Amfenac Increases the Radiosensitivity of Uveal Melanoma Cell Lines
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Eye (2008) 22, 701–706 & 2008 Nature Publishing Group All rights reserved 0950-222X/08 $30.00 www.nature.com/eye 1 1 1 Amfenac increases BF Fernandes , J-C Marshall , S Di Cesare , LABORATORY STUDY P Logan1, S Maloney1 and MN Burnier Jr1,2 the radiosensitivity of uveal melanoma cell lines Abstract with an incidence of seven cases per million.1 Enucleation of the affected eye was the only Purpose To evaluate the proliferation rates of available treatment for UM for most of the last five human uveal melanoma (UM) cell lines 2 after treatment with amfenac, a century. However, with the advent of more cyclooxygenase (COX)-2 inhibitor, and conservative alternatives to treat the primary subsequent radiation exposure. tumour, enucleation rates have substantially Methods Five human UM cell lines (92.1, declined in recent years. The procedure is now SP6.5, MKT-BR, OCM-1, and UW-1) and one reserved for large tumours or those cases where 3 1 human fibroblast cell line (BJ) were incubated there is no hope of regaining vision. Department of Ophthalmology and with amfenac. Treated and non-treated cell Radiation is the preferred form of treatment 4 Pathology, The McGill lines were then exposed to various doses of c for most cases of UM. UMs are typically treated 5 University Health Center & radiation: 0, 2, 4, 6, and 8 Gy. Sulphorhodamine- with a calculated apex dose of 70–85 Gy. Henry C Witelson Ocular B assay was used to assess proliferation rates Iodine-125 is the most commonly used isotope Pathology Laboratory, 48 h post-radiation. for plaque radiotherapy of choroidal Montreal, Canada 6 Results Treatment of UM cell lines with melanomas, although cobalt-60, ruthenium- 2Department of amfenac prior to radiation led to a marked 106, iridium-192, strontium-90, and palladium- 3 Ophthalmology. Federal reduction in proliferation rates. This difference 103 have also been used. Modern techniques University of Sa˜ o was statistically significant in all cell lines at for brachytherapy involve suturing a shielded PauloFUNIFESP/EPM, Sa˜ o every radiation dose (Po0.005), with the plaque containing seeds of the radioactive Paulo, Brazil exception of 92.1 at 2 Gy (P ¼ 0.157). Fibroblasts isotope to the sclera. This remains in place for a treated with amfenac showed significantly specified number of days to deliver the proper Correspondence: BF Fernandes, higher proliferation rates after 2 and 8 Gy, with dose of radiation. Although the rates of tumour Department of no significant differences at 0, 4, and 6 Gy. control are high, visual acuity is compromised Ophthalmology and Conclusions The radiosensitivity of UM cell in 25–35% of cases.6 Some of the complications Pathology, lines was increased by the administration of associated with the treatment are neovascular The McGill University Health amfenac, the active metabolite of nepafenac. glaucoma, cataract, radiation retinopathy, and Center & Henry C Witelson Ocular Pathology There appears to be a radioprotective effect of 7 optic nerve neuropathy. Thus, a substantial Laboratory, amfenac on human fibroblasts. The topical number of eyes are still enucleated due to 3775 University Street, administration of nepafenac may decrease failure of tumour control or radiation-related Room 216, tumour recurrence and radiation-induced complications. Montreal, Quebec, Canada H3A-2B4 complications while broadening the indications There are two forms of the cyclooxygenase Tel: þ 1 514 398 7192 ext. for radiotherapy by treating larger tumours. (COX) enzyme, COX-1 and COX-2. While COX-1 00384; Eye (2008) 22, 701–706; doi:10.1038/sj.eye.6703042; is expressed constitutively in normal tissues,8 Fax: þ 1 514 398 5728. published online 30 November 2007 COX-2 is an inducible enzyme expressed in E-mail: bruno.mtl@ response to a variety of inflammatory and gmail.com Keywords: uveal melanoma; radiation; COX-2; mitogenic stimuli.9 COX-2 expression has been amfenac; cell lines Received: 13 June 2007 reported in a wide variety of malignant Accepted in revised form: tumours,10–12 including uveal melanoma.13 The 27 October 2007 expression of COX-2 has been linked to various Published online: 30 processes, including tumour proliferation,14 November 2007 Introduction 15 16,17 immunosuppression, and metastasis. The authors have no Uveal melanoma (UM) is the most common Specific COX-2 inhibitors are currently in use financial conflict of interest primary intraocular malignant tumour in adults for patients diagnosed with familial regarding this article. Amfenac and uveal melanoma BF Fernandes et al 702 adenomatous polyposis, a genetic disorder, which Irradiation predisposes patients to colon cancer.18 The effectiveness Prior to radiation, each cell line was seeded at a of these selective inhibitors has been