# The Potential Role of Timosaponin-AIII in Cancer Prevention and Treatment
## 4 The Anticancer Effects of TSAIII
### Autophagy Mediation  
Autophagy can be associated with the selective degradation of aggregated protein complexes and damaged organelles, which serve as a cellular quality control system. Attenuated or impaired autophagy has been related to carcinogenesis, and the activation of autophagy has become a therapeutic strategy for cancers [56]. TSAIII can be an inducer of autophagy and stimulate autophagome formation and autophagy flux by inhibiting the mTOR/ULK1 signaling pathway and elevating cytosolic-free calcium. TSAIII pro motes autophagy-mediated protein degradation, compromising the rapid growth of tumor cells [57]. The interaction between autophagy and apoptosis is complex during tumorigenesis progression. Members in the inhibitor of the apoptosis (IAP) family can bind to caspases and inhibit their activity in eukaryotic cells. XIAP, as an identified inhibitor of the apoptosis (IAP) protein, is a potential target for HCC treatment. TSAIII reduces cell viability and exhibits cytotoxicity to HCC cell lines in a p53-independent manner. The inhibition of XIAP promotes caspase-dependent apoptosis in TSAIII-treated HCC cells. Interestingly, the mRNA expression of XIAP is not changed, and TSAIII promotes autophagy-induced XIAP degradation by activating the AMPK\(\alpha\)/mTOR signaling pathway. The ubiquitination of XIAP is required for TSAIII-induced XIAP protein degradation. However, the recruitment of the E3 ligase RING has not been observed, indicating an impaired proteasomal degradation system. Therefore, the inhibition of autophagy switches on necrosis in TSAIII-treated HCC cells [58]. Another study has reported that TSAIII upregulates LC3-II expression and induces autophagy at an early stage in HeLa cancer cells. Prolonged exposure to TSAIII can lead to the overproduction of reactive oxygen species (ROS), a reduction in mitochondrial membrane potential (\(\Delta\)Vm), the release of cytochrome c, and activation of caspase-3 (Table 1) (Figure 4). The inhibition of autophagy by 3-methyladenine (3-MA) or Beclin 1 siRNA enhances the cytotoxic sensitivity of HeLa cells to TSAIII in a caspase-dependent manner. Thus, TSAIII-induced autophagy precedes mitochondria-dependent cell apoptosis [59]. Consistently, TSAIII induces mitochondrial dysfunction, upregulates the protein expression of cleaved caspase-3, cleaved caspase-9, cleaved PARP, and cytochrome c, downregulates the expression of Mcl-1, and enhances the accumulation of p62, LC3-II, and LAMP1 in GBM8401 and M059K cells. The inhibition of autophagy by 3-MA enhances TSAIII-induced apoptosis. In addition, TSAIII has been shown to suppress tumor growth in GBM8401-xenograft-bearing mice and orthotropic glioma mice [60]. In human non-small-cell lung cancer (NSCLC) A549 and H1299 cells, TSAIII at higher concentrations (10 uM and 30 uM) has promoted both autophagy and apoptosis by activating AMPK signaling  
Figure 4: TSAIII inhibits cancer development by mediating various signaling pathways: TSAIII can inhibit the NF-kB, ERK1/2, PI3K/AKT, and Wnt/\(\beta\)-catenin signaling pathways. TSAIII induces mitochondria-dependent apoptosis and suppresses oxidative stress. The NF-kB pathway-mediated upregulation of proinflammatory cytokines and MMPs has been associated with cancer cell apoptosis, inflammation, migration, invasion, and angiogenesis. In addition, the activation of the PI3K/AKT/mTOR pathway inhibits autophagy. The inactivation of the GSK-3\(\beta\) and activated \(\beta\)-catenin/Cyclin D1 pathway could lead to cell proliferation and cell cycle mediation. and inactivating the ERK1/2 pathway (Table 1). At a lower concentration (1 \(\upmu\)M), TSAIII only induces autophagy, protecting cancer cells from apoptosis. In addition, a combination of TSAIII with 3-MA promotes cell apoptosis and enhances their anticancer effects [61]. It has also been reported that TSAIII promotes both apoptosis and autophagy in A375-S2 cells. The activation of autophagy suppresses apoptosis in TSAIII-treated A375-S2 cells.