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Objective To investigate the effect of albumin-bound paclitaxel (ab-PTX) on programmed death ligand 1（PD-L1) expression in lung squamous cell carcinoma H520 cells and its possible mechanism of action. Methods 1 Determination of ab-PTX concentration and duration of effect used in this study： H520 cells were cultured with 0. 001，0. 01，0. 1，1, and 10 μmol/L ab-PTX for 24，36, and 48 h. The cell proliferation inhibition rate was calculated, and finally, the 48 h with the most significant cell proliferation inhibition and its corresponding ab-PTX half maximal inhibitory concentration (IC50) value (1. 221 μmol/L) were selected for subsequent experiments. 2 Observation of PD-L1 expression in H520 cells after administration of different concentrations of ab-PTX in culture： lung squamous carcinoma H520 cells were cultured in vitro with 0，0. 001，0. 01，0. 1，1, and 10 μmol/L ab-PTX in culture medium for 48 h (based on the duration of ab-PTX action determined in 1）, and PD-L1 positive cell rate was measured by flow cytometry and PD-L1 mRNA was detected by qRT-PCR. 3 Observation of extracellular-regulated protein kinase (ERK) and p-ERK expression after administration of ab-PTX, ERK inhibitor alone or in combination in culture： the H520 cells were divided into the Control group, ab-PTX group, PD98059（ERK inhibitor) group, TPA (ERK activator) group, ab-PTX combined with PD98059 group, ab-PTX combined with TPA group; cells in the ab-PTX group were added with 1. 221 μmol/L ab-PTX (based on the ab-PTX concentration determined in 1）; in the PD98059 group，50 μmol/L PD98059 was added; in the TPA group, 50 nmol/L TPA was added; in the ab-PTX combined with PD98059 group，1. 221 μmol/L ab-PTX, and 50 μmol/L PD98059 were added; in the ab-PTX combined with TPA group，1. 221 μmol/L ab-PTX and 50 nmol/L TPA were added, and cells were cultured for 48 h. The PD-L1 positive cell rate was measured by flow cytometry, and ERK and p-ERK were detected by Western blotting. Results 1 PD-L1 expression in H520 cells after different concentrations of ab-PTX culture： The PD-L1 positive cell rates of H520 cell line cultured with 0，0. 001，0. 01，0. 1，1, and 10 μmol/L ab-PTX were 33. 9%±1. 1%，39. 5%±2. 7%，52. 6%±3. 3%，60. 3%±3. 4%，69. 9%±2. 3%, and 77. 8%±2. 2%, respectively, and the relative expression levels of PD-L1 mRNA were 1. 06±0. 07，1. 65±0. 22，2. 12±0. 16，3. 25±0. 11，3. 79±0. 14, and 4. 45±0. 28, respectively; with the increase of ab-PTX concentration, the PD-L1 positive cell rate and the PD-L1 mRNA relative expression gradually increased (all P<0. 05）. 2 Expression of ERK, p-ERK, and PD-L1 positive cell rate after the administration of ab-PTX, ERK inhibitor alone or in combination in culture： Compared with the Control group, the relative expression of p-ERK increased in the ab-PTX group, decreased in the PD98059 group and increased in the TPA group; the relative expression of p-ERK decreased in the ab-PTX combined with PD98059 group compared with the ab-PTX group and increased in the PD98059 group compared with the ab-PTX group; the relative expression of p-ERK was elevated in the abPTX combined with TPA group compared with the ab-PTX group and the TPA group (all P<0. 05). Compared with the Control group, PD-L1 positive cell rate increased in the ab-PTX group; compared with the ab-PTX group, the PD-L1 positive cell rate in the ab-PTX combined with PD98059 group decreased; compared with the PD98059 group, PD-L1 positive cell rate in the ab-PTX combined with PD98059 group increased; compared with the ab-PTX group and the TPA group, PD-L1 positive cell rate in the ab-PTX combined with TPA group increased (all P<0. 05). Conclusion Ab-PTX can upregulate PD-L1 expression in a dose-dependent manner within a certain range. The mechanism of action of ab-PTX up-regulating PD-L1 expression may be partly related to the activation of ERK signaling pathway. [ABSTRACT FROM AUTHOR]

