Neuroblastoma is the most common extracranial solid tumor in children causing death at pre-school age, as no cure has yet been developed. We investigated the proteolytic mechanisms for apoptosis in human malignant (N-type) neuroblastoma SH-SY5Y cells following exposure to flavonoids such as apigenin (APG), (-)-epigallocatechin (EGC), (-)-epigallocatechin-3-gallate (EGCG) and genistein (GST). We found decrease in viability of SH-SY5Y cells with an increase in dose of APG, EGC, EGCG and GST. Predominantly apoptosis occurred following exposure of SH-SY5Y cells to 50 microM APG, 50 microM EGC, 50 microM EGCG and 100 microM GST for 24 hr. Apoptosis was associated with increases in intracellular free [Ca(2+)] and Bax:Bcl-2 ratio, mitochondrial release of cytochrome c and activation of caspase-9, calpain and caspase-3. Induction of apoptosis with APG and GST showed activation of caspase-12 as well. Activation of caspase-3 could cleave the inhibitor-of-caspase-activated DNase (ICAD) to release and translocate caspase-3-activated DNase (CAD) to the nucleus. Activation of caspase-8 cleaved Bid to truncated Bid (tBid) in cells treated with EGC and EGCG. EGC and EGCG induced apoptosis with caspase-8 activation and mitochondria-mediated pathway, whereas APG and GST caused apoptosis via an increase in intracellular free [Ca(2+)] with calpain activation and mitochondria-mediated pathway. Activation of different proteases for cell death was confirmed using caspase-8 inhibitor II, calpeptin (calpain inhibitor), caspase-9 inhibitor I and caspase-3 inhibitor IV. Thus, plant-derived flavonoids cause cell death with activation of proteolytic activities of calpain and caspases in SH-SY5Y cells, and therefore serve as potential therapeutic agents for controlling the growth of neuroblastoma.

Apigenin is a natural dietary flavonoid. It has recently been shown to have anticancer effects on prostate and ovarian cancer cells. However, the molecular basis of the effect of apigenin on cancer cells remains to be elucidated. In this study, we found that apigenin inhibited A549 lung cancer cell proliferation and vascular endothelial growth factor (VEGF) transcriptional activation in a dose-dependent manner. In an attempt to understand the mechanism of apigenin-inhibited VEGF expression, we found that apigenin inhibited VEGF transcriptional activation through the hypoxia-inducible factor 1 (HIF-1) binding site and specifically decreased HIF-1alpha but not HIF-1beta subunit expression in the cells. In our efforts to understand the signaling pathway that mediates VEGF transcriptional activation, we found that apigenin inhibited AKT and p70S6K1 activation. When testing the effect of apigenin in vivo, we found that apigenin significantly inhibited tumor growth in nude mice. Apigenin inhibited HIF-1alpha and VEGF expression in the tumor tissues, suggesting an inhibitory effect of apigenin on angiogenesis. To confirm this, we showed that apigenin inhibited angiogenesis in nude mice using the Matrigel assay. HIF-1alpha and VEGF are well known inducers of angiogenesis. Our data suggested that apigenin may inhibit human lung cancer angiogenesis by inhibiting HIF-1alpha and VEGF expression, thus providing a novel explanation for the anticancer action of apigenin.

Prostate cancer is a commonly diagnosed cancer in men. The ethanolic extract of propolis (EEP) and its phenolic compounds possess immunomodulatory, chemopreventive and antitumor effects. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/APO2L) is a naturally occurring anticancer agent that preferentially induces apoptosis in cancer cells and is not toxic to normal cells. We examined the cytotoxic and apoptotic effects of EEP and phenolic compounds isolated from propolis in combination with TRAIL on two prostate cancer cell lines, hormone-sensitivity LNCaP and hormone-refractory DU145. The cytotoxicity was evaluated by MTT and LDH assays. The apoptosis was determined using flow cytometry with annexin V-FITC/propidium iodide. The prostate cancer cell lines were proved to be resistant to TRAIL-induced apoptosis. Our study demonstrated that EEP and its components significantly sensitize to TRAIL-induced death in prostate cancer cells. The percentage of the apoptotic cells after cotreatment with 50 μg mL(-1) EEP and 100 ng mL(-1) TRAIL increased to 74.9 ± 0.7% for LNCaP and 57.4 ± 0.7% for DU145 cells. The strongest cytotoxic effect on LNCaP cells was exhibited by apigenin, kaempferid, galangin and caffeic acid phenylethyl ester (CAPE) in combination with TRAIL (53.51 ± 0.68-66.06 ± 0.62% death cells). In this work, we showed that EEP markedly augmented TRAIL-mediated apoptosis in prostate cancer cells and suggested the significant role of propolis in chemoprevention of prostate cancer.

