Inhibition Of ERK Pathway Research Articles

Effects of the multi-kinase inhibitor midostaurin in combination with chemotherapy in models of acute myeloid leukaemia.

Recently, several targeted agents have been developed for specific subsets of patients with acute myeloid leukaemia (AML), including midostaurin, the first FDA‐approved FLT3 inhibitor for newly diagnosed patients with FLT3 mutations. However, in the initial Phase I/II clinical trials, some patients without FLT3 mutations had transient responses to midostaurin, suggesting that this multi‐targeted kinase inhibitor might benefit AML patients more broadly. Here, we demonstrate submicromolar efficacy of midostaurin in vitro and efficacy in vivo against wild‐type (wt) FLT3‐expressing AML cell lines and primary cells, and we compare its effectiveness with that of other FLT3 inhibitors currently in clinical trials. Midostaurin was found to synergize with standard chemotherapeutic drugs and some targeted agents against AML cells without mutations in FLT3. The mechanism may involve, in part, the unique kinase profile of midostaurin that includes proteins implicated in AML transformation, such as SYK or KIT, or inhibition of ERK pathway or proviability signalling. Our findings support further investigation of midostaurin as a chemosensitizing agent in AML patients without FLT3 mutations.

Pharmacological Characterizations of anti-Dementia Memantine Nitrate via Neuroprotection and Vasodilation in Vitro and in Vivo.

We have previously designed and synthesized a series of novel memantine nitrates, and some of them have shown neuroprotective effects; however, the detailed mechanisms remain unknown. In this study, we demonstrated that MN-12, one of the memantine nitrates, concentration-dependently protected against glutamate-induced neurotoxicity in rat primary cultured cerebellar granule neurons (CGNs). Western blotting assays revealed that MN-12 might possess neuroprotective effects through the inhibition of ERK pathway and activation of PI3K/Akt pathway concurrently. Moreover, MN-12 concentration-dependently dilated precontracted rat middle cerebral artery through activation of NO-cGMP pathway ex vivo. In the 2-vessel occlusion (2VO) rat model, MN-12 alleviated the impairments of spatial memory and motor dysfunction possibly via neuroprotection and improvement of the cerebral blood flow. Furthermore, the results of preliminary pharmacokinetic studies showed that MN-12 might quickly distribute to the major organs including the brain, indicating that MN-12 could penetrate the blood-brain barrier. Taken together, MN-12 might provide multifunctional therapeutic benefits for dementia associated with Alzheimer's disease, vascular dementia, and ischemic stroke, via neuroprotection and vessel dilation to improve the cerebral blood flow.

Calcium-sensing receptor promotes tumor proliferation and migration in human intrahepatic cholangiocarcinoma by targeting ERK signaling pathway

The Calcium-sensing receptor (CaSR) is functionally expressed in the biliary epithelial cells and it has been verified to possess various regulatory functions in several different forms of human cancers. But its pathological role in human intrahepatic cholangiocarcinoma (ICC) development remains obscure. Here, we confirmed that CaSR expression was up-regulated in ICC tumor specimens and cell lines, which was positively correlated with number of tumors, lymph node metastasis and poor prognosis of ICC patients. CaSR activation induced by CaCl2 or Calindol (a selective CaSR agonist) markedly facilitated cell proliferation and migration in ICC cells, while knockdown of CaSR or NPS2143 treatment (a CaSR antagonist) dramatically suppressed the above effects. We also demonstrated that alteration of CaSR activity mediated tumorigenesis and growth of ICC in vivo. Mechanistically, CaSR activation could promote cell cycle progression and induce an upregulation of MMP-2 and MMP-9 expression partly via the simulation of ERK1/2 signaling pathway. And further inhibition of ERK pathway significantly suppressed ICC cell viability and migration capacity. Together, our findings shed novel light on the role of CaSR as an oncogene in ICC progression and indicated that modulation of CaSR might serve as a preventive or therapeutic strategy for ICC.

