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Evodiamine suppresses Notch3 signaling in lung tumorigenesis via direct binding to γ-secretases
Phytomedicine 68 (2020) 153176
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Original Article
Evodiamine suppresses Notch3 signaling in lung tumorigenesis via direct binding to γ-secretases
T
Xia Yang, Yanmin Zhang, Yanfang Huang, Ying Wang, Xiaoxiao Qi, Tao Su , Linlin Lu ⁎
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Joint Laboratory for Translational Cancer Research of Chinese Medicine of the Ministry of Education of the People's Republic of China, International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong 510006, China
ARTICLE INFO
ABSTRACT
Keywords: Evodiamine Notch3 Γ-secretase Viability Migration Stemness
Background: Notch activation requires proteolytic cleavage of the receptor by γ-secretase protein complex. Inhibition of Notch receptor activation (e.g. Notch3) with γ-secretase inhibitor is a potential new therapeutic approach for the targeted therapy of non-small cell lung cancer (NSCLC). However, only a few safe and effective γ-secretase inhibitors have been discovered. Evodiamine (EVO), a compound derived from Euodiae Fructus (Chinese name, Wu-Zhu-Yu), exhibits remarkable anti-NSCLC activities. However, the underlying mechanisms of action have yet to be fully elucidated. Purpose: We sought to determine the involvement of Notch3 signaling in the anti-NSCLC effects of EVO, and to explore whether EVO suppressed Notch3 signaling by inhibiting γ-secretase in cultured A549 and H1299 NSCLC cells and in urethane-induced lung cancer FVB mouse model. Methods: Cell viability, migration, stemness and cell cycle distribution of EVO were examined by the MTT assay, wound healing assay, soft agar colony assay and flow cytometry analysis, respectively. The binding affinity of EVO and γ-secretase complex was analyzed by molecular docking. Cellular thermal shift assay (CETSA) was performed to study the drug-target interactions in NSCLC cells. Protein levels were determined by Western blotting. Results: EVO dramatically inhibited cell viability, induced G2/M cell cycle arrest, suppressed cell migration, and reduced stemness in NSCLC cells. Mechanistic studies indicated that EVO prevented the γ-secretase cleavage of Notch3 at the cell surface and hence inhibited Notch3 activation. Moreover, EVO notably reduced tumor growth in the mouse model and inhibited Notch3 activity in the tumors. Conclusion: This study provides new insights into the anti-NSCLC action of EVO, and suggests that suppressing Notch3 signaling by inhibiting γ-secretase is a mechanism of action underlying the anti-NSCLC effect of EVO.
Introduction Lung cancer is one of the prevalent cancer types that threatens the lives of millions of people. Its incidence and mortality rates are increasing rapidly worldwide (Mizugaki et al., 2012). Non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC) are the two major types of lung cancer. NSCLC makes up nearly 85% of all the cases (Ye et al., 2013). Although therapies have improved survival and quality of life for patients with advanced NSCLC, it remains as a fatal disease (Allenspach et al., 2002). The 5-year survival rate of patients with NSCLC is less than 20% (Xia et al., 2017). The current
chemotherapeutics for NSCLC are expensive, and with side-effects; moreover, patients often develop drug resistance (Tong et al., 2016). Therefore, exploring effective and safe targeted chemotherapeutic agents for treating NSCLC are urgently needed. Evodiamine (EVO), a naturally occurring indole alkaloid with low toxicity, is one of the main bioactive constituents of the herbal medicine Euodiae Fructus (Chinese name, Wu-Zhu-Yu). Our previous studies and other studies have reported that EVO exerts inhibitory effects on NSCLC cell growth, metastasis and angiogenesis (Ogasawara and Suzuki, 2004; Wang et al., 2014), and potently suppresses Notch3 activity by activating the DNMTs-induced Notch3 methylation in NSCLC cells (Su et al., 2018).
Abbreviations: ADAM, a disintegrin and metalloproteinase; CETSA, cellular thermal shift assay; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; DNMTs, DNA methyltransferase; EMT, epithelial-mesenchymal transition; EVO, evodiamine; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NSCLC, non-small cell lung cancer; PBS, phosphate buffered saline; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate; SDSPAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis ⁎ Corresponding authors. E-mail addresses: [email protected] (T. Su), [email protected] (L. Lu). https://doi.org/10.1016/j.phymed.2020.153176 Received 7 July 2019; Received in revised form 6 January 2020; Accepted 30 January 2020 0944-7113/ © 2020 Elsevier GmbH. All rights reserved.
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EVO is considered as a promising potent anti-tumor drug candidate for treating NSCLC (Hong et al., 2014). However, the underlying mechanism of action is not fully understood. Notch signaling plays critical roles in cell differentiation, proliferation and apoptosis. Increasing evidences support that Notch signaling dysregulation is associated with many cancer types including NSCLC (Zou et al., 2018). Notch3 is expressed in about 40% of NSCLC cases (Konishi et al., 2007). Overexpression of Notch3 is associated with enhanced tumorigenesis (Ye et al., 2013); while, Notch3 silencing in tumor models leads to tumor cell death, tumor regression (Capaccione and Pine, 2013) and inhibition of tumor metastasis (Zhou et al., 2013). Hence, inhibition of Notch receptor activation is a compelling treatment strategy. Notch signaling is initiated via ligandreceptor interactions, and this interaction initiates a series of intramembrane cleavages, the last of which is regulated by the γ-secretase enzyme complex (Luistro et al., 2009). Notch activation requires proteolytic cleavage of the receptor by γ-secretase complex (Konishi et al., 2007). Inhibition of Notch activation using γ-secretase inhibitors could induce apoptosis and suppress NSCLC cells proliferation (Konishi et al., 2007). Several γ-secretase inhibitors have been developed and some have entered the clinical trials, such as MK-0752 (Cook et al., 2018) and BMS-708163 (Coric et al., 2012). However, systemic toxicity and drug resistance greatly limit their clinical application (Coric et al., 2012; Krop et al., 2012). EVO is a bioactive alkaloid compound, which exhibits antitumor activities with tolerable toxicity. Our previous studies have demonstrated that EVO potently suppresses Notch3 signaling via activation of the DNMTs-induced Notch3 methylation in NSCLC cells (Su et al., 2018). In this study, we explored whether EVO inhibited Notch3 activation by suppressing of γ-secretase in NSCLC cells and in urethane-induced lung cancer mouse models.
EdU assay A549 cells were seeded in 96-well plates (2 × 103 cells/well), and treated with vehicle or EVO (4 and 8 μM) for 48 h. Then cells were incubated with EdU labeling medium (50 μM) for 2 h at 37 °C with 5% CO2, fixed with 4% paraformaldehyde (pH 7.4) for 30 min, and incubated with glycine for 5 min. After rinsed with PBS, cells were incubated with anti-EdU working solution at room temperature for 30 min, and then stained with DAPI (1 ×). The images were captured using Leica DMI 3000B (Leica, GER). Percentages of EdU-positive cells were calculated from five random fields in two wells. Cell cycle analysis A549 cells were seeded in 6-well plates (2.5 × 105 cells/well). After treatment with vehicle or EVO (4 and 8 μM) for 48 h, cells were collected, rinsed with PBS, and fixed with ice-cold 70% ethanol at 4 °C overnight. Then the fixed cells were stained with PI (BD Biosciences, San Diego, USA), and then analyzed using a FACS AriaIII (BD Biosciences). Data were analyzed by FlowJo 7.6 software. Soft agar colony assay A549 cells were suspended in medium containing 0.4% agar (1000 cells/well), and then added in 6-well plates filled with 1% agar. After treatment with vehicle or EVO (4 and 8 μM) for two weeks, cell colonies were counted and photographed. Wound healing assay A549 cells were seeded in 6-well plates, when cells were grown to 85% confluence, cell monolayers were scratched using a plastic pipette tip. Then, cells were treated with vehicle or EVO (4 and 8 μM) in serumfree RPMI-1640 medium. Migration of cells into the wound area were photographed at 0, 12, and 24 h time points using Leica DMI 3000B (Leica, GER) at 10 × magnification. Initial and final wound sizes were measured using Image ProPlus 6.0 software.
Materials and methods Reagents and antibodies EVO (purity > 98%) was purchased from Dalian Meilun Biotech Co., Ltd. (Dalian, China). 3-(4,5-dimethylthiazol-2-yl)−2, 5-diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Propidium iodide (PI) staining was purchased from Biosciences (BD Biosciences, NJ, USA). Cdc2, CyclinB1, NICD, JAG2, ADAM9, NUMB, Nicastin, PSEN1, PSEN2 and PEN2 primary antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). β-actin, E-cadherin, N-cadherin and Vimentin primary antibodies were purchased from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA). Notch3 primary antibody was purchased from Abcam (Cambridge, UK).
