Supplementary Material for: Acetylsalicylic Acid Exerts Potent Antitumor and Antiangiogenic Effects in Cutaneous and Uveal Melanoma Cell Lines

<b><i>Introduction:</i></b> Acetylsalicylic acid (ASA) has been investigated for a potential anticancer role in several cancers, such as colorectal, ovarian, and endometrial cancer. Moreover, ASA has been shown to abrogate various processes that contribute to tumor growth and progression. <b><i>Objective:</i></b> The aim of this study was to evaluate the effects of ASA on cutaneous melanoma (CM) and uveal melanoma (UM). <b><i>Methods:</i></b> Human CM and UM cells were treated with 5 mM ASA and assessed for changes in cellular functions. Antiangiogenic effects of ASA were determined using an ELISA-based assay for 10 proangiogenic cytokines, and then validated by Western blot. Finally, proteomic analysis of ASA-treated cells was performed to elucidate the changes that may be responsible for ASA-mediated effects in melanoma cells. <b><i>Results:</i></b> Treatment with ASA significantly inhibited the proliferation, invasion, and migration capabilities, and caused a significant decrease in angiogenin and PIGF secretion in both CM and UM. Mass spectrometry revealed 179 protein changes associated with ASA in the CM and UM cell lines. <b><i>Conclusions:</i></b> These results suggest that ASA may be effective as an adjuvant therapy in metastatic CM and UM. Future studies are needed to determine the regulating targets that are responsible for the antitumor effects of ASA.

**_引言：_** 乙酰水杨酸（ASA）已在结直肠癌、卵巢癌及子宫内膜癌等多种癌症中被研究其潜在的抗肿瘤作用。此外，研究证实ASA可抑制多种促进肿瘤生长与进展的生物学过程。
**_研究目的：_** 本研究旨在评估ASA对皮肤黑色素瘤（cutaneous melanoma，CM）与葡萄膜黑色素瘤（uveal melanoma，UM）的作用效果。
**_研究方法：_** 将人源皮肤黑色素瘤与葡萄膜黑色素瘤细胞以5 mmol/L的ASA进行处理，检测其细胞功能的变化。通过酶联免疫吸附试验（ELISA）检测10种促血管生成细胞因子的水平，以确定ASA的抗血管生成活性，随后通过蛋白质印迹法（Western blot）进行验证。最后，对经ASA处理的细胞开展蛋白质组学分析，以阐明可能介导ASA在黑色素瘤细胞中发挥抗肿瘤作用的分子变化。
**_研究结果：_** ASA处理可显著抑制皮肤黑色素瘤与葡萄膜黑色素瘤细胞的增殖、侵袭与迁移能力，并显著降低两类细胞中血管生成素与胎盘生长因子（PIGF）的分泌水平。质谱分析结果显示，在两种细胞系中，共有179种蛋白质的表达水平在ASA处理后发生了显著改变。
**_研究结论：_** 上述结果表明，ASA可作为转移性皮肤黑色素瘤与葡萄膜黑色素瘤的辅助治疗手段，具备潜在应用价值。未来仍需开展进一步研究，以明确介导ASA发挥抗肿瘤作用的具体调控靶点。