Abulayha, Abdulmunem

Bredan, Amin

Deyab, Mohamed

Elbanani, Abdulrhman

Geriani, Hajer

Lamami, Yosra

Tabal, Salah

English

Ghent University Academic Bibliography

Rituximab Synergizes with Hydroxyurea or Vincristinein the Killing of Ramos Burkitt’s Lymphoma B Cell LineMohamed Deyab,1 Abdulrhman Elbanani,1 Salah Tabal,1 Hajer Geriani,1 Yosra Lamami,1Amin Bredan,2,3 and Abdulmunem Abulayha1AbstractRituximab is an effective immunotherapy for CD20-positive B-cell non-Hodgkin’s lymphoma. However, somepatients show resistance, particularly those suffering from more aggressive lymphoma types, such as Burkitt’slymphoma. Hence, Rituximab is commonly combined with several chemotherapeutic drugs. With a view toreduce the number of such drugs, we examined the effect of combining Rituximab individually with hy-droxyurea, vincristine, or etoposide on the killing of Ramos Burkitt lymphoma cell line type I. Cell death wasexamined by using Annexin-V/propidium iodide staining. Combining Rituximab with hydroxyurea or vin-cristine resulted in a synergistic effect, whereas combining it with etoposide resulted in a subadditive effect. Insingle treatments, the percentage of cell death ranged from 23% (Rituximab) to 36% (hydroxyurea). CombiningRituximab with hydroxyurea or vincristine resulted in a synergistic effect (83% and 74% killing, respectively). Incontrast, only a subadditive effect was noticed with etoposide (36%). We conclude that the synergistic effect ofRituximab with hydroxyurea or vincristine is worthy of further study, and that further in vitro screening ofchemotherapeutics might identify chemo-immunotherapeutic combinations that are effective in vivo but lesstoxic than currently used regimens.Key words: chemotherapy, etoposide, hydroxyurea, Rituximab, vincristineIntroductionRituximab is highly effective in treating a wide range oflymphoproliferative disorders, including many B-celllymphomas (reviewed in Plosker and Figgitt1). Nevertheless,some patients with indolent follicular B cell lymphoma re-lapse repeatedly, and the response rate is still lower in patientswith more aggressive types of B-cell non-Hodgkin’s lym-phoma (NHL).2,3 Rituximab is also combined with chemo-therapy to improve long-term outcome. One of the mostcommonly used immune-chemotherapeutic combinations isR-CHOP, which combines Rituximab with cyclophospha-mide, doxorubicin, vincristine, and prednisone.4 R-CHOPshows a 95% response rate in indolent lymphomas5 and 94%in aggressive lymphomas.6 However, some types of lym-phoma, such as Burkitt’s lymphoma, require even more in-tense chemotherapeutic treatment.Although the introduction of chemo-immunotherapeuticcombinations has improved the outcome of NHL patients,some individuals do not respond to treatment and relapsesremain common. Therefore, new effective strategies for NHLtherapy are needed. The strategies should aim at using asfew chemotherapeutic agents with Rituximab as possible toreduce cytotoxicity while inducing a highly effective syner-gistic effect. We compared the effects of separately combin-ing three different antitumor compounds with Rituximab onthe cell death of a Burkitt’s lymphoma cell line. These com-pounds are commonly used in therapy and research andhave different mechanisms of action: hydroxyurea, a com-petitive inhibitor of ribonucleotide reductase7; vincristine, avinca alkaloid that inhibits the formation of mitotic spindles8;and etoposide, a potent inhibitor of topoisomerase II.9 Weintended to observe whether the chemotherapeutics wouldact synergistically or additively with Rituximab.1Cell Biology Research Group, Biotechnology Research Center, Twisha, Tripoli, Libya.2Inflammation Research Center, VIB, Ghent, Belgium.3Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.Address correspondence to: Abdulmunem Abulayha; Cell Biology Research Group, Biotechnology Research Center; Sedra Road, Twisha,P.O. Box 30313, Tripoli, LibyaE-mail: abdulmunem09@hotmail.comCANCER BIOTHERAPY AND RADIOPHARMACEUTICALSVolume 29, Number 2, 2014ª Mary Ann Liebert, Inc.DOI: 10.1089/cbr.2013.155787Materials and MethodsAntibodies and reagentsRituximab (MabThera; Roche Diagnostics; stock solution10 mg/mL) was a kind gift from the National OncologyInstitute (Sabratah, Libya). Unless stated otherwise, allother reagents were purchased from Sigma ChemicalCompany.Cell linesCD20-expressing human Burkitt’s lymphoma cell line(Ramos) was purchased from the European Collection ofCell Culture (ECACC) (Wiltshire SP4 0JK, UK) and main-tained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 200 mM L-glutamine solutionthat was sterile filtered and cell culture tested, and penicillin-streptomycin that was sterile filtered and cell culture tested.The cells were incubated at 37C in 5% CO2. Viability wasestimated by trypan blue exclusion, and cells were used onlyif their viability exceeded 90%.Cell treatments and Annexin-V-based cell death analysisCells were cultured at 1· 105 cells/mL in 25-cm2 tissueculture plates and treated with 10lg/mL Rituximab, eitheralone or in combination with 1 mM hydroxyurea, 2lM vin-cristine, or 10lM etoposide for 24 or 48 hours. They were thenwashed in phosphate-buffered saline and resuspended inbinding buffer containing 0.25lg/mL Annexin-V and 5lg/mL propidium iodide (PI; BD Biosciences) and incubatedin the dark for 15 min at room temperature before analy-sis. Single-cell suspensions were prepared for cytometricanalysis.Flow cytometry analysisCalibration, parameter optimization, and compensationfor the overlap between the different fluorochrome spectraFIG. 