Oroxylum indicum Stem Bark Extract Reduces Tumor Progression by Inhibiting the EGFR-PI3K-AKT Pathway in an In Vivo 4NQO-Induced Oral Cancer Model.

OBJECTIVES: Oral squamous cell carcinoma (OSCC) is the predominant type of oral cancer. Its incidence is high in certain geographic regions, and it is correlated with chewing tobacco. Epidermal growth factor receptor (EGFR), induced by tobacco carcinogens, is overexpressed in OSCC, leading to poor prognosis. Thus, EGFR inhibitors are promising agents against OSCC. High cost and toxicity of existing EGFR inhibitors necessitate alternative EGFR-targeted therapy. Here, we tested the antitumor potential of ethyl acetate fraction of an ethnomedicinal tree, Oroxylum indicum stem bark extract (OIEA) in a 4-nitroquinoline-1-oxide (4NQO)-induced oral carcinogenesis model. METHODS: OIEA was prepared by solvent extraction method, and subsequently its in vitro radical scavenging activities were measured. High-performance liquid chromatography (HPLC) analysis of OIEA was done to identify the constituent active compounds. Hemolytic, trypan blue exclusion, and MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assays were performed in normal and cancer cells to select an optimum dose of OIEA for antitumor activity study in 4NQO-induced oral cancer in F344 rats. Measurement of tumor volume, weight, and cell count was followed by tumor cell cycle analysis and comet and annexin V/Propidium Iodide (PI) assay. Pro-apoptotic markers were detected by western blot testing. Molecular docking was done to predict the interaction between OIEA active component and EGFR or phosphatidylinositol-3-kinase (PI3K), which was further validated biologically. Finally, hepatic and renal function testing and histopathology were performed. RESULTS: OIEA reduced tumor burden and increased survivability of the tumor-bearing rats significantly as compared to untreated tumor bearers. HPLC revealed oroxylin A as the predominant bioactive component in OIEA. Molecular docking predicted significant binding between oroxylin A and EGFR as well as PI3K, which was confirmed by western blot analysis of in vivo samples. OIEA also ameliorated hepato-, renal- and myelotoxicity induced by 4NQO. CONCLUSION: OIEA reduces 4NQO-induced OSCC by modulating the EGFR/PI3K/AKT signaling cascade and also ameliorated toxicity in tumor bearers.

The PI3K/Akt/FOXO3a pathway regulates regeneration following spinal cord injury in adult rats through TNF-α and p27kip1 expression.

The aim of the present study was to elucidate the expression and role of the phosphatidylinositol 3­kinase (PI3K)/Akt/forkhead box O3 (FOXO3a) pathway in the regeneration of the spinal cord following spinal cord injury (SCI), and its regulatory effect on tumor necrosis factor (TNF)-α and cyclin-dependent kinase inhibitor 1B (p27kip1) expression. Firstly, in a Sprague-Dawley rat model of SCI, western blot analysis revealed that the protein levels of PI3K, phosphorylated Akt and FOXO3a were markedly inhibited compared with those in the sham control group. In vitro experiments were also conducted, in which primary dissociated cultures of rat dorsal spinal cord cells were induced with lipopolysaccharide (LPS; 4 µg/ml). The downregulation of PI3K using LY294002 markedly suppressed cell viability, reduced the protein levels of FOXO3a and p27kip1, and increased TNF-α protein production in the LPS-induced spinal cord cells. In addition, when the LPS-induced spinal cord cells were infected with FOXO3a adenoviral vectors, the overexpression of FOXO3 markedly promoted cell proliferation, activated p27kip1 protein levels and inhibited TNF-α protein production in the spinal cord cells. These results suggest that the PI3K/Akt/FOXO3a pathway regulates regeneration following SCI in adult rats via its modulatory effects on TNF-α and p27kip1 expression.

TbVps15 is required for vesicular transport and cytokinesis in Trypanosoma brucei.

