The effects of carvacrol on oxidative stress, inflammation, and liver function indicators in a systemic inflammation model induced by lipopolysaccharide in rats.

The effect of carvacrol (CAR) on oxidative stress, inflammation, and liver dysfunction induced by lipopolysaccharide (LPS) was explored. The rats (n=40) were daily injected (2 weeks) by saline as control, LPS (1 mg/kg, i.p.), and 25, 50 or 100 mg/kg CAR (i.p.) before LPS. LPS increased aspartate transaminase (AST: 162±13 U/L), alanine aminotransferase (ALT: 74.6±2.15 U/L), alkaline phosphatase (ALK-P: 811±51 U/L), interlukine-1ß (IL-1ß: 1254±51 pg/g tissue), malondialdehyde (MDA: 32±1.09 nM/g tissue), and nitric oxide (NO: 224±13.5 nM/g tissue) (P<0.01-P<0.001) while, decreased total protein(4.08±0.38 g/dl), albumin(2.79±0.16 g/dl), thiol (5.16±0.19 µM/g tissue), superoxide dismutase (SOD: 10.57±0.13 U/g tissue), and catalase (CAT: 0.78±0.02 U/g tissue) compared to control (P<0.001). CAR reversed the effects of LPS (P<0.05-P<0.001). In the rats treated by 100 mg/kg CAR, the indicators were as follows: AST: 118±10.1 U/L, ALT: 42.5±4.13 U/L, ALK-P: 597±39.91 U/L, IL-1ß: 494±15 pg/g tissue, and NO: 141±5.35 nM/g tissue. Both 50 and 100 mg/kg CAR corrected oxidative stress indicators and in the group treated by 100 mg/kg CAR, they were: MDA: 23.4±0.91 nM/g tissue, thiol: 7.98±0.18 µM/g tissue, SOD: 21±0.8 U/g tissue, and CAT: 1.12±0.02 U/g tissue(P<0.05-P<0.001). In conclusion, CAR improved liver function, accompanied with antioxidant and antiinflammatory effects.

Quercetin increases mitochondrial proteins (VDAC and SDH) and downmodulates AXL and PIM-1 tyrosine kinase receptors in NRAS melanoma cells.

Melanoma is a type of skin cancer with low survival rates after it has metastasized. In order to find molecular differences that could represent targets of quercetin in anti-melanoma activity, we have chosen SKMEL-103 and SKMEL-28 melanoma cells and human melanocytes as models. Firstly, we observed that quercetin was able in reducing SKMEL-103 cell viability, but not in SKMEL-28. Besides that, quercetin treatment caused inhibition of AXL in both cell lines, but upregulation of PIM-1 in SKMEL-28 and downregulation in SKMEL-103. Moreover, HIF-1 alpha expression decreased in both cell lines. Interestingly, quercetin was more effective against SKMEL-103 than kinases inhibitors, such as Imatinib, Temsirolimus, U0126, and Erlotinib. Interestingly, we observed that while the levels of succinate dehydrogenase and voltage-dependent anion channel increased in SKMEL-103, both proteins were downregulated in SKMEL-28 after quercetin's treatment. Furthermore, AKT, AXL, PIM-1, ABL kinases were much more active and chaperones HSP90, HSP70 and GAPDH were highly expressed in SKMEL-103 cells in comparison with melanocytes. Our findings indicate, for the first time, that the efficacy of quercetin to kill melanoma cells depends on its ability in inhibiting tyrosine kinase and upregulating mitochondrial proteins, at least when SKMEL-103 and SKMEL-28 cells response were compared.

In vitro and clinical investigations to determine the drug-drug interaction potential of entrectinib, a small molecule inhibitor of neurotrophic tyrosine receptor kinase (NTRK).

