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PubMed1997

Evaluation of properties of apigenin and [G-3H]apigenin and analytic method development.

This study provides baseline data and analytical methods to assist in the evaluation of apigenin, a plant flavonoid with promising chemopreventive activity against skin cancer. Apigenin was freely soluble in dimethylsulfoxide (> 100 mg/mL), but it had low solubility (0.00135-1.63 mg/mL) in all the other solvents and surfactants tested, especially in highly hydrophilic or nonpolar solvents. The partition coefficient (log K) calculated from the solubility ratio of apigenin in n-octanol and water was 2.87. Apigenin strongly absorbed UV light, with three maximum absorption wavelengths at 212, 269, and 337 nm (epsilon = 29,800, 19,020, and 18,930 M-1 cm-1, respectively). Using quercetin as the internal standard, a reversed-phase HPLC method was developed to quantitatively analyze apigenin in epidermal cells obtained from SENCAR mice. Apigenin was labeled at position 6, 8, 3', and 5' with tritium by a platinum-catalyzed proton-tritium exchange as confirmed indirectly by 1H NMR analysis of the deuterated apigenin. The tritium label was stable in aqueous environments, especially under acidic and neutral conditions, so [G-3H]apigenin was considered suitable for subsequent absorption and metabolic studies.

Animals↗
PubMed1996

Influence of vehicle, distant topical delivery, and biotransformation on the chemopreventive activity of apigenin, a plant flavonoid, in mouse skin.

PURPOSE: This study was designed to (a) establish a short-term in vivo system to evaluate topical formulations of apigenin, (b) determine whether apigenin should be topically delivered to the local skin tissue or systemic circulation, (c) investigate if biotransformation was involved in apigenin's chemopreventive activity. METHODS: The effect of topical applied apigenin in acetone/DMSO (A/D, 9:1) on the promotion of skin tumorigenesis was studied. The influence of apigenin in DMSO, A/D (4:1), and propylene glycol/DMSO (PG/D, 4:1) on 12-O-tetradecanoylphorbol-13 acetate (TPA) induced ornithine decarboxylase (ODC) activity was compared. Distant topical delivery of apigenin was conducted on abdominal skin and ODC activity was monitored in dorsal skin. Potential glucuronidation/sulfation of apigenin in intact skin was assessed by measuring isolated apigenin before and after enzyme hydrolysis with glucuronidase/sulfatase. The epidermal extracts from apigenin-treated SENCAR mice were analyzed for metabolites by HPLC. RESULTS: Apigenin (5 mumol) in A/D did not significantly reduce skin tumor incidence in contrast to previous data with DMSO. Inhibition of TPA-induced ODC by apigenin in three vehicles was in the order of DMSO > A/D > PG/D. TPA-induced ODC in dorsal skin was not inhibited by apigenin delivered from abdominal skin. The quantity of apigenin recovered from epidermal extract was not different before and after beta-glucuronidase/sulfatase treatment. Metabolites were not observed in the HPLC profiles of epidermal extracts from apigenin treated mice. CONCLUSIONS: (a) The short-term TPA-induced ODC was validated for evaluating topical formulations of apigenin. (b) Topical delivery of apigenin should target the local skin tissue. (c) Glucuronidation/sulfation appeared not to be involved in apigenin's chemopreventive activity.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1996

In vivo and in vitro percutaneous absorption of cancer preventive flavonoid apigenin in different vehicles in mouse skin.

PURPOSE: In vivo and in vitro percutaneous absorption of apigenin was investigated in three vehicles previously used in cancer prevention studies to determine the drug delivery properties for optimal chemo-preventive activity. METHODS: In vivo percutaneous absorption of apigenin on SENCAR mice was studied with DMSO and acetone/DMSO (A/D, 4:1) vehicle. In vitro percutaneous absorption studies used whole mouse skin, without subcutaneous fat, mounted on Franz diffusion cells with 37 degrees C Dulbecco's phosphate-buffered saline as the receptor fluid. The skin was treated with [G-3H]-apigenin in DMSO, A/D (4:1), or propylene glycol/DMSO (PG/D, 4:1). RESULTS: Apigenin uptake by epidermal cells and distribution in epidermis following in vivo topical treatment in two vehicles was in the order of A/D > DMSO, while apigenin distribution in dermis and subcutaneous fat was not different between DMSO and A/D. Total apigenin absorption in mouse skin in vitro was in the order of A/D > DMSO > PG/D. However, apigenin sub-tissue distribution within epidermis determined by tape-stripping and by determination of apigenin in dermal and epidermal tissue indicated that DMSO delivered more apigenin into viable epidermis than A/D while A/D deposited more apigenin in the stratum corneum. Apigenin absorption in mouse skin with DMSO or A/D showed saturation kinetics while apigenin in PG/D showed very low absorption initially and non-saturated absorption in a period of 6 hr. HPLC-scintillation profiles of in vitro samples showed no evidence of apigenin metabolism in mouse skin. CONCLUSIONS: Delivering apigenin into viable epidermis appears to be a necessary property for an apigenin formulation to be effective in skin cancer prevention.