1. Representative of flow cytometryplots of Ramos cell death induced by ritux-imab or chemotherapy agents; FL1 (Annexin-V) versus FL2 (PI) after 48 h of treatment.Rituximab 10lg/mL; hydroxyurea 1 mM;vincristine 2lM; etoposide 10lM. Annexin-V +ve/PI- ve (early apoptotic cells) are thosein the lower right quadrant, whereasAnnexin-V +ve/PI+ ve (late apoptotic/ne-crotic cells) are in the upper right quadrant.88 DEYAB ET AL.were obtained automatically using FacsComp software(Becton Dickinson). Analysis and calculations were per-formed using CellQuest (Becton Dickinson). A minimum of20,000 events were collected.Statistical analysisAll values are given as the mean – standard deviation(SD). One-sample t-test was used to compare the effect ofcombined treatment with the mathematical sum of the cor-responding individual treatments.ResultsThree chemotherapeutics were evaluated for their abilityto synergize with Rituximab in the induction of cell death inthe Ramos B-cell line. For all treatments, as the time of in-cubation increased (24 and 48 hours) more cells becamepositive for both Annexin-V and PI (results not shown).Figure 1 shows representative plots of flow cytometricanalysis of cell death.Percentages of total apoptosis after 48 hours of incubationare shown in Figure 2. Rituximab alone induced23.5% – 0.74% (SD) apoptosis. Etoposide alone induced29.8% – 0.61%, vincristine 34.6% – 1.92%, and hydroxyurea36.1% – 0.35%. The effect of the Rituximab–etoposide com-bination was subadditive (36.2% – 0.52%). However, hy-droxyurea and vincristine synergized significantly withRituximab to induce 83.0% – 0.49% and 74.4% – 0.10% apo-ptosis, respectively (both P < 0.001 compared to mathemati-cal sum of individual treatments).DiscussionIn the treatment of B-cell NHLs, Rituximab alone inducesapoptosis only in a subpopulation of cells, whereas the restcontinue to proliferate. According to the literature, Ritux-imab is effective against indolent NHL but not against ag-gressive types.3 For that reason, standard chemotherapeuticagents are used to increase the efficacy of treatment. One ofthe most widely used combinations is R-CHOP, in whichRituximab is combined with cyclophosphamide, doxorubi-cin, vincristine, and prednisone. But in the absence of con-vincing in vitro evidence, most clinical trials have often takenan experimental approach with a broad range of combina-tions. Systematic in vitro studies might identify simplerimmune-chemotherapeutic combinations that are as effectiveas current combinations but are less toxic.Burkitt’s lymphoma is highly aggressive, and up to eightdifferent chemotherapeutic drugs, alone or with Rituximab,may be used in its treatment. The Ramos cell line was de-rived from a human Burkitt’s lymphoma and is hence rep-resentative of this type of neoplasm. In vitro, monomericRituximab induces only modest cell death in Ramos cells.10,11In vivo, a recent retrospective study reported that the inten-sive chemotherapy regimens used for adult Burkitt’s lym-phoma were associated with a favorable outcome, but thecontribution of Rituximab in combined therapy was un-certain and the authors suggested that it warrants furtherinvestigation.11We evaluated the effect of combining Rituximab individ-ually with three different chemotherapeutic drugs commonlyused in therapy and research and looked for additive orsynergistic effects. We selected these drugs because theyhave distinct mechanisms of action. Hydroxyurea reducesthe proliferative capacity of cells and induces senescence-likechanges, and eventually causes cells to arrest at G1.12–15Etoposide in vitro arrests Burkitt’s lymphoma cell lines14 andother cells15 in S/G2 by blocking DNA replication. In con-trast, vincristine disrupts cellular microtubules and preventsthe formation of a functional spindle, resulting in the accu-mulation of cells in the mitotic phase.8In vitro, pretreatment of a B lymphoma line with Ritux-imab sensitizes them to cytotoxic drugs,16 and some clinicaltrials have shown that pretreatment with Rituximab beforeeach chemotherapy results in an additive effect.5 However,improved effects have also been achieved in vitro by simul-taneous application of Rituximab and chemotherapeuticagents.10 We observed synergistic effects when CD20-expressingFIG. 