Transforming growth factor β-interacting factor-induced malignant progression of hepatocellular carcinoma cells depends on superoxide production from Nox4

Hepatocellular carcinoma (HCC) is one of the most deadly malignancies worldwide because of its high recurrence rate, high metastatic potential, and resistance to drugs. Elucidation of the mechanisms underlying malignancy in HCC is needed to improve diagnosis, therapy, and prognosis. Previously, we showed that transforming growth factor β-interacting factor (TGIF) antagonizes arsenic trioxide-induced apoptosis of HepG2 cells and is associated with poor prognosis and progression of urothelial carcinoma in patients after radical nephroureterectomy. To determine whether TGIF plays a role in HCC tumorigenesis, we compared the expression of TGIF, its downstream targets, and reactive oxygen species levels between HCC HepG2 cells and the more invasive SK-Hep1 cells. Superoxide production, phosphorylation of c-Src^Y416 and AKT^S473, and expression of TGIF and NADPH oxidase (Nox) were higher in invasive SK-Hep1 cells than in HepG2 cells. TGIF-overexpressing HepG2 xenograft tumors markedly promoted tumor growth and metastasis to the lungs. Overexpression of TGIF in HepG2 cells increased superoxide production from Nox4, matrix metalloproteinase expression, invadopodia formation, and cellular migration/invasion ability. Conversely, knockdown of TGIF in SK-Hep1 cells attenuated these processes. Using gene knockdown and pharmacological inhibitors, we demonstrate that c-Src/AKT is the upstream signaling that regulates TGIF-induced Nox4 activation and subsequent superoxide production. Taken together, our results implicate TGIF as a potential biomarker for prognosis and target for clinical therapy in patients with advanced HCC.     

NAD(P)H oxidase 1, a product of differentiated colon epithelial cells,can partially replace glycoprotein 91phox in the regulated production of superoxide by phagocytes

Reactive oxygen species (ROS) serve several physiological functions; in some settings they act in host defense, while in others they function in cellular signaling or in biosynthetic reactions. We studied the expression and function of a recently described source of ROS, NAD(P)H oxidase 1 or Nox1, which has been associated with cell proliferation. In situ hybridization in mouse colon revealed high Nox1 expression within the lower two-thirds of colon crypts, where epithelial cells undergo proliferation and differentiation. Human multitumor tissue array analysis confirmed colon-specific Nox1 expression, predominantly in differentiated epithelial tumors. Differentiation of Caco2 and HT29 cells with 1alpha,25-dihydroxyvitamin D(3) or IFN-gamma enhances Nox1 expression and decreases cell proliferation, suggesting that Nox1 does not function as a mitogenic oxidase in colon epithelial cells. Transduction with retrovirus encoding Nox1 restored activation and differentiation-dependent superoxide production in gp91(phox)-deficient PLB-985 cells, indicating close functional similarities to the phagocyte oxidase (phox). Furthermore, coexpression of cytosolic components, p47(phox) and p67(phox), augments Nox1 activity in reconstituted K562 cells. Finally, Nox1 partially restores superoxide production in neutrophils differentiating ex vivo from gp91(phox)-deficient CD34(+) peripheral blood-derived stem cells derived from patients with X-linked chronic granulomatous disease. These studies demonstrate a significant functional homology (cofactor-dependent and activation-regulated superoxide production) between Nox1 and its closest homologue, gp91(phox), suggesting that targeted up-regulation of Nox1 expression in phagocytic cells could provide a novel approach in the molecular treatment of chronic granulomatous disease.     

Phosphatidylinositol 3-kinase-dependent membrane recruitment of Rac-1 and p47phox is critical for alpha-platelet-derived growth factor receptor-induced production of reactive oxygen species

