The Aldose Reductase Inhibitor Epalrestat Maintains Blood-Brain Barrier Integrity by Enhancing Endothelial Cell Function during Cerebral Ischemia.

Excessive activation of aldose reductase (AR) in the brain is a risk factor for aggravating cerebral ischemia injury. Epalrestat is the only AR inhibitor with proven safety and efficacy, which is used in the clinical treatment of diabetic neuropathy. However, the molecular mechanisms underlying the neuroprotection of epalrestat remain unknown in the ischemic brain. Recent studies have found that blood-brain barrier (BBB) damage was mainly caused by increased apoptosis and autophagy of brain microvascular endothelial cells (BMVECs) and decreased expression of tight junction proteins. Thus, we hypothesized that the protective effect of epalrestat is mainly related to regulating the survival of BMVECs and tight junction protein levels after cerebral ischemia. To test this hypothesis, a mouse model of cerebral ischemia was established by permanent middle cerebral artery ligation (pMCAL), and the mice were treated with epalrestat or saline as a control. Epalrestat reduced the ischemic volume, enhanced BBB function, and improved the neurobehavior after cerebral ischemia. In vitro studies revealed that epalrestat increased the expression of tight junction proteins, and reduced the levels of cleaved-caspase3 and LC3 proteins in mouse BMVECs (bEnd.3 cells) exposed to oxygen-glucose deprivation (OGD). In addition, bicalutamide (an AKT inhibitor) and rapamycin (an mTOR inhibitor) increased the epalrestat-induced reduction in apoptosis and autophagy related protein levels in bEnd.3 cells with OGD treatment. Our findings suggest that epalrestat improves BBB function, which may be accomplished by reducing AR activation, promoting tight junction proteins expression, and upregulating AKT/mTOR signaling pathway to inhibit apoptosis and autophagy in BMVECs.

Metformin-induced AMPK activation suppresses larval growth and molting probably by disrupting 20E synthesis and glycometabolism in fall webworm, Hyphantria cunea Drury.

Metformin, considered to be a potent AMPK activator, is widely used for clinical therapy of cancer and diabetes due to its distinct function in regulating cell energy balance and body metabolism. However, the effect of metformin-induced AMPK activation on the growth and development of insects remains largely unknown. In the present study, we focused on the role of metformin in regulating the growth and development of Hyphantria cunea, a notorious defoliator in the forestry. Firstly, we obtained the complete coding sequences of HcAMPKα2, HcAMPKß1, HcAMPKγ2 from H. cunea, which encoded a protein of 512, 281, and 680 amino acids respectively. Furthermore, the phylogenetic analysis revealed that these three subunits were highly homologous with the AMPK subunits from other lepidopteran species. According to the bioassay, we found metformin remarkably restrained the growth and development of H. cunea larvae, and caused molting delayed and body weight reduced. In addition, expressions of HcAMPKα2, HcAMPKß1, and HcAMPKγ2 were upregulated 3.30-, 5.93- and 5.92-folds at 24 h after treatment, confirming that metformin activated AMPK signaling at the transcriptional level in H. cunea larvae. Conversely, the expressions of two vital Halloween genes (HcCYP306A1 and HcCYP314A1) in the 20E synthesis pathway were remarkably suppressed by metformin. Thus, we presumed that metformin delayed larval molting probably by impeding 20E synthesis in the H. cunea larvae. Finally, we found that metformin accelerated glycogen breakdown, elevated in vivo trehalose level, promoted chitin synthesis, and upregulated transcriptions of the genes in chitin synthesis pathway. Taken together, the findings provide a new insight into the molecular mechanisms by which AMPK regulates carbohydrate metabolism and chitin synthesis in insects.

3-Bromopyruvate-induced glycolysis inhibition impacts larval growth and development and carbohydrate homeostasis in fall webworm, Hyphantria cunea Drury.

