Doxorubicin and Trifolium pratense L. (Red clover) extract synergistically inhibits brain and lung metastases in 4T1 tumor-bearing BALB/c mice

Abstract

Trifolium pratense L. (Red clover-T. pratense) commonly consumed as a healthy beverage has been demonstrated to have various biological activities including antioxidant and anticancer effects. The aim of this study was to investigate the antimetastasis effects of doxorubicin (DOX) and T. pratense extract in 4T1 tumor-bearing BALB/c mice. In this study, 56 female BALB/c mice were randomly divided into seven groups (n = 8/group) to receive DOX and T. pratense extract in three different doses (100, 200, and 400 mg/kg/day) for 35 days. On day 36 after starting treatments, serum cytokines (IL-8 and IL-6) were measured. Immunohistochemical (IHC) staining was performed for GATA-3 in the brain and lung, and for CK5/6 in tumor tissues. Metastasis-related gene (matrix metalloproteinase-2 [MMP-2] and sirtuin-1 [SIRT-1]) expressions were also measured by real-time PCR. Our results showed that cotreatment with DOX and T. pratense extract improved stereological parameters (i.e., reduction in the volume of metastatic tumors) in the lung and brain and decreased the serum levels of inflammatory cytokines (IL-8 and IL-6). DOX and T. pratense extract synergistically down-regulated MMP-2 and up-regulated SIRT-1 genes, decreased the number of CK5/6-positive cells in tumor tissues, and inhibited metastasis of GATA-3-positive cells into the lung and brain. The combination of T. pratense extract and DOX synergistically inhibited the metastasis of 4T1 xenograft cells in a dose-dependent manner.

Keywords: 4T1; GATA‐3; Trifolium pratense L.; breast cancer; isoflavone; metastasis.

FIGURE 1

4T1 tumor induction and metastasis in lung and brain in BALB/c mice

FIGURE 2

The volume of tumors (top row) and their lung metastasis (bottom row) in C− (A, a), C+ (B, b), t400 (C, c), t200 (D, d), t100 (E, e), t400t (F, f), and normal groups (g)

FIGURE 3

Point probe (50 points) for calculating the volumetric density of lung (a) and brain (b). The total number of points located on each structure is counted in each field of view and is compared to the total number of points in the volume measurement formula: V _v = ∑P _structure/∑P _reference (a: lung, H&E, and b: brain, methylene blue staining, scale bar = 300 μm, ×40)

FIGURE 4

Comparison of serum cytokine levels of (a) IL‐6 and (b) IL‐8 (pg/ml) in negative control (C−), positive control (DOX, C+), the extract groups (t100, t200, and t400) and t400t (mean ± SD). ^$(p < .05) statistically significant between DOX and negative control groups (C−), ^#(p < .05) statistically significant between t400 and DOX groups, *(p < .05) and **(p < .01) statistically significant between treatment and DOX groups, and ^&&(p < .01) statistically significant between C− and normal groups

FIGURE 5

The effect of Trifolium pratense on (a) MMP‐2, (b) sirtuin‐1 (SIRT‐1) gene expression of tumors tissue in negative control (C−), positive control (DOX, C+), the extract groups (t100, t200, and t400) and t400t (mean ± SD). ^$(p < .05) statistically significant between DOX and negative control groups, ^#(p < .05) statistically significant between t400t and negative control groups, and *(p < .05) and **(p < .01) statistically significant between treatment and DOX groups

FIGURE 6

The effect of Trifolium pratense on CK 5/6 expression (CK 5/6‐positive cells) of tumors tissue in positive control (DOX, a), negative control (C−, b), and the extract groups (t100 [c], t200 [d], and t400 [e]) and t400t (f) (mean ± SD). ^$(p < .05) statistically significant between DOX and negative control groups, *(p < .05) and **(p < .01) statistically significant between treatment and DOX groups

FIGURE 7

The effect of Trifolium pratense on GATA‐3 expression (GATA‐3‐positive cells) of brain tissue in negative control (C−, a), positive control (DOX, b), and the extract groups [t100 (c), t200 (d), and t400 (e)] and t400t (f) (mean ± SD). *(p < .05) statistically significant between treatment and DOX groups

FIGURE 8

The effect of Trifolium pratense on GATA‐3 expression (GATA‐3‐positive cells) of lung tissue in negative control (C−, a), positive control (DOX, b), and the extract groups [t100 (c), t200 (d), and t400 (e)] and t400t (f) (mean ± SD). *(p < .05) and **(p < .01) statistically significant between treatment and DOX groups

FIGURE 9

Histopathological examination in lung (1) and brain (2) in normal tissue (a), negative control (C−, b), positive control (DOX, c), and the extract groups (t100 [d], t200 [e], and t400 [f]) and t400t (g). Red square (metastatic brain tumor), and orange circle (metastatic lung tumor); T: tumor and V: vessel (1: H&E and 2: methylene blue staining, scale bar = 300 μm, ×40)

FIGURE 10

The effect of DOX and Trifolium pratense on stereological parameters in (a) brain (nervous tissue, vessels, ventricles, and tumor) and (b) lung (total volume, tissue parenchyma, air parenchyma, vessels, and tumor) in normal tissue (a), negative control (C−, b), positive control (DOX, c), and the extract groups (t100 [d], t200 [e], and t400 [f]) and t400t (g) (mean ± SD). ^&(p < .05) statistically significant between negative and normal control groups, ^$(p < .05) statistically significant between DOX and normal groups, *(p < .05) and **(p < .01) statistically significant between treatment and DOX groups. (a) Final volume (FV), nervous volume (NV), vessels volume (VV), tumor volume (TV), and ventricle volume (VenV). (b) Lung volume (LV), tissue parenchyma volume (TPV), air parenchyma volume (APV), vessels volume (VV), and tumor volume (TV)