Fatty Acids

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Among the saturated and unsaturated fatty acids present in the chrysanthemum flower, linoleic acid, palmitic acid, and stearic acid are believed to have significant antioxidant activities. Stearic acid and palmitic acid also showed apoptosis-inducing potential, while linoleic acid can enhance the growth of tumor cells. Stearic acid suppresses granulosa cell survival by inducing apoptosis, as evidenced by DNA ladder formation and annexin V-EGFP/ propidium iodide staining of the cells (13). Palmitic acid induces apoptosis in cardiomyocytes. The apoptosis is mediated through alterations in mitochondria through cytochrome c release and caspase 3 activation (14) Palmitic acid also induces apoptosis in Chinese hamster ovary cells through generation of reactive oxygen species and a ceramide-independent pathway (15).

Although it has been reported that linoleic acid showed anticancer effect on VX-2 tumor growing in the liver of rabbits (16), another study demonstrated that linoleic acid is a tumor promoter (17). Linoleic acid has a potent growth-promoting effect on several rodent tumors, human tumor xenografts grown in immunodeficient rodents, breast cancer cells, and rat hepatoma 7288CTC (18). Linoleic acid is metabolized to the mitogen 13-hydroxyocta-decadienoic acid and thus enhances tumor growth (19). On the other hand, reduced expression of nm-23-H1, a metastasis-suppressor gene, by linoleic acid may contribute at least partly to its tumor-growth-enhancing activity (20).

B. Triterpenoids

Triterpenoids are the main components of volatile oil in chrysanthemum flower and are believed to be the main effective pharmacological components in chrysanthemum (Fig. 1). Until now, more than 50 triterpenoids have been isolated and identified, among which taraxasterol, sitosterol, and lupeol show both anti-inflammatory and antitumor activity, while lupeol shows antioxidant activity. Stigmasterol and campesterol can inhibit tumor cell growth (discussed below). Five terpenes (helianol, heliantriol C, faradiol, dammar-adienol, and cycloartenol) were found to have anti-inflammatory activity while heliantriol C, faradiol, and elemene show antitumor activity.

1. Anti-Inflammatory Activity

Eleven triterpene alcohols, isolated from chrysanthemum, were tested for their inhibitory effects on TPA-induced inflammation in the ears of mice. All 11 triterpene alcohols showed remarkable inhibitory effect with a 50% inhibitory dose at 0.1-0.8 mg per ear, which was roughly at the level of indomethacin, an anti-inflammatory drug used as positive control (21). Helianol, the most predominant component in the triterpene alcohol fraction, exhibited the strongest inhibitory effect (0.1 mg per ear) among the 11 compounds tested. Since anti-inflammatory activity of the inhibitors is highly related to their anti-cancer-promoting activities, helianol is also expected to be a potent antitumor agent (22). On the other hand, cycloartenol showed anti-inflammatory properties in the mouse carrageenan peritonitis test and also show weak inhibition of phospholipase A2 activity in vitro (23).

2. Antitumor Activity

Fifteen pentacylic triterpenes isolated from chrysanthemum were screened for their anti-tumor-promoting activities. All of the compounds showed inhibi-

Figure 1 Basic structure of triterpenoids isolated from chrysanthemum.

tory effects against Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells (21), which have been demonstrated to closely parallel effects against tumor promotion in vivo. With 50% growth inhibitory (GI) value of less than 10 ||M in sulforhodamine B protein assay, the compounds faradiol, heliantriol B0, heliantriol, arnidiol, faradiol a-epoxide, and mani-ladiol showed significant inhibitory activity against almost all 60 human tumor cell lines derived from seven cancer types (lung, colon, melanoma, renal, ovarian, brain, and leukemia) (4). Arnidiol showed very significant cytotoxicity against HL-60 with a GI 50% at 0.4 ||M.

h-Sitosterol, the most common plant sterol, showed cytotoxicity against cancer cell lines Colo-205, KB, HeLa, HA22T, Hep-2, GBM8401/TSGH, and H1477 (24), Vero cells (25), and colon 26-L5 carcinoma cells (26). As little as 16 |aM sitosterol could induce apoptosis fourfold in LNCaP cells by increasing phosphatase 2A activity and activating the sphingomyelin cycle (27). Incorporation of sitosterol into cell membranes may alter fluidity and thus influence the activation of membrane-bound enzymes such as phosphalipase D (28).

h-Elemene has antitumor activity against HL-60 cells (29). h-Elemene exerts its cytotoxic effect on K562 leukemic cells by the induction of apoptosis, as evidence of the formation of apoptotic bodies and DNA ladder in a dose- and time-dependent manner. It has also been demonstrated that apoptosis in K562 cells induced by elemene is concomitant with decreasing expression of bcl-protein (30). Lupeol showed antitumor activity against lymphocytic leukemia P-388 cells (31). In contrast, stigmasterol inhibits growth of tumor cells Hep-2 and McCoy cells in vitro (32).

3. Antioxidant Activity

Cineol has antioxidant activity and inhibits lipoxygenase against ethanol injury in the rat (33). On the other hand, lupeol can prevent membrane peroxidation in red blood cells (34). This compound is also an effective skin chemopreventive agent that may suppress benzoyl-peroxide-induced cutaneous toxicity (35).

