Dual-Specificity Phosphatase 26 Protects Against Cardiac Hypertrophy Through TAK1.

Background Heart pathological hypertrophy has been recognized as a predisposing risk factor for heart failure and arrhythmia. DUSP (dual-specificity phosphatase) 26 is a member of the DUSP family of proteins, which has a significant effect on nonalcoholic fatty liver disease, neuroblastoma, glioma, and so on. However, the involvement of DUSP26 in cardiac hypertrophy remains unclear. Methods and Results Our study showed that DUSP26 expression was significantly increased in mouse hearts in response to pressure overload as well as in angiotensin II-treated cardiomyocytes. Cardiac-specific overexpression of DUSP26 mice showed attenuated cardiac hypertrophy and fibrosis, while deficiency of DUSP26 in mouse hearts resulted in increased cardiac hypertrophy and deteriorated cardiac function. Similar effects were also observed in cellular hypertrophy induced by angiotensin II. Importantly, we showed that DUSP26 bound to transforming growth factor-ß activated kinase 1 and inhibited transforming growth factor-ß activated kinase 1 phosphorylation, which led to suppression of the mitogen-activated protein kinase signaling pathway. In addition, transforming growth factor-ß activated kinase 1-specific inhibitor inhibited cardiomyocyte hypertrophy induced by angiotensin II and attenuated the exaggerated hypertrophic response in DUSP26 conditional knockout mice. Conclusions Taken together, DUSP26 was induced in cardiac hypertrophy and protected against pressure overload induced cardiac hypertrophy by modulating transforming growth factor-ß activated kinase 1-p38/ c-Jun N-terminal kinase-signaling axis. Therefore, DUSP26 may provide a therapeutic target for treatment of cardiac hypertrophy and heart failure.

USP1-WDR48 deubiquitinase complex enhances TGF-ß induced epithelial-mesenchymal transition of TNBC cells via stabilizing TAK1.

Triple-negative breast cancer (TNBC) is the most aggressive histological subtype of breast cancer and is characterized by poor outcomes and a lack of specific-targeted therapies. Transforming growth factor-ß (TGF-ß) acts as the key cytokine in the epithelial-mesenchymal transition (EMT) and the metastasis of TNBC. However, the regulatory mechanisms of the TGF-ß signaling pathway remain largely unknown. In this study, we identified that the USP1/WDR48 complex could effectively enhance TGF-ß-mediated EMT and migration of TNBC cells. Furthermore, lower phosphorylation of Smad2/3, Erk, Jnk, and p38 was noted on the suppression of the expression of endogenous USP1 or WDR48. Moreover, the USP1-WDR48 complex was found to downregulate the polyubiquitination of TAK1 and mediate its in vitro stability. Therefore, our findings have shed a light on the novel role of the USP1/WDR48 complex in promoting TGF-ß-induced EMT and migration in TNBC via in vitro stabilization of TAK1.

Downregulation of lncRNA NEAT1 Ameliorates LPS-Induced Inflammatory Responses by Promoting Macrophage M2 Polarization via miR-125a-5p/TRAF6/TAK1 Axis.

The lncRNA nuclear enriched abundant transcript 1 (NEAT1) promotes sepsis-inflammatory responses and acute kidney injury (AKI), but little known about the underlying mechanisms. This study aims to investigate the roles of NEAT1 in regulating macrophage polarization and its potential for alleviating inflammatory responses during sepsis pathogenesis. Mouse RAW264.7 macrophages were treated with lipopolysaccharide (LPS) as a cellular inflammatory model. NEAT1 shRNA, miR-125a-5p mimics, and TRAF6-overexpressing vector were used to transfect RAW264.7 cells. NEAT1, miR-125a-5p, and mRNA levels of functional genes were detected by quantitative RT-PCR. Protein abundances were analyzed by western blotting. Macrophage polarization was evaluated by flow cytometry. The bindings of miR-125a-5p with NEAT1 or TRAF6 gene were validated by dual luciferase reporter assay. LPS treatment promoted NEAT1 and suppressed miR-125a-5p expression in mouse macrophage cells. NEAT1 silencing by shRNAs promoted macrophage M2 polarization under LPS treatment, which upregulated miR-125a-5p expression, repressed TRAF6 expression and TAK1 protein phosphorylation in macrophages. These cellular and molecular changes induced by NEAT1 shRNAs were abrogated by miR-125a-5p inhibitors. Moreover, miR-125a-5p mimics suppressed TRAF6 expression and TAK1 protein phosphorylation in LPS-treated macrophages, thus causing macrophage M2 polarization under LPS treatment. TRAF6 overexpression abrogated the miR-125a-5p mimics-induced macrophage M2 polarization. miR-125a-5p could directly bind to NEAT1 or TRAF6 gene in macrophages. lncRNA NEAT1 knockdown ameliorates LPS-induced inflammation by promoting macrophage M2 polarization via miR-125a-5p/TRAF6/TAK1 axis.

