Production of Large Amounts of Hydrogen Peroxide by Human Tumor Cells1

[CANCER RESEARCH 51. 794-798. February 1, 1991] Production of Large Amounts of Hydrogen Peroxide by Human Tumor Cells1

Ted P. Szatrowski2 and Carl F. Nathan

Beatrice and Samuel A. Seaver Laboratory, Division of Hematology-Oncology, Department of Medicine, Cornell University Medical College, New York, New York 10021

ABSTRACT rate typically observed in neutrophils. Pretreatment with 7- interferon augmented Superoxide release from two human tu Few nonphagocytic cells are known to generate reactive oxygen inter mor cell lines from 0.04 nmol up to 0.20 nmol/104 cells/h (20). mediates. Based on horseradish peroxidase-dependent, catalase-inhibitable oxidation of fluorescent scopoletin, seven human tumor cell lines We have identified seven different human tumor cell lines of constitutive!)' elaborated H2O2 at rates (up to 0.5 nmol/104 cells/h) large four histolÃ³gica! types that can produce, without exogenous enough that cumulative amounts at 4 h nere comparable to the amount stimulation, amounts of H2O2 that after 4 h are as large as of IM)., produced by phorbol ester-triggered neutrophils. Superoxide those produced by stimulated PMN, and we have partially dismutase-inhibitable ferricytochrome c reduction was detectable at much characterized the nature and sources of hydrogen peroxide lower rates. IM), production was inhibited by diphenyleneiodonium, a production in these cells. flavoprotein binder (concentration producing 50% inhibition, 0.3 Ã•/M). and diethyldithiocarbamate, a divalent cation chelator (concentration producing 50% inhibition, 3 MM),but not by cyanide or azide, inhibitors MATERIALS AND METHODS of electron transport, or by agents that inhibit xanthine oxidase, pol\ ani- ini1oxidase, or cytochrome P450. Cytochrome Â¿559,presentin human Cell Lines. The human melanoma cell lines SK-Mel 23, 28, 30, and phagocytes and lymphocytes, was undetectable in these tumor cells by a 131 (23-25) were provided by Dr. Alan Houghton (MSKCC, New sensitive spectrophotometric method. Mouse fibroblasts transfected with York, NY). The human colon carcinoma cell lines HT 29 (26) and human tyrosinase complementary DNA made melanin, but not IM).,. HCT 15 (27) and the human pancreatic carcinoma cell line AsPC 1 Constitutive generation of large amounts of reactive oxygen intermedi (28, 29) were obtained from Dr. Sid Weite (MSKCC). Lan 1 human ates, if it occurs in vivo, might contribute to the ability of some tumors neuroblastoma cells (30, 31) were provided by Dr. Nai-Kong Cheung to mutate, inhibit antiproteases, injure local tissues, and therefore pro (MSKCC). The human breast (CAMA-I) (32) and ovarian (SK-Ov 3) mote tumor heterogeneity, invasion, and metastasis. (26, 33) carcinoma cell lines were obtained from the late Dr. J. Fogh (Sloan-Kettering Institute, Rye, NY). Mouse fibroblasts (34) transfected with human tyrosinase cDNA were a gift from Dr. Houghton. All cells INTRODUCTION were negative for Mycoplasma when stained using fluorescent bisben- Appropriately stimulated PMNs3 (1-3) and monocytes (4-8) zimide (Hoechst 33258; Aldrich Chemical Co., Milwaukee, WI). All cells were adherent in culture and were maintained in 25-cm2 produce large amounts of ROI (up to 1.5 nmol of hydrogen peroxide/10" cells/h) in a respiratory burst pattern. In phago tissue culture flasks (Corning Glass Works, Corning, NY) in complete medium consisting of RPMI 1640 medium (Sigma Chemical Co., St. cytes, a membrane-bound oxidase containing an unusual cyto Louis, MO) with 7.5% heat-inactivated fetal bovine serum (HyClone chrome b (cytochrome 6559)is responsible for the generation of Laboratories, Logan, UT), 1% nonessential amino acids (Gibco Labo