Antitumor activity in RAS-driven tumors by blocking AKT and MEK.

PubMed

Tolcher, Anthony W; Khan, Khurum; Ong, Michael; Banerji, Udai; Papadimitrakopoulou, Vassiliki; Gandara, David R; Patnaik, Amita; Baird, Richard D; Olmos, David; Garrett, Christopher R; Skolnik, Jeffrey M; Rubin, Eric H; Smith, Paul D; Huang, Pearl; Learoyd, Maria; Shannon, Keith A; Morosky, Anne; Tetteh, Ernestina; Jou, Ying-Ming; Papadopoulos, Kyriakos P; Moreno, Victor; Kaiser, Brianne; Yap, Timothy A; Yan, Li; de Bono, Johann S

2015-02-15

KRAS is the most commonly mutated oncogene in human tumors. KRAS-mutant cells may exhibit resistance to the allosteric MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) and allosteric AKT inhibitors (such as MK-2206), the combination of which may overcome resistance to both monotherapies. We conducted a dose/schedule-finding study evaluating MK-2206 and selumetinib in patients with advanced treatment-refractory solid tumors. Recommended dosing schedules were defined as MK-2206 at 135 mg weekly and selumetinib at 100 mg once daily. Grade 3 rash was the most common dose-limiting toxicity (DLT); other DLTs included grade 4 lipase increase, grade 3 stomatitis, diarrhea, and fatigue, and grade 3 and grade 2 retinal pigment epithelium detachment. There were no meaningful pharmacokinetic drug-drug interactions. Clinical antitumor activity included RECIST 1.0-confirmed partial responses in non-small cell lung cancer and low-grade ovarian carcinoma. Responses in KRAS-mutant cancers were generally durable. Clinical cotargeting of MEK and AKT signaling may be an important therapeutic strategy in KRAS-driven human malignancies (Trial NCT number NCT01021748). ©2014 American Association for Cancer Research.

FLT3-ITD confers resistance to the PI3K/Akt pathway inhibitors by protecting the mTOR/4EBP1/Mcl-1 pathway through STAT5 activation in acute myeloid leukemia

PubMed Central

Nogami, Ayako; Oshikawa, Gaku; Okada, Keigo; Fukutake, Shusaku; Umezawa, Yoshihiro; Nagao, Toshikage; Kurosu, Tetsuya; Miura, Osamu

2015-01-01

FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in acute myeloid leukemia (AML), with the former associated with poor prognosis. Here, we show that the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-2206 induced apoptosis through the mitochondria-mediated intrinsic pathway more efficiently in hematopoietic 32D cells driven by FLT3-TKD (32D/TKD) than FLT3-ITD (32D/ITD), which robustly activated STAT5. The resistance to GDC-0941 and MK-2206 was gained by expression of the constitutively activated STAT5 mutant STAT5A1*6 in 32D/TKD cells, while it was abrogated by the STAT5 inhibitor pimozide in 32D/ITD cells or FLT3-ITD-expressing human leukemic MV4–11 cells. GDC-0941 or MK-2206 induced dephosphorylation of 4EBP1 more conspicuously in 32D/TKD than in 32D/ITD, which was prevented or augmented by STAT5A1*6 or pimozide, respectively, and correlated with downregulation of the eIF4E/eIF4G complex formation and Mcl-1 expression. Furthermore, exogenous expression of Mcl-1 endowed resistance to GDC-0941 and MK-2206 on 32D/TKD cells. Finally, it was confirmed in primary AML cells with FLT3-ITD that pimozide enhanced 4EBP1 dephosphorylation and Mcl-1 downregulation to augment cytotoxicity of GDC-0941. These data suggest that the robust STAT5 activation by FLT3-ITD protects cells treated with the PI3K/Akt pathway inhibitors from apoptosis by maintaining Mcl-1 expression through the mTORC1/4EBP1/eIF4E pathway. PMID:25826077

FLT3-ITD confers resistance to the PI3K/Akt pathway inhibitors by protecting the mTOR/4EBP1/Mcl-1 pathway through STAT5 activation in acute myeloid leukemia.

PubMed

Nogami, Ayako; Oshikawa, Gaku; Okada, Keigo; Fukutake, Shusaku; Umezawa, Yoshihiro; Nagao, Toshikage; Kurosu, Tetsuya; Miura, Osamu

2015-04-20

FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in acute myeloid leukemia (AML), with the former associated with poor prognosis. Here, we show that the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-2206 induced apoptosis through the mitochondria-mediated intrinsic pathway more efficiently in hematopoietic 32D cells driven by FLT3-TKD (32D/TKD) than FLT3-ITD (32D/ITD), which robustly activated STAT5. The resistance to GDC-0941 and MK-2206 was gained by expression of the constitutively activated STAT5 mutant STAT5A1*6 in 32D/TKD cells, while it was abrogated by the STAT5 inhibitor pimozide in 32D/ITD cells or FLT3-ITD-expressing human leukemic MV4-11 cells. GDC-0941 or MK-2206 induced dephosphorylation of 4EBP1 more conspicuously in 32D/TKD than in 32D/ITD, which was prevented or augmented by STAT5A1*6 or pimozide, respectively, and correlated with downregulation of the eIF4E/eIF4G complex formation and Mcl-1 expression. Furthermore, exogenous expression of Mcl-1 endowed resistance to GDC-0941 and MK-2206 on 32D/TKD cells. Finally, it was confirmed in primary AML cells with FLT3-ITD that pimozide enhanced 4EBP1 dephosphorylation and Mcl-1 downregulation to augment cytotoxicity of GDC-0941. These data suggest that the robust STAT5 activation by FLT3-ITD protects cells treated with the PI3K/Akt pathway inhibitors from apoptosis by maintaining Mcl-1 expression through the mTORC1/4EBP1/eIF4E pathway.

