up-regulation of insulin-like growth factor binding protein-3

©2005 FASEB

The FASEB Journal express article 10.1096/fj.05-3740fje. Published online October 17, 2005.

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 Sanjeev Shukla,*,║ Anil Mishra,† Pingfu Fu,‡,¶ Gregory T. MacLennan,§,║,¶ Martin I. Resnick,*,║,¶ and Sanjay Gupta*,║,¶

*Department of Urology, ‡Epidemiology & Biostatistics, and §Pathology, Case Western Reserve University, ║University Hospitals of Cleveland; and ¶Ireland Cancer Center, Cleveland, Ohio; and †Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania

Correspondence author: Sanjay Gupta, Department of Urology, The James & Eilleen Dicke Research Laboratory, Case Western Reserve University & University Hospitals of Cleveland, 10900 Euclid Avenue, Cleveland, OH 44106. E-mail: [email protected]

ABSTRACT

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 μg/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,

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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.

Key words: insulin-like growth factor-I • insulin-like growth factor receptor-1 • insulin receptor substrate-1

he insulin-like growth factor (IGF) axis is an important modulator of growth and development (1). It comprises six affinity binding proteins, several low-affinity binders, proteases, and receptors (1). The two IGF ligands (IGF-I and IGF-II) modulate a diverse

range of biological activities, including cell growth, differentiation, and apoptosis; imbalance of this growth axis may preferentially favor uncontrolled cell proliferation and malignant transformation (2, 3). IGFs are mostly produced in local tissues, in which they act in an autocrine and paracrine manner. The biological functions of IGFs are mediated primarily by the type I IGF receptor (IGF-IR), a tyrosine kinase transmembrane receptor that binds with the ligand (4). The IGF-IR signal transduction involves activation of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, which promote cell survival and inhibit apoptosis (2–4). The activities of IGFs are modulated by a family of high-affinity specific IGF-binding proteins (IGFBP). The classical role of the IGFBPs involves IGF binding and modulation of the IGF-signaling pathway (4, 5). In fact, IGFBP-3 is the major serum carrier protein for IGF, circulating in the intravascular compartment as a ternary complex composed of IGF, IGFBP-3, and an acid-labile subunit. In addition to its function as a high-affinity binding protein for IGFs, IGFBP-3 has been found to negatively regulate cell proliferation and induce apoptosis in an IGF-independent manner (5). Overexpression of IGFBP-3 has been shown to inhibit cell proliferation and to markedly reduce tumor formation in athymic nude mice (6–8). Exogenous IGFBP-3 inhibits cell growth and induces early apoptosis, presumably by binding to specific cell surface receptors (9). These effects are mediated through tumor suppressor functions of p53 and modulation in the protein expression of Bcl-2 family members (10, 11). Moreover, IGFBP-3 has been shown to be induced by many potent antiproliferative and proapoptotic agents, including TGF-β, retinoids, tumor necrosis factor, antiestrogens, and 1, 25-dihydroxyvitamin D3 (12–15).

A number of laboratory and population-based studies have demonstrated that IGF physiology plays a critical role in the development and progression of prostate cancer (16, 17). Prospective and case control studies have demonstrated a positive correlation between circulating IGF-I levels in healthy men and risk of developing prostate cancer (18, 19). There is evidence that in the normal prostate, IGFs are produced by stromal cells, whereas normal epithelial cells express IGF-IR, suggesting a paracrine mode of regulation (20). Deregulated expression of IGF-I in prostatic epithelium leads to neoplasia in transgenic mice (21). In contrast, blockade of IGF-IR expression inhibits cell proliferation and invasion in human prostate cancer cells (22). Up-regulation of IGF-I expression has been found in neoplastic prostate epithelial cells; it has been postulated that this is an adaptive response that may contribute to the evolution of androgen-independent prostate cancer (23). More recently, IGF/IGFBP-3 plasma levels are being evaluated as potential surrogate biomarkers in patients with risk of prostate cancer (24).

T


Studies from our laboratory and elsewhere have demonstrated that apigenin (4’, 5, 7,-trihydroxyflavone), a common dietary flavonoid abundantly present in fruits and vegetables, may prove useful in the prevention and therapy of prostate cancer (25–28). Similarly, other studies have shown that apigenin suppresses tumorigenesis and angiogenesis in melanoma and carcinoma of the breast, skin and colon (29–32). The effects of apigenin appear to be mediated primarily through suppression of the expression of COX-2, MMP-9, NOS-2, VEGF, and lipoxygenase (33–35). Apigenin has been shown to induce apoptosis in a wide variety of human cancer cells, through activation of caspases, inhibition of fatty acid synthase, topoisomerase inhibition, and modulation in Bax and Bcl-2 expression (29–38). Apigenin has been shown to inhibit activation of phosphatidylinositol 3-kinase, protein kinase B/Akt, mitogen-activated protein kinase: ERK1/2, casein kinase-2 and other upstream kinases involved in prostate cancer development and progression (30, 39, 40). More recently, we have shown that apigenin suppresses both constitutive and TNF-α-induced NF-κB activation, resulting in apoptosis of prostate cancer cells (40). Because apigenin has been shown to inhibit cell proliferation and induces apoptosis in cancer cells, we hypothesized that the IGF axis is involved in this process. We also reasoned whether physiologically attainable concentrations of apigenin could up-regulate IGFBP-3 sufficiently to inhibit prostate cancer growth in vivo. This study presents the first evidence that the in vitro and in vivo growth inhibitory effects of apigenin involve increase in IGFBP-3 gene expression and secretion and decrease in IGF-I levels through modulation of IGF-axis signaling in prostate cancer.

MATERIALS AND METHODS

Cell line and reagents

Human prostate carcinoma 22Rv1 cells were obtained from the American Type Culture Collection (Manassas, VA). Apigenin (>95% purity) was obtained from A.G. Scientific, Inc. (San Diego, CA). Active IGFBP-3 and IGF-I ELISA kits were purchased from Diagnostic Systems Laboratories (Webster, TX). M30-Apoptosense ELISA kit was purchased from Alexis Biochemicals (San Diego, CA). Fetal bovine serum (FBS) was obtained from Gibco BRL (Gaithersburg, MD). Polyclonal antibodies for anti-IGF-I (H-70) and anti-IGFBP-3 (H-98) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies recognizing full-length PARP and its cleaved product (#9542), cleaved PARP (Asp214; #9546), anti-IRS-1(#2382), and anti-phospho-tyrosine (p-Tyr-100; #9411) were purchased from Cell Signaling Technology (Fremont, CA). IGFBP-3 anti-sense oligonucleotides were synthesized from Invitrogen Corporation (Carlsbad, CA). Human recombinant IGF-I was purchased from Sigma Chemical Co. (Saint Louis, MO), whereas recombinant IGFBP-3 protein was obtained from Upstate Biotechnology (Lake Placid, NY).

