Search

Lung cancer is a disease with increasing morbidity worldwide in recent years. Approaches such as chemotherapy and biological targeting for its treatment are urgently needed. Epithelial-mesenchymal transition (EMT) is an important initiation stage for tumor cells to acquire invasive and metastatic abilities. Increasing findings have shown that human schlafen family member 5 (SLFN5) plays a key role in malignant tumors. However, the role of SLFN5 in lung cancer cells is not completely elucidated yet. In this study, overexpression or knockdown of SLFN5 gene were induced by lentiviral transfection in human lung cancer cell line A549, then the EMT of A549 was detected by green fluorescent protein labeling method, the migrative and invasive abilities were evaluated via transwell and wound-healing tests in vitro and chick chorioallantoic membrane inoculation in vivo, and the possible mechanism was studied by quantitative real-time PCR and Western blotting. Our results demonstrated that overexpression of SLFN5 promoted the morphology transformation of A549 from epithelial to mesenchymal, as well as migration and invasion. However, knockdown of SLFN5 resulted in the opposite results. Moreover, with the development of EMT after SLFN5 was overexpressed, A549 exhibited enhanced translocation of β-catenin from membrane to cytoplasm or nucleus, with higher level of EMT-related transcription factor Snail, and lower expression of adhesin E-cadherin. Together these results suggest that SLFN5 may act as a synergist in lung cancer cell tumorigenesis and progression, providing a potential target for developing drugs for lung cancer therapy in future., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Some neuroprotective agents have been used clinically to address the resulting various adverse effects after intracerebral hemorrhage (ICH). Particularly, effectively removing the hematoma is of practical significance to exert neuroprotective effects following ICH. However, such agents are still in need of development. Lithium chloride (LiCl) has shown neuroprotective effects through glycogen synthase kinase-3β (GSK-3β) inhibition in a variety of central nervous system diseases. However, the impact of LiCl on hematoma clearance and the potential molecular mechanisms have not been reported. We hypothesize that LiCl may exert neuroprotective roles after ICH, partly through promoting hematoma resolution. In this study, male Sprague-Dawley rats were subjected to ICH followed by intraperitoneal injection of LiCl (60 mg/kg). The hematoma volumes of ipsilateral hemisphere were determined using Drabkin's method. The sensorimotor deficits were evaluated by neurobehavioral tests. The expressions of target molecules involved in the process of hematoma clearance were assayed using immunofluorescence and Western blot. Our results showed that animals treated with LiCl presented significantly reduced hematoma volume after ICH, which was coupled with enhanced microglia phagocytosis and its differentiation into M2-phenotype within the first 7 days and up-regulated angiogenesis and neurogenesis in the next 7 days. Meanwhile, GSK-3β was inhibited by LiCl and β-catenin became stabilized, which was followed by up-regulation of nuclear factor erythroid 2-related factor 2 and CD36 from days 3 to 7, and increase of vascular endothelial growth factor and brain-derived neurotrophic factor from days 7 to 14. These data suggest that LiCl promotes hematoma resolution via enhancing microglia phagocytosis and M2-phenotype differentiation in the early stage (< 7 days) and angiogenesis and neurogenesis in the chronic phase (days 7-14), thus eventually improving the functional outcomes of ICH rats., (Copyright © 2019. Published by Elsevier Inc.)