Curcumin (CUR) has a range of therapeutic benefits against cancers, but its poor solubility and low bioavailability limit its clinical use. Demethoxycurcumin (DMC) and diphenyl difluoroketone (EF-24) are natural and synthetic curcumin analogues, respectively, with better solubilities and higher anti-carcinogenic activities in various solid tumors than CUR. However, the efficacy of these analogues against non-solid tumors, particularly in acute myeloid leukemia (AML), has not been fully investigated. Herein, we observed that both DMC and EF-24 significantly decrease the proportion of viable AML cells including HL-60, U937, and MV4-11, harboring different NRAS and Fms-like tyrosine kinase 3 (FLT3) statuses, and that EF-24 has a lower half maximal inhibitory concentration (IC50) than DMC. We found that EF-24 treatment induces several features of apoptosis, including an increase in the sub-G1 population, phosphatidylserine (PS) externalization, and significant activation of extrinsic proapoptotic signaling such as caspase-8 and -3 activation. Mechanistically, p38 mitogen-activated protein kinase (MAPK) activation is critical for EF-24-triggered apoptosis via activating protein phosphatase 2A (PP2A) to attenuate extracellular-regulated protein kinase (ERK) activities in HL-60 AML cells. In the clinic, patients with AML expressing high level of PP2A have the most favorable prognoses compared to various solid tumors. Taken together, our results indicate that EF-24 is a potential therapeutic agent for treating AML, especially for cancer types that lose the function of the PP2A tumor suppressor.

Brain-derived neurotrophic factor (BDNF) is a regulator for the formation and maintenance of chronic pain in various chronic disorders and has been shown to increase in the serum of women with endometriosis. However, BDNF expression in the peritoneal fluid (PF) and ectopic lesions and its role in endometriosis pain remain unclear. Thus, this study aims to determine the BDNF concentrations in serum and PFs and BDNF expression levels in ectopic lesions and endometriotic stromal cells (ESCs) of women with endometriosis (n = 60). The obtained results were then compared with those of women without endometriosis (n = 38). Brain-derived neurotrophic factor concentrations in serum and PF, as well as the BDNF expression levels in ectopic lesions and endometriotic cells, were evaluated through enzyme-linked immunosorbent assay, immunohistochemical staining, quantitative real-time polymerase chain reaction, and Western blot analysis. As a result, BDNF concentrations in serum and PF were significantly higher in women with endometriosis with pain (2284.3 ± 51.5 pg/mL, n = 23; 58.8 ± 6.4 pg/mL, n = 16) than in women with endometriosis without pain (1999.8 ± 61.1 pg/mL, n = 37; 31.7 ± 2.9 pg/mL, n = 25; P < .01). Moreover, BDNF messenger RNA (mRNA) expression levels in ectopic lesions (8.97 ± 1.44, n = 29) were significantly higher than eutopic (0.97 ± 0.14, n = 16; P < .01) and control endometrium (1.23 ± 0.19, n = 18; P < .01) and were correlated with endometriosis pain (P < .05). Furthermore, increased BDNF mRNA and protein expression levels in ESCs induced by estradiol or interleukin 1β were removed using a phosphorylated extracellular-regulated protein kinase 1/2 inhibitor. These results suggest that BDNF may play an important role in the pathogenesis of endometriosis pain. [ABSTRACT FROM AUTHOR]

• PD rat model was successfully established by injection of 6-OHDA and showed obvious learning and memory impairments; • Reduced extracellular-regulated protein kinase expression may be associated with. • learning and memory impairments in PD rat model; • BMSCs transplantation ameliorates learning and memory deficits in 6-OHDA induced PD rat model, and its combination treatment with ERK has a synergistic effect; • This supports the potential of targeting ERK-BMSCs for better therapy of learning and memory impairments in PD rat model. The study aimed to investigate bone marrow mesenchymal stem cells (BMSCs) and extracellular signal-regulated kinase (ERK) gene-modified BMSCs (ERK-BMSCs) transplantation in ameliorating cognitive deficits in Parkinson's disease (PD). The PD rat model was built by 6-hydroxydopamine (6-OHDA) injection into the right striatum for 8 weeks, then successful PD rats were randomly divided into three groups and respectively transplanted in the same position of striatum as modeling with PBS, BMSCs and ERK-BMSCs for another 8 weeks. The 6-OHDA-induced PD rat model was successfully established, as demonstrated by reduced active avoidance response (AAR) times, percentage of time exploring in the light area (Ltime%) and platform quadrant time (PQT), as well as p-ERK expression. Compared with PBS rats, both BMSCs and ERK-BMSCs transplantation significantly reduced the left turn number, while increased AAR, Ltime%, PQT and p-ERK expression, suggesting improved cognitive abilities through restoring p-ERK expression. In addition, ERK-BMSCs injection exhibited higher therapeutic efficacy against cognitive deficits compared with BMSCs injection. These results demonstrated that BMSCs transplantation ameliorated cognitive deficits, and ERK-BMSCs exerted synergistic effects, which may prove beneficial against cognitive impairments in PD. [ABSTRACT FROM AUTHOR]