As a typical glycolytic inhibitor, 3-bromopyruvate (3-BrPA) has been extensively studied in cancer therapy in recent decades. However, few studies focused on 3-BrPA in regulating the growth and development of insects, and the relationship and regulatory mechanism between glycolysis and chitin biosynthesis remain largely unknown. The Hyphantria cunea, named fall webworm, is a notorious defoliator, which caused a huge economic loss to agriculture and forestry. Here, we investigated the effects of 3-BrPA on the growth and development, glycolysis, carbohydrate homeostasis, as well as chitin synthesis in H. cunea larvae. To elucidate the action mechanism of 3-BrPA on H. cunea will provide a new insight for the control of this pest. The results showed that 3-BrPA dramatically restrained the growth and development of H. cunea larvae and resulted in larval lethality. Meanwhile, we confirmed that 3-BrPA caused a significant decrease in carbohydrate, adenosine triphosphate (ATP), pyruvic acid (PA), and triglyceride (TG) levels by inhibiting glycolysis in H. cunea larvae. Further studies indicated that 3-BrPA significantly affected the activities of hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), glucose 6-phosphate dehydrogenase (G6PDH) and trehalase, as well as expressions of the genes related to glycolysis, resulting in carbohydrate homeostasis disorder. Moreover, it was found that 3-BrPA enhanced 20-hydroxyecdysone (20E) signaling by upregulating HcCYP306A1 and HcCYP314A1, two critical genes in 20E synthesis pathway, and accelerated chitin synthesis by upregulating transcriptional levels of genes in the chitin synthesis pathway in H. cunea larvae. Taken together, our findings provide a novel insight into the mechanism of glycolytic inhibitor in regulating the growth and development of insects, and lay a foundation for the potential application of glycolytic inhibitors in pest control as well.

The effect of nano-TiO2 photocatalysis on the antioxidant activities of Cu, Zn-SOD at physiological pH.

Security issues of nanoparticles on biological toxicity and potential environmental risk have attracted more and more attention with the rapid development and wide applications of nanotechnology. In this work, we explored the effect and probable mechanism of nano-TiO2 on antioxidant activity of copper, zinc superoxide dismutase (Cu, Zn-SOD) under natural light and mixed light at physiological pH. Nano-TiO2 was prepared by sol-hydrothermal method, and then characterized by X-ray Diffraction (XRD) and Transmission electron micrographs (TEM). The Cu, Zn-SOD was purified by sephadex G75 chromatography and qualitatively analyzed by sodium dodecyl sulfate polypropylene amide gel electrophoresis (SDS-PAGE). The effect and mechanism were elucidated base on Fourier Transform Infrared Spectrometer (FT-IR), Circular Dichroism (CD), zeta potential, and electron spin resonance (ESR) methods. Accompanying the results of FT-IR, CD and zeta potential, it could be concluded that nano-TiO2 had no effect on the antioxidant activity of Cu, Zn-SOD by comparing the relative activity under natural light at physiological pH. But the relative activity of Cu, Zn-SOD significantly decreased along with the increase of nano-TiO2 concentration under the mixed light. The results of ESR showed the cause of this phenomenon was the Cu(II) in the active site of Cu, Zn-SOD was reduced to Cu(I) by H2O2 and decreased the content of active Cu, Zn-SOD. The reduction can be inhibited by catalase. Excess O2·- produced by nano-TiO2 photocatalysis under mixed light accumulated a mass of H2O2 through disproportionation reaction in this experimental condition. The results show that nano-TiO2 cannot affect the antioxidant activity of Cu, Zn-SOD in daily life. The study on the effect of nano-TiO2 on Cu, Zn-SOD will provide a valid theory support for biological safety and the toxicological effect mechanism of nanomaterials on enzyme.

Activation of the adenosine A2A receptor attenuates experimental autoimmune encephalomyelitis and is associated with increased intracellular calcium levels.

