Histone Modification of Colorectal Cancer by Natural Products

Natural products play important roles in the pathogenesis of many human malignancies, including colorectal cancer, and can act as a gene regulator in many cancers. They regulate malignant cell growth through many cellular signal pathways, including Rac family small GTPase 1 (RAC1)/PI3K/AKT (α-serine/threonine-protein kinase), mitogen-activated protein kinase (MAPK), Wnt/β-catenin pathway, transforming growth factor-β (TGF-β), Janus kinase and signal transducer and activator of transcription (JAK-STAT), nuclear factor kappa-B (NF-κB), the Notch pathway, Hippo pathway, and Hedgehog pathway. In this review, we describe the epigenetic roles of several natural products, e.g., platycodin D (PD), ginsenoside Rd, tretinoin, Rutin, curcumin, clove extract, betulinic acid, resveratrol, and curcumin, in colorectal cancer, including their impact on colorectal cancer cell proliferation, apoptosis, invasion, migration, and anti-chemotherapeutic resistance. The aim is to illustrate the epigenetic mechanisms of action of natural products in cancer prevention and treatment, and to provide (1) a theoretical basis for the study of the role of epigenetics in influencing colorectal cancer; (2) new directions for studying the occurrence, development, and prognosis of colorectal cancer; and (3) new targets for treating and preventing colorectal cancer.


Introduction
Colorectal cancer (CRC) is one of the most common malignancies of the gastrointestinal tract, and its incidence is increasing annually [1]. According to cancer statistics from China and the United States for 2022, the age-standardized incidence and mortality rates of lung and CRC in the United States have decreased significantly, However, the incidence and burden of CRC in the global population are increasing. CRC is characterized by insidious onset, inconspicuous early clinical manifestations, difficult diagnosis, and extremely poor prognosis, and most patients with CRC are already in the middle to late stages or have metastatic symptoms at diagnosis. While surgical resection with radiotherapy is currently the main treatment method for CRC, it has no significant therapeutic effect.
CRC has a very complex development mechanism, and the traditional view is that the main mechanism of the tumor is uncontrolled cell growth and differentiation due to DNA sequence variation caused by oncogenic factors. As research has progressed in recent years, it has become apparent that abnormalities in regulatory mechanisms other than DNA sequence are more common in the tumor development process, leading to increasing research on epigenetics. Epigenetics differs from genetics in that heritable changes in gene function can occur, causing changes in phenotype but not in DNA sequence. Changes in epigenetic regulation are inherited along with the replicated DNA in the daughter cells. Epigenetic regulation plays a key role in the diagnosis and treatment of CRC. With the intensive study of human epigenetics, several epigenetic phenomena have been identified; the regulatory mechanisms include DNA methylation, histone modification, non-coding of wildtype p53 protein inhibits abnormal cell proliferation by blocking the cellular G1/G0 phase and preventing the cell from entering the S-phase [7][8][9]. At the same time, the p53 protein can induce programmed cell death. It was found that by transfecting the p53 gene into tumor cell lines cultured in vitro, overexpression of the p53 protein caused tumor cell apoptosis [10,11].
Abnormal epigenetic alterations were first identified in CRC in 1982. Research has subsequently revealed an "epigenetic landscape" comprising a complex set of epigenetic regulatory mechanisms controlling gene expression in normal and cancerous tissues [12]. As research has progressed, it has become evident that there are multiple epigenetic patterns of aberrant expression in CRC, that different epigenetic modifications have various roles in CRC, and that epigenetic modifications are often involved in transcriptional regulation [13]. Epigenetic alterations are a series of heritable changes in gene expression without changes in DNA sequence. They can be divided into several overlapping mechanisms, including DNA methylation, histone modifications, microRNA dysregulation, chromosomal instability, and microsatellite instability.