Animals and diet

Approximately 8-wk-old male athymic nude mice (weighing 28±3 g) were obtained from Ireland Cancer Center and housed at the AAALAC-accredited athymic nude mice facility of Case Western Reserve University. The animals were fed with the autoclaved Teklad 8760 high-protein diet and tap water ad libitum throughout the study. Apigenin (10 mg) was suspended in 1 ml vehicle material (0.5% methyl cellulose and 0.025% Tween 20) by sonication for 30 s at 4°C and further diluted for appropriate concentration. Apigenin, 20 and 50 μg/mouse/day (w/v) was administered by gavage in 0.2 ml of a vehicle consisting of 0.5% methyl cellulose and 0.025%


Tween 20, daily for 8 wk. The per-oral route was chosen in order to simulate dietary consumption of flavonoids in humans. It is important to emphasize that apigenin reaches its maximum concentration in blood 24 h postingestion; therefore, we opted for daily dosing of apigenin in order to maintain peak blood levels in mice during chemoprevention studies (41).

Experimental design for tumor xenograft study

To determine the effect of apigenin on prostate tumor growth, 22Rv1 tumors were grown subcutaneously in athymic nude mice. Briefly, nude mice were randomly divided into two experimental sets using two different protocols of apigenin feeding before and after tumor implantation (Fig. 1). Approximately, ~1 million 22Rv1 cells suspended in 0.05 ml of medium and mixed with 0.05 ml of matrigel were subcutaneously injected in the left and right flank of each mouse to initiate tumor growth. Two weeks after cell inoculation, animals were divided into three equal groups of 6 mice each. The first group received only 0.2 ml of vehicle material by gavage daily and served as a control group. The second and third groups of animals received 20 and 50 μg/mouse/day doses of apigenin in vehicle, respectively, for 8 wk and were assigned to the second protocol. These doses are comparable to the daily consumption of flavonoid in humans as reported in previously published studies (42, 43). In the first protocol, apigenin feeding was started two weeks before cell inoculation and was continued for 8 wk. In both experimental protocols, animals were monitored daily, and their body weights were recorded weekly throughout the studies. Once the tumor xenografts started growing, their sizes were measured twice weekly in two dimensions with calipers. At the termination of the experiment, tumors were excised and weighed to record wet tumor weight.

IGFBP-3 and IGF-I ELISA assay

Active IGFBP-3 and IGF-I ELISA immunoassay kits were used to determine their concentration secreted from 22Rv1 tumors in mouse serum, according to vendor’s protocol. The principle of IGFBP-3 assay was based on an enzymatically amplified “two-step” sandwich-type immunoassay using precoated with anti-IGFBP-3 polyclonal antibody onto microtitration wells. Similarly, IGF-I assay was based on an amplified “one-step” sandwich-type immunoassay, which includes a simple extraction step to separate IGF-I from its binding protein in serum. The absorbance of the developed color was determined at 450 nm with correction wavelength at 620 nm. IGFBP-3 and IGF-I concentration was extrapolated from the standard curve generated using appropriate standards provided with the assay kit.

For the analysis of IGFBP-3 and IGF-I secreted from 22Rv1 cells in culture medium, cells were grown at standard culture conditions in 10% fetal bovine serum-supplemented RPMI 1640. At 70% confluency, cells were treated either with DMSO (final concentration in all treatment groups 0.01% v/v) or 20 and 40 µM apigenin in serum-free medium containing 1 mg/ml BSA and 50 µg/ml ascorbic acid for 12, 24, and 48 h. At the end of treatment, the medium was collected, concentrated for IGFBP-3 and IGF-I assay, and cells were trypsinized, counted and processed for total cell lysate. Final data are presented as IGFBP-3 and IGF-I (ng/ml) for equal number of live cells (106) in each treatment group.


Cell culture and treatment

Human prostate carcinoma 22Rv1 cells were cultured in RPMI 1640 growth medium supplemented with 10% FBS, 1% penicillin, and streptomycin. Cells were maintained at 37°C in a humidified CO2 incubator. Apigenin dissolved in DMSO (maximum concentration 0.1%) was used for the treatment of cells. For determining the effect of apigenin on cell growth, incubation was continued for desired time intervals and cell viability was assessed by trypan blue exclusion assay after a standard protocol, as described previously (25, 26).

For studies assessing the effects of apigenin and/or exogenous recombinant (rh) IGFBP-3 on insulin receptor substrate (IRS)-1, 70% confluent cells were washed twice with PBS and then cultured in serum-deprived and phenol-free medium for 36 h. During the last 3 h of culture, the cells were treated with either vehicle or 40 μM apigenin, or 500 ng/ml rh-IGFBP-3. For the last 15 min of culture, cells were exposed to PBS alone or to IGF-I (100 ng/ml) at 37°C. Monolayers were quickly washed twice with cold PBS and lysed with lysis buffer; protein was estimated and processed for Western blotting.

To determine whether apigenin interferes with the IGF-IR-signaling pathway and whether IGFBP-3 plays a functional role, 70% confluent cultures were washed 3 times with serum and phenol-free RPMI 1640 and later incubated in this media for 48 h with vehicle alone or 40 μM apigenin, or 5.0 μg/ml IGFBP-3 anti-sense oligonucleotide or in combination. The sense (5′-CCC CGG TTG CAG GCG TCA TG-3′) and antisense (5′-CAT GAC GCC TGC AAC CGG GG-3′) IGFBP-3 oligonucleotides were used at a concentration of 5.0 μg/ml, as described previously (44). At the end of these treatments, cultures were exposed to PBS or 100 ng/ml IGF-I and incubated for 15 min at 37°C. Cells were processed for protein estimation, immunoprecipitation, and Western blotting.

Cell cycle analysis

For cell cycle analysis, serum-deprived cells were stimulated with 100 ng/ml IGF-I for 15 min, washed 3 times with serum and phenol-free RPMI 1640, and later incubated in this media for 24 h with vehicle only or 40 μM apigenin, or 5 μg/ml IGFBP-3 anti-sense oligonucleotide alone or in combination. Propidium iodide (PI) staining and flow cytometry were used to examine cell cycle progression, as described previously (26).

