Acetylsalicylic acid (ASA), also known as Aspirin, is a commonly used drug known for more than a century. While its anti-inflammatory and anti-thrombotic effects have been well documented, several newer beneficial effects of ASA, including cardioprotective and tumour growth-inhibiting functions were demonstrated. Recently, several studies showed a lower risk of various cancers in patients regularly using ASA, however, the precise anti-cancer mechanisms of ASA are yet to be elucidated. Melanoma is a highly malignant skin cancer arising from transformed melanocytes. We and others have recently shown that malignant melanocytes produce various bioactive molecules, such as cytokines (e.g., IL-6), chemokines (such as CXCL1 and IL-8) and growth factors that modify the tumour microenvironment with impact on malignant cell growth and spreading. The aim of this study was the explanation of protein changes in malignant melanocytes initiated by ASA. Cells of the human melanoma G361 line were treated with 5 mM ASA. Cell lysates, conditioned medium and exosomes were analysed by multiplex immunoassay for levels of IL-2, IL-6, IL-8, IFNγ, CXCL1, TNFα, HGF, CCL2, SDF1α, and VEGF-A. In addition, global cellular and exosome proteomes were analysed by LC-MS/MS (timsTOF HT) using the dia-PASEF method, followed by data analysis in Spectronaut. ASA treatment led to a reduction of exosome secretion, as documented among others by the decreased amount of isolated exosomes and by the highly significant decrease of all 8 subunits of exocyst complex in exosomes. Moreover, ASA also reduced IL-8 secretion. On the other hand, IL-6 levels in cells, conditioned medium and exosomes were increased upon ASA treatment. The following experiments will focus on the function of selected molecules in ASA-induced anti-inflammatory and anti-cancer effects.

Acknowledgement

This study was supported by the National Institute for Cancer Research (LX22NPO5102) financed by the European Union – Next Generation EU, by the project Center for Tumor Ecology (CZ.02.1.01/0.0/0.0/16_019/0000785), and Charles University Cooperatio Program, research area "Oncology and Haematology".