2. Percent total cell death induced inRamos Burkitt’s lymphoma B cell line byRituximab, hydroxyurea, vincristine, etopo-side, and combinations thereof. Cells wereincubated for 48 hours with various treat-ments: Rituximab 10lg/mL, hydroxyurea1 mM, vincristine 2lM, and etoposide 10lM.‘‘Sum’’ refers to the mathematical sum ofindividual treatments. Values are means(n = 3) and error bars are standard deviations.Asterisks: effect of combined treatment issignificantly higher ( p< 0.001) than mathe-matical sum of corresponding individualeffects.RITUXIMAB SYNERGIZES WITH HYDROXYUREA OR VINCRISTINE 89human Burkitt’s lymphoma cells (Ramos) were incubatedsimultaneously with Rituximab in combination with eithervincristine or hydroxyurea. This synergy seems to be in-dependent of the mode of action of the chemotherapeuticagents, as vincristine and hydroxyurea have differentmechanisms of action. This finding might have clinical rele-vance, particularly in cases of Burkitt’s lymphoma, whereRituximab is not significantly associated with improvedoverall survival. It is worthwhile to evaluate the effect ofcombining Rituximab individually with other chemothera-peutics, and depending on the results, it can also be testedwith pairs of chemotherapeutics. We conclude that in vitrostudies could lay the ground for developing immuno-chemotherapeutic treatments with fewer agents than cur-rently used treatments and hence with fewer side-effects.AcknowledgmentThis work was funded by the Libyan Authority forResearch, Science, and Technology, Tripoli, Libya.Disclosure StatementThe authors declare no conflict of interest and no conflictbetween them.References1. Plosker GL, Figgitt DP. Rituximab: A review of its use innon-Hodgkin’s lymphoma and chronic lymphocytic leu-kaemia. Drugs 2003;63:803.2. Marcus R, Hagenbeek A. The therapeutic use of rituximab innon-Hodgkin’s lymphoma. Eur J Haematol Suppl 2007;67:5.3. van Meerten T, Hagenbeek A. CD20-targeted therapy: Thenext generation of antibodies. Semin Hematol 2010;47:199.4. Rambaldi A, Lazzari M, Manzoni C, et al. Monitoring ofminimal residual disease after CHOP and rituximab inpreviously untreated patients with follicular lymphoma.Blood 2002;99:856.5. Czuczman MS, Grillo-Lopez AJ, White CA, et al. Treatmentof patients with low-grade B-cell lymphoma with the com-bination of chimeric anti-CD20 monoclonal antibody andCHOP chemotherapy. J Clin Oncol 1999;17:268.6. Vose JM, Link BK, Grossbard ML, et al. Long-term update ofa phase II study of rituximab in combination with CHOPchemotherapy in patients with previously untreated, ag-gressive non-Hodgkin’s lymphoma. Leuk Lymphoma2005;46:1569.7. Elledge SJ, Zhou Z, Allen JB. Ribonucleotide reductase:Regulation, regulation, regulation. Trends Biochem Sci1992;17:119.8. Jordan MA, Wilson L. Microtubules as a target for anticancerdrugs. Nat Rev Cancer 2004;4:253.9. Hochhauser D, Harris AL. The role of topoisomerase IIalpha and beta in drug resistance. Cancer Treat Rev1993;19:181.10. Ghetie MA, Bright H, Vitetta ES. Homodimers but notmonomers of Rituxan (chimeric anti-CD20) induce apoptosisin human B-lymphoma cells and synergize with a chemo-therapeutic agent and an immunotoxin. Blood 2001;97:1392.11. Wasterlid T, Brown PN, Hagberg O, et al. Impact of che-motherapy regimen and rituximab in adult Burkitt lym-phoma: A retrospective population-based study from theNordic Lymphoma Group. Ann Oncol 2013;24:1879.12. Ajchenbaum F, Ando K, DeCaprio JA, et al. Independentregulation of human D-type cyclin gene expression duringG1 phase in primary human T lymphocytes. J Biol Chem1993;268:4113.13. Hayashi N, Tsutsui T. Cell cycle dependence of cytotoxicityand clastogenicity induced by treatment of synchronizedhuman diploid fibroblasts with sodium fluoride. Mutat Res1993;290:293.14. Lin CK, Nguyen TT, Morgan TL, et al. Apoptosis may beeither suppressed or enhanced with strategic combina-tions of antineoplastic drugs or anti-IgM. Exp Cell Res 1998;244:1.15. Pocsik E, Mihalik R, Penzes M, et al. Effect of cell cycle onthe regulation of the cell surface and secreted forms of type Iand type II human tumor necrosis factor receptors. J CellBiochem 1995;59:303.16. Demidem A, Lam T, Alas S, Hariharan K, Hanna N, Bona-vida B. Chimeric anti-CD20 (IDEC-C2B8) monoclonal anti-body sensitizes a B cell lymphoma cell line to cell killingby cytotoxic drugs. Cancer Biother Radiopharm 1997;12:177.90 DEYAB ET AL.

Rituximab synergizes with hydroxyurea or vincristine in the killing of Ramos Burkitt's lymphoma B cell line

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Rituximab synergizes with hydroxyurea or vincristine in the killing of Ramos Burkitt's lymphoma B cell line

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