Abstract B074: Sapanisertib in combination with trametinib in glioblastoma models enhances target inhibition and antitumor activity

Abstract Objective: To determine the biological and signaling effects of dual-pathway inhibition with mTORC1/2 and MEK inhibitors in glioblastoma neurosphere cell lines. Background: The PI3K/mTOR signaling pathway is frequently dysregulated in glioblastoma (GBM), making it the focus of active scientific and drug development efforts in brain tumors. Unfortunately, small molecule targeted therapies against EGFR and its downstream effector pathways, while promising in the pre-clinical setting, have been disappointing in glioma clinical trials. The mTOR pathway target ribosomal protein S6 (RPS6) can also be activated by the ERK pathway target p90-RSK. We therefore tested the hypothesis that small molecule inhibition of both ERK and mTOR signaling pathways would result in enhanced anti-tumor activity. Methods: Seven glioblastoma neurosphere lines were tested for sensitivity to the ATP-competitive mTORC1/2 inhibitor sapanisertib (MLN0128) alone or in combination with the allosteric MEK1/2 inhibitor trametinib (GSK1120212) using cell growth assays and immunoblots as indicated. Results: The dual mTORC1/2 inhibitor sapanisertib inhibits mTOR signaling and cell growth in all GBM neurosphere lines, but only partially inhibits phosphorylation of the downstream target ribosomal protein S6 (phospho-RPS6) in these models. The MEK inhibitor, trametinib, inhibited ERK phosphorylation in all neurosphere lines, though phosphorylation of downstream targets and cell growth were inhibited to varying degrees. Inhibition of RPS6 activation was dose-dependent and correlated with cell line sensitivity when treated with trametinib but not with sapanisertib. In combination, sapanisertib plus trametinib showed more profound inhibition of phospho-RPS6, as well as enhanced growth inhibition and induction of apoptosis in sensitive neurosphere lines. Conclusions: The combination of sapanisertib and trametinib may have significant antitumor activity in glioblastoma neurospheres by enhanced inhibition of S6, which serves as the convergence point between these effector pathways. Citation Format: Karisa C Schreck, Amy Allen, Jiawan Wang, Christine A Pratilas. Sapanisertib in combination with trametinib in glioblastoma models enhances target inhibition and antitumor activity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B074. doi:10.1158/1535-7163.TARG-19-B074

Ursonic acid exerts inhibitory effects on matrix metalloproteinases via ERK signaling pathway

Ursonic acid is a pentacyclic triterpenoid compound that can be extracted from Ziziphus jujuba Mill., a traditional medicine. Matrix metalloproteinases (MMPs) are involved in cancer metastasis and skin aging. Regulation of various MMPs is closely associated with mitogen-activated protein kinases (MAPKs), including ERK, p38, and JNK MAPKs. In this study, we investigated the possibility of ursonic acid as an anti-cancer/anti-skin aging agent targeting MMPs. Cytotoxic effects of ursonic acid were analyzed by cell counting kit-8 (CCK-8) assay. Invasive abilities of ursonic acid-treated A549 and H1299 non-small cell lung cancer (NSCLC) cells were tested with Boyden chamber assay. Effects of ursonic acid on MMPs were analyzed by zymography assays and quantitative real time polymerase chain reaction (qRT-PCR). We also conducted flow cytometry and western blot analysis to elucidate the mechanisms of MMP regulation by ursonic acid. Our results revealed that ursonic acid inhibited transcriptional expression of gelatinases (MMP-2 and MMP-9) via inhibition of ERK and CREB signaling pathways in NSCLC cells. Moreover, ursonic acid reduced mRNA levels of collagenase (MMP-1) via suppression of ERK and c-Fos signaling pathways in HaCaT keratinocytes. These results suggest that ursonic acid could be a potential candidate for development of an effective novel anti-cancer and anti-wrinkle agent.

