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Small Molecule Therapeutics

The NADPH Oxidase Inhibitor Imipramine-Blue in theTreatment of Burkitt Lymphoma

Marcel Klingenberg1, J€urgen Becker1, Sonja Eberth2, Dieter Kube2, and J€org Wilting1

AbstractBurkitt lymphoma is a rare malignancy arising from B cells. Current chemotherapeutic regimens achieve

excellent overall survival rates in children, but less impressive rates in adults. There are cases with poor

outcome causedby toxic effects of the therapy, tumor lysis syndrome, ormetastatic spreadof lymphomas to the

central nervous system. Modulators of reactive oxygen species are currently discussed as potential drugs for

the treatment of cancer. The NADPH oxidase 4 inhibitor imipramine-blue might satisfy the aforementioned

requirements, andwas studiedhere.WeusedMTTassay, crystal violet assay, and thymidine 3H-incorporation

assay to analyze the effects of imipramine-blue on Burkitt lymphoma (BL2, BL2B95, BL30B95, BL41B95),

neuroblastoma (KELLY, SH-SY5Y, SMS-KAN), cervix carcinoma (HeLa), breast cancer (MDA-MB231),

angiosarcoma (AS-M), human embryonic kidney (HEK293WT), and nonmalignant (FLP1) cell lines. The

effects of imipramine-blue on BL2B95 cells in vivowere investigated in xenografts on the chick chorioallantoic

membrane (CAM). We report that imipramine-blue is a potent growth inhibitor for several cancer cell lines in

vitro with IC50 values comparable to those of doxorubicin (0.16–7.7 mmol/L). Tumor size of BL2B95 cells

inoculated in the CAMwas reduced significantly (P < 0.05) after treatment with 10 mmol/L imipramine-blue.

Lymphogenic dissemination of BL2B95 and the formation of blood and lymphatic vessels in experimental

tumors were not affected. We show that imipramine-blue can be used to decrease the viability of cancer cell

lines in vitro and in vivo. Imipramine-blue reduces the size of experimental Burkitt lymphoma significantly but

does not affect the dissemination of BL2B95 cells, angiogenesis, and lymphangiogenesis.MolCancer Ther; 13(4);

833–41. �2014 AACR.

IntroductionIn still too many cases, cancer is a fatal diagnosis, and

additional compounds that halt tumor growth andmetas-tasis are needed.Here,we studied the antitumor potentialof the triphenylmethane dye imipramine-blue (1), whichis based on the well-known antidepressant drug imipra-mine. Thereby, we concentrated on the non-Hodgkinlymphoma subtype Burkitt lymphoma,which arises fromB cells. Burkitt lymphoma is a rare disease with an inci-dence rate of approximately 0.2, which means that 2individuals within a population of 1 million are affectedby Burkitt lymphoma per year (2). Because of the lowincidence of the disease, research on novel drugs forBurkitt lymphoma treatment proceeds only slowly. How-ever, as lymphomas are known to form metastases in

several sites, the analysis of their metastatic behavior andthe suppressionof this by imipramine-blue treatmentmayhave implications for other aggressive tumor types aswell.

Treatment of Burkitt lymphoma is based on intensivechemotherapy with cyclophosphamide, doxorubicin,methotrexate, vincristine, cytarabine, and etoposide incombination with immunosuppressive drugs such asprednisolone, dexamethasone, and rituximab. Clinicaltreatment protocols in high-income countries are basedon the FAB LMB study or the Berlin–Frankfurt–Muensterprotocols and achieve outstanding overall survival ratesin children (3). Nevertheless, there are cases of poorclinical outcome caused by the toxicity of the intensivechemotherapy, tumor lysis syndrome, or metastaticspread of lymphoma cells to the central nervous system(4–7). Especially elderly andHIV-positive patients show apoorer overall survival rate because of treatment-relatedmortality (3, 8, 9). Thus, there is the need to developminimally toxic substances to treat patients with high-grade-non-Hodgkin lymphoma efficiently. A novel classof promising chemotherapeutics is the group of reactiveoxygen species (ROS)–modulating substances (10). Theanalysis ofROSand their involvement in the initiation andmaintenance of malignancies is an emerging topic incancer research (11, 12). Until recently, ROS were mainlyregarded as toxic side-products of metabolic reactions, or

Authors' Affiliations: Departments of 1Anatomy and Cell Biology and2Hematology and Oncology, University Medicine Goettingen, Goettingen,Germany

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: J€org Wilting, Department of Anatomy and CellBiology, University Medicine Goettingen, Kreuzbergring 36, 37075 Goet-tingen, Germany. Phone: 49-551-397050; Fax: 49-551-397067; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-13-0688

�2014 American Association for Cancer Research.

