Multiple defects in negative regulation of the PKB/Akt pathway sensitise human cancer cells to the antiproliferative effect of non-steroidal anti-inflammatory drugs

Antitumorigenic effects of non-steroidal anti-inflammatory drugs (NSAIDs) are well established in several types of cancer disease. However, the mechanisms driving these processes are not understood in all details. In our study, we observed significant differences in sensitivity of cancer epithelial cell lines to COX-independent antiproliferative effects of NSAIDs. The prostate cancer cell line LNCaP, lacking both critical enzymes in the negative control of PKB/Akt activation, PTEN and SHIP2, was the most sensitive to these effects, as assessed by analysing the cell cycle profile and expression of cell cycle regulating proteins. We found that p53 protein and its signalling pathway is not involved in early antiproliferative action of the selected NSAID—indomethacin. RNAi provided evidence for the involvement of p21^Cip1/Waf1, but not GDF-15, in antiproliferative effects of indomethacin in LNCaP cells. Interestingly, we also found that indomethacin activated PKB/Akt and induced nuclear localisation of p21^Cip1/Waf1 and Akt2 isoform. Our results are in agreement with other studies and suggest that maintaining of the p21^Cip1/Waf1 level and its intracellular localisation might be influenced by Akt2. Knock-down of SHIP2 by RNAi in PTEN negative prostate and colon cancer cell lines resulted in higher sensitivity to antiproliferative effects of indomethacin. Our data suggest novel mechanisms of NSAIDs antiproliferative action in cancer epithelial cells, which depends on the status of negative regulation of the PKB/Akt pathway and the isoform-specific action of Akt2. Thus, unexpectedly, multiple defects in negative regulation of the PKB/Akt pathway may contribute to increased sensitivity to chemopreventive effects of these widely used drugs.     

E6 variants of human papillomavirus 18 differentially modulate the protein kinase B/phosphatidylinositol 3-kinase (akt/PI3K) signaling pathway

Intra-type genome variations of high risk Human papillomavirus (HPV) have been associated with a differential threat for cervical cancer development. In this work, the effect of HPV18 E6 isolates in Akt/PKB and Mitogen-associated protein kinase (MAPKs) signaling pathways and its implication in cell proliferation were analyzed. E6 from HPV types 16 and 18 are able to bind and promote degradation of Human disc large (hDlg). Our results show that E6 variants differentially modulate hDlg degradation, rebounding in levels of activated PTEN and PKB. HPV18 E6 variants are also able to upregulate phospho-PI3K protein, strongly correlating with activated MAPKs and cell proliferation. Data was supported by the effect of E6 silencing in HPV18-containing HeLa cells, as well as hDlg silencing in the tested cells. Results suggest that HPV18 intra-type variations may derive in differential abilities to activate cell-signaling pathways such as Akt/PKB and MAPKs, directly involved in cell survival and proliferation.     

银屑病皮损中PKB、NF-κBp65、Bcl-xL的表达及相关性研究

目的探讨PKB、NF-κBp65、Bcl-xL与寻常型银屑病发病的相关性。方法采用S-P免疫组织化学法检测30例寻常性银屑病患者皮损及10例正常人皮肤石蜡包埋组织标本中PKB、NF-κBp65、Bcl-xL的表达与定位。结果与正常对照组相比,银屑病皮损角质形成细胞中PKB、NF-κBp65、Bcl-xL的表达均明显上调,其差异有统计学意义(P<0.05)。在银屑病皮损组织角质形成细胞中,随着PASI评分的增加,PKB、NF-κBp65、Bcl-xL的表达逐渐增强,其表达水平与患者PASI评分之间均存在正相关,有统计学意义(P<0.01)。PKB与NF-κBp65、PKB与Bcl-xL、NF-κBp65与Bcl-xL之间的表达水平均存在正相关,有统计学意义(P<0.01)。结论 PKB、NF-κBp65、Bcl-xL可能与银屑病发病及银屑病疾病严重程度有关。     

