Modulation of Multiple Signaling Pathways of the Plant-Derived Natural Products in Cancer

Natural compounds are highly effective anticancer chemotherapeutic agents, and the targets of plant-derived anticancer agents have been widely reported. In this review, we focus on the main signaling pathways of apoptosis, proliferation, invasion, and metastasis that are regulated by polyphenols, alkaloids, saponins, and polysaccharides. Alkaloids primarily affect apoptosis-related pathways, while polysaccharides primarily target pathways related to proliferation, invasion, and metastasis. Other compounds, such as flavonoids and saponins, affect all of these aspects. The association between compound structures and signaling pathways may play a critical role in drug discovery.


INTRODUCTION
In 2018, an estimated 9.6 million deaths were caused by cancer, and cancer is anticipated to be the leading cause of death worldwide in the twenty-first century (1). Therefore, cancer prevention remains an innovative area of anticancer research, in addition to cancer therapy. The mechanisms of aberrant signal transduction pathways in cancer and the impacts of these pathways on tumorigenesis, apoptosis, and metastasis have been increasingly revealed due to intensified study (2). Searching for targeted molecules that can regulate signal transduction has recently emerged as a globally popular research area in biomedicine.
Herbal medicines, such as Chinese medicines, are naturally exceptional at ameliorating many human diseases. Increasing numbers of new drugs with pharmacological activity have been discovered due to the modernization of herbal medicine. The anticancer agents vincristine, taxol, and vinblastine have been used for their anticancer effects in many countries (3). Moreover, other promising anticancer agents are available, including arteannuin (4), quercetin (5), and tetrandrine (6). Alkaloids and polyphenols are significantly dominant among cancer therapeutics (7,8). Recently, the targets and mechanisms of plant-derived anticancer agents have been widely reported (9). In this review, we will focus on advances in knowledge about the signaling pathways affected by plant-derived natural products. chemical structures into several classes such as flavonoids, xanthones, stilbenes, lignans, and curcuminoids ( Table 1) (11)(12)(13)(14). Many natural polyphenols have cytostatic and apoptotic properties because of their antioxidant characteristics (11). The anticancer effects of polyphenols depend not only on their chemical structure and concentration but also on the type of cancer. Lignans considered to be phytoestrogens are bioactive compounds exhibiting various anticancer properties, such as apoptosis induction and tumor growth reduction (15). Xanthones, such as α-mangostin, mediate cytotoxicity mainly via cell cycle arrest and reactive oxygen species (ROS)-induced apoptosis (16). The anticancer effects and molecular mechanisms of polyphenols are reported to be associated with their chemical constitution which is necessary for its anticancer activities, such as the C-3 prenylation of benzoxanthone-type prenylated flavonoids, C-1 hydroxy group and isoprenyl group at C-8 of prenylated xanthones, the C-2 carbonyl group, C-4 prenyl group and pyran ring connected at the C-2 and C-3 of caged xanthones (9). Anticarcinogenic activities of polyphenols include suppressing the proliferation, differentiation, metastasis, and angiogenesis of various kinds of cancer cells through inhibiting several kinases involved in signal transduction (17)(18)(19)(20). Polyphenols can bind and cross cell membranes easily and trigger various pathways involving microRNAs (miRNAs), caspases, B cell lymphoma 2 (Bcl-2) family proteins, nuclear factor (NF)-κB, epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR), phosphatidylinositol-3-kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK) ( Table 2).