To the clinical cognitive impairment following intracerebral hemorrhage, comprehensive neuropsychological assessments and efficacious interventions have rarely been conducted. Lithium chloride, a classical treatment for bipolar disorder, has shown neuroprotective effects through glycogen synthase kinase-3β inhibition in a variety of central nervous system diseases, including stroke. Since neurons that contain glutamate play crucial roles in psychological functions, such as learning and memory, the glutamate-mediated excitotoxicity and consequent neuronal death and cognitive impairment in hippocampus may co-determine the clinical course of intracerebral hemorrhage. However, the potential molecular mechanisms have rarely been demonstrated in intracerebral hemorrhage researches. In this study, Male Sprague-Dawley rats, subjected to intrastriatal blood infusion, were treated with lithium chloride and underwent neurobehavioral test for equivalent injury severity and neurological functional deficits, Morris water maze test for cognitive impairment, high performance liquid chromatography analysis for excitotoxic index determination, immunohistochemistry analysis for neuronal apoptosis, and Western blot analysis for glycogen synthase kinase-3β activity. Our results showed lithium chloride inhibited glycogen synthase kinase-3β activation, which on one hand, suppressed downstream CRMP-2/NR2B, thus diminishing the excitotoxic index level; and on the other, stabilized β-catenin, thus modulating its downstream apoptosis-related factors such as NF-κB, Bcl-2 and Bax. Meanwhile, glycogen synthase kinase-3β inactivation was paralleled by decreased neuronal death, improved neurological functional deficits and ameliorated cognitive deficits in intracerebral hemorrhage animals. These findings indicate that lithium chloride improves glutamate-mediated excitotoxicity-induced cognitive deficits after intracerebral hemorrhage and that lithium chloride might be a potential therapeutic agent for brain damages caused by intracerebral hemorrhage., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Vasogenic cerebral edema formation after blood-brain barrier (BBB) damage aggravates the devastating consequences of intracerebral hemorrhage (ICH). The present study aims to probe into a therapeutic method on BBB preservation after ICH with a glycogen synthase kinase-3β (GSK-3β) inhibitor, lithium. Intrastriatal infusion of semicoagulated autologous whole blood or sham surgery was performed on male Sprague-Dawley (SD) rats (n = 208). Experimental animals received administration of 4,6-disubstitutedpyrrolo-pyrimidine (TWS119), lithium alone or in combination with a phosphatidylinositol 3-kinase inhibitor, wortmannin, after ICH. Behavioral tests, brain edema, and BBB permeability were determined at 24 and 72 h after surgery. Expressions of Akt, GSK-3β, β-catenin, claudin-1 and claudin-3 were evaluated via Western blots. Our results showed lithium alone posttreatment activated GSK-3β, therefore increasing active β-catenin and claudin-1 and claudin-3 expressions, which were accompanied with improved BBB integrity and ameliorated sensorimotor deficits and brain edema in ICH animals. We concluded that lithium alone reduced BBB damage after ICH, likely through regulating Akt/GSK-3β pathway and stabilizing β-catenin., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Overexpression of P-glycoprotein (P-gp) is one of the major problems to successful cancer chemotherapy. To find novel effective P-gp inhibitors, a series of bifendate-chalcone hybrids were synthesized and evaluated. Among them, the most active compound 8g had little intrinsic cytotoxicity (IC(50)>200 μM), and could increase accumulation of Rhodamine 123 in K562/A02 cells more potently than bifendate and verapamil (VRP) by inhibiting P-gp efflux function. And 8g displayed potent chemo-sensitizing effect and persisted for much longer time (>24h) compared with VRP (<6h). In addition, 8g, unlike VRP, showed no stimulation on the P-gp ATPase activity, suggesting it is not a P-gp substrate. Therefore, 8g may represent a promising lead to develop MDR reversal agents for cancer chemotherapy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Hypoxia initiated pulmonary vasoconstriction is due to the inhibition of voltage-gated K(+) (K(V)) channels. But the mechanism is unclear. We have evidence that hypoxia activates 15-lipoxygenase (15-LOX) in distal pulmonary arteries and increases the formation of 15-hydroxyeicosatetraenoate (15-HETE). 15-HETE-induced pulmonary artery constriction to be through the inhibition of K(V) channels (K(V)1.5, K(V)2.1 and K(V)3.4). However, no direct link among hypoxia, 15-HETE and inhibition of K(V) subtypes is established. Therefore, we investigated whether 15-LOX/15-HETE pathway contributes to the hypoxia-induced down-regulation of K(V) channels. As K(V)1.5 channel is O(2)-sensitive, it was chosen in the initial study. We found that inhibition of 15-LOX suppressed the response of hypoxic pulmonary artery rings to phenylephrine. The expressions of K(V)1.5 channel mRNA and protein was robustly up-regulated in cultured PASMC and pulmonary artery after blocking of 15-LOX by lipoxygenase inhibitors in hypoxia. The 15-LOX blockade also partly rescued the voltage-gated K(+) current (I(K(V))). 15-HETE contributes to the down-regulation of K(V)1.5 channel, inhibition of I(K(V)) and increase of native pulmonary artery tension after hypoxia. Hypoxia inhibits K(V)1.5 channel through 15-LOX/15-HETE pathway.

