Mol Cell Biochem DOI 10.1007/s11010-013-1920-3
Neurotensin receptor1 antagonist SR48692 reduces proliferation by inducing apoptosis and cell cycle arrest in melanoma cells Yanli Zhang • Shunqin Zhu • Liang Yi Yaling Liu • Hongjuan Cui
•
Received: 7 August 2013 / Accepted: 6 December 2013 Ó Springer Science+Business Media New York 2013
Abstract Malignant melanoma is highly aggressive, and always resistant to conventional chemo-radiotherapy, which results in poor prognosis. As a specific antagonist of neurotensin receptor 1 (NTSR1), emerging evidences confirmed that SR48692 can reverse the pro-growth effect of neurotensin (NTS) by interrupting the interaction between NTS and NTSR1. A375 melanoma cell line was used in this experiment, and SR48692 was employed as the inhibitor of NTS/NTSR1 pathway. We detected the expression of NTSR1 by NTSR1 immunofluorescence and Western blot. After SR48692 treatment, cell proliferation was determined by cell counting, MTT assay and BrdU incorporation study, the cell cycle and apoptosis were performed by flow cytometry. At last Soft Agar Clonogenic assay and xenograft cancer mice model in vivo were used to confirm our result. In this study, we showed that NTSR1 is commonly high expressed in melanoma cells, but low expressed in normal immortalized human keratinocyte line HaCaT. SR48692 not only reduced cell proliferation and
Y. Zhang Y. Liu (&) Department of Dermatology, the Third Hospital of Hebei Medical University, 139 Ziqiang Rd, Shijiazhuang 050000, Hebei, China e-mail: [email protected] S. Zhu Department of Life Science, Southwest University, Chongqing 400716, China L. Yi Neurosurgery Department, Daping Hospital, the Third Military Medical University, Chongqing, China H. Cui State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China
self-renewal potential in vitro, but also inhibited the tumor growth derived from A375 cells in NOD/SCID mice in vivo. Further, we originally reported that SR48692 inhibited cell proliferation through cell cycle arrest and apoptosis. Considering the favorable toxicity profile in vitro and in vivo though targeting NTS/NTSR1, SR48692 is worthy of further study and exploitation in melanoma treatment. Keywords Malignant melanoma Neurotensin NTSR1 SR48692 Cell proliferation Apoptosis Cell cycle Abbreviations MM Malignant melanoma NTS Neurotensin NTSR1 Neurotensin receptor 1 FBS Fetal bovine serum BrdU 5-bromo-2-deoxyuridine PI Propidium iodide PBS Phosphate-buffered saline MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
Introduction Malignant melanoma (MM) is a highly aggressive tumor and always resistant to traditional chemo-radiotherapy. With highly incidence and increasing mortality, melanoma becomes the leading death of skin neoplasm in developed countries. The majority of patients with early-stage melanoma can be cured by surgical excision, while for the advanced stage, the standard chemotherapeutic approaches
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have been disappointing to improve the prognosis in the last three decades. So it is necessary to identify some new chemotherapeutic regimens with high efficacy and low toxicity for melanoma treatment. Neurotensin (NTS) is a single polypeptide chain containing 13 amino acid residues, which is discovered by Carraway and Leeman in 1973 [1]. NTS works as a neuromodulator in the central nervous system, while it works as an endocrine agent in the periphery, which is released into the blood from the entero-endocrine N cells in response to various biologic activities. NTS plays an important role in some physiologic actions of gastrointestinal tract including the stimulation of gastric acid, pancreatic secretions, and increasing biliary secretion to facilitate the absorption of fatty acid by hormonal and neurocrine regulation [2]. More data also showed that NTS promoted tumor development in various types of cancer including lung cancer [3], prostate cancer [4, 5], colon cancer [6, 7], pancreas cancer [8, 9], pancreatic ductal adenocarcinoma [10], and breast cancer [11]. The biologic activities of NTS are mainly mediated by interaction with specific receptors including two G protein coupled neurotensin receptors NTSR1 and NTSR2, a new type of neurotensin receptor NTSR3 belonging to the sortilin receptor superfamily was also found [12]. Many studies showed NTS and NTSR1 were highly expressed in some types of cancers but not in normal cells and tissues [11, 13–18]. This implicated a functional link between NTS/NTSR1 high expression and neoplastic progression.SR-48692,2-([1-(7-Chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carbonyl]amino)admantane2-carboxylic acid, is a drug which acts as a selective, nonpeptide antagonist at the neurotensin receptor NTSR1, and was the first non-peptide antagonist developed for this receptor [19, 20]. It was used in scientific research to explore the interaction between neurotensin and other neurotransmitters in the brain [19, 21–26], and could inhibit cell growth and tumor development through competing with NTS to interact with NTSR1, which indicated that NTS/NTSR1 could be a potential biomarker and a therapeutic target in some cancers. Nevertheless, there is no report about NTS/NTSR1 in melanoma, and the role of SR48692 in melanoma cells is not clear. Here, we investigated NTSR1 expression, and found that SR48692 inhibited cell proliferation through induced cell cycle arrest and triggered apoptosis in A375 melanoma cell line. Moreover, we also showed that SR48692 decreased self-renewal capacity in vitro and tumor formation in vivo. This revealed that SR48692 is worthy of further study and exploitation in melanoma treatment.
