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Sample records for akt inhibitor ly294002

  1. PI3K inhibitors LY294002 and IC87114 reduce inflammation in carrageenan-induced paw oedema and down-regulate inflammatory gene expression in activated macrophages.

    PubMed

    Eräsalo, Heikki; Laavola, Mirka; Hämäläinen, Mari; Leppänen, Tiina; Nieminen, Riina; Moilanen, Eeva

    2015-01-01

    PI3K/Akt pathway is a well-characterized pathway controlling cellular processes such as proliferation, migration and survival, and its role in cancer is vastly studied. There is also evidence to suggest the involvement of this pathway in the regulation of inflammatory responses. In this study, an attempt was made to investigate the role of PI3Ks in acute inflammation in vivo using pharmacological inhibitors against PI3Ks in the carrageenan-induced paw oedema model. A non-selective PI3K inhibitor LY294002 and a PI3Kδ-selective inhibitor IC87114 were used. Both of these inhibitors reduced inflammatory oedema upon carrageenan challenge in the mouse paw. To explain this result, the effects of the two inhibitors on inflammatory gene expression were investigated in activated macrophages. LY294002 and IC87114 prevented Akt phosphorylation as expected and down-regulated the expression of inflammatory factors IL-6, MCP-1,TNFα and iNOS. These findings suggest that PI3K inhibitors could be used to attenuate inflammatory responses and that the mechanism of action behind this effect is the down-regulation of inflammatory gene expression.

  2. Synergistic inhibition of colon carcinoma cell growth by Hedgehog-Gli1 inhibitor arsenic trioxide and phosphoinositide 3-kinase inhibitor LY294002.

    PubMed

    Cai, Xinyi; Yu, Kun; Zhang, Lijuan; Li, Yunfeng; Li, Qiang; Yang, Zhibin; Shen, Tao; Duan, Lincan; Xiong, Wei; Wang, Weiya

    2015-01-01

    The Hedgehog (Hh) signaling pathway not only plays important roles in embryogenesis and adult tissue homeostasis, but also in tumorigenesis. Aberrant Hh pathway activation has been reported in a variety of malignant tumors including colon carcinoma. Here, we sought to investigate the regulation of the Hh pathway transcription factor Gli1 by arsenic trioxide and phosphoinositide 3-kinase (PI3K) inhibitor LY294002 in colon carcinoma cells. We transfected cells with siGli1 and observed a significant reduction of Gli1 expression in HCT116 and HT29 cells, which was confirmed by quantitative real-time polymerase chain reaction and Western blots. Knocking down endogenous Gli1 reduced colon carcinoma cell viability through inducing cell apoptosis. Similarly, knocking down Gli2 using short interfering RNA impaired colon carcinoma cell growth in vitro. To elucidate the regulation of Gli1 expression, we found that both Gli inhibitor arsenic trioxide and PI3K inhibitor LY294002 significantly reduced Gli1 protein expression and colon carcinoma cell proliferation. Arsenic trioxide treatment also reduced Gli1 downstream target gene expression, such as Bcl2 and CCND1. More importantly, the inhibition of Hedgehog-Gli1 by arsenic trioxide showed synergistic anticancer effect with the PI3K inhibitor LY294002 in colon carcinoma cells. Our findings suggest that the Hh pathway transcription factor Gli1 is involved in the regulation of colon carcinoma cell viability. Inhibition of Hedgehog-Gli1 expression by arsenic trioxide and PI3K inhibitor synergistically reduces colon cancer cell proliferation, indicating that they could be used as an effective anti-colon cancer combination therapy.

  3. LY294002 induces in vitro apoptosis and overexpression of p75NTR in human uterine leiomyosarcoma HTB 114 cells.

    PubMed

    Pistilli, Alessandra; Rende, Mario; Crispoltoni, Lucia; Montagnoli, Claudia; Stabile, Anna Maria

    2015-01-01

    Uterine leiomyosarcoma is a severe neoplasia resistant to conventional therapies. In previous studies, we have shown that human SK-UT-1 (ATCC HTB114) uterine leiomyosarcoma cell line secretes nerve growth factor (NGF) and expresses its receptors tyrosine kinase A receptor (TrKA) and low affinity nerve growth factor receptor (p75NTR). Furthermore, we have demonstrated that direct chemical inhibition or IgG neutralization of TrKA receptor induce apoptosis through p75NTR. In the present study, HTB114 cells were exposed to the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 with and without β-NGF: apoptosis, cell cycle, activation of caspase-3 and protein kinase B (AKT) and TrKA/p75NTR phenotypic expression were evaluated. According to the type of exposure, LY294002 not only induced a relevant increase in apoptosis, but also produced a novel and unexpected phenotypic modulation of the NGF receptors with a downregulation of TrKA and an upregulation of p75NTR. This latter increase enhanced HTB114 apoptosis. Our study confirms that the interference on NGF transduction is a promising therapeutical approach in uterine leiomyosarcoma.

  4. Activation of Telomerase by Ionizing Radiation: Differential Response to the Inhibition of DNA Double-Strand Break Repair by Abrogation of Poly(ADP-ribosyl)ation, by LY294002, or by Wortmannin

    SciTech Connect

    Neuhof, Dirk Zwicker, Felix; Kuepper, Jan-Heiner; Debus, Juergen; Weber, Klaus-Josef

    2007-11-01

    Purpose: Telomerase activity represents a radiation-inducible function, which may be targeted by a double-strand break (DSB)-activated signal transduction pathway. Therefore, the effects of DNA-PK inhibitors (Wortmannin and LY294002) on telomerase upregulation after irradiation were studied. In addition, the role of trans-dominant inhibition of poly(ADP-ribosyl)ation, which strongly reduces DSB rejoining, was assessed in comparison with 3-aminobenzamide. Methods and Materials: COM3 rodent cells carry a construct for the dexamethasone-inducible overexpression of the DNA-binding domain of PARP1 and exhibit greatly impaired DSB rejoining after irradiation. Telomerase activity was measured using polymerase chain reaction ELISA 1 h after irradiation with doses up to 10 Gy. Phosphorylation status of PKB/Akt and of PKC{alpha}/{beta}{sub II} was assessed by western blotting. Results: No telomerase upregulation was detectable for irradiated cells with undisturbed DSB rejoining. In contrast, incubation with LY294002 or dexamethasone yielded pronounced radiation induction of telomerase activity that could be suppressed by Wortmannin. 3-Aminobenzamide not only was unable to induce telomerase activity but also suppressed telomerase upregulation upon incubation with LY294002 or dexamethasone. Phospho-PKB was detectable independent of irradiation or dexamethasone pretreatment, but was undetectable upon incubations with LY294002 or Wortmannin, whereas phospho-PKC rested detectable. Conclusions: Telomerase activation postirradiation was triggered by different treatments that interfere with DNA DSB processing. This telomerase upregulation, however, was not reflected by the phosporylation status of the putative mediators of TERT activation, PKB and PKC. Although an involvement of PKB in TERT activation is not supported by the present findings, a respective role of PKC isoforms other than {alpha}/{beta}{sub II} cannot be ruled out.

  5. Differential modulation of mitogen driven proliferation and homeostasis driven proliferation of T cells by rapamycin, Ly294002 and chlorophyllin.

    PubMed

    Sharma, Deepak; Kumar, Sandur Santosh; Raghu, Rashmi; Khanam, Shazia; Sainis, Krishna Balaji

    2007-04-01

    Homeostasis driven proliferation (HDP) of naïve CD4+ T cells depends upon T cell receptor ligation with self-MHC II along with availability of interleukin-7. But the exact nature of downstream signaling events involved in HDP of helper T cells remains elusive. To identify the specific involvement of signaling molecules in HDP, purified CD4+ T cells were treated with either mTOR inhibitor rapamycin or PI3kinase inhibitor Ly294002 or with an antioxidant chlorophyllin (CHL) in vitro. Rapamycin treated cells failed to proliferate, expressed anergic T cell specific transcription factor genes egr-2 and egr-3 and showed diminished IFN-gamma production in response to Con A stimulation in vitro. Although CHL treated cells also failed to proliferate, they showed a normal IFN-gamma production during primary stimulation and did not upregulate egr-2 and egr-3 genes following restimulation in vitro. Ly294002 treated cells failed to express IL-2 and IFN-gamma and did not divide in response to Con A stimulation in vitro. While all these inhibitors significantly inhibited CD4+ T cell proliferation in response to the mitogen in vitro, only CHL treatment could inhibit their HDP in lymphopenic mice. Our results also demonstrate that combined treatment with rapamycin and Ly294002 did not inhibit HDP of CD4+ T cells. Thus, the present study, for the first time, shows a non-essential role of mTOR and PI3kinase during HDP of CD4+ T cells and also describes its possible regulation by an antioxidant.

  6. Pim-1 ligand-bound structures reveal the mechanism of serine/threonine kinase inhibition by LY294002.

    PubMed

    Jacobs, Marc D; Black, James; Futer, Olga; Swenson, Lora; Hare, Brian; Fleming, Mark; Saxena, Kumkum

    2005-04-08

    Pim-1 is an oncogene-encoded serine/threonine kinase primarily expressed in hematopoietic and germ cell lines. Pim-1 kinase was originally identified in Maloney murine leukemia virus-induced T-cell lymphomas and is associated with multiple cellular functions such as proliferation, survival, differentiation, apoptosis, and tumorigenesis (Wang, Z., Bhattacharya, N., Weaver, M., Petersen, K., Meyer, M., Gapter, L., and Magnuson, N. S. (2001) J. Vet. Sci. 2, 167-179). The crystal structures of Pim-1 complexed with staurosporine and adenosine were determined. Although a typical two-domain serine/threonine protein kinase fold is observed, the inter-domain hinge region is unusual in both sequence and conformation; a two-residue insertion causes the hinge to bulge away from the ATP-binding pocket, and a proline residue in the hinge removes a conserved main chain hydrogen bond donor. Without this hydrogen bond, van der Waals interactions with the hinge serve to position the ligand. The hinge region of Pim-1 resembles that of phosphatidylinositol 3-kinase more closely than it does other protein kinases. Although the phosphatidylinositol 3-kinase inhibitor LY294002 also inhibits Pim-1, the structure of the LY294002.Pim-1 complex reveals a new binding mode that may be general for Ser/Thr kinases.

  7. VEGF secretion by neuroendocrine tumor cells is inhibited by octreotide and by inhibitors of the PI3K/AKT/mTOR pathway.

    PubMed

    Villaume, Karine; Blanc, Martine; Gouysse, Géraldine; Walter, Thomas; Couderc, Christophe; Nejjari, Mimoun; Vercherat, Cécile; Cordier-Bussat, Martine; Roche, Colette; Scoazec, Jean-Yves

    2010-01-01

    Gastroenteropancreatic (GEP) endocrine tumors are hypervascular tumors able to synthesize and secrete high amounts of VEGF. We aimed to study the regulation of VEGF production in GEP endocrine tumors and to test whether some of the drugs currently used in their treatment, such as somatostatin analogues and mTOR inhibitors, may interfere with VEGF secretion. We therefore analyzed the effects of the somatostatin analogue octreotide, the mTOR inhibitor rapamycin, the PI3K inhibitor LY294002, the MEK1 inhibitor PD98059 and the p38 inhibitor SB203850 on VEGF secretion, assessed by ELISA and Western blotting, in three murine endocrine cell lines, STC-1, INS-r3 and INS-r9. Octreotide and rapamycin induced a significant decrease in VEGF production by all three cell lines; LY294002 significantly inhibited VEGF production by STC-1 and INS-r3 only. We detected no effect of PD98059 whereas SB203850 significantly inhibited VEGF secretion in INS-r3 and INS-r9 cells only. By Western blotting analysis, we observed decreased intracellular levels of VEGF and HIF-1alpha under octreotide, rapamycin and LY294002. For rapamycin and LY294002, this effect was likely mediated by the inhibition of the mTOR/HIF-1/VEGF pathway. In addition to its well-known anti-secretory effects, octreotide may also act through the inhibition of the PI3K/Akt pathway, as suggested by the decrease in Akt phosphorylation detected in all three cell lines. In conclusion, our study points out to the complex regulation of VEGF synthesis and secretion in neoplastic GEP endocrine cells and suggests that the inhibition of VEGF production by octreotide and rapamycin may contribute to their therapeutic effects.

  8. The role of the PI3K-Akt signal transduction pathway in Autographa californica multiple nucleopolyhedrovirus infection of Spodoptera frugiperda cells

    SciTech Connect

    Xiao Wei; Yang Yi; Weng Qingbei; Lin Tiehao; Yuan Meijin; Yang Kai; Pang Yi

    2009-08-15

    Many viruses activate the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, thereby modulating diverse downstream signaling pathways associated with antiapoptosis, proliferation, cell cycling, protein synthesis and glucose metabolism, in order to augment their replication. To date, the role of the PI3K-Akt pathway in Baculovirus replication has not been defined. In the present study, we demonstrate that infection of Sf9 cells with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) elevated cellular Akt phosphorylation at 1 h post-infection. The maximum Akt phosphorylation occurred at 6 h post-infection and remained unchanged until 18 h post-infection. The PI3K-specific inhibitor, LY294002, suppressed Akt phosphorylation in a dose-dependent manner, suggesting that AcMNPV-induced Akt phosphorylation is PI3K-dependent. The inhibition of PI3K-Akt activation by LY294002 significantly reduced the viral yield, including a reduction in budded viruses and occlusion bodies. The virus production was reduced only when the inhibitor was added within 24 h of infection, implying that activation of PI3K occurred early in infection. Correspondingly, both viral DNA replication and late (VP39) and very late (POLH) viral protein expression were impaired by LY294002 treatment; LY294002 had no effect on immediate-early (IE1) and early-late (GP64) protein expression. These results demonstrate that the PI3K-Akt pathway is required for efficient Baculovirus replication.

  9. H2O2 treatment or serum deprivation induces autophagy and apoptosis in naked mole-rat skin fibroblasts by inhibiting the PI3K/Akt signaling pathway.

    PubMed

    Zhao, Shanmin; Li, Li; Wang, Shiyong; Yu, Chenlin; Xiao, Bang; Lin, Lifang; Cong, Wei; Cheng, Jishuai; Yang, Wenjing; Sun, Wei; Cui, Shufang

    2016-12-20

    Naked mole-rats (NMR; Heterocephalus glaber) display extreme longevity and resistance to cancer. Here, we examined whether autophagy contributes to the longevity of NMRs by assessing the effects of the PI3K/Akt pathway inhibitor LY294002 and the autophagy inhibitor chloroquine (CQ) on autophagy and apoptosis in NMR skin fibroblasts. Serum starvation, H2O2 treatment, and LY294002 treatment all increased the LC3-II/LC3-I ratio and numbers of double-membraned autophagosomes and autophagic vacuoles, and decreased levels of p70S6K, p-AktSer473, and p-AktThr308. By contrast, CQ treatment decreased p70S6K, AktSer473, and AktThr308 levels. The Bax/Bcl-2 ratio increased after 12 h of exposure to LY294002 or CQ. These data show that inhibiting the Akt pathway promotes autophagy and apoptosis in NMR skin fibroblasts. Furthermore, LY294002 or CQ treatment decreased caspase-3, p53, and HIF1-α levels, suggesting that serum starvation or H2O2 treatment increase autophagy and apoptosis in NMR skin fibroblasts by inhibiting the PI3K/Akt pathway. CQ-induced inhibition of late autophagy stages also prevented Akt activation and induced apoptosis. Finally, the HIF-1α and p53 pathways were involved in serum starvation- or H2O2-induced autophagy in NMR skin fibroblasts.

  10. Activated AKT regulates NF-kappaB activation, p53 inhibition and cell survival in HTLV-1-transformed cells.

    PubMed

    Jeong, Soo-Jin; Pise-Masison, Cynthia A; Radonovich, Michael F; Park, Hyeon Ung; Brady, John N

    2005-10-06

    AKT activation enhances resistance to apoptosis and induces cell survival signaling through multiple downstream pathways. We now present evidence that AKT is activated in HTLV-1-transformed cells and that Tax activation of AKT is linked to NF-kappaB activation, p53 inhibition and cell survival. Overexpression of AKT wild type (WT), but not a kinase dead (KD) mutant, resulted in increased Tax-mediated NF-kappaB activation. Blocking AKT with the PI3K/AKT inhibitor LY294002 or AKT SiRNA prevented NF-kappaB activation and inhibition of p53. Treatment of C81 cells with LY294002 resulted in an increase in the p53-responsive gene MDM2, suggesting a role for AKT in the Tax-mediated regulation of p53 transcriptional activity. Further, we show that LY294002 treatment of C81 cells abrogates in vitro IKKbeta phosphorylation of p65 and causes a reduction of p65 Ser-536 phosphorylation in vivo, steps critical to p53 inhibition. Interestingly, blockage of AKT function did not affect IKKbeta phosphorylation of IkappaBalpha in vitro suggesting selective activity of AKT on the IKKbeta complex. Finally, AKT prosurvival function in HTLV-1-transformed cells is linked to expression of Bcl-xL. We suggest that AKT plays a role in the activation of prosurvival pathways in HTLV-1-transformed cells, possibly through NF-kappaB activation and inhibition of p53 transcription activity.

  11. A new selective AKT pharmacological inhibitor reduces resistance to chemotherapeutic drugs, TRAIL, all-trans-retinoic acid, and ionizing radiation of human leukemia cells.

    PubMed

    Martelli, A M; Tazzari, P L; Tabellini, G; Bortul, R; Billi, A M; Manzoli, L; Ruggeri, A; Conte, R; Cocco, L

    2003-09-01

    It is now well established that the reduced capacity of tumor cells of undergoing cell death through apoptosis plays a key role both in the pathogenesis of cancer and in therapeutic treatment failure. Indeed, tumor cells frequently display multiple alterations in signal transduction pathways leading to either cell survival or apoptosis. In mammals, the pathway based on phosphoinositide 3-kinase (PI3K)/Akt conveys survival signals of extreme importance and its downregulation, by means of pharmacological inhibitors of PI3K, considerably lowers resistance to various types of therapy in solid tumors. We recently described an HL60 leukemia cell clone (HL60AR cells) with a constitutively active PI3K/Akt pathway. These cells were resistant to multiple chemotherapeutic drugs, all-trans-retinoic acid (ATRA), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Treatment with two pharmacological inhibitors of PI3K, wortmannin and Ly294002, restored sensitivity of HL60AR cells to the aforementioned treatments. However, these inhibitors have some drawbacks that may severely limit or impede their clinical use. Here, we have tested whether or not a new selective Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (Akt inhibitor), was as effective as Ly294002 in lowering the sensitivity threshold of HL60 cells to chemotherapeutic drugs, TRAIL, ATRA, and ionizing radiation. Our findings demonstrate that, at a concentration which does not affect PI3K activity, the Akt inhibitor markedly reduced resistance of HL60AR cells to etoposide, cytarabine, TRAIL, ATRA, and ionizing radiation. This effect was likely achieved through downregulation of expression of antiapoptotic proteins such as c-IAP1, c-IAP2, cFLIP(L), and of Bad phosphorylation on Ser 136. The Akt inhibitor did not influence PTEN activity. At variance with Ly294002, the Akt inhibitor did not negatively affect phosphorylation of protein kinase C-zeta and it was less

  12. Co-Inhibition of GLUT-1 Expression and the PI3K/Akt Signaling Pathway to Enhance the Radiosensitivity of Laryngeal Carcinoma Xenografts In Vivo.

    PubMed

    Luo, Xing-Mei; Xu, Bin; Zhou, Min-Li; Bao, Yang-Yang; Zhou, Shui-Hong; Fan, Jun; Lu, Zhong-Jie

    2015-01-01

    In the present study, we investigated the role of GLUT-1 and PI3K/Akt signaling in radioresistance of laryngeal carcinoma xenografts. Volume, weight, radiosensitization, and the rate of inhibition of tumor growth in the xenografts were evaluated in different groups. Apoptosis was evaluated by TUNEL assay. In addition, mRNA and protein levels of GLUT-1, p-Akt, and PI3K in the xenografts were measured. Treatment with LY294002, wortmannin, wortmannin plus GLUT-1 AS-ODN, and LY294002 plus GLUT-1 AS-ODN after X-ray irradiation significantly reduced the size and weight of the tumors, rate of tumor growth, and apoptosis in tumors compared to that observed in the 10-Gy group (p<0.05). In addition, mRNA and protein expression of GLUT-1, p-Akt, and PI3K was downregulated. The E/O values of LY294002, LY294002 plus GLUT-1 AS-ODN, wortmannin, and wortmannin plus GLUT-1 AS-ODN were 2.7, 1.1, 1.8, and 1.8, respectively. Taken together, these data indicate that GLUT-1 AS-ODN as well as the inhibitors of PI3K/Akt signaling may act as radiosensitizers of laryngeal carcinoma in vivo.

  13. Class I PI3-kinase or Akt inhibition do not impair axonal polarization, but slow down axonal elongation.

    PubMed

    Diez, Héctor; Benitez, Ma José; Fernandez, Silvia; Torres-Aleman, Ignacio; Garrido, Juan José; Wandosell, Francisco

    2016-11-01

    PI3K proteins family have multiple and essential functions in most cellular events. This family is composed of class I, class II and class III PI3Ks, which upstream and downstream elements are not completely elucidated. Previous studies using the broad PI3K inhibitor, LY294002 allowed to propose that PI3 kinase>Akt pathway is a key element in the determination of axonal polarity in hippocampal neurons. Recently, new inhibitors with a higher selectivity for class I PI3K have been characterized. In the present study we have examined this widely accepted theory using a new class I PI3K inhibitor (GDC-0941), as well as Akt inhibitors, and PTEN phosphatase constructs to reduce PIP3 levels. Our present data show that both, class I PI3K inhibitor and Akt inhibitor did not alter axon specification in hippocampal neurons, but greatly reduced axon length. However, in the same experiments LY294002 effectively impeded axonal polarization, as previously reported. Our biochemical data show that both, class I PI3K and Akt inhibitors, effectively block downstream elements from Akt to S6K1 activity. Both inhibitors are stable in culture medium along the time period analysed, maintaining the inhibition better than LY294002. Besides, we found evidence that LY294002 directly inhibits mTORC1. However, further analysis using an mTORC1 inhibitor showed no change in neuron polarity. Same result was obtained using a general class III PI3K inhibitor. Interestingly, we found that either, wild-type PTEN, or a phosphatase-dead form of PTEN, disrupted axonal polarization, strongly suggesting that the role of PTEN in axonal polarity can be independent of PIP3.

  14. Role of PI3K/AKT/mTOR signaling in the cell cycle progression of human prostate cancer.

    PubMed

    Gao, Ning; Zhang, Zhuo; Jiang, Bing-Hua; Shi, Xianglin

    2003-10-31

    Prostate cancer is one of the most common cancers among men. Recent studies demonstrated that PI3K signaling is an important intracellular mediator which is involved in multiple cellular functions including proliferation, differentiation, anti-apoptosis, tumorigenesis, and angiogenesis. In the present study, we demonstrate that the inhibition of PI3K activity by LY294002, inhibited prostate cancer cell proliferation and induced the G(1) cell cycle arrest. This effect was accompanied by the decreased expression of G(1)-associated proteins including cyclin D1, CDK4, and Rb phosphorylation at Ser780, Ser795, and Ser807/811, whereas expression of CDK6 and beta-actin was not affected by LY294002. The expression of cyclin kinase inhibitor, p21(CIP1/WAF1), was induced by LY294002, while levels of p16(INK4) were decreased in the same experiment. The inhibition of PI3K activity also inhibited the phosphorylation and p70(S6K), but not MAPK. PI3K regulates cell cycle through AKT, mTOR to p70(S6K). The mTOR inhibitor rapamycin has similar inhibitory effects on G(1) cell cycle progression and expression of cyclin D1, CDK4, and Rb phosphorylation. These results suggest that PI3K mediates G(1) cell cycle progression and cyclin expression through the activation of AKT/mTOR/p70(S6K) signaling pathway in the prostate cancer cells.

  15. PI3K/AKT inhibition induces caspase-dependent apoptosis in HTLV-1-transformed cells.

    PubMed

    Jeong, Soo-Jin; Dasgupta, Arindam; Jung, Kyung-Jin; Um, Jee-Hyun; Burke, Aileen; Park, Hyeon Ung; Brady, John N

    2008-01-20

    The phosphatidylinositol-3-kinase (PI3K) and AKT (protein kinase B) signaling pathways play an important role in regulating cell cycle progression and cell survival. In previous studies, we demonstrated that AKT is activated in HTLV-1-transformed cells and that Tax activation of AKT is linked to p53 inhibition and cell survival. In the present study, we extend these observations to identify regulatory pathways affected by AKT in HTLV-1-transformed cells. We demonstrate that inhibition of AKT reduces the level of phosphorylated Bad, an important member of the pro-apoptotic family of proteins. Consistent with the decrease of phosphorylated Bad, cytochrome c is released from the mitochondria and caspase-9 is activated. Pretreatment of the cells with caspase-9 specific inhibitor z-LEHD-FMK or pan caspase inhibitor Ac-DEVD-CHO prevented LY294002-induced apoptosis. Of interest, p53 siRNA prevents LY294002-induced apoptosis in HTLV-1-transformed cells, suggesting that p53 reactivation is linked to apoptosis. In conclusion, the AKT pathway is involved in targeting multiple proteins which regulate caspase- and p53-dependent apoptosis in HTLV-1-transformed cells. Since AKT inhibitors simultaneously inhibit NF-kappaB and activate p53, these drugs should be promising candidates for HTLV-1-associated cancer therapy.

  16. PI3K/Akt is involved in brown adipogenesis mediated by growth differentiation factor-5 in association with activation of the Smad pathway

    SciTech Connect

    Hinoi, Eiichi; Iezaki, Takashi; Fujita, Hiroyuki; Watanabe, Takumi; Odaka, Yoshiaki; Ozaki, Kakeru; Yoneda, Yukio

    2014-07-18

    Highlights: • Akt is preferentially phosphorylated in BAT and sWAT of aP2-GDF5 mice. • PI3K/Akt signaling is involved in GDF5-induced brown adipogenesis. • PI3K/Akt signaling regulates GDF5-induced Smad5 phosphorylation. - Abstract: We have previously demonstrated promotion by growth differentiation factor-5 (GDF5) of brown adipogenesis for systemic energy expenditure through a mechanism relevant to activating the bone morphological protein (BMP) receptor/mothers against decapentaplegic homolog (Smad)/peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) pathway. Here, we show the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in brown adipogenesis mediated by GDF5. Overexpression of GDF5 in cells expressing adipocyte protein-2 markedly accelerated the phosphorylation of Smad1/5/8 and Akt in white and brown adipose tissues. In brown adipose tissue from heterozygous GDF5{sup Rgsc451} mutant mice expressing a dominant-negative (DN) GDF5 under obesogenic conditions, the basal phosphorylation of Smad1/5/8 and Akt was significantly attenuated. Exposure to GDF5 not only promoted the phosphorylation of both Smad1/5/8 and Akt in cultured brown pre-adipocytes, but also up-regulated Pgc1a and uncoupling protein-1 expression in a manner sensitive to the PI3K/Akt inhibitor Ly294002 as well as retroviral infection with DN-Akt. GDF5 drastically promoted BMP-responsive luciferase reporter activity in a Ly294002-sensitive fashion. Both Ly294002 and DN-Akt markedly inhibited phosphorylation of Smad5 in the nuclei of brown pre-adipocytes. These results suggest that PI3K/Akt signals play a role in the GDF5-mediated brown adipogenesis through a mechanism related to activation of the Smad pathway.

  17. PI3K/AKT Mediated P53 Down-Regulation Participates in CpG DNA Inhibition of Spontaneous B Cell Apoptosis

    PubMed Central

    Zhou, Yongxin; Zhen, Huiling; Mei, Yunqing; Wang, Yongwu; Feng, Jing; Xu, Shuchang; Fu, Xiaoying

    2009-01-01

    The unmethylated CpG DNA can prevent spontaneous apoptosis of B cells. However, the precise mechanisms by which CpG DNA blocks apoptosis remain unclear. In this study, we showed B cell apoptosis was significantly inhibited by addition of CpG DNA. Treatment of CpG DNA could reduce the expression of caspase 3, increase IAP and Bcl-xL expressions, and inhibit p53 protein expression which level was increased in B cell spontaneous apoptosis at 24 h. AKT kinase activity was increased with the incubation of CpG DNA. The wortmannin and Ly294002 could abrogate the protection of B cell from apoptosis by CpG DNA. The up-regulations of Bcl-xL and IAP by CpG DNA were not inhibited when blocking PI3K by specific inhibitor Ly294002, while the inhibition of p53 by CpG DNA could be blocked by Ly294002. These results demonstrated that the inhibition of spontaneous B cell apoptosis by CpG DNA was correlated to up-regulation of Bcl-xL, IAP and down-regulation of p53 and caspase 3. CpG DNA inhibition of p53 is mediated through PI3K/AKT signaling. PMID:19567200

  18. PI3K/AKT mediated p53 down-regulation participates in CpG DNA inhibition of spontaneous B cell apoptosis.

    PubMed

    Zhou, Yongxin; Zhen, Huiling; Mei, Yunqing; Wang, Yongwu; Feng, Jing; Xu, Shuchang; Fu, Xiaoying

    2009-06-01

    The unmethylated CpG DNA can prevent spontaneous apoptosis of B cells. However, the precise mechanisms by which CpG DNA blocks apoptosis remain unclear. In this study, we showed B cell apoptosis was significantly inhibited by addition of CpG DNA. Treatment of CpG DNA could reduce the expression of caspase 3, increase IAP and Bcl-xL expressions, and inhibit p53 protein expression which level was increased in B cell spontaneous apoptosis at 24 h. AKT kinase activity was increased with the incubation of CpG DNA. The wortmannin and Ly294002 could abrogate the protection of B cell from apoptosis by CpG DNA. The up-regulations of Bcl-xL and IAP by CpG DNA were not inhibited when blocking PI3K by specific inhibitor Ly294002, while the inhibition of p53 by CpG DNA could be blocked by Ly294002. These results demonstrated that the inhibition of spontaneous B cell apoptosis by CpG DNA was correlated to up-regulation of Bcl-xL, IAP and down-regulation of p53 and caspase 3. CpG DNA inhibition of p53 is mediated through PI3K/AKT signaling.

  19. Probing the PI3K/Akt/mTor pathway using 31P-NMR spectroscopy: routes to glycogen synthase kinase 3

    PubMed Central

    Phyu, Su M.; Tseng, Chih-Chung; Fleming, Ian N.; Smith, Tim A. D.

    2016-01-01

    Akt is an intracellular signalling pathway that serves as an essential link between cell surface receptors and cellular processes including proliferation, development and survival. The pathway has many downstream targets including glycogen synthase kinase3 which is a major regulatory kinase for cell cycle transit as well as controlling glycogen synthase activity. The Akt pathway is frequently up-regulated in cancer due to overexpression of receptors such as the epidermal growth factor receptor, or mutation of signalling pathway kinases resulting in inappropriate survival and proliferation. Consequently anticancer drugs have been developed that target this pathway. MDA-MB-468 breast and HCT8 colorectal cancer cells were treated with inhibitors including LY294002, MK2206, rapamycin, AZD8055 targeting key kinases in/associated with Akt pathway and the consistency of changes in 31P-NMR-detecatable metabolite content of tumour cells was examined. Treatment with the Akt inhibitor MK2206 reduced phosphocholine levels in MDA-MB-468 cells. Treatment with either the phosphoinositide-3-kinase inhibitor, LY294002 and pan-mTOR inhibitor, AZD8055 but not pan-Akt inhibitor MK2206 increased uridine-5′-diphosphate-hexose cell content which was suppressed by co-treatment with glycogen synthase kinase 3 inhibitor SB216763. This suggests that there is an Akt-independent link between phosphoinositol-3-kinase and glycogen synthase kinase3 and demonstrates the potential of 31P-NMR to probe intracellular signalling pathways. PMID:27811956

  20. The PI3K/Akt signaling pathway exerts effects on the implantation of mouse embryos by regulating the expression of RhoA

    PubMed Central

    LIU, LIYUAN; WANG, YINGXIONG; YU, QIUBO

    2014-01-01

    The aim of this study was to investigate whether the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway affects the implantation of mouse embryos by regulating the expression of RhoA. The expression of PI3K, Akt, phosphorylated (p-)Akt, phosphatase and tensin homolog (PTEN) and RhoA in the uterus of mice on day 5 of pregnancy (D5) and in pseudopregnant mice was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry and western blot analysis. A functional analysis of these genes was also performed by the intrauterine injection with the PI3K inhibitor, LY294002, on day 2 of pregnancy (D2). The expression levels of PI3K, p-Akt, RhoA at the implantation site were higher than those at the inter-implantation site in the endometrium; however, opposite effects were observed for PTEN expression. The expression levels of the above genes in the pseudopregnant group and in the group injected with the PI3K/Akt inhibitor, LY294002, were markedly lower than those in the pregnant group. Functional experiments revealed that the number of implantation sites had been significantly decreased (P<0.05) following the intrauterine injection of the PI3K inhibitor, LY294002, on day 2 of gestation compared with the contralateral injection of phosphate-buffered saline (PBS). These results suggest that the PI3K/Akt signaling pathway affects embryo implantation by regulating the expression of RhoA. PMID:24638941

  1. Akt as a mediator of secretory phospholipase A2 receptor-involved inducible nitric oxide synthase expression.

    PubMed

    Park, Dae-Won; Kim, Jae-Ryong; Kim, Seong-Yong; Sonn, Jong-Kyung; Bang, Ok-Sun; Kang, Shin-Sung; Kim, Jung-Hye; Baek, Suk-Hwan

    2003-02-15

    The induction of inducible NO synthase (iNOS) by group IIA phospholipase A(2) (PLA(2)) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA(2) up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA(2) receptor (sPLA(2)R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA(2) stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA(2), Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA(2)-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA(2), and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA(2)-induced reporter activity of the iNOS promoter. M-type sPLA(2)R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA(2)R enhanced group IIA PLA(2)-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA(2) catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA(2) induces nitrite production by involving of M-type sPLA(2)R, which then mediates signal transduction events that lead to PI3K/Akt activation.

  2. Combination of PI3K/Akt/mTOR inhibitors and PDT in endothelial and tumor cells

    NASA Astrophysics Data System (ADS)

    Fateye, Babasola; Chen, Bin

    2011-02-01

    The PI3/Akt/mTOR kinase signaling pathway is a major signaling pathway in eukaryotic cells, and dysregulation of this signaling pathway has been implicated in tumorigenesis and malignancy in several cancers including prostate cancer. We assessed the effects of combination PI3K pathway inhibition on the efficacy of PDT in human prostate tumor cell line (PC3) and SV40-transformed mouse endothelial cell line (SVEC-40). Combination of PDT and BEZ 235 (BEZ), a pan-PI3/ mTOR kinase inhibitor additively enhanced efficacy of sub-lethal PDT in both cell lines. The combination of the pan-PI3/ mTOR kinase inhibitor LY294002 (LY) with PDT also enhanced efficacy of PDT in PC3 in an additive manner but synergistically in SVEC. In order to determine the mechanism of enhancement of efficacy, we assessed apoptosis and autophagy following PDT. PDT-mediated apoptosis was enhanced in endothelial cells, by both BEZ and LY rapidly after treatment. Compared to SVEC, PC3 cells are apoptosis-deficient and apoptosis was not significantly enhanced by either LY or BEZ. However, lethal PDT of PC3 cells induced a delayed autophagic response which may be enhanced by combination, depending on PI3K inhibitor and dose.

  3. Endothelium-Dependent Relaxation Effect of Apocynum venetum Leaf Extract via Src/PI3K/Akt Signalling Pathway

    PubMed Central

    Lau, Yeh Siang; Ling, Wei Chih; Murugan, Dharmani; Kwan, Chiu Yin; Mustafa, Mohd Rais

    2015-01-01

    Botanical herbs are consumed globally not only as an essential diet but also as medicines or as functional/recreational food supplements. The extract of the Apocynum venetum leaves (AVLE), also known as Luobuma, exerts its antihypertensive effect via dilating the blood vessels in an endothelium- and concentration-dependent manner with optimal effect seen at as low as 10 µg/mL. A commercial Luoboma “antihypertensive tea” is available commercially in the western province of China. The present study seeks to investigate the underlying cellular mechanisms of the nitric oxide (NO)-releasing property of AVLE in rat aortas and human umbilical vein endothelial cells (HUVECs). Endothelium-dependent relaxation induced by AVLE was assessed in organ chambers in the presence or absence of polyethyleneglycol catalase (PP2, 20 µM; inhibitor of Src kinase), wortmannin (30 nM) and LY294002 (20 µM; PI3 (phosphatidylinositol3)-Kinase inhibitor), NG-nitro-l-arginine (L-NAME, 100 µM; endothelial NO synthase inhibitor (eNOS)) and ODQ (1 µM; soluble guanylyl cyclase inhibitor). Total nitrite and nitrate (NOx) level and protein expression of p-Akt and p-eNOS were measured. AVLE-induced endothelium-dependent relaxation was reduced by PP2, wortmannin and LY294002 and abolished by L-NAME and ODQ. AVLE significantly increased total NOx level in rat aortas and in HUVECs compared to control. It also instigated phosphorylation of Akt and eNOS in cultured HUVECs in a concentration-dependent manner and this was markedly suppressed by PP2, wortmannin and LY294002. AVLE also inhibited superoxide generated from both NADPH oxidase and xanthine/xanthine oxidase system. Taken together, AVLE causes endothelium-dependent NO mediated relaxations of rat aortas through Src/PI3K/Akt dependent NO signalling pathway and possesses superoxide scavenging activity. PMID:26133970

  4. Astaxanthin down-regulates Rad51 expression via inactivation of AKT kinase to enhance mitomycin C-induced cytotoxicity in human non-small cell lung cancer cells.

    PubMed

    Ko, Jen-Chung; Chen, Jyh-Cheng; Wang, Tai-Jing; Zheng, Hao-Yu; Chen, Wen-Ching; Chang, Po-Yuan; Lin, Yun-Wei

    2016-04-01

    Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. However, the molecular mechanism of astaxanthin-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination, and studies show that chemo-resistant carcinomas exhibit high levels of Rad51 expression. In this study, astaxanthin treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1703. Astaxanthin treatment (2.5-20 μM) decreased Rad51 expression and phospho-AKT(Ser473) protein level in a time and dose-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vector rescued the decreased Rad51 mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) further decreased the Rad51 expression in astaxanthin-exposed A549 and H1703 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA or cotreatment with LY294002 further enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Additionally, mitomycin C (MMC) as an anti-tumor antibiotic is widely used in clinical NSCLC chemotherapy. Combination of MMC and astaxanthin synergistically resulted in cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced phospho-AKT(Ser473) level and Rad51 expression. Overexpression of AKT-CA or Flag-tagged Rad51 reversed the astaxanthin and MMC-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in astaxanthin and MMC co-treated cells. In conclusion, astaxanthin enhances MMC-induced cytotoxicity by decreasing Rad51 expression and AKT activation. These findings may provide rationale to combine astaxanthin with MMC for the treatment of NSCLC.

  5. Fibroblast growth factor 4-induced migration of porcine trophectoderm cells is mediated via the AKT cell signaling pathway.

    PubMed

    Jeong, Wooyoung; Lee, Jieun; Bazer, Fuller W; Song, Gwonhwa; Kim, Jinyoung

    2016-01-05

    During early pregnancy, a well-coordinated communication network between the conceptus and maternal uterus is especially crucial in pigs in which there is a protracted pre-attachment phase prior to implantation. This network is regulated by an astonishing number of molecules such as growth factors. Fibroblast growth factor 4 (FGF4) is a multipotent growth factor that elicits diverse biological actions on various types of cells and tissues. In pigs, FGF4 and its receptors are expressed in the uterine endometrium and conceptus during early pregnancy, but less is known about the FGF4-mediated regulation of conceptus growth during peri-implantation period of pregnancy. Therefore, the aims of the present study were to investigate: 1) expression of endometrial FGF4 mRNA during early pregnancy; 2) up-regulation of FGF receptor expression in porcine trophectoderm (pTr) cells in response to FGF4; and 3) FGF-induced intracellular signaling and cellular activities in pTr cells. In vitro cultured pTr cells incubated with different concentrations of recombinant FGF4 (0-50 ng/ml) responded with a dose-dependent increase in AKT phosphorylation of 2.9-fold at 20 ng/ml FGF4. Within 30 min after treatment with 20 ng/ml FGF4, the abundances of p-AKT, p-P90RSK and p-RPS6 proteins increased 2.1-, 5.2- and 3.2-fold, respectively, and then returned to basal levels by 120 min. To ensure that the stimulatory effect of FGF4 on AKT signaling was p-AKT-dependent, pTr cells were pre-incubated with an AKT inhibitor (LY294002) for 1 h prior to FGF4 treatment. 20 μM of LY294002 decreased FGF4-induced p-AKT, p-P90RSK and p-RPS6 proteins. Immunofluorescence analyses revealed that p-RPS6 proteins were abundant within the cytoplasm of FGF4-treated cells, but present at basal levels in the presence of LY294002. Furthermore, FGF4 increased migration of pTr cells and LY294002 significantly reduced this effect. Results of the present study suggest that activation of the FGF receptor(s) on trophectoderm

  6. Early changes in [18F]FDG incorporation by breast cancer cells treated with trastuzumab in normoxic conditions: role of the Akt-pathway, glucose transport and HIF-1α.

    PubMed

    Fleming, Ian N; Andriu, Alexandra; Smith, Tim A D

    2014-04-01

    HER-2 overexpression does not guarantee response to HER2-targeting drugs such as trastuzumab, which is cardiotoxic and expensive, so early detection of response status is crucial. Factors influencing [(18)F]FDG incorporation in the timeframe of cell signalling down-regulation subsequent to trastuzumab treatment are investigated to provide a better understanding of the relationship between growth response and modulation of [(18)F]FDG incorporation. HER-2-overexpressing breast tumour cell lines, MDA-MB-453, SKBr3 and BT474 and MDA-MB-468 (HER2 non-over-expressor) were treated with trastuzumab (4 h) and probed for AKT, pAKT, ERK1/2, pERK1/2 and HIF-1α to determine early signalling pathway inhibitory effects of trastuzumab. Cells incubated with trastuzumab and/or PI3K inhibitor LY294002 and ERK1/2 inhibitor U0126 and glucose transport and [(18)F]FDG incorporation measured. Cell lines expressed AKT, pAKT, ERK1/2 and pERK1/2 but not HIF-1α. Trastuzumab treatment decreased pAkt but not pERK1/2 levels. Trastuzumab did not further inhibit AKT when maximally inhibited with LY294002. Treatment with LY294002 and trastuzumab for 4 h decreased [(18)F]FDG incorporation in BT474 and MDA-MB-453 but not SKBr3 cells. LY294002 inhibited glucose transport by each cell line, but the glucose transport rate was tenfold higher by SKBr3 cells than BT474 and MDA-MB-453 cells. AKT-induced uptake of [(18)F]FDG was found to be HIF-1α independent in breast cancer cell lines. AKT inhibition level and tumour cell glucose transport rate can influence whether or not PI3K inhibitors affect [(18)F]FDG incorporation which may account for the variation in preclinical and clinical findings associated with [(18)F]FDG-PET in response to trastuzumab and other HER-2 targeting drugs.

  7. PI3K/Akt pathway regulates retinoic acid-induced Hox gene expression in F9 cells.

    PubMed

    Lee, Youra; Lee, Ji-Yeon; Kim, Myoung Hee

    2014-09-01

    Retinoic acid (RA), the most potent natural form of vitamin A, is a key morphogen in vertebrate development and a potent regulator of both adult and embryonic cell differentiation. Specifically, RA regulates clustered Hox gene expression during embryogenesis and is required to establish the anteroposterior body plan. The PI3K/Akt pathway was also reported to play an essential role in the process of RA-induced cell differentiation. Therefore, we tested whether the PI3K/Akt pathway is involved in RA-induced Hox gene expression in a F9 murine embryonic teratocarcinoma cells. To examine the effect of PI3K/Akt signaling on RA-induced initiation of collinear expression of Hox genes, F9 cells were treated with RA in the presence or absence of PI3K inhibitor LY294002, and time-course gene expression profiles for all 39 Hox genes located in four different clusters-Hoxa, Hoxb, Hoxc, and Hoxd-were analyzed. Collinear expression of Hoxa and -b cluster genes was initiated earlier than that of the -c and -d clusters upon RA treatment. When LY294002 was applied along with RA, collinear expression induced by RA was delayed, suggesting that the PI3K/Akt signaling pathway somehow regulates RA-induced collinear expression of Hox genes in F9 cells. The initiation of Hox collinear expression by RA and the delayed expression following LY294002 in F9 cells would provide a good model system to decipher the yet to be answered de novo collinear expression of Hox genes during gastrulation, which make the gastrulating cells to remember their positional address along the AP body axis in the developing embryo.

  8. Andrographolide Inhibits Nuclear Factor-κB Activation through JNK-Akt-p65 Signaling Cascade in Tumor Necrosis Factor-α-Stimulated Vascular Smooth Muscle Cells

    PubMed Central

    Chen, Yu-Ying; Hsieh, Cheng-Ying; Lee, Lin-Wen; Sheu, Joen-Rong

    2014-01-01

    Critical vascular inflammation leads to vascular dysfunction and cardiovascular diseases, including abdominal aortic aneurysms, hypertension, and atherosclerosis. Andrographolide is the most active and critical constituent isolated from the leaves of Andrographis paniculata, a herbal medicine widely used for treating anti-inflammation in Asia. In this study, we investigated the mechanisms of the inhibitory effects of andrographolide in vascular smooth muscle cells (VSMCs) exposed to a proinflammatory stimulus, tumor necrosis factor-α (TNF-α). Treating TNF-α-stimulated VSMCs with andrographolide suppressed the expression of inducible nitric oxide synthase in a concentration-dependent manner. A reduction in TNF-α-induced c-Jun N-terminal kinase (JNK), Akt, and p65 phosphorylation was observed in andrographolide-treated VSMCs. However, andrographolide affected neither IκBα degradation nor p38 mitogen-activated protein kinase or extracellular signal-regulated kinase 1/2 phosphorylation under these conditions. Both treatment with LY294002, a phosphatidylinositol 3-kinase/Akt inhibitor, and treatment with SP600125, a JNK inhibitor, markedly reversed the andrographolide-mediated inhibition of p65 phosphorylation. In addition, LY294002 and SP600125 both diminished Akt phosphorylation, whereas LY294002 had no effects on JNK phosphorylation. These results collectively suggest that therapeutic interventions using andrographolide can benefit the treatment of vascular inflammatory diseases, and andrographolide-mediated inhibition of NF-κB activity in TNF-α-stimulated VSMCs occurs through the JNK-Akt-p65 signaling cascade, an IκBα-independent mechanism. PMID:25114952

  9. Salinomycin enhances cisplatin-induced cytotoxicity in human lung cancer cells via down-regulation of AKT-dependent thymidylate synthase expression.

    PubMed

    Ko, Jen-Chung; Zheng, Hao-Yu; Chen, Wen-Ching; Peng, Yi-Shuan; Wu, Chia-Hung; Wei, Chia-Li; Chen, Jyh-Cheng; Lin, Yun-Wei

    2016-12-15

    Salinomycin, a polyether antibiotic, acts as a highly selective potassium ionophore and has anticancer activity on various cancer cell lines. Cisplatin has been proved as chemotherapy drug for advanced human non-small cell lung cancer (NSCLC). Thymidylate synthase (TS) is a key enzyme in the pyrimidine salvage pathway, and increased expression of TS is thought to be associated with resistance to cisplatin. In this study, we showed that salinomycin (0.5-2μg/mL) treatment down-regulating of TS expression in an AKT inactivation manner in two NSCLC cell lines, human lung adenocarcinoma A549 and squamous cell carcinoma H1703 cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting AKT activity with PI3K inhibitor LY294002 enhanced the cytotoxicity and cell growth inhibition of salinomycin. A combination of cisplatin and salinomycin resulted in synergistic enhancement of cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced activation of phospho-AKT, and TS expression. Overexpression of a constitutive active AKT (AKT-CA) expression vector reversed the salinomycin and cisplatin-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in salinomycin and cisplatin cotreated cells. Our findings suggested that the down-regulation of AKT-mediated TS expression by salinomycin enhanced the cisplatin-induced cytotoxicity in NSCLC cells. These results may provide a rationale to combine salinomycin with cisplatin for lung cancer treatment.

  10. Lycium barbarum Polysaccharides Protect against Trimethyltin Chloride-Induced Apoptosis via Sonic Hedgehog and PI3K/Akt Signaling Pathways in Mouse Neuro-2a Cells.

    PubMed

    Zhao, Wanyun; Pan, Xiaoqi; Li, Tao; Zhang, Changchun; Shi, Nian

    2016-01-01

    Trimethyltin chloride (TMT) is a classic neurotoxicant that can cause severe neurodegenerative diseases. Some signaling pathways involving cell death play pivotal roles in the central nervous system. In this study, the role of Sonic Hedgehog (Shh) and PI3K/Akt pathways in TMT-induced apoptosis and protective effect of Lycium barbarum polysaccharides (LBP) on mouse neuro-2a (N2a) cells were investigated. Results showed that TMT treatment significantly enhanced apoptosis, upregulated proapoptotic Bax, downregulated antiapoptotic Bcl-2 expression, and increased caspase-3 activity in a dose-dependent manner in N2a cells. TMT induced oxidative stress in cells, performing reactive oxygen species (ROS) and malondialdehyde (MDA) excessive generation, and superoxide dismutase (SOD) activity reduction. TMT significantly decreased phosphorylated glycogen synthase kinase-3β (GSK-3β) and inhibited Shh and PI3K/Akt pathways. However, the addition of LBP upregulated GSK-3β phosphorylation, activated Shh and PI3K/Akt pathways, and eventually reduced apoptosis and oxidative stress caused by TMT. The interaction between Shh and PI3K/Akt pathways was clarified by specific PI3K inhibitor LY294002 or Shh inhibitor GDC-0449. Moreover, LY294002 and GDC-0449 pretreatment both induced phosphorylated GSK-3β downregulation and significantly promoted apoptosis induced by TMT. These results suggest that LBP could reduce TMT-induced N2a cells apoptosis by regulating GSK-3β phosphorylation, Shh, and PI3K/Akt signaling pathways.

  11. Melatonin attenuates sepsis-induced cardiac dysfunction via a PI3K/Akt-dependent mechanism.

    PubMed

    An, Rui; Zhao, Lei; Xi, Cong; Li, Haixun; Shen, Guohong; Liu, Haixiao; Zhang, Shumiao; Sun, Lijun

    2016-01-01

    Myocardial dysfunction is an important manifestation of sepsis. Previous studies suggest that melatonin is protective against sepsis. In addition, activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway has been reported to be beneficial in sepsis. However, the role of PI3K/Akt signaling in the protective effect of melatonin against sepsis-induced myocardial dysfunction remains unclear. Here, LY294002, a PI3K inhibitor, was used to investigate the role of PI3K/Akt signaling in mediating the effects of melatonin on sepsis-induced myocardial injury. Cecal ligation and puncture (CLP) surgery was used to establish a rat model of sepsis. Melatonin was administrated to rats intraperitoneally (30 mg/kg). The survival rate, measures of myocardial injury and cardiac performance, serum lactate dehydrogenase level, inflammatory cytokine levels, oxidative stress level, and the extent of myocardial apoptosis were assessed. The results suggest that melatonin administration after CLP surgery improved survival rates and cardiac function, attenuated myocardial injury and apoptosis, and decreased the serum lactate dehydrogenase level. Melatonin decreased the production of the inflammatory cytokines TNF-α, IL-1β, and HMGB1, increased anti-oxidant enzyme activity, and decreased the expression of markers of oxidative damage. Levels of phosphorylated Akt (p-Akt), unphosphorylated Akt (Akt), Bcl-2, and Bax were measured by Western blot. Melatonin increased p-Akt levels, which suggests Akt pathway activation. Melatonin induced higher Bcl-2 expression and lower Bax expression, suggesting inhibition of apoptosis. All protective effects of melatonin were abolished by LY294002, the PI3K inhibitor. In conclusion, our results demonstrate that melatonin mitigates myocardial injury in sepsis via PI3K/Akt signaling activation.

  12. Salvianolic Acid A Attenuates Cell Apoptosis, Oxidative Stress, Akt and NF-κB Activation in Angiotensin-II Induced Murine Peritoneal Macrophages.

    PubMed

    Li, Ling; Xu, Tongda; Du, Yinping; Pan, Defeng; Wu, Wanling; Zhu, Hong; Zhang, Yanbin; Li, Dongye

    2016-01-01

    We discuss the role of Salvianolic acid A(SAA), one of the main effective components in Salvia Miltiorrhiza (known as 'Danshen' in traditional Chinese medicine), in apoptotic factors, the production of oxidative products, and the expression of Akt and NF-κB in angiotensin II (Ang II)-mediated murine macrophages. In the present study, Ang II was added to mice abdominal macrophages with or without addition of SAA. After cell identification, apoptosis was measured by DNA strand break level with TdT-mediated dUTP nick-end labeling (TUNEL) staining, and the expression of Bcl-2 and Bax. Intracellular concentrations of superoxide dismutase (SOD) and malondialdehyde (MDA) were also measured. Western blotting determined the expression of Akt, p-Akt, NF-κB and p-NF-κB. Ly294002 (the inhibitor of PI3K) was used to determine the mechanism of SAA. Ang II (1 µM) significantly increased the number of TUNEL-positive cells and Bax expression, but reduced Bcl-2 expression. These effects were antagonized when the cells were pretreated with SAA. SAA decreased MDA, but increased SOD in the cell lysis solution treated with Ang II. It markedly reduced the level of p-NF-κB, as also p-Akt, which was partly blocked by Ly294002. SAA prevents Ang IIinduced apoptosis, oxidative stress and related protein expression in the macrophages. It also inhibits the activation of Akt.

  13. 5-AIQ inhibits H{sub 2}O{sub 2}-induced apoptosis through reactive oxygen species scavenging and Akt/GSK-3β signaling pathway in H9c2 cardiomyocytes

    SciTech Connect

    Park, Eun-Seok; Kang, Jun Chul; Kang, Do-Hyun; Jang, Yong Chang; Yi, Kyu Yang; Chung, Hun-Jong; Park, Jong Seok; Kim, Bokyung; Feng, Zhong-Ping; Shin, Hwa-Sup

    2013-04-01

    Poly(adenosine 5′-diphosphate ribose) polymerase (PARP) is a nuclear enzyme activated by DNA strand breaks and plays an important role in the tissue injury associated with ischemia and reperfusion. The aim of the present study was to investigate the protective effect of 5-aminoisoquinolinone (5-AIQ), a PARP inhibitor, against oxidative stress-induced apoptosis in H9c2 cardiomyocytes. 5-AIQ pretreatment significantly protected against H{sub 2}O{sub 2}-induced cell death, as determined by the XTT assay, cell counting, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, and Western blot analysis of apoptosis-related proteins such as caspase-3, Bax, and Bcl-2. Upregulation of antioxidant enzymes such as manganese superoxide dismutase and catalase accompanied the protective effect of 5-AIQ on H{sub 2}O{sub 2}-induced cell death. Our data also showed that 5-AIQ pretreatment protected H9c2 cells from H{sub 2}O{sub 2}-induced apoptosis by triggering activation of Akt and glycogen synthase kinase-3β (GSK-3β), and that the protective effect of 5-AIQ was diminished by the PI3K inhibitor LY294002 at a concentration that effectively abolished 5-AIQ-induced Akt and GSK-3β activation. In addition, inhibiting the Akt/GSK-3β pathway by LY294002 significantly attenuated the 5-AIQ-mediated decrease in cleaved caspase-3 and Bax activation and H9c2 cell apoptosis induction. Taken together, these results demonstrate that 5-AIQ prevents H{sub 2}O{sub 2}-induced apoptosis in H9c2 cells by reducing intracellular reactive oxygen species production, regulating apoptosis-related proteins, and activating the Akt/GSK-3β pathway. - Highlights: ► 5-AIQ, a PARP inhibitor, decreased H{sub 2}O{sub 2}-induced H9c2 cell death and apoptosis. ► 5-AIQ upregulated antioxidant Mn-SOD and catalase, while decreasing ROS production. ► 5-AIQ decreased H{sub 2}O{sub 2}-induced increase in cleaved caspase-3 and Bax and decrease in Bcl2. ► 5-AIQ activated Akt and GSK-3

  14. Snail promotes cell migration through PI3K/AKT-dependent Rac1 activation as well as PI3K/AKT-independent pathways during prostate cancer progression

    PubMed Central

    Henderson, Veronica; Smith, Basil; Burton, Liza J; Randle, Diandra; Morris, Marisha; Odero-Marah, Valerie A

    2015-01-01

    Snail, a zinc-finger transcription factor, induces epithelial-mesenchymal transition (EMT), which is associated with increased cell migration and metastasis in cancer cells. Rac1 is a small G-protein which upon activation results in formation of lamellipodia, the first protrusions formed by migrating cells. We have previously shown that Snail promotes cell migration through down-regulation of maspin tumor suppressor. We hypothesized that Snail's regulation of cell migration may also involve Rac1 signaling regulated by PI3K/AKT and/or MAPK pathways. We found that Snail overexpression in LNCaP and 22Rv1 prostate cancer cells increased Rac1 activity associated with increased cell migration, and the Rac1 inhibitor, NSC23766, could inhibit Snail-mediated cell migration. Conversely, Snail downregulation using shRNA in the aggressive C4–2 prostate cancer cells decreased Rac1 activity and cell migration. Moreover, Snail overexpression increased ERK and PI3K/AKT activity in 22Rv1 prostate cancer cells. Treatment of Snail-overexpressing 22Rv1 cells with LY294002, PI3K/AKT inhibitor or U0126, MEK inhibitor, decreased cell migration significantly, but only LY294002 significantly reduced Rac1 activity, suggesting that Snail promotes Rac1 activation via the PI3K/AKT pathway. Furthermore, 22Rv1 cells overexpressing Snail displayed decreased maspin levels, while inhibition of maspin expression in 22Rv1 cells with siRNA, led to increased PI3K/AKT, Rac1 activity and cell migration, without affecting ERK activity, suggesting that maspin is upstream of PI3K/AKT. Overall, we have dissected signaling pathways by which Snail may promote cell migration through MAPK signaling or alternatively through PI3K/AKT-Rac1 signaling that involves Snail inhibition of maspin tumor suppressor. This may contribute to prostate cancer progression. PMID:26207671

  15. Inhibition of autophagy via activation of PI3K/Akt pathway contributes to the protection of ginsenoside Rb1 against neuronal death caused by ischemic insults.

    PubMed

    Luo, Tianfei; Liu, Guiying; Ma, Hongxi; Lu, Bin; Xu, Haiyang; Wang, Yujing; Wu, Jiang; Ge, Pengfei; Liang, Jianmin

    2014-09-01

    Lethal autophagy is a pathway leading to neuronal death caused by transient global ischemia. In this study, we examined the effect of Ginsenoside Rb1 (GRb1) on ischemia/reperfusion-induced autophagic neuronal death and investigated the role of PI3K/Akt. Ischemic neuronal death in vitro was induced by using oxygen glucose deprivation (OGD) in SH-SY5Y cells, and transient global ischemia was produced by using two vessels occlusion in rats. Cellular viability of SH-SY5Y cells was assessed by MTT assay, and CA1 neuronal death was evaluated by Hematoxylin-eosin staining. Autophagic vacuoles were detected by using both fluorescent microscopy in combination with acridine orange (AO) and Monodansylcadaverine (MDC) staining and transmission electronic microscopy. Protein levels of LC3II, Beclin1, total Akt and phosphor-Akt at Ser473 were examined by western blotting analysis. GRb1 inhibited both OGD and transient ischemia-induced neuronal death and mitigated OGD-induced autophagic vacuoles in SH-SY5Y cells. By contrast, PI3K inhibitor LY294002 counteracted the protection of GRb1 against neuronal death caused by either OGD or transient ischemia. LY294002 not only mitigated the up-regulated protein level of phosphor Akt at Ser473 caused by GRb1, but also reversed the inhibitory effect of GRb1 on OGD and transient ischemia-induced elevation in protein levels of LC3II and Beclin1.

  16. Glimepiride promotes osteogenic differentiation in rat osteoblasts via the PI3K/Akt/eNOS pathway in a high glucose microenvironment.

    PubMed

    Ma, Pan; Gu, Bin; Xiong, Wei; Tan, Baosheng; Geng, Wei; Li, Jun; Liu, Hongchen

    2014-01-01

    Our previous studies demonstrated that glimepiride enhanced the proliferation and differentiation of osteoblasts and led to activation of the PI3K/Akt pathway. Recent genetic evidence shows that endothelial nitric oxide synthase (eNOS) plays an important role in bone homeostasis. In this study, we further elucidated the roles of eNOS, PI3K and Akt in bone formation by osteoblasts induced by glimepiride in a high glucose microenvironment. We demonstrated that high glucose (16.5 mM) inhibits the osteogenic differentiation potential and proliferation of rat osteoblasts. Glimepiride activated eNOS expression in rat osteoblasts cultured with two different concentrations of glucose. High glucose-induced osteogenic differentiation was significantly enhanced by glimepiride. Down-regulation of PI3K P85 levels by treatment with LY294002 (a PI3K inhibitor) led to suppression of P-eNOS and P-AKT expression levels, which in turn resulted in inhibition of RUNX2, OCN and ALP mRNA expression in osteoblasts induced by glimepiride at both glucose concentrations. ALP activity was partially inhibited by 10 µM LY294002. Taken together, our results demonstrate that glimepiride-induced osteogenic differentiation of osteoblasts occurs via eNOS activation and is dependent on the PI3K/Akt signaling pathway in a high glucose microenvironment.

  17. Protection afforded by quercetin against H2O2-induced apoptosis on PC12 cells via activating PI3K/Akt signal pathway.

    PubMed

    Chen, Liang; Sun, Lejin; Liu, Zhene; Wang, Hongxia; Xu, Cunli

    2016-01-01

    Cell damage and apoptosis induced by oxidative stress have been involved in various neurodegenerative diseases. This study aims to explore the neuro-protective effects of quercetin on PC12 cells apoptosis induced by hydrogen peroxide (H(2)O(2)) and the underlying mechanisms. The cell viability was detected, as well as the production of reactive oxygen species (ROS), lactate dehydrogenase (LDH) leakage, and the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) of the cells in control, H(2)O(2) and quercetin groups. It finally turned out that quercetin might protect PC12 cells against the negative effect of H(2)O(2) by decreasing of LDH release, ROS concentration and MDA level and regaining the GSH-Px and SOD activities. To investigate the mechanism, LY294002 was introduced, the phosphatidylinositol-3-kinase (PI3K) inhibitor. Bax/Bcl-2 ratio and Akt phosphorylation (p-Akt) were examined by Western blot analysis. The data showed that LY294002 almost had the same effects with H(2)O(2), which was also significantly reversed by quercetin could enhance Bax/Bcl-2 ratio and adjust the p-Akt expression, which indicated quercetin might protect PC12 cells against the negative effect of H(2)O(2) via activating the PI3K/Akt signal pathway.

  18. Increased basal level of Akt-dependent insulin signaling may be responsible for the development of insulin resistance

    PubMed Central

    Liu, Hui-Yu; Hong, Tao; Wen, Ge-Bo; Han, Jianmin; Zuo, Degen; Liu, Zhenqi

    2009-01-01

    A majority of subjects with insulin resistance and hyperinsulinemia can maintain their blood glucose levels normal for the whole life presumably through protein kinase B (Akt)-dependent insulin signaling. In this study, we found that the basal Akt phosphorylation level was increased in liver and gastrocnemius of mice under the high-fat diet (HFD). Levels of mitochondrial DNA and expression of some mitochondrion-associated genes were decreased by the HFD primarily in liver. Triglyceride content was increased in both liver and gastrocnemius by the HFD. Oxidative stress was induced by the HFD in both liver and gastrocnemius. Insulin sensitivity was decreased by the HFD. All of these changes were largely or completely reversed by treatment of animals with the phosphatidylinositol 3-kinase inhibitor LY-294002 during the time when animals usually do not eat. Consequently, the overall insulin sensitivity was increased by treatment with LY-294002. Together, our results indicate that increased basal Akt-dependent insulin signaling suppresses mitochondrial production, increases ectopic fat accumulation, induces oxidative stress, and desensitizes insulin signaling in subjects with insulin resistance and hyperinsulinemia. PMID:19638508

  19. Lipoteichoic acid promotes nuclear accumulation of β-catenin via AKT in human gingival fibroblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Cardoso-Jiménez, Patricia

    2011-09-01

    Treatment of human gingival fibroblasts (HGFs) with lipoteichoic acid (LTA) results in the activation of multiple signaling pathways. Exposure of HGF to LTA has been shown to result in the activation of phosphatidylinositol 3-kinase (PI3K). The aim of this study was to evaluate the effects of LTA-induced PI3K activation in HGFs. We found that LTA treatment results in the phosphorylation of AKT and glycogen synthase kinase (GSK-3). Inactivation of GSK-3 promotes the nuclear accumulation of β-catenin and expression of connexin43. Treatment with PI3K inhibitors, wortmannin and LY294002, inhibited LTA-induced phosphorylation of AKT and GSK-3, demonstrating that these events require PI3K activation. This report is the first demonstration that LTA treatment activates AKT in HGFs.

  20. Restoration of Akt activity by the bisperoxovanadium compound bpV(pic) attenuates hippocampal apoptosis in experimental neonatal pneumococcal meningitis.

    PubMed

    Sury, Matthias D; Vorlet-Fawer, Lorianne; Agarinis, Claudia; Yousefi, Shida; Grandgirard, Denis; Leib, Stephen L; Christen, Stephan

    2011-01-01

    Pneumococcal meningitis causes apoptosis of developing neurons in the dentate gyrus of the hippocampus. The death of these cells is accompanied with long-term learning and memory deficits in meningitis survivors. Here, we studied the role of the PI3K/Akt (protein kinase B) survival pathway in hippocampal apoptosis in a well-characterized infant rat model of pneumococcal meningitis. Meningitis was accompanied by a significant decrease of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and of phosphorylated (i.e., activated) Akt in the hippocampus. At the cellular level, phosphorylated Akt was decreased in both the granular layer and the subgranular zone of the dentate gyrus, the region where the developing neurons undergo apoptosis. Protein levels and activity of PTEN, the major antagonist of PI3K, were unaltered by infection, suggesting that the observed decrease in PIP(3) and Akt phosphorylation is a result of decreased PI3K signaling. Treatment with the PTEN inhibitor bpV(pic) restored Akt activity and significantly attenuated hippocampal apoptosis. Co-treatment with the specific PI3K inhibitor LY294002 reversed the restoration of Akt activity and attenuation of hippocampal apoptosis, while it had no significant effect on these parameters on its own. These results indicate that the inhibitory effect of bpV(pic) on apoptosis was mediated by PI3K-dependent activation of Akt, strongly suggesting that bpV(pic) acted on PTEN. Treatment with bpV(pic) also partially inhibited the concentration of bacteria and cytokines in the CSF, but this effect was not reversed by LY294002, indicating that the effect of bpV(pic) on apoptosis was independent of its effect on CSF bacterial burden and cytokine levels. These results indicate that the PI3K/Akt pathway plays an important role in the death and survival of developing hippocampal neurons during the acute phase of pneumococcal meningitis.

  1. Neuroprotection of Sevoflurane Against Ischemia/Reperfusion-Induced Brain Injury Through Inhibiting JNK3/Caspase-3 by Enhancing Akt Signaling Pathway.

    PubMed

    Wen, Xiang-Ru; Fu, Yan-Yan; Liu, Hong-Zhi; Wu, Jian; Shao, Xiao-Ping; Zhang, Xun-Bao; Tang, Man; Shi, Yue; Ma, Kai; Zhang, Fang; Wang, Yi-Wen; Tang, Hui; Han, Dong; Zhang, Pu; Wang, Shu-Ling; Xu, Zhou; Song, Yuan-Jian

    2016-04-01

    In this study, we investigated the neuroprotective effect of sevoflurane against ischemic brain injury and its underlying molecular mechanisms. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The rats were pretreated with sevoflurane alone or sevoflurane combined with LY294002/wortmannin (selective inhibitor of PI3K) before ischemia. Cresyl violet staining was used to examine the survival of hippocampal CA1 pyramidal neurons. Immunoblotting and immunoprecipitation were performed to measure the phosphorylation of Akt1, PRAS40, ASK1, and JNK3 and the expression of cleaved-caspase-3. The results demonstrated that a moderate dose of sevoflurane inhalation of 2% for 2 h had significant neuroprotective effects against ischemia/reperfusion induced hippocampal neuron death. Sevoflurane significantly increased Akt and PRAS40 phosphorylation and decreased the phosphorylation of ASK1 at 6 h after reperfusion and the phosphorylation of JNK3 at 3 days after reperfusion following 15 min of transient global brain ischemia. Conversely, LY294002 and wortmannin significantly inhibited the effects of sevoflurane. Taken together, the results suggest that sevoflurane could suppress ischemic brain injury by downregulating the activation of the ASK1/JNK3 cascade via increasing the phosphorylation of Akt1 during ischemia/reperfusion.

  2. Cervical spinal erythropoietin induces phrenic motor facilitation via ERK and Akt signaling

    PubMed Central

    Dale, Erica A.; Satriotomo, Irawan; Mitchell, Gordon S.

    2012-01-01

    Erythropoietin (EPO) is typically known for its role in erythropoiesis, but is also a potent neurotrophic/neuroprotective factor for spinal motor neurons. Another trophic factor regulated by Hypoxia-Inducible Factor-1, vascular endothelial growth factor (VEGF), signals via ERK and Akt activation to elicit long-lasting phrenic motor facilitation (pMF). Since EPO also signals via ERK and Akt activation, we tested the hypothesis that EPO elicits similar pMF. Using retrograde labeling and immunohistochemical techniques, we demonstrate in adult, male, Sprague-Dawley rats that EPO and its receptor, EPO-R, are expressed in identified phrenic motor neurons. Intrathecal EPO at C4 elicits long-lasting pMF; integrated phrenic nerve burst amplitude increased >90 min post-injection (63±12% baseline 90 min post-injection; p<0.001). EPO increased phosphorylation (and presumed activation) of ERK (1.6 fold vs controls; p<0.05) in phrenic motor neurons; EPO also increased pAkt (1.6 fold vs controls; p<0.05). EPO-induced pMF was abolished by the MEK/ERK inhibitor U0126 and the PI3 kinase/Akt inhibitor LY294002, demonstrating that ERK MAP kinases and Akt are both required for EPO-induced pMF. Pre-treatment with U0126 and LY294002 decreased both pERK and pAkt in phrenic motor neurons (p<0.05), indicating a complex interaction between these kinases. We conclude that EPO elicits spinal plasticity in respiratory motor control. Since EPO expression is hypoxia-sensitive, it may play a role in respiratory plasticity in conditions of prolonged or recurrent low oxygen. PMID:22539857

  3. Oxidized LDL at low concentration promotes in-vitro angiogenesis and activates nitric oxide synthase through PI3K/Akt/eNOS pathway in human coronary artery endothelial cells

    SciTech Connect

    Yu, Shan; Wong, Siu Ling; Lau, Chi Wai; Huang, Yu; Yu, Cheuk-Man

    2011-04-01

    Research highlights: {yields} Low-concentration oxidized LDL enhances angiogenesis through nitric oxide (NO). {yields} Oxidized LDL increases intracellular NO levels via eNOS phosphorylation. {yields} Akt/PI3K signaling mediates oxidized LDL-induced eNOS phosphorylation. -- Abstract: It has long been considered that oxidized low-density lipoprotein (oxLDL) causes endothelial dysfunction and is remarkably related to the development of atherosclerosis. However, the effect of oxLDL at very low concentration (<10 {mu}g/ml) on the endothelial cells remains speculative. Nitric oxide (NO) has a crucial role in the endothelial cell function. In this study, we investigated the effect of oxLDL at low concentration on NO production and proliferation, migration, tube formation of the human coronary artery endothelial cells (HCAEC). Results showed that oxLDL at 5 {mu}g/ml enhanced HCAEC proliferation, migration and tube formation. These phenomena were accompanied by an increased intracellular NO production. L-NAME (a NOS inhibitor), LY294002 and wortmannin (PI3K inhibitors) could abolish oxLDL-induced angiogenic effects and prevent NO production in the HCAEC. The phosphorylation of Akt, PI3K and eNOS were up-regulated by oxLDL, which was attenuated by LY294002. Our results suggested that oxLDL at low concentration could promote in-vitro angiogenesis and activate nitric oxide synthesis through PI3K/Akt/eNOS pathway in HCAEC.

  4. Potential of the Akt inhibitor LY294005 to antagonize the efficacy of Cisplatin against HCT116 tumor cells in a DNA mismatch repair-dependent manner.

    PubMed

    Fedier, Andre; Erdmann, Ruediger; Boulikas, Teni; Fink, Daniel

    2006-11-01

    Human colorectal adenocarcinoma sublines either deficient (HCT116+ch2) or proficient (HCT116+ch3) in the function of MLH1, one of five proteins crucial to DNA mismatch repair (MMR), were used to investigate whether the Akt-specific inhibitor LY294005 could not only increase the efficacy of platinum drugs in HCT116 cells in general but also increase the efficacy of the cisplatinum compounds Cisplatin and Lipoplatin specifically in MLH1-deficient, Cisplatin- and Lipoplatin-resistant HCT116 cells. We report that, under the conditions it increased the efficacy of Docetaxel and did not affect that of 6-thioguanine, LY294005 decreased the sensitivity of both sublines to Cisplatin, Lipoplatin, Oxaliplatin, and Lipoxal. Notably, the LY294005-imposed decrease was significantly higher in the MLH1-proficient than in the MLH1-deficient subline with Cisplatin and Lipoplatin, whereas it was nearly the same in both sublines with Oxaliplatin and Lipoxal. These LY294005-imposed changes in drug sensitivity, i.e. increase with Docetaxel and decreases with platinum compounds, were not associated with the concomitant abrogation in the levels of phospho-Aktser473. Analogous changes in drug sensitivity were also observed with the PI3-kinase inhibitor LY294002, but these changes were associated with complete abrogation of phospho-Aktser473. These observations suggest a possible relationship between MMR-mediated cisplatinum DNA damage signaling and the Akt signaling pathway, e.g. a common target for both pathways. A possibly novel property of Akt in aggravating drug sensitivity may also be proposed.

  5. PI3K/AKT Signaling Regulates Bioenergetics in Immortalized Hepatocytes

    PubMed Central

    Li, Chen; Li, Yang; He, Lina; Agarwal, Amit R.; Zeng, Ni; Cadenas, Enrique; Stiles, Bangyan L.

    2013-01-01

    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 residence 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., decrease of 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. PMID:23376468

  6. Berberine protects endothelial progenitor cell from damage of TNF-α via the PI3K/AKT/eNOS signaling pathway.

    PubMed

    Xiao, Min; Men, Li Na; Xu, Ming Guo; Wang, Guo Bing; Lv, Hai Tao; Liu, Cong

    2014-11-15

    Endothelial progenitor cells (EPCs) dysfunction is closely correlated with the coronary artery injury induced by Kawasaki disease (KD). The level of tumor necrosis factor-α (TNF-α) elevated significantly in acute phase of KD which can damage the functions of EPCs. The aim of this study was to investigate whether berberine (BBR) can protect EPCs from the inhibition caused by TNF-α via the PI3K (Phosphatidyl Inositol 3-kinase) /AKT (Serine/threonine protein kinase B) /eNOS (endothelial Nitric Oxide synthase) signaling pathway. The cell proliferative ability of EPCs was determined by MTT (methyl thiazolyl tetrazolium) assays. Nitric oxide (NO) level was determined in supernatants. The mRNA level of eNOS, PI3K and AKT were measured by Real Time-Polymerase Chain Reaction (RT-PCR), and the protein levels of eNOS, phospho-eNOS (p-eNOS), Akt, phospho-Akt (p-Akt) and PI3K were analyzed using Western-blot. The results demonstrated that TNF-α inhibits the proliferative ability of EPCs. However, BBR improves the proliferative activity of EPCs inhibited by TNF-α. Blockade of PI3K by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (Ly294002) and blockade of eNOS by l-NAME (NG-Nitroarginine Methyl Ester) attenuates the effect of BBR. BBR can increase the level of PI3K/Akt/eNOS mRNA and the protein level of PI3K, p-Akt, eNOS and p-eNOS, which can be blocked by PI3K inhibitor (LY294002) and eNOS inhibitor (l-NAME). Therefore, we concluded that impaired EPCs proliferation could be reversed by BBR via the PI3K/AKT/eNOS signaling pathway.

  7. PI3K/Akt/FoxO3a signaling mediates cardioprotection of FGF-2 against hydrogen peroxide-induced apoptosis in H9c2 cells.

    PubMed

    Liu, Mi-Hua; Li, Guo-Hua; Peng, Li-Jun; Qu, Shun-Lin; Zhang, Yuan; Peng, Juan; Luo, Xin-Yuan; Hu, Heng-Jing; Ren, Zhong; Liu, Yao; Tang, Hui; Liu, Lu-Shan; Tang, Zhi-Han; Jiang, Zhi-Sheng

    2016-03-01

    Cardiovascular disease is a growing major global public health problem. Oxidative stress is regarded as one of the key regulators of pathological physiology, which eventually leads to cardiovascular disease. However, mechanisms by which FGF-2 rescues cells from oxidative stress damage in cardiovascular disease is not fully elucidated. Herein this study was designed to investigate the protective effects of FGF-2 in H2O2-induced apoptosis of H9c2 cardiomyocytes, as well as the possible signaling pathway involved. Apoptosis of H9c2 cardiomyocytes was induced by H2O2 and assessed using methyl thiazolyl tetrazolium assay, Hoechst, and TUNEL staining. Cells were pretreated with PI3K/Akt inhibitor LY294002 to investigate the possible PI3K/Akt pathways involved in the protection of FGF-2. The levels of p-Akt, p-FoxO3a, and Bim were detected by immunoblotting. Stimulation with H2O2 decreased the phosphorylation of Akt and FoxO3a, and induced nuclear localization of FoxO3a and apoptosis of H9c2 cells. These effects of H2O2 were abrogated by pretreatment with FGF-2. Furthermore, the protective effects of FGF-2 were abolished by PI3K/Akt inhibitor LY294002. In conclusion, our data suggest that FGF-2 protects against H2O2-induced apoptosis of H9c2 cardiomyocytes via activation of the PI3K/Akt/FoxO3a pathway.

  8. Lipopolysaccharide induces VCAM-1 expression and neutrophil adhesion to human tracheal smooth muscle cells: Involvement of Src/EGFR/PI3-K/Akt pathway

    SciTech Connect

    Lin, W.-N.; Luo, S.-F.; Wu, C.-B.; Lin, C.-C.; Yang, C.-M.

    2008-04-15

    In our previous study, LPS has been shown to induce vascular cell adhesion molecule-1(VCAM-1) expression through MAPKs and NF-{kappa}B in human tracheal smooth muscle cells (HTSMCs). In addition to these pathways, the non-receptor tyrosine kinases (Src), EGF receptor (EGFR), and phosphatidylinositol 3-kinase (PI3K) have been shown to be implicated in the expression of several inflammatory target proteins. Here, we reported that LPS-induced up-regulation of VCAM-1 enhanced the adhesion of neutrophils onto HTSMC monolayer, which was inhibited by LY294002 and wortmannin. LPS stimulated phosphorylation of protein tyrosine kinases including Src, PYK2, and EGFR, which were further confirmed using specific anti-phospho-Src, PYK2, or EGFR Ab, respectively, revealed by Western blotting. LPS-stimulated Src, PYK2, EGFR, and Akt phosphorylation and VCAM-1 expression were attenuated by the inhibitors of Src (PP1), EGFR (AG1478), PI3-K (LY294002 and wortmannin), and Akt (SH-5), respectively, or transfection with siRNAs of Src or Akt and shRNA of p110. LPS-induced VCAM-1 expression was also blocked by pretreatment with curcumin (a p300 inhibitor) or transfection with p300 siRNA. LPS-stimulated Akt activation translocated into nucleus and associated with p300 and VCAM-1 promoter region was further confirmed by immunofluorescence, immunoprecipitation, and chromatin immunoprecipitation assays. This association of Akt and p300 to VCAM-1 promoter was inhibited by pretreatment with PP1, AG1478, wortmannin, and SH-5. LPS-induced p300 activation enhanced VCAM-1 promoter activity and VCAM-1 mRNA expression. These results suggested that in HTSMCs, Akt phosphorylation mediated through transactivation of Src/PYK2/EGFR promoted the transcriptional p300 activity and eventually led to VCAM-1 expression induced by LPS.

  9. Pharmacologic Profiling of Phosphoinositide 3-Kinase Inhibitors as Mitigators of Ionizing Radiation–Induced Cell Death

    PubMed Central

    Sharlow, Elizabeth R.; Epperly, Michael W.; Lira, Ana; Leimgruber, Stephanie; Skoda, Erin M.; Wipf, Peter; Greenberger, Joel S.

    2013-01-01

    Ionizing radiation (IR) induces genotoxic stress that triggers adaptive cellular responses, such as activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade. Pluripotent cells are the most important population affected by IR because they are required for cellular replenishment. Despite the clear danger to large population centers, we still lack safe and effective therapies to abrogate the life-threatening effects of any accidental or intentional IR exposure. Therefore, we computationally analyzed the chemical structural similarity of previously published small molecules that, when given after IR, mitigate cell death and found a chemical cluster that was populated with PI3K inhibitors. Subsequently, we evaluated structurally diverse PI3K inhibitors. It is remarkable that 9 of 14 PI3K inhibitors mitigated γIR-induced death in pluripotent NCCIT cells as measured by caspase 3/7 activation. A single intraperitoneal dose of LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], administered to mice at 4 or 24 hours, or PX-867 [(4S,4aR,5R,6aS,9aR,Z)-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-2,7,10-trioxo-1-(pyrrolidin-1-ylmethylene)-1,2,4,4a,5,6,6a,7,8,9,9a,10-dodecahydroindeno[4,5-H]isochromen-5-yl acetate (CID24798773)], administered 4 hours after a lethal dose of γIR, statistically significantly (P < 0.02) enhanced in vivo survival. Because cell cycle checkpoints are important regulators of cell survival after IR, we examined cell cycle distribution in NCCIT cells after γIR and PI3K inhibitor treatment. LY294002 and PX-867 treatment of nonirradiated cells produced a marked decrease in S phase cells with a concomitant increase in the G1 population. In irradiated cells, LY294002 and PX-867 treatment also decreased S phase and increased the G1 and G2 populations. Treatment with LY294002 or PX-867 decreased γIR-induced DNA damage as measured by γH2AX, suggesting reduced DNA damage. These results indicate pharmacologic inhibition of PI3K after

  10. Carvedilol protects bone marrow stem cells against hydrogen peroxide-induced cell death via PI3K-AKT pathway.

    PubMed

    Chen, Meihui; Chen, Shudong; Lin, Dingkun

    2016-03-01

    Carvedilol, a nonselective β-adrenergic receptor blocker, has been reported to exert potent anti-oxidative activities. In the present study, we aimed to investigate the effects of carvedilol against hydrogen peroxide (H2O2)-induced bone marrow-derived mesenchymal stem cells (BMSCs) death, which imitate the microenvironment surrounding transplanted cells in the injured spinal cord in vitro. Carvedilol significantly reduced H2O2-induced reactive oxygen species production, apoptosis and subsequent cell death. LY294002, the PI3K inhibitor, blocked the protective effects and up-regulation of Akt phosphorylation of carvedilol. Together, our results showed that carvedilol protects H2O2-induced BMSCs cell death partly through PI3K-Akt pathway, suggesting carvedilol could be used in combination with BMSCs for the treatment of spinal cord injury by improving the cell survival and oxidative stress microenvironments.