Modulating TAK1 Expression Inhibits YAP and TAZ Oncogenic Functions in Pancreatic Cancer.

YAP and TAZ are central determinants of malignancy; however, their functions remain still undruggable. We identified TGFß-activated kinase 1 (TAK1) as a central hub integrating the most relevant signals sustaining pancreatic cancer aggressiveness and chemoresistance. Glycogen synthase kinase (GSK)3 is known to stabilize TAK1, and its inhibition causes a reduction in TAK1 levels. Here, we hypothesized that TAK1 could sustain YAP/TAZ program, and thus, modulation of TAK1 expression through the inhibition of GSK3 could impair YAP/TAZ functions in pancreatic cancer.Differentially expressed transcripts between pancreatic cancer cells expressing scramble or TAK1-specific shRNA were annotated for functional interrelatedness by ingenuity pathway analysis. TAK1 expression was modulated by using different GSK3 inhibitors, including LY2090314. In vivo activity of LY2090314 alone or in combination with nab-paclitaxel was evaluated in an orthotopic nude mouse model.Differential gene expression profiling revealed significant association of TAK1 expression with HIPPO and ubiquitination pathways. We measured a significant downregulation of YAP/TAZ and their regulated genes in shTAK1 cells. TAK1 prevented YAP/TAZ proteasomal degradation in a kinase independent manner, through a complex with TRAF6, thereby fostering their K63-ubiquitination versus K48-ubiquitination. Pharmacologic modulation of TAK1 by using GSK3 inhibitors significantly decreased YAP/TAZ levels and suppressed their target genes and oncogenic functions. In vivo, LY2090314 plus nab-paclitaxel significantly prolonged mice survival duration.Our study demonstrates a unique role for TAK1 in controlling YAP/TAZ in pancreatic cancer. LY2090314 is a novel agent that warrants further clinical development in combination with nab-paclitaxel for the treatment of pancreatic cancer.

MicroRNA-148a regulates the MAPK/ERK signaling pathway and suppresses the development of esophagus squamous cell carcinoma via targeting MAP3K9.

OBJECTIVE: Esophagus squamous cell carcinoma (ESCC) was a dominant histological type of esophagus cancer, which has a very high incidence due to distant metastasis and local invasion. MicroRNA-148a (miR-148a) functioned as a tumor suppressor in a variety of cancers. The purpose of our study was to explore the vital role of miR-148a in esophagus squamous cell carcinoma. PATIENTS AND METHODS: The Kaplan-Meier method was applied to calculate the 5-year overall survival of esophagus squamous cell carcinoma patients. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) and Western blot were conducted to calculate the mRNA levels of miR-148a and genes. The cell counting kit-8 (CCK-8) and transwell assays were performed to measure the proliferative and invasive ability. RESULTS: MiR-148a was observed to be significantly downregulated and the downregulation of miR-148 predicted poor prognosis of esophagus squamous cell carcinoma patients. MAP3K9 was a target gene of miR-148a and its expression was mediated by miR-148a through directly binding to the 3'-untranslated region (3'-UTR) of its mRNA in the esophagus squamous cell carcinoma. Moreover, miR-148a remarkably inhibited the proliferation and invasion through directly targeting to MAP3K9 via extracellular-signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and epithelial-mesenchymal transition (EMT) in the ESCC cells. In addition, overexpression of miR-148a inhibited the growth of ESCC in vivo. CONCLUSIONS: MiR-148a inhibited the proliferation and invasion through directly targeting to MAP3K9 by ERK/MAPK pathway and EMT in ESCC cells. The newly identified miR-148a/MAP3K9 axis provides a novel insight into the pathogenesis of the esophagus squamous cell carcinoma.

