- Email: [email protected]
The effect of indatraline on angiogenesis suppression through HIF-1α-mediated VEGF inhibition
Biochemical and Biophysical Research Communications xxx (2017) 1e6
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
The effect of indatraline on angiogenesis suppression through HIF-1amediated VEGF inhibition Chih-na Yen a, Yoon Sun Cho a, Ho Jeong Kwon a, b, * a
Chemical Genomics Global Research Laboratory, Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science & Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea b Department of Internal Medicine, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 February 2017 Accepted 14 February 2017 Available online xxx
The present research reports a novel biological activity of indatraline, a compound clinically used as an antidepressant. We previously identified indatraline as an autophagy inducer. Autophagy is an intracellular catabolic pathway for degrading or recycling unnecessary organelles in response to cellular stress. Indatraline-mediated autophagy induction results from mTOR inhibition. The mTOR is a negative regulator of autophagy as well as a master regulator of angiogenesis. Angiogenesis defines the process by which new vessels are formed from pre-existing vascular tissues, providing nutrients to cancer cells, allowing rapid tumor progression. Accordingly, targeting angiogenesis to prevent cancer is an attractive therapeutic strategy. Here, we demonstrate that indatraline possibly acts to suppress tumor-mediated angiogenesis via downregulation of HIF-1a-mediated VEGF expression. The effects of indatraline on autophagy and angiogenesis could make it a potential drug candidate toward cancer treatment. © 2017 Elsevier Inc. All rights reserved.
Keywords: Autophagy Angiogenesis mTOR HIF-1a Anticancer
1. Introduction Autophagy is a key cellular catabolic mechanism that removes cellular components, including misfolded, aggregated, and aged proteins. Additionally, malfunctioning organelles such as mitochondria, endoplasmic reticulum, peroxisomes, and even pathogens are processed by autophagy [1]. There are accumulating reports that autophagy has been implicated in numerous diseases such as neurodegenerative diseases, vascular diseases, and more particularly, cancer [2,3]. Moreover, autophagy is known to play dual functions in cancer (pro-tumorigenic effect and antitumorigenic effect). The specific underlying mechanism and its role in cancer progression are largely unclear [4,5].
Abbreviations: AMPK, AMP-activated protein kinase; CAM, chick chorioallantoic membrane; FBS, fetal bovine serum; HeLa, human cervical adenocarcinoma; HIF1a, hypoxia-inducible factor 1-alpha; HUVECs, human umbilical vein endothelial cells; JHDL, Johns Hopkins drug library; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; RA, retinoic acid; VEGF, vascular endothelial growth factor. * Corresponding author. Chemical Genomics Global Research Laboratory, Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science & Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea. E-mail address: [email protected] (H.J. Kwon).
Recently, we adopted forward chemical genetics in order to discover a small molecule that induces autophagy. To discover this autophagy-inducing small molecule, we performed in vitro phenotypic screening using the Johns Hopkins drug library (JHDL). As a result of the screening process, indatraline was identified as a novel autophagy inducer. Indatraline-mediated autophagy was due to the activation of AMPK, followed by mTOR inhibition [6]. The mTOR, a serine/threonine protein kinase, is responsible for autophagy. In addition, it is known as a key regulator of angiogenesis [7,8]. Angiogenesis is a process by which new blood vessels are generated from pre-existing ones. The development of new blood networks is crucial in cancer. As cancer cells progress and mature as a tumor, they require excessive angiogenesis to facilitate their rapid expansion, metastasis, and invasion. Therefore, targeting cancerassociated angiogenesis is a promising strategy toward preventing cancer progression, or treating cancer [9e11]. Since indatraline triggers robust autophagy through mTOR inhibition, we sought to examine if indatraline also negatively regulates angiogenesis. As a result, our study suggests that indatraline can impede angiogenesis by suppressing hypoxia-inducible factor 1-alpha (HIF-1a)-induced vascular endothelial growth factor (VEGF) expression.
http://dx.doi.org/10.1016/j.bbrc.2017.02.077 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: C.-n. Yen, et al., The effect of indatraline on angiogenesis suppression through HIF-1a-mediated VEGF inhibition, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.02.077
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2. Material and methods
2.5. VEGF measurement by ELISA
2.1. Cell culture and hypoxic conditions
HeLa cells were incubated overnight under hypoxic conditions to induce VEGF expression. The medium was collected to determine VEGF concentration using a VEGF Immunoassay kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
Human cervical adenocarcinoma (HeLa) cells were grown and passaged in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco). Human umbilical vein endothelial cells (HUVECs) were harvested in EGM-2 (Lonza) containing 10% FBS (Gibco). Cells were subcultured with indicated medium every 2e3 days. Both cell lines were maintained at 37 C in a humidified 5% CO2 atmosphere at pH 7.4. For hypoxic conditions, cells were incubated at a CO2 level of 5% with 1% O2 balanced with N2 in an anaerobic chamber (Forma, Marietta, OH, USA). 2.2. In vitro capillary tube formation assay Human umbilical vein endothelial cells (HUVECs) were serumstarved overnight at 37 C before their use. Matrigel (10 mg/mL) was coated on a 48-well plate, which was subsequently incubated for 1 h at 37 C. After incubation, the serum-starved HUVECs were seeded into the Matrigel-coated 48-well plate (6 104 cells per well). VEGF (30 ng/mL) was used to stimulate tube-like structures. Following this step, various concentrations of indatraline were added in the presence of VEGF (30 ng/mL) for at least 4e8 h at 37 C. Compound-induced morphological changes in tubular structures were observed using a microscope (IX71, Olympus) and photographed at 100 with a DP70 camera (Olympus). 