Anti-tumour activity in RAS-driven tumours by blocking AKT and MEK

PubMed Central

Tolcher, Anthony W.; Khan, Khurum; Ong, Michael; Banerji, Udai; Papadimitrakopoulou, Vassiliki; Gandara, David R.; Patnaik, Amita; Baird, Richard D.; Olmos, David; Garrett, Christopher R.; Skolnik, Jeffrey M.; Rubin, Eric H.; Smith, Paul D.; Huang, Pearl; Learoyd, Maria; Shannon, Keith A.; Morosky, Anne; Tetteh, Ernestina; Jou, Ying-Ming; Papadopoulos, Kyriakos P.; Moreno, Victor; Kaiser, Brianne; Yap, Timothy A.; Yan, Li; de Bono, Johann S.

2014-01-01

Purpose KRAS is the most commonly mutated oncogene in human tumours. KRAS-mutant cells may exhibit resistance to the allosteric MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) and allosteric AKT inhibitors (such as MK-2206), the combination of which may overcome resistance to both monotherapies. Experimental Design We conducted a dose/schedule-finding study evaluating MK-2206 and selumetinib in patients with advanced treatment-refractory solid tumours. Recommended dosing schedules were defined as MK-2206 135 mg weekly and selumetinib 100 mg once-daily. Results Grade 3 rash was the most common dose-limiting toxicity (DLT); other DLTs included grade 4 lipase increase, grade 3 stomatitis, diarrhoea, and fatigue, and grade 3 and grade 2 retinal pigment epithelium detachment. There were no meaningful pharmacokinetic drug-drug interactions. Clinical anti-tumour activity included RECIST 1.0-confirmed partial responses in non-small cell lung cancer and low-grade ovarian carcinoma. Conclusion Responses in KRAS-mutant cancers were generally durable. Clinical co-targeting of MEK and AKT signalling may be an important therapeutic strategy in KRAS-driven human malignancies (Trial NCT number NCT01021748). PMID:25516890

The novel Akt inhibitor API-1 induces c-FLIP degradation and synergizes with TRAIL to augment apoptosis independent of Akt inhibition.

PubMed

Li, Bo; Ren, Hui; Yue, Ping; Chen, Mingwei; Khuri, Fadlo R; Sun, Shi-Yong

2012-04-01

API-1 (pyrido[2,3-d]pyrimidines) is a novel small-molecule inhibitor of Akt, which acts by binding to Akt and preventing its membrane translocation and has promising preclinical antitumor activity. In this study, we reveal a novel function of API-1 in regulation of cellular FLICE-inhibitory protein (c-FLIP) levels and TRAIL-induced apoptosis, independent of Akt inhibition. API-1 effectively induced apoptosis in tested cancer cell lines including activation of caspase-8 and caspase-9. It reduced the levels of c-FLIP without increasing the expression of death receptor 4 (DR4) or DR5. Accordingly, it synergized with TRAIL to induce apoptosis. Enforced expression of ectopic c-FLIP did not attenuate API-1-induced apoptosis but inhibited its ability to enhance TRAIL-induced apoptosis. These data indicate that downregulation of c-FLIP mediates enhancement of TRAIL-induced apoptosis by API-1 but is not sufficient for API-1-induced apoptosis. API-1-induced reduction of c-FLIP could be blocked by the proteasome inhibitor MG132. Moreover, API-1 increased c-FLIP ubiquitination and decreased c-FLIP stability. These data together suggest that API-1 downregulates c-FLIP by facilitating its ubiquitination and proteasome-mediated degradation. Because other Akt inhibitors including API-2 and MK2206 had minimal effects on reducing c-FLIP and enhancement of TRAIL-induced apoptosis, it is likely that API-1 reduces c-FLIP and enhances TRAIL-induced apoptosis independent of its Akt-inhibitory activity. 2012 AACR

The novel Akt inhibitor API-1 induces c-FLIP degradation and synergizes with TRAIL to augment apoptosis independent of Akt inhibition

PubMed Central

Li, Bo; Ren, Hui; Yue, Ping; Chen, Mingwei; Khuri, Fadlo R.; Sun, Shi-Yong

2012-01-01

API-1 is a novel small molecule inhibitor of Akt, which acts by binding to Akt and preventing its membrane translocation, and has promising preclinical antitumor activity. In this study, we reveal a novel function of API-1 in regulation of c-FLIP levels and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, independent of Akt inhibition. API-1 effectively induced apoptosis in tested cancer cell lines including activation of caspase-8 and caspase-9. It reduced the levels of c-FLIP without increasing the expression of DR4 or DR5. Accordingly, it synergized with TRAIL to induce apoptosis. Enforced expression of ectopic c-FLIP did not attenuate API-1-induced apoptosis, but inhibited its ability to enhance TRAIL-induced apoptosis. These data indicate that downregulation of c-FLIP mediates enhancement of TRAIL-induced apoptosis by API-1, but is not sufficient for API-1-induced apoptosis. API-1-induced reduction of c-FLIP could be blocked by the proteasome inhibitor MG132. Moreover, API-1 increased c-FLIP ubiquitination and decreased c-FLIP stability. These data together suggest that API-1 downregulates c-FLIP by facilitating its ubiquitination and proteasome-mediated degradation. Since other Akt inhibitors including API-2 and MK2206 had minimal effects on reducing c-FLIP and enhancement of TRAIL-induced apoptosis, it is likely that API-1 reduces c-FLIP and enhances TRAIL-induced apoptosis independent of its Akt-inhibitory activity. PMID:22345097

