ABSTRACT: Dimethyl fumarate (DMF) is generally used to treat psoriasis and multiple sclerosis. In the present study, we aimed to investigate the effects of DMF on hepatocellular carcinoma progression and its mechanism of action. In vitro, cell viability was examined using CCK-8 assay; cell cycle was analyzed by flow cytometry; angiogenesis was detected using tube formation assay; and autophagic flux assay results were examined using fluorescence microscopy. We also used western blotting to explore the potential mechanisms. In vivo, tumor xenograft experiment was performed with nude mice, and liver function, renal function, and routine blood counts were assessed using biochemical tests. Dimethyl fumarate inhibited tumor growth and angiogenesis in hepatocellular carcinoma, both in vitro and in vivo. Dimethyl fumarate decreased autophagy in hepatocellular carcinoma cells. Treatment with DMF activated SOCS3, which led to repression of JAK1 and STAT3 phosphorylation. DMF inhibited cell proliferation, angiogenesis, and autophagy via activation of the SOCS3/JAK1/STAT3 signaling pathway. This finding may provide a novel approach for the treatment of hepatocellular carcinoma.
Project description:Janus kinase 1/signal transducers and activators of transcription 3 (JAK1/STAT3) pathway is one of the recognized oncogenic signaling pathways that frequently overactivated in a variety of human tumors. Despite rapid progress in elucidating the molecular mechanisms of activation of JAK/STAT pathway, the processes that regulate JAK/STAT deactivation need to be further clarified. Here we demonstrate that CUE domain-containing 2 (CUEDC2) inhibits cytokine-induced phosphorylation of JAK1 and STAT3 and the subsequent STAT3 transcriptional activity. Further analysis by a yeast two-hybrid assay showed that CUEDC2 could engage in a specific interaction with a key JAK/STAT inhibitor, SOCS3 (suppressors of cytokine signaling 3). The interaction between CUEDC2 and SOCS3 is required for the inhibitory effect of CUEDC2 on JAK1 and STAT3 activity. Additionally, we found CUEDC2 functions collaboratively with SOCS3 to inhibit JAK1/STAT3 signaling by increasing SOCS3 stability via enhancing its association with Elongin C. Therefore, our findings revealed a new biological activity for CUEDC2 as the regulator of JAK1/STAT3 signaling and paved the way to a better understanding of the mechanisms by which SOCS3 has been linked to suppression of the JAK/STAT pathway.
Project description:We determined whether the myelofibrosis drug ruxolitinib, an inhibitor of Janus kinases 1/2 (JAK1 and JAK2), could interact with the multiple sclerosis drug dimethyl-fumarate (DMF) to kill tumor cells; studies used the in vivo active form of the drug, mono-methyl fumarate (MMF). Ruxolitinib interacted with MMF to kill brain, breast, lung and ovarian cancer cells, and enhanced the lethality of standard of care therapies such as paclitaxel and temozolomide. MMF also interacted with other FDA approved drugs to kill tumor cells including Celebrex® and Gilenya®. The combination of [ruxolitinib + MMF] inactivated ERK1/2, AKT, STAT3 and STAT5; reduced expression of MCL-1, BCL-XL, SOD2 and TRX; increased BIM expression; decreased BAD S112 S136 phosphorylation; and enhanced pro-caspase 3 cleavage. Expression of activated forms of STAT3, MEK1 or AKT each significantly reduced drug combination lethality; prevented BAD S112 S136 dephosphorylation and decreased BIM expression; and preserved TRX, SOD2, MCL-1 and BCL-XL expression. The drug combination increased the levels of reactive oxygen species in cells, and over-expression of TRX or SOD2 prevented drug combination tumor cell killing. Over-expression of BCL-XL or knock down of BAX, BIM, BAD or apoptosis inducing factor (AIF) protected tumor cells. The drug combination increased AIF : HSP70 co-localization in the cytosol but this event did not prevent AIF : eIF3A association in the nucleus.