Multiple sclerosis (MS) is a common autoimmune disease that inevitably causes inflammatory nerve demyelination. However, an effective approach to prevent its course is still lacking and urgently needed. Recently, the adenosine A2A receptor (A2AR) has emerged as a novel inflammation regulator. Manipulation of A2AR activity may suppress the MS process and protect against nerve damage. To test this hypothesis, we treated murine experimental autoimmune encephalomyelitis (EAE), a model for MS, with the selective A2AR agonist, CGS21680 (CGS). We evaluated the effects of CGS on the pathological features of EAE progression, including CNS cellular infiltration, inflammatory cytokine expression, lymphocyte proliferation, and cell surface markers. Treatment with CGS significantly suppressed specific lymphocyte proliferation, reduced infiltration of CD4(+) T lymphocytes, and attenuated the expression of inflammatory cytokines, which in turn inhibited the EAE progression. For the first time, we demonstrate that CGS can increase the intracellular calcium concentration ([Ca(2+)]i) in murine lymphocytes, which may be the mechanism underlying the suppressive effects of CGS-induced A2AR activation on EAE progression. Our findings strongly suggest that A2AR is a potential therapeutic target for MS and provide insight into the mechanism of action of A2AR agonists, which may offer a therapeutic option for this disease.

Excess salt exacerbates blood-brain barrier disruption via a p38/MAPK/SGK1-dependent pathway in permanent cerebral ischemia.

High salt diet (HSD) is one of the most important risk factors that contribute to many vascular diseases including ischemic stroke. One proposed mechanism underlying the disruption of blood-brain barrier (BBB) mediated by HSD is indirectly through enhancing blood pressure. The direct role of HSD on BBB integrity is unclear. Our purpose is to determine whether and how HSD might be involved in BBB breakdown during ischemia. To test that, we induced model of cerebral ischemia by permanent middle cerebral artery ligation (pMCAL) in either normal diet or HSD fed mice. We observed that HSD significantly enhanced ischemic brain damage which was associated with enhanced BBB disruption, increased leukocytes infiltration and loss of tight junction (TJ) proteins expression without apparently altering blood pressure. Our in vitro experiment also revealed that sodium chloride (NaCl) treatment down-regulated TJ protein expression by endothelial cells and substantially increased BBB permeability during starvation. Inhibition of p38/MAPK/SGK1 pathway eliminated the effect of NaCl on BBB permeability in vitro. In addition, we noticed a positive correlation between urinary sodium levels and ischemic lesion size in stroke patients. Together, our study demonstrates a hypertension-independent role of HSD during ischemia and provides rationale for post cerebral ischemic attack management.

Sox2 is involved in paclitaxel resistance of the prostate cancer cell line PC-3 via the PI3K/Akt pathway.

Prostate cancer is the most commonly diagnosed type of cancer and the second leading cause of cancer­associated mortality in males. The efficacy of prostate cancer chemotherapy is frequently impaired by drug resistance; however, the underlying mechanisms of this resistance remain elusive. Sex determining region Y-box 2 (Sox2) is of vital importance in the regulation of stem cell proliferation and carcinogenesis. In the present study, using MTT, clone formation, cell cycle and apoptosis assays, over-expression of Sox2 was demonstrated to enhance the paclitaxel (Pac) resistance of the PC-3 prostate cancer cell line, promoting cell proliferation and exhibiting an anti­apoptotic effect. Western blot analysis revealed that the phosphoinositide 3-kinase/Akt signaling pathway was activated in cells overexpressing Sox2, and by targeting cyclin E and survivin, Sox2 promoted G1/S phase transition and prevented apoptosis under Pac treatment. The present study provided an understanding of Pac resistance in prostate cancer and may indicate novel therapeutic methods for chemoresistant prostate cancer.

Hypoxia increases CX3CR1 expression via HIF-1 and NF­κB in androgen-independent prostate cancer cells.