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Materials and methods Cell culture The human melanoma cell line A375 was grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10 % fetal bovine serum (FBS; Gibco) and 1 % penicillin–streptomycin (P/S). Human melanoma cell line MV3 and immortalized human keratinocyte line HaCaT was grown in Roswell Park Memorial Institute-1640 (RPMI-1640; Gibco) supplemented with 10 % FBS and 1 % P/S. Cells were cultured at 37 °C in a 5 % CO2 humidified incubator. Immunofluorescence staining assay Cells were grown on coverslips in 24-well plate, washed with PBS, fixed in 4 % paraformaldehyde (PFA) for 20 min, and permeabilized with 0.3 % TritonX-100 for 5 min. Cells were blocked with 10 % goat serum for 1 h, incubated with a primary rabbit anti-NTSR1 (1:200; Abcam) for 2 h, IgG was used for negative control, then incubated with the secondary antibody goat anti-rabbit IgG (1:500) for 1 h, and counterstained with 300nM DAPI. Cells were examined using a Nikon microscope (80i) with Image-Pro Plus software for image analysis. Western blotting assay A375 and MV3 melanoma cell line were harvested and suspended in RIPA Lysis Buffer. A normal immortalized human keratinocyte line HaCaT was used as a control. Protein concentrations were determined with Enhanced BCA protein assay kit (Beyotime), using bovine serum albumin as reference. Proteins (50 lg) were separated on 10 % sodium dodecyl sulfate polyacrylamide gels (SDSPAGE), transferred to nitrocellulose membranes, and probed with antibodies rabbit anti-NTSR1 (1:1000; Abcam) or mouse anti-a-tubulin (1:5000; Sigma), respectively, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (ICN, CA). Proteins were visualized with BeyoECL Plus (Beyotime, China). Cell proliferation assay Cell proliferation was evaluated by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. After 12 h cell culture, the medium was removed and attached cells were rinsed with PBS, and 200 ll DMEM only w/o FBS was added and separately incubated with 0.01 lM NTS and 0.01 lM ?5 lM SR48692 at
Mol Cell Biochem
indicated concentration, DMSO was used as a control. 20 ll MTT (5 mg/ml) was added to each well after indicated time and incubated at 37 °C for 2 h. The medium was removed, and the formazan complex was dissolved with 200 ll DMSO. Absorbance of the complex was measured with a micro-plate reader at a wavelength of 570 nm in order to detect metabolically intact cells. BrdU feeding and staining Cells were grown on coverslips in 24-well plate, cultured in DMEM without FBS, and treated with either DMSO or 5 lM SR48692 for 3 days. After treatment, the thymidine analog 5-bromo-2- deoxyuridine (BrdU; Sigma) stock solution at 10 mg/ml in saline was diluted 1,0009 in the culture medium and incubated for 1 h. After that, cells were treated and fllowed the protocol as immunofluorescence staining assay, except the antibody is against BrdU (1:200, Abcam). Cell cycle assay Cells were plated in 10 cm plates and treated with either 5 lM SR48692 or DMSO. After 3 days treatment, cells were washed with ice-cold PBS, fixed with 70 % ethanol, stained with propidium iodide (PI) for 30 min, and analyzed by flow cytometry. The data were analyzed with CellQuest Pro software (BD BioSciences). The experiments were repeated at least three times. Apoptosis assay After treated with either DMSO or 5 lM SR48692 for 3 days, cells were stained with Hoechst 33258 and imaged using a Nikon microscope (209). The apoptotic cells were determined with the Annexin V-FITC apoptosis detection kit (Sigma). After treatment, cells were collected and washed with PBS buffer, resuspended in 100 ll of binding buffer, incubated with 5 ll of Annexin V-FITC and 10 ll PI for 15 min, and analyzed by flow cytometry. Annexin V?/PI– cells were recognized as apoptotic, Annexin V?/ PI? cells were defined as late apoptotic, Annexin V–/PI? cells were defined as necrotic, and Annexin V–/PI– cells were recognized as normal cells. The experiments were repeated at least three times.
Next, 8-mm pore size transwell culture inserts (Transwell; Costar) were placed into the wells of 24-well culture plates to separate the upper and the lower chamber. 1.0 9 105 cells were plated into the upper chamber containing 200 ll serum-free medium while the lower chambers contained 700 ll 0.1 % serum medium. After 24 h incubation with 5 lM SR48692 or DMSO, cells were staining with Crystal Violet, and image of the migration cells were collected, and the number of migration cells was quantified by counting ten independent visual fields under the microscope using a 209 objective. Soft agar clonogenic and in vivo tumorigenic assays For soft agar assay, 1,000 cells were mixed with 0.35 % Noble agar in growth medium containing DMSO or 5 lM SR48692 and plated onto six-well plates containing a solidified bottom layer (0.6 % Noble agar in growth medium) per well. After incubation for 14 days, cells were stained with MTT and imaged using a digital camera. Colonies containing more than 50 cells were counted using an inverted microscope. For xenograft mice model, 2 9 106 cells in 200 ll DMEM were harvested and injected into the right lateral flanks of NOD/SCID mice. After 7 days tumor growth, the mice were randomly divided into two groups of three mice each, and received the following treatments with intraperitoneal injections of either 100 ll DMSO, or SR48692 (5 mg/kg) for 14 days daily. Tumor growth was estimated by caliper measurements, and tumor volume was calculated with the ellipsoid formula: volume = tumor length 9 width2 9 0.5236. After treatment, mice were sacrificed and the tumors were removed and weighed. All animal experiments were preapproved by the Institutional Animal Care and Use Committee of the Southwest University. Statistical analysis Quantitative data were reported as the average ± SD. Oneway ANOVA and the two-tailed Student’s t test were used to measure differences using the SPSS17.0 software package. A minimum of three independent experiments were performed. Differences were considered statistically significant at p \ 0.05.