Platelet-derived growth factor (PDGF) plays a critical role in the pathogenesis of proliferative diseases. NAD(P)H oxidase (Nox)-derived reactive oxygen species (ROS) are essential for signal transduction by growth factor receptors. Here we investigated the dependence of PDGF-AA-induced ROS production on the cytosolic Nox subunits Rac-1 and p47(phox), and we systematically evaluated the signal relay mechanisms by which the alphaPDGF receptor (alphaPDGFR) induces ROS liberation. Stimulation of the alphaPDGFR led to a time-dependent increase of intracellular ROS levels in fibroblasts. Pharmacological inhibitor experiments and enzyme activity assays disclosed Nox as the source of ROS. alphaPDGFR activation is rapidly followed by the translocation of p47(phox) and Rac-1 from the cytosol to the cell membrane. Experiments performed in p47(phox)(-/-) cells and inhibition of Rac-1 or overexpression of dominant-negative Rac revealed that these Nox subunits are required for PDGF-dependent Nox activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA-dependent ROS production, we investigated Ph cells expressing mutant alphaPDGFRs that lack specific binding sites for alphaPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase (PI3K), phospholipase Cgamma, and SHP-2). Lack of PI3K signaling (but not Src, phospholipase Cgamma, or SHP-2) completely abolished PDGF-dependent p47(phox) and Rac-1 translocation, increase of Nox activity, and ROS production. Conversely, a mutant alphaPDGFR able to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110alpha (but not p110beta) was identified as the crucial isoform that elicits alphaPDGFR-mediated production of ROS. Finally, bromodeoxyuridine incorporation and chemotaxis assays revealed that the lack of ROS liberation blunted PDGF-AA-dependent chemotaxis but not cell cycle progression. We conclude that PI3K/p110alpha mediates growth factor-dependent ROS production by recruiting p47(phox) and Rac-1 to the cell membrane, thereby assembling the active Nox complex. ROS are required for PDGF-AA-dependent chemotaxis but not proliferation.     

Mechanism of angiotensin II-induced superoxide production in cells reconstituted with angiotensin type 1 receptor and the components of NADPH oxidase

The mechanism of angiotensin II (Ang II)-induced superoxide production was investigated with HEK293 or Chinese hamster ovary cells reconstituted with the angiotensin type 1 receptor (AT(1)R) and NADPH oxidase (either Nox1 or Nox2) along with a pair of adaptor subunits (either NOXO1 with NOXA1 or p47(phox) with p67(phox)). Ang II enhanced the activity of both Nox1 and Nox2 supported by either adaptor pair, with more effective activation of Nox1 in the presence of NOXO1 and NOXA1 and of Nox2 in the presence of p47(phox) and p67(phox). Expression of several AT(1)R mutants showed that interaction of the receptor with G proteins but not that with beta-arrestin or with other proteins (Jak2, phospholipase C-gamma1, SH2 domain-containing phosphatase 2) that bind to the COOH-terminal region of AT(1)R, was necessary for Ang II-induced superoxide production. The effects of constitutively active alpha subunits of G proteins and of various pharmacological agents implicated signaling by a pathway comprising AT(1)R, Galpha(q/11), phospholipase C-beta, and protein kinase C as largely, but not exclusively, responsible for Ang II-induced activation of Nox1 and Nox2 in the reconstituted cells. A contribution of Galpha(12/13), phospholipase D, and phosphatidyl-inositol 3-kinase to Ang II-induced superoxide generation was also suggested, whereas Src and the epidermal growth factor receptor did not appear to participate in this effect of Ang II. In reconstituted cells stimulated with Ang II, Nox2 exhibited a more sensitive response than Nox1 to the perturbation of protein kinase C, phosphatidylinositol 3-kinase, or the small GTPase Rac1.     

The NADPH oxidase Nox3 constitutively produces superoxide in a p22phox-dependent manner: its regulation by oxidase organizers and activators

Nox3, a member of the superoxide-producing NADPH oxidase (Nox) family, participates in otoconia formation in mouse inner ears, which is required for perception of balance and gravity. The activity of other Nox enzymes such as gp91(phox)/Nox2 and Nox1 is known to absolutely require both an organizer protein (p47(phox) or Noxo1) andanactivatorprotein (p67(phox) or Noxa1); for the p47(phox)-dependent activation of these oxidases, treatment of cells with stimulants such as phorbol 12-myristate 13-acetate is also indispensable. Here we show that ectopic expression of Nox3 in various types of cells leads to phorbol 12-myristate 13-acetate-independent constitutive production of a substantial amount of superoxide under the conditions where gp91(phox) and Nox1 fail to generate superoxide, i.e. in the absence of the oxidase organizers and activators. Nox3 likely forms a functional complex with p22(phox); Nox3 physically interacts with and stabilizes p22(phox), and the Nox3-dependent superoxide production is totally dependent on p22(phox). The organizers p47(phox) and Noxo1 are capable of enhancing the superoxide production by Nox3 in the absence of the activators, and the enhancement requires the interaction of the organizers with p22(phox), further indicating a link between Nox3 and p22(phox). The p47(phox)-enhanced Nox3 activity is further facilitated by p67(phox) or Noxa1, whereas the activators cancel the Noxo1-induced enhancement. On the other hand, the small GTPase Rac, essential for the gp91(phox) activity, is likely dispensable to the Nox3 system. Thus Nox3 functions together with p22(phox) as an enzyme constitutively producing superoxide, which can be distinctly regulated by combinatorial use of the organizers and activators.     