We have previously reported that 15-hydroxyeicosatetraenoic acid (15-HETE), a metabolite of arachidonic acid by 15-lipoxygenase, causes pulmonary vasoconstriction via increasing the intracellular Ca(2+) concentration ([Ca(2+)]i). However, the multiple sources of Ca(2+) that contribute to Ca(2+) elevation during and after 15-HETE exposure have not been investigated. In the present study, pulmonary arterial ring technique and confocal laser scanning microscope were used to investigate the origin of Ca(2+). 15-HETE (1 microM) elicited an increase in [Ca(2+)]i in pulmonary artery smooth muscle cells in a time-dependent manner under both normal and hypoxic condition. The increases were composed of an initial rapid rise followed by a slow increase in the present of extracellular Ca(2+). The initial rapid phase was attenuated by inositol 1,4,5-triphosphate (IP(3)) receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) and ryanodine receptor-operated Ca(2+) store depletion agent caffeine; the slow increasing phase and the constriction of pulmonary arterial ring were significantly inhibited by voltage-operated Ca(2+) channel blocker nifedipine or transient receptor potential canonical (TRPC) channel blocker La(3+), and almost completely diminished in Ca(2+)-free external solution, suggesting that the initial phase depends on intracellular Ca(2+) store and the second phase relies on extracellular Ca(2+). Interestingly, the effect of caffeine and La(3+) but not nifedipine were diminished in the present of 2-APB. Thus, these results suggest that 15-HETE mobilizes Ca(2+) signaling through: 1) Ca(2+) release immediately from Ca(2+) stores via activation of IP(3) receptor and, subsequently that of ryanodine receptor, 2) the depletion of Ca(2+) through CCE leading to the activation of TRPC, and 3) Ca(2+) entry through L-type Ca(2+) channels.

20-Hydroxyeicosatetraenoic acid (20-HETE), a omega-hydroxylation product of arachidonic acid catalyzed by cytochrome P450 4A (CYP4A), plays a role in vascular smooth muscle remodeling. Although its effects on angiogenic responses are known, it remains unclear whether 20-HETE acts on apoptosis of pulmonary arterial smooth muscle cells (PASMC), an important step in PASMC remodeling, and what pathways are involved in the process. Here we show evidence for the missing information. The effect of 20-HETE on PASMC apoptosis and the apoptosis-associated signaling pathways were determined with cell viability assay, Annexin V and propidium idodide binding, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), mitochondrial potentials assay, caspase activity assay and Western blots. We found that exogenous 20-HETE suppressed the serum deprivation-induced loss of bovine PASMCs and prevented Annexin V binding, DNA nick end labeling and chromatin condensation. The effect was worsened by 17-octadecynoic acid (17-ODYA), which inhibited the production of endogenous 20-HETE. Furthermore, 20-HETE induced the expression of bcl-2, maintained the stability of mitochondria membrane, and relieved the activation of caspase-9 and caspase-3. Such effects were reversed in the presence of 17-ODYA. Thus, these findings indicate that 20-HETE protects PASMCs against apoptosis by acting on, at least in part, the intrinsic apoptotic pathway.