  11. Gefitinib induces lung cancer cell autophagy and apoptosis via blockade of the PI3K/AKT/mTOR pathway

    PubMed Central

    ZHAO, ZHONG-QUAN; YU, ZHONG-YANG; LI, JIE; OUYANG, XUE-NONG

    2016-01-01

    Gefitinib is a selective inhibitor of the tyrosine kinase epidermal growth factor receptor, which inhibits tumor pathogenesis, metastasis and angiogenesis, as well as promoting apoptosis. Therefore, gefitinib presents an effective drug for the targeted therapy of lung cancer. However, the underlying mechanisms by which gefitinib induces lung cancer cell death remain unclear. To investigate the effects of gefitinib on lung cancer cells and the mechanism of such, the present study analyzed the effect of gefitinib on the autophagy, apoptosis and proliferation of the A549 and A549-gefitinib-resistant (GR) cell lines GR. The regulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) pathway was also investigated. Acridine orange staining revealed that gefitinib induced autophagy of A549 cells but not A549-GR cells. In addition, gefitinib promoted apoptosis and inhibited proliferation of A549 cells but not A549-GR cells. Furthermore, western blot analysis demonstrated that gefitinib treatment led to the downregulation of PI3K, AKT, pAKT, mTOR and phosphorylated-mTOR protein expression in A549 cells but not A549-GR cells. LY294002 blocked the PI3K/AKT/mTOR pathway and induced autophagy and apoptosis of A549 cells, however, no synergistic effect was observed following combined treatment with gefitinib and LY294002. In conclusion, the results of the present study indicate that gefitinib promotes autophagy and apoptosis of lung cancer cells via blockade of the PI3K/AKT/mTOR pathway, which leads to lung cancer cell death. PMID:27347100

  12. Neuroprotective Role of the PI3 Kinase/Akt Signaling Pathway in Zebrafish

    PubMed Central

    Chen, Shuang; Liu, Yunzhang; Rong, Xiaozhi; Li, Yun; Zhou, Jianfeng; Lu, Ling

    2017-01-01

    Neuronal survival and growth in the embryo is controlled partly by trophic factors. For most trophic factors (such as Insulin-like growth factor-1), the ability to regulate cell survival has been attributed to the phosphoinositide 3-kinase (PI3K)/Akt kinase cascade. This study presents data illustrating the role of PI3K/Akt in attainment of normal brain size during zebrafish embryogenesis. Blocking PI3K with inhibitor LY294002 caused a significant reduction in brain size (in addition to global growth retardation) during zebrafish embryogenesis. This PI3 Kinase inhibition-induced brain size decrease was recovered by the overexpression of myristoylated Akt (myr-Akt), a constitutive form of Akt. Further analysis reveals that expressing exogenous myr-Akt significantly augmented brain size. Whole mount in situ hybridization analysis of several marker genes showed that myr-Akt overexpression did not alter brain patterning. Furthermore, the expression of myr-Akt was found to protect neuronal cells from apoptosis induced by heat shock and UV light, suggesting that inhibition of neuronal cell death may be part of the underlying cause of the increased brain size. These data provide a foundation for addressing the role of PI3K/Akt in brain growth during zebrafish embryogenesis. PMID:28228749

  13. Lysophosphatidic acid induces cell migration through the selective activation of Akt1

    PubMed Central

    Kim, Eun Kyoung; Yun, Sung Ji; Do, Kee Hun; Kim, Min Sung; Cho, Mong; Suh, Dong-Soo; Kim, Chi Dae; Kim, Jae Ho; Birnbaum, Morris J.

    2008-01-01

    Akt plays pivotal roles in many physiological responses including growth, proliferation, survival, metabolism, and migration. In the current studies, we have evaluated the isoform-specific role of akt in lysophosphatidic acid (LPA)-induced cell migration. Ascites from ovarian cancer patients (AOCP) induced mouse embryo fibroblast (MEF) cell migration in a dose-dependent manner. On the other hand, ascites from liver cirrhosis patients (ALCP) did not induce MEF cell migration. AOCP-induced MEF cell migration was completely blocked by pre-treatment of cells with LPA receptor antagonist, Ki16425. Both LPA- and AOCP-induced MEF cell migration was completely attenuated by PI3K inhibitor, LY294002. Furthermore, cells lacking Akt1 displayed defect in LPA-induced cell migration. Re-expression of Akt1 in DKO (Akt1-/-Akt2-/-) cells restored LPA-induced cell migration, whereas re-expression of Akt2 in DKO cells could not restore the LPA-induced cell migration. Finally, Akt1 was selectively phosphorylated by LPA and AOCP stimulation. These results suggest that LPA is a major factor responsible for AOCP-induced cell migration and signaling specificity of Akt1 may dictate LPA-induced cell migration. PMID:18779657

  14. Inhibition of PI3K-Akt Signaling Blocks Exercise-Mediated Enhancement of Adult Neurogenesis and Synaptic Plasticity in the Dentate Gyrus

    PubMed Central

    Bruel-Jungerman, Elodie; Veyrac, Alexandra; Dufour, Franck; Horwood, Jennifer; Laroche, Serge; Davis, Sabrina

    2009-01-01

    Background Physical exercise has been shown to increase adult neurogenesis in the dentate gyrus and enhances synaptic plasticity. The antiapoptotic kinase, Akt has also been shown to be phosphorylated following voluntary exercise; however, it remains unknown whether the PI3K-Akt signaling pathway is involved in exercise-induced neurogenesis and the associated facilitation of synaptic plasticity in the dentate gyrus. Methodology/Principal Findings To gain insight into the potential role of this signaling pathway in exercise-induced neurogenesis and LTP in the dentate gyrus rats were infused with the PI3K inhibitor, LY294002 or vehicle control solution (icv) via osmotic minipumps and exercised in a running wheel for 10 days. Newborn cells in the dentate gyrus were date-labelled with BrdU on the last 3 days of exercise. Then, they were either returned to the home cage for 2 weeks to assess exercise-induced LTP and neurogenesis in the dentate gyrus, or were killed on the last day of exercise to assess proliferation and activation of the PI3K-Akt cascade using western blotting. Conclusions/Significance Exercise increases cell proliferation and promotes survival of adult-born neurons in the dentate gyrus. Immediately after exercise, we found that Akt and three downstream targets, BAD, GSK3β and FOXO1 were activated. LY294002 blocked exercise-induced phosphorylation of Akt and downstream target proteins. This had no effect on exercise-induced cell proliferation, but it abolished most of the beneficial effect of exercise on the survival of newly generated dentate gyrus neurons and prevented exercise-induced increase in dentate gyrus LTP. These results suggest that activation of the PI3 kinase-Akt signaling pathway plays a significant role via an antiapoptotic function in promoting survival of newly formed granule cells generated during exercise and the associated increase in synaptic plasticity in the dentate gyrus. PMID:19936256

  15. TC21 mediates transformation and cell survival via activation of phosphatidylinositol 3-kinase/Akt and NF-kappaB signaling pathway.

    PubMed

    Rong, Rong; He, Qin; Liu, Yusen; Sheikh, M Saeed; Huang, Ying

    2002-02-07

    The signaling pathways of TC21-mediated transformation and cell survival are not well-established. In this study, we have investigated the role of PI3-K/Akt signaling pathway in oncogenic-TC21-mediated transformation and cell survival. We found that oncogenic-TC21 stimulated the PI3-K activity. This was associated with the activation of Akt, a key component of PI3-K signaling pathway. We also found that TC21 interacted and formed complex with PI3-K. Mutations in the GTP-binding region of TC21, which enhanced GTP-binding potential of this protein, also stimulated its association with PI3-K, suggesting that PI3-K may preferentially interact with the GTP-bound form. Suppression of PI3-K and Akt by specific inhibitors LY294002 and Wortmannin reversed TC21-induced transformation. Likewise, inhibition of PI3-K activity by the PI3-K phosphotase PTEN reduced TC21-mediated focus formation in NIH3T3 cells. Investigation of TC21's effect on cell survival revealed that mutant-TC21 expressing cells were more resistant to etoposide- and cisplatin-induced cell death, and this was associated with the activation of anti-apoptotic protein NF-kappaB, a downstream target of Akt. Treatment of PI3-K inhibitor LY294002 significantly suppressed TC21-mediated NF-kappaB activation. In conclusion, we have identified PI3-K as an effector of TC21 and demonstrated that the PI3-K/Akt signaling pathway plays important roles in TC21-mediated transformation and cell survival.

  16. MiR-20a Induces Cell Radioresistance by Activating the PTEN/PI3K/Akt Signaling Pathway in Hepatocellular Carcinoma

    SciTech Connect

    Zhang, Yuqin; Zheng, Lin; Ding, Yi; Li, Qi; Wang, Rong; Liu, Tongxin; Sun, Quanquan; Yang, Hua; Peng, Shunli; Wang, Wei; Chen, Longhua

    2015-08-01

    Purpose: To investigate the role of miR-20a in hepatocellular carcinoma (HCC) cell radioresistance, which may reveal potential strategies to improve treatment. Methods and Materials: The expression of miR-20a and PTEN were detected in HCC cell lines and paired primary tissues by quantitative real-time polymerase chain reaction. Cell radiation combined with colony formation assays was administrated to discover the effect of miR-20a on radiosensitivity. Bioinformatics prediction and luciferase assay were used to identify the target of miR-20a. The phosphatidylinositol 3-kinase inhibitor LY294002 was used to inhibit phosphorylation of Akt, to verify whether miR-20a affects HCC cell radioresistance through activating the PTEN/PI3K/Akt pathway. Results: MiR-20a levels were increased in HCC cell lines and tissues, whereas PTEN was inversely correlated with it. Overexpression of miR-20a in Bel-7402 and SMMC-7721 cells enhances their resistance to the effect of ionizing radiation, and the inhibition of miR-20a in HCCLM3 and QGY-7701 cells sensitizes them to it. PTEN was identified as a direct functional target of miR-20a for the induction of radioresistance. Overexpression of miR-20a activated the PTEN/PI3K/Akt signaling pathway. Additionally, the kinase inhibitor LY294002 could reverse the effect of miR-20a–induced radioresistance. Conclusion: MiR-20a induces HCC cell radioresistance by activating the PTEN/PI3K/Akt pathway, which suggests that miR-20a/PTEN/PI3K/Akt might represent a target of investigation for developing effective therapeutic strategies against HCC.

  17. Development of sulfonamide AKT PH domain inhibitors.

    PubMed

    Ahad, Ali Md; Zuohe, Song; Du-Cuny, Lei; Moses, Sylvestor A; Zhou, Li Li; Zhang, Shuxing; Powis, Garth; Meuillet, Emmanuelle J; Mash, Eugene A

    2011-03-15

    Disruption of the phosphatidylinositol 3-kinase/AKT signaling pathway can lead to apoptosis in cancer cells. Previously we identified a lead sulfonamide that selectively bound to the pleckstrin homology (PH) domain of AKT and induced apoptosis when present at low micromolar concentrations. To examine the effects of structural modification, a set of sulfonamides related to the lead compound was designed, synthesized, and tested for binding to the expressed PH domain of AKT using a surface plasmon resonance-based competitive binding assay. Cellular activity was determined by means of an assay for pAKT production and a cell killing assay using BxPC-3 cells. The most active compounds in the set are lipophilic and possess an aliphatic chain of the proper length. Results were interpreted with the aid of computational modeling. This paper represents the first structure-activity relationship (SAR) study of a large family of AKT PH domain inhibitors. Information obtained will be used in the design of the next generation of inhibitors of AKT PH domain function.

  18. Camptothecin inhibits platelet-derived growth factor-BB-induced proliferation of rat aortic vascular smooth muscle cells through inhibition of PI3K/Akt signaling pathway

    SciTech Connect

    Park, Eun-Seok; Kang, Shin-il; Yoo, Kyu-dong; Lee, Mi-Yea; Yoo, Hwan-Soo; Hong, Jin-Tae; Shin, Hwa-Sup; Kim, Bokyung; Yun, Yeo-Pyo

    2013-04-15

    The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial wall is a major cause of vascular disorders such as atherosclerosis and restenosis after angioplasty. In this study, we investigated not only the inhibitory effects of camptothecin (CPT) on PDGF-BB-induced VSMC proliferation, but also its molecular mechanism of this inhibition. CPT significantly inhibited proliferation with IC50 value of 0.58 μM and the DNA synthesis of PDGF-BB-stimulated VSMCs in a dose-dependent manner (0.5–2 μM ) without any cytotoxicity. CPT induced the cell cycle arrest at G0/G1 phase. Also, CPT decreased the expressions of G0/G1-specific regulatory proteins including cyclin-dependent kinase (CDK)2, cyclin D1 and PCNA in PDGF-BB-stimulated VSMCs. Pre-incubation of VSMCs with CPT significantly inhibited PDGF-BB-induced Akt activation, whereas CPT did not affect PDGF-receptor beta phosphorylation, extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and phospholipase C (PLC)-γ1 phosphorylation in PDGF-BB signaling pathway. Our data showed that CPT pre-treatment inhibited VSMC proliferation, and that the inhibitory effect of CPT was enhanced by LY294002, a PI3K inhibitor, on PDGF-BB-induced VSMC proliferation. In addition, inhibiting the PI3K/Akt pathway by LY294002 significantly enhanced the suppression of PCNA expression and Akt activation by CPT. These results suggest that the anti-proliferative activity of CPT is mediated in part by downregulating the PI3K/Akt signaling pathway. - Highlights: ► CPT inhibits proliferation of PDGF-BB-induced VSMC without cytotoxicity. ► CPT arrests the cell cycle in G0/G1 phase by downregulation of cyclin D1 and CDK2. ► CPT significantly attenuates Akt phosphorylation in PDGF-BB signaling pathway. ► LY294002 enhanced the inhibitory effect of CPT on VSMC proliferation. ► Thus, CPT is mediated by downregulating the PI3K/Akt signaling pathway.

  19. Id-1 promotes tumorigenicity and metastasis of human esophageal cancer cells through activation of PI3K/AKT signaling pathway.

    PubMed

    Li, Bin; Tsao, Sai Wah; Li, Yuk Yin; Wang, Xianghong; Ling, Ming Tat; Wong, Yong Chuan; He, Qing Yu; Cheung, Annie L M

    2009-12-01

    Id-1 (inhibitor of differentiation or DNA binding) is a helix-loop-helix protein that is overexpressed in many types of cancer including esophageal squamous cell carcinoma (ESCC). We previously reported that ectopic Id-1 expression activates the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in human esophageal cancer cells. In this study, we confirmed a positive correlation between Id-1 and phospho-AKT (Ser473) expressions in ESCC cell lines, as well as in ESCC on a tissue microarray. To investigate the significance of Id-1 in esophageal cancer progression, ESCC cells with stable ectopic Id-1 expression were inoculated subcutaneously into the flank of nude mice and were found to form larger tumors that showed elevated Ki-67 proliferation index and increased angiogenesis, as well as reduced apoptosis, compared with control cells expressing the empty vector.The Id-1-overexpressing cells also exhibited enhanced metastatic potential in the experimental metastasis assay. Treatment with the PI3K inhibitor LY294002 attenuated the tumor promotion effects of Id-1, indicating that the effects were mediated by the PI3K/AKT signaling pathway. In addition, our in vitro experiments showed that ectopic Id-1 expression altered the expression levels of markers associated with epithelial-mesenchymal transition and enhanced the migration ability of esophageal cancer cells. The Id-1-overexpressing ESCC cells also exhibited increased invasive potential, which was in part due to PI3K/AKT-dependent modulation of matrix metalloproteinase-9 expression. In conclusion, our results provide the first evidence that Id-1 promotes tumorigenicity and metastasis of human esophageal cancer in vivo and that the PI3K inhibitor LY294002 can attenuate these effects.

  20. Quercetin attenuates cell apoptosis in focal cerebral ischemia rat brain via activation of BDNF-TrkB-PI3K/Akt signaling pathway.

    PubMed

    Yao, Rui-Qin; Qi, Da-Shi; Yu, Hong-Li; Liu, Jing; Yang, Li-Hua; Wu, Xiu-Xiang

    2012-12-01

    Many studies have demonstrated that apoptosis play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. Neuroprotective effect of quercetin has been shown in a variety of brain injury models including ischemia/reperfusion. It is not clear whether BDNF-TrkB-PI3K/Akt signaling pathway mediates the neuroprotection of quercetin, though there has been some reports on the quercetin increased brain-derived neurotrophic factor (BDNF) level in brain injury models. We therefore first examined the neurological function, infarct volume and cell apoptosis in quercetin treated middle cerebral artery occlusion (MCAO) rats. Then the protein expression of BDNF, cleaved caspase-3 and p-Akt were evaluated in either the absence or presence of PI3K inhibitor (LY294002) or tropomyosin receptor kinase B (TrkB) receptor antagonist (K252a) by immunohistochemistry staining and western blotting. Quercetin significantly improved neurological function, while it decreased the infarct volume and the number of TdT mediated dUTP nick end labeling positive cells in MCAO rats. The protein expression of BDNF, TrkB and p-Akt also increased in the quercetin treated rats. However, treatment with LY294002 or K252a reversed the quercetin-induced increase of BDNF and p-Akt proteins and decrease of cleaved caspase-3 protein in focal cerebral ischemia rats. These results demonstrate that quercetin can decrease cell apoptosis in the focal cerebral ischemia rat brain and the mechanism may be related to the activation of BDNF-TrkB-PI3K/Akt signaling pathway.

  1. SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation

    PubMed Central

    Hayashi, Moyuru; Kaneda, Mizuho; Iida, Kazuko; Shimonaka, Motoyuki; Hara, Takahiko; Arai, Morio; Koike, Yuichi; Yamamoto, Naomasa; Kasahara, Kohji

    2017-01-01

    Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation. PMID:28072855

  2. SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation.

    PubMed

    Ohtsuka, Hiroko; Iguchi, Tomohiro; Hayashi, Moyuru; Kaneda, Mizuho; Iida, Kazuko; Shimonaka, Motoyuki; Hara, Takahiko; Arai, Morio; Koike, Yuichi; Yamamoto, Naomasa; Kasahara, Kohji

    2017-01-01

    Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation.

  3. Resveratrol inhibits TNF-α-induced IL-1β, MMP-3 production in human rheumatoid arthritis fibroblast-like synoviocytes via modulation of PI3kinase/Akt pathway.

    PubMed

    Tian, Jing; Chen, Jin-wei; Gao, Jie-sheng; Li, Len; Xie, Xi

    2013-07-01

    Resveratrol (trans-3,4'-trihydroxystilbene), a natural phytoalexin, possesses anti-inflammatory, anti-proliferative, and immunomodulatory properties and has the potential for treating inflammatory disorders. The present study was designed to investigate the effects of resveratrol on TNF-α-induced inflammatory cytokines production of IL-1β and MMP3 in Rheumatoid arthritis (RA) Fibroblast-like synoviocytes (FLS) and further to explore the role of PI3K/Akt signaling pathway by which resveratrol modulates those cytokines production. The levels of IL-1β, MMP-3 in cultural supernatants among groups were measured by enzyme-linked immunosorbent assay. Messenger RNA expression of IL-1β and MMP-3 in RA FLS was analyzed using a reverse transcription-polymerase chain reaction. Western blot analysis was used to detect proteins expression in RA FLS intervened by resveratrol. Resveratrol inhibited both mRNA and proteins expressions of IL-1β and MMP-3 on RA FLS in a dose-dependent manner. Resveratrol also decreased significantly the expression of phosphorylated Akt dose dependently. Activation of PI3K/Akt signaling pathway exists in TNF-α-induced production of IL-1β and MMP3 on RA FLS, which is hampered by PI3K inhibitor LY294002. Immunofluorescence staining showed that TNF-α alone increased the production of P-Akt, whereas LY294002 and 50 μM resveratrol suppressed the TNF-α-stimulated expression of P-Akt. Resveratrol attenuates TNF-α-induced production of IL-1β and MMP-3 via inhibition of PI3K-Akt signaling pathway in RA FLS, suggesting that resveratrol plays an anti-inflammatory role and might have beneficial effects in preventing and treating RA.

  4. Blood -brain barrier disruption was less under isoflurane than pentobarbital anesthesia via a PI3K/Akt pathway in early cerebral ischemia.

    PubMed

    Chi, Oak Z; Mellender, Scott J; Kiss, Geza K; Liu, Xia; Weiss, Harvey R

    2017-02-24

    One of the important factors altering the degree of blood-brain barrier (BBB) disruption in cerebral ischemia is the anesthetic used. The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway has been reported to be involved in modulating BBB permeability and in isoflurane induced neuroprotection. This study was performed to compare the degree of BBB disruption in focal cerebral ischemia under isoflurane vs pentobarbital anesthesia and to determine whether inhibition of PI3K/Akt would affect the disruption in the early stage of focal cerebral ischemia. Permanent middle cerebral artery (MCA) occlusion was performed in rats under 1.4% isoflurane or pentobarbital (50mg/kg i.p.) anesthesia with controlled ventilation. In half of each group LY294002, which is a PI3K/Akt inhibitor, was applied on the ischemic cortex immediately after MCA occlusion. After one hour of MCA occlusion, the transfer coefficient (Ki) of (14)C-α-aminoisobutyric acid ((14)C-AIB) was determined to quantify the degree of BBB disruption. MCA occlusion increased the Ki both in the isoflurane and pentobarbital anesthetized rats. However, the value of Ki was lower under isoflurane (11.5±6.0μL/g/min) than under pentobarbital (18.3±7.1μL/g/min) anesthesia. The Ki of the contralateral cortex of the pentobarbital group was higher (+74%) than that of the isoflurane group. Application of LY294002 on the ischemic cortex increased the Ki (+99%) only in the isoflurane group. The degree of BBB disruption by MCA occlusion was significantly lower under isoflurane than pentobarbital anesthesia in the early stage of cerebral ischemia. Our data demonstrated the importance of choice of anesthetics and suggest that PI3K/Akt signaling pathway plays a significant role in altering BBB disruption in cerebral ischemia during isoflurane but not during pentobarbital anesthesia.

  5. Effect of the PI3K/AKT signaling pathway on hypoxia-induced proliferation and differentiation of bone marrow-derived mesenchymal stem cells

    PubMed Central

    Sheng, Lingling; Mao, Xiyuan; Yu, Qingxiong; Yu, Dong

    2017-01-01

    Bone marrow-derived mesenchymal stem cell (BM-MSC) transplantation has been demonstrated to be an effective way of augmenting angiogenesis of ischemic tissue. The low oxygen conditions in ischemic tissue directly affect the biological behavior of engrafted cells. However, to date, the mechanism through which hypoxia regulates self-renewal, differentiation and paracrine function of BM-MSCs remains unclear. Clarification of this mechanism would be beneficial to the use of stem cell-based therapy. The PI3K/AKT pathway has been extensively investigated for its role in cell proliferation, cell transformation, paracrine function and angiogenesis. The present study aimed to analyze the role of PI3K/AKT pathway in hypoxia-induced proliferation of BM-MSCs and their differentiation into endothelial cells in vitro by the application of LY294002, a PI3K/AKT pathway inhibitor, with cells cultured in normoxia serving as a control. The results showed that rat BM-MSCs at passage 3 and 4 displayed only few phenotypical differences in the expression of surface antigens as detected by flow cytometry. When compared with the cells treated in normoxia, the proliferation of BM-MSCs in hypoxia was promoted, a greater number of cells expressed CD31 and a higher expression of vascular endothelial growth factor was observed after culture in hypoxic conditions. However, by inhibiting with LY294002, these changes induced by hypoxia were partly inhibited. In conclusion, the present study showed that the PI3K/AKT pathway served an important role in hypoxia-enhanced in vitro proliferation of BM-MSCs and their differentiation into endothelial cells and paracrine vascular endothelial growth factor. PMID:28123468

  6. Dual regulation of glucocorticoid-induced leucine zipper (GILZ) by the glucocorticoid receptor and the PI3-kinase/AKT pathways in multiple myeloma.

    PubMed

    Grugan, Katharine D; Ma, Chunguang; Singhal, Seema; Krett, Nancy L; Rosen, Steven T

    2008-06-01

    Glucocorticoids (GCs) are effective therapeutics commonly used in multiple myeloma (MM) treatment. Clarifying the pathway of GC-induced apoptosis is crucial to understanding the process of drug resistance and to the development of new targets for MM treatment. We have previously published results of a micro-array identifying glucocorticoid-induced leucine zipper (GILZ) as GC-regulated gene in MM.1S cells. Consistent with those results, GCs increased GILZ in MM cell lines and patient samples. Reducing the levels of GILZ with siRNA decreased GC-induced cell death suggesting GILZ may mediate GC-killing. We conducted a screen to identify other pathways that affect GILZ regulation and report that inhibitors of PI3-kinase/AKT enhanced GILZ expression in MM cell lines and clinical samples. The combination of dexamethasone (Dex) and LY294002, wortmannin, triciribine, or AKT inhibitor VIII dramatically up regulated GILZ levels and enhanced apoptosis. Addition of interleukin-6 (IL-6) or insulin-like growth factor (IGF1), both which activate the PI3-kinase/AKT pathway and inhibit GC killing, blocked up regulation of GILZ by GC and PI3-kinase/AKT inhibitors. In summary, these results identify GILZ as a mediator of GC killing, indicate a role of PI3-kinase/AKT in controlling GILZ regulation and suggest that the combination of PI3-kinase/AKT inhibitors and GCs may be a beneficial MM treatment.

  7. Tomosyn is a novel Akt substrate mediating insulin-dependent GLUT4 exocytosis.

    PubMed

    Nagano, Koki; Takeuchi, Hiroshi; Gao, Jing; Mori, Yoshihide; Otani, Takahito; Wang, DaGuang; Hirata, Masato

    2015-05-01

    Insulin triggers glucose uptake into skeletal muscle and adipose tissues by gaining the available number of glucose transporter 4 (GLUT4) on the cell surface. GLUT4-loaded vesicles are targeted to plasma membrane from the intracellular reservoir through multiple trafficking and fusion processes that are mainly regulated by Akt. However, it is still largely unknown how GLUT4 expression in the cell surface is promoted by insulin. In the present study, we identified tomosyn at Ser-783 as a possible Akt-substrate motif and examined whether the phosphorylation at Ser-783 is involved in the regulation of GLUT4 expression. Both Akt1 and Akt2 phosphorylated the wild-type tomosyn, but not the mutant tomosyn in which Ser-783 was replaced with Ala. Phosphorylation of tomosyn at Ser-783 was also observed in the intact cells by insulin stimulation, which was blocked by PI3K inhibitor, LY294002. In vitro pull-down assay showed that phosphorylation of tomosyn at Ser-783 by Akt inhibited the interaction with syntaxin 4. Insulin stimulation increased GLUT4 in the cell surface of CHO-K1 cells to promote glucose uptake, however exogenous expression of the mutant tomosyn attenuated the increase by insulin. These results suggest that Ser-783 of tomosyn is a target of Akt and is implicated in the interaction with syntaxin 4.

  8. Effect of 2-hydroxyethyl methacrylate on human pulp cell survival pathways ERK and AKT.

    PubMed

    Spagnuolo, Gianrico; D'Antò, Vincenzo; Valletta, Rosa; Strisciuglio, Caterina; Schmalz, Gottfried; Schweikl, Helmut; Rengo, Sandro

    2008-06-01

    Previous investigations have revealed that dental monomers could affect intracellular pathways leading to cell survival or cell death. Mitogen-activated protein kinase (MAPK) and protein kinase B (AKT) might mediate cell responses as well as cell survival and apoptosis. The purpose of this study was to evaluate the effects of 2-hydroxyethyl methacrylate (HEMA) on the ERK1/2 and AKT pathways in human primary pulp fibroblasts (HPCs). HPCs were treated with various concentrations of HEMA, after which viability and reactive oxygen species levels were determined by flow cytometry with Annexin V-PI staining and 2,7-dichlorofluorescine diacetate, respectively. Whole-cell extracts were immunoblotted with anti-P-Akt or anti-P-ERK1/2. Cell viability decreased in a dose-dependent manner after HEMA exposure, showing a significant decrease with 10 mmol/L HEMA (p < .05). HEMA treatment resulted in a 4-fold increase in reactive oxygen species formation (p < .05). A short HEMA exposure (30-90 minutes) increased ERK1/2 phosphorylation, whereas a decrease in the AKT phosphorylation was observed. Selective inhibitors of the ERK (PD98059) and AKT (LY294002) pathways amplified HPC cell damage after HEMA exposure. Our findings demonstrated that HEMA exposure modulates the ERK and AKT pathways in different manners, and that in turn, they function in parallel to mediate pro-survival signaling in pulp cells subjected to HEMA cytotoxicity.

  9. Transcriptional and post-transcriptional control of DNA methyltransferase 3B is regulated by phosphatidylinositol 3 kinase/Akt pathway in human hepatocellular carcinoma cell lines.

    PubMed

    Mei, Chuanzhong; Sun, Lidong; Liu, Yonglei; Yang, Yong; Cai, Xiumei; Liu, Mingzhu; Yao, Wantong; Wang, Can; Li, Xin; Wang, Liying; Li, Zengxia; Shi, Yinghong; Qiu, Shuangjian; Fan, Jia; Zha, Xiliang

    2010-09-01

    DNA methyltransferases (DNMTs) are essential for maintenance of aberrant methylation in cancer cells and play important roles in the development of cancers. Unregulated activation of PI3K/Akt pathway is a prominent feature of many human cancers including human hepatocellular carcinoma (HCC). In present study, we found that DNMT3B mRNA and protein levels were decreased in a dose- and time-dependent manner in HCC cell lines with LY294002 treatment. However, we detected that LY294002 treatment did not induce increase of the degradation of DNMT3B protein using protein decay assay. Moreover we found that Akt induced alteration of the expression of DNMT3B in cells transfected with myristylated variants of Akt2 or cells transfected with small interfering RNA respectively. Based on DNMT3B promoter dual-luciferase reporter assay, we found PI3K pathway regulates DNMT3B expression at transcriptional level. And DNMT3B mRNA decay analysis suggested that down-regulation of DNMT3B by LY294002 is also post-transcriptional control. Furthermore, we demonstrated that LY294002 down-regulated HuR expression in a time-dependent manner in BEL-7404. In summary, we have, for the first time, demonstrate that PI3K/Akt pathway regulates the expression of DNMT3B at transcriptional and post-transcriptional levels, which is particularly important to understand the effects of PI3K/Akt and DNMT3B on hepatocarcinogenesis.

  10. Effects of AFP-activated PI3K/Akt signaling pathway on cell proliferation of liver cancer.

    PubMed

    Zheng, Lu; Gong, Wei; Liang, Ping; Huang, XiaoBing; You, Nan; Han, Ke Qiang; Li, Yu Ming; Li, Jing

    2014-05-01

    This study aims to investigate effects of alpha-fetoprotein (AFP)-activated phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway on hepatocellular carcinoma cell proliferation. Active cirrhosis patients after hepatitis B infection (n = 20) and viral hepatitis patients with hepatocellular carcinoma (HCC) (n = 20) were selected as the subjects of the present study. Another 20 healthy subjects were selected as the control group. The serum AFP expression and liver tissue PI3K and Akt gene mRNA expression were detected. The hepatoma cell model HepG2 which had a stable expression of AFP gene was used. Real-time quantitative PCR and Western blot and other methods were used to analyze the intracellular PI3K and Akt protein levels. Compared with control group and cirrhosis group, the serum AFP levels in HCC group significantly increased, and the tissue PI3K and Akt mRNA expression also significantly increased. HepG2 cells were intervened using AFP, in which the PIK and Akt protein expression significantly increased. After intervention by use of AFP monoclonal antibodies or LY294002 inhibitor, the PIK and Akt protein expression in HepG2 cell was significantly decreased (P < 0.05). AFP can promote the proliferation of hepatoma cells via activation of PI3K/Akt signaling pathway.

  11. Synthesis and evaluation of indazole based analog sensitive Akt inhibitors.

    PubMed

    Okuzumi, Tatsuya; Ducker, Gregory S; Zhang, Chao; Aizenstein, Brian; Hoffman, Randy; Shokat, Kevan M

    2010-08-01

    The kinase Akt is a key signaling node in regulating cellular growth and survival. It is implicated in cancer by mutation and its role in the downstream transmission of aberrant PI3K signaling. For these reasons, Akt has become an increasingly important target of drug development efforts and several inhibitors are now reaching clinical trials. Paradoxically it has been observed that active site kinase inhibitors of Akt lead to hyperphosphorylation of Akt itself. To investigate this phenomenon we here describe the application of a chemical genetics strategy that replaces native Akt with a mutant version containing an active site substitution that allows for the binding of an engineered inhibitor. This analog sensitive strategy allows for the selective inhibition of a single kinase. In order to create the inhibitor selective for the analog sensitive kinase, a diversity of synthetic approaches was required, finally resulting in the compound PrINZ, a 7-substituted version of the Abbott Labs Akt inhibitor A-443654.

  12. Role of the phosphatidylinositol 3-kinase/Akt and mTOR/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma.

    PubMed

    Pene, Frédéric; Claessens, Yann-Erick; Muller, Odile; Viguié, Franck; Mayeux, Patrick; Dreyfus, François; Lacombe, Catherine; Bouscary, Didier

    2002-09-26

    Multiple myeloma (MM) is a plasma cell malignancy preliminary localized in the bone marrow and characterized by its capacity to disseminate. IL-6 and IGF-1 have been shown to mediate proliferative and anti-apoptotic signals in plasmocytes. However, in primary plasma-cell leukemia (PCL) and in end-stage aggressive extramedullar disease, the cytokine requirement for both effects may be not mandatory. This suggests that constitutive activation of signaling pathways occurs. One of the signaling pathways whose deregulation may play an oncogenic role in MM is the phosphatidylinositol 3-kinase (PI 3-K) pathway. In human growth factor-independent MM cell lines OPM2 and RPMI8226, we show that the PI 3-K inhibitors LY294002 and Wortmannin strongly inhibited cell proliferation, whereas inhibition of the mammalian Target Of Rapamycin (mTOR)/P70-S6-kinase (P70(S6K)) pathway with rapamycin or of the Mitogen-Activated Protein Kinase (MAPK) pathway with PD98059 had minimal effect on proliferation. In both cell lines, constitutive activation of the PI 3-K/Akt/FKHRL-1, mTOR/P70(S6K) and MAPK pathways was detected. LY294002 inhibited phosphorylation of Akt, FKHRL-1 and P70(S6K) but had no effect on ERK1/2 phosphorylation, indicating that the PI 3-K and MAPK pathways are independent. IGF-1 but not IL-6 increased phosphorylation of Akt, FKHRL-1 and P70(S6K). Purified plasmocytes from four patients with MM and two patients with primary PCL were studied. In three of them including the two patients with PCL, constitutive phosphorylation of Akt, FKHRL-1 and P70(S6K) was present, inhibited by LY294002 and enhanced by IGF-1. In these patients with constitutive Akt activation, normal PTEN expression was detected. PI 3-K inhibition induced caspase-dependent apoptosis as confirmed by inhibition with the large spectrum caspase inhibitor Z-VAD-FMK and cleavage of pro-caspase-3. Both cell lines spontaneously expressed Skp2 and cyclin D1 proteins at high levels but no p27(Kip1) protein. In the

  13. L-F001, a Multifunction ROCK Inhibitor Prevents 6-OHDA Induced Cell Death Through Activating Akt/GSK-3beta and Nrf2/HO-1 Signaling Pathway in PC12 Cells and Attenuates MPTP-Induced Dopamine Neuron Toxicity in Mice.

    PubMed

    Luo, Liting; Chen, Jingkao; Su, Dan; Chen, Meihui; Luo, Bingling; Pi, Rongbiao; Wang, Lan; Shen, Wei; Wang, Rikang

    2017-02-01

    Amounting evidences demonstrated that Rho/Rho-associated kinase (ROCK) might be a novel target for the therapy of Parkinson's disease (PD). Recently, we synthesized L-F001 and revealed it was a potent ROCK inhibitor with multifunctional effects. Here we investigated the effects of L-F001 in PD models. We found that L-F001 potently attenuated 6-OHDA-induced cytotoxicity in PC12 cells and significantly decreased intracellular reactive oxygen species (ROS), prevented the 6-OHDA-induced decline of mitochondrial membrane potential and intracellular GSH levels. In addition, L-F001 increased Akt and GSK-3beta phosphorylation and induced the nuclear Nrf2 and HO-1 expression in a time- and concentration-dependent manner. Moreover, L-F001 restored the levels of p-Akt and p-GSK-3beta (Ser9) as well as HO-1 expression reduced by 6-OHDA. Those effects were blocked by the specific PI3K inhibitor, LY294002, indicating the involvement of Akt/GSK-3beta pathway in the neuroprotective effect of L-F001. In addition, L-F001 significantly attenuated the tyrosinehydroxylase immunoreactive cell loss in 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP)-induced mice PD model. Together, our findings suggest that L-F001 prevents 6-OHDA-induced cell death through activating Akt/GSK-3beta and Nrf2/HO-1 signaling pathway and attenuates MPTP-induced dopaminergic neuron toxicity in mice. L-F001 might be a promising drug candidate for PD.

  14. Andrographolide suppresses endothelial cell apoptosis via activation of phosphatidyl inositol-3-kinase/Akt pathway.

    PubMed

    Chen, Jiun-Han; Hsiao, George; Lee, An-Rong; Wu, Chin-Chen; Yen, Mao-Hsiung

    2004-04-01

    Andrographolide (Andro), an active component isolated from the Chinese official herbal Andrographis paniculata, which has been reported to prevent oxygen radical production and thus prevent inflammatory diseases. In this study, we investigated the molecular mechanisms and signaling pathways by which Andro protects human umbilical vein endothelial cells (HUVECs) from growth factor (GF) deprivation-induced apoptosis. Results demonstrated that HUVECs undergo apoptosis after 18 hr of GF deprivation but that this cell death was suppressed by the addition of Andro in a concentration-dependent manner (1-100 microM). Andro suppresses the mitochondrial pathway of apoptosis by inhibiting release of cytochrome c into the cytoplasm and dissipation of mitochondrial potential (Deltapsi(m)), as a consequence, prevented caspase-3 and -9 activation. Treatment of endothelial cells with Andro-induced activation of the protein kinase Akt, an anti-apoptotic signal, and phosphorylation of BAD, a down-stream target of Akt. Suppression of Akt activity by wortmannin, by LY-294002 and by using a dominant negative Akt mutant abolished the anti-apoptotic effect of Andro. In contrast, the ERK1/2 activities were not affected by Andro. The ERK1/2 inhibitor, PD98059 failed to antagonize the protective effect of Andro. In conclusion, Andro exerts its anti-apoptotic potential via activation of the Akt-BAD pathway in HUVECs and thus may represent a candidate of therapeutic agent for atherosclerosis.

  15. Vascular neuroprotection via TrkB- and Akt-dependent cell survival signaling.

    PubMed

    Guo, Shuzhen; Som, Angel T; Waeber, Christian; Lo, Eng H

    2012-11-01

    The cerebral endothelium can be a vital source of signaling factors such as brain-derived neurotrophic factor that defends the neuronal parenchyma against stress and injury. But the underlying mechanisms remain to be fully defined. Here, we use cell models to ask how vascular neuroprotection is sustained. Human brain endothelial cells were grown in culture, and conditioned media were transferred to primary rat cortical neurons. Brain endothelial cell-conditioned media activated neuronal Akt signaling and protected neurons against hypoxia and oxygen-glucose deprivation. Blockade of Akt phosphorylation with the PI3-kinase inhibitor LY294002 negated this vascular neuroprotective effect. Upstream of Akt signaling, the brain-derived neurotrophic factor receptor TrkB (neurotrophic tyrosine kinase receptor, type 2) was involved because depletion with TrkB/Fc eliminated the ability of endothelial-conditioned media to protect neurons against hypoxia. Downstream of Akt signaling, activation of GSK-3β (glycogen synthase kinase 3 beta), caspase 9, caspase 3 and Bad pathways were detected. Taken together, these findings suggest that the molecular basis for vascular neuroprotection involves TrkB-Akt signaling that ameliorates neuronal apoptosis. Further investigation of these mechanisms may reveal new approaches for augmenting endogenous vascular neuroprotection in stroke, brain injury, and neurodegeneration.

  16. High fat diet reduces neuroprotection of isoflurane post-treatment: role of carboxyl-terminal modulator protein-Akt signaling

    PubMed Central

    Yu, Hai; Deng, Jiao; Zuo, Zhiyi

    2014-01-01

    Objective High fat diet (HFD) contributes to the increased prevalence of obesity and hyperlipidemia in young adults, a possible cause for their recent increase in stroke. Isoflurane post-treatment provides neuroprotection. We determined whether isoflurane post-treatment induced neuroprotection in HFD-fed mice. Design and Methods Six-week old CD-1 male mice were fed HFD or regular diet (RD) for 5 or 10 weeks. Their hippocampal slices (400 µm) were subjected to oxygen-glucose deprivation (OGD). Some slices were exposed to isoflurane for 30 min immediately after OGD. Some mice had a 90-min middle cerebral arterial occlusion and were post-treated with 2% isoflurane for 30 min. Results OGD time-dependently induced cell injury. This injury was dose-dependently reduced by isoflurane. The effect was apparent at 1% or 2% isoflurane in RD-fed mice but required 3% isoflurane in HFD-fed mice. HFD influenced the isoflurane effects in DG. OGD increased carboxyl-terminal modulator protein (CTMP), an Akt inhibitor, and decreased Akt signaling. Isoflurane reduced these effects. LY294002, an Akt activation inhibitor, attenuated the isoflurane effects. HFD increased CTMP and reduced Akt signaling. Isoflurane improved neurological outcome in the RD-fed mice but not in the HFD-fed mice. Conclusions HFD attenuated isoflurane post-treatment-induced neuroprotection possibly due to decreased prosurvival Akt signaling. PMID:25142024

  17. Deciphering Combinations of PI3K/AKT/mTOR Pathway Drugs Augmenting Anti-Angiogenic Efficacy In Vivo

    PubMed Central

    Sasore, Temitope; Kennedy, Breandán

    2014-01-01

    Ocular neovascularization is a common pathology associated with human eye diseases e.g. age-related macular degeneration and proliferative diabetic retinopathy. Blindness represents one of the most feared disabilities and remains a major burden to health-care systems. Current approaches to treat ocular neovascularisation include laser photocoagulation, photodynamic therapy and anti-VEGF therapies: Ranibizumab (Lucentis) and Aflibercept (Eylea). However, high clinical costs, frequent intraocular injections, and increased risk of infections are challenges related with these standards of care. Thus, there is a clinical need to develop more effective drugs that overcome these challenges. Here, we focus on an alternative approach by quantifying the in vivo anti-angiogenic efficacy of combinations of phosphatidylinositol-3-kinase (PI3K) pathway inhibitors. The PI3K/AKT/mTOR pathway is a complex signalling pathway involved in crucial cellular functions such as cell proliferation, migration and angiogenesis. RT-PCR confirms the expression of PI3K target genes (pik3ca, pik3r1, mtor and akt1) in zebrafish trunks from 6 hours post fertilisation (hpf) and in eyes from 2 days post fertilisation (dpf). Using both the zebrafish intersegmental vessel and hyaloid vessel assays to measure the in vivo anti-angiogenic efficacy of PI3K/Akt/mTOR pathway inhibitors, we identified 5 µM combinations of i) NVP-BEZ235 (dual PI3K-mTOR inhibitor) + PI-103 (dual PI3K-mTOR inhibitor); or ii) LY-294002 (pan-PI3K inhibitor) + NVP-BEZ235; or iii) NVP-BEZ235 + rapamycin (mTOR inhibitor); or iv) LY-294002 + rapamycin as the most anti-angiogenic. Treatment of developing larvae from 2–5 dpf with 5 µM NVP-BEZ235 plus PI-103 resulted in an essentially intact ocular morphology and visual behaviour, whereas other combinations severely disrupted the developing retinal morphology and visual function. In human ARPE19 retinal pigment epithelium cells, however, no significant difference in cell number was

  18. Multiple host kinases contribute to Akt activation during Salmonella infection.

    PubMed

    Roppenser, Bernhard; Kwon, Hyunwoo; Canadien, Veronica; Xu, Risheng; Devreotes, Peter N; Grinstein, Sergio; Brumell, John H

    2013-01-01

    SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4) P2/PI(3-5) P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4) P2/PI(3-5) P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

  19. MiR-221 promotes IgE-mediated activation of mast cells degranulation by PI3K/Akt/PLCγ/Ca(2+) pathway.

    PubMed

    Xu, Hong; Gu, Li-Na; Yang, Qian-Yuan; Zhao, De-Yu; Liu, Feng

    2016-06-01

    Mast cells play a pivotal role in the immediate reaction in asthma. In a previous study, it was found that MicroRNA-221 (miR-221) was associated with asthma. Hence, in the present study, the role and the potential mechanisms of miR-221 on immunoglobulin E (IgE)-mediated activation of mast cells degranulation were investigated. MiR-221 expression was first quantified by qRT-PCR in IgE-mediated activation of mast cells. RBL-2H3 cells were then transfected with miR-221 mimic or miR-221 inhibitor, the IgE-mediated degranulation was detected in mast cells. The influence of miR-221 on expression of phospholipase C gamma (PLCγ1), p-PLCγ1, protein kinase B (Akt), phospho-Akt (p-Akt), inhibitor of kappa B (IκB-α), and phospho-IκB-α (p-IκB-α) were examined by Western blot, whereas free calcium ion (Ca(2+)) level was measured by flow cytometry and NF-κB expression was determined by EMSA. Phosphoinositide 3-kinase (PI3K)-inhibitor (LY294002) and NF-κB-inhibitor [pyrrolidine dithiocarbamate (PDTC)] were used to investigate the role of PI3K/Akt pathway and NF-κB in miR-221 promoting IgE-mediated activation of mast cells degranulation. The expression of miR-221 was upregulated in IgE-mediated activation of mast cells, and it was overexpressed in miR-221 mimic transfected cells. The degranulation was found to be significantly increased in miR-221 overexpressed cells while it was found to be significantly decreased in miR-221 downregulated cells. The expression of p-PLCγ1, p-Akt, p-IκB-α as well as NF-κB and Ca(2+) release were increased in miR-221 overexpressed cells. PI3K-inhibitor (LY294002) could rescue the promotion of degranulation caused by miR-221 in IgE-mediated activation of mast cells. However, NF-κB-inhibitor (PDTC) could not rescue the promotion of degranulation caused by miR-221 in IgE-mediated activation of mast cells. MiR-221 promotes IgE-mediated activation of mast cells degranulation by PI3K/Akt/PLCγ/Ca(2+) signaling pathway, in a non

  20. PI3K/Akt contributes to increased expression of Toll-like receptor 4 in macrophages exposed to hypoxic stress

    SciTech Connect

    Kim, So Young; Jeong, Eunshil; Joung, Sun Myung; Lee, Joo Young

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer Hypoxic stress-induced TLR4 expression is mediated by PI3K/Akt in macrophages. Black-Right-Pointing-Pointer PI3K/Akt regulated HIF-1 activation leading to TLR4 expression. Black-Right-Pointing-Pointer p38 mitogen-activated protein kinase was not involved in TLR4 expression by hypoxic stress. Black-Right-Pointing-Pointer Sulforaphane suppressed hypoxia-mediated TLR4 expression by inhibiting PI3K/Akt. -- Abstract: Toll-like receptors (TLRs) play critical roles in triggering immune and inflammatory responses by detecting invading microbial pathogens and endogenous danger signals. Increased expression of TLR4 is implicated in aggravated inflammatory symptoms in ischemic tissue injury and chronic diseases. Results from our previous study showed that TLR4 expression was upregulated by hypoxic stress mediated by hypoxia-inducible factor-1 (HIF-1) at a transcriptional level in macrophages. In this study, we further investigated the upstream signaling pathway that contributed to the increase of TLR4 expression by hypoxic stress. Either treatment with pharmacological inhibitors of PI3K and Akt or knockdown of Akt expression by siRNA blocked the increase of TLR4 mRNA and protein levels in macrophages exposed to hypoxia and CoCl{sub 2}. Phosphorylation of Akt by hypoxic stress preceded nuclear accumulation of HIF-1{alpha}. A PI3K inhibitor (LY294002) attenuated CoCl{sub 2}-induced nuclear accumulation and transcriptional activation of HIF-1{alpha}. In addition, HIF-1{alpha}-mediated upregulation of TLR4 expression was blocked by LY294002. Furthermore, sulforaphane suppressed hypoxia- and CoCl{sub 2}-induced upregulation of TLR4 mRNA and protein by inhibiting PI3K/Akt activation and the subsequent nuclear accumulation and transcriptional activation of HIF-1{alpha}. However, p38 was not involved in HIF-1{alpha} activation and TLR4 expression induced by hypoxic stress in macrophages. Collectively, our results demonstrate that PI3K/Akt

  1. Isoorientin induces apoptosis through mitochondrial dysfunction and inhibition of PI3K/Akt signaling pathway in HepG2 cancer cells

    SciTech Connect

    Yuan, Li; Wang, Jing; Xiao, Haifang; Xiao, Chunxia; Wang, Yutang; Liu, Xuebo

    2012-11-15

    Isoorientin (ISO) is a flavonoid compound that can be extracted from several plant species, such as Phyllostachys pubescens, Patrinia, and Drosophyllum lusitanicum; however, its biological activity remains poorly understood. The present study investigated the effects and putative mechanism of apoptosis induced by ISO in human hepatoblastoma cancer (HepG2) cells. The results showed that ISO induced cell death in a dose-dependent manner in HepG2 cells, but no toxicity in human liver cells (HL-7702) and buffalo rat liver cells (BRL-3A) treated with ISO at the indicated concentrations. ISO-induced cell death included apoptosis which characterized by the appearance of nuclear shrinkage, the cleavage of poly (ADP-ribose) polymerase (PARP) and DNA fragmentation. ISO significantly (p < 0.01) increased the Bax/Bcl-2 ratio, disrupted the mitochondrial membrane potential (MMP), increased the release of cytochrome c, activated caspase-3, and enhanced intracellular levels of reactive oxygen species (ROS) and nitric oxide (NO). In addition, ISO effectively inhibited the phosphorylation of Akt and increased FoxO4 expression. The PI3K/Akt inhibitor LY294002 enhanced the apoptosis-inducing effect of ISO. However, LY294002 markedly quenched ROS and NO generation and diminished the protein expression of heme peroxidase enzyme (HO-1) and inducible nitric oxide synthase (iNOS). Furthermore, the addition of a ROS inhibitor (N-acetyl cysteine, NAC) or iNOS inhibitor (N-[3-(aminomethyl) benzyl] acetamidine, dihydrochloride, 1400W) significantly diminished the apoptosis induced by ISO and also blocked the phosphorylation of Akt. These results demonstrated for the first time that ISO induces apoptosis in HepG2 cells and indicate that this apoptosis might be mediated through mitochondrial dysfunction and PI3K/Akt signaling pathway, and has no toxicity in normal liver cells, suggesting that ISO may have good potential as a therapeutic and chemopreventive agent for liver cancer. Highlights:

  2. PHA665752 inhibits the HGF-stimulated migration and invasion of cells by blocking PI3K/AKT pathway in human cell line uveal melanoma.

    PubMed

    Wang, Z; He, C; Liu, L; Ma, N; Chen, X; Zheng, D; Qiu, G H

    2017-03-03

    HGF/c-MET is frequently associated with tumor metastasis in many cancers, including uveal melanoma (UM). PHA665752, a selective c-MET inhibitor, exhibits anticancer activity through inhibiting cell motility in some cancers. In this study, we investigated the effects of PHA665752 on UM cell lines M17 and SP6.5. Our data show that HGF stimulated the motility of UM cells, and induced the activation of both c-MET and PI3K/AKT, but not ERK1/2. Moreover, consistent with the amount of c-MET within the nucleus, PHA665752 significantly inhibited HGF-promoted cell motility and suppressed the phosphorylation of c-MET and PI3K/AKT, but not ERK1/2 induced by HGF. Additionally, the effects of PHA665752 on both the inhibition of HGF-induced cell motility and the suppression of active AKT are similar to those of PI3K inhibitor LY294002. In xenograft models, PHA665752 significantly inhibited tumor growth in nude mice and similarly suppressed the phosphorylation of c-MET and PI3K/AKT. Our current findings, combined with previous results, demonstrate that PHA665752 inhibits HGF-induced motility via the inhibition of PI3K/AKT. This study suggests that targeting HGF/c-MET could be a promising therapeutic strategy for UM by preventing cell motility.

  3. Tricin, flavonoid from Njavara reduces inflammatory responses in hPBMCs by modulating the p38MAPK and PI3K/Akt pathways and prevents inflammation associated endothelial dysfunction in HUVECs.

    PubMed

    Shalini, V; Pushpan, Chithra K; G, Sindhu; A, Jayalekshmy; A, Helen

    2016-02-01

    Previous studies revealed the potent anti-inflammatory activity of tricin, the active component of Njavara rice bran. Here, we report the involvement of specific signaling pathways in the protective effect of tricin against LPS induced inflammation in hPBMCs and the role of tricin in modulating endothelial dysfunction in LPS induced HUVECs. Pretreatment with tricin (15μM) significantly inhibited the release of TNF-α and was comparable to the specific pathway blockers like ERK inhibitor (PD98059), JNK inhibitor (SP600125) and p38 inhibitor (SB203580), whereas an increased release of TNF-α was observed in PI3K/Akt inhibitor (LY294002) treated cells. Tricin alone and combination treatment of tricin and SB203580 showed more significant inhibition of activation of COX-2 and TNF-α than that of SB203580 alone treated group. Combination treatment of tricin and LY294002 showed increased activation of COX-2 and TNF-α, proved that PI3K activation is essential for the anti-inflammatory effect of tricin. Studies conducted on HUVECs revealed the protective effect of tricin against endothelial dysfunction associated with LPS induced inflammation by inhibiting the activation of proinflammatory mediators like TNF-α, IFN-γ, MCP 1 by modulating NF-κB and MAPK signaling pathways. ELISA and flow cytometric analysis again confirmed the protection of tricin against endothelial damage, especially from the decreased activation of cell adhesion molecules like ICAM-1, VCAM-1 and E-Selectin upon tricin treatment. This work establishes the mechanism behind the potent anti-inflammatory activity of the flavonoid tricin.

  4. Over-Expression of PDGFR-β Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway

    PubMed Central

    Li, Wei; Zhao, Xiaohui; Yu, Yang; Zhu, Jinkun; Qin, Zhexue; Wang, Qiang; Wang, Kui; Lu, Wei; Liu, Jie; Huang, Lan

    2012-01-01

    The proliferation, migration, and angiogenesis of endothelial progenitor cells (EPCs) play critical roles in postnatal neovascularization and re-endothelialization following vascular injury. Here we evaluated whether the over-expression of platelet-derived growth factor receptor-β (PDGFR-β) can enhance the PDGF-BB-stimulated biological functions of EPCs through the PDGFR-β/phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. We first confirmed the expression of endogenous PDGFR-β and its plasma membrane localization in spleen-derived EPCs. We then demonstrated that the PDGFR-β over-expression in EPCs enhanced the PDGF-BB-induced proliferation, migration, and angiogenesis of EPCs. Using AG1295 (a PDGFR kinase inhibitor), LY294002 (a PI3K inhibitor), and sc-221226 (an Akt inhibitor), we further showed that the PI3K/Akt signaling pathway participates in the PDGF-BB-induced proliferation, migration, and angiogenesis of EPCs. In addition, the PI3K/Akt signaling pathway is required for PDGFR-β over-expression to enhance these PDGF-BB-induced phenotypes. PMID:22355314

  5. Allosteric Small-Molecule Inhibitors of the AKT Kinase

    NASA Astrophysics Data System (ADS)

    Dalafave, D. S.

    This research addresses computational design of small druglike molecules for possible anticancer applications. AKT and SGK are kinases that control important cellular functions. They are highly homologous, having similar activators and targets. Cancers with increased SGK activity may develop resistance to AKT-specific inhibitors. Our goal was to design new molecules that would bind both AKT and SGK, thus preventing the development of drug resistance. Most kinase inhibitors target the kinase ATP-binding site. However, the high similarity in this site among kinases makes it difficult to target specifically. Furthermore, mutations in this site can cause resistance to ATP-competitive kinase inhibitors. We used existing AKT inhibitors as initial templates to design molecules that could potentially bind the allosteric sites of both AKT and SGK. Molecules with no implicit toxicities and optimal drug-like properties were used for docking studies. Binding energies of the stable complexes that the designed molecules formed with AKT and SGK were calculated. Possible applications of the designed putative inhibitors against cancers with overexpressed AKT/SGK is discussed.

  6. Tetrandrine inhibits migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes through down-regulating the expressions of Rac1, Cdc42, and RhoA GTPases and activation of the PI3K/Akt and JNK signaling pathways.

    PubMed

    Lv, Qi; Zhu, Xian-Yang; Xia, Yu-Feng; Dai, Yue; Wei, Zhi-Feng

    2015-11-01

    Tetrandrine (Tet), the main active constituent of Stephania tetrandra root, has been demonstrated to alleviate adjuvant-induced arthritis in rats. The present study was designed to investigate the effects of Tet on the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and explore the underlying mechanisms. By using cultures of primary FLS isolated from synoviums of RA patients and cell line MH7A, Tet (0.3, 1 μmol·L(-1)) was proven to significantly impede migration and invasion of RA-FLS, but not cell proliferation. Tet also greatly reduced the activation and expressions of matrix degrading enzymes MMP-2/9, the expression of F-actin and the activation of FAK, which controlled the morphologic changes in migration process of FLS. To identify the key signaling pathways by which Tet exerts anti-migration effect, the specific inhibitors of multiple signaling pathways LY294002, Triciribine, SP600125, U0126, SB203580, and PDTC (against PI3K, Akt, JNK, ERK, p38 MAPK and NF-κB-p65, respectively) were used. Among them, LY294002, Triciribine, and SP600125 were shown to obviously inhibit the migration of MH7A cells. Consistently, Tet was able to down-regulate the activation of Akt and JNK as demonstrated by Western blotting assay. Moreover, Tet could reduce the expressions of migration-related proteins Rho GTPases Rac1, Cdc42, and RhoA in MH7A cells. In conclusion, Tet can impede the migration and invasion of RA-FLS, which provides a plausible explanation for its protective effect on RA. The underlying mechanisms involve the reduction of the expressions of Rac1, Cdc42, and RhoA, inhibition of the activation of Akt and JNK, and subsequent down-regulation of activation and/or expressions of MMP-2/9, F-actin, and FAK.

  7. Role of PI3K/Akt signaling in insulin-like growth factor-1 (IGF-1) skin tumor promotion.

    PubMed

    Wilker, Erik; Lu, Jerry; Rho, Okkyung; Carbajal, Steve; Beltrán, Linda; DiGiovanni, John

    2005-10-01

    Overexpression of human IGF-1 with the bovine keratin 5 (BK5) promoter (BK5.IGF-1 transgenic mice) induces persistent epidermal hyperplasia and leads to spontaneous skin tumor formation. In previous work, PI3K and Akt activities were found to be elevated in the epidermis of BK5.IGF-1 transgenic mice compared to nontransgenic littermates. In the present study, we examined the importance of the PI3K/Akt signaling pathway in mediating the skin phenotype and the skin tumor promoting action of IGF-1 in these mice. Western blot analyses with epidermal lysates showed that signaling components downstream of PI3K/Akt were altered in epidermis of BK5.IGF-1 mice. Increased phosphorylation of GSK-3 (Ser(9/21)), TSC2(Thr(1462)), and mTOR(Ser(2448)) was observed. In addition, hypophosphorylation and increased protein levels of beta-catenin were observed in the epidermis of BK5.IGF-1 mice. These data suggested that components downstream of Akt might be affected, including cell cycle machinery in the epidermis of BK5.IGF-1 mice. Protein levels of cyclins (D1, E, A), E2F1, and E2F4 were all elevated in the epidermis of BK5.IGF-1 mice. Also, immunoprecipitation experiments demonstrated an increase in cdk4/cyclin D1 and cdk2/cyclin E complex formation, suggesting increased cdk activity in the epidermis of transgenic mice. In further studies, the PI3K inhibitor, LY294002, significantly blocked IGF-1-mediated epidermal proliferation and skin tumor promotion in DMBA-initiated BK5.IGF-1 mice. In addition, inhibition of PI3K/Akt with LY294002 reversed many of the cell cycle related changes observed in untreated transgenic animals. Collectively, the current results supported the hypothesis that elevated PI3K/Akt activity and subsequent activation of one or more downstream effector pathways contributed significantly to the tumor promoting action of IGF-1 in the epidermis of BK5.IGF-1 mice.

  8. Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.

    PubMed Central

    King, W G; Mattaliano, M D; Chan, T O; Tsichlis, P N; Brugge, J S

    1997-01-01

    Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases. PMID:9234699

  9. Inhibition of PI3K/Akt pathway impairs G2/M transition of cell cycle in late developing progenitors of the avian embryo retina.

    PubMed

    Ornelas, Isis Moraes; Silva, Thayane Martins; Fragel-Madeira, Lucianne; Ventura, Ana Lucia Marques

    2013-01-01

    PI3K/Akt is an important pathway implicated in the proliferation and survival of cells in the CNS. Here we investigated the participation of the PI3K/Akt signal pathway in cell cycle of developing retinal progenitors. Immunofluorescence assays performed in cultures of chick embryo retinal cells and intact tissues revealed the presence of phosphorylated Akt and 4E-BP1 in cells with typical mitotic profiles. Blockade of PI3K activity with the chemical inhibitor LY 294002 (LY) in retinal explants blocked the progression of proliferating cells through G2/M transition, indicated by an expressive increase in the number of cells labeled for phosphorylated histone H3 in the ventricular margin of the retina. No significant level of cell death could be detected at this region. Retinal explants treated with LY for 24 h also showed a significant decrease in the expression of phospho-Akt, phospho-GSK-3 and the hyperphosphorylated form of 4E-BP1. Although no change in the expression of cyclin B1 was detected, a significant decrease in CDK1 expression was noticed after 24 h of LY treatment both in retinal explants and monolayer cultures. Our results suggest that PI3K/Akt is an active pathway during proliferation of retinal progenitors and its activity appears to be required for proper CDK1 expression levels and mitosis progression of these cells.

  10. Arctigenin, a Natural Lignan Compound, Induces Apoptotic Death of Hepatocellular Carcinoma Cells via Suppression of PI3-K/Akt Signaling.

    PubMed

    Jiang, Xiaoxin; Zeng, Leping; Huang, Jufang; Zhou, Hui; Liu, Yubin

    2015-04-28

    In this study, we explored the cytotoxic effects of arctigenin, a natural lignan compound, on human hepatocellular carcinoma (HCC) cells and check the involvement of phosphatidylinositol 3-kinase (PI3-K)/Akt signaling. HCC cells were treated with different concentrations of arctigenin and cell viability and apoptosis were assessed. Manipulating Akt signaling was used to determine its role in the action of arctigenin. Arctigenin significantly inhibited the viability of HCC cells in a concentration-dependent manner. Arctigenin induced apoptosis and activation of caspase-9 and -3. Overexpression of a constitutively active Akt mutant blocked arctigenin-induced apoptosis. Combinational treatment with arctigenin and the PI3-K inhibitor LY294002 significantly enhanced apoptosis. Arctigenin reduced the expression of Bcl-xL, Mcl-1, and survivin and the phosphorylation of mTOR and S6K, which were significantly reversed by overexpression of constitutively active Akt. This is the first report about the anticancer activity of arctigenin in HCC cells, which is mediated by inactivation of PI3-K/Akt signaling.

  11. Hydrogen Sulfide Prevents Formation of Reactive Oxygen Species through PI3K/Akt Signaling and Limits Ventilator-Induced Lung Injury

    PubMed Central

    Spassov, Sashko Georgiev; Donus, Rosa; Ihle, Paul Mikael; Engelstaedter, Helen; Hoetzel, Alexander

    2017-01-01

    The development of ventilator-induced lung injury (VILI) is still a major problem in mechanically ventilated patients. Low dose inhalation of hydrogen sulfide (H2S) during mechanical ventilation has been proven to prevent lung damage by limiting inflammatory responses in rodent models. However, the capacity of H2S to affect oxidative processes in VILI and its underlying molecular signaling pathways remains elusive. In the present study we show that ventilation with moderate tidal volumes of 12 ml/kg for 6 h led to an excessive formation of reactive oxygen species (ROS) in mice lungs which was prevented by supplemental inhalation of 80 parts per million of H2S. In addition, phosphorylation of the signaling protein Akt was induced by H2S. In contrast, inhibition of Akt by LY294002 during ventilation reestablished lung damage, neutrophil influx, and proinflammatory cytokine release despite the presence of H2S. Moreover, the ability of H2S to induce the antioxidant glutathione and to prevent ROS production was reversed in the presence of the Akt inhibitor. Here, we provide the first evidence that H2S-mediated Akt activation is a key step in protection against VILI, suggesting that Akt signaling limits not only inflammatory but also detrimental oxidative processes that promote the development of lung injury. PMID:28250891

  12. Early single Aspirin-triggered Lipoxin blocked morphine anti-nociception tolerance through inhibiting NALP1 inflammasome: Involvement of PI3k/Akt signaling pathway.

    PubMed

    Tian, Yu; Liu, Ming; Mao-Ying, Qi-Liang; Liu, Huan; Wang, Zhi-Fu; Zhang, Meng-Ting; Wang, Jun; Li, Qian; Liu, Shen-Bin; Mi, Wen-Li; Ma, Hong-Jian; Wu, Gen-Cheng; Wang, Yan-Qing

    2015-11-01

    Clinical usage of opioids in pain relief is dampened by analgesic tolerance after chronic exposure, which is related to opioid-associated neuroinflammation. In the current study, which is based on a chronic morphine tolerance rat model and sustained morphine treatment on primary neuron culture, it was observed that Akt phosphorylation, cleaved-Caspase-1-dependent NALP1 inflammasome activation and IL-1β maturation in spinal cord neurons were significantly enhanced by morphine. Moreover, treatment with LY294002, a specific inhibitor of PI3k/Akt signaling, significantly reduced Caspase-1 cleavage, NALP1 inflammasome activation and attenuated morphine tolerance. Tail-flick tests demonstrated that pharmacological inhibition on Caspase-1 activation or antagonizing IL-1β dramatically blocked the development of morphine tolerance. The administration of an exogenous analogue of lipoxin, Aspirin-triggered Lipoxin (ATL), caused a decline in Caspase-1 cleavage, inflammasome activation and mature IL-1β production and thus attenuated the development of morphine tolerance by inhibiting upstream Akt phosphorylation. Additionally, treatment with DAMGO, a selective μ-opioid receptor peptide, significantly induced Akt phosphorylation, Caspase-1 cleavage and anti-nociception tolerance, all of which were attenuated by ATL treatment. Taken together, the present study revealed the involvement of spinal NALP1 inflammasome activation in the development of morphine tolerance and the role of the μ-receptor/PI3k-Akt signaling/NALP1 inflammasome cascade in this process. By inhibiting this signaling cascade, ATL blocked the development of morphine tolerance.

  13. IL-10 Protects Neurites in Oxygen-Glucose-Deprived Cortical Neurons through the PI3K/Akt Pathway.

    PubMed

    Lin, Longzai; Chen, Hongbin; Zhang, Yixian; Lin, Wei; Liu, Yong; Li, Tin; Zeng, Yongping; Chen, Jianhao; Du, Houwei; Chen, Ronghua; Tan, Yi; Liu, Nan

    2015-01-01

    IL-10, as a cytokine, has an anti-inflammatory cascade following various injuries, but it remains blurred whether IL-10 protects neurites of cortical neurons after oxygen-glucose deprivation injury. Here, we reported that IL-10, in a concentration-dependent manner, reduced neuronal apoptosis and increased neuronal survival in oxygen-glucose-deprived primary cortical neurons, producing an optimal protective effect at 20ng/ml. After staining NF-H and GAP-43, we found that IL-10 significantly protected neurites in terms of axon length and dendrite number by confocal microscopy. Furthermore, it induced the phosphorylation of AKT, suppressed the activation of caspase-3, and up-regulated the protein expression of GAP-43. In contrast, LY294002, a specific inhibitor of PI3K/AKT, reduced the level of AKT phosphorylation and GAP-43 expression, increased active caspase-3 expression and thus significantly weakened IL-10-mediated protective effect in the OGD-induced injury model. IL-10NA, the IL-10 neutralizing antibody, reduced the level of p-PI3K phosphorylation and increased the expression of active caspase-3. These findings suggest that IL-10 provides neuroprotective effects by protecting neurites through PI3K/AKT signaling pathway in oxygen-glucose-deprived primary cortical neurons.

  14. 2,3,7,8-Tetrachlorodibenzo-p-dioxin stimulates proliferation of HAPI microglia by affecting the Akt/GSK-3β/cyclin D1 signaling pathway.

    PubMed

    Xu, Guangfei; Li, Yuanye; Yoshimoto, Katsuhiko; Wu, Qiyun; Chen, Gang; Iwata, Takeo; Mizusawa, Noriko; Wan, Chunhua; Nie, Xiaoke

    2014-01-30

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxin that induces apoptosis of neurons and a pro-inflammatory response in microglial cells. First, we found that TCDD induced proliferation of HAPI microglial cells in a dose- and time-dependent manner. Flow cytometry analysis showed that this proliferation by TCDD was due to mainly enhancing the G1 to S phase transition. Next, it was found that TCDD treatment led to up-regulation of cyclin D1, which induces cell cycle progression from G1 to S phase, in a time-dependent manner. As for molecular mechanism, we revealed that TCDD was capable of inducing Akt phosphorylation and activation, resulting in phosphorylation and inactivation of glycogen synthase kinase-3β (GSK-3β). Inactivated GSK-3β attenuated proteasomal degradation of cyclin D1 by reducing Thr(286)-phosphorylated cyclin D1 levels. Moreover, inactivated GSK-3β increased cyclin D1 gene transcription by increasing its transcription factor β-catenin in the nucleus. Further, blockage of phosphoinositide 3-kinase/Akt kinase with their specific inhibitors, LY294002 and Akt 1/2 kinase inhibitor, significantly reduced TCDD-enhanced proliferation of HAPI microglial cells. In conclusion, TCDD stimulates proliferation of HAPI microglial cells by affecting the Akt/GSK-3β/cyclin D1 signaling pathway.

  15. Serine 1179 phosphorylation of endothelial nitric oxide synthase caused by 2,4,6-trinitrotoluene through PI3K/Akt signaling in endothelial cells

    SciTech Connect

    Sun Yang; Sumi, Daigo; Kumagai, Yoshito . E-mail: yk-em-tu@md.tsukuba.ac.jp

    2006-07-01

    Although 2,4,6-trinitrotoluene (TNT) has been found to uncouple nitric oxide synthase (NOS), thereby leading to reactive oxygen species (ROS), cellular response against TNT still remains unclear. Exposure of bovine aortic endothelial cells (BAECs) to TNT (100 {mu}M) resulted in serine 1179 phosphorylation of endothelial NOS (eNOS). With specific inhibitors (wortmannin and LY294002), we found that PI3K/Akt signaling participated in the eNOS phosphorylation caused by TNT, whereas the ERK pathway did not. ROS were generated following exposure of BAECs to TNT. However, TNT-mediated phosphorylation of either eNOS or Akt was drastically blocked by NAC and PEG-CAT. Interestingly, pretreatment with apocynin, a specific inhibitor for NADPH oxidase, diminished the phosphorylation of eNOS and Akt. These results suggest that TNT affects NADPH oxidase, thereby generating hydrogen peroxide, which is capable of activating PI3K/Akt signaling associated with eNOS Ser 1179 phosphorylation.

  16. Atorvastatin attenuates cognitive deficits through Akt1/caspase-3 signaling pathway in ischemic stroke.

    PubMed

    Yang, Jie; Pan, Ying; Li, Xuejing; Wang, Xianying

    2015-12-10

    Neuronal damage in the hippocampal formation is more sensitive to ischemic stimulation and easily injured, causing severe learning and memory impairment. Therefore, protection of hippocampal neuronal damage is the main contributor for learning and memory impairment during cerebral ischemia. Atorvastatin has been reported to ameliorate ischemic brain damage after ischemia reperfusion (I/R). However, its molecular mechanism has not been elucidated clearly. In this study, we established four-vessel occlusion model in rats with cerebral ischemia. Here, we demonstrated that atorvastatin significantly improves the behavior of I/R-rat in open field tasks. We also found that atorvastatin significantly shortens the distance and time of loading onto the hidden platform in the positioning navigation process, decreases the latency in the space exploration process when cognitive testing with Morris water maze was performed during ischemic stroke in rats. Furthermore, the survival rate of neurons in the CA1 area of the hippocampus and the phosphorylation of Akt (Ser473) in the neurons are increased, whereas the expression of caspase-3 are inhibited by atorvastatin. However, after an intracerebroventricular injection of LY294002 (an inhibitor of Akt1), the above neuroprotective effects of atorvastatin are attenuated. In summary, our results imply atorvastatin may improve the survival rate of hippocampal neurons and reduce the impairment of learning and memory by downregulating the activation of the caspase-3 via increasing the phosphorylation of Akt1 during ischemia/reperfusion.

  17. Bilirubin exerts pro-angiogenic property through Akt-eNOS-dependent pathway.

    PubMed

    Ikeda, Yasumasa; Hamano, Hirofumi; Satoh, Akiho; Horinouchi, Yuya; Izawa-Ishizawa, Yuki; Kihira, Yoshitaka; Ishizawa, Keisuke; Aihara, Ken-Ichi; Tsuchiya, Koichiro; Tamaki, Toshiaki

    2015-11-01

    Low serum bilirubin levels are associated with the risk of cardiovascular diseases including peripheral artery disease. Bilirubin is known to exert its property such as antioxidant effect or the enhancement of flow-mediated vasodilation, however, bilirubin action on angiogenesis remains unclear. To investigate the molecular mechanism of bilirubin on angiogenic effect, we first employed C57BL/6J mice with unilateral hindlimb ischemia surgery and divided the mice into two groups (vehicle-treated group and bilirubin-treated group). The analysis of laser speckle blood flow demonstrated the enhancement of blood flow recovery in response to ischemia of mice with bilirubin treatment. The density of capillaries was significantly higher in ischemic-adductor muscles of bilirubin-treated mice. The phosphorylated levels of endothelial nitric oxide synthase (eNOS) and Akt were increased in ischemic skeletal muscles of mice with bilirubin treatment compared with vehicle treatment. In in vitro experiments by using human aortic endothelial cells, bilirubin augmented eNOS and Akt phosphorylation, cell proliferation, cell migration and tube formation. These bilirubin actions on endothelial cell activation were inhibited by LY294002, a phosphatidylinositol 3-kinase inhibitor. In conclusion, bilirubin promotes angiogenesis through endothelial cells activation via Akt-eNOS-dependent manner.

  18. Chlorogenic Acid Prevents Osteoporosis by Shp2/PI3K/Akt Pathway in Ovariectomized Rats

    PubMed Central

    Zuo, Hui Ling; Yao, Fen Fen; Ruan, Hui Bing; Xu, Jin; Song, Wei; Zhou, Yi Cheng; Wen, Shi Yao; Dai, Jiang Hua; Zhu, Mei Lan; Luo, Jun

    2016-01-01

    Cortex Eucommiae is used worldwide in traditional medicine, various constituents of Cortex Eucommiae, such as chlorogenic acid (CGA), has been reported to exert anti-osteoporosis activity in China, but the mechanism about their contribution to the overall activity is limited. The aims of this study were to determine whether chlorogenic acid can prevent estrogen deficiency-induced osteoporosis and to analyze the mechanism of CGA bioactivity. The effect of CGA on estrogen deficiency-induced osteoporosis was performed in vivo. Sixty female Sprague-Dawley rats were divided randomly among a sham-operated group and five ovariectomy (OVX) plus treatment subgroups: saline vehicle, 17α-ethinylestradiol (E2), or CGA at 9, 27, or 45 mg/kg/d. The rats’ femoral metaphyses were evaluated by micro-computed tomography (μCT). The mechanism of CGA bioactivity was investigated in vitro. Bone mesenchymal stem cells (BMSCs) were treated with CGA, with or without phosphoinositide 3-kinase (PI3K) inhibitor LY294002. BMSCs proliferation and osteoblast differentiation were assessed with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and alkaline phosphatase, with or without Shp2 interfering RNA (RNAi). The results display that CGA at 27 and 45 mg/kg/day inhibited the decrease of bone mineral density (BMD) that induced by OVX in femur (p< 0.01), significantly promoted the levels of bone turnover markers, and prevented bone volume fraction (BV/TV), connectivity density (CoonD), trabecular number (Tb.N), trabecular thickness (Tb.Th) (all p< 0.01) to decrease and prevented the trabecular separation (Tb.Sp), structure model index (SMI)(both p< 0.01) to increase. CGA at 1 or 10 μM enhanced BMSC proliferation in a dose-dependent manner. CGA at 0.1 to 10 μM increased phosphorylated Akt (p-Akt) and cyclin D1. These effects were reversed by LY294002. CGA at 1 or 10 μM increased BMSC differentiation to osteoblasts (p< 0.01), Shp2 RNAi suppressed CGA-induced osteoblast

  19. Tetrandrine inhibits migration and invasion of human renal cell carcinoma by regulating Akt/NF-κB/MMP-9 signaling

    PubMed Central

    Wang, Ke; Fan, Yizeng; Chen, Jiaqi; Ma, Jianbin; Wang, Xinyang; He, Dalin; Zeng, Jin; Li, Lei

    2017-01-01

    Renal cell carcinoma (RCC) is known as one of the most lethal malignancies in the urological system because of its high incidence of metastasis. Tetrandrine (Tet), a traditional Chinese herbal medicine, exerts a potent anti-cancer effect in a variety of cancer cells. However, the anti-metastatic effect of Tet and its possible mechanism in RCC is still unclear. The present study revealed that Tet significantly suppressed the migration and invasion of RCC 786-O and 769-P cells in vitro. Mechanistically, the protein levels of matrix metalloproteinases 9 (MMP-9), phosphorylated PI3K, PDK1, Akt and NF-κB were markedly reduced after Tet treatment. Moreover, co-treatment with LY294002 (PI3K inhibitor) could further enhance the Tet-inhibited migration and invasion, and the NF-κB and MMP-9 protein levels were further decreased. Similar results were observed after PDTC (NF-κB inhibitor) co-treatment. Conversely, SC79, an Akt activator, could partially reverse the anti-metastatic effects of Tet, accompanied by the restoration of NF-κB and MMP-9 protein levels. In conclusion, the current results indicated that Tet inhibited migration and invasion of RCC partially by regulating Akt/NF-κB/MMP-9 signaling pathway, suggesting that Tet may be a potential therapeutic candidate against metastatic RCC. PMID:28288190

  20. PDGF stimulation of Mueller cell proliferation: Contributions of c-JNK and the PI3K/Akt pathway

    SciTech Connect

    Moon, Sang Woong; Chung, Eun Jee; Jung, Sun-Ah; Lee, Joon H.

    2009-10-09

    Platelet-derived growth factor (PDGF) has a critical role in proliferative vitreoretinopathy (PVR) as a chemoattractant and mitogen for retinal pigment epithelial cells and retinal glial cells. Here, we investigated the potential effects of PDGF on the proliferation of Mueller cells and the intracellular signaling pathway mediating these changes. PDGF induced Mueller cell proliferation and increased phosphorylation of the PDGF receptor (PDGFR), as shown by an MTT assay and immunoprecipitation analyses. Both effects were blocked by JNJ, a PDGFR-selective tyrosine kinase inhibitor. PDGF also stimulated phosphorylation of c-JNK and Akt. PDGF-induced Mueller cell proliferation was significantly reduced by pre-treatment with SP600125 and LY294002, inhibitors of c-JNK and Akt phosphorylation, respectively. Our findings collectively indicate that PDGF-stimulated Mueller cell proliferation occurs via activation of the c-JNK and PI3K/Akt signaling pathways. These data provide useful information in establishing the role of Mueller cells in the development of proliferative vitreoretinopathy.

  1. Luteolin enhances cholinergic activities in PC12 cells through ERK1/2 and PI3K/Akt pathways.

    PubMed

    El Omri, Abdelfatteh; Han, Junkyu; Kawada, Kiyokazu; Ben Abdrabbah, Manef; Isoda, Hiroko

    2012-02-09

    Luteolin, a 3', 4', 5, 7-tetrahydroxyflavone, is an active compound in Rosmarinus officinalis (Lamiacea), and has been reported to exert several benefits in neuronal cells. However cholinergic-induced activities of luteolin still remain unknown. Neuronal differentiation encompasses an elaborate developmental program which plays a key role in the development of the nervous system. The advent of several cell lines, like PC12 cells, able to differentiate in culture proved to be the turning point for gaining and understanding of molecular neuroscience. In this work, we investigated the ability of luteolin to induce PC12 cell differentiation and its effect on cholinergic activities. Our findings showed that luteolin treatment significantly induced neurite outgrowth extension, enhanced acetylcholinesterase (AChE) activity, known as neuronal differentiation marker, and increased the level of total choline and acetylcholine in PC12 cells. In addition, luteolin persistently, activated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt; while the addition of pharmacological MEK/ERK1/2 inhibitor (U0126) and PI3k/Akt inhibitor (LY294002) attenuated luteolin-induced AChE activity and neurite outgrowth in PC12 cells. The above findings suggest that luteolin induces neurite outgrowth and enhanced cholinergic activities, at least in part, through the activation of ERK1/2 and Akt signaling.

  2. Tetrandrine inhibits migration and invasion of human renal cell carcinoma by regulating Akt/NF-κB/MMP-9 signaling.

    PubMed

    Chen, Shurui; Liu, Wei; Wang, Ke; Fan, Yizeng; Chen, Jiaqi; Ma, Jianbin; Wang, Xinyang; He, Dalin; Zeng, Jin; Li, Lei

    2017-01-01

    Renal cell carcinoma (RCC) is known as one of the most lethal malignancies in the urological system because of its high incidence of metastasis. Tetrandrine (Tet), a traditional Chinese herbal medicine, exerts a potent anti-cancer effect in a variety of cancer cells. However, the anti-metastatic effect of Tet and its possible mechanism in RCC is still unclear. The present study revealed that Tet significantly suppressed the migration and invasion of RCC 786-O and 769-P cells in vitro. Mechanistically, the protein levels of matrix metalloproteinases 9 (MMP-9), phosphorylated PI3K, PDK1, Akt and NF-κB were markedly reduced after Tet treatment. Moreover, co-treatment with LY294002 (PI3K inhibitor) could further enhance the Tet-inhibited migration and invasion, and the NF-κB and MMP-9 protein levels were further decreased. Similar results were observed after PDTC (NF-κB inhibitor) co-treatment. Conversely, SC79, an Akt activator, could partially reverse the anti-metastatic effects of Tet, accompanied by the restoration of NF-κB and MMP-9 protein levels. In conclusion, the current results indicated that Tet inhibited migration and invasion of RCC partially by regulating Akt/NF-κB/MMP-9 signaling pathway, suggesting that Tet may be a potential therapeutic candidate against metastatic RCC.

  3. The sonic hedgehog signaling pathway stimulates anaplastic thyroid cancer cell motility and invasiveness by activating Akt and c-Met.

    PubMed

    Williamson, Ashley J; Doscas, Michelle E; Ye, Jin; Heiden, Katherine B; Xing, Mingzhao; Li, Yi; Prinz, Richard A; Xu, Xiulong

    2016-03-01

    The sonic hedgehog (Shh) pathway is highly activated in thyroid neoplasms and promotes thyroid cancer stem-like cell phenotype, but whether the Shh pathway regulates thyroid tumor cell motility and invasiveness remains unknown. Here, we report that the motility and invasiveness of two anaplastic thyroid tumor cell lines, KAT-18 and SW1736, were inhibited by two inhibitors of the Shh pathway (cyclopamine and GANT61). Consistently, the cell motility and invasiveness was decreased by Shh and Gli1 knockdown, and was increased by Gli1 overexpression in KAT-18 cells. Mechanistic studies revealed that Akt and c-Met phosphorylation was decreased by a Gli1 inhibitor and by Shh and Gli1 knockdown, but was increased by Gli1 overexpression. LY294002, a PI-3 kinase inhibitor, and a c-Met inhibitor inhibited the motility and invasiveness of Gli1-transfected KAT-18 cells more effectively than the vector-transfected cells. Knockdown of Snail, a transcription factor regulated by the Shh pathway, led to decreased cell motility and invasiveness in KAT-18 and SW1736 cells. However, key epithelial-to-mesenchymal transition (EMT) markers including E-cadherin and vimentin as well as Slug were not affected by cyclopamine and GANT61 in either SW1736 or WRO82, a well differentiated follicular thyroid carcinoma cell line. Our data suggest that the Shh pathway-stimulated thyroid tumor cell motility and invasiveness is largely mediated by AKT and c-Met activation with little involvement of EMT.

  4. CCN1/Cyr61-PI3K/AKT signaling promotes retinal neovascularization in oxygen-induced retinopathy.

    PubMed

    Di, Yu; Zhang, Yiou; Nie, Qingzhu; Chen, Xiaolong

    2015-12-01

    Retinal neovascularization (RNV) is a characteristic pathological finding of retinopathy of prematurity (ROP). Cysteine-rich 61 [Cyr61, also known as CCN family member 1 (CCN1)] has been reported to mediate angiogenesis. The aim of the present study was to investigate the mechanisms of CCN1/Cyr61-phosphoinositide 3-kinase (PI3K)/AKT signaling in ROP. The contribution of CCN1 to human umbilical vein endothelial cell (HUVEC) proliferation and apoptosis under hypoxic conditions was determined using a cell counting kit‑8 (CCK-8) and Annexin V/propidium iodide (PI) staining, respectively, as well as using siRNA targeting CCN1 (CCN1 siRNA). The cells exposed to hypoxia were also treated with the PI3K/AKT inhibitor, LY294002. In addition, mouse pups with oxygen-induced retinopathy (OIR) were administered an intravitreal injection of CCN1 siRNA. RNV was assessed by magnesium-activated adenosine diphosphate-ase (ADPase) staining. RT-qPCR, western blot analysis, immunofluorescence staining and immunohistochemistry were used to detect the distribution and expression of CCN1, PI3K and AKT. Exposure to hypoxia increased the neovascularization clock hour scores (from 1.23±0.49 to 5.60±0.73, P<0.05) and the number of preretinal neovascular cells, as well as the mRNA and protein expression levels of CCN1, PI3K and AKT (all P<0.05). The injection of CCN1 siRNA decreased the neovascularization clock hour scores and the number of preretinal neovascular cells (1.53±0.72 vs. 4.76±1.04; 12.0±2.8 vs. 31.4±2.6, respectively, both P<0.05), as well as the mRNA and protein expression levels of CCN1, PI3K and AKT (protein, -45.3, -22.5 and -28.4%; mRNA, -43.7, -58.7 and -42.9%, respectively, all P<0.05) compared to the administration of scrambled siRNA under hypoxic conditions. Treatment with LY294002 decreased the mRNA and protein expression levels of CCN1 in the cells exposed to hypoxia (both P<0.05). The administration of CCN1 siRNA resulted in less severe neovascularization in the

  5. Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway

    SciTech Connect

    Liang, Qiu-Hua; Liu, Yuan; Wu, Shan-Shan; Cui, Rong-Rong; Yuan, Ling-Qing Liao, Er-Yuan

    2013-11-01

    Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. - Highlights: • We explored the effects of ghrelin on serum deprivation-induced MC3T3-E1 cells apoptosis. • Both ELISA and TUNEL were used to detect the apoptosis. • The receptor of ghrelin, GHSR, was expressed in MC3T3-E1

  6. Protective effect of rutaecarpine against t-BHP-induced hepatotoxicity by upregulating antioxidant enzymes via the CaMKII-Akt and Nrf2/ARE pathways.