Preparation of cytoplasmic and nuclear fractions A549 and H1299 cells were seeded as described in Section “Cell cycle analysis”. After incubation for 48 h, EVO-treated and vehicletreated cells were collected. Cytoplasmic and nuclear extracts were prepared as described previously (Cao et al., 2014). Western blotting A549 and H1299 cells were treated with vehicle or EVO (4 and 8 μM for A549 cells; 1 and 2 μM for H1299 cells) for 48 h, respectively. Then, the cells were lysed using RIPA lysis buffer. Tumors in each group , which collected from our previous study (Su et al., 2018) were rinsed with PBS for three times, and then extracted with RIPA lysis buffer using Tissuelyser-24 (Shanghai Jingxin Industrial Development Co., Ltd., Shanghai, China). All protein samples were quantified using Coomassie Brilliant Blue Kit (Bio-Rad, Hercules, CA, USA), and then separated by SDS-PAGE, transferred onto PVDF membranes, blocked with 5% skimmed milk for 1 h, and incubated with the primary antibodies at 4 °C overnight, subsequently, membranes were washed and then incubated with secondary antibodies for another 1h. ECL chemiluminescence reagent was applied to detect for fluorescent signals using FluorChem E (Santa Clara, CA, USA). Protein bands were quantified using Quantity One software (Bio-Rad, Hercules, CA, USA).
Cell culture Two human NSCLC A549 and H1299 cell lines were purchased from ATCC (Manassas, Virginia, USA). Cells were cultured in complete RPMI1640 medium (GIBCO, USA) containing 10% (v/v) FBS (GIBCO, USA) and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO2 atmosphere. Cell viability assay MTT assay was used to determine the cytotoxic effect of EVO on A549 and H1299 cells. Cells were seeded in 96-well plates (4 × 103 cells/well), cultured overnight, and then treated with various concentrations of EVO (1, 2, 4, 8, 16 μM) or vehicle for 24, 48 and 72 h. After that, cells were treated with the MTT solution (5 mg/ml) and incubated for an additional 3 h. The formazan crystal formed was dissolved with 100 μl of DMSO; absorbance was detected at 490 nm by a microplate reader Victor X3 (PerkinElmer, Waltham, MA, USA).
Cellular thermal shift assay (CETSA) A549 cells were lysed using Tissuelyser-24 (Shanghai Jingxin 2
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adapted for researches on stemness of the malignant cells, which is considered as a hallmark of carcinogenesis (Jin et al., 2017). Here, we found that EVO significantly reduced the colony number and reduced the colony size in A549 cells (Fig. 2A).
Industrial Development Co., Ltd., Shanghai, China). After centrifugated at 14,000 rpm for 20 min, the supernatants were collected and divided into two groups, one group treated with EVO (100 μM) and the other group treated with the same volume of solvent (DMSO) for 30 min at room temperature. Then, the respective lysates were divided into six aliquots and heated individually at 45, 49, 53, 57, 61 and 65 °C for 7 min (Veriti thermal cycler, Applied Biosystems/Life Technologies), followed by cooling for 3 min at room temperature. After centrifuged at 20,000 × g for 20 min at 4 °C, the heated lysates were separated and analyzed by SDS-PAGE. The melting curves were fitted by GraphPad Prism 5.0 software.
EVO inhibited NSCLC cells metastasis Metastasis, which involves migration of tumor cells, is a critical process for NSCLC progression and development (Zhang et al., 2017). To test the effects of EVO on NSCLC cells migration, wound healing assay was performed. As shown in Fig. 2B, A549 cells migrated to the wound area after incubation for 24 h, while EVO dose-dependently inhibited the cells migration. Epithelial-mesenchymal transition (EMT) is a physiological process in which epithelial cells acquire the motile and invasive characteristics of mesenchymal cells. It is thought to be a key event for tumor metastasis (Huang and Zong, 2017). Here, we determined the protein levels of EMT-related molecules, such as N-cadherin, Vimentin and E-cadherin. As expected, EVO treatment significantly reduced the protein levels of N-cadherin and Vimentin (mesenchymal markers) in A549 cells, whereas, the protein level of Ecadherin (epithelial marker) was increased (Fig. 2C). All these results suggested that EVO suppresses NSCLC cells metastasis.
Molecular docking The docking study of γ-secretase and EVO was performed by Autodock vina software. The 3D structure of γ-secretase (PDB: 5FN2) was downloaded from PDB database, ligand was removed via PyMOL and prepared in ADT for docking. EVO structure (PUBCHEMCID: 442088) was converted to 3D coordinates by open Babel 2.3. MK-0752, a well-known γ-secretase inhibitor (Cook et al., 2018), was used as a reference ligand for verifying the binding ability of EVO. The size of space was set as 70 × 90 × 70 point and other parameters were performed with default of the docking software. Docking results were viewed by PyMOL v1.7 software.
EVO inhibited Notch3 signaling in NSCLC cells Notch signaling, a highly conserved pathway, regulates many key cellular processes, such as cell proliferation, apoptosis, metastasis and so on (Ranganathan et al., 2011). Notch3 is mainly expressed in NSCLC (Li et al., 2011). In our previous study, we have demonstrated that EVO potently inhibited Notch3 activation (Su et al., 2018). However, whether it could affect other molecules that are involved in this signaling pathway is not known. NUMB is a membrane-associated protein, which negatively regulates Notch signaling (Bolos et al., 2007). Studies suggest that silence or mutation of NUMB activates Notch signaling and leads to tumorigenesis (Bolos et al., 2007). As expected, EVO significantly increased the protein levels of NUMB in NSCLC cells (Fig. 3A). JAG1 and JAG2 are the two canonical Notch ligands that promote the tumorigenesis and metastasis in NSCLC (Kangsamaksin et al., 2015; Vaish et al., 2017). Here, we found that EVO dose-dependently reduced the protein levels of JAG2 (Fig. 3A) but not JAG1 in both A549 and H1299 cells, suggesting that EVO inhibites the JAG2Notch3 pathway in NSCLC cells. Upon the ligand binding, Notch receptors undertake two cleavage processes mediated by a member of a disintegrin and metalloproteinase (ADAM) family, as well as the γ-secretase complex (a membrane-embedded protease that controls a series of important cellular functions through substrate cleavage) (Lu et al., 2014), leading to the Notch intracellular domain (NICD) release. The released NICD translocates to the nucleus and regulates the expressions of downstream targets (Andersson and Lendahl, 2014; Rizzo et al., 2008). As shown in Fig. 3A, EVO notably decreased the protein levels of ADAM9 and the γ-secretase complex including Nicastrin, PSEN1, and PSEN2 in a dose-dependent manner. Moreover, the protein levels of NICD in nuclear fractions of both A549 and H1299 cells were significantly reduced by EVO in a dose-dependent manner (Fig. 3B).
Statistical analysis All data were presented as the mean ± standard deviation (SD). Significant differences were analyzed by one-way analysis of variance (ANOVA) followed by the LSD post-hoc test using SPSS 19.0 software. Statistical analyses were carried out using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). Statistical differences were considered significant at p < 0.05. Results EVO reduced viability and inhibited proliferation in NSCLC cells The MTT assay was used for determining the cytotoxicity of EVO on A549 and H1299 cells. Cells were treated with vehicle or various concentrations of EVO for 24, 48 and 72 h. As shown in Fig. 1A, EVO reduced A549 and H1299 cells viability in both time- and dose-dependent manners (Fig. 1A), which was consistent with our previous observations (Su et al., 2018). To determine whether EVO inhibited cell proliferation, the EdU assay was performed. In contrast to the abundant EdU-positive cells observed in control, EVO treatments significantly suppressed NSCLC cells proliferation (Fig. 1B). EVO induced G2/M cell cycle arrest in A549 cells Flow cytometric analysis of cell cycle distribution demonstrated that EVO dose-dependently increased the percentage of A549 cells at G2/M phase from 21.78% in control to 41.90% and 76.04% in EVO treatment groups (4 and 8 μM, respectively) (Fig. 1C). Cdc2 and CyclinB1 are two key proteins required for cell cycle regulation and cell progression from G2 to M phase. Both of them play an important role in this phase transition during the cell cycle (Zhang et al., 2011). In agreement with the cell cycle distribution results, EVO treatment notably reduced the protein levels of CyclinB1 and Cdc2 in the A549 cells in a dose-dependent manner (Fig. 1D).
EVO showed potent binding affinity with γ-secretase Since EVO has potent inhibitory effect on Notch3 signaling in NSCLC cells, we sought to explore whether EVO directly binds to Notch3 (the target protein). CETSA is a commonly used assay to verify the binding activity of a drug to its target protein in cells (Molina et al., 2013). It is noteworthy that no liner affinity was observed on Notch3 (Fig. 4A), suggesting that EVO and Notch3 may not have direct binding effect. Notch activation requires proteolytic cleavage of the receptor by γ-secretase complex (Konishi et al., 2007), hence, we further
EVO reduced stemness of A549 cells Soft agar colony formation assay is a well-established method to evaluate cellular anchorage-independent growth. It has been widely 3
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Fig. 1. EVO reduced viability, inhibited proliferation and induced G2/M cell cycle arrest in NSCLC cells. (A) Cell viability was measured by the MTT assay. Both A549 and H1299 cells were treated with various concentrations of EVO or vehicle for 24, 48 and 72 h, respectively. *p < 0.05, **p < 0.01 vs. vehicle (24 h); #p < 0.05, ##p < 0.01 vs. vehicle (48 h); &&p < 0.01 vs. vehicle (72 h). (B) A549 cells were treated with vehicle or EVO (4 and 8 μM) for 48 h. Cells were fixed, incubated with anti-EdU working solution, and then stained with DAPI. The images were captured using Leica DMI 3000B (Leica, GER). Representative images (left panel) and relative fluorescent levels (right panel) were shown. The number of EdU-labeled cells in vehicle group was regarded as 100. (C) After fixation, cells were stained with PI and then analyzed using a flow cytometry. Representative images (left panel) and the statistical analysis of cell cycle data in three independent experiments (right panel) were shown. (D) Protein levels of Cdc2 and CyclinB1 were determined by Western blotting. Data are shown as mean ± SD from three independent experiments, *p < 0.05, **p < 0.01, vs. corresponding control.
