Protein Expression Of phospho-Akt Research Articles

Knockdown of Bcl-3 alleviates psoriasis and dyslipidemia comorbidity by regulating Akt pathway.

Psoriasis is considered as an inflammatory skin disease accompanied by dyslipidemia comorbidity. B-cell leukemia-3 (Bcl-3) belongs to IκB (inhibitor of nuclear factor kappa B [NF-κB]) family, and regulates inflammatory response through associating with NF-κB. The role of Bcl-3 in psoriasis was investigated in this study. Apolipoprotein E (ApoE)-deficient mice were treated with imiquimod to induce psoriasis and dyslipidemia. Mice were injected intradermally in the back with lentiviral particles encoding Bcl-3 small hairpin RNA (shRNA). Hematoxylin and eosin were used to detect pathological characteristics. The blood lipid levels were determined by automatic biochemical analyzer, and inflammation was assessed by enzyme-linked-immunosorbent serologic assay and real-time quantitative reverse transcription polymerase chain reaction. Bcl-3 was elevated in imiquimod-induced ApoE-deficient mice. Injection with lentiviral particles encoding Bcl-3 shRNA reduced Psoriasis area and severity index (PASI) score in ApoE-deficient psoriatic mice. Knockdown of Bcl-3 also ameliorated imiquimod-induced psoriasiform skin lesions in ApoE-deficient mice. Moreover, loss of Bcl-3 enhanced expression of loricrin, an epidermal barrier protein, reduced expression of proliferating cell nuclear antigen (PCNA) and lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1) in imiquimod-induced ApoE-deficient mice. The enhanced levels of blood lipid in ApoE-deficient mice were attenuated by silencing of Bcl-3 with increase of high-density lipoprotein, and reduction of total cholesterol, triglycerides, and low-density lipoprotein cholesterol. Knockdown of Bcl-3 attenuated imiquimod-induced decrease of transforming growth factor beta (TGF-β), and increase of Interleukin (IL)-17A, IL-23, IL-6, and tumor necrosis factor-α (TNF-α) in ApoE-deficient mice. Protein expression of phospho-Akt (p-Akt) and p-GSK3β in ApoE-deficient psoriatic mice was decreased by silencing of Bcl-3. Loss of Bcl-3 exerted anti-inflammatory effect on psoriasis and dyslipidemia comorbidity through inactivation of Akt/GSK3β pathway.

AAV1-Mediated shRNA Knockdown of SASH1 in Rat Bronchus Attenuates Hypoxia-Induced Pulmonary Artery Remodeling.

Pulmonary hypertension (PH) is a proliferative disease characterized by pulmonary arterial remodeling (PAR). SAM and SH3 domain containing 1 (SASH1) is a novel tumor suppressor gene whose biological function in PH is unclear. In this study, a hypoxia-induced pulmonary hypertension (HPH) rat model was constructed to explore the role of SASH1 in PAR. Histopathological changes in the lung tissue and hemodynamic alteration were detected in SASH1-knockdown rats through adeno-associated virus type-1 (AAV1) infection. In vitro, primary human pulmonary arterial smooth muscle cells (HPASMCs) were transfected with SASH1siRNA to investigate the effects of SASH1 on hypoxia-induced proliferation and migration. The molecular mechanisms associated with SASH1 were explored through knockdown and overexpression approaches. We found that SASH1 expression was significantly increased in rat pulmonary arteries and HPASMCs after hypoxia exposure. In vivo, silencing the SASH1 gene expression improved HPH in rats. The SASH1 downregulation inhibited proliferation and migration of hypoxia-induced HPASMCs. The protein expression of phospho-AKT (known as protein kinase B), proliferating cell nuclear antigen, and matrix metalloproteinase 9 (MMP9) in HPASMCs were increased after SASH1 overexpression, whereas these effects were inhibited by SASH1 knockdown. In conclusion, SASH1 downregulation improved hypoxia-induced PAR and PH. SASH1 may be a novel target for PH gene therapy in the era of precision medicine.

Benzyl Butyl Phthalate Induced Early lncRNA H19 Regulation in C3H10T1/2 Stem Cell Line.

