The process of the cell cycle is divided into interphase (G1 phase, S phase, G2 phase), division phase (M phase), and stationary phase (G0 phase), and the normal progression of the cell cycle is regulated by various mechanisms of action to ensure orderly cell division (32); if any of the related factors are dysregulated, the cell cycle is terminated (32,33). The central cell cycle regulation mechanism is the Cyclin-CDK-CDI mechanism, and CDK inhibitor (CKI) genes have positive regulators such as cyclin and p16 and negative regulators such as p53 and p21 (34,35). The overexpression of positive regulators or the absence of negative regulators results in the downregulation of the cell cycle threshold and weakening of cell cycle function, resulting in reduced sensitivity of cells to exogenous regulatory signals (32,35). CDC genes (cell division cycle genes) are a class of genes whose expression is cell cycle-dependent or directly involved in cell cycle regulation; these genes mainly include Cyclin genes, CDK genes and CKI genes (36,37). In addition, DNA polymerase and DNA ligase genes related to DNA replication are also part of the CDC gene family (38).

Several studies have reported that in some tumour cells, DHM can inhibit cell proliferation by blocking the G2/M phase and G1/S phase of the cell cycle (29,39,40). For example, DHM can inactivate the CDK1/cell cycle protein B1 complex by phosphorylating CDK1, thus inducing G2/M phase cell cycle arrest and inhibiting the growth of hepatocellular carcinoma cells (29). In SK-MEL-28 melanoma cells, DHM downregulates the expression of the CDC25A, CDC2, and phosphorylated (p-)CDC2 proteins to induce cell cycle arrest in the G1/S phase (39). DHM also induces G2-M arrest of the U2OS cell cycle in osteosarcoma cells by increasing the levels of p21 protein and RNA and inhibiting cell proliferation (40).

In addition to inhibiting the proliferation of tumour cells by blocking the cell cycle, DHM can also target the activation of ERK1/2 and Akt to inhibit the proliferative potential of fibroblasts in lung cancer cells (41) and upregulate the expression of p53 to inhibit the proliferation of hepatocellular carcinoma cells (42). In animal studies using an in situ prostate tumour model, Ni et al (43) reported that DHM inhibited the proliferation of PC-3 tumours in a dose-dependent manner, which was associated with a decrease in the CXCR4 protein. In BGC-823 gastric cancer cells, DHM induced a decrease in the expression of Cyclin D1, Cyclin E1 and N-cadherin; increased the expression of E-cadherin; inhibited the phosphorylation of Akt and STAT3; and downregulated the expression of HMGB1 in cells. Therefore, DHM inhibits BGC-823 cell proliferation and migration by regulating the activation of the Akt/STAT3 signalling pathway and the expression of HMGB1(44). In cholangiocarcinoma (CAA) cells, DHM upregulated the expression of miR-455-3p, which in turn inhibited the expression of its downstream target ZEB1, thereby suppressing the proliferation of CAA cells (45). ZEB1 is a transcription factor that promotes metastasis and stem cell characteristics, and it is aberrantly expressed in CAA cells (46) (Fig. 1).

The apoptotic pathway includes both exogenous and endogenous pathways, the former being mediated by receptors such as TNF-α and FAS-L, while the latter uses mitochondria as the main site of apoptosis induction by regulating the permeability of their outer membrane (47). When various stress responses, DNA damage and abnormal cell signalling occur, Bax is activated, and under the regulation of this protein, the permeability of the mitochondrial outer membrane increases, and Cytochrome C is released into the cytoplasm, where it binds to Apaf-1 in the presence of dATP, causing Apaf-1 to expose its Card structural domain and bind to the Card of Procaspase-9 to form an apoptotic complex; this complex in turn activates Caspase-9, which further activates Caspase-3, thus causing apoptosis (48-50). By contrast, Bcl-2 plays the opposite role as Bax in inhibiting Cytochrome C release, thus inhibiting the process of apoptosis; therefore, regulation of the expression of Bcl-2 has an important impact on the apoptosis of tumour cells (51). Because of the inverse effects of Bcl-2 and Bax, when cells are stimulated, the ratio of the two determines cell survival (52). Liu et al (42) discovered that DHM significantly increases p53 protein expression in four types of hepatocellular carcinoma cells (HepG2, QGY7701, Hepal-6, and MHcc97L), which activated Bax and Bak, and inhibited Bcl-2 expression, which in turn activated Caspase-3 and eventually led to the apoptosis of tumour cells; the regulatory effect was dose-dependent. Ji et al (53) revealed that DHM significantly (P<0.05) inhibited AGS cell proliferation and induced cell cytotoxicity in a dose- and time-dependent manner; DHM also regulated the expression of apoptosis-related genes such as p53 and Bcl-2 in a dose- and time-dependent manner in AGS cells treated with DHM, as determined by western blotting (53).