Background Entrectinib is a CNS-active, potent inhibitor of tyrosine receptor kinases A/B/C, ROS1 and anaplastic lymphoma kinase approved for use in patients with solid tumors. We describe the in vitro and clinical studies investigating potential entrectinib drug-drug interactions. Methods In vitro studies with human biomaterials assessed the enzymes involved in entrectinib metabolism, and whether entrectinib modulates the activity of the major cytochrome P450 (CYP) enzymes or drug transporter P-glycoprotein. Clinical studies investigated the effect of a strong CYP3A4 inhibitor (itraconazole) and inducer (rifampin) on single-dose entrectinib pharmacokinetics. The effect of entrectinib on sensitive probe substrates for CYP3A4 (midazolam) and P-glycoprotein (digoxin) were also investigated. Results Entrectinib is primarily metabolized by CYP3A4. In vitro, entrectinib is a CYP3A4/5 inhibitor (IC50 2 µM) and a weak CYP3A4 inducer. Entrectinib inhibited P-glycoprotein (IC50 1.33 µM) but is a poor substrate. In healthy subjects, itraconazole increased entrectinib Cmax and AUC by 73% and 504%, respectively, and rifampin decreased entrectinib Cmax and AUC by 56% and 77%, respectively. Single dose entrectinib did not affect midazolam AUC, although Cmax decreased by 34%. Multiple dose entrectinib increased midazolam AUC by 50% and decreased Cmax by 21%. Single dose entrectinib increased digoxin AUC and Cmax by 18% and 28%, respectively, but did not affect digoxin renal clearance. Conclusions Entrectinib is a CYP3A4 substrate and is sensitive to the effects of coadministered moderate/strong CYP3A4 inhibitors and strong inducers, and requires dose adjustment. Entrectinib is a weak inhibitor of CYP3A4 and P-glycoprotein and no dose adjustments are required with CYP3A4/P- glycoprotein substrates.Registration Number (Study 2) NCT03330990 (first posted online November 6, 2017) As studies 1 and 3 are phase 1 trials in healthy subjects, they are not required to be registered.

1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-(2-(dimethylamino)ethyl)imidazolidin-2-one (ZX-42) inhibits cell proliferation and induces apoptosis via inhibiting ALK and its downstream pathways in Karpas299 cells.

Anaplastic lymphoma kinase (ALK) belongs to the family of receptor tyrosine kinases. Recently, the incidence of anaplastic large cell lymphoma (ALCL) with ALK rearrangement has raised considerably. The application of ALK-targeted inhibitors such as ceritinib provides an effective therapy for the treatment of ALK-positive cancers. However, with the prolongation of treatment time, the emergence of resistance is inevitable. We found that 1-(4-((5-chloro-4-((2-(isopropylsulfonyl)phenyl)amino)pyrimidin-2-yl)amino)-3-methoxyphenyl)-3-(2-(dimethylamino)ethyl)imidazolidin-2-one (ZX-42), a novel ceritinib derivative, could inhibit the proliferation of ALK-positive ALCL cells, induce the apoptosis of Karpas299 cells through the mitochondrial pathway in a caspase-dependent manner. In addition, ZX-42 could suppress ALK and downstream pathways including PI3K/Akt, Erk and JAK3/STAT3 and reduce the nuclear translocation of NFκB by inhibiting TRAF2/IKK/IκB pathway. Taken together, our findings indicate that ZX-42 shows more effective activity than ceritinib against ALK-positive ALCL. We hope this study can provide a direction for the structural modification of ceritinib and lay the foundation for the further development of clinical research in ALK-positive ALCL.

Soluble TAM receptor tyrosine kinases in rheumatoid arthritis: correlation with disease activity and bone destruction.