Wound-healing and cell migration assays
Results
Cells were plated and reached full of confluence, scarification was done with a pipette tip. Cells were maintained and treated with 5 lM SR48692 or DMSO. Pictures were taken at indicated time, cells treated with DMSO were used as a control.
NTSR1 is remarkably expressed in A375 melanoma cell line Since there is no any report about NTSR1 expression in melanoma cells, we investigated the NTSR1 expression
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Fig. 1 NTSR1 expression was detected in A375 melanoma cell line through immune-staining assay a Immunofluorescent staining was carried out to detect the expression of NTSR1 in A375 melanoma cell line (green), and the nuclei were counterstained with DAPI (blue).
IgG was used for negative control. b A normal immortalized human keratinocyte line HaCaT and two melanoma cell lines A375 and MV3 were harvested and performed Western blotting assay. a-tubulin was used as a loading control
and found that NTSR1 was significantly expressed in A375 cells, and which is located to membrane (Fig. 1a). Western blot analysis also confirmed that NTSR1 was significantly high expressed in melanoma A375 and MV3 cells but low expressed in normal HaCaT cells (Fig. 1b).
We assessed the apoptosis by Hoechst 33258 staining and flow cytometry analysis. Cell treated with SR48692 exhibited remarkable cell shrinkage changes, suggesting cell apoptosis (Fig. 3c top panel). The Hoechst33258 nuclear staining showed the condensation of chromatin and the dissociation of the nuclear membrane after SR48692 treatment, and the number of apoptotic nuclei increased (Fig. 3c lower panel). As shown in Fig. 3d, Annexin V-FITC/PI staining and flow cytometry analysis also confirmed that SR48692 induced apoptosis from 7.35 to 63.24 % compared with control group (P = 0.001).
SR48692 inhibited cell growth and proliferation in A375 melanoma cells Cells treated with 5 lM SR48692 for 3 days resulted in a significant reduction in the viable cell number compared with DMSO-treated cells (Fig. 2a), and the IC50 of SR48692 for growth inhibition is 3.2243 uM. We further investigated cell growth curve by MTT assay, the result showed that exogenous 0.01 lM NTS immediately increased the cell growth compared with DMSO control, and then we added the inhibitor SR48692 together, which inhibited the cell proliferation which caused by NTS (Fig. 2b). BrdU staining assay also demonstrated that SR48692 significantly inhibited cell proliferation. After treatment with 5 lM SR48692 for 3 days, the percentage of BrdU-positive cells was down-regulated more than half compared with the control group (P = 0.003; Fig. 2c). SR48692 induced the cell cycle arrest and apoptosis As shown in Fig. 3a, b, flow cytometric analysis revealed a significant increase in S phase (P = 0.014) and G2/M phases (P = 0.046), along with reduction in G0/G1 phases (P = 0.004) compared with control cells. These data showed that SR48692 blocked the G2/M transition and induced cell cycle S and G2/M phase arrest in A375 melanoma cells.
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NTS/NTSR1 activation is required for A375 cell migration As shown in Fig. 4a, 24 h after scratching, cell migration in SR48692 group was blocked compared with control group. Transwell migration assay further verified that SR48692 significantly reduced the cell migration (P = 0.004; Fig. 4b). SR48692 inhibited colony formation in vitro and tumor growth in vivo Soft agar assay showed A375 cells treated with SR48692 gave rise to few and small colonies (Fig. 5a). The colony formation was significantly reduced from 11.1 to 5.5 % after SR48692 treatment (P = 0.03) (Fig. 5b) compared with control group. As shown in Fig. 5c and d, the NOD/ SCID xenografted mice model showed that SR48692 treatment was accompanied by a significant 57.6 % decrease in tumor volume and 51.8 % decrease in tumor weight compared with DMSO control group.
Mol Cell Biochem Fig. 2 SR48692 inhibited cell growth and proliferation in melanoma cells. a A375 melanoma cells were treated with DMSO or 5 lM, 10 lM SR48692 for 3 days. Cell morphology was observed (920), and the cell number was determined by trypan blue assay. Data represent the average ± SD of at least three independent experiments. Scale bar, 50 mm. b A375 melanoma cells were treated with 0.01 lM NTS and 0.01 lM ?5 lM SR48692 seprately, DMSO was used as a control. OD570 nm was measured daily from 1 to 4 days. Data are presented as mean ± SD. * P \ 0.05; ** P \ 0.01. c After treated with DMSO and 5 lM SR48692 for 3 days, BrdU was added and incubated for 1 h, cells were stained with an antibody against Brdu (green), counterstained with DAPI (blue), 920. The percentage of Brdu-positive cells was calculated. Cells were counted from at least five randomly selected fields. Data are presented as mean ± SD
Discussion The results presented in this study showed that NTSR1 is significantly high expressed in human melanoma cell line but low expressed in normal HaCaT cell line, which is consistent with reports that NTSR1 is highly expressed in tumor tissue but low-level in normal tissue [11], suggesting that NTS/NTSR1 may be act as a biomarker and a novel drug target in melanoma. We also found that not only exogenous NTS immediately increased the cell growth, and SR48692 could reverse the cell proliferation which caused by NTS, but also
SR48692 significantly abolished self-renewal and colony formation in vitro and tumor growth in vivo, which suggested that SR48692 may serve as an effective therapeutic agent in melanoma treatment. Our studies also demonstrated that SR48692 induced S phase and G2/M cell phase arrest and triggered apoptosis. So inhibition of cell proliferation by SR48692 may be through cell cycle arrest and/or apoptosis in A375 melanoma cells. As we have seen, the changes in cell cycle caused by the addition of SR48692 to A375 cells while significant average about 10 %, so the apoptosis may be the main reason rather than cell cycle arrest to reduce cell growth and proliferation.