Low Phosphorylated AKT Expression in Laryngeal Cancer: Indications for a Higher Metastatic Risk

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nijkamp, Monique M.; Span, Paul N.; Stegeman, Hanneke

2013-10-01

Purpose: To validate the association of phosphorylated (p)AKT with lymph node metastasis in an independent, homogeneous cohort of patients with larynx cancer. Methods and Materials: Seventy-eight patients with laryngeal cancer were included. Epidermal growth factor receptor, pAKT, vimentin, E-cadherin, hypoxia, and blood vessels were visualized in biopsy material using immunohistochemistry. Positive tumor areas and spatial relationships between markers were assessed by automated image analysis. In 6 laryngeal cancer cell lines, E-cadherin and vimentin messenger RNA was quantified by real-time polymerase chain reaction and by immunohistochemistry before and after treatment with the pAKT inhibitor MK-2206. Results: A significant correlation was foundmore » between low pAKT in the primary tumor and positive lymph node status (P=.0005). Tumors with lymph node metastases had an approximately 10-fold lower median pAKT value compared with tumors without lymph node metastases, albeit with large intertumor variations, validating our previous results. After inhibition of pAKT in laryngeal cancer cells with MK-2206, up-regulation of vimentin and a downregulation of E-cadherin occurred, consistent with epithelial–mesenchymal transition. Conclusion: Low pAKT expression in larynx tumors is associated with lymph node metastases. Further, inhibition of pAKT in laryngeal cancer induces epithelial–mesenchymal transition, predisposing for an increased metastatic risk.« less

EZH2 promotes tumor progression via regulating VEGF-A/AKT signaling in non-small cell lung cancer.

PubMed

Geng, Jian; Li, Xiao; Zhou, Zhanmei; Wu, Chin-Lee; Dai, Meng; Bai, Xiaoyan

2015-04-10

Enhancer of Zeste Homologue 2 (EZH2) accounts for aggressiveness and unfavorable prognosis of tumor. We investigated the mechanisms and signaling pathways of EZH2 in non-small cell lung carcinoma (NSCLC) progression. Increased expression of EZH2, vascular endothelial growth factor-A (VEGF-A) and AKT phosphorylation correlated with differentiation, lymph node metastasis, size and TNM stage in NSCLC. There was a positive correlation between EZH2 and VEGF-A expression and high EZH2 expression, as an independent prognostic factor, predicted a shorter overall survival time for NSCLC patients. The expression of VEGF-A and phosphorylated Ser(473)-AKT, cell proliferation, migration and metastasis were enhanced in EZH2-overexpressing A549 cells, but inhibited in parental H2087 cells with EZH2 silencing or GSK126 treatment. AKT activity was enhanced by recombinant human VEGF-165 but suppressed by bevacizumab. An AKT inhibitor MK-2206 blocked VEGF-A expression and AKT phosphorylation in parental H2087 and EZH2-overexpressing A549 cells. EZH2 activity was not affected by either VEGF-A stimulation/depletion or MK-2206 inhibition. These results demonstrate that EZH2 promotes lung cancer progression via the VEGF-A/AKT signaling pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Novel ATP-competitive Akt inhibitor afuresertib suppresses the proliferation of malignant pleural mesothelioma cells.

PubMed

Yamaji, Masayuki; Ota, Akinobu; Wahiduzzaman, Md; Karnan, Sivasundaram; Hyodo, Toshinori; Konishi, Hiroyuki; Tsuzuki, Shinobu; Hosokawa, Yoshitaka; Haniuda, Masayuki

2017-11-01

Malignant pleural mesothelioma (MPM), an asbestos-related occupational disease, is an aggressive and incurable tumor of the thoracic cavity. Despite recent advances in MPM treatment, overall survival of patients with MPM is very low. Recent studies have implicated that PI3K/Akt signaling is involved in MPM cell survival and development. To investigate the effects of Akt inhibitors on MPM cell survival, we examined the effects of nine selective Akt inhibitors, namely, afuresertib, Akti-1/2, AZD5363, GSK690693, ipatasertib, MK-2206, perifosine, PHT-427, and TIC10, on six MPM cell lines, namely, ACC-MESO-4, Y-MESO-8A, MSTO-211H, NCI-H28, NCI-H290, and NCI-H2052, and a normal mesothelial cell line MeT-5A. Comparison of IC 50 values of the Akt inhibitors showed that afuresertib, an ATP-competitive specific Akt inhibitor, exerted tumor-specific effects on MPM cells. Afuresertib significantly increased caspase-3 and caspase-7 activities and apoptotic cell number among ACC-MESO-4 and MSTO-211H cells. Moreover, afuresertib strongly arrested the cell cycle in the G 1 phase. Western blotting analysis showed that afuresertib increased the expression of p21 WAF 1/ CIP 1 and decreased the phosphorylation of Akt substrates, including GSK-3β and FOXO family proteins. These results suggest that afuresertib-induced p21 expression promotes G 1 phase arrest by inducing FOXO activity. Furthermore, afuresertib significantly enhanced cisplatin-induced cytotoxicity. Interestingly, results of gene set enrichment analysis showed that afuresertib modulated the expression E2F1 and MYC, which are associated with fibroblast core serum response. Together, these results suggest that afuresertib is a useful anticancer drug for treating patients with MPM. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Hsp27 regulates Akt activation and polymorphonuclear leukocyte apoptosis by scaffolding MK2 to Akt signal complex.

