Anticancer Activity of Triterpene Glycosides Cucumarioside A0-1 and Djakonovioside A Against MDA-MB-231 as A2B Adenosine Receptor Antagonists

Breast cancer is the most prevalent cancer in women worldwide and presents a major therapeutic challenge, particularly triple-negative breast cancer (TNBC), a subtype characterized by an aggressive clinical course but heightened sensitivity to chemotherapy. Natural products, such as triterpene glycosides derived from sea cucumbers, have emerged as promising candidates with high anticancer potential against TNBC. This study investigated the pathways of anticancer action of cucumarioside A0-1 (Cuc A0-1) and djakonovioside A (Dj A), isolated from the sea cucumber Cucumaria djakonovi, triggered and regulated in MDA-MB-231 cells (triple-negative breast cancer cell line). We employed functional assays (cAMP level, Ca2+ influx, control of cell proliferation and colony formation), Western blotting for mitogen-activated protein kinase MAPK) signaling, and in silico molecular docking. A2B adenosine receptor (A2BAR) was identified as a novel target for both glycosides. As antagonists, they reduced cAMP production and inhibited NECA (5-(N-ethylcarboxamido)adenosine)-induced Ca2+ influx. This A2BAR blockade suppressed the MAPK pathway, profoundly inhibiting phospho-ERK1/2, p38, and JNK1/2, which led to the activation of the intrinsic apoptotic pathway and strong inhibition of cell proliferation and colony formation. Surprisingly, co-treatment with the NECA agonist enhanced the antiproliferative effects of the glycosides. It was supposed that the interaction of glycosides with the NECA-preactivated receptor may bias signaling toward the Gi and Gq/PLC/ERK1/2 pathways, underscoring the central role of the MAPK pathway in controlling cell growth. Molecular docking confirmed binding to the A2BAR orthosteric site, revealing that Cuc A0-1 and Dj A employ distinct interaction modes. To our knowledge, this is the first report to define A2BAR as a target for sea cucumber glycosides. Their potent antitumor effects, mediated through the antagonism of A2BAR and subsequent MAPK pathway inhibition, position them as promising lead compounds for cancer types with high expression A2BAR.