    PubMed

    Jin, Sun Woo; Hwang, Yong Pil; Choi, Chul Yung; Kim, Hyung Gyun; Kim, Se Jong; Kim, Yongan; Chung, Young Chul; Lee, Kyung Jin; Jeong, Tae Cheon; Jeong, Hye Gwang

    2017-02-01

    Rutaecarpine, an indolopyridoquinazolinone alkaloid isolated from the unripe fruit of Evodia rutaecarpa, has been shown to have cytoprotective potential, but the molecular mechanism underlying this activity remains unclear. Our study was designed to investigate the cytoprotective effect of rutaecarpine against tert-butyl hydroperoxide (t-BHP) and to elucidate its action mechanism of action of rutaecarpine in a cultured HepG2 cell line and in mouse liver. Rutaecarpine decreased t-BHP-induced reactive oxygen species (ROS) production, cytotoxicity, and apoptosis in HepG2 cells. Pretreatment with rutaecarpine prior to the injection of t-BHP significantly prevented the increase in serum levels of AST, ALT, and lipid peroxidation in mice liver. It increased the transcriptional activity of NF-E2-related factor 2 (Nrf2) as well as the products of the Nrf2 target genes hemeoxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), and glutamate cysteine ligase (GCL). Moreover, rutaecarpine also enhanced the phosphorylation of Akt and Ca(2+)/calmodulin-dependent protein kinase-II (CaMKII). The pharmaceutical inhibitors, such as KN-93 (CaMKII inhibitor) and LY294002 (Akt inhibitor) suppressed rutaecarpine-induced HO-1 expression and cytoprotection. Our findings identify the CaMKII-PI3K/Akt-Nrf2 cascade as an antioxidant pathway mediating rutaecarpine signaling and leading to HO-1 expression in hepatocytes.

  7. ROS-Dependent Activation of Autophagy through the PI3K/Akt/mTOR Pathway Is Induced by Hydroxysafflor Yellow A-Sonodynamic Therapy in THP-1 Macrophages

    PubMed Central

    Jiang, Yueqing; Kou, Jiayuan; Han, Xiaobo; Li, Xuesong; Zhong, Zhaoyu; Liu, Zhongni; Zheng, Yinghong; Tian, Ye

    2017-01-01

    Monocyte-derived macrophages participate in infaust inflammatory responses by secreting various types of proinflammatory factors, resulting in further inflammatory reactions in atherosclerotic plaques. Autophagy plays an important role in inhibiting inflammation; thus, increasing autophagy may be a therapeutic strategy for atherosclerosis. In the present study, hydroxysafflor yellow A-mediated sonodynamic therapy was used to induce autophagy and inhibit inflammation in THP-1 macrophages. Following hydroxysafflor yellow A-mediated sonodynamic therapy, autophagy was induced as shown by the conversion of LC3-II/LC3-I, increased expression of beclin 1, degradation of p62, and the formation of autophagic vacuoles. In addition, inflammatory factors were inhibited. These effects were blocked by Atg5 siRNA, the autophagy inhibitor 3-methyladenine, and the reactive oxygen species scavenger N-acetyl cysteine. Moreover, AKT phosphorylation at Ser473 and mTOR phosphorylation at Ser2448 decreased significantly after HSYA-SDT. These effects were inhibited by the PI3K inhibitor LY294002, the AKT inhibitor triciribine, the mTOR inhibitor rapamycin, mTOR siRNA, and N-acetyl cysteine. Our results demonstrate that HSYA-SDT induces an autophagic response via the PI3K/Akt/mTOR signaling pathway and inhibits inflammation by reactive oxygen species in THP-1 macrophages. PMID:28191279

  8. Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase Pathway

    DTIC Science & Technology

    2014-10-01

    kinase . This grant proposal will explore the resistance to small molecule AKT protein kinase inhibitors mediated by the... molecule AKT protein kinase inhibitors is potentially mediated by the Pim-1 protein kinase , and that unique Pim protein kinase inhibitors that can in...application is essential for the development of this combined chemotherapeutic strategy. 15. SUBJECT TERMS Small Molecule AKT Inhibitors ,

  9. HPV16 E6-E7 induces cancer stem-like cells phenotypes in esophageal squamous cell carcinoma through the activation of PI3K/Akt signaling pathway in vitro and in vivo

    PubMed Central

    Xi, Ruxing; Pan, Shupei; Chen, Xin; Hui, Beina; Zhang, Li; Fu, Shenbo; Li, Xiaolong; Zhang, Xuanwei; Gong, Tuotuo; Guo, Jia; Zhang, Xiaozhi; Che, Shaomin

    2016-01-01

    High-risk human papillomavirus (HPV), especially HPV16, correlates with cancerogenesis of human esophageal squamous cell carcinoma (ESCC) and we have reported that HPV16 related with a poor prognosis of ESCC patients in China. We aim to investigate the potential role and mechanism of HPV16 in ESCC development and progress. Our following researches demonstrated that ESCC cells which were stably transfected by HPV16 E6-E7 lentiviral vector showed a remarkable cancer stem-like cells (CSCs) phenotype, such as: migration, invasion, spherogenesis, high expression of CSCs marker in ESCC---p75NTR, chemoresistance, radioresistance, anti-apoptosis ability in vitro and cancerogenesis in vivo. HPV16 E6-E7 induced PI3K/Akt signaling pathway activation and this affect could be effectively inhibited by LY294002, a specific PI3K inhibitor. It was also indicated that the inhibition of PI3K/Akt signaling pathway by PI3K and Akt siRNA reverse the effect which induced by HPV16 E6-E7 in ESCC cells. Taken together, the present study demonstrates that HPV16 E6-E7 promotes CSCs phenotype in ESCC cells through the activation of PI3K/Akt signaling pathway. Targeting the PI3K/Akt signaling pathway in HPV16 positive tissues is an available therapeutic for ESCC patients. PMID:27489353

  10. HPV16 E6-E7 induces cancer stem-like cells phenotypes in esophageal squamous cell carcinoma through the activation of PI3K/Akt signaling pathway in vitro and in vivo.

    PubMed

    Xi, Ruxing; Pan, Shupei; Chen, Xin; Hui, Beina; Zhang, Li; Fu, Shenbo; Li, Xiaolong; Zhang, Xuanwei; Gong, Tuotuo; Guo, Jia; Zhang, Xiaozhi; Che, Shaomin

    2016-08-30

    High-risk human papillomavirus (HPV), especially HPV16, correlates with cancerogenesis of human esophageal squamous cell carcinoma (ESCC) and we have reported that HPV16 related with a poor prognosis of ESCC patients in China. We aim to investigate the potential role and mechanism of HPV16 in ESCC development and progress. Our following researches demonstrated that ESCC cells which were stably transfected by HPV16 E6-E7 lentiviral vector showed a remarkable cancer stem-like cells (CSCs) phenotype, such as: migration, invasion, spherogenesis, high expression of CSCs marker in ESCC---p75NTR, chemoresistance, radioresistance, anti-apoptosis ability in vitro and cancerogenesis in vivo. HPV16 E6-E7 induced PI3K/Akt signaling pathway activation and this affect could be effectively inhibited by LY294002, a specific PI3K inhibitor. It was also indicated that the inhibition of PI3K/Akt signaling pathway by PI3K and Akt siRNA reverse the effect which induced by HPV16 E6-E7 in ESCC cells. Taken together, the present study demonstrates that HPV16 E6-E7 promotes CSCs phenotype in ESCC cells through the activation of PI3K/Akt signaling pathway. Targeting the PI3K/Akt signaling pathway in HPV16 positive tissues is an available therapeutic for ESCC patients.

  11. Rapid-acting antidepressant-like effects of acetyl-l-carnitine mediated by PI3K/AKT/BDNF/VGF signaling pathway in mice.

    PubMed

    Wang, W; Lu, Y; Xue, Z; Li, C; Wang, C; Zhao, X; Zhang, J; Wei, X; Chen, X; Cui, W; Wang, Q; Zhou, W

    2015-01-29

    The possible involvement of the PI3K/AKT/BDNF/VGF signaling in rapid-acting antidepressant-like effects of antidepressants has been explored progressively by more studies. However, whether this signaling participates in the antidepressant-like effects of acetyl-l-carnitine (ALC) has not been examined. Herein, we assessed the antidepressant-like effects of ALC using the forced swimming test (FST). Our results demonstrated the dose-effect relationship of acute administration of ALC (5, 25, 50 and 100mg/kg, i.p.) and showed that it dose-dependently decreased the immobility time on FST of mice. In addition, ALC (100 mg/kg, i.p.) also reversed depressive-like behavior and the down-regulation of phosphorylated AKT (pAKT), brain-derived neurotrophic factor (BDNF) and neuropeptide VGF in the hippocampus and prefrontal cortex of mice induced by chronic unpredictable mild stress (CUMS) paradigm. Further, intra-cerebroventricular (i.c.v.) infusions of LY294002 (10 nmol/side), a specific phosphatidylinositol 3-kinase (PI3K) inhibitor, significantly prevented the antidepressant-like effect of ALC (100mg/kg, i.p.). In conclusion, our results demonstrated that ALC exerts rapid-acting antidepressant-like effects that might be mediated by the PI3K/AKT/BDNF/VGF signaling pathway.

  12. HspB8 mediates neuroprotection against OGD/R in N2A cells through the phosphoinositide 3-kinase/Akt pathway.

    PubMed

    Hu, Zhiping; Yang, Binbin; Mo, Xiaoye; Zhou, Fangfang

    2016-08-01

    In a previous study, we found that Heat shock protein B8 (HspB8) overexpression could prevent the apoptosis and reduced cell viability induced by OGD/R and showed that the neuroprotective effect of HspB8 was mediated by inhibition of the mitochondrial apoptotic pathway. In recent study, HspB8 has been shown to protect the heart against ischemia/reperfusion (I/R) injury via activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. However, whether this protective effect applied to brain I/R injury remained unexplored. To further test the mechanism of HspB8's effects in brain, we used oxygen-glucose deprivation followed by reperfusion (OGD/R), an in vitro model of ischemia to examine the involvement of PI3K/Akt signaling by treating mouse neuroblastoma cells (N2A cells) (untransfected or transfected with an HspB8 expression vector) with the PI3K inhibitor LY294002 before OGD/R. Our results revealed that the apoptosis-suppressing effect of HspB8 was mediated by the PI3K/Akt pathway. Therefore, HspB8 protected the N2A cells against OGD/R insult, possibly by activating the PI3K/Akt signaling pathway.

  13. Putative Phosphatidylinositol 3-Kinase (PI3K) Binding Motifs in Ovine Betaretrovirus Env Proteins Are Not Essential for Rodent Fibroblast Transformation and PI3K/Akt Activation

    PubMed Central

    Liu, Shan-Lu; Lerman, Michael I.; Miller, A. Dusty

    2003-01-01

    Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are simple betaretroviruses that cause epithelial cell tumors in the lower and upper airways of sheep and goats. The envelope (Env) glycoproteins of both viruses can transform rodent and chicken fibroblasts, indicating that they play an essential role in oncogenesis. Previous studies found that a YXXM motif in the Env cytoplasmic tail, a putative docking site for phosphatidylinositol 3-kinase (PI3K) after tyrosine phosphorylation, was necessary for rodent cell transformation but was not required for transformation of DF-1 chicken fibroblasts. Here we show that JSRV and ENTV Env proteins with tyrosine or methionine mutations in the YXXM motif can still transform rodent fibroblasts, albeit with reduced efficiency. Akt was activated in cells transformed by JSRV or ENTV Env proteins and in cells transformed by the proteins with tyrosine mutations. Furthermore, the PI3K-specific inhibitor LY294002 could inhibit Akt activation and cell transformation in all cases, indicating that Akt activation and transformation is PI3K dependent. However, we could not detect tyrosine phosphorylation of JSRV or ENTV Env proteins or an interaction between the Env proteins and PI3K in the transformed cells. We found no evidence for mitogen-activated protein kinase activation in cells that were transformed by the JSRV or ENTV Env proteins. We conclude that ovine betaretrovirus Env proteins transform the rodent fibroblasts by indirectly activating the PI3K/Akt pathway. PMID:12829832

  14. Phosphatidylinositol 3-kinase/Akt signaling pathway mediates acupuncture-induced dopaminergic neuron protection and motor function improvement in a mouse model of Parkinson's disease.

    PubMed

    Kim, Seung-Nam; Kim, Seung-Tae; Doo, Ah-Reum; Park, Ji-Yeun; Moon, Woongjoon; Chae, Younbyoung; Yin, Chang Shik; Lee, Hyejung; Park, Hi-Joon

    2011-10-01

    It has been reported that acupuncture treatment reduced dopaminergic neuron degeneration in Parkinson's disease (PD) models. However, the mechanistic pathways underlying, such neuroprotection, are poorly understood. Here, we investigated the effects and the underlying mechanism of acupuncture in a mouse model of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). First, we observed that MPTP-induced impairment of Akt activation, but not MPTP-induced c-Jun activation, was effectively restored by acupuncture treatment in the substantia nigra. Furthermore, we demonstrated for the first time that the brain-specific blockade of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, by intranasal administration of LY294002, a specific inhibitor of PI3K/Akt signaling pathway, significantly blocked acupuncture-induced dopaminergic neuron protection and motor function improvement. Our results provide evidence that PI3K/Akt signaling pathway may play a central role in the mechanism underlying acupuncture-induced benefits in Parkinsonian mice.

  15. CCL21 Facilitates Chemoresistance and Cancer Stem Cell-Like Properties of Colorectal Cancer Cells through AKT/GSK-3β/Snail Signals

    PubMed Central

    Lu, Lin-Lin; Chen, Xiao-Hui; Zhang, Ge; Liu, Zong-Cai; Wu, Nong; Wang, Hao; Qi, Yi-Fei; Wang, Hong-Sheng; Cai, Shao Hui; Du, Jun

    2016-01-01

    Some evidence indicated that chemoresistance associates with the acquisition of cancer stem-like properties. Recent studies suggested that chemokines can promote the chemoresistance and stem cell properties in various cancer cells, while the underling mechanism is still not completely illustrated. In our study, we found that CCL21 can upregulate the expression of P-glycoprotein (P-gp) and stem cell property markers such as Bmi-1, Nanog, and OCT-4 in colorectal cancer (CRC) HCT116 cells and then improve the cell survival rate and mammosphere formation. Our results suggested that Snail was crucial for CCL21-mediated chemoresistance and cancer stem cell property in CRC cells. Further, we observed that CCL21 treatment increased the protein but not mRNA levels of Snail, which suggested that CCL21 upregulates Snail via posttranscriptional ways. The downstream signals AKT/GSK-3β mediated CCL21 induced the upregulation of Snail due to the fact that CCL21 treatment can obviously phosphorylate both AKT and GSK-3β. The inhibitor of PI3K/Akt, LY294002 significantly abolished CCL21 induced chemoresistance and mammosphere formation of HCT116 cells. Collectively, our results in the present study revealed that CCL21 can facilitate chemoresistance and stem cell property of CRC cells via the upregulation of P-gp, Bmi-1, Nanog, and OCT-4 through AKT/GSK-3β/Snail signals, which suggested a potential therapeutic approach to CRC patients. PMID:27057280

  16. Relaxin attenuates aristolochic acid induced human tubular epithelial cell apoptosis in vitro by activation of the PI3K/Akt signaling pathway.

    PubMed

    Xie, Xiang-Cheng; Zhao, Ning; Xu, Qun-Hong; Yang, Xiu; Xia, Wen-Kai; Chen, Qi; Wang, Ming; Fei, Xiao

    2017-04-06

    Aristolochic acid nephropathy remains a leading cause of chronic kidney disease (CKD), however few treatment strategies exist. Emerging evidence has shown that H2 relaxin (RLX) possesses powerful antifibrosis and anti-apoptotic properties, therefore we aimed to investigate whether H2 relaxin can be employed to reduce AA-induced cell apoptosis. Human proximal tubular epithelial (HK-2) cells exposed to AA-I were treated with or without administration of H2 RLX. Cell viability was examined using the WST-8 assay. Apoptotic morphologic alterations were observed using the Hoechst 33342 staining method. Apoptosis was detected using flow cytometry. The expression of caspase 3, caspase 8, caspase 9, ERK1/2, Bax, Bcl-2, and Akt proteins was determined by Western blot. Co-treatment with RLX reversed the increased apoptosis observed in the AA-I only treated group. RLX restored expression of phosphorylated Akt which found to be decreased in the AA-I only treated cells. RLX co-treatment led to a decrease in the Bax/Bcl-2 ratio as well as the cleaved form of caspase-3 compared to the AA-I only treated cells. This anti-apoptotic effect of RLX was attenuated by co-administration of the Akt inhibitor LY294002. The present study demonstrated H2 RLX can decrease AA-I induced apoptosis through activation of the PI3K/Akt signaling pathway.

  17. ets-2 Is a Target for an Akt (Protein Kinase B)/Jun N-Terminal Kinase Signaling Pathway in Macrophages of motheaten-viable Mutant Mice

    PubMed Central

    Smith, James L.; Schaffner, Alicia E.; Hofmeister, Joseph K.; Hartman, Matthew; Wei, Guo; Forsthoefel, David; Hume, David A.; Ostrowski, Michael C.

    2000-01-01

    The transcription factor ets-2 was phosphorylated at residue threonine 72 in a colony-stimulating factor 1 (CSF-1)- and mitogen-activated protein kinase-independent manner in macrophages isolated from motheaten-viable (me-v) mice. The CSF-1 and ets-2 target genes coding for Bcl-x, urokinase plasminogen activator, and scavenger receptor were also expressed at high levels independent of CSF-1 addition to me-v cells. Akt (protein kinase B) was constitutively active in me-v macrophages, and an Akt immunoprecipitate catalyzed phosphorylation of ets-2 at threonine 72. The p54 isoform of c-jun N-terminal kinase–stress-activated kinase (JNK- SAPK) coimmunoprecipitated with Akt from me-v macrophages, and treatment of me-v cells with the specific phosphatidylinositol 3-kinase inhibitor LY294002 decreased cell survival, Akt and JNK kinase activities, ets-2 phosphorylation, and Bcl-x mRNA expression. Therefore, ets-2 is a target for phosphatidylinositol 3-kinase–Akt–JNK action, and the JNK p54 isoform is an ets-2 kinase in macrophages. Constitutive ets-2 activity may contribute to the pathology of me-v mice by increasing expression of genes like the Bcl-x gene that promote macrophage survival. PMID:11027273

  18. miR-21 and KLF4 jointly augment epithelial-mesenchymal transition via the Akt/ERK1/2 pathway

    PubMed Central

    Liu, Chen-Hai; Huang, Qiang; Jin, Zhi-Yuan; Zhu, Cheng-Lin; Liu, Zhen; Wang, Chao

    2017-01-01

    miR-21 induces epithelial-mesenchymal transition (EMT) of human cholangiocarcinoma (CCA) cells. However, the mechanism by which this occurs remains unclear. In the present study, high throughput platform was employed to detect the genes that are differential expressed in QBC939 cells transfected with a hsa-miR-21 antagomir or control vectors. The EMT-related Krüppel-like factor 4 (KLF4) gene was down-regulated after miR-21 was knocked down. Overexpression of miR-21 upregulated KLF4, Akt, ERK and mesenchymal cell markers (N-cadherin and vimentin), downregulated the expression of epithelial cell marker E-cadherin and reduced cell migration and invasion. Immunohistochemistry showed that KLF4, pAkt and pERK were upregulated in tumor xenografts transfected with miR-21 mimics. Inhibitors of the PI3K-Akt and ERK1/2 pathways, LY294002 and U0126, significantly suppressed the EMT phenotype. The present data demonstrated that overexpression of miR-21, accompanied with KLF4, augmented the EMT via inactivation of Akt and ERK1/2 pathways. In conclusion, we have identified a novel mechanism that may be targeted in an attempt to relieve the malignant biological behavior of CCA cells. PMID:28197636

  19. Chicoric acid induces apoptosis in 3T3-L1 preadipocytes through ROS-mediated PI3K/Akt and MAPK signaling pathways.

    PubMed

    Xiao, Haifang; Wang, Jing; Yuan, Li; Xiao, Chunxia; Wang, Yutang; Liu, Xuebo

    2013-02-20

    Chicoric acid has been reported to possess various bioactivities. However, the antiobesity effects of chicoric acid remain poorly understood. In this study, we investigated the effects of chicoric acid on 3T3-L1 preadipocytes and its molecular mechanisms of apoptosis. Chicoric acid inhibited cell viability and induced apoptosis in 3T3-L1 preadipocytes which was characterized by chromatin condensation and poly ADP-ribose-polymerase (PARP) cleavage. Mitochondrial membrane potential (MMP) loss, Bax/Bcl-2 dysregulation, cytochrome c release, and caspase-3 activation were observed, indicating mitochondria-dependent apoptosis induced by chicoric acid. Furthermore, PI3K/Akt and MAPK (p38 MAPK, JNK, and ERK1/2) signaling pathways were involved in chicoric acid-induced apoptosis. The employment of protein kinase inhibitors LY294002, SB203580, SP600125, and U0126 revealed that PI3K/Akt signaling pathway interplayed with MAPK signaling pathways. Moreover, chicoric acid induced reactive oxygen species (ROS) generation. Pretreatment with the antioxidant N-acetylcysteine (NAC) significantly blocked cell death and changes of Akt and MAPK signalings induced by chicoric acid. In addition, chicoric acid down regulated HO-1 and COX-2 via the PI3K/Akt pathway.

  20. Lithium protects against methamphetamine-induced neurotoxicity in PC12 cells via Akt/GSK3β/mTOR pathway

    SciTech Connect

    Wu, Jintao; Zhu, Dexiao; Zhang, Jing; Li, Guibao; Liu, Zengxun; Sun, Jinhao

    2015-09-25

    Methamphetamine (MA) is neurotoxic, especially in dopaminergic neurons. Long-lasting exposure to MA causes psychosis and increases the risk of Parkinson's disease. Lithium (Li) is a known mood stabilizer and has neuroprotective effects. Previous studies suggest that MA exposure decreases the phosphorylation of Akt/GSK3β pathway in vivo, whereas Li facilitates the phosphorylation of Akt/GSK3β pathway. Moreover, GSK3β and mTOR are implicated in the locomotor sensitization induced by psychostimulants and mTOR plays a critical role in MA induced toxicity. However, the effect of MA on Akt/GSK3β/mTOR pathway has not been fully investigated in vitro. Here, we found that MA exposure significantly dephosphorylated Akt/GSK3β/mTOR pathway in PC12 cells. In addition, Li remarkably attenuated the dephosphorylation effect of MA exposure on Akt/GSK3β/mTOR pathway. Furthermore, Li showed obvious protective effects against MA toxicity and LY294002 (Akt inhibitor) suppressed the protective effects of Li. Together, MA exposure dephosphorylates Akt/GSK3β/mTOR pathway in vitro, while lithium protects against MA-induced neurotoxicity via phosphorylation of Akt/GSK3β/mTOR pathway. - Highlights: • Lithium protects against methamphetamine-induced neurotoxicity in vitro. • Methamphetamine exposure dephosphorylates Akt/GSK3β/mTOR pathway. • Lithium attenuates methamphetamine-induced toxicity via phosphorylating Akt/GSK3β/mTOR pathway.

  1. Static magnetic field enhances the viability and proliferation rate of adipose tissue-derived mesenchymal stem cells potentially through activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) pathway.

    PubMed

    Marędziak, Monika; Tomaszewski, Krzysztof; Polinceusz, Paulina; Lewandowski, Daniel; Marycz, Krzysztof

    2017-01-01

    The aim of this work was to investigate the effects of 0.5T static magnetic field (sMF) on the viability and proliferation rate of human adipose-derived mesenchymal stromal stem cells (hASCs) via activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) signaling pathway. In a 7-d culture we examined cell growth kinetic and population doubling time (PDT). We also examined cell morphology and the cellular senescence markers level. Exposure to sMF enhanced the viability of these cells. However, the effect was blocked by treating the cells with LY294002, a P13K inhibitor. We compared this effect by Western Blot analysis of Akt protein expression. We also examined whether the cell response on sMF stimulation is dependent on integrin engagement and we measured integrin gene expression. Our results suggest that stimulation using sMF is a viable method to improve hASC viability. sMF is involved in mechanisms associated with controlling cell proliferative potential signaling events.

  2. PI3 kinase/Akt activation mediates estrogen and IGF-1 nigral DA neuronal neuroprotection against a unilateral rat model of Parkinson's disease.

    PubMed

    Quesada, Arnulfo; Lee, Becky Y; Micevych, Paul E

    2008-04-01

    Recently, using the medial forebrain bundle (MFB) 6-hydroxydopmaine (6-OHDA) lesion rat model of Parkinson's disease (PD), we have demonstrated that blockade of central IGF-1 receptors (IGF-1R) attenuated estrogen neuroprotection of substantia nigra pars compacta (SNpc) DA neurons, but exacerbated 6-OHDA lesions in IGF-1 only treated rats (Quesada and Micevych [2004]: J Neurosci Res 75:107-116). This suggested that the IGF-1 system is a central mechanism through which estrogen acts to protect the nigrostriatal DA system. Moreover, these results also suggest that IGF-1R-induced intracellular signaling pathways are involved in the estrogen mechanism that promotes neuronal survival. In vitro, two convergent intracellular signaling pathways used by estrogen and IGF-1, the mitogen-activated protein kinase (MAPK/ERK), and phosphatidyl-inositol-3-kinase/Akt (PI3K/Akt), have been demonstrated to be neuroprotective. Continuous central infusions of MAPK/ERK and PI3K/Akt inhibitors were used to test the hypothesis that one or both of these signal transduction pathways mediates estrogen and/or IGF-1 neuroprotection of SNpc DA neurons after a unilateral administration of 6-OHDA into the MFB of rats. Motor behavior tests and tyrosine hydroxylase immunoreactivity revealed that the inhibitor of the PI3K/Akt pathway (LY294002) blocked the survival effects of both estrogen and IGF-1, while an inhibitor of the MAPK/ERK signaling (PD98059) was ineffective. Western blot analyses showed that estrogen and IGF-1 treatments increased PI3K/Akt activation in the SN; however, MAPK/ERK activation was decreased in the SN. Indeed, continuous infusions of inhibitors blocked phosphorylation of PI3K/Akt and MAPK/ERK. These findings indicate that estrogen and IGF-1-mediated SNpc DA neuronal protection is dependent on PI3K/Akt signaling, but not on the MAPK/ERK pathway.

  3. Transcriptional activation of peroxisome proliferator-activated receptor-{gamma} requires activation of both protein kinase A and Akt during adipocyte differentiation

    SciTech Connect

    Kim, Sang-pil; Ha, Jung Min; Yun, Sung Ji; Kim, Eun Kyoung; Chung, Sung Woon; Hong, Ki Whan; Kim, Chi Dae; Bae, Sun Sik

    2010-08-13

    Research highlights: {yields} Elevated cAMP activates both PKA and Epac. {yields} PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. {yields} Akt modulates PPAR-{gamma} transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-{gamma} is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-{gamma}. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-{gamma} was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-{gamma} transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-{gamma} transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-{gamma}, suggesting post-translational activation of PPAR-{gamma} might be critical step for adipogenic gene expression.

  4. K6PC-5, a novel sphingosine kinase 1 (SphK1) activator, alleviates dexamethasone-induced damages to osteoblasts through activating SphK1-Akt signaling.

    PubMed

    Ji, Feng; Mao, Li; Liu, Yuanyuan; Cao, Xiaojian; Xie, Yue; Wang, Shouguo; Fei, Haodong

    2015-03-13

    Long-term glucocorticoid usage is a common cause of non-traumatic femoral head osteonecrosis. Glucocorticoids (i.e. dexamethasone (Dex)) could directly induce damages to osteoblasts. In the current study, we investigated the potential activity of K6PC-5 [N-(1,3-dihydroxyisopropyl)-2-hexyl-3-oxo-decanamide], a novel sphingosine kinase 1 (SphK1) activator, against this process. Our data revealed that both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts were responsible to K6PC-5. K6PC-5 activated SphK1, increased sphingosine-1-phosphate (S1P) production and induced Akt phosphorylation in cultured osteoblasts. Functionally, K6PC-5 protected osteoblasts from Dex-induced apoptosis and necrosis. Such signaling and functional effects by K6PC-5 were prevented by the SphK1 inhibitor N,N-dimethylsphingosine (DMS), and by SphK1-siRNAs. On the other hand, exogenously-added S1P activated Akt and reduced Dex-induced osteoblast damages. LY294002 and MK-2206, two established Akt inhibitors, alleviated K6PC-5- or S1P-mediated osteoblast protection against Dex. Together, our results suggest that K6PC-5 alleviates Dex-induced osteoblast injuries through activating SphK1-Akt signaling. K6PC-5 might be further investigated in animal or clinical studies for its anti-glucocorticoids-associated osteonecrosis potential.

  5. Context-dependent antagonism between Akt inhibitors and topoisomerase poisons.

    PubMed

    Gálvez-Peralta, Marina; Flatten, Karen S; Loegering, David A; Peterson, Kevin L; Schneider, Paula A; Erlichman, Charles; Kaufmann, Scott H

    2014-05-01

    Signaling through the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, which is aberrantly activated in >50% of carcinomas, inhibits apoptosis and contributes to drug resistance. Accordingly, several Akt inhibitors are currently undergoing preclinical or early clinical testing. To examine the effect of Akt inhibition on the activity of multiple widely used classes of antineoplastic agents, human cancer cell lines were treated with the Akt inhibitor A-443654 [(2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan-2-amine; ATP-competitive] or MK-2206 (8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one;dihydrochloride; allosteric inhibitor) or with small interfering RNA (siRNA) targeting phosphoinositide-dependent kinase 1 (PDK1) along with cisplatin, melphalan, camptothecin, or etoposide and assayed for colony formation. Surprisingly different results were observed when Akt inhibitors were combined with different drugs. Synergistic effects were observed in multiple cell lines independent of PI3K pathway status when A-443654 or MK-2206 was combined with the DNA cross-linking agents cisplatin or melphalan. In contrast, effects of the Akt inhibitors in combination with camptothecin or etoposide were more complicated. In HCT116 and DLD1 cells, which harbor activating PI3KCA mutations, A-443654 over a broad concentration range enhanced the effects of camptothecin or etoposide. In contrast, in cell lines lacking activating PI3KCA mutations, partial inhibition of Akt signaling synergized with camptothecin or etoposide, but higher A-443654 or MK-2206 concentrations (>80% inhibition of Akt signaling) or PDK1 siRNA antagonized the topoisomerase poisons by diminishing DNA synthesis, a process that contributes to effective DNA damage and killing by these agents. These results indicate that the effects of combining inhibitors of the PI3K/Akt pathway with certain classes of chemotherapeutic agents might be more

  6. An integrative genomic and proteomic analysis of PIK3CA, PTEN and AKT mutations in breast cancer

    SciTech Connect

    Stemke-Hale, Katherine; Gonzalez-Angulo, Ana Maria; Lluch, Ana; Neve, Richard M.; Kuo, Wen-Lin; Davies, Michael; Carey, Mark; Hu, Zhi; Guan, Yinghui; Sahin, Aysegul; Symmans, W. Fraser; Pusztai, Lajos; Nolden, Laura K.; Horlings, Hugo; Berns, Katrien; Hung, Mien-Chie; van de Vijver, Marc J.; Valero, Vicente; Gray, Joe W.; Bernards, Rene; Mills, Gordon B.; Hennessy, Bryan T.

    2008-05-06

    Phosphatidylinositol-3-kinase (PI3K)/AKT pathway aberrations are common in cancer. By applying mass spectroscopy-based sequencing and reverse phase protein arrays to 547 human breast cancers and 41 cell lines, we determined the subtype specificity and signaling effects of PIK3CA, AKT and PTEN mutations, and the effects of PIK3CA mutations on responsiveness to PI3K inhibition in-vitro and on outcome after adjuvant tamoxifen. PIK3CA mutations were more common in hormone receptor positive (33.8%) and HER2-positive (24.6%) than in basal-like tumors (8.3%). AKT1 (1.4%) and PTEN (2.3%) mutations were restricted to hormone receptor-positive cancers with PTEN protein levels also being significantly lower in hormone receptor-positive cancers. Unlike AKT1 mutations, PIK3CA (39%) and PTEN (20%) mutations were more common in cell lines than tumors, suggesting a selection for these but not AKT1 mutations during adaptation to culture. PIK3CA mutations did not have a significant impact on outcome in 166 hormone receptor-positive breast cancer patients after adjuvant tamoxifen. PIK3CA mutations, in comparison with PTEN loss and AKT1 mutations, were associated with significantly less and indeed inconsistent activation of AKT and of downstream PI3K/AKT signaling in tumors and cell lines, and PTEN loss and PIK3CA mutation were frequently concordant, suggesting different contributions to pathophysiology. PTEN loss but not PIK3CA mutations rendered cells sensitive to growth inhibition by the PI3K inhibitor LY294002. Thus, PI3K pathway aberrations likely play a distinct role in the pathogenesis of different breast cancer subtypes. The specific aberration may have implications for the selection of PI3K-targeted therapies in hormone receptor-positive breast cancer.

  7. TGF-β effects on prostate cancer cell migration and invasion are mediated by PGE2 through activation of PI3K/AKT/mTOR pathway.

    PubMed

    Vo, Baohan T; Morton, Derrick; Komaragiri, Shravan; Millena, Ana C; Leath, Chelesie; Khan, Shafiq A

    2013-05-01

    TGF-β plays an important role in the progression of prostate cancer. It exhibits both tumor suppressor and tumor-promoting activities. Correlations between cyclooxygenase (COX)-2 overexpression and enhanced production of prostaglandin (PG)E2 have been implicated in cancer progression; however, there are no studies indicating that TGF-β effects in prostate cancer cells involve PGE2 synthesis. In this study, we investigated TGF-β regulation of COX-1 and COX-2 expression in prostate cancer cells and whether the effects of TGF-β on cell proliferation and migration are mediated by PGE2. COX-1 protein was ubiquitously expressed in prostate cells; however, COX-2 protein levels were detected only in prostate cancer cells. TGF-β treatment increased COX-2 protein levels and PGE2 secretion in PC3 cells. Exogenous PGE2 and PGF2α had no effects on cell proliferation in LNCaP, DU145, and PC3 cells whereas PGE2 and TGF-β induced migration and invasive behavior in PC3 cells. Only EP2 and EP4 receptors were detected at mRNA levels in prostate cells. The EP4-targeting small interfering RNA inhibited PGE2 and TGF-β-induced migration of PC3 cells. TGF-β and PGE2 induce activation of PI3K/AKT/mammalian target of rapamycin pathway as indicated by increased AKT, p70S6K, and S6 phosphorylation. Rapamycin completely blocked the effects of TGF-β and PGE2 on phosphorylation of p70S6K and S6 but not on AKT phosphorylation. PGE2 and TGF-β induced phosphorylation of AKT, which was blocked by antagonists of PGE2 (EP4) receptors (L161982, AH23848) and PI3K inhibitor (LY294002) in PC3 cells. Pretreatment with L161982 or AH23848 blocked the stimulatory effects of PGE2 and TGF-β on cell migration, whereas LY294002 or rapamycin completely eliminated PGE2, TGF-β, and epidermal growth factor-induced migration in PC3 cells. We conclude that TGF-β increases COX-2 levels and PGE2 secretion in prostate cancer cells which, in turn, mediate TGF-β effects on cell migration and invasion through

  8. Molecular neuro-oncology and development of targeted therapeutic strategies for brain tumors. Part 2: PI3K/Akt/PTEN, mTOR, SHH/PTCH and angiogenesis.

    PubMed

    Newton, Herbert B

    2004-02-01

    Brain tumors are a diverse group of malignancies that remain refractory to conventional treatment approaches. Molecular neuro-oncology has now begun to clarify the transformed phenotype of brain tumors and identify oncogenic pathways that might be amenable to targeted therapy. Activity of the phosphoinositide 3; kinase (PI3K)/Akt pathway is often upregulated in brain tumors due to excessive stimulation by growth factor receptors and Ras. Loss of function of the tumor suppressor gene PTEN also frequently contributes to upregulation of PI3K/Akt. Several compounds, such as wortmannin and LY-294002, can target PI3K and inhibit activity of this pathway. The mammalian target of rapamycin (mTOR) is an important regulator of cell growth and metabolism and is often upregulated by Akt. Clinical trials of CCI-779, an inhibitor of mTOR, are ongoing in recurrent malignant glioma patients. The sonic hedgehog/PTCH pathway is involved in the tumorigenesis of some familial and sporadic medulloblastomas. This pathway can be targeted by cyclopamine, which is under evaluation in preclinical studies. Angiogenesis is a critical process for development and progression of brain tumors. Targeted approaches to inhibit angiogenesis include monoclonal antibodies, receptor tyrosine kinase inhibitors, antisense oligonucleotides and gene therapy. Clinical trials are ongoing for numerous angiogenesis inhibitors, including thalidomide, CC-5103 and PTK 787/ZK 222584. Further development of targeted therapies and evaluation of these new agents in clinical trials will be needed to improve survival and quality of life of patients with brain tumors.

  9. PI3K/Akt Pathway Contributes to Neurovascular Unit Protection of Xiao-Xu-Ming Decoction against Focal Cerebral Ischemia and Reperfusion Injury in Rats

    PubMed Central

    Xiang, Jun; Zhang, Yong; Wang, Guo-Hua; Bao, Jie; Li, Wen-Wei; Zhang, Wen; Xu, Li-Li; Cai, Ding-Fang

    2013-01-01

    In the present study, we used a focal cerebral ischemia and reperfusion rat model to investigate the protective effects of Xiao-Xu-Ming decoction (XXMD) on neurovascular unit and to examine the role of PI3K (phosphatidylinositol 3-kinase)/Akt pathway in this protection. The cerebral ischemia was induced by 90 min of middle cerebral artery occlusion. Cerebral infarct area was measured by tetrazolium staining, and neurological function was observed at 24 h after reperfusion. DNA fragmentation assay, combined with immunofluorescence, was performed to evaluate apoptosis of neuron, astrocyte, and vascular endothelial cell which constitute neurovascular unit. The expression levels of proteins involved in PI3K/Akt pathway were detected by Western blot. The results showed that XXMD improved neurological function, decreased cerebral infarct area and neuronal damage, and attenuated cellular apoptosis in neurovascular unit, while these effects were abolished by inhibition of PI3K/Akt with LY294002. We also found that XXMD upregulated p-PDKl, p-Akt, and p-GSK3β expression levels, which were partly reversed by LY294002. In addition, the increases of p-PTEN and p-c-Raf expression levels on which LY294002 had no effect were also observed in response to XXMD treatment. The data indicated the protective effects of XXMD on neurovascular unit partly through the activation of PI3K/Akt pathway. PMID:23781261

  10. Effects of nicorandil in neuroprotective activation of PI3K/AKT pathways in a cellular model of Alzheimer's disease.

    PubMed

    Kong, Jingjing; Ren, Guiru; Jia, Ning; Wang, Yanfu; Zhang, Hua; Zhang, Wei; Chen, Bingkun; Cao, Yunpeng

    2013-01-01

    Nicorandil, an ATP-sensitive potassium (KATP) channel opener, is known to have protective effects on ischemic injury in heart and brain. One of the most important protective mechanisms is the anti-apoptotic effect on cardiomyocytes and neurons. This study explored the anti-apoptotic effect of nicorandil against neurotoxicity in SH-SY5Y cells overexpressing the Swedish mutant APP (APPsw) and the possible mechanisms involved. We used SH-SY5Y cells transiently transfected with APPsw as a cellular model of Alzheimer's disease. Cells were treated with nicorandil (0.1, 0.5, 1 mM) for 24 h with and without glibenclamide (10 μM), a KATP channel inhibitor. The cells were then collected for MTT, apoptosis assay, and Western blot. In addition, we also investigated the potential involvement of the PI3K/Akt pathway in nicorandil-mediated neuroprotection of APPsw cells. Our results showed that nicorandil dose-dependently increased cell viability and reduced the rate of apoptosis as measured by MTT assay and annexin V/PI staining. Western blot showed that nicorandil could upregulate Bcl-2 levels and downregulate Bax and caspase-3 expression. Further studies showed that nicorandil increased the levels of phospho-Akt and upregulated element-binding protein activity by PI3K activation. Applying a PI3K inhibitor, LY294002 blocked the protection. All these findings suggest that nicorandil might be a potential treatment option for Alzheimer's disease.

  11. Role of Akt signaling in resistance to DNA-targeted therapy

    PubMed Central

    Avan, Abolfazl; Narayan, Ravi; Giovannetti, Elisa; Peters, Godefridus J

    2016-01-01

    The Akt signal transduction pathway controls most hallmarks of cancer. Activation of the Akt cascade promotes a malignant phenotype and is also widely implicated in drug resistance. Therefore, the modulation of Akt activity is regarded as an attractive strategy to enhance the efficacy of cancer therapy and irradiation. This pathway consists of phosphatidylinositol 3 kinase (PI3K), mammalian target of rapamycin, and the transforming serine-threonine kinase Akt protein isoforms, also known as protein kinase B. DNA-targeted agents, such as platinum agents, taxanes, and antimetabolites, as well as radiation have had a significant impact on cancer treatment by affecting DNA replication, which is aberrantly activated in malignancies. However, the caveat is that they may also trigger the activation of repairing mechanisms, such as upstream and downstream cascade of Akt survival pathway. Thus, each target can theoretically be inhibited in view of improving the potency of conventional treatment. Akt inhibitors, e.g., MK-2206 and perifosine, or PI3K modulators, e.g., LY294002 and Wortmannin, have shown some promising results in favor of sensitizing the cancer cells to the therapy in vitro and in vivo, which have provided the rationale for incorporation of these novel agents into multimodality treatment of different malignancies. Nevertheless, despite the acceptable safety profile of some of these agents in the clinical studies, with regard to the efficacy, the results are still too preliminary. Hence, we need to wait for the upcoming data from the ongoing trials before utilizing them into the standard care of cancer patients. PMID:27777878

  12. Forskolin increases angiogenesis through the coordinated cross-talk of PKA-dependent VEGF expression and Epac-mediated PI3K/Akt/eNOS signaling.

    PubMed

    Namkoong, Seung; Kim, Chun-Ki; Cho, Young-Lai; Kim, Ji-Hee; Lee, Hansoo; Ha, Kwon-Soo; Choe, Jongseon; Kim, Pyeung-Hyeun; Won, Moo-Ho; Kwon, Young-Geun; Shim, Eun Bo; Kim, Young-Myeong

    2009-06-01

    Forskolin, a potent activator of adenylyl cyclases, has been implicated in modulating angiogenesis, but the underlying mechanism has not been clearly elucidated. We investigated the signal mechanism by which forskolin regulates angiogenesis. Forskolin stimulated angiogenesis of human endothelial cells and in vivo neovascularization, which was accompanied by phosphorylation of CREB, ERK, Akt, and endothelial nitric oxide synthase (eNOS) as well as NO production and VEGF expression. Forskolin-induced CREB phosphorylation, VEGF promoter activity, and VEGF expression were blocked by the PKA inhibitor PKI.Moreover, phosphorylation of ERK by forskolin was inhibited by the MEK inhibitor PD98059, but not PKI. The forskolin-induced Akt/eNOS/NO pathway was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, but not significantly suppressed by PKI. These inhibitors and a NOS inhibitor partially inhibited forskolin-induced angiogenesis. The exchange protein directly activated by cAMP (Epac) activator, 8CPT-2Me-cAMP, promoted the Akt/eNOS/NO pathway and ERK phosphorylation,but did not induce CREB phosphorylation and VEGF expression. The angiogenic effect of the Epac activator was diminished by the inhibition of PI3K and MEK, but not by the PKA inhibitor. Small interfering RNA-mediated knockdown of Epac1 suppressed forskolin-induced angiogenesis and phosphorylation of ERK, Akt, and eNOS, but not CREB phosphorylation and VEGF expression. These results suggest that forskolin stimulates angiogenesis through coordinated cross-talk between two distinct pathways, PKA-dependent VEGF expression and Epac-dependent ERKactivation and PI3K/Akt/eNOS/NO signaling.

  13. Andrographolide inhibits growth of human T-cell acute lymphoblastic leukemia Jurkat cells by downregulation of PI3K/AKT and upregulation of p38 MAPK pathways.

    PubMed

    Yang, Tingfang; Yao, Shuluan; Zhang, Xianfeng; Guo, Yan

    2016-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) as a prevalent hematologic malignancy is one of the most common malignant tumors worldwide in children. Andrographolide (Andro), the major active component from Andrographis paniculata, has been shown to possess antitumor activities in several types of cancer cells. However, whether Andro would inhibit T-ALL cell growth remains unclear. In this study, we investigated the cytotoxic effect of Andro on human T-ALL Jurkat cells and explored the mechanisms of cell death. Cell apoptosis was assayed by flow cytometry, and the signaling transduction for Andro was analyzed by Western blotting. The results indicated 10 μg/mL Andro could significantly induce Jurkat cells' apoptosis, depending on the inhibition of PI3K/AKT pathway. Moreover, Andro-induced apoptosis is enhanced by AKT-selective inhibitor LY294002. ERK- or JNK-selective inhibitors PD98059 and SP600125 had no effect on Andro-induced apoptosis. In addition, p38 inhibitor SB203580 could reverse Andro-induced apoptosis in Jurkat cells. We also found that the protein expression of p-p53 and p-p38 were increased after Andro treatments. The result of an in vivo study also demonstrated Andro's dose-dependent inhibition in subcutaneous Jurkat xenografts. In conclusion, our findings explained a novel mechanism of drug action by Andro in Jurkat cells and suggested that Andro might be developed into a new candidate therapy for T-ALL patients in the coming days.

  14. SDF-1/CXCR4 axis induces human dental pulp stem cell migration through FAK/PI3K/Akt and GSK3β/β-catenin pathways

    PubMed Central

    Li, Mingwei; Sun, Xuefei; Ma, Liang; Jin, Lu; Zhang, Wenfei; Xiao, Min; Yu, Qing

    2017-01-01

    SDF-1 (stromal cell derived factor-1) has been found to be widely expressed during dental pulp inflammation, while hDPSCs (human dental pulp stem cells) contribute to the repair of dental pulp. We showed that the migration of hDPSCs was induced by SDF-1 in a concentration-dependent manner and could be inhibited with siCXCR4 (C-X-C chemokine receptor type 4) and siCDC42 (cell division control protein 42), as well as drug inhibitors such as AMD3100 (antagonist of CXCR4), LY294002 (inhibitor of PI3K) and PF573228 (inhibitor of FAK). It was also confirmed that SDF-1 regulated the phosphorylation of FAK (focal adhesion kinases) on cell membranes and the translocation of β-catenin into the cell nucleus. Subsequent experiments confirmed that the expression of CXCR4 and β-catenin and the phosphorylation of FAK, PI3K (phosphoinositide 3-kinase), Akt and GSK3β (glycogen synthase kinase-3β) were altered significantly with SDF-1 stimulation. FAK and PI3K worked in coordination during this process. Our findings provide direct evidence that SDF-1/CXCR4 axis induces hDPSCs migration through FAK/PI3K/Akt and GSK3β/β-catenin pathways, implicating a novel mechanism of dental pulp repair and a possible application of SDF-1 for the treatment of pulpitis. PMID:28067275

  15. SDF-1/CXCR4 axis induces human dental pulp stem cell migration through FAK/PI3K/Akt and GSK3β/β-catenin pathways.

    PubMed

    Li, Mingwei; Sun, Xuefei; Ma, Liang; Jin, Lu; Zhang, Wenfei; Xiao, Min; Yu, Qing

    2017-01-09

    SDF-1 (stromal cell derived factor-1) has been found to be widely expressed during dental pulp inflammation, while hDPSCs (human dental pulp stem cells) contribute to the repair of dental pulp. We showed that the migration of hDPSCs was induced by SDF-1 in a concentration-dependent manner and could be inhibited with siCXCR4 (C-X-C chemokine receptor type 4) and siCDC42 (cell division control protein 42), as well as drug inhibitors such as AMD3100 (antagonist of CXCR4), LY294002 (inhibitor of PI3K) and PF573228 (inhibitor of FAK). It was also confirmed that SDF-1 regulated the phosphorylation of FAK (focal adhesion kinases) on cell membranes and the translocation of β-catenin into the cell nucleus. Subsequent experiments confirmed that the expression of CXCR4 and β-catenin and the phosphorylation of FAK, PI3K (phosphoinositide 3-kinase), Akt and GSK3β (glycogen synthase kinase-3β) were altered significantly with SDF-1 stimulation. FAK and PI3K worked in coordination during this process. Our findings provide direct evidence that SDF-1/CXCR4 axis induces hDPSCs migration through FAK/PI3K/Akt and GSK3β/β-catenin pathways, implicating a novel mechanism of dental pulp repair and a possible application of SDF-1 for the treatment of pulpitis.

  16. Activation of sonic hedgehog signaling enhances cell migration and invasion by induction of matrix metalloproteinase-2 and -9 via the phosphoinositide-3 kinase/AKT signaling pathway in glioblastoma.

    PubMed

    Chang, Liang; Zhao, Dan; Liu, Hui-Bin; Wang, Qiu-Shi; Zhang, Ping; Li, Chen-Long; Du, Wen-Zhong; Wang, Hong-Jun; Liu, Xing; Zhang, Zhi-Ren; Jiang, Chuan-Lu

    2015-11-01

    Aberrant hedgehog signaling contributes to the development of various malignancies, including glioblastoma (GBM). However, the potential mechanism of hedgehog signaling in GBM migration and invasion has remained to be elucidated. The present study showed that enhanced hedgehog signaling by recombinant human sonic hedgehog N‑terminal peptide (rhSHH) promoted the adhesion, invasion and migration of GBM cells, accompanied by increases in mRNA and protein levels of matrix metalloproteinase‑2 (MMP‑2) and MMP‑9. However, inhibition of hedgehog signaling with cyclopamine suppressed the adhesion, invasion and migration of GBM cells, accompanied by decreases in mRNA and protein levels of MMP‑2 and ‑9. Furthermore, it was found that MMP‑2- and MMP‑9-neutralizing antibodies or GAM6001 reversed the inductive effects of rhSHH on cell migration and invasion. In addition, enhanced hedgehog signaling by rhSHH increased AKT phosphorylation, whereas blockade of hedgehog signaling decreased AKT phosphorylations. Further experiments showed that LY294002, an inhibitor of phosphoinositide-3 kinase (PI3K), decreased rhSHH‑induced upregulation of MMP‑2 and ‑9. Finally, the protein expression of glioblastoma-associated oncogene 1 was positively correlated with levels of phosphorylated AKT as well as protein expressions of MMP‑2 and ‑9 in GBM tissue samples. In conclusion, the present study indicated that the hedgehog pathway regulates GBM-cell migration and invasion by increasing MMP-2 and MMP-9 production via the PI3K/AKT pathway.

  17. Involvement of PI 3 kinase/Akt-dependent Bad phosphorylation in Toxoplasma gondii-mediated inhibition of host cell apoptosis.

    PubMed

    Quan, Juan-Hua; Cha, Guang-Ho; Zhou, Wei; Chu, Jia-Qi; Nishikawa, Yoshifumi; Lee, Young-Ha

    2013-04-01

    Toxoplasma gondii-infected cells are resistant to various apoptotic stimuli, however, the role of the pro-apoptotic BH3-only Bad protein in T. gondii-imposed inhibition of host cell apoptosis in connection with the phosphoinositide 3-kinase (PI3K)-PKB/Akt pathway was not well delineated. Here, we investigated the signaling patterns of Bad, Bax and PKB/Akt in T. gondii-infected and uninfected THP-1 cells treated with staurosporine (STS) or PI3K inhibitors. STS treatment, without T. gondii infection, reduced the viability of THP-1 cells in proportion to STS concentration and triggered many cellular death events such as caspase-3 and -9 activation, Bax translocation, cytochrome c release from host cell mitochondria into cytosol, and PARP cleavage in the host cell. However, T. gondii infection eliminated the STS-triggered mitochondrial apoptotic events described above. Additionally, T. gondii infection in vitro and in vivo induced the phosphorylation of PKB/Akt and Bad in a parasite-load-dependent manner which subsequently inhibited Bax translocation. The PI3K inhibitors, LY294002 and Wortmannin, both blocked parasite-induced phosphorylation of PKB/Akt and Bad. Furthermore, THP-1 cells pretreated with these PI3K inhibitors showed reduced phosphorylation of Bad in a dose-dependent manner and subsequently failed to inhibit the Bax translocation, also these cells also failed to overcome the T. gondii-imposed inhibition of host cell apoptosis. These data demonstrate that the PI3K-PKB/Akt pathway may be one of the major route for T. gondii in the prevention of host cell apoptosis and T. gondii phosphorylates the pro-apoptotic Bad protein to prevent apoptosis.

  18. Activation of the PI3K/Akt pathway by oxidative stress mediates high glucose-induced increase of adipogenic differentiation in primary rat osteoblasts.

    PubMed

    Zhang, Yu; Yang, Jian-Hong

    2013-11-01

    Diabetes mellitus is associated with increased risk of osteopenia and bone fracture that may be related to hyperglycemia. However, the mechanisms accounting for diabetic bone disorder are unclear. Here, we showed that high glucose significantly promoted the production of reactive oxygen species (ROS) in rat primary osteoblasts. Most importantly, we reported for the first time that ROS induced by high glucose increased alkaline phosphatase activity, inhibited type I collagen (collagen I) protein level and cell mineralization, as well as gene expression of osteogenic markers including runt-related transcription factor 2 (Runx2), collagen I, and osteocalcin, but promoted lipid droplet formation and gene expression of adipogenic markers including peroxisome proliferator-activated receptor gamma, adipocyte fatty acid binding protein (aP2), and adipsin, which were restored by pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger. Moreover, high glucose-induced oxidative stress activated PI3K/Akt pathway to inhibited osteogenic differentiation but stimulated adipogenic differentiation. In contrast, NAC and a PI3K inhibitor, LY-294002, reversed the down-regulation of osteogenic markers and the up-regulation of adipogenic markers as well as the activation of Akt under high glucose. These results indicated that oxidative stress played a key role in high glucose-induced increase of adipogenic differentiation, which contributed to the inhibition of osteogenic differentiation. This process was mediated by PI3K/Akt pathway in rat primary osteoblasts. Hence, suppression of oxidative stress could be a potential therapeutic approach for diabetic osteopenia.

  19. FBI-1 Is Overexpressed in Gestational Trophoblastic Disease and Promotes Tumor Growth and Cell Aggressiveness of Choriocarcinoma via PI3K/Akt Signaling.

    PubMed

    Mak, Victor C Y; Wong, Oscar G W; Siu, Michelle K Y; Wong, Esther S Y; Ng, Wai-Yan; Wong, Richard W C; Chan, Ka-Kui; Ngan, Hextan Y S; Cheung, Annie N Y

    2015-07-01

    Human placental trophoblasts can be considered pseudomalignant, with tightly controlled proliferation, apoptosis, and invasiveness. Gestational trophoblastic disease (GTD) represents a family of heterogeneous trophoblastic lesions with aberrant apoptotic and proliferative activities and dysregulation of cell signaling pathways. We characterize the oncogenic effects of factor that binds to the inducer of short transcripts of HIV-1 [FBI-1, alias POZ and Krüppel erythroid myeloid ontogenic factor (POKEMON)/ZBTB7A] in GTD and its role in promoting cell aggressiveness in vitro and tumor growth in vivo. IHC studies showed increased nuclear expression of FBI-1, including hydatidiform moles, choriocarcinoma (CCA), and placental site trophoblastic tumor, in GTD. In JAR and JEG-3 CCA cells, ectopic FBI-1 expression opposed apoptosis through repression of proapoptotic genes (eg, BAK1, FAS, and CASP8). FBI-1 overexpression also promoted Akt activation, as indicated by Akt-pS473 phosphorylation. FBI-1 overexpression promoted mobility and invasiveness of JEG-3 and JAR, but not in the presence of the phosphoinositide 3-kinase inhibitor LY294002. These findings suggest that FBI-1 could promote cell migration and invasion via phosphoinositide 3-kinase/Akt signaling. In vivo, nude mice injected with CCA cells with stable FBI-1 knockdown demonstrated reduced tumor growth compared with that in control groups. These findings suggest that FBI-1 is clinically associated with the progression of, and may be a therapeutic target in, GTD, owing to its diverse oncogenic effects on dysregulated trophoblasts.

  20. Salidroside inhibits oxygen glucose deprivation (OGD)/re-oxygenation-induced H9c2 cell necrosis through activating of Akt-Nrf2 signaling.

    PubMed

    Zheng, Koulong; Sheng, Zhenqiang; Li, Yefei; Lu, Huihe

    2014-08-15

    Oxygen glucose deprivation (OGD)/re-oxygenation has been applied to cultured cardiomyocytes to create a cellular model of ischemic heart damage. In the current study, we explored the potential role of salidroside against OGD/re-oxygenation-induced damage in H9c2 cardiomyocytes, and studied the underlying mechanisms. We found that OGD/re-oxygenation primarily induced necrosis in H9c2 cells, which was inhibited by salidroside. Salidroside suppressed OGD/re-oxygenation-induced reactive oxygen species (ROS) production, p53 mitochondrial translocation and cyclophilin D (Cyp-D) association as well as mitochondrial membrane potential (MMP) decrease in H9c2 cells. Meanwhile, salidroside activated Akt and promoted transcription of NF-E2-related factor 2 (Nrf2)-regulated genes (heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO-1)). Significantly, Nrf2 shRNA knockdown or Akt inhibitors (LY 294002 and wortmannin) not only prevented salidroside-induced HO-1/NQO-1 transcription, but also alleviated salidroside-mediated cytoprotective effect against OGD/re-oxygenation in H9c2 cells. These observations suggest that salidroside activates Nrf2-regulated anti-oxidant signaling, and protects against OGD/re-oxygenation-induced H9c2 cell necrosis via activation of Akt signaling.

  1. CXCL13-CXCR5 axis promotes the growth and invasion of colon cancer cells via PI3K/AKT pathway.

    PubMed

    Zhu, Zhenyu; Zhang, Xukui; Guo, Hongliang; Fu, Ling; Pan, Ganlin; Sun, Yinggang

    2015-02-01

    CXCL13, an inflammatory factor in the microenvironment, plays a vital role in the progression of inflammatory diseases and tumors. CXCL13 and its receptor CXCR5 have been reported to be associated with poor prognosis of advanced colon cancer. However, the molecular mechanisms of CXCL13-CXCR5 axis in colon cancer remain elusive. The aim of this study was to investigate the role of CXCR5-CXCL13 axis in the growth and invasion of colon cancer cells. Our results showed that CXCL13 promoted the growth, migration, and matrigel invasion of colon cancer cells. Furthermore, CXCL13 increased the expression and secretion of MMP-13, and stimulated the activation of PI3K/AKT pathway. After knockdown of CXCR5 by siRNA, the biological functions of colon cancer cells regulated by CXCL13 were significantly inhibited. In addition, inhibition of PI3K/AKT pathway by specific inhibitor LY294002 suppressed the CXCL13-mediated growth, migration, and invasion of colon cancer cells. Together, our findings suggest that CXCL13-CXCR5 axis promotes the growth, migration, and invasion of colon cancer cells, probably via PI3K/AKT pathway. Thus, CXCL13 may be a useful biomarker for the detection and treatment of colon cancer.

  2. Licochalcone A induces autophagy through PI3K/Akt/mTOR inactivation and autophagy suppression enhances Licochalcone A-induced apoptosis of human cervical cancer cells.

    PubMed

    Tsai, Jen-Pi; Lee, Chien-Hsing; Ying, Tsung-Ho; Lin, Chu-Liang; Lin, Chia-Liang; Hsueh, Jung-Tsung; Hsieh, Yi-Hsien

    2015-10-06

    The use of dietary bioactive compounds in chemoprevention can potentially reverse, suppress, or even prevent cancer progression. However, the effects of licochalcone A (LicA) on apoptosis and autophagy in cervical cancer cells have not yet been clearly elucidated. In this study, LicA treatment was found to significantly induce the apoptotic and autophagic capacities of cervical cancer cells in vitro and in vivo. MTT assay results showed dose- and time-dependent cytotoxicity in four cervical cancer cell lines treated with LicA. We found that LicA induced mitochondria-dependent apoptosis in SiHa cells, with decreasing Bcl-2 expression. LicA also induced autophagy effects were examined by identifying accumulation of Atg5, Atg7, Atg12 and microtubule-associated protein 1 light chain 3 (LC3)-II. Treatment with autophagy-specific inhibitors (3-methyladenine and bafilomycin A1) enhanced LicA-induced apoptosis. In addition, we suggested the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of mTOR pathway by LicA. Furthermore, the inhibition of PI3K/Akt by LY294002/si-Akt or of mTOR by rapamycin augmented LicA-induced apoptosis and autophagy. Finally, the in vivo mice bearing a SiHa xenograft, LicA dosed at 10 or 20 mg/kg significantly inhibited tumor growth. Our findings demonstrate the chemotherapeutic potential of LicA for treatment of human cervical cancer.

  3. Transforming growth factor-β1 induces epithelial-to-mesenchymal transition in human lung cancer cells via PI3K/Akt and MEK/Erk1/2 signaling pathways.

    PubMed

    Chen, Xiao-Feng; Zhang, Hui-Jun; Wang, Hai-Bing; Zhu, Jun; Zhou, Wen-Yong; Zhang, Hui; Zhao, Ming-Chuan; Su, Jin-Mei; Gao, Wen; Zhang, Lei; Fei, Ke; Zhang, Hong-Tao; Wang, He-Yong

    2012-04-01

    Metastasis of tumor cells is associated with epithelial-to-mesenchymal transition (EMT), which is a process whereby epithelial cells lose their polarity and acquire new features of mesenchyme. EMT has been reported to be induced by transforming growth factor-β1 (TGF-β1), but its mechanism remains elusive. In this study, we performed a study to investigate whether PI3K/Akt and MAPK/Erk1/2 signaling pathways involved in EMT in the human lung cancer A549 cells. The results showed that after treated with TGF-β1 for 48 h, A549 cells displayed more fibroblast-like shape, lost epithelial marker E-cadherin and increased mesenchymal markers Vimentin and Fibronectin. Moreover, TGF-β1-induced EMT after 48 h was accompanied by increased of cell migration and change of Akt and Erk1/2 phosphorylation. In addition, EMT was reversed by PI3K inhibitor LY294002 and MEK1/2 inhibitor U0126, which suggested that A549 cells under stimulation of TGF-β1 undergo a switch into mesenchymal cells and PI3K/Akt and MAPK/Erk1/2 signaling pathways serve to regulate TGF-β1-induced EMT of A549 cells.

  4. {delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    SciTech Connect

    Heiss, Anika; Ammer, Hermann; Eisinger, Daniela A.

    2009-07-15

    {delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  5. PI3K/Akt pathway restricts epithelial adhesion of Dr+ Escherichia coli by down-regulating the expression of Decay Accelerating Factor (DAF)

    PubMed Central

    Banadakoppa, Manu; Goluszko, Pawel; Liebenthal, Daniel; Nowicki, Bogdan J.; Nowicki, Stella; Yallampalli, Chandra

    2014-01-01

    The urogenital microbial infection in pregnancy is an important cause of maternal and neonatal morbidity and mortality. Uropathogenic Escherichia coli strains which express Dr fimbriae (Dr+) are associated with unique gestational virulence and they utilize cell surface decay accelerating factor (DAF or CD55) as one of the cellular receptor before invading the epithelial cells. Previous studies in our laboratory established that nitric oxide reduces the rate of E. coli invasion by delocalizing the DAF protein from cell surface lipid rafts and down-regulating its expression. The phosphoinositide 3-kinase/ protein kinase B (PI3K/Akt) cell signal pathway plays an important role in host-microbe interaction because many bacteria including E. coli activate this pathway in order to establish infection. In the present study we showed that the PI3K/Akt pathway negatively regulates the expression of DAF on the epithelial cell surface and thus inhibits the adhesion of Dr+ E. coli to epithelial cells. Initially, using two human cell lines Ishikawa and HeLa which differ in constitutive activity of PI3K/Akt we showed that DAF levels were associated with the PI3K/Akt pathway. We then showed that the DAF gene expression was up-regulated and the Dr+ E. coli adhesion increased after the suppression of PI3K/Akt pathway in Ishikawa cells using inhibitor LY-294002, and a plasmid which allowed the expression of PI3K/Akt regulatory protein PTEN. The down-regulation of PTEN protein using PTEN-specific siRNA activated the PI3K/Akt pathway, down-regulated the DAF and decreased the adhesion of Dr+ E. coli. We conclude that the PI3K/Akt pathway regulated the DAF expression in a nitric oxide independent manner. PMID:24599886

  6. Arctigenin Increases Hemeoxygenase-1 Gene Expression by Modulating PI3K/AKT Signaling Pathway in Rat Primary Astrocytes.

    PubMed

    Jeong, Yeon-Hui; Park, Jin-Sun; Kim, Dong-Hyun; Kim, Hee-Sun

    2014-11-01

    In the present study, we found that the natural compound arctigenin inhibited hydrogen peroxide-induced reactive oxygen species (ROS) production in rat primary astrocytes. Since hemeoxygenase-1 (HO-1) plays a critical role as an antioxidant defense factor in the brain, we examined the effect of arctigenin on HO-1 expression in rat primary astrocytes. We found that arctigenin increased HO-1 mRNA and protein levels. Arctigenin also increases the nuclear translocation and DNA binding of Nrf2/c-Jun to the antioxidant response element (ARE) on HO-1 promoter. In addition, arctigenin increased ARE-mediated transcriptional activities in rat primary astrocytes. Further mechanistic studies revealed that arctigenin increased the phosphorylation of AKT, a downstream substrate of phosphatidylinositol 3-kinase (PI3K). Treatment of cells with a PI3K-specific inhibitor, LY294002, suppressed the HO-1 expression, Nrf2 DNA binding and ARE-mediated transcriptional activities in arctigenin-treated astrocyte cells. The results collectively suggest that PI3K/AKT signaling pathway is at least partly involved in HO-1 expression by arctigenin via modulation of Nrf2/ARE axis in rat primary astrocytes.

  7. High-glucose and advanced glycosylation end products increased podocyte permeability via PI3-K/Akt signaling.

    PubMed

    Ha, Tae-Sun

    2010-04-01

    Regardless of the underlying disease, the proteinuric condition demonstrates ultrastructural changes in podocytes with retraction and effacement of the highly specialized interdigitating foot processes. To investigate how high-glucose (HG) and advanced glycosylation end products (AGE) induce podocyte phenotypical changes, including quantitative and distributional changes of zonula occludens (ZO)-1 protein and search for the signaling mechanisms, we cultured rat glomerular epithelial cells (GEpC) and mouse podocytes under: (1) normal glucose (5 mM, control); (2) HG (30 mM); (3) AGE-added; or (4) HG plus AGE-added conditions. HG plus AGE increased the permeability of monolayered GEpCs and induced ultrastructural separation between confluent GEpCs. ZO-1 moved to inner actin filament complexes in both AGE- and/or HG by confocal imaging. HG plus AGE-added condition also decreased ZO-1 protein amount and mRNA expression compared to normal glucose or osmotic control conditions. We could also confirm the induction of RAGE (receptor for AGE) and PI3-K/Akt signaling pathway by AGE and HG. In addition, LY294002, a PI3-K inhibitor, could prevent the quantitative and distributional changes of ZO-1 and RAGE and the increased permeability induced by HG and AGE. These findings suggest that diabetic conditions induce the podocyte ZO-1 changes via RAGE and PI3-K/Akt signaling, leading to increased permeability.

  8. IL-8 induces the epithelial-mesenchymal transition of renal cell carcinoma cells through the activation of AKT signaling

    PubMed Central

    Zhou, Nan; Lu, Fuding; Liu, Cheng; Xu, Kewei; Huang, Jian; Yu, Dexin; Bi, Liangkuan

    2016-01-01

    The epithelial-mesenchymal transition (EMT) process has increasingly been examined due to its role in the progression of human tumors. Renal cell carcinoma (RCC) is one of the most common urological tumors that results in patient mortality. Previous studies have demonstrated that the EMT process is closely associated with the metastasis of RCC; however, the underlying molecular mechanism has not been determined yet. The present study revealed that interleukin (IL)-8 was highly expressed in metastatic RCC. IL-8 could induce the EMT of an RCC cell line by enhancing N-cadherin expression and decreasing E-cadherin expression. Furthermore, IL-8 could induce AKT phosphorylation, and the phosphatidylinositol-4,5-bisphosphate 3-kinase inhibitor LY294002 could inhibit the EMT of RCC cells that was induced by IL-8. Therefore, these results suggest that IL-8 is able to promote the EMT of RCC through the activation of the AKT signal transduction pathway, and this may provide a possible molecular mechanism for RCC metastasis. PMID:27588140

  9. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers.

    PubMed

    Goda, Jayant S; Pachpor, Tejaswini; Basu, Trinanjan; Chopra, Supriya; Gota, Vikram

    2016-02-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies.

  10. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers

    PubMed Central

    Goda, Jayant S.; Pachpor, Tejaswini; Basu, Trinanjan; Chopra, Supriya; Gota, Vikram

    2016-01-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies. PMID:27121513

  11. Selaginella tamariscina (Beauv.) possesses antimetastatic effects on human osteosarcoma cells by decreasing MMP-2 and MMP-9 secretions via p38 and Akt signaling pathways.

    PubMed

    Yang, Jia-Sin; Lin, Chiao-Wen; Hsieh, Yih-Shou; Cheng, Hsin-Lin; Lue, Ko-Huang; Yang, Shun-Fa; Lu, Ko-Hsiu

    2013-09-01

    Selaginella tamariscina is a traditional medicinal plant for treatment of some advanced cancers in the Orient. However, the effect of S. tamariscina on metastasis of osteosarcoma and the underlying mechanism remain unclear. We tested the hypothesis that S. tamariscina suppresses cellular motility, invasion and migration and also investigated its signaling pathways. This study demonstrates that S. tamariscina, at a range of concentrations (from 0 to 50 μg/mL), concentration-dependently inhibited the migration/invasion capacities of three osteosarcoma cell lines without cytotoxic effects. Zymographic and western blot analyses revealed that S. tamariscina inhibited the matrix metalloproteinase (MMP)-2 and MMP-9 enzyme activity, as well as protein expression. Western blot analysis also showed that S. tamariscina inhibits phosphorylation of p38 and Akt. Furthermore, SB203580 (p38 inhibitor) and LY294002 (PI3K inhibitor) showed the similar effects as S. tamariscina in U2OS cells. In conclusion, S. tamariscina possesses an antimetastatic activity in osteosarcoma cells by down-regulating MMP-2 and MMP-9 secretions and increasing TIMP-1 and TIMP-2 expressions through p38 and Akt-dependent pathways. S. tamariscina may be a powerful candidate to develop a preventive agent for osteosarcoma metastasis.

  12. Jaceosidin, a natural flavone, promotes angiogenesis via activation of VEGFR2/FAK/PI3K/AKT/NF-κB signaling pathways in endothelial cells.

    PubMed

    Lee, Tae Hoon; Jung, Hana; Park, Keun Hyung; Bang, Myun Ho; Baek, Nam-In; Kim, Jiyoung

    2014-10-01

    Angiogenesis, the growth of new blood vessels from pre-existing vasculature, plays an important role in physiological and pathological processes such as embryonic development wound healing and revascularization of tissues after exposure to ischemia. We investigated the effects of jaceosidin, a main constituent of medicinal herbs of the genus Artemisia, on angiogenesis and signaling pathways in endothelial cells. Jaceosidin stimulated proliferation, migration and tubulogenesis of ECs as well as ex vivo sprouting from aorta rings, which are phenomena typical of angiogenesis. Jaceosidin activated vascular endothelial growth factor receptor 2 (VEGFR2, FLk-1/KDR) and angiogenic signaling molecules such as focal adhesion kinase, phosphatidylinositol 3-kinase, and its downstream target, the serine-threonine kinase AKTWe also demonstrated that jaceosidin activated the NF-κB-driven expression of a luciferase reporter gene and NF-κB binding to DNA. Jaceosidin-induced proliferation and migration of human umbilical vascular endothelial cells were strongly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002 and NF-κB inhibitor BAY11-7082, indicating that the PI3K/AKT/NF-κB signaling pathway is involved in jaceosidin-induced angiogenesis. Our results suggest that jaceosidin stimulates angiogenesis by activating the VEGFR2/FAK/PI3K/AKT/NF-κB signaling pathway and that it may be useful in developing angiogenic agents to promote the growth of collateral blood vessels in ischemic tissues.

  13. Sodium tanshinone IIA sulfonate protects rat myocardium against ischemia-reperfusion injury via activation of PI3K/Akt/FOXO3A/Bim pathway

    PubMed Central

    Zhang, Mei-qi; Zheng, Yue-liang; Chen, Huan; Tu, Jian-feng; Shen, Ye; Guo, Jun-ping; Yang, Xiang-hong; Yuan, Shu-ren; Chen, Liang-zhong; Chai, Jing-jie; Lu, Jian-hong; Zhai, Chang-lin

    2013-01-01

    Aim: To investigate the mechanisms underlying the protective effects of sodium tanshinone IIA sulfonate (STS) in an ischemia-reperfusion (I/R)-induced rat myocardial injury model. Methods: Male SD rats were iv injected with STS, STS+LY294002 or saline (NS) for 15 d. Then the hearts were subjected to 30 min of global ischemia followed by 2 h of reperfusion. Cardiac function, infarction size and area at risk were assessed. Cell apoptosis was evaluated with TUNEL staining, DNA laddering and measuring caspase-3 activity. In addition, isolated cardiomyocytes of neonatal rats were pretreated with the above drugs, then exposed to H2O2 (200 mol/L) for 1 h. Cell apoptosis was detected using flow cytometric assay. The levels of p-Akt, p-FOXO3A and Bim were examined with immunoblotting. Results: Compared to NS group, administration of STS (20 mg/kg) significantly reduced myocardial infarct size (40.28%±5.36% in STS group vs 59.52%±7.28% in NS group), and improved the myocardial function as demonstrated by the increased values of dp/dtmax, LVDP and coronary flow at different reperfusion time stages. Furthermore, STS significantly decreased the rate of apoptotic cells (15.11%±3.71% in STS group vs 38.21%±7.83% in NS group), and reduced caspase-3 activity to nearly a quarter of that in NS group. Moreover, STS significantly increased the phosphorylation of Akt and its downstream target FOXO3A, and decreased the expression of pro-apoptotic gene Bim. Co-treatment with the PI3K inhibitor LY294002 (40 mg/kg) partially countered the protective effects induced by STS treatment. In isolated cardiomyocytes, STS exerted similar protective effects as shown in the ex vivo I/R model. Conclusion: STS pretreatment reduces infarct size and improves cardiac function in an I/R-induced rat myocardial injury model via activation of Akt/FOXO3A/Bim-mediated signal pathway. PMID:24077633

  14. Titration of signalling output: insights into clinical combinations of MEK and AKT inhibitors

    PubMed Central

    Stewart, A.; Thavasu, P.; de Bono, J. S.; Banerji, U.

    2015-01-01

    Background We aimed to understand the relative contributions of inhibiting MEK and AKT on cell growth to guide combinations of these agents. Materials and methods A panel of 20 cell lines was exposed to either the MEK inhibitor, PD0325901, or AKT inhibitor, AKT 1/2 inhibitor. p-ERK and p-S6 ELISAs were used to define degrees of MEK and AKT inhibition, respectively. Growth inhibition to different degrees of MEK and AKT inhibition, either singly or in combination using 96-h sulphorhodamine assays was then studied. Results A significantly greater growth inhibition was seen in BRAFM and PIK3CAM cells upon maximal MEK (P = 0.004) and AKT inhibition (P = 0.038), respectively. KRASM and BRAF/PIK3CA/KRASWT cells were not significantly more likely to be sensitive to MEK or AKT inhibition. Significant incremental growth inhibition of the combination of MEK + AKT over either MEK or AKT inhibition alone was seen when MEK + AKT was inhibited maximally and not when sub-maximal inhibition of both MEK + AKT was used (11/20 cell lines versus 1/20 cell lines; P = 0.0012). Conclusions KRASM cells are likely to benefit from combinations of MEK and AKT inhibitors. Sub-maximally inhibiting both MEK and AKT within a combination, in a majority of instances, does not significantly increase growth inhibition compared with maximally inhibiting MEK or AKT alone and alternative phase I trial designs are needed to clinically evaluate such combinations. PMID:25908604

  15. EGF-Induced VEGF Exerts a PI3K-Dependent Positive Feedback on ERK and AKT through VEGFR2 in Hematological In Vitro Models

    PubMed Central

    Kassem, Nouhad; Badran, Bassam; El-Zein, Nabil

    2016-01-01

    EGFR and VEGFR pathways play major roles in solid tumor growth and progression, however, little is known about these pathways in haematological tumors. This study investigated the crosstalk between EGFR and VEGFR2 signaling in two hematological in vitro models: THP1, a human monocytic leukemia, and Raji, a Burkitt’s lymphoma, cell lines. Results showed that both cell lines express EGFR and VEGFR2 and responded to EGF stimulation by activating EGFR, triggering VEGF production and phosphorylating ERK, AKT, and p38 very early, with a peak of expression at 10–20min. Blocking EGFR using Tyrphostin resulted in inhibiting EGFR induced activation of ERK, AKT, and p38. In addition, EGF stimulation caused a significant and immediate increase, within 1min, in pVEGFR2 in both cell lines, which peaked at ~5–10 min after treatment. Selective inhibition of VEGFR2 by DMH4, anti-VEGFR2 antibody or siRNA diminished EGF-induced pAKT and pERK, indicating a positive feedback exerted by EGFR-induced VEGF. Similarly, the specific PI3K inhibitor LY294002, suppressed AKT and ERK phosphorylation showing that VEGF feedback is PI3K-dependent. On the other hand, phosphorylation of p38, initiated by EGFR and independent of VEGF feedback, was diminished using PLC inhibitor U73122. Moreover, measurement of intracellular [Ca2+] and ROS following VEGFR2 inhibition and EGF treatment proved that VEGFR2 is not implicated in EGF-induced Ca2+ release whereas it boosts EGF-induced ROS production. Furthermore, a significant decrease in pAKT, pERK and p-p38 was shown following the addition of the ROS inhibitor NAC. These results contribute to the understanding of the crosstalk between EGFR and VEGFR in haematological malignancies and their possible combined blockade in therapy. PMID:27806094

  16. Maximising the potential of AKT inhibitors as anti-cancer treatments.

    PubMed

    Brown, Jessica S; Banerji, Udai

    2017-04-01

    PI3K/AKT signalling is commonly disrupted in human cancers, with AKT being a central component of the pathway, influencing multiple processes that are directly involved in tumourigenesis. Targeting AKT is therefore a highly attractive anti-cancer strategy with multiple AKT inhibitors now in various stages of clinical development. In this review, we summarise the role and regulation of AKT signalling in normal cellular physiology. We highlight the mechanisms by which AKT signalling can be hyperactivated in cancers and discuss the past, present and future clinical strategies for AKT inhibition in oncology.

  17. COMP-angiopoietin 1 increases proliferation, differentiation, and migration of stem-like cells through Tie-2-mediated activation of p38 MAPK and PI3K/Akt signal transduction pathways

    SciTech Connect

    Kook, Sung-Ho; Lim, Shin-Saeng; Cho, Eui-Sic; Lee, Young-Hoon; Han, Seong-Kyu; Lee, Kyung-Yeol; Kwon, Jungkee; Hwang, Jae-Won; Bae, Cheol-Hyeon; Seo, Young-Kwon; Lee, Jeong-Chae

    2014-12-12

    Highlights: • COMP-Ang1 induces Tie-2 activation in BMMSCs, but not in primary osteoblasts. • Tie-2 knockdown inhibits COMP-Ang1-stimulated proliferation and osteoblastogenesis. • Tie-2 knockdown prevents COMP-Ang1-induced activation of PI3K/Akt and p38 MAPK. • COMP-Ang1 induces migration of cells via activation of PI3K/Akt and CXCR4 pathways. • COMP-Ang1 stimulates in vivo migration of PDLSCs into a calvarial defect site of rats. - Abstract: Recombinant COMP-Ang1, a chimera of angiopoietin-1 (Ang1) and a short coiled-coil domain of cartilage oligomeric matrix protein (COMP), is under consideration as a therapeutic agent capable of inducing the homing of cells with increased angiogenesis. However, the potentials of COMP-Ang1 to stimulate migration of mesenchymal stem cells (MSCs) and the associated mechanisms are not completely understood. We examined the potential of COMP-Ang1 on bone marrow (BM)-MSCs, human periodontal ligament stem cells (PDLSCs), and calvarial osteoblasts. COMP-Ang1 augmented Tie-2 induction at protein and mRNA levels and increased proliferation and expression of runt-related transcription factor 2 (Runx2), osterix, and CXCR4 in BMMSCs, but not in osteoblasts. The COMP-Ang1-mediated increases were inhibited by Tie-2 knockdown and by treating inhibitors of phosphoinositide 3-kinase (PI3K), LY294002, or p38 mitogen-activated protein kinase (MAPK), SB203580. Phosphorylation of p38 MAPK and Akt was prevented by siRNA-mediated silencing of Tie-2. COMP-Ang1 also induced in vitro migration of BMMSCs and PDLSCs. The induced migration was suppressed by Tie-2 knockdown and by CXCR4-specific peptide antagonist or LY294002, but not by SB203580. Furthermore, COMP-Ang1 stimulated the migration of PDLSCs into calvarial defect site of rats. Collectively, our results demonstrate that COMP-Ang1-stimulated proliferation, differentiation, and migration of progenitor cells may involve the Tie-2-mediated activation of p38 MAPK and PI3K/Akt pathways.

  18. Activation of phosphatidylinositol 3-kinase/Akt-mammalian target of Rapamycin signaling pathway in the hippocampus is essential for the acquisition of morphine-induced place preference in rats.

    PubMed

    Cui, Yue; Zhang, X Q; Cui, Y; Xin, W J; Jing, J; Liu, X G

    2010-11-24

    Hippocampus is a critical structure for the acquisition of morphine-induced conditioned place preference (CPP), which is a usual learning paradigm for assessing drug reward. However, the precise mechanisms remain largely unknown. Phosphatidylinositol 3-kinase (PI3K) and its downstream targets, including Akt, mammalian target of Rapamycin (mTOR) and 70-kDa ribosomal S6 kinase (p70S6K), are critical molecules implicated in learning and memory. Here, we tested the role of PI3K/Akt-mTOR-p70S6K signaling pathway in morphine-induced CPP in the hippocampus. Our results showed that the acquisition of morphine CPP increased phosphorylation of Akt in the hippocampal CA3, but not in the nucleus accumbens (NAc), the ventral tegmental area (VTA) or the CA1. Moreover, the phosphorylated Akt exclusively expressed in the CA3 neurons. Likewise, levels of phosphorylated mTOR and p70S6K were significantly enhanced in the CA3 following morphine CPP. The alterations of these phosphorylated proteins are positively correlated with the acquisition of morphine CPP. More importantly, microinjection of PI3K inhibitor (LY294002) or mTOR inhibitor (Rapamycin) into the CA3 prevented the acquisition of CPP and inhibited the activation of PI3K-Akt signaling pathway. In addition, pre-infusion of β-FNA (β-funaltrexamine hydrochloride), a selective irreversible μ opioid receptor antagonist, into CA3 significantly prevented the acquisition of CPP and impaired Akt phosphorylation. All these results strongly implied that the PI3K-Akt signaling pathway activated by μ opioid receptor in hippocampal CA3 plays an important role in acquisition of morphine-induced CPP.