In addition to inducing the apoptosis in tumour cells through the p53-mediated signalling pathway, DHM is associated with the Nuclear Factor Kappa B Subunit 1(NF-κB) signalling pathway (22,23,54). Han et al (54) reported that in the leukemic cell lines HL60 and K562, DHM induced apoptosis through nuclear condensation, induced loss of mitochondrial membrane potential, increased ROS production, activated Caspase-9, Caspase-3 and poly ADP-ribose polymerase, and regulated the expression of Bcl-2 family members; these authors also reported that DHM induced apoptosis in the leukaemia cell lines HL60 and K562, possibly through the NF-κB signalling pathway. Li et al (22) examined the expression of phosphorylated nuclear factor kappa B kinase subunit β (p-IKKβ), phosphorylated nuclear factor kappa B kinase subunit α (p-IKKα), nuclear factor kappa Bα (IκB-α) inhibitor, and NF-κB/p65 in a nasopharyngeal carcinoma CNE-2 cell line via western blot analysis and confocal laser scanning microscopy; the observed nuclear translocation of NF-κB/p65 indicated that DHM promotes the inactivation of p-IKKβ and p-IKKα and blocks the nuclear translocation of the NF-κB subunit p65, promoting the apoptosis of CNE-2 cells in nasopharyngeal carcinoma. Guo et al (23) treated a rat model of fatty liver disease with DHM and found that DHM inhibited the protein expression of NF-κB, p53 and Bax, acting as hepatoprotective agent.

In addition, DHM promotes the apoptosis of tumour cells through several other pathways (55-59). For example, DHM inhibits the activation of Akt, which in turn inhibits the formation of the mTOR complex and promotes apoptosis in breast cancer cells (55). DHM is a biologically active natural chemopreventive agent and a potent mTOR inhibitor that may be a useful chemotherapeutic agent for breast cancer treatment (55). Lu et al (56) revealed that Notch1 is involved in the development of HCC and that DHM inhibits cell proliferation and promotes apoptosis by downregulating Notch1 expression. A study by Ye et al (57) demonstrated that in cisplatin-resistant nasopharyngeal carcinoma cell lines (Hone1/Cis and CNE1/Cis), cotreatment with DHM increased the growth-inhibitory effect of cisplatin by blocking the Wnt/β-catenin signalling pathway to increase the antitumour activity of cisplatin in nasopharyngeal carcinoma. In a mouse model of cerebral ischaemia-reperfusion injury, DHM inhibited oxidative stress and apoptosis in mouse hippocampal neuronal HT22 cells by activating the Nrf2/HO-1 signalling pathway (58). Treatment of colon cancer cells with DHM resulted in dose- and time-dependent apoptosis through the activation of endoplasmic reticulum (ER) stress, 5'-adenosine monophosphate-activated protein kinase (AMPK) and JNK/p38 MAPKs, and AMPK/MAPK/XAF1 signalling initiated by DHM through the ER stress pathway, which induces apoptosis in colon cancer cells (59) (Fig. 2).

The invasion-migration cascade is a complex biological process that includes the following major events: (i) Cell migration and local invasion of the basement membrane, (ii) invasion of the vasculature and/or lymphatic system, (iii) survival in the circulation, (iv) arrest and extravasation at distant organ sites, and (v) colonization at metastatic sites (60,61). According to previous case reports, >80% of cancer patients succumb to tumour invasion/metastasis, making it one of the major causes of death in cancer patients. The decrease in 5-year survival upon metastasis is particularly severe in patients with osteosarcoma (62). DHM can suppress the invasion and metastasis of osteosarcoma cells by blocking the TNF-α/p38MAPK/MMP-2 signalling pathway (30). Moreover, Chou et al (63) reported that DHM regulates osteosarcoma metastasis through the ERK pathway; it also inhibits metastasis by suppressing the expression of the downstream urokinase plasminogen activator through the inhibition of SP-1 and NF-κB.