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Fig. 3 SR48692 induced cell cycle arrest and triggered apoptosis. a After treated with DMSO and 5 lM SR48692 for 3 days, respectively, cells were stained with PI, and DNA content was determined by flow cytometry. b Statistical analysis of the cell cycle phase. Data represent the average ± SD of at least three independent experiments. Statistical analysis was performed using the 2-tailed
Student t test. c Representative morphologic change of apoptosis (the top panel) and Hoechst 33258 nuclear staining (the lower panel), 920. d After treated with either DMSO or 5 lM SR48692 for 3 days, respectively, apoptosis was determined using Annexin V-FITC/PI staining and flow cytometry. The graph was statistical analysis of the percentage of apoptosis
In addition, we also confirmed that SR48692 could inhibit cell migration, which is consistent with the report in breast cancer cell [11]. But the nature about SR48692 effects remains to be clarified. In summary, this article originally reported that NTSR1 antagonist SR48692 inhibited cell proliferation through
inducing apoptosis and cell cycle arrest in melanoma cells in vitro and in vivo, suggesting that NTSR1 may be a biomarker and a novel drug target in melanoma, and SR48692 may be a potential new drug for targeted therapy in melanoma.
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Mol Cell Biochem Fig. 4 Cell migration was inhibited by SR48692 in melanoma cells through woundhealing assay and transwell migration assays. a A375 melanoma cells were treated with SR48692 for 0, 24, 48 h, and detected the migration by wound-healing assay (910). b Transwell migration assays: Images of the migrating cells were collected after stained with crystal violet; the migration cells on the bottom surface of each membrane were counted. Data represent the average ± SD of three independent experiments. Statistical analysis was performed using the two-tailed Student’s t test
Fig. 5 SR48692 inhibited the colony formation in vitro and tumor growth in vivo. a After treated with 5 lM SR48692 or DMSO in a soft agar culture system for 2 weeks, the colonies were stained with MTT and imaged. SR48692 treatment gave rise to smaller and fewer colonies than control group. (94). b The colony number was counted, data represented the average ± SD of at least three independent
experiments. Statistical analysis was performed using the two-tailed Student’s t test. c The xenograft tumor after treated by DMSO or 5 mg/kg SR48692 was weighted. Data were analyzed with 2-tailed Student t test. d Xenograft tumor growth curve was checked. Data were analyzed with 2-tailed Student t test
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Mol Cell Biochem Acknowledgments This study was supported by the National Basic Research Program of China (No. 2012cb114603), the National Natural Science Foundation of China (No. 31271462, 31172268, 81201551). Disclosure of interest The authors declare no conflict of proprietary, commercial, or financial interest in the products or companies described in this article.
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11. Souaze´ F, Dupouy S, Viardot-Foucault V, Bruyneel E, Attoub S, Gespach C et al (2006) Expression of neurotensin and NT1 receptor in human breast cancer: a potential role in tumor progression. Cancer Res 66(12):6243–6249 12. Dal Farra C, Sarret P, Navarro V, Botto JM, Mazella J, Vincent JP (2001) Involvement of the neurotensin receptor subtype NTR3 in the growth effect of neurotensin on cancer cell lines. Int J Cancer 92(4):503–509 13. Collier N, Bloom S, Hodgson H, Weinbren K, Lee Y, Blumgart L (1984) Neurotensin secretion by fibrolamellar carcinoma of the liver. The Lancet. 323(8376):538–540 14. Moody TW, Carney DN, Korman LY, Gazdar AF, Minna JD (1985) Neurotensin is produced by and secreted from classic small cell lung cancer cells. Life Sci 36(18):1727–1732 15. Evers BM, Ishizuka J, Chung D, Townsend CM Jr, Thompson JC (1992) Neurotensin expression and release in human colon cancers. Ann Surg 216(4):423 16. Di Sant’Agnese Anthony (1992) P. Neuroendocrine differentiation in human prostatic carcinoma. Hum Pathol 23(3):287–296 17. Reubi JC, Waser B, Schaer JC, Laissue JA (1999) Neurotensin receptors in human neoplasms: high incidence in Ewing’s sarcomas. Int J Cancer 82(2):213–218 18. Alifano M, Souaze´ F, Dupouy S, Camilleri-Broe¨t S, Younes M, Ahmed-Zaı¨d S-M et al (2010) Neurotensin receptor 1 determines the outcome of non-small cell lung cancer. Clin Cancer Res 16(17):4401–4410 19. Gully D, Canton M, Boigegrain R, Jeanjean F, Molimard J-C, Poncelet M et al (1993) Biochemical and pharmacological profile of a potent and selective nonpeptide antagonist of the neurotensin receptor. Proc Natl Acad Sci 90(1):65–69 20. Gully D, Jeanjean F, Poncelet M, Steinberg R, Soubrie P, Fur GL et al (1995) Neuropharmacological profile of non-peptide neurotensin antagonists. Fundam Clin Pharmacol 9(6):513–521 21. Rostene W, Azzi M, Boudin H, Lepee I, Souaze F, MENDEZUBACH M et al (1997) Use of nonpeptide antagonists to explore the physiological roles of neurotensin. Ann N Y Acad Sci 814(1):125–141 22. Jolas T, Aghajanian GK (1997) Neurotensin and the serotonergic system. Prog Neurobiol 