  19. Targeting AKT1-E17K and the PI3K/AKT Pathway with an Allosteric AKT Inhibitor, ARQ 092

    PubMed Central

    Yu, Yi; Savage, Ronald E.; Eathiraj, Sudharshan; Meade, Justin; Wick, Michael J.; Hall, Terence; Abbadessa, Giovanni; Schwartz, Brian

    2015-01-01

    As a critical component in the PI3K/AKT/mTOR pathway, AKT has become an attractive target for therapeutic intervention. ARQ 092 and a next generation AKT inhibitor, ARQ 751 are selective, allosteric, pan-AKT and AKT1-E17K mutant inhibitors that potently inhibit phosphorylation of AKT. Biochemical and cellular analysis showed that ARQ 092 and ARQ 751 inhibited AKT activation not only by dephosphorylating the membrane-associated active form, but also by preventing the inactive form from localizing into plasma membrane. In endometrial PDX models harboring mutant AKT1-E17K and other tumor models with an activated AKT pathway, both compounds exhibited strong anti-tumor activity. Combination studies conducted in in vivo breast tumor models demonstrated that ARQ 092 enhanced tumor inhibition of a common chemotherapeutic agent (paclitaxel). In a large panel of diverse cancer cell lines, ARQ 092 and ARQ 751 inhibited proliferation across multiple tumor types but were most potent in leukemia, breast, endometrial, and colorectal cancer cell lines. Moreover, inhibition by ARQ 092 and ARQ 751 was more prevalent in cancer cell lines containing PIK3CA/PIK3R1 mutations compared to those with wt-PIK3CA/PIK3R1 or PTEN mutations. For both ARQ 092 and ARQ 751, PIK3CA/PIK3R1 and AKT1-E17K mutations can potentially be used as predictive biomarkers for patient selection in clinical studies. PMID:26469692

  20. GM3 suppresses anchorage-independent growth via Rho GDP dissociation inhibitor beta in melanoma B16 cells.

    PubMed

    Wang, Pu; Xu, Su; Wang, Yinan; Wu, Peixing; Zhang, Jinghai; Sato, Toshinori; Yamagata, Sadako; Yamagata, Tatsuya

    2011-08-01

    Ly-GDI, Rho GTPase dissociation inhibitor beta, was found to be expressed parallel to the GM3 level in mouse B16 cells whose GM3 contents were modified by B4galt6 sense, B4galt6 antisense cDNA, or St3galt5 siRNA transfection. Ly-GDI expression was increased on GM3 addition to these cells and decreased with D-PDMP treatment, a glucosylceramide synthesis inhibitor. Suppression of GM3 or Ly-GDI by RNAi was concomitantly associated with an increase in anchorage-independent growth in soft agar. These results clearly indicate that GM3 suppresses anchorage-independent growth through Ly-GDI. GM3 signals regulating Ly-GDI expression was inhibited by LY294002, siRNA against Akt1 and Akt2 and rapamycin, showing that GM3 signals are transduced via the PI3K/Akt/mTOR pathway. Either siRNA towards Rictor or Raptor suppressed Ly-GDI expression. The Raptor siRNA suppressed the effects of GM3 on Ly-GDI expression and Akt phosphorylation at Thr(308) , suggesting GM3 signals to be transduced to mTOR-Raptor and Akt-Thr(308) , leading to Ly-GDI stimulation. siRNA targeting Pdpk1 reduced Akt phosphorylation at Thr(308) and rendered the cells insensitive to GM3 stimulation, indicating that Akt-Thr(308) plays a critical role in the pathway. The components aligned in this pathway showed similar effects on anchorage-independent growth as GM3 and Ly-GDI. Taken together, GM3 signals are transduced in B16 cells through PI3K, Pdpk1, Akt(Thr308) and the mTOR/Raptor pathway, leading to enhanced expression of Ly-GDI mRNA, which in turn suppresses anchorage-independent growth in melanoma B16 cells.

  1. Hepatic stellate cell is activated by microRNA-181b via PTEN/Akt pathway.

    PubMed

    Zheng, Jianjian; Wu, Cunzao; Xu, Ziqiang; Xia, Peng; Dong, Peihong; Chen, Bicheng; Yu, Fujun

    2015-01-01

    Activation of hepatic stellate cells (HSCs) is an essential event in the initiation and progression of liver fibrosis. MicroRNAs have been shown to play a pivotal role in regulating HSC functions such as cell proliferation, differentiation, and apoptosis. Recently, miR-181b has been reported to promote HSCs proliferation by targeting p27. But whether alpha-smooth muscle actin (α-SMA) or collagens could be promoted by miR-181b in activated HSCs is still not clear. Therefore, the understanding of the role of miR-181b in liver fibrosis remains limited. Our results showed that miR-181b expression was increased much higher than miR-181a expression in vitro in transforming growth factor-β1-induced HSC activation as well as in vivo in carbon tetrachloride-induced rat liver fibrosis. Of note, overexpression of miR-181b significantly increased the expressions level of α-SMA and type I collagen, and further promoted HSCs proliferation. Furthermore, phosphatase and tensin homologs deleted on chromosome 10 (PTEN), a negative regulator of PI3K/Akt pathway, were confirmed as a direct target of miR-181b. We demonstrated that miR-181b could suppress PTEN expression and increase Akt phosphorylation in HSCs. Interestingly, the effects of miR-181b on the activation of HSCs were blocked down by Akt inhibitor LY294002. Our results revealed a profibrotic role of miR-181b in HSC activation and demonstrated that miR-181b could activate HSCs, at least in part, via PTEN/Akt pathway.

  2. Interleukin-6 upregulates paraoxonase 1 gene expression via an AKT/NF-κB-dependent pathway

    SciTech Connect

    Cheng, Chi-Chih; Hsueh, Chi-Mei; Chen, Chiu-Yuan; Chen, Tzu-Hsiu; Hsu, Shih-Lan

    2013-07-19

    Highlights: •IL-6 could induce PON1 gene expression. •IL-6 increased NF-κB protein expression and NF-κB-p50 and -p65 subunits nuclear translocation. •IL-6-induced PON1 up-regulation was through an AKT/NF-κB pathway. -- Abstract: The aim of this study is to investigate the relationship between paraoxonase 1 (PON1) and atherosclerosis-related inflammation. In this study, human hepatoma HepG2 cell line was used as a hepatocyte model to examine the effects of the pro-inflammatory cytokines on PON1 expression. The results showed that IL-6, but not TNF-α and IL-1β, significantly increased both the function and protein level of PON1; data from real-time RT-PCR analysis revealed that the IL-6-induced PON1 expression occurred at the transcriptional level. Increase of IκB kinase activity and IκB phosphorylation, and reduction of IκB protein level were also observed in IL-6-treated HepG2 cells compared with untreated culture. This event was accompanied by increase of NF-κB-p50 and -p65 nuclear translocation. Moreover, treatment with IL-6 augmented the DNA binding activity of NF-κB. Furthermore, pharmacological inhibition of NF-κB activation by PDTC and BAY 11-7082, markedly suppressed the IL-6-mediated PON1 expression. In addition, IL-6 increased the levels of phosphorylated protein kinase B (PKB, AKT). An AKT inhibitor LY294002 effectively suppressed IKK/IκB/NF-κB signaling and PON1 gene expression induced by IL-6. Our findings demonstrate that IL-6 upregulates PON1 gene expression through an AKT/NF-κB signaling axis in human hepatocyte-derived HepG2 cell line.

  3. A novel compound DSC suppresses lipopolysaccharide-induced inflammatory responses by inhibition of Akt/NF-κB signalling in macrophages.

    PubMed

    Liu, Xin-Hua; Pan, Li-Long; Jia, Yao-Ling; Wu, Dan; Xiong, Qing-Hui; Wang, Yang; Zhu, Yi-Zhun

    2013-05-15

    A novel compound [4-(2-acetoxy-3-((R)-3-(benzylthio)-1-methoxy-1-oxopropan-2-ylamino)-3-oxopropyl)-1,2-phenylene diacetate (DSC)], derived from Danshensu, exerted cytoprotective effects by anti-oxidative and anti-apoptotic activities in vitro. Herein, we reported the protective effects of DSC on lipopolysaccharide (LPS)-induced inflammatory responses in murine RAW264.7 macrophages and the underlying mechanisms. We showed that DSC concentration-dependently attenuated nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression with less cytotoxicity. Signal transduction studies indicated that DSC significantly inhibited LPS-induced phosphorylation of Akt, but not c-Jun N-terminal kinase 1/2, p38, or extracellular signal-regulated kinase 1/2. Meanwhile, LPS-induced nuclear translocation of nuclear factor-κB (NF-κB) p65 was decreased by DSC. Furthermore, a phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 significantly suppressed LPS-induced NF-κB p65 nuclear translocation, iNOS expression, and NO production, which was also mimicked by pretreatment with DSC. These results suggested that DSC attenuated LPS-induced inflammatory response in macrophages, at least in part, through suppression of PI3K/Akt signaling and NF-κB activation.

  4. EPO-dependent activation of PI3K/Akt/FoxO3a signalling mediates neuroprotection in in vitro and in vivo models of Parkinson's disease.

    PubMed

    Jia, Yu; Mo, Shi-Jing; Feng, Qi-Qi; Zhan, Ma-Li; OuYang, Li-Si; Chen, Jia-Chang; Ma, Yu-Xin; Wu, Jia-Jia; Lei, Wan-Long

    2014-05-01

    Erythropoietin (EPO) may become a potential therapeutic candidate for the treatment of the neurodegenerative disorder -- Parkinson's disease (PD), since EPO has been found to prevent neuron apoptosis through the activation of cell survival signalling. However, the underlying mechanisms of how EPO exerts its neuroprotective effect are not fully elucidated. Here we investigated the mechanism by which EPO suppressed 6-hydroxydopamine (6-OHDA)-induced neuron death in in vitro and in vivo models of PD. EPO knockdown conferred 6-OHDA-induced cytotoxicity. This effect was reversed by EPO administration. Treatment of PC12 cells with EPO greatly diminished the toxicity induced by 6-OHDA in a dose- and time-dependent manner. EPO effectively reduced apoptosis of striatal neurons and induced a significant improvement on the neurological function score in the rat models of PD. Furthermore, EPO increased the expression of phosphorylated Akt and phosphorylated FoxO3a, and abrogated the 6-OHDA-induced dysregulation of Bcl-2, Bax and Caspase-3 in PC12 cells and in striatal neurons. Meanwhile, the EPO-dependent neuroprotection was notably reversed by pretreatment with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). Our data suggest that PI3K/Akt/FoxO3a signalling pathway may be a possible mechanism involved in the neuroprotective effect of EPO in PD.

  5. Protective Effect of Minocycline Against Ketamine-Induced Injury in Neural Stem Cell: Involvement of PI3K/Akt and Gsk-3 Beta Pathway

    PubMed Central

    Lu, Yang; Lei, Shan; Wang, Ning; Lu, Pan; Li, Weisong; Zheng, Juan; Giri, Praveen K.; Lu, Haixia; Chen, Xinlin; Zuo, Zhiyi; Liu, Yong; Zhang, Pengbo

    2016-01-01

    It has been suggested that ketamine cause injury during developing brain. Minocycline (MC) could prevent neuronal cell death through the activation of cell survival signals and the inhibition of apoptotic signals in models of neurodegenerative diseases. Here we investigated the protective effect of MC against ketamine-induced injury in neural stem cells (NSCs) from neonatal rat. Ketamine (100 μM/L) significantly inhibited NSC proliferation, promoted their differentiation into astrocytes and suppressed neuronal differentiation of NSCs. Moreover, the apoptotic level was increased following ketamine exposure. MC pretreatment greatly enhanced cell viability, decreased caspase-3-like activity, even reversed the differentiation changes caused by ketamine. To elucidate a possible mechanism of MC’ neuroprotective effect, we investigated the phosphatidylinositol 3-kinase (PI3K) pathway using LY294002, a specific PI3K inhibitor. Immunoblotting revealed that MC enhanced the phosphorylation/activation of Akt and phosphorylation/inactivation of glycogen synthase kinase-3beta (Gsk-3β). Our results suggest that PI3K/Akt and Gsk-3β pathway are involved in the neuroprotective effect of MC. PMID:28066173

  6. Activation of IL-8 via PI3K/Akt-dependent pathway is involved in leptin-mediated epithelial-mesenchymal transition in human breast cancer cells

    PubMed Central

    Wang, Lin; Tang, Cuiping; Cao, Hong; Li, Kuangfa; Pang, Xueli; Zhong, Liang; Dang, Weiqi; Tang, Hao; Huang, Yunxiu; Wei, Lan; Su, Min; Chen, Tingmei

    2015-01-01

    Background Information: Previous studies have revealed that leptin may be involved in epithelial-mesenchymal transition (EMT), a crucial initiator of cancer progression to facilitate metastatic cascade, increase tumor recurrence, and ultimately cause poor prognosis. However, the underlying mechanism remains unclear. The aim of our present study was to investigate the effect of leptin on EMT of breast cancer cells and the underlying mechanism. Results: Our data demonstrated that leptin significantly increased the phosphorylation of STAT3, Akt, and ERK1/2, elevated the expression of IL-8, and induced breast cancer cells to undergo EMT. The effect of leptin on IL-8 could visibly abolished by the inhibitor of PI3K LY294002. In addition, leptin-induced EMT of breast cancer cells was blocked by anti-IL-8 antibodies. Examination of the expression of ObR, leptin, IL-8 and EMT-related biomarkers in patient specimens demonstrated that malignant breast carcinoma with lymph node metastases (LNM), which represents poor prognosis, expressed higher levels of ObR, leptin, IL-8 than other types of breast cancer, and displayed more obvious EMT transversion. In vivo xenograft experiment revealed that leptin signally promoted tumor growth and metastasis and increased the expressions of IL-8 and EMT-related biomarkers. Conclusions: Our results support that leptin-induced EMT in breast cancer cells requires IL-8 activation via the PI3K/Akt signal pathway. PMID:26121010

  7. Radiosensitization of EGFR/HER2 positive pancreatic cancer is mediated by inhibition of Akt independent of Ras mutational status

    PubMed Central

    Kimple, Randall J.; Vaseva, Angelina V.; Cox, Adrienne D.; Baerman, Kathryn M.; Calvo, Benjamin F.; Tepper, Joel E.; Shields, Janiel M.; Sartor, Carolyn I.

    2009-01-01

    Purpose Epidermal growth factor receptor family members (e.g., EGFR, HER2, HER3, and HER4) are commonly overexpressed in pancreatic cancer. We investigated the effects of inhibition of EGFR/HER2 signaling on pancreatic cancer to elucidate the role(s) of EGFR/HER2 in radiosensitization and to provide evidence in support of further clinical investigations. Experimental Design Expression of EGFR family members in pancreatic cancer lines was assessed by qRT-PCR. Cell growth inhibition was determined by MTS assay. The effects of inhibition of EGFR family receptors and downstream signaling pathways on in vitro radiosensitivity were evaluated using clonogenic assays. Growth delay was used to evaluate the effects of nelfinavir on in vivo tumor radiosensitivity. Results Lapatinib inhibited cell growth in four pancreatic cancer cell lines, but radiosensitized only wild-type K-ras-expressing T3M4 cells. Akt activation was blocked in a wild-type K-ras cell line, whereas constitutive phosphorylation of Akt and ERK was seen in lines expressing mutant K-ras. Overexpression of constitutively-active K-ras(G12V) abrogated lapatinib-mediated inhibition of both Akt phosphorylation and radiosensitization. Inhibition of MEK/ERK signaling with U0126 had no effect on radiosensitization, whereas inhibition of activated Akt with LY294002 (enhancement ratio 1.2–1.8) or nelfinavir (enhancement ratio 1.2–1.4) radiosensitized cells regardless of K-ras mutation status. Oral nelfinavir administration to mice bearing mutant K-ras-containing Capan-2 xenografts resulted in a greater than additive increase in radiation-mediated tumor growth delay (synergy assessment ratio of 1.5). Conclusions Inhibition of EGFR/HER2 enhances radiosensitivity in wild-type K-ras pancreatic cancer. Nelfinavir, and other PI3K/Akt inhibitors, are effective pancreatic radiosensitizers regardless of K-ras mutation status. PMID:20103665

  8. Neuroprotective effect of miR-665 against sevoflurane anesthesia-induced cognitive dysfunction in rats through PI3K/Akt signaling pathway by targeting insulin-like growth factor 2

    PubMed Central

    Lu, Xihua; Lv, Shuaiguo; Mi, Yan; Wang, Lei; Wang, Gensheng

    2017-01-01

    The aim of this study was to investigate the in vivo and in vitro effects of miR-665 on sevoflurane anesthesia-induced cognitive dysfunction. SH-SY5Y cells and male SD rats were treated with sevoflurane to simulate anesthesia-induced cognitive dysfunction. The cells and rats both were transfected with a miR-665 mimic, inhibitor, scramble, IGF-2 siRNA, or treated with P13K/Akt inhibitor LY294002. The cell apoptosis, autophagy, growth related proteins, and mRNA levels were measured using different methods. The motor performance was assessed using the Morris water maze (MWM) test. Finally, the differences were statistically analyzed. It was noted that sevoflurane-induced miR-665 downregulation accompanied with the upregulation of IGF-2 in vivo and motor deficits in vitro. Moreover, sevoflurane also induced hippocampal neuroapoptosis; reduced regular autophagy; increased Bax/Bcl-2 ratio; decreased the expression of Beclin 1, PSD95, and p-CREB; and activated P13K/Akt signaling pathway. However, the treatment by miR-665 mimics significantly reversed all the molecular changes and improved motor performance. Our data demonstrate the neuroprotective effect of miR-665 against sevoflurane anesthesia-induced cognitive impairment. This study suggests that miR-665 might be explored as a potential target of therapy for sevoflurane-induced cognitive impairment. PMID:28386360

  9. Fungiform papilla pattern: EGF regulates inter-papilla lingual epithelium and decreases papilla number by means of PI3K/Akt, MEK/ERK, and p38 MAPK signaling.

    PubMed

    Liu, Hong-Xiang; Henson, Bradley S; Zhou, Yanqiu; D'Silva, Nisha J; Mistretta, Charlotte M

    2008-09-01

    Fungiform papillae are epithelial taste organs that form on the tongue, requiring differentiation of papillae and inter-papilla epithelium. We tested roles of epidermal growth factor (EGF) and the receptor EGFR in papilla development. Developmentally, EGF was localized within and between papillae whereas EGFR was progressively restricted to inter-papilla epithelium. In tongue cultures, EGF decreased papillae and increased cell proliferation in inter-papilla epithelium in a concentration-dependent manner, whereas EGFR inhibitor increased and fused papillae. EGF preincubation could over-ride disruption of Shh signaling that ordinarily would effect a doubling of fungiform papillae. With EGF-induced activation of EGFR, we demonstrated phosphorylation in PI3K/Akt, MEK/ERK, and p38 MAPK pathways; with pathway inhibitors (LY294002, U0126, SB203580) the EGF-mediated decrease in papillae was reversed, and synergistic actions were shown. Thus, EGF/EGFR signaling by means of PI3K/Akt, MEK/ERK, and p38 MAPK contributes to epithelial cell proliferation between papillae; this biases against papilla differentiation and reduces numbers of papillae.

  10. WISP1 overexpression promotes proliferation and migration of human vascular smooth muscle cells via AKT signaling pathway.

    PubMed

    Lu, Shun; Liu, Hao; Lu, Lihe; Wan, Heng; Lin, Zhiqi; Qian, Kai; Yao, Xingxing; Chen, Qing; Liu, Wenjun; Yan, Jianyun; Liu, Zhengjun

    2016-10-05

    Proliferation and migration of vascular smooth muscle cells (VSMCs) play crucial roles in the development of vascular restenosis. Our previous study showed that CCN4, namely Wnt1 inducible signaling pathway protein 1 (WISP1), significantly promotes proliferation and migration of rat VSMCs, but its mechanism remains unclear. This study aims to investigate whether and how WISP1 stimulates proliferation and migration of human VSMCs. Western blot analysis showed that FBS treatment increased WISP1 protein levels in human VSMCs in a dose-dependent manner. Overexpression of WISP1 using adenovirus encoding WISP1 (AD-WISP1) significantly increased proliferation rate of human VSMCs by 2.98-fold compared with empty virus (EV)-transfected cells, shown by EdU incorporation assay. Additionally, Scratch-induced wound healing assay revealed that adenovirus-mediated overexpression of WISP1 significantly increased cell migration compared with EV-transfected cells from 6h (4.56±1.14% vs. 11.23±2.25%, P<0.05) to 48h (25.25±5.51% vs. 97.54±13.12%, P<0.01) after injury. Transwell Migration Assay confirmed that WISP1 overexpression significantly promoted human VSMC migration by 2.25-fold compared with EV. Furthermore, WISP1 overexpression stimulated Akt signaling activation in human VSMCs. Blockage of Akt signaling by Akt inhibitor AZD5363 or PI3K inhibitor LY294002, led to an inhibitory effect of WISP1-induced proliferation and migration in human VSMCs. Moreover, we found that WISP1 overexpression stimulated GSK3α/β phosphorylation, and increased expression of cyclin D1 and MMP9 in human VSMCs, and this effect was abolished by AZD5363. Collectively, we demonstrated that Akt signaling pathway mediates WISP1-induced migration and proliferation of human VSMCs, suggesting that WISP1 may act as a novel potential therapeutic target for vascular restenosis.

  11. Hydroxysafflor yellow A cardioprotection in ischemia-reperfusion (I/R) injury mainly via Akt/hexokinase II independent of ERK/GSK-3β pathway.

    PubMed

    Min, Jia; Wei, Cui

    2017-03-01

    Hydroxysafflor yellow A (HSYA) is the main active component of Carthamus tinctorius L which has been used for hundreds of years in Chinese folk medicine in the treatment cardiovascular disease. This study was designed to investigate whether HSYA exerts cardioprotection in ischemia-reperfusion (I/R) injury heart and the mechanisms involved. The protective effect and mechanisms in myocardial ischemia reperfusion injury of HSYA was evaluated by hypoxia-recover (H/R) injury cell model which induced by hypoxia and recovered with oxygen in H9c2 cells. PI3K/Akt and ERK as the reperfusion injury salvage kinase (RISK) pathway and Hexokinase II (HKII) were both examined. In H/R cell model, HSYA significantly reduced dehydrogenase (LDH), Caspase 3 level, alleviated oxidative stress injury and apoptosis, meanwhile restored mitochondrial energy metabolism. Pretreatment with PI3K inhibitor (LY294002) or hexokinase II inhibitor (3-BrPA), the protective effect of HSYA was significantly attenuated. On the contrary, pretreatment with ERK inhibitor (PD98059), the protective effect of HSYA on myocardial cells was decreased slightly, not as significant as PI3K inhibitor or hexokinase II inhibitor. ERK play a protective role in myocardial protection by phosphorylation of GSK3-β, but the effect of HSYA on phosphorylation of GSK3-β is weakly, however the effect of HSYA on Akt and hexokinase II were significantly up-regulated. Meanwhile, the phosphorylation of GSK3-β by HSYA was significantly reduced after gave the ERK inhibitor and had no significant difference between the model group. The cardioprotection effect of HSYA appears to be mainly mediated via the PI3K/Akt/hexokinase II.

  12. Metastatic function of BMP-2 in gastric cancer cells: The role of PI3K/AKT, MAPK, the NF-{kappa}B pathway, and MMP-9 expression

    SciTech Connect

    Kang, Myoung Hee; Oh, Sang Cheul; Kang, Han Na; Kim, Jung Lim; Kim, Jun Suk

    2011-07-15

    Bone morphogenetic proteins (BMPs) have been implicated in tumorigenesis and metastatic progression in various types of cancer cells, but the role and cellular mechanism in the invasive phenotype of gastric cancer cells is not known. Herein, we determined the roles of phosphoinositide 3-kinase (PI3K)/AKT, extracellular signal-regulated protein kinase (ERK), nuclear factor (NF)-{kappa}B, and matrix metalloproteinase (MMP) expression in BMP-2-mediated metastatic function in gastric cancer. We found that stimulation of BMP-2 in gastric cancer cells enhanced the phosphorylation of AKT and ERK. Accompanying activation of AKT and ERK kinase, BMP-2 also enhanced phosphorylation/degradation of I{kappa}B{alpha} and the nuclear translocation/activation of NF-{kappa}B. Interestingly, blockade of PI3K/AKT and ERK signaling using LY294002 and PD98059, respectively, significantly inhibited BMP-2-induced motility and invasiveness in association with the activation of NF-{kappa}B. Furthermore, BMP-2-induced MMP-9 expression and enzymatic activity was also significantly blocked by treatment with PI3K/AKT, ERK, or NF-{kappa}B inhibitors. Immunohistochemistry staining of 178 gastric tumor biopsies indicated that expression of BMP-2 and MMP-9 had a significant positive correlation with lymph node metastasis and a poor prognosis. These results indicate that the BMP-2 signaling pathway enhances tumor metastasis in gastric cancer by sequential activation of the PI3K/AKT or MAPK pathway followed by the induction of NF-{kappa}B and MMP-9 activity, indicating that BMP-2 has the potential to be a therapeutic molecular target to decrease metastasis.

  13. Inhibition of p21 and Akt potentiates SU6656-induced caspase-independent cell death in FRO anaplastic thyroid carcinoma cells.

    PubMed

    Kim, S H; Kang, J G; Kim, C S; Ihm, S-H; Choi, M G; Yoo, H J; Lee, S J

    2013-06-01

    SU6656 is a small-molecule indolinone that selectively inhibits Src family kinase and induces death of cancer cells. The aim of the present study was to investigate the influence of SU6656 on cell survival and to assess the role of p21 and PI3K/Akt signaling in cell survival resulting from SU6656 treatment in anaplastic thyroid carcinoma (ATC) cells. When 8505C, CAL62, and FRO ATC cells were treated with SU6656, the viability of 8505C and CAL62 ATC cells decreased only after treatment with SU6656 at a dosage of 100 μM for 72 h, while the viability of FRO ATC cells decreased after treatment with SU6656 in a concentration- and time-dependent manner. Cell viability was not changed by pretreatment with the broad-spectrum caspase inhibitor z-VAD-fmk. Phospho-Src protein levels were reduced, and p21 protein levels were elevated. Phospho-ERK1/2 protein levels were multiplied without alteration of total ERK1/2, total Akt, and phospho-Akt protein levels. Regarding FRO ATC cells, the decrement of cell viability, the increment of cleaved PARP-1 protein levels, and the decrement of phospho-Src protein levels were shown in p21 siRNA- or LY294002-pretreated cells compared to SU6656-treated control cells. ERK1/2 siRNA transfection did not affect cell viability and protein levels of cleaved PARP-1, p21, and Akt. In conclusion, these results suggest that SU6656 induces caspase-independent death of FRO ATC cells by overcoming the resistance mechanism involving p21 and Akt. Suppression of p21 and Akt enhances the cytotoxic effect of SU6656 in FRO ATC cells.

  14. Vitamin A (retinol) up-regulates the receptor for advanced glycation endproducts (RAGE) through p38 and Akt oxidant-dependent activation.

    PubMed

    Gelain, Daniel Pens; de Bittencourt Pasquali, Matheus Augusto; Caregnato, Fernanda Freitas; Moreira, José Claudio Fonseca

    2011-10-28

    Retinol (vitamin A) is believed to exert preventive/protective effects against malignant, neurodegenerative and cardiovascular diseases by acting as an antioxidant. However, later clinical and experimental data show a pro-oxidant action of retinol and other retinoids at specific conditions. The receptor for advanced glycation endproducts (RAGE) is a pattern recognition receptor, being activated by different ligands such as S100 proteins, HMGB1 (amphoterin), β-amyloid peptide and advanced glycation endproducts (AGE). RAGE activation influences a wide range of pathological conditions such as diabetes, pro-inflammatory states and neurodegenerative processes. Here, we investigated the involvement of different mitogen-activated protein kinases (MAPK: ERK1/2, p38 and JNK), PKC, PKA and Akt in the up-regulation of RAGE by retinol. As previously reported, we observed that the increase in RAGE immunocontent by retinol is reversed by antioxidant co-treatment, indicating the involvement of oxidative stress in this process. Furthermore, the p38 inhibitor SB203580 and the Akt inhibitor LY294002 also decreased the effect of retinol on RAGE levels, suggesting the involvement of these protein kinases in such effect. Both p38 and Akt phosphorylation were increased by treatment with pro-oxidant concentrations of retinol, and the antioxidant co-treatment blocked this effect, indicating that activation of p38 and Akt during retinol treatment is dependent on reactive species production. The 2',7'-dichlorohydrofluorescein diacetate (DCFH) assay also indicated that retinol treatment enhances cellular reactive species production. Altogether, these data indicate that RAGE up-regulation by retinol is mediated by the free radical-dependent activation of p38 and Akt.

  15. Sonic hedgehog promotes proliferation and differentiation of adult muscle cells: Involvement of MAPK/ERK and PI3K/Akt pathways.

    PubMed

    Elia, Dafna; Madhala, Dorit; Ardon, Eti; Reshef, Ram; Halevy, Orna

    2007-09-01

    Sonic hedgehog (Shh) has been reported to act as a mitogen and survival factor for muscle satellite cells. However, its role in their differentiation remains ambiguous. Here, we provide evidence that Shh promotes the proliferation and differentiation of primary cultures of chicken adult myoblasts (also termed satellite cells) and mouse myogenic C2 cells. These effects are reversed by cyclopamine, a specific chemical inhibitor of the Shh pathway. In addition, we show that Shh and its downstream molecules are expressed in adult myoblast cultures and localize adjacent to Pax7 in muscle sections. These gene expressions are regulated during postnatal muscle growth in chicks. Most importantly, we report that Shh induces MAPK/ERK and phosphoinositide 3-kinase (PI3K)-dependent Akt phosphorylation and that activation of both signaling pathways is essential for Shh's signaling in muscle cells. However, the effect of Shh on Akt phosphorylation is more robust than that on MAPK/ERK, and data suggest that Shh influences these pathways in a manner similar to IGF-I. By exploiting specific chemical inhibitors of the MAPK/ERK and PI3K/Akt signaling pathways, UO126 and Ly294002, respectively, we demonstrate that Shh-induced Akt phosphorylation, but not that of MAPK/ERK, is required for its promotive effects on muscle cell proliferation and differentiation. Taken together, we suggest that Shh acts in an autocrinic manner in adult myoblasts, and provide first evidence of a role for PI3K/Akt in Shh signaling during myoblast differentiation.

  16. miR-21 increases c-kit+ cardiac stem cell proliferation in vitro through PTEN/PI3K/Akt signaling

    PubMed Central

    Long, Xianping; Zhao, Ranzun; Wang, Yan; Chen, Wenming; Xu, Guanxue; Sheng, Jin; Wang, Dongmei; Cao, Song

    2017-01-01

    The low survival rate of cardiac stem cells (CSCs) in the ischemic myocardium is one of the obstacles in ischemic cardiomyopathy cell therapy. The MicroRNA (miR)-21 and one of its target protein, the tensin homolog deleted on chromosome ten (PTEN), contributes to the proliferation of many kinds of tissues and cell types. It is reported that miR-21 promotes proliferation through PTEN/PI3K/Akt pathway, but its effects on c-kit+ CSC remain unclear. The authors hypothesized that miR-21 promotes the proliferation in c-kit+ CSC, and evaluated the involvement of PTEN/PI3K/Akt pathway in vitro. miR-21 up-regulation with miR-21 efficiently mimics accelerated cell viability and proliferation in c-kit+ CSC, which was evidenced by the CCK-8, EdU and cell cycle analyses. In addition, the over-expression of miR-21 in c-kit+ CSCs notably down-regulated the protein expression of PTEN although the mRNA level of PTEN showed little change. Gain-of-function of miR-21 also increased the phosphor-Akt (p-Akt) level. Phen, the selective inhibitor of PTEN, reproduced the pro-proliferation effects of miR-21, while PI3K inhibitor, LY294002, totally attenuated the pro-survival effect of miR-21. These results indicate that miR-21 is efficient in promoting proliferation in c-kit+ CSCs, which is contributed by the PTEN/PI3K/Akt pathway. miR-21 holds the potential to facilitate CSC therapy in ischemic myocardium. PMID:28168101

  17. Canstatin inhibits hypoxia-induced apoptosis through activation of integrin/focal adhesion kinase/Akt signaling pathway in H9c2 cardiomyoblasts

    PubMed Central

    Yamawaki, Hideyuki

    2017-01-01

    A hypoxic stress which causes apoptosis of cardiomyocytes is the main problem in the ischemic heart disease. Canstatin, a non-collagenous fragment of type IV collagen α2 chain, is an endogenous anti-angiogenic factor. We have previously reported that canstatin has a cytoprotective effect on cardiomyoblasts. In the present study, we examined the effects of canstatin on hypoxia-induced apoptosis in H9c2 cardiomyoblasts. Cell counting assay was performed to determine a cell viability. Western blotting was performed to detect expression of cleaved casepase-3 and phosphorylation of focal adhesion kinase (FAK) and Akt. Immunocytochemical staining was performed to observe a distribution of αv integrin. Hypoxia (1% O2, 48 h) significantly decreased cell viability and increased cleaved caspase-3 expression. Canstatin (10–250 ng/ml) significantly inhibited these changes in a concentration-dependent manner. Cilengitide (1 μM), an αvβ3 and αvβ5 integrin inhibitor, significantly prevented the protective effects of canstatin on cell viability. Canstatin significantly increased phosphorylation of FAK and Akt under hypoxic condition, which were inhibited by cilengitide. LY294002, an inhibitor of phosphatidylinositol-3 kinase/Akt pathway, suppressed the canstatin-induced Akt phosphorylation and reversed the protective effects of canstatin. It was observed that hypoxia caused a localization of αv integrin to focal adhesion. In summary, we for the first time clarified that canstatin inhibits hypoxia-induced apoptosis via FAK and Akt pathways through activating integrins in H9c2 cardiomyoblasts. PMID:28235037

  18. Isoorientin induces apoptosis and autophagy simultaneously by reactive oxygen species (ROS)-related p53, PI3K/Akt, JNK, and p38 signaling pathways in HepG2 cancer cells.

    PubMed

    Yuan, Li; Wei, Shuping; Wang, Jing; Liu, Xuebo

    2014-06-11

    Cell death is closely related to autophagy under some circumstances; however, the effect of isoorientin (ISO) on autophagy and the interplay between apoptosis and autophagy in human hepatoblastoma cancer (HepG2) cells remains poorly understood. The present study showed that ISO induced autophagy, which was correlated with the formation of autophagic vacuoles and the overexpression of Beclin-1 and LC3-II. The autophagy inhibitor 3-methyladenine (3-MA) markedly inhibited apoptosis, and the apoptosis inhibitor ZVAD-fmk also decreased ISO-induced autophagy. In addition, the PI3K/Akt inhibitor LY294002 enhanced Beclin-1, LC3-II, and poly(ADP-ribose) polymerase (PARP) cleavage levels. Also, the reactive oxygen species (ROS) inhibitor N-acetyl-L-cysteine (NAC), the JNK inhibitor SP600125, and the p38 inhibitor SB203580 efficiently downregulated the levels of these proteins. Moreover, the p53 inhibitor pifithrin-α and the nuclear factor (NF)-κB inhibitor pyrrolidinedithiocarbamic acid (PDTC) clearly suppressed Beclin-1 and LC3-II and increased cytochrome c release, caspase-3 activation, and PARP cleavage. These results demonstrated for the first time that ISO simultaneously induced apoptosis and autophagy by ROS-related p53, PI3K/Akt, JNK, and p38 signaling pathways. Furthermore, ISO-induced apoptosis by activating the Fas receptor-mediated apoptotic pathway and suppressing the p53 and PI3K/Akt-dependent NF-κB signaling pathway, with the subsequent increase in the release of cytochrome c, caspase-3 activation, and PARP cleavage.

  19. Blockade of the phosphatidylinositol-3-kinase-Akt signaling pathway enhances the induction of apoptosis by microtubule-destabilizing agents in tumor cells in which the pathway is constitutively activated.

    PubMed

    Fujiwara, Yusuke; Hosokawa, Yoshihisa; Watanabe, Kazushi; Tanimura, Susumu; Ozaki, Kei-ichi; Kohno, Michiaki

    2007-03-01

    Constitutive activation of the phosphatidylinositol-3-kinase (PI3K)-Akt signaling pathway is associated with the neoplastic phenotype in many human tumor cell types. Given the antiapoptotic role of this pathway, we examined whether its specific blockade might sensitize human tumor cells to the induction of apoptosis by various anticancer drugs. Although specific blockade of the PI3K-Akt pathway alone with inhibitors such as LY294002 did not induce cell death, it resulted in marked and selective enhancement of the induction of apoptosis by microtubule-destabilizing agents such as vincristine. This effect was apparent only in tumor cells in which the PI3K-Akt pathway is constitutively activated. Blockade of the PI3K-Akt pathway induced the activation of glycogen synthase kinase-3beta, which phosphorylates microtubule-associated proteins such as tau and thereby reduces their ability to bind and stabilize microtubules. The consequent destabilization of microtubules induced by the inhibition of PI3K-Akt signaling appeared to increase their sensitivity to low concentrations of microtubule-destabilizing agents that alone do not lead to the disruption of cytoplasmic microtubules in tumor cells. Such a synergistic effect on microtubule integrity was not apparent for stable microtubules in the neurites of neuronal cells. These results suggest that the administration of a combination of a PI3K-Akt pathway inhibitor and a microtubule-destabilizing agent is a potential chemotherapeutic strategy for the treatment of tumor cells in which this signaling pathway is constitutively activated.

  20. Protein O-fucosyltransferase 1 promotes trophoblast cell proliferation through activation of MAPK and PI3K/Akt signaling pathways.

    PubMed

    Liu, Chang; Liang, Xiaohua; Wang, Jiao; Zheng, Qin; Zhao, Yue; Khan, Muhammad Noman; Liu, Shuai; Yan, Qiu

    2017-04-01

    Protein O-fucosylation is an important glycosylation modification and plays an important role in embryonic development. Protein O-fucosyltransferase 1 (poFUT1) is an essential enzyme that catalyzes the synthesis of protein O-fucosylation. Our previous studies showed that poFUT1 promoted trophoblast cell migration and invasion at the fetal-maternal interface, but the role of poFUT1 in trophoblast cells proliferation remains unclear. Here, immunohistochemistry data showed that poFUT1 and PCNA levels were decreased in abortion patient's trophoblasts compared with women with normal pregnancies. Our results also showed that poFUT1 promoted trophoblast cell proliferation by CCK-8 assay and cell cycle analysis. PoFUT1 increased the phosphorylation of ERK1/2, p38 MAPK, and PI3K/Akt, while inhibitors of ERK1/2(PD98059), p38 MAPK(SB203580), and PI3K (LY294002) prevented ERK1/2, p38 MAPK, and Akt phosphorylation. Moreover, poFUT1 stimulation of trophoblast cells proliferation correlated with increased cell cycle progression by promoting cells into S-phase. The underlying mechanism involved increased cyclin D1, cyclin E, CDK 2, CDK 4, and pRb expression and decreased levels of the cyclin-dependent kinase inhibitors p21 and p27, which were blocked by inhibitors of the upstream signaling molecules MAPK and PI3K/Akt. In conclusion, poFUT1 promotes trophoblast cell proliferation by activating MAPK and PI3K/Akt signaling pathways.

  1. Curcumin inhibited HGF-induced EMT and angiogenesis through regulating c-Met dependent PI3K/Akt/mTOR signaling pathways in lung cancer

    PubMed Central

    Jiao, Demin; Wang, Jian; Lu, Wei; Tang, Xiali; Chen, Jun; Mou, Hao; Chen, Qing-yong

    2016-01-01

    The epithelial-mesenchymal transition (EMT) and angiogenesis have emerged as two pivotal events in cancer progression. Curcumin has been extensively studied in preclinical models and clinical trials of cancer prevention due to its favorable toxicity profile. However, the possible involvement of curcumin in the EMT and angiogenesis in lung cancer remains unclear. This study found that curcumin inhibited hepatocyte growth factor (HGF)-induced migration and EMT-related morphological changes in A549 and PC-9 cells. Moreover, pretreatment with curcumin blocked HGF-induced c-Met phosphorylation and downstream activation of Akt, mTOR, and S6. These effects mimicked that of c-Met inhibitor SU11274 or PI3 kinase inhibitor LY294002 or mTOR inhibitor rapamycin treatment. c-Met gene overexpression analysis further demonstrated that curcumin suppressed lung cancer cell EMT by inhibiting c-Met/Akt/mTOR signaling pathways. In human umbilical vein endothelial cells (HUVECs), we found that curcumin also significantly inhibited PI3K/Akt/mTOR signaling and induced apoptosis and reduced migration and tube formation of HGF-treated HUVEC. Finally, in the experimental mouse model, we showed that curcumin inhibited HGF-stimulated tumor growth and induced an increase in E-cadherin expression and a decrease in vimentin, CD34, and vascular endothelial growth factor (VEGF) expression. Collectively, these findings indicated that curcumin could inhibit HGF-promoted EMT and angiogenesis by targeting c-Met and blocking PI3K/Akt/mTOR pathways. PMID:27525306

  2. Pu-erh tea supplementation suppresses fatty acid synthase expression in the rat liver through downregulating Akt and JNK signalings as demonstrated in human hepatoma HepG2 cells.

    PubMed

    Chiang, Chun-Te; Weng, Meng-Shih; Lin-Shiau, Shoei-Yn; Kuo, Kuan-Li; Tsai, Yao-Jen; Lin, Jen-Kun

    2005-01-01

    Fatty acid synthase (FAS) is a key enzyme of lipogenesis. Overexpression of FAS is dominant in cancer cells and proliferative tissues. The expression of FAS in the livers of rats fed pu-erh tea leaves was significantly suppressed. The gains in body weight, levels of triacylglycerol, and total cholesterol were also suppressed in the tea-treated rats. FAS expression in hepatoma HepG2 cells was suppressed by the extracts of pu-erh tea at both the protein and mRNA levels. FAS expression in HepG2 cells was strongly inhibited by PI3K inhibitor LY294002 and JNK inhibitor II and slightly inhibited by p38 inhibitor SB203580 and MEK inhibitor PD98059, separately. Based on these findings, we suggest that the suppression of FAS in the livers of rats fed pu-erh tea leaves may occur through downregulation of the PI3K/AKt and JNK signaling pathways. The major components of tea that have been demonstrated to be responsible for the antiobesity and hypolipidemic effects are catechins, caffeine, and theanine. The compositions of catechins, caffeine, and theanine varied dramatically in pu-erh, black, oolong, and green teas. The active principles and molecular mechanisms that exerted these biological effects in pu-erh tea deserve future exploration.

  3. PKR promotes choroidal neovascularization via upregulating the PI3K/Akt signaling pathway in VEGF expression

    PubMed Central

    Zhu, Manhui; Liu, Xiaojuan; Wang, Shengcun; Miao, Jin; Wu, Liucheng; Yang, Xiaowei; Wang, Ying; Kang, Lihua; Li, Wendie; Cui, Chen; Sang, Aimin

    2016-01-01

    Purpose The aim of this study was to investigate the functions of dsRNA-activated protein kinase (PKR) in choroidal neovascularization (CNV) and related signaling pathways in the production of vascular endothelial growth factor (VEGF). Methods A chemical hypoxia model of in vitro RF/6A cells, a rhesus choroid-retinal endothelial cell line, was established by adding cobalt chloride (CoCl2) to the culture medium. PKR, phosphophosphatidylinositol 3-kinase (p-PI3K), phosphoprotein kinase B (p-Akt), and VEGF protein levels in RF/6A cells were detected with western blotting. PKR siRNA and the PI3K inhibitor LY294002 were used to evaluate the roles of the PKR and PI3K signaling pathways in VEGF expression with western blotting. In an ARPE-19 (RPE cell line) and RF/6A cell coculture system, proliferation, migration, and tube formation of RF/6A cells under hypoxic conditions were measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Transwell, and Matrigel Transwell assays, respectively. In vivo CNV lesions were induced in C57BL/6J mice using laser photocoagulation. The mice were euthanized in a timely manner, and the eyecups were dissected from enucleated eyes. PKR, p-PI3K, p-Akt, and VEGF protein levels in tissues were detected with western blotting. To evaluate the leakage area, fundus fluorescein angiography and choroidal flat mount were performed on day 7 after intravitreal injection of an anti-PKR monoclonal antibody. Results The in vitro RF/6A cell chemical hypoxia model showed that PKR expression was upregulated in parallel with p-PI3K, p-Akt, and VEGF expression, peaking at 12 h. PKR siRNA downregulated PKR, p-PI3K, p-Akt, and VEGF expression. In addition, the PI3K inhibitor LY294002 greatly decreased the p-PI3K, p-Akt, and VEGF protein levels, but PKR expression was unaffected, indicating that Akt was a downstream molecule of PKR that upregulated VEGF expression. In the ARPE-19 (RPE cell line) and RF/6A cell coculture system, PKR si

  4. Functional Effects of AKT3 on Aurora Kinase Inhibitor-induced Aneuploidy.

    PubMed

    Noguchi, Kohji; Hongama, Keita; Hariki, Shiori; Nonomiya, Yuma; Katayama, Kazuhiro; Sugimoto, Yoshikazu

    2017-02-03

    The suppression of mitotic Aurora kinases (AURKs) by AURK inhibitors frequently causes cytokinetic failure, leading to polyploidy or aneuploidy, indicating the critical role of AURK-mediated phosphorylation during cytokinesis. We demonstrate the deregulated expression of AKT3 in Aurora kinase inhibitor (AURKi)-resistant cells, which we established from human colorectal cancer HCT 116 cells. The AKT family, which includes AKT1, -2, and -3, plays multiple roles in antiapoptotic functions and drug resistance and is involved in cell growth and survival pathways. We found that an AKT inhibitor, AZD5363, showed synergistic effect with an AURKi, VX-680, on two AKT3-expressing AURKi-resistant cell lines, and AKT3 knockdown sensitized cells to VX-680. Consistent with these activities, AKT3 expression suppressed AURKi-induced apoptosis and conferred resistance to AURKi. Thus, AKT3 expression affects cell sensitivity to AURKi. Moreover, we found that AKT3 expression suppressed AURKi-induced aneuploidy, and inversely AKT3 knockdown enhanced it. In addition, partial co-localization of AKT3 with AURKB was observed during anaphase. Overall, this study suggests that AKT3 could repress the antiproliferative effects of AURKi, with a novel activity particularly suppressing the aneuploidy induction.

  5. Promotion of melanoma cell invasion and tumor metastasis by microcystin-LR via phosphatidylinositol 3-kinase/AKT pathway.

    PubMed

    Xu, Pengfei; Zhang, Xu-Xiang; Miao, Chen; Fu, Ziyi; Li, Zhengrong; Zhang, Gen; Zheng, Maqing; Liu, Yuefang; Yang, Liuyan; Wang, Ting

    2013-08-06

    Recently, we have indicated that microcystin-LR, a cyanobacterial toxin produced in eutrophic lakes or reservoirs, can increase invasive ability of melanoma MDA-MB-435 cells; however, the stimulatory effect needs identification by in vivo experiment and the related molecular mechanism is poorly understood. In this study, in vitro and in vivo experiments were conducted to investigate the effect of microcystin-LR on invasion and metastasis of human melanoma cells, and the underlying molecular mechanism was also explored. MDA-MB-435 xenograft model assay showed that oral administration of nude mice with microcystin-LR at 0.001-0.1 mg/kg/d posed no significant effect on tumor weight. Histological examination demonstrated that microcystin-LR could promote lung metastasis, which is confirmed by Matrigel chamber assay suggesting that microcystin-LR treatment at 25 nM can increase the invasiveness of MDA-MB-435 cells. In vitro and in vivo experiments consistently showed that microcystin-LR exposure increased mRNA and protein levels of matrix metalloproteinases (MMP-2/-9) by activating phosphatidylinositol 3-kinase (PI3-K)/AKT. Additionally, microcystin-LR treatment at low doses (≤25 nM) decreased lipid phosphatase PTEN expression, and the microcystin-induced invasiveness enhancement and MMP-2/-9 overexpression were reversed by the PI3-K/AKT chemical inhibitor LY294002 and AKT siRNA, indicating that microcystin-LR promotes invasion and metastasis of MDA-MB-435 cells via the PI3-K/AKT pathway.

  6. Thioredoxin-interacting protein regulates lipid metabolism via Akt/mTOR pathway in diabetic kidney disease.

    PubMed

    Du, Chunyang; Wu, Ming; Liu, Huan; Ren, Yunzhuo; Du, Yunxia; Wu, Haijiang; Wei, Jinying; Liu, Chuxin; Yao, Fang; Wang, Hui; Zhu, Yan; Duan, Huijun; Shi, Yonghong

    2016-10-01

    Abnormal lipid metabolism contributes to the renal lipid accumulation, which is associated with diabetic kidney disease, but its precise mechanism remains unclear. The growing evidence demonstrates that thioredoxin-interacting protein is involved in regulating cellular glucose and lipid metabolism. Here, we investigated the effects of thioredoxin-interacting protein on lipid accumulation in diabetic kidney disease. In contrast to the diabetic wild-type mice, the physical and biochemical parameters were improved in the diabetic thioredoxin-interacting protein knockout mice. The increased renal lipid accumulation, expression of acetyl-CoA carboxylase, fatty acid synthase and sterol regulatory element binding protein-1, and phosphorylated Akt and mTOR associated with diabetes in wild-type mice was attenuated in diabetic thioredoxin-interacting protein knockout mice. Furthermore, thioredoxin-interacting protein knockout significantly increased the expression of peroxisome proliferator-activated receptor-α, acyl-coenzyme A oxidase 1 and carnitine palmitoyltransferaser 1 in diabetic kidneys. In vitro experiments, using HK-2 cells, revealed that knockdown of thioredoxin-interacting protein inhibited high glucose-mediated lipid accumulation, expression of acetyl-CoA carboxylase, fatty acid synthase and sterol regulatory element binding protein-1, as well as activation of Akt and mTOR. Moreover, knockdown of thioredoxin-interacting protein reversed high glucose-induced reduction of peroxisome proliferator-activated receptor-α, acyl-coenzyme A oxidase 1 and carnitine palmitoyltransferaser 1 expression in HK-2 cells. Importantly, blockade of Akt/mTOR signaling pathway with LY294002, a specific PI3K inhibitor, replicated these effects of thioredoxin-interacting protein silencing. Taken together, these data suggest that thioredoxin-interacting protein deficiency alleviates diabetic renal lipid accumulation through regulation of Akt/mTOR pathway, thioredoxin

  7. Is There a Future for AKT Inhibitors in the Treatment of Cancer?

    PubMed

    Jansen, Valerie M; Mayer, Ingrid A; Arteaga, Carlos L

    2016-06-01

    An AKT inhibitor plus an antiestrogen exhibited no significant clinical activity in patients with ER(+)/HER2(-) breast cancer despite laboratory studies supporting an antitumor effect for both drugs combined. These results raise concerns about the development of AKT inhibitors in unselected patients whose tumors have unknown dependence on the PI3K/AKT pathway. Clin Cancer Res; 22(11); 2599-601. ©2016 AACRSee related article by Ma et al., p. 2650.

  8. Effects of intravitreal insulin and insulin signaling cascade inhibitors on emmetropization in the chick

    PubMed Central

    Penha, Alexandra Marcha; Burkhardt, Eva; Schaeffel, Frank

    2012-01-01

    Purpose Intravitreal insulin has been shown to be a powerful stimulator of myopia in chickens, in particular if the retinal image is degraded or defocused. In most tissues, the insulin receptor activates two main signaling pathways: a) the mitogen-activated protein kinase (MAPK) cascade (e.g., mitogen-activated protein kinasem kinase [MEK] and extracellular regulated kinase [ERK]) and b) the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. In the current study, insulin was injected, and these pathways were separately inhibited to determine which is activated when the retinal image is defocused by spectacle lenses. Methods Chicks were treated with either +7 D, −7 D, or no lenses. They were intravitreally injected with insulin, the MEK inhibitor U0126, the PI3K inhibitor Ly294002, or a combination of insulin and one of the inhibitors. Refractions and ocular dimension were measured at the beginning and after four days of treatment. The retinal proteins of the chicks were measured with western blots after 2 h and four days of treatment. Incubation occurred with anti-Akt1, anti-Erk1/2, anti-phospho-AktThr308, and anti-phospho-Erk1/2(Thr202/Tyr204) antibodies, and the ratio between the relative intensity of the phospho-form and the total-form was calculated. Results Chicks wearing positive lenses and injected with saline and with PI3K inhibitor compensated for the imposed defocus and became hyperopic. Insulin injections and insulin plus PI3K inhibitor injections prevented lens-induced hyperopia, whereas the MEK inhibitor alone and insulin plus MEK inhibitor had no effect. Obviously, the MEK inhibitor suppressed the effect of insulin on eye growth in the plus lens–treated animals. Chicks treated with negative lenses and injected with insulin, or with insulin plus MEK inhibitor, overcompensated for the imposed defocus. This effect of insulin was not detected in eyes injected with PI3K inhibitor plus insulin, suggesting that the PI3K inhibitor

  9. Cox-2 Inhibition Protects against Hypoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis via Akt-Dependent Enhancement of iNOS Expression

    PubMed Central

    Cai, Yin; Tang, Eva Hoi Ching; Yan, Dan; Kosuru, Ramoji

    2016-01-01

    The present study explored the potential causal link between ischemia-driven cyclooxygenase-2 (COX-2) expression and enhanced apoptosis during myocardial ischemia/reperfusion (I/R) by using H9C2 cardiomyocytes and primary rat cardiomyocytes subjected to hypoxia/reoxygenation (H/R). The results showed that H/R resulted in higher COX-2 expression than that of controls, which was prevented by pretreatment with Helenalin (NFκB specific inhibitor). Furthermore, pretreatment with NS398 (COX-2 specific inhibitor) significantly attenuated H/R-induced cell injury [lower lactate dehydrogenase (LDH) leakage and enhanced cell viability] and apoptosis (higher Bcl2 expression and lower level of cleaved caspases-3 and TUNEL-positive cells) in cardiomyocytes. The amelioration of posthypoxic apoptotic cell death was paralleled by significant attenuation of H/R-induced increases in proinflammatory cytokines [interleukin 6 (IL6) and tumor necrosis factor (TNFα)] and reactive oxygen species (ROS) production and by higher protein expression of phosphorylated Akt and inducible nitric oxide synthase (iNOS) and enhanced nitric oxide production. Moreover, the application of LY294002 (Akt-specific inhibitor) or 1400W (iNOS-selective inhibitor) cancelled the cellular protective effects of NS398. Findings from the current study suggest that activation of NFκB during cardiomyocyte H/R induces the expression of COX-2 and that higher COX-2 expression during H/R exacerbates cardiomyocyte H/R injury via mechanisms that involve cross talks among inflammation, ROS, and Akt/iNOS/NO signaling. PMID:27795807

  10. Bcl-2 and caspase-3 are major regulators in Agaricus blazei-induced human leukemic U937 cell apoptosis through dephoshorylation of Akt.

    PubMed

    Jin, Cheng-Yun; Moon, Dong-Oh; Choi, Yung Hyun; Lee, Jae-Dong; Kim, Gi-Young

    2007-08-01

    Agaricus blazei is a medicinal mushroom that possesses antimetastatic, antitumor, antimutagenic, and immunostimulating effects. However, the molecular mechanisms involved in A. blazei-mediated apoptosis remain unclear. In the present study, to elucidate the role of the Bcl-2 in A. blazei-mediated apoptosis, U937 cells were transfected with either empty vector (U937/vec) or vector containing cDNA encoding full-length Bcl-2 (U937/Bcl-2). As compared with U937/vec, U937/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2. Treatment of U937/vec with 1.0-4.0 mg/ml of A. blazei extract (ABE) for 24 h resulted in a significant induction of morphologic features indicative of apoptosis. In contrast, U937/Bcl-2 exposed to the same ABE treatment only exhibited a slight induction of apoptotic features. ABE-induced apoptosis was accompanied by downregulation of antiapoptotic proteins such as X-linked inhibitor of apoptosis protein (XIAP), inhibitor of apoptosis protein (cIAP)-2 and Bcl-2, activation of caspase-3, and cleavage of poly(ADP-ribose)polymerase (PARP). Ectopic expression of Bcl-2 was associated with significantly induced expression of antiapoptotic proteins, such as cIAP-2 and Bcl-2, but not XIAP. Ectopic expression of Bcl-2 also reduced caspase-3 activation and PARP cleavage in ABE treated U937 cells. Furthermore, treatment with the caspase-3 inhibitor z-DEVD-fmk was sufficient to restore cell viability following ABE treatment. This increase in viability was ascribed to downregulation of caspase-3 and blockage of PARP and PLC-gamma cleavage. ABE also triggered the downregulation of Akt, and combined treatment with LY294002 (an inhibitor of Akt) significantly decreased cell viability. The results indicated that major regulators of ABE-induced apoptosis in human leukemic U937 cells are Bcl-2 and caspase-3, which are associated with dephosphorylation of the Akt signal pathway.

  11. Polyphyllin G induce apoptosis and autophagy in human nasopharyngeal cancer cells by modulation of AKT and mitogen-activated protein kinase pathways in vitro and in vivo.

    PubMed

    Chen, Jui-Chieh; Hsieh, Ming-Ju; Chen, Chih-Jung; Lin, Jen-Tsun; Lo, Yu-Sheng; Chuang, Yi-Ching; Chien, Su-Yu; Chen, Mu-Kuan

    2016-10-25

    Polyphyllin G (also call polyphyllin VII), extract from rhizomes of Paris yunnanensis Franch, has been demonstrated to have strong anticancer activities in a wide variety of human cancer cell lines. Previous studies found that Polyphyllin G induced apoptotic cell death in human hepatoblastoma cancer and lung cancer cells. However, the underlying mechanisms of autophagy in human nasopharyngeal carcinoma (NPC) remain unclear. In this study, Polyphyllin G can potently induced apoptosis dependent on the activations of caspase-8, -3, and -9 and the changes of Bcl-2, Bcl-xL and Bax protein expression in different human NPC cell lines (HONE-1 and NPC-039). The amount of both LC3-II and Beclin-1 was intriguingly increased suggest that autophagy was induced in Polyphyllin G-treated NPC cells. To further clarify whether Polyphyllin G-induced apoptosis and autophagy depended on AKT/ERK/JNK/p38 MAPK signaling pathways, cells were combined treated with AKT inhibitor (LY294002), ERK1/2 inhibitor (U0126), p38 MAPK inhibitor (SB203580), or JNK inhibitor (SP600125). These results demonstrated that Polyphyllin G induced apoptosis in NPC cells through activation of ERK, while AKT, p38 MAPK and JNK were responsible for Polyphyllin G-induced autophagy. Finally, an administration of Polyphyllin G effectively suppressed the tumor growth in the NPC carcinoma xenograft model in vivo. In conclusion, our results reveal that Polyphyllin G inhibits cell viability and induces apoptosis and autophagy in NPC cancer cells, suggesting that Polyphyllin G is an attractive candidate for tumor therapies. Polyphyllin G may promise candidate for development of antitumor drugs targeting nasopharyngeal carcinoma.

  12. Macrophage migration inhibitory factor promotes cardiac stem cell proliferation and endothelial differentiation through the activation of the PI3K/Akt/mTOR and AMPK pathways

    PubMed Central

    CUI, JINJIN; ZHANG, FENGYUN; WANG, YONGSHUN; LIU, JINGJIN; MING, XING; HOU, JINGBO; LV, BO; FANG, SHAOHONG; YU, BO

    2016-01-01

    Macrophage migration inhibitory factor (MIF) has pleiotropic immune functions in a number of inflammatory diseases. Recent evidence from expression and functional studies has indicated that MIF is involved in various aspects of cardiovascular disease. In this study, we aimed to determine whether MIF supports in vitro c-kit+CD45− cardiac stem cell (CSC) survival, proliferation and differentiation into endothelial cells, as well as the possible mechanisms involved. We observed MIF receptor (CD74) expression in mouse CSCs (mCSCs) using PCR and immunofluorescence staining, and MIF secretion by mCSCs using PCR and ELISA in vitro. Increasing amounts of exogenous MIF did not affect CD74 expression, but promoted mCSC survival, proliferation and endothelial differentiation. By contrast, treatment with an MIF inhibitor (ISO-1) or siRNA targeting CD74 (CD74-siRNA) suppressed the biological changes induced by MIF in the mCSCs. Increasing amounts of MIF increased the phosphorylation of Akt and mammalian target of rapamycin (mTOR), which are known to support cell survival, proliferation and differentiation. These effects of MIF on the mCSCs were abolished by LY294002 [a phosphoinositide 3-kinase (PI3K) inhibitor] and MK-2206 (an Akt inhibitor). Moreover, adenosine monophosphate-activated protein kinase (AMPK) phosphorylation increased following treatment with MIF. The AMPK inhibitor, compound C, partly blocked the pro-proliferative effects of MIF on the mCSCs. In conclusion, our results suggest that MIF promotes mCSC survival, proliferation and endothelial differentiation through the activation of the PI3K/Akt/mTOR and AMPK signaling pathways. Thus, MIF may prove to be a potential therapeutic factor in the treatment of heart failure and myocardial infarction by activating CSCs. PMID:27035848

  13. Polyphyllin G induce apoptosis and autophagy in human nasopharyngeal cancer cells by modulation of AKT and mitogen-activated protein kinase pathways in vitro and in vivo

    PubMed Central

    Chen, Chih-Jung; Lin, Jen-Tsun; Lo, Yu-Sheng; Chuang, Yi-Ching; Chien, Su-Yu; Chen, Mu-Kuan

    2016-01-01

    Polyphyllin G (also call polyphyllin VII), extract from rhizomes of Paris yunnanensis Franch, has been demonstrated to have strong anticancer activities in a wide variety of human cancer cell lines. Previous studies found that Polyphyllin G induced apoptotic cell death in human hepatoblastoma cancer and lung cancer cells. However, the underlying mechanisms of autophagy in human nasopharyngeal carcinoma (NPC) remain unclear. In this study, Polyphyllin G can potently induced apoptosis dependent on the activations of caspase-8, -3, and -9 and the changes of Bcl-2, Bcl-xL and Bax protein expression in different human NPC cell lines (HONE-1 and NPC-039). The amount of both LC3-II and Beclin-1 was intriguingly increased suggest that autophagy was induced in Polyphyllin G-treated NPC cells. To further clarify whether Polyphyllin G-induced apoptosis and autophagy depended on AKT/ERK/JNK/p38 MAPK signaling pathways, cells were combined treated with AKT inhibitor (LY294002), ERK1/2 inhibitor (U0126), p38 MAPK inhibitor (SB203580), or JNK inhibitor (SP600125). These results demonstrated that Polyphyllin G induced apoptosis in NPC cells through activation of ERK, while AKT, p38 MAPK and JNK were responsible for Polyphyllin G-induced autophagy. Finally, an administration of Polyphyllin G effectively suppressed the tumor growth in the NPC carcinoma xenograft model in vivo. In conclusion, our results reveal that Polyphyllin G inhibits cell viability and induces apoptosis and autophagy in NPC cancer cells, suggesting that Polyphyllin G is an attractive candidate for tumor therapies. Polyphyllin G may promise candidate for development of antitumor drugs targeting nasopharyngeal carcinoma. PMID:27602962

  14. Cross-Talk between NFkB and the PI3-Kinase/AKT Pathway Can Be Targeted in Primary Effusion Lymphoma (PEL) Cell Lines for Efficient Apoptosis

    PubMed Central

    Hussain, Azhar R.; Ahmed, Saeeda O.; Ahmed, Maqbool; Khan, Omar S.; Al AbdulMohsen, Sally; Platanias, Leonidas C.; Al-Kuraya, Khawla S.; Uddin, Shahab

    2012-01-01

    Background A number of constitutively activated signaling pathways play critical roles in the survival and growth of primary effusion lymphoma cells (PELs) including NFkB and PI3/AKT kinase cascades. NFkBis constitutively activated in a number of malignancies, including multiple myeloma, Burkitt’s lymphoma and diffuse large cell B-cell lymphoma. However, its role in primary effusion lymphoma has not been fully explored. Methodology/Principal Findings We used pharmacological inhibition and gene silencing to define the role of NFkB in growth and survival of PEL cells. Inhibition of NFkB activity by Bay11-7085 resulted in decreased expression of p65 in the nuclear compartment as detected by EMSA assays. In addition, Bay11-7085 treatment caused de-phosphorylation of AKT and its downstream targets suggesting a cross-talk between NFkB and the PI3-kinase/AKT pathway. Importantly, treatment of PEL cells with Bay11-7085 led to inhibition of cell viability and induced apoptosis in a dose dependent manner. Similar apoptotic effects were found when p65 was knocked down using specific small interference RNA. Finally, co-treatment of PEL cells with suboptimal doses of Bay11-7085 and LY294002 led to synergistic apoptotic responses in PEL cells. Conclusion/Significance These data support a strong biological-link between NFkB and the PI3-kinase/AKT pathway in the modulation of anti-apoptotic effects in PEL cells. Synergistic targeting of these pathways using NFKB- and PI3-kinase/AKT- inhibitors may have a therapeutic potential for the treatment of PEL and possibly other malignancies with constitutive activation of these pathways. PMID:22768179

  15. TNF-{alpha} promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

    SciTech Connect

    Wang, Cheng-hu; Cao, Guo-Fan; Jiang, Qin; Yao, Jin

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer TNF-{alpha} induces MMP-9 expression and secretion to promote RPE cell migration. Black-Right-Pointing-Pointer MAPK activation is not critical for TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer Akt and mTORC1 signaling mediate TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-{alpha}. -- Abstract: Tumor necrosis factor-alpha (TNF-{alpha}) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-{alpha} promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-{alpha}-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-{alpha}-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-{alpha} promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

  16. Regulation of cellular growth, apoptosis, and Akt activity in human U251 glioma cells by a combination of cisplatin with CRM197.

    PubMed

    Wang, Lifei; Wang, Ping; Liu, Yunhui; Xue, Yixue

    2012-01-01

    The aberrantly activated antiapoptotic phospatidyl-3-inositol-kinase (PI3K)/Akt signaling induced by cisplatin limits the effectiveness of chemotherapy; inhibition of this pathway may augment the sensitivity of tumor cells to cisplatin-induced toxicity and promote apoptosis. Cross-reacting material 197 (CRM197), the nontoxic mutant of diphtheria toxin, could act as an heparin-binding epidermal growth factor inhibitor and has been shown to have some anticancer effects, but the effect of CRM197 on glioma cells remains unclear. The aim of this study was to investigate the effects of a combination of cisplatin with CRM197 on the growth and apoptosis of human U251 glioma cells and the possible mechanism. In this study, we demonstrated that cisplatin or CRM197 induced a dose-dependent growth inhibition in U251 cells, but cisplatin at 5 µg/ml and CRM197 at 1 µg/ml did not affect the viability of human astrocytes. Cisplatin induced a time-dependent growth inhibition in U251 cells, whereas the growth-inhibitory effects induced by CRM197 alone or combined with cisplatin reached a peak at 24 h after treatment. Compared with the administration of cisplatin or CRM197 alone, CRM197 combined with cisplatin significantly enhanced U251 cell growth inhibition and apoptosis. Cisplatin induced sustained activation of Akt, whereas CRM197 markedly suppressed the Akt phosphorylation induced by cisplatin. The effects of growth inhibition and apoptosis were markedly enhanced after a combination of cisplatin with CRM197 plus the PI3K inhibitor LY294002 or wortmannin. Therefore, CRM197 combined with cisplatin could enhance growth inhibition and apoptosis of glioma cells by inhibiting the cisplatin-induced PI3K/Akt pathway.

  17. Salvianolic Acid B Inhibits Hydrogen Peroxide-Induced Endothelial Cell Apoptosis through Regulating PI3K/Akt Signaling

    PubMed Central

    Liu, Chen-Li; Xie, Li-Xia; Li, Min; Durairajan, Siva Sundara Kumar; Goto, Shinya; Huang, Jian-Dong

    2007-01-01

    Background Salvianolic acid B (Sal B) is one of the most bioactive components of Salvia miltiorrhiza, a traditional Chinese herbal medicine that has been commonly used for prevention and treatment of cerebrovascular disorders. However, the mechanism responsible for such protective effects remains largely unknown. It has been considered that cerebral endothelium apoptosis caused by reactive oxygen species including hydrogen peroxide (H2O2) is implicated in the pathogenesis of cerebrovascular disorders. Methodology and Principal Findings By examining the effect of Sal B on H2O2-induced apoptosis in rat cerebral microvascular endothelial cells (rCMECs), we found that Sal B pretreatment significantly attenuated H2O2-induced apoptosis in rCMECs. We next examined the signaling cascade(s) involved in Sal B-mediated anti-apoptotic effects. We showed that H2O2 induces rCMECs apoptosis mainly through the PI3K/ERK pathway, since a PI3K inhibitor (LY294002) blocked ERK activation caused by H2O2 and a specific inhibitor of MEK (U0126) protected cells from apoptosis. On the other hand, blockage of the PI3K/Akt pathway abrogated the protective effect conferred by Sal B and potentated H2O2-induced apoptosis, suggesting that Sal B prevents H2O2-induced apoptosis predominantly through the PI3K/Akt (upstream of ERK) pathway. Significance Our findings provide the first evidence that H2O2 induces rCMECs apoptosis via the PI3K/MEK/ERK pathway and that Sal B protects rCMECs against H2O2-induced apoptosis through the PI3K/Akt/Raf/MEK/ERK pathway. PMID:18091994

  18. Opposite regulation by PI3K/Akt and MAPK/ERK pathways of tissue factor expression, cell-associated procoagulant activity and invasiveness in MDA-MB-231 cells

    PubMed Central

    2012-01-01

    Background Tissue factor (TF), an initiator of blood coagulation, participates in cancer progression and metastasis. We recently found that inhibition of MAPK/ERK upregulated both full length TF (flTF) and soluble isoform TF (asTF) gene expression and cell-associated TF activity in breast cancer MDA-MB-231 cells. We explored the possible mechanisms, especially the possible interaction with EGFR and PI3K/Akt pathways. Methods A plasmid containing TF promoter −2174 ~ +128 plus luciferase reporter gene was introduced into MDA-MB-231 cells to evaluate TF promoter activity. In order to study the interaction of these pathways, ERK inhibitor (PD98059), PI3K inhibitors (LY294002, wortmannin), Akt inhibitor (A6730), and EGFR inhibitor (erlotinib) as well as the corresponding siRNAs were used to treat MDA-MB-231 cells, and ovarian cancer OVCAR-3 and SKOV-3 cells. Quantitative PCR and western blot were used to determine TF expression. One stage clotting assays were used to measure pro-coagulation activity of the MDA-MB-231 cells. Results We show that PI3K inhibitors LY294002, wortmannin and A6730 significantly inhibited TF promoter activity, and reduced TF mRNA and protein levels due to the inhibition of Akt phosphorylation. In contrast, ERK inhibitor PD98059 and ERK siRNA enhanced TF promoter activity by 2.5 fold and induced an increase in TF mRNA and protein levels in a dose dependent manner in these cells. The PI3K/Akt pathway was shown to be involved in PD98059-induced TF expression because the induction was inhibited by PI3K/Akt inhibitors. Most interestingly, the EGFR inhibitor erlotinib and EGFR siRNA also significantly suppressed PD98059- or ERK siRNA-induced TF promoter activity and TF protein expression. Similar results were found with ovarian cancer cells SKOV-3 and OVCAR-3. Furthermore, in MDA-MB-231, mRNA levels of asTF were regulated in a similar way to that of TF in response to the cell treatment. Conclusions This study showed a regulatory mechanism in

  19. Ethanol Extracts of Fruiting Bodies of Antrodia cinnamomea Suppress CL1-5 Human Lung Adenocarcinoma Cells Migration by Inhibiting Matrix Metalloproteinase-2/9 through ERK, JNK, p38, and PI3K/Akt Signaling Pathways

    PubMed Central

    Chen, Ying-Yi; Liu, Fon-Chang; Chou, Pei-Yu; Chien, Yi-Chung; Chang, Wun-Shaing Wayne; Huang, Guang-Jhong; Wu, Chieh-Hsi; Sheu, Ming-Jyh

    2012-01-01

    Cancer metastasis is a primary cause of cancer death. Antrodia cinnamomea (A. cinnamomea), a medicinal mushroom in Taiwan, has shown antioxidant and anticancer activities. In this study, we first observed that ethanol extract of fruiting bodies of A. cinnamomea (EEAC) exerted a concentration-dependent inhibitory effect on migration and motility of the highly metastatic CL1-5 cells in the absence of cytotoxicity. The results of a gelatin zymography assay showed that A. cinnamomea suppressed the activities of matrix metalloproteinase-(MMP-) 2 and MMP-9 in a concentration-dependent manner. Western blot results demonstrated that treatment with A. cinnamomea decreased the expression of MMP-9 and MMP-2; while the expression of the endogenous inhibitors of these proteins, that is, tissue inhibitors of MMP (TIMP-1 and TIMP-2) increased. Further investigation revealed that A. cinnamomea suppressed the phosphorylation of ERK1/2, p38, and JNK1/2. A. cinnamomea also suppressed the expressions of PI3K and phosphorylation of Akt. Furthermore, treatment of CL1-5 cells with inhibitors specific for PI3K (LY 294002), ERK1/2 (PD98059), JNK (SP600125), and p38 MAPK (SB203580) decreased the expression of MMP-2 and MMP-9. This is the first paper confirming the antimigration activity of this potentially beneficial mushroom against human lung adenocarcinoma CL1-5 cancer cells. PMID:22454661

  20. IPD-196, a novel phosphatidylinositol 3-kinase inhibitor with potent anticancer activity against hepatocellular carcinoma.

    PubMed

    Lee, Ju-Hee; Lee, Hyunseung; Yun, Sun-Mi; Jung, Kyung Hee; Jeong, Yujeong; Yan, Hong Hua; Hong, Sungwoo; Hong, Soon-Sun

    2013-02-01

    As the activation of phosphatidylinositol 3-kinase (PI3K) is associated with a wide variety of human malignancies, it is emerging as an attractive target for cancer treatment. In this study we synthesized a novel PI3Kα inhibitor, IPD-196 [ethyl 6-(5-(2,4-difluorophenylsulfonamido)pyridin-3-yl)imidazo[1,2-a]pyridine-3-carboxylate], and evaluated its anticancer effects on human hepatocellular carcinoma (HCC) cells. IPD-196 effectively inhibited the phosphorylation of downstream PI3K effectors such as Akt, mTOR, p70S6K, and 4E-BP1, and its antiproliferative effect was more potent than that of sorafenib or LY294002. It also induced cell cycle arrest at the G0/G1 phase as well as apoptosis by increasing the proportion of sub-G1 apoptotic cells, and the levels of cleaved PARP, caspase-3, and caspase-9. Furthermore, it decreased the expression of HIF-1α and VEGF in Huh-7 cells, and inhibited tube formation and migration of human umbilical vein endothelial cells, which was confirmed by a Matrigel plug assay in mice. Taken together, IPD-196 exhibited its anticancer activity through disruption of the PI3K/Akt pathway that caused cell cycle arrest, apoptosis induction, and inhibition of angiogenesis in human HCC cells. We therefore suggest that IPD-196 may be a potential candidate drug for targeted HCC therapy.

  1. Acid fibroblast growth factor preserves blood-brain barrier integrity by activating the PI3K-Akt-Rac1 pathway and inhibiting RhoA following traumatic brain injury

    PubMed Central

    Wu, Fenzan; Chen, Zaifeng; Tang, Chonghui; Zhang, Jinjing; Cheng, Li; Zuo, Hongxia; Zhang, Hongyu; Chen, Daqing; Xiang, Liping; Xiao, Jian; Li, Xiaokun; Xu, Xinlong; Wei, Xiaojie

    2017-01-01

    The blood-brain barrier (BBB) plays important roles in the recovery of traumatic brain injury (TBI) which is a major factor contributing to cerebral edema. Acid fibroblast growth factor (aFGF) contributes to maintain vascular integrity and restores nerve function. However, whether aFGF protects BBB following TBI remains unknown. The purpose of this study was to determine whether exogenous aFGF preserves BBB integrity by activating the PI3K-Akt-Rac1 pathway and inhibiting RhoA after TBI. BBB permeability was assessed using evans blue dye and fluorescein isothiocyanate dextran fluorescence. Neurofunctional tests, such as the garcia test, were conducted in a blinded fashion, and protein expression was evaluated via western blotting and immunofluorescence staining. Our results showed that aFGF improved neurofunctional deficits, preserved BBB integrity, and up-regulated tight junction proteins and adherens junction proteins 24 h after experimental TBI. However, the PI3K/Akt inhibitor LY294002 reversed the protective effects of aFGF on neurofunctional deficits and junction protein expression and significantly suppressed p-Akt and GTP-Rac1 activity. Furthermore, aFGF administration significantly decreased GTP-RhoA expression in the treated group compared with the vehicle group, while PI3K/Akt inhibition increased GTP-RhoA expression. Similar results were observed in vitro, as aFGF exerted protective effects on endothelial cell integrity by up-regulating junction proteins and PI3K-Akt-Rac1 pathway and down-regulating RhoA expression under oxygen-glucose deprivation/reoxygenation (OGD) conditions. These data suggest that exogenous aFGF reduces RhoA activity in part by activating the PI3K-Akt-Rac1 signaling pathway, thus improving neurofunctional deficits and preserving BBB integrity after TBI. PMID:28386321

  2. Potential role of O-GlcNAcylation and involvement of PI3K/Akt1 pathway in the expression of oncogenic phenotypes of gastric cancer cells in vitro.

    PubMed

    Zhang, Nuobei; Chen, Xin

    2016-11-01

    O-GlcNAcylation is a monosaccharide modification by a residue of N-acetylglucosamine (GlcNAc) attached to serine or threonine moieties on nuclear and cytoplasmic proteins. O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Increasing evidence suggests that O-GlcNAcylation is involved in a variety of human cancers. However, the exact role of O-GlcNAcylation in tumor progression remains unclear. Here, we show that O-GlcNAcylation accelerates oncogenic phenotypes of gastric cancer. First, cell models with increased or decreased O-GlcNAcylation were constructed by OGT overexpression, downregulation of OGA activity with specific inhibitor Thiamet-G, or silence of OGT. MTT assays indicated that O-GlcNAcylation increased proliferation of gastric cancer cells. Soft agar assay and Transwell assays showed that O-GlcNAcylation significantly enhanced cellular colony formation, migration, and invasion in vitro. Akt1 activity was stimulated by upregulation of phosphorylation at Ser473 mediated by elevated O-GlcNAcylation. The enhanced cell invasion by Thiamet-G treatment was suppressed by PI3K inhibitor LY294002. Although the cell invasion induced by Thiamet-G was reduced by Akt1 shRNA, it was still higher in comparison with that to the control (cells with Akt1 shRNA alone). And Akt1 overexpression promoted Thiamet-G-induced cell invasion. These results suggested that O-GlcNAcylation enhanced oncogenic phenotypes possibly partially involving PI3K/Akt signaling pathway.

  3. Japanese encephalitis virus induces matrix metalloproteinase-9 expression via a ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent AP-1 pathway in rat brain astrocytes

    PubMed Central

    2012-01-01

    Background Japanese encephalitis virus (JEV) infection is a major cause of acute encephalopathy in children, which destroys central nervous system (CNS) cells, including astrocytes and neurons. Matrix metalloproteinase (MMP)-9 has been shown to degrade components of the basal lamina, leading to disruption of the blood-brain barrier (BBB) and to contribute to neuroinflammatory responses in many neurological diseases. However, the detailed mechanisms of JEV-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) are largely unclear. Methods In this study, the effect of JEV on expression of MMP-9 was determined by gelatin zymography, western blot analysis, RT-PCR, and promoter assay. The involvement of AP-1 (c-Jun and c-Fos), c-Src, PDGFR, PI3K/Akt, and MAPKs in these responses were investigated by using the selective pharmacological inhibitors and transfection with siRNAs. Results Here, we demonstrate that JEV induces expression of pro-form MMP-9 via ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent, AP-1 activation in RBA-1 cells. JEV-induced MMP-9 expression and promoter activity were inhibited by pretreatment with inhibitors of AP-1 (tanshinone), c-Src (PP1), PDGFR (AG1296), and PI3K (LY294002), and by transfection with siRNAs of c-Jun, c-Fos, PDGFR, and Akt. Moreover, JEV-stimulated AP-1 activation was inhibited by pretreatment with the inhibitors of c-Src, PDGFR, PI3K, and MAPKs. Conclusion From these results, we conclude that JEV activates the ROS/c-Src/PDGFR/PI3K/Akt/MAPKs pathway, which in turn triggers AP-1 activation and ultimately induces MMP-9 expression in RBA-1 cells. These findings concerning JEV-induced MMP-9 expression in RBA-1 cells imply that JEV might play an important role in CNS inflammation and diseases. PMID:22251375

  4. Lycium barbarum polysaccharides inhibit proliferation and migration of bladder cancer cell lines BIU87 by suppressing Pi3K/AKT pathway

    PubMed Central

    Zhang, Xian-Jun; Yu, Hong-Yuan; Cai, Yong-jian; Ke, Mang

    2017-01-01

    The aim of this study was to verify whether Lycium barbarum polysaccharides inhibits proliferation and migration of BIU87 cells through Pi3K/AKT pathway. Different concentrations of Lycium barbarum polysaccharides were used to incubate with BIU87cells. LY-294002 and IGF-1 were used to inhibit and activate Pi3K/AKT pathway respectively. MTT were used to investigate the proliferation of BIU87cells. Transwell chambers and wound healing were used to test the migratory ability of BIU87cells. Western blotting were used to investigate the expressions of P21,P27,MMP-2, MMP-9, AKT and p-AKT in BIU87cells. Compared with the control group, the proliferation and migration of BIU87cells and the expression of p-AKT were significantly decreased in the study group; the inhibitory effect of the downregulation of p-AKT by LY-294002on the induction of BIU87cells proliferation and migration was identical to that of Lycium barbarum polysaccharides; upregulation of p-AKT by IGF-1 reversed the Lycium barbarum polysaccharides-induced inhibition of BIU87cells dedifferentiation. In conclusion, LBP inhibits the proliferation and migration of BIU87 cells by suppressing Pi3K/AKT signaling pathway. PMID:27992374

  5. Insulin as a Potent Stimulator of Akt, ERK and Inhibin-βE Signaling in Osteoblast-Like UMR-106 Cells

    PubMed Central

    Ramalingam, Mahesh; Kwon, Yong-Dae; Kim, Sung-Jin

    2016-01-01

    Insulin is a peptide hormone of the endocrine pancreas and exerts a wide variety of physiological actions in insulin sensitive tissues, such as regulation of glucose homeostasis, cell growth, differentiation, learning and memory. However, the role of insulin in osteoblast cells remains to be fully characterized. In this study, we demonstrated that the insulin (100 nM) has the ability to stimulate the phosphorylation of protein kinase B (Akt/PKB) and extracellular signal-regulated kinase (ERK) and the levels of inhibin-βE in the osteoblast-like UMR-106 cells. This insulin-stimulated activities were abolished by the PI3K and MEK1 inhibitors LY294002 and PD98059, respectively. This is the first report proving that insulin is a potential candidate that enables the actions of inhibin-βE subunit of the TGF-β family. The current investigation provides a foundation for the realization of insulin as a potential stimulator in survival signaling pathways in osteoblast-like UMR-106 cells. PMID:27302964

  6. Zinc-induced downregulation of Notch signaling is associated with cytoplasmic retention of Notch1-IC and RBP-Jk via PI3k-Akt signaling pathway.

    PubMed

    Baek, Sang-Hyun; Kim, Mi-Yeon; Mo, Jung-Soon; Ann, Eun-Jung; Lee, Kyu Shik; Park, Ji-Hye; Kim, Jin-Young; Seo, Mi-Sun; Choi, Eui-Ju; Park, Hee-Sae

    2007-09-18

    The Notch signaling pathway appears to perform an important function in the determination of cell fate and in differentiation, in a wide variety of organisms and cell types. In this study, we provide evidence that the inactivation of Notch signaling by zinc is achieved via a PI3K-Akt-dependent, cytoplasmic retention of Notch1-IC and RBP-Jk. Extracellular zinc has been determined to inhibit constitutive active mutants of both Notch1 (DeltaEN1) and Notch1-IC-mediated transcription. However, in such cases, neither the cleavage pattern of Notch nor the protein stability of Notch1-IC and RBP-Jk was found to have significantly changed. With regard to the modulation of Notch signaling, zinc appears to exert a significant negative influence on the binding occurring between Notch1 and RBP-Jk, both in vivo and in vitro. The zinc-induced inhibition of Notch signaling can be rescued via pretreatment with wortmannin or LY294002, both of which are specific PI3K signaling pathway inhibitors. Furthermore, we ascertained that zinc triggers the cytoplasmic retention of Notch1-IC and RBP-Jk, and that cytoplasmic retention could be rescued via treatment with wortmannin. Overall, we have determined that an important relationship exists between zinc and the Notch1 signaling pathway, and that this relationship is intimately involved with the cytoplasmic retention of Notch and RBP-Jk.

  7. 17β-estradiol impedes Bax-involved mitochondrial apoptosis of retinal nerve cells induced by oxidative damage via the phosphatidylinositol 3-kinase/Akt signal pathway.

