Apigenin is a dietary flavonoid with known antioxidant and antitumor effects against several types of cancers by promoting cell death and inducing cell cycle arrest. Apigenin also regulates a variety of intracellular signal transduction pathways during apoptosis or autophagy. However, the precise mechanism underlying the anticancer effects of apigenin in liver cancer remains poorly understood. In this study, we demonstrated that apigenin has anticancer activity against hepatocellular carcinoma cells. Apigenin inhibited the cell growth and induced cell death in a dose- and time-dependent manner in HepG2 cells. We found that apigenin treatment increased the expression of LC3-II and the number of GFP-LC3 puncta. Moreover, inhibition of autophagy with 3-MA and Atg5 gene silencing strengthened apigenin-induced proliferation inhibition and apoptosis. Our data has indicated that apigenin-induced autophagy has a protective effect against cell death. Additionally, apigenin induced apoptosis and autophagy through inhibition of PI3K/Akt/mTOR pathway. Most importantly, in vivo data showed that administration of apigenin decreased tumor growth and autophagy inhibition by 3-MA significantly enhanced the anticancer effect of apigenin. Collectively, our results reveal that apigenin inhibits cell proliferation and induces autophagy via suppressing the PI3K/Akt/mTOR pathway. Our results also suggest combination of autophagy inhibitors and apigenin would be a potential chemotherapeutic strategy against hepatocellular carcinoma.

The effect of various natural flavonoids, cinnamic acid derivatives, and a series of synthetic flavones on cell proliferation was evaluated in vitro in a panel of established human and murine tumor cell lines. The most potent antiproliferative agents were caffeic acid n-butyl ester (12)>> 2'-nitroflavone (26)>> caffeic acid ethyl ester (11) approximately = 2',6-dinitroflavone (27)>> apigenin (3)>> 3'-bromoflavone (20) approximately = 2'-fluoro-6-bromoflavone (31). Some compounds showed a moderate effect, the order of cytotoxic activities being chrysin (2)>> 2'-fluoro-6-chloroflavone (30) approximately = 2'-chlorochrysin (32)>> alpha-naphthoflavone (7)>> beta-naphthoflavone (8) approximately = 6-chloroflavone (14) approximately = 6-bromoflavone (15) approximately = 4'-nitroflavone (23). A structure-activity relationship analysis of each group of compounds was performed. None of the natural or synthetic compounds tested affected the proliferation of epithelial cells derived from normal mammary gland of mice or fibroblastic cells from mouse embryo, suggesting a selective action against tumor cells.

Progression of cancer leads to hypoxic solid tumors that mount specific cell signaling responses to low oxygen conditions. An important objective of anti-cancer therapy is the development of new drugs that suppress hypoxic responses in solid tumors. Apigenin is a natural flavone that has been shown to have chemopreventive and/or anti-cancer properties against a number of tumor types. However, the mechanisms underlying apigenin's chemopreventive properties are not yet completely understood. In this study, we have investigated the effects of apigenin on expression of hypoxia-inducible factor-1 (HIF-1) in human metastatic prostate PC3-M cancer cells. We found that hypoxia induced a time-dependent increase in the level of HIF-1alpha subunit protein in PC3-M cells, and treatment with apigenin markedly decreased HIF-1alpha expression under both normoxic and hypoxic conditions. Further, apigenin prevented the activation of the HIF-1 downstream target gene vascular endothelial growth factor (VEGF). We then showed that apigenin inhibited expression of HIF-1alpha by reducing stability of the protein as well as by reducing the level of HIF-1alpha mRNA. We also found that apigenin inhibited Akt and GSK-3beta phosphorylation in PC3-M cells. Further experiments demonstrated that constitutively active Akt blunted the effect of apigenin on HIF-1alpha expression. Taken together, our results identify apigenin as a bioflavonoid that inhibits hypoxia-activated pathways linked to cancer progression in human prostate cancer, in particular the PI3K/Akt/GSK-3 pathway. Further studies on the mechanism of action of apigenin will likely provide new insight into its applicability for pharmacologic targeting of HIF-1alpha for cancer therapeutic or chemopreventive purposes.

