## 4 Discussion
In this context, WST is likely to exert its anti-mutagenic effects via intracellular, extracellular, or synergistic interactions, thus playing a role in preventing base-pair substitutions and shift mutations. Cellular apoptosis is a spontaneous process that presents as the autonomous, orderly death of cells controlled by specific genes. Different from cell necrosis, apoptosis is an active process involving the activation, expression, and regulation of a series of genes. It is not a phenomenon of autologous injury under pathological conditions, but a death process that is actively induced for adapting to the living environment and maintaining internal environmental stability. This system is capable of removing defective or damaged cells before they are abnormally divided and replicated during the early stages of cancer development. Therefore, it is an effective mechanism for preventing cancer [39]. Liver cancer is one of the most common malignant tumors, with a high incidence and mortality, and there is currently no effective treatment. Some ingredients in food have been shown to prevent liver cancer, and these active ingredients prevent cancer by inducing apoptosis and death of cancer cells by stimulating apoptosis [40]. As typical apoptosis-related factors, the bcl-2 family of proteins play an important role in the regulation of apoptosis and includes the anti-apoptotic factor bcl-2 and the pro-apoptotic factor bax. Bcl-2 inhibits apoptosis by preventing cells from releasing cytochrome C (Cyt-C) from the mitochondria and limiting its entry into the cytosol [41]. In contrast, bax has an inducing effect on apoptosis, and the depolarization of the mitochondrial inner membrane potential leads to the release of Cyt-C and apoptotic protease-activating factor-1 (Apaf-1) into the cytosol, which is combined with the caspase-9 zymogen to form the apoptosome. The latter interaction promotes the activation of the caspase-9 zymogen and ultimately activates the apoptotic effectors caspase-3 and caspase-7, leading to apoptosis [42]. The proportional balance between anti-apoptotic factors and pro-apoptotic factors affects the occurrence of apoptosis. In this study, WST extracts were shown to increase the expression of bax, reduce the expression of bcl-2, especially enhancing the expression level of proapoptotic factor bax, and subsequently increase the mRNA levels of caspase-9, caspase-3, and caspase-7, thus inducing apoptosis of liver cancer cells. These results indicated that WST has a strong anticancer activity against liver cancer cells. The caspase signaling cascade is a key event in endogenous and exogenous apoptosis, and caspase-3, -7, -8, and -9 are the main proteases involved in apoptosis [43]. Caspase-8 and -9 are the starting caspase enzymes, located upstream of the apoptotic pathway. After activation by different pathways, they are responsible for activating other caspases, such as caspase-3 and caspase-7, downstream of the apoptotic pathway. As an executor of apoptosis, this cascade induces apoptosis [44]. During this process, proteins such as calpain, cathepsin, and endonuclease perform programmed cell death and enhance cancer cell apoptosis through their interactions with caspases [45]. WST extracts enhanced the expression of caspase-3, -7, -8, and -9 mRNA levels by activating endogenous and exogenous apoptotic factors, thus inducing apoptosis in HepG2 cancer cells. The tumor suppressor p53 monitors and regulates the DNA damage repair process in the G1 phase of the cell cycle, and it serves to induce multiple pathways to initiate apoptosis if the repair process fails [46].