Y-27632 preserves epidermal integrity in a human skin organ-culture (hSOC) system by regulating AKT and ERK signaling pathways

BackgroundThe human skin organ culture (hSOC) developed a century ago has been widely used to study various aspects of human skin development, differentiation, function, disease as well as skin appendages biology, however, maintaining the integrity of epidermal structure in long-term culture, has remained a challenge. ObjectivesHere we tried to establish a culture system using supplemented William’s E medium in the presence of a ROCK inhibitor Y-27632 to maintain epidermal architecture in the long-term hSOC and to investigate the underlying mechanisms. MethodsHuman breast skins, cut into 5 mm × 5 mm pieces, were cultured in supplemented William’s E medium in the presence of 30μM Y-27632. The cultured skin tissues were collected at different time points for analysis of epidermal cell proliferation and differentiation by real time qRT-PCR and immunofluorescence (IF) staining. The keratinocyte suspension assay and in vivo treatment of Y-27632 on mouse were also carried out to study that the regulation of Y-27632 on keratinocyte proliferation and differentiation. ResultsWe found Y-27632 not only enhanced both basal cell proliferation and expression of suprabasal cell differentiation markers, but also maintained the balance of keratinocyte proliferation and differentiation through activation of AKT pathways on one hand and inhibition of ERK pathways on the other hand. The AKT inhibitor MK-2206 blocked the epidermal preservation effect of Y-27632, while the MEK/ERK inhibitor U0126 enhanced the preservation of epidermal structure in the hSOC. ConclusionsY-227632 can maintain skin epidermal integrity through regulation of AKT and ERK activity in the hSOC.

Baicalin Protects against Thrombin-Induced Cell Injury in Human Umbilical Vein Endothelial Cells.

Thrombin plays a pivotal role in the pathogenesis of atherosclerosis. Baicalin, an active flavonoid compound, was shown to attenuate the development of atherosclerosis, but the mechanism remains elusive. In the present study, the role and mechanism of baicalin in thrombin-induced cell injury was investigated in human umbilical vein endothelial cells (HUVECs). Our results showed that baicalin significantly reduced thrombin-induced apoptosis of HUVECs. Additional experiments showed that baicalin inhibited thrombin-induced NF-κB activation and PAR-1 expression. In addition, baicalin decreased thrombin-induced PAR-1 expression by inhibiting ERK pathway. These results indicated that baicalin has protective effects on thrombin-induced cell injury in HUVECs possibly through inhibition of PAR-1 expression and its downstream NF-κB activation, which was mediated by ERK1/2 activation.

Down-regulation of insulin-like growth factor binding protein 5 is involved in intervertebral disc degeneration via the ERK signalling pathway.

It is obvious that epigenetic processes influence the evolution of intervertebral disc degeneration (IDD). However, its molecular mechanisms are poorly understood. Therefore, we tested the hypothesis that IGFBP5, a potential regulator of IDD, modulates IDD via the ERK signalling pathway. We showed that IGFBP5 mRNA was significantly down‐regulated in degenerative nucleus pulposus (NP) tissues. IGFBP5 was shown to significantly promote NP cell proliferation and inhibit apoptosis in vitro, which was confirmed by MTT, flow cytometry and colony formation assays. Furthermore, IGFBP5 was shown to exert its effects by inhibiting the ERK signalling pathway. The effects induced by IGFBP5 overexpression on NP cells were similar to those induced by treatment with an ERK pathway inhibitor (PD98059). Moreover, qRT‐PCR and Western blot analyses were performed to examine the levels of apoptosis‐related factors, including Bax, caspase‐3 and Bcl2. The silencing of IGFBP5 up‐regulated the levels of Bax and caspase‐3 and down‐regulated the level of Bcl2, thereby contributing to the development of human IDD. Furthermore, these results were confirmed in vivo using an IDD rat model, which showed that the induction of Igfbp5 mRNA expression abrogated the effects of IGFBP5 silencing on intervertebral discs. Overall, our findings elucidate the role of IGFBP5 in the pathogenesis of IDD and provide a potential novel therapeutic target for IDD.