Acetone↗
PubMed2004

Individual and interactive effects of apigenin analogs on G2/M cell-cycle arrest in human colon carcinoma cell lines.

Apigenin has been previously shown to induce G2/M cell-cycle arrest in human colon cancer cell lines. The present study assessed the individual and interactive influence of seven apigenin analogs on cell cycle, cell number, and cell viability in human SW480 and Caco-2 colonic carcinoma cells. Cellular concentration of selected apigenin analogs was further assessed by high-performance liquid chromatography to assess cellular availability. The apigenin analogs studied were acacetin, chrysin, kampherol, luteolin, myricetin, naringenin, and quercetin. DNA flow cytometric analysis indicated that treatment with either chrysin or acacetin at 0 to 80 microM for 48 h resulted in cell-cycle arrest at the G2/M phase in a dose-dependent manner in the SW480 cells but not in the Caco-2 cells. The percentage of SW480 cells at G2/M also increased when cells were treated with kampherol, luteolin, or quercetin between 5 and 30 microM, but the percentage of cells in G2/M decreased at doses greater than 40 microM. Cell number was significantly decreased in a time- and dose-dependent manner following the treatments with each analog except for naringenin and myricetin. The interactive effects of these analogs with apigenin were further assessed by combining each analog at doses from 0 to 80 microM with apigenin at 20 microM, a dose at which apigenin was found to double the proportion of SW480 cells in G2/M. When either acacetin, chrysin, luteolin, kampherol, or quercetin at doses between 5 and 30 microM were combined with apigenin at 20 microM, there was an increase of 22% in the proportion of G2/M cells over that observed with 20 microM apigenin alone (P < 0.05). At doses higher than 40 microM, however, the interaction became antagonistic, and the proportion of cells in G2/M decreased below that observed with apigenin alone. Cell viability, as assessed by Trypan blue exclusion assay, significantly decreased by treatments with high doses of each agent or each agent combined with apigenin. Cellular concentration of apigenin, chrysin, or naringenin in SW480 cells significantly increased at doses of 40 microM or greater, but it was not correlated with their impact on G2/M cell-cycle arrest. The induction of cell-cycle arrest by five of seven tested apigenin analogs and the additive induction by the combination of flavonoids at low doses suggest that apigenin-related flavonoids may cooperatively protect against colorectal cancer through conjoint blocking of cell-cycle progression.

Antineoplastic Agents↗
PubMed1997

Induction of p21/WAF1 and G1 cell-cycle arrest by the chemopreventive agent apigenin.