MolecularCancer

Therapeutics

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on July 6, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst January 30, 2014; DOI: 10.1158/1535-7163.MCT-13-0688

http://mct.aacrjournals.org/

as part of the cellular defense machinery needed for thekilling ofmicrobes. But accumulating evidence suggests amore complex role for ROS in cellular pathways, forexample an involvement in proliferation, apoptosis,angiogenesis, and metastasis (13).

It has been shown that tumor cell lines possess highROS levels, but, depending on the investigated cell line,the experimental alteration of the ROS homeostasis hasrevealed opposing effects (14–16). A recent study provid-ed evidence for an anti-invasive effect of the new ROS-modulating drug imipramine-blue in a highly invasiveRT2 syngeneic astrocytoma ratmodel. Imipramine-blue issynthesized from the antidepressant drug imipramineand Michler ketone (Fig. 1F). Treatment with imipra-mine-blue induced the formation of tumors with a morecompact morphology. It reduced invasion of tumor cellsinto healthy tissues and prolonged survival after combi-nation therapywith liposomal doxorubicin (1). The targetprotein of imipramine-blue,NADPHoxidase 4 (NOX4), isinvolved in antiapoptotic signaling, including the NF-kBsignaling pathway, which is highly active in many non-Hodgkin lymphomas (15, 17–19).

To evaluate the antitumor and antimetastatic effects ofimipramine-blue, we treated numerous tumor and nor-mal cell lines in vitro, and lymphoma cells in a xenograftchick chorioallantoicmembrane (CAM) tumormodel.Wechose the BL2B95 lymphoma cell line for the CAM assay,because our previous experiments showed that the EBVþ

BL2B95 cells exhibited a high potential for lymphogenicdissemination in the CAM (20), and the infection withEBV has been shown to increase ROS levels (21). Further-more, the cell line was isolated from a patient withmultiple metastases, which involved the central nervoussystem, the bones, the abdomen and the bone marrow(22). This fact illustrates BL2’s highly aggressive metas-tasis formation in a living organism and makes this cellline a highly suitable model for in vivo experiments like inour study.

Materials and MethodsCell culture

HEK293WT, HeLa, KELLY, MD-MBA 231 cell lineswere obtained from Leibniz Institute DSMZ-GermanCollection of Microorganisms and Cell Cultures (Bra-unschweig). SH-SY5Y cell line was obtained from Amer-ican Type Culture Collection. SMS-KAN cell line waskindly provided byProf. P. Reynolds (School ofMedicine,Texas Tech University, Lubbock, TX). AS-M cells werekindly provided by Prof. Dr. P. Str€obel (Department ofPathology, University Medicine Goettingen, Goettingen,Germany). The aforementioned cell lineswerenot authen-ticated in the authors’ laboratory. Burkitt lymphomacell lines (BL2B95, BL2, BL30B95, BL41B95)were obtainedfrom IARC Lyon trough G. Bornkamm in Munich, Ger-many. Burkitt lymphoma cell lines are regularly authen-ticated via immunoglobulin PCR once a year. Burkittlymphoma cell lines (BL2B95, BL2, BL30B95, BL41B95)

were cultured in RPMI-1640 medium (Lonza) with 10%fetal calf serum (FCS), 1% penicillin/streptomycin(biochrome), 10 mmol/L HEPES (GIBCO), 1 mmol/Lsodium-pyruvate (GIBCO), 50 mmol/L a-thioglycerol(Sigma-Aldrich), and 20 nmol/L BCS (bathocuproinedisulfonic acid; Sigma-Aldrich). AS-M cells (angiosar-coma) were cultured in EGM-2 (Clonetics). Normalfibroblasts (FLP1), as well as HEK293WT, MD-MBA231 (breast cancer), KELLY, SH-SY5Y, and SMS-KAN(neuroblastoma), were cultured in RPMI-1640 with 10%FCS and 1% penicillin/streptomycin. All cell lines wereincubated at 37�C and 5% CO2.

