• Trifolium pratense L. (Red Clover) Extract and Doxorubicin Synergistically Inhibits Proliferation of 4T1 Breast Cancer in Tumor-Bearing BALB/c Mice Through Modulation of Apoptosis and Increase Anti-Oxidant and Anti-Inflammatory Related Pathways
  • Mohsen Akbaribazm ,1,*
    1. Fertility and Infertility Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran


  • Introduction: Therapeutic strategies against triple negative breast cancer (TNBC) are associated with drug-induced toxicities. Trifolium pratense L. (T. pratense) is rich in polyphenolic compounds which confer the plant potential anti-cancer properties. The aim of this study was to investigate the effects of T. pratense and doxorubicin (DOX) on the apoptosis and proliferation of 4T1 tumor cells in an allograft model of tumor-bearing BALB/c mice.
  • Methods: Fifty-six female 4T1-tumor bearing-BALB/c mice were randomly divided into 7 groups (n=8/group) to receive different doses and combinations of DOX and T. pratense extract for 35 days. On the 36th day, serum estradiol (E2), IL-12 and IFN-γ cytokines, and glutathione peroxidase (GPx) activity were measured. Tumor's ferric reducing antioxidant power (FRAP) and the expressions of apoptosis-related genes (p53, Bax, Bcl-2, and caspase-3) were also evaluated. Immunohistochemically staining for Ki-67 and p53 were performed.
  • Results: Our results showed that the co-treatment of DOX and T. pratense (100–400 mg/kg) inhibited the proliferation of 4T1 tumor cells in dose- and time-dependent manners by decreasing the serum level of E2 (as a stimulant for breast tumor growth) and increasing the serum levels of IL-12 and IFN-γ along with significant increments in serum GPx and tumor FRAP activities. The co-administration of DOX and T. pratense also decreased the expression of Ki-67 proliferation marker and increased the number p-53 positive (i.e. apoptotic) cells within tumors. This was accompanied with the upregulation of pro-apoptotic and down-regulation of anti-apoptotic genes.
  • Conclusion: The key findings indicated the synergistic effects of DOX and T. pretense against TNBC xenografts.
  • Keywords: Trifolium pratense L., Apoptosis, Breast cancer, Doxorubicin, 4T1 cell.