OVERCOMING CHEMORESISTANCE IN NON-HODGKIN LYMPHOMA PRELIMINARY STUDIES OF APOPTOSIS AND NECROSIS BY P-GLYCOPROTEIN REVERSAL AGENTS
Objective: The in vitro measurement of drug-induced apoptosis provides a mechanism-based test for the chemosensitivity of tumor cells isolated from a patient or from a specific cell line. The goal of this study was to investigate the effects of p-glycoprotein reversal agents on apoptosis and necrosis in Burkitt lymphoma cells. These effects were determined by microscopic observation and by electrophoretic separation of DNA fragments.
Methods: We demonstrated induction of apoptosis in Burkitt lymphoma Raji Thymidine Kinase+ (TK+)and TK- cells using different subclasses of p-glycoprotein reversal agents. A low dose of doxorubicin was also used. The selective clonal expansion of mutant lymphocytes is based upon the phenotypic properties of TK-deficient cells. The first phase of the present study involved morphological analyses and DNA degradation on agarose gel electrophoresis. The second phase analyzed DNA damage using the Comet assay and tail moments calculated with Komet imaging software.
Results: Electrophoretic separation resulted in a ladder pattern, indicating that the p-glycoprotein reversal agents were able to induce apoptosis and necrosis. The morphological frequency of apoptosis and necrosis in the cells was significantly increased. Most p-glycoprotein reversal agents showed an increase in tail moments in the Comet assay.
Conclusion: The results indicate that indomethacin and quercetin may help to overcome chemoresistance in Burkittâ€™s lymphoma.
2. Singh N. Apoptosis in health and disease and modulation of apoptosis for therapy: an overview. Indian J Clin Biochem 2007;22:6-16.
3. Dlamini Z, Mbita Z, Zungu M. Genealogy, expression, and molecular mechanisms in apoptosis. Pharmacol Ther 2004;101:1-15.
4. Hicks SD, Lewis L, Ritchie J, Burke P, Abdul-Malak Y, Adackapara N, et al. Evaluation of cell proliferation, apoptosis, and DNA-repair genes as potential biomarkers for ethanol-induced CNS alterations. BMC Neurosci 2012;13:128.
5. Chatzitolios A, Venizelos I, Tripsiannis G, Anastassopoulos G, Papadopoulos N. Prognostic significance of CD95, P53, and BCL2 expression in extranodal non-Hodgkin's lymphoma. Ann Hematol 2010;89:889-96.
6. Kerr JF, Winterford CM, Harmon BV. Apoptosis. Its significance in cancer and cancer therapy. Cancer 1994;73:2013-26.
7. Trump BF, Berezesky IK, Chang SH, Phelps PC. The pathways of cell death: oncosis, apoptosis, and necrosis. Toxicol Pathol 1997;25:82-8.
8. Barry MA, Behnke CA, Eastman A. Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem Pharmacol 1990;40:2353-62.
9. Fadeel B, Zhivotovsky B, Orrenius S. All along the watchtower: on the regulation of apoptosis regulators. FASEB J 1999;13:1647-57.
10. Cocco RE, Ucker DS. Distinct modes of macrophage recognition for apoptotic and necrotic cells are not specified exclusively by phosphatidylserine exposure. Mol Biol Cell 2001;12:919-30.
11. Foroutan B, Zitzelsberger H, Bauer V, Ruf AA, Baumgartner A, Anderson D. Molecular cytogenetic analysis of chemoresistant non-Hodgkin's lymphoma patients with p53 abnormalities using fluorescence in situ hybridisation and comparative genomic hybridisation. Arch Iran Med 2011;14:321-6.
12. Xu-Monette ZY, Medeiros LJ, Li Y, Orlowski RZ, Andreeff M, Bueso-Ramos CE, et al. Dysfunction of the TP53 tumor suppressor gene in lymphoid malignancies. Blood 2012;119:3668-83.
13. Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, Yamashita T, et al. Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Sci 2000;288:1053-8.
14. Owen-Schaub LB, Zhang W, Cusack JC, Angelo LS, Santee SM, Fujiwara T, et al. Wild-type human p53 and a temperature-sensitive mutant induce Fas/APO-1 expression. Mol Cell Biol 1995;15:3032-40.
15. Donald SP, Sun XY, Hu CA, Yu J, Mei JM, Valle D, et al. Proline oxidase, encoded by p53-induced gene-6, catalyzes the generation of proline-dependent reactive oxygen species. Cancer Res 2001;61:1810-5.