2.3. In vitro chemoinvasion assay HUVECs were serum-starved overnight at 37 C before use. The invasiveness of HUVECs was examined in vitro using a Transwell chamber system with polycarbonate filter inserts with 8.0-mm pores. Briefly, the lower side of the filter was coated with gelatin (1 mg/mL), and the upper side was coated with Matrigel (3 mg/mL). Serum-starved HUVECs were seeded at a density of 7 104 cells per well. Cells were placed in the upper part of the filter drop wise. Indatraline was added to the lower part in the presence of VEGF (30 ng/mL) stimulation. Following this step, the chamber was incubated at 37 C for 18 h. Cells were fixed with 70% methanol. Then, cells were stained with hematoxylin and eosin and dehydrated/fixed with 90% ethanol. After drying at 25 C for at least 2 h, invasiveness of cells was examined by visualizing the lower side of the filter using a microscope at 100. Cells were photographed using a DP70 camera. 2.4. In vivo CAM assay An in vivo chick chorioallantoic membrane (CAM) assay was performed as described previously [12]. Briefly, fertilized chick eggs were incubated in a 37 C humidified chamber for 3 days. Approximately 4 mL egg albumin was eliminated using a hypodermic needle, permitting the CAM and yolk sac to drop away from the shell membrane. On the following fourth day, a hole with a diameter of 3 cm was created with a razor and tweezers to peal the shell membrane away. On the fifth day, indatraline and retinoic acid-loaded Thermanox coverslips (NUNC, Rochester, NY) were placed on the CAM surface. Two days after placing the testing compounds-coated coverslips on the CAM surface, approximately 3e4 mL intralipose (Green Cross Co., Yongin, Korea) was injected beneath the CAM to visualize the vessels. One microgram of retinoic acid (RA) was used as a positive control. The images were photographed using a DP70 camera.
2.6. Western blotting and reagents Soluble proteins were harvested from indatraline-treated HUVECs using SDS lysis buffer (50 mM TrisHCl, pH 6.8, containing 10% glycerol, 2% SDS, 10 mM dithiothreitol, and 0.005% bromophenol blue). Lysed protein samples were loaded. Samples were separated on 8e12.5% SDS-polyacrylamide gels and transferred to polyvinylidenefluoride membranes (Millipore). The membranes were blocked using 1e3% skim milk. Then, the membranes were incubated overnight at 4 C with primary antibodies against LC3 (MBL), p-mTOR, mTOR, p-S6K, S6K, p-AMPK, AMPK, HIF-1a (Cell Signaling), tubulin (Abcam). After overnight incubation, immunolabeled membranes were washed with TBS-T for 5 min three times and then washed with TBS once for 5 min before detection. The protein levels within each sample were visualized using an Enhanced Chemiluminescence Kit (Amersham Life Science, Inc.) according to the manufacturer's instructions. Tubulin was used as the loading control. Rapamycin was purchased from Sigma. 2.7. MTT assay and viability assay Cell proliferation was measured by a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT, Amresco) colorimetric assay. HUVECs were seeded at a density of 3 103 cells per well in 96-well plates and incubated for 24 h. The cells were treated with various doses of indatraline, namely 0, 0.5, 1, 2, 5, and 10 mM. A 2 mg/mL MTT solution was added to each well and incubated for 3 h. MTT formazan in each well was dissolved in 150 mL DMSO and the absorbance was measured at 595 nm with a microplate reader (Bio-Tek Instrument Inc.). This process was repeated for three days. Relative cell growth was measured by calculating the ratio between the signals obtained from indatraline-treated and control wells. Cell viability was completed with trypan blue staining. HUVECs were seeded at a concentration of 1 104 cells per well in 24-well plates. Seeded cells were treated with indatraline and cell viability was analyzed at each time course. The cells were harvested and stained with trypan blue (Gibco). The number of live and dead cells was counted and the ratio between these was calculated. 2.8. Statistical analysis Results are expressed as mean ± standard error (±S.E.M.) and all statistical analyses were performed with GraphPad Prism (ver. 5.00 for Windows, GraphPad Software, San Diego, CA, www.graphpad. com). Student's t-tests were used to determine statistical significance between control and test groups. A p-value lower than 0.05 was considered statistically significant (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001). 3. Results 3.1. Effect of indatraline on autophagy of human umbilical vein endothelial cells (HUVECs) The anti-depressive agent, indatraline, was previously reported as a novel autophagy inducer through phenotypic screening. In our previous study, we identified AMPK activation and mTOR/S6K inhibition as mechanisms responsible for indatraline-mediated
Please cite this article in press as: C.-n. Yen, et al., The effect of indatraline on angiogenesis suppression through HIF-1a-mediated VEGF inhibition, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.02.077
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autophagy [6]. To verify this, we re-tested the effect of indatraline on LC3 conversion of human umbilical vein endothelial cells (HUVECs) which is the most widely used marker to monitor autophagy. The conversion of LC3-I to LC3-II occurred in a dosedependent manner upon indatraline treatment (Fig. 1A and B). In addition, indatraline decreased the phosphorylation level of mTOR/ S6K, consistent with previous observations (Fig. 1C). According to many reports, numerous autophagy inducers inhibiting mTOR also exhibit a negative effect on angiogenesis. One of the classical examples is rapamycin. Rapamycin is a direct lipid kinase inhibitor of mTOR, which induces autophagy and inhibits angiogenesis [13e15]. This led us to examine the effect of indatraline on angiogenesis in more detail. 3.2. Effect of indatraline on angiogenesis in vitro and in vivo Before examining the effect of indatraline on angiogenesis, we investigated the effect of indatraline on cell proliferation and viability of HUVECs. HUVECs were treated with various doses of indatraline for 72 h and their cell growth was measured by an MTT colorimetric assay. Indatraline inhibited the cell growth with an IC50 value of 9e10 mM (Fig. 1D). After treating the cells with the indicated doses for 3 days, viable cells were counted using the trypan blue assay. Indatraline did not show any cytotoxicity on HUVECs at doses up to 10 mM for 3 days, except for cells treated with 10 mM indatraline at 72 h (Fig. 1E). Accordingly, a concentration of indatraline <5 mM was used in the subsequent studies. Next, we observed the effect of indatraline on angiogenesis through in vitro assays because indatraline suppresses the phosphorylation of mTOR, as previously mentioned. To determine its effect on angiogenesis, VEGF (30 ng/mL), an angiogenesis promoting growth factor, was used to stimulate capillary-like