CRAF mutations in lung cancer can be oncogenic and predict sensitivity to combined type II RAF and MEK inhibition

Two out of 41 non-small cell lung cancer patients enrolled in a clinical study were found with a somatic CRAF mutation in their tumor, namely CRAFP261A and CRAFP207S. To our knowledge, both mutations are novel in lung cancer and CRAFP261A has not been previously reported in cancer. Expression of CRAFP261A in HEK293T cells and BEAS-2B lung epithelial cells led to increased ERK pathway activation in a dimer-dependent manner, accompanied with loss of CRAF phosphorylation at the negative regulatory S259 residue. Moreover, stable expression of CRAFP261A in mouse embryonic fibroblasts and BEAS-2B cells led to anchorage-independent growth. Consistent with a previous report, we could not observe a gain-of-function with CRAFP207S. Type II but not type I RAF inhibitors suppressed the CRAFP261A-induced ERK pathway activity in BEAS-2B cells, and combinatorial treatment with type II RAF inhibitors and a MEK inhibitor led to a stronger ERK pathway inhibition and growth arrest. Our findings suggest that the acquisition of a CRAFP261A mutation can provide oncogenic properties to cells, and that such cells are sensitive to combined MEK and type II RAF inhibitors. CRAF mutations should be diagnostically and therapeutically explored in lung and perhaps other cancers.

Notch signaling regulates osteosarcoma proliferation and migration through Erk phosphorylation

We used a murine spontaneous osteosarcoma cell line with high metastatic potential, the K7M2 cell line to study the role of Notch signaling in the biological manifestations of osteosarcoma, to understand its underlying mechanism in the regulation of cell proliferation and migration, and to improve patient prognosis in cases of osteosarcoma through the discovery of novel therapeutic targets, First, Notch expression in K7M2 was determined by immunostaining, and the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) was used to inhibit proteolytic cleavage of the Notch intracellular domain (NICD), resulting in the inhibition of Notch activation. By using the Sulforhodamine B assay, colony-forming units assay, Brdu and Ki67 staining, and flow cytometry assays of apoptosis and cell cycle stage, DAPT was found to inhibit K7M2 proliferation in a dose-dependent manner. By using wound healing and transwell migration assays, DAPT was found to inhibit K7M2 migration in a dose-dependent manner as well. By using a combination of micro-Raman spectroscopy and K-means clustering analysis, we found that DAPT inhibit a variety of important cell metabolism-related components in most K7M2 cell structures. Then, DAPT was found to inhibit Notch1ICD expression in a concentration-dependent manner, and this expression was directly correlated with Phospho-Erk1/2 (p-Erk) by using Western blotting. To confirm this finding, we used the Notch signaling ligand Jagged1 to activate the Notch signaling pathway, which in turn up-regulated p-Erk, resulting in increased proliferation and migration of K7M2. Using the Erk pathway inhibitor U0126, we showed that p-Erk was downregulated and the proliferation and migration of K7M2 decreased along with it. Finally, we constructed a K7M2 mouse para-tibial tumor model and lung metastatic model. We found DAPT inhibits p-Erk in vivo, effectively controls tumor growth, reduces angiogenesis, reduces metastasis to the lungs, and improves overall survival. In summary, Notch signaling plays an oncogene role and promotes metastasis in osteosarcoma through p-Erk. DAPT effectively inhibits osteosarcoma proliferation and metastasis in vivo and in vitro by inhibiting Erk phosphorylation. Therefore, the inhibition of Notch activation resulted the down-regulation of phosphorylation of Erk pathway can be used as potential therapeutic targets in clinical treatment to improve osteosarcoma prognosis.

