The anthocyanin used in our experiments was extracted from the seed coat of black soybean and was supplied by the Rural Development Administration, Suwon, Republic of Korea. The anthocyanin was extracted and analyzed as previous described.16 A normal prostate cell line, RWPE-1 (ATCC, Manassas, VA, USA) and an androgen-independent prostate cancer cell line, DU-145 (Korean Cell Line Bank, Seoul, Korea) were cultured in RPMI 1640 supplemented with 10% FBS, L-glutamine (300 mg/L), 25 mM HEPES, and 25 mM NaHCO₃.

Cell growth and viability were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO, USA) assay. RWPE-1 and DU-145 cells were seeded in 96-well plates (5×10³ cells per well in 100 mL). After growing for 24 h in RPMI-1640 medium, the cells were treated with various concentrations (0, 30, 60, or 120 µM) of anthocyanin for 24, 48, or 72 hours to evaluate the effect of the anthocyanin on cell viability. At the end of the treatment, the medium was removed and 0.5 mg·mL^-1 of MTT was added to the medium. After 4 h, DMSO (200 mL) was added to each well and the optical density was read at 570 nm. Cell sensitivity to drug treatment was expressed as the drug concentration that yielded 50% cell inhibition (IC₅₀). All experiments were performed in triplicate.

DU-145 cells (5×10⁵ cells mL^-1) treated with vehicle only or anthocyanin (60 or 120 µM for 24 or 48 hours) and were washed with PBS. Two hundred µL of binding/lysis buffer was added, the total volume was adjusted to 400 µL, and mixed. After incubating at 15-25℃ for 10 minutes, 100 µL of isopropanol was added, and shaken. The samples were added to a filter tube attached to a collection tube and centrifuged at 800 rpm for 1 minute. Washing buffer (500 µL) was added and centrifuged at 800 rpm for 1 minute twice, and a third time at 13000 rpm for 10 seconds. Elution buffer (100 µL) was added, and centrifuged at 800 rpm for 1 deminute. Finally, electrophoresis was performed on a 1% agarose gel at 75 V for 1.5 hours, and viewed under ultraviolet light. Apoptosis of transfected and control cells was confirmed by DNA laddering assay as described.17

DU-145 cells (5×10⁵ cells mL^-1) were treated with with vehicle only or anthocyanin (60 or 120 µM for 24 or 48 hours) were extracted by centrifuging at 2000 rpm, for 5 minutes at 4℃, and lysed with 100 µL of lysis buffer for 30 minutes at room temperature (RT). The supernatant was extracted, quantified using a bicinchonic acid assay (Pierce, Rockford, IL, USA), and electrophoresed on an SDS-PAGE gel. After transfering to a nitrocellulose membrane, the membrane was blocked with 5% skim milk and then incubated overnight at 4℃ with an anti-p53 antibody (1:1000; Abcam, Cambridge, UK), an anti-Bcl-2 antibody (1:1000; Abcam, Cambridge, UK), an anti-Bax antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) an anti-PSA antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or an anti-AR antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were washed with TBS-T (TBS, 0.1% Tween 20) and secondary anti-mouse or anti-rabbit antibodies (Invitrogen Corporation, Paisley, UK) conjugated to horseradish peroxidase were added at RT for 1 h. To adjust for loading differences, membranes were reprobed with a β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The blots were analyzed using a chemiluminescence detection system (GE Healthcare, Pittsburgh, PA, USA). Densitometric analyses were performed using Image J software (NIH, Bethesda, MD, USA).

Nicotinamide adenine dinucleotide (NAD⁺)/NADH ratio was quantitatively assessed by enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (BioVision, Milpitas, CA, USA). Cells were pelleted in a micro-centrifuge tube and extracted with 400 µL of NADH/NAD⁺ extraction buffer by freeze/thaw two cycles or by homogenization. After centrifugation, the extracted NADH/NAD⁺ supernatant was transferred into a labeled tube. To detect total NADH (NADH and NAD⁺), 50 µL of extracted samples were transferred into labeled 96-well plate. To detect NADH, NAD⁺ needs to be decomposed before the reaction. Thus, to decompose NAD⁺, 200 µL aliquot of extracted samples were transferred to eppendorf tubes and heated to 60℃ for 30 min, and then samples were cooled on ice. The samples were spinned to remove precipitates if they occurred. Fifty µL of NAD⁺ decomposed samples were transferred to labeled 96-well plate. After the final color was developed with the addition of 10 µL NADH developer into each well, absorbance was measured at 450 nm.