CAM assayFertilized white Leghorn chick eggs were incubated at

80% relative humidity and 37.8�C. The eggs were win-dowed at day 3 and the window was sealed with cello-tape. At day 10 of chick development, 1 � 106 BL2B95cells/egg were applied on the CAM. Cells were resus-pended in 50% Burkitt lymphoma medium (with andwithout imipramine-blue) and 50%Matrigel and incubat-ed for 30 minutes at 37�C, 5% CO2 before applying themon the CAM. The tumors were surgically removed on day17 of chick development. Tumors were fixed in 4% para-formaldehyde for 15minutes, washed 3 times in PBS, andtransferred into 10% sucrose for 3 hours at 4�C and 30%sucrose overnight at 4�C. Tumors were then embedded inpolyfreeze tissue freezing medium and cut with a cryo-tome into 4 to 12 mm sections.

Thymidine 3H-incorporation assayThymidine 3H-incorporation assays were performed in

duplicate in 96-well plates. Cells were seeded at a con-centration of 3 � 105 cells/mL in a final volume of 100 mLBurkitt lymphoma medium per well. Imipramine-bluedissolved in Burkitt lymphoma medium was added toa final concentration of 5, 10, and 20 mmol/L. Dimethylsulfoxide (DMSO) was added in the control groups to afinal concentration of 0.2%. Cells were incubated for 24hours at 37�C and 5% CO2. After 32 hours, 0.5 mCi thy-midine-5-3H solved in 30 mL Burkitt lymphoma mediumwas added to each well. The cells were then incubated for16 hours at 37�C and 5% CO2 and transferred onto amembrane with Inotech cell harvester. The membranewas air dried and 5 mL liquid scintillation cocktail wasadded. The scintillation was measured with a 1450MicroBeta Trilux Liquid Scintillation and LuminescenceCounter (Wallac).

Methyl violet proliferation assayThe methyl violet proliferation assays were at least

performed as sextuplicates in 96-well plates. Cells wereseeded at a concentration of 105/mL in a final volume of50 mL. After 24 hours, imipramine-blue, dissolved in therespective media, was added to a final concentration of0.1, 1, 5, 10, and 25 mmol/L. DMSO was added in thecontrol groups to a final concentration of 0.25%, whichcorresponded to the highest amount of solvent in 25

Klingenberg et al.

Mol Cancer Ther; 13(4) April 2014 Molecular Cancer Therapeutics834




mmol/L imipramine-blue group. Cells were then incubat-ed at 37�C and 5% CO2 for 24 or 72 hours. Subsequently,cells were fixed with 50 mL of 5% glutaraldehyde per well

and incubated at room temperature (RT) for 30minutes ona rocking table. Then cells were washed 3 times withpurified water and air dried for at least 60 minutes.

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Figure 1. Effect of imipramine-blue onBurkitt lymphomacell line viability. A, 24-hourMTTassay of Burkitt lymphomacell lines treatedwith imipramine-blue. B, effectof 1 mmol/L imipramine-blue for 24 hours on Burkitt lymphoma cell lines (statistical analysis). C, 72-hour MTT assay of Burkitt lymphoma cell lines treatedwith imipramine-blue. D, effect of 1 mmol/L imipramine-blue for 72 hours on Burkitt lymphoma cell lines (statistical analysis). E, thymidine 3H-incorporation assay ofBurkitt lymphoma cells incubated with imipramine-blue for 24 hours. F, chemical structure of imipramine-blue. This image is reproduced with permission from TheAmerican Association for the Advancement of Science (1). �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

Imipramine-Blue Treatment of Burkitt Lymphoma

www.aacrjournals.org Mol Cancer Ther; 13(4) April 2014 835




Subsequently, 100 mL 0.1% methyl violet solution wasadded and incubated for 20 minutes at RT on a rockingtable. Cells were washed 3 times with purified water andair dried for at least 30 minutes. After that, 100 mL of 10%acetic acid was added and plates were agitated until themethyl violet was redissolved. Extinction was measuredwith ThermoMAXmicroplate reader (Molecular Devices)at 630 nm.