PubMed

Wu, Rui; Kausar, Hina; Johnson, Paul; Montoya-Durango, Diego E; Merchant, Michael; Rane, Madhavi J

2007-07-27

We have shown previously that Akt exists in a signal complex with p38 MAPK, MAPK-activated protein kinase-2 (MK2), and heat shock protein 27 (Hsp27) and MK2 phosphorylates Akt on Ser-473. Additionally, dissociation of Hsp27 from Akt, prior to Akt activation, induced polymorphonuclear leukocyte (PMN) apoptosis. However, the role of Hsp27 in regulating Akt activation was not examined. This study tested the hypothesis that Hsp27 regulates Akt activation and promotes cell survival by scaffolding MK2 to the Akt signal complex. Here we show that loss of Akt/Hsp27 interaction by anti-Hsp27 antibody treatment resulted in loss of Akt/MK2 interaction, loss of Akt-Ser-473 phosphorylation, and induced PMN apoptosis. Transfection of myristoylated Akt (AktCA) in HK-11 cells induced Akt-Ser-473 phosphorylation, activation, and Hsp27-Ser-82 phosphorylation. Cotransfection of AktCA with Hsp27 short interfering RNA, but not scrambled short interfering RNA, silenced Hsp27 expression, without altering Akt expression in HK-11 cells. Silencing Hsp27 expression inhibited Akt/MK2 interaction, inhibited Akt phosphorylation and Akt activation, and induced HK-11 cell death. Deletion mutagenesis studies identified acidic linker region (amino acids 117-128) on Akt as an Hsp27 binding region. Deletion of amino acids 117-128 on Akt resulted in loss of its interaction with Hsp27 and MK2 but not with Hsp90 as demonstrated by immunoprecipitation and glutathione S-transferase pulldown studies. Co-transfection studies demonstrated that constitutively active MK2 (MK2EE) phosphorylated Aktwt (wild type) on Ser-473 but failed to phosphorylate Akt(Delta117-128) mutant in transfixed cells. These studies collectively define a novel role of Hsp27 in regulating Akt activation and cellular apoptosis by mediating interaction between Akt and its upstream activator MK2.

Inactivation of AKT Induces Cellular Senescence in Uterine Leiomyoma

PubMed Central

Xu, Xiaofei; Lu, Zhenxiao; Qiang, Wenan; Vidimar, Vania; Kong, Beihua

2014-01-01

Uterine leiomyomas (fibroids) are a major public health problem. Current medical treatments with GnRH analogs do not provide long-term benefit. Thus, permanent shrinkage or inhibition of fibroid growth via medical means remains a challenge. The AKT pathway is a major growth and survival pathway for fibroids. We propose that AKT inhibition results in a transient regulation of specific mechanisms that ultimately drive cells into cellular senescence or cell death. In this study, we investigated specific mechanisms of AKT inhibition that resulted in senescence. We observed that administration of MK-2206, an allosteric AKT inhibitor, increased levels of reactive oxygen species, up-regulated the microRNA miR-182 and several senescence-associated genes (including p16, p53, p21, and β-galactosidase), and drove leiomyoma cells into stress-induced premature senescence (SIPS). Moreover, induction of SIPS was mediated by HMGA2, which colocalized to senescence-associated heterochromatin foci. This study provides a conceivable molecular mechanism of SIPS by AKT inhibition in fibroids. PMID:24476133

AML sensitivity to YM155 is modulated through AKT and Mcl-1

PubMed Central

de Necochea-Campion, Rosalia; Diaz Osterman, Carlos J.; Hsu, Heng-Wei; Fan, Junjie; Mirshahidi, Saied; Wall, Nathan R.; Chen, Chien-Shing

2015-01-01

HL60 and U937 (acute myeloid leukemia (AML) cell lines) were assessed for sensitivity to YM155, and found to have distinct sensitive and resistant phenotypes, respectively. In HL60 cells, YM155 inhibition of growth proliferation was due to apoptosis which was measured by annexin V/PI staining. YM155 induced apoptosis through activation of intrinsic and extrinsic pathways that also culminated in caspase-3 activity and PARP cleavage. YM155 sensitivity was partially associated with this compound’s ability to downregulate survivin transcription since this was more pronounced in the HL60 cell line. However, marked differences were also observed in XIAP, Bcl-2, and Mcl-1L, and Mcl-1s. Furthermore, YM155 treatment completely inhibited production of total Akt protein in HL60, but not U937 cells. Importantly, Akt activity (pAkt-Ser473) levels were maintained in YM155 treated U937 cells which may help stabilize other anti-apoptotic proteins. Combination treatments with an Akt inhibitor, MK-2206, reduced levels of pAkt-Ser473 in U937 cells and synergistically sensitized them to YM155 cytotoxicity. Collectively our results indicate that Akt signaling may be an important factor mediating YM155 response in AML, and combinatorial therapies with Akt inhibitors could improve treatment efficacy in YM155-resistant cells. PMID:26118775

SC79 protects retinal pigment epithelium cells from UV radiation via activating Akt-Nrf2 signaling