The experimental animals were 6-week-old male BALB/c nude mice, weighing 18-22 g, provided by Samtaco Bio Co. (Osan, Korea). After a 1-week adjustment period, the animals were maintained in plastic cages, with 5 animals per cage. This study was approved by the Institutional Animal Care and Use Committee of the Catholic University of Korea, Seoul St. Mary's Hospital (IRB approval no. CUMC-2011-0026-02). Experimental animals were randomly allocated into 2 groups (control and anthocyanin), each consisting of 10 animals. Animal in the anthocyanin group were treated with daily oral anthocyanin (8 mg/kg), dissolved in 1 mL of distilled water and administered through an 8 F red Rob-Nel catheter once daily for 14 weeks. Animal in the control group received vehicle in the same manner and period as the experimental group. After 2 weeks of treatment, to establish tumor xenografts, DU-145 cells (2×10⁶) were suspended in 0.1 mL of medium and inoculated subcutaneously into the right flank of the mice in both groups. Body weights and tumor volumes were monitored twice weekly using calipers and calculated according to the formula (L×W²)/2, where L and W are the length and width, respectively.18

All experiments were performed on 3 separate cultures. All data are presented as mean±standard deviation, where p<0.05 is considered to be statistically significant. Overall comparisons between groups were performed using SPSS program (version 12.0, SPSS Inc., Chicago, IL, USA). Student's t-test and ANOVA followed by Tukey's test were needed to detect differences between groups in multiple comparisons.

To evaluate the effect of anthocyanin on the viability of human DU-145 cells, we used an MTT assay. Anthocyanin treatment significantly inhibited cell growth in a dose-dependent manner at all time points tested (24, 48, and 72 hours) (Fig. 1A). The IC50 value for anthocyanin at 24 hours after treating DU-145 cells was 60-90 µM. Whereas similar anthocyanin doses had insignificant effects on the viability of a normal prostate cell line, RWPE-1 (Fig. 1B). To test whether the anthocyanin-mediated decrease in cell growth was due to apoptosis, we carried out a DNA laddering assay to assess the fragmentation of cellular DNA, a characteristic of apoptotic cells. Agarose gel electrophoreses of the total DNA isolated from DU-145 cells treated with specified anthocyanin concentrations (60 or 120 µM for 24 or 48 hours) showed the characteristic DNA laddering patterns (Fig. 1C).

To elucidate the mechanism by which anthocyanin induces apoptosis in DU-145 cells, we next evaluated the effect of anthocyanin on the levels of apoptotic markers. Western blot analysis showed a significant increase in p53 expression in cells treated with anthocyanin (Fig. 2A and E). We also observed that anthocyanin treatment significant decreased the level of Bcl-2 protein with a concomitant increase in Bax protein (Fig. 2A, B, and C). These changes substantially increased the Bax/Bcl-2 ratio (Fig. 2D), which favors apoptosis, suggesting that the change in p53 and Bax/Bcl-2 ratio following anthocyanin treatment might trigger apoptosis.

To determine the effects of anthocyanin on AR expression, a critical target for treating prostate cancer, and on PSA production, a western blot analysis was performed on DU-145 cells treated with specified anthocyanin concentration. AR and PSA were constitutively expressed in untreated DU-145 cells. However, PSA levels were considerably reduced in anthocyanin-treated cells compared to controls (Fig. 3A and B). Similar to PSA, AR expression was decreased in cells treated with anthocyanin (Fig. 3A and C).

The effect of anthocyanin on the cellular NAD⁺/NADH ratio in DU-145 cells was quantitatively assessed by ELISA. NAD⁺/NADH ratio in DU-145 cells were 1.684±0.406, 3.177±0.351, and 2.069±0.311 in the control, and treated with 60 or 120 µM for 24 or 48 hours, respectively. As shown in Fig. 4, NAD⁺/NADH ratio was genrally increased after anthocyanin treatment. A significant increase in NAD⁺/NADH ratio was found in the 120 µM-treated for 24 and 48 hours groups compared to the control group (p<0.05).

To study the inhibitory effects of anthocyanin on tumor growth in vivo, DU-145 tumor xenografts were established in athymic nude mice. The animals did not exhibit any considerable changes in body weight during the experiment. The in vivo anti-tumorigenic effects of anthocyanin were analyzed every 4 weeks for a total of 12 weeks. At 4 weeks post-inoculation, the initial detection time and average tumor volume did not differ significantly between the 2 groups. At 8 weeks postinoculation, however, the average tumor volume in vehicle-treated mice increased, reaching 317.3 mm³. At 8 weeks, the average tumor volume was only 119.5 mm³ in mice treated with anthocyanin. At 12 weeks post-inoculation, the average tumor volume in the control group increased to 831.3 mm³, while the anthocyanin group grew to 288.4 mm³ (Fig. 5). In addition, one mouse in the control group died. The differences in tumor development between anthocyanin- and vehicle-treated mice were statistically significant at 8 and 12 weeks post-inoculation (p<0.01) (Fig. 5).