Chemopreventive and anti-angiogenic effects of dietary phenethyl isothiocyanate in an N-methyl nitrosourea-induced breast cancer animal model

Abstract

The effect of phenethyl isothiocyanate (PEITC), a component of cruciferous vegetables, on the initiation and progression of cancer was investigated in a chemically induced estrogen-dependent breast cancer model. Breast cancer was induced in female Sprague Dawley rats (8 weeks old) by the administration of N-methyl nitrosourea (NMU). Animals were administered 50 or 150 µmol/kg oral PEITC and monitored for tumor appearance for 18 weeks. The PEITC treatment prolonged the tumor-free survival time and decreased the tumor incidence and multiplicity. The time to the first palpable tumor was prolonged from 69 days in the control, to 84 and 88 days in the 50 and 150 µmol/kg PEITC-treated groups. The tumor incidence in the control, 50 µmol/kg, and 150 µmol/kg PEITC-treated groups was 56.6%, 25.0% and 17.2%, while the tumor multiplicity was 1.03, 0.25 and 0.21, respectively. Differences were statistically significant (p < 0.05) from the control, but there were no significant differences between the two dose levels. The intratumoral capillary density decreased from 4.21 ± 0.30 vessels per field in the controls to 2.46 ± 0.25 in the 50 µmol/kg and 2.36 ± 0.23 in the 150 µmol/kg PEITC-treated animals. These studies indicate that supplementation with PEITC prolongs the tumor-free survival, reduces tumor incidence and burden, and is chemoprotective in NMU-induced estrogen-dependent breast cancer in rats. For the first time, it is reported that PEITC has anti-angiogenic effects in a chemically induced breast cancer animal model, representing a potentially significant mechanism contributing to its chemopreventive activity.

Figure 1

(A) Effect of phenethyl isothiocyanate (PEITC) on tumor-free survival in NMU-treated rats. N-methyl nitrosourea was administered at a dose of 50 mg/kg i.p. at day 0 and between 12 and 15 days. Rats were administered 150 µmol/kg or 50 µmol/kg PEITC in corn oil every 2 days or corn oil alone in the vehicle-treated control group. The black line represents the control group (n = 30), the green dashed line represents the 50 µmol/kg PEITC-treated group (n=20) and the red dotted line represents the 150 µmol/kg PEITC-treated group (n = 29). Curves were determined to be significantly different between the control and treatment groups (Log rank test, p < 0.05). (B) Effect of PEITC on tumor incidence and (C) average number of tumors per group. In panels B and C, the open bar represents the controls, while the gray bar represents the 50 µmol/kg PEITC-treated group, and the black bar represents the 150 µmol/kg PEITC-treated group. *Statistically significant differences from the control group (p < 0.05)

Figure 2

Effect of PEITC on estradiol concentrations. The open bar represents the controls, while the gray bar represents the 50 µmol/kg PEITC-treated group, and the black bar represents the 150 µmol /kg PEITC-treated group. Data are presented ± SEM, n = 12 in each group. *Statistically significant increase compared with control, with p < 0.05

Figure 3

Images of tumor phenotypes. Panel (A): Lobular carcinoma in situ, showing multilayered, nonpolarized epithelial cells (arrowhead) inside lobular ductules (large arrows). Small arrows indicate apoptotic bodies in the lumen of a lesion. Original magnification with 20× objective. Panel (B): Ductal carcinoma in situ, showing multilayered nonpolarized epithelial cells in a cribriform arrangement (arrowheads) within the lumen of a ductule (arrow). Original magnification, 40× objective. Panel (C): Invasive lobular carcinoma, with epithelial cells arranged in a single file embedded within the interstitial stroma (between arrows). Original magnification, 20× objective. Panel (D) enlargement of panel (C) showing nuclear heterogeneity within the invasive lobular carcinoma lesion (between arrows). Original magnification, 40× objective

Figure 4

Effect of PEITC on intratumor capillary density. The open bar represents the vehicle controls (n = 9 tumors), the gray bar represents the 50 µmol/kg PEITC-treated group (n=5 tumors), while the black bar represents the 150 µmol/kg PEITC-treated group (n = 3 tumors). The data are presented as mean ± SEM. *Statistically significant differences from controls (p < 0.05) by Kruskal Wallis test followed by Dunn’s multiple comparison test