Silencing of Long Noncoding RNA Nespas Aggravates Microglial Cell Death and Neuroinflammation in Ischemic Stroke.

Background and Purpose- Ischemic stroke is one of the leading causes of morbidity and mortality worldwide and a major cause of long-term disability. Recently, long noncoding RNAs have been revealed, which are tightly associated with several human diseases. However, the functions of long noncoding RNAs in ischemic stroke still remain largely unknown. In the current study, for the first time, we investigated the role of long noncoding RNA Nespas in ischemic stroke. Methods- We used in vivo models of middle cerebral artery occlusion and in vitro models of oxygen-glucose deprivation to illustrate the effect of long noncoding RNA Nespas on ischemic stroke. Results- We found expression of Nespas was significantly increased in ischemic cerebral tissues and oxygen-glucose deprivation-treated BV2 cells in a time-dependent manner. Silencing of Nespas aggravated middle cerebral artery occlusion operation-induced IR injury and cell death. In addition, proinflammatory cytokine production and NF-κB (nuclear factor-κB) signaling activation were inhibited by Nespas overexpression. TAK1 (transforming growth factor-ß-activated kinase 1) was found to directly interact with Nespas, and TAK1 activation was significantly suppressed by Nespas. At last, we found Nespas-inhibited TRIM8 (tripartite motif 8)-induced K63-linked polyubiquitination of TAK1. Conclusions- We showed that Nespas played anti-inflammatory and antiapoptotic roles in cultured microglial cells after oxygen-glucose deprivation stimulation and in mice after ischemic stroke by inhibiting TRIM8-related K63-linked polyubiquitination of TAK1.

TAK1 suppresses RIPK1-dependent cell death and is associated with disease progression in melanoma.

Melanoma cells are highly resistant to conventional genotoxic agents, and BRAFV600/MEK-targeted therapies as well as immunotherapies frequently remain inefficient. Alternative means to treat melanoma, in particular through the induction of programmed cell death modalities such as apoptosis or necroptosis, therefore still need to be explored. Here, we report that melanoma cell lines expressing notable amounts of RIPK1, RIPK3 and MLKL, the key players of necroptosis signal transduction, fail to execute necroptotic cell death. Interestingly, the activity of transforming growth factor ß-activated kinase 1 (TAK1) appears to prevent RIPK1 from contributing to cell death induction, since TAK1 inhibition by (5Z)-7-Oxozeaenol, deletion of MAP3K7 or the expression of inactive TAK1 were sufficient to sensitize melanoma cells to RIPK1-dependent cell death in response to TNFα or TRAIL based combination treatments. However, cell death was executed exclusively by apoptosis, even when RIPK3 expression was high. In addition, TAK1 inhibitor (5Z)-7-Oxozeaenol suppressed intrinsic or treatment-induced pro-survival signaling as well as the secretion of cytokines and soluble factors associated with melanoma disease progression. Correspondingly, elevated expression of TAK1 correlates with reduced disease free survival in patients diagnosed with primary melanoma. Overall, our results therefore demonstrate that TAK1 suppresses the susceptibility to RIPK1-dependent cell death and that high expression of TAK1 indicates an increased risk for disease progression in melanoma.

Novel bone morphogenetic protein receptor inhibitor JL5 suppresses tumor cell survival signaling and induces regression of human lung cancer.

