Reduced CENPU expression inhibits lung adenocarcinoma cell proliferation and migration through PI3K/AKT signaling

CENPU (centromere protein U), a centromere component essential for mitosis, relates with some cancers progression. However, it is not well illustrated in lung adenocarcinoma (LAC). Here, we aimed to investigate the potential effect of CENPU on LAC progression and prognosis. In this experiment, expression level of CENPU and association between its expression and LAC patients’ clinicopathological characteristics and prognosis were analyzed. The proliferation, migration and invasive abilities of LAC cells were determined by CCK-8, colony formation, transwell assays. Western blot was used to detect PI3K/AKT signaling key proteins. We found CENPU level was overexpressed in LAC tissues on comparing normal tissues. Moreover, CENPU overexpression correlated with clinicopathological variables and predicted an independent prognostic indicator in LAC patients. Functionally, CENPU downregulation significantly inhibited LAC cell proliferation, migration and invasion in, which was possibly mediated by PI3K/AKT pathway inactivation. Our findings insinuate targeting CENPU may be a potential therapeutic strategy for LAC.     

PI3K is required for insulin-stimulated but not EGF-stimulated ERK1/2 activation

The Ras/Raf/extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway is known to cross-talk with other signaling pathways, including phosphatidylinositol 3-kinase (PI3K)/Akt pathway. However, the role of PI3K in ERK-1/2 activation induced by tyrosine kinase receptors was not fully understood. Here, we report that two structurally distinct PI3K inhibitors, wortmannin and LY294002, inhibited insulin-induced activation of ERK1/2 but had no effect on EGF-induced activation of ERK1/2 in hepatocellular carcinoma BEL-7402 and SMMC-7721 cells, breast cancer MCF-7 cells, and prostate cancer LNCaP cells. Although protein kinase C could act as a mediator between PI3K and ERK1/2, protein kinase C inhibitor chelerythrine chloride did not inhibit insulin-induced ERK1/2 activation. Both insulin- and EGF-induced ERK1/2 activation are strictly dependent on Ras activation, however, wortmannin only inhibited insulin-induced, but not EGF-induced Ras activation. These results indicate that PI3K plays different roles in the activation of Ras/ERK1/2 signaling by insulin and EGF, and that insulin-stimulated, but not EGF-stimulated, ERK1/2 and Akt signalings diverge at PI3K.     

Lysophosphatidic acid induces MDA-MB-231 breast cancer cells migration through activation of PI3K/PAK1/ERK signaling

Background

Enhanced motility of cancer cells is a critical step in promoting tumor metastasis. Lysophosphatidic acid (LPA), representing the major mitogenic activity in serum, stimulates migration in various types of cancer cells. However, the underlying signaling mechanisms for LPA-induced motility of cancer cells remain to be elucidated.

Methodology/Principal Findings

In this study, we found that LPA dose-dependently stimulated migration of MDA-MB-231 breast cancer cells, with 10 µM being the most effective. LPA also increased ERK activity and the MEK inhibitor U0126 could block LPA-induced ERK activity and cell migration. In addition, LPA induced PAK1 activation while ERK activation and cell migration were inhibited by ectopic expression of an inactive mutant form of PAK1 in MDA-MB-231 cells. Furthermore, LPA increased PI3K activity, and the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 inhibited both LPA-induced PAK1/ERK activation and cell migration. Moreover, in the breast cancer cell, LPA treatment resulted in remarkable production of reactive oxygen species (ROS), while LPA-induced ROS generation, PI3K/PAK1/ERK activation and cell migration could be inhibited by N-acetyl-L-Cysteine, a scavenger of ROS.

Conclusions/Significance

Taken together, this study identifies a PI3K/PAK1/ERK signaling pathway for LPA-stimulated breast cancer cell migration. These data also suggest that ROS generation plays an essential role in the activation of LPA-stimulated PI3K/PAK1/ERK signaling and breast cancer cell migration. These findings may provide a basis for designing future therapeutic strategy for blocking breast cancer metastasis.     