The unique CX3C chemokine CX3CL1 and its cognate receptor CX3CR1 have been implicated in organ-specific metastasis of various types of tumors. Hypoxia, a common phenomenon in solid tumors, is associated with a malignant cancer phenotype. Previous studies have proved that hypoxia facilitates cancer cell metastasis through upregulation of specific chemokine receptors. We hypothesized that hypoxia could upregulate CX3CR1 expression and lead to an increased chemotactic response to CX3CL1 in prostate cancer cells. In the present study, we found that CX3CR1 expression was significantly increased in androgen-independent prostate cancer cells, including DU145, PC-3 and PC-3M, following exposure to hypoxia. This upregulation of CX3CR1 corresponded to a significant increase in migration and invasion of prostate cancer cells under hypoxic conditions, which was attenuated after knocking down CX3CR1 expression. In addition, we examined the possible role of HIF-1 and NF-κB in the process of hypoxia-induced CX3CR1 expression and hypoxia-mediated metastasis. Attenuation of HIF-1 and NF-κB transcriptional activity by siRNAs or pharmacological inhibitors, abrogated hypoxia-induced upregulation of CX3CR1, and also prevented the migration and invasion of DU145 cells under a hypoxic environment. In summary, our study demonstrated that HIF-1 and NF-κB are essential for hypoxia-regulated CX3CR1 expression, which is associated with increased migratory and invasive potential of prostate cancer cells. CX3CR1 signaling is a potential therapeutic target in the adjuvant treatment of prostate cancer.

[Inhibitory effect of siRNA targeting ADAM17 on the proliferation of prostate cancer PC-3 cells].

OBJECTIVE: To study the effect of siRNA targeting ADAM17 (ADAM17-siRNA) on the proliferation of prostate cancer PC-3 cells. METHODS: After transfecting PC-3 cells with ADAM17-siRNA 1 and ADAM17-siRNA 2, we detected the expressions of ADAM17 mRNA and protein by RT- PCR and Western blotting, respectively. We measured the changes in the proliferation and DNA synthesis of PC-3 cells by MTT and bromodeoxyuridine (BrdU) incorporation assay, examined the cell cycle profile by flow cytometry, and determined the expressions of the genes associated with PC-3 cell proliferation by Western blotting. RESULTS: Both ADAM17-siRNA 1 and 2 effectively reduced the expressions of ADAM17 mRNA and protein in the PC-3 cells. Knockdown of ADAM17 with the two siRNAs significantly inhibited cell proliferation as compared with the control group (0.43 +/- 0.57 and 0.44 +/- 0.64 vs 0.80 +/- 0.51, P < 0.05) and down-regulated DNA synthesis (0.48 +/- 0.43 and 0.54 +/- 0.59 vs 0.79 +/- 0.72, P < 0.05). The cell cycle profile showed that the cell population of the G1 phase was markedly higher in both the ADAM17-siRNA groups than in the control ([61.83 +/- 2.41]% and [59.78 +/- 1.92]% vs [41.38 +/- 1.53]%, P < 0.05), but that of the S phase remarkably lower in the former two than in the latter ([23.64 +/- 2.56]% and [25.24 +/- 1.86]% vs [33.51 +/- 1.47]%, P < 0.05), with a concomitant decrease in the expression of the cell cycle protein cyclin D1 and increase in the cyclin-dependent kinase inhibitor p21. CONCLUSION: ADAM17-siRNA can effectively inhibit the proliferation of PC-3 cells by up-regulating cyclin D1 and down-regulating p21 protein, and ADAM17 has a potential value in the gene therapy of prostate cancer.

ADAM17 regulates prostate cancer cell proliferation through mediating cell cycle progression by EGFR/PI3K/AKT pathway.