Cancer metastasis refers to the spread of cancer cells from the primary neoplasm to distant sites, where secondary tumors are formed, and is the major cause of death from cancer. Natural phytochemicals containing phenolic compounds have been widely demonstrated to have the capability to prevent cancer metastasis. Among phenolic compounds, flavonoids are a very large subclass, and they are abundant in food and nutraceuticals. The number of reports demonstrating that flavonoids are an effective natural inhibitor of cancer invasion and metastasis is increasing in the scientific literature. Catechin derivatives, (−)-epigallocatechin-3-gallate, (−)-epigallocatechin, (−)-epicatechin-3-gallate,and (−)-epicatechin, are the most studied compounds in this topic so far; genistein/genistin, silibinin, quercetin, and anthocyanin have also been widely investigated for their inhibitory activities on invasion/metastasis. Other flavonoids in dietary vegetable foods that are responsible for anti-invasive and anti-metastatic activities of tumors include luteolin,apigenin, myricetin, tangeretin, kaempferol, glycitein, licoricidin,daidzein, and naringenin. To effectively overcome the metastatic cascade, including cell-cell attachment, tissue barrier degradation, migration, invasion, cell-matrix adhesion,and angiogenesis, it is essential that a bioactive compound prevent tumor cells from metastasizing. This review summarizes the effects of flavonoids on the metastatic cascade and the related proteins, the in vitro anti-invasive activity of flavonoids against cancer cells, and the effects of flavonoids on antiangiogenic and in vivo anti-metastatic models. The available scientific evidence indicates that flavonoids are a ubiquitous dietary phenolics subclass and exert extensive in vitro anti-invasive and in vivo anti-metastatic activities.

Evidence suggests that tumor microenvironment is critically involved in supporting survival of chronic lymphocytic leukemia (CLL) cells. However, the molecular mechanisms of this effect and the clinical significance are not fully understood. We applied a microenvironment model to explore the interaction between CLL cells and stromal cells and to elucidate the role of phosphatidylinositol 3 kinase (PI3-K)/Akt/phosphatase and tensin homolog detected on chromosome 10 (PTEN) cascade in this process and its in vivo relevance. Primary human stromal cells from bone marrow, lymph nodes, and spleen significantly inhibited spontaneous apoptosis of CLL cells. Pan-PI3-K inhibitors (LY294002, wortmannin, PI-103), isotype-specific inhibitors of p110α, p110β, p110γ, and small interfering RNA against PI3-K and Akt1 counteracted the antiapoptotic effect of the stromal cells. Induction of apoptosis was associated with a decrease in phosphatidylinositol-3,4,5-triphosphate, PI3-K-p85, and dephosphorylation of phosphatidylinositol-dependent kinase-1 (PDK-1), Akt1, and PTEN. Freshly isolated peripheral blood mononuclear cells from patients with CLL (n = 44) showed significantly higher levels of phosphorylated Akt1, PDK-1, PTEN, and CK2 than healthy persons (n = 8). CK2 inhibitors (4,5,6,7-tetrabromo-1H-benzotriazole, apigenin, and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazol) decreased phosphorylation of PTEN and Akt, induced apoptosis in CLL cells, and enhanced the response to fludarabine. In conclusion, bone marrow microenvironment modulates the PI3-K/Akt/PTEN cascade and prevents apoptosis of CLL cells. Combined inhibition of PI3-K/Akt and recovery of PTEN activity may represent a novel therapeutic concept for CLL.