Integrity of the cortical actin ring is required for activation of the PI3K/Akt and p38 MAPK signaling pathways in redifferentiation of chondrocytes on chitosan

Cell shape alterations and accompanying cytoskeletal changes have diverse effects on cell function. We have already shown that dedifferentiated chondrocytes have a round cell morphology and undergo redifferentiation when cultured on chitosan membrane. In the present study, we investigate the role of the cytoskeleton in chondrocyte redifferentiation. Chondrocytes obtained from a micromass culture of chick limb bud mesenchymal cells were subcultured four times. Immunofluorescence analysis of F-actin showed cortical distribution of the actin cytoskeleton upon subculture of dedifferentiated chondrocytes on chitosan membrane. Treatment with cytochalasin D disrupted the cortical actin ring formed during cultivation of chondrocytes on the chitosan membrane, and inhibited chondrocyte redifferentiation. Moreover, cytochalasin D inhibited the phosphorylation of Akt and p38 mitogen activated protein kinase (MAPK), induced during redifferentiation on chitosan membrane. LY294002, an inhibitor of phosphatidylinositol-3-OH-kinase (PI3K), suppressed chondrocyte redifferentiation. These findings suggest that integrity of the actin cytoskeleton is a crucial requirement for PI3K/Akt and p38 MAPK in chondrocyte redifferentiation.     

Cytoglobin inhibits migration through PI3K/AKT/mTOR pathway in fibroblast cells

Cell proliferation and migration are crucial in many physiological processes including development, cancer, tissue repair, and wound healing. Cell migration is regulated by several signaling molecules. Identification of genes related to cell migration is required to understand molecular mechanism of non-healing chronic wounds which is a major concern in clinics. In the current study, the role of cytoglobin (CYGB) gene in f?broblast cell migration and proliferation was described. L929 mouse fibroblast cells were transduced with lentiviral particles for CYGB and GFP, and analyzed for cell proliferation and migration ability. Fibroblast cells overexpressing CYGB displayed decreased cell proliferation, colony formation capacity, and cell migration. Phosphorylation levels of mTOR and two downstream effectors S6 and 4E-BP1 which take part in PI3K/AKT/mTOR signaling declined in CYGB-overexpressing cells. Microarray analysis indicated that CYGB overexpression leads to downregulation of cell proliferation, migration, and tumor growth associated genes in L929 cell line. This study demonstrated the role of CYGB in fibroblast cell motility and proliferation. CYGB could be a promising candidate for further studies as a potential target for diseases related to cell migration such as cancer and chronic wound treatment.     

Mollugin induces tumor cell apoptosis and autophagy via the PI3K/AKT/mTOR/p70S6K and ERK signaling pathways

Mollugin, a bioactive phytochemical isolated from Rubia cordifolia L., has shown preclinical anticancer efficacy in various cancer models. However the effects of mollugin in regulating cancer cell survival and death remains undefined. In the present study we found that mollugin exhibited cytotoxicity on various cancer models. The suppression of cell viability was due to the induction of mitochondria apoptosis. In addition, the presence of autophagic hallmarks was observed in mollugin-treated cells. Notably, blockade of autophagy by a chemical inhibitor or RNA interference enhanced the cytotoxicity of mollugin. Further experiments demonstrated that phosphatidylinositide 3-kinases/protein kinase B/mammalian target of rapamycin/p70S6 kinase (PI3K/AKT/mTOR/p70S6K) and extracellular regulated protein kinases (ERK) signaling pathways participated in mollugin-induced autophagy and apoptosis. Together, these findings support further studies of mollugin as candidate for treatment of human cancer cells.     