Superoxide aniÃ³n (9-11). The latter ROI dismutates to H2O2 ratories, Grand Island, NY), 1% L-glutamine (Hazleton Biologies, spontaneously or upon catalysis by Superoxide dismutase. In Lenexa, KS), and 1% penicillin-streptomycin (Hazleton). Cells were contrast, few nonphagocytic cells can generate ROI. Rat glo- grown in a humidified atmosphere of 5% CO2 in air at 37Â°C.Cellswere merular mesangial cells triggered by serum-treated zymosan detached with trypsin (Flow Laboratories, McLean, VA) except Lan 1 (12), sea urchin ova triggered by fertilization (13), and porcine cells, which could be detached in phosphate-buffered saline (Gibco) thyroid cells in the presence of NADPH (14) generate ROI. alone. PMN Isolation. Human blood drawn into heparinized Vacutainer Human platelets (15), human endothelial cells stimulated by tubes using standard venipuncture techniques was layered onto Neutro- interleukin 1 or 7-interferon (16), human fibroblasts stimulated phil Isolation Medium (Los Alamos Diagnostics, Los Alamos, NM) and by interleukin 1 or tumor necrosis factor-Â«(17), and human B- spun at 400 x g at 25Â°Cfor30 min. The neutroph.il layer was removed cells (18) have also been shown to generate ROI. Recently, and washed twice in KRPG. This procedure (35) yielded >96% PMNs. human B-lymphocytes were found to contain cytochrome A5S9 ROI Assays. Tumor cells were seeded in complete medium (contain (19). Constitutive generation of ROI by human tumor cells (20) ing phenol-free RPMI; Sigma) at 2xl05 cells/ml, 0.1 ml/well in 96- and by fMLP-stimulated rat Walker carcinosarcoma cells (21, well microculture plates (Falcon Primaria; Becton-Dickson and Co., 22) has been demonstrated but only at rates <0.05 nmol of Lincoln Park, NJ) and allowed to adhere overnight. The cells were then H2O2/104 cells/h, approximately 25-30 times slower than the washed twice with warm KRPG and H2O2 measured using a scopoletin fluorescence assay, in which H2O2 oxidizes up to 35 ^M scopoletin Received 8/16/90; accepted 11/20/90. (Sigma) to a nonfluorescent state in a reaction catalyzed by 1 purpu- The costs of publication of this article were defrayed in part by the payment rogallin unit/ml horseradish peroxidase (Sigma). The presence of 1 mivi of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. sodium azide (Fisher Scientific Co., Fair Lawn, NJ) in the reaction ' This work was supported by National Cancer Institute Grant 5 PDI mixture prevents the competitive action of catalase and myeloperoxi- CA33049-08. dase on H2O2. Fluorescence was measured using an automated plate 2To whom requests for reprints should be addressed, at Division of Hematol reader (MicroFluor MR6000; Dynatech Laboratories, Alexandria, VA) ogy-Oncology, Cornell University Medical College, 1300 York Avenue. Box 57, as previously described (36). Superoxide was measured using IO5cells New York, NY 10021. 3The abbreviations used are: PMN, polymorphonuclear leukocyte; DDC, with or without preincubation with 0.1 mivi diethyldithiocarbamate diethyldithiocarbamate; cDNA, complementary DNA; DPI, diphenyleneiodon (Sigma) by reduction of ferricytochrome c (Sigma) as previously de ium; fMLP, formyl-methionyl-leucyl-phenylalanine; HPO, horseradish peroxi- scribed (37). The modified method of Ding and Nathan (38) was used dase; KRPG, Krebs-Ringer phosphate buffer with 5.5 mivi glucose; MSKCC, Memorial Sloan-Kettering Cancer Center; PMA, phorbol myristate acetate; ROI, to measure cytochrome Â¿>559content.Both of the latter two assays were reactive oxygen intermediates; rTNF-a, recombinant human tumor necrosis fac- carried out using a Perkin-Elmer/Hitachi model 557 spectrophotome- tor-a; ICso, concentration producing 50% inhibition. ter with a 2-nm slit width. 794