BMP receptor inhibitors induce death of cancer cells through the downregulation of antiapoptotic proteins XIAP, pTAK1, and Id1-Id3. However, the current most potent BMP receptor inhibitor, DMH2, does not downregulate BMP signaling in vivo because of metabolic instability and poor pharmacokinetics. Here we identified the site of metabolic instability of DMH2 and designed a novel BMP receptor inhibitor, JL5. We show that JL5 has a greater volume of distribution and suppresses the expression of Id1 and pTak1 in tumor xenografts. Moreover, we demonstrate JL5-induced tumor cell death and tumor regression in xenograft mouse models without immune cells and humanized with adoptively transferred human immune cells. In humanized mice, JL5 additionally induces the infiltration of immune cells within the tumor microenvironment. Our studies show that the BMP signaling pathway is targetable in vivo and BMP receptor inhibitors can be developed as a therapeutic to treat cancer patients.

The MAPKKK and MAPKK gene families in banana: identification, phylogeny and expression during development, ripening and abiotic stress.

The mitogen-activated protein kinase (MAPK) cascade, which is a major signal transduction pathway widely distributed in eukaryotes, has an important function in plant development and stress responses. However, less information is known regarding the MAPKKK and MAPKK gene families in the important fruit crop banana. In this study, 10 MAPKK and 77 MAPKKK genes were identified in the banana genome, and were classified into 4 and 3 subfamilies respectively based on phylogenetic analysis. Majority of MAPKKK and MAPKK genes in the same subfamily shared similar gene structures and conserved motifs. The comprehensive transcriptome analysis indicated that MAPKKK-MAPKK genes is involved in tissue development, fruit development and ripening, and response to abiotic stress of drought, cold and salt in two banana genotypes. Interaction networks and co-expression assays demonstrated that MAPK signaling cascade mediated network participates in multiple stress signaling, which was strongly activated in Fen Jiao (FJ). The findings of this study advance understanding of the intricately transcriptional control of MAPKKK-MAPKK genes and provide robust candidate genes for further genetic improvement of banana.

DLK silencing attenuated neuron apoptosis through JIP3/MA2K7/JNK pathway in early brain injury after SAH in rats.

OBJECTIVE: Dual leucine zipper kinase (DLK/MA3K12) has been reported involved in apoptosis and neuronal degeneration during neural development and traumatic brain injury. This study was designed to investigate the role of DLK with its adaptor protein JNK interacting protein-3 (JIP3), and its downstream MA2K7/JNK signaling pathway in early brain injury (EBI) after subarachnoid hemorrhage (SAH) in a rat model. DESIGN: Controlled in vivo laboratory study. SETTING: Animal research laboratory. SUBJECTS: Two hundred and twenty-three adult male Sprague-Dawley rats weighing 280-320g. INTERVENTIONS: SAH was induced by endovascular perforation in rats. The SAH grade, neurological score, and brain water content were measured at 24 and 72h after SAH. Immunofluorescence staining was used to detect the cells that expressed DLK. The terminal deoxynucleotid transferase-deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) was used to detect the neuronal apoptosis. In mechanism research, the expression of DLK, JIP3, phosphorylated-JNK (p-JNK)/JNK, and cleaved caspase-3 (CC-3) were analyzed by western blot at 24h after SAH. The DLK small interfering RNA (siRNA), JIP3 siRNA, MA2K7 siRNA and recombinant DLK protein which injected intracerebroventricularly were given as the interventions. MEASUREMENTS AND MAIN RESULTS: The DLK expression was increased in the left cortex neurons and peaked at 24h after SAH. DLK siRNA attenuated brain edema, reduced neuronal apoptosis, and improved the neurobehavioral functions after SAH, but the recombinant DLK protein deteriorated neurobehavioral functions and brain edema. DLK siRNA decreased and recombinant DLK protein increased the expression of MA2K7/p-JNK/CC-3 at 24h after SAH. The JIP3 siRNA reduced the level of JIP3/MA2K7/p-JNK/CC-3, combined DLK siRNA and JIP3 siRNA further decreased the expression of DLK/MA2K7/p-JNK/CC-3, and MA2K7 siRNA lowered the amount of MA2K7/p-JNK/CC-3 at 24h after SAH. CONCLUSIONS: As a negative role, DLK was involved in EBI after SAH, possibly mediated by its adaptor protein JIP3 and MA2K7/JNK signaling pathways. To reduce the level of DLK may be a new target as intervention for SAH.

