Citrus is a kind of common fruit and contains multiple beneficial nutrients for human beings. Flavonoids, as a class of plant secondary metabolites, exist in citrus fruits abundantly. Due to their broad range of pharmacological properties, citrus flavonoids have gained increased attention. Accumulative in vitro and in vivo studies indicate protective effects of polymethoxyflavones (PMFs) against the occurrence of cancer. PMFs inhibit carcinogenesis by mechanisms like blocking the metastasis cascade, inhibition of cancer cell mobility in circulatory systems, proapoptosis, and antiangiogenesis. This review systematically summarized anticarcinogenic effect of citrus flavonoids in cancer therapy, together with the underlying important molecular mechanisms, in purpose of further exploring more effective use of citrus peel flavonoids.
In our daily diet, the average intake of flavonoids of every day ranges from 150 mg to 300 mg [
In recent years, epidemiological studies have shown that there is a connection that flavonoid intake may reduce the risk of developing colon cancer [
Cancer is the life threatening and dreadful disease characterized by the abnormal proliferation of cells that invade the adjacent tissues and cause the destruction of these tissues. It is the second leading cause of death all over the world. More than six million deaths each year occurring in the world are due to cancer. Several lines of evidence indicated that tumorigenesis in humans is a multistep process and these steps reflect genetic alterations that drive the progressive transformation of normal human cells into highly malignant derivatives [
In the tumor microenvironment, from cancer cells initiation to promotion and eventually progression, compelling evidence indicates the potential activities of flavonoids in citrus peel cover inhibiting oncogenesis, proliferation, neovascularization, and metastasis and inducing apoptosis. Figure
Anticarcinogenic activity of citrus polymethoxyflavonoids and their derivatives.
Polymethoxyflavone | Functions | Mechanisms | References |
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Naringin | Cell cycle arrest | G1 cycle arrest by increasing p21 and decreasing survivin in MDA-MB-231 xenograft mice | [ |
Anticancer metastasis | Suppressed the upregulation of metallopeptidase-9 (MMP-9) and repressed the PI3K/AKT/mTOR/p70S6K signaling pathway | [ | |
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Hesperetin | Cell cycle arrest | G1-phase cell cycle arrest in human breast cancer MCF-7 cells by downregulating CDK2 and CDK4 together with cyclin D and upregulating p21Cip1 and p27Kip1 | [ |
Induced the G2/M phase and increased expression of caspase-3, caspase-8, caspase-9, p53, Bax, and Fas death receptor and its adaptor protein Fas-associated death domain-containing protein (FADD) in human cervical cancer SiHa cells | [ | ||
Suppress proliferation | Exerted significant inhibitory effect on proliferating cell nuclear antigen in ACF in 1,2-dimethylhydrazine induced colon cancer model in rats | [ | |
Inhibited growth of aromatase-expressing MCF-7 tumor in ovariectomized athymic mice by reducing cyclin D1, CDK4, and Bcl-x(L), while upregulating the level of p57Kip2 | [ | ||
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Nobiletin | Cell cycle arrest | Blocked cell cycle progression at G1 breast cancer cell lines MDA-MB-435 and MCF-7 and human colon cancer line HT-29 | [ |
Antiangiogenesis | Inhibited angiogenic differentiation induced by VEGF and FGF by downregulation of ERK1/2 and c-JNK and activation of the caspase pathway | [ | |
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Tangeretin | Cell cycle arrest | Blocked cell cycle progression at G1 breast cancer cell lines MDA-MB-435 and MCF-7 and human colon cancer line HT-29 | [ |
Suppress proliferation | Led to caspase-3 activation and elevated surface phosphatidylserine in human cocon LoVo/Dx cells | [ | |
Anticancer metastasis | Inhibited PGDF-BB-induced proliferation and migration of aortic smooth muscle cells by blocking AKT activation | [ | |
Scavenging of ROS | Inhibited cancer cell proliferation by SOD, CAT, GPx, and nonenzymatic antioxidants and phase II detoxification in 7,12-dimethyl benz(a)anthracene induced mammary carcinoma in rats | [ | |
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5-Demethyltangeretin (5DT) | Cell cycle arrest | Upregulated p53 and p21Cip1/Waf1 and downregulated cdc-2 and cyclin B1 leading to G2/M cell cycle arrest | [ |
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Sinensetin | Antiangiogenesis | Inhibited angiogenesis by inducing cell cycle arrest in the G0/G1 phase in HUVEC culture; in zebrafish embryos, it downregulated the mRNA expressions of angiogenesis genes |
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5HTMF | Suppress proliferation | Induced cellular apoptosis in human colon cancer cells by p53- and Bax-dependent mechanisms in HCT116 colon cancer cells | [ |
Cell cycle arrest | Induced cell cycle arrest at G0/G1 phase through a p53- and p21Cip1/Waf-dependent mechanism in HCT116 colon cancer cells | [ | |
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5HPMF | Suppress proliferation | Induced cellular apoptosis in human colon cancer cells by p53- and Bax-dependent mechanisms in HCT116 colon cancer cells | [ |