    PubMed

    Li, Hongbo; Wang, Baoying; Zhu, Chunhui; Feng, Yan; Wang, Shaolan; Shahzad, Muhammad; Hu, Chenghu; Mo, Mingshu; Du, Fangying; Yu, Xiaorui

    2013-07-01

    Oxidative stress leading to retinal nerve cells (RNCs) apoptosis is a major cause of neurodegenerative disorders of the retina. 17β-Estradiol (E2) has been suggested to be a neuroprotective agent in the central nervous system; however, at present, the underlying mechanisms are not well understood, and the related research on the RNCs is less reported. Here, in order to investigate the protective role and mechanism of E2 against oxidative stress-induced damage on RNCs, the transmission electron microscopy and annexin V-FITC/propidium iodide assay were applied to detect the RNCs apoptosis. Western blot and real-time PCR were used to determine the expression of the critical molecules in Bcl-2 and caspase family associated with apoptosis. The transmission electron microscopy results showed that H(2)O(2) could induce typical features of apoptosis in RNCs, including formation of the apoptosome. E2 could, however, suppress the H(2)O(2)-induced morphological changes of apoptosis. Intriguingly, we observed E2-mediated phagocytic scavenging of apoptosome. In response to H(2)O(2)-induced apoptosis, Bax, acting as one of the pivotal pro-apoptotic members of Bcl-2 family, increased significantly, which directly resulted in an increased ratio of Bax to anti-apoptotic protein Bcl-2 (Bax/Bcl-2). Additionally, caspases 9 and 3, which are the critical molecules of the mitochondrial apoptosis pathway, were activated by H(2)O(2). In contrast, E2 exerted anti-apoptotic effects by reducing the expression of Bax to decrease the ratio of Bax/Bcl-2 and impeded the caspases 9/3 activation. Moreover, LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, could sharply block the effect of E2 in reducing the percentage of apoptotic cells resistance to H(2)O(2). And the attenuation of Bax, the reduced activities of caspases 9/3 and the impeded release of mitochondrial cytochrome c mediated by E2 resistance to H(2)O(2) damage were significantly retrieved by LY294002 administration. Taken

  8. Isoquercitrin Inhibits Hydrogen Peroxide-Induced Apoptosis of EA.hy926 Cells via the PI3K/Akt/GSK3β Signaling Pathway.

    PubMed

    Zhu, Meixia; Li, Jiankuan; Wang, Ke; Hao, Xuliang; Ge, Rui; Li, Qingshan

    2016-03-21

    Oxidative stress plays a critical role in endothelial injury and the pathogenesis of diverse cardiovascular diseases, including atherosclerosis. Isoquercitrin (quercetin-3-glucoside), a flavonoid distributed widely in plants, exhibits many biological activities, including anti-allergic, anti-viral, anti-inflammatory, and anti-oxidative effects. In the present study, the inhibitory effect of isoquercitrin on H2O2-induced apoptosis of EA.hy926 cells was evaluated. MTT assays showed that isoquercitrin significantly inhibited H2O2-induced loss of viability in EA.hy926 cells. Hoechst33342/PI and Annexin V-FITC/PI fluorescent double staining indicated that isoquercitrin inhibited H2O2-induced apoptosis of EA.hy926 cells. Western blotting demonstrated that isoquercitrin prevented H2O2-induced increases in cleaved caspase-9 and cleaved caspase-3 expression, while increasing expression of anti-apoptotic protein Mcl-1. Additionally, isoquercitrin significantly increased the expression of p-Akt and p-GSK3β in a dose-dependent manner in EA.hy926 cells. LY294002, a PI3K/Akt inhibitor, inhibited isoquercitrin-induced GSK3β phosphorylation and increase of Mcl-1 expression, which indicated that regulation of isoquercitrin on Mcl-1 expression was likely related to the modulation of Akt activation. These results demonstrated that the anti-apoptotic effect of isoquercitrin on H2O2-induced EA.hy926 cells was likely associated with the regulation of isoquercitrin on Akt/GSK3β signaling pathway and that isoquercitrin could be used clinically to interfere with the progression of endothelial injury-associated cardiovascular disease.

  9. Non-tumor tissue derived interleukin-17B activates IL-17RB/AKT/β-catenin pathway to enhance the stemness of gastric cancer

    PubMed Central

    Bie, Qingli; Sun, Caixia; Gong, Aihua; Li, Chunye; Su, Zhaoliang; Zheng, Dong; Ji, Xiaoyun; Wu, Yumin; Guo, Qi; Wang, Shengjun; Xu, Huaxi

    2016-01-01

    Inflammation is a critical component involved in tumor progression. Interleukin-17 (IL-17) belongs to a relatively new family of cytokines that has been associated with the progression of cancers. However, the role of IL-17B/IL-17RB (IL-17 receptor B) signaling to stemness of gastric cancer remains unknown. Here, we confirmed that the expression of IL-17RB in gastric cancer tissues was significantly increased, that overexpression was associated with poor prognosis of gastric cancer patients, and that overexpression was positively correlated with some stemness markers. Interestingly, the expression of IL-17B was upregulated in patient serum rather than gastric tumor tissues. Furthermore, exogenous rIL-17B significantly promoted the stemness of gastric cancer cells depending on IL-17RB and induced the expression of IL-17RB. Simultaneously, the expression of phosphorylated AKT, GSK-3β, and β-catenin as well as the nuclear translocation of β-catenin were significantly increased in the MGC-803 cell in a dose-dependent manner, when treated with rIL-17B. The AKT inhibitor, LY294002, and the knockdown of AKT expression reversed the rIL-17B-induced upregulation of β-catenin and some stemness markers. Together, our results indicate that the IL-17B/IL-17RB signal can promote the growth and migration of tumor cells, and upregulate cell stemness through activating the AKT/β-catenin pathway in gastric cancer, suggesting that IL-17RB may be a novel target in human gastric cancer therapy. PMID:27146881

  10. Phosphocreatine protects endothelial cells from oxidized low-density lipoprotein-induced apoptosis by modulating the PI3K/Akt/eNOS pathway.

    PubMed

    Ahsan, Anil; Han, Guozhu; Pan, Junfang; Liu, Shumin; Padhiar, Arshad Ahmed; Chu, Peng; Sun, Zhengwu; Zhang, Zonghui; Sun, Bin; Wu, Jingjun; Irshad, Aisha; Lin, Yuan; Peng, Jinyong; Tang, Zeyao

    2015-12-01

    Endothelial apoptosis triggered by oxidized low-density lipoprotein (oxLDL) can accelerate the progression of endothelial dysfunction atherosclerosis. Phosphocreatine (PCr) is a natural compound, which has been used in cardiac disease and cardiopulmonary resuscitation. However, its protective effects on atherosclerosis and its mechanism have not been clarified. In the present study, we investigated the anti-apoptotic effect of phosphocreatine in human umbilical vein endothelial cells (HUVECs) exposed to oxLDL and explored the possible mechanisms. HUVECs were pre-treated with 10-30 mM PCr and then stimulated with oxLDL. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, CCK assay, and flow cytometry respectively. Levels of Bax, Bcl-2, protein expression of protein kinase B (Akt), eNOS and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Lactate dehydrogenase (LDH), malondialdehyde (MDA), nitric oxide (NO) and superoxide dismutase (SOD) contents were determined by spectrophotometer. Our results showed that PCr dose-dependently prevented oxLDL associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, LDH and MDA leakage and loss of SOD, decrease of Bcl-2/Bax protein ratio, activation of caspase-3 and 9, and ROS generation. In addition, the antiapoptotic effect of PCr was partially inhibited by a PI3K inhibitor (LY294002) and also enhanced p-Akt/Akt protein ratio, eNOS activation and NO production. In conclusion, our data show that the inhibition of oxLDL-induced endothelial apoptosis by PCr is due, at least in part to its anti-oxidant activity and its ability to modulate the PI3K/Akt/eNOS signaling pathway.

  11. Tanshinone IIA Prevents Leu27IGF-II-Induced Cardiomyocyte Hypertrophy Mediated by Estrogen Receptor and Subsequent Akt Activation.

    PubMed

    Weng, Yueh-Shan; Wang, Hsueh-Fang; Pai, Pei-Ying; Jong, Gwo-Ping; Lai, Chao-Hung; Chung, Li-Chin; Hsieh, Dennis Jine-Yuan; HsuanDay, Cecilia; Kuo, Wei-Wen; Huang, Chih-Yang

    2015-01-01

    IGF-IIR plays important roles as a key regulator in myocardial pathological hypertrophy and apoptosis, which subsequently lead to heart failure. Salvia miltiorrhiza Bunge (Danshen) is a traditional Chinese medicinal herb used to treat cardiovascular diseases. Tanshinone IIA is an active compound in Danshen and is structurally similar to 17[Formula: see text]-estradiol (E[Formula: see text]. However, whether tanshinone IIA improves cardiomyocyte survival in pathological hypertrophy through estrogen receptor (ER) regulation remains unclear. This study investigates the role of ER signaling in mediating the protective effects of tanshinone IIA on IGF-IIR-induced myocardial hypertrophy. Leu27IGF-II (IGF-II analog) was shown in this study to specifically activate IGF-IIR expression and ICI 182,780 (ICI), an ER antagonist used to investigate tanshinone IIA estrogenic activity. We demonstrated that tanshinone IIA significantly enhanced Akt phosphorylation through ER activation to inhibit Leu27IGF-II-induced calcineurin expression and subsequent NFATc3 nuclear translocation to suppress myocardial hypertrophy. Tanshinone IIA reduced the cell size and suppressed ANP and BNP, inhibiting antihypertrophic effects induced by Leu27IGF-II. The cardioprotective properties of tanshinone IIA that inhibit Leu27IGF-II-induced cell hypertrophy and promote cell survival were reversed by ICI. Furthermore, ICI significantly reduced phospho-Akt, Ly294002 (PI3K inhibitor), and PI3K siRNA significantly reduced the tanshinone IIA-induced protective effect. The above results suggest that tanshinone IIA inhibited cardiomyocyte hypertrophy, which was mediated through ER, by activating the PI3K/Akt pathway and inhibiting Leu27IGF-II-induced calcineurin and NFATC3. Tanshinone IIA exerted strong estrogenic activity and therefore represented a novel selective ER modulator that inhibits IGF-IIR signaling to block cardiac hypertrophy.

  12. Mutant p53 promotes ovarian cancer cell adhesion to mesothelial cells via integrin β4 and Akt signals.

    PubMed

    Lee, Jong-Gyu; Ahn, Ji-Hye; Jin Kim, Tae; Ho Lee, Jae; Choi, Jung-Hye

    2015-07-30

    Missense mutations in the TP53 gene resulting in the accumulation of mutant proteins are extremely common in advanced ovarian cancer, which is characterised by peritoneal metastasis. Attachment of cancer cells to the peritoneal mesothelium is regarded as an initial, key step for the metastatic spread of ovarian cancer. In the present study, we investigated the possible role of a p53 mutant in the mesothelial adhesion of ovarian cancer cells. We found that OVCAR-3 cells with the R248 TP53 mutation (p53(R248)) were more adhesive to mesothelial Met5A cells than were A2780 cells expressing wild-type p53. In addition, ectopic expression of p53(R248) in p53-null SKOV-3 cells significantly increased adhesion to Met5A cells. Knockdown of mutant p53 significantly compromised p53(R248)-induced cell adhesion to Met5A cells. Microarray analysis revealed that several adhesion-related genes, including integrin β4, were markedly up-regulated, and certain signalling pathways, including PI3K/Akt, were activated in p53(R248) transfectants of SKOV-3 cells. Inhibition of integrin β4 and Akt signalling using blocking antibody and the inhibitor LY294002, respectively, significantly attenuated p53(R248)-mediated ovarian cancer-mesothelial adhesion. These data suggest that the p53(R248) mutant endows ovarian cancer cells with increased adhesiveness and that integrin β4 and Akt signalling are associated with the mutation-enhanced ovarian cancer-mesothelial cell adhesion.

  13. Combined effects of PI3K and SRC kinase inhibitors with imatinib on intracellular calcium levels, autophagy, and apoptosis in CML-PBL cells.

    PubMed

    Ciarcia, Roberto; Damiano, Sara; Montagnaro, Serena; Pagnini, Ugo; Ruocco, Antonio; Caparrotti, Giuseppe; d'Angelo, Danila; Boffo, Silvia; Morales, Fátima; Rizzolio, Flavio; Florio, Salvatore; Giordano, Antonio

    2013-09-01

    Imatinib induces a complete cytogenetic regression in a large percentage of patients affected by chronic myeloid leukemia (CML) until mutations in the kinase domain of BCR-ABL appear. Alternative strategies for CML patients include the inhibition of phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway, which is constitutively activated in leukemia cells and seems important for the regulation of cell proliferation, viability, and autophagy. In this study, we verified the effect of imatinib mesylate (IM), alone or in association with LY294002 (LY) (a specific PI3K protein tyrosine kinase inhibitor) or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1) (a Src tyrosine kinase inhibitor), on viability, intracellular calcium mobilization, apoptosis, and autophagy, in order to verify possible mechanisms of interaction. Our data demonstrated that PP1 and LY interact synergistically with IM by inducing apoptosis and autophagy in Bcr/Abl+ leukemia cells and this mechanism is related to the stress of the endoplasmic reticulum (ER). Our findings suggest a reasonable relationship between apoptotic and autophagic activity of tyrosine kinase inhibitors (TKIs) and the functionality of smooth ER Ca (2+)-ATPase and inositol triphosphate receptors, independently of intracellular calcium levels. Therapeutic strategies combining imatinib with PI3K and/or Src kinase inhibitors warrant further investigations in Bcr/Abl+ malignancies, particularly in the cases of imatinib mesylate-resistant disease.

  14. Isobutyrylshikonin inhibits lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in BV2 microglial cells by suppressing the PI3K/Akt-mediated nuclear transcription factor-κB pathway.

    PubMed

    Jayasooriya, Rajapaksha Gedara Prasad Tharanga; Lee, Kyoung-Tae; Kang, Chang-Hee; Dilshara, Matharage Gayani; Lee, Hak-Ju; Choi, Yung Hyun; Choi, Il-Whan; Kim, Gi-Young

    2014-12-01

    Microglia are important macrophages to defend against pathogens in the central nervous system (CNS); however, persistent or acute inflammation of microglia lead to CNS disorders via neuronal cell death. Therefore, we theorized that a good strategy for the treatment of CNS disorders would be to target inflammatory mediators from microglia in disease. Consequently, we investigated whether isobutyrylshikonin (IBS) attenuates the production of proinflammatory mediators, such as nitric oxide (NO) and prostaglandin E2, in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Treatment with IBS inhibited the secretion of NO and prostaglandin E2 (as well as the expression of their key regulatory genes), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2). Isobutyrylshikonin also suppressed LPS-induced DNA-binding activity of nuclear transcription factor-κB (NF-κB), by inhibiting the nuclear translocation of p50 and p65 in addition to blocking the phosphorylation and degradation of IκBα. Pretreatment with pyrrolidine dithiocarbamate, a specific NF-κB inhibitor, showed the down-regulation of LPS-induced iNOS and COX-2 messenger RNA by suppressing NF-κB activity. This indirectly suggests that IBS-mediated NF-κB inhibition is the main signaling pathway involved in the inhibition of iNOS and COX-2 expression. In addition, IBS attenuated LPS-induced phosphorylation of PI3K and Akt, which are upstream molecules of NF-κB, in LPS-stimulated BV2 microglial cells. The functional aspects of the PI3K/Akt signaling pathway were analyzed with LY294002, which is a specific PI3K/Akt inhibitor that attenuated LPS-induced iNOS and COX-2 expression by suppressing NF-κB activity. These data suggest that an IBS-mediated anti-inflammatory effect may be involved in suppressing the PI3K/Akt-mediated NF-κB signaling pathway.

  15. 2,2',4,4'-Tetrabromodiphenyl ether promotes human neuroblastoma SH-SY5Y cells migration via the GPER/PI3K/Akt signal pathway.

    PubMed

    Tian, P-C; Wang, H-L; Chen, G-H; Luo, Q; Chen, Z; Wang, Y; Liu, Y-F

    2016-02-01

    Neuroblastoma is the predominant tumor of early childhood. 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) has the highest concentration among all polybrominated diphenyl ether (PBDE) congeners in human body, particularly for children. Considering that accumulating evidences showed developmental neurotoxicity of PBDE, there is an urgent need to investigate the effects of BDE-47 on the development of neuroblastoma. This study revealed that BDE-47 had limited effects on the cytotoxicity while significantly increased the in vitro migration and invasion of human neuroblastoma SH-SY5Y cells. This was further confirmed by the results that BDE-47 treatment significantly downregulated the expression of E-cadherin and zona occludin-1 and upregulated the expression of matrix metalloproteinase-9 (MMP-9). Silencing of MMP-9 by specific small interfering RNA significantly abolished the BDE-47-induced migration and invasion of SH-SY5Y cells. Further, the signals G protein-coupled estrogen receptor 1 (GPER)/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (Akt) mediated the BDE-47-induced upregulation of MMP-9 and in vitro migration of SH-SY5Y cells since G15 (GPER inhibitor) and LY 294002 (PI3K/Akt inhibitor) significantly abolished the effects of BDE-47. Our results revealed that BDE-47 significantly triggered the metastasis of human neuroblastoma SH-SY5Y cells via upregulation of MMP-9 by the GPER/PI3K/Akt signal pathway. This study revealed for the first time that BDE-47 can promote the migration of SH-SY5Y cells. It also provided a better understanding about the metastasis of human neuroblastoma induced by environmental endocrine disruptors.

  16. Piceatannol inhibits MMP-9-dependent invasion of tumor necrosis factor-α-stimulated DU145 cells by suppressing the Akt-mediated nuclear factor-κB pathway

    PubMed Central

    JAYASOORIYA, RAJAPAKSHA GENDARA PRASAD THARANGA; LEE, YONG-GAB; KANG, CHANG-HEE; LEE, KYOUNG-TAE; CHOI, YUNG HYUN; PARK, SUNG-YONG; HWANG, JAE-KWAN; KIM, GI-YOUNG

    2013-01-01

    Piceatannol has potent anti-inflammatory, immunomodulatory, anticancer and antiproliferative effects. However, little is known about the mechanism by which piceatannol inhibits invasion and metastasis. The aim of the current study was to investigate the effects of piceatannol on the expression of matrix metalloproteinase-9 (MMP-9) in DU145 human prostate cancer cells. The results revealed that MMP-9 activity was significantly increased in response to tumor necrosis factor-α (TNF-α). However, treatment with piceatannol reversed TNF-α- and MMP-9-induced gelatin zymography and its gene expression. In addition, a Matrigel invasion assay determined that piceatannol reduces the TNF-α-induced invasion of DU145 cells. Nuclear factor-κ B (NF-κB) is a significant transcription factor that regulates numerous genes involved in tumor cell invasion and metastasis. Therefore, whether piceatannol acts on NF-κB to regulate MMP-9 gene expression was analyzed. The results revealed that piceatannol attenuates MMP-9 gene expression via the suppression of NF-κB activity. Using a specific NF-κB inhibitor, pyrrolidine dithiocarbamate, it was confirmed that TNF-α-induced MMP-9 gene expression is primarily regulated by NF-κB activation. Piceatannol inhibited NF-κB activity by suppressing nuclear translocation of the NF-κB p65 and p50 subunits. Furthermore, TNF-α-induced Akt phosphorylation was significantly downregulated in the presence of piceatannol. The Akt inhibitor LY294002 caused a significant decrease in TNF-α-induced NF-κB activity and MMP-9 gene expression. Overall, these data suggest that piceatannol inhibits TNF-α-induced invasion by suppression of MMP-9 activation via the Akt-mediated NF-κB pathway in DU145 prostate cancer cells. PMID:23255946

  17. Activating PTEN by COX-2 inhibitors antagonizes radiation-induced AKT activation contributing to radiosensitization

    SciTech Connect

    Meng, Zhen; Gan, Ye-Hua

    2015-05-01

    Radiotherapy is still one of the most effective nonsurgical treatments for many tumors. However, radioresistance remains a major impediment to radiotherapy. Although COX-2 inhibitors can induce radiosensitization, the underlying mechanism is not fully understood. In this study, we showed that COX-2 selective inhibitor celecoxib enhanced the radiation-induced inhibition of cell proliferation and apoptosis in HeLa and SACC-83 cells. Treatment with celecoxib alone dephosphorylated phosphatase and tensin homolog deleted on chromosome ten (PTEN), promoted PTEN membrane translocation or activation, and correspondingly dephosphorylated or inactivated protein kinase B (AKT). By contrast, treatment with radiation alone increased PTEN phosphorylation, inhibited PTEN membrane translocation and correspondingly activated AKT in the two cell lines. However, treatment with celecoxib or another COX-2 selective inhibitor (valdecoxib) completely blocked radiation-induced increase of PTEN phosphorylation, rescued radiation-induced decrease in PTEN membrane translocation, and correspondingly inactivated AKT. Moreover, celecoxib could also upregulate PTEN protein expression by downregulating Sp1 expression, thereby leading to the activation of PTEN transcription. Our results suggested that COX-2 inhibitors could enhance radiosensitization at least partially by activating PTEN to antagonize radiation-induced AKT activation. - Highlights: • COX-2 inhibitor, celecoxib, could enhance radiosensitization. • Radiation induced PTEN inactivation (phosphorylation) and AKT activation. • COX-2 inhibitor induced PTEN expression and activation, and inactivated AKT. • COX-2 inhibitor enhanced radiosensitization through activating PTEN.

  18. miR-222 induces Adriamycin resistance in breast cancer through PTEN/Akt/p27(kip1) pathway.

    PubMed

    Wang, Dan-Dan; Yang, Su-Jin; Chen, Xiu; Shen, Hong-Yu; Luo, Long-Ji; Zhang, Xiao-Hui; Zhong, Shan-Liang; Zhao, Jian-Hua; Tang, Jin-Hai

    2016-11-01

    The high resistant rate of Adriamycin (Adr) is associated with a poor prognosis of breast cancer in women worldwide. Since miR-222 might contribute to chemoresistance in many cancer types, in this study, we aimed to investigate its efficacy in breast cancer through PTEN/Akt/p27 (kip1) pathway. Firstly, in vivo, we verified that miR-222 was upregulated in chemoresistant tissues after surgery compared with the paired preneoadjuvant samples of 21 breast cancer patients. Then, human breast cancer Adr-resistant cell line (MCF-7/Adr) was constructed to validate the pathway from the parental sensitive cell line (MCF-7/S). MCF-7/Adr and MCF-7/S were transfected with miR-222 mimics, miR-222 inhibitors, or their negative controls, respectively. The results showed that inhibition of miR-222 in MCF-7/Adr significantly increased the expressions of PTEN and p27 (kip1) and decreased phospho-Akt (p-Akt) both in mRNA and protein levels (p < 0.05) by using quantitative real-time PCR (qRT-PCR) and western blot. MTT and flow cytometry suggested that lower expressed miR-222 enhanced apoptosis and decreased the IC50 of MCF-7/Adr cells. Additionally, immunofluorescence demonstrated that the subcellular location of p27 (kip1) was dislocated resulting from the alteration of miR-222. Conversely, in MCF-7/S transfected with miR-222 mimics, upregulation of miR-222 is associated with decreasing PTEN and p27 (kip1) and increasing Akt accompanied by less apoptosis and higher IC50. Importantly, Adr resistance induced by miR-222 overexpression through PTEN/Akt/p27 was completely blocked by LY294002, an Akt inhibitor. Taken together, these data firstly elucidated that miR-222 could reduce the sensitivity of breast cancer cells to Adr through PTEN/Akt/p27 (kip1) signaling pathway, which provided a potential target to increase the sensitivity to Adr in breast cancer treatment and further improved the prognosis of breast cancer patients.

  19. PARP1 inhibitors attenuate AKT phosphorylation via the upregulation of PHLPP1

    SciTech Connect

    Wang, Shuai; Wang, Huibo; Davis, Ben C.; Liang, Jiyong; Cui, Rutao; Chen, Sai-Juan; Xu, Zhi-Xiang

    2011-08-26

    Highlights: {yields} PARP1 inhibitors cause a cytotoxic effect independent of DNA repair impairment. {yields} PARP1 inhibitors attenuated AKT-FOXO3A signaling by activating PHLPP1. {yields} PHLPP1 regulates the sensitivity of cancer cells to PARP1 inhibitors. -- Abstract: Poly(ADP-ribose) polymerase-1 (PARP1) inhibitors are emerging as an important class of drugs for treating BRCA-deficient cancers. Recent discoveries have shown that PARP1 inhibitors may treat other cancer patients in addition to the relatively small proportion of patients carrying BRCA mutations. However, the additional targets by which PARP1 inhibitor-mediated tumor suppression remain poorly understood. In this study, we show that two PARP1 inhibitors, PJ-34 and 3-AB, attenuate AKT phosphorylation at serine 473 (S473) independent of DNA repair impairment. These inhibitors decrease the AKT-associated phosphorylation of FOXO3A, enhance the nuclear retention of FOXO3A, and activate its transcriptional activity. We further demonstrate that treatment with PJ-34 or 3-AB dramatically increases the level of PHLPP1. Overexpression of PHLPP1 enhances the PARP1 inhibitor-induced downregulation of AKT phosphorylation and increases tumor cell death. In contrast, knockdown of PHLPP1 abrogates the PARP1 inhibitor-mediated AKT inhibition and desensitizes cells to its treatment. Therefore, our findings not only show the robust role of PARP1 inhibitors in AKT inhibition but also develop a novel strategy to increase the effectiveness of cancer treatment via PARP1 inhibitor-induced PHLPP1 upregulation.

  20. Active form of AKT controls cell proliferation and response to apoptosis in hepatocellular carcinoma

    PubMed Central

    KUNTER, IMGE; ERDAL, ESRA; NART, DENIZ; YILMAZ, FUNDA; KARADEMIR, SEDAT; SAGOL, OZGUL; ATABEY, NESE

    2014-01-01

    Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality worldwide. Deregulation of the AKT signaling pathway has been found in HCC. However, the effect of AKT activation on the proliferation and apoptosis in HCC is not clear. Herein, expression of phosphorylated form of AKT (Ser 473) was investigated in HCC tumor (n=73), cirrhosis (n=17), normal liver (n=22) samples and in HCC cell lines (n=8). The results showed that expression of p-AKT was higher in tumor (53%) than in cirrhotic tissues (12%) while it was absent in normal liver (p<0.0001). p-AKT expression was also associated with number of tumor nodules and differentiation status (p<0.05). LY294002 induced cell cycle arrest at G0/G1 in SNU-449 and Mahlavu cells by decreasing expression of CDK2, CDK4, CycD1, CycD3, CycE, CycA and increasing expression of p21 and p27 as well; it also caused a decrease in the E2F1 transcriptional activity through declining phosphorylated Rb. LY294002 did not affect the basal level of apoptosis; however, it amplified cisplatin-induced apoptosis in SNU-449 cells. When the p-AKT level was decreased specifically after transfection with the DN-AKT plasmid, SNU-449 cells became more sensitive to cisplatin-induced apoptosis. HuH-7 cells with no basal p-AKT, were markedly affected by the treatment of doxorubicin. Thus, Akt signaling controls growth and chemical-induced apoptosis in HCC and p-AKT may be a potential target for therapeutic interventions in HCC patients. PMID:24337632

  1. Hepatocyte growth factor promoting the proliferation of human eccrine sweat gland epithelial cells is relative to AKT signal channel and β-catenin.

    PubMed

    Lei, Xia; Wu, Jinjin; Liu, Bo; Lu, Yuangang

    2012-01-01

    Hepatocyte growth factor (HGF) is a multi-effective molecule, playing important roles in organ growth, tumorigenesis and trauma healing. This experiment aims at studying the promoting function of HGF on the proliferation of human eccrine sweat gland epithelial cells (hESGc) and its relative signal channels. After HGF at different concentrations were added into cells, MTT was adopted to detect the cell proliferations, Annexin-V/PI the cell apoptosis, and Westernblot the expressions of p-AKT, AKT, p-ERK, p-GSK3β, p-IKBα, and β-catenin in hESGc. After adding siRNA c-Met to block HGF or LY294002 to inhibit p-AKT, we used MTT to detect the proliferation of hESGc and Westernblot to detect the expression of β-catenin. As a result, 20-40 ng/mL HGF could promote the proliferation of hESGc and inhibit its apoptosis. HGF could promote the expressions of p-AKT1/2/3, p-ERK, p-GSK3β, p-IKBα, and β-catenin. The additions of siRNA c-Met to block HGF or LY294002 to inhibit p-AKT could downregulate β-catenin and inhibit the proliferation promotion caused by HGF. Consequently, we concluded HGF can promote the proliferation of human eccrine sweat gland epithelial cells, which is relative to AKT signal channel and β-catenin.

  2. Andrographolide inhibits growth of human T-cell acute lymphoblastic leukemia Jurkat cells by downregulation of PI3K/AKT and upregulation of p38 MAPK pathways

    PubMed Central

    Yang, Tingfang; Yao, Shuluan; Zhang, Xianfeng; Guo, Yan

    2016-01-01

    T-cell acute lymphoblastic leukemia (T-ALL) as a prevalent hematologic malignancy is one of the most common malignant tumors worldwide in children. Andrographolide (Andro), the major active component from Andrographis paniculata, has been shown to possess antitumor activities in several types of cancer cells. However, whether Andro would inhibit T-ALL cell growth remains unclear. In this study, we investigated the cytotoxic effect of Andro on human T-ALL Jurkat cells and explored the mechanisms of cell death. Cell apoptosis was assayed by flow cytometry, and the signaling transduction for Andro was analyzed by Western blotting. The results indicated 10 μg/mL Andro could significantly induce Jurkat cells’ apoptosis, depending on the inhibition of PI3K/AKT pathway. Moreover, Andro-induced apoptosis is enhanced by AKT-selective inhibitor LY294002. ERK- or JNK-selective inhibitors PD98059 and SP600125 had no effect on Andro-induced apoptosis. In addition, p38 inhibitor SB203580 could reverse Andro-induced apoptosis in Jurkat cells. We also found that the protein expression of p-p53 and p-p38 were increased after Andro treatments. The result of an in vivo study also demonstrated Andro’s dose-dependent inhibition in subcutaneous Jurkat xenografts. In conclusion, our findings explained a novel mechanism of drug action by Andro in Jurkat cells and suggested that Andro might be developed into a new candidate therapy for T-ALL patients in the coming days. PMID:27114702

  3. Akt inhibitors in cancer treatment: The long journey from drug discovery to clinical use (Review)

    PubMed Central

    NITULESCU, GEORGE MIHAI; MARGINA, DENISA; JUZENAS, PETRAS; PENG, QIAN; OLARU, OCTAVIAN TUDOREL; SALOUSTROS, EMMANOUIL; FENGA, CONCETTINA; SPANDIDOS, DEMETRIOS A.; LIBRA, MASSIMO; TSATSAKIS, ARISTIDIS M.

    2016-01-01

    Targeted cancer therapies are used to inhibit the growth, progression, and metastasis of the tumor by interfering with specific molecular targets and are currently the focus of anticancer drug development. Protein kinase B, also known as Akt, plays a central role in many types of cancer and has been validated as a therapeutic target nearly two decades ago. This review summarizes the intracellular functions of Akt as a pivotal point of converging signaling pathways involved in cell growth, proliferation, apoptotis and neo-angiogenesis, and focuses on the drug design strategies to develop potent anticancer agents targeting Akt. The discovery process of Akt inhibitors has evolved from adenosine triphosphate (ATP)-competitive agents to alternative approaches employing allosteric sites in order to overcome the high degree of structural similarity between Akt isoforms in the catalytic domain, and considerable structural analogy to the AGC kinase family. This process has led to the discovery of inhibitors with greater specificity, reduced side-effects and lower toxicity. A second generation of Akt has inhibitors emerged by incorporating a chemically reactive Michael acceptor template to target the nucleophile cysteines in the catalytic activation loop. The review outlines the development of several promising drug candidates emphasizing the importance of each chemical scaffold. We explore the pipeline of Akt inhibitors and their preclinical and clinical examination status, presenting the potential clinical application of these agents as a monotherapy or in combination with ionizing radiation, other targeted therapies, or chemotherapy. PMID:26698230

  4. Biological Effect of Licochalcone C on the Regulation of PI3K/Akt/eNOS and NF-κB/iNOS/NO Signaling Pathways in H9c2 Cells in Response to LPS Stimulation.

    PubMed

    Franceschelli, Sara; Pesce, Mirko; Ferrone, Alessio; Gatta, Daniela Maria Pia; Patruno, Antonia; Lutiis, Maria Anna De; Quiles, José Luis; Grilli, Alfredo; Felaco, Mario; Speranza, Lorenza

    2017-03-23

    Polyphenols compounds are a group molecules present in many plants. They have antioxidant properties and can also be helpful in the management of sepsis. Licochalcone C (LicoC), a constituent of Glycyrrhiza glabra, has various biological and pharmacological properties. In saying this, the effect of LicoC on the inflammatory response that characterizes septic myocardial dysfunction is poorly understood. The aim of this study was to determine whether LicoC exhibits anti-inflammatory properties on H9c2 cells that are stimulated with lipopolysaccharide. Our results have shown that LicoC treatment represses nuclear factor-κB (NF-κB) translocation and several downstream molecules, such as inducible nitric oxide synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Moreover, LicoC has upregulated the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) signaling pathway. Finally, 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), a specific PI3K inhibitor, blocked the protective effects of LicoC. These findings indicate that LicoC plays a pivotal role in cardiac dysfunction in sepsis-induced inflammation.

  5. Tianeptine sodium salt suppresses TNF-α-induced expression of matrix metalloproteinase-9 in human carcinoma cells via suppression of the PI3K/Akt-mediated NF-κB pathway.

    PubMed

    Jayasooriya, Rajapaksha Gedara Prasad Tharanga; Dilshara, Matharage Gayani; Choi, Yung Hyun; Moon, Sung-Kwon; Kim, Wun-Jae; Kim, Gi-Young

    2014-09-01

    Tianeptine sodium salt (TSS) is a selective facilitator of serotonin, but there are no reports regarding anti-invasive effects of TSS. Therefore, we investigated the effect of TSS on the expression of matrix metalloproteinase-9 (MMP-9) and invasion in three different human carcinoma cell lines. Our findings showed that MMP-9 activity was significantly increased in response to tumor necrosis factor-α (TNF-α), and that TSS reduced TNF-α-induced MMP-9 activity in a dose-dependent manner. TSS also downregulated both MMP-9 expression and TNF-α-induced MMP-9 promoter activity. Using a matrigel invasion assay, we showed that TSS significantly attenuated invasive rates in TNF-α-stimulated LNCaP prostate carcinoma cells. Furthermore, TSS suppressed TNF-α-induced NF-κB activity, which is a potential transcriptional factor for regulating many invasive genes, including MMP-9, by suppressing IκB degradation and nuclear translocation of NF-κB subunits in LNCaP prostate carcinoma cells. TSS also downregulated TNF-α-induced phosphorylation of phosphatidyl-inositol 3 kinase (PI3K) and Akt, and a selective PI3K/Akt inhibitor, LY294002, diminished TNF-α-induced NF-κB activation followed by levels of MMP-9, suggesting that TSS also reduces MMP-9 expression by inhibiting the PI3K/Akt-mediated NF-κB pathway. These results indicate that TSS is a potential anti-invasive agent by suppression of TNF-α-induced MMP-9 expression via inhibition of PI3K/Akt-mediated NF-κB activity.

  6. The inflammatory cytokine TNF-α promotes the premature senescence of rat nucleus pulposus cells via the PI3K/Akt signaling pathway

    PubMed Central

    Li, Pei; Gan, Yibo; Xu, Yuan; Song, Lei; Wang, Liyuan; Ouyang, Bin; Zhang, Chengmin; Zhou, Qiang

    2017-01-01

    Premature senescence of nucleus pulposus (NP) cells and inflammation are two common features of degenerated discs. This study investigated the effects of the inflammatory cytokine TNF-α on the premature senescence of NP cells and the molecular mechanism behind this process. Rat NP cells were cultured with or without different concentrations of TNF-α for 1 and 3 days. The inhibitor LY294002 was used to determine the role of the PI3K/Akt pathway. NP cells that were incubated with TNF-α for 3 days followed by 3 days of recovery in the control medium were used to analyze cellular senescence. Results showed that TNF-α promoted premature senescence of NP cells, as indicated by decreased cell proliferation, decreased telomerase activity, increased SA-β-gal staining, the fraction of cells arrested in the G1 phase of the cell cycle, the attenuated ability to synthesize matrix proteins and the up-regulated expression of the senescence marker p16 and p53. Moreover, a high TNF-α concentration produced greater effects than a low TNF-α concentration on day 3 of the experiment. Further analysis indicated that the inhibition of the PI3K/Akt pathway attenuated the TNF-α-induced premature senescence of NP cells. Additionally, TNF-α-induced NP cell senescence did not recover after TNF-α was withdrawn. In conclusion, TNF-α promotes the premature senescence of NP cells, and activation of the PI3K/Akt pathway is involved in this process. PMID:28211497

  7. The antipsychotic agent chlorpromazine induces autophagic cell death by inhibiting the Akt/mTOR pathway in human U-87MG glioma cells.

    PubMed

    Shin, Soon Young; Lee, Kyoung Sun; Choi, Yang-Kyu; Lim, Hyunjung Jade; Lee, Hong Ghi; Lim, Yoongho; Lee, Young Han

    2013-09-01

    2-Chloro-10-[3(-dimethylamino)propyl]phenothiazine mono hydrochloride (chlorpromazine; CPZ) is an antipsychotic agent that was originally developed to control psychotic disorders. The cytotoxic properties of the CPZ are well known, but its mechanism of action is poorly understood. In this study, we investigated the role of apoptosis and autophagy in CPZ-induced cytotoxicity in U-87MG glioma cells. CPZ treatment inhibited cell proliferation and long-term clonogenic survival. Additionally, CPZ triggered autophagy, as indicated by electron microscopy and accumulation of the membrane form of microtubule-associated protein 1 light chain 3 (LC3-II); however, CPZ did not induce apoptosis. Inhibition of autophagy by expression of Beclin 1 small interfering RNA (siRNA) in U-87MG cells attenuated CPZ-induced LC3-II formation. Furthermore, U-87MG cells expressing Beclin 1 siRNA attenuated CPZ-induced cell death. CPZ inhibited phosphatidylinositol 3-kinase (PI3K)/AKT/ mTOR pathway in U-87MG cells. Treatment with LY294002, a PI3K inhibitor, alone increased the accumulation of LC3-II and potentiated the effect of CPZ. In contrast, exogenous expression of AKT partially inhibited CPZ-induced LC3-II formation. When U-87MG cells were implanted into the brain of athymic nude mouse, CPZ triggered autophagy and inhibited xenograft tumor growth. These results provided the first evidence that CPZ-induced cytotoxicity is mediated through autophagic cell death in PTEN (phosphatase and tensin homolog deleted on chromosome 10)-null U-87MG glioma cells by inhibiting PI3K/AKT/mTOR pathway.

  8. Licochalcone A induces autophagy through PI3K/Akt/mTOR inactivation and autophagy suppression enhances Licochalcone A-induced apoptosis of human cervical cancer cells

    PubMed Central

    Ying, Tsung-Ho; Lin, Chu-Liang; Lin, Chia-Liang; Hsueh, Jung-Tsung; Hsieh, Yi-Hsien

    2015-01-01

    The use of dietary bioactive compounds in chemoprevention can potentially reverse, suppress, or even prevent cancer progression. However, the effects of licochalcone A (LicA) on apoptosis and autophagy in cervical cancer cells have not yet been clearly elucidated. In this study, LicA treatment was found to significantly induce the apoptotic and autophagic capacities of cervical cancer cells in vitro and in vivo. MTT assay results showed dose- and time-dependent cytotoxicity in four cervical cancer cell lines treated with LicA. We found that LicA induced mitochondria-dependent apoptosis in SiHa cells, with decreasing Bcl-2 expression. LicA also induced autophagy effects were examined by identifying accumulation of Atg5, Atg7, Atg12 and microtubule-associated protein 1 light chain 3 (LC3)-II. Treatment with autophagy-specific inhibitors (3-methyladenine and bafilomycin A1) enhanced LicA-induced apoptosis. In addition, we suggested the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of mTOR pathway by LicA. Furthermore, the inhibition of PI3K/Akt by LY294002/si-Akt or of mTOR by rapamycin augmented LicA-induced apoptosis and autophagy. Finally, the in vivo mice bearing a SiHa xenograft, LicA dosed at 10 or 20 mg/kg significantly inhibited tumor growth. Our findings demonstrate the chemotherapeutic potential of LicA for treatment of human cervical cancer. PMID:26311737

  9. Bone marrow-derived mesenchymal stem cells enhance autophagy via PI3K/AKT signalling to reduce the severity of ischaemia/reperfusion-induced lung injury.

    PubMed

    Li, Jing; Zhou, Jian; Zhang, Dan; Song, Yuanlin; She, Jun; Bai, Chunxue

    2015-10-01

    Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow-derived mesenchymal stem cells (BM-MSCs) have great potential for cell therapy. However, the mechanisms underlying the BM-MSC activation of autophagy to provide a therapeutic effect in ischaemia/reperfusion-induced lung injury (IRI) remain unclear. Thus, we investigate the activation of autophagy in IRI following transplantation with BM-MSCs. Seventy mice were pre-treated with BM-MSCs before they underwent lung IRI surgery in vivo. Human pulmonary micro-vascular endothelial cells (HPMVECs) were pre-conditioned with BM-MSCs by oxygen-glucose deprivation/reoxygenation (OGD) in vitro. Expression markers for autophagy and the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signalling pathway were analysed. In IRI-treated mice, administration of BM-MSCs significantly attenuated lung injury and inflammation, and increased the level of autophagy. In OGD-treated HPMVECs, co-culture with BM-MSCs attenuated endothelial permeability by decreasing the level of cell death and enhanced autophagic activation. Moreover, administration of BM-MSCs decreased the level of PI3K class I and p-Akt while the expression of PI3K class III was increased. Finally, BM-MSCs-induced autophagic activity was prevented using the inhibitor LY294002. Administration of BM-MSCs attenuated lung injury by improving the autophagy level via the PI3K/Akt signalling pathway. These findings provide further understanding of the mechanisms related to BM-MSCs and will help to develop new cell-based therapeutic strategies in lung injury.

  10. Cinnamaldehyde affects the biological behavior of human colorectal cancer cells and induces apoptosis via inhibition of the PI3K/Akt signaling pathway.

    PubMed

    Li, Jiepin; Teng, Yuhao; Liu, Shenlin; Wang, Zifan; Chen, Yan; Zhang, Yingying; Xi, Songyang; Xu, Song; Wang, Ruiping; Zou, Xi

    2016-03-01

    Cinnamaldehyde (CA) is a bioactive compound isolated from the stem bark of Cinnamomum cassia, that has been identified as an antiproliferative substance with pro-apoptotic effects on various cancer cell lines in vitro. In the present study, the effects of CA on human colon cancer cells were investigated at both the molecular and cellular levels. Three types of colorectal cancer cells at various stages of differentiation and invasive ability (SW480, HCT116 and LoVo) were treated with CA at final concentrations of 20, 40 and 80 µg/ml for 24 h. Compared with the control group, the proliferation inhibition rate of the human colorectal cancer cells following treatment with CA increased in a dose- and time-dependent manner. The invasion and adhesion abilities of the cells were significantly inhibited as indicated by Transwell and cell-matrix adhesion assays. Meanwhile, CA also upregulated the expression of E-cadherin and downregulated the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9. CA also elevated the apoptotic rate. The levels of pro-apoptotic genes were upregulated while the levels of apoptosis inhibitory genes were decreased which further confirmed the pro-apoptotic effect of CA. In order to explore the mechanism of CA-induced apoptosis, insulin-like growth factor-1 (IGF-1) and PI3K inhibitor (LY294002) were used to regulate the phosphoinositide 3-kinase (PI3K)/AKT pathway. The transcription activity of PI3K/AKT was markedly inhibited by CA, as well as IGF-1 which functions as an anti-apoptotic factor. In conclusion, CA has the potential to be developed as a new antitumor drug. The mechanisms of action involve the regulation of expression of genes involved in apoptosis, invasion and adhesion via inhibition of the PI3K/Akt signaling pathway.

  11. Bone marrow-derived mesenchymal stem cells enhance autophagy via PI3K/AKT signalling to reduce the severity of ischaemia/reperfusion-induced lung injury

    PubMed Central

    Li, Jing; Zhou, Jian; Zhang, Dan; Song, Yuanlin; She, Jun; Bai, Chunxue

    2015-01-01

    Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow-derived mesenchymal stem cells (BM-MSCs) have great potential for cell therapy. However, the mechanisms underlying the BM-MSC activation of autophagy to provide a therapeutic effect in ischaemia/reperfusion-induced lung injury (IRI) remain unclear. Thus, we investigate the activation of autophagy in IRI following transplantation with BM-MSCs. Seventy mice were pre-treated with BM-MSCs before they underwent lung IRI surgery in vivo. Human pulmonary micro-vascular endothelial cells (HPMVECs) were pre-conditioned with BM-MSCs by oxygen-glucose deprivation/reoxygenation (OGD) in vitro. Expression markers for autophagy and the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signalling pathway were analysed. In IRI-treated mice, administration of BM-MSCs significantly attenuated lung injury and inflammation, and increased the level of autophagy. In OGD-treated HPMVECs, co-culture with BM-MSCs attenuated endothelial permeability by decreasing the level of cell death and enhanced autophagic activation. Moreover, administration of BM-MSCs decreased the level of PI3K class I and p-Akt while the expression of PI3K class III was increased. Finally, BM-MSCs-induced autophagic activity was prevented using the inhibitor LY294002. Administration of BM-MSCs attenuated lung injury by improving the autophagy level via the PI3K/Akt signalling pathway. These findings provide further understanding of the mechanisms related to BM-MSCs and will help to develop new cell-based therapeutic strategies in lung injury. PMID:26177266

  12. Agmatine Reduces Lipopolysaccharide-Mediated Oxidant Response via Activating PI3K/Akt Pathway and Up-Regulating Nrf2 and HO-1 Expression in Macrophages

    PubMed Central

    Chai, Jianshen; Luo, Li; Hou, Fengyan; Fan, Xia; Yu, Jing; Ma, Wei; Tang, Wangqi; Yang, Xue; Zhu, Junyu; Kang, Wenyuan; Yan, Jun; Liang, Huaping

    2016-01-01

    Macrophages are key responders of inflammation and are closely related with oxidative stress. Activated macrophages can enhance oxygen depletion, which causes an overproduction of reactive oxygen species (ROS) and leads to further excessive inflammatory response and tissue damage. Agmatine, an endogenous metabolite of L-arginine, has recently been shown to have neuroprotective effects based on its antioxidant properties. However, the antioxidant effects of agmatine in peripheral tissues and cells, especially macrophages, remain unclear. In this study we explored the role of agmatine in mediating antioxidant effects in RAW 264.7 cells and studied its antioxidant mechanism. Our data demonstrate that agmatine is an activator of Nrf2 signaling that markedly enhances Nrf2 nuclear translocation, increases nuclear Nrf2 protein level, up-regulates the expression of the Nrf2 downstream effector HO-1, and attenuates ROS generation induced by Lipopolysaccharide (LPS). We further demonstrated that the agmatine-induced activation of Nrf2 is likely through the PI3K/Akt pathway. LY294002, a specific PI3K/Akt inhibitor, abolished agmatine-induced HO-1 up-regulation and ROS suppression significantly. Inhibiting HO-1 pathway significantly attenuated the antioxidant effect of agmatine which the products of HO-1 enzymatic activity contributed to. Furthermore, the common membrane receptors of agmatine were evaluated, revealing that α2-adrenoceptor, I1-imidazoline receptor or I2-imidazoline receptor are not required by the antioxidant properties of agmatine. Taken together, our findings revealed that agmatine has antioxidant activity against LPS-induced ROS accumulation in RAW 264.7 cells involving HO-1 expression induced by Nrf2 via PI3K/Akt pathway activation. PMID:27685463

  13. NLS‑RARα modulates acute promyelocytic leukemia NB4 cell proliferation and differentiation via the PI3K/AKT pathway.

    PubMed

    Song, Hao; Li, Liu; Zhong, Liang; Yang, Rong; Jiang, Kailing; Yang, Xiaoqun; Liu, Beizhong

    2016-12-01

    In patients with acute promyelocytic leukemia (APL), ~98% express the promyelocytic leukemia (PML)‑retinoic acid receptor α (RARα) fusion protein. Previous studies have shown that, in primary leukemia cells of patients with APL, the cleavage of PML‑RARα by neutrophil elastase is important for its ability to initiate APL. This cleavage separates the nuclear localization signal (NLS) from PML, leading to the formation of a novel protein, NLS‑RARα, although its underlying mechanism in APL remains to be fully elucidated. In the present study, the role of NLS‑RARα on the proliferation and differentiation of APL NB4 cells was investigated. Lentiviral vectors were constructed and transfected NLS‑RARα in NB4 cells, puromycin was used to select the stable transfected cell lines. Cell Counting Kit‑8 and flow cytometry analysis revealed that the efficient overexpression of NLS‑RARα significantly promoted NB4 cell proliferation and inhibited all‑trans retinoic acid‑induced cell differentiation. Furthermore, the NLS‑RARα protein promoted a significant increase in AKT and glycogen synthase kinase 3β (GSK‑3β) phosphorylation. The protein levels of phosphorylated (p) AKT and pGSK‑3β were decreased following pretreatment with the phosphatidylinositol 3‑kinase (PI3K) inhibitor, LY294002. These findings suggested that NLS‑RARα was an important molecule associated with the occurrence of APL via the PI3K‑AKT signaling pathway, and indicated that the NLS‑RARα protein may be a novel target for the treatment of APL.

  14. Activation of Akt/FKHR in the medulla oblongata contributes to spontaneous respiratory recovery after incomplete spinal cord injury in adult rats.

    PubMed

    Felix, M S; Bauer, S; Darlot, F; Muscatelli, F; Kastner, A; Gauthier, P; Matarazzo, V

    2014-09-01

    After incomplete spinal cord injury (SCI), patients and animals may exhibit some spontaneous functional recovery which can be partly attributed to remodeling of injured neural circuitry. This post-lesion plasticity implies spinal remodeling but increasing evidences suggest that supraspinal structures contribute also to the functional recovery. Here we tested the hypothesis that partial SCI may activate cell-signaling pathway(s) at the supraspinal level and that this molecular response may contribute to spontaneous recovery. With this aim, we used a rat model of partial cervical hemisection which injures the bulbospinal respiratory tract originating from the medulla oblongata of the brainstem but leads to a time-dependent spontaneous functional recovery of the paralyzed hemidiaphragm. We first demonstrate that after SCI the PI3K/Akt signaling pathway is activated in the medulla oblongata of the brainstem, resulting in an inactivation of its pro-apoptotic downstream target, forkhead transcription factor (FKHR/FOXO1A). Retrograde labeling of medullary premotoneurons including respiratory ones which project to phrenic motoneurons reveals an increased FKHR phosphorylation in their cell bodies together with an unchanged cell number. Medulla infusion of the PI3K inhibitor, LY294002, prevents the SCI-induced Akt and FKHR phosphorylations and activates one of its death-promoting downstream targets, Fas ligand. Quantitative EMG analyses of diaphragmatic contractility demonstrate that the inhibition of medulla PI3K/Akt signaling prevents spontaneous respiratory recovery normally observed after partial cervical SCI. Such inhibition does not however affect either baseline contractile frequency or the ventilatory reactivity under acute respiratory challenge. Together, these findings provide novel evidence of supraspinal cellular contribution to the spontaneous respiratory recovery after partial SCI.

  15. Ankyrin repeat and suppressor of cytokine signaling (SOCS) box-containing protein (ASB) 15 alters differentiation of mouse C2C12 myoblasts and phosphorylation of mitogen-activated protein kinase and Akt.

    PubMed

    McDaneld, T G; Spurlock, D M

    2008-11-01

    activator, in ASB15+ was able to partially override the previously observed phenotype of delayed differentiation, whereas administration of the PI3K/ Akt inhibitor, LY294002, decreased phosphorylation of Akt and differentiation of all cell lines similar to the untreated ASB15+ myoblasts. These results provide initial evidence that ASB15 has a role in early myoblast differentiation and that its effects may be mediated in part by the PI3K/Akt signal transduction pathway.

  16. Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate.

    PubMed

    Navrátilová, Jarmila; Karasová, Martina; Kohutková Lánová, Martina; Jiráková, Ludmila; Budková, Zuzana; Pacherník, Jiří; Šmarda, Jan; Beneš, Petr

    2017-02-28

    Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis-activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti-cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down-regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells.

  17. DNA–PKcs–SIN1 complexation mediates low-dose X-ray irradiation (LDI)-induced Akt activation and osteoblast differentiation

    SciTech Connect

    Xu, Yong; Fang, Shi-ji; Zhu, Li-juan; Zhu, Lun-qing; Zhou, Xiao-zhong

    2014-10-24

    Highlights: • LDI increases ALP activity, promotes type I collagen (Col I)/Runx2 mRNA expression. • LDI induces DNA–PKcs activation, which is required for osteoblast differentiation. • Akt activation mediates LDI-induced ALP activity and Col I/Runx2 mRNA increase. • DNA–PKcs–SIN1 complexation mediates LDI-induced Akt Ser-473 phosphorylation. • DNA–PKcs–SIN1 complexation is important for osteoblast differentiation. - Abstract: Low-dose irradiation (LDI) induces osteoblast differentiation, however the underlying mechanisms are not fully understood. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA–PKcs)–Akt signaling in LDI-induced osteoblast differentiation. We confirmed that LDI promoted mouse calvarial osteoblast differentiation, which was detected by increased alkaline phosphatase (ALP) activity as well as mRNA expression of type I collagen (Col I) and runt-related transcription factor 2 (Runx2). In mouse osteoblasts, LDI (1 Gy) induced phosphorylation of DNA–PKcs and Akt (mainly at Ser-473). The kinase inhibitors against DNA–PKcs (NU-7026 and NU-7441) or Akt (LY294002, perifosine and MK-2206), as well as partial depletion of DNA–PKcs or Akt1 by targeted-shRNA, dramatically inhibited LDI-induced Akt activation and mouse osteoblast differentiation. Further, siRNA-knockdown of SIN1, a key component of mTOR complex 2 (mTORC2), also inhibited LDI-induced Akt Ser-473 phosphorylation as well as ALP activity increase and Col I/Runx2 expression in mouse osteoblasts. Co-immunoprecipitation (Co-IP) assay results demonstrated that LDI-induced DNA–PKcs–SIN1 complexation, which was inhibited by NU-7441 or SIN1 siRNA-knockdown in mouse osteoblasts. In summary, our data suggest that DNA–PKcs–SIN1 complexation-mediated Akt activation (Ser-473 phosphorylation) is required for mouse osteoblast differentiation.

  18. CCN1 acutely increases nitric oxide production via integrin αvβ3-Akt-S6K-phosphorylation of endothelial nitric oxide synthase at the serine 1177 signaling axis.

    PubMed

    Hwang, Soojin; Lee, Hyeon-Ju; Kim, Gyungah; Won, Kyung-Jong; Park, Yoon Shin; Jo, Inho

    2015-12-01

    Although CCN1 (also known as cysteine-rich, angiogenic inducer 61, CYR61) has been reported to promote angiogenesis and neovascularization in endothelial cells (ECs), its effects on endothelial nitric oxide (NO) production have never been studied. Using human umbilical vein ECs, we investigated whether and how CCN1 regulates NO production. CCN1 acutely increased NO production in a time- and dose-dependent manner, which was accompanied by increased phosphorylation of endothelial NO synthase (eNOS) at serine 1177 (eNOS-Ser(1177)), but not that of eNOS-Thr(495) or eNOS-Ser(114). The level of total eNOS expression was unaltered. Treatment with either LY294002, a selective inhibitor of phosphoinositide 3-kinase known as an upstream kinase of Akt, or H-89, an inhibitor of protein kinase A, mitogen- and stress-activated protein kinase 1, Rho-associated protein kinase 2, and ribosomal protein S6 kinase (S6K), inhibited CCN1-stimulated eNOS-Ser(1177) phosphorylation and subsequent NO production. Ectopic expression of small interfering RNA against Akt and S6K significantly inhibited the effects of CCN1. Consistently, CCN1 increased the phosphorylation of Akt-Ser(473) and S6K-Thr(389). However, CCN1 did not alter the expression or secretion of VEGF, a known downstream factor of CCN1 and a potential upstream factor of Akt-mediated eNOS-Ser(1177) phosphorylation. Furthermore, neutralization of integrin αvβ3 with corresponding antibody completely reversed all of the observed effects of CCN1. Moreover, CCN1 increased acetylcholine-induced relaxation in the rat aortas. Finally, we also found that CCN1-stimulated eNOS-Ser(1177) phosphorylation and NO production are true for other types of EC tested. In conclusion, CCN1 acutely increases NO production via activation of a signaling axis in integrin αvβ3-Akt-S6K-eNOS-Ser(1177) phosphorylation, suggesting an important role for CCN1 in vasodilation.

  19. Procyanidins from wild grape (Vitis amurensis) seeds regulate ARE-mediated enzyme expression via Nrf2 coupled with p38 and PI3K/Akt pathway in HepG2 cells.

    PubMed

    Bak, Min-Ji; Jun, Mira; Jeong, Woo-Sik

    2012-01-01

    Procyanidins, polymers of flavan-3-ol units, have been reported to exhibit many beneficial health effects such as antioxidant and anti-carcinogenic effects. In this study, we investigated the cancer chemopreventive properties of procyanidins from wild grape (Vitis amurensis) seeds in particular their roles in inducing phase II detoxifying/antioxidant enzymes as well as in modulating the upstream kinases. Ethanolic extract of V. amurensis seeds was fractionated with a series of organic solvents and finally separated into six fractions, F1-F6. Chemical properties of the procyanidins were analyzed by vanillin assay, BuOH-HCl test, and depolymerization with phloroglucinol followed by LC/MS analysis. The F5 had the highest procyanidin content among all the fractions and strongly induced the reporter activity of antioxidant response element as well as the protein expression of nuclear factor E2-related factor (Nrf2) in HepG2 human hepatocarcinoma cells. The procyanidin-rich F5 also strongly induced the expression of the phase II detoxifying and antioxidant enzymes such as NAD(P)H:quinone oxidoreductase1 and hemeoxygenase1. Phosphorylations of the upstream kinases such as MAPKs and PI3K/Akt were significantly increased by treatment with procyanidin fraction. In addition, the procyanidin-mediated Nrf2 expression was partly attenuated by PI3K inhibitor LY294002, and almost completely by p38 inhibitor SB202190, but neither by JNK inhibitor SP600125 nor by MEK1/2 inhibitor U0126. Taken together, the procyanidins from wild grape seeds could be used as a potential natural chemopreventive agent through Nrf2/ARE-mediated phase II detoxifying/antioxidant enzymes induction via p38 and PI3K/Akt pathway.

  20. PI3K/AKT, JNK, and ERK pathways are not crucial for the induction of cholesterol biosynthesis gene transcription in intestinal epithelial cells following treatment with the potato glycoalkaloid alpha-chaconine.

    PubMed

    Mandimika, Tafadzwa; Baykus, Hakan; Poortman, Jenneke; Garza, Cutberto; Kuiper, Harry; Peijnenburg, Ad

    2008-09-24

    We previously reported that exposure of the intestinal epithelial Caco-2 cell line to noncytotoxic concentrations of potato glycoalkaloids resulted in increased expression of cholesterol biosynthesis genes. Genes involved in mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homologue (AKT) pathways and their downstream effectors such as Jun, c-Myc, and Fos also were induced. MAPK and PI3K/AKT pathways have been described to regulate the activity of sterol regulatory element binding transcription factors (SREBPs) and consequently the expression of cholesterol biosynthesis genes. In this study, to understand the mechanism of induction of cholesterol biosynthesis upon alpha-chaconine treatment, its effect on SREBP-2 protein levels was investigated. We also examined whether MAPK and PI3K/AKT pathways are required for the observed induction of these genes following exposure of cells to alpha-chaconine. Differentiated Caco-2 cells were pretreated with LY294002 (PI3K inhibitor), PD98059 (MEK1 inhibitor), or SP600125 (JNK inhibitor) or a combination of all inhibitors for 24 h prior to coincubation with 10 microM alpha-chaconine for 6 h. Significant increases in precursor and mature protein levels of SREBP-2 were observed after alpha-chaconine exposure. We also observed that alpha-chaconine treatment resulted in significant phosphorylation of AKT, extracellular signal related protein kinase (ERK), and c-jun N terminal protein kinase (JNK) but not that of p38. In general, the kinase inhibitor experiments revealed that phosphorylation of kinases of PI3K/AKT, ERK, and JNK pathways was not crucial for the induction of expression of cholesterol biosynthesis genes, with the exception of SC5DL. The transcription of this later gene was reduced when all three pathways were inhibited. On the basis of these results, it can be postulated that other mechanisms, which may be independent of the MAPK and PI3K/AKT pathways

  1. Insulin-like growth factor-1 (IGF-1) promotes myoblast proliferation and skeletal muscle growth of embryonic chickens via the PI3K/Akt signalling pathway.

    PubMed

    Yu, Minli; Wang, Huan; Xu, Yali; Yu, Debing; Li, Dongfeng; Liu, Xiuhong; Du, Wenxing

    2015-08-01

    During embryonic development, IGF-1 fulfils crucial roles in skeletal myogenesis. However, the involvement of IGF-1-induced myoblast proliferation in muscle growth is still unclear. In the present study, we have characterised the role of IGF-1 in myoblast proliferation both in vitro and in vivo and have revealed novel details of how exogenous IGF-1 influences myogenic genes in chicken embryos. The results show that IGF-1 significantly induces the proliferation of cultured myoblasts in a dose-dependent manner. Additionally, the IGF-1 treatment significantly promoted myoblasts entering a new cell cycle and increasing the mRNA expression levels of cell cycle-dependent genes. However, these effects were inhibited by the PI3K inhibitor LY294002 and the Akt inhibitor KP372-1. These data indicated that the pro-proliferative effect of IGF-1 was mediated in response to the PI3K/Akt signalling pathway. Moreover, we also showed that exogenous IGF-1 stimulated myoblast proliferation in vivo. IGF-1 administration obviously promoted the incorporation of BrdU and remarkably increased the number of PAX7-positive cells in the skeletal muscle of chicken embryos. Administration of IGF-1 also significantly induced the upregulation of myogenic factors gene, the enhancement of c-Myc and the inhibition of myostatin (Mstn) expression. These findings demonstrate that IGF-1 has strong activity as a promoter of myoblast expansion and muscle fiber formation during early myogenesis. Therefore, this study offers insight into the mechanisms responsible for IGF-1-mediated stimulation of embryonic skeletal muscle development, which could have important implications for the improvement of chicken meat production.

  2. Single Agent and Synergistic Activity of the "First-in-Class" Dual PI3K/BRD4 Inhibitor SF1126 with Sorafenib in Hepatocellular Carcinoma

    PubMed Central

    Singh, Alok R.; Joshi, Shweta; Burgoyne, Adam M.; Sicklick, Jason K.; Ikeda, Sadakatsu; Kono, Yuko; Garlich, Joseph R.; Morales, Guillermo A.; Durden, Donald L.

    2017-01-01

    Deregulated PI3K/AKT/mTOR, Ras/Raf/MAPK, and c-Myc signaling pathways are of prognostic significance in hepatocellular carcinoma (HCC). Sorafenib, the only drug clinically approved for patients with advanced HCC, blocks the Ras/Raf/MAPK pathway but it does not inhibit the PI3K/AKT/mTOR pathway or c-Myc activation. Hence, there is an unmet medical need to identify potent PI3K/BRD4 inhibitors, which can be used either alone or in combination with sorafenib to treat patients with advanced HCC. Herein, we show that SF1126 (pan PI3K/BRD4 inhibitor) as single agent or in combination with sorafenib inhibited proliferation, cell cycle, apoptosis, and multiple key enzymes in PI3K/AKT/mTOR and Ras/Raf/MAPK pathway in Hep3B, HepG2, SK-Hep1, and Huh7 HCC cell lines. We demonstrate that the active moiety of the SF1126 prodrug LY294002 binds to and blocks BRD4 interaction with the acetylated histone-H4 chromatin mark protein and displaced BRD4 coactivator protein from the transcriptional start site of MYC in Huh7 and SK-Hep1 HCC cell lines. Moreover, SF1126 blocked expression levels of c-Myc in HCC cells. Treatment of SF1126 either alone or in combination with sorafenib showed significant antitumor activity in vivo. Our results establish that SF1126 is a dual PI3K/BRD4 inhibitor. This agent has completed a phase I clinical trial in humans with good safety profile. Our data support the potential future consideration of a phase II clinical trial of SF1126, a clinically relevant dual "first-in-class" PI3K/BRD4 inhibitor in advanced HCC, and a potential combination with sorafenib. PMID:27496136

  3. Coprinus comatus Cap Inhibits Adipocyte Differentiation via Regulation of PPARγ and Akt Signaling Pathway

    PubMed Central

    Jang, Sun-Hee; Kang, Suk Nam; Jeon, Beong-Sam; Ko, Yeoung-Gyu; Kim, Hong-Duck; Won, Chung-Kil; Kim, Gon-Sup; Cho, Jae-Hyeon

    2014-01-01

    This study assessed the effects of Coprinus comatus cap (CCC) on adipogenesis in 3T3-L1 adipocytes and the effects of CCC on the development of diet-induced obesity in rats. Here, we showed that the CCC has an inhibitory effect on the adipocyte differentiation of 3T3-L1 cells, resulting in a significant decrease in lipid accumulation through the downregulation of several adipocyte specific-transcription factors, including CCAAT/enhancer binding protein β, C/EBPδ, and peroxisome proliferator-activated receptor gamma (PPARγ). Moreover, treatment with CCC during adipocyte differentiation induced a significant down-regulation of PPARγ and adipogenic target genes, including adipocyte protein 2, lipoprotein lipase, and adiponectin. Interestingly, the CCC treatment of the 3T3-L1 adipocytes suppressed the insulin-stimulated Akt and GSK3β phosphorylation, and these effects were stronger in the presence of an inhibitor of Akt phosphorylation, LY294002, suggesting that CCC inhibited adipocyte differentiation through the down-regulation of Akt signaling. In the animal study, CCC administration significantly reduced the body weight and adipose tissue weight of rats fed a high fat diet (HFD) and attenuated lipid accumulation in the adipose tissues of the HFD-induced obese rats. The size of the adipocyte in the epididymal fat of the CCC fed rats was significantly smaller than in the HFD rats. CCC treatment significantly reduced the total cholesterol and triglyceride levels in the serum of HFD rats. These results strongly indicated that the CCC-mediated decrease in body weight was due to a reduction in adipose tissue mass. The expression level of PPARγ and phospho-Akt was significantly lower in the CCC-treated HFD rats than that in the HFD obesity rats. These results suggested that CCC inhibited adipocyte differentiation by the down-regulation of major transcription factor involved in the adipogenesis pathway including PPARγ through the regulation of the Akt pathway in 3T3

  4. Identification of natural allosteric inhibitor for Akt1 protein through computational approaches and in vitro evaluation.

    PubMed

    Pragna Lakshmi, T; Kumar, Amit; Vijaykumar, Veena; Natarajan, Sakthivel; Krishna, Ramadas

    2017-03-01

    Akt, a serine/threonine protein kinase, is often hyper activated in breast and prostate cancers, but with poor prognosis. Allosteric inhibitors regulate aberrant kinase activity by stabilizing the protein in inactive conformation. Several natural compounds have been reported as inhibitors for kinases. In this study, to identify potential natural allosteric inhibitor for Akt1, we generated a seven-point pharmacophore model and screened it through natural compound library. Quercetin-7-O-β-d-glucopyranoside or Q7G was found to be the best among selected molecules based on its hydrogen bond occupancy with key allosteric residues, persistent polar contacts and salt bridges that stabilize Akt1 in inactive conformation and minimum binding free energy during molecular dynamics simulation. Q7G induced dose-dependent inhibition of breast cancer cells (MDA MB-231) and arrested them in G1 and sub-G phase. This was associated with down-regulation of anti-apoptotic protein Bcl-2, up-regulation of cleaved caspase-3 and PARP. Expression of p-Akt (Ser473) was also down-regulated which might be due to Akt1 inhibition in inactive conformation. We further confirmed the Akt1 and Q7G interaction which was observed to have a dissociation constant (Kd) of 0.246μM. With these computational, biological and thermodynamic studies, we suggest Q7G as a lead molecule and propose for its further optimization.

  5. Tetraarsenic hexoxide induces G2/M arrest, apoptosis, and autophagy via PI3K/Akt suppression and p38 MAPK activation in SW620 human colon cancer cells

    PubMed Central

    Nagappan, Arulkumar; Lee, Won Sup; Yun, Jeong Won; Lu, Jing Nan; Chang, Seong-Hwan; Jeong, Jae-Hoon; Kim, Gon Sup; Jung, Jin-Myung; Hong, Soon Chan

    2017-01-01

    Tetraarsenic hexoxide (As4O6) has been used in Korean folk medicines for the treatment of cancer, however its anti-cancer mechanisms remain obscured. Here, this study investigated the anti-cancer effect of As4O6 on SW620 human colon cancer cells. As4O6 has showed a dose-dependent inhibition of SW620 cells proliferation. As4O6 significantly increased the sub-G1 and G2/M phase population, and Annexin V-positive cells in a dose-dependent manner. G2/M arrest was concomitant with augment of p21 and reduction in cyclin B1, cell division cycle 2 (cdc 2) expressions. Nuclear condensation, cleaved nuclei and poly (adenosine diphosphate‑ribose) polymerase (PARP) activation were also observed in As4O6-treated SW620 cells. As4O6 induced depolarization of mitochondrial membrane potential (MMP, ΔΨm) but not reactive oxygen species (ROS) generation. Further, As4O6 increased death receptor 5 (DR5), not DR4 and suppressed the B‑cell lymphoma‑2 (Bcl-2) and X-linked inhibitor of apoptosis protein (XIAP) family proteins. As4O6 increased the formation of AVOs (lysosomes and autophagolysosomes) and promoted the conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3)-I to LC3-II in a dose- and time- dependent manner. Interestingly, a specific phosphoinositide 3-kinase (PI3K)/Akt inhibitor (LY294002) augmented the As4O6 induced cell death; whereas p38 mitogen-activated protein kinases (p38 MAPK) inhibitor (SB203580) abrogated the cell death. Thus, the present study provides the first evidence that As4O6 induced G2/M arrest, apoptosis and autophagic cell death through PI3K/Akt and p38 MAPK pathways alteration in SW620 cells. PMID:28355296

  6. Therapeutic ultrasound reverses peripheral ischemia in type 2 diabetic mice through PI3K-Akt-eNOS pathway

    PubMed Central

    Lu, Zhao-Yang; Li, Rui-Lin; Zhou, Hong-Sheng; Huang, Jing-Juan; Su, Zhi-Xiao; Qi, Jia; Zhang, Lan; Li, Yue; Shi, Yi-Qin; Hao, Chang-Ning; Duan, Jun-Li

    2016-01-01

    Therapeutic ultrasound (TUS) has been demonstrated to improve endothelial nitric oxide synthase (eNOS) activity, which played a crucial role in the regulation of angiogenesis. Diabetes Mellitus (DM) impairs eNOS activity. We tested the hypothesis that DM may retard unilateral hindlimb ischemia-induced angiogenesis by inhibiting eNOS in high-fat diet (HFD)/streptozocin (STZ) induced diabetic mice, and that TUS may reverse DM-related impairment of angiogenesis. C57BL/6 mice were allocated to four groups: (A) mice were fed standard diet (control); (B) mice were fed standard diet and treated with TUS (control+TUS); (C) type-2 DM mice were induced by HFD/STZ (diabetic); and (D) type-2 DM mice and treated with TUS (dabetic+TUS). All mice were surgically induced unilateral limb ischemia. The ischemic skeletal muscles in groups B and D were irradiated with extracorporeal TUS for 9 minutes/day (frequency of 1 MHz, intensity of 0.3 W/cm2) for 14 consecutive days. The result showed that TUS augmented the blood perfusion, increased capillary density accompanied by an upregulation of angiogenic factors and a downregulation of apoptotic proteins in group D relative to group C. In vitro, TUS inhibited the apoptosis, promoted tubule formation, proliferation and migration capacities, increased angiogenic factors expression and reduced apoptotic protein levels in human umbilical vein endothelial cells (HUVECs). Furthermore, TUS can robust reverse the inhibiting effect induced by high glucose (HG) on HUVECs, and these benefits could be blocked by phosphoinositide 3-kinase (PI3K) inhibitor (LY294002) or eNOS inhibitor (L-NAME). Together, TUS restored type-2 DM-mediated inhibition of ischemia-induced angiogenesis, partially via PI3K-Akt-eNOS signal pathway. PMID:27725849

  7. Differential sensitivities of trastuzumab (Herceptin)-resistant human breast cancer cells to phosphoinositide-3 kinase (PI-3K) and epidermal growth factor receptor (EGFR) kinase inhibitors.

    PubMed

    Chan, Carmel T; Metz, Marianne Z; Kane, Susan E

    2005-05-01

    Her2 (erbB2/neu) is overexpressed in 25-30% of human breast cancers. Herceptin is a recombinant humanized Her2 antibody used to treat breast cancer patients with Her2 overexpression. Over a 5-month selection process, we isolated clones of BT474 (BT) human breast carcinoma cells (BT/Her(R)) that were resistant to Herceptin in vitro. In BT/Her(R) subclones, cell-surface, phosphorylated and total cellular Her2 protein remained high in the continuous presence of Herceptin. Likewise, the levels of cell-surface, phosphorylated, and total cellular Her3 and EGFR were either unchanged or only slightly elevated in BT/Her(R) subclones relative to BT cells. One BT/Her(R) subclone had substantially upregulated cell-surface EGFR, but this did not correlate with a higher relative resistance to Herceptin. In looking at the downstream PI-3K/Akt signaling pathway, phosphorylated and total Akt levels and Akt kinase activities were all sustained in BT/Her(R) subclones in the presence of Herceptin, but significantly downregulated in BT cells exposed to Herceptin. Whereas BT cells lost sensitivity to the PI-3K inhibitor LY294002 in the presence of Herceptin, BT/Her(R) subclones were equally sensitive to this agent in the presence and absence of Herceptin. This suggests that BT/Her(R) subclones acquired a Herceptin-resistant mechanism of PI-3K signaling. BT/Her(R) subclones were also sensitive to the EGFR kinase inhibitor AG1478 in the presence of Herceptin, to the same extent as BT cells. The BT/Her(R) subclones provide new insights into mechanisms of Herceptin resistance and suggest new treatment strategies in combination with other inhibitors targeted to signal transduction pathways.

  8. Cucurbitacin E inhibits TNF-α-induced inflammatory cytokine production in human synoviocyte MH7A cells via suppression of PI3K/Akt/NF-κB pathways.

    PubMed

    Jia, Qingyun; Cheng, Wenxiang; Yue, Ye; Hu, Yipping; Zhang, Jian; Pan, Xiaohua; Xu, Zhanwang; Zhang, Peng

    2015-12-01

    Increasing studies indicated that Cucurbitacin E (CuE), a compound isolated from Cucurbitaceae, has been shown anti-inflammatory effect. However, the effect of CuE on rheumatoid arthritis (RA) inflammatory response and its potential molecular mechanism are still unknown. In this study, we demonstrated that CuE significantly suppressed TNF-α-induced inflammatory cytokines production interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8) mRNA and protein expression in human synoviocyte MH7A cells. Furthermore, we found that CuE also inhibited TNF-α-induced phosphorylation of NF-κBp65, IKKα/β, and IκBα in a dose-and time-dependent manner as well as NF-κBp65 nuclear translocation. Finally, we showed that CuE blocked the upstream targets of NF-κB pathway RIP1/PI3K/Akt. Interestingly, PI3K inhibitor LY294002 completely blocked the TNF-α-induced activation of p85, Akt and the whole cascade of the NF-κB signaling components and suppressed inflammatory cytokines production in mRNA and protein levels similarly as CuE. Our studies provided the first evidence that CuE inhibited TNF-α-induced inflammatory cytokine production in human synoviocyte MH7A cells via modulation of PI3K/Akt/NF-κB pathway. These findings indicated that CuE is a potential candidate for RA therapy.

  9. Rutin attenuates H2O2-induced oxidation damage and apoptosis in Leydig cells by activating PI3K/Akt signal pathways.

    PubMed

    Sun, Jianhua; Wang, Heng; Liu, Bei; Shi, Wenhao; Shi, Juanzi; Zhang, Zhou; Xing, Junping

    2017-04-01

    Oxidative stress is a primary factor in the pathology of male infertility. The strong antioxidative capacity of rutin has been proven by numerous studies, but a protective role in the context of male reproduction remains to be elucidated. To explore the biological role of rutin in protecting male reproductive function and the potential underlying mechanism, H2O2-induced Leydig cells were used as a cell model of oxidation damage. Our findings showed that rutin at concentrations of 10, 20, and 40μmol/L remarkably increased cell survival rate of H2O2-induced Leydig cells to 70.1%, 86.8%, and 80.3% respectively. Next, rutin with concentrations of 10, 20, and 40μmol/L decreased reactive oxygen species (ROS) and malondialdehyde (MDA) levels but increased the levels of glutathione (GSH) and testosterone in H2O2-induced Leydig cells. The activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were remarkably increased by rutin treatment with concentrations of 20 and 40μmol/L, but glutathione peroxidase (GSH-Px) activity was notably decreased. Moreover, rutin with concentrations of 10, 20, and 40μmol/L increased Bcl-2 protein levels but decreased protein levels of Bax and caspase-3. Furthermore, 20μmol/L rutin significantly abrogated the decrease in levels of phosphoinositide 3-kinase (PI3K) and phosphorylated serine/threonine kinase (p-AKT) induced by H2O2. Pretreatment with LY294002, a PI3K inhibitor, antagonized protective action of 20μmol/L rutin against H2O2-induced cell activities, intracellular oxidant, testosterone, antioxidant enzyme activities, and the apoptosis related protein expression. Taken together, these results suggest that rutin attenuates H2O2-induced oxidation damage and apoptosis in Leydig cells by activating PI3K/Akt signal pathways, providing a promising strategy to decrease oxidative stress associated with male infertility.

  10. Crocin Inhibits Oxidative Stress and Pro-inflammatory Response of Microglial Cells Associated with Diabetic Retinopathy Through the Activation of PI3K/Akt Signaling Pathway.

    PubMed

    Yang, Xinguang; Huo, Fuquan; Liu, Bei; Liu, Jing; Chen, Tao; Li, Junping; Zhu, Zhongqiao; Lv, Bochang

    2017-02-25

    Diabetic retinopathy (DR) is a serious microvascular complication of diabetes mellitus that is closely associated with the degeneration and loss of retinal ganglion cells (RGCs) caused by diabetic microangiopathy and subsequent oxidative stress and an inflammatory response. Microglial cells are classed as neurogliocytes and play a significant role in neurodegenerative diseases. Over-activated microglial cells may cause neurotoxicity and induce the death and apoptosis of RGCs. Crocin is one of the two most pharmacologically bioactive constituents in saffron. In the present study, we focused on the role of microglial cells in DR, suggesting that DR may cause the over-activation of microglial cells and induce oxidative stress and the release of pro-inflammatory factors. Microglial cells BV-2 and N9 were cultured, and high-glucose (HG) and free fatty acid (FFA) were used to simulate diabetes. The results showed that HG-FFA co-treatment caused the up-regulated expression of CD11b and Iba-1, indicating that BV-2 and N9 cells were over-activated. Moreover, oxidative stress markers and pro-inflammatory factors were significantly enhanced by HG-FFA treatment. We found that crocin prevented the oxidative stress and pro-inflammatory response induced by HG-FFA co-treatment. Moreover, using the PI3K/Akt inhibitor LY294002, we revealed that PI3K/Akt signaling plays a significant role in blocking oxidative stress, suppressing the pro-inflammatory response, and maintaining the neuroprotective effects of crocin. In total, these results provide a new insight into DR and DR-induced oxidative stress and the inflammatory response, which provide a potential therapeutic target for neuronal damage, vision loss, and other DR-induced complications.

  11. Involvement of PI3K/Akt/FoxO3a and PKA/CREB Signaling Pathways in the Protective Effect of Fluoxetine Against Corticosterone-Induced Cytotoxicity in PC12 Cells.

    PubMed

    Zeng, Bingqing; Li, Yiwen; Niu, Bo; Wang, Xinyi; Cheng, Yufang; Zhou, Zhongzhen; You, Tingting; Liu, Yonggang; Wang, Haitao; Xu, Jiangping

    2016-08-01

    The selective serotonin reuptake inhibitor fluoxetine is neuroprotective in several brain injury models. It is commonly used to treat major depressive disorder and related conditions, but its mechanism of action remains incompletely understood. Activation of the phosphatidylinositol-3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FoxO3a) and protein kinase A/cAMP-response element binding protein (PKA/CREB) signaling pathways has been strongly implicated in the pathogenesis of depression and might be the downstream target of fluoxetine. Here, we used PC12 cells exposed to corticosterone (CORT) to study the neuroprotective effects of fluoxetine and the involvement of the PI3K/Akt/FoxO3a and PKA/CREB signaling pathways. Our results show that CORT reduced PC12 cells viability by 70 %, and that fluoxetine showed a concentration-dependent neuroprotective effect. Neuroprotective effects of fluoxetine were abolished by inhibition of PI3K, Akt, and PKA using LY294002, KRX-0401, and H89, respectively. Treatment of PC12 cells with fluoxetine resulted in increased phosphorylation of Akt, FoxO3a, and CREB. Fluoxetine also dose-dependently rescued the phosphorylation levels of Akt, FoxO3a, and CREB, following administration of CORT (from 99 to 110, 56 to 170, 80 to 170 %, respectively). In addition, inhibition of PKA and PI3K/Akt resulted in decreased levels of p-CREB, p-Akt, and p-FoxO3a in the presence of fluoxetine. Furthermore, fluoxetine reversed CORT-induced upregulation of p53-upregulated modulator of apoptosis (Puma) and Bcl-2-interacting mediator of cell death (Bim) via the PI3K/Akt/FoxO3a signaling pathway. H89 treatment reversed the effect of fluoxetine on the mRNA level of brain-derived neurotrophic factor, which was decreased in the presence of CORT. Our data indicate that fluoxetine elicited neuroprotection toward CORT-induced cell death that involves dual regulation from PI3K/Akt/FoxO3a and PKA/CREB pathways.

  12. Statins upregulate cystathionine γ-lyase transcription and H2S generation via activating Akt signaling in macrophage.

    PubMed

    Xu, Yuan; Du, Hua-Ping; Li, Jiaojiao; Xu, Ran; Wang, Ya-Li; You, Shou-Jiang; Liu, Huihui; Wang, Fen; Cao, Yong-Jun; Liu, Chun-Feng; Hu, Li-Fang

    2014-09-01

    Hydrogen sulfide (H2S), the third gaseous transmitter, is implicated in various pathophysiologic processes. In the cardiovascular system, H2S exerts effects of cardioprotection, vascular tone regulation, and atherogenesis inhibition. Recent studies demonstrated that atorvastatin, the inhibitor of 3-hydroxyl-3-methyl coenzyme A reductase, affected H2S formation in kidney and other organs. However, the underlying mechanisms are not fully understood. In this study, we examined the effects of three different statins (fluvastatin, atorvastatin and pravastatin) on H2S formation in raw264.7 macrophages. There was a remarkable rise in H2S level in fluvastatin- and atorvastatin-stimulated macrophages, while pravastatin failed to show any significant effect on it. Moreover, fluvastatin and atorvastatin enhanced the mRNA and protein expression of cystathionine γ-lyase (CSE) in dose- and time-dependent manners. Fluvastatin also markedly enhanced the CSE activity. However, fluvastatin did not alter the mRNA or protein expression of another H2S-producing enzyme 3-mercaptopyruvate sulfurtransferase. Blockade of CSE with its inhibitor dl-propargylglycine (PAG) or siRNA markedly reduced the H2S level in fluvastatin-stimulated macrophages. In addition, fluvastatin elevated Akt phosphorylation, which occurred as early as 15 min after treatment, peaked at 1h, and lasted at least 3h. Both PI3K inhibitor LY294002 (10 μM) and Akt inhibitor perifosine (10μM) were able to reverse the increases of CSE mRNA and H2S production in fluvastatin-stimulated macrophages. Last, we showed that fluvastatin reduced the mRNA levels of pro-inflammatory molecules such as IL-1β and MCP-1 in LPS-treated macrophages, which were completely reversed by CSE inhibitor PAG. Taken together, the findings demonstrate that statins may up-regulate CSE expression/activity and subsequently elevate H2S generation by activating Akt signaling pathway and also imply that CSE-H2S pathway plays a critical role in the anti

  13. Blocks to thyroid cancer cell apoptosis can be overcome by inhibition of the MAPK and PI3K/AKT pathways.

    PubMed

    Gunda, V; Bucur, O; Varnau, J; Vanden Borre, P; Bernasconi, M J; Khosravi-Far, R; Parangi, S

    2014-03-06

    Current treatment for recurrent and aggressive/anaplastic thyroid cancers is ineffective. Novel targeted therapies aimed at the inhibition of the mutated oncoprotein BRAF(V600E) have shown promise in vivo and in vitro but do not result in cellular apoptosis. TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a tumor-selective manner by activating the extrinsic apoptotic pathway. Here, we show that a TRAIL-R2 agonist antibody, lexatumumab, induces apoptosis effectively in some thyroid cancer cell lines (HTh-7, TPC-1 and BCPAP), while more aggressive anaplastic cell lines (8505c and SW1736) show resistance. Treatment of the most resistant cell line, 8505c, using lexatumumab in combination with the BRAF(V600E) inhibitor, PLX4720, and the PI3K inhibitor, LY294002, (triple-drug combination) sensitizes the cells by triggering both the extrinsic and intrinsic apoptotic pathways in vitro as well as 8505c orthotopic thyroid tumors in vivo. A decrease in anti-apoptotic proteins, pAkt, Bcl-xL, Mcl-1 and c-FLIP, coupled with an increase in the activator proteins, Bax and Bim, results in an increase in the Bax to Bcl-xL ratio that appears to be critical for sensitization and subsequent apoptosis of these resistant cells. Our results suggest that targeting the death receptor pathway in thyroid cancer can be a promising strategy for inducing apoptosis in thyroid cancer cells, although combination with other kinase inhibitors may be needed in some of the more aggressive tumors initially resistant to apoptosis.