MicroRNA-155 targets MAP3K10 and regulates osteosarcoma cell growth.

Osteosarcoma is the most common type of bone cancer prevalent in young adults. Recent studies suggested that aberrant microRNA expression plays an essential role in osteosarcoma pathogenesis. In this study, we found miR-155 up-regulation in different osteosarcoma cell lines U2OS, Saos-2 and MG-63. Through bioinformatic prediction and analysis, we identified its target MAP3K10 that involved in cell proliferation and apoptosis. This work demonstrates novel interaction between microRNA, intercellular MAPK signaling and apoptosis in osteosarcoma, which will provide targets for therapeutic development.

Enhanced Expression of miR-199b-5p Promotes Proliferation of Pancreatic ß-Cells by Down-Regulation of MLK3.

BACKGROUND: The initiation of ß-cell proliferation to recover reduced ß-cell mass is considered as one of the attractive therapeutic approaches for type 1 and 2 diabetes. In this study, we investigated the involvement of miRNAs in ß-cell proliferation. METHODS: Global miRNA array analysis of pancreas tissue was carried out using a 60% partial pancreatectomy (PPx) rodent model, which is a well-characterized model for pancreatic regeneration with accelerated proliferation of ß-cells. To explore miRNAs with mitogenic activity on ß-cells, precursors and antisense oligonucleotides (ASOs) for miRNAs were transfected into a primary islet monolayer cell cultures isolated from adult rats in order to modify their expression and proliferation of ß-cells. RESULTS: We found that miR-199b-5p, which was up-regulated 2.6 times in the pancreas of the PPx treated group, significantly enhanced the proliferation of ß-cells when its precursor was over-expressed. Target genes of miR-199b-5p were investigated and Mixed lineage kinase-3 (MLK3) was identified as one of the candidates since its expression was down-regulated through an interaction with miR-199b-5p and siRNA treatment for MLK3 enhanced the proliferation of ß-cells. CONCLUSION: Our data suggest that the up-regulation of miR-199b-5p enhances proliferation of ß-cells at least in part through down-regulation of MLK3.

Tozasertib attenuates neuronal apoptosis via DLK/JIP3/MA2K7/JNK pathway in early brain injury after SAH in rats.

BACKGROUND AND PURPOSE: Since tozasertib is neuroprotective for injured optic nerve, this study is intended to test whether tozasertib reduces early brain injury after subarachnoid hemorrhage (SAH) in a rat model. METHODS: Two hundred sixteen (216) male Sprague-Dawley rats were randomly subjected to endovascular perforation model of SAH and sham group. SAH grade, neurological score, and brain water content were measured at 24 and 72 h after SAH. Dual leucine zipper kinase (DLK) and its downstream factors, JNK-interacting protein 3 (JIP3), MA2K7, p-JNK/JNK (c-Jun N-terminal kinase), and apoptosis related proteins cleaved caspase-3 (CC-3), Bim, Bcl-2, and cleaved caspase-9 (CC-9) were analyzed by western blot at 24 h after SAH. Apoptotic cells were detected by terminal deoxynucleotid transferase-deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL). DLK small interfering RNA (siRNA), JIP3 siRNA and MA2K7 siRNA, the JNK, p38MAPK, and MEK inhibitors SP600125, SB203580, and PD98059 were used for intervention. RESULTS: Tozasertib reduced neuronal apoptosis, attenuated brain edema and improved neurobehavioral deficits 24 and 72 h after SAH. At 24 h After SAH, DLK/JIP3/MA2K7/p-JNK/CC-3 expressions were elevated markedly and tozasertib reduced DLK, MA2K7/p-JNK/CC-3 expressions but enhanced JIP3 expression. In the presence of tozasertib, DLK/JIP3/MA2K7 siRNA and SP600125, SB203580 and PD98059 deteriorated the neurobehavioral deficits, brain edema and increased the expression of CC-3. SAH potentiated the expression of Bim, CC-9, and CC-3 but reduced Bcl-2, while tozasertib reduced expression of Bim, CC-9, and CC-3 but enhanced Bcl-2. CONCLUSIONS: Tozasertib reduced neuronal apoptosis and improved outcome possibly via DLK/JIP3/MA2K7/JNK pathways after SAH.