MTT assayMTTcell viability assayswere performed in triplicate in

96-well plates. Cells were seeded at a density of 3 � 105

cells/well for 24hoursMTTassay and104 cells/well for 72hoursMTT assay. Imipramine-bluewas diluted in Burkittlymphoma medium to a final concentration of 0.05, 0.1,0.3, 1, 2.5, 5, 10, or 20 mmol/L per well. Cells wereincubated for 24 or 72 hours at 37�C and 7% CO2. After20 or 68 hours, 10 mL of 0.5% MTT in PBS was added toeach well and incubated for 4 hours at 37�C and 5% CO2.Then the plates were centrifuged at 1,000 rpm for 10minutes, supernatant was discarded and 100 mL of 33%DMSO, 5% formic acid, and 62% isopropanolwere added.The plates were shaken until the precipitate was resolvedand the extinctionwasmeasuredwith SynergyMxmicro-plate reader (BioTek) at 540 nm.

Immunofluorescence analysesImmunohistological staining of specimens was per-

formed by incubation for 1 hour with blocking reagent(PBS, 1% BSA, 5% goat serum, 0.2% Triton X-100), 1-hourincubation of primary antibody diluted in antibody solu-tion [TBS (0.05mol/L, pH7.2–7.4), 1%BSA, 0.5%TritonX-100] and 1-hour incubation of secondary antibody dilutedin antibody solution mixed with DAPI (1:10,000). Aftereach step, specimens were rinsed 3 times with PBS. Sam-ples weremountedwith Fluoromount-G (Sigma-Aldrich)and dried overnight at room temperature. Specimenswere studied with Zeiss Axio Imager.Z1 (Zeiss) and filtersets 38HE, 43, 49, and 50. Primary antibodies were rabbit-anti-human Prox1 (ReliaTech) at 1:500 dilution, mouse-anti-humanHLAA,B,C (BioLegend) at 1:200dilution, andmouse-anti-Mep21 (chick CD34 homolog; M. Williams,AbLab) at dilution of 1:100.

Determination of tumor size and vesselsPicturesof tumorcryosections (H&Estained)were taken

with a Leica MZ 16 FA stereo microscope at bright-fieldmode and tumor area was quantified with ImageJ 1.46rsoftware. Numbers of intratumoral Mep21-positive bloodvessels and Prox1-positive lymphatic endothelial cellswere counted in immunofluorescence specimens at �20magnification.

Statistical analyses and IC50-value calculationStatistical analyses were performed using the program

GraphPad Prism 5. Data of methyl violet andMTT assayswere normalized to the internal DMSO control, concen-trations (mmol/L) were log-transformed and absorption

values were transformed with the term Y¼ 100� Y. Datawere plotted in log-dose versus response and analyzedwith nonlinear regression (log inhibitor vs. normalizedresponse-variable slope). IC50 values and 95% confidenceintervals (CI) were calculated by the software automati-cally. Data of tumor area were normalized to the internaltest series DMSO control. Statistical significance was ana-lyzed with an unpaired 2-tailed Student t test at a signif-icance level of 0.05.

ResultsImipramine-blue causes a reduction of Burkittlymphoma viability and proliferation in vitro

The treatment with imipramine-blue caused a signifi-cant decrease of Burkitt lymphoma cell viability in vitro(Fig. 1A–D). Twenty-four-hour incubation with 1 mmol/Limipramine-blue showed a reduction to less than 25%viability (compared with the DMSO-treated controls) forthe Burkitt lymphoma cell lines BL2B95 (P < 0.001), BL2(P < 0.001), and BL30B95 (P < 0.001). The viability of theBL41B95 (P < 0.01) cell line was only reduced to approx-imately 75% after 24-hour incubation with 1 mmol/Limipramine-blue (Fig. 1B). Treatment for 72 hours causeda significant decrease of viability to less than 15% inBL2B95 (P < 0.001), BL2 (P < 0.001), BL30B95 (P <0.001), and BL41B95 (P < 0.05) cell lines (Fig. 1C and D).The thymidine 3H-incorporation assay showed that theproliferative activity of the analyzed Burkitt lymphomacell lines was completely (<1%) inhibited (Fig. 1E).