Neuropeptide Y binds to G-protein coupled receptors whose action results in inhibition of adenylyl cyclase activity. Using HEK293 cells stably expressing the native neuropeptide Y Y 1 receptors, we found that the NPY agonist elicits a transient phosphorylation of the extracellular signal-regulated kinases (ERK1/2). We first show that ERK1/2 activation following Y 1 receptor stimulation is dependent on heterotrimeric G i/o since it is completely inhibited by pre-treatment with pertussis toxin. In addition, ERK1/2 activation is internalization-independent since mutant Y 1 receptors unable to recruit β-arrestins, can still activate ERK signaling to the same extent as wild-type receptors. We next show that this activation of the MAPK pathway is inhibited by the MEK inhibitor U0126, is not dependent on calcium signaling at the Y 1 receptor (no effect upon inhibition of phospholipase C, protein kinase C or protein kinase D) but instead dependent on Gβ/γ and associated signaling pathways that activate PI3-kinase. Although inhibition of the epidermal-growth factor receptor tyrosine kinase did not influence NPY-induced ERK1/2 activation, we show that the inhibition of insulin growth factor receptor IGFR by AG1024 completely blocks activation of ERK1/2 by the Y 1 receptor. This Gβ/γ-PI3K-AG1024-sensitive pathway does not involve activation of IGFR through the release of a soluble ligand by metalloproteinases since it is not affected by the metalloproteinase inhibitor marimastat. Finally, we found that a similar pathway, sensitive to wortmannin-AG1024 but insensitive to marimastat, is implicated in activation of ERK signaling in HEK293 cells by endogenously expressed GPCRs coupled to Gq-protein (muscarinic M3 receptors) or coupled to Gs-protein (endothelin ETB receptors). Our analysis is the first to show that β-arrestin recruitment to the NPY Y 1 receptor is not necessary for MAPK activation by this receptor but that transactivation of the IGFR receptor is required. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Chronic hyperglycaemia promotes the progressive failure of pancreatic beta cells in patients with type 2 diabetes mellitus, a clinically highly relevant phenomenon known as glucotoxicity. The intracellular metabolic consequences of a chronically high availability of glucose in beta cells are, as yet, poorly understood in its full complexity. Methods: An unbiased metabolite profiling analysis (GC-time-of-flight-MS) was used to identify the time course of core metabolite patterns in rat beta cell line INS-1E during exposure to high glucose concentrations and its relation to insulin expression. Results: We report here that pentose phosphate pathway (PPP) metabolites accumulate remarkably during chronic but not acute glucose treatment, indicating altered processing of glucose through the pentose phosphate pathway. Subsequent functional studies in INS-1E cells and human islets revealed that a disturbance in this pathway contributes to decreases in insulin gene expression and a lack of glucose-stimulated insulin secretion. These effects were found to depend on the activation of extracellular-regulated-kinase (ERK1/2). Long-term inhibition of 6-phosphogluconic acid dehydrogenase resulted in accumulation of PPP metabolites, induced ERK1/2 activation independently of high glucose and impaired beta cell function. In turn, inhibition of ERK1/2 overstimulation during chronic glucose exposure partly inhibited metabolite accumulation and restored beta cell function. Conclusions/interpretation: Based on unbiased metabolite analyses, the data presented here provide novel targets, namely the inhibition of PPP metabolite accumulation towards the therapeutic goal to preserve and potentially improve beta cell function in diabetes. [ABSTRACT FROM AUTHOR]