The effect of some flavonoids, which are components of fruits, vegetables, and peas, on the cell cycle progression of human LNCaP prostate cancer cells has been investigated in this study. Genistein arrested the cell cycle at the G2/M phases, which is attributed to the suppression of cyclin B expression. In addition, genistein induced the cyclin-dependent kinase inhibitor p21, which does not depend on p53 activation. Apigenin and luteolin also increased p21 levels, but quercetin did not. Apigenin induced p21 production through a p53-dependent pathway, but luteolin did so in a p53-independent manner. These results suggest that flavonoids are potent regulators of cyclin B and p21 for cell cycle progression, which may play some roles in prevention of carcinogenesis.

Hepatocyte growth factor (HGF) and its receptor, Met, known to control invasive growth program have recently been shown to play crucial roles in the survival of breast cancer patients. The diet-derived flavonoids have been reported to possess anti-invasion properties; however, knowledge on the pharmacological and molecular mechanisms in suppressing HGF/Met-mediated tumor invasion and metastasis is poorly understood. In our preliminary study, we use HGF as an invasive inducer to investigate the effect of flavonoids including apigenin, naringenin, genistein and kaempferol on HGF-dependent invasive growth of MDA-MB-231 human breast cancer cells. Results show that apigenin presents the most potent anti-migration and anti-invasion properties by Boyden chamber assay. Furthermore, apigenin represses the HGF-induced cell motility and scattering and inhibits the HGF-promoted cell migration and invasion in a dose-dependent manner. The effect of apigenin on HGF-induced signaling activation involving invasive growth was evaluated by immunoblotting analysis, it shows that apigenin blocks the HGF-induced Akt phosphorylation but not Met, ERK, and JNK phosphorylation. In addition to MDA-MB-231 cells, apigenin exhibits inhibitory effect on HGF-induced Akt phosphorylation in hepatoma SK-Hep1 cells and lung carcinoma A549 cells. By indirect immunofluorescence microscopy assay, apigenin inhibits the HGF-induced clustering of beta 4 integrin at actin-rich adhesive site and lamellipodia through PI3K-dependent manner. Treatment of apigenin inhibited HGF-stimulated integrin beta 4 function including cell-matrix adhesion and cell-endothelial cells adhesion in MDA-MB-231 cells. By Akt-siRNA transfection analysis, it confirmed that apigenin inhibited HGF-promoted invasive growth involving blocking PI3K/Akt pathway. Finally, we evaluated the effect of apigenin on HGF-promoted metastasis by lung colonization of tumor cells in nude mice and organ metastasis of tumor cells in chick embryo. By histological and gross examination of mouse lung and real-time PCR analysis of human alu in host tissues, it showed that apigenin, wortmannin, as well as anti-beta 4 antibody all inhibit HGF-promoted metastasis. These data support the inhibitory effect of apigenin on HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and integrin beta 4 function.

Topoisomerases are involved in many aspects of DNA metabolism such as replication and transcription reactions. Camptothecins, which stabilize the covalent intermediate of topoisomerase I and DNA are effective, though toxic, drugs for cancer therapy. In this study, a new class of topoisomerase I inhibitors was identified, and their mode of action was characterized using recombinant human topoisomerase I preparations and human HL-60 leukemic cells. Quercetin and the related natural flavones, acacetin, apigenin, kaempferol, and morin, inhibit topoisomerase I-catalyzed DNA religation. In contrast to camptothecin, these compounds do not act directly on the catalytic intermediate and also do 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. 3,3',4',7-Tetrahydroxy-substituted flavones stabilize the covalent topoisomerase I-DNA intermediate most efficiently. Enhanced formation of covalent topoisomerase I-DNA complexes was also demonstrated in human HL-60 cells. In contrast, synthetic 3,5'-dibromo- 4'-hydroxy-3-methylflavones bind selectively to topoisomerase I in its non-DNA-bound form and block the following DNA binding step. As a consequence, these synthetic flavonoids are capable of counteracting topoisomerase I-directed effects of camptothecin. Inhibition of DNA binding is obtained by voluminous hydrophobic substituents in 6-position of the flavone structure. Our data show that selective inhibitors of both half-reactions of topoisomerase I can be derived from the flavone structure.