The TAM receptor tyrosine kinases (TAM RTK) are a subfamily of receptor tyrosine kinases, the role of which in autoimmune diseases such as systemic lupus erythematosus has been well explored, while their functions in rheumatoid arthritis (RA) remain largely unknown. In this study, we investigated the role of soluble TAM receptor tyrosine kinases (sAxl/sMer/sTyro3) in patients with RA. A total of 306 RA patients, 100 osteoarthritis (OA) patients and 120 healthy controls (HCs) were enrolled into this study. The serum concentrations of sAxl/sMer/sTyro3 were measured by enzyme-linked immunosorbent assay (ELISA), then the associations between sAxl/sMer/sTyro3 levels and clinical features of RA patients were analysed. We also investigated whether sTyro3 could promote osteoclast differentiation in vitro in RA patients. The results showed that compared with healthy controls (HCs), sTyro3 levels in the serum of RA patients were elevated remarkably and sMer levels were decreased significantly, whereas there was no difference between HCs and RA patients on sAxl levels. The sTyro3 levels were correlated weakly but positively with white blood cells (WBC), immunoglobulin (Ig)M, rheumatoid factor (RF), swollen joint counts, tender joint counts, total sharp scores and joint erosion scores. Conversely, there were no significant correlations between sMer levels and the above indices. Moreover, RA patients with high disease activity also showed higher sTyro3 levels. In-vitro osteoclast differentiation assay showed further that tartrate-resistant acid phosphatase (TRAP)+ osteoclasts were increased significantly in the presence of sTyro3. Collectively, our study indicated that serum sTyro3 levels were elevated in RA patients and correlated positively with disease activity and bone destruction, which may serve as an important participant in RA pathogenesis.

Culture methods of diffuse intrinsic pontine glioma cells determine response to targeted therapies.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive type of brainstem cancer occurring mainly in children, for which there currently is no effective therapy. Current efforts to develop novel therapeutics for this tumor make use of primary cultures of DIPG cells, maintained either as adherent monolayer in serum containing medium, or as neurospheres in serum-free medium. In this manuscript, we demonstrate that the response of DIPG cells to targeted therapies in vitro is mainly determined by the culture conditions. We show that particular culture conditions induce the activation of different receptor tyrosine kinases and signal transduction pathways, as well as major changes in gene expression profiles of DIPG cells in culture. These differences correlate strongly with the observed discrepancies in response to targeted therapies of DIPG cells cultured as either adherent monolayers or neurospheres. With this research, we provide an argument for the concurrent use of both culture conditions to avoid false positive and false negative results due to the chosen method.

An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis.

Aberrant signaling through the Axl receptor tyrosine kinase has been associated with a myriad of human diseases, most notably metastatic cancer, identifying Axl and its ligand Gas6 as important therapeutic targets. Using rational and combinatorial approaches, we engineered an Axl 'decoy receptor' that binds Gas6 with high affinity and inhibits its function, offering an alternative approach from drug discovery efforts that directly target Axl. Four mutations within this high-affinity Axl variant caused structural alterations in side chains across the Gas6-Axl binding interface, stabilizing a conformational change on Gas6. When reformatted as an Fc fusion, the engineered decoy receptor bound Gas6 with femtomolar affinity, an 80-fold improvement compared to binding of the wild-type Axl receptor, allowing effective sequestration of Gas6 and specific abrogation of Axl signaling. Moreover, increased Gas6 binding affinity was critical and correlative with the ability of decoy receptors to potently inhibit metastasis and disease progression in vivo.

Tumor angiogenesis is caused by single melanoma cells in a manner dependent on reactive oxygen species and NF-κB.

Melanomas have a high angiogenic potential, but respond poorly to medical treatment and metastasize very early. To understand the early events in tumor angiogenesis, animal models with high tumor resolution and blood vessel resolution are required, which provide the opportunity to test the ability of small molecule inhibitors to modulate the angiogenic tumor program. We have established a transgenic melanoma angiogenesis model in the small laboratory fish species Japanese medaka. Here, pigment cells are transformed by an oncogenic receptor tyrosine kinase in fish expressing GFP throughout their vasculature. We show that angiogenesis occurs in a reactive oxygen species (ROS)- and NF-κB-dependent, but hypoxia-independent manner. Intriguingly, we observed that blood vessel sprouting is induced even by single transformed pigment cells. The oncogenic receptor as well as human melanoma cells harboring other oncogenes caused the production of pro-angiogenic factors, most prominently angiogenin, through NF-κB signaling. Inhibiting NF-κB prevented tumor angiogenesis and led to the regression of existing tumor blood vessels. In conclusion, our high-resolution medaka melanoma model discloses that ROS and NF-κB signaling from single tumor cells causes hypoxia-independent angiogenesis, thus, demonstrating that the intrinsic malignant tumor cell features are sufficient to initiate and maintain a pro-angiogenic signaling threshold.