DHM inhibits the invasion and migration of human retinal pigment epithelial cells (ARPE-19) by decreasing the expression of MMP-2(64). By contrast, Zhang et al (65) reported that DHM significantly inhibited the migration and invasion of SK-Hep-1 and MHCC97L hepatocellular carcinoma cells by decreasing MMP-9 protein expression, while downregulation of MMP-9 protein expression was closely associated with increased PKC-δ protein levels and decreased phosphorylation of p38, ERK1/2 and JNK in SK-Hep-1 and MHCC97L cells. In the ovarian cancer cell line A2780, DHM upregulated E-cadherin and downregulated N-cadherin and Vimentin in the Snail signalling pathway in a concentration- and time-dependent manner, inhibiting the nuclear translocation of NF-κB; these results suggested that DHM inhibits epithelial-mesenchymal transition (EMT) via the NF-κB/Snail pathway and suppresses ovarian cancer cell invasion (66). DHM also was also demonstrated to inhibit the migration and invasion of human gastric cancer MKN45 cells and reverse EMT through the downregulation of MMP-2 expression via the JNK signalling pathway (67). In an in vivo experiment on mice transplanted with B16 melanoma cells, Zheng et al (68) reported that mice given DHM at doses of 150, 200 and 250 mg/kg exhibited a significant reduction in the number of metastatic tumours compared with those in the control group, demonstrating the anti-invasive and anti-metastatic effects of DHM on B16 melanoma. Moreover, DHM inhibits the migration and invasion of nasopharyngeal carcinoma cells by suppressing the ERK1/2 signalling pathway and suppressing MMP-2 expression (69); it also inhibits the proliferation, migration and invasion of CAA HCCC9810 and TFK-1 cells by regulating miR-21 and promoting apoptosis (70) (Fig. 3).

ROS are highly reactive substances containing oxygen radicals, and ROS are produced in various biochemical reactions in cellular organelles, such as the ER, mitochondria and peroxisomes, as a by-product of normal oxygen metabolism (71). ROS are cytotoxic molecules that stimulate apoptosis, but high levels of ROS can induce tumorigenesis, leading to uncontrolled cancer cell proliferation (72). Autophagy is an evolutionarily conserved catabolic mechanism by which eukaryotic cells recycle or degrade internal components through a membrane transport pathway (73). The process of autophagy is divided into four key steps: Initiation, nucleation, maturation and degradation (74). In cancer, autophagy plays a dichotomous role, that is, it inhibits tumorigenesis and supports tumour development (75).

DHM induces autophagy in head and neck squamous cell carcinoma (HNSCC) through the phosphorylation and activation of the STAT3 transcription factor (31). In HNSCC cells, the increase in ROS levels was proportional to the increase in DHM concentration, and DHM activated p-STAT3-dependent autophagy through ROS production in HNSCC (31). Zhou et al (76) demonstrated that in the human breast cancer cell lines MCF-7 and MDA-MB-231, DHM had antitumour effects through ROS production, and the ER stress pathway had antitumour effects on breast cancer cells. However, in human melanoma cells, autophagy has a protective role in DHM-induced apoptosis, and pharmacological inhibition or genetic blockade of autophagy increases DHM-induced cell death and apoptosis (77). Liu et al (78) evaluated the effects of DHM on the induction of ROS accumulation and activation of mitochondrial signalling pathways in human hepatocellular carcinoma HepG2 cells. DHM reduced the accumulation of ROS in a concentration-dependent manner, while the expression of proteins involved in the apoptotic program increased in a concentration-dependent manner. This suggests that ROS can act as redox signalling messengers that regulate DHM-induced apoptosis (78). In a pharmacobiochemical study of the effect of DHM on inflammation in a RAW264.7 macrophage model, DHM was discovered to inhibit the accumulation of ROS, suppress the release of NO and the proinflammatory cytokines IL-1β, IL-6 and TNF-α, and suppress lipopolysaccharide-induced inducible nitric oxide synthase by inhibiting nuclear factor-κB (NF-κB) activation (79). In cutaneous squamous cell carcinoma (CSCC), DHM induced TFEB (Ser142) dephosphorylation, activated TFEB nuclear translocation, increased TFEB reporter activity, decreased lncRNA MALAT1 expression, and induced CSCC cell death by inducing excessive autophagy via the MALAT1-TFEB pathway (80) (Fig. 4).