# 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
It has been reported that TSAIII strengthens the suppressive activity of paclitaxel (PTX) on the proliferation of nasopharyngeal carcinoma CNE-1 and HNE-2 cells, upregulates the expression of Bad and RAP1 GAP, downregulates the expression of Bcl-2, RAP1, and RasGRP2, and promotes PTX-induced apoptosis. TSAIII can synergize with PTX to protect against tumor growth in a xenograft mouse model [45] (Table 1). The cGMP/PKG signaling pathway is downregulated in the glioblastoma multiforme (GBM), and the activation of the cGMP/PKG pathway has been shown to inhibit the progression of GBM [46]. TSAIII promotes cell apoptosis and inhibits the growth of human glioblastoma U87 MG cells, which is the deadliest malignant tumor in the brain. Mechanically, TSAIII downregulates the expression of cGMP-specific phosphodiesterase 5 (PDE5), upregulates the expression of guanylate cyclases (sGC\(\beta\)), cGMP, PKG, and VASP-Ser239 phosphorylation, and suppresses the Wnt/\(\beta\)-catenin signaling pathway [47]. Sterol regulatory element-binding proteins (SREBPs) mediate the synthesis of fatty acids and cholesterol. In the endoplasmic reticulum (ER), SREBP can be inactivated by forming a complex with INSIG-1 and the SREBP cleavage-activating protein (SCAP). Active SREBP-1 mediates fatty acid synthesis, and SREBP-2 regulates cholesterol synthesis by activating target genes, such as HMG-CoA reductase (HMGCR) [48; 49]. It has been reported that TSAIII inhibits the expression of SREBP-1, FASN, ACC, and HMGCR and suppresses the growth of pancreatic cancer BxPC-3 cells. In addition, TSAIII causes cell cycle arrest at the G0/G1 phase, decreases the expression of cyclin E1, cyclin D1, CDK2, and CDK6, increases the expression of p21 and p27, and upregulates the expression of cleaved caspase-3, cleaved caspase-9, cleaved PARP, and Bid [50] (Table 1). Cholesterol-based liposomes in the formulation have been approved for drug development. However, cholesterol is vulnerable to oxidation, which negatively affects the stability and quality of liposomes. TSAIII has a similar structure to cholesterol. It has been reported that TSAIII-based TSAIII/DOX codelivery liposomes have been developed, and TSAIII acts as a bilayer stabilizer, increasing the doxorubicin hydrochloride (DOX) uptake and synergizing with DOX to induce apoptosis in HepG2 and HCC-LM3 cells [51]. CCR4-NOT2 (CNOT2) plays a key role in apoptosis, autophagy, proliferation, and angiogenesis in various tumor cells [52]. Midline1 interacting protein 1 (MID1 IP1), an upstream factor of AMPK, mediates acetyl-CoA carboxylase activity and acts as an oncogene [53]. It has been reported that deficiency of both MID1 IP1 and CNOT2 can promote cell growth and inhibit the apoptosis of hepatocellular carcinoma HepG2 and Huh7 cells by downregulated the expression of c-Myc [54]. TSAIII can induce colon cancer HCT116 cell apoptosis by suppressing the expression of MID1 IP1, CNOT2, and c-Myc, interrupting the interaction between MID1 IP1 and c-Myc and decreasing the stability of c-Myc. The cotreatment of TSAIII with 5-FU or doxorubicin can attenuate c-Myc and caspase-3 expression and increase cleaved-PARP production, potentiating the apoptotic effects of 5-FU or doxorubicin in HCT116 cells [55] (Table 1).