Role of the small GTPase Rac in p22phox-dependent NADPH oxidases

The superoxide-producing phagocyte NADPH oxidase gp91(phox)/Nox2 and the non-phagocytic oxidases Nox1 and Nox3 each form a complex in the membrane with p22(phox), which provides both stabilization and a docking site for organizer proteins. The p22(phox)-complexed Nox2 and Nox1 are dormant on their own, and their activation requires soluble supportive proteins such as a Nox organizer (p47(phox) or Noxo1) and a Nox activator (p67(phox) or Noxa1). The small GTPase Rac directly binds to the activators, and thus plays an essential role in the Nox2-based oxidase containing p47(phox) and p67(phox) or a positive role in Nox1 activity supported by Noxo1 and Noxa1. Although Nox3 complexed with p22(phox) constitutively produce superoxide, the production can be enhanced by supportive proteins. Here we compare the roles of Rac in these p22(phox)-dependent oxidases using the organizer and activator in different combinations. Expression of constitutively active Rac1(Q61L) is essential for activation of the Nox2- or Nox1-based oxidase containing the organizer p47(phox) and either p67(phox) or Noxa1. When these oxidases use Noxo1 as an organizer instead of p47(phox), they produce a small but significant amount of superoxide without expression of Rac1(Q61L), although the production is enhanced by Rac1(Q61L). Thus p47(phox) is likely related to strict dependence on Rac. The Nox3-based oxidase has a similar tendency in the change of the dependence: Rac plays a positive role in Nox3 activation in the presence of p47(phox) and either p67(phox) or Noxa1, whereas Rac fails to upregulate Nox3 activity when p47(phox) is replaced with Noxo1. We also demonstrate that, in the Nox3-based oxidase containing solely p67(phox) as supportive protein, expression of Rac1(Q61L) enhances not only superoxide production but also membrane translocation of p67(phox). Since the enhancements are not observed with a mutant p67(phox) defective in binding to Rac, this GTPase appear to directly recruit p67(phox) to the membrane.     

Role of PI3K and AKT specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway

Ovarian cancer is the leading cause of death from gynecological malignancy for women. The amplification of the PI3K catalytic subunit (p110) and the lost function of PTEN are frequently detected in ovarian cancer cells. PI3K plays an important role in tumorigenesis. To specifically inhibit PI3K activity in ovarian cancer cells, we constructed small interfering RNA (siRNA) against p110. The expression of p110 siRNA significantly decreased cell migration, invasion, and proliferation compared to the siSCR control cells. The expression of p110 siRNA induced CDK inhibitor p27^KIP1 levels, and decreased levels of cyclin D1, CDK4, and phosphorylated retinoblastoma protein. PI3K transmits the mytogenic signal through AKT. AKT has three isoforms in the cells: AKT1, AKT2 and AKT3. We found that inhibition of AKT1 is sufficient to affect cell migration, invasion, and proliferation. Expression of AKT1 siRNA had a similar effect as p110 siRNA in the cells. We showed the roles of specific PI3K and AKT isoforms in the cells, which are important to understanding the mechanism of PI3K/AKT signaling in ovarian cancer cells. Both p110 and AKT1 siRNA-expressing cells decreased the activation of p70S6K1. Inhibition of p70S6K1 activity by its siRNA also decreased cell migration, invasion, and proliferation associated with the induction of p27^KIP1 levels, and with the inhibition of cell cycle-associated proteins including cyclin D1, CDK2, and phosphorylated retinoblastoma protein. This study demonstrates the important role of the PI3K/AKT/mTOR/p70S6K1 pathway in cell proliferation, migration, and invasion in ovarian cancer cells by using siRNA-mediated gene silencing as a reverse genetic method.     