Reagents. Superoxide dismutase, metyrapone, a-naphthoflavone, chloramphenicol, ellipticene, PMA, and fMLP were obtained from Sigma. Sodium dithionite and potassium cyanide were from Fisher. to4 x SK-MÂ«I30 cellÂ» rTNF-a was provided by Genentech Inc. (South San Francisco, CA). â€¢4.e DPI, a nucleoside oxidase inhibitor and selective flavoprotein inhibitor (39), was a gift from Dr. Andrew R. Cross (University of Bristol, Bristol, United Kingdom). N O

RESULTS Production of H2O2 by Tumor Cells. Seven adherent human tumor cell lines (two pigmented and two nonpigmented mela nomas, one neuroblastoma, one colon carcinoma, and one ovarian carcinoma) elaborated hydrogen peroxide constitutively at rates ranging from 0.2 nmol to 0.5 nmol/10" cells/h (Fig. 1). One colon, one breast, and one pancreatic carcinoma cell line 120 100 240 produced <0.1 nmol of H2O2/104 cells/h. Human PMNs, when minutes Fig. 2. H2O2 Production as a function of time and tumor cell number. , stimulated by 10 ng/ml PMA, generated H2O2 at a rate of cumulative amounts of H2O2 produced as a function of time for five different about 1.4 nmol/h (averaged over the first 2 h of the respiratory tumor cell densities: , representative PMA-stimulated PMN respiratory burst for comparison. Insel, Ilo accumulation at 3 h is plotted versus tumor cell burst) when tested simultaneously. density on a log scale. Results are the means of triplicates. SD at all points ranged Fig. 2 shows that elaboration of hydrogen peroxide by SK- between 0.01 and 0.09 nmol H2O2. Mel 30 cells depended on the number of cells plated and was continuous at a constant rate over several hours until the nmol H202/104 cells/h scopoletin indicator became limiting. Such a continuous rate of H2O2 production was observed in all experiments performed 0.0 0.2 0.4 0.6 0.8 with the tumor cell lines shown in Fig. 1 (data not shown). Dependency of H2O2 production on cell number was also ob served for SK-Mel 28, SK-Mel 131, and Lan 1 cell lines (data Complete system not shown). While the maximal rate of hydrogen peroxide generation by tumor cells was less than that of similar numbers of PMNs, cumulative amounts released by tumor cells sur No azide passed those of triggered neutrophils after 4 h. Neither PMA (dose range, 10-300 ng/ml), fMLP (0.5-10 MM),nor rTNF-a (10-300 ng/ml), agents capable of triggering CÃ¡talasÂ«,noazide a respiratory burst in PMNs in this system (35), could augment the observed H2O2 production rates in tumor cells as tested in SK-Mel 28, SK-Mel 30, SK-Mel 131, Lan 1, and HCT 15 cell No cells lines (data not shown). The omission of 1 HIM azide from the assay medium, per mitting endogenous catalase and peroxidase activity (8), re NoHPO sulted in a decrease in the H2O2 detected (Fig. 3). The addition

nmol H202/104 cells/h Fig. 3. Evidence that tumor cell-dependent changes in fluorescence are due to 0.5 1.0 1.5 2.0 H2O2. Plotted is the effect on measured H2O2production by SK-Mel 30 melanoma cells of azide and catalase in the scopoletin-based assay. The change in fluores cence in the presence of tumor cells in the absence of horseradish peroxidase (14) (HPO) is defined as 0 nmol H2O2/h/10* cells. Columns, means of triplicates; error bars, SD.

of 3000 units/ml of exogenous catalase to the system resulted in the suppression of detected H2O2 to baseline, as defined by the change in fluorescence in the absence of horseradish per Lan 1 oxidase. HCT15HT (6)iho)S^(2)HID Production of Superoxide AniÃ³n.Table 1 shows Superoxide 29AsPC aniÃ³ngeneration as detected by a superoxide-inhibitable, ferricytochrome c reduction assay. The amounts of Superoxide 1SK-Ov generated constitutively appeared to be about one-tenth those 3CAMA-I223- (2) of H2O2 on a per cell per hour basis. To test the hypothesis that (2) endogenous Superoxide dismutase might rapidly convert superoxide to hydrogen peroxide, tumor cells were preincubated with Fig. 1. Rates of H2O2 production by tumor cell lines. The rates of H2O2 production/IO4 cells are shown for 10 different cell lines. Columns, means; 100 MMDDC (37) for 2 h and then washed with KRPG. This numbers in parentheses, numbers of experiments; error bars, SD calculated across experiments. Tumor cell lines are shaded according to cell type (see "Methods"). augmented modestly the amounts of Superoxide aniÃ³n meas The rapid generation of H2O2 by PMNs in the first 2 h of a PMA-stimulated ured in the medium. respiratory burst is displayed for comparison. In contrast, when cells were preincubated with varying con- 795