Participation of PI3K and ERK1/2 pathways are required for human brain vascular smooth muscle cell migration

Human brain vascular smooth muscle cell (HBVSMC) migration contributes to angiogenesis and several pathological processes in the brain. However, the molecular mechanism of angiogenesis, in which smooth muscle cell contributes, remains unclear. Our study investigates the role of vascular endothelial growth factor (VEGF) in the HBVSMC migration and elucidates the chemotactic signaling pathway mediating this action. We used the in vitro 'scratch' wound method to detect the HBVSMC migration. VEGF(165) (1-40ng/ml) induced the HBVSMC migration in a dose-dependent manner (P<0.05). VEGF(165) does not induce HBVSMC proliferation. Wortmannin, a specific phosphatidylinositol 3-kinase (PI3K) inhibitor, significantly inhibited serine/threonine kinase Akt/protein kinase B (PKB) phosphorylation and reduced HBVSMC migration into the wound edge following VEGF(165) stimulation (P<0.05). PD98059, an extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor, also significantly inhibited ERK1/2 phosphorylation and reduced the numbers of SMC migration. Parallel distance measurement showed that VEGF(165) induced HBVSMC migration significantly reduced due to inhibition of PI3K or ERK1/2 phosphorylation (P<0.05). Our results demonstrate that VEGF(165) could induce HBVSMC migration but not proliferation in vitro. Inhibiting Akt/PKB or ERK1/2 phosphorylation could reduce VEGF(165) induced HBVSMC migration. We provide the first evidence that activation of PI3K or ERK1/2 pathways are a crucial event in VEGF(165) mediated signal transduction leading to HBVSMC migration.     

Leptin Inhibits Glucose Intestinal Absorption via PKC,p38MAPK,PI3K and MEK/ERK

The role of leptin in controlling food intake and body weight is well recognized, but whether this is achieved by modulating nutrient absorption is still a controversial issue. The aim of this work was to investigate the direct effect of luminal leptin on glucose intestinal absorption and elucidate for the first time its signaling pathway. Fully differentiated Caco-2 cells grown on transwell filters were used for glucose transport studies. Leptin caused a significant reduction in glucose absorption. Individual and simultaneous inhibition of ERK, p38MAPK, PI3K or PKC abrogated completely the inhibitory effect of leptin. Activating PKC, lead to a stimulatory effect that appeared only when ERK, p38MAPK, or PI3K was inactive. Moreover, leptin increased the phosphorylation of ERK, Akt and p38MAPK. This increase changed into a decrease when p38MAPK and PKC were inactivated individually. Inhibiting ERK maintained the leptin-induced up-regulation of p-Akt and p-p38MAPK while inhibiting PI3K reduced the level of p-ERK and p-Akt but maintained the increase in p-p38MAPK. These results suggest that leptin reduces glucose absorption by activating PKC. Although the latter modulates glucose absorption via a stimulatory and an inhibitory pathway, only the latter is involved in leptin’s action. Active PKC leads to a sequential activation of p38MAPK, PI3K and ERK which exerts an inhibitory effect on glucose absorption. The results reveal a modulatory role of leptin in nutrient absorption in addition to its known satiety inducing effect.     

PI3K/AKT and ERK regulate retinoic acid-induced neuroblastoma cellular differentiation

Thymoquinone (TQ), a bioactive component of black caraway seed (Nigella sativa) oil, is reported to have antineoplastic properties. In this study we investigated the effect of TQ on a panel of human breast cancer cell lines. Cell viability assays showed that TQ killed T-47D, MDA-MB-231, and MDA-MB-468 cells via p53-independent induction of apoptosis; however, MCF-7 cells were refractory to the cytotoxic action of TQ. Western Blot analysis showed that MCF-7 cells expressed high levels of cytoprotective NADPH quinone oxidoreductase 1 (NQO1), which was responsible for TQ-resistance since inhibition of NQO1 with dicoumarol rendered MCF-7 cells TQ-sensitive. These findings may be clinically important when considering TQ as a possible adjunct treatment for breast cancer since a high percentage of breast tumors express NQO1.     