C. Flavonoids

Flavonoids are well-known natural antioxidant widely distributed in most edible plants. Flavonoids are believed to have putative beneficial effects due to their antioxidant activities. The evidence came from in vivo animal tests, in vitro cell culture tests, and a cohort epidemiological study. Numerous epidemiological studies show a clear correlation between fruit and vegetable consumption and lower risk of cancer of the gastrointestinal tract (36). A

cohort study in Finland also supported that flavonoid intake in some circumstances may be involved in slowing the cancer process, resulting in lowered risk (37). Several studies suggested an inverse correlation with stroke and cardiovascular disease, while no relationship between flavonol intake and reduced risk of cancers was found (36).

Flavonoids are ubiquitous secondary plant metabolites with a common C6-C3-C6 structure (Fig. 2). They exist more often in the form of glycoside derivatives but less often in the aglycone form. Eleven flavonoids and flavonoid glycosides have been identified in chrysanthemum since the first report in 1974. All of them are also widely distributed in other plants, for example green tea, red wine, vegetables, and fruits. Nearly all flavonoids (luteolin, diosmetin, chrysin, apigenin, quecertin, and their glycosides) that could be isolated from chrysanthemum showed antioxidant activity and antimutagenic activity or inhibited tumor cell proliferation. Luteolin, chrysin, quecertin, and apigenin were proved to induce apoptosis and cell cycle arrest at G1 phase or G2/M or both. Flavonoids may block several points in the process of tumor promotion, including inhibiting kinases, reducing transcription factors, and regulating cell cycle (38).



Figure 2 Structures of apigenin and luteolin, two major flavonoids in chrysanthemum. Apigenin and luteolin are two major flavonoids isolated from chrysanthemum. Both have the typical basic C6-C3-C6 structure with different hydroxyl substitution at different positions.

1. Antioxidant Activity

Reactive oxygen species (ROS), which are constantly produced mainly in mitochondria of cells, are believed to be potent harmful agents in the cells. An elevated level of ROS is damaging to the cells because ROS can readily react with many molecules, such as DNA, proteins, and phospholipids, and hence resulted in dysfunctions of these important molecules, resulting in many diseases. Therefore, it was speculated that antioxidants would be beneficial in reducing the damaging ROS. Further research on functions of ROS proved that ROS are inevitable in our cells and ROS, as second messengers, are necessary and important factors in mediating normal cell signaling. So the effects of antioxidants are probably related to their other effects, such as cytotoxicity, inducing cell cycle arrest, and inducing apoptosis.

Flavonoids have antioxidant activity in many cell-free systems. Lu-teolin, one of the major flavonoids of C. morifolium Ramat (39), inhibits lipoxygenase activity, cyclooxygenase activity, and inhibition of ascorbic-acid-stimulated malonaldehyde formation in liver lipids (40). Both luteolin and apigenin can inhibit CCl4-induced peroxidation of rat liver microsomes (41). Further reports demonstrated that luteolin and apigenin showed remarkable antiperoxidative activity against lipid peroxidation induced in liver cell membranes by either nonenzymic or enzymic method (42). Both luteolin and apigenin also inhibit DNA damage induced by hydrogen peroxide or singlet molecular oxygen in human cells (43,44). The glycosylated form of luteolin, luteolin-7-O-glucoside, demonstrated a dose-dependent reduction of LDL oxidation with less effectiveness than luteolin. Studies of the copper-chelating properties of luteolin-7-O-glucoside and luteolin suggest that both act as hydrogen donors and metal ion chelators (45). Apigenin also inhibits xanthine oxidase and monoamine oxidase, two potent ROS-producing enzymes (46).

Oxidative stress is pivotal in many pathological processes and reduced oxidative stress is believed to prevent many diseases. Glutathione (GSH) is an important antioxidant in all cell types. Its main function is to maintain the intracellular redox balance and to eliminate xenobiotica and ROS. As well-known antioxidants, luteolin and apigenin are speculated to decrease the oxidative stress in the cells. However, the effect of luteolin and apigenin in regulating the redox balance in the cell is equivocal. In a cell-free system, apigenin can oxidize GSH to GSSH via a thiyl radical when incubated with H2O2 and horseradish peroxidase (HRP). Luteolin can also deplete GSH but without forming a thiyl radical or GSSG. Luteolin forms mono-GSH or bis-GSH conjugates while apigenin does not form GSH conjugates. On the contrary, quercetin increased the transcription of g-glutamylcysteine synthetase (GCS) in COS-1 cells. The two-step synthesis of GSH is catalyzed by

GCS and glutathione synthetase. These inconsistent results suggested that the effects of flavonoids on ROS balance are far more complex than defining them as antioxidant or prooxidant agents.

2. Anti-Inflammatory Activity

Apigenin and luteolin inhibit interleukin (IL)-5, which promotes the growth and survival of eosinophils and plays an important role in eosinophilia-associated allergic inflammation (47). These two flavonoids also inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-activated RAW 264.7 cells by reducing inducible nitric oxide synthase (iNOS) expression but not inhibiting iNOS enzyme activity. NO produced by iNOS is one of the inflammatory mediators (48).