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1991 American Association for Cancer Research. PRODUCTION OF HYDROGEN PEROXIDE BY TUMOR CELLS centrations of DDC prior to assay with scopoletin, measured hydrogen peroxide production was markedly inhibited, with an ICso of about 100 pM (Fig. 4). When DDC was present in the scopoletin assay medium, the degree of suppression of detect able H2O2 was even larger (IC5o, about l /UM)and considerably 0.002AU larger than one would expect from Superoxide dismutase inhi bition (since Superoxide dismutates rapidly to H2O2 even with out catalysis) or from weak interference by DDC in the scopo letin assay demonstrated in Fig. 4, inset. Thus, DDC may have inhibited Superoxide dismutase-mediated conversion of superoxide to hydrogen peroxide, but there may be an additional 500 600 mechanism by which DDC can affect H2O2 production. Wavelength (nm) Mechanism of ROI Production. The best known enzymatic Fig. 5. Lack of detectable cytochrome ft!59in tumor cells. The absorbance peak source of large amounts of ROI in mammalian cells is the at 559 nm demonstrated for 3 x IO7 PMNs is not detectable for IO7 Lan 1 NADPH:O2 oxidoreductase that contains cytochrome Â¿Â»559(9- neuroblastoma cells, representing an equivalent amount of cell protein. 11). The upper-most spectrophotometric tracing in Fig. 5 shows a peak at 559 nm when the technique was applied to 3 x IO7 PMNs, after double reduction by dithionite to eliminate the absorbance contributed by hemoglobin and by mitochondria! cytochromes (38). The lower tracings reflect the same technique when applied to IO7neuroblastoma cells (an equivalent amount o o Lan 1 of cellular protein). No peak corresponding to cytochrome Â¿>559 â€¢ Â»PMN+PMA was detectable; the lower limit of sensitivity of the technique was estimated to be about 1 pmol of cytochrome A559(38). Cytochrome Â¿Â»559waslikewise not detectable in SK-Mel 28 or in SK-Mel 30 cells (tracings not shown).

Table I Production of Superoxide aniÃ³n The amount of Superoxide aniÃ³ngenerated was measured by ferricytochrome c reduction with and without Superoxide dismutase. Shown is the effect of preincubation with 100 Â»JMDDCfor 2 h prior to assay, for seven different tumor cell lines. 0.0 Superoxide (nmol/105 cells/h)

DPI Concentration Fig. 6. Effect of DPI on tumor cell HÂ¡OÂ¡production.HjOÂ¡generated by Lan SK-Mel30SK-Mel Â±0.07'0.1 Â±0.030.23 1 neuroblastoma cells (O) or by PMA-stimulated PMN (â€¢)isshown as a function 28SK-Mel 5Â±0.020.17 Â±0.010.24 of DPI concentration in the assay mixture. Points, means of triplicates; error bars, 131SK-Mel Â±0.140.28 Â±0.13Not SD. 23HT Â±0.090.13 done0.14 29HCT Â±0.120.01 Â±0.110.13 15Un Â±0.070.07 Â±0.120.13 In the scopoletin-based assay system, DPI inhibited tumor 1No Â±0.040.02 Â±0.120.02 cell H2O2 production with an IC50 of about 0.3 pM (Fig. 6), cellsControl0.24 Â±0.03DDCpreincubation0.30Â±0.03 suggesting a role for a flavoprotein in the generation of H2O2 1Mean Â±SD. by tumor cells. DPI also inhibited the neutrophil respiratory burst (IC5o, about 0.1 MM),as has been previously reported (39). 20 - Incubation with 10 MMDPI was not toxic to cells, as judged by their ability to exclude trypan blue. â€¢ â€¢DDCin osioy lysttm O OprÂ«-incubation with DOC Inhibitors of electron transport (potassium cyanide, sodium 15 azide), xanthine oxidase (allopurinol), monoamine oxidase (pargyline, clorgyline, nialamide, phenylzine), polyamine oxi dase (diphenhydramine, quinacrine), or cytochrome P450 (metyrapone, a-naphthoflavone, chloramphenicol, ellipticene) at concentrations up to 10 mM were all unable to block the CM detection of tumor cell-generated H2O2 in the scopoletin assay O CM (data not shown).