Molecular Mechanisms of Tyrosine Kinase Inhibitor Resistance Induced by Membranous/Cytoplasmic/Nuclear Translocation of Epidermal Growth Factor Receptor

IntroductionThe molecular mechanism underlying the induction of resistance to tyrosine kinase inhibitors (TKIs) via the membranous/cytoplasmic/nuclear translocation of EGFR has not yet been reported. MethodsWe performed immunohistochemistry to detect the distribution of EGFR in lung adenocarcinoma specimens after TKI treatment and analyzed the relationship between different EGFR locations and patient survival duration. Mass spectrometry analysis and immunoprecipitation were performed to show the interaction of cytosolic EGFR with YY1 associated protein 1 (YAP) and salt inducible kinase 2 (SIK2). Dual-luciferase assays, immunoblotting, real-time polymerase chain reaction, and functional experiments were used to elucidate the role of EGFR cytoplasmic/nuclear translocation in Hippo pathway dysregulation. ResultsPatients with advanced lung adenocarcinoma with membranous mutant EGFR (19del or 21 L858R) showed significantly longer progression-free survival than those with cytoplasmic mutant EGFR after gefitinib treatment. The concentration that inhibits 50% in PC-9 with cytoplasmic EGFR was higher than that in hunman non-small cell lung cancer 827 with membranous EGFR. During first-generation TKI resistance induction, membrane EGFR translocated to the cytoplasm/nucleus, accompanied by the Hippo pathway inhibition. Cytoplasmic EGFR and SIK2 interaction inhibited large tumor suppressor kinase 1 (LATS1) and macrophage stimulating 1 (MST1) interaction, promoting YAP nuclear translocation. However, cells with osimertinib-induced resistance also showed EGFR translocation and lower phospho-EGF receptor but did not show Hippo pathway inhibition. Moreover, osimertinib and erlotinib could restore sensitivity to each other in resistant cells. ConclusionsPlasma/nuclear translocation of EGFR and inhibition of the Hippo pathway are some of the important mechanisms underlying the resistance induced by first-generation TKIs. Membrane/plasma translocation of EGFR induced by osimertinib may be another resistance phenomenon besides MNNG HOS transforming gene (c-MET) amplification, C797S mutation, and ERK pathway inhibition.

SHOC2 complex-driven RAF dimerization selectively contributes to ERK pathway dynamics

Despite the crucial role of RAF kinases in cell signaling and disease, we still lack a complete understanding of their regulation. Heterodimerization of RAF kinases as well as dephosphorylation of a conserved "S259" inhibitory site are important steps for RAF activation but the precise mechanisms and dynamics remain unclear. A ternary complex comprised of SHOC2, MRAS, and PP1 (SHOC2 complex) functions as a RAF S259 holophosphatase and gain-of-function mutations in SHOC2, MRAS, and PP1 that promote complex formation are found in Noonan syndrome. Here we show that SHOC2 complex-mediated S259 RAF dephosphorylation is critically required for growth factor-induced RAF heterodimerization as well as for MEK dissociation from BRAF. We also uncover SHOC2-independent mechanisms of RAF and ERK pathway activation that rely on N-region phosphorylation of CRAF. In DLD-1 cells stimulated with EGF, SHOC2 function is essential for a rapid transient phase of ERK activation, but is not required for a slow, sustained phase that is instead driven by palmitoylated H/N-RAS proteins and CRAF. Whereas redundant SHOC2-dependent and -independent mechanisms of RAF and ERK activation make SHOC2 dispensable for proliferation in 2D, KRAS mutant cells preferentially rely on SHOC2 for ERK signaling under anchorage-independent conditions. Our study highlights a context-dependent contribution of SHOC2 to ERK pathway dynamics that is preferentially engaged by KRAS oncogenic signaling and provides a biochemical framework for selective ERK pathway inhibition by targeting the SHOC2 holophosphatase.

Total flavonoids from sea buckthorn ameliorates lipopolysaccharide/cigarette smoke-induced airway inflammation.