Apigenin is a plant flavonoid that has been shown to significantly inhibit ultraviolet-induced mouse skin tumorigenesis when applied topically and may be an alternative sunscreen agent for humans. A long-term goal of our laboratory is to elucidate the molecular mechanism or mechanism by which apigenin inhibits skin tumorigenesis. In a previous publication, we characterized the mechanism by which apigenin induced G2/M arrest in keratinocytes. More recent studies in our laboratory have provided evidence that apigenin can induce G1 arrest in addition to arresting cells at G2/M. Here we describe the mechanism of the apigenin-induced G1 arrest in human diploid fibroblasts (HDF). Treatment of asynchronous HDF for 24 h with 10-50 microM apigenin resulted in dose-dependent cell-cycle arrest at both the G0/G1 and G2/M phases as measured by flow cytometry. The G0/G1 arrest was more clearly defined by using HDF that were synchronized in G0 and then released from quiescence by replating at subconfluent densities in medium containing 10-70 microM apigenin. The cells were analyzed for cell-cycle progression or cyclin D1 expression 24 h later. A dose of apigenin as low as 10 microM reduced the percentage of cells in S phase by 20% compared with control cultures treated with solvent alone. Western blot analysis of apigenin-treated HDF indicated that cyclin D1 was expressed at higher levels than in untreated cells, which signifies that they were arrested in G1 phase rather than in a G0 quiescent state. The G1 arrest was further studied by cyclin-dependent kinase 2 (cdk2) immune complex-kinase assays of apigenin-treated asynchronous HDF, which demonstrated a dose-dependent inhibition of cdk2 by apigenin. Inhibition of cdk2 kinase activity in apigenin-treated cells was associated with the accumulation of the hypophosphorylated form of the retinoblastoma (Rb) protein as measured by western blot analysis. The cdk inhibitor p21/WAF1 was also induced in a dose-dependent manner, with a 22-fold induction of p21/WAF1 in 70 microM apigenin-treated cells. In conclusion, apigenin treatment produced a G1 cell-cycle arrest by inhibiting cdk2 kinase activity and the phosphorylation of Rb and inducing the cdk inhibitor p21/WAF1, all of which may mediate its chemopreventive activities in vivo. To our knowledge this is the first report of a chemopreventive agent inducing p21/WAF1, a known downstream effector of the p53 tumor suppressor protein.

Anticarcinogenic Agents↗
PubMed2005

Up-regulation of insulin-like growth factor binding protein-3 by apigenin leads to growth inhibition and apoptosis of 22Rv1 xenograft in athymic nude mice.

Epidemiological studies suggest that increased intake of fruits and vegetables may be associated with a reduced risk of prostate cancer. Apigenin (4', 5, 7,-trihydroxyflavone), a common dietary flavonoid abundantly present in fruits and vegetables, has shown remarkable anti-proliferative effects against various malignant cell lines. However, the mechanisms underlying these effects remain to be elucidated. We investigated the in vivo growth inhibitory effects of apigenin on androgen-sensitive human prostate carcinoma 22Rv1 tumor xenograft subcutaneously implanted in athymic male nude mice. Apigenin was administered to mice by gavage at doses of 20 and 50 mug/mouse/day in 0.2 ml of a vehicle containing 0.5% methyl cellulose and 0.025% Tween 20 in two different protocols. In the first protocol, apigenin was administered for 2 wk before inoculation of tumor and was continued for 8 wk, resulting in significant inhibition of tumor volume by 44 and 59% (P<0.002 and 0.0001), and wet weight of tumor by 41 and 53% (P<0.05), respectively. In the second protocol, administration of apigenin began 2 wk after tumor inoculation and continued for 8 wk; tumor volume and wet weights of tumor were reduced by 39 and 53% (P<0.01 and 0.002) and 31 and 42% (P<0.05), respectively. The tumor inhibitory effect of apigenin was more pronounced in the first protocol of extended treatment, which was associated with increased accumulation of human IGFBP-3 in mouse serum along with significant increase in IGFBP-3 mRNA and protein expression in tumor xenograft. Apigenin intake by these mice also resulted in simultaneous decrease in serum IGF-I levels and induction of apoptosis in tumor xenograft. Importantly, tumor growth inhibition, induction of apoptosis, and accumulation of IGFBP-3 correlated with increasing serum and tumor apigenin levels. In both studies, animals did not exhibit any signs of toxicity or reduced food consumption. In cell culture studies, apigenin treatment resulted in cell growth inhibition and induction of apoptosis, which correlated with increased accumulation of IGFBP-3 in culture medium and cell lysate. These effects were associated with significant reduction in IGF-I secretion; inhibition of IGF-I-induced cell cycle progression and insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, along with an increase in sub-G1 peak by apigenin. Further, treatment of cells with IGFBP-3 antisense oligonucleotide reversed these effects and attenuated apigenin-mediated inhibition of IRS-1 phosphorylation conferring inhibitory effects of apigenin on IGF-signaling. This study presents the first evidence that the in vitro and in vivo growth inhibitory effects of apigenin involve modulation of IGF-axis signaling in prostate cancer.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Apigenin inhibits expression of vascular endothelial growth factor and angiogenesis in human lung cancer cells: implication of chemoprevention of lung cancer.

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.

Animals↗
PubMed2000

Cell-cycle arrest at G2/M and growth inhibition by apigenin in human colon carcinoma cell lines.