Tumor formation and size in a xenograft CAMmodelBL2B95 cells treated with 10 mmol/L imipramine-blue

and DMSO formed lentiform tumors in the CAM, whichvaried in color between reddish (highly vascularized) andwhitish (sparsely vascularized).Hemorrhage in the centerof the tumors was found in both imipramine-blue– andDMSO-treated groups (Fig. 2A–C). DMSO (N¼ 4) and 10mmol/L imipramine-blue–treated (N ¼ 7) tumors werecross-sectioned and the sizes (mm2)weredetermined. Theanalysis showed a significant decrease of the tumor size inthe 10 mmol/L imipramine-blue group to 60% comparedwith the DMSO-treated group (P < 0.05; Fig. 2D).

Lymphogenic dissemination of BL2B95 cellsThe treatment with imipramine-blue had no obvious

effect on the dissemination of the BL2B95 cells via thelymphatic system. Tumor cells were detectable withinlymphatic vessels in all 3 groups (DMSO, 1 mmol/L imip-ramine-blue, 10 mmol/L imipramine-blue). BL2B95 cellswere found within lymphatics both in the vicinity of thetumors and at distances of 5- to 10-mmapart (Fig. 2E, a–h).

Lymphatic and blood vessel formation and densityNext, we analyzedwhether imipramine-blue treatment

had an effect on the vascularization of the experimentaltumors. We determined the blood and lymphatic vesseldensity with an immunofluorescence approach, and

Klingenberg et al.





found that imipramine-blue had no effect on the forma-tion of vessels in the tumor. The number of Prox1þ

lymphatic endothelial cells was unchanged (Supplemen-tary Fig. S1A) as was the number of blood vessels in thetreated tumors compared with DMSO control tumors(Supplementary Fig. S1B).

Effects of imipramine-blue on the viability of othercancer cell linesTo determine if the effects of imipramine-blue were

restricted to Burkitt lymphoma cells, we tested the via-bility of neuroblastoma (SH-SY5Y, KELLY, SMS-KAN),angiosarcoma (AS-M), cervix carcinoma (HeLa), humanembryonic kidney (HEK-293WT), and breast cancer(MDA-MB231) cell lines. The responses of the cancer celllines to imipramine-blue treatment were compared withthe nontumorigenic FLP1 fibroblast cell line (Fig. 3A–C,a–f). This was done to determine if the observed effects

were restricted to cancer cell lines or if there were non-specific cytotoxic effects. The incubation with imipra-mine-blue caused clearly visible morphologic changes ofthe cells. FLP-1 cells showed a slight decrease of theirdiameter, whereas KELLY cells detached from the culturedish most likely by apoptosis (Fig. 3a–f). The cancer celllines showed distinct responses to imipramine-blue treat-ment. The highest sensitivity toward imipramine-bluetreatment was observed in the neuroblastoma cell lineKELLY. These cells responded to the treatment with aviability reduction to less than 10% after 72-hour treat-ment with 1 mmol/L imipramine-blue (Fig. 3A and B).Incubation with 5 mmol/L imipramine-blue for 72 hoursdecreased the viability of all analyzed cancer cell lines toless than 25% (Fig. 3C). In contrast, the viability of thenonmalignant cell line FLP-1 remained nearly unaltered(97%; Fig. 3A–C). The statistical analyses of 2 imipramine-blue concentrations (1 and 5 mmol/L) showed significant