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5HHMF | Cell cycle arrest | Induced G2/M arrest through p53- and p21-independent mechanisms in HCT116 colon cancer cells | [ |
Suppress proliferation | Induced cellular apoptosis in human colon cancer cells by p53- and Bax-dependent mechanisms in HCT116 colon cancer cells | [ | |
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Naringenin | Cell cycle arrest | Partly formed an accumulation of cells in the G0/G1 and G2/M phases of the cell cycle in human hepatocellular carcinoma HepG2 cells | [ |
Anticancer metastasis | Induced heme oxygenase-1(HO-1) expression and subsequently decreased ROS generation and VSMC activation induced by TNF-α | [ | |
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Hesperidin | Suppress proliferation | Promoted apoptosis via conducting the expression of p53 and PPAR |
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Flavonoids extracted from Korean |
Cell cycle arrest | Induced non-small-cell lung cancer (NSCLC) A549 cells arrest at the G2/M checkpoint | [ |
Suppress proliferation | Induced caspase-dependent apoptosis through AKT pathway by inhibiting expression of XIAP and Bcl-2 in human leukemia cells U937 | [ | |
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Gold lotion | Suppress proliferation | In azoxymethane-induced aberrant crypt foci formation, it downregulated the protein levels of iNOS, COX-2, ornithine decarboxylase, VEGF, and matrix metallopeptidase 9 in colonic tissues of mice | [ |
Anticancer metastasis | Downregulated MMP-2 and MMP-9 protein expression levels and reduced tumor volumes and weights in human prostate tumor xenograft mouse model | [ | |
Antiangiogenesis | Significantly suppressed the protein expression level of VEGF | [ | |
Reduced the protein levels of VEGF in AOM-induced colonic tissues | [ |
Polymethoxyflavones exert beneficial effects through antigrowth, antiangiogenesis and cell cycle arrest commands or mediate signals to live or die by apoptosis. At one level, this depiction is simplistic, as different cancer cells are exposed to a specific complex microenvironment, each of these pathways regulated by PMFs is connected with signals originating from other cells in the tumor microenvironment. Schematic representation of the main molecular mechanisms of flavonoids in citrus peel on anticancer.
Cell cycle abnormalities are closely associated with cancer, and citrus peel flavonoids substantially influence on cell cycle arrest. Cell cycle is an important regulatory mechanism of cell growth, development, and differentiation. In mammals, the cell cycle comprises the G1, S, G2, and M phases. Cell cycle checkpoints keep the maintenance of genomic integrity by inhibiting damaged or incomplete DNA. G2/M checkpoint ensures that the cells do not initiate mitosis before repairing damaged DNA after replication. The cell cycle progression depends on a cascade of enzymes by sequential activation and inactivation of cyclin, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CDKIs) [
The chemical structures of citrus peel flavonoids molecules that are discussed in this paper.
One of the most basic features of cancer cells is their ability to proliferate chronically. Apart from blocking cell cycle, flavonoids in citrus peel can also inhibit cell proliferation and promote apoptosis, especially in triple-negative (ER-/PR-/HER2-) breast cancer (TNBC). PMFs triggered influx of Ca2+ and mobilization of intracellular Ca2+ store, accompanied by activation of calpain and caspase-12 [
In the research of mice, oral feeding of gold lotion (GL), a formulated product made from the peels of six citrus fruits, decreased the number of aberrant crypt foci (ACF) in mice colonic tissues [
Our latest research showed that, in MCF-7 human breast cancer cells, 5-acetyl-6,7,8,4′-tetramethylnortangeretin (5-ATAN), which replaces the methyl groups of tangeretin with acetyl groups at the C5 position of tangeretin, showed more powerful abilities than its parent compound. Then, we looked for evidence of 5-ATAN on apoptosis. Translocation of apoptosis-inducing factor (AIF) and phosphorylation of H2AX are commonly used for evaluating the impact of natural compounds-induced caspase-independent apoptosis pathway [
Traditional treatment of cancer has been facing a huge number of problems, in view of its complex molecular pathophysiology that varies according to each type. Several ways in the treatment of breast cancer have been developed that are surgery, chemotherapy, hormonal therapy, and radiation. Doxorubicin, a chemotherapeutic agent commonly used in breast cancer treatment, showed low effectivity, rendering its resistance and toxicity on normal tissues [
Invasion and metastasis are a multistep process and are described as a series of discrete steps, usually called invasion—the metastatic cascade [
Angiogenesis is a physiological process of forming new blood vessels from preexisting vessels, which involves the induction of new sprouts, coordinated and directed endothelial cell migration, proliferation, sprout fusion, and lumen formation [
As one of the angiogenesis inducers, vascular endothelial growth factor-A (VEGF) can be used as a marker of angiogenesis.