The total flavonoids from sea buckthorn (TFSB) exhibit a potent anti-inflammatory activity; however, the effect of TFSB on respiratory inflammatory disease is not fully known. The present study evaluated the potential of TFSB to prevent airway inflammation and the underlying mechanism. The results showed that TFSB remarkably inhibited lipopolysaccharide/cigarette smoke extract (LPS/CSE)-induced expression of IL-1β, IL-6, CXCL1, and MUC5AC at both mRNA and protein levels in HBE16 bronchial epithelial cells. TFSB also decreased the production of PGE2 through inhibition the expression of COX2 in LPS/CSE-stimulated HBE16 cells. Furthermore, bronchoalveolar fluid and histological analyses revealed that LPS/cigarette smoke exposure-induced elevated cell numbers of neutrophils and macrophages in bronchoalveolar fluid, inflammatory cell infiltration, and airway remodeling were remarkably attenuated by TFSB in mice. Immunohistochemical results also confirmed that TFSB decreased the expression of IL-1β, IL-6, COX2, CXCL1, and MUC5AC in LPS/CS-exposed mice. Mechanistically, TFSB blocked LPS/CSE-induced activation of ERK, Akt, and PKCα. Molecular docking further confirmed that the main components in TFSB including quercetin and isorhamnetin showed potent binding affinities to MAPK1 and PIK3CG, two upstream kinases of ERK and Akt, respectively. In summary, TFSB exerts a potent protective effect against LPS/CS-induced airway inflammation through inhibition of ERK, PI3K/Akt, and PKCα pathways, suggesting that TFSB may be a novel therapeutic agent for respiratory diseases.

Targeting ERK beyond the boundaries of the kinase active site in melanoma

Extracellular signal-regulated kinase 1/2 (ERK1/2) constitute a point of convergence for complex signaling events that regulate essential cellular processes, including proliferation and survival. As such, dysregulation of the ERK signaling pathway is prevalent in many cancers. In the case of BRAF-V600E mutant melanoma, ERK inhibition has emerged as a viable clinical approach to abrogate signaling through the ERK pathway, even in cases where MEK and Raf inhibitor treatments fail to induce tumor regression due to resistance mechanisms. Several ERK inhibitors that target the active site of ERK have reached clinical trials, however, many critical ERK interactions occur at other potentially druggable sites on the protein. Here we discuss the role of ERK signaling in cell fate, in driving melanoma, and in resistance mechanisms to current BRAF-V600E melanoma treatments. We explore targeting ERK via a distinct site of protein-protein interaction, known as the D-recruitment site (DRS), as an alternative or supplementary mode of ERK pathway inhibition in BRAF-V600E melanoma. Targeting the DRS with inhibitors in melanoma has the potential to not only disrupt the catalytic apparatus of ERK but also its noncatalytic functions, which have significant impacts on spatiotemporal signaling dynamics and cell fate.

The Protective Role of Inhibition of Keratin on Rheumatoid Arthritis

Objective: Rheumatoid arthritis (RA) is a common inflammatory disease. Studies showed that keratin type II cuticular Hb4 (KRT84) was highly expressed in the synovial membrane of patients with RA. However, the function and mechanism of KRT84 in RA is still unclear. Methods: In this study, we isolated fibroblast-like synoviocytes (FLS) from the mixed knee joint synovial tissues from five patients with RA and the cells were treated with KRT84 siRNA. After transfection of 24 h and 36 h, the knockdown efficiency and expression of relevant genes was detected by RT-PCR. MTT assay, transwell assay, wound scratch assays, flow cytometric analysis and ELISA were used to assess cell proliferation, invasive and migratory capacity. Results: We found that the invasion and migration of RAFLS were significantly decreased after transfection of KRT84 siRNA. ELISA showed a remarkably decrease in TNF-α secretion after KRT84 knockdown. We also explore stimulatory factors for high expression of KRT84 in RA. The inhibitors of ERK, STAT3 and NF-κ B pathways were employed. Our results showed that the expression of KRT84 in RAFLS was evidently increased after treatment with ERK and STAT3 pathway inhibitor. Conclusions: These results imply a protective role of KRT84 knockdown on RA and lay a foundation for further studies on the pathogenetic mechanisms of RA.