The class III phosphatidylinositol 3-kinase (PI3K) Vps34 is an important regulator of key cellular functions, including cell growth, survival, intracellular trafficking, autophagy and nutrient sensing. In yeast, Vps34 is associated with the putative serine/threonine protein kinase Vps15, however, its role in signaling has not been deeply evaluated. Here, we have identified the Vps15 orthologue in Trypanosoma brucei, named TbVps15. Knockdown of TbVps15 expression by interference RNA resulted in inhibition of cell growth and blockage of cytokinesis. Scanning electron microcopy revealed a variety of morphological abnormalities, with enlarged parasites and dividing cells that often exhibited a detached flagellum. Transmission electron microscopy analysis of TbVps15 RNAi cells showed an increase in intracellular vacuoles of the endomembrane system and some cells displayed an enlargement of the flagellar pocket, a common feature of cells defective in endocytosis. Moreover, uptake of dextran, transferrin and Concanavalin A was impaired. Finally, TbVps15 downregulation affected the PI3K activity, supporting the hypothesis that TbVps15 and TbVps34 form a complex as occurs in other organisms. In summary, we propose that TbVps15 has a role in the maintenance of cytokinesis, endocytosis and intracellular trafficking in T. brucei.

Inhibition of Phosphatidylinositol 3-kinease suppresses formation and progression of experimental abdominal aortic aneurysms.

Accumulating evidence suggests an important role of Phosphatidylinositol 3-kinease (PI3K) pathway in inflammatory cells infiltration. Given the essential role of inflammatory cells infiltration during the formation and progression of abdominal aortic aneurysm (AAA), to investigate the possibility of preventing AAA formation and progression via targeting PI3K is anticipated. Here, experimental AAAs was created in rats by transient intraluminal porcine pancreatic elastase (PPE) infusion into the infrarenal aorta firstly. AAAs rats were administrated with vehicle or Wortmannin during the period of day 0 to day 28 after PPE infusion. The aortic diameter of rats treated with Wortmannin was significantly smaller than those treated with vehicle. Meanwhile, Elastin destruction score and SMC destruction score were significantly decreased in rats treated with Wortmannin. Furthermore, histological analysis revealed infiltration of inflammatory cells were significantly reduced in rats treated with Wortmannin. Finally, the mRNA expression of PI3K and protein expression of pAKT in human abdominal aneurismal aorta tissues was elevated as compare to normal aorta. Our study revealed that PI3K inhibitor suppresses experimental AAAs formation and progression, through mechanisms likely related to impairing inflammation cells infiltration and median elastin degradation. These findings indicated that PI3K inhibitor may hold substantial translation value for AAA diseases.

Protective effect of cyanidin-3-O-glucoside on neonatal porcine islets.

Oxidative stress is a major cause of islet injury and dysfunction during isolation and transplantation procedures. Cyanidin-3-O-glucoside (C3G), which is present in various fruits and vegetables especially in Chinese bayberry, shows a potent antioxidant property. In this study, we determined whether C3G could protect neonatal porcine islets (NPI) from reactive oxygen species (H2O2)-induced injury in vitro and promote the function of NPI in diabetic mice. We found that C3G had no deleterious effect on NPI and that C3G protected NPI from damage induced by H2O2 Significantly higher hemeoxygenase-1 (HO1) gene expression was detected in C3G-treated NPI compared to untreated islets before and after transplantation (P < 0.05). Western blot analysis showed a significant increase in the levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K/Akt) proteins in C3G-treated NPI compared to untreated islets. C3G induced the nuclear translocation of nuclear erythroid 2-related factor 2 (NRF2) and the significant elevation of HO1 protein. Recipients of C3G-treated NPI with or without C3G-supplemented drinking water achieved normoglycemia earlier compared to recipients of untreated islets. Mice that received C3G-treated islets with or without C3G-supplemented water displayed significantly lower blood glucose levels at 5-10 weeks post-transplantation compared to mice that received untreated islets. Mice that received C3G-treated NPI and C3G-supplemented drinking water had significantly (P < 0.05) lower blood glucose levels at 7 and 8 weeks post-transplantation compared to mice that received C3G-treated islets. These findings suggest that C3G has a beneficial effect on NPI through the activation of ERK1/2- and PI3K/AKT-induced NRF2-mediated HO1 signaling pathway.

Gastric electrical stimulation improves enteric neuronal survival.