Exposure to endocrine-disrupting chemicals used in plastic manufacturing may contribute to the current obesity and diabetes epidemic. Our previous study demonstrated that benzyl butyl phthalate (BBP) induced adipogenesis in the C3H10T1/2 stem cell line. Here we investigated if BBP deregulated long noncoding RNA H19 and its downstream pathway and whether BBP plays a role in the insulin signaling pathway during adipocyte diiferentiation. Cells treated with an 8 day BBP regimen showed that H19 expression was decreased at day 2 with 50 μM BBP exposure (p < 0.05). However, no significant changes were observed from day 4 to day 8. Expression of miRNA-103/107, H19 regulated miRNAs, was upregulated at day 2 (p < 0.05) but not from day 4 to day 8. Similarly, expression of the let-7 family members (a, b, c, d, f, and g) was also significantly increased at day 2 (p < 0.05 or p < 0.01), except for let-7e. Both let-7 and miRNA-103/107 are targets of H19 and play roles in insulin signaling. Insulin receptor substrate (IRS)-1, one of the key insulin signal transduction regulators, was significantly downregulated from day 2 to day 8 (p < 0.05). Gene expression of insulin receptor (IR) and IRS-2 were not altered by BBP exposure. The ratio of IRS1/IRS2 was significantly decreased from day 2 to day 8. On day 4, phospho-Akt protein expression was significantly decreased (p < 0.05). In conclusion, BBP exposure may lead to metabolic dysregulation by altering vital epigenetic regulators such as lncRNA H19 and its target microRNAs at an earlier stage, which further regulates insulin signaling.

TRIM31 enhances chemoresistance in glioblastoma through activation of the PI3K/Akt signaling pathway.

Temozolomide (TMZ) resistance is a complication of treatment of glioma, and new strategies are urgently required to overcome chemoresistance in glioma cells. In the present study, it was demonstrated that tripartite motif-containing 31 (TRIM31) was abnormally upregulated in glioma tissues and cell lines compared with normal samples. Furthermore, the role of TRIM31 was assessed by overexpressing and knocking down its expression. Overexpression of TRIM31 increased cell viability, increased TMZ IC50 values and inhibited apoptosis in A172 and U251 cells; whereas overexpression of TRIM31 decreased the expression of the apoptosis-associated protein p53. Knockdown of TRIM31 increased apoptosis in cells treated with TMZ. Additionally, the mechanisms by which TRIM31 affected glioma cells treated with TMZ were determined. Overexpression of TRIM31 increased phosphorylation of AKT and inhibiting the PI3K/AKT signaling pathway abolished the increase in cell viability and decreased phospho-Akt protein expression in TRIM31 overexpressing A172 cells treated with TMZ. Together, the findings suggest that TRIM31 may be a potentially novel target for glioma chemotherapy.

Resveratrol attenuates brain damage in permanent focal cerebral ischemia via activation of PI3K/Akt signaling pathway in rats

ABSTRACTObjective: The aim of this study was to evaluate the potential molecular mechanism of resveratrol (RSV) that attenuates brain damage from focal cerebral ischemia.Methods and materials: To investigate whether phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway was involved in RSV anti-inflammatory and neuroprotective properties. Middle cerebral artery occlusion (MCAO) animal model was used in this study. Adult male Sprague–Dawley (SD) rats underwent MCAO, and then received treatment with RSV or vehicle after the onset of ischemia. PI3K inhibitor LY294002 was injected intracerebroventricularly to inhibit the PI3K/Akt signaling pathway. Neurological deficit scores and cerebral water content were assessed 24 h after MCAO. The inflammatory factors interleukin (IL)-1β, tumor necrosis factor (TNFα), and cyclooxygenase-2 (COX2) mRNA level were examined by real-time PCR. The enzymatic activity of myeloperoxidase (MPO) was measured 24 h after MCAO. The protein expression of phospho-Akt and COX2 in ischemic brain were determined by western blot.Results: RSV significantly reduced neurological deficit scores, cerebral water content and the enzymatic activity of MPO, all of which were abolished by LY294002 administration. Real-time PCR showed that RSV significantly suppressed the upregulation of the inflammatory factors IL-1β, TNFα, COX2 mRNA levels. RSV significantly inhibited upregulated the protein expression of COX2 24 h after MCAO, which effect was abolished by LY294002 administration.Conclusion: RSV attenuated ischemic brain damage induced by cerebral artery occlusion mainly through PI3K/Akt signaling pathway.Abbreviation: MCAO: Middle cerebral artery occlusion; RSV: resveratrol; PI3K/Akt: phosphatidylinositol 3-kinase/Akt; TNF: tumor necrosis factor; COX2: cyclooxygenase-2; MPO: myeloperoxidase; IL: interleukin.

Dihydroartemisinin-induced apoptosis in human acute monocytic leukemia cells.

