Ziyu II induces autophagosome formation in CRC cells
As increasing evidence has highlighted the important roles of drug-induced autophagy in anticancer therapies [24-27], we thus investigated whether autophagy is involved in Ziyu II induced CRC cell death. We first examined the protein levels of autophagy related proteins in Ziyu II-treated CRC cells, and found that Ziyu II treatment promoted the turnover of LC3-I to lipidated LC3-II in a dose-dependent manner in CRC cells (Fig.3A). Also, we evaluated the expression levels of ATG5 and ATG7, two autophagy related proteins, which are notably increased in a dose dependent manner after Ziyu II treatment (Fig.3A). When combined with 3-MA, an inhibitor of class III PI3K, the elevated LC3-II levels were prominently inhibited in Ziyu II-treated CRC cells, suggesting that Ziyu II induces autophagy initiation (Fig. 3B). This was further confirmed by siRNA-mediated ATG5 silencing, as evidenced by decreased LC3 puncta in combinatorial treatment group (Fig. S3A-E). Furthermore, both the endogenous LC3 and exogenous GFP-LC3 puncta, representing the number of autophagic vacuoles [28], were remarkably increased in Ziyu II-treated CRC cells (Fig. 3C-F). In addition, Ziyu II-treated xenografts displayed stronger LC3 staining than that of the control group (Fig. 3G and H). Taken together, these results show that Ziyu II promotes the initiation process of autophagy in CRC cells.