Fig. 2. EVO suppressed stemness and metastasis in NSCLC cells. (A) Representative images of colony formation were captured after EVO (4 and 8 μM) treatment for two weeks (scale bar: 100 μm), and the quantification of colony numbers were shown. (B) A549 cells were plated in 6-well plates, and 1 day later, wounds were created and then followed by vehicle or EVO (4 and 8 μM) treatments. Each scratch was photographed after 12- and 24-h treatment. Relative migration ratio (%) ==Treatment group [Gap width (0 h) - Gap width (12 or 24 h)]/Control group [Gap width (0 h) - Gap width (12 or 24 h)] × 100%. (C) Protein levels of EMTrelated markers (E-cadherin, N-cadherin and Vimentin) were determined by Western blotting. Data are shown as mean ± SD from three independent experiments, **p < 0.01, vs. corresponding control.
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Fig. 3. EVO suppressed Notch3 signaling in NSCLC cells. (A) A549 cells were treated with various concentrations of EVO or vehicle for 48 h, then total cell lysates were extracted for immunoblotting by using antibodies specific to Notch3, JAG1, JAG2, NUMB, ADAM9 and γ-secretase complex. The representative figure (left panel) and the relative protein levels analyzed by Image J software (right panel) were shown. (B) Protein levels of NICD in cytoplasmic and nucleus extracts were examined by immunoblotting (left panel); and quantitative results were analyzed using Image J software (right panel). β-actin and Histone H3 served as loading controls of cytoplasmic and nuclear extractions, respectively. Data are shown as mean ± SD from three independent experiments, *p < 0.05, **p < 0.01, vs. corresponding control.
determined whether EVO could affect the γ-secretase complex. Molecular docking assay was performed to find out the binding energy. MK0752, a γ-secretase inhibitor, was selected as reference ligand. The docking studies of γ-secretase complex with EVO or MK-0752 were performed using the same parameters. Optimal binding conformation of the γ-secretase-EVO complexes were presented in Fig. 4B, as well as the binding affinity of each complex were shown in Fig. 4C. Results showed that EVO had similar binding affinity to MK-0752 in γ-secretase docking domain (PDB ID 5FN2). To further confirm the binding effect of EVO to γ-secretase complex, we used the CETSA assay. Interestingly, EVO notably shifted Tm50 of PSEN2, whereas there is no liner affinity on PSEN1 and PEN2 (Fig. 4D). Furthermore, we found that the band intensity of PSEN2 was dose-dependently increased after EVO treatment (Fig. 4E). PSEN2 is a polytopic transmembrane protein, which functions as a part of the γ-secretase protein complex (Konishi et al., 2007). These results strongly suggested that EVO effectively inhibited the activation of Notch3 in NSCLC cells, and this suppression may be attributed to the inhibition of the cleavage by γ-secretase. It is well-known that inactivation of the Notch pathway by targeting γ-secretase is a viable strategy for lung cancer treatment (Rizzo et al., 2008). Thus, EVO which inhibits γ-secretase activity, is a promising anti-NSCLC agent.
EVO restrained tumor growth and inhibited Notch3 activation in urethaneinduced tumorigenesis of FVB mice In our previous study, we found that EVO significantly reduced the tumor growth in urethane-induced lung cancer mouse model (Supplementary Fig. 1A). Furthermore, no obvious toxicity and organ weight loss (Supplementary Fig. 1B) were shown in each treatment group. Here, to determine whether EVO affects the Notch3 signaling pathway in vivo, we examined the protein levels of Notch3, NUMB, ADAM9 and the γ-secretase complex in tumors by using Western blotting. As shown in Fig. 5, protein levels of Notch3, JAG2, ADAM9, PSEN1, PSEN2 were remarkably decreased, while the level of NUMB was increased in the lung tumors of the EVO-treated group when compared with that of the vehicle control group, these results are consistent with our in vivo observations. Discussion In the last decades, Chinese medicinal herb has been increasingly used, because it is well-known for its significant role in cancer prevention and treatment. In this study, we investigated the anti-NSCLC activities of EVO, an alkaloid isolated from a Chinese medicinal herb 6
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Fig. 4. EVO showed potent binding activity with γ-secretase. (A) A549 cells were treated with EVO (100 μM) for 30 min, and then heated by individual temperature (45–65 °C) for 7 min. Representative Notch3 bands were shown on the upper panel and the melting curve fitted by GraphPad with Boltzmann sigmoidal were shown on the lower panel. E: EVO; D: DMSO. Relative band intensity (%) = Band intensity in each tempeture/Band intensity in 45 °C × 100%. Band intensity in 45 °C of each group was regarded as 100. Data are shown as mean ± SD from three independent experiments. (B) The optimal binding conformation of the γsecretase-EVO complex. (C) The binding affinity of each complex. Docking studies of γ-secretase with EVO or reference ligand (MK-0752) were performed using the same parameters. (D) Respective lysates were divided into six aliquots and heated individually at 45, 49, 53, 57, 61 and 65 °C for 7 min. Representative bands of PSEN1 and PSEN2 were shown on the upper panel and the melting curve fitted by Graphpad with Boltzmann sigmoidal were shown on the lower panel. E: EVO; D: DMSO. Relative band intensity (%) = Band intensity in each tempeture/Band intensity in 45 °C × 100%. Band intensity in 45 °C of each group was regarded as 100. Data are shown as mean ± SD from three independent experiments. **p < 0.01 vs. corresponding vehicle. (E) A549 cells were treated with different concentrations of EVO (0.1, 1, 10, 100 and 1000 μM) for 30 min, and then heated by 57 °C for 7 min. Representative PSEN2 bands were shown on the upper panel and the relative protein level was shown on the lower panel. Relative band intensity (%) = Band intensity of each concentration of EVO-treated cells/Band intensity in 1000 μM of EVO-treated cells × 100%. The band intensity of 1000 μM of EVO-treated cells was regarded as 100. Data are shown as mean ± SD for three individual experiments. *p < 0.05, **p < 0.01, vs. 0.1 μM of EVO treatment.
Euodiae Fructus. EVO possesses many biological and pharmacological activities that are beneficial to human health. Increasing evidence indicates that EVO has cytotoxic effects on several human cancer cells, such as melanoma cells (Wang et al., 2005), prostate cancer cells (Kan et al., 2007), colon cancer cells (Ogasawara et al., 2001), breast cancer cells (Liao et al., 2005) and lung cancer cells (Lin et al., 2016). EVO inhibits NSCLC cell growth and metastasis (Ogasawara and Suzuki, 2004). However, up to present, the underlying molecular mechanism of the anti-NSCLC action of EVO remains poorly illustrated. In this study, we examined the anti-NSCLC activity of EVO both in vitro and in vivo. In agreement with previous studies, EVO inhibited NSCLC cell viability (Fig. 1A), suppressed cell proliferation (Fig. 1B), induced G2/M cell cycle arrest (Fig. 1C), reduced stemness (Fig. 2A) and inhibited cell migration (Fig. 2B). Importantly, EVO exerted potent antilung cancer effects in urethane-induced lung cancer mouse model (Supplementary Fig. 1A). EVO has advantages over synthetic
compounds, EVO is noncarcinogenic and with low intrinsic toxicity properties. EVO is considered to be a potential candidate agent for NSCLC prevention and treatment. In the in vitro study, based on the different cytotoxicity and IC50 of EVO in A549 and H1299 cells, we used different doses to treat these cell lines. Interestingly, A549 and H1299 cells have distinct genetic backgrounds. Although both A549 and H1299 cells are human epithelial carcinoma cells, A549 is p53 wide-type, while H1299 is p53-null (Li et al., 2004). EVO has different cytotoxicity in these two cell lines, implying that p53 signaling may be involved in the anti-NSCLC effects of EVO, which deserves further investigation. Many effective therapeutics approaches, such as surgery, radiation therapy and chemotherapy are being used for NSCLC treatment. Recently, targeted therapy becomes an important part of the management for advanced NSCLC patients. Targeted therapy includes tyrosine kinase inhibitors of the active epidermal growth factor receptor (EGFR) 7
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Fig. 5. EVO suppressed Notch3 signaling in vivo. Protein levels of Notch3, NUMB, JAG2, ADAM9 and γ-secretase complex in the lung tumors of each group were detected by Western blotting. The representative figure (left panel) and the relative protein levels analyzed by Image J software (right panel) were shown. Data are shown as mean ± SD from three independent experiments, *p < 0.05, **p < 0.01, vs. vehicle-treated group.
