بررسی اثر افزایش cAMP بر فسفوریلاسیون پروتئین BAD در رده‌ی سلولی لوسمی لنفوبلاستیک حاد پیش سازB- (NALM-6) تیمارشده با دوکسوروبیسین

Kashiri M1, Safa M2, Kazemi A3 1Dept. of Hematology, Allied Medical School, Tehran University of Medical Sciences, Tehran, Iran 2Cellular and Molecular Research Center, Dept. of Hematology and Blood Banking, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran 3Dept. of Hematology & Blood Banking, School of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran Corresponding Author: Safa M, Cellular and Molecular Research Center, Dept. of Hematology and Blood Banking, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran E-mail: [email protected] Received: 8 Feb 2014 Accepted: 22 Aug 2014 Background and Objective: BAD (Bcl-2-associated death promoter) is a proapoptotic protein which in phosphorylated state is inactivated in terms of its apoptotic function while in dephosphorylated status causes proceeding of apoptosis via inhibition of BCL-XL. cAMP elevating agents attenuate Doxorubicin induced-apoptosis. The aim of this study was to assess the effects of cAMP elevating agents via Forskolin and IBMX on phosphorylation of BAD in Pre-B acute lymphoblastic leukemia (pre-B ALL) cells. Materials and Methods: NALM-6 cells were incubated with cAMP increasing agents along with two concentrations of Doxorubicin 250 and 500 for 24 hours. Assessment of cell death was performed by Trypan blue staining and apoptosis was assessed by Annexin V via flowcytometry. Western blot was used for evaluating the expression of BAD protein and phosphorylation changes. Data was analyzed by Paired t-test. Results: The flowcytometry analysis of Annexin V revealed that cAMP increasing agents in combination with Doxorubicin lead to decreased rate of apoptosis in comparison to Doxorubicin alone. Analysis of western blotting showed that cAMP increasing agents cause phosphorylate serine residues of BAD. The study also indicated that Doxorubicin alone is unable to alter the phosphorylation status of BAD. Conclusion: Results of this study indicate that cAMP increasing agents attenuate doxorubicin-induced apoptosis via phosphorylation of BAD in NALM-6 cells. References 1- Jeha S, Razzouk B, Rytting M, et al. Phase II study of clofarabine in pediatric patients with refractory or relapsed acute myeloid leukemia. J Clin Oncol. 2009 27: 4392-7. 2- Martin A, Morgan E, Hijiya N. Relapsed or refractory pediatric acute lymphoblastic leukemia: current and emerging treatments. Paediatric Drugs. 2012 14: 377-87. 3- S Stanulla M, Schrappe M. Treatment of childhood acutelymphoblastic leukemia. Semin Hematol. 2009 46: 52-63. 4- Rai NK, Tripathi K, Sharma D, Shukla VK. Apoptosis: a basic physiologic process in wound healing. Int J Lower Extremity Wounds. 2005 4(3): 138-44. 5- Arends M, Wyllie A. Apoptosis: mechanism and roles in pathology. Pathol. 1991 32: 223-54. 6- Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L). Cell. 1996 87: 619-28. 7- Datta SR, Katsov A, Hu L, Petros A, Fesik SW, Yaffe MB. 14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation. Mol Cell. 2000 6: 41-51. 8- Zha J, Harada H, Osipov K, Jockel J, Waksman G, Korsmeyer SJ. BH3 domain of BAD is required for heterodimerization with BCL-XL and pro-apoptotic activity. J Biol Chem. 1997 272: 24101-4. 9- Condorelli F, Salomoni P, Cotteret S, Cesi V, Srinivasula SM, Alnemri ES. Caspase cleavage enhances the apoptosis-inducing effects of BAD. Mol and Cell Biology. 2001 21: 3025-36. 10- Danial NN. BAD: undertaker by night, candyman by day. Oncogene. 2008 27: 53-70. 11- Tasken K, Aandahl EM: Localized effects of cAMP mediated by distinct routes of protein kinase A. Physiol Rev. 2004, 84: 137-67. 