Lung and Lung Tumors1

NAD(P)H:quinone oxidoreductase (NQO1) is a flavoenzyme that catalyzes the two-electron reduction of quinones and related compounds. With the use of biochemical assays, NQO1 has been shown to be overexpressed in many types of cancer, including non-small cell lung cancer (NSCLC). NQO1 can bioactlvate antitumor qulnones such as mitomycm C, and new qwnone-based drugs are currently being developed to target this enzyme in tumors such as NSCLC. Because there is no information on the cell-specific expression of NQO1 in lung, the purpose of this study was to examine the expression of NQO1 in human NSCLC, small cell lung cancer, carcinoid lung tumors, and normal lung tising immunohistochemistry. A high level of NQO1 protein expression was detected by immunohistochemistry in NSCLC (adenocarcinoma, squamous cell carcinoma, and bronchoalveolar carcinoma), but no NQO1 protein could be detected in small cell lung cancer or carcinoid lung tumors. In addition, NQO1 protein expression was examined by lmmunohistochemlstry in normal lung tissue. A high level of NQO1 protein expression was detected by immunohistochemistry in normal lung respiratory epithelium, with the highest levels of expression observed in cifiated columnar epithelial cells. Significant amounts of NQO1 protein were also detected in the vascular endothelium and adipocytes. These data demonstrate that NQO1 Is overexpressed in NSCLC. Cells in normal lung also contain marked NQO1 protein and may be damaged by drugs activated by NQO1. These data validate NSCLC as a target for NQO1-directed agents and suggest that the potential for lung toxicity be considered in the preclinical development of quinone-based antitumor drugs. Received 4/21/98; revised 6/23/98; accepted 6/24/98. The costs of publication of this article were defrayed in part by the payment 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. I This work was supported in part by NIH Grant CA 51210. D. S. was supported by a Lung SPORE Career Developmental Award from the University of Colorado Health Sciences Center. 2 To whom requests for reprints should be addressed, at School of Pharmacy C238, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. Phone: (303) 315-3678; Fax: (303) 315-6281 ; E-mail: [email protected]. Introduction NQO13 (DT-diaphorase, EC 1.6.99.2) is a flavoenzyme catalyzing NADHor NADPH-dependent two-electron reduction of a broad range of substrates. Whether reduction by NQO1 results in activation or deactivation of quinones depends upon the properties of the hydroquinone generated. Evidence suggests that simple quinones, such as benzoquinone and napthoquinone, are deactivated following reduction by NQO1 (1, 2). In contrast to the role of NQO1 in detoxifying quinones, NQO1 has also been shown to play a role in the activation of many quinone antitumor compounds via reduction to their hydroquinone forms. The hydroquinone may autoxidize to produce reactive oxygen species or undergo rearrangement to produce a reactive alkylating species. NQO1 can bioactivate many antitumor quinones such as mitomycin C (3, 4), EO9 (5), streptonignin (6), MeDZQ (7), and diaziquone (8) via the production of reductionoxidation-labile hydroquinones and/or reactive alkylating species. The ability ofNQOl to activate these agents has implicated NQO1 as a target enzyme for quinone antitumor drug design (9). Aziridnylbenzoquinones, which are highly efficient substrates for NQO1, are currently undergoing evaluations in clinical trials (10). With the use of biochemical assays and immunoblot analysis, NQO1 has been shown to be expressed in many human tissues, and many human tumors have elevated NQO 1 activities, including liver (1 1), colon (12, 13), breast (14, 15), brain (16), and lung (12-14, 17) carcinomas. The overexpression of NQO1 in tumors has led to increased interest in the use of compounds bioactivated by NQO1 for chemotherapy. In the case of lung cancer, NQO1 activity is low in normal human lung tissue, whereas NQO1 activity in lung neoplasia is dependent upon the histological type of tumor (17). NSCLCs and mesotheliomas have markedly higher NQO1 activities than does normal lung tissue, whereas SCLCs have NQO1 activities similar to that of normal lung. Although the lung is a complex organ with over 40 different cell types, there is no information on which of these cell types express NQO1. The purpose of this work was to examine human lung tumors and normal lung for cell-specific expression of NQO1 by immunohistochemistry. Materials and Methods Lung Samples. Normal lung and lung tumors were obmined from the University of Colorado Health Sciences Center lung tissue bank. All samples were genotyped as either wild type or heterozygous for the NQO1 #{176} C-to-T mutation using PCRRFLP with restriction endonuclease Hinfl and genomic DNA 3 The abbreviations used are: NQO1, NAD(P)H:quinone oxidoreductase 1; NSCLC, non-small cell lung cancer; SCLC, small cell lung cancer; TBSTM, 10 mM Tris-HC1 (pH 8.0), 150 mM NaCl, 0.2% Tween 20, and 5% nonfat dry milk; DAB, 