Looking at the blood-brain barrier: Molecular anatomy and possible investigation approaches

The blood-brain barrier (BBB) is a dynamic and complex interface between blood and the central nervous system that strictly controls the exchanges between the blood and brain compartments, therefore playing a key role in brain homeostasis and providing protection against many toxic compounds and pathogens. In this review, the unique properties of brain microvascular endothelial cells and intercellular junctions are examined. The specific interactions between endothelial cells and basement membrane as well as neighboring perivascular pericytes, glial cells and neurons, which altogether constitute the neurovascular unit and play an essential role in both health and function of the central nervous system, are also explored. Some relevant pathways across the endothelium, as well as mechanisms involved in the regulation of BBB permeability, and the emerging role of the BBB as a signaling interface are addressed as well. Furthermore, we summarize some of the experimental approaches that can be used to monitor BBB properties and function in a variety of conditions and have allowed recent advances in BBB knowledge. Elucidation of the molecular anatomy and dynamics of the BBB is an essential step for the development of new strategies directed to maintain or restore BBB integrity and barrier function and ultimately preserve the delicate interstitial brain environment.     

Inhibition of ROS production in peripheral blood mononuclear cells from type 2 diabetic patients by autologous plasma depends on Akt/PKB signaling pathway

OBJECTIVE: To compare the role of Akt/PKB signaling pathway in the modulation of reactive oxygen species (ROS) production by autologous plasma in peripheral blood mononuclear cells (PBMNC) from type 2 diabetic patients and healthy subjects. MATERIALS AND METHODS: This study was approved by Santa Casa Ethical Committee and has included patients diagnosed with diabetes type 2 (DM2) and control group (non-diabetic) (ND). PBMNC were purified utilizing Ficoll-hypaque gradient. ROS was quantified by luminol-dependent chemiluminescence. The Akt/PKB phosphorylation was measured using a CASE kit. Statistical analyses were made with t Student test and chi-square (chi(2)). p<0.05 was considered significant. RESULTS: 12, 13-Phorbol dibutyrate (PDB) stimulated the production of higher levels of ROS in PBMNC from type 2 diabetic patients than that from healthy subjects. Autologous plasma, however, inhibited induced or not ROS production in PBMNC in both groups. The inhibition of PBMNC-ROS derived by autologous plasma from healthy subjects was higher than that from type 2 diabetic patients. Plasma phosphorylated (activated) Akt/PKB. The percentage of phosphorylation induced by autologous plasma in PBMNC from patients and healthy control were 14% and 93%, respectively. Inhibition of ROS production in PBMNC from DM2 were similar for PBMNC+plasma; PBMNC+Akti; and PBMNC+plasma+Akti. However, in ND control, plasma showed a higher ROS inhibition than Akti or plasma plus Akti. CONCLUSIONS: Our results suggest that the low antioxidant capacity observed in autologous plasma from DM2 patients in conjunction with the decreased activation of PKB may cause an imbalance in the oxidizing/reducing responses, possible inducing an oxidative stress state, which could be associated with tissular damage.     

Oncogenic HRAS mutations cause prolonged PI3K signaling in response to epidermal growth factor in fibroblasts of patients with Costello syndrome

Costello syndrome (CS) is a rare congenital disorder characterized by failure to thrive, craniofacial dysmorphisms, cardiac and skin abnormalities, mental retardation, and predisposition to malignancies. CS is caused by heterozygous gain‐of‐function mutations in HRAS that also occur as somatic alterations in human tumors. HRAS is one of the three classical RAS proteins and cycles between an active, GTP‐ and an inactive, GDP‐bound conformation. We used primary human skin fibroblasts from patients with CS as a model system to study the functional consequences of HRAS mutations on endogenous signaling pathways. The GTP‐bound form of HRAS was significantly enriched in CS compared with normal fibroblasts. Active HRAS is known to stimulate both the RAF‐MEK‐ERK and the PI3K–AKT signaling cascade. Phosphorylation of MEK and ERK was normal in CS fibroblasts under basal conditions and slightly prolonged after epidermal growth factor (EGF) stimulation. Interestingly, basal phosphorylation of AKT was increased yet more in CS fibroblasts. Moreover, AKT phosphorylation was diminished in the early and enhanced in the late phase of EGF stimulation. Taken together, these results document that CS‐associated HRAS mutations result in prolonged signal flux in a ligand‐dependent manner. Our data suggest that altered cellular response to growth factors rather than constitutive activation of HRAS downstream signaling molecules may contribute to some of the clinical features in patients with CS. Hum Mutat 0,1–11, 2008. © 2008 Wiley‐Liss, Inc.     