PGC-1ß suppresses saturated fatty acid-induced macrophage inflammation by inhibiting TAK1 activation.

Inflammation of infiltrated macrophages in adipose tissue is a key contributor to the initiation of adipose insulin resistance. These macrophages are exposed to high local concentrations of free fatty acids (FFAs) and can be proinflammatory activated by saturated fatty acids (SFAs). However, the regulatory mechanisms on SFA-induced macrophage inflammation are still elusive. Peroxisome proliferator-activated receptor γ coactivator-1ß (PGC-1ß) is a member of the PGC-1 family of transcriptional coactivators and has been reported to play a key role in SFAs metabolism and in the regulation of inflammatory signaling. However, it remains unclear whether PGC-1ß is involved in SFA-induced macrophage inflammation. In this study, we found that PGC-1ß expression was significantly decreased in response to palmitic acid (PA) in macrophages in a dose dependent manner. PGC-1ß inhibited PA induced TNFα, MCP-1, and IL-1ß mRNA and protein expressions. Furthermore, PGC-1ß significantly antagonized PA induced macrophage nuclear factor-κB (NF-κB) p65 and JUN N-terminal kinase activation. Mechanistically, we revealed that TGF-ß-activated kinase 1 (TAK1) and its adaptor protein TAK1 binding protein 1 (TAB1) played a dominant role in the regulatory effects of PGC-1ß. We confirmed that PGC-1ß inhibited downstream inflammatory signals via binding with TAB1 and thus preventing TAB1/TAK1 binding and TAK1 activation. Finally, we showed that PGC-1ß overexpression in PA treated macrophages improved adipocytes PI3K-Akt insulin signaling in a paracrine fashion. Collectively, our results uncovered a novel mechanism on how macrophage inflammation induced by SFAs was regulated and suggest a potential target in the treatment of obesity induced insulin resistance.

Picroside II Inhibits the MEK-ERK1/2-COX2 Signal Pathway to Prevent Cerebral Ischemic Injury in Rats.

The objective of this study is to explore the neuroprotective effect and mechanism of picroside II on ERK1/2-COX2 signal transduction pathway after cerebral ischemic injury in rats. Focal cerebral ischemic models were established by inserting monofilament threads into the middle cerebral artery in 200 Wistar rats. Twenty four rats were randomly selected into control group, while the other rats were randomly divided into six groups: model group, picroside group, lipopolysaccharide (LPS) with picroside group, U0126 with picroside group, LPS group, and U0126 group with each group containing three subgroups with ischemia at 6, 12, and 24 h. Neurobehavioral function in the rats was evaluated by modified neurological severity score points (mNSS) test; structure of neurons was observed using hematoxylin-eosin (HE) staining; apoptotic cells were counted using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay; expressions of phosphorylated mitogen/extracellular signal-regulated kinase kinas1/2 (pMEK1/2), phosphorylated extracellular signal-regulated protein kinase1/2 (pERK1/2), and cyclooxygenase (COX2) in the cortex were determined using immunohistochemistry (IHC) and Western blot (WB); and real-time PCR was used to determine the level of COX2 mRNA. The neurological behavioral malfunction appeared in all rats with middle cerebral artery occlusion (MCAO). In the model group, neuron damage was extensive, while the neurobehavioral function score, apoptotic cell index, expression of pMEK1/2, pERK1/2, and COX2 and the level of COX2 mRNA increased significantly when compared to the control group. The peak COX2 mRNA level was in ischemia 12 h, prior to the peak in COX2 protein expression. In the picroside and U0126 groups, the neurological behavioral function was improved, and the number of apoptotic cells and the expression of pMEK1/2, pERK1/2, and COX2 decreased significantly when compared to the model group. In the LPS with picroside group, at ischemia 6 h neuron damage was extensive, and pMEK1/2, pERK1/2, and COX2 expression were much higher than in the model group. But at ischemia 12 and 24 h, the expression of pMEK1/2, pERK1/2, and COX2 decreased slightly, and the neurobehavioral function also improved slightly. In LPS group, neuron damage was extensive, pMEK1/2, pERK1/2, and COX2 expression was still at a high level, and COX2 mRNA peak arrived at ischemic 12 h. Picroside II downregulates COX2 expression after MCAO by inhibiting MEK-ERK1/2 in rats to protect neurons from apoptosis and inflammation.