NF-κB Pathways
NF-κB can regulate the transcription of genes associated with the inflammatory response, cell death, and proliferation (90,91). NF-κB pathways participating in the development of various cancers can be disrupted by polyphenols. The PI3K/Akt signaling pathway and MAPK signaling pathways are related to the activation of NF-κB in numerous tumor cell lines (92).
The flavonoid component chrysin (5,7-dihydroxyflavone, Figure 1) has been shown to suppress the growth of colon cancer cells via direct inhibition of NF-κB expression and activity, according to computational docking experiments (24). In addition, 30 µM chrysin activates NF-κB/p65 by inducing p38 MAPK signaling pathways in HeLa cells (33). Quercetin (Figure 1) has a potential role in inhibiting processes in human oral cancer cells through the NF-κB pathway (93). The results of Western blot and flow cytometric assays indicate that the flavonoid fisetin (3,3 ′ ,4 ′ ,7-tetrahydroxyflavone, Figure 1) effectively suppresses the apoptosis, metastasis, angiogenesis and invasion of cancer cells via ERK1/2-, Akt/NF-κB/mTORand p38 MAPK-dependent NF-κB signaling pathways (94,95). Furthermore, fisetin is not cytotoxic to normal cells (94). Genistein has a potential role in inhibiting cell division and apoptosis via Akt and NF-κB (28). Wogonin (Figure 1), extracted from Scutellaria baicalensis Georgi, can decrease the phosphorylation levels of IκB and p65. Modulation of the NF-κB/Bcl-2 signaling pathway has been shown by Western blot analysis to play a critical role in both of the invasion and proliferation of hepatocellular carcinoma (HCC) in a dosedependent manner (96). Wogonin is shown to decrease the protein and mRNA levels of cyclooxygenase (COX)-2 in skin fibroblast NIH/3T3 cells and in animal experiments (97). The stilbene pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene, Figure 2), the dimethylated analog of resveratrol, is a highly bioactive natural polyphenolic compound that is mainly found in grapes, blueberries, tomatoes, and other berries (98). According to the results of COX-2 activity assays and enzymatic immunoassays, both resveratrol ( Figure 2) and pterostilbene cause COX-2 inactivation via the NF-κB signaling pathway (31, 99).
Matrix Metalloproteinase (MMP)-2 and MMP-9 The MMPs are a group of metal-dependent proteolytic enzymes that are involved in matrix remodeling and facilitate the migration of cancer cells through degradation of the extracellular matrix (100). MMP-2 and MMP-9 can degrade type IV collagen in the basement membrane and facilitate tumor cell metastasis (101).  104,105). MMP-2 expression is downregulated in human prostate cancer cells by genistein treatment (28). In addition, treatment with 5 µM quercetin and chrysin decreases the expression of MMP-9 in A549 cells (106). Still other polyphenols affect both the activity and expression of MMPs. For example, naringin (4' ,5,7trihydroxyflavanone 7-rhamnoglucoside, Figure 1) can inhibit the adhesion and invasion of human glioblastoma U87 cells and  U251 cells via dose-dependent reductions in both the activity and expression of MMP-2 and MMP-9, according to zymograohy and Western blotting results, this effect is associated with the p38 MAPK signaling pathway (107,108). EGCG (20 µM) reduce the activity of MMP-2 and MMP-9 in prostate cancer cells (109) and decrease the expression of MMP-9 in bladder cancer cells (110).

Caspases
Caspases, which are activated by other caspases, are cysteinyl aspartate-specific proteases and are divided into two groups. One group comprises initiators (caspase-8, -9, and -10); the others, executioners (caspase-3, -6, and -7). Caspase -3 is considered the major downstream target of caspase-4, -8, and -9. Overexpression of caspases is a common alteration in cancer cells that can be exploited therapeutically. Activation of caspase-3 by fisetin treatment associated with induction of the proapoptotic proteins Bad, Bax, Bim, and inhibition of the antiapoptotic proteins Bcl-2 and Mcl-1(L) (35). Genistein has also been shown to increase the expression of caspase-3,-9 and Bax in vitro (28). Chrysin-induced apoptosis was associated with induction of caspase-3 and-8 and downregulation of phospholipase C-gamma-1 (PLC-gamma1) and XIAP. This finding suggests that the mechanism of apoptosis induced by chrysin is associated with Akt dephosphorylation in the PI3K signaling pathway (33). EGCG can induce apoptosis and reduce cancer cell proliferation by decreasing the mitochondrial membrane potential ( m) and stimulating caspase-3, -9 and c-Jun N-terminal kinase 1 (JNK1) expression in human glioblastoma T98G and U87MG cells but does not induce apoptosis in human normal astrocytes (111). The flavonoid baicalein (Figure 1), found in Scutellaria baicalensis Georgi, participates in apoptosis by increasing the expression of caspase-3 and -8 (112). The lignan phillygenin (Figure 3) induces apoptosis by increasing the mitochondrial membrane potential due to increased ROS levels in human esophageal cancer SH-1-V1 cells. Concurrent upregulation of Bax and cleaved caspase-3 and -9, along with dose-dependent downregulation of Bcl-2, was found by propidium iodide staining and Western blotting (15). The anticancer effects of arctigenin (Figure 3), the active component of Arctium lappa, are mainly directed toward cancer   cell growth inhibition and apoptosis through the peroxisome proliferation-activated receptor α (PPARα)/gankyrin, Bax and caspase pathways (36). The xanthone α-mangostin (Figure 4) increases the activity of caspase-3 and causes late apoptosis in ovarian adenocarcinoma SKOV3 cells after 12 h and 72 h of treatment, respectively (113).