12- de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A. Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature. 1998 396: 474-7. 13- Monahan TM, Marchand NW, Fritz RR, Abell CW. Cyclic adenosine 3’:5’ monophosphate levels and activities of related enzymes in normal and leukemic lymphocytes. Cancer Res. 1975 35: 2540-7. 14- Garcia-Bermejo L, Perez C, Vilaboa NE, de Blas E, Aller P. cAMP increasing agents attenuate the generation of apoptosis by etoposide in promonocytic leukemia cells. J Cell Sci. 1998 111: 637-44. 15- Linet MS, Cartwright RA. The leukemias. In: Schottenfeld D Fraumeni JF Jr. Cancer epidemiology and prevention, 2nd ed. New York: Oxford University Press. 1996. 16- Mantovani G, Bondioni S, Lania A, et al. High expression of PKA regulatory subunit 1A protein is related to proliferation of human melanoma cells. Oncogene. 2008, 27: 1834-43. 17- Neary CL, Nesterova M, Cho YS, Cheadle C, Becker KG, Cho-Chung YS. Protein kinase A isozyme switching: eliciting differential cAMP signaling and tumor reversion. Oncogene. 2004 23: 8847-56. 18- Safa M, Zand H, Mousavizadeh K, Kazemi A, Bakhshayesh M, Hayat P. Elevation of cyclic AMP causes an imbalance between NF-kappaB and p53 in NALM-6 cells treated by doxorubicin. FEBS lett. 2010 584: 3492-8. 19- Safa M, Kazemi A, Zand H, Azarkeivan A, Zaker F, Hayat P. Inhibitory role of cAMP on doxorubicin-induced apoptosis in pre-B ALL cells through dephosphorylation of p53 serine residues. Apoptosis. 2010 15: 196-203. 20- Safa M, Kazemi A, Zaker F, Razmkhah F. Cyclic AMP-induced p53 destabilization is independent of EPAC in pre-B acute lymphoblastic leukemia cells in vitro. J Recept Signal Transduct Res. 2011 31: 256-63. 21- Naderi EH, Findley HW, Ruud E, Blomhoff HK, Naderi S. Activation of cAMP signaling inhibits DNA damage-induced apoptosis in BCP-ALL cells through abrogation of p53 accumulation. Blood. 2009 114: 608-18. 22- von Knethen A, Brune B. Attenuation of macrophage apoptosis by the cAMP-signaling system. Mol Cell Biochem. 2000 212: 35-43. 23- Geier A, Weiss C, Beery R, Haimsohn M, Hemi R, Malik Z. Multiple pathways are involved in protection of MCF-7 cells against death due to protein synthesis inhibition. J Cell Physiol. 1995 163: 570-576. 24- Follin-Arbelet V, Hofgaard PO, Hauglin H, et al. Cyclic AMP induces apoptosis in multiple myeloma cells and inhibits tumor development in a mouse myeloma model. BMC Cancer. 2011 11: 301. 25- Lomo J, Blomhoff HK, Beiske K, Stokke T, Smeland EB. TGF-beta 1 and cyclic AMP promote apoptosis in resting human B lymphocytes. J Immunol. 1995 154: 1634-43. 26- Mentz F, Merle-Beral H, Dalloul AH. Theophyllineinduced B-CLL apoptosis is partly dependent on cyclic AMP production but independent of CD38 expression and endogenous IL-10 production. Leukemia. 1999 13: 78-84. 27- Mentz F, Mossalayi MD, Ouaaz F, et al. Theophylline synergizes with chlorambucil in inducing apoptosis of B-chronic lymphocytic leukemia cells. Blood. 1996 88: 2172-2182 28- Webster CR, Usechak P, Anwer MS. cAMP inhibits bile acid-induced apoptosis by blocking caspase activation and cytochrome c release. Am J Gastroenterol. 2002 283: 727-G38. 29- Blume-Jensen P, Janknecht R, Hunter T. The kit receptor promotes cell survival via activation of PI 3-kinase and subsequent Akt-mediated phosphorylation of Bad on Ser136. CB. 1998 8(13): 779-82. 30- Fang X, Yu S, Eder A, et al. Regulation of BAD phosphorylation at serine 112 by the Ras-mitogen-activated protein kinase pathway. Oncogene. 1999 18: 6635-40. 31- del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science. 1997 278: 687-9.