Protease inhibitor cocktail for use with mammalian cells and tissue extracts was obtained from Sigma-Aldrich (St. Louis, MO, USA). Chrysin was purchased from Alexis Biochemicals (San Diago, CA, USA). Specific inhibitors of STAT3 (Cucurbitacin-I), PI3K (LY294002), ERK (PD98059), and NF-κB (IKK-2 inhibitor IV) were obtained from Calbiochem (San Diego, CA, USA). For in vitro experiments, test compounds were dissolved in dimethyl sulfoxide (DMSO) and kept as a stock solution at −20°C. The final concentration of DMSO was kept below 0.2% throughout the study. Primary antibodies specific to Tubulin, STAT3, Akt, ERK1/2, and p65 NF-κB and phosphorylated forms of STAT3 (Tyr705), Akt (Ser⁴⁷³), ERK1/2 (Thr²⁰²/Tyr²⁰⁴), p65 NF-κB (Ser⁵³⁶) were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibody specific to HIF-1α (H1α67) was obtained from Novus Biologicals (Littleton, CO, USA). Agonistic monoclonal antibody targeting mouse DR5 TRAIL receptor (DR5 mAb or MD5-1) was obtained from the Department of Immunology, Juntendo University School of Medicine, Japan.

Thai propolis was harvested from a hive of Apis mellifera collected in Chiangmai province, Thailand, in August 2006. A voucher specimen (SWU 0212) was deposited at the Faculty of Pharmacy, Srinakharinwirot University, Nakhon Nayok province, Thailand. Two major compounds, chrysin and tectochrysin, were isolated. Propolis (1 kg) was extracted with methanol (MeOH) under sonication yielding 517 g extract. Part of the extract (140 g) was fractionated by silica gel column chromatography using dichloromethane (CH₂Cl₂), with increasing concentration of MeOH, to obtain 16 fractions. Fraction 5 (3.0 g, 1–1.5% MeOH-CH₂Cl₂ eluate) and fraction 8 (9.5 g, 2% MeOH-CH₂Cl₂ eluate) was left overnight to obtain the crystals of tectochrysin (314 mg) and chrysin (732 mg), respectively. Their structures were identified by comparison of physical and spectroscopic data with authentic standard and the published data (17,18).

4T1 murine mammary carcinoma cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in DMEM supplemented with 2 mM L-glutamine, 10% fetal bovine serum (FBS) and antibiotics, and maintained at 37°C in a humidified atmosphere of 5% CO₂: these conditions were used as normoxic condition (21% O₂). For hypoxic condition (1% O₂), the cells were incubated at 37°C, in a humidified atmosphere of 1% O₂ and 5% CO₂ maintained by a regulated supply of N₂ and CO₂ gas inside a hypoxic chamber (C-Chamber with ProOX and ProCO₂ controllers, Biospherix, NY, USA).

Cells were seeded into 96-well plates (5×10³ cells/well) and left overnight in a CO₂ incubator. The cells were pretreated by adding medium containing test compound to the wells, and incubated for 30 min, then the plates were further incubated under hypoxia (1% O₂) for 48 h. Number of surviving cells after treatment was determined by crystal violet staining method as previously described (19). Briefly, the cells were washed three times with phosphate buffer saline (PBS), fixed with 95% ethanol, and stained with crystal violet solution (0.5% w/v in 25% MeOH). After extensive washing by tap water, the stained cells were lysed with 0.1 N HCl in MeOH, after which absorbance was determined at 550 nm using a microplate reader. Assay was performed in triplicate wells, and results were expressed as percent survival compared with control (DMSO).

After treatment, cells were rinsed with cold PBS, scraped and lysed in RIPA lysis buffer containing phosphatase inhibitors (20 mM β-glycerophosphate, 1 mM Na₃VO₄, 10 mM NaF) and protease inhibitor cocktail. Cell lysates were fractionated by electrophoresis in 7.5% or 10% SDS-PAGE, and electrophoretically transferred to PVDF membrane (Immobilon-P nylon, Millipore, Bedford, MA, USA). After blocking with BlockAce (Dainippon Pharmaceutical, Co. Ltd., Osaka, Japan), the membranes were probed with the indicated primary antibodies overnight, followed by horseradish peroxidase-conjugated secondary antibodies. Bands were visualized using WesternBright ECL reagent (Advansta, Menlo Park, CA, USA).

