Complementary roles in cancer prevention: protease inhibitor makes the cancer preventive peptide lunasin bioavailable

Abstract

Background: The lower incidence of breast cancer among Asian women compared with Western countries has been partly attributed to soy in the Asian diet, leading to efforts to identify the bioactive components that are responsible. Soy Bowman Birk Inhibitor Concentrate (BBIC) is a known cancer preventive agent now in human clinical trials.

Methodology/principal findings: The objectives of this work are to establish the presence and delineate the in vitro activity of lunasin and BBI found in BBIC, and study their bioavailability after oral administration to mice and rats. We report that lunasin and BBI are the two main bioactive ingredients of BBIC based on inhibition of foci formation, lunasin being more efficacious than BBI on an equimolar basis. BBI and soy Kunitz Trypsin Inhibitor protect lunasin from in vitro digestion with pancreatin. Oral administration of (3)H-labeled lunasin with lunasin-enriched soy results in 30% of the peptide reaching target tissues in an intact and bioactive form. In a xenograft model of nude mice transplanted with human breast cancer MDA-MB-231 cells, intraperitoneal injections of lunasin, at 20 mg/kg and 4 mg/kg body weight, decrease tumor incidence by 49% and 33%, respectively, compared with the vehicle-treated group. In contrast, injection with BBI at 20 mg/kg body weight shows no effect on tumor incidence. Tumor generation is significantly reduced with the two doses of lunasin, while BBI is ineffective. Lunasin inhibits cell proliferation and induces cell death in the breast tumor sections.

Conclusions/significance: We conclude that lunasin is actually the bioactive cancer preventive agent in BBIC, and BBI simply protects lunasin from digestion when soybean and other seed foods are eaten by humans.

Figure 1. Lunasin is the main bioactive component in BBIC.

A: Western-Blot of BBIC using antibodies specific for BBI and lunasin. (1) Marker, (2) 6.25 µg protein, (3) 12 µg protein, (4) 25 µg protein, (5) Standard BBI (3 µg) or standard lunasin (200 ng). B: Foci formation inhibitory activity in DMBA-induced NIH3T3 cells. (a) Vehicle-treated cells (b) positive control or DMBA-treated cells, (c) BBIC (100 nM lunasin +1160 nM BBI), (d) BBI (100 nM) from BBIC, (e) Lunasin (100 nM) from BBIC, (f) Standard BBI (100 nM), (g) Synthetic lunasin (100 nM). Bars with different lower case letter designations are statistically significantly different from each other (P<0.05, n = 6).

Figure 2. Soy-derived BBI and KTI protect synthetic lunasin from in vitro digestion.

The upper, lighter bands are Coomassie Blue protein stains and the lower, darker bands are Western blots. (M) Markers, (1) Lunasin (600 ng) + pancreatin (600 ng) incubated for 0 h at 37°C, (2) Lunasin (600 ng) + pancreatin (600 ng) incubated for 1 h at 37°C, (3) Lunasin (600 ng) + BBI (18,000 ng, unboiled) + pancreatin (600 ng) incubated for 1 h at 37°C, (4). Lunasin (600 ng) + KTI (18,000 ng, unboiled) + pancreatin (600 ng) incubated for 1 h at 37°C, (5) Lunasin (600 ng) + BBI (18,000 ng, boiled) + pancreatin (600 ng) incubated for 1 h at 37°C, (6) Lunasin (600 ng)+KTI (18,000 ng, boiled) + pancreatin (600 ng) incubated for 1 h at 37°C, (7) BBI (unboiled), (8) KTI (unboiled).

Figure 3. Lunasin is absorbed through gastrointestinal barrier and reach the target tissues and organs in an intact and bioactive state.

A: Distribution of ³H-lunasin activity in various tissues and gastrointestinal contents of mice after () 3 h, () 6 h, () 9 h, () 12 h and () 24 h of oral administration of LES. Error bars shown for 3 h-results. B: Foci formation inhibitory activity in DMBA-induced NIH3T3 cells. (a) Vehicle-treated cells, (b) DMBA-treated cells, (c) synthetic lunasin, (d) lunasin extracted from liver of rats fed control diet, (e) lunasin extracted from liver of rats fed LES diet. Bars with different lower case letter designations are statistically significantly different from each other (P<0.05, n = 6).

Figure 4. Lunasin reduces breast tumor development in MDA-MB-231 tumor xenografts in athymic nude mice.

A: Body weight of mice treated with (♦) PBS (control), (□) 20 mg BBI/kg body weight, (▴) 20 mg lunasin/kg body weight, and (×) 4 mg lunasin/kg body weight, and injected with 1×10⁷ MDA-MB-231 cells in the flank of 15-week-old nude mice. No significant differences were observed among four groups. B: Tumor incidence in mice treated with PBS, BBI and two doses of lunasin. Lunasin reduced number of mice that showed tumors and delayed the appearance of these breast tumors. C: Tumor generation in mice treated with PBS, BBI and two doses of lunasin. Low dose of lunasin (4 mg/kg body weight) significantly reduced tumor generation compared to control group (*P = 0.029). Data are shown: mean ± SEM (n = 8).

Figure 5. Effects of BBI and lunasin on the growth of MDA-MB-231 tumors in athymic nude mice.

A: Tumor volume (mm³) of breast tumors induced by the administration of (♦) PBS (control), (□) 20 mg BBI/kg body weight, (▴) 20 mg lunasin/kg body weight and (×) 4 mg/kg of lunasin. B: Breast tumors weight induced by PBS (control), 20 mg BBI/kg body weight, 20 mg lunasin/kg body weight (P = 0.2134 vs control group and 0.0909 vs BBI group) and 4 mg lunasin/kg body weight (P = 0.1880 vs control group and 0.0569 vs BBI-group). Data are shown mean ± SEM (n = 8).

Figure 6. Histological features of MDA-MB-231 tumors treated with PBS (control), BBI and lunasin in athymic nude mice (400× magnification).

A: Tumors were collected at 7 week after breast cancer cells injection, and processed for H&E staining. Representative pictures are shown for H&E-stained sections (upper column), immunohistochemical staining for Ki-67 (middle column) and in situ TUNEL apoptotic indices (bottom column). B: Percentage of Ki-67 expression in tumors of mice. Ki-67 labeling revealed significantly lower levels of proliferating cells in lunasin groups versus control group (*P = 0.0062 in high lunasin group; *P = 0.0158 in low lunasin group). C: Percentage of apoptotic cells in tumors of mice measured with the TUNEL assay. Staining revealed significantly higher levels of apoptotic tumor cells in lunasin groups versus control group. (*P = 0.0088 in high lunasin group; 0.0141 in low lunasin group). Data are shown mean ± SEM.