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.