PubMed Central

Cao, Guo-fan; Cao, Cong; Jiang, Qin

2016-01-01

Excessive Ultra-violet (UV) radiation causes oxidative damages and apoptosis in retinal pigment epithelium (RPE) cells. Here we tested the potential activity of SC79, a novel small molecule activator of Akt, against the process. We showed that SC79 activated Akt in primary and established (ARPE-19 line) RPE cells. It protected RPE cells from UV damages possibly via inhibiting cell apoptosis. Akt inhibition, via an Akt specific inhibitor (MK-2206) or Akt1 shRNA silence, almost abolished SC79-induced RPE cytoprotection. Further studies showed that SC79 activated Akt-dependent NF-E2-related factor 2 (Nrf2) signaling and inhibited UV-induced oxidative stress in RPE cells. Reversely, Nrf2 shRNA knockdown or S40T mutation attenuated SC79-induced anti-UV activity. For the in vivo studies, we showed that intravitreal injection of SC79 significantly protected mouse retina from light damages. Based on these results, we suggest that SC79 protects RPE cells from UV damages possibly via activating Akt-Nrf2 signaling axis. PMID:27517753

Antiangiogenic treatment diminishes renal injury and dysfunction via regulation of local AKT in early experimental diabetes.

PubMed

Bai, Xiaoyan; Li, Xiao; Tian, Jianwei; Zhou, Zhanmei

2014-01-01

In view of increased vascular endothelial growth factor-A (VEGF-A) expression and renal dysfunction in early diabetes, we designed a study to test whether VEGF-A inhibition can prevent early renal injury and dysfunction. We investigated the relationship and mechanism between VEGF-A and AKT regulation. In vitro, VEGF-A small interfering RNA (siRNA) and AKT inhibitor MK-2206 were employed to podocytes and NRK-52 cells cultured in high glucose (30 mM). In vivo, the antiangiogenic drug endostatin was administered in 12 week-old streptozotocin-induced male Sprague Dawley rats. The levels of VEGF-A, AKT, phosphorylated Ser⁴⁷³-AKT, phosphorylated Thr³⁰⁸-AKT, nephrin, angiotensin II (Ang II), angiotensin type II receptor 1 (ATR1) were examined using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunohistochemistry. Interactions between phosphorylated Thr³⁰⁸-AKT and either nephrin in podocytes or Ang II in renal tubules were studied, respectively, using confocal immunofluorescence microscopy and immunoprecipitation. Silencing VEGF-A in podocytes upregulated phosphorylated Thr³⁰⁸-AKT and nephrin. Silencing VEGF-A in NRK-52E cells upregulated phosphorylated Thr³⁰⁸-AKT while downregulated Ang II and ATR1. MK-2206 enhanced VEGF-A expression in both podocytes and NRK-52E cells by inhibiting AKT activities. In diabetic rat kidneys, VEGF-A was upregulated and phosphorylated Thr³⁰⁸-AKT colocalized with either nephrin in podocytes or Ang II in renal tubules. With the endostatin treatment, the level of VEGF-A decreased while phosphorylated Thr³⁰⁸-AKT increased in both glomeruli and renal tubules. Treatment with endostatin upregulated nephrin in podocytes while downregulated Ang II and AT1R in renal tubules. Glomerular mesangial expansion was attenuated by the endostatin treatment, however, differences did not reach statistical significance. Endostatin ameliorated the interstitial fibrosis

Antiangiogenic Treatment Diminishes Renal Injury and Dysfunction via Regulation of Local AKT in Early Experimental Diabetes

PubMed Central

Zhou, Zhanmei

2014-01-01

In view of increased vascular endothelial growth factor-A (VEGF-A) expression and renal dysfunction in early diabetes, we designed a study to test whether VEGF-A inhibition can prevent early renal injury and dysfunction. We investigated the relationship and mechanism between VEGF-A and AKT regulation. In vitro, VEGF-A small interfering RNA (siRNA) and AKT inhibitor MK-2206 were employed to podocytes and NRK-52 cells cultured in high glucose (30 mM). In vivo, the antiangiogenic drug endostatin was administered in 12 week-old streptozotocin-induced male Sprague Dawley rats. The levels of VEGF-A, AKT, phosphorylated Ser473-AKT, phosphorylated Thr308-AKT, nephrin, angiotensin II (Ang II), angiotensin type II receptor 1 (ATR1) were examined using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunohistochemistry. Interactions between phosphorylated Thr308-AKT and either nephrin in podocytes or Ang II in renal tubules were studied, respectively, using confocal immunofluorescence microscopy and immunoprecipitation. Silencing VEGF-A in podocytes upregulated phosphorylated Thr308-AKT and nephrin. Silencing VEGF-A in NRK-52E cells upregulated phosphorylated Thr308-AKT while downregulated Ang II and ATR1. MK-2206 enhanced VEGF-A expression in both podocytes and NRK-52E cells by inhibiting AKT activities. In diabetic rat kidneys, VEGF-A was upregulated and phosphorylated Thr308-AKT colocalized with either nephrin in podocytes or Ang II in renal tubules. With the endostatin treatment, the level of VEGF-A decreased while phosphorylated Thr308-AKT increased in both glomeruli and renal tubules. Treatment with endostatin upregulated nephrin in podocytes while downregulated Ang II and AT1R in renal tubules. Glomerular mesangial expansion was attenuated by the endostatin treatment, however, differences did not reach statistical significance. Endostatin ameliorated the interstitial fibrosis, urine albumin excretion rate

AKT inhibition mitigates GRP78 (glucose-regulated protein) expression and contribution to chemoresistance in endometrial cancers.