Mesenchymal stem cells alleviate hypoxia-induced oxidative stress and enhance the pro-survival pathways in porcine islets.

The utilization of mesenchymal stem cells (MSCs) is a promising approach to serve as adjuvant therapy for islet transplantation. But the inability to translate promising preclinical results into sound therapeutic effects in human subjects indicates a lack of key knowledge of MSC-islet interactions that warrant further research. Hypoxia and oxidative stress are critical factors which lead to a tremendous loss of islet grafts. However, previous studies mainly focused on other aspects of MSC protection such as inducing revascularization, enhancing insulin secretion, and reducing islet apoptosis. In this study, we aim to investigate whether MSC can protect islet cells from hypoxic damage by inhibiting ROS production and the potential underlying pathways involved. We also explore the effects of MSC-derived exosomes and IL-6 on hypoxia-injured islets. Our data provide new molecular targets for developing MSC applications, and this may ultimately promote the efficiency of clinical islet transplantation.

Sargassum horneri mitigates particulate matter induced inflammatory response in MLE-12 cells via MAPK pathways

Abstract Rise of ambient particulate matter (PM) exhibits positive correlation with increased respiratory diseases since PM triggers an inflammatory response in lung. As approaches aiming at limiting PM exposure provide only partial solutions, we investigated a therapeutic approach to mitigate harmful effects of PM in this study. Specifically, we examined the anti-inflammatory potential of the 80% ethanol extract of Sargassum honeri (SHE) against PM exposed murine lung epithelial cells (MLE-12). PM exhibited a cytotoxic effect on MLE-12 from rather low concentrations (concentrations > 15.6 μg/mL; IC50 44.1 μg/mL), and the cytotoxic concentration of PM utilized in this study was 125 μg/mL. But, SHE (0–125 μg/mL) was not cytotoxic on MLE-12 cells, and concentrations 62.5 and 125 μg/mL were tested in this study. SHE reversed the PM induced lactate dehydrogenase (LDH) release and cell proliferation loss of MLE-12 cells. SHE also decreased the PM induced secretion of pro-inflammatory cytokines (IL-1β, TNF-α), pro-inflammatory mediator COX-2, anti-inflammatory cytokines (IL-6, IL-8) and inflammatory chemokines (MCP-1, CCL5). Furthermore, SHE decreased PM induced phosphorylation of p38 mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase 1/2 (Erk1/2) and c-Jun NH (2)-terminal kinase (JNK). Taken together, we conclude that SHE’s anti-inflammatory effect was regulated probably via the inhibition of p38, ERK and JNK MAP kinase signaling pathway in MLE-12 cells. This research is a part of the project, “Development of functional food products with natural materials derived from marine resources”, funded by the Ministry of Oceans and Fisheries, Korea in 2017 (Project NO. 20172085).

<p>Disulfiram inhibits epithelial–mesenchymal transition through TGFβ–ERK–Snail pathway independently of Smad4 to decrease oral squamous cell carcinoma metastasis</p>