Apigenin, a common dietary flavonoid, has been shown to induce cell cycle arrest in both epidermal and fibroblast cells and inhibit skin tumorigenesis in murine models. The present study assessed the influence of apigenin on cell growth and the cell cycle in the human colon carcinoma cell lines SW480, HT-29, and Caco-2. Treatment of each cell line with apigenin (0-80 microM) resulted in a dose-dependent reduction in both cell number and cellular protein content, compared with untreated control cultures. DNA flow cytometric analysis indicated that treatment with apigenin resulted in G2/M arrest in all three cell lines in a time- and dose-dependent manner. Apigenin treatment (80 microM) for 48 h produced maximum G2/M arrest of 64%, 42%, and 26% in SW480 cells, HT-29 cells, and Caco-2 cells, respectively, in comparison with control cells (15%). The proportion of S-phase cells was not altered by apigenin treatment in each of the three cell lines. The G2/M arrest was reversible after 48 h of apigenin treatment in the most sensitive cell line SW480. The degree of G2/M arrest by apigenin was inversely correlated with the corresponding inhibition of cell growth measurements in all three cell lines (r = -0.626 to -0.917, P</=0. 005). Moreover, an immune complex kinase assay demonstrated an inhibition of p34(cdc2) kinase activity, a critical enzyme in G2/M transition, in each cell line after treatment with apigenin (50-80 microM). Western blot analyses indicated that both p34(cdc2) and cyclin B1 proteins were also decreased after apigenin treatment. These results indicate that apigenin inhibits colon carcinoma cell growth by inducing a reversible G2/M arrest and that this arrest is associated, at least in part, with inhibited activity of p34(cdc2) kinase and reduced accumulation of p34(cdc2) and cyclin B1 proteins. Differences in induction of G2/M arrest by apigenin in the three colon carcinoma cell lines suggest that dietary apigenin may be differentially effective against tumors with specific mutational spectra. Mol. Carcinog. 28:102-110, 2000.

Apigenin↗
PubMed2005

Induction of caspase-dependent, p53-mediated apoptosis by apigenin in human neuroblastoma.

Neuroblastoma is a pediatric tumor accounting for 15% of childhood cancer deaths and has a poor prognosis in children >1 year of age. We investigated the ability of apigenin, a nonmutagenic dietary flavonoid that has been shown to have antitumor effects in various tumor cell lines, to inhibit growth and induce apoptosis of the human neuroblastoma cell lines NUB-7, LAN-5, and SK-N-BE(2). Apigenin inhibited colony-forming ability and survival, and induced apoptosis of NUB-7 and LAN-5 cells. The presence of the C2-C3 double bond and the 4'-OH group on the flavonoid structure correlated with the growth-inhibitory potential of apigenin. Furthermore, apigenin inhibited NUB-7 xenograft tumor growth in anonobese diabetic/severe combined immunodeficiency mouse model, likely by inducing apoptosis. Apigenin did not inhibit survival of primary sympathetic neurons, suggesting that it is not toxic to nontransformed cells. The mechanism of action of apigenin seems to involve p53, as it increased the levels of p53 and the p53-induced gene products p21WAF1/CIP1 and Bax. Furthermore, apigenin (15-60 micromol/L) induced cell death and apoptosis of neuroblastoma cells expressing wild-type but not mutant p53. Apigenin increased caspase-3 activity and PARP cleavage, and Z-VAD-FMK, a broad-spectrum caspase-3 inhibitor, rescued NUB-7 cells from apigenin-mediated apoptosis indicating that apigenin induced apoptosis in acaspase-dependent manner. Overexpression of Bcl-X(L) rescued NUB-7 from apigenin-induced cell death, suggesting that Bax activity is important for the action of apigenin. Apigenin is thus a candidate therapeutic for neuroblastoma that likely acts by regulating a p53-Bax-caspase-3 apoptotic pathway.

Amino Acid Chloromethyl Ketones↗
PubMed2002

A p21(waf1)-independent pathway for inhibitory phosphorylation of cyclin-dependent kinase p34(cdc2) and concomitant G(2)/M arrest by the chemopreventive flavonoid apigenin.