Proliferation assay

The proliferation assay was performed in cells fixed with 10% formaldehyde in phosphate-buffered saline for 1 h and 2% fresh methylene blue in 0.1 M borate buffer (pH 8.5) was added. After elution of dye from the cells, the plates were washed with 1:1 (v/v) ethanol and 0.1 M HCl and were photographed.

Western blot analysis

For Western blotting, appropriate amounts of cell lysates (25 μg protein) were resolved over 4–20% Tris-glycine polyacrylamide gel and then transferred onto the nitrocellulose membrane. The blots were blocked using 5% nonfat dry milk and probed using appropriate primary antibodies in blocking buffer overnight at 4°C. The membrane was then incubated with appropriate secondary


antibody horseradish peroxidase (HRP) conjugate (Amersham Life Sciences, Arlington Heights, IL) followed by detection using chemiluminescence ECL kit (Amersham Life Sciences). For equal loading of protein, the membrane was stripped and reprobed with anti-β-actin antibody.

Immunoprecipitation

Cell lysate (400 μg) was immunoprecipitated with 2 μg of anti-IRS-1 antibody and was incubated at 4°C for 3 h. Protein A-agarose beads (20 μl) were added and incubated overnight at 4°C. Immunoprecipitated proteins were washed 4 times with lysis buffer, electrophoresed by SDS-PAGE, and analyzed by Western blotting, as described previously (40).

Reverse transcriptase-PCR

Total RNA (1 μg) was reverse-transcribed for cDNA synthesis using M-MLV Reverse Transcriptase and Oligo(dT)12-18 primer (Gibco BRL, Grand Island, NY). PCR was performed using gene specific primers for IGFBP-3. Primer sequences used for IGFBP-3 are: sense 5′- TCC AGG AAA TGC TAG TGA GTC GGA G-3′, anti-sense 5′-CTT GCT CTG CAT GCT GTA GCA GTG C-3′ (475 bp). The cDNA was amplified with an initial denaturation at 94°C for 9 min followed by the sequential cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min for 35 cycles with final extension at 72°C for 1 min. A constitutively expressed β-actin sense 5′- AAG AGA GGC ATC CTC ACC CT-3′, anti-sense 5′- TAC ATG GCT GGG GTG TTG AA-3′ (218 bp) was coamplified to confirm the equal loading of the RT-product. The annealing PCR products were electrophoresed on 1.5% agarose gel containing 0.5 μg/ml ethidium bromide.

Apoptosis by ELISA

Apoptosis was assessed by M30-Apoptosense ELISA kit (Alexis Biochemicals, San Diego, CA) according to the manufacturer’s protocol and color developed was read at 450 nm against the blank, and values were plotted against standards provided and expressed as units per liter.

Immunohistochemistry and immunofluorescence

Immunohistochemistry for IGFBP-3 was performed on formalin-fixed, paraffin-embedded tumor xenograft sections using a standard protocol, as described previously using IGFBP-3 (H-98) antibody obtained from Santa Cruz Biotechnology (45). Immunoreactive complexes were detected using 3, 3′-diaminobenzidene. Slides were then counterstained in Mayer’s hematoxylin, mounted in crystal mount media, and dried overnight on a level surface. For fluorescence staining of cleaved PARP (Asp214), (Cell Signaling Technology, Fremont, CA) Alexa Fluor 488-fluorescence labeled-goat anti-mouse IgG antibody was used (Caltag Laboratories, Burlingame, CA) and imaged under an inverted Olympus BH2 microscope equipped with a fluorescent light source.

HPLC analysis of serum and tumor samples for apigenin levels

Serum samples (0.2 ml) and tissue homogenate from the various experimental groups were deproteinized by adding 0.4 ml of methanol, vortex-mixed for 60 s, and centrifuged at 2200 g for 15 min at 4°C. The supernatant (0.6 ml) was collected into the tube and evaporated to dryness by


vacuum freeze-drying. The residue was dissolved in 200 μl of methanol and chromatographically analyzed by HPLC system.

Analytical RP-HPLC was performed for apigenin estimation on a Waters 600 system, Amphotech. (Beverly, MA) connected to a Waters UV detector set at 349 nm using a Vydac 250 mm, 5 μm C18 columns. The mobile phase consisting of 2% formic acid-acetonitrile-methanol (40:35:25) was run at a flow rate of 1 ml/min. Apigenin concentrations in serum samples were calculated based on the sum of the area of each diastereomer peak and compared with the standard curve.

Statistical analysis

Changes in tumor volume and body weight during the course of the experiments were visualized by scatterplot. Differences in tumor volume (mm3) and body weight at the termination of the experiment among three treatment groups were examined using ANOVA followed by Tukey’s multiple comparison procedure. All tests were two-tailed and P values less than 0.05 were considered to be statistically significant.

RESULTS

Apigenin inhibits growth of human prostate cancer 22Rv1 xenograft in nude mice

Our previous studies demonstrated growth inhibition and induction of apoptosis by apigenin in various human prostate cancer cells (25, 26). In the present study, we have demonstrated that daily oral intake of apigenin at doses of 20 and 50 μg/mouse/day (w/v) administered to nude mice exhibited a dose-dependent inhibition of tumor xenograft growth as measured by tumor volume, as well as by wet tumor weight (Fig. 1). We used two specific protocols whose designs simulate prevention and therapeutic regimens. In the first protocol, we studied whether two different doses of apigenin feeding 2 weeks before tumor implantation and was continued for 8 weeks in mice had any efficacy on the growth of prostate cancer xenograft. As shown in Fig. 1A, tumor volume was inhibited by 44 and 59% (P<0.002 and 0.0001), and wet weight of tumor by 41 and 53% (P<0.05), respectively, at the termination of the experiment. In this experimental protocol, prior intake of apigenin delayed the establishment of tumor xenograft at both doses of apigenin.

In the second protocol, in which mice received apigenin for 8 wk beginning 2 wk after tumor implantation, tumor volumes were reduced by 39 and 53% (P<0.01 and 0.002), and wet weights of tumor were reduced by 31 and 42% (P<0.05), respectively, at the end of the studies (Fig. 1B). In this experimental protocol, a marked dose-dependent inhibitory effect of apigenin was observed on tumor xenograft that remained constant up to the termination of the experiment.

