Inhibition of the Otub1/c-Maf axis by the herbal acevaltrate induces myeloma cell apoptosis.

BACKGROUND: The oncogenic transcript factor c-Maf is stabilized by the deubiquitinase Otub1 and promotes myeloma cell proliferation and confers to chemoresistance. Inhibition of the Otub1/c-Maf axis is a promising therapeutic target, but there are no inhibitors reported on this specific axis. METHODS: A luciferase assay was applied to screen potential inhibitors of Otub1/c-Maf. Annexin V staining/flow cytometry was applied to evaluate cell apoptosis. Immunoprecipitation was applied to examine protein ubiquitination and interaction. Xenograft models in nude mice were used to evaluate anti-myeloma activity of AVT. RESULTS: Acevaltrate (AVT), isolated from Valeriana glechomifolia, was identified based on a bioactive screen against the Otub1/c-Maf/luciferase system. AVT disrupts the interaction of Otub1/c-Maf thus inhibiting Otub1 activity and leading to c-Maf polyubiquitination and subsequent degradation in proteasomes. Consistently, AVT inhibits c-Maf transcriptional activity and downregulates the expression of its target genes key for myeloma growth and survival. Moreover, AVT displays potent anti-myeloma activity by triggering myeloma cell apoptosis in vitro and impairing myeloma xenograft growth in vivo but presents no marked toxicity. CONCLUSIONS: The natural product AVT inhibits the Otub1/c-Maf axis and displays potent anti-myeloma activity. Given its great safety and efficacy, AVT could be further developed for MM treatment. Video Abstract.

Tryptanthrin inhibits Th2 development, and IgE-mediated degranulation and IL-4 production by rat basophilic leukemia RBL-2H3 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Tryptanthrin is a compound isolated from Polygonum tinctorium, which is a known folk medicine with various biological activities. AIM OF THE STUDY: Allergic diseases are initiated by the development of allergen-specific T helper type 2 (Th2) cells and amplified by the degranulation of and cytokine release from basophils and mast cells during an effector phase. We found that Tryptanthrin could down-regulate IL-4 production by Th2 cells, while IFN-γ production by Th1 cells was not affected. Since IL-4 produced by basophils and effector Th2 cells has been shown to play important roles in the development and amplification of Th2-dominated allergic responses, we examined the effects of Tryptanthrin on the initiation and effector phase responses of Type I allergy in vitro. MATERIALS AND METHODS: To determine the mechanisms of Tryptanthrin-induced down-regulation of IL-4 production, the expression of Th2-specific transcription factors, c-Maf and GATA-3, was analyzed by RT-PCR. The effects of Tryptanthrin on Th cell differentiation were evaluated using CD4(+) T cells purified from spleen cells of Sugi basic protein (SBP)-immunized BALB/c mice. In primary cultures, cells were stimulated with SBP and antigen-presenting cells under neutral or Th2-skewing conditions in the presence or absence of Tryptanthrin. Cytokines produced by differentiated Th cells in secondary cultures were analyzed by ELISA. The effects of Tryptanthrin on IgE-mediated degranulation and IL-4 production were determined using rat basophilic leukemia (RBL-2H3) cells. Phosphorylation of ERK1/2 and Akt in Tryptanthrin-treated RBL-2H3 cells was analyzed to determine the mechanism of Tryptanthrin actions. RESULTS: Tryptanthrin suppressed c-Maf mRNA expression in Th2 clone cells, and even under Th2-skewing conditions, Tryptanthrin inhibited differentiation toward the Th2 phenotype, which is an essential event for the initiation phase of allergic diseases. Tryptanthrin also inhibited the IgE-mediated degranulation of and IL-4 production by RBL-2H3 cells, probably due to inhibiting IgE-mediated signaling pathways, including the phosphorylation of ERK1/2 and Akt. CONCLUSION: These findings suggest that Tryptanthrin effectively inhibits the effector and exacerbation responses, as well as the initiator responses, of Type I allergy. Thus, Tryptanthrin may have beneficial effects for immediate-type allergic responses.