PubMed

Gray, Michael J; Mhawech-Fauceglia, Paulette; Yoo, Eunjeong; Yang, Wangrong; Wu, Eijean; Lee, Amy S; Lin, Yvonne G

2013-07-01

Overexpression of the unfolded protein response master regulator GRP78 is associated with poor prognosis and therapeutic resistance in numerous human cancers, yet its role in endometrial cancers (EC) is undefined. To better understand the contribution of GRP78 to EC, we examined its expression levels in EC patient samples and EC cell lines. We demonstrate that GRP78 overexpression occurs more frequently in EC tissues compared with that found in normal endometrium, and that GRP78 expression occurs in most EC cell lines examined. Functional analysis demonstrated that GRP78 is inducible by cisplatin in EC cells, and siRNA knockdown of GRP78 augments chemotherapy-mediated cell death. Examination of AKT and GRP78 expression demonstrated that inhibition of AKT activity by MK2206 blocks GRP78 expression in EC cells. SiRNA studies also revealed that knockdown of GRP78 reduces but does not abrogate AKT activity, demonstrating that GRP78 is required for optimal AKT activity. In the presence of MK2206, siRNA knockdown of GRP78 does not augment AKT mediated survival in response to cisplatin treatment, suggesting that GRP78's antiapoptosis functions are part of the AKT survival pathway. Targeted therapies that reduce GRP78 expression or activity in cancers may serve to increase the effectiveness of current therapies for EC patients. Copyright © 2012 UICC.

Icariside II activates EGFR-Akt-Nrf2 signaling and protects osteoblasts from dexamethasone.

PubMed

Liu, Weidong; Mao, Li; Ji, Feng; Chen, Fengli; Wang, Shouguo; Xie, Yue

2017-01-10

The potential effect of icariside II on dexamethasone-induced osteoblast cell damages was evaluated here. In MC3T3-E1 osteoblastic cells and the primary murine osteoblasts, co-treatment with icariside II dramatically attenuated dexamethasone- induced cell death and apoptosis. Icariside II activated Akt signaling, which is required for its actions in osteoblasts. Akt inhibitors (LY294002, perifosine and MK-2206) almost abolished icariside II-induced osteoblast cytoprotection against dexamethasone. Further studies showed that icariside II activated Nrf2 signaling, downstream of Akt, to inhibit dexamethasone-induced reactive oxygen species (ROS) production in MC3T3-E1 cells and primary osteoblasts. On the other hand, Nrf2 shRNA knockdown inhibited icariside II-induced anti-dexamethasone cytoprotection in MC3T3-E1 cells. Finally, we showed that icariside II induced heparin-binding EGF (HB-EGF) production and EGFR trans-activation in MC3T3-E1 cells. EGFR inhibition, via anti-HB-EGF antibody, EGFR inhibitor AG1478 or EGFR shRNA knockdown, almost blocked icariside II-induced Akt-Nrf2 activation in MC3T3-E1 cells. Collectively, we conclude that icariside II activates EGFR-Akt-Nrf2 signaling and protects osteoblasts from dexamethasone. Icariside II might have translational value for the treatment of dexamethasone-associated osteoporosis/osteonecrosis.

FLT3-ITD induces expression of Pim kinases through STAT5 to confer resistance to the PI3K/Akt pathway inhibitors on leukemic cells by enhancing the mTORC1/Mcl-1 pathway.

PubMed

Okada, Keigo; Nogami, Ayako; Ishida, Shinya; Akiyama, Hiroki; Chen, Cheng; Umezawa, Yoshihiro; Miura, Osamu

2018-02-06

FLT3-ITD is the most frequent tyrosine kinase mutation in acute myeloid leukemia (AML) associated with poor prognosis. We previously reported that activation of STAT5 confers resistance to PI3K/Akt inhibitors on the FLT3-ITD-positive AML cell line MV4-11 and 32D cells driven by FLT3-ITD (32D/ITD) but not by FLT3 mutated in the tyrosine kinase domain (32D/TKD). Here, we report the involvement of Pim kinases expressed through STAT5 activation in acquisition of this resistance. The specific pan-Pim kinase inhibitor AZD1208 as well as PIM447 in combination with the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-2206 cooperatively downregulated the mTORC1/4EBP1 pathway, formation of the eIF4E/eIF4G complex, and Mcl-1 expression leading to activation of Bak and Bax to induce caspase-dependent apoptosis synergistically in these cells. These cooperative effects were enhanced or inhibited by knock down of mTOR or expression of its activated mutant, respectively. Overexpression of Mcl-1 conferred the resistance on 32D/ITD cells to combined inhibition of the PI3K/Akt pathway and Pim kinases, while the Mcl-1-specific BH3 mimetic A-1210477 conquered the resistance of MV4-11 cells to GDC-0941. Furthermore, overexpression of Pim-1 in 32D/TKD enhanced the mTORC1/Mcl-1 pathway and partially protected it from the PI3K/Akt inhibitors or the FLT3 inhibitor gilteritinib to confer the resistance to PI3K/Akt inhibitors. Finally, AZD1208 and GDC-0941 cooperatively inhibited the mTORC1/Mcl-1 pathway and reduced viable cell numbers of primary AML cells from some FLT3-ITD positive cases. Thus, Pim kinases may protect the mTORC1/4EBP1/Mcl-1 pathway to confer the resistance to the PI3K/Akt inhibitors on FLT3-ITD cells and represent promising therapeutic targets.

