Induction of p۵۳-dependent Apoptosis in pre-B Acute Lymphoblastic Leukemia Cell Line )NALM-۶) by Small Molecule RITA

Background and Objective: The use of low-molecular-weight, nonpeptidic molecules that degrade the interaction between the p۵۳ protein and its negative regulator MDM۲ (Murine- double minute colon ۲) is a new therapeutic strategy for treatment of various types of cancer. One of these agents is RITA (reactivation of p۵۳ and induction of tumor cell apoptosis) which binds to p۵۳ protein and inhibits formation of p۵۳-MDM۲ complex while induces apoptosis in tumor cells by increasing p۵۳ protein levels. The aim of this study was to assess the possible in vitro apoptotic effects of RITA on pre-B ALL NALM-۶ cells. Materials and Methods: NALM-۶ cells were treated with different concentrations of RITA at different time intervals. The viability of NALM-۶ cells and apoptosis was measured by MTT assay and PI staining, respectively. The level of p۵۳, acetylated p۵۳ as well as cleaved PARP and procaspase-۳ were determined by Western blot in RITA-treated cells. The results were analyzed using Paired t test. Results: RITA has cytotoxic effects on NALM-۶ cells. Flow cytometry analysis indicated increased apoptotic cells in sub G۱ region in treated cells (P < ۰.۰۵). The Western blot analysis revealed that protein expression levels of p۵۳, and its acetylation increased in response to RITA. In addition, RITA-induced apoptosis associated with activation of caspase-۳ and PARP cleavage. Conclusion: The results of this study showed that RITA induced p۵۳-dependent apoptosis in pre-B ALL NALM-۶ cells. Key words: RITA, p۵۳, MDM۲, Apoptosis, ALL ۱- Inaba H, Greaves M, Mullighan CG. Acute lymphoblastic leukaemia. Lancet. ۲۰۱۳ ۳۸۱: ۱۹۴۳-۵۵. ۲- Schrappe M, Hunger SP, Pui C-H, et al. Outcomes after induction failure in childhood acute lymphoblastic leukemia. New Eng J Med. ۲۰۱۲ ۳۶۶: ۱۳۷۱-۸۱. ۳- Schultz KR, Bowman WP, Aledo A, et al. Improved early event-free survival with imatinib in Philadelphia chromosome–positive acute lymphoblastic leukemia: A childrenchr('۳۹')s oncology group study. J Clin Oncol. ۲۰۰۹ ۲۷: ۵۱۷۵-۸۱. ۴- Shah NN, Dave H, Wayne AS. Immunotherapy for pediatric leukemia. Frontiers in Oncology. ۲۰۱۳ ۳. ۵- Bai L, Zhu W-G. p۵۳: structure, function and therapeutic applications. J Cancer Mol. ۲۰۰۶ ۲: ۱۴۱-۵۳. ۶- Allen P, Newland A, Bustin S. The role of apoptosis (programmed cell death) in haemopoiesis and the immune system. Blood Reviews. ۱۹۹۳ ۷: ۶۳-۷۳. ۷- Inoue K, Kurabayashi A, Shuin T, Ohtsuki Y, Furihata M. Overexpression of p۵۳ protein in human tumors. Med Mol Morpho. ۲۰۱۲ ۴۵: ۱۱۵-۲۳. ۸- Saha MN, Qiu L, Chang H. Targeting p۵۳ by small molecules in hematological malignancies. J Hematol Oncol. ۲۰۱۳ ۶: ۲۳. ۹- Gaidano G, Ballerini P, Gong JZ, et al. p۵۳ mutations in human lymphoid