3. Antimutagenic Activity

Mutagenesis is usually the first step of several stages involved in carcinogen-esis, which is a long-term, multistage process that results from accumulation of mutation and dysfunction of important molecules regulating cell proliferation. During the initiation stage, a potential carcinogen is transformed into a mutagen product by Phase I enzyme such as cytochrome P450. The mutagen product may react with cellular molecules including DNA and result in genetic mutation. The mutagen product may be detoxified by Phase II enzymes, which transform the mutagen into a form easily eliminated from the body. During the promotion stage, overgeneration of ROS resulting from overexpression of pro-oxidant enzymes will lead to accumulation of DNA mutations. Cell-proliferation-related functions are elevated to a high degree; for example, DNA synthesis is increased by overexpression or activation of DNA polymerase or topoisomerase. Cell proliferation is finally increased by changes in cell-proliferation-regulating pathways such as protein kinase C and A, mitogen-activated protein kinases (MAPK), and cyclin-dependent kinases (CDK). During the final stage, the mutations are fixed and the cells are proliferating uncontrollably (49).

Flavonoids of chrysanthemum have been demonstrated to inhibit mutagenesis and carcinogenesis by targeting various molecules at different stages. The induction of cell cycle arrest and apoptosis and interaction of several pathways will be reviewed below.

Luteolin suppresses formation of mutagenic and carcinogenic hetero-cyclic amines, such as 2-amino-3,8-dimethylimidazo(4,5-f) quinoxaline and 2-amino-1-methyl-6-phenylimidazo(4,5-b) pyridine (50). Luteolin also inhibits the mutagenic activity resulting from metabolic activation of benzo-pyrene and trans-7,8-dihydroxy-7,8-dihydrobenzo-pyrene by rat liver microsomes (51). Luteolin showed significant inhibitory effect on tumor expression of fibrosarcoma induced by 20-methylcholanthrene in male Swiss albino mice (52). On the other hand, apigenin is a potent inhibitor of epidermal ornithine decarboxylase inducted by TPA in a dose-dependent manner (53).

A carbonyl group at C-4 of the flavone nucleus seems to be essential for the antimutagenicity. Increasing polarity by introduction of hydroxyl groups reduced antimutagenic potency. Reducing polarity of hydroxyl flavonoids by methyl etherification, however, increased antimutagenic potency again. Of the 11 flavonoid glycosides all compounds showed antimutagenic activity except that apigenin and luteolin-7-glucoside are inactive or only weakly antimutagenic (54).

4. Estrogenic and Antiestrogenic Activity

Estrogens are involved in the proliferation and differentiation of target cells and are among the main risk factors for breast and uterine cancer (55). Antiestrogens, which inhibit estrogen action by competing for its receptors, have been used as therapy against breast cancer and have been proposed to prevent cancers.

Many flavonoids, including luteolin and apigenin, were found to have very weak estrogenic activity, 103-105-fold less than 17-h-estradial, through binding to estrogen receptor (ER) (56,57). They also show antiestrogenic activity, which is closely related to their antiproliferation activity. Luteolin, as well as other flavonoids such as daidzein, genistein, and quercetin, is able to inhibit the proliferation-stimulating activity in MCF-7 cells caused by 1 ||M environmental estrogens such as diethylstilbestrol, clopmiphene, bisphenol, etc. (58,59). The suppressive effect of flavonoids suggests that these compounds have antiestrogenic and anticancer activities. Wang and Kurzer also found that 10 ||M luteolin or apigenin inhibited estradiol-induced DNA synthesis (60). In an in vivo test, Holland and Roy proved that luteolin inhibited estrogen-stimulated proliferation in mammary epithelial cells of female Noble rats, which suggests it may play a protective role in estrogen-induced mammary carcinogenesis (61).

The concentration of flavonoids determines their effects on cell growth. At 0.1-10 |aM, luteolin induces DNA synthesis in estrogen-depedent (MCF-7) breast cancer cells but not in estrogen-independent (MDA-MB-231) human breast cancer cells; however, at 20-90 |M, luteolin inhibits DNA synthesis in both types of cells. This suggests that additional mechanisms may be involved in the effect of high concentrations of flavonoids (60).

It is, however, important to point out that the antiestrogenicity of flavonoids does not correlate with their estrogen receptor (ER)-binding capacity, suggesting alternative signaling mechanisms may be involved in their antagonistic effects (62). Mammalian cells contain two classes of estradiol-binding sites, type I (Kd ~1.0 nM) and type II (Kd ~20 nM), designed according to their affinity (63). Luteolin competes for estradiol binding to cytosol and nuclear type II sites in rat uterine preparations but it does not interact with the rat uterine estrogen receptor (64). In an in vivo study, injection of luteolin blocked estradiol stimulation of nuclear type II sites in the immature rat uterus and this correlated with an inhibition of uterine growth. Further studies showed that luteolin could bind to nuclear type II sites irreversibly owing to covalent attachment (65). These studies suggest luteolin, through an interaction with type II sites, may be involved in cell growth regulation.

A lot of evidence demonstrated cross-talk between the estrogen pathway and the insulin-like growth factor (IGF) pathway. The IGFs can activate the ER, while the ER transcriptionally regulates genes required for IGF action. Moreover, blockade of ER function can inhibit IGF-mediated mito-genesis and interruption of IGF action can similarly inhibit estrogenic stimulation of breast cancer cells (66,67).