Oral intake of apigenin does not exhibit any apparent toxic symptoms in nude mice

Because toxicity and/or side effects of a test agent are important factors that must be assessed before initiation of a clinical trial, we observed the mice for evidence of apigenin toxicity throughout the study. Apigenin feeding to mice did not appear to induce any adverse effects as judged by monitoring body weight gain, dietary intake, and prostate weight. As shown in Fig. 2, oral intake of apigenin at doses of 20 and 50 μg/mouse/day was associated with a decrease in total body weight, which was more pronounced in the first protocol at the higher dose of


apigenin feeding. Lower body weights in apigenin-fed groups were attributed to reduced tumor volume and weight as compared with control group. Moreover, the animals from all the groups did not exhibit reduction in food consumption (data not shown). We evaluated the prostate weights of all mice at the termination of the experiment to determine whether apigenin might play a role in prostatic hyperplasia. There was no significant difference in prostate weights among the various groups at the end of the study, although a modest decrease in the prostate weight was observed in mice administered higher doses of apigenin in both treatment protocols (data not shown).

Oral intake of apigenin induces IGFBP-3 and reduces IGF-I levels in mouse serum

On the basis of previous cell culture studies (25), in which apigenin was shown to have antiproliferative and apoptotic effects on 22Rv1 cells, we hypothesized that these responses may be due to involvement of the IGF axis, associated with up-regulation of IGFBP-3 and/or decrease in IGF-I levels. Therefore, we assessed the levels of IGFBP-3 and IGF-I in the serum of nude mice with and without 22Rv1 xenograft implants and further studied the effects of apigenin. As shown in Fig. 3A, oral intake of apigenin at 20 and 50 μg/mouse/day for 8 weeks after tumor implantation resulted in 1.8- and 2.4-fold increases (P<0.0001) in IGFBP-3 levels in the first experimental protocol. Similar effects were observed in the second protocol, where 1.2- and 2.0-fold increases were observed (P<0.0001) in IGFBP-3, levels were observed after 20 and 50 μg/mouse/day apigenin feeding for 8 wk. These results were consistent with the IGFBP-3 protein expression observed after apigenin intake in both treatment regimens (Fig. 3A, B). A modest increase in IGFBP-3 level (1.06-fold) was observed after 50 μg/mouse/day apigenin feeding for 8 wk to nude mice without 22Rv1 tumors.

We next determined the serum IGF-I levels in mouse serum. As shown in Fig. 3A, oral intake of apigenin at 20 and 50 mg/mouse/day for 8 wk after tumor implantation resulted in 0.38- and 0.85-fold decreases (P<0.001) in IGF-I levels in the first experimental protocol. Similar effects were observed in the second protocol, where 0.34- and 0.66-fold decreases were observed after apigenin feeding (Fig. 3B). These results were consistent with IGF-I protein expression in both treatment protocols (Fig. 3A, B).

Tumor growth inhibition and up-regulation of IGFBP-3 levels correlates with mouse serum and tumor apigenin levels

Next, we determined the levels of apigenin in serum and evaluated whether these levels correlate with IGFBP-3 levels and tumor growth inhibition. To assess the levels of apigenin in the serum of apigenin-fed mice, a standard HPLC profile of apigenin was developed and its retention time was determined. The apigenin concentration in mouse serum was determined under a linear range of detection using area under curve of apigenin peak in HPLC profiles of these samples (Fig. 4A). As shown in Fig. 4A–B, control animals receiving vehicle material only showed undetectable levels of apigenin in their serum, whereas apigenin feeding resulted in sharp dose-dependent peaks in serum levels in mice in both the first and second protocols as determined by HPLC profiles of these samples. Similar effects in apigenin levels were observed in 22Rv1 tumors in vivo (Fig. 4C). As shown in Fig. 4D, compared with control mice, showing undetectable serum levels of apigenin, 20 or 50 μg/mouse/day apigenin administration (w/v) resulted in 0.84 ± 0.03 and 1.26 ± 0.06 μM apigenin in serum and 0.42 ± 0.02 and 0.68 ± 0.004 μMoles/g tumor tissue in the first protocol groups (P<0.0001). Similar observations of 0.78 ±


0.02 and 1.06 ± 0.04 μM apigenin in serum and 0.30 ± 0.02 and 0.62 ± 0.04 μMoles/g tumor tissue was noted at 20 or 50 μg/mouse/day dose of apigenin in the second protocol groups (P<0.0001), respectively. This increase in apigenin concentration in the serum and tumors of apigenin-fed mice positively correlated with induction of IGFBP-3 levels and inhibition of tumor growth in the apigenin-fed group.

Oral intake of apigenin induces IGFBP-3 mRNA and protein expression in tumor xenograft

Next, we evaluated whether oral intake of apigenin could induce IGFBP-3 levels in 22Rv1 tumor xenograft. As shown in Fig. 5, apigenin feeding at 20 and 50 μg/mouse/day dose for 8 wk resulted in induction of IGFBP-3 mRNA expression in a dose-dependent fashion in mice in the first protocol group. Compared with the control group, apigenin feeding for 8 wk after tumor implantation to mice in the first protocol group resulted in 1.6- and 2.8-fold increases in IGFBP-3 mRNA expression at doses of 20 and 50 μg/mouse/day (Fig. 5A). Similar observations were noted in mice in the second protocol group, in which apigenin feeding resulted in 1.4- and 1.8-fold increases in IGFBP-3 mRNA expression at doses of 20 and 50 μg/mouse/day, compared with expression levels in mice in the corresponding control group (Fig. 5B). These results were comparable with increases in protein expression in the apigenin-fed group. Compared with the control group, apigenin feeding at 20 and 50 μg/mouse/day dose resulted in 1.5- and 2.0-fold increase in IGFBP-3 protein expression in mice in the first protocol group, whereas a 1.3- and 1.7-fold increase in IGFBP-3 levels was observed in mice in the second protocol group after apigenin feeding. Similar results were noted for IGFBP-3 protein with immunohistochemical analysis in both treatment groups (Fig. 5A, B).