Flavonoids have been intensively studied on their pharmacological activities such as anti-cancer, anti-oxidant and anti-inflammation. However, little is known about their neuroprotective effects. Recent studies suggest that inflammation mediated by microglia may play a role in neurodegenerative diseases. In this study, we evaluated the anti-inflammatory effect of various flavonoid compounds by using BV-2, a murine microglia cell line. Of the compounds that were evaluated, apigenin inhibited the production of nitric oxide and prostaglandin E(2) by suppressing the expression of inducible nitric oxide synthase and cyclooxygenase-2 protein, respectively. Moreover, apigenin suppressed p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) phosphorylation without affecting the activity of extracellular signal-regulated kinase (ERK). Apigenin was also found to protect neuronal cells from injury in middle cerebral artery occlusion.

Poor oral bioavailability has been a major limitation for the successful use of dietary flavonoids as cancer chemopreventive agents. In this study, we examined fully methylated flavones as promising improved agents. In the human oral SCC-9 cancer cells, 5,7-dimethoxyflavone and 5,7,4'-trimethoxyflavone were both 10 times more potent inhibitors of cell proliferation (IC(50) values 5-8 microM) than the corresponding unmethylated analogs chrysin and apigenin. Flow cytometry indicated that both methylated flavones arrested the SCC-9 cells in the G1 phase with a concomitant decrease in the S phase, dramatically different from the unmethylated analogs, which promoted G2/M phase arrest. Both methylated compounds inhibited the proliferation of two other cancer cell lines with very little effect on two immortalized normal cell lines. Examination of additional flavone structures indicated that methylated flavones in general have antiproliferative properties. Finally, we demonstrated that 5,7-dimethoxyflavone, in contrast to its unmethylated analog chrysin, was well absorbed and had high oral bioavailability as well as tissue accumulation in vivo in the rat. Thus, fully methylated flavones appear to have great potential as cancer chemopreventive/chemotherapeutic agents, in particular in oral cancer.

BACKGROUND

Flavonoids may protect against cancer development through several biological mechanisms. However, epidemiologic studies on dietary flavonoids and cancer risk have yielded inconsistent results.

OBJECTIVE

We prospectively investigated the association between the intake of selected flavonoids and flavonoid-rich foods and risk of cancers in the Women's Health Study.

METHODS

A total of 3234 incident cancer cases were identified during 11.5 y of follow-up among 38,408 women aged>> or = 45 y. Intake of individual flavonols (quercetin, kaempferol, and myricetin) and flavones (apigenin and luteolin) was assessed from food-frequency questionnaires. Cox regression models were used to estimate the relative risk (RR) of total and site-specific cancer across increasing intakes of total and individual selected flavonoids and flavonoid-rich foods (tea, apple, broccoli, onion, and tofu).

RESULTS

The multivariate RRs of total cancer across increasing quintiles of total quantified flavonoid intake were 1.00, 1.00, 0.93, 0.94, and 0.97 (P for trend = 0.72). For site-specific cancers, the multivariate RRs in the highest quintile of total quantified flavonoid intake compared with the lowest quintile were 1.03 for breast cancer, 1.01 for colorectal cancer, 1.03 for lung cancer, 1.15 for endometrial cancer, and 1.09 for ovarian cancer (all P>> 0.05). The associations for the individual flavonoid intakes were similar to those for the total intake. There was also no significant association between intake of flavonoid-rich foods and the incidence of total and site-specific cancers.

CONCLUSIONS

Our results do not support a major role of 5 common flavonols and flavones or selected flavonoid-rich foods in cancer prevention.