Dissecting signalling by individual Akt/PKB isoforms, three steps at once.

PubMed

Osorio-Fuentealba, Cesar; Klip, Amira

2015-09-01

The serine/threonine kinase Akt/PKB (protein kinase B) is key for mammalian cell growth, survival, metabolism and oncogenic transformation. The diverse level and tissue expression of its three isoforms, Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ, make it daunting to identify isoform-specific actions in vivo and even in isolated tissues/cells. To date, isoform-specific knockout and knockdown have been the best strategies to dissect their individual overall functions. In a recent article in the Biochemical Journal, Kajno et al. reported a new strategy to study isoform selectivity in cell lines. Individual Akt/PKB isoforms in 3T3-L1 pre-adipocytes are first silenced via shRNA and stable cellular clones lacking one or the other isoform are selected. The stably silenced isoform is then replaced by a mutant engineered to be refractory to inhibition by MK-2206 (Akt1(W80A) or Akt2(W80A)). Akt1(W80A) or Akt2(W80A) are functional and effectively recruited to the plasma membrane in response to insulin. The system affords the opportunity to acutely control the activity of the endogenous non-silenced isoform through timely addition of MK-2206. Using this approach, it is confirmed that Akt1/PKBα is the preferred isoform sustaining adipocyte differentiation, but both Akt1/PKBα and Akt2/PKBβ can indistinctly support insulin-dependent FoxO1 (forkhead box O1) nuclear exclusion. Surprisingly, either isoform can also support insulin-dependent glucose transporter (GLUT) 4 translocation to the membrane, in contrast with the preferential role of Akt2/PKBβ assessed by knockdown studies. The new strategy should allow analysis of the plurality of Akt/PKB functions in other cells and in response to other stimuli. It should also be amenable to high-throughput studies to speed up advances in signal transmission by this pivotal kinase. © 2015 Authors; published by Portland Press Limited.

Targeting the PI3K/Akt pathway in murine MDS/MPN driven by hyperactive Ras.

PubMed

Akutagawa, J; Huang, T Q; Epstein, I; Chang, T; Quirindongo-Crespo, M; Cottonham, C L; Dail, M; Slusher, B S; Friedman, L S; Sampath, D; Braun, B S

2016-06-01

Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are myelodysplastic/myeloproliferative neoplasia (MDS/MPN) overlap syndromes that respond poorly to conventional treatments. Aberrant Ras activation because of NRAS, KRAS, PTPN11, CBL and NF1 mutations is common in CMML and JMML. However, no mechanism-based treatments currently exist for cancers with any of these mutations. An alternative therapeutic strategy involves targeting Ras-regulated effector pathways that are aberrantly activated in CMML and JMML, which include the Raf/MEK/ERK and phosphoinositide-3'-OH kinase (PI3K)/Akt cascades. Mx1-Cre, Kras(D12) and Mx1-Cre, Nf1(flox/)(-) mice accurately model many aspects of CMML and JMML. Treating Mx1-Cre, Kras(D12) mice with GDC-0941 (also referred to as pictilisib), an orally bioavailable inhibitor of class I PI3K isoforms, reduced leukocytosis, anemia and splenomegaly while extending survival. However, GDC-0941 treatment attenuated activation of both PI3K/Akt and Raf/MEK/ERK pathways in primary hematopoietic cells, suggesting it could be acting through suppression of Raf/MEK/ERK signals. To interrogate the importance of the PI3K/Akt pathway specifically, we treated mice with the allosteric Akt inhibitor MK-2206. This compound had no effect on Raf/MEK/ERK signaling, yet it also induced robust hematologic responses in Kras and Nf1 mice with MPN. These data support investigating PI3K/Akt pathway inhibitors as a therapeutic strategy in JMML and CMML patients.

Lapatinib-resistant cancer cells possessing epithelial cancer stem cell properties develop sensitivity during sphere formation by activation of the ErbB/AKT/cyclin D2 pathway.

PubMed

Ohnishi, Yuichi; Yasui, Hiroki; Kakudo, Kenji; Nozaki, Masami

2016-11-01

Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR)/ErbB2, has antiproliferative effects and is used to treat patients with ErbB2-positive metastatic breast cancer. In the present study, we examined the effects of lapatinib on growth of oral and prostate cancer cells. Oral squamous cell carcinoma (OSCC) cell lines HSC3, HSC4 and Ca9-22 were sensitive to the antiproliferative effects of lapatinib in anchorage-dependent culture, but the OSCC cell lines KB and SAS and the prostate cancer cell line DU145 were resistant to lapatinib. Phosphorylation levels of EGFR in all cell lines decreased during lapatinib treatment in anchorage‑dependent culture. Furthermore, the phosphorylation levels of ErbB2, ErbB3 and Akt and the protein levels of cyclin D1 were decreased by lapatinib treatment of HSC3, HSC4 and Ca9-22 cells. ErbB3 was not expressed and cyclin D1 protein levels were not altered by lapatinib treatment in KB, DU145 and SAS cells. The phosphorylation of ErbB2 and AKT was not affected by lapatinib in SAS cells and was not detected in KB and DU145 cells. Lapatinib-resistant cell lines exhibited sphere-forming ability, and SAS cells developed sensitivity to lapatinib during sphere formation. The phosphorylation levels of ErbB2 and AKT and protein levels of cyclin D2 increased during sphere formation of SAS cells and decreased with lapatinib treatment. In addition, sphere formation of SAS cells was inhibited by the AKT inhibitor MK2206. AKT phosphorylation and cyclin D2 levels in SAS spheres were decreased by MK2206 treatment. SAS cells expressed E-cadherin, but not vimentin and KB cells expressed vimentin, but not E-cadherin. DU145 cells expressed vimentin and E-cadherin. These results suggested that phosphorylation of EGFR and ErbB2 by cell detachment from the substratum induces the AKT pathway/cyclin D2-dependent sphere growth in SAS epithelial cancer stem-like cells, thereby rendering SAS spheres sensitive to lapatinib treatment.

