Myricetin exhibit selective anti-lymphoma activity by targeting BTK and is effective via oral administration in vivo

Abstract

Background: Myricetin (MYR) is a polyhydroxy flavone originally isolated from Myrica rubra, and is widely distributed in a variety of medicinal plants and delicious food. MYR has been proven to have inhibitory effects against various types of cancer. However, the exact role of MYR in lymphoma development is still unclear.

Methods: In vitro, the MTT assay was performed to evaluate the activity of human diffuse large B lymphoma cell TMD-8 and other tumor cells. Homogeneous time-resolved fluorescence (HTRF) and molecular docking were used to detect the target of MYR inhibiting TMD-8 cells. In addition, flow cytometry, Annexin V-FITC/PI assays, Hoechst 33258, and mondansylcadaverine (MDC) fluorescent standing were used to detect the cell cycle, apoptosis, and autophagy, respectively. Moreover, Western blot analysis was conducted to analyze related signaling pathways. In TMD-8 cell xenotransplanted mice, immunohistochemistry, histopathology, and blood biochemical tests were used to evaluate the effectiveness and safety of oral administration of MYR.

Results: Here, we found that MYR is more sensitive to TMD-8 cells than other tumor cells by targeting bruton tyrosine kinase (BTK). BTK is an attractive target for the treatment of B-cell malignancies. The HTRF assay showed that MYR inhibited BTK kinase with an IC₅₀ of 1.82 μM. Furthermore, the HTRF assay and Western blot analysis demonstrated that MYR could bind to key residues (Ala478, Leu408, Thr474) in the BTK active pocket, inhibit the autophosphorylation on tyrosine 223, and block BTK/ERK and BTK/AKT signal transduction cascades (including downstream substrates GSK-3β, IKK, STAT3, and NF-κb). The results of cell cycle, apoptosis, and autophagy showed that MYR could induce G1/G0 cycle arrest by regulating cyclinB1/D1 expression, induce apoptosis by increasing the Bax/Bcl-2 ratio, and trigger autophagy by inhibiting mTOR activation. In vivo, oral administration of MYR significantly inhibited the growth of TMD-8 xenograft tumora without toxic side effects. Furthermore, Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis showed that MYR could inhibit proliferation and induce apoptosis of tissue lymphoma cells.

Conclusion: Taken together, MYR is an oral available natural BTK inhibitor that effectively inhibits the growth of lymphoma TMD-8 cells both in vitro and in vivo. In addition, our findings support that the use of MYR is a novel and promising therapeutic strategy for the treatment of lymphoma.

Keywords: B-cell malignancies; BTK inhibitors; Myricetin; TMD-8 cells; Xenotransplantation.