ALKALOIDS
Alkaloids are the secondary biologically active components found in many plants. Alkaloids have various biological activities that render them important sources for drug discovery. The presence of nitrogen in their molecular architecture is critical to the biological activity of this class of compounds. Many studies have shown that alkaloids inhibit the growth of human breast, liver, colon, prostate, and liver cancer cells (114).

Bcl-2 Protein Family
Bcl-2 proteins are divided into two groups. Bcl-2 and Bcl-xL are antiapoptotic proteins, while Bax and Bad are multidomain proapoptotic proteins. The balance of antiapoptotic proteins to proapoptotic proteins, for example, the ratio of Bax to Bcl-2 is crucial to the regulation of apoptotic pathways (115). The balance between Bcl-2 family proteins is a potential target of alkaloids for inducing cell death (116). Oxymatrine (Figure 5), derived from Sophora flavescens Aiton, significantly increases p53 and Bax expression and decreases Bcl-2 expression dose-dependently, as evidenced by A Western blot assay, in osteosarcoma cancer cells via dephosphorylation of PI3K and Akt in the PI3K/Akt signaling pathway (117).
Treatment with crude alkaloid extractof Rhazya stricta (CAERS) induced apoptosis and suppressed the proliferation of HCT116 cells. Downregulation of Bcl-2, survivin, Bcl-X and XIAP expression and upregulation of Bad and Noxa expression were examined by qRT-PCR and Western blot analyses and coincided with the increase in the Bax/Bcl-2 ratio (118).
Various alkaloids induce apoptosis via an increase in the Bax/Bcl-2 ratio. Cancer cells treated with nitidine chloride (NC, Figure 5

PI3K/Akt/mTOR Signaling Pathway
Autophagy is a critical process for maintaining intracellular homeostasis. Generally, autophagy may play a critical role in cancer prevention (124). The PI3K/Akt/mTOR pathway is critical for autophagy induction and is a latent target in cancer therapeutics and control (101).

ERK Signaling Pathway
The MAPK/ERK pathway participates in multiple processes in cancer including growth, invasion, metastasis, angiogenesis, and inhibition of apoptosis (131,132). Because of these multiaspect effects, the MAPK/ERK pathway plays a critical role in the promotion of cancer cell growth and the inhibition of apoptosis (133,134).
β-carboline alkaloids extracted from the seeds of Peganum harmala inhibit the proliferation and induce the apoptosis of SGC-7901 cells, possibly because β-carboline alkaloids can disrupt the balance between PTEN and ERK, inhibit the MAPK/ERK signaling pathway and induce apoptosis in cancer cells (135). Berberine can suppress the senescence of human glioblastoma cells by inhibiting the EGFR/Raf/MEK/ERK pathway (136). Sinomenine, extracted from Sinomenium acutum, is reported to inhibit various types of cancer cells. Sinomenine hydrochloride (SH) increases the phosphorylation of ERK1/2, p38 and JNK but does not affect the total levels of the abovementioned cytokines (137). The benzo phenanthridine alkaloid chelerythrine chloride (CC, Figure 5) (5 and 10 µM) significantly enhances ERK1/2 phosphorylation and dose-dependently decreases Akt phosphorylation, as detected by Western blot analysis (138).
The other anticancer targets of alkaloids are summarized in Table 3.