Flavonoids have beneficial activities which modulate oxidative stress, allergy, tumor growth and viral infection, and which stimulate apoptosis of tumor cells. In addition to these activities, dietary flavonoids are able to regulate acute and chronic inflammatory responses. Here we describe new aspects of regulatory mechanisms by which flavonoids suppress production of tumor necrosis factor-alpha (TNF-alpha) by macrophages, microglial cells and mast cells stimulated with lipopolysaccharide (LPS) and others via toll-like receptors (TLRs), and TNF-alpha-mediated acute and chronic inflammatory responses. Treatment with flavonoids such as luteolin, apigenin, quercetin, genistein, (-)-epigallocatechin gallate, and anthocyanidin resulted in significant downregulation of LPS-elicited TNF-alpha and nitric oxide (NO) production and diminished lethal shock. In chronic diseases, pathogenesis of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis which is triggered by TNF-alpha, was improved by the oral administration of flavonoids after the onset of CIA. Here, we discuss that inhibitory effects of flavonoids on acute and chronic inflammation are due to regulation of signaling pathways, including the nuclear factor kappaB (NF-kappaB) activation and mitogen-activated protein (MAP) kinase family phosphorylation. FcetaRI expression by NF-kappaB activation was also reduced by flavonoids; while accumulation of lipid rafts, which is the critical step for signaling, was blocked by flavonoids. The intake of dietary flavonoids reduces acute and chronic inflammation due to blocking receptor accumulation and signaling cascades, and would assist individuals at high-risk from life-style related diseases.

Apigenin, a dietary plant derived flavone subclass of flavonoid is expected to play a role in cancer chemoprevention and cancer chemotherapy. Here we designed our experiment to establish whether treatment of apigenin (25 mg/kg body weight) for 14 consecutive days to (N-nitrosodiethylamine) DEN induced (200 mg/kg body weight; by single ip. injection) and phenobarbital promoted (0.05% through drinking water for 14 successive weeks) rats provide protection against the oxidative stress caused by the carcinogen. The level of lipid peroxidation (LPO) markedly increased in carcinogen administered animals, which was brought back to near normal by apigenin treatment. In contrast the activities/levels of the antioxidant status both in liver and kidney were decreased in carcinogen administered animals, which was recouped back to near normal upon apigenin administration. From our findings we concluded that apigenin prevents LPO and protects antioxidant system in DEN induced and phenobarbital promoted hepatocellular carcinogenesis.

Ethanolic extract of propolis (EEP) is one of the richest sources of phenolic acids and flavonoids. EEP and its phenolic compounds have been known for various biological activities including immunopotentiation, chemopreventive and antitumor effects. Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is a naturally occurring anticancer agent that preferentially induces apoptosis in cancer cells and is not toxic toward normal cells. We examined the cytotoxic and apoptotic effect of EEP and phenolic compounds identified in propolis in combination with TRAIL on HeLa cancer cells. HeLa cells were resistant to TRAIL-induced apoptosis. Our study demonstrated that EEP and its components significantly sensitize to TRAIL induced death in cancer cells. The percentage of the apoptotic cell after exposure to 50 microg/mL EEP and 100 ng/mL TRAIL increased to 71.10 +/- 1.16%. The strongest cytotoxic effect in combination with TRAIL on HeLa cells exhibited apigenin and CAPE at the concentration of 50 microM (58.87 +/- 0.75% and 49.59 +/- 0.39%, respectively). In this report, we show for the first time that EEP markedly augmented TRAIL mediated apoptosis in cancer cells and confirmed the importance of propolis in chemoprevention of malignant tumors.