52(6):455–468 23. Dobner PR, Deutch AY, Fadel J (2003) Neurotensin: dual roles in psychostimulant and antipsychotic drug responses. Life Sci 73(6):801–811 24. Chen L, Yung K, Yung W (2006) Neurotensin selectively facilitates glutamatergic transmission in globus pallidus. Neuroscience 141(4):1871–1878 25. Petkova-Kirova P, Rakovska A, Della Corte L, Zaekova G, Radomirov R, Mayer A (2008) Neurotensin modulation of acetylcholine, GABA, and aspartate release from rat prefrontal cortex studied in vivo with microdialysis. Brain Res Bull 77(2):129–135 26. Petkova-Kirova P, Rakovska A, Zaekova G, Ballini C, Corte LD, Radomirov R et al (2008) Stimulation by neurotensin of dopamine and 5-hydroxytryptamine (5-HT) release from rat prefrontal cortex: possible role of NTR1 receptors in neuropsychiatric disorders. Neurochem Int 53(6):355–361
Neurotensin receptor1 antagonist SR48692 reduces proliferation by inducing apoptosis and cell cycle arrest in melanoma cells Yanli Zhang • Shunqin Zhu • Liang Yi Yaling Liu • Hongjuan Cui
•
Received: 7 August 2013 / Accepted: 6 December 2013 Ó Springer Science+Business Media New York 2013
Abstract Malignant melanoma is highly aggressive, and always resistant to conventional chemo-radiotherapy, which results in poor prognosis. As a specific antagonist of neurotensin receptor 1 (NTSR1), emerging evidences confirmed that SR48692 can reverse the pro-growth effect of neurotensin (NTS) by interrupting the interaction between NTS and NTSR1. A375 melanoma cell line was used in this experiment, and SR48692 was employed as the inhibitor of NTS/NTSR1 pathway. We detected the expression of NTSR1 by NTSR1 immunofluorescence and Western blot. After SR48692 treatment, cell proliferation was determined by cell counting, MTT assay and BrdU incorporation study, the cell cycle and apoptosis were performed by flow cytometry. At last Soft Agar Clonogenic assay and xenograft cancer mice model in vivo were used to confirm our result. In this study, we showed that NTSR1 is commonly high expressed in melanoma cells, but low expressed in normal immortalized human keratinocyte line HaCaT. SR48692 not only reduced cell proliferation and
Y. Zhang Y. Liu (&) Department of Dermatology, the Third Hospital of Hebei Medical University, 139 Ziqiang Rd, Shijiazhuang 050000, Hebei, China e-mail: [email protected] S. Zhu Department of Life Science, Southwest University, Chongqing 400716, China L. Yi Neurosurgery Department, Daping Hospital, the Third Military Medical University, Chongqing, China H. Cui State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China
self-renewal potential in vitro, but also inhibited the tumor growth derived from A375 cells in NOD/SCID mice in vivo. Further, we originally reported that SR48692 inhibited cell proliferation through cell cycle arrest and apoptosis. Considering the favorable toxicity profile in vitro and in vivo though targeting NTS/NTSR1, SR48692 is worthy of further study and exploitation in melanoma treatment. Keywords Malignant melanoma Neurotensin NTSR1 SR48692 Cell proliferation Apoptosis Cell cycle Abbreviations MM Malignant melanoma NTS Neurotensin NTSR1 Neurotensin receptor 1 FBS Fetal bovine serum BrdU 5-bromo-2-deoxyuridine PI Propidium iodide PBS Phosphate-buffered saline MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
Introduction Malignant melanoma (MM) is a highly aggressive tumor and always resistant to traditional chemo-radiotherapy. With highly incidence and increasing mortality, melanoma becomes the leading death of skin neoplasm in developed countries. The majority of patients with early-stage melanoma can be cured by surgical excision, while for the advanced stage, the standard chemotherapeutic approaches
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have been disappointing to improve the prognosis in the last three decades. So it is necessary to identify some new chemotherapeutic regimens with high efficacy and low toxicity for melanoma treatment. Neurotensin (NTS) is a single polypeptide chain containing 13 amino acid residues, which is discovered by Carraway and Leeman in 1973 [1]. NTS works as a neuromodulator in the central nervous system, while it works as an endocrine agent in the periphery, which is released into the blood from the entero-endocrine N cells in response to various biologic activities. NTS plays an important role in some physiologic actions of gastrointestinal tract including the stimulation of gastric acid, pancreatic secretions, and increasing biliary secretion to facilitate the absorption of fatty acid by hormonal and neurocrine regulation [2]. More data also showed that NTS promoted tumor development in various types of cancer including lung cancer [3], prostate cancer [4, 5], colon cancer [6, 7], pancreas cancer [8, 9], pancreatic ductal adenocarcinoma [10], and breast cancer [11]. The biologic activities of NTS are mainly mediated by interaction with specific receptors including two G protein coupled neurotensin receptors NTSR1 and NTSR2, a new type of neurotensin receptor NTSR3 belonging to the sortilin receptor superfamily was also found [12]. Many studies showed NTS and NTSR1 were highly expressed in some types of cancers but not in normal cells and tissues [11, 13–18]. This implicated a