Apigenin, as well as chalcone and flavone, stimulates transcription factor activator protein-1 activity (AP-1). They also activate Elk-1, c-Jun, or C/EBP homologous protein (CHOP), the downstream targets of various MAPK pathways, suggesting flavonoids may affect multiple signaling pathways that converge at the level of transcriptional regulation (68).

5. Antiangiogenesis

Angiogenesis, the generation of new capillaries, occurs during many physiological processes, including development, wound healing, and formation of the corpus luteum, endometrium, and placenta (69). Angiogenesis also occurs in some pathological processes, such as solid tumor growth and metastasis, which require nutrition and oxygen owing to the fact that avascular tumors do not grow beyond a diameter of 1-2 mm (70). In response to secretion of angiogenic stimuli, such as vascular endothelial growth factor (VEGF) and matrix metalloproteases (MMP), which degrade the extracellular matrix, the endothelial cell basal membrane is degraded by the action of protease and then the endothelial cells migrate and proliferate and finally organize into capillary tubes. Several important enzymes are involved in the process. (1) VEGF is one of the most potent and specific known angiogenic factors in vivo. It increases microvascular permeability in response to hypoxia, multiple growth factors, cytokines, and estradiol (70). (2) Hyaluronic acid (HA) is one of the most abundant constituents of the extracellular matrix and acts as a barrier to neovascularization (71). Fragments of HA, as a result of catalytic activity of hyaluronidase, bind to CD44 receptor exposed on the membrane of endothelial cells and then are responsible for endothelial cell proliferation, migration, and finally angiogenesis. (3) Matrix metalloproteases (MMP) also promotes angiogenesis by degrading the extracellular matrix.

Both apigenin and luteolin inhibit the proliferation of normal and tumor cells as well as in vitro angiogenesis (72). Luteolin, on the other hand, significantly inhibits corneal neovascularization induced by basic fibroblast growth factor (bFGF), and is a potent inhibitor of corneal angiogenesis in vivo (73).

Flavonoids, including luteolin and apigenin, are competitive inhibitors of hyaluronidase (74). As inhibitor of hyaluronidase, apigenin inhibits the proliferation and the migration of endothelial cells and capillary formation in vitro. In contrast, it stimulates vascular smooth-muscle-cell proliferation. Apigenin inhibits endothelial-cell proliferation by blocking the cells in the G2/ M phase. Apigenin stimulation of smooth-muscle cells is attributed to the reduced expression of two cyclin-dependent kinase inhibitors, p21 and p27, which negatively regulate the G1-phase cyclin-dependent kinase (75).

6. Antiproliferation

Many flavonoids, including luteolin and apigenin, were found to inhibit proliferation of several normal cells and cancer cells derived from nearly all tissues, such as human fibroblast HFK2, human keratinocytes HaCaT, human breast cancer MCF-7, human neuroblastoma cell SHEP and WAC2 (59,72), Raji lymphoma cell (76), pancreatic cancer cell MiaPaCa-2 (77), human leukemia HL-60 (78), hepatic stellate cells (79), human thyroid carcinoma cell lines (80), human melanoma cells OCM-1 (81), human epidermoid carcinoma A431 (82), and human prostatic tumor cells (83). Apigenin also has an antiproliferation effect in mouse erythroleukemia cells (84), B104 rat neuronal cells (85), and rat aortic vascular smooth-muscle cells (86). This exhaustive list suggests that flavonoids play an essential role in antiproliferation of numerous cancer cells.

Luteolin, as well as quercetin, kaempferol, genistein, apigenin, and myricetin, suppresses the proliferation ofhuman prostatic tumor cells (PC-3), androgen-independent cells, indicating that flavonoids show their antiprolif-eration activity in an androgen-independent manner (83).

The antiproliferation effect of flavonoids may be related to their estrogenic and/or antiestrogenic activity. Luteolin exhibits cell-proliferation-inhibiting activity and is able to inhibit the proliferation-stimulating activity in MCF-7 cells caused by environmental estrogens (58,59). Both apigenin and luteolin inhibit proliferation of several human thyroid carcinoma cell lines, UCLA NPA-87-1 (with estrogen receptor ER), UCLA RO-82W-1 (with anti-estrogen-binding site AEBS), and UCLA RO-81A-1 (lacking both the AEBS and the ER), suggesting that the inhibitory activity of flavonoids may be mediated via the antiestrogen binding site (AEBS and/or type II estrogen receptor, EBS), but other mechanisms of action are involved as well (80).

Further studies showed that the inhibitory effect of apigenin on UCLA RO-81-A-1 cell proliferation is associated with an inhibition of epidermal growth factor receptor (EGFR) tyrosine autophosphorylation, phosphorylation of its downstream effector mitogen-activated protein (MAP) kinase, and phosphorylated c-Myc, a nuclear substrate for MAPK. However, protein levels of these signaling molecules were not affected (Fig. 3) (87).