Polyphenols present in foods and supplements may contribute to human health by preventing cardiovascular diseases and cancer. Drug-food or drug-herb interactions have recently come into focus but, except for some phytochemicals, few components of food or herbs participate in such interactions. In this study, we systematically evaluated the inhibitory effects of 60 polyphenols and related compounds on human cytochrome P450 (CYP) 3A4 and CYP2C9 activity by in vitro assay to investigate whether some polyphenols induce drug interactions. In addition, the kinetics of potent CYP inhibitors was investigated by Lineweaver-Burk plot analysis. Three coumarins and 12 flavonoids significantly suppressed CYP3A4 or CYP2C9 activities. Lineweaver-Burk plot analysis indicated that apigenin and its dimer amentoflavone and imperatorin displayed a mixed type of inhibition on CYP3A4 or CYP2C9. Among the inhibitors, amentoflavone was the most potent inhibitor of CYP3A4 and CYP2C9 activities with IC(50) values of 0.07 and 0.03 microM, respectively. The K(i) value of amentoflavone was significantly lower than that of the CYP2C9 inhibition positive control sulfaphenazole. These findings suggest that some dietary polyphenols may have the potential to inhibit the metabolism of clinical drugs.

Chronic inflammation can augment tumor development in various types of cancers, including prostate cancer (PCa). Reduction of inflammation is therefore an important anticancer therapeutic opportunity. Here, we report four anti-proliferative phytocompounds in Wedelia chinensis, an oriental herbal medicine, identified through their ability to modulate the androgen receptor (AR) activation of transcription from prostate-specific antigen promoter in PCa cells. The 50% inhibition concentration values of indole-3-carboxylaldehyde, wedelolactone, luteolin and apigenin, were 34.9, 0.2, 2.4 and 9.8 muM, respectively. A formula that combined the phytocompounds in the same proportions as in the herbal extract decreased the dosage of each compound required to achieve maximal AR inhibition. In correlation with the AR suppression effect, these active compounds specifically inhibited the growth of AR-dependent PCa cells and as a combination formula they also synergistically suppressed growth in AR-dependent PCa cells. Our study has identified synergistic effects of active compounds in W. chinensis and demonstrated their potential in PCa prevention and therapy. The paradigm of multiple activities and synergism is a useful framework to investigate the therapeutic effects of whole extracts from assorted medicinal plant species.

High levels of cytokines, such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6, are associated with chronic diseases like rheumatoid arthritis, asthma, atherosclerosis, Alzheimer's disease and cancer; therefore cytokine inhibition might be an important target for the treatment of these diseases. Most drugs used to alleviate some inflammation-related symptoms act by inhibiting cyclooxygenases activity or by blocking cytokine receptors. Nevertheless, these drugs have secondary effects when used on a long-term basis. It has been mentioned that flavonoids, namely quercetin, apigenin and luteolin, reduce cytokine expression and secretion. In this regard, flavonoids may have therapeutical potential in the treatment of inflammation-related diseases as cytokine modulators. This review is focused on current research about the effect of flavonoids on cytokine modulation and the description of the way these compounds exert their effect.

Scutellaria is a traditional herbal remedy with potential anti-cancer activity. The purpose of this study was to evaluate anticancer mechanisms of thirteen Scutellaria species and analyze their leaf, stem and root extracts for levels of common biologically active flavonoids: apigenin, baicalein, baicalin, chrysin, scutellarein, and wogonin. Malignant glioma, breast carcinoma and prostate cancer cells were used to determine tumor-specific effects of Scutellaria on cell proliferation, apoptosis and cell cycle progression, via the MTT assay and flow cytometry-based apoptosis and cell cycle analysis. The extracts and individual flavonoids inhibited the proliferation of malignant glioma and breast carcinoma cells without affecting primary or non-malignant cells. The flavonoids exhibited different mechanisms of anti-tumor activity as well as positive interactions. The antitumor mechanisms involved induction of apoptosis and cell cycle arrest at G1/G2. Of the extracts tested, leaf extracts of S. angulosa, S. integrifolia, S. ocmulgee and S. scandens were found to have strong anticancer activity. This study provides basis for further mechanistic and translational studies into adjuvant therapy of malignant tumors using Scutellaria leaf tissues.