functional link between NTS/NTSR1 high expression and neoplastic progression.SR-48692,2-([1-(7-Chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carbonyl]amino)admantane2-carboxylic acid, is a drug which acts as a selective, nonpeptide antagonist at the neurotensin receptor NTSR1, and was the first non-peptide antagonist developed for this receptor [19, 20]. It was used in scientific research to explore the interaction between neurotensin and other neurotransmitters in the brain [19, 21–26], and could inhibit cell growth and tumor development through competing with NTS to interact with NTSR1, which indicated that NTS/NTSR1 could be a potential biomarker and a therapeutic target in some cancers. Nevertheless, there is no report about NTS/NTSR1 in melanoma, and the role of SR48692 in melanoma cells is not clear. Here, we investigated NTSR1 expression, and found that SR48692 inhibited cell proliferation through induced cell cycle arrest and triggered apoptosis in A375 melanoma cell line. Moreover, we also showed that SR48692 decreased self-renewal capacity in vitro and tumor formation in vivo. This revealed that SR48692 is worthy of further study and exploitation in melanoma treatment.
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Materials and methods Cell culture The human melanoma cell line A375 was grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10 % fetal bovine serum (FBS; Gibco) and 1 % penicillin–streptomycin (P/S). Human melanoma cell line MV3 and immortalized human keratinocyte line HaCaT was grown in Roswell Park Memorial Institute-1640 (RPMI-1640; Gibco) supplemented with 10 % FBS and 1 % P/S. Cells were cultured at 37 °C in a 5 % CO2 humidified incubator. Immunofluorescence staining assay Cells were grown on coverslips in 24-well plate, washed with PBS, fixed in 4 % paraformaldehyde (PFA) for 20 min, and permeabilized with 0.3 % TritonX-100 for 5 min. Cells were blocked with 10 % goat serum for 1 h, incubated with a primary rabbit anti-NTSR1 (1:200; Abcam) for 2 h, IgG was used for negative control, then incubated with the secondary antibody goat anti-rabbit IgG (1:500) for 1 h, and counterstained with 300nM DAPI. Cells were examined using a Nikon microscope (80i) with Image-Pro Plus software for image analysis. Western blotting assay A375 and MV3 melanoma cell line were harvested and suspended in RIPA Lysis Buffer. A normal immortalized human keratinocyte line HaCaT was used as a control. Protein concentrations were determined with Enhanced BCA protein assay kit (Beyotime), using bovine serum albumin as reference. Proteins (50 lg) were separated on 10 % sodium dodecyl sulfate polyacrylamide gels (SDSPAGE), transferred to nitrocellulose membranes, and probed with antibodies rabbit anti-NTSR1 (1:1000; Abcam) or mouse anti-a-tubulin (1:5000; Sigma), respectively, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (ICN, CA). Proteins were visualized with BeyoECL Plus (Beyotime, China). Cell proliferation assay Cell proliferation was evaluated by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. After 12 h cell culture, the medium was removed and attached cells were rinsed with PBS, and 200 ll DMEM only w/o FBS was added and separately incubated with 0.01 lM NTS and 0.01 lM ?5 lM SR48692 at
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indicated concentration, DMSO was used as a control. 20 ll MTT (5 mg/ml) was added to each well after indicated time and incubated at 37 °C for 2 h. The medium was removed, and the formazan complex was dissolved with 200 ll DMSO. Absorbance of the complex was measured with a micro-plate reader at a wavelength of 570 nm in order to detect metabolically intact cells. BrdU feeding and staining Cells were grown on coverslips in 24-well plate, cultured in DMEM without FBS, and treated with either DMSO or 5 lM SR48692 for 3 days. After treatment, the thymidine analog 5-bromo-2- deoxyuridine (BrdU; Sigma) stock solution at 10 mg/ml in saline was diluted 1,0009 in the culture medium and incubated for 1 h. After that, cells were treated and fllowed the protocol as immunofluorescence staining assay, except the antibody is against BrdU (1:200, Abcam). Cell cycle assay Cells were plated in 10 cm plates and treated with either 5 lM SR48692 or DMSO. After 3 days treatment, cells were washed with ice-cold PBS, fixed with 70 % ethanol, stained with propidium iodide (PI) for 30 min, and analyzed by flow cytometry. The data were analyzed with CellQuest Pro software (BD BioSciences). The experiments were repeated at least three times. Apoptosis assay After treated with either DMSO or 5 lM SR48692 for 3 days, cells were stained with Hoechst 33258 and imaged using a Nikon microscope (209). The apoptotic cells were determined with the Annexin V-FITC apoptosis detection kit (Sigma). After treatment, cells were collected and washed with PBS buffer, resuspended in 100 ll of binding buffer, incubated with 5 ll of Annexin V-FITC and 10 ll PI for 15 min, and analyzed by flow cytometry. Annexin V?/PI– cells were recognized as apoptotic, Annexin V?/ PI? cells were defined as late apoptotic, Annexin V–/PI? cells were defined as necrotic, and Annexin V–/PI– cells were recognized as normal cells. The experiments were repeated at least three times.