Dihydroartemisinin (DHA) is a derivative of artemisinin. The present study aimed to investigate whether DHA induces apoptosis in the THP-1 human acute monocytic leukemia cell line (AMoL), and to identify the relative molecular mechanisms. The results of the present study demonstrated that the viability of THP-1 cells were inhibited by DHA in a dose- and time-dependent manner, which was accompanied by morphological characteristics associated with apoptosis. After 24 h of 200 µM DHA treatment, the proportion of apoptotic cells was significantly increased compared with the untreated controls (P<0.01). In addition, DHA downregulated the levels of B-cell lymphoma (Bcl)-2, protein kinase B (Akt)1, Akt2 and Akt3 gene expression, and increased the expression of the Bcl-2-associated X protein apoptosis regulator. The protein expression of phospho-Akt and phospho-extracellular signal-regulated kinase (ERK) was also decreased, and the protein expression level of cleaved caspase-3 was increased following treatment with DHA. Therefore, DHA may induce apoptosis in the AMoL THP-1 cell line via currently unknown underlying molecular mechanisms, including the downregulation of ERK and Akt, and the activation of caspase-3.

Telluric Acid Ameliorates Endotoxemic Kidney Injury in Mice: Involvement of TLR4, Nrf2, and PI3K/Akt Signaling Pathways.

Being one of the most abundant trace elements in the human body, the therapeutic potential of tellurium-based compounds has been a target of interest. Recent reports denoted their redox-modulating and anti-inflammatory activities in experimental endotoxemia. However, their potential nephroprotective effect against endotoxemic kidney injury is yet to be elucidated. This study investigated the possible renoprotective effect of telluric acid (TEL) against lipopolysaccharide (LPS)-induced acute kidney injury (AKI) in mice, targeting toll-like receptor 4 (TLR4), phosphoinositide 3-kinase (PI3K)/Akt, and nuclear factor-erythroid 2-related factor-2 (Nrf2) pathways as possible mechanistic contributors to TEL's effect. AKI was induced by LPS (2mg/kg). TEL (60μg/kg; i.p.) was administered once daily for seven consecutive days before LPS injection. Pretreatment with TEL alleviated LPS-induced AKI as evidenced by the hampered serum levels of creatinine and cystatin C. TEL also opposed LPS-induced elevation in renal kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, nuclear factor-kappa B p65, interleukin-1β, and thiobarbituric acid-reactive substance contents. This was accompanied by a replenishment of renal glutathione, transcriptional upregulation of Nrf2, enhancement of heme oxygenase-1 activity, and a marked upregulation of phospho-PI3K and phospho-Akt protein expressions. Histopathological findings corroborated with the amendment of biochemical parameters. In view of these findings, we may conclude that TEL pretreatment purveyed novel nephroprotective effects against endotoxemic kidney injury, which might be partly attributed to the modulation of TLR4, PI3K/Akt, and Nrf2 signaling pathways and may hence provide a valuable asset for the management of endotoxemic renal complications.

Conditioned Media From Adipose Tissue Derived Mesenchymal Stem Cells Reverse Insulin Resistance in Cellular Models.

The link between insulin resistance (IR) and type 2 diabetes has been recognized for a long time. Type 2 diabetes is often associated with basal hyperinsulinemia, reduced sensitivity to insulin, and disturbances in insulin release. There are evidences showing the reversal of IR by mesenchymal stem cells. However, the effect of conditioned media from adipose derived mesenchymal stem cells (ADSCs-CM) in reversal of IR has not been established. We established an insulin resistant model of 3T3L1 and C2C12 cells and treated with ADSCs-CM. 2-NBDG (2-[N-[7-Nitrobenz-2-oxa-1,3-diazol-4-yl]Amino]-2-Deoxyglucose) uptake was performed to assess improvement in glucose uptake. Genes involved in glucose transport and in inflammation were also analysed. Western blot for glucose transporter-4 and Akt was performed to evaluate translocation of Glut4 and insulin signaling respectively. We found that the ADSCs-CM treated cells restored insulin, stimulated glucose uptake as compared to the untreated control indicating the insulin sensitizing effect of the CM. The treated cells also showed inhibition adipogenesis in 3T3L1 cells and significant reduction of intramuscular triglyceride accumulation in C2C12 cells. Gene expressions studies revealed the drastic upregulation of GLUT4 gene and significant reduction in IL6 and PAI1 gene in both 3T3L1 and C2C12 cells, indicating possible mechanism of glucose uptake with concomitant decrease in inflammation. Enhancement of GLUT4 and phospho Akt protein expression seems to be responsible for the increment in glucose uptake and enhanced insulin signaling, respectively. Our study revealed for the first time that ADSCs-CM acts as an alternative insulin sensitizer providing stem cell solution to IR. J. Cell. Biochem. 118: 2037-2043,2017. © 2016 Wiley Periodicals, Inc.

