Nrf2/Keap1 Pathway and Expression of Oxidative Stress Lesions 8-hydroxy-2’-deoxyguanosine and Nitrotyrosine in Melanoma

Materials and methods

The study included 121 patient samples collected from paraffin block archives stored in the Department of Pathology of Oulu University Hospital between 1999 and 2011. All samples were fixed in neutral buffered formalin and embedded in paraffin. Cases were randomly collected based on the diagnosis and the adequacy of the samples. Only primary melanomas without prior biopsies were included. The series consisted of 23 benign nevi (8 junctional, 7 compositus, 8 intradermal), 13 dysplastic nevi, 14 lentigo maligna, 15 lentigo malignant melanomas, 28 nodular melanomas, 20 superficially spreading melanoma and 8 acral melanoma samples (Table I). Diagnoses were according to the current WHO classification (11) using S100, HMB45 and/or Melan A in addition to morphology when necessary.

The clinical data and the data of the pathological diagnosis of the patients were retrospectively collected from the patient records of the Oulu University Hospital. Survival data were collected and complemented with ulceration, Breslow and Clark when reported (Table I). Median survival was 34.0 months and 24 patients died of melanoma during follow-up.

Immunohistochemistry. Primary antibodies were mouse monoclonal anti-8-OHdG (clone N45.1, Japan Institute for the Control of Aging, Fukuroi, Japan), rabbit polyclonal anti-nitrotyrosine (AB5411, Millipore, Darmstadt, Germany), rabbit polyclonal anti- Nrf2 (C-20), goat polyclonal anti- Keap1 (E-20):sc-15246, Santa Cruz Biotechnology, Inc, TX, USA) and mouse monoclonal anti-BRAF V600E-specific antibody (VE1; Spring Bioscience, Pleasanton, CA, USA) designed for formalin-fixed paraffin-embedded tissue sections for immunohistochemistry. Hematoxylin-eosin staining was performed to all samples.

Sections of 3-4 μm thickness were de-paraffinised and rehydrated in graded alcohol. Sections were first heated in a microwave oven in citrate for 10-15 min and then incubated with the primary antibody anti-8-OHdG dilution 1:50 for 1 h, anti-nitrotyrosine dilution 1:750 overnight, anti-Nrf2 dilution 1:200 for 2 h and anti-Keap1 dilution 1:100 1 h. VE1 was used at a dilution of 1:2,000 for 16 min (diaminobenzidine) and 1:100 for 24 min (The Fast Red chromogen).

The Invitrogen kit (8-OHdG and Nitrotyrosine), Novolink Polymer Detection kit (Nrf2) and Biocare goat-on-rodent HRP-polymer kit (Keap1) were used according to the supplier's instructions for the detection of primary antibody. The colour was developed by diaminobenzidine (Dako, Glostrup, Denmark) and the sections were counterstained with haematoxylin. The staining protocol for BRAF V600E with the Fast Red chromogen and diaminobenzidine is described by Thiel et al. (9).

Immunoreactivities of Nrf2, Keap1, 8-OHdG and nitrotyrosine were assessed as negative, very weak, weak, moderate or strong intensity by two investigators, the first author and an experienced dermatopathologist (K-M.H). The expression of melanocytic cells, keratinocytes, fibroblasts, endothelial structures, sweat gland and apocrine gland structures and leukocytes were separately assessed. BRAF was determined as negative or positive in melanocytic cells according to Thiel et al. (9). Negative control samples were used in all processes and were handled as described previously, but with the primary antibody replaced by serum or phosphate buffered saline.

Statistical analysis. In the statistical evaluation of immunoreactivity intensity groups were pooled into two categories: i) negative to very weak and ii) weak, moderate to strong staining. Breslow's thickness was used as a continuous variable and Clark I-II was pooled to present a horizontal growth and Clark III-V to present a vertical growth. Statistical analyses were performed by IBM SPSS Statistics 22 (IBM Corporation, Armonk, NY, USA). The significance of associations was defined using a 2-sided Chi-square test and a Mann-Whitney test. The Kaplan-Meier curves with a log-rank test were applied in the survival analysis and a Cox regression model with the ulceration status and the Breslow's thickness was used in a multivariate analysis. Only malignant melanomas (Lentigo maligna melanoma, nodular, superficially spreading and acral melanoma, N=71) were included to the survival analysis and to the analysis against clinicopathological factors. Only deaths to melanoma were considered as an endpoint in the survival analysis. p-Values ≤0.05 were considered significant.