Epigenetic alterations come in two forms. One involves epigenetic factors directly affecting epigenetic enzymes, changing their bioavailability in the cell. The other involves epigenetic factors interfering with biochemical pathways, changing the availability of the metabolites required to create the epigenetic label. Both forms may result in abnormal or inadequate alteration of epigenetic tag recruitment to non-specific promoters in a stochastic manner, ultimately producing epigenetic aberrations [14]. Among others, epigenetic factors indirectly affect the epigenome by first interfering with cellular signal pathways, including PI3K/AKT, MAPK, Wnt/β-catenin, TGF-β, JAK-STAT, NF-κB, Notch, and Hedgehog, which can lead to altered expression of growth factors, receptors, and ion channels, resulting in non-homeostatic cellular processes. This process may in turn lead to alterations in the state of the transcriptional machinery and its bioavailability in the cell, which has implications for CRC development and progression. Among others, the Wnt/β-catenin signal pathway is considered the initial CRC marker (i.e., high expression of Wnt-related genes). The Wnt signaling pathway includes the adenomatous polyposis coli (APC) protein and the low-density lipoprotein receptor-related protein 5 (LRP5). Among them, β-catenin is a key component of the Wnt signal pathway, which plays an important role in maintaining intercellular adhesion and the morphological structure of adjacent tissues [15]. Histone modifications can mediate the transcription and translation of proteins associated with the Wnt signaling pathway by acetylating N-methylated histone tails, thereby inhibiting CRC cellular replication.
As a form of epigenetic regulation, histone modifications have been studied in treating and preventing CRC, mainly by modifying chromatin structure to regulate gene expression in CRC. Histones are highly conserved proteins comprising five core protein types (H1, H3, H2A, H2B, and H4) [16] that are usually covalently modified at their N-terminal tail, exposing the surface of the nucleosome to exert a regulatory effect on gene expression. Because different histones (H3 and H4) can undergo different types of modifications at different amino acids (7Lys and 2Ser at the end of H3; 5Lys and 1Ser at the end of H4), including acetylation, methylation, phosphorylation, ubiquitination, glycosylation, poly-ADP-ribosylation, carbonylation, and biotin(acyl)ation of lysine residues, they are all basic elements of histone modifications [17]. Histone modifications are catalyzed by enzymes such as histone methyltransferases (HMTs), demethylases (HDMs), acetyltransferases (HATs), and HDACs. They can act on related target proteins and genes downstream of histone-modifying enzymes to influence the activation and silencing of signal pathways, thereby exerting epistatic regulation.

Histone Methylation Regulates CRC
Histone methylation is a post-translational modification of protein side chain amino acids to acquire different numbers of methyl groups as methyl donors using S-adenosylmethionine catalyzed by various methylation enzymes [16]. It is considered a marker of chromatin activity. The main methylation modification sites are the lysine and arginine residues of histones H3 and H4, as shown in Figure 1. A single lysine or arginine can be modified with up to three methyl groups. Depending on the amino acid residues modified, HMTs can be divided into arginine and lysine methyltransferases. Histone arginine methylation is a common post-translational modification, and abnormal histone arginine methylation is closely associated with carcinogenesis and metastasis [18]. HMTs and HDMs mainly catalyze histone methylation. There are >50 methylesterases with HMT activity and >24 HDMs. Histone methylation is a post-translational modification of protein side chain amino acids to acquire different numbers of methyl groups as methyl donors using S-adenosylmethionine catalyzed by various methylation enzymes [16]. It is considered a marker of chromatin activity. The main methylation modification sites are the lysine and arginine residues of histones H3 and H4, as shown in Figure 1. A single lysine or arginine can be modified with up to three methyl groups. Depending on the amino acid residues modified, HMTs can be divided into arginine and lysine methyltransferases. Histone arginine methylation is a common post-translational modification, and abnormal histone arginine methylation is closely associated with carcinogenesis and metastasis [18]. HMTs and HDMs mainly catalyze histone methylation. There are >50 methylesterases with HMT activity and >24 HDMs. Histone and DNA methylation can act together to induce tumors by silencing oncogenes. A frequently mutated gene in CRC is the tumor suppressor gene p53, known as the guardian of the genome [19]. Various stress signals can activate p53, such as DNA damage or oncogene activation. When activated, it mediates various cellular responses, including DNA repair, cell cycle arrest, senescence, apoptosis, ferroptosis, stem cell reprogramming, invasive metastasis, autophagy, metabolism, and cell death [20,21]. P53 mainly acts as a transcription factor, controlling the expression of hundreds of target genes [22]. Methylation modification of histones tightens chromatin and inhibits gene expression, while HDMs antagonize this process. The histone lysine methyltransferases SET domain containing 9, histone lysine methyltransferase (Set9), SET and MYND domain containing 2 (SMYD2), and SET domain containing 8/histone lysine methyltransferase (Set8) have been found to methylate the p53 protein and regulate its function. Its methylation sites are K372, K370, and K382, respectively [23][24][25]. Histone lysine-specific demethylase 1 (LSD1) was found to inhibit p53 function by specifically demethylating its K370 site, leaving CRC cells uninhibited and able to continue proliferating and differentiating [26].