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Figure 2. Treatment of xenograftedBL2B95 with imipramine-blue in aCAM assay. A–C, tumors on CAMformed by BL2B95 cells after 7days. Scale bar, 5 mm. D, tumorarea quantification of DMSO (n¼ 4)and 10 mmol/L imipramine-blue(n ¼ 7); �, P < 0.05. E, imagecomposition of 9 single picturestaken from aBL2B95 tumor treatedwith 10 mmol/L imipramine-blue.Bv, blood vessel; �, hemorrhagewith BL2B95 cells. Scale bar, 100mm. a–d, higher magnification ofthe white rectangle in E. Scale bar,50 mm. e–h, longitudinallysectioned lymphatic vessel filledwith BL2B95 cells. Picture takenfrom a tumor treated with 1 mmol/Limipramine-blue. Scale bar, 100mm.






differences in the response between the FLP1 cell line andthe cell lines HeLa (P < 0.001; P < 0.001), HEK-293WT (P <0.001;P < 0.001), SH-SY5Y (P < 0.01;P < 0.001), KELLY (P <0.001; P < 0.001), SMS-KAN (P < 0.001; P < 0.001), AS-M(P < 0.001; P < 0.001), and MDA-MB231 (P < 0.001; P <0.001) after 72-hour incubation. The transformation of themethyl violet and MTT data produced a typical logarith-mic dose versus response curve (Fig. 3A). IC50 values andCIswere calculated for each concentration and time-point.Data are summarized in Table 1.

DiscussionThe most important finding of this study is the reduc-

tion of Burkitt lymphoma tumor size after the treatmentwith imipramine-blue in the chick CAMmodel. Althoughwe initially assumed to reduce the metastatic behavior ofthe lymphoma cells, our findings may rather suggest apossible use of imipramine-blue as a cytotoxic drug forBurkitt lymphoma treatment. The dissemination of

BL2B95 cells via the lymphatics was unaltered, as wasthe raggedappearance of the tumor borders. This seems tobe in contrast to the findings of the anti-invasive effects ofimipramine-blue in a rodent astrocytoma (RT2)model (1).However, these differences may reside in inherent differ-ences of the chickCAMmodel and the ratmodel, differentmodes of migration of the analyzed tumor cells, or in thedifferent application forms of the drug. The RT2 cell line,whichwas used in the astrocytoma study, has been shownto migrate mainly amoeboid-like (MMP-independent) invitro (23). This is the samemigratory mode as observed inBL2B95 cells. But against expectations the 2 cell lines showa different response to imipramine-blue treatment. Apossible explanation may be a switch of the RT2 cellsfrom amoeboid to mesenchymal migration in the in vivorat model. If this transition took place, RT2 migrationwould be sensitive to imipramine-blue treatment viainhibition of theNAPDH oxidase complex and the down-stream RACprotein pathway.Whether this amoeboid-to-mesenchymal transition occurs in the BL2B95 cells in the

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Figure 3. Effect of imipramine-blue on cancer cell line viability. A, logarithmic dose vs. response (% inhibition of proliferation) curve of cancer cell lines treatedwith imipramine-blue for 72 hours. B, effect of 1mmol/L imipramine-blue for 72 hours on cancer cell lines (statistical analysis). C, effect of 5mmol/L imipramine-blue for 72 hours on cancer cell lines (statistical analysis). a–f, phase-contrast imaging of FLP1 and KELLY cells treated with imipramine-blue for 72 hours.�, P < 0.05; ��, P < 0.001.

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CAMmodel is not known. However, the main metastaticroute for BL2B95 cells is the dissemination via the lym-phatic system, which we found unaltered by imipramine-blue in our experiments. Furthermore, in the CAM studywe applied imipramine-blue with a pre-incubation pro-tocol, whereas Munson and colleagues used intravenousimipramine-blue application in the rat model, starting 1week after the inoculation of the tumor cells (1). Intrave-nous imipramine-blue application starting 1 week aftertumor cell inoculation in the CAM model is impossiblebecause of the limited time window for tumor growth inthe egg. We therefore decided to use the preincubationprotocol, knowing that this does not reflect the patientsituation.ROS inhibitors obviously act in a context-depen-dent manner. Therefore, the different findings of the invivo models may also reside in the fact that BL2B95 cellsrespond to imipramine-bluewith apoptosis rather than aninhibition of movement. This is supported by our obser-vation that the viability of Burkitt lymphoma cell line isefficiently inhibited already at very low imipramine-blueconcentrations [BL2B95 IC50 (24 hours imipramine-blue)¼ 0.16 mmol/L], whereas other cell lines, like HeLa, areless sensitive to imipramine-blue (HeLa IC50 (24 hoursimipramine-blue) ¼ 3.92 mmol/L). This suggests thatBL2B95 cells are highly dependent on basal ROS levelsto prevent apoptosis. Other cell lines, such as rat RT2 andHeLa, seem to depend to aminor extent onROS-mediatedantiapoptotic mechanisms, and react with an altered cel-lular movement.Here, we were able to show that imipramine-blue