Oral intake of apigenin induces apoptosis in 22Rv1 xenograft in nude mice

Previous studies (6–8) have demonstrated that IGFBP-3 can modulate the apoptotic process; therefore, we evaluated the effect of apigenin feeding on induction of apoptosis in tumor xenografts. As shown in Fig. 6, oral intake of apigenin at doses of 20 and 50 μg/mouse/day for 8 wk after tumor implantation resulted in marked induction of apoptosis in tumor lysate in mice in the first protocol groups as shown by M-30 apoptosense reactivity (Fig. 6A). Compared with control group, 1.7- and 2.9-fold increases (P<0.0001) in induction of apoptosis were observed in mice in the first protocol group, whereas 1.9- and 3.0-fold increases (P<0.0001) in induction of apoptosis were observed in mice in the second protocol group after intake of 20 and 50 μg/mouse/day of apigenin (Fig. 6A, B). Similar results were noted in tumor xenografts, where apigenin feeding led to significant increases in cleaved product of poly-ADP ribose polymerase (PARP; Asp214) in both experimental protocols by immunofluorescence analysis (Fig. 6A, B).

Apigenin induces cell growth inhibition and apoptosis in 22Rv1 cells

The effects of apigenin on cell growth inhibition and apoptosis and their correlation with serum apigenin levels after oral intake were additionally assessed for their biological relevance and mechanisms of action in 22Rv1 cell culture. As shown in Fig. 7A, treatment of 22Rv1 cells with 20 μM concentration of apigenin for 12, 24, and 48 h, resulted in 8.0, 28.8, and 47.6% inhibition of cell growth, compared with vehicle-treated control. A high dose of 40 μM apigenin resulted in 22.5, 39.4, and 67.5% cell growth inhibition in these cells. We also studied the effect of low doses of apigenin on cell growth inhibition. Compared with control, treatment of 22Rv1 cells


with 1 μM concentration of apigenin for 12, 24, and 48 h resulted in 0.8, 2.8, and 3.5% cell growth inhibition; whereas a 5 μM concentration of apigenin resulted in 2.1, 4.6, and 8.7% cell growth inhibition. The inhibition in cell proliferation was further demonstrated by methylene blue assay after treatment with 20 and 40 μM apigenin (Fig. 7B).

Next, we determined whether cell growth inhibition by apigenin correlates with induction of apoptosis in these cells. As shown in Fig. 7C, treatment of 22Rv1 cells with apigenin resulted in dose- and time- dependent induction of apoptosis as shown by M-30 apoptosense ELISA assay. Treatment of 22Rv1 cells with 1-μM concentration of apigenin resulted in 1.06- and 1.1-fold increase in induction of apoptosis at 24 and 48 h of treatment. No significant induction of apoptosis was observed at 12 h of treatment with apigenin. Compared with vehicle-treated control, treatment with 5 μM concentration of apigenin for 12, 24, and 48 h resulted in 1.1-, 1.3-, and 1.6-fold increase in induction of apoptosis. Similarly, treatment with 20 μM concentration of apigenin for 12, 24, and 48 h, resulted in 1.4-, 2.0-, and 2.4-fold increase in induction of apoptosis, compared with vehicle-treated control. A higher dose of apigenin (40 μM) produced 1.6-, 2.5-, and 3.7-fold increases in induction of apoptosis in these cells. This apoptotic effect was further confirmed by cleavage of PARP by apigenin in dose- and time- dependent fashion (Fig. 7D).

Apigenin treatment results in induction of IGFBP-3 and decrease in IGF-I levels in cell culture medium and lysate of 22Rv1 cells

Consistent with the effect of apigenin on up-regulation of IGFBP-3 levels in 22Rv1 tumors in nude mice, apigenin treatment resulted in significant increases in IGFBP-3 levels in 22Rv1 cell culture medium. Compared with vehicle-treated control, a significant increase of 2.5- to 6.2-fold at 12 h, 2.4- to 5.0-fold at 24 h, and 2.2- to 4.5-fold at 48 h in IGFBP-3 levels were observed in cell culture medium after 20 and 40 μM apigenin treatment, respectively (Fig. 8A). Similar results were observed in cell lysate, in which 20 and 40 μM apigenin treatment resulted in a significant increase of 2.1- to 3.2-fold at 12 h, 2.2- to 3.4-fold at 24 h, and 2.6- to 3.8-fold at 48 h in IGFBP-3 levels, respectively (Fig. 8B). These results were further confirmed by Western blot analysis in which apigenin treatment significantly increased IGFBP-3 protein expression with simultaneous decrease in IGF-I protein expression in dose- and time-dependent fashion in cell culture medium and cell lysate (Fig. 8A, B).

Apigenin treatment inhibits IGF-I-stimulated cell cycle progression in 22Rv1 cells

We next determined the effect of apigenin on IGF-I induced cell cycle progression. As shown in Fig. 9A, serum deprivation of cells for 36 h arrested 22Rv1 cells in G0/G1 phase (77%) of the cell cycle. IGF-I (100 ng/ml) stimulation for 15 min to serum deprived 22Rv1 cells significantly increased the proportion of cells in S phase (10.5 vs. 29.1%) of the cell cycle after 24 h. Treatment of 22Rv1 cells with 40 μM apigenin to IGF-I-stimulated cells resulted in a marked increase of cells in G0/G1 phase (51.8 vs. 68.8%) and sub-G1 phase (10.2 vs. 18.8%) of the cell cycle, which may be an indication for apoptosis. Further, treatment of cells with IGFBP-3 antisense oligonucleotide led to increased accumulation of cells in G0/G1 phase (68.8 vs. 79.2%) along with significant reduction of cells in sub-G1 phase (18.8 vs. 3.1%) of the cell cycle. Compared with apigenin-treated group, simultaneous treatment of 22Rv1 cells with apigenin and IGFBP-3 antisense oligonucleotide led to decreased accumulation of cells in Sub-G1 phase (18.8


vs. 11.3%) and S phase (20.1 vs. 12.7%) of the cell cycle. These results suggest that antisense IGFBP-3 partially rescues tumor cells from apigenin-mediated apoptosis.