mutations (e.g. erlotinib, gefinitib and afatinib), monoclonal antibodies against vascular endothelial growth factor (VEGF) (e.g. bevacizumab) and anaplastic lymphoma kinase (ALK) rearrangement (e.g. crizotinib). In 2014, the European Medicines Agency approved five of them for NSCLC treatment, which include afatinib, bevacizumab, crizotinib, gefinitib and erlotinib (Sculier et al., 2015). Unfortunately, drug resistance is often developed in less than one year (Chong and Janne, 2013). Therefore, identification of the novel molecular targets for treating NSCLC are urgently needed. Notch3 is overexpressed in about 40% of NSCLC cases, and has been proposed as a therapeutic target of NSCLC. Inhibition of Notch3 activation represents a compelling treatment strategy. Notch3 is one of the mammalian Notch family receptors. All the four members of the Notch family have been implicated in various types of cancers including lung cancer (Motooka et al., 2017). It has been reported that Notch3 signaling is highly context-dependent in solid tumors, which can promote or inhibit tumor growth. Multivariate analysis demonstrated that Notch3 is a potential biomarker for predicting the prognosis of NSCLC patients (Ye et al., 2013). Inhibiting Notch3 activation could inhibit tumor growth in NSCLC (Konishi et al., 2007). Consistently, in this study, we found that EVO exerted anti-NSCLC effects and potently inhibited Notch3 signaling in cultured NSCLC cells (Fig. 3) and in tumor of the lung cancer mouse model (Fig. 5). To further elucidate the mechanisms of EVO-induced Notch3 inhibition, we used molecular docking assay to predict the binding affinity, and the CETSA assay to verity the binding affinity of EVO to its target proteins. Interestingly, EVO showed more potent binding activity with γ-secretase than with Notch3 (Fig. 4). γ-secretase, a large protease complex, is composed of a catalytic subunit (PSEN1 and PSEN2) and accessory subunits (Aph1, PEN2 and nicastrin) (Kaether et al., 2006). γ-secretase has been considered as a molecular switch for Notch3 signaling; and it is one of the most promising targets to inhibit the Notch3 signaling, which has been considered as a molecular switch for Notch3 signaling activation (Shih Ie and Wang, 2007). More and more preclinical studies have suggested that γ-secretase inhibitors hold promise as a new targetbased therapy for the tumors with aberrant Notch activation (Shih Ie and Wang, 2007). Nevertheless, these inhibitors have substantial side effects that hinder their clinical application (Capaccione and Pine, 2013). The main adverse toxicity of γ-secretase inhibitors shown
in patients is dose-limiting secretory diarrhea (Wong et al., 2004). In addition, some pharmacological studies show that γ-secretase inhibitors cause reversible thymic suppression (Wong et al., 2004), reversible hair depigmentation and body weight loss in mice (Rizzo et al., 2008). Hence, exploring new γ-secretase inhibitors for treating NSCLC are critically needed. EVO with low toxicity but exerts anti-NSCLC activities, it inhibits Notch3 signaling by inhibiting the γ-secretase cleavage of Notch3 (Fig. 4). It is expected that EVO has a great potential to be developed as a novel γ-secretase inhibitor for lung cancer prevention or treatment. Currently, increasing evidences highlight natural products with antitumor activities (Wang et al., 2013). In Chinese medicinal herb Euodiae Fructus, more than twenty alkaloids have been isolated, and some of them are shown to have potent anti-lung cancer activities (Lin et al., 2016). EVO is a major active component in the total alkaloids. Further studies are warranted to identify more active constituents from Euodiae Fructus that can serve as anti-cancer agents. Conclusion In summary, we demonstrate that EVO inhibits cell viability, induces G2/M cell cycle arrest, suppresses cell migration and reduces stemness in human NSCLC cells. Further studies show that EVO potently inhibits Notch3 signaling via preventing its cleavage at the cell surface by the γ-secretase complex. Moreover, EVO reduces tumor numbers and inhibits the Notch3 in vivo. These findings suggest that EVO exerts anti-NSCLC effects and these effects are at least in part attributed to the inhibition of Notch 3 signaling via direct binding to γsecretases. This study provides a pharmacological basis for developing EVO as a novel phytotherapeutic agent against NSCLC. Author contributions X.Y., Y.M.Z. and Y.F.H. performed the majority experiments. X.X.Q. and Y.W. participated in some experiments. T.S.interpreted the data and drafted the manuscript. T.S. and L.L.L. supervised the study, reviewed the original data and finalized the manuscript. All authors have read and approved the final manuscript. 8
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Declaration of Competing Interest
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The authors declare that they have no competing interests. Acknowledgments This work was supported by the projects of National Natural Science Foundation of China (81874367 and 81703705), Natural Science Foundation for Distinguished Young Scholars of Guangdong Province, China (2017A030306033), Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2016), Project of Educational Commission of Guangdong Province of China (2016KTSCX012), Pearl River Nova Program of Guangzhou, China (201710010108). Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2020.153176. References Allenspach, E.J., Maillard, I., Aster, J.C., Pear, W.S., 2002. Notch signaling in cancer. Cancer Biol. Ther. 1, 466–476. Andersson, E.R., Lendahl, U., 2014. Therapeutic modulation of Notch signalling-are we there yet? Nat. Rev. Drug Discov. 13, 357–378. Bolos, V., Grego-Bessa, J., de la Pompa, J.L., 2007. Notch signaling in development and cancer. Endocr. Rev. 28, 339–363. Capaccione, K.M., Pine, S.R., 2013. The Notch signaling pathway as a mediator of tumor survival. Carcinogenesis 34, 1420–1430. Chong, C.R., Janne, P.A., 2013. The quest to overcome resistance to EGFR-targeted therapies in cancer. Nat. Med. 19, 1389–1400. Cook, N., Basu, B., Smith, D.M., Gopinathan, A., Evans, T.J., Steward, W.P., Hagemann, T., Venugopal, B., Tuveson, D.A., Hategan, M., Anthoney, D.A., Hampson, L.V., Nebozhyn, M., Tuveson, D., Farmer-Hall, H., Turner, H., McLeod, R., Halford, S., Jodrell, D., 2018. A phase I trial of the gamma-secretase inhibitor (GSI) MK-0752 in combination with gemcitabine in patients with pancreatic ductal adenocarcinoma. Br. J. Cancer 118 (6), 793–801. Coric, V., van Dyck, C.H., Salloway, S., Andreasen, N., Brody, M., Richter, R.W., Soininen, H., Thein, S., Shiovitz, T., Pilcher, G., Colby, S., Rollin, L., Dockens, R., Pachai, C., Portelius, E., Andreasson, U., Blennow, K., Soares, H., Albright, C., Feldman, H.H., Berman, R.M., 2012. Safety and tolerability of the gamma-secretase inhibitor avagacestat in a phase 2 study of mild to moderate alzheimer disease. Arch. Neurol. 69, 1430–1440. Cao, H.H., Tse, A.K., Kwan, H.Y., Yu, H., Cheng, C.Y., Su, T., Yu, Z.L., 2014. Quercetin exerts anti-melanoma activities and inhibits STAT3 signaling. Biochem. Pharmacol. 87 (3), 424–434. Hong, J.Y., Park, S.H., Min, H.Y., Park, H.J., Lee, S.K., 2014. Anti-proliferative effects of evodiamine in human lung cancer cells. J. Cancer Prev. 19, 7–13. Huang, R., Zong, X.Y., 2017. Aberrant cancer metabolism in epithelial-mesenchymal transition and cancer metastasis: mechanisms in cancer progression. Crit. Rev. Oncol. Hemat. 115, 13–22. Jin, Z., Feng, W., Ji, Y., Jin, L., 2017. Resveratrol mediates cell cycle arrest and cell death in human esophageal squamous cell carcinoma by directly targeting the EGFR signaling pathway. Oncol. Lett. 13, 347–355. Kaether, C., Haass, C., Steiner, H., 2006. Assembly, trafficking and function of gammasecretase. Neurodegener. Dis. 3, 275–283. Kan, S.F., Yu, C.H., Pu, H.F., Hsu, J.M., Chen, M.J., Wang, P.S., 2007. Anti-proliferative effects of evodiamine on human prostate cancer cell lines DU145 and PC3. J. Cell Biochem. 101, 44–56. Kangsamaksin, T., Murtomaki, A., Kofler, N.M., Cuervo, H., Chaudhri, R.A., Tattersall, I.W., Rosenstiel, P.E., Shawber, C.J., Kitajewski, J., 2015. NOTCH decoys yhat selectively block DLL/NOTCH or JAG/NOTCH disrupt angiogenesis by unique mechanisms to inhibit tumor growth. Cancer Discov. 5, 182–197. Konishi, J., Kawaguchi, K.S., Vo, H., Haruki, N., Gonzalez, A., Carbone, D.P., Dang, T.P., 2007. Gamma-secretase inhibitor prevents Notch3 activation and reduces proliferation in human lung cancers. Cancer Res. 67, 8051–8057. Krop, I., Demuth, T., Guthrie, T., Wen, P.Y., Mason, W.P., Chinnaiyan, P., Butowski, N., Groves, M.D., Kesari, S., Freedman, S.J., Blackman, S., Watters, J., Loboda, A., Podtelezhnikov, A., Lunceford, J., Chen, C., Giannotti, M., Hing, J., Beckman, R., Lorusso, P., 2012. Phase I pharmacologic and pharmacodynamic study of the gamma secretase (Notch) inhibitor MK-0752 in adult patients with advanced solid tumors. J. Clin. Oncol. 30, 2307–2313. Li, M., Brooks, C.L., Kon, N., Gu, W., 2004. A dynamic role of HAUSP in the p53-Mdm2 pathway. Mol. Cell 13, 879–886.