Apigenin, a widely distributed plant flavonoid, was previously found to inhibit chemically induced ornithine decarboxylase (ODC) activity and skin tumor promotion. The purpose of the present research was to determine if apigenin is effective in the prevention of ultraviolet-B light (UVB) induced skin carcinogenesis. Further studies ascertained if apigenin would be expected to absorb UVB light in a manner to prevent DNA damage in a cell free system. ODC activity was induced with 0.45 J/cm2 ultraviolet A and B (UVA/B) light. Apigenin (5 mumoles/200 microliters DMSO:acetone, 1:9) treatment from 12 hours before until 1 hour following UVA/B exposure was effective in inhibition (25-45% inhibition) of ODC activity measured at 28 hours following UVA/B exposure. Mouse skin carcinogenesis was induced by exposure to a total dose of 40 J/cm2 UVB over 11 weeks. Treatment with 10 mumoles apigenin in 200 microliters DMSO:acetone (1:9) prior to each UVB exposure resulted in reduction in cancer incidence (52% inhibition) and an increase in tumor free survival in comparison with control mice (P < 0.01). Apigenin (0-100 microM) did not prevent the in vitro production of photoproducts in salmon sperm DNA suggesting that apigenin did not inhibit UVA/B induced ODC activity or UVB induced skin carcinogenesis by simply absorbing ultraviolet light or decreasing DNA damage.

The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser(2). This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology.

Since reactive oxygen radicals play an important role in carcinogenesis and other human disease states, antioxidants present in consumable fruits, vegetables, and beverages have received considerable attention as cancer chemopreventive agents. Thus, in order to identify antioxidants in plant extracts, test materials were assessed for potential to scavenge stable 1,2-diphenyl-2-picrylhydrazyl (DPPH) free radicals, reduce TPA-induced free radical formation in cultured HL-60 human leukemia cells, and inhibit responses observed with a xanthine/xanthine oxidase assay system. Approximately 700 plant extracts were evaluated, and 28 were found to be active in the DPPH free radical scavenging assay. Based on secondary analyses performed to assess inhibition of 7,12-dimethylbenz(a)anthracene-induced preneoplastic lesion formation with a mouse mammary organ culture model, Chorizanthe diffusa Benth. (Polygonaceae), Mezoneuron cucullatum Roxb. (Leguminosae), Cerbera manghas L. (Apocynaceae) and Daphniphyllum calycinum Benth. (Daphniphyllaceae) were selected and subjected to bioassay-guided fractionation. 5,7,3',5'-Tetrahydroxy-8,4'-dimethoxyflavonol, 5,8,4'-trihydroxy-7,3'-dimethoxyflavonol, 5,3',4'-trihydroxy-7-methoxyflavonol, and 6,3',4'-trihydroxy-7-methoxyflavonol were identified as active principles from C. diffusa. Piceatannol, trans-resveratrol, apigenin and scirpusin A were found as the active principles of M. cucullatum, olivil, (-)-carinol, and (+)-cycloolivil were active principles from C. manghas, and 5,6,7,4'-tetrahydroxyflavone 3-O-rutinoside and kaempferol 3-O-neohesperidoside were active principles from D. calycinum. Of these substances, the hydroxystilbenes piceatannol and transresveratrol have thus far been shown to inhibit carcinogen-induced preneoplastic lesion formation in the mouse mammary gland organ culture model.

OBJECTIVE

Development of androgen independence and resistance to apoptosis in prostate cancer are often correlated with high levels of serum tumor necrosis factor (TNF)-alpha in these patients. The loss of sensitivity to TNF-alpha-induced apoptosis in androgen-insensitive prostate carcinoma cells is due in part to constitutive activation of Rel/nuclear factor (NF)-kappaB transcription factors that regulate several cell survival and antiapoptotic genes. Our previous studies have demonstrated growth inhibitory and apoptotic effects of apigenin, a common plant flavonoid, in a variety of human prostate carcinoma cells. Here we examined whether apigenin is effective in inhibiting NF-kappaB expression in androgen-insensitive human prostate carcinoma cells exhibiting high constitutive levels of NF-kappaB.

METHODS

Using androgen-insensitive human prostate carcinoma PC-3 cells, the effect of apigenin was assessed on NF-kappaB activation by electrophoretic mobility shift assay and reporter gene assay. Expression of NF-kappaB subunits p65 and p50, IkappaBalpha, p-IkappaBalpha, in-beads kinase assay and NF-kappaB-regulated genes were determined by Western blot analysis. Apoptosis was determined by annexin V/propidium iodide staining after fluorescence-activated cell-sorting analysis.