Human breast carcinoma cells are induced to apoptosis by samsum ant venom through an IGF-1-dependant pathway, PI3K/AKT and ERK signaling

In the present study we evaluated the anti-tumor potential of samsum ant venom (SAV) from Pachycondyla sennaarensis on the human breast carcinoma cell line MCF-7. We found that SAV induced growth arrest of MCF-7 cells without affecting the viability of MCF-10 (non-tumorigenic normal breast epithelial cells) and normal PBMCs. We then analyzed its impact on IGF-1-mediated MCF-7 cell proliferation and its effect on the underlying IGF-1 signaling pathways. Using flow cytometry analysis, we showed that the percentage of apoptotic cells was fourfold higher in SAV-treated cells as compared to untreated cells. More importantly, treatment with SAV induced a marked reduction in actin polymerization and a subsequent marked reduction in IGF-1-mediated cell proliferation. In addition to growth-inhibitory and pro-apoptotic effects, significant reductions were also observed in the phosphorylation of AKT and ERK, but not p38MAPK, in SAV-treated cells as compared to untreated cells. Our data reveal unique anti-tumor effects of samsum ant venom.     

PI3K/AKT signaling regulates bioenergetics in immortalized hepatocytes

Regulation of cellular bioenergetics by PI3K/AKT signaling was examined in isogenic hepatocyte cell lines lacking the major inhibitor of PI3K/AKT signaling, PTEN (phosphatase and tensin homolog deleted on chromosome 10). PI3K/AKT signaling was manipulated using the activator (IGF-1) and the inhibitor (LY 294002) of the PI3K/AKT pathway. Activation of PI3K/AKT signaling resulted in an enhanced anaerobic glycolysis and mitochondrial respiration. AKT, when phosphorylated and activated, translocated to mitochondria and localized within the membrane structure of mitochondria, where it phosphorylated a number of mitochondrial-resident proteins including the subunits α and β of ATP synthase. Inhibition of GSK3β by either phosphorylation by AKT or lithium chloride resulted in activation of pyruvate dehydrogenase, i.e., a decrease in its phosphorylated form. AKT-dependent phosphorylation of ATP synthase subunits α and β resulted in an increased complex activity. AKT translocation to mitochondria was associated with an increased expression and activity of complex I. These data suggest that the mitochondrial signaling pathway AKT/GSK3β/PDH, AKT-dependent phosphorylation of ATP synthase, and upregulation of mitochondrial complex I expression and activity are involved in the control of mitochondrial bioenergetics by increasing substrate availability and regulating the mitochondrial catalytic/energy-transducing capacity.     

PI3K/PTEN/AKT signaling regulates prostate tumor angiogenesis

PI3K pathway exerts its function through its downstream molecule AKT in regulating various cell functions including cell proliferation, cell transformation, cell apoptosis, tumor growth and angiogenesis. PTEN is an inhibitor of PI3K, and its loss or mutation is common in human prostate cancer. But the direct role and mechanism of PI3K/PTEN signaling in regulating angiogenesis and tumor growth in vivo remain to be elucidated. In this study, by using chicken chorioallantoic membrane (CAM) and in nude mice models, we demonstrated that inhibition of PI3K activity by LY294002 decreased PC-3 cells-induced angiogenesis. Reconstitution of PTEN, the molecular inhibitor of PI3K in PC-3 cells inhibited angiogenesis and tumor growth. Immunohistochemical staining indicated that PTEN expression suppressed HIF-1, VEGF and PCNA expression in the tumor xenographs. Similarly, expression of AKT dominant negative mutant also inhibited angiogenesis and tumor growth, and decreased the expression of HIF-1 and VEGF in the tumor xenographs. These results suggest that inhibition of PI3K signaling pathway by PTEN inhibits tumor angiogenesis and tumor growth. In addition, we found that AKT is the downstream target of PI3K in controlling angiogenesis and tumor growth, and PTEN could inhibit angiogenesis by regulating the expression of HIF-1 and VEGF expression through AKT activation in PC-3 cells.     