inhibits growth of various Burkitt lymphoma cell lines invitro and in vivo. Imipramine-blue possesses a high ther-apeutic potency in vitro, comparable to that of doxorubi-cin, for the cell lines SH-SY5Y, MDA-MB231, HeLa, andHEK-293WT (24–27). The most sensitive cell lines toward

imipramine-blue treatment were BL30B95, BL2, BL2B95,and the neuroblastoma cell line KELLY with IC50(24hours) values lower than 0.8 mmol/L (0.79, 0.49, 0.16, and0.78 mmol/L). The aforementioned most sensitive celllines (BL30B95, BL2, BL2B95, KELLY) are considered tobelong to the "reactive oxygen driven tumor" phenotype.This tumor phenotype is characterized by high ROS andAKT levels, and wild-type p53 (28–30). Therefore it is notsurprising that the tumor cell lines of this phenotype arevery sensitive to imipramine-blue treatment. Our in vitrostudies were extended to several cancer types, which arecharacterized by metastasis formation and partiallyknown to spreadvia the lymphatics (31–33). This includedangiosarcoma, neuroblastoma, and breast cancer celllines. There was no correlation between imipramine-bluesensitivity and the growth rate of the treated cancer celllines. Nevertheless, all analyzed cell lines responded tothe imipramine-blue treatment effectively. Whether theobserved effects rely mainly on NOX4 inhibition or if off-target effects are involved is still unclear. It was shown byMunson and colleagues (1) that imipramine-blue appli-cation in vivo influences a number of genes in RT2 tumors(1). Among these, MFHAS1 may be of special interest.MFHAS1 is a potential oncogene that may cause B-celllymphoma and is downregulated at the transcriptionallevel by imipramine-blue treatment (1,34). The inhibitionofMFHAS1may explain the high sensitivity of the Burkittlymphoma cell lines toward imipramine-blue treatment.

Of note, the nonmalignant FLP1 fibroblasts were theleast sensitive [IC50 (24 hours)¼ 7.7 mmol/L] of all studiedcell lines. To determine whether this is an incidentalfinding or if it hints toward a general lower sensitivity ofnonmalignant cells toward NOX4-inhibition, additionalstudies, for example on endothelial cells, are needed toclarify the therapeutic window for imipramine-blue.

Table 1. IC50 values of cell lines treated with imipramine-blue

Cell lineCancer/cell type(viral infection)

IC50 24-hour imipramine-blue/(95% CI), mmol/L

IC5072-hour imipramine-blue/(95% Cl), mmol/L

FLP1 Fibroblast cell line 7.70/(6.57–9.04) 7.16/(6.13–8.37)HEK-293WT Embryonic kidney (HAdV-5þ) 4.11/(3.49–4.84) 2.36/(2.24–2.50)HeLa Cervix carcinoma (HPV18þ) 3.92/(3.41–4.49) 2.02/(1.83–2.23)MDA-MB-231 Breast carcinoma 1.91/(1.64–2.22) 1.11/(0.88–1.40)AS-M Angiosarcoma 1.81/(1.56–2.10) 0.98/(0.91–1.06)SH-SY5Y Neuroblastoma 1.74/(1.17–2.58) 1.79/(1.13–2.84)SMS-KAN Neuroblastoma 1.60/(0.97–2.64) 1.17/(0.61–2.24)KELLY Neuroblastoma 0.78/(0.52–1.17) 0.14/(0.11–0.18)BL41B95a Burkitt lymphoma (EBVþ) 1.11/aD 0.78/aDBL30B95a Burkitt lymphoma (EBVþ) 0.79/aD 0.47/(0.31–0.70)BL2a Burkitt lymphoma 0.49/(0.42–0.57) 0.75/aDBL2B95a Burkitt lymphoma (EBVþ) 0.16/(0.13–0.21) 0.45/(0.35–0.59)

NOTE: IC50 values were calculated on the basis of methyl violet assay data; IC50 is the concentration that inhibits 50% of proliferation;aD is the ambiguous data, which means no reliable calculation of 95% CIs was possible.aIC50 values calculated on the basis of MTT cell viability assay data instead of the methyl violet assay.