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structures and invasiveness of HUVECs. Indatraline inhibited VEGFinduced invasion of HUVECs in a dose-dependent manner as well as tube formation (Fig. 2A and B). VEGF-stimulated tube-like structures of HUVECs were suppressed upon indatraline treatment. In both assays, a low dose of indatraline (0.5 mM) was sufficient to suppress invasiveness and the ability to form the capillary structures in HUVECs. These results demonstrated that indatraline suppresses angiogenic activity along with an autophagy-inducing ability of HUVECs. Indatraline effect on angiogenesis was further investigated with in vivo CAM assay. Robust neovascularization was shown in nontreated CAM, whereas apparent suppression of angiogenesis was observed in indatraline (1 mg)-treated CAM. Indatraline effect on angiogenesis activity was similar to that of the positive control (RAtreated CAM) without causing any hemolysis. Consistent with the in vitro results, indatraline exerted suppressive influences on the growth of new blood vessels in vivo CAM assay (Fig. 3). 3.3. Indatraline suppresses tumor cell-induced angiogenesis HIF-1a activates the transcription of genes that play key roles in tumorigenesis, including angiogenesis and invasion [16]. HIF-1a is sensitive to cellular oxygen levels and is stabilized in hypoxic conditions to activate its target genes, particularly VEGF. Therefore, overexpression of HIF-1a is common in many types of cancers, suggesting that HIF-1a could represent as a key target for cancer therapy [17e19]. Here, we performed western blotting to assess the effect of indatraline on HIF-1a stability. HIF-1a was stimulated after 4 h of incubation in hypoxic conditions. Indatraline dosedependently decreased the stabilized HIF-1a protein levels. A dose as low as 1 mM was sufficient to reduce the HIF-1a level, and 5 mM of indatraline reduced the HIF-1a protein expression down to
Fig. 1. Effect of indatraline on autophagy and proliferation of human umbilical vein endothelial cells (HUVECs). (A) Structure of indatraline. (Inda: indatraline) (B) Indatraline induces the conversion of LC3-1 to LC3-II, detected by western blotting. (C) Effect of indatraline on the phosphorylation of mTOR and S6K was assayed by western blotting. (D) Effect of indatraline on proliferation of HUVECs was examined by the MTT assay following 72-h indatraline treatment at the indicated concentrations. (E) Viable cells were measured by the trypan blue assay.
Please cite this article in press as: C.-n. Yen, et al., The effect of indatraline on angiogenesis suppression through HIF-1a-mediated VEGF inhibition, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.02.077
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Fig. 2.. Effects of indatraline on angiogenesis. (A) Effect of indatraline on HUVECs invasiveness, determined by an in vitro chemoinvasion assay. (B) Indatraline inhibits VEGF-induced tube formation. Arrows indicates broken tube-like structures. In (A) and (B), 30 ng/mL of VEGF was used to induce HUVEC invasion and tube formation. Quantified bar graphs are presented as mean (±SEM) compared to VEGF-treated control (** indicates p < 0.001 and *** indicates p < 0.0001).
Fig. 3. Validation of anti-angiogenic activity of indatraline in in vivo assay. Inhibitory effect of indatraline on angiogenesis was retested by the chick chorioallantoic membrane (CAM) assay. Calculations were completed based on the proportion of positive eggs relative to the total number of eggs tested. Arrow represents inhibition of neovascularization. Retinoic acid (RA) was used as a positive control.
nearly 50% of that observed in the non-treated control (Fig. 4A). Next, we investigated the effect of indatraline on the hypoxiainduced VEGF level using a VEGF ELISA. The VEGF level was increased by approximately 40% in hypoxic conditions compared to that observed in the normoxic control. Indatraline reduced the hypoxia-stimulated VEGF level in a dose-dependent manner
(Fig. 4B). Due to the inhibitory effect of indatraline on hypoxiainduced HIF-1a and VEGF levels, we examined whether indatraline suppresses tumor-mediated invasion of HUVECs. HeLa cells were incubated under hypoxic conditions and their conditioned medium was used to stimulate the invasion of HUVECs. Indatraline inhibited HUVECs invasion, which was stimulated by the
Please cite this article in press as: C.-n. Yen, et al., The effect of indatraline on angiogenesis suppression through HIF-1a-mediated VEGF inhibition, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.02.077
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Fig. 4.. Mechanism of indatraline on tumor angiogenesis. (A) Destabilization of HIF-1a protein upon indatraline treatment was analyzed via immunoblotting. (B) Indatraline decreased the hypoxia-induced VEGF level, detected by VEGF ELISA. In (A) and (B), HIF-1a and VEGF expressions of HeLa cells were stimulated under hypoxic conditions. After hypoxic induction, cells were treated with the indicated doses of indatraline. Quantified bar graphs are presented as mean (±SEM) compared to the VEGF-treated control (** indicates p < 0.001 and *** indicates p < 0.0001). (C) Effect of indatraline on tumor-induced angiogenesis. Conditioned medium (CM) was derived from HeLa cells. (D) Schematic representation of indatraline-induced biological activity. Indatraline triggers autophagy through AMPK activation, followed by mTOR and S6K inhibition. Indatraline also exerts negative effects on angiogenesis through the reduction in HIF-1a and VEGF levels.