Histone methylation was considered irreversible until LSD1 was discovered in 2004, which confirmed that histone methylation was a reversible genetic marker [27]. Studies have shown that LSD1 has an important role in the progression of human cancers, and research on LSD1 inhibitors has emerged as a new target for therapy [28,29]. HMTs can Histone and DNA methylation can act together to induce tumors by silencing oncogenes. A frequently mutated gene in CRC is the tumor suppressor gene p53, known as the guardian of the genome [19]. Various stress signals can activate p53, such as DNA damage or oncogene activation. When activated, it mediates various cellular responses, including DNA repair, cell cycle arrest, senescence, apoptosis, ferroptosis, stem cell reprogramming, invasive metastasis, autophagy, metabolism, and cell death [20,21]. P53 mainly acts as a transcription factor, controlling the expression of hundreds of target genes [22]. Methylation modification of histones tightens chromatin and inhibits gene expression, while HDMs antagonize this process. The histone lysine methyltransferases SET domain containing 9, histone lysine methyltransferase (Set9), SET and MYND domain containing 2 (SMYD2), and SET domain containing 8/histone lysine methyltransferase (Set8) have been found to methylate the p53 protein and regulate its function. Its methylation sites are K372, K370, and K382, respectively [23][24][25]. Histone lysine-specific demethylase 1 (LSD1) was found to inhibit p53 function by specifically demethylating its K370 site, leaving CRC cells uninhibited and able to continue proliferating and differentiating [26].
Histone methylation was considered irreversible until LSD1 was discovered in 2004, which confirmed that histone methylation was a reversible genetic marker [27]. Studies have shown that LSD1 has an important role in the progression of human cancers, and research on LSD1 inhibitors has emerged as a new target for therapy [28,29]. HMTs can catalyze the methylation of the promoter region of the E-cadherin (ECAD) gene at histone H3 lysine 27 (H3K27) or lysine 9 (H3K9) [30], promoting the epithelial-mesenchymal transition (EMT) process in tumor cells. Arginine methylation transferases (PRMTs) can affect the expression of cancer-related genes by methylating histone arginine residues. Curcumin can reduce the mRNA and protein levels of PRMT5 to inhibit cancer cell proliferation. HMT enhancer of zeste-2polycomb repressive complex 2 subunit (EZH2) was found to promote tumor proliferation and transformation when overexpressed in various tumors. The polycomb repressive complex (PRC) repressed the expression of oncogenes by regulating the distribution of H3K27me3 across the genome. Cadherin transcription promotes the EMT process in CRC cells [31].
HDM lysine demethylase 4D (KDM4D), which belongs to the JMJD2 subfamily of HDMs containing the JMJC structural domain, can be recruited to the β-catenin promoter to induce β-catenin transcription and can act as a coactivator to enhance the expression of β-catenin target genes. KDM4D interacts with LSD1 to synergistically remove monomethyl dimethyl and trimethyl groups from H3K9 [32]. H3 lysine 4 (H3K4) methylation is usually associated with gene activation and transcriptional prolongation. It is mediated by HMTs such as SET domain containing 1A, histone lysine methyltransferase (SET1), and SET domain containing 7/histone lysine methyltransferase (SET7/9). In contrast, H3K9 is usually associated with gene repression, and is catalyzed by HMTs such as SUV39H1, G9a, and SETDB1/ESET [33]. As an HMT that promotes cancer development by repressing oncogenes, Drosophila zeste homolog 2 (EZH2) is a key marker of CRC. It is responsible for the methylation of histone H3K27 [34]. Among the many EZH2 inhibitors, CPI-169 and GSK126 have similar oncogenic effects [35]. Treating mice loaded with KARPAS-42 xenografts with both CPI-169 and GSK126 effectively reduced the level of H3K27 trimethylation, causing tumor regression. Another inhibitor [36], EPZ011989, showed significant tumor suppression in a mouse model with human B-cell lymphoma. PF-06726304 inhibited both wildtype EZH2 and Y641N-mutated EZH2 [37].
