Antiproliferative and Apoptotic effects of Aprepitant on Human Glioblastoma U87MG Cells

Year 2017, Volume: 21 Issue: 1, 156 - 164, 20.09.2016

Miriş Dikmen

https://doi.org/10.12991/marupj.259893

Abstract

Glioblastoma multiforme, is the
most malignant glioma among  other
gliomas with high incidence. It has aggresive properties  such as high proliferation, invasion and
migration rates.  Therefore, recently
studies have focused on the development  of
new therapeutic targets in glioblastoma chemotherapy.  Substance P, neurokinin-1 (SP/NK-1) receptor
system has an  important role in cancer
development and metastasis. Therefore,  NK-1
receptors are new therapeutic targets for glioblastoma  therapy and development of NK-1 receptor
antagonists. 

In this study, antiproliferative
and apoptotic effects of aprepitant  which
is an antiemetic drug commonly using in chemotherapy  on human U87MG glioblastoma cells were
investigated.  Antiproliferative effects
of aprepitant were evaluated by  MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium  bromide) and Real Time Analyses System (RTCA
DP). Also  apoptotic effects of
aprepitant were evaluated by annexin-VPI 
and caspase-3 activity by using flow cytometer. According  to the cell index values obtained from
real-time cell analysis  system, with 50
μM and higher concentrations, cell proliferation  was decreased and IC50 values were determined
as 76.9, 62.5  and 59.2 μM for 24, 48 and
72 hours respectively. Aprepitant  showed
significant antiproliferative effects on U87MG cells.  Depending on increasing aprepitant
concentrations, both  early and late
apoptotic cell ratios were increased. In addition,  caspase-3 activation was slightly increased
with IC25 and IC50  concentrations of
aprepitant according to the control group. 
As a result, aprepitant showed important antiproliferative and  apoptotic effects on U87MG cells. In
conclusion, NK-1 receptor  antagonists
may have a potential therapeutic role for human 
glioblastoma chemotherapy.   

Keywords

aprepitant, U87MG cell, antiproliferative, apoptosis, neurokinin‑1 receptor

Aprepitantın İnsan Glioblastoma U87MG Hücreleri üzerinde Antiproliferatif ve Apoptotik Etkileri

Abstract

Glioblastoma multiforme, gliomalar arasında en malign özellikte olup, görülme sıklığı  oldukça yüksektir. Hızlı çoğalma, beyin dokusuna invazyon ve migrasyon gibi agresif özelliklere sahiptir. Bu nedenle son yıllarda glioblastoma kemoterapisinde, yeni tedavi hedeflerinin gelişmesini üzerine çalışmalar yoğunlaşmıştır. Substans P, nörokinin-1 (SP/NK-1) reseptör sistemi, kanser gelişimi ve kanser metastazında önemli bir rol oynamaktadır. Bu nedenle NK-1 reseptörleri, glioblastoma tedavisinde ve NK-1 reseptör antagonistlerinin gelişiminde yeni tedavi hedefleri arasında yer almaktadır.

Bu çalışmada,  klinikte özellikle kemoterapi sırasında antiemetik amaçla kullanılan,  bir NK-1 reseptör antagonisti olan aprepitantın, insan malignant glioblastoma U87-MG hücre hattında antiproliferatif ve apoptotik etkileri araştırılmıştır. Çalışmada MTT yöntemi ve gerçek zamanlı hücre analiz sistemi (RTCA DP)  ile hücre proliferasyon analizleri, anneksin V-PI ve kaspaz 3 flow sitometri analizleri ile de apoptotik etkiler çalışılmıştır. MTT analizine göre Aprepitantın konsantrasyon ve zamana artışına bağlı olarak U87MG hücre önemli derecede sitotoksik etki yaptığı ve U87MG hücre proliferasyonunu azalttığı görülmüştür. Gerçek zamanlı analiz sisteminden elde edilen cell index değerlerine göre de, 50 µM ve üzeri aprepitant konsantrasyonlarında hücre proliferasyonunun azaldığı ve sırasıyla IC₅₀ değerlerinin 24. saatte 76.9 µM,  48. saatte 62.5 µM ve 72. saatte de 59.2 µM olarak belirlenmiştir. Flow sitometri analiz sonuçlarına göre aprepitantın 24. saatteki erken ve geç apoptotik etkisi konsantrasyon artışına bağlı olarak artış göstermiştir. Özellikle IC₂₅ ve IC₅₀ aprepitant değerlerinde aktif kaspaz-3 değeri kontrole göre, az da olsa artmıştır.  Sonuç olarak aprepitantın malignant glioblastoma U87-MG hücrelerinde önemli antiproliferatif ve apoptotik etkileri belirlenmiş olup, aprepitant ve yeni NK-1 reseptörler antagonistlerinin, kemoterapi sırasında antikanser ilaçlar ile birlikte umut vadeden yeni bir terapötik hedef olabileceği görülmektedir

