Hendrian D. SOEBAGJO, Susy FATMARİYANTİ, Delfitri LUTFİ

Cells Culture

Anahtar Kelimeler:

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Effectiveness of Natural Killer (NK) Cells in Peripheral Blood Stem-Cell towards Expression of EZH2, Ki-67 and Apoptosis in Retinoblastoma (RB) Cells Culture

AbstractA natural killer (NK) cell is a large granular lymphocyte having a cytotoxic activity against malignant tumor cells in the cellular immune response. NK cells play an important role to inhibit the metastasis of tumor and express glikoprotein molecule antigen (IgG) and variable cytotoxic factors like perforin, granzyme, cytolysin such as TNF-α against retinoblastoma cells. The aim of the study was to investigate the role of Natural Killer Cells (NK cells) as an alternative therapy, especially in cases of retinoblastoma. It is highly expected that the study can be used as a starting point in determining the strategic therapy, e.g. the cell targeted therapy to inhibit EZH2 and retinoblastoma cell proliferation. There were 2 groups of retinoblastoma cell cultures in this study, the groups of control (n=10) and exposure (n=10). The control group was a group of retinoblastoma cell cultures that did not get any therapy, while the exposure group was a group of retinoblastoma cell cultures with NK Cell exposure in vitro for 3 days. The data obtained were analyzed using the T- test and Pearson Correlation. In T-test, the results of the two groups indicated a significant difference in the positive expression of cell count of EZH2, Ki-67 and Apoptotic cells. The Pearson Correlation test showed that the expression of EZH2 was statistically negative significant (α: -0.930; p<0.05) towards the apoptotic cells expression. While in the proliferation cell expression (Ki-67) towards the apoptotic cell expression, the correlation was significantly negative (α: -0,868; p<0.05). The study showed that NK cells played an important role in the aggressiveness of RB cell cultures by changing the expression of EZH2, Ki-67 and apoptosis in RB cells. It is expected that the research can be used as a reference to determine the strategic therapy in retinoblastoma. Keywords: NK cells, retinoblastoma, EZH2, proliferation, apoptosis

Keywords:

NK cells, retinoblastoma, EZH2, proliferation apoptosis,

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References
Abbas AK, Lichtman AH. Immunity to Tumors. In: Celluler and Molecular Immunology. 5th edition. Philadelphia; WB Saunders Co. 2003:391-410.
Ishikawa E, Tsuboi K, Saijo K, Harada H, Takano S, Nose T, Ohno T. Autologous natural killer cell therapy for human recurrent malignant glioma. Anticancer Res. 2004; 24(3b):1861-71.
Fehniger TA, Cooper MA, Nuovo GJ, Cella M, Facchetti F, Colonna M, Caligiuri M. CD56bright natural killer cells are present in human lymph nodes and are activated by T cell-derived IL-2: a potential new link between adaptive and innate immunity. Blood. 2000;101(8):3052-7.
Marcenaro E, Carlomagno S, Pesce S, Chiesa MD, Parolini S, Moretta A, Sivori S. NK Cells and Their Receptors During Viral Infections. Immunotherapy. 2011;3(9):1075-86.
Gerosa F, Baldani-Guerra B, Nisii C, Marchesini V, Carra G, Trinchieri G. Reciprocal activating interaction between natural killer cells and dendritic cells. J Exp Med. 2002;195(3):327-33.
Piccioli D, Sbrana S, Melandri E, Valiante NM. Contact dependent stimulation and inhibition of dendritic cells by natural killer cells. J Exp Med. 2002;195(3):335-341.
Abe H, Akiyama S, Okamoto M. Clinical Cancer Immunotherapy: Molecular Targeting Immunotherapy. J Integ Med. 2008;1(1):38-46.
Pradeu T, Jaeger S, Vivier E. The speed change: towards a discontinuity theory of immunity? Nat Rev Immunol. 2013;13(10):764-9.
Navarro, AG. Activated NK cells are potent effectors against glioblastoma cells due to activating KIR2DS2 and KIR2DS4 - HLA ligand interactions-In vitro study. Thesis, University of Bergen, Norway, 2013.
Saito H, Osaki T, Ikeguchi M. Decreased NKG2D expression on NK cells correlates with impaired NK cell function in patients with gastric cancer. Gastric Cancer. 2012;15(1):27-33.
Lim SA, Kim TJ, Lee JE, Sonn CH, Kim K, Kim J, Choi JG, Choi IK, Yun CO, Kim JH, Yee C, Kumar V, Lee KM. Ex Vivo Expansion of Highly Cytotoxic human NK Cells by Cocultivation with Irradiated Tumor Cells for Adoptive Immunotherapy. Therapeutics, Targets and Chemical Biology. Cancer Res. 2013;73(8):2598-607.
Divan A, Lawry J, Dunsmore IR, Parsons MA, Royds JA. p53 and p21waf-1 expression correlates with apoptosis or cell survival in poorly differentiated, but not well-differentiated, retinoblastomas. Cancer Res. 2001;61(7):3157-63.
Catoi C, Baba A. Comparative Oncology. The Publishing House of the Romanian Academy. 2007; Chapter 3.
Wahyuhadi J. Ekspresi Sel NK, Sel T Sitotoksik Il-2 dan IFN-Ɣ Intratumoral dan Pengaruh Terhadap Agresivitas Astrositoma. Dissertation, Program Studi Ilmu Kedokteran, Program Pascasarjana Universitas Airlangga, 2010;1-75.
Ito Y, Matsuura N, Sakon M, Takeda T, Umeshita K, Nagano H, Nakamori S, Dono K, Tsujimoto M, Nakahara M, Nakao K, Monden M. Both cell proliferation and apoptosis significantly predict shortened disease-free survival in hepatocellular carcinoma. British Journal of Cancer. 1999;81(4):747-51.
Xie W, Wong YC, Tsao SW. Correlation of increased apoptosis and proliferation with development of prostatic intraepithelial neoplasia (PIN) in ventral prostate of the Noble rat. Prostate. 2000;44(1):31-9.
Liu S, Edgerton SM, Moore II DH, Thor AD. Measures of Cell Turnover (Proliferation and Apoptosis) and Their Association with Survival in Breast cancer. Clin Cancer Res. 2001;7(6):1716-23.
Sitorus RS, Gumay S, van der Valk P. The apoptotic paradox in retinoblastoma. Ann N Y Acad Sci. 2009;1171:77-86.
Urruticoechea A, Smith IE and Dowsett M. Proliferation Marker Ki-67 in Early Breast cancer. J Clin Oncol. 2005;23(28):7212-20.
Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100(1):57-70.