Role of nicotinamide adenine dinucleotide phosphate oxidase 1 in oxidative burst response to Toll-like receptor 5 signaling in large intestinal epithelial cells

The NADPH oxidase 1 (Nox1) is a gp91(phox) homologue preferentially expressed in the colon. We have established primary cultures of guinea pig large intestinal epithelial cells giving 90% purity of surface mucous cells. These cells spontaneously released superoxide anion (O(2)(-)) of 160 nmol/mg protein/h and expressed the Nox1, p22(phox), p67(phox), and Rac1 mRNAs, but not the gp91(phox), Nox4, p47(phox), p40(phox), and Rac2 mRNAs. They also expressed novel homologues of p47(phox) and p67(phox) (p41(nox) and p51(nox), respectively). Human colon cancer cell lines (T84 and Caco2 cells) expressed the Nox1, p22(phox), p51(nox), and Rac1 mRNAs, but not the other NADPH component mRNAs, and secreted only small amounts of O(2)(-) (<2 nmol/mg protein/h). Cotransfection of p41(nox) and p51(nox) cDNAs in T84 cells enhanced PMA-stimulated O(2)(-) release 5-fold. Treatment of the transfected T84 cells with recombinant flagellin (rFliC) from Salmonella enteritidis further augmented the O(2)(-) release in association with the induction of Nox1 protein. The enhanced O(2)(-) production by cotransfection of p41(nox) and p51(nox) vectors further augmented the rFliC-stimulated IL-8 release from T84 cells. T84 cells expressed the Toll-like receptor 5, and rFliC rapidly phosphorylated TGF-beta-activated kinase 1 and TGF-beta-activated kinase 1-binding protein 1. A potent inhibitor for NF-kappaB (pyrrolidine dithiocarbamate) significantly blocked the rFliC-primed increase in O(2)(-) production and induction of Nox1 protein. These results suggest that p41(nox) and p51(nox) are involved in the Nox1 activation in surface mucous cells of the colon, and besides that, epithelial cells discern pathogenicities among bacteria to appropriately operate Nox1 for the host defense.     

Retracted: MicroRNA-99b suppresses human cervical cancer cell activity by inhibiting the PI3K/AKT/mTOR signaling pathway

Cervical cancer is common cancer among women with high morbidity. MicroRNAs (miRs) are involved in the progression and development of cervical cancer. This study aimed to explore the effect of miR-99b-5p (miR-99b) on invasion and migration in cervical cancer through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) signaling pathway. The microarray-based analysis was used to screen out differentially expressed miRNAs. Expression of miR-99b, PI3K, AKT, mTOR, and ribosomal protein S6 kinase (p70S6K) was determined in both cervical cancer tissues and paracancerous tissues. Next, alteration of miR-99b expression in cervical cancer was conducted to evaluate levels of PI3K, AKT, mTOR, p70S6K matrix metallopeptidase 2, epithelial cell adhesion molecule, and intercellular adhesion molecule 1, as well as the effect of miR-99b on cell proliferation, invasion, migration, cell cycle distribution, and apoptosis. The results demonstrated that miR-99b expression was decreased and levels of PI3K, AKT, mTOR, and p70S6K were elevated in cervical cancer tissues. More important, overexpressed miR-99b repressed the PI3K/AKT/mTOR signaling pathway, inhibited cell proliferation, invasion, and migration, blocked cell cycle entry, and promoted apoptosis in cervical cancer. These results indicate that miR-99b attenuates the migration and invasion of human cervical cancer cells through downregulation of the PI3K/AKT/mTOR signaling pathway, which provides a therapeutic approach for cervical cancer treatment.     