3,3-diaminobenzidine. Research. on July 15, 2017. © 1998 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 2066 Detection of NQO1 in Human Lung and Lung Tumors 4 S. Chen and D. Siegel, unpublished results. Table 1 Immunostaining of NQO1 in human lung tumors Immunostaining of Case no. Tumor type tumor for NQOV’ Intensity of immunostaiing 22l4” ” Adenocarcinoma Positive (95) +4 (cytoplasmic) 2326 et Adenocarcinoma Positive (90) +3 (cytoplasmic) 2357k 5255 c 5229ce Adenocarcinoma Bronchioalveolar Squamous Positive (95) Positive (75) Positive (80) +4 (cytoplasmic) +3-4 (cytoplasmic) +4 (cytoplasmic) 5058cet Squamous Positive (95) +4 (cytoplasmic) Squamous Positive (80) +4 (cytoplasmic) 2297g Squamous Positive (60) +3 (cytoplasmic) 5243 e Squamous Negative 5072 ,e Small cell Negative Tumor negative, +4 staining of normal epitheium 5115c.d.e Small cell Negative Tumor negative, +4 staining of normal epitheium 5092 e Small cell Negative 33 bf.i Carcinoid Negative Tumor negative, +4 staining of normal epithelium 5433e.h.s Carcinoid Negative 2665’ ’j Carcinoid Negative a Numbers in parentheses represent percentage of tumor cells staining positive for NQOI. b Female. C Smoker. d No chemotherapy history available. e Male. 1Chemotherapy prior to specimen collection. g No medical history available. h No chemotherapy prior to specimen collection. I Nonsmoker. j No smoking history available. obtained from either blood or lung tissue, as described previously (18). Genotyping for the #{176} C-to-T mutation was performed on tissue samples because previous data have shown that cell lines genotyped as homozygous for the #{176} C-to-T mutation have decreased NQO1 protein expression (18). Anti-NQO1 and Control Monodonal Antibodies. AntiNQO1 monoclonal antibody (IgGl)-secreting hybridomas (clones Al80 and B771) were derived from a BALB-c mouse immunized with purified recombinant human NQO1 protein. Antibodies from these hybridoma clones will react with both wild-type and mutant NQO1 proteins (18). These antibodies, however, do not cross-react with purified human NQO2.4 A control (nonspecific IgGl secreting) hybridoma (clone C 100) was derived from a BALB-c mouse. All hybridoma cell lines were grown in spinner flasks in RPMI containing 50 units/mi penicillin, 50 p.g/ml streptomycin, 1% L-glutanMne (Life Technologies, Inc., Gaithersburg, MD), and 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) in 5% CO2 at 37#{176}C to a concentration of 106 cells/nil. Hybridoma tissue culture supernatants were prepared by centnifugation at 1800 rpm for 10 mm and then stored at -80#{176}C. Prior to use, supernatants were centrifuged at 14,000 rpm for 5 mm. Immunohistochemistry. Lung sections (4 i.m) were cut from archival paraffm blocks. Sections were heated to 80#{176}C for 30 mm and then deparaffinized in xylene and rehydrated through graded alcohols to distilled water. Sections were then placed in a 10 mr i citric acid solution (pH 6.0) and microwaved for two 3-mn cycles. Sections were cooled to room temperature and then transferred to PBS for 10 mm. Endogenous peroxidase activity was eliminated by placing sections in 3% hydrogen peroxide for 20 mm. Sections were rinsed in PBS for 10 mm and then blocked in 5% horse serum in TBSTM for 30 mm. Immunodetection of NQO1 was performed using tissue culture supernatants from hybridoma clones A180 and B77l mixed 1 :1 and then further diluted with an equal part of TBSTM. Negative controls were performed using tissue culture supernatant from control hybridoma clone Cl00 diluted with an equal part of TBSTM. Serial sections of each tissue sample were incubated with either anti-NQO1 or control antibodies. Sections were incubated with 1 ml of diluted hybridoma supematant for 30 rain at 27#{176}C. Sections were washed in TBSTM for three 10-rain cycles. Immunodetection was performed using a horseradish peroxidase-based Vectastain Elite ABC Kit (Vector Laboratolies, Burlingame, CA). Biotinylated horse antimouse IgG (H+L) secondary was diluted 1:200 in TBSTM containing 3% horse serum, and 1 ml of diluted secondary antibody was added to each section for 30 rain. Sections were washed in TBSTM as described above. Avidin-horseradish peroxidase complex was prepared in TBSTM as described by the manufacturer, and 1 ml of complex was added to each section for 30 mm. Sections were washed in TBSTM as described above, followed by a 1-mm wash in 10 mM Tris-HC1 (pH 8.0), 150 nmi NaCl, and 0.2% Tween 20. DAB and hydrogen peroxide (DAB Staining Kit; Vector Laboratories) were used as the horseradish peroxidase substrates. DAB/hydrogen peroxide solution was prepared according to the manufacturer, and 1 ml of solution was added to each section for 5-7 mm. Sections were then rinsed in distilled water, counterstained with hematoxylin or toluidine blue, dehydrated, and mounted. Sections were photographed using a Nikon FX microscope with Agfa RSX-50 film. The intensity of NQO1 Research. on July 15, 2017. © 1998 American Association for Cancer clincancerres.aacrjournals.org Downloaded from