The EGFR or MAPK inhibition effect offlavonoids in several other cell lines indicates that flavonoids may inhibit cell proliferation through a rather general mechanism, inhibiting activity of crucial enzymes involved in cell signaling prone to cell proliferation. The antiproliferation activity of luteolin (20 |aM) in pancreatic cancer cell MiaPaCa-2 is closely related to the decreases

Figure 3 Mechanisms that could have been involved in the antiproliferation effect by flavonoids of chrysanthemum, in particular luteolin and apigenin. Growth factors, such as EGF and PDGF, promote cell proliferation through the MAP kinase pathway, in which phosphorylation and dephosphorylation of enzymes play a role in regulating activity of important molecules in cell signaling and cell proliferation. Flavonoids may inhibit cell proliferation through inhibiting EGFR autophosphorylation, phosphorylation of MAP kinase, and its nuclear substrate c-myc. As inhibitors of topoisomerase, flavonoids may show their antiproliferation activity by suppresing DNA replication.

Figure 3 Mechanisms that could have been involved in the antiproliferation effect by flavonoids of chrysanthemum, in particular luteolin and apigenin. Growth factors, such as EGF and PDGF, promote cell proliferation through the MAP kinase pathway, in which phosphorylation and dephosphorylation of enzymes play a role in regulating activity of important molecules in cell signaling and cell proliferation. Flavonoids may inhibit cell proliferation through inhibiting EGFR autophosphorylation, phosphorylation of MAP kinase, and its nuclear substrate c-myc. As inhibitors of topoisomerase, flavonoids may show their antiproliferation activity by suppresing DNA replication.

in cellular protein phosphorylation by effectively modulating protein tyrosine kinase (PTK) activities, including that of EGFR, but not modifying the protein synthesis (88). The same group found that luteolin inhibits proliferation of human epidermoid carcinoma A431 by a similar mechanism (82). Apigenin was found to inhibit fetal bovine serum (FBS)- and platelet-derived growth factor PDGF-induced rat aortic vascular smooth muscle cell proliferation. The activity may be mediated, at least in part, by downregulation of extracellular-signal-regulated kinase ERK 1/2 and its downstream c-fos mRNA (86).

The antiproliferation effect of flavonoids is concomitant with cell cycle arrest or apoptosis in specific cell lines. For example, luteolin inhibits the proliferation of human leukemia HL-60 at 15 ||M and is concomitant with apoptosis-inducing effect (78). Both apigenin and luteolin inhibit proliferation of human melanoma cells OCM-1 (89). The inhibition is mediated by arresting the cell at phase G1 or G2/M. More details about cell-cycle-arrest-or apoptosis-inducing effects of flavonoids will be elaborated below. As inhibitor of topoisomerase I and II, flavonoids may also suppress cell proliferation by inhibiting DNA synthesis (see below).

Although it seems that flavonoids inhibit proliferation of many cell lines, the antiproliferation activity is cell-type-specific as well as flavonoid-structure-specific; for example, neither apigenin nor luteolin shows significant growth-inhibitory activity in B16 melanoma 4A5 cells (81).

7. Induction of Cell Cycle Arrest

Cell cycle progression is timely regulated by cyclin-dependent kinases (CDKs) and their cyclin subunits. G1 progression and G1/S transition are regulated by CDK4-cyclin D, CDK6-cyclin D, and later CDK2-cyclin E, while CDK2 controls S-phase when associated with cyclin A and G2/M transition is regulated by CDK1 in combination with cyclins A and B (90).

Activation of CDKs is regulated by cyclin synthesis and degradation, phosphorylations and dephosphorylations on specific residues, CDK inhibitor (CKIs) synthesis, and degradation. For example, dephosphorylation of CDK1 on Thr14 and Tyr15 residues by the phosphatase CDC25C is required for the activation of CDK1. Two families of mammalian CKIs have been identified: the INK4 family, comprising p16INK4a, p15INK4b, p18INK4c, and p18INK4d, which specifically inhibit CDK4 and CDK6, and the CIP/ KIP family, including p21cip1/waf1, p27kip1, and p57kip2, which have a broad range of inhibition (Fig. 4).

Recently, cell cycle checkpoints have been the targets for chemothera-peutic and chemopreventive agents. Inhibition of the cell cycle at two major checkpoints, G1 and G2/M, may be important in the antitumor activities of the flavonoids (91,92). A large number of epidemiological studies as well as

Figure 4 Pathways involved in the cell-cycle-arresting effect of flavonoids. Cell cycle progress is regulated directly by CDKs and cyclins, activation of which is regulated by CKIs, cdc25c, pBR, p53, etc. Flavonoids of chrysanthemum may arrest the cell cycle at G1/S transition by increasing CKIs, decreasing CDK2, or pRb activation. They also induce G2/M arrest by decreasing the protein level of CDK1 and cyclin A and B.

Figure 4 Pathways involved in the cell-cycle-arresting effect of flavonoids. Cell cycle progress is regulated directly by CDKs and cyclins, activation of which is regulated by CKIs, cdc25c, pBR, p53, etc. Flavonoids of chrysanthemum may arrest the cell cycle at G1/S transition by increasing CKIs, decreasing CDK2, or pRb activation. They also induce G2/M arrest by decreasing the protein level of CDK1 and cyclin A and B.

experiments on animal models suggest that flavonoids can prevent or inhibit cancer development. In several in vitro experiments, flavonoids have been found to inhibit the proliferation of many cancer cells by arresting cell cycle progression at either G1 or G2/M phase. The site of checkpoint arrest with a specific flavonoid can vary with cell type although apigenin and luteolin are very similar in structure.