Glycated albumin activates NADPH oxidase in rat mesangial cells through up-regulation of p47phox

Glycated albumin, an early-glycation Amadori-modified protein, stimulates transforming growth factor-β (TGF-β) expression and increases the production of the extracellular matrix proteins in mesangial cells, contributing to the pathogenesis of diabetic nephropathy. Glycated albumin has been shown to increase NADPH oxidase-dependent superoxide formation in mesangial cells. However, the mechanisms are not well understood. Therefore, in the present studies, we determined the mechanisms by which glycated albumin activates NADPH oxidase in primary rat mesangial cells and its contribution to glycated albumin-induced TGF-β expression and extracellular matrix protein production. Our data showed that glycated albumin treatment stimulated NADPH oxidase activity and increased the formation of superoxide formation in rat mesangial cells. Moreover, glycated albumin treatment stimulated the expression and phosphorylation of p47phox, one of the cytosolic regulatory subunits of the NADPH oxidase. However, the levels of other NADPH oxidase subunits including Nox1, Nox2, Nox4, p22phox, and p67phox were not altered by glycated albumin. Moreover, siRNA-mediated knockdown of p47phox inhibited glycated albumin-induced NADPH oxidase activity and superoxide formation. Glycated albumin-induced TGF-β expression and extracellular matrix production (fibronectin) was also inhibited by p47phox knock down. Taken together, these data suggest that up-regulation of p47phox is involved in glycated albumin-mediated activation of NADPH oxidase, leading to glycated albumin-induced expression of TGF-β and extracellular matrix proteins in mesangial cells and contributing to the development of diabetic nephropathy.     

Blockade of class IA phosphoinositide 3-kinase in neutrophils prevents NADPH oxidase activation- and adhesion-dependent inflammation

We examined the role of class IA phosphoinositide 3-kinase (PI3K) in the regulation of activation of NADPH oxidase in PMNs and the mechanism of PMN-dependent lung inflammation and microvessel injury induced by the pro-inflammatory cytokine TNF-alpha. TNF-alpha stimulation of PMNs resulted in superoxide production that was dependent on CD11b/CD18-mediated PMN adhesion. Additionally, TNF-alpha induced the association of CD11b/CD18 with the NADPH oxidase subunit Nox2 (gp91(phox)) and phosphorylation of p47(phox), indicating the CD11b/CD18 dependence of NADPH oxidase activation. Transduction of wild-type PMNs with Deltap85 protein, a dominant-negative form of the class IA PI3K regulatory subunit, p85alpha, fused to HIV-TAT (TAT-Deltap85) prevented (i) CD11b/CD18-dependent PMN adhesion, (ii) interaction of CD11b/CD18 with Nox2 and phosphorylation of p47(phox), and (iii) PMN oxidant production. Furthermore, studies in mice showed that i.v. infusion of TAT-Deltap85 significantly reduced the recruitment of PMNs in lungs and increase in lung microvascular permeability induced by TNF-alpha. We conclude that class IA PI3K serves as a nodal point regulating CD11b/CD18-integrin-dependent PMN adhesion and activation of NADPH oxidase, and leads to oxidant production at sites of PMN adhesion, and the resultant lung microvascular injury in mice.     

Identification of NCF2/p67phox as a novel p53 target gene

Analysis of microarrays performed in p53-, TAp63α- and ΔNp63α-inducible SaOs-2 cell lines allowed the identification of NCF2 mRNA upregulation in response to p53 induction. NCF2 gene encodes for p67phox, the cytosolic subunit of the NADPH oxidase enzyme complex. The recruitment of p67phox to the cell membrane causes the activation of the NADPH oxidase complex followed by the generation of NADP+ and superoxide from molecular oxygen. The presence of three putative p53 binding sites on the NCF2 promoter was predicted, and the subsequent luciferase and chromatin immunoprecipitation assays showed the activation of NCF2 promoter by p53 and its direct binding in vivo to at least one of the sites, thus confirming the hypothesis. NCF2 upregulation was also confirmed by real-time PCR in several cell lines after p53 activation. NCF2 knockdown by siRNA results in a significant reduction of ROS production and stimulates cell death, suggesting a protective function of Nox2-generated ROS in cells against apoptosis. These results provide insight into the redox-sensitive signaling mechanism that mediates cell survival involving p53 and its novel target NCF2/p67phox.     

