Abstract
Purpose :
Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults. It is characterized by frequent driver mutations in GNAQ and GNA11 genes. Both genes are activated by cysteinyl leukotriene (CysLT) receptors and are upstream effectors of MAPK/MEK/ERK signalling pathway. Previous studies have demonstrated that CysLT receptors play a role in human tumorigenesis. The anti-asthmatic drug MK571, acts as a CysLT1 inverse agonist, was shown to induce apoptosis in glioblastoma, prostate and colon cancers. MK571 has a well-established toxicity profile tested in humans, which could be available for UM patients. The aim of this study is to analyze the expression of CysLT1 receptor and test the biological effects of MK571 in the context of UM.
Methods :
In this study, we used four UM cell lines (MP41, MP46, MEL270, OMM2.5). We determined the expression of CysLT1 receptor by immunocytochemistry. Cells were then treated with increasing concentrations of MK571 (25, 50, 100, 150μM) and our control were untreated cells. Afterwards, cell proliferation was assessed using live cell imaging microscopy, and dose-dependent cytotoxic effects were evaluated using the Cell Counting Kit 8 (CCK8) viability assay. In parallel, we analysed cell apoptosis using Annexin V/PI staining and flow cytometry. The expression of CysLT1 receptor was also determined in UM patient samples (N=31) by immunohistochemistry. Statistical analysis was performed using Χ2 test, one-way ANOVA tests and two-way ANOVA; P<0.05 was considered significant.
Results :
All four UM cell lines were positive for the expression of CysLT1 receptor. When treated with MK571, UM cells displayed a dose-dependent reduction in cell proliferation (P<0.0001) and cell viability (P=0.049). Notably, after 24 hours of treatment, MK571 75μM induced an apoptotic cell death (Annexin V positive and PI negative), while at 100μM it induced cell necrosis (Annexin V negative and PI positive). The expression of CysLT1 receptor was confirmed in all 31 UM cases, Χ2 (1, N = 31, P<0.001).
Conclusions :
Our results demonstrate that MK571 reduces cell viability, cell proliferation and induces apoptosis of UM cell lines in an in vitro model. The high expression of CysLT1 receptor observed in UM patient samples suggest that targeting this receptor could provide an alternative therapeutic approach for a more efficient treatment of uveal melanoma.
This is a 2020 ARVO Annual Meeting abstract.