  14. Blocks to thyroid cancer cell apoptosis can be overcome by inhibition of the MAPK and PI3K/AKT pathways

    PubMed Central

    Gunda, V; Bucur, O; Varnau, J; Vanden Borre, P; Bernasconi, M J; Khosravi-Far, R; Parangi, S

    2014-01-01

    Current treatment for recurrent and aggressive/anaplastic thyroid cancers is ineffective. Novel targeted therapies aimed at the inhibition of the mutated oncoprotein BRAFV600E have shown promise in vivo and in vitro but do not result in cellular apoptosis. TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a tumor-selective manner by activating the extrinsic apoptotic pathway. Here, we show that a TRAIL-R2 agonist antibody, lexatumumab, induces apoptosis effectively in some thyroid cancer cell lines (HTh-7, TPC-1 and BCPAP), while more aggressive anaplastic cell lines (8505c and SW1736) show resistance. Treatment of the most resistant cell line, 8505c, using lexatumumab in combination with the BRAFV600E inhibitor, PLX4720, and the PI3K inhibitor, LY294002, (triple-drug combination) sensitizes the cells by triggering both the extrinsic and intrinsic apoptotic pathways in vitro as well as 8505c orthotopic thyroid tumors in vivo. A decrease in anti-apoptotic proteins, pAkt, Bcl-xL, Mcl-1 and c-FLIP, coupled with an increase in the activator proteins, Bax and Bim, results in an increase in the Bax to Bcl-xL ratio that appears to be critical for sensitization and subsequent apoptosis of these resistant cells. Our results suggest that targeting the death receptor pathway in thyroid cancer can be a promising strategy for inducing apoptosis in thyroid cancer cells, although combination with other kinase inhibitors may be needed in some of the more aggressive tumors initially resistant to apoptosis. PMID:24603332

  15. PM2.5-induced oxidative stress increases adhesion molecules expression in human endothelial cells through the ERK/AKT/NF-κB-dependent pathway.

    PubMed

    Rui, Wei; Guan, Longfei; Zhang, Fang; Zhang, Wei; Ding, Wenjun

    2016-01-01

    The aim of this study was to explore the intracellular mechanisms underlying the cardiovascular toxicity of air particulate matter (PM) with an aerodynamic diameter of less than 2.5 µm (PM2.5) in a human umbilical vein cell line, EA.hy926. We found that PM2.5 exposure triggered reactive oxygen species (ROS) generation, resulting in a significant decrease in cell viability. Data from Western blots showed that PM2.5 induced phosphorylation of Jun N-terminal kinase (JNK), extracellular signal regulatory kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and protein kinase B (AKT), and activation of nuclear factor kappa B (NF-κB). We further observed a significant increase in expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) in a time- and dose-dependent manner. Moreover, the adhesion of monocytic THP-1 cells to EA.hy926 cells was greatly enhanced in the presence of PM2.5 . However, N-acetylcysteine (NAC), a scavenger of ROS, prevented the increase of ROS generation, attenuated the phosphorylation of the above kinases, and decreased the NF-κB activation as well as the expression of ICAM-1 and VCAM-1. Furthermore, ERK inhibitor (U0126), AKT inhibitor (LY294002) and NF-κB inhibitor (BAY11-7082) significantly down-regulated PM2.5 -induced ICAM-1 and VCAM-1 expression as well as adhesion of THP-1 cells, but not JNK inhibitor (SP600125) and p38 MAPK inhibitor (SB203580), indicating that ERK/AKT/NF-κB is involved in the signaling pathway that leads to PM2.5 -induced ICAM-1 and VCAM-1 expression. These findings suggest PM2.5 -induced ROS may function as signaling molecules triggering ICAM-1 and VCAM-1 expressions through activating the ERK/AKT/NF-κB-dependent pathway, and further promoting monocyte adhesion to endothelial cells.

  16. Involvement of PI3K/Akt/GSK-3β and mTOR in the antidepressant-like effect of atorvastatin in mice.

    PubMed

    Ludka, Fabiana Kalyne; Constantino, Leandra Celso; Dal-Cim, Tharine; Binder, Luisa Bandeira; Zomkowski, Andréa; Rodrigues, Ana Lúcia S; Tasca, Carla Inês

    2016-11-01

    Atorvastatin is a cholesterol-lowering statin that has been shown to exert several pleiotropic effects in the nervous system as a neuroprotective and antidepressant-like agent. Antidepressant-like effect of atorvastatin in mice is mediated by glutamatergic and serotoninergic receptors, although the precise intracellular signaling pathways involved are unknown. PI3K/Akt/GSK-3β/mTOR signaling pathway has been associated to neurobiology of depression and seems to be modulated by some pharmacological antidepressant strategies. The present study investigated the participation of the PI3K/Akt/GSK-3β/mTOR signaling pathway in the antidepressant-like effect of an acute atorvastatin treatment in mice. Atorvastatin sub-effective (0.01 mg/kg) or effective (0.1 mg/kg) doses in the tail suspension test (TST) was administered orally alone or in combination with PI3K, GSK-3β or mTOR inhibitors. The administration of PI3K inhibitor, LY294002 (10 nmol/site, i.c.v) completely prevented the antidepressant-like effect of atorvastatin (0.1 mg/kg, p.o.). The participation of GSK-3β in the antidepressant-like effect of atorvastatin was demonstrated by co-administration of a sub-effective dose of atorvastatin (0.01 mg/kg, p.o.) with AR-A014418 (0.01 μg/site, i.c.v., a selective GSK-3β inhibitor) or with lithium chloride (10 mg/kg, p.o., a non-selective GSK-3β inhibitor). The mTOR inhibitor, rapamycin (0.2 nmol/site, i.c.v.) was also able to prevent atorvastatin (0.1 mg/kg, p.o.) antidepressant-like effect. These behavioral findings were supported by neurochemical observations, as atorvastatin treatment increased the immunocontent of the phosphorylated isoforms of Akt, GSK-3β and mTOR in the hippocampus of mice. Taken together, our results suggest an involvement of the PI3K/Akt/GSK-3β/mTOR signaling pathway in the antidepressant-like effect of atorvastatin in mice.

  17. Follicle-stimulating hormone and insulin-like growth factor I synergistically induce up-regulation of cartilage link protein (Crtl1) via activation of phosphatidylinositol-dependent kinase/Akt in rat granulosa cells.

    PubMed

    Sun, Guang Wei; Kobayashi, Hiroshi; Suzuki, Mika; Kanayama, Naohiro; Terao, Toshihiko

    2003-03-01

    FSH and IGF-I are both important determinants of follicle development and the process of cumulus cell-oocyte complex expansion. FSH stimulates the phosphorylation of Akt by mechanisms involving phosphatidylinositol 3-kinase (PI3-K), a pattern of response mimicking that of IGF-I. Cartilage link protein (Crtl1) is confined to the cartilaginous lineage and is assembled into a macroaggregate complex essential for hyaluronan-rich matrix stabilization. The present studies were performed to determine the actions of FSH and IGF-I on Crtl1 production in rat granulosa cells. Primary cultures of granulosa cells were prepared from 24-d-old rats. After treatments, cell extracts and media were prepared, and the Crtl1 level was determined by immunoblotting analysis using anti-Crtl1 antibodies. Here we showed that 1) treatment with FSH (> or = 25 ng/ml) or IGF-I (> or = 25 ng/ml) for 4 h increased Crtl1 production; 2) maximal stimulatory effects of FSH or IGF-I were observed at 100 or 50 ng/ml, respectively; 3) FSH caused a concentration-dependent increase in IGF-I-induced Crtl1 production and vice versa; 4) FSH and IGF-I also up-regulate the expression of Crtl1 mRNA; 5) FSH- and IGF-I-dependent Crtl1 production were abrogated by PI3-K inhibitors (LY294002 and wortmannin), and inhibition of Crtl1 production by p38 mitogen-activated protein kinase inhibitor (SB202190) was partial (approximately 30%), suggesting that PI3-K and, to a lesser extent, p38 mitogen-activated protein kinase are critical for the response. Our study represents the first report that FSH amplifies IGF-I-mediated Crtl1 production, possibly via PI3-K-Akt signaling cascades in rat granulosa cells.

  18. Simvastatin prevents β-amyloid(25-35)-impaired neurogenesis in hippocampal dentate gyrus through α7nAChR-dependent cascading PI3K-Akt and increasing BDNF via reduction of farnesyl pyrophosphate.

    PubMed

    Wang, Conghui; Chen, Tingting; Li, Guoxi; Zhou, Libin; Sha, Sha; Chen, Ling

    2015-10-01

    Simvastatin (SV) is reported to improve cognition and slow progression of Alzheimer's disease (AD), however underlying mechanism still remains unclear. In hippocampal dentate gyrus (DG), β-amyloid (Aβ) selectively impairs survival and neurite growth of newborn neurons in the 2(nd) week after birth. The aim of this study was to examine the effects of SV on the impairment of neurogenesis and the spatial cognitive deficits in Aβ25-35 (3 nmol)-injected (i.c.v.) mice (Aβ25-35-mice). Herein, we reported that the SV-treatment (20 mg/kg) on days 2-14 after BrdU-injection could dose-dependently protect the survival and neurite growth of newborn neurons, which was blocked by the α7nAChR antagonist MLA or the farnesol (FOH) that can convert to farnesyl pyrophosphate (FPP), but not the α4β2nAChR antagonist DHβE. The SV-treatment in Aβ25-35-mice rescued the decline of Akt phosphorylation and increased the ERK1/2 phosphorylation in hippocampus, which was sensitive to MLA and FOH. The PI3K inhibitor LY294002 could abolish the SV-protected neurogenesis in Aβ25-35-mice, but the MEK inhibitor U0126 had no effects. The SV-treatment could correct the decline of hippocampal BDNF concentration in Aβ25-35-mice, which was blocked by MLA and FOH. Using Morris water maze and Y-maze tasks, we further observed that the SV-treatment in Aβ25-35-mice could improve their spatial cognitive deficits, which was sensitive to the application of FOH. The results indicate that the SV-treatment in Aβ25-35-mice via reduction of FPP can protect neurogenesis through α7nAChR-cascading PI3K-Akt and increasing BDNF, which may improve spatial cognitive function.

  19. Cudraflavone C Induces Tumor-Specific Apoptosis in Colorectal Cancer Cells through Inhibition of the Phosphoinositide 3-Kinase (PI3K)-AKT Pathway

    PubMed Central

    Soo, Hsien-Chuen; Chung, Felicia Fei-Lei; Lim, Kuan-Hon; Yap, Veronica Alicia; Bradshaw, Tracey D.; Hii, Ling-Wei; Tan, Si-Hoey; See, Sze-Jia; Tan, Yuen-Fen; Leong, Chee-Onn

    2017-01-01

    Cudraflavone C (Cud C) is a naturally-occurring flavonol with reported anti-proliferative activities. However, the mechanisms by which Cud C induced cytotoxicity have yet to be fully elucidated. Here, we investigated the effects of Cud C on cell proliferation, caspase activation andapoptosis induction in colorectal cancer cells (CRC). We show that Cud C inhibits cell proliferation in KM12, Caco-2, HT29, HCC2998, HCT116 and SW48 CRC but not in the non-transformed colorectal epithelial cells, CCD CoN 841. Cud C induces tumor-selective apoptosis via mitochondrial depolarization and activation of the intrinsic caspase pathway. Gene expression profiling by microarray analyses revealed that tumor suppressor genes EGR1, HUWE1 and SMG1 were significantly up-regulated while oncogenes such as MYB1, CCNB1 and GPX2 were down-regulated following treatment with Cud C. Further analyses using Connectivity Map revealed that Cud C induced a gene signature highly similar to that of protein synthesis inhibitors and phosphoinositide 3-kinase (PI3K)-AKT inhibitors, suggesting that Cud C might inhibit PI3K-AKT signaling. A luminescent cell free PI3K lipid kinase assay revealed that Cud C significantly inhibited p110β/p85α PI3K activity, followed by p120γ, p110δ/p85α, and p110α/p85α PI3K activities. The inhibition by Cud C on p110β/p85α PI3K activity was comparable to LY-294002, a known PI3K inhibitor. Cud C also inhibited phosphorylation of AKT independent of NFκB activity in CRC cells, while ectopic expression of myristoylated AKT completely abrogated the anti-proliferative effects, and apoptosis induced by Cud C in CRC. These findings demonstrate that Cud C induces tumor-selective cytotoxicity by targeting the PI3K-AKT pathway. These findings provide novel insights into the mechanism of action of Cud C, and indicate that Cud C further development of Cud C derivatives as potential therapeutic agents is warranted. PMID:28107519

  20. Combination treatment of prostate cancer with FGF receptor and AKT kinase inhibitors

    PubMed Central

    Feng, Shu; Shao, Longjiang; Castro, Patricia; Coleman, Ilsa; Nelson, Peter S; Smith, Paul D; Davies, Barry R; Ittmann, Michael

    2017-01-01

    Activation of the PI3K/AKT pathway occurs in the vast majority of advanced prostate cancers (PCas). Activation of fibroblast growth factor receptor (FGFR) signaling occurs in a wide variety of malignancies, including PCa. RNA-Seq of castration resistant PCa revealed expression of multiple FGFR signaling components compatible with FGFR signaling in all cases, with multiple FGF ligands expressed in 90% of cases. Immunohistochemistry confirmed FGFR signaling in the majority of xenografts and advanced PCas. AZD5363, an AKT kinase inhibitor and AZD4547, a FGFR kinase inhibitor are under active clinical development. We therefore sought to determine if these two drugs have additive effects in PCa models. The effect of both agents, singly and in combination was evaluated in a variety of PCa cell lines in vitro and in vivo. All cell lines tested responded to both drugs with decreased invasion, soft agar colony formation and growth in vivo, with additive effects seen with combination treatment. Activation of the FGFR, AKT, ERK and STAT3 pathways was examined in treated cells. AZD5363 inhibited AKT signaling and increased FGFR1 signaling, which partially compensated for decreased AKT kinase activity. While AZD4547 could effectively block the ERK pathway, combination treatment was needed to completely block STAT3 activation. Thus combination treatment with AKT and FGFR kinase inhibitors have additive effects on malignant phenotypes in vitro and in vivo by inhibiting multiple signaling pathways and mitigating the compensatory upregulation of FGFR signaling induced by AKT kinase inhibition. Our studies suggest that co-targeting these pathways may be efficacious in advanced PCa. PMID:28008155

  1. Synthesis and biological evaluation of analogues of AKT (protein kinase B) inhibitor-IV.

    PubMed

    Sun, Qi; Wu, Runzhi; Cai, Sutang; Lin, Yuan; Sellers, Llewlyn; Sakamoto, Kaori; He, Biao; Peterson, Blake R

    2011-03-10

    Inhibitors of the PI3-kinase/AKT (protein kinase B) pathway are under investigation as anticancer and antiviral agents. The benzimidazole derivative AKT inhibitor-IV (ChemBridge 5233705) affects this pathway and exhibits potent anticancer and antiviral activity. To probe its biological activity, we synthesized AKT inhibitor-IV and 21 analogues using a novel six-step route based on ZrCl(4)-catalyzed cyclization of 1,2-arylenediamines with α,β-unsaturated aldehydes. We examined effects on viability of HeLa carcinoma cells, viability of normal human cells (NHBE), replication of recombinant parainfluenza virus 5 (PIV5) in HeLa cells, and replication of the intracellular bacterium Mycobacterium fortuitum in HeLa cells. Replacement of the benzimidazole N-ethyl substitutent of AKT inhibitor-IV with N-hexyl and N-dodecyl groups enhanced antiviral activity and cytotoxicity against the cancer cell line, but these compounds showed substantially lower toxicity (from 6-fold to >20-fold) against NHBE cells and no effect on M. fortuitum, suggesting inhibition of one or more host protein(s) required for proliferation of cancer cells and PIV5. The key structural elements identified here may facilitate identification of targets of this highly biologically active scaffold.

  2. Novel B55α-PP2A mutations in AML promote AKT T308 phosphorylation and sensitivity to AKT inhibitor-induced growth arrest

    PubMed Central

    Shouse, Geoffrey; de Necochea-Campion, Rosalia; Mirshahidi, Saied; Liu, Xuan; Chen, Chien-Shing

    2016-01-01

    Activation of the Protein Kinase B (PKB), or AKT pathway has been shown to correlate with acute myeloid leukemia (AML) prognosis. B55α-Protein Phosphatase 2A (PP2A) has been shown to dephosphorylate AKT at Thr-308 rendering it inactive. In fact, low expression of the PP2A regulatory subunit B55α was associated with activated phospho-AKT and correlated with inferior outcomes in AML. Despite this fact, no studies have specifically demonstrated a mechanism whereby B55α expression is regulated in AML. In this study, we demonstrate novel loss of function mutations in the PPP2R2A gene identified in leukemic blasts from three AML patients. These mutations eliminate B55α protein expression thereby allowing constitutive AKT activation. In addition, leukemic blasts with PPP2R2A gene mutation were more sensitive to treatment with the AKT inhibitor MK2206, but less responsive to the PP2A activator FTY720. Using leukemia cell lines, we further demonstrate that B55α expression correlates with AKT Thr-308 phosphorylation and predicts responsiveness to AKT inhibition and PP2A activation. Together our data illustrate the importance of the B55α-PP2A-AKT pathway in leukemogenesis. Screening for disruptions in this pathway at initial AML diagnosis may predict response to targeted therapies against AKT and PP2A. PMID:27531894

  3. Snail regulates Nanog status during the epithelial–mesenchymal transition via the Smad1/Akt/GSK3β signaling pathway in non-small-cell lung cancer

    PubMed Central

    Liu, Chen-Wei; Li, Ching-Hao; Yi-Jen, Peng; Cheng, Yu-Wen; Chen, Huei-Wen; Liao, Po-Lin; Kang, Jaw-Jou; Yeng, Mao-Hsiung

    2014-01-01

    The epithelial–mesenchymal transition (EMT), a crucial step in cancer metastasis, is important in transformed cancer cells with stem cell-like properties. In this study, we established a Snail-overexpressing cell model for non-small-cell lung cancer (NSCLC) and investigated its underlying mechanism. We also identified the downstream molecular signaling pathway that contributes to the role of Snail in regulating Nanog expression. Our data shows that high levels of Snail expression correlate with metastasis and high levels of Nanog expression in NSCLC. NSCLC cells expressing Snail are characterized by active EMT characteristics and exhibit an increased ability to migrate, chemoresistance, sphere formation, and stem cell-like properties. We also investigated the signals required for Snail-mediated Nanog expression. Our data demonstrate that LY294002, SB431542, LDN193189, and Noggin pretreatment inhibit Snail-induced Nanog expression during EMT. This study shows a significant correlation between Snail expression and phosphorylation of Smad1, Akt, and GSK3β. In addition, pretreatment with SB431542, LDN193189, or Noggin prevented Snail-induced Smad1 and Akt hyperactivation and reactivated GSK3β. Moreover, LY294002 pretreatment prevented Akt hyperactivation and reactivated GSK3β without altering Smad1 activation. These findings provide a novel mechanistic insight into the important role of Snail in NSCLC during EMT and indicate potentially useful therapeutic targets for NSCLC. PMID:25003810

  4. Hydrogen sulfide protects H9c2 cardiac cells against doxorubicin-induced cytotoxicity through the PI3K/Akt/FoxO3a pathway.

    PubMed

    Liu, Mi-Hua; Zhang, Yuan; He, Jun; Tan, Tian-Ping; Wu, Shao-Jian; Guo, Dong-Ming; He, Hui; Peng, Juan; Tang, Zhi-Han; Jiang, Zhi-Sheng

    2016-06-01

    Doxorubicin (DOX) is an efficient drug used in cancer therapy that also produces reactive oxygen species (ROS) that induces severe cytotoxicity, which limits its clinical application. Hydrogen sulfide (H2S), a novel gasotransmitter, has been shown to exert cardioprotective effects. The present study aimed to determine whether exogenous H2S protects H9c2 cardiac cells against DOX-induced cytotoxicity and whether these protective effects are mediated through the PI3K/Akt/FoxO3a pathway. The H9c2 cardiac cells were exposed to 5 µM DOX for 24 h to establish a model of DOX-induced cardiotoxicity. The results showed that the treatment of H9c2 cardiac cells with sodium hydrosulfide (NaHS) for 30 min prior to DOX exposure markedly attenuated the phosphorylation of Akt and FoxO3a. Notably, pre-treatment of the H9c2 cells with NaHS significantly attenuated the nuclear localization of FoxO3a as well as the apoptosis of H9c2 cells induced by DOX. The treatment of H9c2 cells with N-acetyl-L-cysteine (NAC), a scavenger of ROS, prior to DOX exposure, also markedly increased the phosphorylation of Akt and FoxO3a which was inhibited by DOX alone. Furthermore, pre-treatment with LY294002, a selective inhibitor of PI3K/Akt, reversed the protective effect of H2S against DOX-induced injury of cardiomyocytes, as demonstrated by an increased number of apoptotic cells, a decrease in cell viability and the reduced phosphorylation of Akt and FoxO3a. These findings suggested that exogenous H2S attenuates DOX-induced cytotoxic effects in H9c2 cardiac cells through the PI3K/Akt/FoxO3a pathway.

  5. Bisdemethoxycurcumin protects endothelial cells against t-BHP-induced cell damage by regulating the phosphorylation level of ERK1/2 and Akt.

    PubMed

    Li, Ying-Bo; Gao, Jian-Li; Lee, Simon Ming-Yuen; Zhang, Qing-Wen; Hoi, Pui-Man; Wang, Yi-Tao

    2011-02-01

    Curcuminoids are the major active components extracted from Curcuma longa and are well known for their antioxidant effects. Previous studies have reported that the antioxidant properties of curcuminoids are mainly attributed to their free radical scavenging abilities. However, whether there are other mechanisms besides the non-enzymatic process and how they are involved, still remains unknown. In the present study, we explored the protective effects of bisdemethoxycurcumin (Cur3) against tert-butyl hydroperoxide (t-BHP)-induced cytotoxicity in human umbilical vein endothelial cells (HUVECs), focusing on the effect of Cur3 on the regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt and the mitogen-activated protein kinase (MAPK) pathways. The pre-treatment with Cur3 inhibited t-BHP-induced cell damage dose-dependently, which was evident by the increased cell viability and the corresponding decrease in lactate dehydrogenase release. The pre-treatment with Cur3 also attenuated t-BHP-induced cell morphological changes and apoptosis. MAPKs, including p38, c-Jun N-terminal kinase (JNK), extracellular signal-regulated protein kinase 1/2 (ERK1/2), as well as PI3K/Akt have been reported to be involved in proliferation, apoptosis and differentiation under various stress stimulations. The pre-treatment with Cur3 decreased t-BHP-induced ERK1/2 phosphorylation and increased t-BHP-induced Akt phosporylation but did not affect the phosphorylation of p38 or JNK. In addition, the Cur3-induced increase in cell viability was attenuated by the treatment with wortmannin or LY294002, the upstream inhibitors of Akt, and was enhanced by the treatment with 2-[2'-amino-3'-methoxyphenyl]-oxanaphthalen-4-one (PD98059), an upstream inhibitor of ERK1/2. These results suggest that the ERK1/2 and PI3K/Akt signaling pathways could be involved in the protective effects of Cur3 against t-BHP-induced damage in HUVECs.

  6. Rumex acetosa L. induces vasorelaxation in rat aorta via activation of PI3-kinase/Akt- AND Ca(2+)-eNOS-NO signaling in endothelial cells.

    PubMed

    Sun, Y Y; Su, X H; Jin, J Y; Zhou, Z Q; Sun, S S; Wen, J F; Kang, D G; Lee, H S; Cho, K W; Jin, S N

    2015-12-01

    Rumex acetosa L. (RA) (Polygonaceae) is an important traditional Chinese medicine (TCM) commonly used in clinic for a long history in China and the aerial parts of RA has a wide variety of pharmacological actions such as diuretic, anti-hypertensive, anti-oxidative, and anti-cancer effects. However, the mechanisms involved are to be defined. The purpose of the present study was to evaluate the vasorelaxant effect and define the mechanism of action of the ethanol extract of Rumex acetosa L. (ERA) in rat aorta. ERA was examined for its vascular relaxant effect in isolated phenylephrine-precontracted rat thoracic aorta and its acute effects on arterial blood pressure. In addition, the roles of the nitric oxide synthase (NOS)-nitric oxide (NO) signaling in the ERA-induced effects were tested in human umbilical vein endothelial cells (HUVECs). The phosphorylation levels of Akt and eNOS were assessed by Western blot analysis in the cultured HUVECs. ERA induced endothelium-dependent vasorelaxation. The ERA-induced vasorelaxation was abolished by L-NAME (an NOS inhibitor) or ODQ (a sGC inhibitor), but not by indomethacin. Inhibition of PI3-kinase/Akt signaling pathway markedly reduced the ERA-induced vasorelaxation. In HUVECs, ERA increased NO formation in a dose-dependent manner, which was inhibited by L-NAME and by removing extracellular Ca(2+). In addition, ERA promoted phosphorylation of Akt and eNOS, which was prevented by wortmannin and LY294002, indicating that ERA induces eNOS phosphorylation through the PI3-kinase/Akt pathway. Further, in anesthetized rats, intravenously administered ERA decreased arterial blood pressure in a dose-dependent manner through an activation of the NOS-NO system. In summary, the ERA- induced vasorelaxation was dependent on endothelial integrity and NO production, and was mediated by activation of both the endothelial PI3-kinase/Akt- and Ca(2+)-eNOS-NO signaling and muscular NO-sGC-cGMP signaling.

  7. Involvement of cyclin D1/CDK4 and pRb mediated by PI3K/AKT pathway activation in Pb{sup 2+}-induced neuronal death in cultured hippocampal neurons

    SciTech Connect

    Li Chenchen Xing Tairan Tang Mingliang Yong Wu Yan Dan Deng Hongmin Wang Huili Wang Ming Chen Jutao Ruan Diyun

    2008-06-15

    Lead (Pb) is widely recognized as a neurotoxicant. One of the suggested mechanisms of lead neurotoxicity is apoptotic cell death. And the mechanism by which Pb{sup 2+} causes neuronal death is not well understood. The present study sought to examine the obligate nature of cyclin D1/cyclin-dependent kinase 4 (CDK4), phosphorylation of its substrate retinoblastoma protein (pRb) and its select upstream signal phosphoinositide 3-kinase (PI3K)/AKT pathway in the death of primary cultured rat hippocampal neurons evoked by Pb{sup 2+}. Our data showed that lead treatment of primary hippocampal cultures results in dose-dependent cell death. Inhibition of CDK4 prevented Pb{sup 2+}-induced neuronal death significantly but was incomplete. In addition, we demonstrated that the levels of cyclin D1 and pRb/p107 were increased during Pb{sup 2+} treatment. These elevated expression persisted up to 48 h, returning to control levels after 72 h. We also presented pharmacological and morphological evidences that cyclin D1/CDK4 and pRb/p107 were required for such kind of neuronal death. Addition of the PI3K inhibitor LY294002 (30 {mu}M) or wortmannin (100 nM) significantly rescued the cultured hippocampal neurons from death caused by Pb{sup 2+}. And that Pb{sup 2+}-elicited phospho-AKT (Ser473) participated in the induction of cyclin D1 and partial pRb/p107 expression. These results provide evidences that cell cycle elements play a required role in the death of neurons evoked by Pb{sup 2+} and suggest that certain signaling elements upstream of cyclin D1/CDK4 are modified and/or required for this form of neuronal death.

  8. Akt inhibitor MK2206 prevents influenza pH1N1 virus infection in vitro.

    PubMed

    Denisova, Oxana V; Söderholm, Sandra; Virtanen, Salla; Von Schantz, Carina; Bychkov, Dmitrii; Vashchinkina, Elena; Desloovere, Jens; Tynell, Janne; Ikonen, Niina; Theisen, Linda L; Nyman, Tuula A; Matikainen, Sampsa; Kallioniemi, Olli; Julkunen, Ilkka; Muller, Claude P; Saelens, Xavier; Verkhusha, Vladislav V; Kainov, Denis E

    2014-07-01

    The influenza pH1N1 virus caused a global flu pandemic in 2009 and continues manifestation as a seasonal virus. Better understanding of the virus-host cell interaction could result in development of better prevention and treatment options. Here we show that the Akt inhibitor MK2206 blocks influenza pH1N1 virus infection in vitro. In particular, at noncytotoxic concentrations, MK2206 alters Akt signaling and inhibits endocytic uptake of the virus. Interestingly, MK2206 is unable to inhibit H3N2, H7N9, and H5N1 viruses, indicating that pH1N1 evolved specific requirements for efficient infection. Thus, Akt signaling could be exploited further for development of better therapeutics against pH1N1 virus.

  9. Angiogenin-induced protein kinase B/Akt activation is necessary for angiogenesis but is independent of nuclear translocation of angiogenin in HUVE cells

    SciTech Connect

    Kim, Hye-Mi; Kang, Dong-Ku; Kim, Hak Yong; Kang, Sang Sun; Chang, Soo-Ik . E-mail: sichang@cbnu.ac.kr

    2007-01-12

    Angiogenin, a potent angiogenic factor, binds to endothelial cells and is endocytosed and rapidly translocated to and concentrated in the nucleolus where it binds to DNA. In this study, we report that angiogenin induces transient phosphorylation of protein kinase B/Akt in cultured human umbilical vein endothelial (HUVE) cells. LY294002 inhibits the angiogenin-induced protein kinase B/Akt activation and also angiogenin-induced cell migration in vitro as well as angiogenesis in chick embryo chorioallantoic membrane in vivo without affecting nuclear translocation of angiogenin in HUVE cells. These results suggest that cross-talk between angiogenin and protein kinase B/Akt signaling pathways is essential for angiogenin-induced angiogenesis in vitro and in vivo, and that angiogenin-induced PKB/Akt activation is independent of nuclear translocation of angiogenin in HUVE cells.

  10. Novel Kinase Inhibitors Targeting the PH Domain of AKT for Preventing and Treating Cancer | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute's Medical Oncology Branch is seeking statements of capability or interest from parties interested in licensing and co-development collaborative research to further develop, evaluate, or commercialize novel kinase inhibitors targeting the PH domain of AKT.

  11. Ginsenoside-Rd Promotes Neurite Outgrowth of PC12 Cells through MAPK/ERK- and PI3K/AKT-Dependent Pathways

    PubMed Central

    Wu, Song-Di; Xia, Feng; Lin, Xue-Mei; Duan, Kang-Li; Wang, Fang; Lu, Qing-Li; Cao, Huan; Qian, Yi-Hua; Shi, Ming

    2016-01-01

    Panax ginseng is a famous herbal medicine widely used in Asia. Ginsenosides have been identified as the principle active ingredients for Panax ginseng’s biological activity, among which ginsenoside Rd (Rd) attracts extensive attention for its obvious neuroprotective activities. Here we investigated the effect of Rd on neurite outgrowth, a crucial process associated with neuronal repair. PC12 cells, which respond to nerve growth factor (NGF) and serve as a model for neuronal cells, were treated with different concentrations of Rd, and then their neurite outgrowth was evaluated. Our results showed that 10 μM Rd significantly increased the percentages of long neurite- and branching neurite-bearing cells, compared with respective controls. The length of the longest neurites and the total length of neurites in Rd-treated PC12 cells were much longer than that of respective controls. We also showed that Rd activated ERK1/2 and AKT but not PKC signalings, and inhibition of ERK1/2 by PD98059 or/and AKT by LY294002 effectively attenuated Rd-induced neurite outgrowth. Moreover, Rd upregulated the expression of GAP-43, a neuron-specific protein involved in neurite outgrowth, while PD98059 or/and LY294002 decreased Rd-induced increased GAP-43 expression. Taken together, our results provided the first evidence that Rd may promote the neurite outgrowth of PC12 cells by upregulating GAP-43 expression via ERK- and ARK-dependent signaling pathways. PMID:26840295

  12. Synergistic cardioprotective effects of Danshensu and hydroxysafflor yellow A against myocardial ischemia-reperfusion injury are mediated through the Akt/Nrf2/HO-1 pathway

    PubMed Central

    HU, TIANXIN; WEI, GUO; XI, MIAOMIAO; YAN, JIAJIA; WU, XIAOXIAO; WANG, YANHUA; ZHU, YANRONG; WANG, CHAO; WEN, AIDONG

    2016-01-01

    In clinical practice, the traditional Chinese medicinal herbs, Radix Salvia Miltiorrhiza and Carthamus tinctorius L., are usually prescribed in combination due to their significant cardioprotective effects. However, the mechanisms responsible for these combined effects remain unknown. Thus, in this study, we investigated the mechanisms responsible for the combined effects of Danshensu (DSS) and hydroxysafflor yellow A (HSYA) by establishing a rat model of myocardial ischemia/reperfusion (MI/R), as well as a model of hypoxia/reoxygenation (H/R) using H9c2 cells. The combination index (CI) was calculated using the median-effect method. DSS and HSYA in combination led to a CI value of <1 as regards infarct size in vivo and cell viability in vitro. The rats with MI/R injury that were treated with DSS and/or HSYA were found to have significantly lower levels of creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) and malondialdehyde (MDA), and a lower expressoin of 8-hydroxydeoxyguanosine (8-OHdG), and markedly enhanced superoxide dismutase (SOD) activity. Our in vitro experiments revealed that the cells treated with DSS and/or HSYA had a reduced lactate dehydrogenase (LDH) activity and a decreased percentage of cell apoptosis (increased Bcl-2/Bax ratio, decreased expression of cleaved caspase-3). DSS and HSYA increased the expression of heme oxygenase-1 (HO-1), the phosphorylation of Akt and the trans-location of nuclear factor erythroid 2-related factor 2 (Nrf2). Furthermore, the Akt inhibitor, LY294002, partially hampered the expression of Nrf2 and HO-1. The HO-1 inhibitor, zinc protoporphyrin IX (ZnPP-IX), did not decrease the expression of p-Akt and Nrf2, although it abolished the anti-apoptotic and antioxidant effects of DSS and HSYA. The findings of our study thus demonstrate that DSS and HSYA confer synergistic cardioprotective effects through the Akt/Nrf2/HO-1 signaling pathway, to certain extent, by enhancing the antioxidant defense system and exerting

  13. The neuroprotective effect of a novel agent N2 on rat cerebral ischemia associated with the activation of PI3K/Akt signaling pathway.

    PubMed

    Huang, Jinru; Kodithuwakku, Nandani Darshika; He, Wei; Zhou, Yi; Fan, Wenxiang; Fang, Weirong; He, Guangwei; Wu, Qiang; Chu, Shaoxing; Li, Yunman

    2015-08-01

    Ischemic stroke is the third leading cause of death and the main reason for severe disabilities in the world today. N2, 4 - (2 - (1H - imidazol - 1 - yl) ethoxy) - 3 - methoxybenzoic acid is considered as a novel potent agent for cerebral ischemia due to its effect in preventing neuronal cell death after ischemic stroke. In the present study, we investigated the post-ischemic neuroprotective effect of N2 and its underlying mechanisms. Using a MCAO rat model, we found that N2 reversed brain infarct size, reduced cerebral edema and decreased the neurological deficit score significantly. Moreover, N2 diminished TUNEL positive cells, down-regulated bax expression and up-regulated bcl-2 expression notably. In addition, we evaluated the oxygen glucose deprivation/reoxygenation (OGD/R) injury induced neuron cell death in rat primary cortical neuron and assessed the neuroprotective effect of our drug. N2 increased cell viability, ameliorated neuron cell injury by decreasing LDH activity, and inhibited cell apoptotic rate while suppressed apoptotic signaling via inhibiting the bax expression, and elevating the bcl-2 expression. Furthermore, the neuroprotective effect of N2 was associated with the PI3K/Akt pathway which was proved by the use of PI3K inhibitor LY294002. The combination of our findings disclosed that N2 can be used as an effective neuroprotective agent for ischemic stroke due to its significant effect on preventing neuronal cell death after cerebral ischemia both in vivo and in vitro and the effectiveness was dose dependent.

  14. In search of AKT kinase inhibitors as anticancer agents: structure-based design, docking and molecular dynamics studies of 2,4,6-trisubstituted pyridines.

    PubMed

    Trejo-Soto, Pedro Josué; Hernández-Campos, Alicia; Romo-Mancillas, Antonio; Medina-Franco, José L; Castillo, Rafael

    2017-02-02

    The AKT isoforms are a group of key kinases that play a critical role in tumorigenesis. These enzymes are overexpressed in different types of cancers, such as breast, colon, prostate, ovarian and lung. Because of its relevance the AKT isoforms are attractive targets for the design of anticancer molecules. However, it has been found that AKT1 and AKT3 isoforms have a main role in tumor progression and metastasis; thus, the identification of AKT isoforms specific inhibitors seems to be a challenge. Previously, we identified an ATP binding pocket pan-AKT inhibitor, this compound is a 2,4,6-trisubstituted pyridine (compound 11), which represents a new interesting scaffold for the developing of AKT inhibitors. Starting from the 2,4,6-trisubstituted pyridine scaffold, and guided by structure-based design technique, 42 new inhibitors were designed and further evaluated in the three AKT isoforms by multiple docking approach and molecular dynamics. Results showed that seven compounds presented binding selectivity for AKT1 and AKT3, better than for AKT2. The binding affinities of these seven compounds on AKT1 and AKT3 isoforms were mainly determined by hydrophobic contributions between the aromatic portion at position 4 of the pyridine ring with residues Phe236/234, Phe237/235, Phe438/435 and Phe442/439 in the ATP binding pocket. Results presented in this work provide an addition knowledge leading to promising selective AKT inhibitors.

  15. Novel Cancer Chemotherapy Hits by Molecular Topology: Dual Akt and Beta-Catenin Inhibitors

    PubMed Central

    Morell, Cecilia; Rodríguez-Henche, Nieves; Recio-Iglesias, Maria Carmen; Garcia-Domenech, Ramon

    2015-01-01

    Background and Purpose Colorectal and prostate cancers are two of the most common types and cause of a high rate of deaths worldwide. Therefore, any strategy to stop or at least slacken the development and progression of malignant cells is an important therapeutic choice. The aim of the present work is the identification of novel cancer chemotherapy agents. Nowadays, many different drug discovery approaches are available, but this paper focuses on Molecular Topology, which has already demonstrated its extraordinary efficacy in this field, particularly in the identification of new hit and lead compounds against cancer. This methodology uses the graph theoretical formalism to numerically characterize molecular structures through the so called topological indices. Once obtained a specific framework, it allows the construction of complex mathematical models that can be used to predict physical, chemical or biological properties of compounds. In addition, Molecular Topology is highly efficient in selecting and designing new hit and lead drugs. According to the aforementioned, Molecular Topology has been applied here for the construction of specific Akt/mTOR and β-catenin inhibition mathematical models in order to identify and select novel antitumor agents. Experimental Approach Based on the results obtained by the selected mathematical models, six novel potential inhibitors of the Akt/mTOR and β-catenin pathways were identified. These compounds were then tested in vitro to confirm their biological activity. Conclusion and Implications Five of the selected compounds, CAS n° 256378-54-8 (Inhibitor n°1), 663203-38-1 (Inhibitor n°2), 247079-73-8 (Inhibitor n°3), 689769-86-6 (Inhibitor n°4) and 431925-096 (Inhibitor n°6) gave positive responses and resulted to be active for Akt/mTOR and/or β-catenin inhibition. This study confirms once again the Molecular Topology’s reliability and efficacy to find out novel drugs in the field of cancer. PMID:25910265

  16. Effects of AKT inhibitor therapy in response and resistance to BRAF inhibition in melanoma

    PubMed Central

    2014-01-01

    Background The clinical use of BRAF inhibitors for treatment of metastatic melanoma is limited by the development of drug resistance. In this study we investigated whether co-targeting the MAPK and the PI3K-AKT pathway can prevent emergence of resistance or provide additional growth inhibitory effects in vitro. Methods Anti-tumor effects of the combination of the BRAF inhibitor (BRAFi) dabrafenib and GSK2141795B (AKTi) in a panel of 23 BRAF mutated melanoma cell lines were evaluated on growth inhibition by an ATP-based luminescent assay, on cell cycle and apoptosis by flow cytometry and on cell signaling by western blot. Moreover, we investigated the possibilities of delaying or reversing resistance or achieving further growth inhibition by combining AKTi with dabrafenib and/or the MEK inhibitor (MEKi) trametinib by using long term cultures. Results More than 40% of the cell lines, including PTEN-/- and AKT mutants showed sensitivity to AKTi (IC50 < 1.5 μM). The combination of dabrafenib and AKTi synergistically potentiated growth inhibition in the majority of cell lines with IC50 > 5 nM dabrafenib. Combinatorial treatment induced apoptosis only in cell lines sensitive to AKTi. In long term cultures of a PTEN-/- cell line, combinatorial treatment with the MAPK inhibitors, dabrafenib and trametinib, and AKTi markedly delayed the emergence of drug resistance. Moreover, combining AKTi with the MAPK inhibitors from the beginning provided superior growth inhibitory effects compared to addition of AKTi upon development of resistance to MAPK inhibitors in this particular cell line. Conclusions AKTi combined with BRAFi-based therapy may benefit patients with tumors harboring BRAF mutations and particularly PTEN deletions or AKT mutations. PMID:24735930

  17. Intracellular Networks of the PI3K/AKT and MAPK Pathways for Regulating Toxoplasma gondii-Induced IL-23 and IL-12 Production in Human THP-1 Cells

    PubMed Central

    Choi, In-Wook; Ismail, Hassan Ahmed Hassan Ahmed; Zhou, Wei; Cha, Guang-Ho; Zhou, Yu; Yuk, Jae-Min; Jo, Eun-Kyeong; Lee, Young-Ha

    2015-01-01

    Interleukin (IL)-23 and IL-12 are closely related in structure, and these cytokines regulate both innate and adaptive immunity. However, the precise signaling networks that regulate the production of each in Toxoplasma gondii-infected THP-1 monocytic cells, particularly the PI3K/AKT and MAPK signaling pathways, remain unknown. In the present study, T. gondii infection upregulated the expression of IL-23 and IL-12 in THP-1 cells, and both cytokines increased with parasite dose. IL-23 secretion was strongly inhibited by TLR2 monoclonal antibody (mAb) treatment in a dose-dependent manner and by TLR2 siRNA transfection, whereas IL-12 secretion was strongly inhibited by TLR4 mAb treatment dose-dependently and by TLR4 siRNA transfection. IL-23 production was dose-dependently inhibited by the PI3K inhibitors LY294002 and wortmannin, whereas IL-12 production increased dose-dependently. THP-1 cells exposed to live T. gondii tachyzoites underwent rapid p38 MAPK, ERK1/2 and JNK activation. IL-23 production was significantly upregulated by the p38 MAPK inhibitor SB203580 dose-dependently, whereas pretreatment with 10 μM SB203580 significantly downregulated IL-12 production. ERK1/2 inhibition by PD98059 was significantly downregulated IL-23 production but upregulated IL-12 production. JNK inhibition by SP600125 upregulated IL-23 production, but IL-12 production was significantly downregulated dose-dependently. T. gondii infection resulted in AKT activation, and AKT phosphorylation was inhibited dose-dependently after pretreatment with PI3K inhibitors. In T. gondii-infected THP-1 cells, ERK1/2 activation was regulated by PI3K; however, the phosphorylation of p38 MAPK and JNK was negatively modulated by the PI3K signaling pathway. Collectively, these results indicate that IL-23 production in T. gondii-infected THP-1 cells was regulated mainly by TLR2 and then by PI3K and ERK1/2; however, IL-12 production was mainly regulated by TLR4 and then by p38 MAPK and JNK. Our findings

  18. 2-Methoxy-5((3,4,5-trimethosyphenyl)seleninyl) phenol (SQ0814061), a novel microtubule inhibitor, evokes G2/M cell cycle arrest and apoptosis in human breast cancer cells.

    PubMed

    Xu, Jingwen; Zuo, Daiying; Qi, Huan; Shen, Qirong; Bai, Zhaoshi; Han, Mengting; Li, Zengqiang; Zhang, Weige; Wu, Yingliang

    2016-03-01

    Breast cancer is the leading cause of cancer death in women worldwide, and novel chemotherapeutic drugs with high activity and no drug resistance for treating breast cancer are needed urgently. In this study, we investigated the antitumor effect of 2-methoxy-5((3,4,5-trimethosyphenyl)seleninyl) phenol (SQ0814061), which has a strong inhibition of cell growth in MCF-7 and MDA-MB-231 cells. We demonstrated that SQ0814061 (SQ) time-dependently induced cell cycle arrest at G2/M phase and subsequently progressed into apoptosis, which is associated with microtubule depolymerization. Western blot analysis revealed that up-regulation of cyclin B1 and Aurora A was related with G2/M phase arrest in MCF-7 and MDA-MB-231 cells treatment with SQ. However, the formation of multinucleated cells after a long time exposed to SQ of MCF-7 cells delayed the cell death. In addition, apoptosis induced by SQ is correlated with the down-regulation of the PI3K-Akt-MDM2 pathway in MCF-7 and MDA-MB-231 cells. Treatment with the PI3K specific inhibitor, LY294002, increased SQ-induced cell growth inhibitory rate and apoptosis rate of MCF-7 and MDA-MB-231 cells. Moreover, SQ induced MCF-7 and MDA-MB-231 cells to generate reactive oxygen species (ROS), and the SQ-induced cell death was ROS dependent. In conclusion, all the data demonstrated that SQ exhibited its antitumor activity through disrupting the microtubule assembly, inducing cell cycle arrest and eventually apoptosis which is associated with PI3K-Akt-MDM2 pathway in MCF-7 and MDA-MB-231 cells. Therefore, the novel compound SQ is a promising microtubule inhibitor that has tremendous potentials for therapeutic treatment of human mastocarcinoma.

  19. Computational modeling of novel inhibitors targeting the Akt pleckstrin homology domain.

    PubMed

    Du-Cuny, Lei; Song, Zuohe; Moses, Sylvestor; Powis, Garth; Mash, Eugene A; Meuillet, Emmanuelle J; Zhang, Shuxing

    2009-10-01

    Computational modeling continues to play an important role in novel therapeutics discovery and development. In this study, we have investigated the use of in silico approaches to develop inhibitors of the pleckstrin homology (PH) domain of AKT (protein kinase B). Various docking/scoring schemes have been evaluated, and the best combination was selected to study the system. Using this strategy, two hits were identified and their binding behaviors were investigated. Robust and predictive QSAR models were built using the k nearest neighbor (kNN) method to study their cellular permeability. Based on our in silico results, long flexible aliphatic tails were proposed to improve the Caco-2 penetration without affecting the binding mode. The modifications enhanced the AKT inhibitory activity of the compounds in cell-based assays, and increased their activity as in vivo antitumor testing.

  20. HER2-induced metastasis is mediated by AKT/JNK/EMT signaling pathway in gastric cancer

    PubMed Central

    Choi, Yiseul; Ko, Young San; Park, Jinju; Choi, Youngsun; Kim, Younghoon; Pyo, Jung-Soo; Jang, Bo Gun; Hwang, Douk Ho; Kim, Woo Ho; Lee, Byung Lan

    2016-01-01

    AIM To investigated the relationships between HER2, c-Jun N-terminal kinase (JNK) and protein kinase B (AKT) with respect to metastatic potential of HER2-positive gastric cancer (GC) cells. METHODS Immunohistochemistry was performed on tissue array slides containing 423 human GC specimens. Using HER2-positve GC cell lines SNU-216 and NCI-N87, HER2 expression was silenced by RNA interference, and the activations of JNK and AKT were suppressed by SP600125 and LY294002, respectively. Transwell assay, Western blot, semi-quantitative reverse transcription-polymerase chain reaction and immunofluorescence staining were used in cell culture experiments. RESULTS In GC specimens, HER2, JNK, and AKT activations were positively correlated with each other. In vitro analysis revealed a positive regulatory feedback loop between HER2 and JNK in GC cell lines and the role of JNK as a downstream effector of AKT in the HER2/AKT signaling pathway. JNK inhibition suppressed migratory capacity through reversing EMT and dual inhibition of JNK and AKT induced a more profound effect on cancer cell motility. CONCLUSION HER2, JNK and AKT in human GC specimens are positively associated with each other. JNK and AKT, downstream effectors of HER2, co-operatively contribute to the metastatic potential of HER2-positive GC cells. Thus, targeting of these two molecules in combination with HER2 downregulation may be a good approach to combat HER2-positive GC. PMID:27895401

  1. Regulation of different components from Ophiopogon japonicus on autophagy in human lung adenocarcinoma A549Cells through PI3K/Akt/mTOR signaling pathway.

    PubMed

    Chen, Juan; Yuan, Jiarui; Zhou, Liqiang; Zhu, Maomao; Shi, Ziqi; Song, Jie; Xu, Qingyu; Yin, Guowen; Lv, You; Luo, Yi; Jia, Xiaobin; Feng, Liang

    2017-03-01

    Autophagy plays a dual role in the development of cancer, acting as both a tumor suppressor and a cell survival inducer. Ophiopogon japonicus (L.f) Ker-Gawl (OJ), as a traditional Chinese medicine, specially possesses remarkable anti-cancer activity in the clinical. Previously, studies have indicated that flavonoids (FOJ) and steroidal saponins (SSOJ) are the main active substances of OJ. However, the effects of FOJ and SSOJ on autophagy of A549 cells have not been fully elucidated. In this study, we found that the expressions of autophagy-related mediators (LC3-II/LC3-I ratio, Atg-3, Atg-7 and Beclin-1) were increased in A549 cells by the treatment with FOJ (7.9mg crude drug/mL) and SSOJ (12.2mg crude drug/mL). Meanwhile, FOJ or SSOJ could induce the up-regulation of LC3-II at both protein and mRNA levels. Moreover, we observed the cytoplasmic vaculoes which formed double-layered membranes and only some cytoplasmic organelles or myelin figures remained in FOJ or SSOJ-treated A549 cells for 24h by Transmission Electron Microscopy (TEM). Further detection about the PI3K/Akt/mTOR signaling pathway showed that the levels of PI3K, Akt and mTOR were significantly suppressed with the FOJ or SSOJ treatment. The 3-MA (an autophagy inhibitor) and LY294002 (a PI3K inhibitor) further confirmed the underlying mechanism in the FOJ or SSOJ-induced autophagy of A549 cells. Additionally, the pretreatment with FOJ and SSOJ increased the level of p53, whereas decreased the expression of Ki67. These findings suggested that FOJ or SSOJ could activate the autophagy of A549 cells, wherein the mechanism might be associated with their inhibition of PI3K/Akt/mTOR signaling pathway. Thus, FOJ or SSOJ could be a potential autophagy inducer to prevent the process of lung cancer.

  2. A novel and promising therapeutic approach for NSCLC: recombinant human arginase alone or combined with autophagy inhibitor.

    PubMed

    Shen, Weitao; Zhang, Xuyao; Fu, Xiang; Fan, Jiajun; Luan, Jingyun; Cao, Zhonglian; Yang, Ping; Xu, Zhongyuan; Ju, Dianwen

    2017-03-30

    Recombinant human arginase (rhArg), an enzyme capable of depleting arginine, has been shown to be an effective therapeutic approach for various cancers. Non-small-cell lung cancer (NSCLC), a histological subtype of pulmonary carcinoma, has a high rate of morbidity and mortality in the world. Thus, the need for novel and more effective treatment is urgent. In this study, it is the first time to report that rhArg could induce significant cytotoxicity and caspase-dependent apoptosis in NSCLC cells. Subsequently, our research revealed that rhArg dramatically stimulated autophagic response in NSCLC cells, which was proved by the formation and accumulation of autophagosomes and the conversion of microtubule-associated protein light chain 3 (LC3) from LC3-I to LC3-II. Furthermore, blocking autophagy by chloroquine or LY294002 remarkably enhanced rhArg-induced cytotoxicity and caspase-dependent apoptosis, suggesting that autophagy acted a cytoprotective role in rhArg-treated NSCLC cells. Further experiments showed that two signaling pathways including the Akt/mTOR and extracellular signal-regulated kinase pathway, and mitochondrial-derived reactive oxygen species (ROS) production were involved in rhArg-induced autophagy and apoptosis. Meanwhile, N-acetyl-L-cysteine, a common antioxidant, was employed to scavenge ROS, and we detected that it could significantly block rhArg-induced autophagy and cytotoxicity, indicating that ROS played a vital role in arginine degradation therapy. Besides, xenograft experiment showed that combination with autophagy inhibitor potentiated the anti-tumor efficacy of rhArg in vivo. Therefore, these results provided a novel prospect and viewpoint that autophagy acted a cytoprotective role in rhArg-treated NSCLC cells, and treatment with rhArg alone or combined with autophagy inhibitor could be a novel and promising therapeutic approach for NSCLC in vivo and in vitro.

  3. Fisetin inhibits TNF-α-induced inflammatory action and hydrogen peroxide-induced oxidative damage in human keratinocyte HaCaT cells through PI3K/AKT/Nrf-2-mediated heme oxygenase-1 expression.

    PubMed

    Seo, Seung-Hee; Jeong, Gil-Saeng

    2015-12-01

    Oxidative skin damage and skin inflammation play key roles in the pathogenesis of skin-related diseases. Fisetin is a naturally occurring flavonoid abundantly found in several vegetables and fruits. Fisetin has been shown to exert various positive biological effects, such as anti-cancer, anti-proliferative, neuroprotective and anti-oxidative effects. In this study, we investigate the skin protective effects and anti-inflammatory properties of fisetin in hydrogen peroxide- and TNF-α-challenged human keratinocyte HaCaT cells. When HaCaT cells were treated with non-cytotoxic concentrations of fisetin (1-20μM), heme oxygenase (HO)-1 mRNA and protein expression increased in a dose-dependent manner. Furthermore, fisetin dose-dependently increased cell viability and reduced ROS production in hydrogen peroxide-treated HaCaT cells. Fisetin also inhibited the production of NO, PGE2 IL-1β, IL-6, expression of iNOS and COX-2, and activation of NF-κB in HaCaT cells treated with TNF-α. Fisetin induced Nrf2 translocation to the nuclei. HO-1 siRNA transient transfection reversed the effects of fisetin on cytoprotection, ROS reduction, NO, PGE2, IL-1β, IL-6, and TNF-α production, and NF-κB DNA-binding activity. Moreover, fisetin increased Akt phosphorylation and a PI3K pathway inhibitor (LY294002) abolished fisetin-induced cytoprotection and NO inhibition. Taken together, these results provide evidence for a beneficial role of fisetin in skin therapy.

  4. Cobalt chloride stimulates phosphoinositide 3-kinase/Akt signaling through the epidermal growth factor receptor in oral squamous cell carcinoma.

    PubMed

    Ryu, Mi Heon; Park, Jeong Hee; Park, Ji Eun; Chung, Jin; Lee, Chang Hun; Park, Hae Ryoun

    2010-04-01

    Tumor cells are often found under hypoxic conditions due to the rapid outgrowth of their vascular supply, and, in order to survive hypoxia, these cells induce numerous signaling factors. Akt is an important kinase in cell survival, and its activity is regulated by the upstream phosphoinositide 3-kinase (PI3K) and receptor tyrosine kinases (RTKs). In this study, we examined Akt activation and RTKs/PI3K/Akt signaling using the hypoxia-mimetic cobalt chloride in oral squamous carcinoma cells. Cobalt chloride increases Akt phosphorylation in both a dose- and time-dependent manner. Blocking the activation of the PI3K/Akt pathway using LY294002 abolished Akt activation in response to cobalt chloride, suggesting that Akt phosphorylation by cobalt chloride is dependent on PI3K. In addition, activation of the PI3K/Akt pathway seems to rely on the epidermal growth factor receptor (EGFR), since the inhibition of EGFR attenuated cobalt chloride-induced Akt activation. The results in this study also demonstrate that cobalt chloride increases EGFR protein levels and induces oral squamous cell carcinoma cells to enter S phase.

  5. The novel Akt inhibitor Palomid 529 (P529) enhances the effect of radiotherapy in prostate cancer.

    PubMed

    Diaz, R; Nguewa, P A; Diaz-Gonzalez, J A; Hamel, E; Gonzalez-Moreno, O; Catena, R; Serrano, D; Redrado, M; Sherris, D; Calvo, A

    2009-03-24

    Radiotherapy (RT) is a common treatment for localised prostate cancer, but can cause important side effects. The therapeutic efficacy of RT can be enhanced by pharmacological compounds that target specific pathways involved in cell survival. This would elicit a similar therapeutic response using lower doses of RT and, in turn, reducing side effects. This study describes the antitumour activity of the novel Akt inhibitor 8-(1-Hydroxy-ethyl)-2-methoxy-3-(4-methoxy-benzyloxy)-benzo[c]chromen-6-one (Palomid 529 or P529) as well as its ability to decrease radiation-activated phospho-Akt (p-Akt) signalling in a prostate cancer model. P529 showed a potent antiproliferative activity in the NCI-60 cell lines panel, with growth inhibitory 50 (GI50) <35 microM. In addition, P529 significantly enhanced the antiproliferative effect of radiation in prostate cancer cells (PC-3). Analysis of signalling pathways targeted by P529 exhibited a decrease in p-Akt, VEGF, MMP-2, MMP-9, and Id-1 levels after radiation treatment. Moreover, the Bcl-2/Bax ratio was also reduced. Treatment of PC-3 tumour-bearing mice with 20 mg kg(-1) P529 or 6 Gy radiation dose decreased tumour size by 42.9 and 53%, respectively. Combination of both treatments resulted in 77.4% tumour shrinkage. Decreased tumour growth was due to reduced proliferation and increased apoptosis (as assessed by PCNA and caspase-3 immunostaining). Our results show the antitumour efficacy of P529 alone, and as a radiosensitiser, and suggest that this compound could be used in the future to treat human prostate cancer.

  6. The novel Akt inhibitor Palomid 529 (P529) enhances the effect of radiotherapy in prostate cancer

    PubMed Central

    Diaz, R; Nguewa, P A; Diaz-Gonzalez, J A; Hamel, E; Gonzalez-Moreno, O; Catena, R; Serrano, D; Redrado, M; Sherris, D; Calvo, A

    2009-01-01

    Radiotherapy (RT) is a common treatment for localised prostate cancer, but can cause important side effects. The therapeutic efficacy of RT can be enhanced by pharmacological compounds that target specific pathways involved in cell survival. This would elicit a similar therapeutic response using lower doses of RT and, in turn, reducing side effects. This study describes the antitumour activity of the novel Akt inhibitor 8-(1-Hydroxy-ethyl)-2-methoxy-3-(4-methoxy-benzyloxy)-benzo[c]chromen-6-one (Palomid 529 or P529) as well as its ability to decrease radiation-activated phospho-Akt (p-Akt) signalling in a prostate cancer model. P529 showed a potent antiproliferative activity in the NCI-60 cell lines panel, with growth inhibitory 50 (GI50) <35 μM. In addition, P529 significantly enhanced the antiproliferative effect of radiation in prostate cancer cells (PC-3). Analysis of signalling pathways targeted by P529 exhibited a decrease in p-Akt, VEGF, MMP-2, MMP-9, and Id-1 levels after radiation treatment. Moreover, the Bcl-2/Bax ratio was also reduced. Treatment of PC-3 tumour-bearing mice with 20 mg kg−1 P529 or 6 Gy radiation dose decreased tumour size by 42.9 and 53%, respectively. Combination of both treatments resulted in 77.4% tumour shrinkage. Decreased tumour growth was due to reduced proliferation and increased apoptosis (as assessed by PCNA and caspase-3 immunostaining). Our results show the antitumour efficacy of P529 alone, and as a radiosensitiser, and suggest that this compound could be used in the future to treat human prostate cancer. PMID:19240717

  7. Autophagy Inhibitors as a Potential Antiamoebic Treatment for Acanthamoeba Keratitis

    PubMed Central

    Moon, Eun-Kyung; Kim, So-Hee; Hong, Yeonchul; Chung, Dong-Il; Goo, Youn-Kyoung

    2015-01-01

    Acanthamoeba cysts are resistant to extreme physical and chemical conditions. Autophagy is an essential pathway for encystation of Acanthamoeba cells. To evaluate the possibility of an autophagic Acanthamoeba encystation mechanism, we evaluated autophagy inhibitors, such as 3-methyladenine (3MA), LY294002, wortmannin, bafilomycin A, and chloroquine. Among these autophagy inhibitors, the use of 3MA and chloroquine showed a significant reduction in the encystation ratio in Acanthamoeba cells. Wortmannin also inhibited the formation of mature cysts, while LY294002 and bafilomycin A did not affect the encystation of Acanthamoeba cells. Transmission electron microscopy revealed that 3MA and wortmannin inhibited autophagy formation and that chloroquine interfered with the formation of autolysosomes. Inhibition of autophagy or autolysosome formation resulted in a significant block in the encystation in Acanthamoeba cells. Clinical treatment with 0.02% polyhexamethylene biguanide (PHMB) showed high cytopathic effects on Acanthamoeba trophozoites and cysts; however, it also revealed high cytopathic effects on human corneal epithelial cells. In this study, we investigated effects of the combination of a low (0.00125%) concentration of PHMB with each of the autophagy inhibitors 3MA, wortmannin, and chloroquine on Acanthamoeba and human corneal epithelial cells. These new combination treatments showed low cytopathic effects on human corneal cells and high cytopathic effects on Acanthamoeba cells. Taken together, these results provide fundamental information for optimizing the treatment of Acanthamoeba keratitis. PMID:25896709

  8. Autophagy inhibitors as a potential antiamoebic treatment for Acanthamoeba keratitis.

    PubMed

    Moon, Eun-Kyung; Kim, So-Hee; Hong, Yeonchul; Chung, Dong-Il; Goo, Youn-Kyoung; Kong, Hyun-Hee

    2015-07-01

    Acanthamoeba cysts are resistant to extreme physical and chemical conditions. Autophagy is an essential pathway for encystation of Acanthamoeba cells. To evaluate the possibility of an autophagic Acanthamoeba encystation mechanism, we evaluated autophagy inhibitors, such as 3-methyladenine (3MA), LY294002, wortmannin, bafilomycin A, and chloroquine. Among these autophagy inhibitors, the use of 3MA and chloroquine showed a significant reduction in the encystation ratio in Acanthamoeba cells. Wortmannin also inhibited the formation of mature cysts, while LY294002 and bafilomycin A did not affect the encystation of Acanthamoeba cells. Transmission electron microscopy revealed that 3MA and wortmannin inhibited autophagy formation and that chloroquine interfered with the formation of autolysosomes. Inhibition of autophagy or autolysosome formation resulted in a significant block in the encystation in Acanthamoeba cells. Clinical treatment with 0.02% polyhexamethylene biguanide (PHMB) showed high cytopathic effects on Acanthamoeba trophozoites and cysts; however, it also revealed high cytopathic effects on human corneal epithelial cells. In this study, we investigated effects of the combination of a low (0.00125%) concentration of PHMB with each of the autophagy inhibitors 3MA, wortmannin, and chloroquine on Acanthamoeba and human corneal epithelial cells. These new combination treatments showed low cytopathic effects on human corneal cells and high cytopathic effects on Acanthamoeba cells. Taken together, these results provide fundamental information for optimizing the treatment of Acanthamoeba keratitis.

  9. LEOPARD-type SHP2 mutant Gln510Glu attenuates cardiomyocyte differentiation and promotes cardiac hypertrophy via dysregulation of Akt/GSK-3β/β-catenin signaling.

    PubMed

    Ishida, Hidekazu; Kogaki, Shigetoyo; Narita, Jun; Ichimori, Hiroaki; Nawa, Nobutoshi; Okada, Yoko; Takahashi, Kunihiko; Ozono, Keiichi

    2011-10-01

    LEOPARD syndrome (LS) is an autosomal dominant inherited multisystemic disorder. Most cases involve mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase Src homology 2-containing protein phosphatase 2 (SHP2). LS frequently causes severe hypertrophic cardiomyopathy (HCM), even from the fetal period. However, the molecular pathogenesis has not been clearly elucidated. Here, we analyzed the roles of the LS-type SHP2 mutant Gln510Glu (Q510E), which showed the most severe type of HCM in LS, in cardiomyocyte differentiation, and in morphological changes. We generated mutant P19CL6 cell lines, the most convenient cardiomyocyte differentiation model, which continuously expressed SHP2-Q510E, SHP2-D61N (Noonan-type mutant), wild-type SHP2, and green fluorescent protein (native SHP2 expression only). SHP2-Q510E mutant P19CL6 cells showed significant attenuation of myofibrillogenesis, with increased proliferative activity. Mature cardiomyocytes from the SHP2-Q510E mutant were significantly larger than those of controls and the other mutants. However, expression of cardiac-specific transcriptional factors (Gata4, Tbx5, and Nkx2.5) did not differ significantly between the LS-type SHP2-Q510E mutants and the other mutants and controls. Our results indicate that SHP2-Q510E mutants can differentiate into cardiac progenitors but are inhibited from undergoing terminal differentiation into mature cardiomyocytes. In contrast, Akt and glycogen synthase kinase (GSK)-3β phosphorylation were upregulated, and nuclear β-catenin at the late stage of differentiation was highly accumulated in SHP2-Q510E mutant P19CL6 cells. Supplementation with the phosphoinositide 3-kinase/Akt inhibitor LY-294002 during the late stage of differentiation was found to partially restore myofibrillogenesis while suppressing the increase in size of individual mature cardiomyocytes derived from the SHP2-Q510E mutants. Our findings suggest that dysregulation of the Akt/GSK-3

  10. Donepezil, an acetylcholine esterase inhibitor, and ABT-239, a histamine H3 receptor antagonist/inverse agonist, require the integrity of brain histamine system to exert biochemical and procognitive effects in the mouse.

    PubMed

    Provensi, Gustavo; Costa, Alessia; Passani, M Beatrice; Blandina, Patrizio

    2016-10-01

    Histaminergic H3 receptors (H3R) antagonists enhance cognition in preclinical models and modulate neurotransmission, in particular acetylcholine (ACh) release in the cortex and hippocampus, two brain areas involved in memory processing. The cognitive deficits seen in aging and Alzheimer's disease have been associated with brain cholinergic deficits. Donepezil is one of the acetylcholinesterase (AChE) inhibitor approved for use across the full spectrum of these cognitive disorders. We addressed the question if H3R antagonists and donepezil require an intact histamine neuronal system to exert their procognitive effects. The effect of the H3R antagonist ABT-239 and donepezil were evaluated in the object recognition test (ORT), and on the level of glycogen synthase kinase 3 beta (GSK-3β) phosphorylation in normal and histamine-depleted mice. Systemic administration of ABT-239 or donepezil ameliorated the cognitive performance in the ORT. However, these compounds were ineffective in either genetically (histidine decarboxylase knock-out, HDC-KO) or pharmacologically, by means of intracerebroventricular (i.c.v.) injections of the HDC irreversible inhibitor a-fluoromethylhistidine (a-FMHis), histamine-deficient mice. Western blot analysis revealed that ABT-239 or donepezil systemic treatments increased GSK-3β phosphorylation in cortical and hippocampal homogenates of normal, but not of histamine-depleted mice. Furthermore, administration of the PI3K inhibitor LY294002 that blocks GSK-3β phosphorylation, prevented the procognitive effects of both drugs in normal mice. Our results indicate that both donepezil and ABT-239 require the integrity of the brain histaminergic system to exert their procognitive effects and strongly suggest that impairments of PI3K/AKT/GSK-3β intracellular pathway activation is responsible for the inefficacy of both drugs in histamine-deficient animals.

  11. Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

    SciTech Connect

    Kizhakkayil, Jaleel; Thayyullathil, Faisal; Chathoth, Shahanas; Hago, Abdulkader; Patel, Mahendra; Galadari, Sehamuddin

    2010-04-09

    Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3{beta} (GSK3{beta}), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3{beta}. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.

  12. Reactivation of ERK and Akt confers resistance of mutant BRAF colon cancer cells to the HSP90 inhibitor AUY922.

    PubMed

    Wang, Chun Yan; Guo, Su Tang; Wang, Jia Yu; Yan, Xu Guang; Farrelly, Margaret; Zhang, Yuan Yuan; Liu, Fen; Yari, Hamed; La, Ting; Lei, Fu Xi; Jin, Lei; Zhang, Xu Dong; Jiang, Chen Chen

    2016-08-02

    Oncogenic mutations of BRAF occur in approximately 10% of colon cancers and are associated with their resistance to clinically available therapeutic drugs and poor prognosis of the patients. Here we report that colon cancer cells with mutant BRAF are also resistant to the heat shock protein 90 (HSP90) inhibitor AUY922, and that this is caused by rebound activation of ERK and Akt. Although AUY922 triggered rapid reduction in ERK and Akt activation in both wild-type and mutant BRAF colon cancer cells, activation of ERK and Akt rebounded shortly in the latter leading to resistance of the cells to AUY922-induced apoptosis. Reactivation of ERK was associated with the persistent expression of mutant BRAF, which, despite being a client of HSP90, was only partially degraded by AUY922, whereas reactivation of Akt was related to the activity of the HSP90 co-chaperone, cell division cycle 37 (CDC37), in that knockdown of CDC37 inhibited Akt reactivation in mutant colon cancer cells treated with AUY922. In support, as a HSP90 client protein, Akt was only diminished by AUY922 in wild-type but not mutant BRAF colon cancer cells. Collectively, these results reveal that reactivation of ERK and Akt associated respectively with the activity of mutant BRAF and CDC37 renders mutant BRAF colon cancer cells resistant to AUY922, with implications of co-targeting mutant BRAF and/or CDC37 and HSP90 in the treatment of mutant BRAF colon cancers.

  13. CHRNA7 inhibits cell invasion and metastasis of LoVo human colorectal cancer cells through PI3K/Akt signaling.

    PubMed

    Xiang, Tao; Yu, Feng; Fei, Rushan; Qian, Jing; Chen, Wenbin

    2016-02-01

    The α7 neuronal nicotinic receptor gene (CHRNA7) is widely expressed in both the brain and periphery whereas its encoding protein of α7 neuronal acetylcholine receptor (α7nAChR) belongs to the nicotinic acetylcholine receptor family. Considerable evidence suggests that α7nAChR plays an important role in chronic inflammatory and neuropathic pain signaling and thus has been proposed as a potential target for treating cognitive deficits in patients with schizophrenia, attention deficit hyperactivity disorder (ADHD) and Alzheimer's disease. The aim of the present study was to determine the role of endogenous α7nAChR signaling in human colorectal cancer growth and metastasis. pLVX‑CHRNA7 encoding the full length of CHRNA7 was constructed and transfected into LoVo human colorectal cancer cells. Cell proliferation was measured by Cell Counting Kit‑8 (CCK‑8), and cell migration and invasion were detected by Transwell chamber assays. Expression and activity of metastasis‑related metalloproteinases (MMPs) were analyzed by western blotting and gelatin zymography, respectively. Activation of metastasis-related signaling molecules was detected by western blotting. LY294002 was used to specifically block the phosphatidylinositol 3‑kinase/v‑akt murine thymoma viral oncogene homologue (PI3K/Akt) pathway. We showed that concomitantly with an increase in α7nAChR expression after transfection, LoVo cells presented reduced abilities for migration and invasion, which was accompanied by reduced expression levels of MMP‑1 and MMP‑9 as well as activation of the PI3K/Akt signaling pathway. The application of LY294002 restored the migration and invasion abilities of the LoVo cells bearing CHRNA7. Collectively, we conclude that overexpression of CHRNA7 negatively controls colorectal cancer LoVo cell invasion and metastasis via PI3K/Akt pathway activation and may serve as either a diagnostic marker or a therapeutic target for colorectal cancer metastasis.

  14. Activation of the phosphatidylinositol 3-kinase/Akt pathway is involved in lipocalin-2-promoted human pulmonary artery smooth muscle cell proliferation.

    PubMed

    Wang, Guoliang; Ma, Ning; Meng, Liukun; Wei, Yingjie; Gui, Jingang

    2015-12-01

    Over-activated PI3K/Akt signaling, a pathway strongly related to cancer survival and proliferation, has been reported recently to be involved in pulmonary artery smooth muscle cell apoptosis and proliferation in pulmonary hypertension (PH). In this study, we observed greatly increased lipocalin-2 (Lcn2) expression accompanied with over-activated PI3K/Akt signaling in a standard rat model of PH induced by monocrotaline. In view of the close relationship between Lcn2 and PI3K/Akt pathway, we hypothesized that the up-regulated Lcn2 might be a trigger of over-activated PI3K/Akt signaling in PH. Our results showed that Lcn2 significantly activated the PI3K/Akt pathway (determined by augmented Akt phosphorylation and up-regulated Mdm2) and significantly promoted proliferation (assessed by Ki67 staining) in cultured human pulmonary artery smooth muscle cells. Furthermore, we demonstrated that inhibition of Akt phosphorylation (LY294002) abrogated the Lcn2-promoted proliferation in cultured human pulmonary artery smooth muscle cells. In conclusion, Lcn2 significantly promoted human pulmonary artery smooth muscle cell proliferation by activating PI3K/Akt pathway. Further study on the role and mechanism of Lcn2 will help explore novel therapeutic strategies based on attenuating over-activated PI3K/Akt signaling in PH.

  15. NGF increases VEGF expression and promotes cell proliferation via ERK1/2 and AKT signaling in Müller cells

    PubMed Central

    Wang, Jing; He, Chang; Zhou, Tian; Huang, Zijing; Zhou, Lingli

    2016-01-01

    Purpose Nerve growth factor (NGF) is a classic neuroprotective factor that contributes to angiogenesis under pathological conditions, which might be mediated by the upregulation of vascular endothelial growth factor (VEGF). Retinal Müller cells are a critical source of growth factors, including NGF and VEGF, and express the receptor for NGF, indicating the functional significance of NGF signaling in Müller cells. The aim of this study is to explore the effect of NGF on the production of other growth factors and cellular proliferation in Müller cells and to further detect the potential mechanism of these effects. Methods Primary Müller cells from C57BL/6J mice were isolated and identified with glutamine synthetase (GS) immunofluorescence (IF), a specific marker for Müller cells. TrkA, a high affinity receptor for NGF, was detected with IF staining in the primary Müller cells. Then, the cultured cells were stimulated with recombinant mouse NGF, and the supernatants and the cellular lysate were collected at different time points. VEGF secretion in the supernatant was detected with an enzyme-linked immunosorbent assay (ELISA). The signaling activation in the Müller cells was accessed by western blot using specific phosphorylated antibodies. In addition, cell proliferation was analyzed with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Furthermore, K252a, U0126, and LY294002, the inhibitors for TrkA, extracellular signal-regulated kinases 1/2 (ERK1/2), and phosphatidylinositol 3-kinase (PI3K)/AKT, respectively, were used in combination with NGF in the assays analyzing VEGF expression and cell proliferation. Results Primary mouse Müller cells were successfully cultured and confirmed with GS positive staining. The IF results showed that the TrkA receptor was abundantly expressed on Müller cells. The ELISA results revealed that NGF significantly promoted the production and secretion of VEGF in Müller cells after 12 or 24 h of

  16. ApoSOD1 lacking dismutase activity neuroprotects motor neurons exposed to beta-methylamino-L-alanine through the Ca2+/Akt/ERK1/2 prosurvival pathway

    PubMed Central

    Petrozziello, Tiziana; Secondo, Agnese; Tedeschi, Valentina; Esposito, Alba; Sisalli, MariaJosè; Scorziello, Antonella; Di Renzo, Gianfranco; Annunziato, Lucio

    2017-01-01

    Amyotrophic lateral sclerosis (ALS) is a severe human adult-onset neurodegenerative disease affecting lower and upper motor neurons. In >20% of cases, the familial form of ALS is caused by mutations in the gene encoding Cu,Zn-superoxide dismutase (SOD1). Interestingly, administration of wild-type SOD1 to SOD1G93A transgenic rats ameliorates motor symptoms through an unknown mechanism. Here we investigated whether the neuroprotective effects of SOD1 are due to the Ca2+-dependent activation of such prosurvival signaling pathway and not to its catalytic activity. To this aim, we also examined the mechanism of neuroprotective action of ApoSOD1, the metal-depleted state of SOD1 that lacks dismutase activity, in differentiated motor neuron-like NSC-34 cells and in primary motor neurons exposed to the cycad neurotoxin beta-methylamino-L-alanine (L-BMAA). Preincubation of ApoSOD1 and SOD1, but not of human recombinant SOD1G93A, prevented cell death in motor neurons exposed to L-BMAA. Moreover, ApoSOD1 elicited ERK1/2 and Akt phosphorylation in motor neurons through an early increase of intracellular Ca2+ concentration ([Ca2+]i). Accordingly, inhibition of ERK1/2 by siMEK1 and PD98059 counteracted ApoSOD1- and SOD1-induced neuroprotection. Similarly, transfection of the dominant-negative form of Akt in NSC-34 motor neurons and treatment with the selective PI3K inhibitor LY294002 prevented ApoSOD1- and SOD1-mediated neuroprotective effects in L-BMAA-treated motor neurons. Furthermore, ApoSOD1 and SOD1 prevented the expression of the two markers of L-BMAA-induced ER stress GRP78 and caspase-12. Collectively, our data indicate that ApoSOD1, which is devoid of any catalytic dismutase activity, exerts a neuroprotective effect through an early activation of Ca2+/Akt/ERK1/2 pro-survival pathway that, in turn, prevents ER stress in a neurotoxic model of ALS. PMID:28085149

  17. The Effects of IGF-1 on TNF-α-Treated DRG Neurons by Modulating ATF3 and GAP-43 Expression via PI3K/Akt/S6K Signaling Pathway.

    PubMed

    Zhang, Lei; Yue, Yaping; Ouyang, Meishuo; Liu, Huaxiang; Li, Zhenzhong

    2017-02-16

    Upregulation of the pro-inflammatory cytokine tumor necrosis factor α (TNF-α) is involved in the development and progression of numerous neurological disorders. Recent reports have challenged the concept that TNF-α exhibits only deleterious effects of pro-inflammatory destruction, and have raised the awareness that it may play a beneficial role in neuronal growth and function in particular conditions, which prompts us to further investigate the role of this cytokine. Insulin-like growth factor-1 (IGF-1) is a cytokine possessing powerful neuroprotective effects in promoting neuronal survival, neuronal differentiation, neurite elongation, and neurite regeneration. The association of IGF-1 with TNF-α and the biological effects, produced by interaction of IGF-1 and TNF-α, on neuronal outgrowth status of primary sensory neurons are still to be clarified. In the present study, using an in vitro model of primary cultured rat dorsal root ganglion (DRG) neurons, we demonstrated that TNF-α challenge at different concentrations elicited diverse biological effects. Higher concentration of TNF-α (10 ng/mL) dampened neurite outgrowth, induced activating transcription factor 3 (ATF3) expression, reduced growth-associated protein 43 (GAP-43) expression, and promoted GAP-43 and ATF3 coexpression, which could be reversed by IGF-1 treatment; while lower concentration of TNF-α (1 ng/mL) promoted neurite sprouting, decreased ATF3 expression, increased GAP-43 expression, and inhibited GAP-43 and ATF3 coexpression, which could be potentiated by IGF-1 supplement. Moreover, IGF-1 administration restored the activation of Akt and p70 S6 kinase (S6K) suppressed by higher concentration of TNF-α (10 ng/mL) challenge. In contrast, lower concentration of TNF-α (1 ng/mL) had no significant effect on Akt or S6K activation, and IGF-1 administration activated these two kinases. The effects of IGF-1 were abrogated by phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. These data

  18. Insulin-like growth factor-I stimulates H{sub 4}II rat hepatoma cell proliferation: Dominant role of PI-3'K/Akt signaling

    SciTech Connect

    Alexia, Catherine; Fourmatgeat, Pascal; Delautier, Daniele; Groyer, Andre . E-mail: groyer@bichat.inserm.fr

    2006-04-15

    Although hepatocytes are the primary source of endocrine IGF-I and -II in mammals, their autocrine/paracrine role in the dysregulation of proliferation and apoptosis during hepatocarcinogenesis and in hepatocarcinomas (HCC) remains to be elucidated. Indeed, IGF-II and type-I IGF receptors are overexpressed in HCC cells, and IGF-I is synthesized in adjacent non-tumoral liver tissue. In the present study, we have investigated the effects of type-I IGF receptor signaling on H{sub 4}II rat hepatoma cell proliferation, as estimated by {sup 3}H-thymidine incorporation into DNA. IGF-I stimulated the rate of DNA synthesis of serum-deprived H{sub 4}II cells, stimulation being maximal 3 h after the onset of IGF-I treatment and remaining elevated until at least 6 h. The IGF-I-induced increase in DNA replication was abolished by LY294002 and only partially inhibited by PD98059, suggesting that phosphoinositol-3' kinase (PI-3'K) and to a lesser extent MEK/Erk signaling were involved. Furthermore, the 3- to 19-fold activation of the Erks in the presence of LY294002 suggested a down-regulation of the MEK/Erk cascade by PI-3'K signaling. Finally, the effect of IGF-I on DNA replication was almost completely abolished in clones of H{sub 4}II cells expressing a dominant-negative form of Akt but was unaltered by rapamycin treatment of wild-type H{sub 4}II cells. Altogether, these data support the notion that the stimulation of H{sub 4}II rat hepatoma cell proliferation by IGF-I is especially dependent on Akt activation but independent on the Akt/mTOR signal0009i.

  19. In vitro and in vivo activity of novel small-molecule inhibitors targeting the pleckstrin homology domain of protein kinase B/AKT.

    PubMed

    Moses, Sylvestor A; Ali, M Ahad; Zuohe, Song; Du-Cuny, Lei; Zhou, Li Li; Lemos, Robert; Ihle, Nathan; Skillman, A Geoffrey; Zhang, Shuxing; Mash, Eugene A; Powis, Garth; Meuillet, Emmanuelle J

    2009-06-15

    The phosphatidylinositol 3-kinase/AKT signaling pathway plays a critical role in activating survival and antiapoptotic pathways within cancer cells. Several studies have shown that this pathway is constitutively activated in many different cancer types. The goal of this study was to discover novel compounds that bind to the pleckstrin homology (PH) domain of AKT, thereby inhibiting AKT activation. Using proprietary docking software, 22 potential PH domain inhibitors were identified. Surface plasmon resonance spectroscopy was used to measure the binding of the compounds to the expressed PH domain of AKT followed by an in vitro activity screen in Panc-1 and MiaPaCa-2 pancreatic cancer cell lines. We identified a novel chemical scaffold in several of the compounds that binds selectively to the PH domain of AKT, inducing a decrease in AKT activation and causing apoptosis at low micromolar concentrations. Structural modifications of the scaffold led to compounds with enhanced inhibitory activity in cells. One compound, 4-dodecyl-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide, inhibited AKT and its downstream targets in cells as well as in pancreatic cancer cell xenografts in immunocompromised mice; it also exhibited good antitumor activity. In summary, a pharmacophore for PH domain inhibitors targeting AKT function was developed. Computer-aided modeling, synthesis, and testing produced novel AKT PH domain inhibitors that exhibit promising preclinical properties.