RNA extraction and first-strand cDNA synthesis were performed as previously described (20). Real-time PCR was run on a LightCycler^® 2.0 Instrument (Roche), using QuantiTect™ SYBR Green PCR Master mix (Qiagen, Valencia, CA, USA). Sequences of PCR primers were obtained from following references; mouse VEGF (21); mouse β-actin (22). VEGF primer (fwd) 5′-GAGATGAGCTTCCTACAGCAC-3′; and VEGF primer (rev) 5′-TCACCGCCTTGGCTTGTCAC-3′. β-actin primer (fwd) 5′-AAG TGT GAC GTT GAC ATC CGT AA-3′; and β-actin primer (rev) 5′-TGC CTG GGT ACA TGG TGG TA-3′. PCR reaction was started by an initial activation step at 95°C for 15 min, followed by 40 cycles comprising denaturation at 94°C/15 sec, annealing at 55°C/30 sec, and extension at 72°C/30 sec. Expression level of the VEGF gene was normalized to β-actin gene, and calculated from crossing point (Cp) value of the sample, relative gene expression level = 2^{(Cpactin− Cptarget)}. The relative change in gene expression compared with untreated normoxic cells was expressed as fold change, calculated by 2^−ΔΔCp method.

Balb/c mice (6-week-old female) were purchased from Sankyo Lab Service Co. (Hamamatsu, Japan). Experiments were conducted in accordance with institutional guidelines and approval of University of Toyama Animal Experimental Ethics Committee. Cell suspensions (5×10⁵ cells) were injected into mammary fat pads on day 0. Tumors were measured every other day (length and width) using a caliper. Daily oral administration (p.o.) of chrysin suspended in 0.5% carboxymethyl cellulose began on day 1 after tumor inoculation. DR5 mAb (10 μg/mouse) was intraperitoneally (i.p.) injected on day 10 and day 14 after tumor inoculation. Primary tumors were excised on day 18, weighed and fixed in 10% formalin. After sacrificing the mice on day 32, lungs were collected and fixed in Bouin’s solution (0.9% picric acid, 9% formaldehyde, 5% acetic acid). Number of metastatic colonies on surface of the lungs was counted. Size of primary tumors and metastatic colonies was measured using a caliper square along the longer axes (a) and shorter axes (b), and tumor volume was calculated by the following formula; tumor volume (mm³) = ab²/2. The volume of metastatic colonies was categorized into 3 sizes; small (<0.5 mm³), medium (0.5–1.5 mm³), and large (>1.5 mm³). Metastatic burden was determined from total volume of lung metastatic colonies. Data are expressed as mean ± SD of 10 mice per group.

For in vitro experiments, data are expressed as mean ± SD, and analyzed by Student’s two-tailed t-test to determine the significance of differences between groups. For in vivo experiments, the significance of differences between groups was analyzed by analysis of variance (ANOVA), and the means were compared by using Tukey, with a p-value <0.05 being considered significant.

We first examined the effect of Thai propolis extract against hypoxic survival of 4T1 cells. Pretreatment with propolis extract (10–100 μg/ml) for 30 min before exposure to hypoxia (1% O₂) for 2 days decreased hypoxic cell survival in a concentration-dependent manner (Fig. 1A). Next, the two major constituents present in Thai propolis extract, chrysin and tectochrysin (Fig. 1B) were examined. Chrysin treatment showed a concentration-dependent decrease in hypoxic cell survival, with 28–74% inhibition observed at 40–100 μM chrysin, while tectochrysin did not show inhibition at the highest soluble concentration (60 μM) (Fig. 1C). These results indicate that chrysin contributes to the inhibitory effect of Thai propolis extract on hypoxic cell survival.

We further explored effect of chrysin on hypoxia-induced activation of multiple cell signaling pathways that contribute to cell survival, including HIF-1, STAT3, Akt, ERK, and NF-κB. 4T1 cells were pretreated with 60–100 μM chrysin for 30 min before exposure to hypoxia for 6–24 h. HIF-1α protein stabilization and hypoxia-induced phosphorylation of ERK and p65 NF-κB were not inhibited by chrysin treatment (Fig. 2A). In contrast, hypoxia-induced STAT3 tyrosine phosphorylation was markedly inhibited by chrysin, in a dose- and time-dependent manner, while marginal decrease in Akt phosphorylation was observed only at the highest concentration test (100 μM) (Fig. 2A), suggesting that STAT3 was primary target of chrysin rather than Akt.

