Activated Akt and Erk Expression and Survival After Surgery in Pancreatic Carcinoma

Background Long-term survival of surgically resectable pancreatic cancer patients is uncommon. The epidermal growth factor receptor (EGFR) and the phosphoinositol-3-kinase pathways are often activated in pancreatic cancer, and an understanding of their role in resected cases may help refine adjuvant therapy. Methods We investigated the expression of EGFR, Erk, Akt, and their phosphoforms (p-) in pancreatectomy specimens and correlated these with survival. Thirty-nine consecutive surgically resected pancreatic adenocarcinoma cases were included. Immunohistochemical staining of paraffin-embedded blocks was performed by using monoclonal antibodies against EGFR, Erk, p-Erk, Akt, and p-Akt. A standard immunoperoxidase technique was used to detect the avidin-biotin peroxidase complex. Immunostaining was visually scored with the histoscore method by two surgical pathologists. Results Patient characteristics were as follows: 17 men and 22 women; median age, 66 years; and American Joint Committee on Cancer stage I, 5 patients; stage II, 4 patients; stage III, 27 patients; and stage IV, 3 patients. The tumor was World Health Organization grade 1 in 4, grade 2 in 17, and grade 3 in 18 cases. Adjuvant therapies were chemotherapy (n = 6), radiotherapy (n = 1), and chemoradiotherapy (n = 17). Immunohistochemistry revealed positive expression of EGFR in 30.8%, Erk in 92.3%, p-Erk in 45.9%, Akt in 71.8%, and p-Akt in 20.5% of cases. On univariate analyses, tumor grade (P = .0098), p-Akt (P = .0003), and p-Erk (P = .0052) expression correlated with survival. On multivariate analyses, age (P = .0002; hazard ratio [HR], 1.8), grade (P = .00318; HR, 3.0), Akt (P = .0433; HR, .4), p-Akt (P = .0002; HR, .2), and p-Erk (P = .0003; HR, 3.5) expression correlated significantly with survival. Conclusions p-Erk and p-Akt expression may have prognostic and therapeutic implications in pancreatic cancer.     

Sulindac sulfide and caffeic acid phenethyl ester suppress the motility of lung adenocarcinoma cells promoted by transforming growth factor-β through Akt inhibition

Cell migration is essential for invasive and metastatic phenotypes of cancer cells. Potential chemopreventive agents of cancer—sulindac sulfide, caffeic acid phenethyl ester (CAPE), curcumin, and (+)-catechin—have been reported to interfere with several types of intracellular signaling. In this study, we examined the effects of these agents on transforming growth factor-(TGF-)-induced motility and Akt phosphorylation in A549 cells. Judged by gold particle phagokinesis assay, sulindac sulfide, CAPE, and curcumin suppressed the motility of A549 cells promoted by TGF-. LY294002, a specific inhibitor of phosphatidylinositol 3-kinase(PI3K)/Akt signaling, also suppressed TGF--induced motility and Akt phosphorylation. Sulindac sulfide and CAPE, but not curcumin, suppressed TGF--induced Akt phosphorylation. We conclude that sulindac sulfide and CAPE suppress the motility promoted by TGF- in lung adenocarcinoma cells through the suppression of Akt. Our observations raise the possibility that these agents, except for (+)-catechin, can be applied not only as chemopreventive agents but also as anti-metastatic therapy.Abbreviations CAPE Caffeic acid phenethyl ester - TGF- Transforming growth factor- - FAK Focal adhesion kinase - MMP-2 Matrix metalloproteinase-2     