  20. Reactivation of ERK and Akt confers resistance of mutant BRAF colon cancer cells to the HSP90 inhibitor AUY922

    PubMed Central

    Wang, Chun Yan; Guo, Su Tang; Wang, Jia Yu; Yan, Xu Guang; Farrelly, Margaret; Zhang, Yuan Yuan; Liu, Fen; Yari, Hamed; La, Ting; Lei, Fu Xi; Jin, Lei; Zhang, Xu Dong; Jiang, Chen Chen

    2016-01-01

    Oncogenic mutations of BRAF occur in approximately 10% of colon cancers and are associated with their resistance to clinically available therapeutic drugs and poor prognosis of the patients. Here we report that colon cancer cells with mutant BRAF are also resistant to the heat shock protein 90 (HSP90) inhibitor AUY922, and that this is caused by rebound activation of ERK and Akt. Although AUY922 triggered rapid reduction in ERK and Akt activation in both wild-type and mutant BRAF colon cancer cells, activation of ERK and Akt rebounded shortly in the latter leading to resistance of the cells to AUY922-induced apoptosis. Reactivation of ERK was associated with the persistent expression of mutant BRAF, which, despite being a client of HSP90, was only partially degraded by AUY922, whereas reactivation of Akt was related to the activity of the HSP90 co-chaperone, cell division cycle 37 (CDC37), in that knockdown of CDC37 inhibited Akt reactivation in mutant colon cancer cells treated with AUY922. In support, as a HSP90 client protein, Akt was only diminished by AUY922 in wild-type but not mutant BRAF colon cancer cells. Collectively, these results reveal that reactivation of ERK and Akt associated respectively with the activity of mutant BRAF and CDC37 renders mutant BRAF colon cancer cells resistant to AUY922, with implications of co-targeting mutant BRAF and/or CDC37 and HSP90 in the treatment of mutant BRAF colon cancers. PMID:27391062

  1. Reduction of metastasis, cell invasion, and adhesion in mouse osteosarcoma by YM529/ONO-5920-induced blockade of the Ras/MEK/ERK and Ras/PI3K/Akt pathway

    SciTech Connect

    Tsubaki, Masanobu; Satou, Takao; Itoh, Tatsuki; Imano, Motohiro; Ogaki, Mitsuhiko; Yanae, Masashi; Nishida, Shozo

    2012-03-15

    Osteosarcoma is one of the most common primary malignant bone tumors in children and adolescents. Some patients continue to have a poor prognosis, because of the metastatic disease. YM529/ONO-5920 is a nitrogen-containing bisphosphonate that has been used for the treatment of osteoporosis. YM529/ONO-5920 has recently been reported to induce apoptosis in various tumors including osteosarcoma. However, the mode of metastasis suppression in osteosarcoma by YM529/ONO-5920 is unclear. In the present study, we investigated whether YM529/ONO-5920 inhibited tumor cell migration, invasion, adhesion, or metastasis in the LM8 mouse osteosarcoma cell line. We found that YM529/ONO-5920 significantly inhibited metastasis, cell migration, invasion, and adhesion at concentrations that did not have antiproliferative effects on LM8 cells. YM529/ONO-5920 also inhibited the mRNA expression and protein activities of matrix metalloproteinases (MMPs). In addition, YM529/ONO-5920 suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and the serine/threonine protein kinase B (Akt) by the inhibition of Ras prenylation. Moreover, U0126, a mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, also inhibited LM8 cell migration, invasion, adhesion, and metastasis, as well as the mRNA expression and protein activities of MMP-1, MMP-2, MMP-9, and MT1-MMP. The results indicated that YM529/ONO-5920 suppressed the Ras/MEK/ERK and Ras/PI3K/Akt pathways, thereby inhibiting LM8 cell migration, invasion, adhesion, and metastasis. These findings suggest that YM529/ONO-5920 has potential clinical applications for the treatment of tumor cell metastasis in osteosarcoma. -- Highlights: ► We investigated whether YM529/ONO-5920 inhibited tumor metastasis in osteosarcoma. ► YM529/ONO-5920 inhibited metastasis, cell migration, invasion, and adhesion. ► YM529/ONO-5920 suppressed Ras signalings. ► YM529/ONO-5920

  2. Nanoparticle delivery of an AKT/PDK1 inhibitor improves the therapeutic effect in pancreatic cancer

    PubMed Central

    Lucero-Acuña, Armando; Jeffery, Justin J; Abril, Edward R; Nagle, Raymond B; Guzman, Roberto; Pagel, Mark D; Meuillet, Emmanuelle J

    2014-01-01

    The K-ras mutation in pancreatic cancer can inhibit drug delivery and increase drug resistance. This is exemplified by the therapeutic effect of PH-427, a small molecule inhibitor of AKT/PDK1, which has shown a good therapeutic effect against a BxPC3 pancreatic cancer model that has K-ras, but has a poor therapeutic effect against a MiaPaCa-2 pancreatic cancer model with mutant K-ras. To increase the therapeutic effect of PH-427 against the MiaPaCa-2 pancreatic cancer model with mutant K-ras, we encapsulated PH-427 into poly(lactic-co-glycolic acid) nanoparticles (PNP) to form drug-loaded PH-427-PNP. PH-427 showed a biphasic release from PH-427-PNP over 30 days during studies in sodium phosphate buffer, and in vitro studies revealed that the PNP was rapidly internalized into MiaPaCa-2 tumor cells, suggesting that PNP can improve PH-427 delivery into cells harboring mutant K-ras. In vivo studies of an orthotopic MiaPaCa-2 pancreatic cancer model showed reduced tumor load with PH-427-PNP as compared with treatment using PH-427 alone or with no treatment. Ex vivo studies confirmed the in vivo results, suggesting that PNP can improve drug delivery to pancreatic cancer harboring mutant K-ras. PMID:25516710

  3. Effects of RAF inhibitors on PI3K/AKT signalling depend on mutational status of the RAS/RAF signalling axis

    PubMed Central

    Fritsche-Guenther, Raphaela; Witzel, Franziska; Kempa, Stefan; Brummer, Tilman; Sers, Christine; Blüthgen, Nils

    2016-01-01

    Targeted therapies within the RAS/RAF/MEK/ERK signalling axis become increasingly popular, yet cross-talk and feedbacks in the signalling network lead to unexpected effects. Here we look systematically into how inhibiting RAF and MEK with clinically relevant inhibitors result in changes in PI3K/AKT activation. We measure the signalling response using a bead-based ELISA, and use a panel of three cell lines, and isogenic cell lines that express mutant forms of the oncogenes KRAS and BRAF to interrogate the effects of the MEK and RAF inhibitors on signalling. We find that treatment with the RAF inhibitors have opposing effects on AKT phosphorylation depending on the mutational status of two important oncogenes, KRAS and BRAF. If these two genes are in wildtype configuration, RAF inhibitors reduce AKT phosphorylation. In contrast, if BRAF or KRAS are mutant, RAF inhibitors will leave AKT phosphorylation unaffected or lead to an increase of AKT phosphorylation. Down-regulation of phospho-AKT by RAF inhibitors also extends to downstream transcription factors, and correlates with apoptosis induction. Our results show that oncogenes rewire signalling such that targeted therapies can have opposing effects on parallel pathways, which depend on the mutational status of the cell. PMID:26799289

  4. Discovery of a novel class of AKT pleckstrin homology domain inhibitors.

    PubMed

    Mahadevan, Daruka; Powis, Garth; Mash, Eugene A; George, Benjamin; Gokhale, Vijay M; Zhang, Shuxing; Shakalya, Kishore; Du-Cuny, Lei; Berggren, Margareta; Ali, M Ahad; Jana, Umasish; Ihle, Nathan; Moses, Sylvestor; Franklin, Chloe; Narayan, Satya; Shirahatti, Nikhil; Meuillet, Emmanuelle J

    2008-09-01

    AKT, a phospholipid-binding serine/threonine kinase, is a key component of the phosphoinositide 3-kinase cell survival signaling pathway that is aberrantly activated in many human cancers. Many attempts have been made to inhibit AKT; however, selectivity remains to be achieved. We have developed a novel strategy to inhibit AKT by targeting the pleckstrin homology (PH) domain. Using in silico library screening and interactive molecular docking, we have identified a novel class of non-lipid-based compounds that bind selectively to the PH domain of AKT, with "in silico" calculated K(D) values ranging from 0.8 to 3.0 micromol/L. In order to determine the selectivity of these compounds for AKT, we used surface plasmon resonance to measure the binding characteristics of the compounds to the PH domains of AKT1, insulin receptor substrate-1, and 3-phosphoinositide-dependent protein kinase 1. There was excellent correlation between predicted in silico and measured in vitro K(D)s for binding to the PH domain of AKT, which were in the range 0.4 to 3.6 micromol/L. Some of the compounds exhibited PH domain-binding selectivity for AKT compared with insulin receptor substrate-1 and 3-phosphoinositide-dependent protein kinase 1. The compounds also inhibited AKT in cells, induced apoptosis, and inhibited cancer cell proliferation. In vivo, the lead compound failed to achieve the blood concentrations required to inhibit AKT in cells, most likely due to rapid metabolism and elimination, and did not show antitumor activity. These results show that these compounds are the first small molecules selectively targeting the PH domain of AKT.

  5. Embryonic liver fordin is involved in glucose glycolysis of hepatic stellate cell by regulating PI3K/Akt signaling

    PubMed Central

    Tu, Wei; Ye, Jin; Wang, Zhi-Jun

    2016-01-01

    AIM To investigate the role of embryonic liver fordin (ELF) in liver fibrosis by regulating hepatic stellate cells (HSCs) glucose glycolysis. METHODS The expression of ELF and the glucose glycolysis-related proteins were evaluated in activated HSCs. siRNA was used to silence ELF expression in activated HSCs in vitro and the subsequent changes in PI3K/Akt signaling and glucose glycolysis-related proteins were observed. RESULTS The expression of ELF increased remarkably in HSCs of the fibrosis mouse model and HSCs that were cultured for 3 wk in vitro. Glucose glycolysis-related proteins showed an obvious increase in the activated HSCs, such as phosphofructokinase, platelet and glucose transporter 1. ELF-siRNA, which perfectly silenced the expression of ELF in activated HSCs, led to the induction of glucose glycolysis-related proteins and extracellular matrix (ECM) components. Moreover, pAkt, which is an important downstream factor in PI3K/Akt signaling, showed a significant change in response to the ELF silencing. The expression of glucose glycolysis-related proteins and ECM components decreased remarkably when the PI3K/Akt signaling was blocked by Ly294002 in the activated HSCs. CONCLUSION ELF is involved in HSC glucose glycolysis by regulating PI3K/Akt signaling. PMID:27784964

  6. AKT-aro and HER2-aro, models for de novo resistance to aromatase inhibitors; molecular characterization and inhibitor response studies.

    PubMed

    Wong, Cynthie; Wang, Xin; Smith, David; Reddy, Kaladhar; Chen, Shiuan

    2012-07-01

    Aromatase inhibitors (AI) are currently the first line therapy for estrogen receptor (ER)-positive postmenopausal women. De novo AI resistance is when a patient intrinsically does not respond to an AI therapy as well as other targeted endocrine therapy. To characterize this type of resistance and to examine potential therapies for treatment, we have generated two cell models for de novo resistance. These models derive from MCF-7 cells that stably overexpress aromatase and Akt (AKT-aro) or HER2 (HER2-aro). Evaluation of these cell lines revealed that the activities of aromatase and ER were inhibited by AI and ICI 187280 (ICI) treatment, respectively; however, cell growth was resistant to therapy. Proliferation in the presence of the pure anti-estrogen ICI, indicates that these cells do not require ER for cell growth and distinguishes these cells from the acquired AI resistant cells. We further determined that the HSP90 inhibitor 17-DMAG suppressed the growth of the AI-resistant cell lines studied. Our analysis revealed 17-DMAG-mediated decreased expression of growth promoting signaling proteins. It was found that de novo AI resistant AKT-aro and HER2-aro cells could not be resensitized to letrozole or ICI by treatment with 17-DMAG. In summary, we have generated two cell lines which display the characteristics of de novo AI resistance. Together, these data indicate the possibility that HSP90 inhibitors may be a viable therapy for endocrine therapy resistance although additional clinical evaluation is needed.

  7. Histone Deacetylase Inhibitors Inhibit the Proliferation of Gallbladder Carcinoma Cells by Suppressing AKT/mTOR Signaling.

    PubMed

    Zhang, Peng; Guo, Zhiyong; Wu, Ying; Hu, Ronglin; Du, Jun; He, Xiaoshun; Jiao, Xingyuan; Zhu, Xiaofeng

    2015-01-01

    Gallbladder carcinoma is an aggressive malignancy with high mortality mainly due to the limited potential for curative resection and its resistance to chemotherapeutic agents. Here, we show that the histone deacetylase inhibitors (HDACIs) trichostatin-A (TSA) and suberoylanilide hydroxamic acid (SAHA) reduce the proliferation and induce apoptosis of gallbladder carcinoma cells by suppressing the AKT/mammalian target of rapamycin (mTOR) signaling. Gallbladder carcinoma SGC-996 cells were treated with different concentrations of TSA and SAHA for different lengths of time. Cell proliferation and morphology were assessed with MTT assay and microscopy, respectively. Cell cycle distribution and cell apoptosis were analyzed with flow cytometry. Western blotting was used to detect the proteins related to apoptosis, cell cycle, and the AKT/mTOR signaling pathway. Our data showed that TSA and SAHA reduced SGC-996 cell viability and arrested cell cycle at the G1 phase in a dose- and time-dependent manner. TSA and SAHA promoted apoptosis of SGC-996 cells, down-regulated the expression of cyclin D1, c-Myc and Bmi1, and decreased the phosphorylation of AKT, mTOR p70S6K1, S6 and 4E-BP1. Additionally, the mTOR inhibitor rapamycin further reduced the cell viability of TSA- and SAHA-treated SGC-996 cells and the phosphorylation of mTOR, whereas the mTOR activator 1,2-dioctanoyl-sn-glycero-3-phosphate (C8-PA) exerted the opposite influence. Our results demonstrate that histone deacetylase inhibitors (HDACIs) suppress the proliferation of gallbladder carcinoma cell via inhibition of AKT/mTOR signaling. These findings offer a mechanistic rationale for the application of HDACIs in gallbladder carcinoma treatment.

  8. Blockade of autophagy enhances proapoptotic potential of BI-69A11, a novel Akt inhibitor, in colon carcinoma.

    PubMed

    Pal, Ipsita; Parida, Sheetal; Prashanth Kumar, B N; Banik, Payel; Kumar Dey, Kaushik; Chakraborty, Sandipan; Bhutia, Sujit K; Mandal, Mahitosh

    2015-10-15

    BI-69A11, novel Akt inhibitor, is currently drawing much attention due to its intriguing effect in inducing apoptosis in melanoma, breast, prostate and colon cancer. However, earlier reports reveal that PI3K/Akt/mTOR inhibitors promote autophagy at the early stage as a survival mechanism that might affect its apoptotic potential. It is necessary to investigate whether BI-69A11 mediated apoptosis is associated with autophagy for enhancing its therapeutic efficacy. Here, we found that BI-69A11 induced autophagy at earlier time point through the inhibition of Akt/mTOR/p70S6kinase pathway. Dose-dependent and time-dependent conversion of LC3-I to LC3-II, increased accumulation of LC3-GFP dots in cytoplasm and increase in other autophagic markers such as Beclin-1, firmly supported the fact that BI-69A11 induces autophagy. Atg5, Atg7 and Beclin-1 siRNA mediated genetic attenuation and pre-treatment with pharmacological inhibitor 3-MA and CQ diminished the autophagy and increased the propensity of cell death towards apoptosis. It was also suggested that BI-69A11 mediated interaction between Akt, HSP-90 and Beclin-1 maintained the fine balance between autophagy and apoptosis. Interaction between Beclin-1 and HSP90 is one of the prime causes of induction of autophagy. Here, we also generated a novel combination therapy by pretreatment with CQ that inhibited the autophagy and accelerated the apoptotic potential of BI-69A11. In summary; our findings suggest that induction of autophagy lead to the resistance of colon cancer towards BI-69A11 mediated apoptosis.

  9. Phase 0 Clinical Chemoprevention Trial of the AKT Inhibitor SR13668

    PubMed Central

    Reid, Joel M.; Walden, Chad; Qin, Rui; Allen Ziegler, Katie L.; Haslam, John L.; Rajewski, Roger A.; Warndahl, Roger; Fitting, Cindy L.; Boring, Daniel; Szabo, Eva; Crowell, James; Perloff, Marjorie; Jong, Ling; Mandrekar, Sumithra J.; Ames, Matthew M.; Limburg, Paul J.

    2011-01-01

    Purpose SR13668, an orally active AKT pathway inhibitor, has demonstrated cancer chemopreventive potential in preclinical studies. To accelerate the clinical development of this promising agent, we designed and conducted the first-ever phase 0 chemoprevention trial to evaluate and compare the effects of food and formulation on SR13668 bioavailability. Patients and Methods Healthy adult volunteers were randomly assigned to receive a single, 38 mg oral dose of SR13668 in one of five different formulations, with or without food. Based on existing animal data, SR13668 in a PEG400/Labrasol® oral solution was defined as the reference formulation. Blood samples were obtained pre- and post-agent administration for pharmacokinetic analyses. Area under the plasma concentration-time curve (AUC0-∞) was defined as the primary endpoint. Data were analyzed and compared using established statistical methods for phase 0 trials with a limited sample size. Results Participants (N=20) were rapidly accrued over a 5-month period. Complete pharmacokinetic data were available for 18 randomized participants. AUC0-∞ values were highest in the fed state (range = 122–439 ng/mL × hours) and were statistically significantly different across formulations (p = 0.007), with Solutol® HS15 providing the highest bioavailability. SR13668 time to peak plasma concentration (3 hours; range, 2 – 6 hours) and half-life were (11.2 ± 3.1 hours) were not formulation dependent. Conclusions Using a novel, highly efficient study design, we rapidly identified a lead formulation of SR13668 for further clinical testing. Our findings support application of the phase 0 trial paradigm to accelerate chemoprevention agent development. PMID:21372034

  10. DC120, a novel AKT inhibitor, preferentially suppresses nasopharyngeal carcinoma cancer stem-like cells by downregulating Sox2

    PubMed Central

    Tang, Jun; Yang, Fen; Feng, Gong-Kan; Chen, Wen-Dan; Wu, Xiao-Qi; Qian, Xiao-Jun; Ding, Ke; Zhu, Xiao-Feng

    2015-01-01

    Side population (SP) contains cancer stem-like cells (CSLCs). In this study, we characterized SP cells from nasopharyngeal carcinoma (NPC) cell lines and found that SP cells had a higher self-renewal ability in vitro and greater tumorigenicity in vivo. The AKT pathway was activated in NPC SP cells. DC120, a 2-pyrimidyl-5-amidothiazole inhibitor of the ATP binding site of AKT, inhibited phosphorylation of FKHRL1 and GSK-3β. DC120 inhibited SP fraction, the sphere-forming ability in vitro and growth of primary xenografts as well as secondary xenografts’ tumor recurrence. This inhibition was accompanied by reduced expression of stem-related gene Sox2 due to induction of p27 and miR-30a. A combination of DC120 and CDDP more effectively inhibited NPC cells compared with monotherapy in vitro and in vivo. Clinical evaluation of DC120 is warranted. PMID:25749514

  11. Syntheses of potent, selective, and orally bioavailable indazole-pyridine series of protein kinase B/Akt inhibitors with reduced hypotension.

    PubMed

    Zhu, Gui-Dong; Gandhi, Viraj B; Gong, Jianchun; Thomas, Sheela; Woods, Keith W; Song, Xiaohong; Li, Tongmei; Diebold, R Bruce; Luo, Yan; Liu, Xuesong; Guan, Ran; Klinghofer, Vered; Johnson, Eric F; Bouska, Jennifer; Olson, Amanda; Marsh, Kennan C; Stoll, Vincent S; Mamo, Mulugeta; Polakowski, James; Campbell, Thomas J; Martin, Ruth L; Gintant, Gary A; Penning, Thomas D; Li, Qun; Rosenberg, Saul H; Giranda, Vincent L

    2007-06-28

    Compound 7 was identified as a potent (IC50 = 14 nM), selective, and orally bioavailable (F = 70% in mouse) inhibitor of protein kinase B/Akt. While promising efficacy was observed in vivo, this compound showed effects on depolarization of Purkinje fibers in an in vitro assay and CV hypotension in vivo. Guided by an X-ray structure of 7 bound to protein kinase A, which has 80% homology with Akt in the kinase domain, our efforts have focused on structure-activity relationship (SAR) studies of the phenyl moiety, in an attempt to address the cardiovascular liability and further improve the Akt potency. A novel and efficient synthetic route toward diversely substituted phenyl derivatives of 7 was developed utilizing a copper-mediated aziridine ring-opening reaction as the key step. To improve the selectivity of these Akt inhibitors over other protein kinases, a nitrogen atom was incorporated into selected phenyl analogues of 7 at the C-6 position of the methyl indazole scaffold. These modifications resulted in the discovery of inhibitor 37c with greater potency (IC50 = 0.6 nM vs Akt), selectivity, and improved cardiovascular safety profile. The SARs, pharmacokinetic profile, and CV safety of selected Akt inhibitors will be discussed.

  12. Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascade inhibitors: how mutations can result in therapy resistance and how to overcome resistance.

    PubMed

    McCubrey, James A; Steelman, Linda S; Chappell, William H; Abrams, Stephen L; Franklin, Richard A; Montalto, Giuseppe; Cervello, Melchiorre; Libra, Massimo; Candido, Saverio; Malaponte, Grazia; Mazzarino, Maria C; Fagone, Paolo; Nicoletti, Ferdinando; Bäsecke, Jörg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Milella, Michele; Tafuri, Agostino; Chiarini, Francesca; Evangelisti, Camilla; Cocco, Lucio; Martelli, Alberto M

    2012-10-01

    The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Targeting these pathways is often complex and can result in pathway activation depending on the presence of upstream mutations (e.g., Raf inhibitors induce Raf activation in cells with wild type (WT) RAF in the presence of mutant, activated RAS) and rapamycin can induce Akt activation. Targeting with inhibitors directed at two constituents of the same pathway or two different signaling pathways may be a more effective approach. This review will first evaluate potential uses of Raf, MEK, PI3K, Akt and mTOR inhibitors that have been investigated in pre-clinical and clinical investigations and then discuss how cancers can become insensitive to various inhibitors and potential strategies to overcome this resistance.

  13. Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Cascade Inhibitors: How Mutations Can Result in Therapy Resistance and How to Overcome Resistance

    PubMed Central

    McCubrey, James A.; Steelman, Linda S.; Chappell, William H.; Abrams, Stephen L.; Franklin, Richard A.; Montalto, Giuseppe; Cervello, Melchiorre; Libra, Massimo; Candido, Saverio; Malaponte, Grazia; Mazzarino, Maria C.; Fagone, Paolo; Nicoletti, Ferdinando; Bäsecke, Jörg; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Milella, Michele; Tafuri, Agostino; Chiarini, Francesca; Evangelisti, Camilla; Cocco, Lucio; Martelli, Alberto M.

    2012-01-01

    The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Targeting these pathways is often complex and can result in pathway activation depending on the presence of upstream mutations (e.g., Raf inhibitors induce Raf activation in cells with wild type (WT) RAF in the presence of mutant, activated RAS) and rapamycin can induce Akt activation. Targeting with inhibitors directed at two constituents of the same pathway or two different signaling pathways may be a more effective approach. This review will first evaluate potential uses of Raf, MEK, PI3K, Akt and mTOR inhibitors that have been investigated in pre-clinical and clinical investigations and then discuss how cancers can become insensitive to various inhibitors and potential strategies to overcome this resistance. PMID:23085539

  14. Subcutaneous adipocytes promote melanoma cell growth by activating the Akt signaling pathway: role of palmitic acid.

    PubMed

    Kwan, Hiu Yee; Fu, Xiuqiong; Liu, Bin; Chao, Xiaojuan; Chan, Chi Leung; Cao, Huihui; Su, Tao; Tse, Anfernee Kai Wing; Fong, Wang Fun; Yu, Zhi-Ling

    2014-10-31

    Tumorigenesis involves constant communication between tumor cells and neighboring normal cells such as adipocytes. The canonical function of adipocytes is to store triglyceride and release fatty acids for other tissues. This study was aimed to find out if adipocytes promoted melanoma cell growth and to investigate the underlying mechanism. Here we isolated adipocytes from inguinal adipose tissue in mice and co-cultured with melanoma cells. We found that the co-cultured melanoma had higher lipid accumulation compared with mono-cultured melanoma. In addition, fluorescently labeled fatty acid BODIPY® FLC16 signal was detected in melanoma co-cultured with the adipocytes that had been loaded with the fluorescent dye, suggesting that the adipocytes provide fatty acids to melanoma cells. Compared with mono-cultured melanoma, co-cultured melanoma cells had a higher proliferation and phospho-Akt (Ser-473 and Thr-450) expression. Overexpression of Akt mutants in melanoma cells reduced the co-culture-enhanced proliferation. A lipidomic study showed that the co-cultured melanoma had an elevated palmitic acid level. Interestingly, we found that palmitic acid stimulated melanoma cell proliferation, changed the cell cycle distribution, and increased phospho-Akt (Ser-473 and Thr-450) and PI3K but not phospho-PTEN (phosphophosphatase and tensin homolog) expressions. More importantly, the palmitic acid-stimulated proliferation was further enhanced in the Akt-overexpressed melanoma cells and was reduced by LY294002 or knockdown of endogenous Akt or overexpression of Akt mutants. We also found that palmitic acid-pretreated B16F10 cells were grown to a significantly larger tumor in mice compared with control cells. Taken together, we suggest that adipocytes may serve as an exogenous source of palmitic acid that promotes melanoma cell growth by activating Akt.

  15. Subcutaneous Adipocytes Promote Melanoma Cell Growth by Activating the Akt Signaling Pathway

    PubMed Central

    Kwan, Hiu Yee; Fu, Xiuqiong; Liu, Bin; Chao, Xiaojuan; Chan, Chi Leung; Cao, Huihui; Su, Tao; Tse, Anfernee Kai Wing; Fong, Wang Fun; Yu, Zhi-Ling

    2014-01-01

    Tumorigenesis involves constant communication between tumor cells and neighboring normal cells such as adipocytes. The canonical function of adipocytes is to store triglyceride and release fatty acids for other tissues. This study was aimed to find out if adipocytes promoted melanoma cell growth and to investigate the underlying mechanism. Here we isolated adipocytes from inguinal adipose tissue in mice and co-cultured with melanoma cells. We found that the co-cultured melanoma had higher lipid accumulation compared with mono-cultured melanoma. In addition, fluorescently labeled fatty acid BODIPY® FLC16 signal was detected in melanoma co-cultured with the adipocytes that had been loaded with the fluorescent dye, suggesting that the adipocytes provide fatty acids to melanoma cells. Compared with mono-cultured melanoma, co-cultured melanoma cells had a higher proliferation and phospho-Akt (Ser-473 and Thr-450) expression. Overexpression of Akt mutants in melanoma cells reduced the co-culture-enhanced proliferation. A lipidomic study showed that the co-cultured melanoma had an elevated palmitic acid level. Interestingly, we found that palmitic acid stimulated melanoma cell proliferation, changed the cell cycle distribution, and increased phospho-Akt (Ser-473 and Thr-450) and PI3K but not phospho-PTEN (phosphophosphatase and tensin homolog) expressions. More importantly, the palmitic acid-stimulated proliferation was further enhanced in the Akt-overexpressed melanoma cells and was reduced by LY294002 or knockdown of endogenous Akt or overexpression of Akt mutants. We also found that palmitic acid-pretreated B16F10 cells were grown to a significantly larger tumor in mice compared with control cells. Taken together, we suggest that adipocytes may serve as an exogenous source of palmitic acid that promotes melanoma cell growth by activating Akt. PMID:25228694

  16. Inhibitors beta-amyloid-induced toxicity by modulating the Akt signaling pathway.

    PubMed

    Nakagami, Yasuhiro

    2004-12-01

    The Akt signaling pathway plays a crucial role in neuronal survival, leading to inhibition of apoptosis. Many stimulants including neurotrophins are reported to activate this pathway in preclinical studies; however, there are no drugs for neurodegenerative diseases adopting such a concept on the market so far. Among neurodegenerative diseases, Alzheimer's disease is the most common and characterized by senile plaques and neurofibrillary tangles, which consist of beta-amyloid and hyperphosphorylated tau, respectively. Recent studies suggest that activation of Akt inhibits toxicity of beta-amyloid and formation of neurofibrillary tangles, leading to protection of neurons against apoptosis. This review discusses the possibility of treatment of Alzheimer's disease by activating the Akt signaling pathway.

  17. Preclinical pharmacology of AZD5363, an inhibitor of AKT: pharmacodynamics, antitumor activity, and correlation of monotherapy activity with genetic background.

    PubMed

    Davies, Barry R; Greenwood, Hannah; Dudley, Phillippa; Crafter, Claire; Yu, De-Hua; Zhang, Jingchuan; Li, Jing; Gao, Beirong; Ji, Qunsheng; Maynard, Juliana; Ricketts, Sally-Ann; Cross, Darren; Cosulich, Sabina; Chresta, Christine C; Page, Ken; Yates, James; Lane, Clare; Watson, Rebecca; Luke, Richard; Ogilvie, Donald; Pass, Martin

    2012-04-01

    AKT is a key node in the most frequently deregulated signaling network in human cancer. AZD5363, a novel pyrrolopyrimidine-derived compound, inhibited all AKT isoforms with a potency of 10 nmol/L or less and inhibited phosphorylation of AKT substrates in cells with a potency of approximately 0.3 to 0.8 μmol/L. AZD5363 monotherapy inhibited the proliferation of 41 of 182 solid and hematologic tumor cell lines with a potency of 3 μmol/L or less. Cell lines derived from breast cancers showed the highest frequency of sensitivity. There was a significant relationship between the presence of PIK3CA and/or PTEN mutations and sensitivity to AZD5363 and between RAS mutations and resistance. Oral dosing of AZD5363 to nude mice caused dose- and time-dependent reduction of PRAS40, GSK3β, and S6 phosphorylation in BT474c xenografts (PRAS40 phosphorylation EC(50) ~ 0.1 μmol/L total plasma exposure), reversible increases in blood glucose concentrations, and dose-dependent decreases in 2[18F]fluoro-2-deoxy-D-glucose ((18)F-FDG) uptake in U87-MG xenografts. Chronic oral dosing of AZD5363 caused dose-dependent growth inhibition of xenografts derived from various tumor types, including HER2(+) breast cancer models that are resistant to trastuzumab. AZD5363 also significantly enhanced the antitumor activity of docetaxel, lapatinib, and trastuzumab in breast cancer xenografts. It is concluded that AZD5363 is a potent inhibitor of AKT with pharmacodynamic activity in vivo, has potential to treat a range of solid and hematologic tumors as monotherapy or a combinatorial agent, and has potential for personalized medicine based on the genetic status of PIK3CA, PTEN, and RAS. AZD5363 is currently in phase I clinical trials.

  18. In-vitro and in-vivo combined effect of ARQ 092, an AKT inhibitor, with ARQ 087, a FGFR inhibitor.

    PubMed

    Yu, Yi; Hall, Terence; Eathiraj, Sudharshan; Wick, Michael J; Schwartz, Brian; Abbadessa, Giovanni

    2017-02-24

    The PI3K/AKT pathway plays an important role in the initiation and progression of cancer, and the drug development efforts targeting this pathway with therapeutic interventions have been advanced by academic and industrial groups. However, the clinical outcome is moderate. Combination of inhibition of PI3K/AKT and other targeted agents became a feasible approach. In this study we assessed the combined effect of ARQ 092, a pan-AKT inhibitor, and ARQ 087, a pan-FGFR inhibitor, in vitro and in vivo. In a panel of 45 cancer cell lines, on 24% (11 out of 45) the compounds showed synergistic effect, on 62% (28 out of 45) additive, and on 13% (6 out of 45) antagonistic. The highest percentage of synergism was found on endometrial and ovarian cancer cell lines. Mutational analysis revealed that PIK3CA/PIK3R1 mutations and aberrant activation of FGFR2 predicted synergism, whereas Ras mutations showed a reverse correlation. Pathway analysis revealed that a combination of ARQ 092 and ARQ 087 enhanced the inhibition of both the AKT and FGFR pathways in cell lines in which synergistic effects were found (AN3CA and IGROV-1). Cell cycle arrest and apoptotic response occurred only in AN3CA cell, and was not seen in IGROV-1 cells. Furthermore, enhanced antitumor activity was observed in mouse models with endometrial cancer cell line and patient-derived tumors when ARQ 092 and ARQ 087 were combined. These results from in-vitro and in-vivo studies provide a strong rationale in treating endometrial and other cancers with the activated PI3K/AKT and FGFR pathways.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0/.

  19. Inhibition of autophagy enhances the effects of the AKT inhibitor MK-2206 when combined with paclitaxel and carboplatin in BRAF wild-type melanoma

    PubMed Central

    Rebecca, Vito W.; Massaro, Renato R.; Fedorenko, Inna V.; Sondak, Vernon K.; Anderson, Alexander R.A.; Kim, Eunjung; Amavaradi, Ravi K.; Maria-Engler, Silvya Stuchi; Messina, Jane L.; Gibney, Geoffrey T.; Kudchadkar, Ragini R.; Smalley, Keiran S. M.

    2014-01-01

    Summary This study investigates the mechanism of action behind the long-term responses (12–16 months) of two BRAF WT melanoma patients to the AKT inhibitor MK-2206 in combination with paclitaxel and carboplatin. Although single agent MK-2206 inhibited phospho-AKT signaling, it did not impact in vitro melanoma growth or survival. The combination of MK-2206 with paclitaxel and carboplatin was cytotoxic in long-term colony formation and 3D spheroid assays, and induced autophagy. Autophagy was initially protective with autophagy inhibitors and deletion of ATG5 found to enhance cytotoxicity. Although prolonged autophagy induction (>6 days) led to caspase-dependent apoptosis, drug resistant clones still emerged. Autophagy inhibition enhanced the cell death response through reactive oxygen species and could be reversed by anti-oxidants. We demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma and show that an autophagy inhibitor may prevent resistance to these drugs. Significance Approximately 30% of all cutaneous melanomas are wild-type for both BRAF and NRAS. As yet, no targeted therapy strategies exist for this sub-set of tumors. Constitutive signaling through the PI3K/AKT pathway is a common occurrence in cutaneous melanoma, irrespective of the driver mutation. Here we report durable responses to the AKT inhibitor MK-2206 in combination with carboplatin and paclitaxel in two patients with BRAF wild-type melanoma. Through mechanistic studies, we demonstrate a role for autophagy induction in the response to the AKT inhibitor/chemotherapy combination and suggest that autophagy inhibitors may be one strategy to enhance efficacy in the clinical setting. PMID:24490764

  20. Inhibition of autophagy enhances apoptosis induced by the PI3K/AKT/mTor inhibitor NVP-BEZ235 in renal cell carcinoma cells.

    PubMed

    Li, Hongyan; Jin, Xuefei; Zhang, Zhuo; Xing, Yuanyuan; Kong, Xiangbo

    2013-07-01

    The PI3K/AKT/mTOR pathway plays a key role in the development of the hypervascular tumor renal cell carcinoma (RCC). NVP-BEZ235 (NVP), a novel dual PI3K/mTOR inhibitor, showed great antitumor benefit and provided a treatment strategy in RCC. In this study, we test the effect of NVP on survival rate, apoptosis and autophagy in the RCC cell line, 786-0. We also explore the hypothesis that NVP, in combination with autophagy inhibitors, leads to apoptosis enhancement in 786-0 cells. The results showed that the PI3K/AKT/mTOR pathway proteins p-AKT and p-P70S6K were highly expressed in RCC tissue. We also showed that NVP inhibited cell growth and induced apoptosis and autophagy in RCC cells. The combination treatment of NVP with autophagy inhibitors enhanced the effect of NVP on suppressing 786-0 growth and induction of apoptosis. This study proposes a novel treatment paradigm where combining PI3K/AKT/mTOR pathway inhibitors and autophagy inhibitors lead to enhanced RCC cell apoptosis.

  1. Presence of both alterations in FGFR/FGF and PI3K/AKT/mTOR confer improved outcomes for patients with metastatic breast cancer treated with PI3K/AKT/mTOR inhibitors.

    PubMed

    Wheler, Jennifer J; Atkins, Johnique T; Janku, Filip; Moulder, Stacy L; Stephens, Philip J; Yelensky, Roman; Valero, Vicente; Miller, Vincent; Kurzrock, Razelle; Meric-Bernstam, Funda

    2016-01-01

    There is limited data on co-expression of FGFR/FGR amplifications and PI3K/ AKT/mTOR alterations in breast cancer. Tumors from patients with metastatic breast cancer referred to our Phase I Program were analyzed by next generation sequencing (NGS). Genomic libraries were selected for all exons of 236 (or 182) cancer-related genes sequenced to average depth of >500× in a CLIA laboratory (Foundation Medicine, Cambridge, MA, USA) and analyzed for all classes of genomic alterations. We report genomic profiles of 112 patients with metastatic breast cancer, median age 55 years (range, 27-78). Twenty-four patients (21%) had at least one amplified FGFR or FGF. Fifteen of the 24 patients (63%) also had an alteration in the PI3K/ AKT/mTOR pathway. There was no association between alterations in FGFR/FGF and PI3K/AKT/mTOR (P=0.49). Patients with simultaneous amplification in FGFR/FGF signaling and the PI3K/AKT/mTOR pathway had a higher rate of SD≥6 months/PR/ CR when treated with therapies targeting the PI3K/AKT/mTOR pathway than patients with only alterations in the PI3K/AKT/mTOR pathway (73% vs. 34%; P=0.0376) and remained on treatment longer (6.8 vs. 3.7 months; P=0.053). Higher response rates were seen in patients with simultaneous amplification in FGFR/FGF signaling and alterations in the PI3K/AKT/mTOR pathway who were treated with inhibitors of that pathway.

  2. Presence of both alterations in FGFR/FGF and PI3K/AKT/mTOR confer improved outcomes for patients with metastatic breast cancer treated with PI3K/AKT/mTOR inhibitors

    PubMed Central

    Wheler, Jennifer J.; Atkins, Johnique T.; Janku, Filip; Moulder, Stacy L.; Stephens, Philip J.; Yelensky, Roman; Valero, Vicente; Miller, Vincent; Kurzrock, Razelle; Meric-Bernstam, Funda

    2016-01-01

    There is limited data on co-expression of FGFR/FGR amplifications and PI3K/ AKT/mTOR alterations in breast cancer. Tumors from patients with metastatic breast cancer referred to our Phase I Program were analyzed by next generation sequencing (NGS). Genomic libraries were selected for all exons of 236 (or 182) cancer-related genes sequenced to average depth of >500× in a CLIA laboratory (Foundation Medicine, Cambridge, MA, USA) and analyzed for all classes of genomic alterations. We report genomic profiles of 112 patients with metastatic breast cancer, median age 55 years (range, 27-78). Twenty-four patients (21%) had at least one amplified FGFR or FGF. Fifteen of the 24 patients (63%) also had an alteration in the PI3K/ AKT/mTOR pathway. There was no association between alterations in FGFR/FGF and PI3K/AKT/mTOR (P=0.49). Patients with simultaneous amplification in FGFR/FGF signaling and the PI3K/AKT/mTOR pathway had a higher rate of SD≥6 months/PR/ CR when treated with therapies targeting the PI3K/AKT/mTOR pathway than patients with only alterations in the PI3K/AKT/mTOR pathway (73% vs. 34%; P=0.0376) and remained on treatment longer (6.8 vs. 3.7 months; P=0.053). Higher response rates were seen in patients with simultaneous amplification in FGFR/FGF signaling and alterations in the PI3K/AKT/mTOR pathway who were treated with inhibitors of that pathway. PMID:27489863

  3. Sulfated polysaccharide isolated from the sea cucumber Stichopus japonicas promotes the SDF-1α/CXCR4 axis-induced NSC migration via the PI3K/Akt/FOXO3a, ERK/MAPK, and NF-κB signaling pathways.

    PubMed

    Cui, Chao; Wang, Peng; Cui, Ningshan; Song, Shuliang; Liang, Hao; Ji, Aiguo

    2016-03-11

    The present study describes a positive regulatory loop between SJP and the SDF-1α/CXCR4 axis in NSC migration. The treatment of NSCs with SJP and SDF-1α increases the cell migration capacity and promotes cell migration from the neurospheres. These effects are accompanied by the up-regulation of Nestin, N-cadherin, TLR4, TNF-α, Cyclin D1, EGFR, Alpha 6 integrin, MMP-2, MMP-9, and iNOS, including SDF-1α and CXCR4 themselves. However, these effects are blocked by AMD3100, LY294002, U0126, and PDTC. SJP enhances the SDF-1α/CXCR4 axis-induced MMP-2 and MMP-9 secretion and NO release. Results demonstrate that interaction of SJP with the SDF-1α/CXCR4 axis regulates NSC migration via the PI3K/Akt/FOXO3a, ERK-MAPK, and NF-κB signaling pathways.

  4. Effects of an oral allosteric AKT inhibitor (MK-2206) on human nasopharyngeal cancer in vitro and in vivo

    PubMed Central

    Zhao, Yuan-Yuan; Tian, Ying; Zhang, Jing; Xu, Fei; Yang, Yun-Peng; Huang, Yan; Zhao, Hong-Yun; Zhang, Jian-Wei; Xue, Cong; Lam, Michael H; Yan, Li; Hu, Zhi-Huang; Dinglin, Xiao-Xiao; Zhang, Li

    2014-01-01

    Aim Protein kinase B (AKT) signaling frequently is deregulated in human cancers and plays an important role in nasopharyngeal carcinoma (NPC). This preclinical study investigated the effect of MK-2206, a potent allosteric AKT inhibitor, on human NPC cells in vitro and in vivo. Methods The effect of MK-2206 on the growth and proliferation of CNE-1, CNE-2, HONE-1, and SUNE-1 cells was assessed by Cell Counting Kit 8 and colony formation assay. Flow cytometry was performed to analyze cell cycle and apoptosis. The effects of MK-2206 on the AKT pathway were analyzed by Western blotting. Autophagy induction was evaluated via electron microscopy and Western blot. To test the effects of MK-2206 in vivo, CNE-2 cells were subcutaneously implanted into nude mice. Tumor-bearing mice were treated orally with MK-2206 or placebo. Tumors were harvested for immunohistochemical analysis. Results In vitro, MK-2206 inhibited the four NPC cell line growths and reduced the sizes of the colonies in a dose-dependent manner. At 72 and 96 hours, the half maximal inhibitory concentration (IC50) values of MK-2206 in CNE-1, CNE-2, and HONE-1 cell lines were 3–5 μM, whereas in SUNE-1, IC50 was less than 1 μM, and MK-2206 induced cell cycle arrest at the G1 phase. However, our study found no evidence of apoptosis. MK-2206 induced autophagy in NPC cells, as evidenced by electron microscopy and Western blot, and inhibited the growth of tumors that were subcutaneously implanted in mice. Inhibition of downstream phosphorylation through the PRAS40 and S6 pathways seems to be the main mechanism for the MK-2206-induced growth inhibition. Conclusion Our preclinical study suggests that MK-2206’s antiproliferative effect may be useful for NPC treatment; however, strategies for reinforcing this effect are needed to maximize clinical benefit. PMID:25336925

  5. ARQ 092, an orally-available, selective AKT inhibitor, attenuates neutrophil-platelet interactions in sickle cell disease

    PubMed Central

    Kim, Kyungho; Li, Jing; Barazia, Andrew; Tseng, Alan; Youn, Seock-Won; Abbadessa, Giovanni; Yu, Yi; Schwartz, Brian; Andrews, Robert K.; Gordeuk, Victor R.; Cho, Jaehyung

    2017-01-01

    Previous studies identified the Ser/Thr protein kinase, AKT, as a therapeutic target in thrombo-inflammatory diseases. Here we report that specific inhibition of AKT with ARQ 092, an orally-available AKT inhibitor currently in phase Ib clinical trials as an anti-cancer drug, attenuates the adhesive function of neutrophils and platelets from sickle cell disease patients in vitro and cell-cell interactions in a mouse model of sickle cell disease. Studies using neutrophils and platelets isolated from sickle cell disease patients revealed that treatment with 50–500 nM ARQ 092 significantly blocks αMβ2 integrin function in neutrophils and reduces P-selectin exposure and glycoprotein Ib/IX/V-mediated agglutination in platelets. Treatment of isolated platelets and neutrophils with ARQ 092 inhibited heterotypic cell-cell aggregation under shear conditions. Intravital microscopic studies demonstrated that short-term oral administration of ARQ 092 or hydroxyurea, a major therapy for sickle cell disease, diminishes heterotypic cell-cell interactions in venules of sickle cell disease mice challenged with tumor necrosis factor-α. Co-administration of hydroxyurea and ARQ 092 further reduced the adhesive function of neutrophils in venules and neutrophil transmigration into alveoli, inhibited expression of E-selectin and intercellular adhesion molecule-1 in cremaster vessels, and improved survival in these mice. Ex vivo studies in sickle cell disease mice suggested that co-administration of hydroxyurea and ARQ 092 efficiently blocks neutrophil and platelet activation and that the beneficial effect of hydroxyurea results from nitric oxide production. Our results provide important evidence that ARQ 092 could be a novel drug for the prevention and treatment of acute vaso-occlusive complications in patients with sickle cell disease. PMID:27758820

  6. ARQ 092, an orally-available, selective AKT inhibitor, attenuates neutrophil-platelet interactions in sickle cell disease.

    PubMed

    Kim, Kyungho; Li, Jing; Barazia, Andrew; Tseng, Alan; Youn, Seock-Won; Abbadessa, Giovanni; Yu, Yi; Schwartz, Brian; Andrews, Robert K; Gordeuk, Victor R; Cho, Jaehyung

    2017-02-01

    Previous studies identified the Ser/Thr protein kinase, AKT, as a therapeutic target in thrombo-inflammatory diseases. Here we report that specific inhibition of AKT with ARQ 092, an orally-available AKT inhibitor currently in phase Ib clinical trials as an anti-cancer drug, attenuates the adhesive function of neutrophils and platelets from sickle cell disease patients in vitro and cell-cell interactions in a mouse model of sickle cell disease. Studies using neutrophils and platelets isolated from sickle cell disease patients revealed that treatment with 50-500 nM ARQ 092 significantly blocks αMβ2 integrin function in neutrophils and reduces P-selectin exposure and glycoprotein Ib/IX/V-mediated agglutination in platelets. Treatment of isolated platelets and neutrophils with ARQ 092 inhibited heterotypic cell-cell aggregation under shear conditions. Intravital microscopic studies demonstrated that short-term oral administration of ARQ 092 or hydroxyurea, a major therapy for sickle cell disease, diminishes heterotypic cell-cell interactions in venules of sickle cell disease mice challenged with tumor necrosis factor-α. Co-administration of hydroxyurea and ARQ 092 further reduced the adhesive function of neutrophils in venules and neutrophil transmigration into alveoli, inhibited expression of E-selectin and intercellular adhesion molecule-1 in cremaster vessels, and improved survival in these mice. Ex vivo studies in sickle cell disease mice suggested that co-administration of hydroxyurea and ARQ 092 efficiently blocks neutrophil and platelet activation and that the beneficial effect of hydroxyurea results from nitric oxide production. Our results provide important evidence that ARQ 092 could be a novel drug for the prevention and treatment of acute vaso-occlusive complications in patients with sickle cell disease.

  7. Discovery of 14-3-3 protein-protein interaction inhibitors that sensitize multidrug-resistant cancer cells to doxorubicin and the Akt inhibitor GSK690693.

    PubMed

    Mori, Mattia; Vignaroli, Giulia; Cau, Ylenia; Dinić, Jelena; Hill, Richard; Rossi, Matteo; Colecchia, David; Pešić, Milica; Link, Wolfgang; Chiariello, Mario; Ottmann, Christian; Botta, Maurizio

    2014-05-01

    14-3-3 is a family of highly conserved adapter proteins that is attracting much interest among medicinal chemists. Small-molecule inhibitors of 14-3-3 protein-protein interactions (PPIs) are in high demand, both as tools to increase our understanding of 14-3-3 actions in human diseases and as leads to develop innovative therapeutic agents. Herein we present the discovery of novel 14-3-3 PPI inhibitors through a multidisciplinary strategy combining molecular modeling, organic synthesis, image-based high-content analysis of reporter cells, and in vitro assays using cancer cells. Notably, the two most active compounds promoted the translocation of c-Abl and FOXO pro-apoptotic factors into the nucleus and sensitized multidrug-resistant cancer cells to apoptotic inducers such as doxorubicin and the pan-Akt inhibitor GSK690693, thus becoming valuable lead candidates for further optimization. Our results emphasize the possible role of 14-3-3 PPI inhibitors in anticancer combination therapies.

  8. Novel agents and associated toxicities of inhibitors of the pi3k/Akt/mtor pathway for the treatment of breast cancer

    PubMed Central

    Chia, S.; Gandhi, S.; Joy, A.A.; Edwards, S.; Gorr, M.; Hopkins, S.; Kondejewski, J.; Ayoub, J.P.; Califaretti, N.; Rayson, D.; Dent, S.F.

    2015-01-01

    The pi3k/Akt/mtor (phosphatidylinositol 3 kinase/ Akt/mammalian target of rapamycin) signalling pathway is an established driver of oncogenic activity in human malignancies. Therapeutic targeting of this pathway holds significant promise as a treatment strategy. Everolimus, an mtor inhibitor, is the first of this class of agents approved for the treatment of hormone receptor–positive, human epidermal growth factor receptor 2–negative advanced breast cancer. Everolimus has been associated with significant improvements in progression-free survival; however, it is also associated with increased toxicity related to its specific mechanism of action. Methods A comprehensive review of the literature conducted using a focused medline search was combined with a search of current trials at http://ClinicalTrials.gov/. Summary tables of the toxicities of the various classes of pi3k/Akt/mtor inhibitors were created. A broad group of Canadian health care professionals was assembled to review the data and to produce expert opinion and summary recommendations for possible best practices in managing the adverse events associated with these pathway inhibitors. Results Differing toxicities are associated with the various classes of pi3k/Akt/mtor pathway inhibitors. The most common unique adverse events observed in everolimus clinical trials in breast cancer include stomatitis (all grades: approximately 60%), noninfectious pneumonitis (15%), rash (40%), hyperglycemia (15%), and immunosuppression (40%). To minimize grades 3 and 4 toxicities and to attempt to attain optimal outcomes, effective management of those adverse events is critical. Management should be interdisciplinary and should use approaches that include education, early recognition, active intervention, and potentially prophylactic strategies. Discussion Everolimus likely represents the first of many complex oral targeted therapies for the treatment of breast cancer. Using this agent as a template, it is essential to

  9. TNF-α induces cytosolic phospholipase A2 expression via Jak2/PDGFR-dependent Elk-1/p300 activation in human lung epithelial cells.

    PubMed

    Yang, Chuen-Mao; Lee, I-Ta; Chi, Pei-Ling; Cheng, Shin-Ei; Hsiao, Li-Der; Hsu, Chih-Kai

    2014-03-15

    Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid release for prostaglandin (PG) synthesis during inflammation triggered by tumor necrosis factor-α (TNF-α). However, the mechanisms underlying TNF-α-induced cPLA2 expression in human lung epithelial cells (HPAEpiCs) were not completely understood. Here, we demonstrated that TNF-α induced cPLA2 mRNA and protein expression, promoter activity, and PGE2 secretion in HPAEpiCs. These responses induced by TNF-α were inhibited by pretreatment with the inhibitor of Jak2 (AG490), platelet-derived growth factor receptor (PDGFR) (AG1296), phosphoinositide 3 kinase (PI3K) (LY294002), or MEK1/2 (PD98059) and transfection with siRNA of Jak2, PDGFR, Akt, or p42. We showed that TNF-α markedly stimulated Jak2, PDGFR, Akt, and p42/p44 MAPK phosphorylation, which were attenuated by their respective inhibitors. Moreover, TNF-α stimulated Akt activation via a Jak2/PDGFR pathway in HPAEpiCs. In addition, TNF-α-induced p42/p44 MAPK phosphorylation was reduced by AG1296 or LY294002. On the other hand, TNF-α could induce Akt and p42/p44 MAPK translocation from the cytosol into the nucleus, which was inhibited by AG490, AG1296, or LY294002. Finally, we showed that TNF-α stimulated Elk-1 phosphorylation, which was reduced by LY294002 or PD98059. We also observed that TNF-α time dependently induced p300/Elk-1 and p300/Akt complex formation in HPAEpiCs, which was reduced by AG490, AG1296, or LY294002. The activity of cPLA2 protein upregulated by TNF-α was reflected on the PGE2 release, which was reduced by AG490, AG1296, LY294002, or PD98059. Taken together, these results demonstrated that TNF-α-induced cPLA2 expression and PGE2 release were mediated through a Jak2/PDGFR/PI3K/Akt/p42/p44 MAPK/Elk-1 pathway in HPAEpiCs.

  10. Tumor necrosis factor-alpha enhances neutrophil adhesiveness: induction of vascular cell adhesion molecule-1 via activation of Akt and CaM kinase II and modifications of histone acetyltransferase and histone deacetylase 4 in human tracheal smooth muscle cells.

    PubMed

    Lee, Chiang-Wen; Lin, Chih-Chung; Luo, Shue-Fen; Lee, Hui-Chun; Lee, I-Ta; Aird, William C; Hwang, Tsong-Long; Yang, Chuen-Mao

    2008-05-01

    Up-regulation of vascular cell adhesion molecule-1 (VCAM-1) involves adhesions between both circulating and resident leukocytes and the human tracheal smooth muscle cells (HTSMCs) during airway inflammatory reaction. We have demonstrated previously that tumor necrosis factor (TNF)-alpha-induced VCAM-1 expression is regulated by mitogen-activated protein kinases, nuclear factor-kappaB, and p300 activation in HTSMCs. In addition to this pathway, phosphorylation of Akt and CaM kinase II has been implicated in histone acetyltransferase and histone deacetylase 4 (HDAC4) activation. Here, we investigated whether these different mechanisms participated in TNF-alpha-induced VCAM-1 expression and enhanced neutrophil adhesion. TNF-alpha significantly increased HTSMC-neutrophil adhesions, and this effect was associated with increased expression of VCAM-1 on the HTSMCs and was blocked by the selective inhibitors of Src [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1)], epidermal growth factor receptor [EGFR; 4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline, (AG1478)], phosphatidylinositol 3-kinase (PI3K) [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride(LY294002) and wortmannin],calcium[1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester; BAPTA-AM], phosphatidylinositol-phospholipase C (PLC) [1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122)], protein kinase C (PKC) [12-(2-cyanoethyl)-6,7,12, 13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö6976), rottlerin, and 3-1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide (bisindolylmaleimide IX) (Ro 31-8220)], CaM (calmidazolium chloride), CaM kinase II [(8R(*),9S(*),11S(*))-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3,9, 10-tetrahydro-8,11-epoxy, 1H,8H, 11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinden-1-one (KT5926) and 1-[N,O-bis(5-isoquinolinesulfonyl

  11. Compound library screening identified Akt/PKB kinase pathway inhibitors as potential key molecules for the development of new chemotherapeutics against schistosomiasis.

    PubMed

    Morel, Marion; Vanderstraete, Mathieu; Cailliau, Katia; Lescuyer, Arlette; Lancelot, Julien; Dissous, Colette

    2014-12-01

    Protein kinases (PKs) are one of the largest protein families in most eukaryotic organisms. These enzymes are involved in the control of cell proliferation, differentiation and metabolism and a large number of the anticancer drugs currently used are directed against PKs. The structure and function of PKs are well conserved throughout evolution. In schistosome parasites, PKs were shown to be involved in essential functions at every stage of the parasite life cycle, making these enzymes promising anti-parasite drug targets. In this study, we tested a panel of commercial inhibitors for various PKs and analyzed their effects on pairing and egg production by schistosomes as well as their toxicity towards schistosomula larvae. Results obtained confirmed the deleterious effect of PK targeting on Schistosoma mansoni physiology and the important function of different tyrosine and serine/threonine kinases in the biology and reproduction of this parasite. They also indicated for the first time that the Protein kinase B (also called Akt) which is a major downstream target of many receptor tyrosine kinases and a central player at the crossroads of signal transduction pathways activated in response to growth factors and insulin, can constitute a novel target for anti-schistosome chemotherapy. Structural and functional studies have shown that SmAkt is a conserved kinase and that its activity can be inhibited by commercially available Akt inhibitors. In treated adult worms, Akt/PKB kinase pathway inhibitors induced profound alterations in pairing and egg laying and they also greatly affected the viability of schistosomula larvae.

  12. miR-221/222 confers radioresistance in glioblastoma cells through activating Akt independent of PTEN status.

    PubMed

    Li, W; Guo, F; Wang, P; Hong, S; Zhang, C

    2014-01-01

    Glioblastoma is highly resistant to radiation therapy. The underlying molecular mechanism is not completely understood. The DNA damage response (DDR) pathway plays a crucial role in radioresistance of glioablastoma cells. Growing evidence has demonstrated that radiation induces alterations in microRNA (miR) profiles. However, how radiation induces specific miRs and how they might regulate the DDR remain elusive. In our study, we found that radiation induced c-jun transcription of miR-221 and miR-222. miR-221 and miR- 222 modulated DNA-PKcs expression to affect DNA damage repair by activating Akt independent of PTEN status. Knocking down of miR-221/222 significantly increased radiosensitivity of glioblastoma cells. Inhibition of Akt by RNAi or LY294002 treatment may overcome miR-221/222 induced radioresistance. Notably, combined anti-miR-221/222 and radiotherapy has remarkably inhibited tumor growth compared with anti-miR-221/222 or radiotherapy alone in a subcutaneous mouse model. Our results suggest that radio-induced c-jun promotes transcription of miR-221/222, which mediates DNA damage repair of glioblastoma cells independent of PTEN. These data indicate for the first time that miR-221/222 play an important role in mediating radio-induced DNA damage repair and that miR-221/222 could serve as potential therapeutic targets for increasing radiosensitivity of glioblastoma cells.

  13. Withaferin A-Caused Production of Intracellular Reactive Oxygen Species Modulates Apoptosis via PI3K/Akt and JNKinase in Rabbit Articular Chondrocytes

    PubMed Central

    2014-01-01

    Withaferin A (WFA) is known as a constituent of Ayurvedic medicinal plant, Withania somnifera, and has been used for thousands of years. Although WFA has been used for the treatment of osteoarthritis (OA) and has a wide range of biochemical and pharmacologic activities, there are no findings suggesting its properties on chondrocytes or cartilage. The aim of the present study is to investigate the effects of WFA on apoptosis with focus on generation of intracellular reactive oxygen species (ROS). Here we showed that WFA significantly increased the generation of intracellular ROS in a dose-dependent manner. We also determined that WFA markedly leads to apoptosis as evidenced by accumulation of p53 by Western blot analysis. N-Acetyl-L-Cystein (NAC), an antioxidant, prevented WFA-caused expression of p53 and inhibited apoptosis of chondrocytes. We also found that WFA causes the activation of PI3K/Akt and JNKinase. Inhibition of PI3K/Akt and JNKinase with LY294002 (LY)/triciribine (TB) or SP600125 (SP) in WFA-treated cells reduced accumulation of p53 and inhibited fragmented DNA. Our findings suggested that apoptosis caused by WFA-induced intracellular ROS generation is regulated through PI3K/Akt and JNKinase in rabbit articular chondrocytes. Graphical Abstract PMID:25120312

  14. Naoxintong Protects Primary Neurons from Oxygen-Glucose Deprivation/Reoxygenation Induced Injury through PI3K-Akt Signaling Pathway

    PubMed Central

    Zhao, Pei; Zhu, Jinqiang; Yan, Chen; Li, Lin; Zhang, Han; Zhang, Meng; Gao, Xiumei

    2016-01-01

    Naoxintong capsule (NXT), developed from Buyang Huanwu Decoction, has shown the neuroprotective effects in cerebrovascular diseases, but the neuroprotection mechanisms of NXT on ischemia/reperfusion injured neurons have not yet been well known. In this study, we established the oxygen-glucose deprivation/reoxygenation (OGD/R) induced neurons injury model and treat the neurons with cerebrospinal fluid containing NXT (BNC) to investigate the effects of NXT on OGD/R induced neurons injury and potential mechanisms. BNC improved neuron viability and decreased apoptotic rate induced by OGD/R. BNC attenuated OGD/R induced cytosolic and mitochondrial Ca2+ overload, ROS generation, intracellular NO levels and nNOS mRNA increase, and cytochrome-c release when compared with OGD/R group. BNC significantly inhibited both mPTP opening and ΔΨm depolarization. BNC increased Bcl-2 expression and decreased Bax expression, upregulated the Bcl-2/Bax ratio, downregulated caspase-3 mRNA and caspase-9 mRNA expression, and decreased cleaved caspase-3 expression and caspase-3 activity. BNC increased phosphorylation of Akt following OGD/R, while LY294002 attenuated BNC induced increase of phosphorylated Akt expression. Our study demonstrated that NXT protected primary neurons from OGD/R induced injury by inhibiting calcium overload and ROS generation, protecting mitochondria, and inhibiting mitochondrial apoptotic pathway which was mediated partially by PI3K-Akt signaling pathway activation. PMID:26949405

  15. miR-222 attenuates cisplatin-induced cell death by targeting the PPP2R2A/Akt/mTOR Axis in bladder cancer cells.

    PubMed

    Zeng, Li-Ping; Hu, Zheng-Mao; Li, Kai; Xia, Kun

    2016-03-01

    Increased miR-222 levels are associated with a poor prognosis in patients with bladder cancer. However, the role of miR-222 remains unclear. In the present study, we found that miR-222 enhanced the proliferation of both the T24 and the 5637 bladder cancer cell lines. Overexpression of miR-222 attenuated cisplatin-induced cell death in bladder cancer cells. miR-222 activated the Akt/mTOR pathway and inhibited cisplatin-induced autophagy in bladder cancer cells by directly targeting protein phosphatase 2A subunit B (PPP2R2A). Blocking the activation of Akt with LY294002 or mTOR with rapamycin significantly prevented miR-222-induced proliferation and restored the sensitivity of bladder cancer cells to cisplatin. These findings demonstrate that miR-222 modulates the PPP2R2A/Akt/mTOR axis and thus plays a critical role in regulating proliferation and chemotherapeutic drug resistance. Therefore, miR-222 may be a novel therapeutic target for bladder cancer.

  16. A bioinformatic and mechanistic study elicits the antifibrotic effect of ursolic acid through the attenuation of oxidative stress with the involvement of ERK, PI3K/Akt, and p38 MAPK signaling pathways in human hepatic stellate cells and rat liver

    PubMed Central

    He, Wenhua; Shi, Feng; Zhou, Zhi-Wei; Li, Bimin; Zhang, Kunhe; Zhang, Xinhua; Ouyang, Canhui; Zhou, Shu-Feng; Zhu, Xuan

    2015-01-01

    NADPH oxidases (NOXs) are a predominant mediator of redox homeostasis in hepatic stellate cells (HSCs), and oxidative stress plays an important role in the pathogenesis of liver fibrosis. Ursolic acid (UA) is a pentacyclic triterpenoid with various pharmacological activities, but the molecular targets and underlying mechanisms for its antifibrotic effect in the liver remain elusive. This study aimed to computationally predict the molecular interactome and mechanistically investigate the antifibrotic effect of UA on oxidative stress, with a focus on NOX4 activity and cross-linked signaling pathways in human HSCs and rat liver. Drug–drug interaction via chemical–protein interactome tool, a server that can predict drug–drug interaction via chemical–protein interactome, was used to predict the molecular targets of UA, and Database for Annotation, Visualization, and Integrated Discovery was employed to analyze the signaling pathways of the predicted targets of UA. The bioinformatic data showed that there were 611 molecular proteins possibly interacting with UA and that there were over 49 functional clusters responding to UA. The subsequential benchmarking data showed that UA significantly reduced the accumulation of type I collagen in HSCs in rat liver, increased the expression level of MMP-1, but decreased the expression level of TIMP-1 in HSC-T6 cells. UA also remarkably reduced the gene expression level of type I collagen in HSC-T6 cells. Furthermore, UA remarkably attenuated oxidative stress via negative regulation of NOX4 activity and expression in HSC-T6 cells. The employment of specific chemical inhibitors, SB203580, LY294002, PD98059, and AG490, demonstrated the involvement of ERK, PI3K/Akt, and p38 MAPK signaling pathways in the regulatory effect of UA on NOX4 activity and expression. Collectively, the antifibrotic effect of UA is partially due to the oxidative stress attenuating effect through manipulating NOX4 activity and expression. The results

  17. A bioinformatic and mechanistic study elicits the antifibrotic effect of ursolic acid through the attenuation of oxidative stress with the involvement of ERK, PI3K/Akt, and p38 MAPK signaling pathways in human hepatic stellate cells and rat liver.

    PubMed

    He, Wenhua; Shi, Feng; Zhou, Zhi-Wei; Li, Bimin; Zhang, Kunhe; Zhang, Xinhua; Ouyang, Canhui; Zhou, Shu-Feng; Zhu, Xuan

    2015-01-01

    NADPH oxidases (NOXs) are a predominant mediator of redox homeostasis in hepatic stellate cells (HSCs), and oxidative stress plays an important role in the pathogenesis of liver fibrosis. Ursolic acid (UA) is a pentacyclic triterpenoid with various pharmacological activities, but the molecular targets and underlying mechanisms for its antifibrotic effect in the liver remain elusive. This study aimed to computationally predict the molecular interactome and mechanistically investigate the antifibrotic effect of UA on oxidative stress, with a focus on NOX4 activity and cross-linked signaling pathways in human HSCs and rat liver. Drug-drug interaction via chemical-protein interactome tool, a server that can predict drug-drug interaction via chemical-protein interactome, was used to predict the molecular targets of UA, and Database for Annotation, Visualization, and Integrated Discovery was employed to analyze the signaling pathways of the predicted targets of UA. The bioinformatic data showed that there were 611 molecular proteins possibly interacting with UA and that there were over 49 functional clusters responding to UA. The subsequential benchmarking data showed that UA significantly reduced the accumulation of type I collagen in HSCs in rat liver, increased the expression level of MMP-1, but decreased the expression level of TIMP-1 in HSC-T6 cells. UA also remarkably reduced the gene expression level of type I collagen in HSC-T6 cells. Furthermore, UA remarkably attenuated oxidative stress via negative regulation of NOX4 activity and expression in HSC-T6 cells. The employment of specific chemical inhibitors, SB203580, LY294002, PD98059, and AG490, demonstrated the involvement of ERK, PI3K/Akt, and p38 MAPK signaling pathways in the regulatory effect of UA on NOX4 activity and expression. Collectively, the antifibrotic effect of UA is partially due to the oxidative stress attenuating effect through manipulating NOX4 activity and expression. The results suggest that

  18. Rho GDP dissociation inhibitor beta promotes cell proliferation and invasion by modulating the AKT pathway in hepatocellular carcinoma.

    PubMed

    Fang, Yang; Yi, Jiang; Lizhi, Lv; Qiucheng, Cai

    2014-11-01

    Rho GDP dissociation inhibitor (GDI) beta, (RhoGDI2), has been identified as a proto-oncogene that is upregulated in human cancers, but the role of RhoGDI2 in hepatocellular carcinoma (HCC) remains unclear. In the present study, we investigated the RhoGDI2 expression level in HCC tissues and the function of RhoGDI2 in HCC cell growth and metastasis. We examined the RhoGDI2 mRNA expression level in 64 sets of HCC tissue and their adjacent nontumor tissue counterparts using quantitative real-time polymerase chain reaction. In vitro proliferation and invasion assays were conducted to determine the effect of RhoGDI2 on the ability of HCC cells to proliferate and invade, respectively. Western blot analysis was conducted to examine expression levels of RhoGDI2p-AKT, MMP-2, and MMP-9 in HCC cells. RhoGDI2 mRNA was significantly overexpressed in the HCC specimens compared with the nonneoplastic liver specimens, and the RhoGDI2 mRNA and protein levels were higher in the HCC cell lines, especially the highly metastatic cell lines 97L and 97H. To further investigate the role that RhoGDI2 plays in HCC, we overexpressed RhoGDI2 using a lentivirus-mediated overexpression technique in two HCC cell lines (Huh7 and 7721) that endogenously express a low level of RhoGDI2. Stable cells overexpressing RhoGDI2 demonstrated a significant increase in cell proliferation and invasion. Furthermore, our additional findings indicated that RhoGDI2-mediated cellular invasion requires the PI3K/Akt signaling-dependent expression of matrix metalloproteinases (MMPs). Our findings suggest that RhoGDI2 plays an important role in HCC growth and invasion and should be considered a novel HCC therapeutic target candidate.

  19. PTEN Regulates Beta-Catenin in Androgen Signaling: Implication in Prostate Cancer Progression

    DTIC Science & Technology

    2007-03-01

    of androgens, whereas purified Wnt3a showed a pronounced effect in the presence of low concentrations of ligands. We also showed that Wnt3a-CM and the...that the effect of PI3K/Akt in prostate cells is mediated through androgen signaling. The PI3K inhibitor, LY294002, and a tumor suppressor, PTEN...progression of prostate cancer remain largely unknown. Androgen ablation is an effective treatment for the majority of advanced prostate cancer patients

  20. IL-7 splicing variant IL-7δ5 induces EMT and metastasis of human breast cancer cell lines MCF-7 and BT-20 through activation of PI3K/Akt pathway.

    PubMed

    Yang, Jie; Zeng, Zhi; Peng, Yuyu; Chen, Jianhua; Pan, Ling; Pan, Deshun

    2014-10-01

    Our previous study has confirmed that IL-7δ5 (an IL-7 variant lacking exon 5) promotes breast cancer growth. However, whether IL-7δ5 is involved in tumor cell EMT and metastasis remains unclear. In this study, we investigated the preclinical effects and molecular mechanisms of IL-7δ5 on EMT and metastasis in human MCF-7 and BT-20 breast cancer cells in vitro and in vivo. The results showed that IL-7δ5 induced EMT and invasion in tumor cells, associated with up-regulation of N-cadherin and the down-regulation of E-cadherin. Furthermore, we found that IL-7δ5 induced the activation of Akt. Inhibition of PI3K/Akt pathway by LY294002 reversed the EMT transition in breast cancer cell lines MCF-7 and BT-20 induced by IL-7δ5. In addition, IL-7δ5 enhanced cancer metastasis and shortened survival time, with increased level changes of activated Akt in nude mice with breast cancer. In conclusion, our findings demonstrate that IL-7δ5 induces human breast cancer cell lines EMT and metastasis via activation of PI3K/Akt pathway. Thus, IL-7δ5 may be a potential target against human breast cancer.

  1. RES-529: a PI3K/AKT/mTOR pathway inhibitor that dissociates the mTORC1 and mTORC2 complexes

    PubMed Central

    2016-01-01

    RES-529 (previously named Palomid 529, P529) is a phosphoinositide 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) pathway inhibitor that interferes with the pathway through both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) dissociation. This compound is currently being developed in oncology and ophthalmology. The oncology focus is for the treatment of glioblastoma, where it has received orphan designation by the US Food and Drug Administration, and prostate cancer. We present a review of the PI3K/AKT/mTOR pathway, its role in tumorigenesis, and the potential of RES-529 in cancer treatment. RES-529 inhibits mTORC1/mTORC2 activity in various cancer cell lines, as noted by decreased phosphorylation of substrates including ribosomal protein S6, 4E-BP1, and AKT, leading to cell growth inhibition and death, with activity generally in the range of 5–15 μmol/l. In animal tumor models where the PI3K/AKT/mTOR pathway is abnormally activated (i.e. glioblastoma, prostate cancer, and breast cancer), RES-529 reduces tumor growth by as much as 78%. RES-529 treatment is synergistic with radiation therapy, chemotherapy, and hormonal therapy in reducing tumor growth, potentially by preventing PI3K/AKT/mTOR pathway activation associated with these treatments. Furthermore, this compound has shown antiangiogenic activity in several animal models. mTORC1 and mTORC2 have redundant and distinct activities that contribute toward oncogenesis. Current inhibitors of this pathway have primarily targeted mTORC1, but have shown limited clinical efficacy. Inhibitors of mTORC1 and mTORC2 such as RES-529 may therefore have the potential to overcome the deficiencies found in targeting only mTORC1. PMID:26918392

  2. RES-529: a PI3K/AKT/mTOR pathway inhibitor that dissociates the mTORC1 and mTORC2 complexes.

    PubMed

    Weinberg, Mark A

    2016-07-01

    RES-529 (previously named Palomid 529, P529) is a phosphoinositide 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) pathway inhibitor that interferes with the pathway through both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) dissociation. This compound is currently being developed in oncology and ophthalmology. The oncology focus is for the treatment of glioblastoma, where it has received orphan designation by the US Food and Drug Administration, and prostate cancer. We present a review of the PI3K/AKT/mTOR pathway, its role in tumorigenesis, and the potential of RES-529 in cancer treatment. RES-529 inhibits mTORC1/mTORC2 activity in various cancer cell lines, as noted by decreased phosphorylation of substrates including ribosomal protein S6, 4E-BP1, and AKT, leading to cell growth inhibition and death, with activity generally in the range of 5-15 μmol/l. In animal tumor models where the PI3K/AKT/mTOR pathway is abnormally activated (i.e. glioblastoma, prostate cancer, and breast cancer), RES-529 reduces tumor growth by as much as 78%. RES-529 treatment is synergistic with radiation therapy, chemotherapy, and hormonal therapy in reducing tumor growth, potentially by preventing PI3K/AKT/mTOR pathway activation associated with these treatments. Furthermore, this compound has shown antiangiogenic activity in several animal models. mTORC1 and mTORC2 have redundant and distinct activities that contribute toward oncogenesis. Current inhibitors of this pathway have primarily targeted mTORC1, but have shown limited clinical efficacy. Inhibitors of mTORC1 and mTORC2 such as RES-529 may therefore have the potential to overcome the deficiencies found in targeting only mTORC1.

  3. Resistance after Chronic Application of the HDAC-Inhibitor Valproic Acid Is Associated with Elevated Akt Activation in Renal Cell Carcinoma In Vivo

    PubMed Central

    Juengel, Eva; Makarević, Jasmina; Tsaur, Igor; Bartsch, Georg; Nelson, Karen; Haferkamp, Axel; Blaheta, Roman A.

    2013-01-01

    Targeted drugs have significantly improved the therapeutic options for advanced renal cell carcinoma (RCC). However, resistance often develops, negating the benefit of these agents. In the present study, the molecular mechanisms of acquired resistance towards the histone deacetylase (HDAC) inhibitor valproic acid (VPA) in a RCC in vivo model were investigated. NMRI:nu/nu mice were transplanted with Caki-1 RCC cells and then treated with VPA (200 mg/kg/day). Controls remained untreated. Based on tumor growth dynamics, the mice were divided into “responders” and “non-responders” to VPA. Histone H3 and H4 acetylation and expression of cell signaling and cell cycle regulating proteins in the RCC mouse tumors were evaluated by Western blotting. Tumor growth of VPA responders was significantly diminished, whereas that of VPA non-responders even exceeded control values. Cdk1, 2 and 4 proteins were strongly enhanced in the non-responders. Importantly, Akt expression and activity were massively up-regulated in the tumors of the VPA non-responders. Chronic application (12 weeks) of VPA to Caki-1 cells in vitro evoked a distinct elevation of Akt activity and cancer cells no longer responded with cell growth reduction, compared to the short 2 week treatment. We assume that chronic use of an HDAC-inhibitor is associated with (re)-activation of Akt, which may be involved in resistance development. Consequently, combined blockade of both HDAC and Akt may delay or prevent drug resistance in RCC. PMID:23372654

  4. FOXM1 confers resistance to gefitinib in lung adenocarcinoma via a MET/AKT-dependent positive feedback loop.

    PubMed

    Wang, Yu; Zhang, Weiwei; Wen, Li; Yang, Huiling; Wen, Mingling; Yun, Yuyu; Zhao, Lisheng; Zhu, Xiaofei; Tian, Li; Luo, Erping; Li, Yu; Liu, Wenchao; Wen, Ning

    2016-09-13

    Gefitinib resistance remains a major problem in the treatment of lung adenocarcinoma. However, the molecular mechanisms of gefitinib resistance are not fully understood. In this study, we characterized the critical role of transcription factor Forkhead box protein M1 (FOXM1) in gefitinib resistance of lung adenocarcinoma cells. In vitro drug sensitivity assays demonstrated that FOXM1 inhibition sensitized PC9/GR and HCC827/GR cells to gefitinib, whereas FOXM1 overexpression enhanced PC9 and HCC827 cell resistance to gefitinib. Increased FOXM1 resulted in the upregulation of hepatocyte growth factor receptor (MET), which led to activation of the protein kinase B (AKT) pathway, whereas knockdown of FOXM1 did the opposite. FOXM1 bound directly to the MET promoter regions and regulated the promoter activities and the expression of MET at the transcriptional level. Moreover, MET/AKT pathway upregulated the expression of FOXM1 in lung adenocarcinoma cells. Inhibition of pAKT by LY294002 or inhibition of pMET by PHA-665752 significantly inhibited the expression of FOXM1 in lung adenocarcinoma cells. Importantly, we further demonstrated that the expression levels of FOXM1, pAKT and MET were significantly increased in lung adenocarcinoma tissues relative to normal lung tissues, and these three biomarkers were concomitantly overexpressed in lung adenocarcinoma tissues. Taken together, our results indicate that FOXM1 promotes acquired resistance to gefitinib of lung adenocarcinoma cells, and FOXM1 crosstalks with MET/AKT signaling to form a positive feedback loop to promote lung adenocarcinoma development.

  5. FOXM1 confers resistance to gefitinib in lung adenocarcinoma via a MET/AKT-dependent positive feedback loop

    PubMed Central

    Wen, Mingling; Yun, Yuyu; Zhao, Lisheng; Zhu, Xiaofei; Tian, Li; Luo, Erping; Li, Yu; Liu, Wenchao; Wen, Ning

    2016-01-01

    Gefitinib resistance remains a major problem in the treatment of lung adenocarcinoma. However, the molecular mechanisms of gefitinib resistance are not fully understood. In this study, we characterized the critical role of transcription factor Forkhead box protein M1 (FOXM1) in gefitinib resistance of lung adenocarcinoma cells. In vitro drug sensitivity assays demonstrated that FOXM1 inhibition sensitized PC9/GR and HCC827/GR cells to gefitinib, whereas FOXM1 overexpression enhanced PC9 and HCC827 cell resistance to gefitinib. Increased FOXM1 resulted in the upregulation of hepatocyte growth factor receptor (MET), which led to activation of the protein kinase B (AKT) pathway, whereas knockdown of FOXM1 did the opposite. FOXM1 bound directly to the MET promoter regions and regulated the promoter activities and the expression of MET at the transcriptional level. Moreover, MET/AKT pathway upregulated the expression of FOXM1 in lung adenocarcinoma cells. Inhibition of pAKT by LY294002 or inhibition of pMET by PHA-665752 significantly inhibited the expression of FOXM1 in lung adenocarcinoma cells. Importantly, we further demonstrated that the expression levels of FOXM1, pAKT and MET were significantly increased in lung adenocarcinoma tissues relative to normal lung tissues, and these three biomarkers were concomitantly overexpressed in lung adenocarcinoma tissues. Taken together, our results indicate that FOXM1 promotes acquired resistance to gefitinib of lung adenocarcinoma cells, and FOXM1 crosstalks with MET/AKT signaling to form a positive feedback loop to promote lung adenocarcinoma development. PMID:27494877

  6. MiR-21 inhibitor suppressed the progression of retinoblastoma via the modulation of PTEN/PI3K/AKT pathway.

    PubMed

    Gui, Fu; Hong, Zhengdong; You, Zhipeng; Wu, Hongxi; Zhang, Yulan

    2016-12-01

    MicroRNA-21 (miR-21) was reported to act as an oncogene during the development of many human tumors. However, little was revealed about the function of miR-21 in retinoblastoma (RB). In this study, we examined the expression of miR-21 in RB tissues and explored the relationship between miR-21 and phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3-OH kinase (PI3K)/AKT signal. Quantitative real-time PCR (qRT-PCR) results showed that the level of miR-21 in RB tissues was higher than that in retinal normal tissues. In Weri-Rb-1 cells, miR-21 inhibitor suppressed the expression of miR-21 and cell viability, but improved cell apoptotic rates by modulating the levels of PDCD4, Bax, and Bcl-2. Meanwhile, miR-21 inhibitor suppressed cell migration and invasion via inhibiting the protein levels of MMP2 and MMP9 and significantly affected the expression of PTEN, PI3K, and p-AKT. Taken together, miR-21 inhibitor suppressed cell proliferation, migration, and invasion via the PTEN/PI3K/AKT signal. These findings revealed the molecular basis of miR-21 functioning in the progression of RB and provided a new means for cell therapy in RB.

  7. Dissociation of ERK and Akt signaling in endothelial cell angiogenic responses to {beta}-amyloid

    SciTech Connect

    Magrane, Jordi; Christensen, Rial A.; Rosen, Kenneth M.; Veereshwarayya, Vimal; Querfurth, Henry W. . E-mail: hquerf01@granite.tufts.edu

    2006-04-15

    Cerebrovascular deposits of {beta}-amyloid (A{beta}) peptides are found in Alzheimer's disease and cerebral amyloid angiopathy with stroke or dementia. Dysregulations of angiogenesis, the blood-brain barrier and other critical endothelial cell (EC) functions have been implicated in aggravating chronic hypoperfusion in AD brain. We have used cultured ECs to model the effects of {beta}-amyloid on the activated phosphorylation states of multifunctional serine/threonine kinases since these are differentially involved in the survival, proliferation and migration aspects of angiogenesis. Serum-starved EC cultures containing amyloid-{beta} peptides underwent a 2- to 3-fold increase in nuclear pyknosis. Under growth conditions with sublethal doses of {beta}-amyloid, loss of cell membrane integrity and inhibition of cell proliferation were observed. By contrast, cell migration was the most sensitive to A{beta} since inhibition was significant already at 1 {mu}M (P = 0.01, migration vs. proliferation). In previous work, intracellular A{beta} accumulation was shown toxic to ECs and Akt function. Here, extracellular A{beta} peptides do not alter Akt activation, resulting instead in proportionate decreases in the phosphorylations of the MAPKs: ERK1/2 and p38 (starting at 1 {mu}M). This inhibitory action occurs proximal to MEK1/2 activation, possibly through interference with growth factor receptor coupling. Levels of phospho-JNK remained unchanged. Addition of PD98059, but not LY294002, resulted in a similar decrease in activated ERK1/2 levels and inhibition of EC migration. Transfection of ERK1/2 into A{beta}-poisoned ECs functionally rescued migration. The marked effect of extracellular A{beta} on the migration component of angiogenesis is associated with inhibition of MAPK signaling, while Akt-dependent cell survival appears more affected by cellular A{beta}.

  8. PI3K-Akt1 expression and its significance in liver tissues with chronic fluorosis.

    PubMed

    Fan, Bin; Yu, Yanni; Zhang, Ying

    2015-01-01

    This study was to explore the effect and significance of PI3K signal pathway on mechanism of liver injury in chronic fluorosis. We used 48 Sprague-Dawley rats which were randomly divided into 4 groups according to the body weight, 12 in each group, half of male and female. The control group was fed with the solid feed (the fluorine content was 1.5 mg/kg). The fluorosis animals were fed with the corn containing fluorine content of 17 mg/kg from the endemic fluorosis areas. Blocking agent LY294002 was injected in the blocking group and phosphate buffer solution was injected in the blocking control in the caudal vein with 10 mg/kg once every other day in the one week before the end of the experiment. The animals were drunk by tap water freely. The fluoride contents of urinary and skeletal were determined by the F-ion selective electrode method. The mRNA and protein expressions of PI3K, Akt1 in the liver tissues were determined by real-time polymerase chain reaction, and streptavidin-perosidase and Western blot, respectively. Results showed that fluoride contents of the urine and bone were increased in the fluorosis compared to those in the control. The expression of PI3K and Akt1 mRNA and proteins was significantly increased in fluorosis hepatocytes, and lower than that of the fluorosis in the blocking. The apoptosis and the intracellular calcium concentration were increased. Therefore, we conclude that PI3K-Akt signaling pathway may be one of the signaling pathways in the pathogenesis of liver injury caused by fluorosis.

  9. PI3K-Akt1 expression and its significance in liver tissues with chronic fluorosis

    PubMed Central

    Fan, Bin; Yu, Yanni; Zhang, Ying

    2015-01-01

    This study was to explore the effect and significance of PI3K signal pathway on mechanism of liver injury in chronic fluorosis. We used 48 Sprague-Dawley rats which were randomly divided into 4 groups according to the body weight, 12 in each group, half of male and female. The control group was fed with the solid feed (the fluorine content was 1.5 mg/kg). The fluorosis animals were fed with the corn containing fluorine content of 17 mg/kg from the endemic fluorosis areas. Blocking agent LY294002 was injected in the blocking group and phosphate buffer solution was injected in the blocking control in the caudal vein with 10 mg/kg once every other day in the one week before the end of the experiment. The animals were drunk by tap water freely. The fluoride contents of urinary and skeletal were determined by the F-ion selective electrode method. The mRNA and protein expressions of PI3K, Akt1 in the liver tissues were determined by real-time polymerase chain reaction, and streptavidin-perosidase and Western blot, respectively. Results showed that fluoride contents of the urine and bone were increased in the fluorosis compared to those in the control. The expression of PI3K and Akt1 mRNA and proteins was significantly increased in fluorosis hepatocytes, and lower than that of the fluorosis in the blocking. The apoptosis and the intracellular calcium concentration were increased. Therefore, we conclude that PI3K-Akt signaling pathway may be one of the signaling pathways in the pathogenesis of liver injury caused by fluorosis. PMID:25973007

  10. Molecular pharmacology and antitumor activity of PHT-427, a novel Akt/phosphatidylinositide-dependent protein kinase 1 pleckstrin homology domain inhibitor.

    PubMed

    Meuillet, Emmanuelle J; Zuohe, Song; Lemos, Robert; Ihle, Nathan; Kingston, John; Watkins, Ryan; Moses, Sylvestor A; Zhang, Shuxing; Du-Cuny, Lei; Herbst, Roy; Jacoby, Jörg J; Zhou, Li Li; Ahad, Ali M; Mash, Eugene A; Kirkpatrick, D Lynn; Powis, Garth

    2010-03-01

    Phosphatidylinositol 3-kinase/phosphatidylinositide-dependent protein kinase 1 (PDPK1)/Akt signaling plays a critical role in activating proliferation and survival pathways within cancer cells. We report the molecular pharmacology and antitumor activity of PHT-427, a compound designed to bind to the pleckstrin homology (PH) binding domain of signaling molecules important in cancer. Although originally designed to bind the PH domain of Akt, we now report that PHT-427 also binds to the PH domain of PDPK1. A series of PHT-427 analogues with variable C-4 to C-16 alkyl chain length were synthesized and tested. PHT-427 itself (C-12 chain) bound with the highest affinity to the PH domains of both PDPK1 and Akt. PHT-427 inhibited Akt and PDPK1 signaling and their downstream targets in sensitive but not resistant cells and tumor xenografts. When given orally, PHT-427 inhibited the growth of human tumor xenografts in immunodeficient mice, with up to 80% inhibition in the most sensitive tumors, and showed greater activity than analogues with C4, C6, or C8 alkyl chains. Inhibition of PDPK1 was more closely correlated to antitumor activity than Akt inhibition. Tumors with PIK3CA mutation were the most sensitive, and K-Ras mutant tumors were the least sensitive. Combination studies showed that PHT-427 has greater than additive antitumor activity with paclitaxel in breast cancer and with erlotinib in non-small cell lung cancer. When given >5 days, PHT-427 caused no weight loss or change in blood chemistry. Thus, we report a novel PH domain binding inhibitor of PDPK1/Akt signaling with significant in vivo antitumor activity and minimal toxicity.