Project description:Suppressor of cytokine signaling 3 (SOCS3) is regarded as a vital repressor in the liver carcinogenesis mainly by inhibiting signal transducer and activator of transcription 3 (STAT3) activity. Farnesoid X Receptor (FXR), highly expressed in liver, has an important role in protecting against hepatocellular carcinoma (HCC). However, it is unclear whether the tumor suppressive activity of FXR involves the regulation of SOCS3. In the present study, we found that activation of FXR by its specific agonist GW4064 in HCC cells inhibited cell growth, induced cell cycle arrest at G1 phase, elevated p21 expression and repressed STAT3 activity. The above anti-tumor effects of FXR were dramatically alleviated by knockdown of SOCS3 with siRNA. Reporter assay revealed that FXR activation enhanced the transcriptional activity of SOCS3 promoter. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay displayed that FXR directly bound to IR9 DNA motif within SOCS3 promoter region. The in vivo study in nude mice showed that treatment with FXR ligand GW4064 could decelerate the growth of HCC xenografts, up-regulate SOCS3 and p21 expression and inhibit STAT3 phosphorylation in the xenografts. These results suggest that induction of SOCS3 may be a novel mechanism by which FXR exerts its anti-HCC effects, and the FXR-SOCS3 signaling may serve as a new potential target for the prevention/treatment of HCC.
Project description:Inflammatory hepatocellular adenomas (IHCAs) are benign liver lesions that can be characterized histologically by the presence of an inflammatory infiltrate and at the molecular level by the overexpression of acute phase inflammatory response genes. Recurrent somatic mutations of the interleukin-6 (IL-6) signal transducer (IL6ST) locus, encoding the critical component of the IL-6 signal transduction machinery gp130, are present in 60% of IHCAs and in a subset (2%) of hepatocellular carcinoma (HCCs). By screening of 256 human hepatic adenoma specimens (the largest genetic analysis of IL6ST performed to date in this setting), we identified 24 distinct somatic IL6ST mutations among 66 mutant adenomas. The functional analysis of nine different gp130 mutants expressed in hepatic cancer cell lines consistently revealed the constitutive and IL-6-independent activation of the JAK/STAT signaling pathway. We further demonstrated that the signaling activity of mutant gp130 in IHCA remains responsive to suppressor of cytokine signaling 3 (SOCS3), a physiological gp130 inhibitor. Specifically, cells expressing a double mutant variant of gp130 with a disrupted SOCS3-binding site at residue 759 (Y186/Y759F) displayed a hyperactivation of signal transducer and activator of transcription 3 (STAT3) as compared with cells expressing the endogenous IHCA-associated Y186 gp130 mutant. Notably, we identified that constitutive signaling via gp130 in IHCA requires the Janus kinase family member JAK1, but not JAK2 or tyrosine kinase 2. In support of this notion, AG490, a tyrosine kinase inhibitor that selectively blocks JAK2, had no effect on gp130 activity. In stark contrast, we showed that ruxolitinib, a JAK1/JAK2-selective tyrosine kinase inhibitor used to treat patients with myelofibrosis, dramatically impaired JAK1-STAT signaling downstream of all IHCA-associated gp130 mutants. In conclusion, our findings provide a rationale for the use of JAK1 inhibitors for the treatment of HCAs expressing mutant gp130 as well as a subset of HCCs that bear similar mutations.
Project description:Pulmonary arterial hypertension (PAH) is a fatal condition for which there is no cure. Dimethyl Fumarate (DMF) is an FDA approved anti-oxidative and anti-inflammatory agent with a favorable safety record. The goal of this study was to assess the effectiveness of DMF as a therapy for PAH using patient-derived cells and murine models. We show that DMF treatment is effective in reversing hemodynamic changes, reducing inflammation, oxidative damage, and fibrosis in the experimental models of PAH and lung fibrosis. Our findings indicate that effects of DMF are facilitated by inhibiting pro-inflammatory NF?B, STAT3 and cJUN signaling, as well as ?TRCP-dependent degradation of the pro-fibrogenic mediators Sp1, TAZ and ?-catenin. These results provide a novel insight into the mechanism of its action. Collectively, preclinical results demonstrate beneficial effects of DMF on key molecular pathways contributing to PAH, and support its testing in PAH treatment in patients.
Project description:Delayed-release dimethyl fumarate (also known as gastro-resistant dimethyl fumarate), an oral therapeutic containing dimethyl fumarate (DMF) as the active ingredient, is currently approved for the treatment of relapsing multiple sclerosis. DMF is also a component in a distinct mixture product with 3 different salts of monoethyl fumarate (MEF), which is marketed for the treatment of psoriasis. Previous studies have provided insight into the pharmacologic properties of DMF, including modulation of kelch-like ECH-associated protein 1 (KEAP1), activation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway, and glutathione (GSH) modulation; however, those of MEF remain largely unexplored. Therefore, the aim of this study was to evaluate the in vitro effects of DMF and MEF on KEAP1 modification, activation of the NRF2 pathway, and GSH conjugation. Using mass spectrometry, DMF treatment resulted in a robust modification of specific cysteine residues on KEAP1. In comparison, the overall degree of KEAP1 modification following MEF treatment was significantly less or undetectable. Consistent with KEAP1 cysteine modification, DMF treatment resulted in nuclear translocation of NRF2 and a robust transcriptional response in treated cells, as did MEF; however, the responses to MEF were of a lower magnitude or distinct compared to DMF. DMF was also shown to produce an acute concentration-dependent depletion of GSH; however, GSH levels eventually recovered and rose above baseline by 24 hours. In contrast, MEF did not cause acute reductions in GSH, but did produce an increase by 24 hours. Overall, these studies demonstrate that DMF and MEF are both pharmacologically active, but have differing degrees of activity as well as unique actions. These differences would be expected to result in divergent effects on downstream biology.