The present studies compared the effects of selected bioflavonoids on the proliferation of androgen-independent human prostatic tumor cells (PC-3). Complete growth retardation was observed in PC-3 cells treated with 100 microM quercetin, kaempferol, and luteolin, while isomolar genistein, apigenin, and myricetin suppressed PC-3 proliferation by 73%, 70%, and 59%, respectively (p < 0.05). Naringenin and rutin were not as effective and inhibited growth by < 25%. Exposure to increasing concentrations of quercetin and kaempferol led to a dose-dependent decrease in proliferation. Refeeding kaempferol-treated cells (50 microM) complete medium without the flavonoid resulted in a return toward control growth rates. Similar growth recovery was not observed in quercetin-treated cells. The antiproliferative response of PC-3 cells to quercetin and kaempferol was additive when supplemented to the medium at 25 microM. A block in G2-to-M phase progression was observed after the addition of 25 microM kaempferol. When quercetin reached 100 microM, an increase in the proportion of cells in the S phase became apparent within 24 hours. Apoptosis was not evident, even when concentrations of quercetin or kaempferol were raised to 100 microM. The present studies suggest that alterations in cell cycle progression contribute significantly to the antiproliferative effects of quercetin and kaempferol in PC-3 cells.

Flavonoids are among the best candidates for mediating the protective effect of diets rich in fruits and vegetables with respect to colorectal cancer. To gain additional information about their growth effects on colorectal tumors and their cellular mechanisms of action, a series of related flavonoids was added to cultures of colonic tumor cells. Most compounds induced growth inhibition and cell loss at concentrations of 1-100 microM, relative effectivity being quercetin>> apigenin>> fisetin>> robinetin and kaempferol. Myricetin was only slightly effective. Quercetin was the strongest inducer of apoptosis in a process that was reversible until 10 hours by flavonoid removal and until 24 hours by fetal calf serum. Cells were preferentially retained in the S phase. On the cellular level, quercetin sensitivity was correlated with epidermal growth factor (EGF) receptor levels, rapid growth, and poor differentiation, indicating the possibility of targeting those cells most harmful for the organism. The flavonoid transiently inhibited EGF receptor phosphorylation but had only little effect on other signaling molecules. Even after recovery of receptor phosphorylation, cells remained resistant to EGF stimulation. In summary, the data indicate that inhibition of EGF receptor kinase is an integral part of quercetin-induced growth inhibition, but additional mechanisms also contribute to the overall effect.

Flavonoids are polyphenolic antioxidants naturally present in vegetable foods. Some flavonoids, such as quercetin, inhibit carcinogenesis in rodents, but their effect in humans is unknown. We measured the flavonoids quercetin, kaempferol, myricetin, apigenin, and luteolin in foods and assessed flavonoid intake in 1985 by dietary history in 738 men aged 65-84 years without a history of cancer, who were then followed for five years. Mean flavonoid intake was 25.9 mg/day. The major sources of flavonoid intake were tea at 61% and vegetables and fruits (mainly onions, kale, endive, and apples) at 38%. Between 1985 and 1990, 75 men developed cancer (all sites) and 34 men died from cancer. Flavonoid intake in 1985 was not associated with incidence of all-cause cancer (p for trend = 0.54) or with mortality from all-cause cancer (p for trend = 0.51). Flavonoid intake was also not associated with risk of cancers of the alimentary and respiratory tract (p for trend = 0.92). Adjustment for age, body mass index, smoking, physical activity, and vitamin C, vitamin E, beta-carotene, and dietary fiber intake did not change the relative risks. A high intake of flavonoids from vegetables and fruits only was inversely associated with risk of cancer of the alimentary and respiratory tract (relative risk of highest vs. lowest tertile = 0.51, 95% confidence interval 0.25-1.05); these results suggest the presence of other nonvitamin components with anticarcinogenic potential in these foods. We conclude that intake of flavonoids, mainly from tea, apples, and onions, does not predict a reduced risk of all-cause cancer or of cancer of the alimentary and respiratory tract in elderly men. The effect of flavonoids on risk of cancer at specific sites needs further investigation in prospective cohort studies.

