Total flavonoids of Litchi chinensis Sonn. seed inhibit prostate cancer growth in bone by regulating the bone microenvironment via inactivation of the HGFR/NF-cB signaling pathway

Ethnopharmacological relevance: Litchi chinensis Sonn. (Litchi) seed, a traditional Chinese medicine, is habitually used in the clinical treatment of prostate cancer (PCa)-induced bone pain. In our previous study, flavonoids have been identified as the active ingredient of litchi seed against PCa. However, its anti-tumor activities in bone and associated molecular mechanisms are still unclear. Aim of the study: To investigate the effects and underlying mechanisms of total flavonoids of litchi seed (TFLS) on the growth of PCa in bone. Materials and methods: The effect of TFLS on the growth of PCa in bone was observed using a mouse model constructed with tibial injection of luciferase-expressing RM1-luc cells. Conditioned medium (CM) from bone marrow stromal cells OP9 and CM treated with TFLS (T-CM) was used to investigate the effect on the prolif-eration, colony formation, and apoptosis of PCa cells (LNCaP, PC3, RM1). An antibody microarray was per-formed to detect cytokine expression in the supernatant fraction of OP9 cell cultures treated with TFLS or left untreated. Western blot assay was employed to determine the expression and activity of HGFR and its key downstream proteins, Akt, mTOR, NF-cB, and Erk, in PCa cells. The potential target was further verified using immunofluorescence and immunohistochemistry assays. Results: Treatment with TFLS (80 mg/kg, 24 days) significantly suppressed the growth of RM1 cells in bone. CM from bone marrow stromal cells OP9 stimulated the proliferation and colony formation of the PCa cells as well as inhibited the apoptosis of PC3 cells, while T-CM reversed the effects mediated by OP9 cells in vitro. In an antibody array assay, TFLS regulated the majority of cytokines in OP9 cell culture supernatant, among which HGF, HGFR, IGF-1R, and PDGF-AA showed the greatest fold changes. Mechanistically, CM upregulated HGFR and promoted phosphorylation of NF-cB while T-CM induced reduction of HGFR and dephosphorylation of NF-cB in PC3 cells. Moreover, T-CM inhibited NF-cB entry into PC3 cell nuclei. Data from in vivo experiments further confirmed the inhibitory effects of TFLS on NF-cB. Conclusion: TFLS suppresses the growth of PCa in bone through regulating bone microenvironment and the underlying mechanism potentially involves attenuation of the HGFR/NF-cB signaling axis.