Supplementary Material for: Citrus Limonin Lacks the Antichemotherapeutic Effect in Human Models of Breast Cancer

Background/Aims: Chemicals that interfere with reactive oxygen species metabolism can act as potential candidates for the treatment of cancer. Some of the glucosides of citrus limonin inhibit the endogenously generated reactive oxygen species. The aim is to study the interactions of limonin with chemotherapy. Methods: Breast cancer cell lines MCF-7 (p53 wild type) and MDA-MB-231 (p53 mutant) as well as the nontumorigenic epithelial cell line MCF-10 were used to screen the effect of limonin at 1-, 5- and 10-µM concentrations with camptothecin for apoptosis and NFĸB, p38 and ERK-MAPK signaling kinase assays. The effect of cyclophosphamide and limonin on MDA MB 231 xenografts was also studied. Results: Our results indicate that limonin did not inhibit camptothecin-induced apoptosis in human breast cancer cells in vitro through noninterference of camptothecin-induced phosphorylation of p38 MAPK and ERK-MAPK. Using an in vivo model of human breast cancer, limonin in combination with cyclophosphamide was not found to inhibit the cyclophosphamide-induced tumor regression through a reduced mitotic index of tumor xenograft cells when compared to treatment with cyclophosphamide alone. Conclusion: Both in vitro and in vivo results suggest that limonin could be beneficial for breast cancer patients undergoing chemotherapy.