RESULTS

Treatment of cells with 10-40- micro M doses of apigenin inhibited DNA binding and reduced nuclear levels of the p65 and p50 subunits of NF-kappaB. Apigenin inhibited IkappaBalpha degradation and IkappaBalpha phosphorylation and significantly decreased IKKalpha kinase activity. Apigenin also inhibited TNF-alpha-induced activation of NF-kappaB via the IkappaBalpha pathway, thereby sensitizing the cells to TNF-alpha-induced apoptosis. The inhibition of NF-kappaB activation correlated with a decreased expression of NF-kappaB-dependent reporter gene and suppressed expression of NF-kappaB-regulated genes [specifically, Bcl2, cyclin D1, cyclooxygenase-2, matrix metalloproteinase 9, nitric oxide synthase-2 (NOS-2), and vascular endothelial growth factor].

CONCLUSIONS

Our results indicate that inhibition of NF-kappaB by apigenin may lead to prostate cancer suppression by transcriptional repression of NF-kappaB-responsive genes as well as selective sensitization of prostate carcinoma cells to TNF-alpha-induced apoptosis.

Dietary supplements are not subject to the same pre-market approval as conventional drugs, thus the true efficacy and, in cases, safety of these products is not known. The objective of this study was to evaluate the potential anti-inflammatory properties of three herbal constituents, apigenin (chamomile), ginsenoside Rb(1) (ginseng) and parthenolide (feverfew) on lipopolysaccaharide (LPS)-induced proinflammatory cytokine production, and to determine if effects in cell culture could predict results in an intact animal model. Murine macrophage cells and mice were treated with the stimulant LPS and herbal constituents, and resultant culture supernatant and serum, respectively, were evaluated for interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha by ELISA. All three constituents inhibited LPS-induced IL-6 and/or TNF-alpha production in culture. Inhibition of these two cytokines was observed in mice, but did not display the same patterns of inhibition as cell culture data. The results suggest that all three constituents possessed anti-inflammatory properties, but that cell culture data can only be used to approximate potential effects in animals, and must be confirmed using appropriate animal models.

Apigenin is a common dietary flavonoid that is abundantly present in many fruits, vegetables and Chinese medicinal herbs and serves multiple physiological functions, such as strong anti-inflammatory, antioxidant, antibacterial and antiviral activities and blood pressure reduction. Therefore, apigenin has been used as a traditional medicine for centuries. Recently, apigenin has been widely investigated for its anti-cancer activities and low toxicity. Apigenin was reported to suppress various human cancers in vitro and in vivo by multiple biological effects, such as triggering cell apoptosis and autophagy, inducing cell cycle arrest, suppressing cell migration and invasion, and stimulating an immune response. In this review, we focus on the most recent advances in the anti-cancer effects of apigenin and their underlying mechanisms, and we summarize the signaling pathways modulated by apigenin, including the PI3K/AKT, MAPK/ERK, JAK/STAT, NF-κB and Wnt/β-catenin pathways. We also discuss combinatorial strategies to enhance the anti-cancer effect of apigenin on various cancers and its use as an adjuvant chemotherapeutic agent to overcome cancer drug resistance or to alleviate other adverse effects of chemotherapy. The functions of apigenin against cancer stem cells are also summarized and discussed. These data demonstrate that apigenin is a promising reagent for cancer therapy. Apigenin appears to have the potential to be developed either as a dietary supplement or as an adjuvant chemotherapeutic agent for cancer therapy.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox- sensitive transcription factor regulating expression of a number of cytoprotective genes. Recently, Nrf2 has emerged as an important contributor to chemoresistance in cancer therapy. In the present study, we found that non-toxic dose of apigenin (APG) significantly sensitizes doxorubicin-resistant BEL-7402 (BEL-7402/ADM) cells to doxorubicin (ADM) and increases intracellular concentration of ADM. Mechanistically, APG dramatically reduced Nrf2 expression at both the messenger RNA and protein levels through downregulation of PI3K/Akt pathway, leading to a reduction of Nrf2-downstream genes. In BEL-7402 xenografts, APG and ADM cotreatment inhibited tumor growth, reduced cell proliferation and induced apoptosis more substantially when compared with ADM treatment alone. These results clearly demonstrate that APG can be used as an effective adjuvant sensitizer to prevent chemoresistance by downregulating Nrf2 signaling pathway.

