Raddeanin A (RA) Inhibited EMT and Stemness in Glioblastoma via downregulating Skp2

<b>Background:</b> Glioblastoma (GBM), notorious for its poor prognosis, stands as a formidable challenge within the central nervous system tumor category, primarily due to its intricate pathology that encompasses stemness and the epithelial-mesenchymal transition (EMT). The ubiquity of S phase kinase-associated protein 2 (Skp2) overexpression in GBM, a protein implicated in both EMT and stemness traits, correlates with increased drug resistance, elevated tumor grades, and adverse outcomes. This investigation delves into the impact of Raddeanin A (RA), a triterpenoid compound extracted from Anemone raddeana Regel, on GBM, with a special focus on its influence over Skp2 expression levels. <b>Method:</b> The study assessed RA's influence on GBM cell lines U87 and U251 via CCK-8 and colony formation assays to gauge cell proliferation, alongside Transwell assays for evaluating migration and invasion capabilities. mRNA expression was detected by RT-PCR. Protein expression alterations were examined through western blotting and immunofluorescence techniques. The therapeutic potential of RA <i>in vivo</i> was also evaluated using subcutaneous and intracranial xenograft model in mice, developed using U87 cells. The molecular docking experiment was performed to evaluate the binding of RA to Skp2. <b>Results:</b> RA markedly curtailed the proliferation of U87 and U251 cells in a concentration-dependent manner, alongside diminishing sphere formation in glioblastoma stem-like cells (GSCs). A significant suppression of Skp2 expression was observed in both cell lines and GSCs following RA treatment. This reduction in Skp2 was associated with a decrease in stemness markers (Sox2, Nestin) and the inhibition of EMT markers (Vimentin, N-cadherin, Snail). Moreover, Skp2 overexpression was found to mitigate RA's suppressive effects on EMT and stemness, highlighting Skp2's crucial role in these processes. The <i>in vivo</i> experiments supported these findings, indicating that RA not only thwarted tumor growth but also substantially lowered the expression of Skp2, EMT markers, and stemness indicators. Additionally, molecular docking experiments demonstrated that RA exhibits a notable binding affinity to Skp2. <b>Conclusion:</b> This study elucidates RA's significant antitumor efficacy against GBM <i>in vitro</i> and <i>in vivo</i> by targeting pathways linked to stemness and EMT, chiefly via the downregulation of Skp2. These findings underscore RA's therapeutic promise in GBM management, offering insights into its mechanism of action and laying the groundwork for subsequent clinical investigations.