conditioned medium of cancer cells (Fig. 4C). These results indicate that indatraline affects hypoxia-mediated tumor angiogenesis by suppressing HIF-1a-mediated VEGF expression. 4. Discussion We have previously reported that indatraline, originally known as an antidepressant, (monoamine neurotransmitter blocker), induces autophagy [6]. Enhanced autophagy by indatraline was due to the negative regulation of mTOR as a result of AMPK activation. The mTOR has been identified as a negative regulator of autophagy; however, it has also been recognized as ‘angiogenic switch’ over the last two decades [20]. Therefore, our previous observations prompted us to determine the effect of indatraline on angiogenesis due to its suppressive effect on the mTOR signaling pathway. A recent study has identified the anti-angiogenic activity of indatraline via the inhibition of Rho- and calcium-mediated glioblastoma cell motility [21]. In reference to this study, we further determined whether indatraline could affect angiogenesis by exploring in vitro and in vivo angiogenesis using HUVECs. Indatraline blocked the tube-forming ability and invasiveness of HUVECs. Similarly, indatraline suppressed neovascularization in the in vivo CAM assay. The effect of indatraline on angiogenesis was due to the suppression of HIF-1a-associated VEGF secretion. Additionally, indatraline suppressed the invasiveness of HUVECs when stimulated with the conditioned medium of cells cultured in hypoxic
conditions. These results suggest that indatraline can negatively regulates angiogenesis in cancer by controlling cell growth. The relationship between indatraline-induced autophagy and its effect on angiogenesis is still not clear. However, further investigations on monoamine neurotransmitter inhibition, autophagy induction, and angiogenesis suppression by indatraline might possibly reveal the mechanisms underlying the relationship between indatralineassociated autophagy and angiogenesis suppression. Autophagy is progressively recognized as an alternative approach for apoptosis-resistant cancer treatment. Likewise, antiangiogenesis drugs are regarded as promising anti-cancer agents because blocking angiogenesis prevents not only invasion of tumors to nearby regions, but also limits the enlargement of tumor size. Therefore, indatraline-associated autophagy induction and anti-angiogenic activity may act synergistically in treating cancer. Identification of additional protein targets of indatraline will be helpful to gain a clear understanding of its poly-biological activities on autophagy and angiogenesis. In summary, we demonstrated for the first time that autophagy inducer indatraline controls angiogenesis via HIF-1a pathway. This finding suggests the potential of indatraline as an anti-cancer drug candidate. Together with our previous observations, the present findings on the poly-biological activities of indatraline may allow expansion of its application from anti-depressive agent into cancer therapeutics (Fig. 4D).
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Acknowledgements This work was partly supported by grants from the National Research Foundation of Korea, funded by the Korean government (MSIP; 2015K1A1A2028365, 2015M3A9B6027818, 2015M3A9C4676321, 2012M3A9D1054520) and Brain Korea 21 Plus Project, Republic of Korea. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.02.077. References [1] M. Tsukada, Y. Ohsumi, Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae, FEBS Lett. 333 (1993) 169e174. [2] M. Martinez-Vicente, Z. Talloczy, E. Wong, G. Tang, H. Koga, S. Kaushik, R. de Vries, E. Arias, S. Harris, D. Sulzer, A.M. Cuervo, Cargo recognition failure is responsible for inefficient autophagy in Huntington's disease, Nat. Neurosci. 13 (2010) 567e576. [3] S. Verheye, W. Martinet, M.M. Kockx, M.W. Knaapen, K. Salu, J.P. Timmermans, J.T. Ellis, D.L. Kilpatrick, G.R. De Meyer, Selective clearance of macrophages in atherosclerotic plaques by autophagy, J. Am. Coll. Cardiol. 49 (2007) 706e715. [4] Y. Kondo, T. Kanzawa, R. Sawaya, S. Kondo, The role of autophagy in cancer development and response to therapy, Nat. Rev. Cancer 5 (2005) 726e734. [5] P. Boya, F. Reggiori, P. Codogno, Emerging regulation and functions of autophagy, Nat. Cell Biol. 15 (2013) 713e720. [6] Y.S. Cho, C.N. Yen, J.S. Shim, D.H. Kang, S.W. Kang, J.O. Liu, H.J. Kwon, Antidepressant indatraline induces autophagy and inhibits restenosis via suppression of mTOR/S6 kinase signaling pathway, Sci. Rep. 3 (6) (2016) 34655. [7] K. Tsuji-Tamura, M. Ogawa, Inhibition of the PI3K/Akt and mTORC1 signaling pathways promotes the elongation of vascular endothelial cells, J. Cell Sci. 129