G1 Arrest. Luteolin arrests the cell cycle at G1 phase in human gastric cancer HGC-27 cells, human melanoma cells OCM-1 (89), and human prostate cancer cells LNCaP (93). G1 cell cycle arrest induced by luteolin on OCM-1 is mediated by inhibiting the activity of CDK2 but not CDK1. Upregulation of the CDK inhibitors p27/kip1 and p21/waf1 is responsible for the inhibition of activity of CDK2 (89).

Only two cell lines, human diploid fibroblast and LNCaP, were found to be arrested at G1 phase by apigenin, while it could arrest more than 10 cell lines at G2/M phase. Apigenin induced G1 arrest on these two cell lines by inhibiting cdk2 kinase activity too (94,95). As for LNCaP cells, apigenin decreases the protein expression of cyclin D1, D2, and E and their activating partner cdk2, 4, and 6 with concomitant induction of CDK inhibitor p21/

wafl and p27/kip1. The induction of p21/waf1 appears to be transcriptionally upregulated and is tumor-suppressor-gene-p53-dependent. In addition, apigenin inhibited the hyperphosphorylation of the pRb protein in these cells

G2/M Arrest. Since the first report that apigenin inhibited the proliferation of malignant tumor cells by G2-M arrest in 1994 (85), apigenin has been found to induce G2/M arrest in 16 cell lines, including human prostate cancer cells LNCaP, human melanoma cells OCM-1, human leukemia HL-60, human colorectal cancer cells SW480, HT-29, and Caco-2, human breast cancer MCF7 and MDA-MB-468, rat hepatoma 5L and BP8, rat neuronal cells B104 (85), human prostate adenocarcinoma CA-HPV-10

(96), human endothelial cells, mouse skin cell line C50 and 308, and mouse keratinocytes (97).

G2/M arrest on most cells induced by apigenin is concomitant with change in CDK kinase activity or kinase protein level. Apigenin inhibits colon carcinoma cell SW480, HT-29, and Caco-2, mouse-skin-derived cell lines C50 and 308, and human leukemia HL-60 cell growth by inducing G2/M arrest with inhibited activity of cdc2 kinase and reduced accumulation of cyclin B1 proteins in a dose-dependent manner (97,98). Cdc2 protein level was reduced by apigenin in three colon cells while apigenin treatment did not change the steady-state level of cdc2 protein in C50, 308, and HL-60 cell lines.

Similarly, G2/M arrest in mouse keratinocytes induced by apigenin was accompanied by inhibition of both cdc2 kinase protein level and activity in a p21/waf1-independent manner (99). In contrast, apigenin arrested LNCaP prostate cancer cells at the G2/M phase by increasing p21 levels through a p53-dependent pathway (93).

Cyclin B1 and CDK1 protein levels decreased significantly in G2/M arrest on breast carcinoma cells MCF-7 and MDA-MB-468 after apigenin treatment. Apigenin reduced the protein levels of CDK4 and cyclins D1 and A, but did not affect cyclin E, CDK2, or CDK6 protein expression. Apigenin markedly reduced Rb phosphorylation in MCF-7 cells while inhibiting ERK MAP kinase phosphorylation and activation in MDA-MB-468 cells (100). On the contrary, accumulation of the hyperphosphorylated form of the retino-blastoma protein results in G2/M arrest in endothelial cell after apigenin treatment (75).

Apigenin induces G2 arrest on human melanoma cells OCM-1 by inhibiting CDK1 by 50-70% owing to the phosphorylation of the kinase on Tyr15 residue (89). Aryl hydrocarbon receptor (AHR) is believed to be a possible receptor of flavonoid in the cells. Apigenin arrests both AHR-containing rat hepatoma 5L and AHR-deficient cell line BP8 primarily in G2/M, which indicates that the cytostatic activities of apigenin do not require the AHR (101).

Only two prostate cell lines, LNCaP and PC-3, have been shown to be arrested by luteolin at the G2/M phase. Furthermore, it has been shown that luteolin can induce p21/waf1 production in a p53-independent manner (93).

8. Induction of Apoptosis

Apoptosis is a tightly regulated cell death characterized by specific morphological and biochemical changes including cell shrinkage, nuclear fragmentation, chromatin condensation, membrane blebbing, and formation of apoptotic bodies. Mitochondria are the pivotal mediator ofapoptosis, which, like a big ''funnel,'' accumulate cell death signals from both ligand-receptor interaction and stresses and then release the molecules leading to cell death such as cytochrome C, Apaf-1, procaspase-9, Smac, and apoptosis-inducing factors (AIFs). Cytochrome C with Apaf-1 and procaspase-9 trigger a caspase cascade that results in morphological and biochemical changes. The activation of mitochondria is tightly regulated by bcl-2 family proteins; for example, Bid, Bad, and Bax can induce mitochondria to release cytochrome C while Bcl-2 prevents the induction (Fig. 5). On the other hand, NF-kB is an important transcriptional factor that prevents cell death. The translocation and activation of NF-kB is regulated by the inhibitor of NF-kB (IkB).