Apigenin treatment inhibits IGF-induced IRS-1 tyrosine phosphorylation in 22Rv1 cells

The IGF axis mediates its cell growth effects by interaction of IGF-I with the IGF-1R receptor, which activates IRS-1, a key signaling molecule activated by IGF-I (22, 23). Increased tyrosine phosphorylation by IGF-I is directly correlated with increased activation of the downstream signaling pathway that includes PI3K-Akt and MAPK (2–4). Therefore, we next evaluated the influence of apigenin on the effects of exogenously added IGFBP-3 on IGF-I-induced IRS tyrosine phosphorylation. For these studies, 22Rv1 cells were grown under serum-deprived medium for 36 h and later exposed to vehicle or 40 μM apigenin or 500 ng/ml rh IGFBP-3 for 3 h. Later, the cells were incubated for 15 min with or without 100 ng/ml IGF-I. As shown in Fig. 9B, lane1, cells grown under serum-deprived conditions exhibit very low or undetected levels of IRS-1 tyrosine phosphorylation, as evidenced by the absence of reactivity of immunoprecipitated IRS-1 with anti-phosphotyrosine antibody by Western blotting. In contrast, treatment of IGF-I for 15 min resulted in a significant activation of IRS-1, as evidenced by very strong reactivity with the anti-phosphotyrosine antibody (lane 2). A 3-h pretreatment with apigenin resulted in a modest decrease in IGF-I-induced IRS-1 tyrosine phosphorylation (lane 3 vs. lane 2), whereas significant inhibition (75%) of IGFI-induced IRS-1 phosphorylation was observed with a 3 h pretreatment with 500 ng/ml rh-IGFBP-3 (lane 4 vs. lane 2). These results suggest that exogenous IGFBP-3 addition had an important inhibitory effect on IRS-1 tyrosine phosphorylation, whereas apigenin did not exhibit any significant changes.

Because our previous results have demonstrated that apigenin induces IGFBP-3 expression and that IGFBP-3 reduces IRS-1 tyrosine phosphorylation, we next conducted an experiment to determine whether prolonged exposure to apigenin could reduce IRS-1 tyrosine phosphorylation and whether this was dependent on IGFBP-3 induction. As shown in Fig. 9C, incubation of 22Rv1 cells with 100 ng/ml IGF-I for 15 min led to a significant activation of IRS-1 compared with vehicle-treated control (lane 2 vs. lane 1). In cells pretreated with 40 μM apigenin for 48 h, the 15 min IGF-1 incubation resulted in a significant decrease (82%) in IRS-1 activation (lane 3 vs. lane 2). However, in cells pretreated with both 40 μM apigenin and IGFBP-3 anti-sense oligonucleotide for 48 h, the reduction in IRS-1 tyrosine phosphorylation was attenuated (lane 4 vs. lane 3). None of these treatments affected IRS-1 protein levels, as shown in Fig. 9B, C, bottom. These data provide evidence that the anti-proliferative effects of apigenin in 22Rv1 cells involve inhibition of IGF-I-induced IRS-1 phosphorylation.

IGFBP-3 antisense oligonucleotide treatment reverses the apoptotic effects of apigenin in 22Rv1 cells

Next, we determined whether apigenin-mediated cell growth, inhibition, and apoptosis were mediated by increased IGFBP-3 secretion. For these studies, log phase growing cells were treated with either 40 μM apigenin or 5 μg/ml IGFBP-3 antisense oligonucleotide alone or in combination for 24 h. As shown in Fig. 9D, treatment of 22Rv1 cells with 40 μM concentration of apigenin for 24 h resulted in significant decrease (41%) in number of viable cells, whereas IGFBP-3 antisense oligonucleotide exhibited an increase (9%) in cell growth, compared with vehicle-treated control. Further, combination of apigenin and IGFBP-3 antisense oligonucleotide reversed cell growth inhibitory effects of apigenin by 31%. Further, treatment of 22Rv1 cells


with 40 μM concentration of apigenin for 24 h resulted in increased apoptosis as evidenced by a 89 kDa fragment, a large fragment of cleaved product of PARP (lane 2 vs. lane 1). In cells treated with IGFBP-3 antisense oligonucleotide, no evidence of PARP cleavage was ascertained (lane 3 vs. lane 2). However, combination treatments of apigenin and IGFBP-3 antisense oligonucleotide for 24 h attenuated apigenin-mediated PARP cleavage (lane 4 vs. lane 2). These results correlated with IGFBP-3 protein expression (Fig. 9D, bottom) and provide evidence that the cell growth inhibitory and apoptotic effects of apigenin in 22Rv1 cells are associated with increased IGFBP-3 expression.

DISCUSSION

In recent years chemoprevention has gained increasing attention as a potential means of reducing the incidence of clinical malignancies (46, 47). Chemopreventive agents are especially attractive in the management of prostate cancer, because of its long latency period and high incidence (46, 47). Prospective studies have suggested that regular consumption of fruits and vegetables protects against various types of human cancers (48). This concept is supported by observations that Asian men who consume low-fat, high-fiber, plant-based diets rich in flavonoids have the lowest prostate cancer incidence in the world. Migration studies have further strengthened these observations (49). Asian men who migrate to the United States and adopt a Western diet acquire a risk of developing prostate cancer that approaches the average U.S. incidence within one generation (49). Therefore, there is an increased impetus to provide scientific support for the use of dietary agents in chemoprevention strategies. On the basis of epidemiologic observations and our findings with apigenin exhibiting promising results in cell culture system, we evaluated the chemoprotective potential of apigenin in preclinical models of prostate cancer. In the present study, we provide the first in vivo evidence for the efficacy of apigenin in inhibiting prostate cancer growth in athymic nude mice.

Epidemiological and other studies have demonstrated that higher circulating IGF-I levels and/or lower IGFBP-3 levels are strongly and positively correlated with increased risk of prostate cancer (18, 19, 24). The importance of IGF-I signaling in deregulated cellular growth has been established in prostate cancer cells and transgenic mice (21–23). Increased IGF signaling stimulates proliferation and inhibits apoptosis in prostate cancer cells (16, 17). In contrast, IGFBP-3 has been shown to induce apoptosis in prostate cancer cells through IGF-I-independent pathways (50). Further evidence for these effects was provided by studies showing that IGFBP-3 mutants that do not bind IGFs stimulate apoptosis in human prostate cancer cells (51). Together, these studies suggested that mitogenic signaling, as well as cell survival signaling via the IGF/IGFBP-3 pathway, is constitutively activated in human prostate cancer cells. Therefore, sustained inhibition of this pathway may inhibit the development and/or progression of prostate cancer. Recent reports have suggested that several natural and synthetic agents are capable of IGFBP-3/IGF-I modulation (12–15, 52, 53). In the present study, apigenin was able to induce elevated IGFBP-3 and decrease in IGF-I levels both in cell culture and in vivo environments, which correlated with cell growth inhibition and apoptosis of cancer cells. These results suggest that prostate cancer inhibition by apigenin may, in part, be due to modulation of IGF-axis signaling. These studies support the concept that circulating IGFBP-3 and/or IGF-I levels in serum may serve as a surrogate marker to monitor chemoprevention with apigenin.