Research and clinical experience with vagotomy have confirmed that damage to the central nervous system severely affects physiological movement in the gastrointestinal system. The aim of this study was to investigate the effects of synchronized dual-pulse gastric electrical stimulation (SGES) on the apoptosis of enteric neurons and the possible pathways involved in these effects in vagotomized rats. For this purpose, Male Sprague-Dawley (SD) rats were randomized into a control group, an early subdiaphragmatic vagotomized group (ESDV group), an early subdiaphragmatic vagotomized group with short-term SGES (ESDV + SSGES group), a terminal subdiaphragmatic vagotomized group (TSDV group) and a terminal subdiaphragmatic vagotomized group with long-term SGES (TSDV + LSGES group). The expression levels of connexin 43 (Cx43), glial cell line-derived neurotrophic factor (GDNF), p-Akt, pan-Akt and PGP9.5 were assessed by RT-qPCR, western blot analysis and immunofluorescence staining. Apoptosis was determined by terminal-deoxynucleoitidyl transferase­mediated nick-end labeling (TUNEL) assay. We found that Cx43 expression was decreased in the ESDV and TSDV groups, but was significantly upregulated in the SSGES and LSGES groups. In addition, the GDNF and PGP9.5 expression levels were significantly decreased in the ESDV group compared with the control and TSDV groups and were upregulated in both the SSGES and LSGES groups. The LSGES group exhibited a clear increase in p-Akt expression compared with the TSDV group. Fewer TUNEL-positive cells were observed in the SSGES and LSGES groups than in the ESDV and TSDV groups. More TUNEL-positive cells were found in the stomach of rats subjected to subdiaphragmatic vagotomy. On the whole, our data indicate that SGES improved enteric neuronal survival, possibly through GDNF and the phosphatidylinositol 3-kinase (PI3K)/Akt pathways.

PTEN/PI3K/AKT protein expression is related to clinicopathological features and prognosis in breast cancer with axillary lymph node metastases.

We explored the relations between PTEN/PI3K/AKT expression and clinicopathological characteristics and prognosis in breast cancer patients with and without axillary lymph node metastasis (LNM). Tissues and follow-up data from 142 patients with (LNM group) and 154 without (non-LNM group) metastases were collected. Expression of PTEN/PI3K/AKT was detected using immunohistochemistry staining. With axillary LNM, the positive rate of PTEN was reduced, whereas that of PI3K and AKT was increased. Expression of AKT was negatively correlated with PTEN expression but positively correlated with PI3K expression. Apparent correlations were detected between AKT and axillary LNM with a tumor size of 2 cm or less; between PTEN, PI3K, and AKT and axillary LNM in stage T1 or T2 breast cancer and invasive carcinoma of a nonspecial type; and between PTEN and AKT and axillary LNM of histologic grade I or II tumors and non-triple-negative breast cancer (all P<.05). In the LNM group, the 5-year survival rate of patients with PTEN-positive tumors was higher than that of patients with PTEN-negative lesions; whereas in the non-LNM group, the 5-year survival rate of patients with AKT-positive tumors was lower than that of patients with AKT-negative lesions (both P<.05). Cox regression analysis showed that PTEN expression was an independent prognostic factor for patients with LNM; AKT expression, tumor diameter, pathologic grade, and pathologic type were independent prognostic factors for patients without LNM. In conclusion, TEN/PI3K/AKT proteins are related to the clinicopathological features and prognosis of breast cancer with axillary LNM.

Effect of atorvastatin on wound healing in rats.

Skin-wound healing is a complex and dynamic biological process involving inflammation, proliferation, and remodeling. Recent studies have shown that statins are new therapeutical options because of their actions, such as anti-inflammatory and antioxidant activity, on vasodilation, endothelial dysfunction and neoangiogenesis, which are independent of their lipid-lowering action. Our aim was to investigate the effect of atorvastatin on tissue repair after acute injury in healthy animals. Rats were divided into four groups: placebo-treated (P), topical atorvastatin-treated (AT), oral atorvastatin-treated (AO), topical and oral atorvastatin-treated (ATO). Under anesthesia, rats were wounded with an 8-mm punch in the dorsal region. Lesions were photographed on Days 0, 1, 3, 7, 10, 12, and 14 post-injury and samples taken on Days 1, 3, 7, and 14 for protein-expression analysis of insulin receptor substrate (IRS)-1, phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), glycogen synthase kinase (GSK)-3, endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), extracellular signal-regulated kinase (ERK), interleukin (IL)-10, IL-1ß, IL-6, and tumor necrosis factor (TNF)-α. Upon macroscopic examination, we observed significant reductions of lesion areas in groups AT, AO, and ATO compared to the P group. Additionally, AT and AO groups showed increased expression of IRS-1, PI3K, Akt, GSK-3, and IL-10 on Days 1 and 3 when compared with the P group. All atorvastatin-treated groups showed higher expression of IRS-1, PI3K, Akt, GSK-3, IL-10, eNOS, VEGF, and ERK on Day 7. On Days 1, 3, and 7, all atorvastatin-treated groups showed lower expression of IL-6 and TNF-α when compared with the P group. We conclude that atorvastatin accelerated tissue repair of acute lesions in rats and modulated expressions of proteins and cytokines associated with cell-growth pathways.