Morey L and Helin K. Polycomb group protein-mediated repression of transcription. Trends Biochem Sci. 2010;35(6):323-32.
O'Meara MM, Simon JA. Inner workings and regulatory inputs that control Polycomb repressive complex 2. Chromosoma. 2012;121(3):221-34.
Yamaguchi H, Hung MC. Regulation and Role of EZH2 in Cancer. Review Article. Cancer Res Treat. 2014;46(3):209-22.
Kleer CG, Cao Q, Varambally S, Shen R, Ota I, Tomlins SA. EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells. Proc Natl Acad Sci USA. 2003;100(20):11606-11.
Varambally S, Cao Q, Mani RS, Shankar S, Wang X, Ateeq B. Genomic loss of microRNA-101 leads to overexpression of histone methyltransferase EZH2 in cancer. Science. 2008;322(5908):1695-9.
Chang CJ, Yang JY, Xia W, Chen CT, Xie X, Chao CH, Woodward WA, Hsu JM, Hortobagyi GN, Hung MC. EZH2 Promotes Expansion of Breast Tumor Initiating Cells through Activation of RAF1-b-Catenin Signaling. Cancer Cell. 2011;19(1):86-100.
Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-Sinha C, Sanda MG, Ghosh D, Pienta KJ, Sewalt RG, Otte AP, Rubin MA, Chinnaiyan AM. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature. 2002;419(6907):624-9.
Yang YA and Yu J. EZH2, an epigenetic driver of prostate cancer. Protein Cell. 2013; 4(5):331-41.
Kodach LL, Jacobs RJ, Heijmans J, van NoeselCJ, Langers AM, Verspaget HW, Hommes DW, Offerhaus GJ, van den Brink GR and HardwickJC. The role of EZH2 and DNA methylation in the silencing of the tumour suppressor RUNX3 in colorectal cancer. Carcinogenesis. 2010;31(9):1567-75.
Ciarapica R, Miele L, Giordano A, Locatelli F, Rota R. Enhancer of zeste homolog 2 (EZH2) in pediatric soft tissue sarcomas: first implications. BMC Med. 2011;9:63.
Hajósi-Kalcakosz, S, Dezső K, Bugyik E, Bödör C, Paku S, Pávai Z, Halász J, Schlachter K, Schaff Z, Nagy P. Enhancer of zeste homologue 2 (EZH2) is a reliable immunohistochemical marker to differentiate malignant and benign hepatic tumors. Diagnostic Pathology. 2012;7(86):1-7.
Sastroasmoro S and Ismael S. Dasar-Dasar Metodologi Penelitian Klinis, Sagung Seto. Edisi 4. 2011.
Setiawan A, Sunyoto D, Buku Ajar: Statistik Kesehatan Parametrik, Non Paramatik, Validitas, dan Reliabilitas, Nuha Medika, Yogyakarta. 2011.
Böyum A. Isolation of leucocytes from human blood. Further observations. Methylcellulose, dextran, and ficoll as erythrocyteaggregating agents. Scand J Clin Lab Invest Suppl. 1968;97:31-50.
Rose NR, de Macario EC, Fahey JL, Friedman H, Penn GM. Manual of Clinical Laboratory Immunology. American Society for Microbiology, Washington, D.C.1994.
Rantam FA. Metode Imunologi. Surabaya: Airlangga University Press. Hal 83-84. 2003.
Bancroft JD and Gamble M. Theory and Practice of Histological Techniques. 6th Edition. Churchill Livingstone Elsevier, 2008;161-86.
Chan W, Cheung K, Schorge JO, Huang L, Welch WR, Bell DA, Berkowitz RS, Mok SC. Bcl-2 and p53 Protein Expression, Apoptosis and p53 Mutation in Human Epithelial Ovarian Cancers. Am J Pathol. 2000;156(2):409-17.
Eskander RN, Ji T, Huynh B, Wardeh R, Randall LM, Hoang B. Inhibition of Enhancer of Zeste Homolog 2 (EZH2) expression is associated with decreased tumor cell proliferation, migration and invasion in endometrial cancer cell lines. Int J Gynecol Cancer. 2013;(6):997-1005.
Jia N, Li Q, Tao X, Wang J, Hua K, Feng W. Enhancer of zeste homolog 2 is involved in the proliferation of endometrial carcinoma. Oncol Lett. 2014;8(5):2049-54.
Imai C, Iwamoto S, Campana D. Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood. 2005;106(1):376-83.
Geller MA, Cooley S, Judson PL, Ghebre R, Carson LF, Argenta PA. A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer. Cytotherapy. 2011;13(1):98-107.
Sauvageau M. and Sauvageau G. Polycomb group proteins: multi-faceted regulators of somatic stem cells and cancer. J Stem Cell. 2010;7(3):299-313.
Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, Helin K. EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. EMBO J. 2003;22(20)5323-35.
Tan D, Tan S, Zhang J, Tang P, Huang J, Zhou W, Wu S. Histone trimethylation of the p53 gene by expression of a constitutively active prolactin receptor in prostate cancer cells. Chin J Physiol. 2013;56(5):282-90.
Kotake Y, Cao R, Viatour P, Sage J, Zhang Y, Xiong Y. pRB family proteins are required for H3K27 trimethylation and Polycomb repression complexes binding to and silencing p16INK4alpha tumor suppressor gene. Genes Dev. 2007;21(1):49-54.
Mehlen P, Puisieux A. Metastasis: a question of life or death. Nat Rev Cancer. 2006;6(6):449-58.
Paholyuk TD, Zacharzeva LM, Koshel KV, Oliynichenko GP, Berezhnaya NM. Stage of differentiation, proliferative index of tumor cells and cytotoxic activity of peripheral blood lymphocytes in colorectal cancer patients. Exp Oncol. 2004;26(2):161-3.
Trihia H, Murray S, Price K, Gelber RD, Golouh R, Goldhirsch A, Coates AS, Collins J, Castiglione-Gertsch M, Gusterson BA; International Breast Cancer Study Group. Ki-67 expression in breast carcinoma: its association with grading systems, clinical parameters, and other prognostic factors--a surrogate marker? Cancer. 2003;97(5):1321-31.
Dyer MA, Cepko CL. Regulating proliferation during retinal development. Nat Rev Neurosci. 2001;2(5):333-42.
Soebagjo HD. Korelasi antara Asam Hialuronat, Matrix Metalloproteinase-14 (MMP-14), dan Enhancer of Zeste Homolog 2 (EZH2) dalam Perkembangan Agresivitas Retinoblastoma. Dissertation, Universitas Brawijaya, Malang, 2014.
Madani SH, Ameli S, Khazaei S, Kanani M, Izadi B. Frequency of Ki-67 (MIB-1) and P53 expressions among patients with prostate cancer. Indian J Pathol Microbiol. 2011;54(4):688-91.
Dranoff G. Cytokines in cancer pathogenesis and cancer therapy. Nat Rev Cancer. 2004;4(1):11-22.
Abbas AK, Lichtman AH. Basic Immunology: Functions and Disorders of the Immune System. 3rd edition. Philadelphia; WB Saunders Co, 2011;32-36.
Wood SM, Meeths M, Chiang SC, Bechensteen AG, Boelens JJ, Heilmann C, Horiuchi H, Rosthøj S, Rutynowska O, Winiarski J, Stow JL, Nordenskjöld M, Henter JI, Ljunggren HG, Bryceson YT. Different NK cell-activating receptors preferentially recruit Rab27a or Munc13-4 to perforin-containing granules for cytotoxicity. Blood. 2009;114(19):4117-27.
Liu D, Meckel T, Long EO. Distinct role of rab27a in granule movement at the plasma membrane and in the cytosol of NK cells. PLoS One. 2010;5(9):e12870.
Krzewki K. and Coligan JE. Human NK cell lytic granules and regulation of their exocytosis. Review Article. Front Immunol. 2012;3(335):1-16.
Keefe D, Shi L, Feske S, Massol R, Navarro F, Kirchhausen T. Perforin triggers a plasma membrane-repair response that facilitates CTL induction of apoptosis. Immunity 2005;23(3):249-62.
Thiery J, Keefe D, Saffarian S, Martinvalet D, Walch M, Boucrot E, Kirchhausen T, Lieberman J. Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis. Blood. 2010;115(8):1582-93.
Praper T, Sonnen A, Viero G, Kladnik A, Froelich CJ, Anderluh G, Dalla Serra M, Gilbert RJ. Human perforin employs different avenues to damage membranes. J Biol Chem. 2011;286(4):2946-55.
Lumongga F. Apoptosis. Departemen Patologi Anatomi Fakultas Kedokteran Universitas Sumatera Utara. USU Repository. 2008.
Houston A and O’Connell J. The Fas signalling pathway and its role in the pathogenesis of cancer. Current Opinion in Pharmacology 2004;4(4):321-6.
Shimizu M, Kondo M, Ito Y, Kume H, Suzuki R, Yamaki K. Soluble Fas and Fas ligand provide new information on metastasis and response to chemotherapy in SCLC patients. Cancer Detect Prev. 2005;29(2):175-80.