TAS0612, a Novel RSK, AKT, and S6K Inhibitor, Exhibits Antitumor Effects in Preclinical Tumor Models.

The MAPK and PI3K pathways are involved in cancer growth and survival; however, the clinical efficacy of single inhibitors of each pathway is limited or transient owing to resistance mechanisms, such as feedback signaling and/or reexpression of receptor-type tyrosine kinases (RTK). This study identified a potent and novel kinase inhibitor, TAS0612, and characterized its properties. We found that TAS0612 is a potent, orally available compound that can inhibit p90RSK (RSK), AKT, and p70S6K (S6K) as a single agent and showed a strong correlation with the growth inhibition of cancer cells with PTEN loss or mutations, regardless of the presence of KRAS and BRAF mutations. Additional RSK inhibitory activity may differentiate the sensitivity profile of TAS0612 from that of signaling inhibitors that target only the PI3K pathway. Moreover, TAS0612 demonstrated broad-spectrum activity against tumor models wherein inhibition of MAPK or PI3K pathways was insufficient to exert antitumor effects. TAS0612 exhibited a stronger growth-inhibitory activity against the cancer cell lines and tumor models with dysregulated signaling with the genetic abnormalities described above than treatment with inhibitors against AKT, PI3K, MEK, BRAF, and EGFR/HER2. In addition, TAS0612 demonstrated the persistence of blockade of downstream growth and antiapoptotic signals, despite activation of upstream effectors in the signaling pathway and FoxO-dependent reexpression of HER3. In conclusion, TAS0612 with RSK/AKT/S6K inhibitory activity may provide a novel therapeutic strategy for patients with cancer to improve clinical responses and overcome resistance mechanisms.

Angiopoietin-1 promotes endothelial differentiation from embryonic stem cells and induced pluripotent stem cells.

Angiopoietin-1 (Ang1) plays a crucial role in vascular and hematopoietic development, mainly through its cognate receptor Tie2. However, little is known about the precise role of Ang1 in embryonic stem cell (ESC) differentiation. In the present study, we used COMP-Ang1 (a soluble and potent variant of Ang1) to explore the effect of Ang1 on endothelial and hematopoietic differentiation of mouse ESCs in an OP9 coculture system and found that Ang1 promoted endothelial cell (EC) differentiation from Flk-1(+) mesodermal precursors. This effect mainly occurred through Tie2 signaling and was altered in the presence of soluble Tie2-Fc. We accounted for this Ang1-induced expansion of ECs as enhanced proliferation and survival. Ang1 also had an effect on CD41(+) cells, transient precursors that can differentiate into both endothelial and hematopoietic lineages. Intriguingly, Ang1 induced the preferential differentiation of CD41(+) cells toward ECs instead of hematopoietic cells. This EC expansion promoted by Ang1 was also recapitulated in induced pluripotent stem cells (iPSCs) and human ESCs. We successfully achieved in vivo neovascularization in mice by transplantation of ECs obtained from Ang1-stimulated ESCs. We conclude that Ang1/Tie2 signaling has a pivotal role in ESC-EC differentiation and that this effect can be exploited to expand EC populations.

Molecular profiling of TAM tyrosine kinase receptors and ligands in endometrial carcinoma: An in silico-study.