Natural products provide various chemical scaffolds with unique activity and relatively mild toxicity. Natural products with LSD1 inhibitory activity have been identified, including baicalin, resveratrol, and guanylic acid (GGA). Baicalin was the first LSD1 inhibitor discovered to have a non-covalent bond to the flavonoid group. Resveratrol, an irreversible LSD1 inhibitor, is believed to inhibit LSD1 activity in cells in vitro by directly binding to LSD1 [28]. GGA and its derivatives inhibit LSD1 activity by disrupting the protein-protein interaction between LSD1 and H3K4me2, leading to transcriptional neurotrophic receptor tyrosine kinase 2 (NTRK2) upregulation in SH-SY5Y cells [38]. The oligopeptide transporter solute carrier family 15 member 1 (SLC15A1) is an intestine-specific transporter protein, and DNA methyltransferase 1 (DNMT1) mediates hypermethylation of the proximal promoter region of the PEPT1 gene, which in turn leads to transcriptional repression and increased sensitivity of CRC to anticancer drugs [39].
Other natural products may complete histone methylation by regulating epistasis factors. For example, kaempferol reduces A4CT2 methylation levels by inhibiting the expression of DNA methyltransferases (DNMTs) in HCT-116 and HT-29 colon cancer cells, thereby promoting gene transcription. Kaempferol significantly downregulated the expression of key genes in the Wnt signal pathway downstream of A4CT2 [40] and the β-catenin signaling pathway in HCT-116 cells, thereby blocking the cells in the G1 phase and inducing apoptosis [41]. Rhubarbic acid can exert anti-CRC effects by inhibiting cell proliferation, promoting apoptosis, and inhibiting invasion and metastasis through the STAT3 signaling pathway [42]. PD can upregulate the activity of caspase 3 (CASP3) and shear its substrate PARP, inducing SW620 cell death [43]. Radicicol could inhibit β-catenin entry into the nucleus to disrupt the Wnt/β-catenin signaling pathway, reduce downstream target gene LEF/TCF expression, inhibit cell transcription and translation, cause G1 phase block in colon cancer HT29 and SW480 cells, and inhibit cell proliferation in a dose-and time-dependent manner [44]. Geniposide, shikonin, costunolide, asterolide, ursolic acid, and curcumin inhibit CRC development or induce its apoptosis (Table 1).

Histone Acetylation Regulates CRC
Histone acetylation refers to the addition of acetyl groups carried by acetyl coenzyme A to histone residues in the presence of HATs. Histone acetylation usually occurs on lysine residues at positions 9, 14, 18, 23, 27, 36, and 56 of H3 and positions 5, 8, 12, and 16 of H4. Histone acetylation counteracts the positive charge carried by these residues, increasing repulsion from DNA and leading to a looser chromatin structure, thereby activating gene expression. The histone acetylation level is regulated by the dynamic balance of HAT and HDAC enzymatic activities [59], as shown in Figure 2. Histones can be deacetylated by HDACs, allowing them to carry a positive charge again, which enhances the interaction between histones and negatively charged DNA, leading to a tighter chromatin structure and consequent transcription repression. Eighteen HDACs have been identified in humans; based on their homology to yeast HDACs, they can be classified into four classes: class I Pharmaceuticals 2023, 16, 1095 7 of 21 (HDAC1, HDAC2, HDAC3, and HDAC8), class II (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10), class III (sirtuin, including SIRT1-SIRT7), and class IV (HDAC11).
Histone acetylation counteracts the positive charge carried by these residues, increasing repulsion from DNA and leading to a looser chromatin structure, thereby activating gene expression. The histone acetylation level is regulated by the dynamic balance of HAT and HDAC enzymatic activities [59], as shown in Figure 2. Histones can be deacetylated by HDACs, allowing them to carry a positive charge again, which enhances the interaction between histones and negatively charged DNA, leading to a tighter chromatin structure and consequent transcription repression. Eighteen HDACs have been identified in humans; based on their homology to yeast HDACs, they can be classified into four classes: class I (HDAC1, HDAC2, HDAC3, and HDAC8), class II (HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10), class III (sirtuin, including SIRT1-SIRT7), and class Ⅳ (HDAC11). Sirtuins 1 to 7 (SIRT1-SIRT7) are HDACs essential for normal cell proliferation and growth. CRC development is closely associated with HDACs, with HDAC1 accounting for 36.4%, HDAC2 for 57.9%, and HDAC3 for 72.9% of CRC cases. HDACs are a family of proteins responsible for removing acetyl groups from the N-acetyl group of histone lysine residues [60,61]. Abnormal expression of these genes leads to cell cycle arrest, chemosensitization, and apoptosis. In addition, during cellular stress HDAC1 can deacetylate the tumor suppressor p53 [62], reducing the cellular stress response [63,64].