Next, 8-mm pore size transwell culture inserts (Transwell; Costar) were placed into the wells of 24-well culture plates to separate the upper and the lower chamber. 1.0 9 105 cells were plated into the upper chamber containing 200 ll serum-free medium while the lower chambers contained 700 ll 0.1 % serum medium. After 24 h incubation with 5 lM SR48692 or DMSO, cells were staining with Crystal Violet, and image of the migration cells were collected, and the number of migration cells was quantified by counting ten independent visual fields under the microscope using a 209 objective. Soft agar clonogenic and in vivo tumorigenic assays For soft agar assay, 1,000 cells were mixed with 0.35 % Noble agar in growth medium containing DMSO or 5 lM SR48692 and plated onto six-well plates containing a solidified bottom layer (0.6 % Noble agar in growth medium) per well. After incubation for 14 days, cells were stained with MTT and imaged using a digital camera. Colonies containing more than 50 cells were counted using an inverted microscope. For xenograft mice model, 2 9 106 cells in 200 ll DMEM were harvested and injected into the right lateral flanks of NOD/SCID mice. After 7 days tumor growth, the mice were randomly divided into two groups of three mice each, and received the following treatments with intraperitoneal injections of either 100 ll DMSO, or SR48692 (5 mg/kg) for 14 days daily. Tumor growth was estimated by caliper measurements, and tumor volume was calculated with the ellipsoid formula: volume = tumor length 9 width2 9 0.5236. After treatment, mice were sacrificed and the tumors were removed and weighed. All animal experiments were preapproved by the Institutional Animal Care and Use Committee of the Southwest University. Statistical analysis Quantitative data were reported as the average ± SD. Oneway ANOVA and the two-tailed Student’s t test were used to measure differences using the SPSS17.0 software package. A minimum of three independent experiments were performed. Differences were considered statistically significant at p \ 0.05.
Wound-healing and cell migration assays
Results
Cells were plated and reached full of confluence, scarification was done with a pipette tip. Cells were maintained and treated with 5 lM SR48692 or DMSO. Pictures were taken at indicated time, cells treated with DMSO were used as a control.
NTSR1 is remarkably expressed in A375 melanoma cell line Since there is no any report about NTSR1 expression in melanoma cells, we investigated the NTSR1 expression
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Fig. 1 NTSR1 expression was detected in A375 melanoma cell line through immune-staining assay a Immunofluorescent staining was carried out to detect the expression of NTSR1 in A375 melanoma cell line (green), and the nuclei were counterstained with DAPI (blue).
IgG was used for negative control. b A normal immortalized human keratinocyte line HaCaT and two melanoma cell lines A375 and MV3 were harvested and performed Western blotting assay. a-tubulin was used as a loading control
and found that NTSR1 was significantly expressed in A375 cells, and which is located to membrane (Fig. 1a). Western blot analysis also confirmed that NTSR1 was significantly high expressed in melanoma A375 and MV3 cells but low expressed in normal HaCaT cells (Fig. 1b).
We assessed the apoptosis by Hoechst 33258 staining and flow cytometry analysis. Cell treated with SR48692 exhibited remarkable cell shrinkage changes, suggesting cell apoptosis (Fig. 3c top panel). The Hoechst33258 nuclear staining showed the condensation of chromatin and the dissociation of the nuclear membrane after SR48692 treatment, and the number of apoptotic nuclei increased (Fig. 3c lower panel). As shown in Fig. 3d, Annexin V-FITC/PI staining and flow cytometry analysis also confirmed that SR48692 induced apoptosis from 7.35 to 63.24 % compared with control group (P = 0.001).
SR48692 inhibited cell growth and proliferation in A375 melanoma cells Cells treated with 5 lM SR48692 for 3 days resulted in a significant reduction in the viable cell number compared with DMSO-treated cells (Fig. 2a), and the IC50 of SR48692 for growth inhibition is 3.2243 uM. We further investigated cell growth curve by MTT assay, the result showed that exogenous 0.01 lM NTS immediately increased the cell growth compared with DMSO control, and then we added the inhibitor SR48692 together, which inhibited the cell proliferation which caused by NTS (Fig. 2b). BrdU staining assay also demonstrated that SR48692 significantly inhibited cell proliferation. After treatment with 5 lM SR48692 for 3 days, the percentage of BrdU-positive cells was down-regulated more than half compared with the control group (P = 0.003; Fig. 2c). SR48692 induced the cell cycle arrest and apoptosis As shown in Fig. 3a, b, flow cytometric analysis revealed a significant increase in S phase (P = 0.014) and G2/M phases (P = 0.046), along with reduction in G0/G1 phases (P = 0.004) compared with control cells. These data showed that SR48692 blocked the G2/M transition and induced cell cycle S and G2/M phase arrest in A375 melanoma cells.