Several mechanisms facilitate the progression of hormone-sensitive prostate cancer to castration-resistant prostate cancer (CRPC). At present, the approved chemotherapies for CRPC include systemic drugs (docetaxel and cabazitaxel) and agents that target androgen signaling, including enzalutamide and abiraterone. While up to 30% of patients have primary resistance to these treatments, each of these drugs confers a significant survival benefit for many. Over time, however, all patients inevitably develop resistance to treatment and their disease will continue to progress. Several key mechanisms have been identified that give rise to drug resistance. Expression of constitutively active variants of the androgen receptor, such as AR-V7, intracrine androgens and overexpression of androgen synthesis enzymes like AKR1C3, and increased drug efflux through ABCB1 are just some of the many discovered mechanisms of drug resistance. Treatment strategies are being developed to target these pathways and reintroduce drug sensitivity. Niclosamide has been discovered to reduce AR-V7 activity and synergized to enzalutamide. Indomethacin has been explored to inhibit AKR1C3 activity and showed to be able to reverse resistance to enzalutamide. ABCB1 transport activity can be mitigated by the phytochemical apigenin and by antiandrogens such as bicalutamide, with each improving cellular response to chemotherapeutics. By better understanding the mechanisms by which drug resistance develops improved treatment strategies will be made possible. Herein, we review the existing knowledge of CRPC therapies and resistance mechanisms as well as methods that have been identified which may improve drug sensitivity.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that plays a major role in cellular adaptation to hypoxia. The mechanisms regulating HIF-1 activity occurs at multiple levels in vivo. The HIF-1alpha subunit is highly sensible to oxygen and is rapidly degraded by the proteasome 26S in normoxia. Activation in hypoxia occurs through a multistep process including inhibition of HIF-1alpha degradation, but also increase in the transactivation activity of HIF-1. Several data indicate that phosphorylation could play a role in this regulation. In this report, we investigated the role of casein kinase 2 (CK2), an ubiquitous serine/threonine kinase, in the regulation of HIF-1 activity. Hypoxia was capable of increasing the expression of the beta subunit of CK2, of inducing a relocalization of this subunit at the plasma membrane, of inducing nuclear translocation of the alpha subunit and of increasing CK2 activity. Three inhibitors of this kinase, DRB (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole), TBB (4,5,6,7-tetrabromotriazole) and apigenin, as well as overexpression of a partial dominant negative mutant of CK2alpha, were shown to inhibit HIF-1 activity as measured by a reporter assay and through hypoxia-induced VEGF and aldolase expression. This does not occur at the stabilization process since they did not affect HIF-1alpha protein level. DNA-binding activity was also not inhibited. We conclude that CK2 is an important regulator of HIF-1 transcriptional activity but the mechanism of this regulation remains to be determined. Since HIF-1 plays a major role in tumor angiogenesis and since CK2 has been described to be overexpressed in tumor cells, this new pathway of regulation can be one more way for tumor cells to survive.