Differential distribution of PI3K isoforms in spinal cord and dorsal root ganglia: potential roles in acute inflammatory pain.

PI3-kinases (PI3Ks) participate in nociception within spinal cord, dorsal root ganglion (DRG), and peripheral nerves. To extend our knowledge, we immunohistochemically stained for each of the 4 class I PI3K isoforms along with several cell-specific markers within the lumbar spinal cord, DRG, and sciatic nerve of naive rats. Intrathecal and intraplantar isoform specific antagonists were given as pretreatments before intraplantar carrageenan; pain behavior was then assessed over time. The α-isoform was localized to central terminals of primary afferent fibers in spinal cord laminae IIi to IV as well as to neurons in ventral horn and DRG. The PI3Kß isoform was the only class I isoform seen in dorsal horn neurons; it was also observed in DRG, Schwann cells, and axonal paranodes. The δ-isoform was found in spinal cord white matter oligodendrocytes and radial astrocytes, and the γ-isoform was seen in a subpopulation of IB4-positive DRG neurons. No isoform co-localized with microglial markers or satellite cells in naive tissue. Only the PI3Kß antagonist, but none of the other antagonists, had anti-allodynic effects when administered intrathecally; coincident with reduced pain behavior, this agent completely blocked paw carrageenan-induced dorsal horn 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (AMPA) receptor trafficking to plasma membranes. Intraplantar administration of the γ-antagonist prominently reduced pain behavior. These data suggest that each isoform displays specificity with regard to neuronal type as well as to specific tissues. Furthermore, each PI3K isoform has a unique role in development of nociception and tissue inflammation.

Human sperm anatomy: different expression and localization of phosphatidylinositol 3-kinase in normal and varicocele human spermatozoa.

Abstract Recent reports support the possible role of PI3K in sperm capacitation and acrosome reaction, although studies regarding PI3K identity in human sperm, under certain disease states such as varicocele, are still lacking. The authors, therefore, examined the expression profile and ultrastructural localization of PI3K in human semen samples, comparing healthy donors and patients with varicocele. The results obtained performing western blotting assay showed decreased PI3K expression in varicocele with respect to the "healthy" sperm. Immunogold labeling revealed human sperm cellular compartments containing PI3K, evidencing it in the head at both the membrane and nucleus and the entire tail, from the middle to the end piece of normal sperm. In varicocele PI3K label was confined to the head, with a strong reduction of specific reaction in the neck, middle piece, and tail. In conclusion, the data suggest that PI3K may play a role in the maintenance of male factor infertility associated with varicocele, and it may be further exploited as an additional molecular marker for the diagnosis of male infertility disorders.

Localization and signaling patterns of vascular endothelial growth factors and receptors in human periapical lesions.

INTRODUCTION: Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key players in vasculogenesis and are also involved in pathologic conditions with bone destruction. Vasculogenesis is critical for disease progression, and bone resorption is a hallmark of apical periodontitis. However, the localization of VEGFs and VEGFRs and their gene signaling pathways in human apical periodontitis have not been thoroughly investigated. The aim of this study was to localize VEGFs and VEGFRs and analyze their gene expression as well as signaling pathways in human periapical lesions. METHODS: Tissue was collected after endodontic surgery from patients diagnosed with chronic apical periodontitis. Periodontal ligament samples from extracted healthy wisdom teeth was also collected and used as control tissue. In lesion cryosections, VEGFs/VEGFRs were identified by immunohistochemistry/double immunofluorescence by using specific antibodies. A human VEGF signaling polymerase chain reaction array system was used for gene expression analysis comparing lesions with periodontal ligament samples. RESULTS: The histologic evaluation revealed heterogeneous morphology of the periapical lesions with various degrees of inflammatory infiltrates. In the lesions, all investigated factors and receptors were identified in blood vessels and various immune cells. No lymphatic vessels were detected. Gene expression analysis revealed up-regulation of VEGF-A and VEGFR-3, although not significant. Phosphatidylinositol-3-kinases, protein kinase C, mitogen-activated protein kinases, and phospholipases, all known to be involved in VEGF-mediated angiogenic activity, were significantly up-regulated. CONCLUSIONS: The cellular and vascular expressions of VEGFs and VEGFRs in chronic apical periodontitis, along with significant alterations of genes mediating VEGF-induced angiogenic responses, suggest ongoing vascular remodeling in established chronic periapical lesions.