16. Bennett M, Macdonald K, Chan SW, Luzio JP, Simari R, Weissberg P. Cell surface trafficking of Fas: a rapid mechanism of p53-mediated apoptosis. Sci 1998;282:290-3.
17. Mizutani T, Masuda M, Nakai E, Furumiya K, Togawa H, Nakamura Y, et al. Genuine functions of P-glycoprotein (ABCB1). Curr Drug Metab 2008,9:167-74.
18. Ambudkar SV, Dey S, Hrycyna CA, Ramachandra M, Pastan I, Gottesman MM. Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu Rev Pharmacol Toxicol 1999;39:361-98.
19. Ganguly A, Banerjee K, Chakraborty P, Das S, Sarkar A, Hazra A, et al. Overcoming multidrug resistance (MDR) in cancer in vitro and in vivo by a quinoline derivative. Biomed Pharmacother 2011;65:387-94.
20. Fojo T, Bates S. Strategies for reversing drug resistance. Oncogene 2003;22:7512-23.
21. Pulvertaft JV. Cytology of burkitt's tumour (African Lymphoma). Lancet 1964;1:238-40.
22. Lehne G. P-glycoprotein as a drug target in the treatment of multidrug resistant cancer. Curr Drug Targets 2000;1:85-99.
23. Lehne G, Morkrid L, den Boer M, Rugstad HE. Diverse effects of P-glycoprotein inhibitory agents on human leukemia cells expressing the multidrug resistance protein (MRP). Int J Clin Pharmacol Ther 2000;38:187-95.
24. Wyllie AH, Kerr JF, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 1980;68:251-306.
25. Rydberg B, Johanson KJ. Estimation of DNA strand breaks in single mammalian cells. In: DNA repair mechanisms. New York: Academic Press; 1978.
26. Ostling O, Johanson KJ. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 1984;123:291-8.
27. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988;175:184-91.
28. Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, et al. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 2000;35:206-21.
29. Pool-Zobel BL, Lotzmann N, Knoll M, Kuchenmeister F, Lambertz R, Leucht U, et al. Detection of genotoxic effects in human gastric and nasal mucosa cells isolated from biopsy samples. Environ Mol Mutagen 1994;24:23-45.
30. Henderson L, Wolfreys A, Fedyk J, Bourner C, Windebank S. The ability of the Comet assay to discriminate between genotoxins and cytotoxins. Mutagen 1998;13:89-94.
31. Searle J, Kerr JF, Bishop CJ. Necrosis and apoptosis: distinct modes of cell death with fundamentally different significance. Pathol Annu 1982;17(2):229-59.
32. Singh NP. Microgels for estimation of DNA strand breaks, DNA protein crosslinks and apoptosis. Mutat Res 2000;455:111-27.
33. Plaumann B, Fritsche M, Rimpler H, Brandner G, Hess RD. Flavonoids activate wild-type p53. Oncogene 1996;13:1605-14.
34. Anensen N, Hjelle SM, Van Belle W, Haaland I, Silden E, Bourdon JC, et al. Correlation analysis of p53 protein isoforms with NPM1/FLT3 mutations and therapy response in acute myeloid leukemia. Oncogene 2012;31:1533-45.
35. Muller C, Chatelut E, Gualano V, De Forni M, Huguet F, Attal M, et al. Cellular pharmacokinetics of doxorubicin in patients with chronic lymphocytic leukemia: comparison of bolus administration and continuous infusion. Cancer Chemother Pharmacol 1993;32:379-84.
36. Ye CG, Wu WK, Yeung JH, Li HT, Li ZJ, Wong CC, et al. Indomethacin and SC236 enhance the cytotoxicity of doxorubicin in human hepatocellular carcinoma cells via inhibiting P-glycoprotein and MRP1 expression. Cancer Lett 2011;304:90-6.
37. Arisawa M, Kasaya Y, Obata T, Sasaki T, Nakamura T, Araki T, et al. Design and synthesis of indomethacin analogues that inhibit P-glycoprotein and/or multidrug resistant protein without COX inhibitory activity. J Med Chem 2012,55:8152-63.
38. Li SZ, Li K, Zhang JH, Dong Z. The effect of quercetin on doxorubicin cytotoxicity in human breast cancer cells. Anticancer Agents Med Chem 2013;13:352-5.