualate enhanced HIF-1α protein expression and HIF activity and secretion of VEGF and IL-8 in supernatants, which was abolished by co-incubation with MK2206. 3.5. Role of mGlu1 receptor/AKT in HUVECs HUVECs were pre-treated for 30 min with BAY36-7620 (25 μM) or not, then they were stimulated with VEGF (100 ng/ml). VEGF stimulation led to increased AKT phosphorylation levels. However, pre-treatment with BAY36-7620 prior to VEGF stimulation blocked this increase in AKT phosphorylation levels (Fig. 5(A)), indicating that BAY36-7620 treatment blocked VEGF/AKT signaling in HUVECs. HUVECs were plated onto Matrigel coated 24 well plates at 2 105 cells per well and incubated in the presence of VEGF (100 ng/ml) and treated with BAY36-7620 (25 μM), L-quisqualate (10 μM) or MK2206 (5 μM), either alone or in combination. About
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Fig. 7. Effects of mGlu1 receptor knockdown on cell proliferation of A549 and H1299, and tumor growth and survival of athymic mice with A549 and H1299 lung tumors. Cell numbers (A and B) were determined after 1, 3, 5, 7 days of incubation. 5 105 cells were injected s.c. into the flank of athymic mice. Tumor volumes (C and D) were measured every six days (n¼ 8 in each group). Overall survival was calculated from the day of inoculation to end of survival. Survival (E and F) was estimated using the Kaplan-Meier product limit method. n ¼8 in each group with A549 lung tumors; n¼ 9 in each group with H1299 lung tumors; Microvessel density was determined by CD31 immunohistochemisty (G and H). mGlu1 receptor, metabotropic glutamate receptor 1; *P o0.05 versus control group.
48 h later, it was found that treatment with BAY36-7620 led to a reduction in capillary tube formation. Treatment of HUVECs with L-quisqualate resulted in enhancement of capillary tube formation, which was blocked by co-incubation with MK2206 (Fig. 5(B)). When compared to tumors of control group, microvessel density marked by positive CD31 staining was significantly less in tumors treated with BAY36-7620 (Fig. 5(C)), indicating that inhibition of mGlu1 receptor by BAY36-7620 suppressed
angiogenesis in A549 lung tumors. 3.6. Effect of mGlu1 receptor knockdown on NSCLC A549 and H1299 cells were transfected with mGlu1 receptor shRNA lentiviral particles or negative lentiviral particles according to manufacturer's instructions for 12 h. After transfection, cells were washed out with PBS and changed with fresh medium and cultured
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Fig. 8. Effect of mGlu1 receptor knockdown on expression of several proteins in A549 tumors. 5 105 A549 cells were injected s.c. into the flank of athymic mice. 24 days after implantation, tumors were removed for determination of p-AKT, AKT, cleaved PARP, PARP, Bcl-2, and HIF-1α by Western blotting (A, B), and VEGF by ELISA (C). HIF-1α, hypoxia-inducible factor 1 alpha; VEGF, vascular endothelial growth factor; mGlu1 receptor, metabotropic glutamate receptor1; PARP, poly(ADP-ribose) polymerase; bcl-2, B-cell lymphoma-2; mGlu1 receptor, metabotropic glutamate receptor1; n ¼ 8 in each group; * P o 0.05 versus control group.
for 48 h. Transfection of A549 cells and H1299 cells with mGlu1 receptor shRNA lentiviral particles reduced protein expression (Fig. 6) of mGlu1 receptor. When compared to the Blank group (cells with no transfection), transfection of cells with control lentiviral particles had no significant effect on mGlu1 receptor expression. A549 and H1299 cells transfected with mGlu1 receptor shRNA lentiviral particles exhibited a significant decrease in cellular proliferation after about 5 days of incubation (Fig. 7(A) and (B)). Athymic nude mice were injected in the right dorsal flank with 5 105 control or mGlu1 receptor-RNAi cells. 24 days after implantation, volumes of both A549 (Fig. 7(C)) and H1299 tumors (Fig. 7(D)) were lower in mGlu1 receptor-RNAi group than that in control group, indicating that mGlu1 receptor knockdown inhibited NSCLC tumor growth in vivo. According to Kaplan–Meier analysis, the survival curves for the control and mGlu1 receptor-RNAi mice differed significantly. The mGlu1 receptor knockdown markedly prolonged the survival (Fig. 7(E) and (F)) of inoculated mice when compared to control group. Microvessel density (Fig. 7(G) and (H)) marked by positive CD31 staining was significantly less in tumors of mGlu1 receptorRNAi group than that in control group. In A549 tumors, ratio of p-AKT/AKT (Fig. 8(A) and (B)) and protein expression Bcl-2 (Fig. 8(A) and (B)), HIF-1α (Fig. 8(A) and (B)), and VEGF (Fig. 8(C)) were lower, and ratio of cleaved PARP/ PARP (Fig. 8(A) and (B)) was higher in mGlu1 receptor-RNAi group than that in control group.