  11. Insufficient radiofrequency ablation promotes epithelial-mesenchymal transition of hepatocellular carcinoma cells through Akt and ERK signaling pathways

    PubMed Central

    2013-01-01

    Background Residual tumor progression after insufficient radiofrequency ablation (RFA) has been recently reported. However, whether epithelial-mesenchymal transition (EMT), which is a key process that drives cancer metastasis, is involved in the tumor progression after insufficient RFA is not well understood. Methods Human hepatocellular carcinoma (HCC) cell lines SMMC7721 and Huh7 were used. Insufficient RFA was simulated using a water bath (47°C 5 min, 10 min, 15 min, 20 min and 25 min gradually). MTT assay was used to evaluate the proliferation of HCC cells in vitro. Migration and invasion of HCC cells were determined by transwell assay. The molecular changes in HCC cells after insufficient RFA were evaluated by western blot. LY294002 and PD98059 were used to treat HCC cells. An ectopic nude mice model and a tail vein metastatic assay were used to evaluate the growth and metastatic potential of SMMC7721 cells in vivo after insufficient RFA. Results SMMC7721 and Huh7 cells after insufficient RFA (named as SMMC7721-H and Huh7-H respectively) exhibited enhanced proliferation, migration and invasion (6.4% and 23.6%, 33.2% and 66.1%, and 44.1% and 57.4% increase respectively) in vitro. Molecular changes of EMT were observed in SMMC7721-H and Huh7-H cells. LY294002 and PD98059 inhibited the EMT of SMMC7721-H and Huh7-H cells. SMMC7721-H cells also exhibited larger tumor size (1440.8 ± 250.3 mm3 versus 1048.56 ± 227.6 mm3) and more lung metastasis (97.4% increase) than SMMC7721 cells in vivo. Higher expression of PCNA, N-cadherin and MMP-2 and MMP-9, was also observed in SMMC7721-H tumors. Conclusions Insufficient RFA could directly promote the invasiveness and metastasis of HCC cells. Insufficient RFA may promote the EMT of HCC cells through Akt and ERK signaling pathways. PMID:24168056

  12. Synergistic effects of selective inhibitors targeting the PI3K/AKT/mTOR pathway or NUP214-ABL1 fusion protein in human Acute Lymphoblastic Leukemia

    PubMed Central

    Martelli, Alberto M.; Zauli, Giorgio; Milani, Daniela; McCubrey, James A.; Capitani, Silvano; Neri, Luca M.

    2016-01-01

    Philadelphia chromosome-positive (Ph+) Acute Lymphoblastic Leukemia (ALL) accounts for 25–30% of adult ALL and its incidence increases with age in adults >40 years old. Irrespective of age, the ABL1 fusion genes are markers of poor prognosis and amplification of the NUP214-ABL1 oncogene can be detected mainly in patients with T-ALL. T cell malignancies harboring the ABL1 fusion genes are sensitive to many cytotoxic agents, but up to date complete remissions have not been achieved. The PI3K/Akt/mTOR signaling pathway is often activated in leukemias and plays a crucial role in leukemogenesis. We analyzed the effects of three BCR-ABL1 tyrosine kinase inhibitors (TKIs), alone and in combination with a panel of selective PI3K/Akt/mTOR inhibitors, on three NUP214-ABL1 positive T-ALL cell lines that also displayed PI3K/Akt/mTOR activation. Cells were sensitive to anti BCR-ABL1 TKIs Imatinib, Nilotinib and GZD824, that specifically targeted the ABL1 fusion protein, but not the PI3K/Akt/mTOR axis. Four drugs against the PI3K/Akt/mTOR cascade, GSK690693, NVP-BGT226, ZSTK474 and Torin-2, showed marked cytotoxic effects on T-leukemic cells, without affecting the NUP214-ABL1 kinase and related pathway. Dephosphorylation of pAkt and pS6 showed the cytotoxicity of these compounds. Either single or combined administration of drugs against the different targets displayed inhibition of cellular viability associated with a concentration-dependent induction of apoptosis, cell cycle arrest in G0/G1 phase and autophagy, having the combined treatments a significant synergistic cytotoxic effect. Co-targeting NUP214-ABL1 fusion gene and PI3K/Akt/mTOR signaling pathway could represent a new and effective pharmacological strategy to improve the outcome in NUP214-ABL1 positive T-ALL. PMID:27821800

  13. Compound library screening identified Akt/PKB kinase pathway inhibitors as potential key molecules for the development of new chemotherapeutics against schistosomiasis

    PubMed Central

    Morel, Marion; Vanderstraete, Mathieu; Cailliau, Katia; Lescuyer, Arlette; Lancelot, Julien; Dissous, Colette

    2014-01-01

    Protein kinases (PKs) are one of the largest protein families in most eukaryotic organisms. These enzymes are involved in the control of cell proliferation, differentiation and metabolism and a large number of the anticancer drugs currently used are directed against PKs. The structure and function of PKs are well conserved throughout evolution. In schistosome parasites, PKs were shown to be involved in essential functions at every stage of the parasite life cycle, making these enzymes promising anti-parasite drug targets. In this study, we tested a panel of commercial inhibitors for various PKs and analyzed their effects on pairing and egg production by schistosomes as well as their toxicity towards schistosomula larvae. Results obtained confirmed the deleterious effect of PK targeting on Schistosoma mansoni physiology and the important function of different tyrosine and serine/threonine kinases in the biology and reproduction of this parasite. They also indicated for the first time that the Protein kinase B (also called Akt) which is a major downstream target of many receptor tyrosine kinases and a central player at the crossroads of signal transduction pathways activated in response to growth factors and insulin, can constitute a novel target for anti-schistosome chemotherapy. Structural and functional studies have shown that SmAkt is a conserved kinase and that its activity can be inhibited by commercially available Akt inhibitors. In treated adult worms, Akt/PKB kinase pathway inhibitors induced profound alterations in pairing and egg laying and they also greatly affected the viability of schistosomula larvae. PMID:25516836

  14. The promotion of angiogenesis induced by three-dimensional porous beta-tricalcium phosphate scaffold with different interconnection sizes via activation of PI3K/Akt pathways

    NASA Astrophysics Data System (ADS)

    Xiao, Xin; Wang, Wei; Liu, Dong; Zhang, Haoqiang; Gao, Peng; Geng, Lei; Yuan, Yulin; Lu, Jianxi; Wang, Zhen

    2015-03-01

    The porous architectural characteristics of biomaterials play an important role in scaffold revascularization. However, no consensus exists regarding optimal interconnection sizes for vascularization and its scaffold bioperformance with different interconnection sizes. Therefore, a series of disk-type beta-tricalcium phosphates with the same pore sizes and variable interconnections were produced to evaluate how the interconnection size influenced biomaterial vascularization in vitro and in vivo. We incubated human umbilical vein endothelial cells on scaffolds with interconnections of various sizes. Results showed that scaffolds with a 150 μm interconnection size ameliorated endothelial cell function evidenced by promoting cell adhesion and migration, increasing cell proliferation and enhancing expression of platelet-endothelial cell adhesion molecules and vascular endothelial growth factor. In vivo study was performed on rabbit implanted with scaffolds into the bone defect on femoral condyles. Implantation with scaffolds with 150 μm interconnection size significantly improved neovascularization as shown by micro-CT as compared to scaffolds with 100 and 120 μm interconnection sizes. Moreover, the aforementioned positive effects were abolished by blocking PI3K/Akt/eNOS pathway with LY-294002. Our study explicitly demonstrates that the scaffold with 150 μm interconnection size improves neovascularization via the PI3K/Akt pathway and provides a target for biomaterial inner structure modification to attain improved clinical performance in implant vascularization.

  15. 7-Ketocholesterol induces ATM/ATR, Chk1/Chk2, PI3K/Akt signalings, cytotoxicity and IL-8 production in endothelial cells

    PubMed Central

    Chang, Mei-Chi; Chen, Yi-Jane; Liou, Eric Jein-Wein; Tseng, Wan-Yu; Chan, Chiu-Po; Lin, Hseuh-Jen; Liao, Wan-Chuen; Chang, Ya-Ching; Jeng, Po-Yuan; Jeng, Jiiang-Huei

    2016-01-01

    Cardiovascular diseases (atherosclerosis, stroke, myocardiac infarction etc.) are the major systemic diseases of elder peoples in the world. This is possibly due to increased levels of oxidized low-density lipoproteins (oxLDLs) such as 7-ketocholesterol (7-KC) and lysophosphatidylcholine (LPC) that damage vascular endothelial cells, induce inflammatory responses, to elevate the risk of cardiovascular diseases, Alzheimer's disease, and age-related macular degeneration. However the toxic effects of 7-KC on endothelial cells are not known. In this study, 7-KC showed cytotoxicity to endothelial cells at concentrations higher than 10 μg/ml. 7-KC stimulated ATM/Chk2, ATR-Chk1 and p53 signaling pathways in endothelial cells. 7-KC also induced G0/G1 cell cycle arrest and apoptosis with an inhibition of Cyclin dependent kinase 1 (Cdk1) and cyclin B1 expression. Secretion and expression of IL-8 in endothelial cells were stimulated by 7-KC. 7-KC further induced intracellular ROS production as shown by increase in DCF fluorescence and Akt phosphorylation. LY294002 attenuated the 7-KC-induced apoptosis and IL-8 mRNA expression of endothelial cells. These results indicate that oxLDLs such as 7-KC may contribute to the pathogenesis of atherosclerosis, thrombosis and cardiovascular diseases by induction of endothelial damage, apoptosis and inflammatory responses. These events are associated with ROS production, activation of ATM/Chk2, ATR/Chk1, p53 and PI3K/Akt signaling pathways. PMID:27740938

  16. Human Phosphatidylethanolamine-Binding Protein 4 Promoted the Radioresistance of Human Rectal Cancer by Activating Akt in an ROS-Dependent Way

    PubMed Central

    Qiu, Jianming; Yang, Guangen; Lin, Ali; Shen, Zhong; Wang, Dong; Ding, Lei

    2014-01-01

    Human phosphatidylethanolamine-binding protein 4(hPEBP4) is a novel anti-apoptosis molecule associated with the resistance of tumors to apoptotic agents. Here we sought to investigate the role of hPEBP4 in the radioresistance of rectal cancer. Immunohistochemistry analysis showed hPEBP4 was expressed in 27/33 of rectal cancer specimens, but only in 2/33 of neighboring normal mucosa. Silencing the expression of hPEBP4 with siRNA significantly reduced the clonogenic survival and enhanced the apoptosis of rectal cancer cells on irradiation. Instead, forced overexpression of hPEBP4 promoted its survival and decreased the apoptosis. Western blot showed hPEBP4 could increase the radiation-induced Akt activation, for which reactive oxygen specimen(ROS) was required. The radioresistance effect of hPEBP4 was reversed after given LY-294002 to inhibit Akt activation or antioxidant to abolish the ROS production. We also confirmed that effect of hPEBP4 in vivo with nude mice. Thus we concluded that hPEBP4, specifically expressed in rectal cancer cells, is associated with radioresistance of rectal cancer, implying that modulation of hPEBP4 may have important therapeutic implications in radiotherapy of rectal cancer. PMID:24594691

  17. The promotion of angiogenesis induced by three-dimensional porous beta-tricalcium phosphate scaffold with different interconnection sizes via activation of PI3K/Akt pathways

    PubMed Central

    Xiao, Xin; Wang, Wei; Liu, Dong; Zhang, Haoqiang; Gao, Peng; Geng, Lei; Yuan, Yulin; Lu, Jianxi; Wang, Zhen

    2015-01-01

    The porous architectural characteristics of biomaterials play an important role in scaffold revascularization. However, no consensus exists regarding optimal interconnection sizes for vascularization and its scaffold bioperformance with different interconnection sizes. Therefore, a series of disk-type beta-tricalcium phosphates with the same pore sizes and variable interconnections were produced to evaluate how the interconnection size influenced biomaterial vascularization in vitro and in vivo. We incubated human umbilical vein endothelial cells on scaffolds with interconnections of various sizes. Results showed that scaffolds with a 150 μm interconnection size ameliorated endothelial cell function evidenced by promoting cell adhesion and migration, increasing cell proliferation and enhancing expression of platelet-endothelial cell adhesion molecules and vascular endothelial growth factor. In vivo study was performed on rabbit implanted with scaffolds into the bone defect on femoral condyles. Implantation with scaffolds with 150 μm interconnection size significantly improved neovascularization as shown by micro-CT as compared to scaffolds with 100 and 120 μm interconnection sizes. Moreover, the aforementioned positive effects were abolished by blocking PI3K/Akt/eNOS pathway with LY-294002. Our study explicitly demonstrates that the scaffold with 150 μm interconnection size improves neovascularization via the PI3K/Akt pathway and provides a target for biomaterial inner structure modification to attain improved clinical performance in implant vascularization. PMID:25797242

  18. 7-Ketocholesterol induces ATM/ATR, Chk1/Chk2, PI3K/Akt signalings, cytotoxicity and IL-8 production in endothelial cells.

    PubMed

    Chang, Mei-Chi; Chen, Yi-Jane; Liou, Eric Jein-Wein; Tseng, Wan-Yu; Chan, Chiu-Po; Lin, Hseuh-Jen; Liao, Wan-Chuen; Chang, Ya-Ching; Jeng, Po-Yuan; Jeng, Jiiang-Huei

    2016-11-15

    Cardiovascular diseases (atherosclerosis, stroke, myocardiac infarction etc.) are the major systemic diseases of elder peoples in the world. This is possibly due to increased levels of oxidized low-density lipoproteins (oxLDLs) such as 7-ketocholesterol (7-KC) and lysophosphatidylcholine (LPC) that damage vascular endothelial cells, induce inflammatory responses, to elevate the risk of cardiovascular diseases, Alzheimer's disease, and age-related macular degeneration. However the toxic effects of 7-KC on endothelial cells are not known. In this study, 7-KC showed cytotoxicity to endothelial cells at concentrations higher than 10 µg/ml. 7-KC stimulated ATM/Chk2, ATR-Chk1 and p53 signaling pathways in endothelial cells. 7-KC also induced G0/G1 cell cycle arrest and apoptosis with an inhibition of Cyclin dependent kinase 1 (Cdk1) and cyclin B1 expression. Secretion and expression of IL-8 in endothelial cells were stimulated by 7-KC. 7-KC further induced intracellular ROS production as shown by increase in DCF fluorescence and Akt phosphorylation. LY294002 attenuated the 7-KC-induced apoptosis and IL-8 mRNA expression of endothelial cells. These results indicate that oxLDLs such as 7-KC may contribute to the pathogenesis of atherosclerosis, thrombosis and cardiovascular diseases by induction of endothelial damage, apoptosis and inflammatory responses. These events are associated with ROS production, activation of ATM/Chk2, ATR/Chk1, p53 and PI3K/Akt signaling pathways.

  19. Intranasal Administration of GDNF Protects Against Neural Apoptosis in a Rat Model of Parkinson's Disease Through PI3K/Akt/GSK3β Pathway.

    PubMed

    Yue, Peijian; Gao, Lin; Wang, Xuejing; Ding, Xuebing; Teng, Junfang

    2017-02-28

    Glial cell line-derived neurotrophic factor (GDNF) plays important roles in protecting the damaged or dying dopamine neurons in the animal models of Parkinson's disease (PD). This study was to determine the effect and mechanisms of GDNF on the apoptosis of neurons in 6-hydroxydopamine (6-OHDA) induced Parkinson's disease model of rats. Healthy male Sprague-Dawley rats (220-240 g) were randomly divided into six groups (n = 10). 6-OHDA was used to establish the PD rat model. Tyrosine hydroxylase (TH) immunohistochemistry was used to assess the neuron loss in 6-OHDA-lesioned rats. TUNEL and western blot were used to identify the effects and mechanisms of GDNF in the rat model of PD. The numbers of TH-positive neurons in the 6-OHDA-injected lesioned substantia nigra (SN) decreased significantly compared with the Sham group. GDNF treatment effectively ameliorated the apoptosis of neuronal cells in SN induced by 6-OHDA. In addition, GDNF significantly increased serine protein kinase B (Akt) and glycogen synthase kinase 3 beta (GSK3β) phosphorylation induced by 6-OHDA. In contrast, application of LY294002 or triciribine reversed the roles of GDNF in PD models. The results implicated that the anti-apoptosis effects of GDNF in neurons might be mediated through PI3K/Akt/GSK3β pathway. Therefore, GDNF may be a promising agent for PD treatment.

  20. Asperosaponin VI promotes bone marrow stromal cell osteogenic differentiation through the PI3K/AKT signaling pathway in an osteoporosis model

    PubMed Central

    Ke, Ke; Li, Qi; Yang, Xiaofeng; Xie, Zhijian; Wang, Yu; Shi, Jue; Chi, Linfeng; Xu, Weijian; Hu, Lingling; Shi, Huali

    2016-01-01

    Asperosaponin VI (ASA VI), a natural compound isolated from the well-known traditional Chinese herb Radix Dipsaci, has an important role in promoting osteoblast formation. However, its effects on osteoblasts in the context of osteoporosis is unknown. This study aimed to investigate the effects and mechanism of ASA VI action on the proliferation and osteogenic differentiation of bone marrow stromal cells isolated from the ovariectomized rats (OVX rBMSCs). The toxicity of ASA VI and its effects on the proliferation of OVX rBMSCs were measured using a CCK-8 assay. Various osteogenic differentiation markers were also analyzed, such as ALP activity, calcified nodule formation, and the expression of osteogenic genes, i.e., ALP, OCN, COL 1 and RUNX2. The results indicated that ASA VI promoted the proliferation of OVX rBMSCs and enhanced ALP activity and calcified nodule formation. In addition, while ASA VI enhanced the expression of ALP, OCN, Col 1 and RUNX2, treatment with LY294002 reduced all of these osteogenic effects and reduced the p-AKT levels induced by ASA VI. These results suggest that ASA VI promotes the osteogenic differentiation of OVX rBMSCs by acting on the phosphatidylinositol—3 kinase/AKT signaling pathway. PMID:27756897

  1. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals

    PubMed Central

    Kureishi, Yasuko; Luo, Zhengyu; Shiojima, Ichiro; Bialik, Ann; Fulton, David; Lefer, David J.; Sessa, William C.; Walsh, Kenneth

    2010-01-01

    Recent studies suggest that statins can function to protect the vasculature in a manner that is independent of their lipid-lowering activity. We show here that statins rapidly activate the protein kinase Akt/PKB in endothelial cells. Accordingly, simvastatin enhanced phosphorylation of the endogenous Akt substrate endothelial nitric oxide synthase (eNOS), inhibited apoptosis and accelerated vascular structure formation in vitro in an Akt-dependent manner. Similar to vascular endothelial growth factor (VEGF) treatment, both simvastatin administration and enhanced Akt signaling in the endothelium promoted angiogenesis in ischemic limbs of normocholesterolemic rabbits. Therefore, activation of Akt represents a mechanism that can account for some of the beneficial side effects of statins, including the promotion of new blood vessel growth. PMID:10973320

  2. Matrix metalloproteinase-9 is up-regulated by CCL19/CCR7 interaction via PI3K/Akt pathway and is involved in CCL19-driven BMSCs migration.

    PubMed

    Zhang, Wei; Tu, Guanjun; Lv, Chen; Long, Jun; Cong, Lin; Han, Yaxin

    2014-08-22

    C-C chemokine receptor 7 (CCR7) and its ligands CCL19 contributes to the directional migration of certain cancer cell lines, but its role in the migration of BMSCs remains vague. The aim of this study was to determine the possible interaction between CCL19-induced conditions and matrix metalloproteinases-9 (MMP9) expression in BMSCs. Cell migration using Transwell assay indicated that activation of CCR7 by its specific ligand, exogenous chemokine ligand 19 (CCL19), was associated with a significant linear increase. Western blot and real-time PCR indicated that CCL19/CCR7 significantly upregulated expression of MMP9, which is related to metastasis-associated genes. The CCL19/CCR7 interaction significantly enhanced phosphorylation of Akt, as measured by Western blot. P-Akt and MMP9 protein expression exhibited a time-dependent pattern, and the peak was at 48h. LY294002 significantly abolished the effects of exogenous CCL19. These results suggest that CCL19/CCR7 contributes to the migration of BMSCs by upregulating MMP9 potentially via the PI3K/Akt pathway.

  3. PI3K/Akt/mTOR pathway inhibitors enhance radiosensitivity in radioresistant prostate cancer cells through inducing apoptosis, reducing autophagy, suppressing NHEJ and HR repair pathways.

    PubMed

    Chang, L; Graham, P H; Hao, J; Ni, J; Bucci, J; Cozzi, P J; Kearsley, J H; Li, Y

    2014-10-02

    The PI3K/Akt/mTOR pathway has a central role in cancer metastasis and radiotherapy. To develop effective therapeutics to improve radiosensitivity, understanding the possible pathways of radioresistance involved and the effects of a combination of the PI3K/Akt/mTOR inhibitors with radiotherapy on prostate cancer (CaP) radioresistant cells is needed. We found that compared with parent CaP cells, CaP-radioresistant cells demonstrated G0/G1 and S phase arrest, activation of cell cycle check point, autophagy and DNA repair pathway proteins, and inactivation of apoptotic proteins. We also demonstrated that compared with combination of single PI3K or mTOR inhibitors (BKM120 or Rapamycin) and radiation, low-dose of dual PI3K/mTOR inhibitors (BEZ235 or PI103) combined with radiation greatly improved treatment efficacy by repressing colony formation, inducing more apoptosis, leading to the arrest of the G2/M phase, increased double-strand break levels and less inactivation of cell cycle check point, autophagy and non-homologous end joining (NHEJ)/homologous recombination (HR) repair pathway proteins in CaP-radioresistant cells. This study describes the possible pathways associated with CaP radioresistance and demonstrates the putative mechanisms of the radiosensitization effect in CaP-resistant cells in the combination treatment. The findings from this study suggest that the combination of dual PI3K/Akt/mTOR inhibitors (BEZ235 or PI103) with radiotherapy is a promising modality for the treatment of CaP to overcome radioresistance.

  4. IL-29 and IFN-α regulate the expression of MxA, 2',5'-OAS and PKR genes in association with the activation of Raf-MEK-ERK and PI3K-AKT signal pathways in HepG2.2.15 cells.

    PubMed

    Chai, Yu; Huang, Hai-Liang; Hu, Dao-Jun; Luo, Xin; Tao, Qian-Shan; Zhang, Xiao-Ling; Zhang, Sheng-Quan

    2011-01-01

    Interferons (IFNs) can activate the PI3K-AKT and Raf-MEK-ERK signal pathways and induce antiviral proteins (MxA, 2',5'-OAS and PKR) expression in specific cell lines. However, the relationship between those antiviral proteins expression and signal pathways remains unknown at present. Thus our experiments were designed to determine the exact relationship in HepG2.2.15 cell line. The results demonstrated that IFN-α and IL-29 were both able to activate PI3K-AKT and Raf-MEK-ERK signal pathways, and IFN-α up-regulated the expression of MxA, 2',5'-OAS and PKR whereas IL-29 increased mRNA expression of MxA and 2',5'-OAS and had no influence on PKR. Furthermore, MxA, 2',5'-OAS and PKR expression were down-regulated while PI3K-AKT signal pathway was blocked by LY294002. And MxA was up-regulated after Raf-MEK-ERK signal pathway being blocked by PD98059. These findings indicate that the expression of MxA, 2',5'-OAS and PKR are up-regulate by PI3K-AKT signal pathway, and Raf-MEK-ERK signal pathway has a negative regulatory effect on the expression of MxA and no significant effect on 2',5'-OAS and PKR.

  5. Exogenous hydrogen sulfide exerts proliferation, anti-apoptosis, migration effects and accelerates cell cycle progression in multiple myeloma cells via activating the Akt pathway.

    PubMed

    Zheng, Dong; Chen, Ziang; Chen, Jingfu; Zhuang, Xiaomin; Feng, Jianqiang; Li, Juan

    2016-10-01

    Hydrogen sulfide (H2S), regarded as the third gaseous transmitter, mediates and induces various biological effects. The present study investigated the effects of H2S on multiple myeloma cell progression via amplifying the activation of Akt pathway in multiple myeloma cells. The level of H2S produced in multiple myeloma (MM) patients and healthy subjects was measured using enzyme-linked immunosorbent assay (ELISA). MM cells were treated with 500 µmol/l NaHS (a donor of H2S) for 24 h. The expression levels of phosphorylated-Akt (p-Akt), Bcl-2 and caspase-3 were measured by western blot assay. Cell viability was detected by Cell Counting Kit 8 (CCK-8). The cell cycle was analyzed by flow cytometry. Our results show that the concentration of H2S was higher in MM patients and that it increased in parallel with disease progression. Treating MM cells with 500 µmol/l NaHS for 24 h markedly increased the expression level of Bcl-2 and the activation of p-Akt, however, the expression level of caspase-3 was decreased, cell viability was increased, and cell cycle progression was accelerated in MM cells. NaHS also induced migration in MM cells in transwell migration assay. Furthermore, co-treatment of MM cells with 500 µmol/l NaHS and 50 µmol/l LY294002 for 24 h significantly overset these effects. In conclusion, our findings demonstrate that the Akt pathway contributes to NaHS-induced cell proliferation, migration and acceleration of cell cycle progression in MM cells.

  6. The neuroprotective action of pyrroloquinoline quinone against glutamate-induced apoptosis in hippocampal neurons is mediated through the activation of PI3K/Akt pathway

    SciTech Connect

    Zhang Qi; Shen Mi; Ding Mei; Shen Dingding; Ding Fei

    2011-04-01

    Pyrroloquinoline quinone (PQQ), a cofactor in several enzyme-catalyzed redox reactions, possesses a potential capability of scavenging reactive oxygen species (ROS) and inhibiting cell apoptosis. In this study, we investigated the effects of PQQ on glutamate-induced cell death in primary cultured hippocampal neurons and the possible underlying mechanisms. We found that glutamate-induced apoptosis in cultured hippocampal neurons was significantly attenuated by the ensuing PQQ treatment, which also inhibited the glutamate-induced increase in Ca2+ influx, caspase-3 activity, and ROS production, and reversed the glutamate-induced decrease in Bcl-2/Bax ratio. The examination of signaling pathways revealed that PQQ treatment activated the phosphorylation of Akt and suppressed the glutamate-induced phosphorylation of c-Jun N-terminal protein kinase (JNK). And inhibition of phosphatidylinositol-3-kinase (PI3K)/Akt cascade by LY294002 and wortmannin significantly blocked the protective effects of PQQ, and alleviated the increase in Bcl-2/Bax ratio. Taken together, our results indicated that PQQ could protect primary cultured hippocampal neurons against glutamate-induced cell damage by scavenging ROS, reducing Ca2+ influx, and caspase-3 activity, and suggested that PQQ-activated PI3K/Akt signaling might be responsible for its neuroprotective action through modulation of glutamate-induced imbalance between Bcl-2 and Bax. - Research Highlights: >PQQ attenuated glutamate-induced cell apoptosis of cultured hippocampal neurons. >PQQ inhibited glutamate-induced Ca{sup 2+} influx and caspase-3 activity. >PQQ reduced glutamate-induced increase in ROS production. >PQQ affected phosphorylation of Akt and JNK signalings after glutamate injury. >PI3K/Akt was required for neuroprotection of PQQ by modulating Bcl-2/Bax ratio.

  7. Downregulation of Fes inhibits VEGF-A-induced chemotaxis and capillary-like morphogenesis by cultured endothelial cells

    PubMed Central

    Kanda, Shigeru; Kanetake, Hiroshi; Miyata, Yasuyoshi

    2007-01-01

    Abstract The aim of this study was to determine whether the downregulation of endogenous Fes by siRNA in cultured endothelial cells affects vascular endothelial growth factor-A (VEGF-A)-induced chemotaxis and capillary-like morphogenesis, which are considered as angiogenic cellular responses in vitro. VEGF-A-treatment induced autophosphorylation of Fes in cultured endothelial cells.LY294002, a phosphoinositide 3-kinase inhibitor, significantly inhibited VEGF-A-induced chemotaxis and capillary-like morphogenesis.Downregulation of Fes attenuated these VEGF-A-induced cellular responses but LY294002 did not produce further inhibition of these responses. Downregulation of Fes neither affected VEGF-A-induced autophosphorylation of VEGF receptor 2 nor mitogen-activated protein kinase activation, but markedly decreased Akt activation.Taken together, our novel results indicate the involvement of Fes in VEGF-A-induced cellular responses by cultured endothelial cells. PMID:17521372

  8. Synthesis and Docking Analysis of New Heterocyclic System N1, N4-bis (2-chloroquinolin-3-yl) methylene) benzene-1, 4-diamine as Potential Human AKT1 Inhibitor

    PubMed Central

    Ghanei, Sohrab; Lari, Jalil; Eshghi, Hossein; Saadatmandzadeh, Mohammad

    2016-01-01

    In recent years, the chemistry of 2-chloroquinoline-3-carbaldehydes have received considerable attention owing to their synthetic and effective biological importance which exhibits a wide variety of biological activity, N1,N4-bis((2-chloroquinolin-3-yl)methylene)benzene-1,4-diamine derivatives that synthesized from 2-chloroquinoline-3-carbaldehydes may have biological effects. As the inhibitor of AKT1 (RAC-alpha serine/threonine-protein kinase is an enzyme that in humans is encoded by the AKT1), the aforementioned compounds may have implication in preventing complications of cancers. A group of N1, N4-bis ((2-chloroquinolin-3-yl) methylene) benzene-1, 4-diamine derivatives (3a-3i) (H, 6-Me, 6-OMe, 6-OEt, 6-Cl, 7-Me, 6-Et, 6-Isopropyl, 7-Cl) were synthesized, and theoretically evaluated for their inhibitory as Potential Human AKT1 Inhibitors via docking process. The docking calculation was done in GOLD 5.2.2 software using Genetic algorithm. Compounds 3b (6-Me) and 3d (6-OEt) showed the best inhibitory potency by GOLD score value of 113.76 and 107.58 respectively. Some of the best models formed strong hydrogen bonds with Asn 49, Lys 220, Ser 157, Arg 225 and Trp 76 via quinoline moiety and nitrogen of quinolone ring (Figure 1.). pi-pi interaction between Lys 220, Trp 76, Tyr 224, Arg 225, Ile 80, and Asn 49 quinoline moiety was one of the common factor in enzyme-inhibitor junction. It was found that both hydrogen bonding and hydrophobic interactions are important in function of biological molecules, especially for inhibition in a complex. PMID:27980566

  9. Long-term acquired everolimus resistance in pancreatic neuroendocrine tumours can be overcome with novel PI3K-AKT-mTOR inhibitors

    PubMed Central

    Vandamme, Timon; Beyens, Matthias; de Beeck, Ken Op; Dogan, Fadime; van Koetsveld, Peter M; Pauwels, Patrick; Mortier, Geert; Vangestel, Christel; de Herder, Wouter; Van Camp, Guy; Peeters, Marc; Hofland, Leo J

    2016-01-01

    Background: The mTOR-inhibitor everolimus improves progression-free survival in advanced pancreatic neuroendocrine tumours (PNETs). However, adaptive resistance to mTOR inhibition is described. Methods: QGP-1 and BON-1, two human PNET cell lines, were cultured with increasing concentrations of everolimus up to 22 weeks to reach a dose of 1 μM everolimus, respectively, 1000-fold and 250-fold initial IC50. Using total DNA content as a measure of cell number, growth inhibitory dose–response curves of everolimus were determined at the end of resistance induction and over time after everolimus withdrawal. Response to ATP-competitive mTOR inhibitors OSI-027 and AZD2014, and PI3K-mTOR inhibitor NVP-BEZ235 was studied. Gene expression of 10 PI3K-Akt-mTOR pathway-related genes was evaluated using quantitative real-time PCR (RT–qPCR). Results: Long-term everolimus-treated BON-1/R and QGP-1/R showed a significant reduction in everolimus sensitivity. During a drug holiday, gradual return of everolimus sensitivity in BON-1/R and QGP-1/R led to complete reversal of resistance after 10–12 weeks. Treatment with AZD2014, OSI-027 and NVP-BEZ235 had an inhibitory effect on cell proliferation in both sensitive and resistant cell lines. Gene expression in BON-1/R revealed downregulation of MTOR, RICTOR, RAPTOR, AKT and HIF1A, whereas 4EBP1 was upregulated. In QGP-1/R, a downregulation of HIF1A and an upregulation of ERK2 were observed. Conclusions: Long-term everolimus resistance was induced in two human PNET cell lines. Novel PI3K-AKT-mTOR pathway-targeting drugs can overcome everolimus resistance. Differential gene expression profiles suggest different mechanisms of everolimus resistance in BON-1 and QGP-1. PMID:26978006

  10. Momordin Ic induces HepG2 cell apoptosis through MAPK and PI3K/Akt-mediated mitochondrial pathways.

    PubMed

    Wang, Jing; Yuan, Li; Xiao, Haifang; Xiao, Chunxia; Wang, Yutang; Liu, Xuebo

    2013-06-01

    Momordin Ic is a natural triterpenoid saponin enriched in various Chinese and Japanese natural medicines such as the fruit of Kochia scoparia (L.) Schrad. So far, there is little scientific evidence for momordin Ic with regard to the anti-tumor activities. The aim of this work was to elucidate the anti-tumor effect of momordin Ic and the signal transduction pathways involved. We found that momordin Ic induced apoptosis in human hepatocellular carcinoma HepG2 cells, which were supported by DNA fragmentation, caspase-3 activation and PARP cleavage. Meanwhile, momordin Ic triggered reactive oxygen species (ROS) production together with collapse of mitochondrial membrane potential, cytochrome c release, down-regulation of Bcl-2 and up-regulation of Bax expression. The activation of p38 and JNK, inactivation of Erk1/2 and Akt were also demonstrated. Although ROS production rather than NO was stimulated, the expression of iNOS and HO-1 were altered after momordin Ic treatment for 4 h. Furthermore, the cytochrome c release, caspase-3 activation, Bax/Bcl-2 expression and PARP cleavage were promoted with LY294002 and U0126 intervention but were blocked by SB203580, SP600125, PI3K activator, NAC and 1,400 W pretreatment, demonstrating the mitochondrial disruption. Furthermore, momordin Ic combination with NAC influenced MAPK, PI3K/Akt and HO-1, iNOS pathways, MAPK and PI3K/Akt pathways also regulated the expression of HO-1 and iNOS. These results indicated that momordin Ic induced apoptosis through oxidative stress-regulated mitochondrial dysfunction involving the MAPK and PI3K-mediated iNOS and HO-1 pathways. Thus, momordin Ic might represent a potential source of anticancer candidate.

  11. Apigenin Attenuates Atherogenesis through Inducing Macrophage Apoptosis via Inhibition of AKT Ser473 Phosphorylation and Downregulation of Plasminogen Activator Inhibitor-2.

    PubMed

    Zeng, Ping; Liu, Bin; Wang, Qun; Fan, Qin; Diao, Jian-Xin; Tang, Jing; Fu, Xiu-Qiong; Sun, Xue-Gang

    2015-01-01

    Macrophage survival is believed to be a contributing factor in the development of early atherosclerotic lesions. Dysregulated apoptosis of macrophages is involved in the inflammatory process of atherogenesis. Apigenin is a flavonoid that possesses various clinically relevant properties such as anti-inflammatory, antiplatelet, and antitumor activities. Here we showed that apigenin attenuated atherogenesis in apoE (-/-) mice in an in vivo test. In vitro experiments suggested that apigenin induced apoptosis of oxidized low density lipoprotein- (OxLDL-) loaded murine peritoneal macrophages (MPMs). Proteomic analysis showed that apigenin reduced the expression of plasminogen activator inhibitor 2 (PAI-2). PAI-2 has antiapoptotic effects in OxLDL-loaded MPMs. Enhancing PAI-2 expression significantly reduced the proapoptosis effects of apigenin. Molecular docking assay with AutoDock software predicted that residue Ser473 of Akt1 is a potential binding site for apigenin. Lentiviral-mediated overexpression of Akt1 wild type weakened the proapoptosis effect of apigenin in OxLDL-loaded MPMs. Collectively, apigenin executes its anti-atherogenic effects through inducing OxLDL-loaded MPMs apoptosis. The proapoptotic effects of apigenin were at least partly attributed to downregulation of PAI-2 through suppressing phosphorylation of AKT at Ser473.

  12. Akt1-Inhibitor of DNA binding2 is essential for growth cone formation and axon growth and promotes central nervous system axon regeneration

    PubMed Central

    Ko, Hyo Rim; Kwon, Il-Sun; Hwang, Inwoo; Jin, Eun-Ju; Shin, Joo-Ho; Brennan-Minnella, Angela M; Swanson, Raymond; Cho, Sung-Woo; Lee, Kyung-Hoon; Ahn, Jee-Yin

    2016-01-01

    Mechanistic studies of axon growth during development are beneficial to the search for neuron-intrinsic regulators of axon regeneration. Here, we discovered that, in the developing neuron from rat, Akt signaling regulates axon growth and growth cone formation through phosphorylation of serine 14 (S14) on Inhibitor of DNA binding 2 (Id2). This enhances Id2 protein stability by means of escape from proteasomal degradation, and steers its localization to the growth cone, where Id2 interacts with radixin that is critical for growth cone formation. Knockdown of Id2, or abrogation of Id2 phosphorylation at S14, greatly impairs axon growth and the architecture of growth cone. Intriguingly, reinstatement of Akt/Id2 signaling after injury in mouse hippocampal slices redeemed growth promoting ability, leading to obvious axon regeneration. Our results suggest that Akt/Id2 signaling is a key module for growth cone formation and axon growth, and its augmentation plays a potential role in CNS axonal regeneration. DOI: http://dx.doi.org/10.7554/eLife.20799.001 PMID:27938661

  13. The role of PI3K/AKT-related PIP5K1α and the discovery of its selective inhibitor for treatment of advanced prostate cancer.

    PubMed

    Semenas, Julius; Hedblom, Andreas; Miftakhova, Regina R; Sarwar, Martuza; Larsson, Rikard; Shcherbina, Liliya; Johansson, Martin E; Härkönen, Pirkko; Sterner, Olov; Persson, Jenny L

    2014-09-02

    Nitrogen-containing heterocyclic compounds are an important class of molecules that are commonly used for the synthesis of candidate drugs. Phosphatidylinositol-4-phosphate 5-kinase-α (PIP5Kα) is a lipid kinase, similar to PI3K. However, the role of PIP5K1α in oncogenic processes and the development of inhibitors that selectively target PIP5K1α have not been reported. In the present study we report that overexpression of PIP5K1α is associated with poor prognosis in prostate cancer and correlates with an elevated level of the androgen receptor. Overexpression of PIP5K1α in PNT1A nonmalignant cells results in an increased AKT activity and an increased survival, as well as invasive malignant phenotype, whereas siRNA-mediated knockdown of PIP5K1α in aggressive PC-3 cells leads to a reduced AKT activity and an inhibition in tumor growth in xenograft mice. We further report a previously unidentified role for PIP5K1α as a druggable target for our newly developed compound ISA-2011B using a high-throughput KINOMEscan platform. ISA-2011B was discovered during our synthetic studies of C-1 indol-3-yl substituted 1,2,3,4-tetrahydroisoquinolines via a Pictet-Spengler approach. ISA-2011B significantly inhibits growth of tumor cells in xenograft mice, and we show that this is mediated by targeting PIP5K1α-associated PI3K/AKT and the downstream survival, proliferation, and invasion pathways. Further, siRNA-mediated knockdown of PIP5K1α exerts similar effects on PC3 cells as ISA-2011B treatment, significantly inhibiting AKT activity, increasing apoptosis and reducing invasion. Thus, PIP5K1α has high potential as a drug target, and compound ISA-2011B is interesting for further development of targeted cancer therapy.

  14. The role of PI3K/AKT-related PIP5K1α and the discovery of its selective inhibitor for treatment of advanced prostate cancer

    PubMed Central

    Semenas, Julius; Hedblom, Andreas; Miftakhova, Regina R.; Sarwar, Martuza; Larsson, Rikard; Shcherbina, Liliya; Johansson, Martin E.; Härkönen, Pirkko; Sterner, Olov; Persson, Jenny L.

    2014-01-01

    Nitrogen-containing heterocyclic compounds are an important class of molecules that are commonly used for the synthesis of candidate drugs. Phosphatidylinositol-4-phosphate 5-kinase-α (PIP5Kα) is a lipid kinase, similar to PI3K. However, the role of PIP5K1α in oncogenic processes and the development of inhibitors that selectively target PIP5K1α have not been reported. In the present study we report that overexpression of PIP5K1α is associated with poor prognosis in prostate cancer and correlates with an elevated level of the androgen receptor. Overexpression of PIP5K1α in PNT1A nonmalignant cells results in an increased AKT activity and an increased survival, as well as invasive malignant phenotype, whereas siRNA-mediated knockdown of PIP5K1α in aggressive PC-3 cells leads to a reduced AKT activity and an inhibition in tumor growth in xenograft mice. We further report a previously unidentified role for PIP5K1α as a druggable target for our newly developed compound ISA-2011B using a high-throughput KINOMEscan platform. ISA-2011B was discovered during our synthetic studies of C-1 indol-3-yl substituted 1,2,3,4-tetrahydroisoquinolines via a Pictet-Spengler approach. ISA-2011B significantly inhibits growth of tumor cells in xenograft mice, and we show that this is mediated by targeting PIP5K1α-associated PI3K/AKT and the downstream survival, proliferation, and invasion pathways. Further, siRNA-mediated knockdown of PIP5K1α exerts similar effects on PC3 cells as ISA-2011B treatment, significantly inhibiting AKT activity, increasing apoptosis and reducing invasion. Thus, PIP5K1α has high potential as a drug target, and compound ISA-2011B is interesting for further development of targeted cancer therapy. PMID:25071204

  15. Lysine-Specific Demethylase 1 (LSD1) Inhibitor S2101 Induces Autophagy via the AKT/mTOR Pathway in SKOV3 Ovarian Cancer Cells

    PubMed Central

    Feng, Shujun; Jin, Ye; Cui, Mengjiao; Zheng, Jianhua

    2016-01-01

    Background S2101 is one of the most potent LSD1 inhibitors, which can inhibit ovarian cancer cells viability. This study aimed to detect the mechanism behind the anticancer properties of S2101 in SKOV3 ovarian cells. Material/Methods Cell viability was tested by Cell Counting Kit-8 (CCK-8) assay. Cellular apoptosis and autophagy were evaluated by flow cytometric analysis using Annexin-V/PI staining methods and Green fluorescent protein (GFP)-fused-LC3 (GFP-LC3), respectively. Western blotting was performed for analyzing the Bax, Bcl-2, mTOR, p-mTOR, p62, LC3-I, LC3-II, AKT, and p-AKT protein expression. Results Our results show that the proportion of early apoptotic and late apoptotic cells increased significantly for cells treated with S2101 at a concentration of 100 μM for 48 h. Treatment of S2101 in SKOV3 cells resulted in upregulation of Bax and downregulation of Bcl-2 in a time-dependent manner, indicating that S2101 can induce apoptosis in SKOV3. There was a downward trend in the expression of p62 when the SKOV3cells were treated with 100 μm S2101 for 12 h, 24 h and 48 h. The conversion of LC3-I to LC3-II was increased significantly at 24 h and 48 h. Autophagy was induced by S2101 in SKOV3 cells, evidenced by an increase in punctuate localization of GFP-LC3 and a change in expression of autophagy-related proteins. Conclusions S2101 treatment decreased the levels of phosphorylated AKT and mTOR. S2101 inhibits SKOV3 cells viability and induces apoptosis and autophagy. The AKT/mTOR signaling pathway was found to be affected by S2101. PMID:27914215

  16. Interrogating two schedules of the AKT inhibitor MK-2206 in patients with advanced solid tumors incorporating novel pharmacodynamic and functional imaging biomarkers

    PubMed Central

    Yap, Timothy A.; Yan, Li; Patnaik, Amita; Tunariu, Nina; Biondo, Andrea; Fearen, Ivy; Papadopoulos, Kyriakos P.; Olmos, David; Baird, Richard; Delgado, Liliana; Tetteh, Ernestina; Beckman, Robert A.; Lupinacci, Lisa; Riisnaes, Ruth; Decordova, Shaun; Heaton, Simon P.; Swales, Karen; deSouza, Nandita M; Leach, Martin O.; Garrett, Michelle D.; Sullivan, Daniel M.; de Bono, Johann S.; Tolcher, Anthony W.

    2014-01-01

    Purpose Multiple cancers harbor genetic aberrations that impact AKT signaling. MK-2206 is a potent pan-AKT inhibitor with a maximum tolerated dose (MTD) previously established at 60mg on alternate days (QOD). Due to a long half-life (60-80h), a weekly (QW) MK-2206 schedule was pursued to compare intermittent QW and continuous QOD dosing. Experimental Design Patients with advanced cancers were enrolled onto a QW dose-escalation phase I study to investigate the safety and pharmacokinetic-pharmacodynamic profiles of tumor and platelet-rich plasma (PRP). The QOD MTD of MK-2206 was also assessed in patients with ovarian and castration-resistant prostate cancers, and patients with advanced cancers undergoing multiparametric functional magnetic resonance imaging (MRI) studies, including dynamic contrast-enhanced MRI, diffusion-weighted imaging, magnetic resonance spectroscopy and intrinsic susceptibility-weighted MRI. Results Seventy-one patients were enrolled; 38 patients had 60mg MK-2206 QOD, while 33 received MK-2206 at 90mg, 135mg, 150mg, 200mg, 250mg, and 300mg QW. The QW MK-2206 MTD was established at 200mg following dose-limiting rash at 250mg and 300mg. QW dosing appeared to be similarly tolerated to QOD, with toxicities including rash, gastrointestinal symptoms, fatigue, and hyperglycemia. Significant AKT pathway blockade was observed with both continuous QOD and intermittent QW dosing of MK-2206 in serially-obtained tumor and PRP specimens. The functional imaging studies demonstrated that complex multiparametric MRI protocols may be effectively implemented in a phase I trial. Conclusions MK-2206 safely results in significant AKT pathway blockade in QOD and QW schedules. The intermittent dose of 200mg QW is currently used in phase II MK-2206 monotherapy and combination studies. PMID:25239610

  17. Protective effect of carnosic acid against paraquat-induced redox impairment and mitochondrial dysfunction in SH-SY5Y cells: Role for PI3K/Akt/Nrf2 pathway.

    PubMed

    de Oliveira, Marcos Roberto; Ferreira, Gustavo Costa; Schuck, Patrícia Fernanda

    2016-04-01

    Carnosic acid (CA) is a phenolic diterpene isolated from Rosmarinus officinalis and exerts anti-inflammatory, antioxidant, and anticarcinogenic activities in different cell types. It has been reported that CA is able to cause protective effects on experimental models of neurodegeneration. However, the exact mechanism by which CA prevents neuronal degeneration remains to be better studied. We investigated here whether there is a role for CA as a neuroprotective agent in a paraquat (PQ) model of Parkinson's disease (PD) regarding cellular and mitochondrial-related redox parameters. SH-SY5Y cells were treated with CA for 12h and were exposed to 100 μM PQ for 24h. It was found that CA at different concentrations prevented the effects of PQ on cell viability and redox parameters. CA alleviated reactive oxygen and nitrogen species production elicited by PQ, as well as decreased the toxic effect on mitochondrial function. Inhibition of Pi3K/Akt pathway with LY294002 or silencing of Nrf2 expression partially blocked the reversal of redox impairment induced by CA. Therefore, CA activated Nrf2 through modulation of PI3K/Akt pathway resulting in increased levels of antioxidant enzymes and consequent neuroprotection. Thus, CA may be viewed as a potential neuroprotective agent to be used in cases of Parkinson's disease (PD).

  18. A novel PKB/Akt inhibitor, MK-2206, effectively inhibits insulin-stimulated glucose metabolism and protein synthesis in isolated rat skeletal muscle.

    PubMed

    Lai, Yu-Chiang; Liu, Yang; Jacobs, Roxane; Rider, Mark H

    2012-10-01

    PKB (protein kinase B), also known as Akt, is a key component of insulin signalling. Defects in PKB activation lead to insulin resistance and metabolic disorders, whereas PKB overactivation has been linked to tumour growth. Small-molecule PKB inhibitors have thus been developed for cancer treatment, but also represent useful tools to probe the roles of PKB in insulin action. In the present study, we examined the acute effects of two allosteric PKB inhibitors, MK-2206 and Akti 1/2 (Akti) on PKB signalling in incubated rat soleus muscles. We also assessed the effects of the compounds on insulin-stimulated glucose uptake, glycogen and protein synthesis. MK-2206 dose-dependently inhibited insulin-stimulated PKB phosphorylation, PKBβ activity and phosphorylation of PKB downstream targets (including glycogen synthase kinase-3α/β, proline-rich Akt substrate of 40 kDa and Akt substrate of 160 kDa). Insulin-stimulated glucose uptake, glycogen synthesis and glycogen synthase activity were also decreased by MK-2206 in a dose-dependent manner. Incubation with high doses of MK-2206 (10 μM) inhibited insulin-induced p70 ribosomal protein S6 kinase and 4E-BP1 (eukaryotic initiation factor 4E-binding protein-1) phosphorylation associated with increased eEF2 (eukaryotic elongation factor 2) phosphorylation. In contrast, Akti only modestly inhibited insulin-induced PKB and mTOR (mammalian target of rapamycin) signalling, with little or no effect on glucose uptake and protein synthesis. MK-2206, rather than Akti, would thus be the tool of choice for studying the role of PKB in insulin action in skeletal muscle. The results point to a key role for PKB in mediating insulin-stimulated glucose uptake, glycogen synthesis and protein synthesis in skeletal muscle.

  19. Dietary flavonoid fisetin: A novel dual inhibitor of PI3K/Akt and mTOR for prostate cancer management

    PubMed Central

    Adhami, Vaqar Mustafa; Syed, Deeba; Khan, Naghma; Mukhtar, Hasan

    2013-01-01

    Epidemiologic and case control population based studies over the past few decades have identified diet as an important determinant of cancer risk. This evidence has kindled an interest into research on bioactive food components and has till date resulted in the identification of many compounds with cancer preventive and therapeutic potential. Among such compounds has been fisetin (3,7,3’,4’-tetrahydroxyflavone), a flavonol and a member of the flavonoid polyphenols that also include quercetin, myricetin and kaempferol. Fisetin is commonly found in many fruits and vegetables such as apples, persimmons, grapes, kiwis, strawberries, onions and cucumbers. We evaluated the effects of fisetin against melanoma and cancers of the prostate, pancreas and the lungs. Using prostate and lung adenocarcinoma cells, we observed that fisetin acts as a dual inhibitor of the PI3K/Akt and the mTOR pathways. This is a significant finding considering the fact that mTOR is phosphorylated and its activation is more frequent in tumors with overexpression of PI3K/Akt. Dual inhibitors of PI3K/Akt and mTOR signaling have been suggested as valuable agents for treating such cancers. Here, we summarize our findings on the dietary flavonoid fisetin and its effects on cancer with particular focus on prostate cancer. Our observations and findings from other laboratories suggest that fisetin could be a useful chemotherapeutic agent that could be used either alone or as an adjuvant with conventional chemotherapeutic drugs for the management of prostate and other cancers. PMID:22842629

  20. IL-7 splicing variant IL-7{delta}5 induces human breast cancer cell proliferation via activation of PI3K/Akt pathway

    SciTech Connect

    Pan, Deshun; Liu, Bing; Jin, Xiaobao; Zhu, Jiayong

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer This study confirms the role of IL-7{delta}5 in breast cancer cell proliferation. Black-Right-Pointing-Pointer IL-7{delta}5 promotes breast cancer cell proliferation and cell cycle progression. Black-Right-Pointing-Pointer IL-7{delta}5 promotes cell proliferation via activation of PI3K/Akt pathway. -- Abstract: Various tumor cells express interleukin 7 (IL-7) and IL-7 variants. IL-7 has been confirmed to stimulate solid tumor cell proliferation. However, the effect of IL-7 variants on tumor cell proliferation remains unclear. In this study, we evaluated the role of IL-7{delta}5 (an IL-7 variant lacking exon 5) on proliferation and cell cycle progression of human MDA-MB-231 and MCF-7 breast cancer cells. The results showed that IL-7{delta}5 promoted cell proliferation and cell cycle progression from G1 phase to G2/M phase, associated with upregulation of cyclin D1 expression and the downregulation of p27{sup kip1} expression. Mechanistically, we found that IL-7{delta}5 induced the activation of Akt. Inhibition of PI3K/Akt pathway by LY294002 reversed the proliferation and cell cycle progression of MDA-MB-231 and MCF-7 cells induced by IL-7{delta}5. In conclusion, our findings demonstrate that IL-7{delta}5 variant induces human breast cancer cell proliferation and cell cycle progression via activation of PI3K/Akt pathway. Thus, IL-7{delta}5 may be a potential target for human breast cancer therapeutics intervention.

  1. Modulatory effect of curcumin on survival of irradiated human intestinal microvascular endothelial cells: role of Akt/mTOR and NF-κB

    PubMed Central

    Binion, David G.; Wellner, Michael; Behmaram, Behnaz; Floer, Martin; Mitton, Elizabeth; Nie, Linghui; Zhang, Zhihong; Otterson, Mary F.

    2010-01-01

    Radiation therapy is an essential modality in the treatment of colorectal cancers. Radiation exerts an antiangiogenic effect on tumors, inhibiting endothelial proliferation and survival in the tumor microvasculature. However, damage from low levels of irradiation can induce a paradoxical effect, stimulating survival in endothelial cells. We used human intestinal microvascular endothelial cells (HIMEC) to define effects of radiation on these gut-specific endothelial cells. Low-level irradiation (1–5 Gy) activates NF-κB and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is involved in cell cycle reentry and cell survival in HIMEC. A downstream target of PI3K/Akt is mammalian target of rapamycin (mTOR), which contributes to endothelial proliferation and angiogenesis. The aim of this study was to investigate the signaling molecules involved in the radiosensitizing effects of curcumin on HIMEC subjected to low levels of irradiation. We have demonstrated that exposure of HIMEC to low levels of irradiation induced Akt and mTOR phosphorylation, which was attenuated by curcumin, rapamycin, LY294002, and mTOR small interference RNA (siRNA). Activation of NF-κB by low levels of irradiation was inhibited by curcumin, SN-50, and mTOR siRNA. Curcumin also induced apoptosis by induction of caspase-3 cleavage in irradiated HIMEC. In conclusion, curcumin significantly inhibited NF-κB and attenuated the effect of irradiation-induced prosurvival signaling through the PI3K/Akt/mTOR and NF-κB pathways in these gut-specific endothelial cells. Curcumin may be a potential radiosensitizing agent for enhanced antiangiogenic effect in colorectal cancer radiation therapy. PMID:20299603

  2. CD147 modulates androgen receptor activity through the Akt/Gsk-3β/β-catenin/AR pathway in prostate cancer cells.

    PubMed

    Fang, Fang; Qin, Yingxin; Hao, Feng; Li, Qiang; Zhang, Wei; Zhao, Chen; Chen, Shuang; Zhao, Liangzhong; Wang, Liguo; Cai, Jianhui

    2016-08-01

    The androgen signaling pathway serves an important role in the development of prostate cancer. β-Catenin is an androgen receptor (AR) cofactor and augments AR signaling. Glycogen synthase kinase-3β (GSK-3β), a target of phosphorylated serine/threonine protein kinase B (p-Akt), regulates β-catenin stability. In addition, β-catenin, a coregulator of AR, physically interacts with AR and enhances AR-mediated target gene transcription. The multifunctional glycoprotein cluster of differentiation (CD) 147 is highly expressed on the cell surface of the majority of cancer cells, and it promotes tumor invasion, metastasis and growth. In the present study, the molecular effects of CD147 on the Akt/GSK-3β/β-catenin/AR signaling network were investigated in LNCaP cells. Using short hairpin-mediated RNA knockdown of CD147 in LNCaP cells, it was demonstrated that downregulation of CD147 resulted in inhibitory phosphorylation of GSK-3β, and then promoted degeneration of β-catenin and reduced nuclear accumulation of β-catenin. In addition, immunoprecipitation studies demonstrated that CD147 downregulation decreased the formation of a complex between β-catenin and AR. It was shown that CD147 knockdown suppressed the expression of the AR target gene prostate-specific antigen and the growth of AR-positive LNCaP cells. Furthermore, inhibition of PI3K/Akt with LY294002 augmented CD147-mediated function. The present study indicates that the PI3K/Akt pathway may facilitate CD147-mediated activation of the AR pathway.

  3. CD147 modulates androgen receptor activity through the Akt/Gsk-3β/β-catenin/AR pathway in prostate cancer cells

    PubMed Central

    Fang, Fang; Qin, Yingxin; Hao, Feng; Li, Qiang; Zhang, Wei; Zhao, Chen; Chen, Shuang; Zhao, Liangzhong; Wang, Liguo; Cai, Jianhui

    2016-01-01

    The androgen signaling pathway serves an important role in the development of prostate cancer. β-Catenin is an androgen receptor (AR) cofactor and augments AR signaling. Glycogen synthase kinase-3β (GSK-3β), a target of phosphorylated serine/threonine protein kinase B (p-Akt), regulates β-catenin stability. In addition, β-catenin, a coregulator of AR, physically interacts with AR and enhances AR-mediated target gene transcription. The multifunctional glycoprotein cluster of differentiation (CD) 147 is highly expressed on the cell surface of the majority of cancer cells, and it promotes tumor invasion, metastasis and growth. In the present study, the molecular effects of CD147 on the Akt/GSK-3β/β-catenin/AR signaling network were investigated in LNCaP cells. Using short hairpin-mediated RNA knockdown of CD147 in LNCaP cells, it was demonstrated that downregulation of CD147 resulted in inhibitory phosphorylation of GSK-3β, and then promoted degeneration of β-catenin and reduced nuclear accumulation of β-catenin. In addition, immunoprecipitation studies demonstrated that CD147 downregulation decreased the formation of a complex between β-catenin and AR. It was shown that CD147 knockdown suppressed the expression of the AR target gene prostate-specific antigen and the growth of AR-positive LNCaP cells. Furthermore, inhibition of PI3K/Akt with LY294002 augmented CD147-mediated function. The present study indicates that the PI3K/Akt pathway may facilitate CD147-mediated activation of the AR pathway. PMID:27446405

  4. Hemiasterlin derivative (R)(S)(S)-BF65 and Akt inhibitor MK-2206 synergistically inhibit SKOV3 ovarian cancer cell growth.

    PubMed

    Lai, Wei-Ting; Cheng, Kai-Lin; Baruchello, Riccardo; Rondanin, Riccardo; Marchetti, Paolo; Simoni, Daniele; Lee, Ray M; Guh, Jih-Hwa; Hsu, Lih-Ching

    2016-08-01

    We reported previously that a hemiasterlin derivative BF65 is a potent anticancer agent that can inhibit microtubule assembly. Here we show that a more potent stereospecific diastereomer (R)(S)(S)-BF65 can synergize with an allosteric Akt inhibitor MK-2206 to suppress the growth of SKOV3 ovarian cancer cells with constitutively active Akt. (R)(S)(S)-BF65 induced mitotic arrest and MK-2206 caused G0/G1 arrest, while the combination of both induced simultaneous G0/G1 and G2/M cell cycle arrest. (R)(S)(S)-BF65 induced phosphorylation and inactivation of Bcl-2, and downregulated Mcl-1, consequently may lead to apoptosis. (R)(S)(S)-BF65 inhibited mitogen-activated protein kinases (MAPKs), which may stimulate cell proliferation upon activation. (R)(S)(S)-BF65 also induced DNA damage after long-term treatment. MK-2206 is known to inhibit phosphorylation and activation of Akt and suppress cancer cell growth. The combination of (R)(S)(S)-BF65 and MK-2206 also inhibited the Akt pathway. Interestingly, MK-2206 upregulated Bcl-2 and induced activation of MAPKs in SKOV3 cells; however, when combined with (R)(S)(S)-BF65, these prosurvival effects were reversed. The combination also more significantly decreased Mcl-1 protein, increased PARP cleavage, and induced γ-H2AX, a DNA damage marker. Remarkably, MK-2206 enhanced the microtubule depolymerization effect of (R)(S)(S)-BF65. The combination of (R)(S)(S)-BF65 and MK-2206 also markedly inhibited cell migration. Thus, MK-2206 synergizes with (R)(S)(S)-BF65 to inhibit SKOV3 cell growth via downregulating the Akt signaling pathway, and enhancing the microtubule disruption effect of (R)(S)(S)-BF65. (R)(S)(S)-BF65 in turn suppresses Bcl-2 and MAPKs induced by MK-2206. (R)(S)(S)-BF65 and MK-2206 compensate each other leading to increased apoptosis and enhanced cytotoxicity, and may also suppress cancer cell invasion.

  5. Induction of granzyme B expression in T-cell receptor/CD28-stimulated human regulatory T cells is suppressed by inhibitors of the PI3K-mTOR pathway

    PubMed Central

    2009-01-01

    Background Regulatory T cells (Tregs) can employ a cell contact- and granzyme B-dependent mechanism to mediate suppression of bystander T and B cells. Murine studies indicate that granzyme B is involved in the Treg-mediated suppression of anti-tumor immunity in the tumor microenvironment and in the Treg-mediated maintenance of allograft survival. In spite of its central importance, a detailed study of granzyme B expression patterns in human Tregs has not been performed. Results Our data demonstrated that natural Tregs freshly isolated from the peripheral blood of normal adults lacked granzyme B expression. Tregs subjected to prolonged TCR and CD28 triggering, in the presence of IL-2, expressed high levels of granzyme B but CD3 stimulation alone or IL-2 treatment alone failed to induce granzyme B. Treatment of Tregs with the mammalian target of rapamycin (mTOR) inhibitor, rapamycin or the PI3 kinase (PI3K) inhibitor LY294002 markedly suppressed granzyme B expression. However, neither rapamycin, as previously reported by others, nor LY294002 inhibited Treg proliferation or induced significant cell death in TCR/CD28/IL-2 stimulated cells. The proliferation rate of Tregs was markedly higher than that of CD4+ conventional T cells in the setting of rapamycin treatment. Tregs expanded by CD3/CD28/IL-2 stimulation without rapamycin demonstrated increased in vitro cytotoxic activity compared to Tregs expanded in the presence of rapamycin in both short term (6 hours) and long term (48 hours) cytotoxicity assays. Conclusion TCR/CD28 mediated activation of the PI3K-mTOR pathway is important for granyzme B expression but not proliferation in regulatory T cells. These findings may indicate that suppressive mechanisms other than granzyme B are utilized by rapamycin-expanded Tregs. PMID:19930596

  6. Bortezomib induces apoptosis and growth suppression in human medulloblastoma cells, associated with inhibition of AKT and NF-ĸB signaling, and synergizes with an ERK inhibitor.

    PubMed

    Yang, Fan; Jove, Veronica; Chang, Shirley; Hedvat, Michael; Liu, Lucy; Buettner, Ralf; Tian, Yan; Scuto, Anna; Wen, Wei; Yip, M L Richard; Van Meter, Timothy; Yen, Yun; Jove, Richard

    2012-04-01

    Medulloblastoma is the most common brain tumor in children. Here, we report that bortezomib, a proteasome inhibitor, induced apoptosis and inhibited cell proliferation in two established cell lines and a primary culture of human medulloblastomas. Bortezomib increased the release of cytochrome c to cytosol and activated caspase-9 and caspase-3, resulting in cleavage of PARP. Caspase inhibitor (Z-VAD-FMK) could rescue medulloblastoma cells from the cytotoxicity of bortezomib. Phosphorylation of AKT and its upstream regulator mTOR were reduced by bortezomib treatment in medulloblastoma cells. Bortezomib increased the expression of Bad and Bak, pro-apoptotic proteins, and p21Cip1 and p27Kip1, negative regulators of cell cycle progression, which are associated with the growth suppression and induction of apoptosis in these tumor cells. Bortezomib also increased the accumulation of phosphorylated IĸBα, and decreased nuclear translocation of NF-ĸB. Thus, NF-ĸB signaling and activation of its downstream targets are suppressed. Moreover, ERK inhibitors or downregulating ERK with ERK siRNA synergized with bortezomib on anticancer effects in medulloblastoma cells. Bortezomib also inhibited the growth of human medulloblastoma cells in a mouse xenograft model. These findings suggest that proteasome inhibitors are potentially promising drugs for treatment of pediatric medulloblastomas.

  7. A 2D-QSAR and Grid-Independent Molecular Descriptor (GRIND) Analysis of Quinoline-Type Inhibitors of Akt2: Exploration of the Binding Mode in the Pleckstrin Homology (PH) Domain

    PubMed Central

    Akhtar, Noreen; Jabeen, Ishrat

    2016-01-01

    Protein kinase B-β (PKBβ/Akt2) is a serine/threonine-specific protein kinase that has emerged as one of the most important regulators of cell growth, differentiation, and division. Upregulation of Akt2 in various human carcinomas, including ovarian, breast, and pancreatic, is a well-known tumorigenesis phenomenon. Early on, the concept of the simultaneous administration of anticancer drugs with inhibitors of Akt2 was advocated to overcome cell proliferation in the chemotherapeutic treatment of cancer. However, clinical studies have not lived up to the high expectations, and several phase II and phase III clinical studies have been terminated prematurely because of severe side effects related to the non-selective isomeric inhibition of Akt2. The notion that the sequence identity of pleckstrin homology (PH) domains within Akt-isoforms is less than 30% might indicate the possibility of the development of selective antagonists against the Akt2 PH domain. Therefore, in this study, various in silico tools were utilized to explore the hypothesis that quinoline-type inhibitors bind in the Akt2 PH domain. A Grid-Independent Molecular Descriptor (GRIND) analysis indicated that two hydrogen bond acceptors, two hydrogen bond donors and one hydrophobic feature at a certain distance from each other were important for the selective inhibition of Akt2. Our docking results delineated the importance of Lys30 as an anchor point for mapping the distances of important amino acid residues in the binding pocket, including Lys14, Glu17, Arg25, Asn53, Asn54 and Arg86. The binding regions identified complement the GRIND-based pharmacophoric features. PMID:28036396

  8. Tumor necrosis factor receptor 2 promotes growth of colorectal cancer via the PI3K/AKT signaling pathway

    PubMed Central

    Zhao, Tao; Li, Huihui; Liu, Zifeng

    2017-01-01

    Tumor necrosis factor receptor 2 (TNFR2) is the receptor for tumor necrosis factor α (TNF-α). TNFR2 differs from tumor necrosis factor 1 (TNFR1) in various ways and is mainly expressed in hematopoietic and endothelial cells. However, studies about its functions in tumors are limited. The contributions of TNFR2 in colorectal cancer (CRC) remain unknown. In the present study, it was found that TNFR2 was positively associated with Ki67 expression in CRC tissues using immunohistochemistry (IHC), and western blot analysis found that Ki67 was upregulated by overexpressing TNFR2 in SW1116 cells and inhibited by silencing TNFR2 in HT29 cells. Methyl thiazolyl tetrazolium assay found that growth of SW1116 cells overexpressing TNFR2 was significantly increased compared with the control group and that the growth of HT29 cells subsequent to silencing TNFR2 was significantly decreased compared with the control group. Clone formation assay found that more clones were formed in SW1116 cells overexpressing TNFR2 than the control group, and less clones formed in HT29 cells subsequent to silencing TNFR2 than the control group. In addition, western blot analysis found that phosphorylation of protein kinase B (AKT) was activated subsequent to overexpressing TNFR2 in SW1116 cells, and inhibited following silencing of TNFR2 in HT29 cells. Additionally, treatment using LY294002 significantly abrogated the promotion of Ki67 expression, growth and clone formation abilities induced by TNFR2 overexpression in SW1116 cells. All the results suggest that TNFR2 can significantly promote CRC growth via the phosphoinositide 3-kinase/AKT signaling pathway; this provides evidential support for taking TNFR2 as a new target for CRC treatment. PMID:28123565

  9. Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies

    PubMed Central

    Duong, Minh Ngoc; Matera, Eva-Laure; Mathé, Doriane; Evesque, Anne; Valsesia-Wittmann, Sandrine; Clémenceau, Béatrice; Dumontet, Charles

    2015-01-01

    Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated. PMID:25523586

  10. Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies.

    PubMed

    Duong, Minh Ngoc; Matera, Eva-Laure; Mathé, Doriane; Evesque, Anne; Valsesia-Wittmann, Sandrine; Clémenceau, Béatrice; Dumontet, Charles

    2015-01-01

    Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated.

  11. Proton pump inhibitor pantoprazole abrogates adriamycin-resistant gastric cancer cell invasiveness via suppression of Akt/GSK-β/β-catenin signaling and epithelial-mesenchymal transition.

    PubMed

    Zhang, Bin; Yang, Yan; Shi, Xiaoting; Liao, Wanyu; Chen, Min; Cheng, Alfred Sze-Lok; Yan, Hongli; Fang, Cheng; Zhang, Shu; Xu, Guifang; Shen, Shanshan; Huang, Shuling; Chen, Guangxia; Lv, Ying; Ling, Tingsheng; Zhang, Xiaoqi; Wang, Lei; Zhuge, Yuzheng; Zou, Xiaoping

    2015-01-28

    The effect of proton pump inhibitor (PPI) on cancer risk has received much attention recently. In this study, we investigated the mechanism underlying multidrug resistance and the effect of a PPI pantoprazole using an adriamycin-resistant gastric cancer cell model (SGC7901/ADR). Compared with the parental cell line, SGC7901/ADR cells showed reduced proliferation rate, but higher resistance to adriamycin under both anchorage-dependent and -independent conditions. Notably, SGC7901/ADR cells underwent epithelial to mesenchymal transition (EMT) and showed increased migrating and invading capabilities. At molecular level, SGC7901/ADR cells showed strong activation of Wnt/β-catenin signaling pathway compared with parental sensitive cells. Interestingly, we found that a PPI pantoprazole can effectively reverse the aggressiveness and EMT marker expression of SGC7901/ADR cells. Furthermore, pantoprazole treatment resulted in a profound reduction of both total and phosphorylated forms of Akt and GSK-3β, which in turn suppressed the adriamycin-induced Wnt/β-catenin signaling in SGC7901/ADR cells. Taken together, we demonstrate that the aggressive phenotype of adriamycin-resistant SGC7901/ADR cells is mediated by induction of EMT and activation of the canonical Wnt/β-catenin signaling pathway. And for the first time, we show that it is possible to suppress the invasiveness of SGC7901/ADR cells by pantoprazole which targets the EMT and Akt/GSK-3β/β-catenin signaling.

  12. Netrin-1 induces the migration of Schwann cells via p38 MAPK and PI3K-Akt signaling pathway mediated by the UNC5B receptor

    SciTech Connect

    Lv, Jianwei; Sun, Xiaolei; Ma, Jianxiong; Ma, Xinlong; Zhang, Yang; Li, Fengbo; Li, Yanjun; Zhao, Zhihu

    2015-08-14

    Schwann cells (SCs) play an essentially supportive role in the regeneration of injured peripheral nerve system (PNS). As Netrin-1 is crucial for the normal development of nervous system (NS) and can direct the process of damaged PNS regeneration, our study was designed to determine the role of Netrin-1 in RSC96 Schwann cells (an immortalized rat Schwann cell line) proliferation and migration. Our studies demonstrated that Netrin-1 had no effect on RSC96 cells proliferation, while significantly promoted RSC96 cells migration. The Netrin-1-induced RSC96 cells migration was significantly attenuated by inhibition of p38 and PI3K through pretreatment with SB203580 and LY294002 respectively, but not inhibition of MEK1/2 and JNK by U0126-EtOH and SP600125 individually. Treatment with Netrin-1 enhanced the phosphorylation of p38 and Akt. QRT-PCR indicated that Netrin-1 and only its receptors Unc5a, Unc5b and Neogenin were expressed in RSC96 cells, among which Unc5b expressed the most. And UNC5B protein was significantly increased after stimulated by Netrin-1. In conclusion, we show here that Netrin-1-enhanced SCs migration is mediated by activating p38 MAPK and PI3K-Akt signal cascades via receptor UNC5B, which suggests that Netrin-1 could serve as a new therapeutic strategy and has potential application value for PNS regeneration. - Highlights: • Netrin-1 attracts RSC96 Schwann cells migration in a dose dependent manner. • Netrin-1 induced Schwann cells migration is p38 and PI3K-Akt signaling dependent. • UNC5B may be dominant receptor mediating Netrin-1′ effect on RSC96 cells motility. • Netrin-1 may promote peripheral nerve repair by enhancing Schwann cells motility.

  13. Nitric oxide decreases subventricular zone stem cell proliferation by inhibition of epidermal growth factor receptor and phosphoinositide-3-kinase/Akt pathway.

    PubMed

    Torroglosa, Ana; Murillo-Carretero, Maribel; Romero-Grimaldi, Carmen; Matarredona, Esperanza R; Campos-Caro, Antonio; Estrada, Carmen

    2007-01-01

    Nitric oxide (NO) inhibits proliferation of subventricular zone (SVZ) neural precursor cells in adult mice in vivo under physiological conditions. The mechanisms underlying this NO effect have now been investigated using SVZ-derived neural stem cells, which generate neurospheres in vitro when stimulated by epidermal growth factor (EGF). In these cultures, NO donors decreased the number of newly formed neurospheres as well as their size, which indicates that NO was acting on the neurosphere-forming neural stem cells and the daughter neural progenitors. The effect of NO was cytostatic, not proapoptotic, and did not involve cGMP synthesis. Neurosphere cells expressed the neuronal and endothelial isoforms of NO synthase (NOS) and produced NO in culture. Inhibition of NOS activity by N(omega)-nitro-L-arginine methylester (L-NAME) promoted neurosphere formation and growth, thus revealing an autocrine/paracrine action of NO on the neural precursor cells. Both exogenous and endogenous NO impaired the EGF-induced activation of the EGF receptor (EGFR) tyrosine kinase and prevented the EGF-induced Akt phosphorylation in neurosphere cells. Inhibition of the phosphoinositide-3-kinase (PI3-K)/Akt pathway by LY294002 significantly reduced the number of newly formed neurospheres, which indicates that this is an essential pathway for neural stem cell self-renewal. Chronic administration of l-NAME to adult mice enhanced phospho-Akt staining in the SVZ and reduced nuclear p27(Kip1) in the SVZ and olfactory bulb. The inhibition of EGFR and PI3-K pathway by NO explains, at least in part, its antimitotic effect on neurosphere cells and may be a mechanism involved in the physiological role of NO as a negative regulator of SVZ neurogenesis in adult mice.

  14. MLN4924, an NAE inhibitor, suppresses AKT and mTOR signaling via upregulation of REDD1 in human myeloma cells.

    PubMed

    Gu, Yanyan; Kaufman, Jonathan L; Bernal, Leon; Torre, Claire; Matulis, Shannon M; Harvey, R Donald; Chen, Jing; Sun, Shi-Yong; Boise, Lawrence H; Lonial, Sagar

    2014-05-22

    The function and survival of normal and malignant plasma cells depends on the elaborately regulated ubiquitin proteasome system. Proteasome inhibitors such as bortezomib have proved to be highly effective in the treatment of multiple myeloma (MM), and their effects are related to normal protein homeostasis which is critical for plasma cell survival. Many ubiquitin ligases are regulated by conjugation with NEDD8. Therefore, neddylation may also impact survival and proliferation of malignant plasma cells. Here, we show that MLN4924, a potent NEDD8 activating enzyme (NAE) inhibitor, induced cytotoxicity in MM cell lines, and its antitumor effect is associated with suppression of the AKT and mammalian target of rapamycin (mTOR) signaling pathways through increased expression of REDD1. Combining MLN4924 with the proteasome inhibitor bortezomib induces synergistic apoptosis in MM cell lines which can overcome the prosurvival effects of growth factors such as interleukin-6 and insulin-like growth factor-1. Altogether, our findings demonstrate an important function for REDD1 in MLN4924-induced cytotoxicity in MM and also provide a promising therapeutic combination strategy for myeloma.

  15. Computational drugs repositioning identifies inhibitors of oncogenic PI3K/AKT/P70S6K-dependent pathways among FDA-approved compounds

    PubMed Central

    Carrella, Diego; Manni, Isabella; Tumaini, Barbara; Dattilo, Rosanna; Papaccio, Federica; Mutarelli, Margherita; Sirci, Francesco; Amoreo, Carla A.; Mottolese, Marcella; Iezzi, Manuela; Ciolli, Laura; Aria, Valentina; Bosotti, Roberta; Isacchi, Antonella; Loreni, Fabrizio; Bardelli, Alberto; Avvedimento, Vittorio E.; di Bernardo, Diego; Cardone, Luca

    2016-01-01

    The discovery of inhibitors for oncogenic signalling pathways remains a key focus in modern oncology, based on personalized and targeted therapeutics. Computational drug repurposing via the analysis of FDA-approved drug network is becoming a very effective approach to identify therapeutic opportunities in cancer and other human diseases. Given that gene expression signatures can be associated with specific oncogenic mutations, we tested whether a “reverse” oncogene-specific signature might assist in the computational repositioning of inhibitors of oncogenic pathways. As a proof of principle, we focused on oncogenic PI3K-dependent signalling, a molecular pathway frequently driving cancer progression as well as raising resistance to anticancer-targeted therapies. We show that implementation of “reverse” oncogenic PI3K-dependent transcriptional signatures combined with interrogation of drug networks identified inhibitors of PI3K-dependent signalling among FDA-approved compounds. This led to repositioning of Niclosamide (Niclo) and Pyrvinium Pamoate (PP), two anthelmintic drugs, as inhibitors of oncogenic PI3K-dependent signalling. Niclo inhibited phosphorylation of P70S6K, while PP inhibited phosphorylation of AKT and P70S6K, which are downstream targets of PI3K. Anthelmintics inhibited oncogenic PI3K-dependent gene expression and showed a cytostatic effect in vitro and in mouse mammary gland. Lastly, PP inhibited the growth of breast cancer cells harbouring PI3K mutations. Our data indicate that drug repositioning by network analysis of oncogene-specific transcriptional signatures is an efficient strategy for identifying oncogenic pathway inhibitors among FDA-approved compounds. We propose that PP and Niclo should be further investigated as potential therapeutics for the treatment of tumors or diseases carrying the constitutive activation of the PI3K/P70S6K signalling axis. PMID:27542212

  16. The tissue inhibitor of metalloproteinases-1 (TIMP-1) promotes survival and migration of acute myeloid leukemia cells through CD63/PI3K/Akt/p21 signaling.

    PubMed

    Forte, Dorian; Salvestrini, Valentina; Corradi, Giulia; Rossi, Lara; Catani, Lucia; Lemoli, Roberto M; Cavo, Michele; Curti, Antonio

    2017-01-10

    We and others have shown that the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1), a member of the inflammatory network exerting pleiotropic effects in the bone marrow (BM) microenvironment, regulates the survival and proliferation of different cell types, including normal hematopoietic progenitor cells. Moreover, TIMP-1 has been shown to be involved in cancer progression. However, its role in leukemic microenvironment has not been addressed. Here, we investigated the activity of TIMP-1 on Acute Myelogenous Leukemia (AML) cell functions. First, we found that TIMP-1 levels were increased in the BM plasma of AML patients at diagnosis. In vitro, recombinant human (rh)TIMP-1 promoted the survival and cell cycle S-phase entry of AML cells. These kinetic effects were related to the downregulation of cyclin-dependent kinase inhibitor p21. rhTIMP-1 increases CXCL12-driven migration of leukemic cells through PI3K signaling. Interestingly, activation of CD63 receptor was required for TIMP-1's cytokine/chemokine activity. Of note, rhTIMP-1 stimulation modulated mRNA expression of Hypoxia Inducible Factor (HIF)-1α, downstream of PI3K/Akt activation. We then co-cultured AML cells with normal or leukemic mesenchymal stromal cells (MSCs) to investigate the interaction of TIMP-1 with cellular component(s) of BM microenvironment. Our results showed that the proliferation and migration of leukemic cells were greatly enhanced by rhTIMP-1 in presence of AML-MSCs as compared to normal MSCs. Thus, we demonstrated that TIMP-1 modulates leukemic blasts survival, migration and function via CD63/PI3K/Akt/p21 signaling. As a "bad actor" in a "bad soil", we propose TIMP-1 as a potential novel therapeutic target in leukemic BM microenvironment.

  17. ZSTK474, a novel phosphatidylinositol 3-kinase inhibitor identified using the JFCR39 drug discovery system.

    PubMed

    Kong, De-xin; Yamori, Takao

    2010-09-01

    JFCR39 is an informatic anticancer drug discovery system that utilizes a panel of 39 human cancer cells coupled with a drug-activity database. This system not only provides disease-oriented information but can also predict the mechanism of action of a given antitumor agent. Development of a phosphatidylinositol 3-kinase (PI3K) inhibitor as an anticancer drug candidate has attracted a great deal of attention from both academia and industry because PI3K is known to be closely involved in carcinogenesis. ZSTK474 was identified as a PI3K inhibitor using JFCR39 system in combination with COMPARE analysis program. These findings were based on the similar fingerprint (growth inhibition profiles for JFCR39 human cancer cell line panel) with that of a classical PI3K inhibitor LY294002. Biochemical experiments confirmed ZSTK474 to be a potent pan-class I PI3K inhibitor, with high selectivity over other classes of PI3K and protein kinases. We previously reported the in vitro and in vivo antitumor efficacy of ZSTK474, together with the G(0)/G(1) arrest and antiangiogenic activity. Here, we review the JFCR39 system and summarize recent studies on PI3K biology and the development of PI3K inhibitors before discussing ZSTK474 in some detail.

  18. Alterations in microRNA expression profile in HCV-infected hepatoma cells: Involvement of miR-491 in regulation of HCV replication via the PI3 kinase/Akt pathway

    SciTech Connect

    Ishida, Hisashi; Tatsumi, Tomohide; Hosui, Atsushi; Nawa, Takatoshi; Kodama, Takahiro; Shimizu, Satoshi; Hikita, Hayato; Hiramatsu, Naoki; Kanto, Tatsuya; Hayashi, Norio; Takehara, Tetsuo

    2011-08-19

    Highlights: {yields} HCV infection upregulated miR-192, -194, -215, downregulated miR-320, -491. {yields} Transfection of miR-192, -215, and -491 enhanced HCV replication. {yields} Transfection of miR-491 inhibited Akt phosphorylation. {yields} Akt inhibition could be responsible for augmentation of HCV replication by miR-491. -- Abstract: The aim of this study was to investigate the role of microRNA (miRNA) on hepatitis C virus (HCV) replication in hepatoma cells. Using miRNA array analysis, miR-192/miR-215, miR-194, miR-320, and miR-491 were identified as miRNAs whose expression levels were altered by HCV infection. Among them, miR-192/miR-215 and miR-491 were capable of enhancing replication of the HCV replicon as well as HCV itself. HCV IRES activity or cell proliferation was not increased by forced expression of miR-192/miR-215 or miR-491. Investigation of signaling pathways revealed that miR-491 specifically suppressed the phosphoinositol-3 (PI3) kinase/Akt pathway. Under inhibition of PI3 kinase by LY294002, the suppressive effect of miR-491 on HCV replication was abolished, indicating that suppression of HCV replication by miR-491 was dependent on the PI3 kinase/Akt pathway. miRNAs altered by HCV infection would then affect HCV replication, which implies a complicated mechanism for regulating HCV replication. HCV-induced miRNA may be involved in changes in cellular properties including hepatocarcinogenesis.

  19. Invention of a novel photodynamic therapy for tumors using a photosensitizing PI3K inhibitor.

    PubMed

    Hayashida, Yushi; Ikeda, Yuka; Sawada, Koichi; Kawai, Katsuhisa; Kato, Takuma; Kakehi, Yoshiyuki; Araki, Nobukazu

    2016-08-01

    XL147 (SAR245408, pilaralisib), an ATP-competitive pan-class I phosphoinositide 3-kinase (PI3K) inhibitor, is a promising new anticancer drug. We examined the effect of the PI3K inhibitor on PC3 prostate cancer cells under a fluorescence microscope and found that XL147-treated cancer cells are rapidly injured by blue wavelength (430 nm) light irradiation. During the irradiation, the cancer cells treated with 0.2-2 μM XL147 showed cell surface blebbing and cytoplasmic vacuolation and died within 15 min. The extent of cell injury/death was dependent on the dose of XL147 and the light power of the irradiation. These findings suggest that XL147 might act as a photosensitizing reagent in photodynamic therapy (PDT) for cancer. Moreover, the cytotoxic effect of photosensitized XL147 was reduced by pretreatment with other ATP-competitive PI3K inhibitors such as LY294002, suggesting that the cytoto