Furthermore, we plan to test imipramine-blue in combi-nation with classical chemotherapeutic drugs (CHOP/CODOX-M/IVAC) to analyze the possibility of synergis-tic effects that may improve chemotherapeutic regimensfor the treatment of patients with Burkitt lymphoma.Because imipramine-blue encapsulated in PEGylatedliposomes accumulates in brain tumor sites and diffusesinto the brain (1), its application may be of high value forthe treatment of brain metastases of lymphomas.

Our observations are in line with several publicationsthat described the beneficial effects of ROS inhibition invitro as well as in clinical trials (15, 35–37). On the otherhand, numerous publications and clinical trialswith ROS-inducing drugs have shown that an experimental upre-gulation ofROSmaykill cancer cells aswell (14, 38–40).Asnoted above, ROS levels act in a highly context-dependentmanner, and the effects must be determined for eachtumor entity. Therefore, it is still under debate whetherROS decrease or increase may be more beneficial for thetreatment of cancer. Cancer cellsmay either be affected bythe accumulation of ROS above a cytotoxic threshold, orby the inhibition of ROS below a concentration, which isessential for signaling processes (10). Thereby, we assumethat the upregulation of ROS may not be the preferredapproach, as it may severely affect normal cells. A sys-temic treatmentwithdrugs that increase intracellularROSlevels above a thresholdmay induce apoptosis and severeside-effects in healthy tissues. This is supported by obser-vations of unwanted side-effects in a phase II clinical trialof the pro-oxidant drug triapine, where patients devel-oped neutropenia, hypoxia, methemoglobinemia, anddyspnea, without any beneficial effects (41).

In summary, our study shows that the NOX4 inhibitingsmall molecule imipramine-blue is able to inhibit theproliferation of BL2B95 cells in vivo and in vitro. Severalother cancer cell lines are significantly influenced by

imipramine-blue, too. Thereby, imipramine-blue showsa potency that is comparable to that of doxorubicin. Themost sensitive cell lines are the lymphoma cell lines BL2,BL2B95, and BL30B95, as well as the neuroblastoma cellline KELLY. In an animalmodelwith xenografted BL2B95cells, imipramine-blue caused a reduction of the tumor ofsize, but the dissemination of lymphoma cells via thelymphatics and tumor-induced angiogenesis remainedunaltered.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Klingenberg, D. Kube, J. WiltingDevelopment of methodology: M. Klingenberg, J. Becker, J. WiltingAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. KlingenbergAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): M. Klingenberg, J. Becker, S. Eberth,D. Kube, J. WiltingWriting, review, and/or revision of the manuscript: M. Klingenberg,J. Becker, D. Kube, J. WiltingStudy supervision: J. Wilting

AcknowledgmentsThe authors thankN.Walther, S. Hengst, S. Schwoch, Ch. Zelent, andB.

Manshausen for their excellent technical assistance. Imipramine-blue waskindly provided by M.Y. Bonner, Department of Dermatology, EmorySchool of Medicine, Atlanta.

Grant SupportThis study was supported by the Deutsche Forschungsgemeinschaft,

grants FOR942/12-1 and GRK1034 to D. Kube.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 20, 2013; revised January 6, 2014; accepted January 10,2014; published OnlineFirst January 30, 2014.

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2014;13:833-841. Published OnlineFirst January 30, 2014.Mol Cancer Ther Marcel Klingenberg, Jürgen Becker, Sonja Eberth, et al. Burkitt LymphomaThe NADPH Oxidase Inhibitor Imipramine-Blue in the Treatment of

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