Keywords

NK-1, aprepitant, U87MG, apoptoz, antiproliferatif

References

• KAYNAKÇA
• Mut M., Glioblastomalarda Phosphatidylinositol 3-Kinase/Akt Yolu Üzerinden Elk-1 Aktivasyonunun Değerlendirilmesi, Doktora tezi, Hacettepe Üniversitesi, Sağlık bilimleri Enstitüsü, 2007.
• Chen A, Xu J, Johnson AC. Curcumin Inhibits Human Colon Cancer Cell Growth By Suppressing Gene Expression of Epidermal Growth Factor Receptor Through Reducing The Activity of The Transcription Factor Egr-1. Oncogene 2006; 25:(2) 278–287.
• Kleihues P, Cavenee W. World Health Organization Classification of Tumours. Pathology and genetics of tumours of the nervous system, IARC, Lyon, 2000.
• Hökfelt T, Pernow B, Wahren J. Substance P: A Pioneer Amongst Neuropeptides. J Intern Med. 2001; 249:27-40.
• Muñoz M, Rosso M, González-Ortega A, Coveñas R. The NK-1 Receptor Antagonist L-732.138 Induces Apoptosis and Counteracts Substance P-Related Mitogenesis in Human Melanoma Cell Lines, Cancers 2010; 2: 611-623.
• Muñoz M, Rosso M, Casinello F, Coveñas R. Paravertebral Anesthesia:How Substance P And The Nk-1 Receptor Could Be Involved In Regional Block And Breast Cancer Recurrence, Breast Cancer Res Treat. 2010; 122: 601-603.
• Muñoz M, Coveñas R. Neurokinin-1 Receptor: A New Promising Target In The Treatment of Cancer, Discovery Medicine 2010;10:53, 305-13.
• Rupniak N, Kramer M. Substance P and Related Tachykinins, Neuropsychopharmacology: The Fifth Generation of Progress, Chapter 13. 2002, pp169-177.
• Pennefather JN, Lecci A, Candenas ML, Patak E, Pinto FM, Maggi CA. Tachykinins And Tachykinin Receptors: A Growing Family. Life Sciences 2004;74:1445–1463.
• Mengi M, P Maddesinin Emosyonel Aktivite Üzerine Santral ve Periferik Etkileri, Bu etkilerde WIN-51708’in Rolü, Doktora tezi, İstanbul Üniversitesi, Sağlık Bilimleri Enstitüsü, 2009.
• Muñoz M, Rosso M, Coveñas R. A New Frontier in The Treatment of Cancer: NK-1 Receptor Antagonists. Curr Med Chem 2010,17, 504-516.
• Diemunsch P, Grelot L. Potential of Substance P Antagonists As Antiemetics. Drugs, 2000; 60:533-46.
• Lang, K, Drell TL, Lindecke A, Niggemann B, Kaltschmidt C, Zaenker KS. Entschladen F. Induction of a metastatogenic tumor cell type by neurotransmitters and its pharmacological inhibition by established drugs. Int J Cancer 2004;112: 231-238.
• Mosmann T, Rapid colorometric assay for cellular growth and survival: application to proliferation and cytotoxicity assay. J. Immunol. Method 1983; 65:55-63.
• Freshney RI. Culture of Animal Cells, A Manual of Basic Technique, JohnWiley & Sons, 5th Edition, 2005, pp359-372.
• Dikmen M, Canturk Z, Ozturk Y, Tunalı Y. Investigation of the Apoptotic Effect of Curcumin in Human Leukemia HL-60 Cells by Using Flow Cytometry CANCER Biotherapy and Radiopharmaceuticals 2010; 25(6):749-755
• Limame R, Wouters A, Pauwels B, Fransen E, Peeters M, Lardon F, Wever OD, Pauwels P. Comparative Analysis of Dynamic Cell Viability, Migration and Invasion Assessments by Novel Real-Time Technology and Classic Endpoint Assays, Plos One 2012; 7 (10):1-12
• Kaya Tilki E, Dikmen M, Öztürk Y. Effects of DNMT and HDAC Inhibitors (RG108 and Trichostatin A) on NGF-induced Neurite Outgrowth and Cellular Migration. Int J Pharmacol 2016; 12(4);351-360.