OBJECTIVES: TAM Receptors (TYRO3, AXL, and MerTK) and their ligands on tumor-associated macrophages are promising therapeutic targets for most solid cancers. However, in endometrial cancer, the most common invasive gynecologic malignancy, the TAM receptor-mediated activation pathway, its molecular mechanisms, and its pathophysiology are unknown. The goal of this research; to uncover the comprehensive genetic profile of TAM receptors and ligands in endometrial cancer. MATERIAL AND METHODS: Mutation and expression profiles of the Uterine Corpus Endometrial Carcinoma (UCEC) cohort (n = 509) were obtained using bioinformatics tools providing data from The Cancer Genome Atlas (TCGA). PolyPhen-2 and SNAP tools were used to predict the oncogenic/pathogenic properties of the identified mutations for UCEC. STRING network analysis was performed to better understand the functional relationships of the mutant proteins in cellular processes. Furthermore to the mutation profile, gene expression and survival profiles were also determined. Finally, the correlation between target genes and macrophage infiltration was investigated using the tool TIMER. RESULTS: A total of 229 mutations were detected in 6 genes, and 81 missense mutations are pathogenic. In the UCEC cohort, the expression level of MerTK, AXL, GAS6, and PROS1 was statistically significantly lower in the patient group, while the expression level of CD47 was higher in the patient group than in the healthy group (p < 0.01). Protein-protein interaction analysis identified target genes, SRC protein responsible for important cellular mechanisms such as cell proliferation, adhesion and migration, ITGB3, ITGAV and THSB1 proteins involved in endothelial mesenchymal transition and tumor metabolism reprogramming, and FOLR1 involved in DNA replication and damage repair. CONCLUSION: We believe that TAM receptors and their ligands may be attractive molecular targets for the treatment of endometrial carcinoma because they act as pleiotropic inhibitors of immune cells, effectively regulate phagocytic clearance of apoptotic cells, and make the tumor microenvironment a more suitable niche for the tumour.

MiR-140-3p directly targets Tyro3 to regulate OGD/R-induced neuronal injury through the PI3K/Akt pathway.

BACKGROUND AND PURPOSE: MicroRNAs (miRNAs) are highly expressed in the central nervous system and play important roles in ischaemic stroke pathogenesis. However, the role of miRNAs in cerebral ischaemia-reperfusion injury remains unclear. Here, we investigated the role of miR-140-3p in regulating oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal injury in vitro to identify a new biomarker for research on ischaemic stroke. METHODS: The differential expression of miR-140-3p and Tyro3 in OGD/R-exposed N2a cells was verified by qRT-PCR. N2a cells were transfected with miR-140-3p mimic, miR-140-3p inhibitor, Tyro3 or siTyro3, and qRT-PCR, Western blotting, the Cell counting kit-8 (CCK-8) assay, Hoechst 33342/PI staining and flow cytometry analyses were performed to measure miRNA, mRNA and protein expression; cell viability; and apoptosis. RESULTS: OGD/R-exposed N2a cells exhibited increased miR-140-3p expression, decreased viability, reduced Bcl-2 protein expression and increased Bax and Caspase-3 protein expression and apoptosis; the miR-140-3p mimic markedly amplified these changes, exacerbating OGD/R-induced injury to N2a cells, while the miR-140-3p inhibitor reversed these changes and alleviated OGD/R-induced injury. OGD/R-exposed N2a cells expressed less Tyro3, and Tyro3 overexpression increased cell viability and Bcl-2 protein expression, reduced Bax and Caspase-3 protein expression, and alleviated OGD/R-induced injury. However, silencing Tyro3 reversed these changes and exacerbated OGD/R-induced injury. MiR-140-3p directly bound the Tyro3 mRNA 3'UTR. Rescue experiments indicated that the miR-140-3p mimic-induced changes in cell viability and protein expression were alleviated by Tyro3 overexpression and that the miR-140-3p inhibitor-induced changes in cell viability and protein expression were alleviated by silencing Tyro3. Tyro3 overexpression increased cell viability and PI3K and p-Akt protein expression, but these effects were weakened by the addition of LY294002. CONCLUSIONS: MiR-140-3p directly targets Tyro3 to regulate cell viability and apoptosis of OGD/R-exposed N2a cells through the PI3K/Akt pathway, suggesting that miR-140-3p is a novel biomarker and therapeutic target for ischaemic stroke.