Molecular composition and regulation of the Nox family NAD(P)H oxidases

Reactive oxygen species (ROS) are conventionally regarded as inevitable deleterious by-products in aerobic metabolism with a few exceptions such as their significant role in host defense. The phagocyte NADPH oxidase, dormant in resting cells, becomes activated during phagocytosis to deliberately produce superoxide, a precursor of other microbicidal ROS, thereby playing a crucial role in killing pathogens. The catalytic center of this oxidase is the membrane-integrated protein gp91(phox), tightly complexed with p22(phox), and its activation requires the association with p47(phox), p67(phox), and the small GTPase Rac, which normally reside in the cytoplasm. Since recent discovery of non-phagocytic gp91(phox)-related enzymes of the NAD(P)H oxidase (Nox) family--seven homologues identified in humans--deliberate ROS production has been increasingly recognized as important components of various cellular events. Here, we describe a current view on the molecular composition and post-translational regulation of Nox-family oxidases in animals.     

Nox1-dependent superoxide production controls colon adenocarcinoma cell migration

Reactive oxygen species are well-known mediators of various biological responses. Recently, new homologues of the catalytic subunit of NADPH oxidase have been discovered in non-phagocytic cells. These new homologues (Nox1-Nox5) produce low levels of superoxides compared to the phagocytic homologue Nox2/gp91phox. Using Nox1 siRNA, we show that Nox1-dependent superoxide production affects the migration of HT29-D4 colonic adenocarcinoma cells on collagen-I. Nox1 inhibition or down-regulation led to a decrease of superoxide production and alpha 2 beta 1 integrin membrane availability. An addition of arachidonic acid stimulated Nox1-dependent superoxide production and HT29-D4 cell migration. Pharmacological evidences using phospholipase A2, lipoxygenases and protein kinase C inhibitors show that upstream regulation of Nox1 relies on arachidonic acid metabolism. Inhibition of 12-lipoxygenase decreased basal and arachidonic acid induced Nox1-dependent superoxide production and cell migration. Migration and ROS production inhibited by a 12-lipoxygenase inhibitor were restored by the addition of 12(S)-HETE, a downstream product of 12-lipoxygenase. Protein kinase C delta inhibition by rottlerin (and also GO6983) prevented Nox1-dependent superoxide production and inhibited cell migration, while other protein kinase C inhibitors were ineffective. We conclude that Nox1 activation by arachidonic acid metabolism occurs through 12-lipoxygenase and protein kinase C delta, and controls cell migration by affecting integrin alpha 2 subunit turn-over.     

Retracted: Targeting of SPP1 by microRNA-340 inhibits gastric cancer cell epithelial–mesenchymal transition through inhibition of the PI3K/AKT signaling pathway

Gastric cancer (GC) is a common heterogeneous disease. The critical roles of microRNA-340 (miR-340) in the development and progression of GC were emphasized in accumulating studies. This study aims to examine the regulatory mechanism of miR-340 in GC cellular processes. Initially, microarray technology was used to identify differentially expressed genes and regulatory miRs in GC. After that, the potential role of miR-340 in GC was determined via ectopic expression, depletion, and reporter assay experiments. Expression of secreted phosphoprotein 1 (SPP1), miR-340, phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway, and epithelial–mesenchymal transition (EMT)-related genes was measured. Moreover, to further explore the function of miR-340 in vivo and in vitro, proliferation, apoptosis, migration, invasion, and tumorigenic capacity were evaluated. SPP1 was a target gene of miR-340 which could then mediate the PI3K/AKT signaling pathway by targeting SPP1 in GC. Furthermore, miR-340 levels were reduced and SPP1 was enriched in GC tissues and cells, with the PI3K/AKT signaling pathway being activated. Inhibitory effects of upregulated miR-340 on SPP1 and the PI3K/AKT signaling pathway were confirmed in vivo and in vitro. Overexpression of miR-340 or the silencing of SPP1 inhibited GC cell proliferation, invasion, migration, and EMT process, but promoted apoptosis of GC cells. Typically, targeting of SPP1 by miR-340 may contribute to the inhibition of proliferation, migration, invasion, and EMT of GC cells via suppression of PI3K/AKT signaling pathway.     

