Sulforaphane retards the growth of human PC-3 xenografts and inhibits HDAC activity in human subjects

Abstract

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables such as broccoli. This anticarcinogen was first identified as a potent inducer of Phase 2 enzymes, but evidence is mounting that SFN acts through other cancer chemopreventive mechanisms. We recently reported on a novel mechanism of chemoprotection by SFN in human colon cancer cells and prostate epithelial cells, namely the inhibition of histone deacetylase (HDAC). In the present investigation, we sought to test whether SFN also might inhibit HDAC activity in vivo. When consumed in the diet at an average daily dose of 7.5 mumol per animal for 21 days, SFN suppressed the growth of human PC-3 prostate cancer cells by 40% in male nude mice. There was a significant decrease in HDAC activity in the xenografts, as well as in the prostates and mononuclear blood cells (MBC), of mice treated with SFN, compared to controls. There also was a trend towards increased global histone acetylation in the xenografts, prostates, and MBC. In human subjects, a single dose of 68 g BroccoSprouts inhibited HDAC activity significantly in peripheral blood mononuclear cells (PBMC) 3 and 6 hrs following consumption. These findings provide evidence that one mechanism through which SFN acts as a cancer chemopreventive agent in vivo is through the inhibition of HDAC activity. Moreover, the data suggest that HDAC activity in PBMC may be used as a biomarker for assessing exposure to novel dietary HDAC inhibitors in human subjects.

Figure 1

SFN retards the growth of PC-3 xenografts. (A) PC-3 cells were implanted into male nude mice (10 mice per group). SFN was administered in the diet (443 mg/kg; 2.5 μmol/g) beginning on the day of implantation. Tumor volume was determined as described in Materials and Methods. (B) No adverse effect of SFN on mouse body weight. Data are expressed as mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

SFN inhibits HDAC activity in PC-3 xenografts. (A) HDAC activity (arbitrary fluorescent units [AFU]) was determined in xenografts as described in Materials and Methods. Data are expressed as mean ± SE. n = 10. ***P < 0.001. (B) Xenografts were immunoblotted for acetylated H3, acetylated H4, and BAX; β-actin was used as the loading control. Each lane represents a xenograft from an individual animal, and the blot is a representative sample from each group of 10 animals. (C) Quantification of immunoblots using National Institutes of Health Image J, normalized for β–actin. Data are expressed as mean ± SE. *P < 0.05.

Figure 3

SFN increases acetylated histones and induces BAX expression in mouse prostates. (A) HDAC activity was determined in mouse prostate tissues as described in Materials and Methods. Data are expressed as mean ± SE. n = 10. ***P < 0.001. (B) Prostates were immunoblotted for acetylated H3, acetylated H4, and BAX; β-actin was included as a loading control. Each lane represents a prostate from an individual animal, and the blot is a representative sample from each group of 10 animals. (C) Quantification of immunoblots using National Institutes of Health Image J, normalized for β–actin. Data are expressed as mean ± SE.

Figure 4

SFN inhibits HDAC activity in mouse MBC and human PBMC. (A) HDAC activity in mouse MBC was determined as described in Materials and Methods; mean ± SE. **P < 0.01 for SFN versus controls. (B) Human subjects consumed 68 g of BroccoSprouts and blood was obtained at the times indicated in the figure. PBMC were isolated and HDAC activity assays were performed as described in Materials and Methods. Data are expressed as mean ± SE. n = 3. *P < 0.05. (C) Data for HDAC activity in PBMC from each subject.