SAPONINS
Saponins are valuable sources with minimal toxic effects and are found in many dietary plants. Saponins are composed of a triterpenoid or steroidal aglycone attached to one or more sugar chains (145). Saponins are divided into two types: triterpenoid saponins and steroidal saponins. Both types have various biological activities, such as anticancer and immunological adjuvant activities (146).
The steroidal saponin of Paris polyphylla (Chinese name: Chonglou) has long been used for lung cancer treatment (148). Paris saponin I (PSI, Figure 6) and Paris polyphylla steroidal saponins (PPSS) regulate the Bcl-2 family and caspase-3 and -8, inducing apoptosis (149). In addition, PSI and PPSS induce autophagy by the conversion of LC3 I to LC3 II and upregulation of Beclin 1 (150). Paris saponin VII (PS VII, Figure 6), extracted from Trillium tschonoskii Maxim, inhibits the migration and invasion of several types of cancer cells via the downregulation of MMP-2 and -9 expression and p38 MAPK phosphorylation in a dose-and time-dependent manner (151).

POLYSACCHARIDES
Polysaccharides which are abundant in plants, possess anticancer activities, and are being used as immunopotentiators for cancer patients, thus they are relatively ideal anticancer agents (160).
The purified polysaccharide extracted from Caulerpa lentillifera, SP1, composed mainly of sulfated xylogalatan and galactose, showed potent immunostimulatory effects by activating macrophage cells through both the NF-κB and p38 MAPK signaling pathways (168). SP1 decreased the levels of IκBα and the NF-κB p65 subunit and increased p38 MAPK phosphorylation, as determined by Western blot assay.
Polysaccharides extracted from Phellinus linteus (PL) significantly inhibit cell proliferation by decreasing β-catenin and cyclin D1 expression in vitro. In addition, PL inhibits invasion and motility by directly reducing the activity of MMP-2 and -9, with no effect on the gene expression or secretion of MMPs, as indicated by RT-PCR and gelatin zymography (169).
The Radix astragali active extract Astragalus polysaccharide (APS) can enhance the immune response by promoting IL-2, IL-6, and TNF-α in H22 tumor-bearing mice. The effects on the immune response are involved in the inhibition of cancer. In addition to the immune response, the anticancer mechanism involves apoptosis, cell cycle arrest, Akt phosphorylation, Bcl-2 and Bax, caspase-3 and -9, p53 and PTEN (163,170).
The polysaccharides obtained from enzymatic digestion by Celluclast enzyme digest (CCP) suppresses the activation of NF-κB p50 and p65 and the phosphorylation of p38 MAPK in macrophages (171).

CONCLUSION
Natural compounds offer a great diversity of chemical structures that are likely important in cancer therapeutics (18). Many studies have shown that phytochemicals influence targets and signaling pathways involved in oncogenesis and tumor progression such as proliferation, invasion, metastasis and angiogenesis (173). Different components have various anticancer activities. (1) Alkaloids, with low bioavailability and poor water solubility, have difficulty to reaching the intended target. Moreover, the toxicity of alkaloids cannot be ignored, primarily target apoptosis-related pathways (174). (2) Flavonoids can affect the development of colon, lung, esophageal, stomach and endometrial cancer, with minimal acute toxic effects because of their poor water solubility accompanied by their rapid digestion (17,175). Polyphenols primarily target pathways related to proliferation, apoptosis, invasion and metastasis. (3) Polysaccharides and saponins effectively modulate the immune response rather than directly inducing cell death. Polysaccharides primarily affect apoptosis-related pathways, while saponins affect apoptosis-related and invasion-and metastasis-related pathways (176). The anticancer effects of these compounds are associated with multiple targets (Figure 8) (176). Signaling pathways are believed to be associated with specific chemical structures, and this association is critical for continuing drug development.