Cell lines. The cell lines used in this study were: COLO-800 (ACC 193) and SK-MEL-1 (ACC 303) with BRAF mutation, IPC-298 (ACC 251) and SK-MEL-30 (ACC 151) with NRAS-mutation ordered from Leibniz-Institut, DSMZ (Braunschweig, Germany). Cells were cultured in RPMI-1640 with 10% fetal bovine serum and 100 IU/ml penicillin and streptomycin (Pen-Strep solution HyClone laboratories, Inc. UT, USA).

Inhibitors. The following inhibitors were used: CI-1040 (PD 184352) (Alexis Biochemicals, Lausen, Switzerland) and vemurafenib (V-2800) (LC Laboratories, Woburn, MA, USA). Hydrogen peroxide was used for the oxidative stress modeling. Drug solutions were prepared from a 10-mM stock solution before use. Hydrogen peroxide solution was made from a 30% stock solution (J.T. Baker Chemicals, Avantor, PA, USA) and used in gradual concentrations 10 μM, 33 μM, 100 μM and 333 μM. CI-1040 is a specific small-molecule drug inhibiting MEK1/MEK2 by blocking ERK phosphorylation and is known to inhibit growth of many human tumor cell lines. Inhibition of MAPK activation by CI-1040 prevents cell cycle progression and induces a G₁ arrest (12). The dose of CI-1040 was 1 μM in western blot assay and 3.3 μM in cytotoxicity assay. Vemurafenib is an inhibitor of ERK1/2 in the highly sensitive BRAFV^600E/K -mutated cells (13). Its dose was 1 μM.

Cytotoxicity. The cells were trypsinised (Trypsin 0.25%) and plated onto 96-well plates with three parallel wells for each treatment and the experiments were replicated at least three times. Drug treatments were started the following day, and the plates were developed 72 h later using either MTS reagent mix ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt], Promega; Madison, WI, USA) supplemented with phenazine methosulfate (Sigma-Aldrich, St. Louis, MO, USA) or with CCK-8 reagent (Dojindo, Rockville, MD, USA) for SK-MEL-1 cell line according to manufacturer's guidelines. Absorbances were read on a plate reader (Anthos Reader 2001, Athos Labtec Instruments; Salzburg, Austria) at a wavelength of 490 nm or 450nm (CCK-8). Data was displayed on Microsoft Office Excel 2007.

Western blot analysis. The cells were trypsinised and then plated onto 96-well plates and treated with the inhibitors 24 h later for 48 h, after which time they were lysed in RIPA buffer (1% Igepal CA-630, 20 mM Tris HCl pH 8.0, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 10 g/ml Aprotinin, 10 g/ml Leupeptin, and 10 g/ml Pepstatin). The protein concentrations were measured using the Bio-Rad Protein Assay (Bio-Rad; Hercules, CA, USA) and the concentrations in individual samples were equalized before adding 3x Laemmli buffer to a final concentration of 1x. Equal amounts of protein were run on 7.5% SDS-PAGE gels, transferred to PVDF membranes, probed with the antibodies and developed using the ECL chemiluminescence system (Millipore, Billerica, MA, USA) for detection on radiographic films, which were scanned to an electronic format. Nrf2 antibody ((C-20): sc-722) (Santa Cruz Biotechnology) was used as primary antibody and Anti-rabbit HRP conjugated antibody was used as a secondary antibody. B-Actin was used to control total protein levels.

Ethical approval. The study was approved by the Finnish National Supervisory Authority for Welfare and the Health and the Local Ethics Committee of the Northern Ostrobothnia Hospital District.