Lappaconitine, a kind of C₁₈ diterpenoid alkaloids, inhibited the proliferation of lung cancer A549 cells and induced cell apoptosis (Sheng et al., 2014; Fang, 2018). In vivo investigation revealed that the tumor inhibitory rate of mice bearing liver cancer is 11.20–53.08%; meanwhile that of mice bearing S180 sarcoma cells was 29.81–53.96% after the administration of lappaconitine (Lin et al., 2005). Besides, lappaconitine was widely used in the analgesia of various cancers with high safety and no addiction (Zhang et al., 2016; Sun, 2017). Figure 1 displays the structural formula of lappaconitine and the mother nucleus of C₁₈ diterpenoid alkaloids in Fuzi.

Most of the constituents with antineoplastic efficacies in Fuzi are C₁₉ diterpenoid alkaloids. Apart from the bioactivity, these compounds are also poisonous, which lead to cardiotoxicity and neurotoxicity. Three main C₁₉-diester-diterpenoid alkaloids (DDAs) were responsible for the toxicity of Fuzi, namely, aconitine, mesaconitine, and hypaconitine. Studies have shown that the median lethal dose (LD₅₀) of single-dose oral aconitine in mice is 1.0–1.8 mg/kg, while the LD₅₀ of mesaconitine and hypaconitine are 1.9 and 5.8 mg/kg, respectively. The dose–effect relationship of DDAs on clinical therapy remains to be further studied (Wada et al., 2005; Singhuber et al., 2009). So far, nearly 80 kinds of C₁₉ diterpenoid alkaloids have been extracted (Tang et al., 2017; Zhang et al., 2020). The mother nucleus of C₁₉ diterpenoid alkaloids is shown in Figure 2, and natural C₁₉ diterpenoid alkaloids with anticancer activity are listed in Table 1. As shown in Table 1, the common ingredients of Fuzi, such as aconitine, mesaconitine, hypaconitine, benzoylaconitine, and neoline, have been proved to exhibit anticancer functions. Furthermore, the derivatives of C₁₉ diterpenoid alkaloids could also repress the growth of tumor cells (Ren et al., 2017). The antiproliferative effects of C₁₉ diterpene alkaloids and their derivatives on different tumor cell lines are summarized in Table 2. Among the tested cells, the HepG2 cells were highly sensitive to C₁₉ diterpenoid alkaloids as well as their derivatives. By analyzing the structure–activity relationship, it was found that the alkaloids with stronger cytotoxicity, such as aconitine, mesaconitine, hypaconitine, crassicauline A, oxonitine, and dexyaconitine, were associated with the acetoxy group (OAc) on the substituent of R6, suggesting that the positions and species of substituents on the mother nucleus were closely related to the antitumor activity of C₁₉ diterpenoid alkaloid.

C₂₀ diterpenoid alkaloid is another kind of alkaloid in Fuzi. Songorine, a natural C₂₀ diterpenoid alkaloid found in Fuzi, has been proved to be antineoplastic. Studies revealed that songorine suppressed the proliferation of HepG2 cells effectively and increased apoptosis at both early and late stages (Sun et al., 2018). Moreover, songorine manifested other biological functions such as analgesic, anti-arrhythmic, and anti-inflammatory activities (Khan et al., 2018). Figure 3 shows the structural formula of songorine and two types of mother nucleus of C₂₀ diterpenoid alkaloids in Fuzi. Based on the structure of natural C₂₀ diterpenoid alkaloids, researchers synthesized a series of derivatives and tested their antiproliferative activity on different tumor cell lines. The results revealed that most derivatives exhibited considerable cytotoxic effects (Table 3). Malignant glioma A172 cells and lung cancer A549 cells were highly sensitive to most of C₂₀ diterpenoid alkaloid derivatives. Of note, certain synthetic alkaloid derivatives displayed stronger anticancer activity than the natural ones. It was suggested that diterpenoid alkaloids demonstrated great potentiality in cancer treatment.

Apart from C₁₈, C₁₉, and C₂₀ diterpenoid alkaloids, other types of alkaloids in Fuzi such as higenamine and salsolinol also manifested antitumor biological function. Higenamine enhanced the anticancer effects of cucurbitacin B in breast cancer by suppressing the interaction of protein kinase B (AKT) and cyclin-dependent kinase 2 (CDK2) (Jin et al., 2018). In colorectal cancer-bearing nude mice, higenamine inhibited tumor volume by inducing apoptosis (Song et al., 2020). Salsolinol, a water-soluble alkaloid in Fuzi, induced apoptosis of human neuroblastoma SH-SY5Y cells. After incubation with salsolinol, the levels of Bcl-2 decreased, while protein Bax and the release of cytochrome C increased, which led to mitochondrial dysfunction and apoptosis (Wanpen et al., 2007). Figure 4 displays the structures of higenamine and salsolinol.