investigated in a concentration of 500 000 cells per ml in micro petri dishes variety of tumours and shows promise for use as an and incubated overnight with a 150 nM concentration of adjuvant therapy.19 amfenac (Alcon Laboratories Inc., Fort Worth, TX, USA), COX-2 inhibitors have also previously been shown to the active metabolite of nepafenac. The concentration of increase the radiosensitivity of lung20 and breast21 cancer 150 nm was the recommended 50% inhibitory cell lines. Moreover, it has been demonstrated that a concentration (IC ) of COX-2 activity.28 On the following COX-2 inhibitor is capable of minimizing radiation 50 day, the cells were exposed to graded doses of g damage to non-neoplastic tissues.22 Nepafenac is a COX- irradiation: 2, 4, 6, and 8 Gy (137Cs source, g Cell 1000). 2 inhibitor formulated for topical administration to the Controls consisted of cell lines exposed to radiation eye.23,24 The purpose of our study was to determine the without prior incubation with amfenac. effects of amfenac, the active metabolite of nepafenac, on Post radiation, all micro petri dishes containing the radiosensitivity of five human UM cell lines and one 500 000 cells/plate were trypsinized using 0.05% Trypsin human fibroblast cell line. in EDTA, then washed in 5% FBS RPMI solution, and centrifuged for 5 min at 3000 g. The cells were then diluted to a concentration of 50 000 cells/ml in 5% FBS Methods RPMI solution. These dilutions were seeded in a 96-well Cell culture plate format at a concentration of 5000 cells per well and left to incubate at 371C for 48 h to adhere to the bottom of Four previously characterized human UM cell lines (92.1, the wells. After 48 h, we could already see colonies SP6.5, MKT-BR, OCM-1), one transformed human uveal formed. Thus, to guarantee exponential growth, we melanocyte cell line (UW-1) and one human fibroblast chose to run the proliferation assay at this time point. The cell line (BJ, American Type Culture Collection, USA) proliferation assay was done in triplicate per exposure were incubated at 371C in a humidified 5% CO -enriched 2 condition. atmosphere.25 The cell lines used have previously been graded in terms of proliferative and invasive abilities and 25 metastatic potential. The UM cell lines were cultured in Sulphorhodamine-B assay RPMI-1640 medium (Invitrogen, Burlington, ON, Canada), supplemented with 5% heat inactivated fetal The Sulphorhodamine-B assay kit (TOX-6, Sigma-Aldrich bovine serum (FBS), 1% fungizone, and 1% penicillin– Co., St Louis, Missouri, USA) was used to measure total 29 streptomycin purchased from Invitrogen (Burlington, cell material as described previously. Each of the ON, Canada). The fibroblast cell line was cultured in treated and non-treated cell lines were seeded into wells 3 DMEM medium (Invitrogen, Burlington, ON, Canada), at a concentration of 5 Â 10 cells per well, with a supplemented with 10% heat-inactivated FBS, 1% minimum of three wells per cell line/radiation dose. The 1 fungizone, and 1% penicillin-streptomycin purchased cells were then allowed to incubate for 48 h at 37 C. from Invitrogen (Burlington, ON, Canada). Cells were Following the incubation period, the cells were fixed to cultured as a monolayer in 25 cm2 flasks (Fisher, Whitby, the bottom of the wells using a solution of 50% 1 ON, Canada) and observed two times weekly, at every Trichloroacetic acid for 1 h at 4 C. The plates were then media change, for normal growth by phase contrast rinsed with distilled water, to remove trichloroacetic acid microscopy. The cultures were grown to confluence and and medium, and later air dried. The Sulphorhodamine- passaged by treatment with 0.05% trypsin in EDTA B dye solution was next added to each well and allowed (Fisher) at 371C and washed in 7 ml RPMI-1640 media to stain for 25 min. The Sulphorhodamine-B solution was before being centrifuged at 120 g for 10 min to form a subsequently removed by washing with a 10% acetic acid pellet. Cells were then suspended in 1 ml of medium and solution and once more allowed to air dry. The dye that counted using the Trypan Blue dye exclusion test for use had become incorporated into the fixed cells at the in all subsequent assays. bottom of the wells was solubilized in a 10 mM solution The UM cell lines 92.1, SP6.5, and MKT-BR were of Tris. The absorbance of the solute was measured using established by Dr Jager (University Hospital Leiden, The a microplate reader at a wavelength of 510 nm. Netherlands), Dr Pelletier (Laval University, Quebec, Canada) and Dr Belkhou (CJF INSERM, France), Data analysis respectively. Dr Albert (University of Wisconsin- Madison, USA) established the OCM-1 and UW-1 cell The Student’s t-test was used to compare proliferation lines.26,27 results between treated and non-treated cell lines after Eye Amfenac and uveal melanoma BF Fernandes et al 703 various doses of radiation.