• Gatti R, Belletti S, Orlandini G, Bussolati O, Dall’asta V, Gazzola GC. Comparison of Annexin V and calcein-AM as early vital markers of apoptosis in adherent cells by confocal laser microscopy. J. Histochem. Cytochem.1998;46:895–900.
• Kopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, Van Oers NH. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994;84:1415–1420.
• Overbeeke R, Steffens-Nakken H, Vermes I, Reutelingsperger C, Hanen C. Early features of apoptosis detected by four different flow cytometry assays. Apoptosis 1998;3:115.
• Zhang G, Gurtu V, Kain SR, Yan G. Early detection of apoptosis using a fluorescent conjugate of Annexin V. Biotechniques1997; 23:525–531.
• Boğa C. Akım sitometri ile apoptozis tayini. Sözer O, Ed. Klinik ve pratikte akım sitometri, 1. baskı, Haberal Eğitim Vakfı Ankara. 2009; 155-158
• Engür S, Dikmen M, Öztürk Y. Comparison of antiproliferative and apoptotic effects of a novel proteasome inhibitor MLN2238 with bortezomib on K562 chronic myeloid leukemia cells. Immunopharmacol Immunotoxicol, 2016; 38(2): 87–97
• Kaufmann SH, Hengartner MO. Programmed cell death: alive and well in the new millennium. Trends Cell Biol. 2001; 11(12):526-534.
• Slee EA, Adrain C, Martın SJ. Executioner caspases-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis. The Journal of Biological Chemistry. 2001;276 (10):7320-7326.
• Wei-Lun C, Li P, Douglas K, Steven MS, Joanna EB. Silvestrol induces early autophagy and apoptosis in human melanoma cells. Chen et al. BMC Cancer 2016; 16:17
• Muñoz M, Berger M, Rosso M, Gonzalez-Ortega A, Carranza A, Coveñas R. Antitumor Activity of Neurokinin-1 Receptor Antagonists in MG-63 Human Osteosarcoma Xenografts. Internatıonal Journal of Oncology 2014;44: 137-146.
• Muñoz M, Gonzalez-Ortega A, Robles-Frias M, Carranza A, Salinas-Martín M, Coveñas R. The Substance P/Neurokinin-1 Receptor System in Lung Cancer: Focus on The Antitumor Action of Neurokinin-1 Receptor Antagonists. Peptides, 2012:38:318–325.
• Luo W, Sharif TR, Sharif M. Substance P-İnduced Mitogenesis in Human Astrocytoma Cells Correlates With Activation Of The Mitogen-Activated Protein Kinase Signaling Pathway. Cancer Res. 1995; 56,:4983–91.
• DeFea KA, Vaughn ZD, O’Bryan EM, Nishijima D, Dery O, Bunnett NW. The Proliferative And Antiapoptotic Effects Of Substance P Are Facilitated By Formation of A Beta-Arrestin-Dependent Scaffolding Complex. Proc Natl Acad Sci USA. 2000; 97:11086–91.
• Muñoz M, Rosso M, Pérez A, Coveñas R, Zamarriego C, Piruat JI. The NK-1 Receptor is İnvolved in The Antitumoural Action of L-733,060 and in The Mitogenic Action of Substance P on Neuroblastoma and Glioma Cell Lines. Neuropeptides 2005; 39: 427–432.
• Akazawa T, Kwatra SG, Goldsmith LE, Richardson MD, Cox EA, Sampson JH, Kwatra MM. A Constitutively Active Form of Neurokinin 1 Receptor and Neurokinin 1 Receptor-Mediated Apoptosis in Glioblastomas. Journal of Neurochemıstry 2009; 109;1079-1086.
• Spitsin S, Stevens KE, Douglas SD. Expression Of Substance P, Neurokinin-1 Receptor And İmmune markers in the Brains Of İndividuals With HIV-Associated Neuropathology. Journal of the Neurological Sciences 2013;15 (334):18-23.
• Mou L, Kang Y, Zhou Y, Zeng Q, Song H, Wang R. Neurokinin-1 Receptor Directly Mediates Glioma Cell Migration by Up-regulation of Matrix Metalloproteinase-2 (MMP-2) and Membrane Type 1-Matrix Metalloproteinase (MT1-MMP), The Journal Of Biological Chemistry 2013; 288(1): 306–318