Histone H3 lysine 27 acetylation (H3K27ac) [65] is generally enriched in genes regulating cell proliferation and differentiation [66]. H3K27ac is a current marker of transcriptional activation of gene expression [67,68]. In CRC cells, H3K27ac activates the expression of lncRNAEIF3J-AS1 and promotes cell proliferation. HDAC2 expression in CRC metastatic tissues is low, and is closely associated with the survival of patients with CRC. Low HDAC2 expression promotes EMT and CRC metastasis. By binding to the transcription factor SP1, HDAC2 is "anchored" to the H19 promoter region and catalyzes the deacetylation of histone H3K27 to suppress H19 expression. HDAC2 has a negative regulatory effect on CRC metastasis. Low HDAC2 expression promotes EMT and rectal cancer metastasis by upregulating H19/MMP14 [69].
Chen [70] found that the novel HMT SETDB1 was highly expressed in most CRC samples and cell lines. A review of studies on the relationship between histone modifications and cancer recently found that the acetylase activity of hMOF, an important acetyltransferase of human histone H4 lysine 16 (H4K16), increased its acetylation. Mitani et al. [71] studied human colon and rectal cancer cell lines, finding that histone acetylation was significantly upregulated in these cancer cells compared to normal cells. The dynamic balance of histone acetylation in the distal promoter region of the PEPT1 gene is maintained by HDAC1, which is responsible for the deacetylation of histone H3 lysine 18 acetylation (H3K18Ac) and H3K27Ac, and P300, which is responsible for catalyzing H3K18Ac and H3K27Ac to enhance the sensitivity of CRC to anticancer drugs [72,73]. HDAC2 knockdown promotes the expression of CRC EMT marker genes. HDAC2 is negatively regulated for CRC metastasis. Low HDAC2 expression promotes EMT and rectal cancer metastasis by upregulating H19/MMP14 [74,75]. HDAC7 is a member of the HDAC class IIa family, which is an epigenetic regulatory AKT [76], also known as protein kinase B (PKB), a serine/threonine protein kinase with a molecular weight of approximately 60 kDa [77]. AKT regulates several biological processes, including cell survival, proliferation, growth, and glycogen metabolism. The AKT signal pathway is closely associated with the development of many diseases, including malignancies. AKT signal pathway activation has been reported to be inhibited by targeting HDAC7 to suppress tumor development. P21 (CDKN1A) is an important member of the cyclin-dependent kinase inhibitor family that coordinates the cell cycle by inhibiting the activity of the cyclin-dependent kinase (CDK) complexes. Inhibition of AKT signal activation leads to increased p21 expression, leading to cell cycle inhibition. B-cell leukemia/lymphoma 2 (BCL2) [78], an oncogene with a significant inhibitory effect on apoptosis, is a downstream target gene of the AKT signal pathway. Inhibition of AKT signal pathway activation and reduced BCL2 expression promotes apoptosis.
HDAC7 knockdown has been found to increase ATF3 expression, which decreases AKT signaling pathway activation, increases p21 expression, and inhibits cell proliferation.
HDACis inhibit tumor cell proliferation and survival. Their inhibition of HDAC activity enables HATs to increase acetylation levels, turning regions initially unable to engage with promoters into potential or novel target sites for transcription factors, allowing normal expression of oncogenes and thereby inhibiting tumor cell growth and proliferation and promoting apoptosis or necrosis [81]. The HDACi structure usually comprises three parts: a cap structure that interacts with the edge of the HDAC active pocket, a zinc ion binding group (ZBG), and a linker arm that is responsible for the linkage between the cap structure and the ZBG and can interact with the hydrophobic channel in the active site. The primary traditional ZBGs are iso-hydroxamic acid, benzamide, carboxylic acid, and mercaptan. HDACis containing these ZBG structures have been approved for marketing by the FDA or are in clinical trials. Iso-hydroxamic acid is the most widely used ZBG.
The FDA has approved a number of hydroxamic acid inhibitors for cancer treatment, including the well-known SAHA and panobinostat [93]. Romidepsin (FK-228) is the main cyclic peptide inhibitor, directly inhibiting HDAC1 and HDAC2, which has been reported to inhibit PI3K activity as well [94]. The first two have been shown to have tumor-inhibiting effects in vitro, while mangosteen has shown antitumor properties in vitro [95]. Curcumin is already in clinical trials for cancer treatment [96]. Other small molecule inhibitors, such as C646 (a specific inhibitor of p300), have been shown to inhibit the proliferation of various cancer cells in vitro [97].