Apoptosis is crucial for development, organ morphogenesis, tissue homeostasis, and removal of infected or damaged cells. The accumulation of damaged cells in the tissue resulting from lack of proper apoptosis is believed to lead to the generation of tumor. Many molecules involved in the apoptosis pathway, such as NF-kB, IkB, and p53, are good targets to find new chemopreventive or chemotherapic compounds. Luteolin and apigenin can induce apoptosis in both cancer cell lines and nontumor cell lines, including human epidermoid carcinoma A431, human leukemia HL-60, human prostate carcinoma LNCaP, MiaPaCa-2, normal human prostate epithelial cells, and nontumor cell line C3H10T1/2CL8 (96). Extensive research has been carried out on the apoptosis-inducing activity of apigenin and luteolin in HL-60 and LNCaP cells.

By forming a luteolin-topoII-DNA ternary complex, luteolin induces apoptosis in HL-60 cells with no increase in 8-oxodG formation but with inhibited topoisomerase II (topo II) activity and cleaved DNA (102). Apigenin can also induce HL-60 apoptosis through a rapid induction of caspase-3 activity and stimulated proteolytic cleavage ofpoly-(ADP-ribose) polymerase (PARP). Apigenin induced loss of mitochondrial transmembrane potential, elevation of ROS production, release of mitochondrial cytochrome c into the cytosol, and subsequent induction of procaspase-9 processing (103).

Apigenin treatment also resulted in apoptosis in human prostate carcinoma LNCaP cells as determined by DNA fragmentation, PARP cleavage, fluorescence microscopy, and flow cytometry. These effects were


Figure 5 Apoptosis-inducing pathway induced by flavonoids of chrysanthemum. Cytochrome C, Apaf-1, and pro-caspase-9, released from mitochondria in response to apoptosis activators, will activate caspase-9 and then caspase-3 and finally induce apoptosis. Smac, also released from mitochondria, promotes apoptosis by inhibiting the suppressing effect of inhibitor of apoptosis proteins (IAPs). Apoptosis-inducing factors (AIFs) can induce activation of caspase-3 directly, which is believed to be the executor caspase to apoptosis. Bid, Bad, and Bax trigger the mitochondria change, which is prevented by bcl-2 protein. Flavonoids from chrysanthemum may induce apoptosis by inhibiting InB degradation, decreasing bcl-2 protein, and triggering release of several molecules from mitochondria.


Figure 5 Apoptosis-inducing pathway induced by flavonoids of chrysanthemum. Cytochrome C, Apaf-1, and pro-caspase-9, released from mitochondria in response to apoptosis activators, will activate caspase-9 and then caspase-3 and finally induce apoptosis. Smac, also released from mitochondria, promotes apoptosis by inhibiting the suppressing effect of inhibitor of apoptosis proteins (IAPs). Apoptosis-inducing factors (AIFs) can induce activation of caspase-3 directly, which is believed to be the executor caspase to apoptosis. Bid, Bad, and Bax trigger the mitochondria change, which is prevented by bcl-2 protein. Flavonoids from chrysanthemum may induce apoptosis by inhibiting InB degradation, decreasing bcl-2 protein, and triggering release of several molecules from mitochondria.

found to correlate with a shift in Bax/Bcl-2 ratio more toward apoptosis. Apigenin treatment also resulted in downmodulation of the constitutive expression of NF-kappaB/p65 (95).

The antiproliferative effect of luteolin might result from the modulation of the EGF-mediated signaling pathway. Blockade of the EGFR-signaling pathway by the protein tyrosine kinase (PTK) inhibitor luteolin significantly inhibits the growth of MiaPaCa-2 cells and induces apoptosis (88).

Treatment of WEHI 231 cells with apigenin decreased the rate of IkB turnover and nuclear levels of NF-kB (104). Apigenin blocked the LPS-induced activation of nuclear factor-nB (NF-kB) through the prevention of inhibitor kB (IkB) degradation and IkB kinase activity. This suggests that apigenin may be important in the prevention of carcinogenesis and inflammation (105).

Apigenin and luteolin also induced p53 accumulation and apoptosis in nontumor cell line C3H10T1/2CL8. The apoptosis apparently occurs in the p53-dependent G2/M phase of the cell (106).

9. Inhibition of Kinases Involved in MAPK Pathway

MAP kinase signal transduction is a typical and vital mediator of a number of cellular activities, including cell growth, proliferation, and survival. There are three major MAP kinases, extracellular signal-regulated kinases (ERKs), c-jun NH2-terminal protein kinase (JNK), and p38. Each MAP is a member of a three-protein kinase cascade including MAPKKKs, MAPKKs, and MAPK. The most extensively studied MAP kinase pathway is the Raf-MEK-ERK cascade. ERKs pathway is activated in response to the stimuli from growth factors such as insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) (88). Ligand-receptor interaction triggers the dimerization and activation of receptor tyrosine kinases (RTKs) due to autophosphorylation. The activated RTKs activate the Raf-MEK-ERK pathway via the protein kinase C (PKC) or phosphatidylinositol-3-kinase (PI3K) pathway (107). Once ERKs are activated, ERKs can phosphorylate and activate transcription factors, which will have an effect on the cell growth or proliferation. JNKs are activated by two MAPK kinases, MKK4 and MKK7, in response to stimuli such as inflammatory cytokines (TNF, IL-lbeta), ultraviolet light, ROS, and heat shock. The activated JNKs will phosphorylate c-jun, which is an important transcription factor in effecting cell growth, inflammation, apoptosis, or differentiation. p38 is activated by MKK3 and MKK6 in response to stimuli similar to those that trigger JNKs. Chemopreventive or chemotherapeutic agents are targeted on various points along the MAPK pathway, such as inhibition of ligand-receptor interaction, inhibition of activity of receptor tyrosine kinase, PI3K, and PKC, which are also the inhibitory targets of apigenin and luteolin (108-111).