(6) (2016) 1165e1178. [8] C.H. Jung, S.H. Ro, J. Cao, N.M. Otto, D.H. Kim, mTOR regulation of autophagy, FEBS Lett. 584 (2010) 1287e1295. [9] J. Folkman, Clinical applications of research on angiogenesis, N. Engl. J. Med. 235 (1995) 1757e1763. [10] P. Carmeliet, R.K. Jain, Angiogenesis in cancer and other diseases, Nature 407 (2000) 249e257. [11] R. Kalluri, Basement membranes: structure, assembly and role in tumour angiogenesis, Nat. Rev. Cancer 3 (2003) 422e433. [12] P.O. Seglen, P.B. Gordon, 3-Methyladenine: specific inhibitor of autophagic/ lysosomal protein degradation in isolated rat hepatocytes, Proc. Natl. Acad. Sci. U. S. A. 79 (1982) 1889e1892. [13] R. Salido-Vallejo, G. Garnacho-Saucedo, A. Velez, Elucidation of the mTOR pathway and therapeutic applications in dermatology, Actas Dermosifiliogr. 107 (5) (2016) 379e390. [14] S. Ramakrishnan, T.M. Nguyen, I.V. Subramanian, A. Kelekar, Autophagy and angiogenesis inhibition, Autophagy 3 (2007) 512e515. [15] J. Du, R.J. Teng, T. Guan, A. Eis, S. Kaul, G.G. Konduri, Y. Shi, Role of autophagy in angiogenesis in aortic endothelial cells, Am. J. Physiol. Cell Physiol. 302 (2012) C383eC391. [16] J. Pouyssegur, F. Dayan, N.M. Mazure, Hypoxia signalling in cancer and approaches to enforce tumour regression, Nature 441 (2006) 437e443. [17] G.L. Semenza, Targeting HIF-1 for cancer therapy, Nat. Rev. Cancer 3 (2003) 721e732. [18] H. Zhang, G. Ji, P. Hauptmann, Y. Ingham, Cao, Hypoxia-induced pathological angiogenesis mediates tumor cell dissemination, invasion, and metastasis in a zebrafish tumor model, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 19485e19490. [19] J.E. Shay, H.Z. Imtiyaz, S. Sivanand, A.C. Durham, N. Skuli, S. Hsu, V. Mucaj, T.S. Eisinger-Mathason, B.L. Krock, D.N. Giannoukos, M.C. Simon, Inhibition of hypoxia-inducible factors limits tumor progression in a mouse model of colorectal cancer, Carcinogenesis 35 (2014) 1067e1077. [20] H. Mead, M. Zeremski, M. Guba, mTOR signaling in angiogenesis, in: A.V. Polunovsky, J.P. Houghton (Eds.), mTOR Pathway and mTOR Inhibitors in Cancer Therapy, Humana Press, Totowa, NJ, 2010, pp. 49e74. [21] J.C. Heo, T.H. Jung, D.Y. Jung, W.K. Park, H. Cho, Indatraline inhibits Rho- and calcium-mediated glioblastoma cell motility and angiogenesis, Biochem. Biophys. Res. Commun. 443 (2014) 749e755.
Please cite this article in press as: C.-n. Yen, et al., The effect of indatraline on angiogenesis suppression through HIF-1a-mediated VEGF inhibition, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.02.077
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
The effect of indatraline on angiogenesis suppression through HIF-1amediated VEGF inhibition Chih-na Yen a, Yoon Sun Cho a, Ho Jeong Kwon a, b, * a
Chemical Genomics Global Research Laboratory, Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science & Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea b Department of Internal Medicine, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 February 2017 Accepted 14 February 2017 Available online xxx
The present research reports a novel biological activity of indatraline, a compound clinically used as an antidepressant. We previously identified indatraline as an autophagy inducer. Autophagy is an intracellular catabolic pathway for degrading or recycling unnecessary organelles in response to cellular stress. Indatraline-mediated autophagy induction results from mTOR inhibition. The mTOR is a negative regulator of autophagy as well as a master regulator of angiogenesis. Angiogenesis defines the process by which new vessels are formed from pre-existing vascular tissues, providing nutrients to cancer cells, allowing rapid tumor progression. Accordingly, targeting angiogenesis to prevent cancer is an attractive therapeutic strategy. Here, we demonstrate that indatraline possibly acts to suppress tumor-mediated angiogenesis via downregulation of HIF-1a-mediated VEGF expression. The effects of indatraline on autophagy and angiogenesis could make it a potential drug candidate toward cancer treatment. © 2017 Elsevier Inc. All rights reserved.
Keywords: Autophagy Angiogenesis mTOR HIF-1a Anticancer
1. Introduction Autophagy is a key cellular catabolic mechanism that removes cellular components, including misfolded, aggregated, and aged proteins. Additionally, malfunctioning organelles such as mitochondria, endoplasmic reticulum, peroxisomes, and even pathogens are processed by autophagy [1]. There are accumulating reports that autophagy has been implicated in numerous diseases such as neurodegenerative diseases, vascular diseases, and more particularly, cancer [2,3]. Moreover, autophagy is known to play dual functions in cancer (pro-tumorigenic effect and antitumorigenic effect). The specific underlying mechanism and its role in cancer progression are largely unclear [4,5].
Abbreviations: AMPK, AMP-activated protein kinase; CAM, chick chorioallantoic membrane; FBS, fetal bovine serum; HeLa, human cervical adenocarcinoma; HIF1a, hypoxia-inducible factor 1-alpha; HUVECs, human umbilical vein endothelial cells; JHDL, Johns Hopkins drug library; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; RA, retinoic acid; VEGF, vascular endothelial growth factor. * Corresponding author. Chemical Genomics Global Research Laboratory, Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science & Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea. E-mail address: [email protected] (H.J. Kwon).