基于网络药理学和实验验证的黄芪六一汤药效组分促肾小管上皮细胞自噬抗糖尿病肾病的机制研究

目的 通过网络药理学分析及相关的动物实验探讨黄芪六一汤药效组分（HQD）调控肾小管上皮细胞自噬水平抗糖尿病肾病（DN）的机制。方法 采用 DN 大鼠模型考察 HQD 抗 DN 的作用。利用 SwissTargetPrediction 数据库获得 HQD 主要入血成分的相关靶点,并与 GeneCard、DisGeNET 和 OMIM 数据库中筛选的疾病靶点取交集;利用 STRING 数据库和Cytoscape 构建蛋白质-蛋白质相互作用（PPI）网络获得核心靶点;采用 Metascape 数据库对交集靶点进行基因本体（GO）注释及京都基因与基因组百科全书（KEGG）通路富集分析。利用 DN 大鼠模型对预测结果进行验证。结果 与对照组比较,HQD 能显著降低 DN 模型大鼠血糖（FBG）、血肌酐（Scr）、尿素氮（BUN）、总胆固醇（TC）、三酰甘油（TG）、低密度脂蛋白（LDL）、24 h 尿白蛋白（24 h-U-Alb）值（P＜0.05）,抑制肾组织细胞凋亡（P＜0.05）。网络药理学研究显示 HQD 主要通过 STAT3、EGFR、VEGFA、JUN、TNF、AKT1、CASP3、mTOR、IL2 等关键靶点,作用于癌症通路、癌症中的蛋白多糖、PI3K-Akt 信号通路、MAPK 等信号通路;动物验证实验显示 HQD 能下调 PI3K/Akt/mTOR 信号通路相关蛋白的表达,上调肾脏组织自噬相关蛋白 Beclin-1、LC3Ⅱ/Ⅰ的表达（P＜0.05）,增加肾小管上皮细胞自噬体及自噬溶酶体数量。结论 HQD 抗 DN 的分子机制可能与抑制 PI3K/Akt/mTOR 信号通路激活,促进肾小管上皮细胞自噬水平,抑制细胞凋亡,减轻肾小管上皮细胞损伤有关。     

基于PI3K/Akt信号通路探讨白芷颗粒对糖尿病视网膜病变的影响

目的 研究白芷颗粒对早期糖尿病视网膜病变（diabetic retinopathy,DR）的保护作用。方法 采用ip链脲佐菌素（streptozotocin,STZ）法制备糖尿病模型,设置对照组、模型组、白芷（1.25 g/kg）组及羟苯磺酸钙（135 mg/kg）组。连续给药12周后采用苏木素-伊红（HE）和高碘酸-席夫（PAS）染色观察大鼠视网膜病理学改变;采用TUNEL染色法检测视网膜细胞凋亡情况;采用Western blotting检测视网膜组织紧密连接蛋白及磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase,PI3K）/蛋白激酶B（protein kinase B,Akt）通路相关蛋白表达。人视网膜上皮细胞（adult retinal pigment epithelial cell line-19,ARPE-19）分为对照组、高渗组、高糖组、白芷（150 μg/mL）组和羟苯磺酸钙（20 μmol/L）组,对照组在含5.5 mmol/L葡萄糖的培养基中培养;高渗组在含5.5 mmol/L葡萄糖和25 mmol/L甘露醇的培养基中培养;高糖组和各给药组在含30 mmol/L葡萄糖的培养基中培养,并给予相应药物。采用TUNEL染色、免疫荧光、Western blotting法观察细胞凋亡、紧密连接蛋白及PI3K/Akt通路相关蛋白表达。使用PI3K抑制剂LY294002后,观察其下游凋亡相关蛋白的变化。结果 HE染色结果显示,模型组大鼠视网膜组织排列紊乱,视网膜变薄,白芷干预后较模型组有所缓解。TUNEL染色显示视网膜组织中细胞凋亡率增加。Western blotting结果显示,与模型组比较,白芷组咬合蛋白（Occludin）、闭锁小带蛋白1（Zonula occluden-1,ZO-1）、p-PI3K/PI3K、p-Akt/Akt蛋白表达水平明显升高（P＜0.05）,Bcl-2相关X蛋白（Bcl-2 associated X protein,Bax）/ B淋巴细胞瘤-2（B-cell lymphoma-2,Bcl-2）、剪切型半胱氨酸天冬氨酸蛋白酶-3（cleaved cystein-asparate protease-3,cleaved Caspase-3）蛋白表达水平明显降低（P＜0.05）。体外实验结果显示,白芷干预后细胞凋亡率降低（P＜0.05）,Bax/Bcl-2、cleaved Caspase-3蛋白表达水平明显降低（P＜0.05）,Occludin、ZO-1蛋白表达水平明显升高（P＜0.05）;免疫荧光结果显示白芷干预后Occludin、ZO-1表达升高;此外,白芷能够促进高糖环境下PI3K/Akt的磷酸化,在使用PI3K抑制剂干预后抑制了Akt的磷酸化,Bax/Bcl-2、cleaved Caspase-3蛋白表达水平明显升高（P＜0.05）。结论 白芷颗粒能够通过抑制细胞凋亡保护血-视网膜屏障损伤,从而延缓早期DR的发展,其作用机制可能与调节PI3K/Akt信号通路有关。     