A herbal formula comprising Rosae Multiflorae Fructus and Lonicerae Japonicae Flos inhibits the production of inflammatory mediators and the IRAK-1/TAK1 and TBK1/IRF3 pathways in RAW 264.7 and THP-1 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: As documented in the Chinese Materia Medica Grand Dictionary (), a herbal formula (RL) consisting of Rosae Multiflorae Fructus (multiflora rose hips) and Lonicerae Japonicae Flos (Japanese honeysuckle flowers) has traditionally been used in treating inflammatory disorders. RL was previously reported to inhibit the expression of various inflammatory mediators regulated by NF-κB and MAPKs that are components of the TLR4 signalling pathways. AIM OF THE STUDY: This study aims to provide further justification for clinical application of RL in treating inflammatory disorders by further delineating the involvement of the TLR4 signalling cascades in the effects of RL on inflammatory mediators. MATERIALS AND METHODS: RL consisting of Rosae Multiflorae Fructus and Lonicerae Japonicae Flos (in 5:3 ratio) was extracted using absolute ethanol. We investigated the effect of RL on the production of cytokines and chemokines that are regulated by three key transcription factors of the TLR4 signalling pathways AP-1, NF-κB and IRF3 in LPS-stimulated RAW264.7 cells using the multiplex biometric immunoassay. Phosphorylation of AP-1, NF-κB, IRF3, IκB-α, IKKα/ß, Akt, TAK1, TBK1, IRAK-1 and IRAK-4 were examined in LPS-stimulated RAW264.7 cells and THP-1 cells using Western blotting. Nuclear localizations of AP-1, NF-κB and IRF3 were also examined using Western blotting. RESULTS: RL reduced the secretion of various pro-inflammatory cytokines and chemokines regulated by transcription factors AP-1, NF-κB and IRF3. Phosphorylation and nuclear protein levels of these transcription factors were decreased by RL treatment. Moreover, RL inhibited the activation/phosphorylation of IκB-α, IKKα/ß, TAK1, TBK1 and IRAK-1. CONCLUSIONS: Suppression of the IRAK-1/TAK1 and TBK1/IRF3 signalling pathways was associated with the effect of RL on inflammatory mediators in LPS-stimulated RAW264.7 and THP-1 cells. This provides further pharmacological basis for the clinical application of RL in the treatment of inflammatory disorders.

EGFR and MEK Blockade in Triple Negative Breast Cancer Cells.

Although evidence suggests that the RAF/MEK/ERK pathway plays an important role in triple negative breast cancer (TNBC), resistance to MEK inhibitors has been observed in TNBC cells. Different mechanisms have been hypothesized to be involved in this phenomenon, including receptor tyrosine kinase-dependent activation of the PI3K/AKT pathway. In this study, we analyzed the effects of the MEK1/2 inhibitor selumetinib in combination with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib in a panel of TNBC cell lines that showed different levels of sensitivity to single-agent selumetinib: SUM-149 and MDA-MB-231 cells resulted to be sensitive, whereas SUM-159, MDA-MB-468 and HCC70 cells were relatively resistant to the drug. Treatment of TNBC cells with selumetinib produced an increase of the phosphorylation of the EGFR both in selumetinib-sensitive SUM-149, MDA-MB-231 and in selumetinib-resistant MDA-MB-468 TNBC cells. The combination of selumetinib and gefitinib resulted in a synergistic growth inhibitory effect in all the TNBC cell lines, although the IC50 was not reached in SUM-159 and MDA-MB-468 cells. This effect was associated with an almost complete suppression of ERK1/2 activation and a reduction of selumetinib-induced AKT phosphorylation. In addition, in selumetinib-sensitive TNBC cells the combination of selumetinib and gefitinib induced a significant G0/G1 cell cycle arrest and apoptosis. Taken together, our data demonstrated that blockade of the EGFR might efficiently increase the antitumor activity of selumetinib in a subgroup of TNBC and that this phenomenon might be related to the effects of such combination on both ERK1/2 and AKT activation.