Several natural products have HDACi effects, such as radicicol [98], which can exert antitumor effects by inhibiting HDAC activity, and safranin, which can increase histone H3 Pharmaceuticals 2023, 16, 1095 9 of 21 and H4 acetylation. Treating A549 cells with safranin decreased HDAC mRNA and protein levels, leading to cell growth arrest and morphological changes. The inhibitory effect of flavopiridol on HDACs was associated with the downregulation of oncogenes (e.g., tumor necrosis factor [TNF], matrix metalloproteinase 2 [MMP2], and matrix metalloproteinase 9 [MMP9]), upregulation of oncogenes (e.g., p21 and p53), and positive regulation of the Bcl2/Bax family proteins in A549 cells. It triggered the cysteine cascade apoptotic pathway and promoted apoptosis in A549 cells. Resveratrol significantly inhibited class I and II HDACs and enhanced HAT activity [99]. Other natural products may be indirectly involved in histone acetylation modifications by modulating different epigenetic regulators. For example, the Salvia officinalis extract dihydrotanshinone could inhibit colon cancer cell proliferation by downregulating the expression of β-catenin and/or c-Myc, both of which are proteins downstream of the HDAC1/HIF1α/VEGFA signaling pathway, to inhibit Wnt/β-catenin signaling [100].

Histone Phosphorylation Regulates CRC
Histone phosphorylation modifications occur mainly on two amino acids, namely, serine (including threonine) and tyrosine. Serine phosphorylation activates the protein's activity, primarily its enzyme activity. In contrast, tyrosine phosphorylation, in addition to its role in deactivating and activating the protein's activity, has a more important function facilitating its interactions with other proteins to form a multiprotein complex, which further facilitates its phosphorylation [101], as shown in Figure 3. Therefore, tyrosine phosphorylation and the formation of multiprotein complexes represent the basic cell signaling mechanism; almost all peptide cell growth factors activate cells and stimulate cell growth through this pathway. Therefore, the enzymes that catalyze protein tyrosine phosphorylation, called tyrosine kinases, are key proteins in the signal transduction machinery and control of cell growth [102]. Tyrosine kinases and protein tyrosine phosphorylation play a decisive role in tumourigenesis and growth as well. Many antitumor drugs have been developed to target such molecules. phosphorylation and the formation of multiprotein complexes represent the basic cell signaling mechanism; almost all peptide cell growth factors activate cells and stimulate cell growth through this pathway. Therefore, the enzymes that catalyze protein tyrosine phosphorylation, called tyrosine kinases, are key proteins in the signal transduction machinery and control of cell growth [102]. Tyrosine kinases and protein tyrosine phosphorylation play a decisive role in tumourigenesis and growth as well. Many antitumor drugs have been developed to target such molecules. Phosphorylation modifications often occur at threonine and serine residues of histones. Histone phosphorylation is associated with DNA damage and repair, and can increase the negative charge of the target proteins [103,104]. Histone phosphorylation leads to charge rejection between histones and DNA, potentially loosening DNA binding to his- Phosphorylation modifications often occur at threonine and serine residues of histones. Histone phosphorylation is associated with DNA damage and repair, and can increase the negative charge of the target proteins [103,104]. Histone phosphorylation leads to charge rejection between histones and DNA, potentially loosening DNA binding to histones, leading to decreased nucleosome stability, increased exposure of chromatin DNA, and enhanced chromatin accessibility. Phosphorylation is mainly involved in the activation of cellular signal pathways. Activation of the ERK-MAPK and p38-MAPK pathways induces Ser-10 phosphorylation of histone H3, which plays an important role in activating eukaryotic gene transcription [105]. Activation of the ATM/ATR and DNA-PK pathways induces Ser-10 phosphorylation of histone H2A variant H2AX, which is involved in DNA damage repair. In signaling, one group is phosphorylated by protein kinases and covalently binds phosphate groups provided by ATP, while the other binds GTP in response to the signal, usually replacing GDP with GTP. Both intracellular signaling proteins share the ability to be activated by acquiring one or more phosphate groups when the signal is received and inactivated by their removal when the signal is diminished.