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Phytomedicine journal homepage: www.elsevier.com/locate/phymed
Original Article
Evodiamine suppresses Notch3 signaling in lung tumorigenesis via direct binding to γ-secretases
T
Xia Yang, Yanmin Zhang, Yanfang Huang, Ying Wang, Xiaoxiao Qi, Tao Su , Linlin Lu ⁎
⁎
Joint Laboratory for Translational Cancer Research of Chinese Medicine of the Ministry of Education of the People's Republic of China, International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong 510006, China
ARTICLE INFO
ABSTRACT
Keywords: Evodiamine Notch3 Γ-secretase Viability Migration Stemness
Background: Notch activation requires proteolytic cleavage of the receptor by γ-secretase protein complex. Inhibition of Notch receptor activation (e.g. Notch3) with γ-secretase inhibitor is a potential new therapeutic approach for the targeted therapy of non-small cell lung cancer (NSCLC). However, only a few safe and effective γ-secretase inhibitors have been discovered. Evodiamine (EVO), a compound derived from Euodiae Fructus (Chinese name, Wu-Zhu-Yu), exhibits remarkable anti-NSCLC activities. However, the underlying mechanisms of action have yet to be fully elucidated. Purpose: We sought to determine the involvement of Notch3 signaling in the anti-NSCLC effects of EVO, and to explore whether EVO suppressed Notch3 signaling by inhibiting γ-secretase in cultured A549 and H1299 NSCLC cells and in urethane-induced lung cancer FVB mouse model. Methods: Cell viability, migration, stemness and cell cycle distribution of EVO were examined by the MTT assay, wound healing assay, soft agar colony assay and flow cytometry analysis, respectively. The binding affinity of EVO and γ-secretase complex was analyzed by molecular docking. Cellular thermal shift assay (CETSA) was performed to study the drug-target interactions in NSCLC cells. Protein levels were determined by Western blotting. Results: EVO dramatically inhibited cell viability, induced G2/M cell cycle arrest, suppressed cell migration, and reduced stemness in NSCLC cells. Mechanistic studies indicated that EVO prevented the γ-secretase cleavage of Notch3 at the cell surface and hence inhibited Notch3 activation. Moreover, EVO notably reduced tumor growth in the mouse model and inhibited Notch3 activity in the tumors. Conclusion: This study provides new insights into the anti-NSCLC action of EVO, and suggests that suppressing Notch3 signaling by inhibiting γ-secretase is a mechanism of action underlying the anti-NSCLC effect of EVO.
Introduction Lung cancer is one of the prevalent cancer types that threatens the lives of millions of people. Its incidence and mortality rates are increasing rapidly worldwide (Mizugaki et al., 2012). Non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC) are the two major types of lung cancer. NSCLC makes up nearly 85% of all the cases (Ye et al., 2013). Although therapies have improved survival and quality of life for patients with advanced NSCLC, it remains as a fatal disease (Allenspach et al., 2002). The 5-year survival rate of patients with NSCLC is less than 20% (Xia et al., 2017). The current
chemotherapeutics for NSCLC are expensive, and with side-effects; moreover, patients often develop drug resistance (Tong et al., 2016). Therefore, exploring effective and safe targeted chemotherapeutic agents for treating NSCLC are urgently needed. Evodiamine (EVO), a naturally occurring indole alkaloid with low toxicity, is one of the main bioactive constituents of the herbal medicine Euodiae Fructus (Chinese name, Wu-Zhu-Yu). Our previous studies and other studies have reported that EVO exerts inhibitory effects on NSCLC cell growth, metastasis and angiogenesis (Ogasawara and Suzuki, 2004; Wang et al., 2014), and potently suppresses Notch3 activity by activating the DNMTs-induced Notch3 methylation in NSCLC cells (Su et al., 2018).
Abbreviations: ADAM, a disintegrin and metalloproteinase; CETSA, cellular thermal shift assay; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; DNMTs, DNA methyltransferase; EMT, epithelial-mesenchymal transition; EVO, evodiamine; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NSCLC, non-small cell lung cancer; PBS, phosphate buffered saline; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate; SDSPAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis ⁎ Corresponding authors. E-mail addresses: [email protected] (T. Su), [email protected] (L. Lu). https://doi.org/10.1016/j.phymed.2020.153176 Received 7 July 2019; Received in revised form 6 January 2020; Accepted 30 January 2020 0944-7113/ © 2020 Elsevier GmbH. All rights reserved.
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EVO is considered as a promising potent anti-tumor drug candidate for treating NSCLC (Hong et al., 2014). However, the underlying mechanism of action is not fully understood. Notch signaling plays critical roles in cell differentiation, proliferation and apoptosis. Increasing evidences support that Notch signaling dysregulation is associated with many cancer types including NSCLC (Zou et al., 2018). Notch3 is expressed in about 40% of NSCLC cases (Konishi et al., 2007). Overexpression of Notch3 is associated with enhanced tumorigenesis (Ye et al., 2013); while, Notch3 silencing in tumor models leads to tumor cell death, tumor regression (Capaccione and Pine, 2013) and inhibition of tumor metastasis (Zhou et al., 2013). Hence, inhibition of Notch receptor activation is a compelling treatment strategy. Notch signaling is initiated via ligandreceptor interactions, and this interaction initiates a series of intramembrane cleavages, the last of which is regulated by the γ-secretase enzyme complex (Luistro et al., 2009). Notch activation requires proteolytic cleavage of the receptor by γ-secretase complex (Konishi et al., 2007). Inhibition of Notch activation using γ-secretase inhibitors could induce apoptosis and suppress NSCLC cells proliferation (Konishi et al., 2007). Several γ-secretase inhibitors have been developed and some have entered the clinical trials, such as MK-0752 (Cook et al., 2018) and BMS-708163 (Coric et al., 2012). However, systemic toxicity and drug resistance greatly limit their clinical application (Coric et al., 2012; Krop et al., 2012). EVO is a bioactive alkaloid compound, which exhibits antitumor activities with tolerable toxicity. Our previous studies have demonstrated that EVO potently suppresses Notch3 signaling via activation of the DNMTs-induced Notch3 methylation in NSCLC cells (Su et al., 2018). In this study, we explored whether EVO inhibited Notch3 activation by suppressing of γ-secretase in NSCLC cells and in urethane-induced lung cancer mouse models.
EdU assay A549 cells were seeded in 96-well plates (2 × 103 cells/well), and treated with vehicle or EVO (4 and 8 μM) for 48 h. Then cells were incubated with EdU labeling medium (50 μM) for 2 h at 37 °C with 5% CO2, fixed with 4% paraformaldehyde (pH 7.4) for 30 min, and incubated with glycine for 5 min. After rinsed with PBS, cells were incubated with anti-EdU working solution at room temperature for 30 min, and then stained with DAPI (1 ×). The images were captured using Leica DMI 3000B (Leica, GER). Percentages of EdU-positive cells were calculated from five random fields in two wells. Cell cycle analysis A549 cells were seeded in 6-well plates (2.5 × 105 cells/well). After treatment with vehicle or EVO (4 and 8 μM) for 48 h, cells were collected, rinsed with PBS, and fixed with ice-cold 70% ethanol at 4 °C overnight. Then the fixed cells were stained with PI (BD Biosciences, San Diego, USA), and then analyzed using a FACS AriaIII (BD Biosciences). Data were analyzed by FlowJo 7.6 software. Soft agar colony assay A549 cells were suspended in medium containing 0.4% agar (1000 cells/well), and then added in 6-well plates filled with 1% agar. After treatment with vehicle or EVO (4 and 8 μM) for two weeks, cell colonies were counted and photographed. Wound healing assay A549 cells were seeded in 6-well plates, when cells were grown to 85% confluence, cell monolayers were scratched using a plastic pipette tip. Then, cells were treated with vehicle or EVO (4 and 8 μM) in serumfree RPMI-1640 medium. Migration of cells into the wound area were photographed at 0, 12, and 24 h time points using Leica DMI 3000B (Leica, GER) at 10 × magnification. Initial and final wound sizes were measured using Image ProPlus 6.0 software.
Materials and methods Reagents and antibodies EVO (purity > 98%) was purchased from Dalian Meilun Biotech Co., Ltd. (Dalian, China). 3-(4,5-dimethylthiazol-2-yl)−2, 5-diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Propidium iodide (PI) staining was purchased from Biosciences (BD Biosciences, NJ, USA). Cdc2, CyclinB1, NICD, JAG2, ADAM9, NUMB, Nicastin, PSEN1, PSEN2 and PEN2 primary antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). β-actin, E-cadherin, N-cadherin and Vimentin primary antibodies were purchased from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA). Notch3 primary antibody was purchased from Abcam (Cambridge, UK).