Endothelin receptor blockers reduce shunting and angiogenesis in cirrhotic rats.

Angiogenesis plays a pivotal role in splanchnic hyperaemia and portosystemic collateral formation in cirrhosis. Endothelin-1 (ET-1), an endothelium-derived vasoconstrictor, has also been implicated in the pathogenesis of cirrhosis and portal hypertension. This study aimed to survey the influences of ET-1 in cirrhosis-related angiogenesis. Common bile duct ligation was performed on Spraque-Dawley rats to induce cirrhosis. Since the 14th day after the operation, rats randomly received distilled water (DW, control), bosentan [a nonselective ET receptor (ETR) blocker] or ambrisentan (a selective ETA R blocker) for 4weeks. On the 43rd day, portal and systemic haemodynamics, liver biochemistry, portosystemic shunting degree, mesenteric vascular density, mRNA and/or protein expressions of relevant angiogenic factors were evaluated. In cirrhotic rats, bosentan significantly reduced portal pressure. Ambrisentan did not influence haemodynamics and liver biochemistry. Both of them significantly improved the severity of portosystemic collaterals and decreased the mesenteric vascular density. Compared with the DW-treated cirrhotic rats, splenorenal shunt and mesenteric inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), vascular endothelial growth factor mRNA expressions and mesenteric iNOS, COX2, VEGF, phospho-VEGF receptor 2, Akt and phospho-Akt protein expressions were down-regulated in both groups. In rats with liver cirrhosis, both nonselective and selective ETA R blockade ameliorate the severity of portosystemic shunting and mesenteric angiogenesis via the down-regulation of VEGF pathway and relevant angiogenic factors. ET receptors may be targeted to control the severity of portosystemic collaterals and associated complications in cirrhosis.

The Neuroprotective Effect of Puerarin in Acute Spinal Cord Injury Rats

Background: Acute spinal cord injury (SCI) leads to permanent disabilities. This study evaluated the neuroprotective effect of puerarin, a natural extract, in a rat model of SCI. Methods: Acute SCI models were established in rats using a modified Allen's method. Locomotor function was evaluated using the BBB test. The histological changes in the spinal cord were observed by H&E staining. Neuron survival and glial cells activation were evaluated by immunostaining. ELISA and realtime PCR were used to measure secretion and gene expression of cytokines. TUNEL staining was used to examine cell apoptosis and western blot analysis was used to detect protein expression. Results: Puerarin significantly increased BBB score in SCI rats, attenuated histological injury of spinal cord, decreased neuron loss, inhibited glial cells activation, alleviated inflammation, and inhibited cell apoptosis in the injured spinal cords. In addition, the downregulated PI3K and phospho-Akt protein expression were restored by puerarin. Conclusion: Puerarin accelerated locomotor function recovery and tissue repair of SCI rats, which is associated with its neuroprotection, glial cell activation suppression, anti-inflammatory and anti-apoptosis effects. These effects may be associated with the activation of PI3K/Akt signaling pathway.

Increased chemoresistance to paclitaxel in the MCF10AT series of human breast epithelial cancer cells

The MCF10AT cell series of human breast epithelial cancer cells includes normal MCF10A (10A), premalignant MCF10AT (10AT) and MCF10ATG3B (10ATG3B), and fully malignant MCF10CA1a (10CA1a) cells. The series is a unique model system showing progressive tumorigenic potential with the same origin. The effects of paclitaxel, a microtubule inhibitor, were evaluated in this cell system. Paclitaxel inhibited cell proliferation in a time-dependent (24, 48 and 72h) and concentration-dependent (0-10nM) manners with less sensitivity in 10CA1a cells. Treatment with paclitaxel (10nM) for 24h induced apoptosis in 10A, 10AT, 10ATG3B and 10CA1a cells, with 23.6, 26.1, 25.2 and 8.96%, respectively, in the sub-G1phase. Treatment with paclitaxel (0-10nM) for 24h, resulted in the appearance of DNA fragmentation (a hallmark of apoptosis) with less sensitivity in the 10CA1a tumor cells. Paclitaxel increased p53 protein expression in 10A, 10AT, 10ATG3B and 10CA1a cells, by 87, 102, 812 and 84%, respectively. The p21Waf1/Cip1 protein expression increased by 2.57-, 1.53-and 2.48-fold in 10A, 10AT and 10ATG3B cells, respectively, with negligible detection in the 10CA1a cells. Activation of the Akt signaling pathway was observed in the MCF10AT cell lineage and the protein expression of phospho-Akt (Ser473 and Thr308). The downstream targets of this pathway, phospho-p70S6K and phospho-S6RP, were also inhibited by paclitaxel in 10A, 10AT and 10ATG3B cells, but minimally inhibited in 10CA1a cells, suggestive of chemoresistance in 10CA1a cells. The effects of paclitaxel on the multidrug resistance1 (MDR1), MRP1 and breast cancer resistance protein (BCRP) gene expression were not significant in the MCF10AT cell lineage. These results collectively indicated that paclitaxel inhibited cell proliferation and induced apoptosis in the MCF10AT cell lineage, with chemoresistance in 10CA1a tumor cells. The decreased responsiveness to paclitaxel observed in 10CA1a tumor cells was likely due, in part, to activation of the Akt signaling pathway and a high expression of wild-type p53 with lack of p21Waf1/Cip1.

