# The Potential Role of Timosaponin-AIII in Cancer Prevention and Treatment
## 4 The Anticancer Effects of TSAIII
### Proliferation Inhibition, Cell Cycle Arrest, and Apoptosis Induction  
One of the most dominant characteristics of tumor cells is uncontrolled proliferation, which could be due to the dysregulation of the cell cycle and its resistance to apoptotic cell death. The cell cycle at each cellular phase was tightly mediated by cyclin-dependent kinases (CDKs). Specifically, CDK/cyclin complexes and CDK inhibitors govern the progression of the cell cycle at each phase. The induction of cell cycle arrest could result in the inhibition of cell proliferation [35]. In addition, the induction of apoptosis or programmed cell death is also an effective way to control the proliferation of cancer cells [36]. TSAIII has shown regulatory effects against tumor pathogenesis. For example, TSAIII can inhibit proliferation, cause cell cycle arrest at the G1 phase, and promote caspase-dependent apoptosis by mediating the PI3K/AKT signaling pathway in pancreatic cancer cells [37]. TSAIII also induces caspase-dependent apoptosis in promylocytic leukemia HL60 cells by enhancing the phosphorylation levels of JNK1/2 and p38 MAPK [38]. Another study reported that TSAIII downregulates Cyclin B1 Cdc2, and Cdc25C expression, triggers DNA damage, causes cell cycle arrest at the G2/M phase, and induces apoptosis in breast cancer MDA-MB-231 and MCF7 cells by activating the ATM/Chk2 and p38 MAPK signaling pathways [39] (Table 1). The signal transducer and activator of transcription 3 (STAT3) are constantly activated via Ser705 phosphorylation to promote G1/S-phase progression and enhance the proliferation of pancreatic cancer cells. Potentially, TSAIII can suppress the phosphorylation of STAT3 and ERK1/2, decrease cell viability, cause cell cycle arrest at the G1 phase, and promote cell apoptosis in AsPC-1 cells by inhibiting the constitutive expression of c-Src kinase, which is frequently and aberrantly activated in various malignancies. Specifically, TSAIII has been shown to significantly decrease the expression of Bcl-2, Bcl-xL, MMP-9, VEGF-1, and survivin and increase the expression of the cyclin-dependent kinase inhibitor p21 and cell cycle regulator cyclin D1 [40]. In human colorectal cancer HCT-15 cells, TSAIII has shown an inhibitory effect with an IC50 value of 6.1 \(\upmu\)M. TSAIII suppresses proliferation, causes cell cycle arrest at the G0/G1 and G2/M phase, and induces apoptosis, as demonstrated by the downregulated expression of cyclin A, cyclin B1, CDK2, CDK4, the proliferating cell nuclear antigen, c-Myc, Bcl-2, and Bcl-xL and increased DNA fragmentation, caspase activation, and cleaved poly-(ADP ribose) polymerase in HCT-15 cells. Consistently, TSAIII has been shown to inhibit tumor growth in HCT-15-xenograft-bearing mice [41] (Table 1). Further studies have shown that TSAIII induces mitochondria-mediated and caspase-dependent apoptosis in HepG2 cells, as shown by the increased expression of caspase-3,  
Figure 3: The anticancer effects of TSAIII are shown: TSAIII inhibits proliferation, causes cell cycle arrest, induces apoptosis, mediates autophagy, inhibits migration and invasion, attenuates angiogenesis, suppresses multidrug resistance, decreases inflammation, and ameliorates oxidative stress in cancer cells. \(\uparrow\) indicates up-regulation, and \(\downarrow\) indicates down-regulation. caspase-7, caspase-7, and caspase-9, the decreased expression of Bcl-2, Mcl-1, cIAP-1, cIAP-2, XIAP, survivin, and livin, and the enhanced cytosolic production of HtrA2/Omi, Smac/Diablo, and cytochrome c. In addition, TSAIII reduces the viability of HepG2 cells with an IC50 value of 15.41 \(\upmu\)M [42]. The Ras-associated protein 1 (RAP1) is a small GTPase that is responsible for inside-out signaling cascades to trigger integrin [43]. The aberrant expression of RAP1 can be associated with aggressive phenotypes and tumor invasion and metastasis, while the GTPase-activating protein RAP1 GAP can negatively regulate the activity of RAP1 [44].