Andrographis paniculata (Burm. f.) Nees (Acanthaceae) is a medicinal plant used in many countries. Its major constituents are diterpenoids, flavonoids and polyphenols. Among the single compounds extracted from A. paniculata, andrographolide is the major one in terms of bioactive properties and abundance. Among the andrographolide analogues, 14-deoxy-11,12-didehydroandrographolide is immunostimulatory, anti-infective and anti-atherosclerotic; neoandrographolide is anti-inflammatory, anti-infective and anti-hepatotoxic; 14-deoxyandrographolide is immunomodulatory and anti-atherosclerotic. Among the less abundant compounds from A. paniculata, andrograpanin is both anti-inflammatory and anti-infective; 14-deoxy-14,15-dehydroandrographolide is anti-inflammatory; isoandrographolide, 3,19-isopropylideneandrographolide and 14-acetylandrographolide are tumor suppressive; arabinogalactan proteins are anti-hepatotoxic. The four flavonoids from A. paniculata, namely 7-O-methylwogonin, apigenin, onysilin and 3,4-dicaffeoylquinic acid are anti-atherosclerotic.

Evidence from laboratory-based in vitro studies provides compelling evidence supporting the involvement of dietary flavonoid intake in human cancer risk. Associations between intakes of individual flavonoids and disease outcomes at the population level are emerging from recent epidemiological studies. As an important step in the development of methods to assess flavonoid intakes across populations, the major sources of dietary flavonoids in the adult Australian population were identified. Data from a 24-h diet recall questionnaire used in a national nutrition survey (NNS95-comprising a sample of 10,851 subjects aged 19 yr and over) were combined with U.S. Department of Agriculture data on flavonoid content of foods to identify key sources. Black and green teas clearly were the dominant sources of the flavonols kaempferol, myricetin, and quercetin. Other significant flavonol sources included onion (isorhamnetin and quercetin), broccoli (kaempferol and quercetin), apple (quercetin), grape (quercetin), coffee (myrcetin), and beans (quercetin). Black and green teas also were dominant sources of flavon-3-ols, with wine, apples, and pears contributing somewhat. In terms of flavanone consumption, oranges (hesperetin and naringenin), lemon (eriodictyol), mandarin (hesperetin), and grapefruit (naringenin) were the major sources. Parsley (apigenin), celery (apigenin and luteolin), and English spinach (luteolin) were the major flavone sources. Wine was the major anthocyanadin source (delphinidin, malvidin, peonidin and petunidin), with smaller amounts from cherry (peonidin) and blueberry (delphinidin, malvidin, peonidin and petunidin). It is suggested that the relatively small number of aforementioned key foods form the basis of food frequency questionnaires to assess flavonoid intake.

We have previously reported that apigenin inhibits the growth of thyroid cancer cells by attenuating epidermal growth factor receptor (EGF-R) tyrosine phosphorylation and phosphorylation of ERK mitogen-activated protein (MAP) kinase. In this study, we assessed the growth inhibitory effect of apigenin on MCF-7 breast carcinoma cells that express two key cell cycle regulators, wild-type p53 and the retinoblastoma tumor suppressor protein (Rb), and MDA-MB-468 breast carcinoma cells that are mutant for p53 and Rb negative. We found that apigenin potently inhibited growth of both MCF-7 and MDA-MB-468 breast carcinoma cells. The approximate IC50 values determined after 3 days incubation, were 7.8 micrograms/ml for MCF-7 cells, and 8.9 micrograms/ml for MDA-MB-468 cells, respectively. Because the cell cycle studies using FACS showed that both MCF-7 and MDA-MB-468 cells were arrested in G2/M phase after apigenin treatment, we studied the effects of apigenin on cell cycle regulatory molecules. We observed that G2/M arrest by apigenin involved a significant decrease in cyclin B1 and CDK1 protein levels, resulting in a marked inhibition of CDK1 kinase activity. Apigenin reduced the protein levels of CDK4, cyclins D1 and A, but did not affect cyclin E, CDK2 and CDK6 protein expression. In MCF-7 cells, apigenin markedly reduced Rb phosphorylation after 12 h. We also found that apigenin treatment resulted in a dose- and time-dependent inhibition of ERK MAP kinase phosphorylation and activation in MDA-MB-468 cells. These results suggest that apigenin is a promising antibreast cancer agent and its growth inhibitory effects are mediated by targeting different signal transduction pathways in MCF-7 and MDA-MB-468 breast carcinoma cells.