Apigenin, a nonmutagenic flavonoid, has been shown to inhibit ultraviolet light-induced skin tumorigenesis when topically applied to mouse skin. Our previous studies have shown that apigenin treatment of cultured mouse keratinocytes induces G(2)/M arrest accompanied by an increase in p53 protein stability and expression of p21(waf1). In this study, we determined whether the G(2)/M arrest induced by apigenin was dependent upon the presence of the cyclin dependent kinase inhibitor p21(waf1). We exposed WWT.8 (p21(waf1) wild-type) and WKO.16 (p21(waf1) null) mouse keratinocytes to various doses of apigenin for 24 h and observed G(2)/M arrest in both cell lines, thereby establishing that the apigenin-induced G(2)/M arrest was p21(waf1) independent. A 4-h treatment with apigenin induced increases in p53 protein level by sixfold and tenfold in the WWT.8 p21(waf1) wild-type cells and WKO.16 p21(waf1) null cells, respectively. After 24 h in WWT.8 cells, p21(waf1) protein also was induced in a dose-dependent manner, but it was not expressed in WKO.16 keratinocytes. We then measured the effect of apigenin treatment on the mammalian homologue of the yeast cdc2 gene (p34(cdc2)) cyclin-dependent kinase and cyclin B1 (cycB1), because these proteins complex to regulate G(2)/M progression. Apigenin treatment decreased the protein level of p34(cdc2), and p34(cdc2) kinase activity was inhibited in both p21(waf1)(+/+) and p21(waf1)(-/-) cell lines by approximately 40%. The inhibition of p34(cdc2) kinase activity by apigenin treatment correlated with increasing levels of p34(cdc2) phosphorylation at Tyr15, a site in the p34(cdc2) kinase that undergoes inhibitory phosphorylation by Wee1 kinase. Apigenin treatment also had no effect on the protein level or activity of the competing phosphatase, cdc25c, which dephosphorylates p34(cdc2) kinase at Tyr15. Apigenin had little effect on the accumulation of cycB1 protein. These results supported the conclusion that G(2)/M arrest induced by apigenin was accompanied by inhibition of the p34(cdc2) cyclin-dependent kinase protein level and activity in a p21(waf1)-independent manner.

Animals↗
PubMed2000

Endothelium-dependent vasorelaxant and antiproliferative effects of apigenin.

This study was designed to determine whether the relaxant effect of apigenin was endothelium dependent and to examine the possible antiproliferative effect of apigenin. Apigenin relaxed the phenylephrine-precontracted endothelium-intact aortic rings with IC(50) value of 3.7+/-0.5 microM and removal of a functional endothelium significantly attenuated this relaxation (IC(50)=8.2+/-0.9 microM). However, apigenin did not affect the 0.1 microM phorbol 12,13-dibutyrate-induced contraction (IC(50)=34.6+/-1.2 microM) within the concentration range that relaxed the phenylephrine-contracted arteries, suggesting that apigenin did not influence protein kinase C-mediated contractile mechanisms in rat aorta. Pretreatment of apigenin significantly potentiated the relaxant effect of acetylcholine on phenylephrine-induced contraction. Pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME) or methylene blue reduced the relaxant effect of apigenin. Apigenin (10 microM) increased the guanosine 3',5'-cyclic monophosphate (cGMP) content of endothelium-intact tissues. Pretreatment with L-NAME (100 microM) or removal of endothelium significantly suppressed the effect of apigenin on cGMP production. In addition, apigenin significantly inhibited [3H]thymidine incorporation into DNA of primary cultured rat aortic smooth muscle cell in a dose-dependent manner. These findings suggest that besides influx and release of Ca(2+), nitric oxide (NO) and cGMP may account for the apigenin-induced endothelium-dependent relaxation and hypotensive activity. Both vasorelaxant and antiproliferative activities may contribute to a benefit of apigenin in the vascular system.

Animals↗
PubMed2006

Effect of dietary apigenin on colonic ornithine decarboxylase activity, aberrant crypt foci formation, and tumorigenesis in different experimental models.