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NTS/NTSR1 activation is required for A375 cell migration As shown in Fig. 4a, 24 h after scratching, cell migration in SR48692 group was blocked compared with control group. Transwell migration assay further verified that SR48692 significantly reduced the cell migration (P = 0.004; Fig. 4b). SR48692 inhibited colony formation in vitro and tumor growth in vivo Soft agar assay showed A375 cells treated with SR48692 gave rise to few and small colonies (Fig. 5a). The colony formation was significantly reduced from 11.1 to 5.5 % after SR48692 treatment (P = 0.03) (Fig. 5b) compared with control group. As shown in Fig. 5c and d, the NOD/ SCID xenografted mice model showed that SR48692 treatment was accompanied by a significant 57.6 % decrease in tumor volume and 51.8 % decrease in tumor weight compared with DMSO control group.
Mol Cell Biochem Fig. 2 SR48692 inhibited cell growth and proliferation in melanoma cells. a A375 melanoma cells were treated with DMSO or 5 lM, 10 lM SR48692 for 3 days. Cell morphology was observed (920), and the cell number was determined by trypan blue assay. Data represent the average ± SD of at least three independent experiments. Scale bar, 50 mm. b A375 melanoma cells were treated with 0.01 lM NTS and 0.01 lM ?5 lM SR48692 seprately, DMSO was used as a control. OD570 nm was measured daily from 1 to 4 days. Data are presented as mean ± SD. * P \ 0.05; ** P \ 0.01. c After treated with DMSO and 5 lM SR48692 for 3 days, BrdU was added and incubated for 1 h, cells were stained with an antibody against Brdu (green), counterstained with DAPI (blue), 920. The percentage of Brdu-positive cells was calculated. Cells were counted from at least five randomly selected fields. Data are presented as mean ± SD
Discussion The results presented in this study showed that NTSR1 is significantly high expressed in human melanoma cell line but low expressed in normal HaCaT cell line, which is consistent with reports that NTSR1 is highly expressed in tumor tissue but low-level in normal tissue [11], suggesting that NTS/NTSR1 may be act as a biomarker and a novel drug target in melanoma. We also found that not only exogenous NTS immediately increased the cell growth, and SR48692 could reverse the cell proliferation which caused by NTS, but also
SR48692 significantly abolished self-renewal and colony formation in vitro and tumor growth in vivo, which suggested that SR48692 may serve as an effective therapeutic agent in melanoma treatment. Our studies also demonstrated that SR48692 induced S phase and G2/M cell phase arrest and triggered apoptosis. So inhibition of cell proliferation by SR48692 may be through cell cycle arrest and/or apoptosis in A375 melanoma cells. As we have seen, the changes in cell cycle caused by the addition of SR48692 to A375 cells while significant average about 10 %, so the apoptosis may be the main reason rather than cell cycle arrest to reduce cell growth and proliferation.
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Fig. 3 SR48692 induced cell cycle arrest and triggered apoptosis. a After treated with DMSO and 5 lM SR48692 for 3 days, respectively, cells were stained with PI, and DNA content was determined by flow cytometry. b Statistical analysis of the cell cycle phase. Data represent the average ± SD of at least three independent experiments. Statistical analysis was performed using the 2-tailed
Student t test. c Representative morphologic change of apoptosis (the top panel) and Hoechst 33258 nuclear staining (the lower panel), 920. d After treated with either DMSO or 5 lM SR48692 for 3 days, respectively, apoptosis was determined using Annexin V-FITC/PI staining and flow cytometry. The graph was statistical analysis of the percentage of apoptosis
In addition, we also confirmed that SR48692 could inhibit cell migration, which is consistent with the report in breast cancer cell [11]. But the nature about SR48692 effects remains to be clarified. In summary, this article originally reported that NTSR1 antagonist SR48692 inhibited cell proliferation through
inducing apoptosis and cell cycle arrest in melanoma cells in vitro and in vivo, suggesting that NTSR1 may be a biomarker and a novel drug target in melanoma, and SR48692 may be a potential new drug for targeted therapy in melanoma.
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Mol Cell Biochem Fig. 4 Cell migration was inhibited by SR48692 in melanoma cells through woundhealing assay and transwell migration assays. a A375 melanoma cells were treated with SR48692 for 0, 24, 48 h, and detected the migration by wound-healing assay (910). b Transwell migration assays: Images of the migrating cells were collected after stained with crystal violet; the migration cells on the bottom surface of each membrane were counted. Data represent the average ± SD of three independent experiments. Statistical analysis was performed using the two-tailed Student’s t test
Fig. 5 SR48692 inhibited the colony formation in vitro and tumor growth in vivo. a After treated with 5 lM SR48692 or DMSO in a soft agar culture system for 2 weeks, the colonies were stained with MTT and imaged. SR48692 treatment gave rise to smaller and fewer colonies than control group. (94). b The colony number was counted, data represented the average ± SD of at least three independent
experiments. Statistical analysis was performed using the two-tailed Student’s t test. c The xenograft tumor after treated by DMSO or 5 mg/kg SR48692 was weighted. Data were analyzed with 2-tailed Student t test. d Xenograft tumor growth curve was checked. Data were analyzed with 2-tailed Student t test
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Mol Cell Biochem Acknowledgments This study was supported by the National Basic Research Program of China (No. 2012cb114603), the National Natural Science Foundation of China (No. 31271462, 31172268, 81201551). Disclosure of interest The authors declare no conflict of proprietary, commercial, or financial interest in the products or companies described in this article.
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