A disintegrin and metalloprotease 17 (ADAM17) is a transmembrane protein that can cleave membrane anchored proteins to release soluble factors and regulate important biological phenomena in cancers. In the present study, we evaluated the effects of ADAM17 on the proliferation and on the cell cycle distribution of human prostate cancer cells. Experiments were also performed to gain insights into the possible mechanism of action of ADAM17. We used over-expression and RNAi strategy to investigate the function of ADAM17 in human prostate cancer cells. Changes in rate of proliferation and cell cycle profile were measured by growth curve, Cell Counting Kit-8 (CCK-8) assay, bromodeoxyuridine (BrdU) incorporation assay and cell cycle analysis. In addition, changes in expression of associated genes and proteins were studied by semiquantitative RT-PCR, western blotting and ELISA analysis. Ectopic over-expression of ADAM17 resulted in increased cell proliferation. We also showed that ADAM17 promoted G1 to S phase transition concomitantly with upregulation of cyclin E, CDK2 and downregulation of p21 and p27 proteins. ADAM17 over-expression cells showed that more TGF-α released to the supernatant and activated the EGFR/PI3K/AKT pathway. Conversely, silencing ADAM17 led to the opposite effect. Both siRNAs knockdown of ADAM17 and blocking the EGFR/PI3K/AKT pathway using specific inhibitor caused downregulation of cyclin E, CDK2, and upregulation of p21 and p27 in prostate cancer cells. Collectively, this study demonstrates that over-expression of ADAM17 might target cyclin E, CDK2, p21, and p27 to promote prostate cancer cell proliferation through activation of the EGFR/PI3K/AKT pathway.

ADAM17 targets MMP-2 and MMP-9 via EGFR-MEK-ERK pathway activation to promote prostate cancer cell invasion.

ADAM17, also known as tumor necrosis factor-α converting enzyme (TACE), is involved in proteolytic ectodomain shedding of cell surface molecules and cytokines. Although aberrant expression of ADAM17 has been shown in various malignancies, the function of ADAM17 in prostate cancer has not been clarified. In the present study, we sought to elucidate whether ADAM17 contributes to prostate cancer cell invasion, as well as the mechanism involved in the process. The expression pattern of ADAM17 was investigated in human prostate cancer cells. The results showed that ADAM17 expression levels are correlated with the invasive ability of androgen-independent prostate cancer cell lines. Further, ADAM17 was overexpressed in cells showing high invasion characteristics, activation of the EGFR-MEK-ERK pathway, up-regulation of MMP-2, MMP-9, and an increased TGF-α release into the supernatant. However, AG1478, PD98059 and antibody against TGF-α deactivating the EGFR-MEK-ERK signaling pathway, abolished up-regulation of MMP-2, MMP-9 and prevented cell invasion. In addition, cells with knockdown of ADAM17 by siRNA exhibited low invasive ability, deactivated EGFR-MEK-ERK signaling pathway, reduced TGF-α released and down-regulation of MMP-2, MMP-9. However, these effects could be reversed by simultaneous addition of TGF-α. These data demonstrated that ADAM17 contributes to androgen-independent prostate cancer cell invasion by shedding of EGFR ligand TGF-α, which subsequently activates the EGFR-MEK-ERK signaling pathway, leading finally to overexpression of MMP-2 and MMP-9. This study suggests that the ADAM17 expression level may be a new predictive biomarker of invasion and metastasis of prostate cancer, and ADAM17 could provide a target for treating metastatic PCa.

Fbxw8 is involved in the proliferation of human choriocarcinoma JEG-3 cells.

Fbxw8 is the F-box component of a SCF-like E3 ubiquitin ligase complex. Mice lacking Fbxw8 exhibit pathological defects in placenta and embryo similar to fetal growth retardation, suggesting a role of Fbxw8 in placentation. Proliferative capacity of trophoblast cells is very important in placental development. In this context, we revealed that Fbxw8 was expressed in four different human trophoblast cell lines. Silencing of Fbxw8 expression by siRNA inhibited the growth of choriocarcinoma JEG-3 cells. By Western blotting, cell cycle analysis, we showed that down-regulation of Fbxw8 by RNAi induced cell-growth arrest at G2/M phase through decreasing the levels of CDK1, CDK2, cyclin A and cyclin B1 and up-regulation of p27 at protein level. Conversely, over-expression of Fbxw8 led to the opposite effect. These results suggest that Fbxw8 plays an essential role in the proliferation of human trophoblast cells, especially JEG-3 cells, via G2/M phase transition in association with regulation of CDK1, CDK2, cyclin A, cyclin B1 and p27 expression.