We have previously reported that subacute hypoxia activates lung 15-lipoxygenase (15-LOX), which catalyzes arachidonic acid to produce 15-HETE, leading to constriction of neonatal rabbit pulmonary arteries. Subacute hypoxia suppresses Kv3.4 channel expression and results in an inhibition of whole-cell K(+) currents (I(K)). Although the Kv channel inhibition is likely to be mediated through 15-HETE, direct evidence is still lacking. To reveal the role of the 15-LOX/15-HETE pathway in the hypoxia-induced down-regulation of Kv3.4 channel expression and inhibition of I(K), we performed studies using 15-LOX blockers, whole-cell patch-clamp, semi-quantitative PCR, ELISA and Western blot analysis. We found that Kv3.4 channel expression at the mRNA and protein levels was greatly up-regulated in pulmonary arterial smooth muscle cells after blockade of 15-LOX by CDC or NDGA. The 15-LOX blockade also partially restored I(K). In comparison, 15-HETE had a stronger effect than 12-HETE on the expression of Kv3.4 channels. 5-HETE had no noticeable effect on Kv3.4 channel expression. These data indicate that the 15-LOX pathway via its metabolite, 15-HETE, seems to play a role in the down-regulation of Kv3.4 expression and I(K) inhibition after subacute hypoxia.

The aim of the study was to investigate the influence of sodium ozagrel on CYP2D6 (cytochromeP450 2D6) activity. The studies were performed with rat urine and liver microsomes and chemical inhibitors. The metabolism of dextromethorphan (dextrophan/dextromethorphan, dextrophan is a metabolite of dextromethorphan) and phenacetin (paracetamol/phenacetin, paracetamol is a metabolites of phenacetin) was used as probe to measure CYP2D6 and CYP1A2 (cytochromeP450 1A2) activity, respectively, determined by high-performance liquid chromatography (HPLC). The results showed that the metabolism of dextrophan/dextromethorphan in the sodium ozagrel-treated group (37 mg/kg) was higher than that of the control (P<0.05/6) in both in vivo and in vitro studies (r=0.9811). The rate of dextromethorphan metabolism was inhibited by sodium ozagrel and cimetidine in rat liver microsomes prepared from sodium ozagrel-treated rats and control rats group (sodium ozagrel IC(50)=26.5 microM, cimetidine IC(50)=86.3 microM in sodium ozagrel-treated group; sodium ozagrel IC(50)=13.9 microM, cimetidine IC(50)=24.8 microM in control group). The inhibitory effect of sodium ozagrel on CYP2D6 activity was noncompetitive with dextromethorphan with a K(i) of 324.94 microM. Kinetic parameters of the reactions were established by using Lineweaver-Burk with K(m)=0.67 mM and V(max)=2.13 pm/min/mg protein for the sodium ozagrel-treated group and K(m)=0.29 mM, and V(max)=0.91 pm/min/mg protein for the control group, respectively. The expression of CYP2D6 protein in the treated group was higher than that of the control group, as determined by Western blotting. The activity and expression of CYP1A2 did not show obvious differences in the control group and sodium ozagrel treated group. In conclusion, sodium ozagrel metabolism in rats is mediated primarily through CYP2D6, and sodium ozagrel can induce CYP2D6 activity.

The aim of the present study was to investigate the contribution of extracellular signal regulated kinase-1/2 (ERK1/2) to pulmonary artery contraction in response to 4-aminopyridione (4-AP), an inhibitor of a voltage-gated K(+) channels that regulate pulmonary vascular tone. Pulmonary artery rings 1-1.5 mm in diameter from male adult Wistar rat were isolated and cut into 3-mm in length. ERK1/2 up-stream kinase (MEK) inhibitors 2'-amino-3'-methoxyflavone (PD98059) and 1,4-diamino-2,3-dicyano-1,4-bis (2-aminophenylthio)-butadiene (U0126), which block the activation of ERK1/2, were used to test the role of ERK1/2 in 4-AP induced pulmonary arterial vasoconstriction and the influences of 4-AP on expressions of phosphorylated ERK1/2 (p-ERK1/2) in cultured rat pulmonary arterial smooth muscle cells (PASMCs) and whole tissues. Our results show that 4-AP elicited concentration-dependent increases in tension of rat pulmonary artery rings, effects that were reduced by pretreatment of the rings with ERK inhibitors, PD98059 (20 microM) and U0126 (10 microM). Moreover, 4-AP increased the expressions of p-ERK1/2 in cultured PASMCs s and whole tissues, which were prevented by pretreatment of the cells or tissues with U0126. These results indicate that ERK1/2 signaling pathway contributes to pulmonary vasoconstriction induced by 4-AP.