Preparation of cytoplasmic and nuclear fractions A549 and H1299 cells were seeded as described in Section “Cell cycle analysis”. After incubation for 48 h, EVO-treated and vehicletreated cells were collected. Cytoplasmic and nuclear extracts were prepared as described previously (Cao et al., 2014). Western blotting A549 and H1299 cells were treated with vehicle or EVO (4 and 8 μM for A549 cells; 1 and 2 μM for H1299 cells) for 48 h, respectively. Then, the cells were lysed using RIPA lysis buffer. Tumors in each group , which collected from our previous study (Su et al., 2018) were rinsed with PBS for three times, and then extracted with RIPA lysis buffer using Tissuelyser-24 (Shanghai Jingxin Industrial Development Co., Ltd., Shanghai, China). All protein samples were quantified using Coomassie Brilliant Blue Kit (Bio-Rad, Hercules, CA, USA), and then separated by SDS-PAGE, transferred onto PVDF membranes, blocked with 5% skimmed milk for 1 h, and incubated with the primary antibodies at 4 °C overnight, subsequently, membranes were washed and then incubated with secondary antibodies for another 1h. ECL chemiluminescence reagent was applied to detect for fluorescent signals using FluorChem E (Santa Clara, CA, USA). Protein bands were quantified using Quantity One software (Bio-Rad, Hercules, CA, USA).
Cell culture Two human NSCLC A549 and H1299 cell lines were purchased from ATCC (Manassas, Virginia, USA). Cells were cultured in complete RPMI1640 medium (GIBCO, USA) containing 10% (v/v) FBS (GIBCO, USA) and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO2 atmosphere. Cell viability assay MTT assay was used to determine the cytotoxic effect of EVO on A549 and H1299 cells. Cells were seeded in 96-well plates (4 × 103 cells/well), cultured overnight, and then treated with various concentrations of EVO (1, 2, 4, 8, 16 μM) or vehicle for 24, 48 and 72 h. After that, cells were treated with the MTT solution (5 mg/ml) and incubated for an additional 3 h. The formazan crystal formed was dissolved with 100 μl of DMSO; absorbance was detected at 490 nm by a microplate reader Victor X3 (PerkinElmer, Waltham, MA, USA).
Cellular thermal shift assay (CETSA) A549 cells were lysed using Tissuelyser-24 (Shanghai Jingxin 2
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adapted for researches on stemness of the malignant cells, which is considered as a hallmark of carcinogenesis (Jin et al., 2017). Here, we found that EVO significantly reduced the colony number and reduced the colony size in A549 cells (Fig. 2A).
Industrial Development Co., Ltd., Shanghai, China). After centrifugated at 14,000 rpm for 20 min, the supernatants were collected and divided into two groups, one group treated with EVO (100 μM) and the other group treated with the same volume of solvent (DMSO) for 30 min at room temperature. Then, the respective lysates were divided into six aliquots and heated individually at 45, 49, 53, 57, 61 and 65 °C for 7 min (Veriti thermal cycler, Applied Biosystems/Life Technologies), followed by cooling for 3 min at room temperature. After centrifuged at 20,000 × g for 20 min at 4 °C, the heated lysates were separated and analyzed by SDS-PAGE. The melting curves were fitted by GraphPad Prism 5.0 software.
EVO inhibited NSCLC cells metastasis Metastasis, which involves migration of tumor cells, is a critical process for NSCLC progression and development (Zhang et al., 2017). To test the effects of EVO on NSCLC cells migration, wound healing assay was performed. As shown in Fig. 2B, A549 cells migrated to the wound area after incubation for 24 h, while EVO dose-dependently inhibited the cells migration. Epithelial-mesenchymal transition (EMT) is a physiological process in which epithelial cells acquire the motile and invasive characteristics of mesenchymal cells. It is thought to be a key event for tumor metastasis (Huang and Zong, 2017). Here, we determined the protein levels of EMT-related molecules, such as N-cadherin, Vimentin and E-cadherin. As expected, EVO treatment significantly reduced the protein levels of N-cadherin and Vimentin (mesenchymal markers) in A549 cells, whereas, the protein level of Ecadherin (epithelial marker) was increased (Fig. 2C). All these results suggested that EVO suppresses NSCLC cells metastasis.
Molecular docking The docking study of γ-secretase and EVO was performed by Autodock vina software. The 3D structure of γ-secretase (PDB: 5FN2) was downloaded from PDB database, ligand was removed via PyMOL and prepared in ADT for docking. EVO structure (PUBCHEMCID: 442088) was converted to 3D coordinates by open Babel 2.3. MK-0752, a well-known γ-secretase inhibitor (Cook et al., 2018), was used as a reference ligand for verifying the binding ability of EVO. The size of space was set as 70 × 90 × 70 point and other parameters were performed with default of the docking software. Docking results were viewed by PyMOL v1.7 software.
EVO inhibited Notch3 signaling in NSCLC cells Notch signaling, a highly conserved pathway, regulates many key cellular processes, such as cell proliferation, apoptosis, metastasis and so on (Ranganathan et al., 2011). Notch3 is mainly expressed in NSCLC (Li et al., 2011). In our previous study, we have demonstrated that EVO potently inhibited Notch3 activation (Su et al., 2018). However, whether it could affect other molecules that are involved in this signaling pathway is not known. NUMB is a membrane-associated protein, which negatively regulates Notch signaling (Bolos et al., 2007). Studies suggest that silence or mutation of NUMB activates Notch signaling and leads to tumorigenesis (Bolos et al., 2007). As expected, EVO significantly increased the protein levels of NUMB in NSCLC cells (Fig. 3A). JAG1 and JAG2 are the two canonical Notch ligands that promote the tumorigenesis and metastasis in NSCLC (Kangsamaksin et al., 2015; Vaish et al., 2017). Here, we found that EVO dose-dependently reduced the protein levels of JAG2 (Fig. 3A) but not JAG1 in both A549 and H1299 cells, suggesting that EVO inhibites the JAG2Notch3 pathway in NSCLC cells. Upon the ligand binding, Notch receptors undertake two cleavage processes mediated by a member of a disintegrin and metalloproteinase (ADAM) family, as well as the γ-secretase complex (a membrane-embedded protease that controls a series of important cellular functions through substrate cleavage) (Lu et al., 2014), leading to the Notch intracellular domain (NICD) release. The released NICD translocates to the nucleus and regulates the expressions of downstream targets (Andersson and Lendahl, 2014; Rizzo et al., 2008). As shown in Fig. 3A, EVO notably decreased the protein levels of ADAM9 and the γ-secretase complex including Nicastrin, PSEN1, and PSEN2 in a dose-dependent manner. Moreover, the protein levels of NICD in nuclear fractions of both A549 and H1299 cells were significantly reduced by EVO in a dose-dependent manner (Fig. 3B).
Statistical analysis All data were presented as the mean ± standard deviation (SD). Significant differences were analyzed by one-way analysis of variance (ANOVA) followed by the LSD post-hoc test using SPSS 19.0 software. Statistical analyses were carried out using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). Statistical differences were considered significant at p < 0.05. Results EVO reduced viability and inhibited proliferation in NSCLC cells The MTT assay was used for determining the cytotoxicity of EVO on A549 and H1299 cells. Cells were treated with vehicle or various concentrations of EVO for 24, 48 and 72 h. As shown in Fig. 1A, EVO reduced A549 and H1299 cells viability in both time- and dose-dependent manners (Fig. 1A), which was consistent with our previous observations (Su et al., 2018). To determine whether EVO inhibited cell proliferation, the EdU assay was performed. In contrast to the abundant EdU-positive cells observed in control, EVO treatments significantly suppressed NSCLC cells proliferation (Fig. 1B). EVO induced G2/M cell cycle arrest in A549 cells Flow cytometric analysis of cell cycle distribution demonstrated that EVO dose-dependently increased the percentage of A549 cells at G2/M phase from 21.78% in control to 41.90% and 76.04% in EVO treatment groups (4 and 8 μM, respectively) (Fig. 1C). Cdc2 and CyclinB1 are two key proteins required for cell cycle regulation and cell progression from G2 to M phase. Both of them play an important role in this phase transition during the cell cycle (Zhang et al., 2011). In agreement with the cell cycle distribution results, EVO treatment notably reduced the protein levels of CyclinB1 and Cdc2 in the A549 cells in a dose-dependent manner (Fig. 1D).
EVO showed potent binding affinity with γ-secretase Since EVO has potent inhibitory effect on Notch3 signaling in NSCLC cells, we sought to explore whether EVO directly binds to Notch3 (the target protein). CETSA is a commonly used assay to verify the binding activity of a drug to its target protein in cells (Molina et al., 2013). It is noteworthy that no liner affinity was observed on Notch3 (Fig. 4A), suggesting that EVO and Notch3 may not have direct binding effect. Notch activation requires proteolytic cleavage of the receptor by γ-secretase complex (Konishi et al., 2007), hence, we further
EVO reduced stemness of A549 cells Soft agar colony formation assay is a well-established method to evaluate cellular anchorage-independent growth. It has been widely 3
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Fig. 1. EVO reduced viability, inhibited proliferation and induced G2/M cell cycle arrest in NSCLC cells. (A) Cell viability was measured by the MTT assay. Both A549 and H1299 cells were treated with various concentrations of EVO or vehicle for 24, 48 and 72 h, respectively. *p < 0.05, **p < 0.01 vs. vehicle (24 h); #p < 0.05, ##p < 0.01 vs. vehicle (48 h); &&p < 0.01 vs. vehicle (72 h). (B) A549 cells were treated with vehicle or EVO (4 and 8 μM) for 48 h. Cells were fixed, incubated with anti-EdU working solution, and then stained with DAPI. The images were captured using Leica DMI 3000B (Leica, GER). Representative images (left panel) and relative fluorescent levels (right panel) were shown. The number of EdU-labeled cells in vehicle group was regarded as 100. (C) After fixation, cells were stained with PI and then analyzed using a flow cytometry. Representative images (left panel) and the statistical analysis of cell cycle data in three independent experiments (right panel) were shown. (D) Protein levels of Cdc2 and CyclinB1 were determined by Western blotting. Data are shown as mean ± SD from three independent experiments, *p < 0.05, **p < 0.01, vs. corresponding control.