Atherosclerosis, which occurs prematurely in individuals with diabetes, incorporates vascular smooth muscle cell (VSMC) chemotaxis. Glucose, through protein kinase C-βII signalling, increases chemotaxis to low concentrations of platelet-derived growth factor (PDGF)-BB. In VSMC, a biphasic response in PDGF-beta receptor (PDGF-βR) level occurs as PDGF-BB concentrations increase. The purpose of this study was to determine whether increased concentrations of PDGF-BB and raised glucose level had a modulatory effect on the mitogen-activated protein kinase/extracellular-regulated protein kinase pathway, control of PDGF-βR level and chemotaxis. Cultured aortic VSMC, exposed to normal glucose (NG) (5 mmol/l) or high glucose (HG) (25 mmol/l) in the presence of PDGF-BB, were assessed for migration (chemotaxis chamber) or else extracted and immunoblotted. At concentrations of PDGF-BB <540 pmol/l, HG caused an increase in the level of PDGF-βR in VSMC (immunoblotting) versus NG, an effect that was abrogated by inhibition of aldose reductase or protein kinase C-βII. At higher concentrations of PDGF-BB (>540 pmol/l) in HG, receptor level was reduced but in the presence of aldose reductase or protein kinase C-βII inhibitors the receptor levels increased. It is known that phosphatases may be activated at high concentrations of growth factors. At high concentrations of PDGF-BB, the protein phosphatase (PP)2A inhibitor, endothall, caused an increase in PDGF-βR levels and a loss of biphasicity in receptor levels in HG. At higher concentrations of PDGF-BB in HG, the chemoattractant effect of PDGF-BB was lost (chemotaxis chamber). Under these conditions inhibition of PP2A was associated with a restoration of chemotaxis to high concentrations of PDGF-BB. The biphasic response in PDGF-βR level and in chemotaxis to PDGF-BB in HG is due to PP2A activation. [ABSTRACT FROM AUTHOR]

The present study examined in subcellular fractions from rat brain the nature and sensitivity to hydrogen peroxide of constitutively expressed mitogen-activated protein kinase (MAPK) phosphatase activity. MAPK phosphatase activity was defined as the activity directed towards a dual-phosphorylated (pT/pY) peptide corresponding to the activation domain of the extracellular-regulated kinase (ERK) subtype of the MAPKs. The use of phosphatase inhibitors and biochemical analyses demonstrate that the MAPK phosphatase activity, which was highest in the microsomal membrane and soluble fractions, was attributable mainly, if not entirely, to protein phosphatase 2A (PP2A). Moreover, hydrogen peroxide (in the absence and presence of reduced glutathione) and glutathione disulfide inhibited the MAPK phosphatase activity by a dithiothreitol-reversible mechanism. These results provide direct support for mounting evidence that PP2A is a major regulator of MAPK phosphorylation in brain and suggest that inhibition of PP2A activity via reversible oxidation of a cysteine thiol(s) may underlie at least in part the activation of MAPKs occurring in response to hydrogen peroxide and oxidative stress. [Copyright &y& Elsevier]

Curcumin (CUR) has a range of therapeutic benefits against cancers, but its poor solubility and low bioavailability limit its clinical use. Demethoxycurcumin (DMC) and diphenyl difluoroketone (EF-24) are natural and synthetic curcumin analogues, respectively, with better solubilities and higher anti-carcinogenic activities in various solid tumors than CUR. However, the efficacy of these analogues against non-solid tumors, particularly in acute myeloid leukemia (AML), has not been fully investigated. Herein, we observed that both DMC and EF-24 significantly decrease the proportion of viable AML cells including HL-60, U937, and MV4-11, harboring different NRAS and Fms-like tyrosine kinase 3 (FLT3) statuses, and that EF-24 has a lower half maximal inhibitory concentration (IC50) than DMC. We found that EF-24 treatment induces several features of apoptosis, including an increase in the sub-G1 population, phosphatidylserine (PS) externalization, and significant activation of extrinsic proapoptotic signaling such as caspase-8 and -3 activation. Mechanistically, p38 mitogen-activated protein kinase (MAPK) activation is critical for EF-24-triggered apoptosis via activating protein phosphatase 2A (PP2A) to attenuate extracellular-regulated protein kinase (ERK) activities in HL-60 AML cells. In the clinic, patients with AML expressing high level of PP2A have the most favorable prognoses compared to various solid tumors. Taken together, our results indicate that EF-24 is a potential therapeutic agent for treating AML, especially for cancer types that lose the function of the PP2A tumor suppressor. [ABSTRACT FROM AUTHOR]