0 Mouse fibroblasts transfected with human tyrosinase cDNA

C and producing melanin (34) did not produce H2O2 in the scopoletin assay (data not shown).

0.0 DISCUSSION DDC Concentration Fig. 4. Effect of DDC on tumor cell H2O2 production. The effect of preincu It was surprising that several tumor cell lines, representing a bation of Lan I neuroblastoma cells with varying concentrations of DDC (O) variety of tissue types, constitutively produced large amounts compared to like concentrations of DDC in the assay mixture (â€¢).Inset,measured H2O2 at 4 h when DDC and 4 nmol H2O2 are placed in the assay mixture, without of H2O2. Hydrogen peroxide production continued for at least cells. Points, means of triplicates; error bars, SD. 4 h at a constant rate, its detection eventually limited by the 796

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1991 American Association for Cancer Research. PRODUCTION OF HYDROGEN PEROXIDE BY TUMOR CELLS assay indicator; the cumulative amount of H2O2 produced after min inhibitor are susceptible to oxidation at methionine resi 4 h was comparable to the magnitude of the respiratory burst dues of the active site (48, 49). Inactivation of Â«i-proteinase of similar numbers of PMNs. Superoxide aniÃ³n was detected inhibitor, plasminogen activator inhibitor, and/or Â«2-macro- at production rates an order of magnitude smaller than those globulin by oxidation favors unrestrained action of various for H2O2, even when DDC was used to block Superoxide dis- proteases, such as elastase, plasminogen activator, and plasmin, mutase-mediated conversion of Superoxide to H2O2. While it is which are capable of considerable tissue destruction. Plasmin possible that stoichiometric quantities of Superoxide are being ogen activator is produced by many tumors and is strongly made by these cells but are not detected by the assay used, these implicated in tumorigenicity and metastasis (50-52). findings are also consistent with a mechanism by which H2O2 Thus, constitutive generation of hydrogen peroxide by tumor is produced directly. Several enzymes, such as glucose oxidase cells could potentially enhance neoplastic behavior by aug in fungi and uric acid or amino acid oxidase in mammalian menting both genetic instability of a tumor and its capacity to cells, produce H2O2 directly. Likewise, the respiratory' burst of injure and penetrate host tissues. fertilization in sea urchin ova forms H2O2 but not Superoxide (40). ACKNOWLEDGMENTS The source of H2O2 generation is unclear. The degree of inhibition of H2O2 generation by DDC was striking, whether We thank Drs. Alan Houghton, Sid Weite, and Nai-Kong Cheung DDC was present only in the scopoletin assay mixture or when for cell lines. Dr. Andrew R. Cross for diphenyleneiodonium, and cells were pretreated with DDC and washed prior to the sco Genentech, Inc. for supplying rTNF-a. poletin assay. This suggests that DDC may be affecting H2O2 production by a mechanism distinct from inhibition of super- REFERENCES oxide disimilase. Since DDC is a chelator of divalent cations, 1. Babior, B. M. Oxygen-dependent microbial killing by phagocytes. N. Engl. the H2O2 production pathway may be dependent upon a diva J. Med.. 298: 659-725, 1978. lent metal ion. 2. Klebanoff, S. J. Antimicrobial mechanisms in neutrophilic polymorphonu- The ability of DPI, an agent thought to bind to a specific clear leukocytes. Semin. Hematol., 12: 117-142, 1975. component of the PMN membrane oxidase (39), to inhibit 3. Babior, B. M., Kipnes, R. S., and Curnutte, J. T. 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Ted P. Szatrowski and Carl F. Nathan

Cancer Res 1991;51:794-798.

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