The consumption of a plant-based diet can prevent the development and progression of chronic diseases associated with extensive neovascularization, including the progression and growth of solid malignant tumours. We have previously shown that the plant-derived isoflavonoid genistein is a potent inhibitor of cell proliferation and in vitro angiogenesis. Moreover, the concentration of genistein in the urine of subjects consuming a plant-based diet is 30-fold higher than that in subjects consuming a traditional Western diet. We have also reported that certain structurally related flavonoids are more potent inhibitors than genistein. Indeed, 3-hydroxyflavone, 3',4'-dihydroxyflavone, 2',3'-dihydroxyflavone, fisetin, apigenin and luteolin inhibit the proliferation of normal and tumour cells as well as in vitro angiogenesis at half-maximal concentrations in the lower micromolar range. The wide distribution of isoflavonoids and flavonoids in the plant kingdom, together with their anti-angiogenic and anti-mitotic properties, suggest that these phytoestrogens may contribute to the preventive effect of a plant-based diet on chronic diseases, including solid tumours.

Treatment of human endothelial cells with cytokines such as interleukin-1, tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. In the present study we report that cotreatment of human endothelial cells with certain hydroxyflavones and flavanols blocks cytokine-induced ICAM-1, VCAM-1, and E-selectin expression on human endothelial cells. One of the most potent flavones, apigenin, exhibited a dose- and time-dependent, reversible effect on adhesion protein expression as well as inhibiting adhesion protein upregulation at the transcriptional level. Apigenin also inhibited IL-1 alpha-induced prostaglandin synthesis and TNF-alpha-induced IL-6 and IL-8 production, suggesting that the hydroxyflavones may act as general inhibitors of cytokine-induced gene expression. Although apigenin did not inhibit TNF-alpha-induced nuclear translocation of NF-kappa B(p50(NFKB1)/p65(RelA)) we found this flavonoid did inhibit TNF-alpha induced beta-galactosidase activity in SW480 cells stably transfected with a beta-galactosidase reporter construct driven by four NF-kappa B elements, suggesting an action on NF-kappa B transcriptional activation. Adhesion of leukocytes to cytokine-treated endothelial cells was blocked in endothelial cells cotreated with apigenin. Finally, apigenin demonstrated potent anti-inflammatory activity in carrageenan induced rat paw edema and delayed type hypersensitivity in the mouse. We conclude that flavonoids offer important therapeutic potential for the treatment of a variety of inflammatory diseases involving an increase in leukocyte adhesion and trafficking.

Activation of the aryl hydrocarbon receptor (AHR) by agonists and environmental contaminants like dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) leads to many adverse biological effects, including tumor promotion. With this in mind, we propose that agents that block the AHR pathway may be therapeutically beneficial, particularly by exhibiting chemopreventive activities. In our current research, we have focused on the development of an AHR antagonist using a chemical genetic approach called PROTACS (PROteolysis-TArgeting Chimeric moleculeS). PROTACS is a novel approach of tagging small recognition sequences of a specific E3 ubiquitin ligase complex to a known ligand for the receptor of interest (AHR) for targeting its degradation. Here, we present the design and initial characterization of AHR targeting PROTACS (Apigenin-Protac) designed to degrade and inhibit the AHR in epithelial cells. Our results demonstrate the "proof of concept" of this approach in effectively blocking AHR activity in cultured cells.