In the signal relay network, phosphorylation of one signal protein usually causes downstream proteins to be phosphorylated in turn, forming a phosphorylation cascade [106]. Protein phosphorylation is mainly concentrated on tyrosine, serine, and threonine residues in the peptide chain, which have free hydroxyl groups and are uncharged, which is the mechanism of protein phosphorylation [107]. Cinobufagin was intraperitoneally injected into mice with in situ human colon cancer SW480 cell tumors daily. After three weeks, mTOR phosphorylation was inhibited and HIF1α expression was downregulated in the tumor, blocking the endothelial mTOR/HIF-1α pathway and creating a microenvironment in which the tumor vasculature is not easily generated, triggering the apoptosis of vascular endothelial cells and inhibiting neoplastic tumor and vessel formation [47].
Acetylharpagide is expected to be a new candidate for treating tumors. β-catenin phosphorylation by the destruction complex is reduced, resulting in a large accumulation of β-catenin protein in the cytoplasm. The excess β-catenin is transferred from the cytoplasm to the nucleus, where it binds to intranuclear transcription factors such as T-cell transcription factor/lymphoid enhancement factor (TTF/LEF) and other intranuclear transcription factors to activate downstream target genes such as c-Myc and cyclin Dl (CCND1) [108]. Betulinic acid inhibits colon cancer cell growth and metastasis by regulating the TGF-β/Smad signaling pathway. It causes Smad protein receptor phosphorylation [109]. ANP32A is highly expressed in CRC tissues, correlating with their low differentiation, and is accompanied by the over-activation of AKT and reduced phosphorylation of p38. Molecular biology experiments have confirmed that knocking down ANP32A inhibits CRC cell proliferation, inducing over-phosphorylation of p38 and dephosphorylation of AKT [110].
Fan Ruolan and Chen Hailan found that C. rhamnose could bind to rhSTAT3 protein and inhibit STAT3 phosphorylation at the Tyr705 site, with significant anti-CRC activity [111]. Curcumin, a natural compound derived from natural products of the ginger and tannin families, inhibited the survival of human colon cancer HCT116 cells. Curcumin may slow down or even inhibit the development of distant metastasis by inhibiting the EMT of HCT116 cells induced by TNF-α through downregulation of the NF-κBp65 protein [112]. Curcumin inhibited NF-κB's cytokine-induced DNA binding activity, RelA nuclear translocation, IκBα degradation, IκB Ser32 phosphorylation, and IκB kinase activity, thereby blocking NF-κB-induced kinase and IKK upstream signaling and inhibiting NF-κB activity [113].
The drug-containing serum of the Jianshuang Cancer Control Formula inhibited human colon cancer HCT116 cell proliferation [114], which was associated with the regulation of the ANP32A/p38/AKT signaling pathway by inhibiting ANP32A expression, increasing p38 phosphorylation, and inhibiting AKT phosphorylation [115]. Platycodon saponin, one of the active antitumor components in the Chinese medicine Platycodon, inhibits colon cancer cell growth by downregulating the expression of CCND1, c-Myc, and CDK6, blocking cells in the G1 phase, by inducing apoptosis by shear activation of pro-CASP3 and PARP, and by inhibiting JNK, p38 activation, and ERK1/2 dephosphorylation [116]. Acetylharpagide, a cyclic enol ether terpene compound in the natural plant genus Tendrils, inhibits HCT116 cell growth mainly through the Wnt/β-catenin signal pathway.
β-catenin has been identified as a promising target for cancer prevention and treatment. Many natural products can act as β-catenin inhibitors, mainly by regulating phosphorylation, promoting ubiquitination and proteasomal degradation, and inhibiting nuclear translocation. Natural product inhibitors have shown good preventive and therapeutic effects in various in vitro and in vivo tumor models [117]. According to the research statistics in recent years, most (>90%) CRCs show activation of the classical Wnt pathway, as shown in Figure 4. The aberrant activation of Wnt/β-catenin, as one of the classical Wnt signal pathways, plays a crucial role in CRC development. The aberrant activation of this pathway is usually accompanied by the stabilization of β-catenin and a large accumulation of β-catenin in the nucleus [118]. This observation has been clinically demonstrated by the accumulation of β-catenin in the nucleus in >80% of patients with CRC [119]. In addition, clinical results have shown that the poor prognosis of patients with CRC is closely associated with high levels of cytosolic β-catenin [26,120]. Natural products act on Wnt signal pathway proteins to ameliorate CRC proliferation ( Table 2).       Inhibition of Wnt/β-catenin signaling by down-regulation of β-catenin and/or c-Myc expression to suppress proliferation of colon cancer cells. [139] 6. Discussion CRC is a malignant disease with the highest mortality rate of oncological diseases worldwide. It is treated only by surgical resection with radiotherapy, which remains ineffective. In the last decade or so, the clinical status of natural products as preventive and therapeutic agents has become a focus of attention due to their easy availability, low cost, high body tolerance, low toxicity, and diverse biological functions. Indeed, up to 70% of clinical drugs are derived from them. In recent years, epigenetic therapies have been increasingly accepted as effective treatments for cancer and various other diseases. Therefore, the epigenetic mechanisms of natural products have been used to investigate their preventive and therapeutic roles in CRC, where natural products provide a holistic approach to treating CRC.