Tyrosine Protein Kinases and MAP Kinase. Apigenin and luteolin are effective inhibitors of the proliferation of certain human thyroid cancer cell lines. The dose-dependent inhibitory effect of apigenin on a human anaplastic thyroid carcinoma cell line proliferation is associated with an inhibition of both EGFR tyrosine autophosphorylation and phosphorylation of its downstream effector MAP kinase (80). Apigenin is also a proposed inhibitor of the mitogen-activated protein kinase (MAPK) pathway by inhibiting MAP kinase directly (112,113).

Luteolin inhibits the growth and reduces the cellular protein phospho-rylation level by effectively inhibiting epidermal growth factor receptor (EGFR) tyrosine kinase activity of MiaPaCa-2 cancer cells. The inhibition of EGFR activity may partly contribute to the apoptosis in MianPaCa-2 cells

(77). Pretreatment of the cells with luteolin attenuates LPS-induced tyrosine phosphorylation of cellular proteins and then abolishes the decreasing effects of LPS on InB-alpha in murine macrophages (114).

Apigenin suppresses 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-mediated growth of NIH 3T3 cells by inhibiting activity of fibroblast growth factor (FGF) receptor and reducing the level of phosphorylation of cellular proteins (115). Apigenin can also reduce the level of TPA-stimulated phosphorylation of cellular proteins and inhibited TPA-induced c-jun and c-fos expression in mouse skin (111).

Protein Kinase C and Phosphoinositide 3-Kinases. Protein kinase C is a family of serine-threonine protein kinases that are involved in signal transduction pathways that regulate growth factor response, proliferation, differentiation, apoptosis, etc. The C-terminal catalytic domain contains several reactive cysteines that are targets for various chemopreventive agents. Modification of these cysteines decreases cellular PKC activity.

PKC is inhibited in a concentration-dependent manner by many flavonoids, including luteolin and apigenin (110,111). Further study showed that luteolin is a potent inhibitor of human mast cell activation through the inhibition of Ca2+ influx and PKC activation (116). On the other hand, apigenin inhibits PKC by competing with adenosine triphosphate (ATP). This may be an additional mechanism by which apigenin inhibits or prevents tumorigenesis.

Both luteolin and apigenin are also effective inhibitors of phosphoino-sitide 3-kinase PI3K, an enzyme recently shown to play an important role in signal transduction and cell transformation (109).

A. Inhibition of Other Enzymes

Topoisomerase. DNA topoisomerases are essential enzymes that catalyze the interconversion of topological isomers of DNA molecules. Acting by sequential breakage and reunion strands of DNA, two topoisomerases (topoisomerase I and topoisomerase II) are involved in many vital cellular processes, such as DNA replication, transcription, recombination, integration, and chromosomal segregation. Many anticancer agents were designed as inhibitors of these two enzymes. The DNA damage resulting from dysfunction of the vital enzymes may induce cell cycle arrest or apoptosis. Several flavonoids have been shown to exert their action by interacting with DNA topoisomerases and promoting site-specific DNA cleavage (117). Luteolin inhibits topoisomerase II activity of HL-60 cells by forming a luteolin-topo II-DNA ternary complex and then induces apoptosis in the cells (102).

By inhibiting DNA synthesis and promoting topoisomerase-II-mediat-ed cleavage of kinetoplast DNA minicircles, luteolin inhibits the growth of

Leishmania donovani promastigotes and arrests cell cycle progression in L. donovani promastigotes, leading to apoptosis in vitro (118). Apigenin was found to inhibit activity of topoisomerase II and induced death of IL-2-dependent CTLL-2 cells with DNA fragmentation (119).

Other than inducing topoisomerase II-mediated apoptosis, luteolin strongly inhibits the catalytic activity of eukaryotic DNA topoisomerase I with an IC50 of 5 ||M. Although luteolin not only intercalates directly with the enzyme but also intercalates with substrate DNA at very high concentration, the compound does not prevent the assembly of enzyme-DNA complex but stabilizes the topoisomerase-DNA covalent complex and blocks the subsequent rejoining of the DNA break (120). Apigenin inhibits topoisomerase I-catalyzed DNA relegation in a similar way as luteolin. It does not act directly on the catalytic intermediate and also does not interfere with DNA cleavage. However, formation of a ternary complex with topoisomerase I and DNA during the cleavage reaction inhibits the following DNA religation step (121).

Matrix Metalloproteinase (MMP). MMPs play a critical role in tumor cell invasion and metastasis. Luteolin suppresses the secretion of two MMPs, MMP2 and MMP9, in A431 cells (82). Apigenin also significantly reduces expression of MMP9 in SKBR-3 cells. The inhibition of expression of MMPs may be due to the inhibited MAPK activity (122).

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