1. Resveratrol, a polyphenolic compound present in grape and wine, has beneficial effects against cancer and protective effects on the cardiovascular system. Resveratrol is sulphated, and the hepatic and duodenal sulphation might limit the bioavailability of this compound. The aim of this study was to see whether natural flavonoids present in wine, fruits and vegetables inhibit the sulphation of resveratrol in the human liver and duodenum. 2. In the liver, IC50 for the inhibition of resveratrol sulphation was 12+/-2 pM (quercetin), 1.0+/-0.04 microM (fisetin), 1.4+/-0.1 microM (myricetin), 2.2+/-0.1 microM (kaempferol) and 2.8+/-0.2 microM (apigenin). Similarly, in the duodenum, IC50 was 15+/-2 pM (quercetin), 1.3+/-0.1 microM (apigenin), 1.3+/-0.5 microM (fisetin), 2.3+/-0.1 microM (kaempferol) and 2.5+/-0.3 microM (myricetin). 3. The type of inhibition of quercetin on resveratrol sulphation was studied in three liver samples and was determined to be non-competitive and mixed in nature. Km (mean+/-SD; microM) was 0.23+/-0.07 (control), 0.40+/-0.08 (5 pM quercetin) and 0.56+/-0.09 (10 pM quercetin). Vmax (mean+/-SD; pmol min(-1) x mg(-1)) was 99+/-11 (control), 73+/-15 (5 pM quercetin) and 57 +/- 10 (10 pM quercetin). Kj and Kies estimates (mean+/-SD) were 3.7+/-1.8 pM and 12.1+/-1.7 pM respectively (p = 0.010). 4. Chrysin was a substrate for the sulphotransferase(s) and an assay was developed for measuring the chrysin sulphation rate in human liver. The enzyme followed Michaelis-Menten kinetics and Km and Vmax (mean+/-SD) measured in four livers were 0.29+/-0.07 microM and 43.1+/-1.9 pmol x min(-1) x mg(-1) respectively. 5. Catechin was neither an inhibitor of resveratrol sulphation nor a substrate of sulphotransferase. 6. These results are consistent with the view that many, but not all, flavonoids inhibit the hepatic and duodenal sulphation of resveratrol, and such inhibition might improve the bioavailability of this compound.

The p53 tumor suppressor induces cell growth arrest and apoptosis in response to DNA damage. Because these functions are achieved largely by the transcriptional properties of p53, nuclear localization of the protein is essential. Indeed, the tumors with aberrant cytoplasmic localization of wild-type p53 often exhibit an impaired response to DNA damage. In this study, we report that Thr-55 phosphorylation induces the association of p53 with the nuclear export factor CRM1, leading to p53 nuclear export. We further show that MDM2 also promotes the CRM1-p53 association and Thr-55 phosphorylation is required for this process. Interestingly, inhibition of Thr-55 phosphorylation by a dietary flavonoid, apigenin, specifically blocks the CRM1-p53 association, restores p53 nuclear localization, and sensitizes tumor cells with cytoplasm localized wild-type p53 to DNA damage. These data provide insights into the regulation of p53 nuclear localization by post-translational modification and suggest an avenue for targeted therapy for cancers caused by aberrant cytoplasm localization of wild-type p53.

Apigenin, a common dietary flavonoid, has been shown to induce cell growth-inhibition and cell cycle arrest in many cancer cell lines. One important effect of apigenin is to increase the stability of the tumor suppressor p53 in normal cells. Therefore, apigenin is expected to play a large role in cancer prevention by modifying the effects of p53 protein. However, the mechanisms of apigenin's effects on p53-mutant cancer cells have not been revealed yet. We assessed the influence of apigenin on cell growth and the cell cycle in p53-mutant cell lines. Treatment with apigenin resulted in growth-inhibition and G2/M phase arrest in two p53-mutant cancer cell lines, HT-29 and MG63. These effects were associated with a marked increase in the protein expression of p21/WAF1. We have shown that p21/WAF1 mRNA expression was also markedly increased by treatment with apigenin in a dose- and time-dependent manner. However, we could not detect p21/WAF1 promoter activity following treatment with apigenin. Similarly, promoter activity from pG13-Luc, a p53-responsive promoter plasmid, was not activated by treatment with apigenin with or without p53 protein expression. These results suggest that there is a p53-independent pathway for apigenin in p53-mutant cell lines, which induces p21/WAF1 expression and growth-inhibition. Apigenin may be a useful chemopreventive agent not only in wild-type p53 status, but also in cancer with mutant p53.