Specific inhibitors of STAT3 (cucurbitacin-I, 2 μM), PI3K (LY294002, 25 μM), ERK (PD98059, 25 μM), and NF-κB (IKK-2 inhibitor IV, 10 μM), were used to evaluate the contribution of the signaling pathways on hypoxic survival of 4T1 cells. Pretreatment with inhibitors for 30 min before hypoxic exposure for 2 days revealed that hypoxic cell survival was inhibited by inhibitors of STAT3, Akt, and NF-κB pathways, while ERK inhibitor showed marginal effect (Fig. 2B). These results indicate the important role of STAT3, Akt, and NF-κB pathways in hypoxic survival of 4T1 cells. Moreover, STAT3 inhibitor treatment mimics the effect of chrysin, suggesting that STAT3 inhibition is the mechanism underlying inhibitory effect of chrysin on hypoxic survival of 4T1 cells.

VEGF has been shown to be an autocrine survival factor for breast cancer cells under hypoxia (12). We examined the effect of chrysin on hypoxia-induced VEGF gene expression in 4T1 cells, in comparison to a STAT3 inhibitor (cucurbitacin-I). The cells were pretreated with chrysin (60 μM) or STAT3 inhibitor (2 μM) for 30 min before exposure to hypoxia for 24 h. qRT-PCR analysis revealed that hypoxia increased VEGF mRNA levels in 4T1 cells by 16-fold higher than normoxic cells (Fig. 2C). Chrysin decreased VEGF mRNA levels in hypoxic 4T1 cells, similar to that observed with STAT3 inhibitor treatment, by 71 and 81% inhibition, respectively, compared with control hypoxic cells (Fig. 2C). The reduction of VEGF expression by chrysin and STAT3 inhibitor was correlated with decrease in STAT3 tyrosine phosphorylation levels under hypoxia (Fig. 2D). Taken together, the results indicate that STAT3 inhibition is likely to be a mechanism of action of chrysin for decreasing hypoxia-induced VEGF expression in 4T1 cells, and this might contribute to the effect of chrysin on hypoxic survival.

Since hypoxia and angiogenesis contribute to tumor progression and metastatic formation (7), chrysin was further studied in 4T1 spontaneous metastasis model. The experimental treatment protocol (Fig. 3A) did not affect body weight of the mice (data not shown). The effect of treatments was evaluated from both primary tumor growth and metastatic burden. Chrysin (100 and 250 mg/kg daily) had no significant effect on primary tumor growth (Fig. 3B and C). However, metastatic burden was largely decreased by chrysin, in a dose-dependent manner, and significant suppression was observed with the highest dose of chrysin treatment (Fig. 3D). Reduction of metastatic burden might result from either decrease in metastatic rate (number of metastatic colonies) or metastatic growth (size of metastatic colonies), or both. Chrysin tended to decrease metastatic rate but without statistical significance (Fig. 3E). Suppressive effect of chrysin on metastatic growth was observed by decreased number of medium and large size metastatic colonies, in a dose-dependent manner, and significant reduction was clearly observed with large size metastatic colonies (Fig. 3F). These results reveal the antimetastatic activity of chrysin in vivo by inhibiting the growth of metastatic colonies.

In addition to therapeutic effect on primary tumor, DR5 mAb also expresses antimetastatic activity in mouse models (2). We evaluated the potential of chrysin as an adjuvant for antimetastatic activity of DR5 mAb. Since 4T1 cells are sensitive to apoptosis induced by DR5 mAb, low dose treatment of DR5 mAb (10 μg/mouse, ×2) was used instead of therapeutic doses (200 μg/mouse, ×3) used previously (2), in order to avoid metastasis reduction by massive elimination. DR5 mAb alone significantly suppressed primary tumor growth as shown by reduction in primary tumor size and weight, compared with control (Fig. 4B and C). Combination of DR5 mAb + chrysin (250 mg/kg/d) tended to be more suppressive, but no statistical difference between DR5 mAb alone and the combination treatment was observed (Fig. 4C). Antimetastatic activity of DR5 mAb was revealed by significant decrease in metastatic burden and metastatic rate, and the combination treatment resulted in a significantly greater effect (Fig. 4D and E). DR5 mAb also expressed suppressive effect on metastatic growth, as shown by significant decrease in number of large size metastatic colonies, with greater effect being observed in combination treatment (Fig. 4F). Moreover, the effect of DR5 mAb on the number of small size metastatic colonies was significantly potentiated when DR5 mAb was combined with chrysin (Fig. 4F). Collectively, the results suggested that chrysin per se had a suppressive effect on metastatic growth, and could enhance antimetastatic activity of DR5 mAb in combination treatment.