The total alkaloids of Sophora alopecuroides L. improve depression-like behavior in mice via BDNF-mediated AKT/mTOR signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Depression has become a global public health problem and the development of new highly effective, low-toxicity antidepressants is imminent. Sophora alopecuroides L. is a common medicinal plant, which has therapeutic effect on central nervous system diseases. AIM OF THE STUDY: In this study, the antidepressant effect of total alkaloids (ALK) isolated from Sophora alopecuroides L. was explored and the mechanism was further elucidated. MATERIALS AND METHODS: A primary neuronal injury model was established in vitro by corticosterone. ICR mice were then selected to construct an in vivo model of chronic unpredictable mild stress (CUMS)-induced depression, and the ameliorative effects of ALK on depression were examined by various behavioral tests. The antidepressant molecular mechanism of ALK was subsequently revealed by ELISA, Western blot, immunohistochemistry and Golgi staining. RESULTS: BDNF secretion as well as TrkB and ERK phosphorylated protein levels were found to be improved in primary cortical neurons, along with improved dendritic complexity of neurons. The results of in vivo showed that the depression-like behavior of CUMS-induced mice was reversed after 2 weeks of continuous gavage administration of ALK, and the neurotransmitter levels in the plasma of mice were increased. Moreover, the expression levels of key proteins of BDNF-AKT-mTOR pathway and the complexity of neuronal dendrites were improved in the prefrontal cortex of mice. CONCLUSIONS: These findings indicate that ALK of Sophora alopecuroides L. can effectively improve the depressive phenotype of mice, possibly by promoting the expression of BDNF in prefrontal cortex, activating the downstream AKT/mTOR signal pathway, and ultimately enhancing neuronal dendritic complexity.

Discovery of Small-Molecule Inhibitors of the PTK7/ß-Catenin Interaction Targeting the Wnt Signaling Pathway in Colorectal Cancer.

Colorectal cancer (CRC), the second cause of death due to cancer worldwide, is a major public health issue. The discovery of new therapeutic targets is thus essential. Pseudokinase PTK7 intervenes in the regulation of the Wnt/ß-catenin pathway signaling, in part, through a kinase domain-dependent interaction with the ß-catenin protein. PTK7 is overexpressed in CRC, an event associated with metastatic development and reduced survival of nonmetastatic patients. In addition, numerous alterations have been identified in CRC inducing constitutive activation of the Wnt/ß-catenin pathway signaling through ß-catenin accumulation. Thus, targeting the PTK7/ß-catenin interaction could be of interest for future drug development. We have developed a NanoBRET screening assay recapitulating the interaction between PTK7 and ß-catenin to identify compounds able to disrupt this protein-protein interaction. A high-throughput screening allowed us to identify small-molecule inhibitors targeting the Wnt pathway signaling and inducing antiproliferative and antitumor effects in vitro in CRC cells harboring ß-catenin or adenomatous polyposis coli (APC) mutations. Thus, inhibition of the PTK7/ß-catenin interaction could represent a new therapeutic strategy to inhibit cell growth dependent on the Wnt signaling pathway. Moreover, despite a lack of enzymatic activity of its tyrosine kinase domain, targeting the PTK7 kinase domain-dependent functions appears to be of interest for further therapeutic development.

Excessive toxicity of cabozantinib in a phase II study in patients with recurrent and/or metastatic salivary gland cancer.

