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.

Di-(2-ethylhexyl) phthalate adjuvantly induces imbalanced humoral immunity in ovalbumin-sensitized BALB/c mice ascribing to T follicular helper cells hyperfunction.

Di-(2-ethylhexyl) phthalate (DEHP) has been considered as a widespread environmental persistent organic pollutant and its potential concern on human health is raised by previous studies. In particular, children are more likely to be exposed to DEHP through gastrointestinal route and consequently are more susceptible to DEHP hazards. Some reports have uncovered a positive association between DEHP exposure and an increased prevalence of allergic diseases in infants and juveniles. Allergy is a hypersensitive reaction rooted in imbalanced humoral immunity. T follicular helper cell (Tfh), an important CD4(+) Th cell subset, until recently has been identified as a key player in humoral immune response by modifying B cells functions. Tfh cells are therefore perceived as the therapeutic target of immune disorders. In the present study, focusing on the newly confirmed Tfh cells, we examined the effects of DEHP on humoral immunity and investigated the underlying mechanisms. Using ovalbumin (OVA) sensitization weanling mice model under the condition of gastrointestinal exposure to DEHP, we found that DEHP acted as an immunoadjuvant to augment OVA-specific IgE and IgG1 production, amplified germinal center formation in lymphoid nodule, as well as stimulated the expansion of CD4(+)CXCR5(+)ICOS(+)/CD4(+)CXCR5(+)PD-1(+) Tfh cells and CD19(+)CD138(+)GL7(+) plasma cells. Based on the results of immune adoptive transfusion, DEHP-related anaphylactic response was ascribed to Tfh cells hyperfunction directly. We further proved that DEHP gavage together with OVA sensitization adjuvantly promoted the synthesis of cytokines IL-21 and IL-4 and the expression of transcription factors Bcl-6 and c-Maf in Tfh cells. In conclusion, our study demonstrates that DEHP has adjuvant toxic effects on Tfh cells by synthesizing an excess of cytokines IL-21 and IL-4 via over-expression of transcription factors Bcl-6 and c-Maf, leading to an increasing secretion of allergy-related IgE and IgG1.

Shikonin inhibited mitogen-activated IL-4 and IL-5 production on EL-4 cells through downregulation of GATA-3 and c-Maf induction.

AIM: To investigate the effects of shikonin on phorbol myristate acetate (PMA) plus cyclic adenosine monophosphate (cAMP)-induced T helper (T(H)) 2 cell cytokine production, and the underlying mechanism. MAIN METHODS: We used activated EL-4 murine T-lymphoma cells, which produce interleukin (IL)-4 and IL-5, but not interferon (IFN)-γ, as T(H)2 cell-like cells and treated them with PMA+cAMP to investigate the effects of shikonin on T(H)2 cytokines, transcriptional factors, and the related mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB signaling pathway. KEY FINDINGS: The data show that shikonin inhibited the PMA+cAMP-induced mRNA and protein expression of IL-4 and IL-5 via the downregulation of GATA-binding protein-3 (GATA-3) and c-musculoaponeurotic fibrosarcoma (Maf) but not T-box expressed in T cells (T-bet). Moreover, shikonin suppressed the phosphorylation of p38, inhibitor of κB (IκB) kinase (IKK)-ß and IκB-α, and the subsequent IκB-α degradation induced by PMA+cAMP; however, the PMA+cAMP-induced phosphorylation of extracellular signal-related kinase (ERK), which resulted in minor inhibition and phosphorylation of c-Jun N-terminal kinase (JNK), seemed to be unaffected by shikonin treatment. SIGNIFICANCE: This study suggests that downregulation of GATA-3 and c-Maf via the suppression of p38, IKK-ß and IκB-α phosphorylation might contribute to the inhibitory effect of shikonin on mitogen-induced IL-4 and IL-5 production in EL-4T cells. Furthermore, shikonin is a potential drug for treating allergic diseases.

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.

A chemical biology screen identifies glucocorticoids that regulate c-maf expression by increasing its proteasomal degradation through up-regulation of ubiquitin.

The oncogene c-maf is frequently overexpressed in multiple myeloma cell lines and patient samples and contributes to increased cellular proliferation in part by inducing cyclin D2 expression. To identify regulators of c-maf, we developed a chemical screen in NIH3T3 cells stably overexpressing c-maf and the cyclin D2 promoter driving luciferase. From a screen of 2400 off-patent drugs and chemicals, we identified glucocorticoids as c-maf-dependent inhibitors of cyclin D2 transactivation. In multiple myeloma cell lines, glucocorticoids reduced levels of c-maf protein without influencing corresponding mRNA levels. Subsequent studies demonstrated that glucocorticoids increased ubiquitination-dependent degradation of c-maf and up-regulated ubiquitin C mRNA. Moreover, ectopic expression of ubiquitin C recapitulated the effects of glucocorticoids, demonstrating regulation of c-maf protein through the abundance of the ubiquitin substrate. Thus, using a chemical biology approach, we identified a novel mechanism of action of glucocorticoids and a novel mechanism by which levels of c-maf protein are regulated by the abundance of the ubiquitin substrate.