Through combined inhibition of multiple angiogenesis-related endothelial cells (EC) functions, nobiletin had been demonstrated to have concentration-dependent inhibitory effects on angiogenic differentiation induced by VEGF and FGF (fibroblast growth factors). In a chick embryo chorioallantoic membrane assay, nobiletin showed an antiangiogenic activity with the ID50 value being 10 lg (24.9 nmol) per egg [
With human umbilical vein endothelial cells (HUVECs) in vitro and zebrafish in vivo models, PMFs showed different degrees of potency of antiangiogenesis activity. Sinensetin, which showed the most potent antiangiogenesis activity and the lowest toxicity, inhibited angiogenesis by inducing cell cycle arrest in the G0/G1 phase in HUVEC culture and downregulating the mRNA expressions of angiogenesis genes
Flavonoids also exert their chemopreventative effect via inhibition of certain phase I metabolizing enzymes, such as cytochrome P450 which metabolically activates a large number of procarcinogens triggering carcinogenesis. The chemopreventative effects of flavonoids are closely linked to their anticancer properties that involve the scavenging of reactive oxygen species (ROS) and growth promoting oxidants which are the major catalysts for tumor promotion. Tangeretin, a polymethoxylated flavone, can inhibit cancer cell proliferation by improving antioxidant properties such as decreasing the levels of lipid peroxide, enzymatic antioxidants SOD, CAT, and GPx, and nonenzymatic antioxidants such as GSH, vitamin C, and vitamin E in 7,12-dimethyl benz(a)anthracene (DMBA) induced mammary carcinoma in rats [
Pharmacokinetics describes how the body affects a specific drug after administration through mechanisms of absorption and distribution, as well as the chemical changes of the substance in the body. At a practical level, a drug’s bioavailability can be defined as the proportion of the drug that reaches its site of action. Poor absorption and extensive conjugative metabolisms greatly limit bioavailability of dietary flavonoids.
The bioavailability is an overall effect of absorption, distribution, metabolism, and excretion and plays an important role in dictating cancer preventive efficacy of dietary components in humans. Bioavailability testing can be divided into in vitro and in vivo bioavailability. In vitro bioavailability test can be a good predictor of the latter one.
Currently, human colon adenocarcinoma cell line caco-2 cell model is established to simulate the human intestinal absorption so as to test permeability and study absorption mechanism. The caco-2 data of 3′-hydroxy-5,6,7,4′-tetramethoxyflavone, 3,5,6,7,8,3′,4′-heptamethoxyflavone, and 3-hydroxy-5,6,7,8,3′,4′-hexamethoxyflavone showed superb permeability [
Also, in NSCLC A549 cell line, 5-hydroxylated PMFs had much stronger inhibitory effects on cancer cells in comparison with their permethoxylated counterparts, for IC50 value of 5-demethyltangeretin (5DT) was 78.9-fold lower than that of tangeretin. Since cancer cells can pump the cytotoxic agents out via overexpression of multidrug resistant efflux proteins, cells were treated with 5DT or tangeretin at the same concentration. HPLC analysis revealed that the intracellular levels of 5DT in NSCLC cells were 2.7–4.9-fold higher than those of TAN. This suggested that NSCLC cells may have better uptake efflux of 5DT compared with TAN. Additionally, molecular structure showed that 5DT had higher lipophilicity than tangeretin [
Biotransformation of dietary components is crucial for their in vivo biological activities after oral ingestion because the process of drug metabolism notably influences drugs effects and toxicity. In the research of nobiletin metabolites, by comparing supercritical fluid chromatography (SFC) profiles of metabolite mixtures with the synthesized standard compounds, three major metabolites were proved to be 4′-demethylnobiletin, 3′-demethylnobiletin, and 3′,4′-didemethylnobilietin in mouse urine [
Taken all together, a considerable number of well-established lines of evidence have confirmed that flavonoids in citrus peel exhibit a remarkable spectrum of efficacious biological activities, particularly in antitumorigenesis. Excellent permeability through membrane allows citrus flavonoids to possess great bioavailability which consequently attracts researchers to perform scientific studies for effective disease prevention and treatment. There are more modified flavonoids in citrus peel being investigated, which could offer help to improve dose-effect relationship greatly and advance the security and stability of compounds.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was supported by the National Natural Science Foundation of China (Grant nos. 81172837 and 31270050), the Tianjin Research Program of Application Foundation and Advanced Technology (Grant no. 13JCQNJC12200), and the Tianjin Innovative Research Team Grant of Agriculture Storage and Procession (TD-12-5049).