Cumin Prevents 17β-Estradiol-Associated Breast Cancer in ACI Rats

Abstract

Breast cancer (BC) is a leading cause of cancer deaths in women in less developed countries and the second leading cause of cancer death in women in the U.S. In this study, we report the inhibition of E2-mediated mammary tumorigenesis by Cuminum cyminum (cumin) administered via the diet as cumin powder, as well as dried ethanolic extract. Groups of female ACI rats were given either an AIN-93M diet or a diet supplemented with cumin powder (5% and 7.5%, w/w) or dried ethanolic cumin extract (1%, w/w), and then challenged with subcutaneous E2 silastic implants (1.2 cm; 9 mg). The first appearance of a palpable mammary tumor was significantly delayed by both the cumin powder and extract. At the end of the study, the tumor incidence was 96% in the control group, whereas only 55% and 45% animals had palpable tumors in the cumin powder and extract groups, respectively. Significant reductions in tumor volume (660 ± 122 vs. 138 ± 49 and 75 ± 46 mm³) and tumor multiplicity (4.21 ± 0.43 vs. 1.16 ± 0.26 and 0.9 ± 0.29 tumors/animal) were also observed by the cumin powder and cumin extract groups, respectively. The cumin powder diet intervention dose- and time-dependently offset E2-related pituitary growth, and reduced the levels of circulating prolactin and the levels of PCNA in the mammary tissues. Mechanistically, the cumin powder diet resulted in a significant reversal of E2-associated modulation in ERα, CYP1A1 and CYP1B1. Further, the cumin powder diet reversed the expression levels of miRNAs (miR-182, miR-375, miR-127 and miR-206) that were highly modulated by E2 treatment. We analyzed the composition of the extract by GC/MS and established cymene and cuminaldehyde as major components, and further detected no signs of gross or systemic toxicity. Thus, cumin bioactives can significantly delay and prevent E2-mediated mammary tumorigenesis in a safe and effective manner, and warrant continued efforts to develop these clinically translatable spice bioactives as chemopreventives and therapeutics against BC.

Keywords: ACI rats; Cuminum cyminum; breast cancer; cumin; estradiol.

Figure 1

Effect of diet supplemented with cumin powder (7.5%, w/w) (A) and dried ethanolic cumin extract (1%, w/w) (B) on the body weight gain of the August Copenhagen Irish (ACI) rats. Inset in the left and right panels show the average daily diet consumption. Arrow in each figure shows the start of the 17ß-estradiol (E2) treatment.

Figure 2

Effects of diet supplemented with cumin powder (7.5%, w/w) (A) and dried ethanolic cumin extract (1%, w/w) (B) on the following various mammary tumorigenesis indices: tumor incidence and latency (A1,B1), tumor multiplicity (A2,B2), and tumor volume (A3,B3). After acclimatization, ACI rats were subcutaneously grafted with 9 mg E2-containing silastic implants. Animals were palpated starting from week 12 and tumor incidence was recorded weekly and analyzed using a non-parametric log-rank test. Numbers denote the animals without tumor at the end of the study. Tumor multiplicity and tumor volumes were calculated at the time of euthanasia and analyzed using unpaired two-tailed Student’s t-test. Asterisk indicates significant difference between control diet versus cumin diet-fed animals. ** p < 0.01; *** p < 0.001.

Figure 3

Effect of cumin diet on 17ß-estradiol (E2)-mediated mammary cell proliferation in the absence and presence of E2 treatment. (A) Representative photomicrographs (20× magnification) of normal and hyperplastic mammary tissue following immuno-histochemical staining for proliferating cell nuclear antigen (PCNA) after 3 weeks (left panel) and after 26 weeks (right panel). (B) Bar diagrams show the percentage (average ± SD) of deeply stained cells for PCNA in mammary tissues (n = 5). *** p < 0.001.

Figure 4

Effect of cumin powder diet on circulating 17ß-estradiol (E2) and prolactin. Time- (3 and 26 weeks, 7.5%, w/w) and dose-dependent (5% vs. 7.5%, w/w at 3 weeks) effect of cumin diet on circulating E2 levels (A) or prolactin levels (B) was measured in the blood plasma of the animals and compared with control animals. Data represent mean ± SD (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Effect of dietary cumin seed powder (7.5%, w/w) on select microRNAs (A), their downstream targets (B) and 17ß-estradiol (E2)-related markers (C) in E2-mediated mammary tumorigenesis. Total RNA was used to assess relative gene expression using the differences in normalized Ct (^ΔΔCt) method after normalization to 18s rRNA. Fold changes were calculated by ^2-ΔΔCt. Experiments were performed in triplicate for each data point. Data represent mean ± SD of fold change in mRNA expression relative to the untreated control fed with AIN93M control diet (CD) of five animals. Statistical significance was analyzed by Student’s t test where * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 6

Effect of dietary cumin on the liver and kidney function enzymes, hematopoietic parameters and biochemical parameters in ACI rats. ACI rats (5–6 weeks old) were provided control diet (AIN 93M) or diet supplemented with cumin seed powder (7.5%, w/w) in the absence and presence of E2 for 26 weeks. Animals were euthanized and blood plasma was analyzed for (A) protein levels, (B) liver enzyme test, (C) kidney functions/electrolyte, (D) effect on leukocytes and (E) hematological parameters. Data represent mean ± SD. Student’s t-test was used for statistical analysis. * p < 0.05.

Figure 7

Chemical structure of phytocompounds identified in cumin. Ethanolic cumin extract was dissolved in methanol and analyzed by GC-MS to identify the chemical constituents on a GCD 1800A Hewlett Packard apparatus coupled with a HP-1 column. Helium was used as a carrier gas at the rate of 1 mL/min. Identification of major compounds was carried out on the basis of exact mass and compared with MS reference database of NBS75K.

Figure 8

Ground Cuminum cyminum (cumin) seeds powder and derived dried ethanolic extract was used in this study. Schematic representation of animal study plan; table shows the animal study design used in the study.