Fig. 2. EVO suppressed stemness and metastasis in NSCLC cells. (A) Representative images of colony formation were captured after EVO (4 and 8 μM) treatment for two weeks (scale bar: 100 μm), and the quantification of colony numbers were shown. (B) A549 cells were plated in 6-well plates, and 1 day later, wounds were created and then followed by vehicle or EVO (4 and 8 μM) treatments. Each scratch was photographed after 12- and 24-h treatment. Relative migration ratio (%) ==Treatment group [Gap width (0 h) - Gap width (12 or 24 h)]/Control group [Gap width (0 h) - Gap width (12 or 24 h)] × 100%. (C) Protein levels of EMTrelated markers (E-cadherin, N-cadherin and Vimentin) were determined by Western blotting. Data are shown as mean ± SD from three independent experiments, **p < 0.01, vs. corresponding control.
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Fig. 3. EVO suppressed Notch3 signaling in NSCLC cells. (A) A549 cells were treated with various concentrations of EVO or vehicle for 48 h, then total cell lysates were extracted for immunoblotting by using antibodies specific to Notch3, JAG1, JAG2, NUMB, ADAM9 and γ-secretase complex. The representative figure (left panel) and the relative protein levels analyzed by Image J software (right panel) were shown. (B) Protein levels of NICD in cytoplasmic and nucleus extracts were examined by immunoblotting (left panel); and quantitative results were analyzed using Image J software (right panel). β-actin and Histone H3 served as loading controls of cytoplasmic and nuclear extractions, respectively. Data are shown as mean ± SD from three independent experiments, *p < 0.05, **p < 0.01, vs. corresponding control.
determined whether EVO could affect the γ-secretase complex. Molecular docking assay was performed to find out the binding energy. MK0752, a γ-secretase inhibitor, was selected as reference ligand. The docking studies of γ-secretase complex with EVO or MK-0752 were performed using the same parameters. Optimal binding conformation of the γ-secretase-EVO complexes were presented in Fig. 4B, as well as the binding affinity of each complex were shown in Fig. 4C. Results showed that EVO had similar binding affinity to MK-0752 in γ-secretase docking domain (PDB ID 5FN2). To further confirm the binding effect of EVO to γ-secretase complex, we used the CETSA assay. Interestingly, EVO notably shifted Tm50 of PSEN2, whereas there is no liner affinity on PSEN1 and PEN2 (Fig. 4D). Furthermore, we found that the band intensity of PSEN2 was dose-dependently increased after EVO treatment (Fig. 4E). PSEN2 is a polytopic transmembrane protein, which functions as a part of the γ-secretase protein complex (Konishi et al., 2007). These results strongly suggested that EVO effectively inhibited the activation of Notch3 in NSCLC cells, and this suppression may be attributed to the inhibition of the cleavage by γ-secretase. It is well-known that inactivation of the Notch pathway by targeting γ-secretase is a viable strategy for lung cancer treatment (Rizzo et al., 2008). Thus, EVO which inhibits γ-secretase activity, is a promising anti-NSCLC agent.
EVO restrained tumor growth and inhibited Notch3 activation in urethaneinduced tumorigenesis of FVB mice In our previous study, we found that EVO significantly reduced the tumor growth in urethane-induced lung cancer mouse model (Supplementary Fig. 1A). Furthermore, no obvious toxicity and organ weight loss (Supplementary Fig. 1B) were shown in each treatment group. Here, to determine whether EVO affects the Notch3 signaling pathway in vivo, we examined the protein levels of Notch3, NUMB, ADAM9 and the γ-secretase complex in tumors by using Western blotting. As shown in Fig. 5, protein levels of Notch3, JAG2, ADAM9, PSEN1, PSEN2 were remarkably decreased, while the level of NUMB was increased in the lung tumors of the EVO-treated group when compared with that of the vehicle control group, these results are consistent with our in vivo observations. Discussion In the last decades, Chinese medicinal herb has been increasingly used, because it is well-known for its significant role in cancer prevention and treatment. In this study, we investigated the anti-NSCLC activities of EVO, an alkaloid isolated from a Chinese medicinal herb 6
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Fig. 4. EVO showed potent binding activity with γ-secretase. (A) A549 cells were treated with EVO (100 μM) for 30 min, and then heated by individual temperature (45–65 °C) for 7 min. Representative Notch3 bands were shown on the upper panel and the melting curve fitted by GraphPad with Boltzmann sigmoidal were shown on the lower panel. E: EVO; D: DMSO. Relative band intensity (%) = Band intensity in each tempeture/Band intensity in 45 °C × 100%. Band intensity in 45 °C of each group was regarded as 100. Data are shown as mean ± SD from three independent experiments. (B) The optimal binding conformation of the γsecretase-EVO complex. (C) The binding affinity of each complex. Docking studies of γ-secretase with EVO or reference ligand (MK-0752) were performed using the same parameters. (D) Respective lysates were divided into six aliquots and heated individually at 45, 49, 53, 57, 61 and 65 °C for 7 min. Representative bands of PSEN1 and PSEN2 were shown on the upper panel and the melting curve fitted by Graphpad with Boltzmann sigmoidal were shown on the lower panel. E: EVO; D: DMSO. Relative band intensity (%) = Band intensity in each tempeture/Band intensity in 45 °C × 100%. Band intensity in 45 °C of each group was regarded as 100. Data are shown as mean ± SD from three independent experiments. **p < 0.01 vs. corresponding vehicle. (E) A549 cells were treated with different concentrations of EVO (0.1, 1, 10, 100 and 1000 μM) for 30 min, and then heated by 57 °C for 7 min. Representative PSEN2 bands were shown on the upper panel and the relative protein level was shown on the lower panel. Relative band intensity (%) = Band intensity of each concentration of EVO-treated cells/Band intensity in 1000 μM of EVO-treated cells × 100%. The band intensity of 1000 μM of EVO-treated cells was regarded as 100. Data are shown as mean ± SD for three individual experiments. *p < 0.05, **p < 0.01, vs. 0.1 μM of EVO treatment.
Euodiae Fructus. EVO possesses many biological and pharmacological activities that are beneficial to human health. Increasing evidence indicates that EVO has cytotoxic effects on several human cancer cells, such as melanoma cells (Wang et al., 2005), prostate cancer cells (Kan et al., 2007), colon cancer cells (Ogasawara et al., 2001), breast cancer cells (Liao et al., 2005) and lung cancer cells (Lin et al., 2016). EVO inhibits NSCLC cell growth and metastasis (Ogasawara and Suzuki, 2004). However, up to present, the underlying molecular mechanism of the anti-NSCLC action of EVO remains poorly illustrated. In this study, we examined the anti-NSCLC activity of EVO both in vitro and in vivo. In agreement with previous studies, EVO inhibited NSCLC cell viability (Fig. 1A), suppressed cell proliferation (Fig. 1B), induced G2/M cell cycle arrest (Fig. 1C), reduced stemness (Fig. 2A) and inhibited cell migration (Fig. 2B). Importantly, EVO exerted potent antilung cancer effects in urethane-induced lung cancer mouse model (Supplementary Fig. 1A). EVO has advantages over synthetic
compounds, EVO is noncarcinogenic and with low intrinsic toxicity properties. EVO is considered to be a potential candidate agent for NSCLC prevention and treatment. In the in vitro study, based on the different cytotoxicity and IC50 of EVO in A549 and H1299 cells, we used different doses to treat these cell lines. Interestingly, A549 and H1299 cells have distinct genetic backgrounds. Although both A549 and H1299 cells are human epithelial carcinoma cells, A549 is p53 wide-type, while H1299 is p53-null (Li et al., 2004). EVO has different cytotoxicity in these two cell lines, implying that p53 signaling may be involved in the anti-NSCLC effects of EVO, which deserves further investigation. Many effective therapeutics approaches, such as surgery, radiation therapy and chemotherapy are being used for NSCLC treatment. Recently, targeted therapy becomes an important part of the management for advanced NSCLC patients. Targeted therapy includes tyrosine kinase inhibitors of the active epidermal growth factor receptor (EGFR) 7
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Fig. 5. EVO suppressed Notch3 signaling in vivo. Protein levels of Notch3, NUMB, JAG2, ADAM9 and γ-secretase complex in the lung tumors of each group were detected by Western blotting. The representative figure (left panel) and the relative protein levels analyzed by Image J software (right panel) were shown. Data are shown as mean ± SD from three independent experiments, *p < 0.05, **p < 0.01, vs. vehicle-treated group.