Recently, we adopted forward chemical genetics in order to discover a small molecule that induces autophagy. To discover this autophagy-inducing small molecule, we performed in vitro phenotypic screening using the Johns Hopkins drug library (JHDL). As a result of the screening process, indatraline was identified as a novel autophagy inducer. Indatraline-mediated autophagy was due to the activation of AMPK, followed by mTOR inhibition [6]. The mTOR, a serine/threonine protein kinase, is responsible for autophagy. In addition, it is known as a key regulator of angiogenesis [7,8]. Angiogenesis is a process by which new blood vessels are generated from pre-existing ones. The development of new blood networks is crucial in cancer. As cancer cells progress and mature as a tumor, they require excessive angiogenesis to facilitate their rapid expansion, metastasis, and invasion. Therefore, targeting cancerassociated angiogenesis is a promising strategy toward preventing cancer progression, or treating cancer [9e11]. Since indatraline triggers robust autophagy through mTOR inhibition, we sought to examine if indatraline also negatively regulates angiogenesis. As a result, our study suggests that indatraline can impede angiogenesis by suppressing hypoxia-inducible factor 1-alpha (HIF-1a)-induced vascular endothelial growth factor (VEGF) expression.
http://dx.doi.org/10.1016/j.bbrc.2017.02.077 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: C.-n. Yen, et al., The effect of indatraline on angiogenesis suppression through HIF-1a-mediated VEGF inhibition, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.02.077
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C.-n. Yen et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e6
2. Material and methods
2.5. VEGF measurement by ELISA
2.1. Cell culture and hypoxic conditions
HeLa cells were incubated overnight under hypoxic conditions to induce VEGF expression. The medium was collected to determine VEGF concentration using a VEGF Immunoassay kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
Human cervical adenocarcinoma (HeLa) cells were grown and passaged in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco). Human umbilical vein endothelial cells (HUVECs) were harvested in EGM-2 (Lonza) containing 10% FBS (Gibco). Cells were subcultured with indicated medium every 2e3 days. Both cell lines were maintained at 37 C in a humidified 5% CO2 atmosphere at pH 7.4. For hypoxic conditions, cells were incubated at a CO2 level of 5% with 1% O2 balanced with N2 in an anaerobic chamber (Forma, Marietta, OH, USA). 2.2. In vitro capillary tube formation assay Human umbilical vein endothelial cells (HUVECs) were serumstarved overnight at 37 C before their use. Matrigel (10 mg/mL) was coated on a 48-well plate, which was subsequently incubated for 1 h at 37 C. After incubation, the serum-starved HUVECs were seeded into the Matrigel-coated 48-well plate (6 104 cells per well). VEGF (30 ng/mL) was used to stimulate tube-like structures. Following this step, various concentrations of indatraline were added in the presence of VEGF (30 ng/mL) for at least 4e8 h at 37 C. Compound-induced morphological changes in tubular structures were observed using a microscope (IX71, Olympus) and photographed at 100 with a DP70 camera (Olympus). 2.3. In vitro chemoinvasion assay HUVECs were serum-starved overnight at 37 C before use. The invasiveness of HUVECs was examined in vitro using a Transwell chamber system with polycarbonate filter inserts with 8.0-mm pores. Briefly, the lower side of the filter was coated with gelatin (1 mg/mL), and the upper side was coated with Matrigel (3 mg/mL). Serum-starved HUVECs were seeded at a density of 7 104 cells per well. Cells were placed in the upper part of the filter drop wise. Indatraline was added to the lower part in the presence of VEGF (30 ng/mL) stimulation. Following this step, the chamber was incubated at 37 C for 18 h. Cells were fixed with 70% methanol. Then, cells were stained with hematoxylin and eosin and dehydrated/fixed with 90% ethanol. After drying at 25 C for at least 2 h, invasiveness of cells was examined by visualizing the lower side of the filter using a microscope at 100. Cells were photographed using a DP70 camera. 2.4. In vivo CAM assay An in vivo chick chorioallantoic membrane (CAM) assay was performed as described previously [12]. Briefly, fertilized chick eggs were incubated in a 37 C humidified chamber for 3 days. Approximately 4 mL egg albumin was eliminated using a hypodermic needle, permitting the CAM and yolk sac to drop away from the shell membrane. On the following fourth day, a hole with a diameter of 3 cm was created with a razor and tweezers to peal the shell membrane away. On the fifth day, indatraline and retinoic acid-loaded Thermanox coverslips (NUNC, Rochester, NY) were placed on the CAM surface. Two days after placing the testing compounds-coated coverslips on the CAM surface, approximately 3e4 mL intralipose (Green Cross Co., Yongin, Korea) was injected beneath the CAM to visualize the vessels. One microgram of retinoic acid (RA) was used as a positive control. The images were photographed using a DP70 camera.