Epidemiological studies have indicated a significant difference in the incidence of cancers among ethnic groups, who have different lifestyles and have been exposed to different environmental factors. It has been estimated that more than two-thirds of human cancers, which are contributed by mutations in multiple genes, could be prevented by modification of lifestyle including dietary modification. The consumption of fruits, soybean and vegetables has been associated with reduced risk of several types of cancers. The in vitro and in vivo studies have demonstrated that some dietary components such as isoflavones, indole-3-carbinol (I3C), 3,3'-diindolylmethane (DIM), curcumin, (-)-epigallocatechin-3-gallate (EGCG), apigenin, etc., have shown inhibitory effects on human and animal cancers, suggesting that they may serve as chemopreventive agents. Experimental studies have also revealed that these components regulate the molecules in the cell signal transduction pathways including NF-kappaB, Akt, MAPK, p53, AR, and ER pathways. By modulating cell signaling pathways, these components, among other mechanisms, activate cell death signals and induce apoptosis in precancerous or cancer cells, resulting in the inhibition of cancer development and/or progression. This article reviews current studies regarding the effects of natural chemopreventive agents on cancer-related cell signaling pathways and provides comprehensive knowledge of the biological and molecular roles of chemopreventive agents in cancer cells.

Chamomile (Matricaria chamomilla), a popular herb valued for centuries as a traditional medicine, has been used to treat various human ailments; however, its anticancer activity is unknown. We evaluated the anticancer properties of aqueous and methanolic extracts of chamomile against various human cancer cell lines. Exposure of chamomile extracts caused minimal growth inhibitory responses in normal cells, whereas a significant decrease in cell viability was observed in various human cancer cell lines. Chamomile exposure resulted in differential apoptosis in cancer cells but not in normal cells at similar doses. HPLC analysis of chamomile extract confirmed apigenin 7-O-glucoside as the major constituent of chamomile; some minor glycoside components were also observed. Apigenin glucosides inhibited cancer cell growth but to a lesser extent than the parent aglycone, apigenin. Ex vivo experiments suggest that deconjugation of glycosides occurs in vivo to produce aglycone, especially in the small intestine. This study represents the first reported demonstration of the anticancer effects of chamomile. Further investigations of the mechanism of action of chamomile are warranted in evaluating the potential usefulness of this herbal remedy in the management of cancer patients.

OBJECTIVE

The antiproliferative mechanisms of flavonoid drugs inpancreatic cancer cells remain unclear. In this study, we evaluated the effects of the flavonoid apigenin on glucose uptake, on the expression of the glucose transporter 1 (GLUT-1), and on the phosphoinositide 3-kinase (PI3K)/Akt pathway in human pancreatic cancer cells.

METHODS

Human pancreatic cancer cells were treated with apigenin and then underwent glucose uptake assays. Real-time reverse transcription-polymerase chain reaction and Western blot analysis were conducted to evaluate GLUT-1 and pAkt expression in CD18 and S2-013 human pancreatic cancer cells after treatment with apigenin or PI3K inhibitors (LY294002 and wortmannin).

RESULTS

Apigenin (0-100 microM) significantly inhibited, in a dose-dependent fashion, glucose uptake in CD18 and S2-013 human pancreatic cancer cell lines. Apigenin inhibited both GLUT-1 mRNA and protein expression in a concentration- and time-dependent fashion. The PI3K inhibitors, like apigenin, downregulated both GLUT-1 mRNA and protein expression.

CONCLUSIONS

Our results demonstrate that the flavonoid apigenin decreases glucose uptake and downregulates the GLUT-1 glucose transporter in human pancreatic cancer cells. In addition, the inhibitory effects of apigenin and the PI3K inhibitors on GLUT-1 are similar, indicating that the PI3K/Akt pathway is involved in mediating apigenin's effects on downstream targets such as GLUT-1.