Tyrosine phosphorylation of insulin receptor substrates during ischemia/reperfusion-induced apoptosis in rat liver

Background  Phosphoregulation of signal transduction pathways is a complex series of reactions that may modulate the cellular response to ischemia–reperfusion (I–R). The aim of this study was to evaluate the effect of normothermic liver I/R-induced apoptosis on phosphorylation and activation of signal proteins in tyrosine kinase pathways. Materials and methods  In rats, a segmental normothermic ischemia of the liver was induced for 120 min. Liver apoptosis was determined using terminal deoxynucleotide-transferase-mediated deoxyuridine triphosphate nick end labeling assay, and activity of caspases-3 and -7 was determined by fluorescence. Liver tyrosine phosphorylation of proteins was examined by Western blot analysis. Results  Normothermic I–R resulted in increased in vivo caspases-3 and -7 activity and in liver apoptosis. Shc tyrosine phosphorylation and activation of ERK1/2 were increased after reperfusion, while tyrosine phosphorylation of IRS-1 and activation of PKB/Akt were decreased. Conclusions  Normothermic liver I–R leads to increased apoptosis and to modifications in protein tyrosine phosphorylation pathways. Scientific meetings: Presented to the World Transplant Congress, Boston, USA, July 22–27, 2006, the 7th World Congress of the International Hepato-Pancreato-Biliary Association, Edinburg, UK, September 3–7, 2006, and the 31st Congress of the Società Italiana Trapianti d’Organo, Modena, Italy, November 28–38, 2007. Raffaele Cursio and Claudia Miele have contributed equally to this work     

Molecular targets of dietary agents for prevention and therapy of cancer

While fruits and vegetables are recommended for prevention of cancer and other diseases, their active ingredients (at the molecular level) and their mechanisms of action less well understood. Extensive research during the last half century has identified various molecular targets that can potentially be used not only for the prevention of cancer but also for treatment. However, lack of success with targeted monotherapy resulting from bypass mechanisms has forced researchers to employ either combination therapy or agents that interfere with multiple cell-signaling pathways. In this review, we present evidence that numerous agents identified from fruits and vegetables can interfere with several cell-signaling pathways. The agents include curcumin (turmeric), resveratrol (red grapes, peanuts and berries), genistein (soybean), diallyl sulfide (allium), S-allyl cysteine (allium), allicin (garlic), lycopene (tomato), capsaicin (red chilli), diosgenin (fenugreek), 6-gingerol (ginger), ellagic acid (pomegranate), ursolic acid (apple, pears, prunes), silymarin (milk thistle), anethol (anise, camphor, and fennel), catechins (green tea), eugenol (cloves), indole-3-carbinol (cruciferous vegetables), limonene (citrus fruits), beta carotene (carrots), and dietary fiber. For instance, the cell-signaling pathways inhibited by curcumin alone include NF-kappaB, AP-1, STAT3, Akt, Bcl-2, Bcl-X(L), caspases, PARP, IKK, EGFR, HER2, JNK, MAPK, COX2, and 5-LOX. The active principle identified in fruit and vegetables and the molecular targets modulated may be the basis for how these dietary agents not only prevent but also treat cancer and other diseases. This work reaffirms what Hippocrates said 25 centuries ago, let food be thy medicine and medicine be thy food.