Visualization studies of the anti-tumor mechanism of fluorescently labeled ergosterol

Ergosterol (ER) is a steroid compound widely present in fungi and has demonstrated promising potential in tumor therapy. However, the deeper molecular mechanisms underlying the anti-tumor activity of ergosterol have not yet been fully elucidated. Therefore, in this study, a novel ergosterol derivative Cy5-ER with visual fluorescence properties was designed and synthesized to reveal the anti-tumor mechanism of ergosterol. Experimental results showed that Cy5-ER was rapidly taken up by HepG2 cells in a time- and concentration-dependent manner and exhibiting strong colocalization with mitochondria, leading to a significant decrease in MMP. Western blot analysis was conducted to assess the expression of mitochondrial pathway-associated proteins. The results demonstrated that treatment with ergosterol and Cy5-ER significantly upregulated the expression of mitochondrial apoptosis-associated proteins, including Bax, Cytochrome C, Caspase-3, Cleaved Caspase-3, Cleaved Caspase-9 and Cleaved PARP, while downregulating Bcl-2. In this study, the antitumor mechanism of ergosterol targeting mitochondria was elucidated through molecular-level visual imaging, which directly showed a high degree of colocalization relationship between Cy5-ER and mitochondria, and a significant reduction in mitochondrial membrane potential. The alterations in apoptosis-related protein expression further corroborated these findings. This study offers novel approach for visualizing the anti-tumor mechanism of ergosterol.