2.6. Western blotting and reagents Soluble proteins were harvested from indatraline-treated HUVECs using SDS lysis buffer (50 mM TrisHCl, pH 6.8, containing 10% glycerol, 2% SDS, 10 mM dithiothreitol, and 0.005% bromophenol blue). Lysed protein samples were loaded. Samples were separated on 8e12.5% SDS-polyacrylamide gels and transferred to polyvinylidenefluoride membranes (Millipore). The membranes were blocked using 1e3% skim milk. Then, the membranes were incubated overnight at 4 C with primary antibodies against LC3 (MBL), p-mTOR, mTOR, p-S6K, S6K, p-AMPK, AMPK, HIF-1a (Cell Signaling), tubulin (Abcam). After overnight incubation, immunolabeled membranes were washed with TBS-T for 5 min three times and then washed with TBS once for 5 min before detection. The protein levels within each sample were visualized using an Enhanced Chemiluminescence Kit (Amersham Life Science, Inc.) according to the manufacturer's instructions. Tubulin was used as the loading control. Rapamycin was purchased from Sigma. 2.7. MTT assay and viability assay Cell proliferation was measured by a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT, Amresco) colorimetric assay. HUVECs were seeded at a density of 3 103 cells per well in 96-well plates and incubated for 24 h. The cells were treated with various doses of indatraline, namely 0, 0.5, 1, 2, 5, and 10 mM. A 2 mg/mL MTT solution was added to each well and incubated for 3 h. MTT formazan in each well was dissolved in 150 mL DMSO and the absorbance was measured at 595 nm with a microplate reader (Bio-Tek Instrument Inc.). This process was repeated for three days. Relative cell growth was measured by calculating the ratio between the signals obtained from indatraline-treated and control wells. Cell viability was completed with trypan blue staining. HUVECs were seeded at a concentration of 1 104 cells per well in 24-well plates. Seeded cells were treated with indatraline and cell viability was analyzed at each time course. The cells were harvested and stained with trypan blue (Gibco). The number of live and dead cells was counted and the ratio between these was calculated. 2.8. Statistical analysis Results are expressed as mean ± standard error (±S.E.M.) and all statistical analyses were performed with GraphPad Prism (ver. 5.00 for Windows, GraphPad Software, San Diego, CA, www.graphpad. com). Student's t-tests were used to determine statistical significance between control and test groups. A p-value lower than 0.05 was considered statistically significant (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001). 3. Results 3.1. Effect of indatraline on autophagy of human umbilical vein endothelial cells (HUVECs) The anti-depressive agent, indatraline, was previously reported as a novel autophagy inducer through phenotypic screening. In our previous study, we identified AMPK activation and mTOR/S6K inhibition as mechanisms responsible for indatraline-mediated
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autophagy [6]. To verify this, we re-tested the effect of indatraline on LC3 conversion of human umbilical vein endothelial cells (HUVECs) which is the most widely used marker to monitor autophagy. The conversion of LC3-I to LC3-II occurred in a dosedependent manner upon indatraline treatment (Fig. 1A and B). In addition, indatraline decreased the phosphorylation level of mTOR/ S6K, consistent with previous observations (Fig. 1C). According to many reports, numerous autophagy inducers inhibiting mTOR also exhibit a negative effect on angiogenesis. One of the classical examples is rapamycin. Rapamycin is a direct lipid kinase inhibitor of mTOR, which induces autophagy and inhibits angiogenesis [13e15]. This led us to examine the effect of indatraline on angiogenesis in more detail. 3.2. Effect of indatraline on angiogenesis in vitro and in vivo Before examining the effect of indatraline on angiogenesis, we investigated the effect of indatraline on cell proliferation and viability of HUVECs. HUVECs were treated with various doses of indatraline for 72 h and their cell growth was measured by an MTT colorimetric assay. Indatraline inhibited the cell growth with an IC50 value of 9e10 mM (Fig. 1D). After treating the cells with the indicated doses for 3 days, viable cells were counted using the trypan blue assay. Indatraline did not show any cytotoxicity on HUVECs at doses up to 10 mM for 3 days, except for cells treated with 10 mM indatraline at 72 h (Fig. 1E). Accordingly, a concentration of indatraline <5 mM was used in the subsequent studies. Next, we observed the effect of indatraline on angiogenesis through in vitro assays because indatraline suppresses the phosphorylation of mTOR, as previously mentioned. To determine its effect on angiogenesis, VEGF (30 ng/mL), an angiogenesis promoting growth factor, was used to stimulate capillary-like
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structures and invasiveness of HUVECs. Indatraline inhibited VEGFinduced invasion of HUVECs in a dose-dependent manner as well as tube formation (Fig. 2A and B). VEGF-stimulated tube-like structures of HUVECs were suppressed upon indatraline treatment. In both assays, a low dose of indatraline (0.5 mM) was sufficient to suppress invasiveness and the ability to form the capillary structures in HUVECs. These results demonstrated that indatraline suppresses angiogenic activity along with an autophagy-inducing ability of HUVECs. Indatraline effect on angiogenesis was further investigated with in vivo CAM assay. Robust neovascularization was shown in nontreated CAM, whereas apparent suppression of angiogenesis was observed in indatraline (1 mg)-treated CAM. Indatraline effect on angiogenesis activity was similar to that of the positive control (RAtreated CAM) without causing any hemolysis. Consistent with the in vitro results, indatraline exerted suppressive influences on the growth of new blood vessels in vivo CAM assay (Fig. 3). 3.3. Indatraline suppresses tumor cell-induced angiogenesis HIF-1a activates the transcription of genes that play key roles in tumorigenesis, including angiogenesis and invasion [16]. HIF-1a is sensitive to cellular oxygen levels and is stabilized in hypoxic conditions to activate its target genes, particularly VEGF. Therefore, overexpression of HIF-1a is common in many types of cancers, suggesting that HIF-1a could represent as a key target for cancer therapy [17e19]. Here, we performed western blotting to assess the effect of indatraline on HIF-1a stability. HIF-1a was stimulated after 4 h of incubation in hypoxic conditions. Indatraline dosedependently decreased the stabilized HIF-1a protein levels. A dose as low as 1 mM was sufficient to reduce the HIF-1a level, and 5 mM of indatraline reduced the HIF-1a protein expression down to
Fig. 1. Effect of indatraline on autophagy and proliferation of human umbilical vein endothelial cells (HUVECs). (A) Structure of indatraline. (Inda: indatraline) (B) Indatraline induces the conversion of LC3-1 to LC3-II, detected by western blotting. (C) Effect of indatraline on the phosphorylation of mTOR and S6K was assayed by western blotting. (D) Effect of indatraline on proliferation of HUVECs was examined by the MTT assay following 72-h indatraline treatment at the indicated concentrations. (E) Viable cells were measured by the trypan blue assay.
