Loranthus Micranthus Extracts Suppress the Expression and Enzymatic Activity of Indoleamine 2, 3-Dioxygenase in Human Breast Cancer Cells via the Inhibition of JAK/STAT and NF-κB Pathways

Background: Loranthus micranthus (African mistletoe) is a woody parasitic plant. It is found in Africa, Asia, Europe, and America. Its leaves and stem/twigs have been used in traditional medicine in various continents for the treatment of metabolic diseases: diabetes, hypertension, and cancer. The present study is designed to investigate the effect of Loranthus micranthus leaves and stem (LML and LMS) extracts on indoleamine 2,3-dioxygenase (IDO); a key regulator of cancer immunity; and the possible molecular mechanism(s) involved. Methods: Human breast cancer cells; MDA MB-231 and MCF-7 were pre-treated with LML and LMS at doses of 1-30µg/mL for 2 hours. This was followed by interferon-γ (INF-γ) stimulation at 50 ng/mL for 24 hours. After the treatment period, the activity of indoleamine 2,3 dioxygenase 1 (IDO-1) and T cell proliferation were determined using a spectrophotometric assay. Protein expression of IDO-1, IDO-2, tryptophan-2,3-dioxygenase-2 (TDO-2), Bridging inhibitor 1 (BIN-1), Janus kinase/signal transducers and activators of transcription (JAK/STAT), Inhibitor of κ B (IκBα) and Nuclear factor-κB (NF-κB) pathway were analyzed using the western blotting technique. The mRNA expression of IDO-1 was quantified using the qRT-PCR technique.   Results: MDA MB-231 and MCF-7 cells pre-treated with LML and LMS exhibited lower kynurenine synthesis than that of INF-γ treatment. We observed downregulation of IDO-1, IDO-2, and TDO-2 protein expressions, and lower IDO-1 catalytic activity in LML and LMS pretreated cells than those of INF-γ treated cells alone. Cells pretreated with LML and LMS exhibited significant downregulation of phosphorylation of JAK-1, JAK-2, STAT-1, and STAT-3 than those of IFN-γ treatment alone. Furthermore, LML and LMS pre-treated cells exhibited upregulation of IκBα and BIN-1 protein expression compared with IFN-γ treated cells alone. Additionally, LML and LMS pretreatment significantly improved T cell viability when compared with IFN-γ treated cells alone. Conclusion: These findings indicate that LML and LMS extracts regulate immune responses in cancer cells by inhibiting IDO activity/expression in a JAK/STAT and NF-κB-dependent manner. This accounts for their anticancer effects in traditional medicine, hence a promising candidate for future drug development.