Numerous studies have demonstrated that mitogenic, as well as cell survival signaling via the IGF/IGF-IR pathway is constitutively activated and that such signaling provides growth/survival


advantage to prostate cancer cells (2–4, 17, 18). It is widely accepted that the IGF axis activates antiapoptotic signaling, which, in turn, up-regulates phosphatidylinositol-3 kinase and mitogen-activated protein kinase pathway in cancer cells (17, 18). The induction of these transduction pathways occurs through the IGF-IR signaling pathway. Therefore, inhibition of IGF-IR signaling by a strong induction of IGFBP-3 may be one of the major mechanisms of apigenin-induced cell/tumor growth inhibition. Our studies indicate that apigenin is capable of inhibiting IGF-I-induced cell cycle progression by modulating IGF-I-induced tyrosine phosphorylation and IRS-1 inhibition. This inhibitory effect may be due to IGFBP-3 induction by apigenin, thereby reducing the amount of bioavailable ligand available for interaction with IGF-IR and/or also inhibiting IGF-I induced IRS-1 phosphorylation. This study for the first time demonstrates that plant flavonoid apigenin modulates IGF-I/IGFBP-3 physiology in prostate cancer cells. The schematic representation is shown in Fig. 10.

Dietary sources of flavonols and flavones vary between different countries. Average consumption of these plant flavonoids ranges from 6 mg/day in Finland to 64 mg/day in Japan, with intermediate intake in the United States (13 mg/day), Italy (27 mg/day), and Netherlands (33 mg/day), respectively. Naturally occurring sources of apigenin include many fruits and vegetables such as parsley, onions, chamomile, wheat sprouts, and some seasonings (42, 43). The doses of apigenin used in our animal studies are physiologically attainable concentrations in humans (42, 43). The dose of 20 μg/mouse/day relates to the median apigenin intake (40–50 mg/day) and the higher dose of 50 μg/mouse/day apigenin mimics an intake of ~120 mg/day consumption by an adult. In the present studies, oral intake of apigenin at these doses has shown to circulate ~1% of apigenin in the mouse serum, which is consistent with recent studies documenting ~1.2% of radioactivity after a single oral administration of [3H] apigenin to Wistar rats (41). Our observation that a significantly strong prostate cancer xenograft growth inhibitory effect of apigenin at 50 μg/mouse/day dose without any noticeable toxicity in the present study may have direct practical and translational relevance to human prostate cancer patients.

One of the major drawbacks associated with flavonoids, particularly apigenin, is limited bio-availability in its pure form (43). In natural conditions, flavonoids present in food are generally bound to sugar as β-galactosides (42, 43). Upon absorption the flavonoids are metabolized by methylation or by conjugation with gluconate or sulfate via dual recycling involving both enteric and enterohepatic pathways (43, 54). A recent study of apigenin has shown lack of efficacy in tumor-bearing mice that received apigenin in three different galenical formulations (55). The vehicle preparations used in this report were 1) apigenin dissolved in 15% dimethyl sulfoxide diluted in PBS containing 5% polyethylene glycol and 0.5% Tween 80, 2) 0.15 N NaOH and 7.5% dimethyl sulfoxide, and 3) matrigel. In our studies, we used 0.5% methyl cellulose and 0.025% Tween 20 to prepare a suspension of apigenin, which was administered orally to the animals on a daily basis. These observations suggest that vehicle plays an important role in flavonoid absorption. Further detailed studies are needed to identify an ideal vehicle that may increase the efficacy of plant flavonoids in in vivo studies.

An important consideration in cancer chemoprevention research is whether pharmacological levels of the agent being tested relate to biological efficacy. In this study we demonstrated that higher serum and tumor apigenin levels positively correlated with growth inhibition and induction of apoptosis in prostate cancer xenografts. The concentration of apigenin used in these studies exhibited dose- and time- dependent inhibition of cell growth and induction of apoptosis, which correlated with increasing IGFBP-3 levels; inhibition of IGF-I secretion and IGF-I-


induced IRS-1 phosphorylation. These observations indicate that apigenin is capable of modulating cell regulatory events related to cell growth inhibition and is capable of activating apoptosis in preclinical models of prostate cancer. Our findings raise the possibility that apigenin has the potential to target IGF-signaling, a strategy that may be useful in the prevention or treatment of prostate cancer.

ACKNOWLEDGMENTS

The research was supported by the Athymic Animal Core Facility of the Comprehensive Cancer Center of Case Western Reserve University and University Hospitals of Cleveland (P30 CA43703). Financial support was provided by grants from United States Public Health Services RO1 CA108512, RO3 CA094248, RO3 CA099049, and partial support of funds from Cancer Research and Prevention Foundation to S. G.

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Received February 16, 2005; accepted September 1, 2005


Fig. 1


Fig. 1 (cont)

Figure 1. Effect of oral intake of apigenin on 22Rv1 tumor xenograft growth in athymic nude mice. Approximately 1 million cells were injected in both the flanks of each mouse to initiate ectopic prostate tumor growth as described in Materials and Methods. A) First protocol: apigenin was provided to animals 2 wk before cell inoculation. B) Second protocol: apigenin was provided to animals 2 wk after cell inoculation. Mice were fed ad libitum with Teklad 8760 autoclaved high-protein diet. Apigenin was provided with 0.5% methyl cellulose and 0.025% Tween 20 as vehicle, to these animals orally on a daily basis. Group I, control received 0.2 ml vehicle only; Group II, 20 µg/mouse apigenin in 0.2 ml vehicle; Group III, 50 µg/mouse apigenin in 0.2 ml vehicle, daily for 8 weeks. Once the tumor xenograft started growing, their sizes were measured twice weekly in two dimensions throughout the study. Tumor volume (mm3) is represented as the mean of 8–10 tumors in each group. Tumors were excised, weighed, and measured at the termination of the study. Wet weight of tumors is represented as mean of 8–10 tumors from each group. †P < 0.05, ‡P < 0.001 at the termination of the experiment; bars ± SE. Photograph, animals with tumor and tumors from control and apigenin-fed group.


Fig. 2

Figure 2. Effect of oral intake of apigenin on body weight of nude mice during 22Rv1 tumor xenograft study. A) First and (B) second experimental protocol detailed in Figure 1. The body weight of each mouse in different groups was recorded weekly throughout the experiment. The body weights of mice are representative as mean of 5–6 mice in each group, bars ± SE.