mutations (e.g. erlotinib, gefinitib and afatinib), monoclonal antibodies against vascular endothelial growth factor (VEGF) (e.g. bevacizumab) and anaplastic lymphoma kinase (ALK) rearrangement (e.g. crizotinib). In 2014, the European Medicines Agency approved five of them for NSCLC treatment, which include afatinib, bevacizumab, crizotinib, gefinitib and erlotinib (Sculier et al., 2015). Unfortunately, drug resistance is often developed in less than one year (Chong and Janne, 2013). Therefore, identification of the novel molecular targets for treating NSCLC are urgently needed. Notch3 is overexpressed in about 40% of NSCLC cases, and has been proposed as a therapeutic target of NSCLC. Inhibition of Notch3 activation represents a compelling treatment strategy. Notch3 is one of the mammalian Notch family receptors. All the four members of the Notch family have been implicated in various types of cancers including lung cancer (Motooka et al., 2017). It has been reported that Notch3 signaling is highly context-dependent in solid tumors, which can promote or inhibit tumor growth. Multivariate analysis demonstrated that Notch3 is a potential biomarker for predicting the prognosis of NSCLC patients (Ye et al., 2013). Inhibiting Notch3 activation could inhibit tumor growth in NSCLC (Konishi et al., 2007). Consistently, in this study, we found that EVO exerted anti-NSCLC effects and potently inhibited Notch3 signaling in cultured NSCLC cells (Fig. 3) and in tumor of the lung cancer mouse model (Fig. 5). To further elucidate the mechanisms of EVO-induced Notch3 inhibition, we used molecular docking assay to predict the binding affinity, and the CETSA assay to verity the binding affinity of EVO to its target proteins. Interestingly, EVO showed more potent binding activity with γ-secretase than with Notch3 (Fig. 4). γ-secretase, a large protease complex, is composed of a catalytic subunit (PSEN1 and PSEN2) and accessory subunits (Aph1, PEN2 and nicastrin) (Kaether et al., 2006). γ-secretase has been considered as a molecular switch for Notch3 signaling; and it is one of the most promising targets to inhibit the Notch3 signaling, which has been considered as a molecular switch for Notch3 signaling activation (Shih Ie and Wang, 2007). More and more preclinical studies have suggested that γ-secretase inhibitors hold promise as a new targetbased therapy for the tumors with aberrant Notch activation (Shih Ie and Wang, 2007). Nevertheless, these inhibitors have substantial side effects that hinder their clinical application (Capaccione and Pine, 2013). The main adverse toxicity of γ-secretase inhibitors shown
in patients is dose-limiting secretory diarrhea (Wong et al., 2004). In addition, some pharmacological studies show that γ-secretase inhibitors cause reversible thymic suppression (Wong et al., 2004), reversible hair depigmentation and body weight loss in mice (Rizzo et al., 2008). Hence, exploring new γ-secretase inhibitors for treating NSCLC are critically needed. EVO with low toxicity but exerts anti-NSCLC activities, it inhibits Notch3 signaling by inhibiting the γ-secretase cleavage of Notch3 (Fig. 4). It is expected that EVO has a great potential to be developed as a novel γ-secretase inhibitor for lung cancer prevention or treatment. Currently, increasing evidences highlight natural products with antitumor activities (Wang et al., 2013). In Chinese medicinal herb Euodiae Fructus, more than twenty alkaloids have been isolated, and some of them are shown to have potent anti-lung cancer activities (Lin et al., 2016). EVO is a major active component in the total alkaloids. Further studies are warranted to identify more active constituents from Euodiae Fructus that can serve as anti-cancer agents. Conclusion In summary, we demonstrate that EVO inhibits cell viability, induces G2/M cell cycle arrest, suppresses cell migration and reduces stemness in human NSCLC cells. Further studies show that EVO potently inhibits Notch3 signaling via preventing its cleavage at the cell surface by the γ-secretase complex. Moreover, EVO reduces tumor numbers and inhibits the Notch3 in vivo. These findings suggest that EVO exerts anti-NSCLC effects and these effects are at least in part attributed to the inhibition of Notch 3 signaling via direct binding to γsecretases. This study provides a pharmacological basis for developing EVO as a novel phytotherapeutic agent against NSCLC. Author contributions X.Y., Y.M.Z. and Y.F.H. performed the majority experiments. X.X.Q. and Y.W. participated in some experiments. T.S.interpreted the data and drafted the manuscript. T.S. and L.L.L. supervised the study, reviewed the original data and finalized the manuscript. All authors have read and approved the final manuscript. 8
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Declaration of Competing Interest
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The authors declare that they have no competing interests. Acknowledgments This work was supported by the projects of National Natural Science Foundation of China (81874367 and 81703705), Natural Science Foundation for Distinguished Young Scholars of Guangdong Province, China (2017A030306033), Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2016), Project of Educational Commission of Guangdong Province of China (2016KTSCX012), Pearl River Nova Program of Guangzhou, China (201710010108). Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2020.153176. References Allenspach, E.J., Maillard, I., Aster, J.C., Pear, W.S., 2002. Notch signaling in cancer. Cancer Biol. Ther. 1, 466–476. Andersson, E.R., Lendahl, U., 2014. Therapeutic modulation of Notch signalling-are we there yet? Nat. Rev. Drug Discov. 13, 357–378. Bolos, V., Grego-Bessa, J., de la Pompa, J.L., 2007. Notch signaling in development and cancer. Endocr. Rev. 28, 339–363. Capaccione, K.M., Pine, S.R., 2013. The Notch signaling pathway as a mediator of tumor survival. Carcinogenesis 34, 1420–1430. Chong, C.R., Janne, P.A., 2013. The quest to overcome resistance to EGFR-targeted therapies in cancer. Nat. Med. 19, 1389–1400. Cook, N., Basu, B., Smith, D.M., Gopinathan, A., Evans, T.J., Steward, W.P., Hagemann, T., Venugopal, B., Tuveson, D.A., Hategan, M., Anthoney, D.A., Hampson, L.V., Nebozhyn, M., Tuveson, D., Farmer-Hall, H., Turner, H., McLeod, R., Halford, S., Jodrell, D., 2018. A phase I trial of the gamma-secretase inhibitor (GSI) MK-0752 in combination with gemcitabine in patients with pancreatic ductal adenocarcinoma. Br. J. Cancer 118 (6), 793–801. Coric, V., van Dyck, C.H., Salloway, S., Andreasen, N., Brody, M., Richter, R.W., Soininen, H., Thein, S., Shiovitz, T., Pilcher, G., Colby, S., Rollin, L., Dockens, R., Pachai, C., Portelius, E., Andreasson, U., Blennow, K., Soares, H., Albright, C., Feldman, H.H., Berman, R.M., 2012. Safety and tolerability of the gamma-secretase inhibitor avagacestat in a phase 2 study of mild to moderate alzheimer disease. Arch. Neurol. 69, 1430–1440. Cao, H.H., Tse, A.K., Kwan, H.Y., Yu, H., Cheng, C.Y., Su, T., Yu, Z.L., 2014. Quercetin exerts anti-melanoma activities and inhibits STAT3 signaling. Biochem. Pharmacol. 87 (3), 424–434. Hong, J.Y., Park, S.H., Min, H.Y., Park, H.J., Lee, S.K., 2014. Anti-proliferative effects of evodiamine in human lung cancer cells. J. Cancer Prev. 19, 7–13. Huang, R., Zong, X.Y., 2017. Aberrant cancer metabolism in epithelial-mesenchymal transition and cancer metastasis: mechanisms in cancer progression. Crit. Rev. Oncol. Hemat. 115, 13–22. Jin, Z., Feng, W., Ji, Y., Jin, L., 2017. Resveratrol mediates cell cycle arrest and cell death in human esophageal squamous cell carcinoma by directly targeting the EGFR signaling pathway. Oncol. Lett. 13, 347–355. Kaether, C., Haass, C., Steiner, H., 2006. Assembly, trafficking and function of gammasecretase. Neurodegener. Dis. 3, 275–283. Kan, S.F., Yu, C.H., Pu, H.F., Hsu, J.M., Chen, M.J., Wang, P.S., 2007. Anti-proliferative effects of evodiamine on human prostate cancer cell lines DU145 and PC3. J. Cell Biochem. 101, 44–56. Kangsamaksin, T., Murtomaki, A., Kofler, N.M., Cuervo, H., Chaudhri, R.A., Tattersall, I.W., Rosenstiel, P.E., Shawber, C.J., Kitajewski, J., 2015. NOTCH decoys yhat selectively block DLL/NOTCH or JAG/NOTCH disrupt angiogenesis by unique mechanisms to inhibit tumor growth. Cancer Discov. 5, 182–197. Konishi, J., Kawaguchi, K.S., Vo, H., Haruki, N., Gonzalez, A., Carbone, D.P., Dang, T.P., 2007. Gamma-secretase inhibitor prevents Notch3 activation and reduces proliferation in human lung cancers. Cancer Res. 67, 8051–8057. Krop, I., Demuth, T., Guthrie, T., Wen, P.Y., Mason, W.P., Chinnaiyan, P., Butowski, N., Groves, M.D., Kesari, S., Freedman, S.J., Blackman, S., Watters, J., Loboda, A., Podtelezhnikov, A., Lunceford, J., Chen, C., Giannotti, M., Hing, J., Beckman, R., Lorusso, P., 2012. Phase I pharmacologic and pharmacodynamic study of the gamma secretase (Notch) inhibitor MK-0752 in adult patients with advanced solid tumors. J. Clin. Oncol. 30, 2307–2313. Li, M., Brooks, C.L., Kon, N., Gu, W., 2004. A dynamic role of HAUSP in the p53-Mdm2 pathway. Mol. Cell 13, 879–886.
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