The efficacy of dietary apigenin, a dietary flavonoid, in colon cancer prevention was investigated by evaluating the inhibition of the ornithine decarboxylase (ODC) activity and the formation of aberrant crypt foci (ACF) and by studying the ability of apigenin to block colon carcinogenesis in two mouse models. First, the activity of ODC was measured in colon cancer cells (Caco-2) and in the colon epithelium of CF-1 mice. Apigenin at 10 and 30 muM significantly inhibited the ODC activity of Caco-2 cells by 26% and 57%, respectively. Colonic ODC activity in CF-1 mice was reduced with 0.1% dietary apigenin by 42% compared with the control, but this difference was not statistically significant. Second, ACF formation was evaluated in azoxymethane (AOM)-induced CF-1 mice. Female CF-1 mice at 6 wk of age were i.p. injected with 5 mg/kg body weight (BW) AOM once to induce ACF. ACF formation in CF-1 mice was reduced by 50% (P < 0.05) with 0.1% dietary apigenin fed for 6 wk when compared with the control. Dietary apigenin inhibited ACF only in the distal region of the CF-1 mouse colon. Finally, tumorigenesis studies were conducted using two different mouse models: AOM-induced CF-1 mice and Min mice with mutant adenomatous polyposis coli (APC) gene. Female CF-1 mice at 6 wk of age were i.p. injected with 10 mg/kg BW AOM weekly for 6 (AOM Study I) or 4 (AOM Study II) wk to induce tumors. CF-1 mice were fed diets containing 0.025% or 0.1% apigenin for 23-25 wk. Female Min mice were fed diets for 10 wk beginning at 5 wk of age. In two AOM-treated mouse colon tumor studies 0.025% and 0.1% dietary apigenin modestly reduced tumors in the group fed 0.025% apigenin (25% incidence in comparison with 65% in the controls) in a non-dose response manner. Apigenin failed to inhibit adenoma formation in the Min mouse study. These results suggest that dietary apigenin showed promise in cancer prevention by reducing the ODC activity and ACF formation, however, clear evidence of cancer prevention was not obtained in mouse tumor studies. Further investigation of the potential chemopreventive effect of apigenin in carcinogenesis is warranted.

Animals↗
PubMed1996

The chemopreventive flavonoid apigenin induces G2/M arrest in keratinocytes.

Apigenin is a plant flavonoid which has been shown to significantly inhibit UV-induced mouse skin tumorigenesis when applied topically, and may represent an alternative sunscreen agent in humans. We have investigated the molecular mechanism(s) by which apigenin inhibits skin tumorigenesis. Initial studies examined the effects of apigenin on the cell cycle. DNA flow cytometric analysis indicated that culturing cells for 24 h in medium containing apigenin induced a G2/M arrest in two mouse skin derived cell lines, C50 and 308, as well as in human HL-60 cells. The G2/M arrest was fully reversible after an additional 24 h in medium without apigenin. We investigated the effects of apigenin on cyclin B1 and p34cdc2, since cyclin B1/p34cdc2 complexes regulate G2/M progression. Western blot and immune complex kinase assays using whole cell lysates from 308 and C50 cells treated for 24 h with 0-70 microM doses of apigenin demonstrated that apigenin treatment did not change the steady-state level of p34cdc2 protein, but did inhibit p34cdc2 H1 kinase activity in 308 cells. Western blot analysis showed that apigenin treatment of C50 cells and 308 cells inhibited the accumulation of cyclin B1 protein in a dose-dependent manner. The apigenin levels detected in cultured keratinocytes were relevant to those detected in epidermal cells of Sencar mice treated with tumor inhibitory doses of apigenin. In conclusion, we present evidence that apigenin induces a reversible G2/M arrest in cultured keratinocytes, the mechanism of which is in part due to inhibition of the mitotic kinase activity of p34cd2, and perturbation of cyclin B1 levels.

Animals↗
PubMed2000

Increase in wild-type p53 stability and transactivational activity by the chemopreventive agent apigenin in keratinocytes.

Apigenin, a naturally occurring, non-mutagenic flavonoid, has been shown to inhibit UV-induced skin tumorigenesis in mice when topically applied. In this report we have used the mouse keratinocyte 308 cell line, which contains a wild-type p53 gene, to study the effect of apigenin treatment on p53 protein levels and the expression of its downstream partner, p21/waf1. Cells were treated with 70 microM apigenin for various times and levels of p53 and p21/waf1 protein were assessed by western blot analysis. The level of p53 protein was induced 27-fold after 4 h of apigenin treatment and levels remained elevated through 10 h of exposure. After 24 h of exposure to 70 microM apigenin, p53 protein levels returned to control levels. p21/waf1 protein levels increased approximately 1. 5-2-fold after 4 h and remained elevated at 24 h. To investigate the mechanism of p53 protein accumulation, we compared the half-life of p53 protein in vehicle- and apigenin-treated cells. Cells were incubated for 4 h in the presence of apigenin, then cycloheximide was added to inhibit further protein synthesis and p53 protein levels were measured by western blot. The half-life of p53 protein was found to be increased an average of 8-fold in apigenin-treated cells compared with vehicle-treated cells (t(1/2) = 131 min versus 16 min in apigenin- versus vehicle-treated cells, respectively). The mechanism of p53 protein stabilization is currently being investigated. To determine whether p53 was transcriptionally active, we also performed gel mobility shift assays and transient transfection studies using a luciferase plasmid under the control of the p21/waf1 promoter. Both p53 DNA-binding activity and transcriptional activation peaked after 24 h of exposure to apigenin. These studies suggest that apigenin may exert anti-tumorigenic activity by stimulating the p53-p21/waf1 response pathway.