DMBT1 suppresses progression of gallbladder carcinoma through PI3K/AKT signaling pathway by targeting PTEN

ABSTRACT

Gallbladder carcinoma (GBC) is a highly lethal malignancy of the gastrointestinal tract. Despite extensive research, the underlying molecular mechanism of GBC remains largely unclear. Deleted in malignant brain tumors 1 (DMBT1) is low-expression during cancer progression and as a potential tumor-suppressor gene in various types of cancer. However, its role in Gallbladder cancer remains poorly understood. Here, we found that DMBT1 was significantly low-expression and deletion of copy number in GBC tissues by qRT-PCR and Western blot. Overexpression of DMBT1 impaired survival, promoted apoptosis in GBC cells in vitro, and inhibited tumor progression in vivo. Further study of underlying mechanisms demonstrated that DMBT1 combined with PTEN which could stabilize PTEN protein, resulting in inhibiting the activation of PI3K/AKT signaling pathway. Our study revealed a new sight of DMBT1 as a tumor-suppressor gene on the PI3K/AKT pathway in GBC, which may be a potential therapeutic target for improving treatment.     

TWEAK promotes hepatic stellate cell migration through activating EGFR/Src and PI3K/AKT pathways

Activated human hepatic stellate cells (HSCs) showed enhanced ability of migration compared with quiescent HSCs, which is pivotal in liver fibrogenesis. The aim of the present study was to investigate the effects of tumor necrosis factor‐like weak inducer of apoptosis (TWEAK) on the migration of activated HSCs and to explore the relevant potential mechanisms. Human HSCs LX‐2 cells were cultured with TWEAK. TNFRSF12A‐downexpressing lentiviruses were used to infect LX‐2 cells. The specific matrix metalloproteinases inhibitor BB94, the Src family kinase inhibitor, Dasatinib, and the specific inhibitor of phosphoinositide 3‐kinase (PI3K), LY294002 were used to treat LX‐2 cells combined with TWEAK. Cell migration and invasion was tested by the transwell assay. The expression of EGFR/Src, PI3K/AKT, and matrix metallopeptidase 9 (MMP9) was identified by real‐time polymerase chain reaction or western blotting. The result showed TWEAK promoted HSC migration and collagen production. BB94 significantly attenuated the migration of LX‐2 induced by TWEAK. Dasatinib inhibited the ability of cell migration stimulated by TWEAK. TWEAK upregulated the phosphorylation of epidermal growth factor receptor (EGFR) and Src. The phosphorylation of PI3K and AKT was significantly activated by TWEAK stimulation. Inhibition of PI3K/AKT reduced the expression of MMP9 induced by TWEAK. The present study, for the first time, demonstrated that TWEAK promoted HSC migration through the activation of EGFR/Src and PI3K/AKT pathways, and showed a novel potential mechanism of HSC migration regulated by TWEAK.     

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.     

miRNA-30-3p improves myocardial ischemia via the PTEN/PI3K/AKT signaling pathway

The aim of this study was to explain the effect and mechanisms of miRNA-30-3p in myocardial ischemia-induced cell apoptosis in vitro and in vivo studies. In the cell experiment, the H9C2 cells were divided into the normal control (NC), and the model, miRNA, and miRNA + phosphatidylinositol 3-kinase (PI3K) inhibitor groups. The cell survival rates of the different groups were measured with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay kit; the lactate dehydrogenase (LDH), malondialdehyde (MDA) content, and superoxidedimutase (SOD) activity in the bathing medium were assayed for the evaluation of myocardial cell injury. The cell apoptosis rate of different groups was measured with flow cytometry analysis. The relative protein expressions of different cell groups were evaluated by Western blot analysis. In the vivo study, the Sprague-Dawley rats were divided into four groups: the NC group, the model group, miRNA group, and the (miRNA + PI3K inhibitor) group. The pathological observations, cell apoptosis, LDH, SOD, MDA, and relative protein expressions were evaluated with hematoxylin and eosin, enzyme-linked immunosorbent assay, terminal deoxynucleotide transferase dUTP nick-end labeling or immunohistochemical methods. The results show that miRNA-30-3p had the effect of improving cell apoptosis induced by myocardial ischemia in vitro and in vivo studies by the regulation of the PTEN/PI3K/AKT pathway.