Knockdown of Clusterin Expression Increases the In Vitro Sensitivity of Human Prostate Cancer Cells to Paclitaxel

Clusterin/apolipoprotein J is a secreted heterodimeric glycoprotein that is implicated in several pathophysiological processes, including tissue remodeling, reproduction, lipid transport, and apoptosis. Although previous studies demonstrated that clusterin is able to protect against apoptosis, the role of the clusterin in cellular proliferation remains elusive. To determine whether clusterin plays an important role in cellular proliferation, the function of clusterin was examined using a small interfering RNA (siRNA) in PC3 human prostate cancer cells. Transient transfection with clusterin siRNA resulted in significant suppression of clusterin mRNA and protein expression. Clusterin knockdown resulted in a decrease in protein expression of phospho-Akt and an increase in expression of proteins phosphatase type 2AC (PP2AC) and phosphorylation of p38. However, treatment with PP2AC siRNA exerted minimal effects on clusterin expression. Interestingly, clusterin mRNA expression was reduced in paclitaxel-treated cells, and the cytotoxic effect of paclitaxel was more potent when cells were incubated with clusterin siRNA. In addition, co-treatment with paclitaxel and clusterin siRNA significantly enhanced PP2AC levels. Taken together, these results indicate that clusterin plays a crucial role in PC3 cell proliferation and that clusterin depletion may contribute to enhanced sensitivity of PC3 cells to anticancer agents such as paclitaxel.

Follistatin could promote the proliferation of duck primary myoblasts by activating PI3K/Akt/mTOR signalling.

FST (follistatin) is essential for skeletal muscle development, but the intracellular signalling networks that regulate FST-induced effects are not well defined. We sought to investigate whether FST promotes the proliferation of myoblasts through the PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) signalling. In the present study, we transfected the pEGFP-duFST plasmid and added PI3K and mTOR inhibitors to the medium of duck primary myoblasts. Then, we analysed the cellular phenotypic changes that occurred and analysed the expression of target genes. The results showed that FST promoted myoblast proliferation, induced the mRNA expression of PI3K, Akt, mTOR, 70-kDa ribosomal protein S6K (S6 kinase) and the protein expression of phospho-Akt (Thr308), mTOR, phospho-mTOR (serine 2448), phospho-S6K (Ser417), inhibited the mRNA expression of FoxO1, MuRF1 (muscle RING finger-1) and the protein expression of phospho-FoxO1 (Ser256). Moreover, we found that the overexpression of FST could alleviate the inhibitory effect of myoblast proliferation caused by the addition of LY294002, a PI3K inhibitor. Additionally, the overexpression of duck FST also relieved the inhibition of myoblast proliferation caused by the addition of rapamycin (an mTOR inhibitor) through PI3K/Akt/mTOR signalling. In light of the present results, we hypothesize that duck FST could promote myoblast proliferation, which is dependent on PI3K/Akt/mTOR signalling.