Project description:Interleukin-6 (IL-6) has been demonstrated to be involved in Hepatitis B virus (HBV)-associated hepatocarcinogenesis through activation of the STAT3 pathway. The sustained activation of the IL-6/STAT3 pathway is frequently associated with repression of SOCS3, which is both a target gene and a negative regulator of STAT3. However, the silencing mechanism of SOCS3 in hepatocellular carcinoma (HCC) remains to be elucidated. Here, we showed that the repression of SOCS3 and sustained activation of IL-6/STAT3 pathway in HBV-producing HCC cells were caused by HBV-induced mitochondrial ROS accumulation. Mechanistic studies revealed that ROS-mediated DNA methylation resulted in the silencing of SOCS3. Decreased SOCS3 expression significantly promoted the proliferation of HCC cells and growth of tumor xenografts in mice. Further studies revealed that HBV-induced ROS accumulation upregulated the expression of the transcription factor, Snail, which bound to the E-boxes of SOCS3 promoter and mediated the epigenetic silencing of SOCS3 in association with DNMT1 and HDAC1. In addition, we found that the expression of Snail and SOCS3 were inversely correlated in HBV-associated HCC patients, suggesting that SOCS3 and/or Snail could be used as prognostic markers in HCC pathogenesis. Taken together, our data show that HBV-induced mitochondrial ROS production represses SOCS3 expression through Snail-mediated epigenetic silencing, leading to the sustained activation of IL-6/STAT3 pathway and ultimately contributing to hepatocarcinogenesis.
Project description:The fumarate ester dimethyl fumarate (DMF) has been introduced recently as a treatment for relapsing remitting multiple sclerosis (RRMS), a chronic inflammatory condition that results in neuronal demyelination and axonal loss. DMF is known to act by depleting intracellular glutathione and modifying thiols on Keap1 protein, resulting in the stabilization of the transcription factor Nrf2, which in turn induces the expression of antioxidant response element genes. We have previously shown that DMF reacts with a wide range of protein thiols, suggesting that the complete mechanisms of action of DMF are unknown. Here, we investigated other intracellular thiol residues that may also be irreversibly modified by DMF in neurons and astrocytes. Using mass spectrometry, we identified 24 novel proteins that were modified by DMF in neurons and astrocytes, including cofilin-1, tubulin and collapsin response mediator protein 2 (CRMP2). Using an in vitro functional assay, we demonstrated that DMF-modified cofilin-1 loses its activity and generates less monomeric actin, potentially inhibiting its cytoskeletal remodeling activity, which could be beneficial in the modulation of myelination during RRMS. DMF modification of tubulin did not significantly impact axonal lysosomal trafficking. We found that the oxygen consumption rate of N1E-115 neurons and the levels of proteins related to mitochondrial energy production were only slightly affected by the highest doses of DMF, confirming that DMF treatment does not impair cellular respiratory function. In summary, our work provides new insights into the mechanisms supporting the neuroprotective and remyelination benefits associated with DMF treatment in addition to the antioxidant response by Nrf2.
Project description:Through a small scale metabolic-modulator screening, we have identified dimethyl fumarate (DMF), a FDA approved drug for multiple sclerosis, which suppresses neuroblastoma cell growth in vitro and in vivo. Mechanistically, DMF suppresses neuroblastoma cell growth through inducing ROS and subsequently suppressing MYCN expression. Overall design: Biological duplicate samples were treated with 100uM of DMF for 6 and 12 hours.
Project description:Primary adult dermal fibroblasts isolated from forearm skin biopsy of 3 healthy donors were treated with DMF and TGFb. Overall design: 3 cell lines of dermal fibroblasts were derived from skin biopses from healthy donors. All cell lines were subjected to 4 treatments: control (DMSO), dimethyl fumarate (DMF), TGFb, and TGFb+DMF. The goal of the study was to identify sets of genes regulated by different treatments.