Cellular infiltrates and NFκB subunit c-Rel signaling in kidney allografts of patients with clinical operational tolerance.

BACKGROUND: Nuclear factor kappa B (NFκB) plays a potential role in tolerance by orchestrating onset and resolution of inflammation and regulatory T cell differentiation through subunit c-Rel. We characterized cellular infiltrates and expression of NFκB1, c-Rel and its upstream regulators phosphatidylinositol 3-kinase/RAC-alpha serine/threonine kinase, in allograft biopsies from patients with spontaneous clinical operational tolerance (COT). METHODS: Paraffin-fixed kidney allograft biopsies from 40 patients with COT (n=4), interstitial rejection (IR; n=12), borderline changes (BC; n=12), and long-term allograft function without rejection (NR; n=12) were used in the study. Cellular infiltrates and immunohistochemical expression of key proteins of the NFκB pathway were evaluated in the cortical tubulointerstitium and in cellular infiltrates using digital image analysis software. Results were given as mean±SEM. RESULTS: Biopsies from patients with COT exhibited a comparable amount of cellular infiltrate to IR, BC, and NR (COT, 191±81; IR, 291±62; BC, 178±45; and NR, 210±42 cells/mm) but a significantly higher proportion of forkhead box P3-positive cells (COT, 11%±1.7%; IR, 3.5%±0.70%; BC, 3.4%±0.57%; and NR, 3.7%±0.78% of infiltrating cells; P=0.02). c-Rel expression in cellular infiltrates was significantly elevated in IR, BC, and NR when analyzing the number of positive cells per mm (P=0.02) and positive cells per infiltrating cells (P=0.04). In contrast, tubular PI3K and c-Rel expression were significantly higher in IR and BC but not in NR compared with COT (P=0.03 and P=0.006, respectively). With RAC-alpha serine-threonine kinase, similar tendencies were observed (P=0.2). CONCLUSIONS: Allografts from COT patients show significant cellular infiltrates but a distinct expression of proteins involved in the NFκB pathway and a higher proportion of forkhead box P3-positive cells.

HGF/C-MET system pathways in benign and malignant histotypes of thyroid nodules: an immunohistochemical characterization.

OBJECTIVE: Upon binding with HGF, the thyrosine-kinase receptor c-met induces cell growth, scattering and morphogenic effects via the trasducers STAT3 and phosphorylated-STAT3, PI3K/Akt, Rho. HGF, c-met and STAT3 are expressed with very high frequency in papillary thyroid carcinomas (PTC), suggesting a role in PTC. Herein we first investigate the simultaneous expression of HGF, c-met, STAT3, phosphor-STAT3, PI3K, Akt and Rho in thyroid nodules. DESIGN AND METHODS: Using immunohistochemistry, we studied: 30 colloid nodules (CN), 18 hyperplastic nodules (HN), 20 follicular adenomas (FA), 15 oncocytic adenomas (OA), 20 PTC, 16 follicular carcinomas (FTC) and 6 anaplastic carcinomas (ATC). RESULTS: All 7 proteins were expressed in 15% of FA (with HGF, PI3K and Rho stromal reactivity) and 25% of PTC, and the combination HGF/c-met/STAT3/ pSTAT3/PI3K was expressed by all PTC, each protein being expressed by tumor cells. In contrast, 13/16 FTC (81%) exhibited immunoreactivity for PI3K (both epithelial and stromal), and 100% of ATC was PI3K+ (both epithelial and stromal) and Rho+ (epithelial). Epithelial expression of PI3K correlated with the clinical behavior of histotypes and, within FTC, the proportion of PI3K+ cells correlated with both the clinical and pathological stage (r=0.95; p<0.001). As for the shared epithelial expression of PI3K, this concerned approximately one-fourth of tumor cells in FTC and ATC vs one-thirtieth in PTC. CONCLUSIONS: Our data may have practical implications for the targeted medical therapy of thyroid cancer arising from the follicular epithelium.