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

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.