Evidence that AKT and GSK-3β pathway are involved in acute hyperhomocysteinemia

Homocysteine is a neurotoxic amino acid that accumulates in several disorders including homocystinuria, neurodegenerative and neuroinflammatory diseases. In the present study we evaluated the effect of acute and chronic hyperhomocysteinemia on Akt, NF-κB/p65, GSK-3β, as well as Tau protein in hippocampus of rats. For acute treatment, rats received a single injection of homocysteine (0.6 μmol/g body weight) or saline (control). For chronic treatment, rats received daily subcutaneous injections of homocysteine (0.3-0.6 μmol/g body weight) or saline (control) from the 6th to the 28th days-of-age. One or 12h after the last injection, rats were euthanized, the hippocampus was removed and samples were submitted to electrophoresis followed by Western blotting. Results showed that acute hyperhomocysteinemia increases Akt phosphorylation, cytosolic and nuclear immunocontent of NF-κB/p65 subunit and Tau protein phosphorylation, but reduces GSK-3β phosphorylation at 1h after homocysteine injection. However, 12h after acute hyperhomocysteinemia there is no effect on Akt and GSK-3β phosphorylation. Furthermore, chronic hyperhomocysteinemia did not alter Akt and GSK-3β phosphorylation at 1h and 12h after the last administration of this amino acid. Our data showed that Akt, NF-κB/p65, GSK-3β and Tau protein are activated in hippocampus of rats subjected to acute hyperhomocysteinemia, suggesting that these signaling pathways may be, at least in part, important contributors to the neuroinflammation and/or brain dysfunction observed in some hyperhomocystinuric patients.     

Membrane depolarization-mediated survival of sympathetic neurons occurs through both phosphatidylinositol 3-kinase- and CaM kinase II-dependent pathways

It has been well established that the NGF-mediated survival of sympathetic neurons in culture occurs through the phosphatidylinositol (PI) 3-kinase/Akt-dependent pathway. In contrast, the mechanism by which membrane depolarization promotes neuronal survival independently of NGF remains unresolved. Here we show that LY294002, a specific inhibitor of PI 3-kinase, induced cell death of sympathetic neurons under depolarizing conditions with elevated K(+) (IC(50)= approximately 30 microM). Interestingly, lower concentrations of this agent (< or =10 microM) were sufficient to suppress Akt phosphorylation at Ser-473, a putative downstream target of PI 3-kinase, under these conditions. We also show that KN-62, a specific inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) suppressed depolarization-mediated survival in a does-dependent manner (IC(50)= approximately 2 microM) that paralleled attenuation of sustained levels of intracellular Ca(2+) evoked by depolarization. This IC(50) value is greater than that for CaMKII ( approximately 0.8 microM). These findings led us to hypothesize that depolarization-mediated survival occurs through both the PI 3-kinase/Akt and the CaMKII pathways. Indeed, combined treatment with LY294002 (25 microM) and KN-62 (0.5 microM) dramatically abolished depolarization-mediated survival, whereas each alone did not significantly attenuate it. Under these conditions, KN-62 neither impaired sustained levels of intracellular Ca(2+), nor inhibited the phosphorylation of Akt. It is thus likely that PI 3-kinase and CaMKII independently promote the membrane depolarization-mediated survival of sympathetic neurons in culture.     

c-Kit receptor expression in normal human Schwann cells and Schwann cell lines derived from neurofibromatosis type 1 tumors

The growth factor receptor c-Kit has several well-characterized functions during the development of numerous cell types, including red blood cells, mast cells, and melanocytes. Its role in Schwann cells has been described in transformed cells derived from malignant peripheral nerve sheath tumors from patients with neurofibromatosis type 1 (NF1 MPNST; Badache et al. [1998] Oncogene 17:795-800). However, c-Kit functions have not been investigated in normal Schwann cells. We report here that neonatal rat Schwann cells express low c-Kit levels, whereas expression levels for c-Kit are high for Schwann cells derived from MPNST of NF1 patients. In addition, c-Kit expression is not detectable in normal adult human Schwann cells. Although the c-Kit ligand stem cell factor (SCF) induces the phosphorylation of protein kinase B (or Akt) and prevents apoptosis in Schwann cells, SCF has no effect on the proliferation or differentiation of Schwann cells.