AIM: Because the tyrosine kinases c-MET and vascular endothelial growth factor receptors (VEGFR) are often overexpressed in salivary gland cancer (SGC), this study evaluated the efficacy and safety of cabozantinib in patients with recurrent/metastatic (R/M) SGC. PATIENTS AND METHODS: A single-centre phase II study was conducted. Patients with immunohistochemical c-MET-positive R/M SGC were included in three cohorts: adenoid cystic carcinoma (ACC); salivary duct carcinoma (SDC) and other miscellaneous SGCs. No prior systemic treatments were required. Patients started cabozantinib 60 mg once daily. The primary outcome was the objective response rate (ORR). Secondary outcomes included survival, safety and quality of life. Per Simon-two-stage design, depending on efficacy, a maximum of 43 patients would be included. RESULTS: In total, 25 patients were included until premature closure owing to severe toxicity. Six patients (24%) had grade 3-5 wound complications, occurring at a median of 7.1 months on cabozantinib treatment (range 2.1-12.6). Remarkably, four of these six patients developed this complication in the area prior exposed to high-dose radiotherapy. Other grade ≥3 adverse events in >1 patient were hypertension (20%), diarrhoea (8%) and dehydration (8%). Twenty-one patients were evaluable for response; 1/15 ACC (ORR: 7%); 1/4 SDC and 0/2 patients with other miscellaneous SGC responded. Median progression-free survival was 9.4 months (95% confidence interval [CI] 7.4-11.4 months), 7.2 months (95%CI 0.0-15.1) and 6.9 months (95%CI 0.0-15.1), respectively. CONCLUSION: This study showed too many severe cabozantinib-associated wound complications in patients with SGC, especially in prior irradiated areas. Therefore, the study closed prematurely. The efficacy in the limited number of evaluable patients was low to moderate. TRIAL REGISTRATION: This trial was registered on ClinicalTrials.gov: NCT03729297.

Alkannin reverses lipopolysaccharides-induced inflammatory responses by suppressing mitogen-activated protein kinase and nuclear factor kappa-B signalling.

Rheumatoid arthritis (RA) is an inflammatory disease that seriously affects human health worldwide. Meanwhile, inflammation in RAW264.7 cells could lead to the progression of RA. Alkannin (ALK) is derived from Alkanna tinctoria and is known to exert anti-tumor effects. However, the function of ALK in inflammation of RAW264.7 cells remains unclear. Thus, this research sought to investigate the detailed function of ALK in inflammatory responses of RAW264.7 cells. To induce an inflammatory response, RAW264.7 cells were exposed to lipopolysaccharide (LPS). MTT assay was applied to examine cell viability. Enzyme-linked immunosorbent assay (ELISA) was used to assess the levels of inflammatory cytokines. Furthermore, the mechanism underlying ALK function in inflammatory responses was investigated using RT-qPCR and western blotting. The data revealed that LPS significantly increased the expression of cyclooxygenase 2 (COX-2), Interleukin (IL)-1ß, inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), and IL-6, whereas ALK reversed this effect. ALK also restored LPS-induced nuclear factor kappa-B (NF-κB) activation by inhibiting the downregulation of p-inhibitor kappa B alpha (IκBα). LPS elevated p-extracellular regulated protein kinases 1/2 (ERK1/2), phosphorylated p38 (p-p38), and phosphorylated -c-Jun N-terminal kinase (p-JNK) levels, which were markedly decreased in the presence of ALK. In summary, Alkannin attenuated LPS-induced inflammation by inhibiting NF-κB and MAPK signaling. Thus, our research might provide a new theoretical basis for exploring new strategies against RA.

PM2.5 induce the defective efferocytosis and promote atherosclerosis via HIF-1α activation in macrophage.