Animals↗
PubMed2005

Apigenin induces cell cycle arrest and p21/WAF1 expression in a p53-independent pathway.

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.

Anticarcinogenic Agents↗
PubMed2005

Flavonoid apigenin inhibits motility and invasiveness of carcinoma cells in vitro.

Investigations of the mechanisms of the cancer-preventive activity of apigenin (4',5,7,-trihydroxyflavone), a plant-derived, anti-carcinogenic flavonoid, showed its interference with cell proliferation, survival, and gap junctional coupling. We used a model based on non-invasive HeLa wild-type cells and their connexin43 (Cx43) transfected counterparts to correlate the effect of apigenin on tumour cell invasiveness with its influence on cell motility. Both cell lines displayed similar motile properties in control conditions. Apigenin treatment resulted in a significant and reversible inhibition of translocation of both HeLa wild-type cells and HeLa Cx43 transfectants. The effect of apigenin on cell proliferation was less pronounced especially at low apigenin concentration, whereas its influence on cell motility correlated with the reduction of the invasive potential of HeLa Cx43 cells as shown by an invasion assay based on the confrontation of tumour cell spheroids with chick embryo heart fragments. HeLa Cx43 cells were highly invasive in controls, but did not invade the heart tissue at tumour cell aggregate-fibroblast capsule interfaces in the presence of apigenin and failed to fully engulf these heart fragments. Because the motility of chick heart fibroblasts was only slightly affected by apigenin, these observations indicate that apigenin exerts its anti-invasive effect on HeLa cells predominantly via a specific inhibition of tumour cell motility. This inhibitory effect of apigenin on tumour cell invasiveness in vitro demonstrates that apigenin may exert its anti-tumorigenic effect in vivo via inhibition of tumour cell penetration of the healthy tissue.

Antineoplastic Agents↗
PubMed2005

Apigenin drives the production of reactive oxygen species and initiates a mitochondrial mediated cell death pathway in prostate epithelial cells.

BACKGROUND: Phytoestrogens may reduce tumorigenesis in prostate cancer. We screened five phytoestrogens for their effect on cell growth and apoptosis in PWR-1E, LNCaP, PC-3, and DU145 prostate epithelial cells in vitro. METHODS: We assessed cell number, proliferation, and apoptosis using crystal violet assays, flow cytometric analysis, and TUNEL. Focusing specifically on apigenin we assessed the ability of calpain, serine protease, caspase, estrogen receptor, and ceramide synthase inhibitors to block apigenin induced apoptosis. We also analyzed caspase 3, 7, 8, 9, Bcl-2, Bax, Bid, and cytochrome C by Western analysis, and mitochondrial permeability and reactive oxygen species production by flow cytometry using mitosensor(TM) and DCFH-DA, respectively. RESULTS: Apigenin and silybinin significantly reduced cell number, with apigenin inducing apoptosis in PWR-1E, LNCaP, PC-3, and DU145 cells. The PC-3 and DU145 cells were less susceptible to apigenin induced apoptosis then LNCaP and PWR-1E cells. The induction of apoptosis by apigenin was caspase dependent. Apigenin generated reactive oxygen species, a loss of mitochondrial Bcl-2 expression, mitochondrial permeability, cytochrome C release, and the cleavage of caspase 3, 7, 8, and 9 and the concomitant cleavage of the inhibitor of apoptosis protein, cIAP-2. The overexpression of Bcl-2 in LNCaP B10 cells reduced the apoptotic effects of apigenin. CONCLUSIONS: Apigenin induces cell death in prostate epithelial cells using a mitochondrial mediated cell death pathway. Bcl-2 has a role in inhibiting apigenin induced cell death in prostate epithelial cells.

Amino Acid Chloromethyl Ketones↗