Dexmedetomidine Reduces Isoflurane-Induced Neuroapoptosis Partly by Preserving PI3K/Akt Pathway in the Hippocampus of Neonatal Rats

Prolonged exposure to volatile anesthetics, such as isoflurane and sevoflurane, causes neurodegeneration in the developing animal brains. Recent studies showed that dexmedetomidine, a selective α2-adrenergic agonist, reduced isoflurane-induced cognitive impairment and neuroapoptosis. However, the mechanisms for the effect are not completely clear. Thus, we investigated whether exposure to isoflurane or sevoflurane at an equivalent dose for anesthesia during brain development causes different degrees of neuroapoptosis and whether this neuroapoptosis is reduced by dexmedetomidine via effects on PI3K/Akt pathway that can regulate cell survival. Seven-day-old (P7) neonatal Sprague-Dawley rats were randomly exposed to 0.75% isoflurane, 1.2% sevoflurane or air for 6 h. Activated caspase-3 was detected by immunohistochemistry and Western blotting. Phospho-Akt, phospho-Bad, Akt, Bad and Bcl-xL proteins were detected by Western blotting in the hippocampus at the end of exposure. Also, P7 rats were pretreated with various concentrations of dexmedetomidine alone or together with PI3K inhibitor LY294002, and then exposed to 0.75% isoflurane. Terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) and activated caspase-3 were used to detect neuronal apoptosis in their hippocampus. Isoflurane, not sevoflurane at the equivalent dose, induced significant neuroapoptosis, decreased the levels of phospho-Akt and phospho-Bad proteins, increased the expression of Bad protein and reduced the ratio of Bcl-xL/Bad in the hippocampus. Dexmedetomidine pretreatment dose-dependently inhibited isoflurane-induced neuroapoptosis and restored protein expression of phospho-Akt and Bad as well as the Bcl-xL/Bad ratio induced by isoflurane. Pretreatment with single dose of 75 µg/kg dexmedetomidine provided a protective effect similar to that with three doses of 25 µg/kg dexmedetomidine. Moreover, LY294002, partly inhibited neuroprotection of dexmedetomidine. Our results suggest that dexmedetomidine pretreatment provides neuroprotection against isoflurane-induced neuroapoptosis in the hippocampus of neonatal rats by preserving PI3K/Akt pathway activity.

Moscatilin inhibits migration and metastasis of human breast cancer MDA-MB-231 cells through inhibition of Akt and Twist signaling pathway

Breast cancer metastasis is more resistant to chemotherapy and radiotherapy than is cancer of the visceral tissues; therefore, new treatment strategies are urgently needed. Moscatilin, derived from the orchid Dendrobrium loddigesii, has shown anticancer activity. We evaluated the mechanism by which moscatilin suppresses the migration and metastasis of human breast cancer MDA-MB-231 cells in vitro and in vivo. We demonstrated that moscatilin significantly inhibits MDA-MB-231 cell migration by using scratch assays and Boyden chambers. Transcriptional factors inducing epithelial-mesenchymal transition, such as Twist, Snail, and Akt, play important roles in cell migration and cancer metastasis. Moscatilin inhibited the mRNA and protein expression of Twist, but not that of Snail, and subsequently inhibited N-cadherin expression. However, these effects were reversed by constitutively expressing active myristoylated (myr)-Akt and Twist overexpression. Moscatilin also suppressed Akt phosphorylation. However, Akt overexpression reversed the inhibitory effects of moscatilin on phospho-Akt protein expression but not its effects on Twist. The moscatilin-mediated inhibition of cell migration was reversed by Akt and Twist overexpression, demonstrating that moscatilin blocked cell migration by inhibiting Akt and Twist. In an MDA-MB-231 metastatic animal model, moscatilin (100 mg/kg) significantly suppressed breast cancer metastasis to the lungs and reduced the number of metastatic lung nodules and lung weight without causing any toxicity. These results indicated that moscatilin inhibited MDA-MB-231 cell migration via Akt- and Twist-dependent pathways; this finding was consistent with moscatilin's antimetastatic activity in vivo. Therefore, moscatilin may be an effective compound for the prevention of human breast cancer metastasis.

The regulation of DIDS on PI3K/Akt and ROS signal transduction in the ischemia-reperfusion injury myocardium

ObjectiveTo investigate the regulation of DIDS on PI3K/Akt and ROS signal transduction in the ischemia-reperfusion injury myocardium.MethodsMale SD rats were randomised into four groups sham group, I/RI (ischemia-reperfusion injury) group,...