malignancies: association with Burkitt lymphoma and chronic lymphocytic leukemia. Proceedings of the National Academy of Sciences. ۱۹۹۱ ۸۸: ۵۴۱۳-۷. ۱۰- Caligaris-Cappio F, Hamblin TJ. B-cell chronic lymphocytic leukemia: a bird of a different feather. Journal of Clinical Oncology. ۱۹۹۹ ۱۷: ۳۹۹. ۱۱- Lens D, De Schouwer PJ, Hamoudi RA, Abdul-Rauf M, Farahat N, Matutes E, et al. p۵۳ abnormalities in B-cell prolymphocytic leukemia. Blood. ۱۹۹۷ ۸۹: ۲۰۱۵-۲۳. ۱۲- Ashcroft M, Vousden KH. Regulation of p۵۳ stability. Oncogene. ۱۹۹۹ ۱۸: ۷۶۳۷-۴۳. ۱۳- Roh J-L, Ko JH, Moon SJ, Ryu CH, Choi JY, Koch WM. The p۵۳-reactivating small-molecule RITA enhances cisplatin-induced cytotoxicity and apoptosis in head and neck cancer. Cancer letters. ۲۰۱۲ ۳۲۵: ۳۵-۴۱. ۱۴- Issaeva N, Bozko P, Enge M, Protopopova M, Verhoef LG, Masucci M, et al. Small molecule RITA binds to p۵۳, blocks p۵۳–HDM-۲ interaction and activates p۵۳ function in tumors. Nature Medicine. ۲۰۰۴ ۱۰: ۱۲۸-۱۳۲۱. ۱۵- Selivanova G, editor Therapeutic targeting of p۵۳ by small molecules. Seminars in cancer biology ۲۰۱۰: Elsevier. ۱۶- Zhao CY, Szekely L, Bao W, Selivanova G. Rescue of p۵۳ function by small-molecule RITA in cervical carcinoma by blocking E۶-mediated degradation. Cancer Research. ۲۰۱۰ ۷۰: ۳۳۷۲-۸۱. ۱۷- Adachi N, So S, Iiizumi S, Nomura Y, Murai K, Yamakawa C, et al. The human pre-B cell line Nalm-۶ is highly proficient in gene targeting by homologous recombination. DNA and Cell Biology. ۲۰۰۶ ۱۹ ۲۴-۲۵. ۱۸- Fulda S. Inhibitor of apoptosis proteins in hematological malignancies. Leukemia. ۲۰۰۸ ۲۳: ۴۶۷-۷۶. ۱۹- Saha MN, Yang Y, Chang H. Targeting p۵۳ by small molecule p۵۳ activators in multiple myeloma. J Hematol Oncol. ۲۰۱۲ ۵(Suppl ۱): A۷. ۲۰- Ito A, Kawaguchi Y, Lai CH, Kovacs JJ, Higashimoto Y, Appella E, et al. MDM۲–HDAC۱‐mediated deacetylation of p۵۳ is required for its degradation. The EMBO J. ۲۰۰۲ ۲۱: ۶۲۳۶-۴۵. ۲۱- Marine J-C, Lozano G. Mdm۲-mediated ubiquitylation: p۵۳ and beyond. Cell Death & Differentiation. ۲۰۰۹ ۱۷: ۹۳-۱۰۲. ۲۲- Toledo F, Wahl GM. MDM۲ and MDM۴: p۵۳ regulators as targets in anticancer therapy. The international journal of biochemistry & cell biology. ۲۰۰۷ ۳۹: ۱۴۷۶-۸۲. ۲۳- Kastan MB. Wild-type p۵۳: tumors canchr('۳۹')t stand it. Cell. ۲۰۰۷ ۱۲۸: ۸۳۷-۴۰. ۲۴- Saha MN, Jiang H, Mukai A, Chang H. RITA inhibits multiple myeloma cell growth through induction of p۵۳-mediated caspase-dependent apoptosis and synergistically enhances nutlin-induced cytotoxic responses. Molecular Cancer Therapeutics. ۲۰۱۰ ۹: ۳۰۴۱-۵۱. ۲۵- Grinkevich VV, Nikulenkov F, Shi Y, Enge M, Bao W, Maljukova A, et al. Ablation of key oncogenic pathways by RITA-reactivated p۵۳ is required for efficient apoptosis. Cancer Cell. ۲۰۰۹۱۵: ۴۴۱-۵۳. ۲۶- Igney FH, Krammer PH. Death and anti-death: tumour resistance to apoptosis. Nature Reviews Cancer. ۲۰۰۲ ۲: ۲۷۷-۸۸.