As we know, apoptosis is crucial to the development of the body and the stability of internal environment. When carcinogenic driving factors lead to excessive cell proliferation, it usually triggers apoptosis (Green and Evan, 2002). Apoptosis is also called programmed cell death. The process of apoptosis is actually a cascade amplification process of irreversible hydrolysis of substrate by cysteinyl aspartate specific proteinase (caspase) (McIlwain et al., 2015), which could be divided into two major categories (intrinsic pathway and extrinsic pathway) (Carneiro and El-Deiry, 2020). As a lysosome degradation pathway with the function of nutritional circulation and metabolic adaptation, autophagy is considered to be involved in the regulation of cancer (Amaravadi et al., 2019). In many cases, autophagy is closely related to apoptosis, and autophagy-related genes are also involved in the process of apoptosis (Maheswari et al., 2018). Inducing apoptosis and autophagy is one of the primary mechanisms responsible for the anticancer effect of Fuzi alkaloids and polysaccharides.

Proliferation, migration, and invasion are basic biological characteristics of malignancies. Uncontrolled proliferation, rapid migration, and excessive invasion of tumor cells are leading causes for poor prognosis and high recurrence. Accordingly, inhibition of these pathological processes would contribute to the anticancer efficacies of Fuzi. Studies have demonstrated that several processes and targets were involved in these pathological processes, including p38 MAPK, AKT, EMT, β3GnT8, and MMPs.

The immune escape of tumor refers to the phenomenon that tumor cells escape the recognition and attack of immune system through a variety of mechanisms, so as to survive and proliferate in the body. Some cytokines, such as interleukin-2 (IL-2), IL-6, and IL-12, are able to enhance immune response. In contrast, regulatory T cells (Tregs) could rapidly detect and inhibit IL-2 or other cytokines in the early stage of the immune response to suppress autoimmunity, which might hinder the anticancer immunity (Wing et al., 2019). Therefore, inhibiting the production of Tregs is one of the methods of anticancer immunotherapy. Fuzi aqueous extract has been proved to enhance the anticancer effect of radiotherapy in the treatment of lung cancer. The experimental data indicated that ionizing radiation affected the anticancer immune response of mice. However, after the administration of Fuzi aqueous extract, the levels of IL-2, IL-6, and IL-12 in mice serum increased, which might arouse the immune response. Moreover, Fuzi aqueous extract reduced the radiation-induced production of IL-10, TGF-β, and Tregs, revealing the mechanism of Fuzi in modulating immunity and inhibiting tumor growth (Zhang et al., 2017). Another research showed that, in gastric cancer-bearing mice, aconitine was involved in the intervention of Tregs by regulating the prostaglandin E2/cyclooxygenase-2 (PGE2/COX-2) pathway. It was noteworthy that high-dose aconitine reduced PGE2 and Tregs significantly, whose effect was more obvious than that of the celecoxib-positive group. These results suggested that aconitine exerted antitumor efficacy by reversing immune escape through regulating Tregs and mediating the PGE2/COX-2 pathway (Cheng, 2019).

Cancer immunotherapy aims to promote tumor-specific T-cell response. As antigen-presenting cells, dendritic cells (DC) allow antigens to be recognized by CD4⁺ T cells and CD8⁺ T cells, and then CD4⁺ T cells transmit information to CD8⁺ T cells and facilitate the differentiation of effector T cells to kill tumor cells (Borst et al., 2018). Aconitine possessed a direct anticancer activity, but it also had the drawback of damaging immunity to a certain extent (Qian, 2015). FPS increased the number of macrophages, CD4⁺, and CD8⁺ T cells in the spleen of Hepa1-6 tumor-bearing mice, indicating FPS had immune-improving functions. The results suggested that FPS combined with Fuzi alkaloids might achieve better anticancer efficacy (Qian, 2015). Treatment with FPS induced differentiation of peripheral blood monocytes to DC and expressed its mature phenotypes, which acted as the second signal to activate T lymphocytes and stimulated tumor immunity (Gao L. L. et al., 2012). The synergic effect of FPS and adriamycin was evaluated in H22 tumor-bearing mice. The killing activity of NK cells and lymphocyte transformation rate were dramatically improved; meanwhile the expression of IL-2 and IL-12 in splenic lymphocytes increased, indicating that FPS enhanced the anticancer effect of adriamycin by strengthening the immune system (Dong et al., 2003b; Dong et al., 2006). The anticancer immunomodulatory mechanism of Fuzi is shown in Figure 6.