Please cite this article in press as: C.-n. Yen, et al., The effect of indatraline on angiogenesis suppression through HIF-1a-mediated VEGF inhibition, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.02.077
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Fig. 2.. Effects of indatraline on angiogenesis. (A) Effect of indatraline on HUVECs invasiveness, determined by an in vitro chemoinvasion assay. (B) Indatraline inhibits VEGF-induced tube formation. Arrows indicates broken tube-like structures. In (A) and (B), 30 ng/mL of VEGF was used to induce HUVEC invasion and tube formation. Quantified bar graphs are presented as mean (±SEM) compared to VEGF-treated control (** indicates p < 0.001 and *** indicates p < 0.0001).
Fig. 3. Validation of anti-angiogenic activity of indatraline in in vivo assay. Inhibitory effect of indatraline on angiogenesis was retested by the chick chorioallantoic membrane (CAM) assay. Calculations were completed based on the proportion of positive eggs relative to the total number of eggs tested. Arrow represents inhibition of neovascularization. Retinoic acid (RA) was used as a positive control.
nearly 50% of that observed in the non-treated control (Fig. 4A). Next, we investigated the effect of indatraline on the hypoxiainduced VEGF level using a VEGF ELISA. The VEGF level was increased by approximately 40% in hypoxic conditions compared to that observed in the normoxic control. Indatraline reduced the hypoxia-stimulated VEGF level in a dose-dependent manner
(Fig. 4B). Due to the inhibitory effect of indatraline on hypoxiainduced HIF-1a and VEGF levels, we examined whether indatraline suppresses tumor-mediated invasion of HUVECs. HeLa cells were incubated under hypoxic conditions and their conditioned medium was used to stimulate the invasion of HUVECs. Indatraline inhibited HUVECs invasion, which was stimulated by the
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Fig. 4.. Mechanism of indatraline on tumor angiogenesis. (A) Destabilization of HIF-1a protein upon indatraline treatment was analyzed via immunoblotting. (B) Indatraline decreased the hypoxia-induced VEGF level, detected by VEGF ELISA. In (A) and (B), HIF-1a and VEGF expressions of HeLa cells were stimulated under hypoxic conditions. After hypoxic induction, cells were treated with the indicated doses of indatraline. Quantified bar graphs are presented as mean (±SEM) compared to the VEGF-treated control (** indicates p < 0.001 and *** indicates p < 0.0001). (C) Effect of indatraline on tumor-induced angiogenesis. Conditioned medium (CM) was derived from HeLa cells. (D) Schematic representation of indatraline-induced biological activity. Indatraline triggers autophagy through AMPK activation, followed by mTOR and S6K inhibition. Indatraline also exerts negative effects on angiogenesis through the reduction in HIF-1a and VEGF levels.
conditioned medium of cancer cells (Fig. 4C). These results indicate that indatraline affects hypoxia-mediated tumor angiogenesis by suppressing HIF-1a-mediated VEGF expression. 4. Discussion We have previously reported that indatraline, originally known as an antidepressant, (monoamine neurotransmitter blocker), induces autophagy [6]. Enhanced autophagy by indatraline was due to the negative regulation of mTOR as a result of AMPK activation. The mTOR has been identified as a negative regulator of autophagy; however, it has also been recognized as ‘angiogenic switch’ over the last two decades [20]. Therefore, our previous observations prompted us to determine the effect of indatraline on angiogenesis due to its suppressive effect on the mTOR signaling pathway. A recent study has identified the anti-angiogenic activity of indatraline via the inhibition of Rho- and calcium-mediated glioblastoma cell motility [21]. In reference to this study, we further determined whether indatraline could affect angiogenesis by exploring in vitro and in vivo angiogenesis using HUVECs. Indatraline blocked the tube-forming ability and invasiveness of HUVECs. Similarly, indatraline suppressed neovascularization in the in vivo CAM assay. The effect of indatraline on angiogenesis was due to the suppression of HIF-1a-associated VEGF secretion. Additionally, indatraline suppressed the invasiveness of HUVECs when stimulated with the conditioned medium of cells cultured in hypoxic
conditions. These results suggest that indatraline can negatively regulates angiogenesis in cancer by controlling cell growth. The relationship between indatraline-induced autophagy and its effect on angiogenesis is still not clear. However, further investigations on monoamine neurotransmitter inhibition, autophagy induction, and angiogenesis suppression by indatraline might possibly reveal the mechanisms underlying the relationship between indatralineassociated autophagy and angiogenesis suppression. Autophagy is progressively recognized as an alternative approach for apoptosis-resistant cancer treatment. Likewise, antiangiogenesis drugs are regarded as promising anti-cancer agents because blocking angiogenesis prevents not only invasion of tumors to nearby regions, but also limits the enlargement of tumor size. Therefore, indatraline-associated autophagy induction and anti-angiogenic activity may act synergistically in treating cancer. Identification of additional protein targets of indatraline will be helpful to gain a clear understanding of its poly-biological activities on autophagy and angiogenesis. In summary, we demonstrated for the first time that autophagy inducer indatraline controls angiogenesis via HIF-1a pathway. This finding suggests the potential of indatraline as an anti-cancer drug candidate. Together with our previous observations, the present findings on the poly-biological activities of indatraline may allow expansion of its application from anti-depressive agent into cancer therapeutics (Fig. 4D).
Please cite this article in press as: C.-n. Yen, et al., The effect of indatraline on angiogenesis suppression through HIF-1a-mediated VEGF inhibition, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.02.077
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Please cite this article in press as: C.-n. Yen, et al., The effect of indatraline on angiogenesis suppression through HIF-1a-mediated VEGF inhibition, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/j.bbrc.2017.02.077