4. Discussion Prior studies demonstrated that cancer cells released glutamate (Sharma et al., 2010), and increased glutamate efflux occurred in hepatocellular carcinoma cells in response to hypoxic conditions (Hu et al., 2014), indicating the important role of glutamate signaling in cancers. Several groups reported that glutamate receptor antagonists inhibited proliferation of cancer cells in vitro and limited tumor growth in vivo (Luksch et al., 2011; Wu et al., 2010). Stepulak et al. (2009) reported that mGlu1 receptor was expressed in A549 cells and it was shown that A549 cells depolarized in response to application of glutamate agonists and that this effect was reversed by glutamate receptor antagonists, which was similar with TE671 cells (a human rhabdomyosarcoma/medulloblastoma cell line). In this work, we found that mGlu1 receptor protein was detected in all four NSCLC cell lines, and first demonstrated that inhibition of mGlu1 receptor by BAY36-7620 suppressed tumor growth and prolonged the survival of inoculated mice with A549 and H1299 cells. AKT, as a pathway central in regulating cell proliferation,
survival, death, migration, and angiogenesis in NSCLC (Lee et al., 2011), was reported to be the primary downstream mediator of mGlu1 receptor signaling in melanoma (Shin et al., 2010). In this work, we demonstrated that AKT was also one of the downstream targets of mGlu1 in A549 cells and HUVECs. Members of the Bcl-2 protein family are important regulators of apoptosis, contributing to prolonged cell survival through their ability to block apoptosis (Kelly and Strasser, 2011). Down-regulation of Bcl-2 expression induces sensitivity to anti-cancer drugs (Oltersdorf et al., 2005; Li et al., 2009) and increases survival (Letai et al., 2004). In this work, we found that inhibition of mGlu1 receptor by BAY36-7620 treatment reduced bcl-2 protein expression; AKT signaling mediated the effect of mGlu1 receptor on bcl-2 in A549 cells, which was similar the report by Shin et al. (2010). In addition, we found that treatment of BAY36-7620 led to a rise in levels of another apoptotic marker, the cleaved form of PARP; however, it was not dependent on AKT signaling. Pathological angiogenesis is a relatively early event in carcinogenesis, and increased tumor angiogenesis is correlated with invasive tumor growth and metastasis and a poor prognosis (Vamesu, 2008; Vermeulen et al., 2010). Our results revealed that inhibition of mGlu1 receptor by BAY36-7620 reduced angiogenesis in A549 tumors. HIF-1α/VEGF pathway plays a critical role in tumor angiogenesis. VEGF, which is the most extensively characterized endothelial cell-specific angiogenic factor, leads to increased vascular permeability and plays a significant role in physiological and pathological angiogenesis (Tammela et al., 2005; Gabhann and Popel, 2008). It was reported that the expression of HIF-1α and VEGF was up-regulated in NSCLC and was related to a poor prognosis and worse overall survival (Rovina et al., 2011; Park et al., 2011). It was reported that mGlu1 receptor signal transduction promoted angiogenesis in melanoma through activation of the AKT/mTOR/HIF-1α signaling pathway (Wen et al., 2014). In A549 cells, inhibition of mGlu1 receptor reduced protein expression of HIF-1α and secretion of VEGF via inactivation of AKT. It was shown that glutamate caused a loss in human cerebral endothelial barrier integrity through activation of NMDA receptor (Sharp et al., 2003). Recently, it was found that Group I metabotropic glutamate receptors (including mGlu1 receptor and mGlu5 receptor) was also expressed in vascular endothelial cells and they could prevent endothelial programmed cell death independent from MAP kinase p38 activation (Lin et al., 2001). Speyer et al. (2014) detected expression of mGlu1 receptor by human dermal microvascular endothelial cells, HUVECs, and human pulmonary microvascular endothelial cells, and first demonstrated a novel role for mGlu1 receptor in mediating various steps of the angiogenic process including EC proliferation, Matrigel tube formation and vessel formation in vitro. AKT signaling is crucial for the signaling of angiogenic growth factors
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that promote endothelial cell migration, proliferation, microtubule formation, and survival (Shiojima and Walsh, 2002). In this work, we found that inhibition of mGlu1 receptor blocked VEGF-induced activation of AKT in HUVECs and reduced capillary tube formation. Therefore, inhibition of mGlu1 receptor suppressed tumor angiogenesis via suppressing AKT/HIF-1α/VEGF pathway in tumor cells and blocking VEGF/AKT pathway in endothelial cells. In addition, it was reported that non-small cell lung carcinoma cells synthesized and released norepinephrine and epinephrine (Al-Wadei et al., 2012), and exogenous norepinephrine treatment upregulated the expression of VEGF and IL-8 and stimulated tumor growth in vivo (Deng et al., 2014). The specific agonist of Group I mGluRs, DHPG, was able to stimulate the release of norepinephrine and epinephrine in several kinds of cells (Arce et al., 2004; Luccini et al., 2007). Therefore, norepinephrine and epinephrine might be involved in the function of mGlu1 receptor in NSCLC, which required further investigation. In conclusion, inhibition of metabotropic glutamate receptor 1 suppressed tumor growth and angiogenesis in experimental NSCLC, partly via inactivation of AKT signaling.
Conflicts of interest disclosure The authors declare that we have no conflict of interest.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ejphar.2016.04.053.
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