Inhibition of fatty acid synthase suppresses U-2 OS cell invasion and migration via downregulating the activity of HER2/PI3K/AKT signaling pathway in vitro

FASN plays an important role in the malignant phenotype of various tumors. Our previous studies show that inhibition FASN could induce apoptosis and inhibit proliferation in human osteosarcoma (OS) cell in vivo and vitro. The aim in this study was to investigate the effect of inhibition FASN on the activity of HER2/PI3K/AKT axis and invasion and migration of OS cell. The expression of FASN, HER2 and p-HER2(Y1248) proteins was detected by immunohistochemistry in OS tissues from 24 patients with pulmonary metastatic disease, and the relationship between FASN and p-HER2 as well as HER2 was investigated. The results showed that there was a positive correlation between FASN and HER2 as well as p-HER2 protein expression. The U-2 OS cells were transfected with either the FASN specific RNAi plasmid or the negative control RNAi plasmid. FASN mRNA was measured by RT-PCR. Western blot assays was performed to examine the protein expression of FASN, HER2, p-HER2(Y1248), PI3K, Akt and p-Akt (Ser473). Migration and invasion of cells were investigated by wound healing and transwell invasion assays. The results showed that the activity of HER2/PI3K/AKT signaling pathway was suppressed by inhibiting FASN. Meanwhile, the U-2OS cells migration and invasion were also impaired by inhibiting the activity of FASN/HER2/PI3K/AKT. Our results indicated that inhibition of FASN suppresses OS cell invasion and migration via down-regulation of the “HER2/PI3K/AKT” axis in vitro. FASN blocker may be a new therapeutic strategy in OS management.     

Identification of NCF2/p67phox as a novel p53 target gene

Analysis of microarrays performed in p53-, TAp63α- and ΔNp63α-inducible SaOs-2 cell lines allowed the identification of NCF2 mRNA upregulation in response to p53 induction. NCF2 gene encodes for p67phox, the cytosolic subunit of the NADPH oxidase enzyme complex. The recruitment of p67phox to the cell membrane causes the activation of the NADPH oxidase complex followed by the generation of NADP+ and superoxide from molecular oxygen. The presence of three putative p53 binding sites on the NCF2 promoter was predicted, and the subsequent luciferase and chromatin immunoprecipitation assays showed the activation of NCF2 promoter by p53 and its direct binding in vivo to at least one of the sites, thus confirming the hypothesis. NCF2 upregulation was also confirmed by real-time PCR in several cell lines after p53 activation. NCF2 knockdown by siRNA results in a significant reduction of ROS production and stimulates cell death, suggesting a protective function of Nox2-generated ROS in cells against apoptosis. These results provide insight into the redox-sensitive signaling mechanism that mediates cell survival involving p53 and its novel target NCF2/p67phox.     

Cooperation of p40(phox) with p47(phox) for Nox2-based NADPH oxidase activation during Fcγ receptor (FcγR)-mediated phagocytosis: mechanism for acquisition of p40(phox) phosphatidylinositol 3-phosphate (PI(3)P) binding

During activation of the phagocyte (Nox2-based) NADPH oxidase, the cytoplasmic Phox complex (p47(phox)-p67(phox)-p40(phox)) translocates and associates with the membrane-spanning flavocytochrome b(558). It is unclear where (in cytoplasm or on membranes), when (before or after assembly), and how p40(phox) acquires its PI(3)P-binding capabilities. We demonstrated that in addition to conformational changes induced by H(2)O(2) in the cytoplasm, p40(phox) acquires PI(3)P-binding through direct or indirect membrane targeting. We also found that p40(phox) is essential when p47(phox) is partially phosphorylated during FcγR-mediated oxidase activation; however, p40(phox) is less critical when p47(phox) is adequately phosphorylated, using phosphorylation-mimicking mutants in HEK293(Nox2/FcγRIIa) and RAW264.7(p40/p47KD) cells. Moreover, PI binding to p47(phox) is less important when the autoinhibitory PX-PB1 domain interaction in p40(phox) is disrupted or when p40(phox) is targeted to membranes. Furthermore, we suggest that high affinity PI(3)P binding of the p40(phox) PX domain is critical during its accumulation on phagosomes, even when masked by the PB1 domain in the resting state. Thus, in addition to mechanisms for directly acquiring PI(3)P binding in the cytoplasm by H(2)O(2), p40(phox) can acquire PI(3)P binding on targeted membranes in a p47(phox)-dependent manner and functions both as a "carrier" of the cytoplasmic Phox complex to phagosomes and an "adaptor" of oxidase assembly on phagosomes in cooperation with p47(phox), using positive feedback mechanisms.