The abnormal energy metabolism is one of the major characteristics of tumor cells (Hanahan and Weinberg, 2011). The main sources of biological energy depend on central carbon metabolism, including aerobic respiration and glycolysis. In the presence of adequate oxygen, pyruvate, transformed from glucose, is further oxidatively phosphorylated by mitochondrial respiration. However, under hypoxic conditions, pyruvate would be reduced to lactate. Tumor cells tend to obtain ATP through glycolysis even under aerobic conditions, this phenomenon is called Warburg effect (Warburg et al., 1927). PI3K/AKT/mTOR and HIF-1 pathways would influence glycolysis and participate in the energy metabolism of tumor cells. The PI3K/AKT/mTOR pathway could activate glucose transporters on cell membrane and the metabolic enzyme hexokinase 2 (HK2) in glycolysis, sequentially increase glucose uptake and glycolysis rate (Gottlob et al., 2001; Roberts et al., 2013; Abdel-Wahab et al., 2019). The HIF-1α could induce the activation of lactate dehydrogenase A (LDHA) and pyruvate dehydrogenase kinase 1 (PDK1) to catalyze the conversion of pyruvate to lactate, accordingly promote glycolysis to produce ATP in the anoxic tumor microenvironment (Valvona et al., 2016; Zhou et al., 2018; Nagao et al., 2019).

Mitochondria are the most important organelle of energy metabolism. Besides supplying energy for life, mitochondria also regulate cell death, control redox reactions, and provide substrates for anabolism (Porporato et al., 2018). Angiogenesis in the center of many solid tumors is poor, resulting in limited supply of glucose and oxygen. However, electron transport chain (ETC) in the mitochondria could function even under hypoxia, allowing the tumor cells to breathe and produce ATP (Vasan et al., 2020). Complex I, II, III, and IV, located on the mitochondrial inner membrane, are key components of ETC to transfer electrons. Complex I and II respectively oxidize the NADH and FADH₂ to NAD⁺ (nicotinamide adenine dinucleotide) and FAD⁺ (flavin adenine dinucleotide), which keep TCA cycle running. Complex I, II, III, and IV transfer electrons to oxygen, discharge protons into the gap of mitochondrial membrane, and eventually generate ATP by ATP synthase (Letts and Sazanov, 2017). Regulation of cellular metabolism might be a significant clue in developing antineoplastic agents (Stuart et al., 2014; Wheaton et al., 2014; Kuntz et al., 2017). Figure 7 shows glycolysis, TCA cycle, and relevant pathways involved in cellular energy metabolism.

Existing studies found that Fuzi alkaloids regulated cellular metabolism by affecting ETC in the mitochondria. Salsolinol blocked the energy supply of SH-SY5Y cells and induced cell death. It was noteworthy that adding d-glucose to enhance glycolysis could not prevent the cytotoxicity of salsolinol on SH-SY5Y cells. Further research demonstrated that salsolinol inhibited the activity of complex II (succinate-Q reductase) and caused a rapid depletion in intracellular ATP, which might account for the cytotoxic effects of salsolinol (Storch et al., 2000). Moreover, network pharmacology predicted that Fuzi alkaloids might exert antitumor effects by suppressing the PI3K/AKT/mTOR pathway, HIF-1 pathway, and central carbon metabolism in cancer (Lu et al., 2021) (Figure 7). The evidence proved that energy metabolism and its relevant signal pathways might be involved in the anticancer process of Fuzi alkaloids.

Multidrug resistance (MDR) is a principal reason of the therapeutic failure. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) located on the cell membrane control the absorption, distribution, and excretion of various chemicals. These transporters protect cancer cells from high doses of intracellular drugs and lead to the occurrence of MDR (Bukowski et al., 2020). Many inhibitors, such as verapamil and cyclosporine A, are designed to target P-gp. However, due to the low affinity of these inhibitors to P-gp, high concentrations are required and side effects would ensue (Höllt et al., 1992; List et al., 2001; Waghray and Zhang, 2018). Aconitine was shown to reverse the MDR of KB_V200 cells, accordingly the inhibitory rate by aconitine reached 56.15% at 50 μg/ml. The combined use of aconitine and vincristine achieved a better reversal effect of MDR, largely due to downregulating P-gp expression (Liu et al., 2004).

The anticancer active compounds of Fuzi and their mechanisms are summarized in Table 4.