Inhibition of HSP90‐dependent telomerase activity in amyloid β‐induced apoptosis of cerebral endothelial cells

Although apoptosis induced by amyloid β (Aβ) has been identified, the effect of Aβ on telomerase activity in relation to apoptosis induction remains unclear. In the present study, Aβ(1-40) and Aβ(25-35), but not Aβ(1-16) and Aβ(35-25), reduce the viability of primary cerebral endothelial cells (CECs) in accordance with apoptosis induction. Increases in caspase 3 and PARP protein cleavage with reductions of the Bcl-2/Bax protein ratio accompanied by a loss in the mitochondria membrane potential were identified in Aβ(1-40) and Aβ(25-35)-treated CECs. A significant decrease in intracellular telomerase activity by Aβ(1-40) and Aβ(25-35) was detected; meanwhile, reduced telomerase activity by telomerase reverse transcriptase (TERT) siRNA enhanced the cytotoxic effect of Aβ. The addition of serum might block the Aβ(25-35)-induced cytotoxic effect via elevated telomerase activity in according with stimulating phospho-AKT protein expression, which was blocked by adding AKT inhibitor LY294002. Decreases in heat shock protein 90 (HSP90) and its client proteins including TERT, AKT, p53, CDK4 were observed in Aβ(1-40) and Aβ(25-35), but not Aβ(1-16) and Aβ(35-25), -treated CECs. The knockdown of HSP90 gene expression by HSP90 siRNA significantly inhibits telomerase activity with decreasing TERT protein expression. The application of HSP90 activity inhibitor geldanamycin (GA) and radicicol (RD) potentiates the telomerase inhibition and apoptosis induction of Aβ in CECs. An increase in protein ubiquitination by Aβ(25-35), but not Aβ(35-25), treatment was examined, and Aβ-inhibited HSP90 and TERT protein expression and telomerase activity was reversed by adding proteasome inhibitor, MG132. Additionally, increased TERT protein ubiquitination by Aβ(25-35) was detected in CECs via immunoprecipitation/Western blotting analysis. The data of the present study firstly demonstrates that telomerase inhibition contributes to the apoptosis induction of Aβ in CECs.

Evaluation of MAPK and PI3K/AKT Signaling Pathways In Adult Acute Lymphoblastic Leukemia

AbstractAbstract 3240 Introduction:Treatment of adult acute lymphoblastic leukemia (ALL) has shown only modest improvements over the last 2 decades, with overall survival of 15% to 40%. The mitogen-activated protein kinase (MAPK) signaling cascade and the phosphoinosytol-3 phosphate/AKT (PI3K/AKT) pathways are involved in proliferation and differentiation of hematopoietic cells. It has been reported that those pathways are frequently activated in solid tumors and acute myeloid leukemia. However, their role in adult ALL is still uncertain. Better understanding of such pathways is necessary for development of novel therapeutic strategies. Aims:To evaluate the phopho-ERK and phospho-AKT protein expression in ALL at diagnosis and to correlate with biological and clinical parameters. Material and Methods:Twenty eight patients (median age 33y, 14–69y) with ALL at diagnosis were studied. Bone marrow and/or peripheral blood mononuclear cells (PBMC) (10 fresh and 18 cryopreserved cells at diagnosis) were stained using phospho-ERK and phospho-AKT/alexafluor 488 monoclonal antibodies (Cell Signaling Technology, Beverly, MA) and their expression was evaluated by flow cytometry (FACScalibur cytometer and CELL Quest program - BDB, San Jose, CA). The monoclonal antibodies CD19, CD10, CD3, CD45, IgM, CD34, CD7, CD2 were used for leukemic cells characterization by four-colour staining. Healthy donor PBMC and Jurkat cell line were used as controls: normal T lymphocytes are negative for p-ERK and Jurkat cell line express p-ERK and p-AKT in low levels. Samples were analyzed for constitutive expression of p-ERK and p-AKT and also after cell activation by phorbol-myristate-acetate (PMA). The expression of these proteins was evaluated by Kolmogorov-Smirnov test using fluorescence ratio between control isotype and phospho-protein (D). p-ERK and p-AKT expression was also evaluated in fresh and frozen samples of the same patients (2 cases) and similar results were obtained. In addition, patients were evaluated for multidrug resistance (MDR) through p-glycoprotein (PGP) expression and Rhodamine (Rh) efflux test and minimal residual disease (MRD) detection at the end of induction by flow cytometry. Results:Twenty cases were B-ALL (EGIL B-I 3, B-II 9, B-III 8, B-IV 5) and 3 T-ALL. Median WBC count was 25.3×109/L (2.3-373×109/L). The expression of p-ERK and p-AKT varied and the median value of p-ERK expression was D = 0.16 (0.01-0.80) and p-AKT median D = 0.08 (0.00-0.63). Considering these values as cutoff there was no difference regarding the patients' age and WBC count at the diagnosis between the positive and negative groups. In regards to EGIL subtypes, p-ERK expression was higher in T-ALL [median 0.50 (0.18-0.54)] than in B-precursor ALL [median 0.14 (0.01-0.80)] (p=0.03). Conversely p-AKT expression was similar in all cases, although high levels were observed among BIII cases. The frequency of Rh efflux was 88% in pERK negative cases and 66% in positive group but there was no difference on PGP expression. On the contrary, PGP expression and Rh efflux were more frequently seen in p-AKT positive cases (88% and 100%, respectively) than p-AKT negative ones (63% and 60%). MRD analysis was performed in seven patients. Two cases presented detectable MRD (>0.01%) and both were p-ERK positive and p-AKT negative. Interestingly all five MRD (-) cases were p-ERK negative and p-AKT positive. In addition, PMA test showed p-ERK activation of normal T lymphocytes and the expression was increased in 52% of ALL cases upon treatment. Conclusion:MAPK and PI3/AKT activation varied among ALL patients. MAPK pathway showed to be more activated in T-ALL than in precursor-B ALL. The functional analysis of these pathways can address the role in ALL pathogenesis. Both pathways may be potential therapeutic targets for novel therapies. (Support: FAPESP proc.09/51002-8). Disclosures:No relevant conflicts of interest to declare.