Fig. 3

Figure 3. Effect of oral intake of apigenin on serum IGFBP-3 and IGF-I levels in nude mice 22Rv1 tumor xenograft study. A) First and (B) second experimental protocol detailed in Figure 1. At the termination of the study, serum concentration of human IGFBP-3 and IGF-I was estimated using Active IGFBP-3 and IGF-I ELISA kits and by Western blotting as detailed in Materials and Methods. IGFBP-3 and IGF-I concentration (ng/ml) in serum samples were calculated by logistic curve fit generated standard curve and are represented as mean of 5–6 serum samples from individual mouse in each treatment group. †P < 0.05, ‡P < 0.001; bars ± SE; ND, nondetectable.


Fig. 4

Figure 4. Pharmacologically attainable apigenin concentration in serum and tumor of nude mice 22Rv1 tumor xenograft. Apigenin levels in serum and tumors were detected by HPLC analysis as described in Materials and Methods. A) Chromatogram of extracted blank serum and spiking with 1–5 µM apigenin. B) and (C) represents chromatogram of apigenin extracted from serum and tumor tissue from apigenin-fed mice in both first and second protocols. Each chromatogram is representative of individual mouse in each experimental group. D) Apigenin levels in mouse serum and tumors as detected by HPLC analysis described in Materials and Methods. In each case, the data shown are mean of 5–6 serum and tumor samples, ‡P < 0.001; bars ± SE; ND, nondetectable.


Fig. 5

Figure 5. Effect of oral intake of apigenin on IGFBP-3 expression in 22Rv1 tumor xenograft in nude mice. A) First and (B) second experimental protocol detailed in Figure 1. Tumors were harvested for extraction of total RNA and protein and fixed in paraformaldehyde for paraffin-embedded sections to determine IGFBP-3 expression by RT-PCR, Western blotting, and immunohistochemistry, as described in Materials and Methods. Representative results and sections from tumors from mice in each treatment group are shown here.


Fig. 6

Figure 6. Effect of oral intake of apigenin on induction of apoptosis in 22Rv1 tumor xenograft in nude mice. A) First and (B) second experimental protocol detailed in Figure 1. Tumors were harvested for extraction of total protein and fixed in paraformaldehyde for paraffin-embedded sections to determine the extent of apoptosis by M-30 apoptosense ELISA assay and immunofluorescence of cleaved PARP (Asp214) product in various groups. Data in ELISA assay are represented as mean of 5–6 samples from individual mouse in each treatment group. †P < 0.05, ‡P < 0.001; bars ± SE. Representative results and sections from tumors from mice in each treatment group are shown here.


Fig. 7

Figure 7. Effect of apigenin treatment on cell growth inhibition and induction of apoptosis in 22Rv1 cells. The cells were grown in 70% confluence in RPMI 1640 medium and treated with either DMSO alone or 20- and 40-µM apigenin (final concentration in medium) at standard culture conditions described in Materials and Methods. A) Cells were treated with indicated doses of apigenin and were harvested at desired times interval and counted with hemocytometer. The data are represented as % cell inhibition. B) Cells were treated with apigenin, and proliferation was determined by methylene blue assay. C) Cells were treated with apigenin and induction of apoptosis was determined in total cell lysate by M-30 apoptosense ELISA assay. D) Apoptosis by PARP cleavage. For equal protein loading membrane was stripped and reprobed with anti-actin antibody. Data shown in A and C are mean of 2 independent plates, each in duplicate and experiment was repeated with similar results. †P < 0.05, ‡P < 0.001; bars ± SE.


Fig. 8

Figure 8. Effect of apigenin treatment on induction of IGFBP-3 and decrease in IGF-I levels in cell culture media and lysate of 22Rv1 cells. The cells were grown to 70% confluency and treated with indicated doses of apigenin for various time intervals. The media were collected and concentrated by freeze drying, and the total cell lysate were prepared for estimation of IGFBP-3 by ELISA and IGF-I/IGFBP-3 by Western blotting as described in Materials and Methods. A) IGFBP-3 and IGF-I levels in the culture medium and (B) IGFBP-3 and IGF-I levels in total cell lysate. Data shown here are the mean of 2 independent plates, each in duplicate, and the experiment was repeated with similar results. †P < 0.05, ‡P < 0.001; bars ± SE.


Fig. 9

Figure 9. Effect of apigenin on IGF-I-induced cell cycle progression, IRS-1 tyrosine phosphorylation, and apoptosis in 22Rv1 cells. A) After reaching 70% confluency, the cells were washed and grown under serum-deprived conditions for 36 h. Cells were incubated with PBS or 100 ng/ml IGF-I for 15 min at 37°C, washed 3 times with serum and phenol-free RPMI 1640, and later incubated in this media for 24 h with 1) vehicle only; 2) 40 µM apigenin; and 3) 5 µg/ml IGFBP-3 anti-sense oligonucleotide alone or combination and later subjected to cell cycle analysis. B) During last 3 h of serum starvation, 22Rv1 cells were treated either with 1) vehicle only; 2) 40 µM apigenin; 3) 500 ng/ml rh IGFBP-3 alone or combination. After treatments, cell lysates were prepared and IRS-1 was immunoprecipitated using anti-IRS-1 antibody and then SDS-PAGE, and Western blotting was performed. C) Cells were incubated for 48 h in serum and phenol-free medium with 5 µg/ml IGFBP-3-antisense (AS) oligonucleotides in the presence or absence of 40 µM apigenin and then stimulated for 15 min with 100 ng/ml IGF-I. D) Log phase growing cells were treated either with 1) vehicle only; 2) 40 µM apigenin; 3) 5 µg/ml AS IGFBP-3 alone or combination for 24 h. After treatments, cell number was determined; cell lysates were prepared and subjected to SDS-PAGE and Western blotting as detailed in Materials and Methods.


Fig. 10

Figure 10. Schematic representation of the effect of apigenin on IGF-axis signaling in prostate cancer. IGF-I is sequestered as ternary complex with IGFBP-3 and acid labile subunit in the intravascular compartment or as binary complex with IGFBP-3 (or other IGFBPs) in the cellular environment. Modification of IGFBP-3 due to cleavage or phosphorylation releases free IGF to bind to the IGF-IR, which phosphorylates itself and several downstream targets, including the adaptor proteins IRS-1 and SHC, which link the IGF-IR to the PI3K and Ras/MAPK signaling pathways leading to cell survival and proliferation. IGFBP-3 also has IGF-independent antiproliferative and proapoptotic effects. Apigenin has shown to modulate this process (↑ represents activation, ┬ represents inhibition).


up-regulation of insulin-like growth factor binding protein-3

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