5Z-7-Oxozeanol Inhibits the Effects of TGFß1 on Human Gingival Fibroblasts.

Transforming growth factor (TGF)ß acts on fibroblasts to promote the production and remodeling of extracellular matrix (ECM). In adult humans, excessive action of TGFß is associated with fibrotic disease and fibroproliferative conditions, including gingival hyperplasia. Understanding how the TGFß1 signals in fibroblasts is therefore likely to result in valuable insights into the fundamental mechanisms underlying fibroproliferative disorders. Previously, we used the TAK1 inhibitor (5Z)-7-Oxozeaenol to show that, in dermal fibroblasts, the non-canonical TAK1 pathway mediates the ability of TGFß1 to induce genes promoting tissue remodeling and repair. However, the extent to which TAK1 mediates fibroproliferative responses in fibroblasts in response to TGFß1 remains unclear. Herein, we show that, in gingival fibroblasts, (5Z)-7-Oxozeaenol blocks the ability of TGFß1 to induce expression of the pro-fibrotic mediator CCN2 (connective tissue growth factor, CTGF) and type I collagen protein. Moreover, genome-wide expression profiling revealed that, in gingival fibroblasts, (5Z)-7-Oxozeaenol reduces the ability of TGFß1 to induce mRNA expression of essentially all TGFß1-responsive genes (139/147), including those involved with a hyperproliferative response. Results from microarray analysis were confirmed using real time polymerase chain reaction analysis and a functional cell proliferation assay. Our results are consistent with the hypothesis that TAK1 inhibitors might be useful in treating fibroproliferative disorders, including that in the oral cavity.

MicroRNA-509-3p inhibits cancer cell proliferation and migration by targeting the mitogen-activated protein kinase kinase kinase 8 oncogene in renal cell carcinoma.

microRNAs (miRNAs; miR) are a class of small non-coding RNA molecules, which are involved in the pathogenesis of human diseases through the negative regulation of gene expression. Previous studies have demonstrated that miR-509-3p is a novel miRNA associated with cell proliferation and migration in 786-O renal cell carcinoma (RCC) cells. However, the mechanism of action of miR-509-3p in RCC remains to be elucidated. The present study aimed to examine the functional role and mechanism of miR-509-3p in the development of RCC. The results demonstrated that the expression levels of miR-509-3p were downregulated in the 786-O and ACHN RCC cell lines compared with the normal tissues of 10 patients with RCC, as determined by reverse transcription-quantitative polymerase chain reaction. The mRNA expression levels of mitogen-activated protein kinase kinase kinase 8 (MAP3K8) were upregulated in the RCC cell lines. Functional investigations demonstrated that the overexpression of miR-509-3p inhibited the migration and proliferation of the RCC cells, as determined by wound scratch and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Luciferase reporter assays revealed that the overexpression of miR-509-3p reduced the transcriptional activity of MAP3K8. Furthermore, the present study demonstrated that the ectopic transfection of miR-509-3p led to a significant reduction in the mRNA and protein expression levels of MAP3K8 in the RCC cells. Finally, knockdown of MAP3K8 inhibited the migration and proliferation of the RCC cells. Therefore, the results of the present study demonstrated that the miR-509-3p RCC suppressor was a significant regulator of the MAP3K8 oncogene, suggesting that it may have a potential therapeutic role in the treatment of RCC.