Epidemiological studies demonstrate that fine particulate matter (PM2.5) promotes the development of atherosclerosis. However, the mechanism insight of PM2.5-induced atherosclerosis is still lacking. The aim of this study was to explore the biological effects of hypoxia-inducible factor 1α (HIF-1α) on PM2.5-triggered atherosclerosis. The vascular stiffness, carotid intima-media thickness (CIMT), lipid and atherosclerotic lesion were increased when von Hippel-Lindau (VHL)-null mice were exposed to PM2.5. Yet, knockout of HIF-1α markedly decreased the PM2.5-triggered atherosclerotic lesion. We firstly performed microarray analysis in PM2.5-treated bone morrow-derived macrophages (BMDMs), which showed that PM2.5 significantly changed the genes expression patterns and affected biological processes such as phagocytosis, apoptotic cell clearance, cellular response to hypoxia, apoptotic process and inflammatory response. Moreover, the data showed knockout of HIF-1α remarkably relieved PM2.5-induced defective efferocytosis. Mechanistically, PM2.5 inhibited the level of genes and proteins of efferocytosis receptor c-Mer tyrosine kinase (MerTK), especially in VHL-null BMDMs. In addition, PM2.5 increased the genes and proteins of a disintegrin and metallopeptidase domain 17 (ADAM17), which caused the MerTK cleavage to form soluble MerTK (sMer) in plasma and cellular supernatant. The sMer was significantly up-regulated in plasma of VHL-null PM2.5-exposed mice. Moreover, PM2.5 could induce defective efferocytosis and activate inflammatory response through MerTK/IFNAR1/STAT1 signaling pathway in macrophages. Our results demonstrate that PM2.5 could induce defective efferocytosis and inflammation by activating HIF-1α in macrophages, ultimately resulting in accelerating atherosclerotic lesion formation and development. Our data suggest HIF-1α in macrophages might be a potential target for PM2.5-related atherosclerosis.

AXL Inhibitor TP-0903 Reduces Metastasis and Therapy Resistance in Pancreatic Cancer.

Pancreatic cancer is the third leading cause of cancer-related deaths in the United States with a 5-year survival less than 5%. Resistance to standard therapy and limited response to immune checkpoint blockade due to the immunosuppressive and stroma-rich microenvironment remain major challenges in the treatment of pancreatic cancer. A key cellular program involved in therapy resistance is epithelial plasticity, which is also associated with invasion, metastasis, and evasion of immune surveillance. The receptor tyrosine kinase AXL is a key driver of tumor cell epithelial plasticity. High expression and activity of AXL is associated with poor prognosis, metastasis, and therapy resistance in multiple types of cancer including pancreatic. Here, we show that an AXL inhibitor (TP-0903), has antitumor and therapy sensitizing effects in preclinical models of pancreatic ductal adenocarcinoma (PDA). We demonstrate that TP-0903 as a single agent or in combination with gemcitabine and/or anti-programmed cell death protein 1 (PD1) antibody has anti-metastatic and anti-tumor effects in PDA tumor bearing mice, leading to increased survival. In addition, gene expression analysis of tumors demonstrated upregulation of pro-inflammatory and immune activation genes in tumors from TP-0903-treated animals compared with the vehicle, indicating pharmacologic inhibition of AXL activation leads to an immunostimulatory microenvironment. This effect was augmented when TP-0903 was combined with gemcitabine and anti-PD1 antibody. These results provide clear rationale for evaluating TP-0903 in the treatment of pancreatic cancer.

Proteomic and Phosphoproteomic Analyses Reveal the Oncogenic Role of PTK7-NDRG1 Axis in Non-small-cell Lung Cancer Cell Resistance to AZD9291.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the most important chemotherapeutics for non-small-cell lung cancer (NSCLC) therapy. The resistance to EGFR-TKIs is one of the biggest obstacles to NSCLC outcome. In this study, taking advantage of phospho- and proximal proteomic techniques, we analyzed the network rearrangement in cell lines responding to AZD9291 treatment and found that cell-cell adhesion was dramatically enhanced in AZD9291-resistant cells. Further analysis revealed that protein tyrosine kinase 7 (PTK7) expression was significantly elevated. Knockdown or overexpression assays showed that PTK7 played a critical role in improving cell adhesion, which enhanced drug resistance. Because PTK7 is a membrane-localized pseudokinase, the proximal labeling probe BirA* was fused to reveal PTK7-interacting proteins. We found that PTK7 interacted with and stabilized NDRG1, which is located predominantly adjacent to adherens junctions. Downregulation of PTK7 or NDRG1 eliminated the resistance of H1975-resistant (H1975-R) and PC9-resistant (PC9-R) cells to AZD9291, suggesting that the PTK7-NDRG1 axis might be a potential target to eliminate the EGFR-TKI resistance during NSCLC therapy.