Administration of a low dose of sildenafil for 1 week decreases intrahepatic resistance in rats with biliary cirrhosis: the role of NO bioavailability

Increasing NO bioavailability improves hepatic endothelial dysfunction, which ameliorates intrahepatic resistance and portal hypertension. Acute administration of sildenafil increases hepatic production of NO with a reduction in hepatic sinusoid resistance in cirrhotic patients and enhances the vasorelaxation response to NO in cirrhotic rat livers. However, the mechanisms were still unclear. Therefore, our present study aims to evaluate the effects and mechanisms of administration of sildenafil for 1 week on the hepatic microcirculation of cirrhotic rats. Cirrhosis was induced by bile duct ligation with sham-operated rats serving as normal controls. Intrahepatic resistance was evaluated by in situ liver perfusion. Expression of phospho-eNOS (endothelial NO synthase), iNOS (inducible NO synthase), phospho-Akt, PDE-5 (phosphodiesterase-5) and sGC (soluble guanylate cyclase) were determined by Western blot analysis. Biosynthesis of BH4 (tetrahydrobiopterin) and GTPCH-I (GTP cyclohydrolase I) activity were examined by HPLC. Intravital microscopy was used to observe the direct change in hepatic microcirculation. In cirrhotic rat livers, sildenafil treatment increased hepatic sinusoid volumetric flow, NO bioavailability, BH4, GTPCH-I activity, and the protein expression of phospho-Akt, phospho-eNOS and sGC. These events were associated with reduced protein expression of PDE-5, portal perfusion pressure and portal vein pressure. In contrast, sham rats did not produce any significant change in these measurements. In conclusion, sildenafil treatment improves endothelial dysfunction by augmenting NO bioavailability in the hepatic microcirculation.

Improving insulin sensitivity via activation of PPAR-γ increases telomerase activity in the heart of OLETF rats

This study was conducted to examine telomere biology in terms of improving insulin sensitivity in a type 2 diabetic animal model: Otsuka Long-Evans Tokushima fatty (OLETF) rats. To improve insulin sensitivity, pioglitazone (PG; 10 mg.kg(-1).day(-1)) was administrated to OLETF rats from 20 to 40 wk of age, and the effects of treatment were compared with those in untreated OLETF or control Long-Evans Tokushima Otsuka fatty rats. At the end of the study, the homeostasis model assessment of insulin resistance significantly increased in OLETF rats but decreased in OLETF rats treated with PG. No shortening of telomere length was observed in the heart tissue of OLETF rats, whereas telomerase activity was decreased in OLETF heart tissue. The mRNA expression of both telomerase reverse transcriptase and telomere repeat binding factor 2 was downregulated in the hearts of OLETF rats. The protein expression of phospho-Akt, insulin-like growth factor, and endothelial nitric oxide synthase was reduced in OLETF rats. On the other hand, myocardial matrix metalloproteinase-9 expression was elevated in OLETF rats. The changes observed in OLETF rats were inhibited by PG treatment. However, protein and mRNA expression of Sirt1, a lifespan modulator, were attenuated in OLETF rat hearts, although they were enhanced in OLETF rats with PG treatment. Myocardial fibrosis was less extensive and diastolic dysfunction more greatly ameliorated in PG-treated OLETF rats than in OLETF rats. These findings suggest that improving insulin sensitivity via the activation of peroxisom proliferator-activated receptor-gamma may exert regulatory effects on cardiac telomere biology and may have desirable morphological and functional effects on the diabetic heart.