Diet high in vegetables and fruits has been associated with reduced cancer risk. However, the involved mechanisms are unknown. Previously, we reported that the dietary flavonoid apigenin could inhibit the proteasome activity and induce apoptosis in tumor cells. To further investigate the structure-proteasome-inhibitory activity relationships, we chose and tested five dietary flavonoids, including luteolin, apigenin, chrysin, naringenin and eriodictyol. We found that the order of inhibitory potencies and apoptosis-inducing potencies of these five compounds in 20S purified proteasome and tumor cells was: (1) luteolin>> apigenin>> chrysin, and (2) apigenin>>) naringenin, and luteolin>>) eriodictyol. Therefore, flavonoids with hydroxylized B ring and/or unsaturated C ring are natural potent proteasome inhibitors and tumor cell apoptosis inducers. Furthermore, neither apigenin nor luteolin could inhibit the proteasome and induce apoptosis in non-transformed human natural killer cells. This finding may provide a molecular basis for the clinically observed cancer-preventive effects of fruits and vegetables.

Colorectal cancer (CRC) is a major cause of morbidity and mortality throughout the world. Apigenin is a flavonoid that possesses various clinically relevant properties such as anti-tumour, anti-platelet and anti-inflammatory activities. Our results showed that apigenin has anti-proliferation, anti-invasion and anti-migration effects in three kinds of colorectal adenocarcinoma cell lines, namely SW480, DLD-1 and LS174T. Proteomic analysis with SW480 indicated that apigenin up-regulated the expression of transgelin (TAGLN) in mitochondria to exert its anti-tumour growth and anti-metastasis effects. Real-time quantitative polymerase chain reaction (RQ-PCR) and western blot confirm the up-regulation in all the three colorectal adenocarcinoma cells. An inverse correlation was observed between TAGLN expression and CRC metastasis in tissue microarray staining. TAGLN siRNA increased the viability of SW480. Apigenin decreased the expression of MMP-9 in a dose-dependent manner. Transfection of three truncated forms of TAGLN and wild type has identified TAGLN as a repressor of MMP-9 expression. A synergetic effect was observed in overexpression of TAGLN wild type and apigenin treatment which manifested as lowered phosphorylation of AKT Ser473 and ATK Thr308. In an orthotopic CRC model, apigenin inhibited tumour growth and metastasis to liver and lung. In conclusion, our research provided direct evidence that apigenin inhibited tumour growth and metastasis both in vitro and in vivo. Apigenin up-regulated TAGLN and hence down-regulated MMP-9 expression through decreasing phosphorylation of Akt at Ser473 and in particular Thr308 to prevent cell proliferation and migration.

BACKGROUND

Multiple myeloma (MM) is a B-cell malignancy that is largely incurable and is characterized by the accumulation of malignant plasma cells in the bone marrow. Apigenin, a common flavonoid, has been reported to suppress proliferation in a wide variety of solid tumors and hematological cancers; however its mechanism is not well understood and its effect on MM cells has not been determined.

RESULTS

In this study, we investigated the effects of apigenin on MM cell lines and on primary MM cells. Cell viability assays demonstrated that apigenin exhibited cytotoxicity against both MM cell lines and primary MM cells but not against normal peripheral blood mononuclear cells. Together, kinase assays, immunoprecipitation and western blot analysis showed that apigenin inhibited CK2 kinase activity, decreased phosphorylation of Cdc37, disassociated the Hsp90/Cdc37/client complex and induced the degradation of multiple kinase clients, including RIP1, Src, Raf-1, Cdk4 and AKT. By depleting these kinases, apigenin suppressed both constitutive and inducible activation of STAT3, ERK, AKT and NF-κB. The treatment also downregulated the expression of the antiapoptotic proteins Mcl-1, Bcl-2, Bcl-xL, XIAP and Survivin, which ultimately induced apoptosis in MM cells. In addition, apigenin had a greater effects in depleting Hsp90 clients when used in combination with the Hsp90 inhibitor geldanamycin and the histone deacetylase inhibitor vorinostat.

CONCLUSIONS

Our results suggest that the primary mechanisms by which apigenin kill MM cells is by targeting the trinity of CK2-Cdc37-Hsp90, and this observation reveals the therapeutic potential of apigenin in treating multiple myeloma.