• Doxorubicin and Trifolium Pratense L. (Red Clover) Extract Synergistically Inhibits Brain and Lung Metastasis in 4T1 Tumor-Bearing BALB/c Mice
  • Mohsen Akbaribazm ,1,*
    1. Fertility and Infertility Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran


  • Introduction: 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 aims of study were to investigate the anti-metastasis effects of doxorubicin (DOX) and T. pratense extract in 4T1 tumor-bearing BALB/c mice.
  • Methods: In this study, 56 female BALB/c mice were divided randomly into seven 4T1 groups (n=8/group) to receive DOX and T. pratense in 3 different doses (100, 200 and 400 mg/kg/d) for 35 days. On the 36th day, serum cytokines (IL-8 and IL-6) were measured. GATA-3 Immunohistochemical (IHC) staining for brain and lung, and also CK5/6 for tumors tissue were performed. Metastasis-related genes [matrix metalloproteinase-2 (MMP-2) and sirtuin-1 (SIRT-1)] expressions are also measured by the Real- Time PCR.
  • Results: Our results showed that the co-treatment of DOX and T. pratense improved the lung and brain stereological parameters (decreased the volume of metastatic tumors) and decreased the serum levels of inflammatory and mediators of metastasis cytokines (IL-8 and IL-6). DOX and T. pratense synergistically down-regulated the MMP-2 and up-regulated the SIRT-1 genes and also decreased the CK5/6 positive cells in tumor tissues and inhibited metastasis in GATA-3 positive cells into lung and brain.
  • Conclusion: Our results present the first evidence on the anti-metastasis effects of T. pratense hydroalcoholic extract synergistically with doxorubicin as demonstrated by in vivo 4T1 metastasis inhibition in BALB/c mice bearing orthotopic breast cancer tumors.
  • Keywords: Trifolium pratense L., GATA-3, Metastasis, Isoflavone, 4T1