Purpose: Smad4 loss is highly related to poor prognosis and decreased patient survival in oral squamous cell carcinoma (OSCC), suggesting that agents that target both Smad4-mutated and Smad4 wild-type cells could treat OSCC more effectively. Disulfiram (Dsf) has anticancer activity through a variety of mechanisms, including inhibition of epithelial–mesenchymal transition (EMT). It remains unclear whether Dsf has the same effect on Smad4-mutated and Smad4 wild-type OSCC or not and what mechanism is involved.Methods: Effect of Dsf on TGFβ1-induced EMT in CAL27 (Smad4 mutation) and SCC25 (Smad4 wild-type) cells were evaluated through analyzing changes in morphology, expression of EMT markers, and migration and invasion of cells. The ERK-pathway inhibitor U0126 was used to confirm TGFβ–ERK–Snail pathway–mediated cell behavior. Dsf’s effects on tumor growth and metastasis in vivo were examined through a subcutaneous xenograft mouse model and an intravenous tumor mouse model.Results: Dsf inhibited TGFβ1-induced EMT through suppression of morphological change, EMT-marker expression, and cell migration and invasion in both CAL27 and SCC25. Phosphorylation of ERK and expression of Snail were blocked by Dsf treatment. Like Dsf, U0126 had a similar effect on EMT of CAL27 and SCC25. Dsf also reduced tumor growth and metastasis in vivo, accompanied by decreased expression of EMT markers in tumors.Conclusion: These results indicated that Dsf inhibited EMT of OSCC in vitro and in vivo independently of Smad4 through suppression of the TGFβ–ERK–Snail pathway, suggesting the broad-spectrum anticancer potential of Dsf for clinical use against OSCC.

Human vascular cell responses to the circulating bone hormone osteocalcin.

The purpose of this study was to characterize the direct effects of uncarboxylated osteocalcin (ucOCN) on vascular cell biology in vitro, to assess its potential function in pathophysiological conditions such as atherosclerosis. Human aortic endothelial cells (HAECs) and smooth muscle cells (HASMCs) were treated with ucOCN (0.1–50 ng/ml) and changes in phosphorylation of intracellular signaling proteins, angiogenesis, proliferation, migration, monolayer permeability, and protein secretion were measured. In HAECs, phosphorylated JNK and CREB were decreased with ucOCN (p < 0.05). In HASMCs, phosphorylated p70S6K and NF‐ΚB were increased by ucOCN (p < 0.05). Cell proliferation increased in both cell types dose dependently which was blocked by AKT and ERK pathway inhibitors. ucOCN did not affect cell permeability, angiogenesis, or migration. The direct activity of ucOCN on vascular cells is recognized, particularly its proliferative effects. However, at least in physiological settings, it does not appear that osteocalcin may directly promote atherogenesis based on the outcomes measured.

Repression of hepatocyte nuclear factor 4 alpha by AP-1 underlies dyslipidemia associated with retinoic acid

All-trans retinoic acid (atRA) is used to treat certain cancers and dermatologic diseases. A common adverse effect of atRA is hypercholesterolemia; cytochrome P450 (CYP) 7A repression is suggested as a driver. However, the underlying molecular mechanisms remain unclear. We investigated CYP7A1 expression in the presence of atRA in human hepatocytes and hepatic cell lines. In HepaRG cells, atRA increased cholesterol levels dose-dependently alongside dramatic decreases in CYP7A1 expression. Lentiviral-mediated CYP7A1 overexpression reversed atRA-induced cholesterol accumulation, suggesting that CYP7A1 repression mediated cholesterol accumulation. In CYP7A1 promoter reporter assays and gene-knockdown studies, altered binding of hepatocyte nuclear factor 4 α (HNF4α) to the proximal promoter was essential for atRA-mediated CYP7A1 repression. Pharmacologic inhibition of c-Jun N-terminal kinase (JNK) and ERK pathways attenuated atRA-mediated CYP7A1 repression and cholesterol accumulation. Overexpression of AP-1 (c-Jun/c-Fos), a downstream target of JNK and ERK, repressed CYP7A1 expression. In DNA pull-down and chromatin immunoprecipitation assays, AP-1 exhibited sequence-specific binding to the proximal CYP7A1 promoter region overlapping the HNF4α binding site, and atRA increased AP-1 but decreased HNF4α recruitment to the promoter. Collectively, these results indicate that atRA activates JNK and ERK pathways and the downstream target AP-1 represses HNF4α transactivation of the CYP7A1 promoter, potentially responsible for hypercholesterolemia.