With a growing understanding of the fundamental role of epigenetics in CRC initiation, progression, recurrence, and chemoresistance, natural products are very strong candidates for developing new drugs for cancer treatment. However, there remain limitations to using these natural products as chemotherapeutic agents, particularly regarding the range of effective concentrations of single natural product chemicals in preliminary in vitro models, making it difficult to define the most effective and biocompatible concentrations in animal models. In addition, in most cases the in vivo results are not as promising and encouraging as those observed with in vitro studies; therefore, they do not qualify for clinical trials. In addition, due to natural products' confounding nature and reactivity, a thorough analysis of their appropriate validation and administration routes is required. Moreover, the mechanism of action of specific epigenetic factors needs further experimental validation. However, natural products continue to occupy a surprising amount of space in epigenetic drug development.
Understanding the mechanisms and collating the interactions between the various natural product chemicals to understand how they target the multi-step carcinogenesis process can help in developing personalized epigenetic regimens for patients. Drugs that target epigenetic alterations can overcome the immunogenicity of the tumor microenvironment, making these tumors sensitive to chemotherapy. Manipulating the epigenomic landscape through natural products would be an effective way to target immune-related pathways. Establishing this transcription factor pathway of histone modifications, such as acetylation, for key genes using natural products would be fascinating. To date, natural products have shown great potential in sensitizing CRC cells to conventional therapies, though this may lead to complex and unwanted off-target effects. There are many possible reasons for this issue, with a prominent drawback being that targetable enzymes/proteins with multiple biochemical functions in the normal cellular state may be disrupted upon treatment with these natural products. Indeed, detailed knowledge of the role of each protein complex/enzyme in the normal epigenetic landscape is essential for further consideration of natural products in epigenetic therapies. Therefore, natural product epigenetic modulators need to be considered more broadly in order to maximize their clinical benefit for patients with CRC, which may help expand their therapeutic potential in treating and preventing CRC.

Materials and Methods
Data sources and search strategy: in the PubMed database with "Colorectal cancer", "Natural Products" AND "Epigenetics" OR "Histone modification" OR "Histone deacetylation and acetylation" OR "Histone phosphorylation" OR "Histone methylation and demethylation" as the subject of the search, for which there was no time limit and the search remained open until 31 May 2023.
Literature Screening Criteria: 1 Inclusion Criteria: articles investigating natural products to epigenetically regulate the treatment and prevention of colorectal cancer; 2 Exclusion Criteria: non-natural products, non-epigenetic regulation, old or incomplete information, sketchy description of mechanism of action, unclear description of test method, sketchy description of test index, inconsistency between the description of test index and results, conference papers, the same experiment or the same batch of experiments. Data extraction and processing: End Note x20 standard edition was used for literature management. The preliminary search returned 287 PubMed and 211 Web of Science documents, for a total of 498 documents. After checking, 189 were excluded, leaving 309 documents. After reading through the titles and abstracts, 103 documents with incompatible topics were deleted, leaving 206 documents. After reading the full text and deleting 68 articles that did not meet the inclusion criteria, 138 articles were finally included, among which many had only abbreviations for gene and protein descriptions with reference to "the resource for approved human gene nomenclature" for standardizing the names, types, etc. of genes and proteins. Excel 2019 software was applied to enter the information of article year, author, title, impact factor, signaling pathway, epigenetic regulation mode of action, colorectal cancer treatment detection indexes, etc., to establish the data file of natural product epigenetic regulation of colorectal cancer, and the entered data were sorted, statistically and analytically analyzed, and quantitatively summarized.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest:
The authors declare no conflict of interest.