Targeting the PI3K/Akt pathway in murine MDS/MPN driven by hyperactive Ras

PubMed Central

Akutagawa, Jon; Huang, Tannie Q.; Epstein, Inbal; Chang, Tiffany; Quirindongo-Crespo, Maricel; Cottonham, Charisa L.; Dail, Monique; Slusher, Barbara S.; Friedman, Lori S.; Sampath, Deepak; Braun, Benjamin S.

2016-01-01

Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are myelodysplastic/myeloproliferative neoplasia (MDS/MPN) overlap syndromes that respond poorly to conventional treatments. Aberrant Ras activation due to NRAS, KRAS, PTPN11, CBL, and NF1 mutations is common in CMML and JMML. However, no mechanism-based treatments currently exist for cancers with any of these mutations. An alternative therapeutic strategy involves targeting Ras-regulated effector pathways that are aberrantly activated in CMML and JMML, which include the Raf/MEK/ERK and phosphoinositide-3´-OH kinase (PI3K)/Akt cascades. Mx1-Cre, KrasD12 and Mx1-Cre, Nf1flox/− mice accurately model many aspects of CMML and JMML. Treating Mx1-Cre, KrasD12 mice with GDC-0941 (also referred to as pictilisib), an orally bioavailable inhibitor of class I PI3K isoforms, reduced leukocytosis, anemia, and splenomegaly while extending survival. However, GDC-0941 treatment attenuated activation of both PI3K/Akt and Raf/MEK/ERK pathways in primary hematopoietic cells, suggesting it could be acting through suppression of Raf/MEK/ERK signals. To interrogate the importance of the PI3K/Akt pathway specifically, we treated mice with the allosteric Akt inhibitor MK-2206. This compound had no effect on Raf/MEK/ERK signaling, yet it also induced robust hematologic responses in Kras and Nf1 mice with MPN. These data support investigating PI3K/Akt pathway inhibitors as a therapeutic strategy in JMML and CMML patients. PMID:26965285

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

B Cell Receptor Activation Predominantly Regulates AKT-mTORC1/2 Substrates Functionally Related to RNA Processing

PubMed Central

Mohammad, Dara K.; Ali, Raja H.; Turunen, Janne J.; Nore, Beston F.; Smith, C. I. Edvard

2016-01-01

Protein kinase B (AKT) phosphorylates numerous substrates on the consensus motif RXRXXpS/T, a docking site for 14-3-3 interactions. To identify novel AKT-induced phosphorylation events following B cell receptor (BCR) activation, we performed proteomics, biochemical and bioinformatics analyses. Phosphorylated consensus motif-specific antibody enrichment, followed by tandem mass spectrometry, identified 446 proteins, containing 186 novel phosphorylation events. Moreover, we found 85 proteins with up regulated phosphorylation, while in 277 it was down regulated following stimulation. Up regulation was mainly in proteins involved in ribosomal and translational regulation, DNA binding and transcription regulation. Conversely, down regulation was preferentially in RNA binding, mRNA splicing and mRNP export proteins. Immunoblotting of two identified RNA regulatory proteins, RBM25 and MEF-2D, confirmed the proteomics data. Consistent with these findings, the AKT-inhibitor (MK-2206) dramatically reduced, while the mTORC-inhibitor PP242 totally blocked phosphorylation on the RXRXXpS/T motif. This demonstrates that this motif, previously suggested as an AKT target sequence, also is a substrate for mTORC1/2. Proteins with PDZ, PH and/or SH3 domains contained the consensus motif, whereas in those with an HMG-box, H15 domains and/or NF-X1-zinc-fingers, the motif was absent. Proteins carrying the consensus motif were found in all eukaryotic clades indicating that they regulate a phylogenetically conserved set of proteins. PMID:27487157

Study of Akt Inhibitor MK2206 in Patients With Relapsed Lymphoma

ClinicalTrials.gov

2015-10-09

Adult Nasal Type Extranodal NK/T-cell Lymphoma; Anaplastic Large Cell Lymphoma; Angioimmunoblastic T-cell Lymphoma; B-cell Adult Acute Lymphoblastic Leukemia; B-cell Chronic Lymphocytic Leukemia; Cutaneous B-cell Non-Hodgkin Lymphoma; Extranodal Marginal Zone B-cell Lymphoma of Mucosa-associated Lymphoid Tissue; Hepatosplenic T-cell Lymphoma; Intraocular Lymphoma; Nodal Marginal Zone B-cell Lymphoma; Noncutaneous Extranodal Lymphoma; Peripheral T-cell Lymphoma; Recurrent Adult Acute Lymphoblastic Leukemia; Recurrent Adult Diffuse Large Cell Lymphoma; Recurrent Adult Diffuse Mixed Cell Lymphoma; Recurrent Adult Diffuse Small Cleaved Cell Lymphoma; Recurrent Adult Grade III Lymphomatoid Granulomatosis; Recurrent Adult Hodgkin Lymphoma; Recurrent Adult Immunoblastic Large Cell Lymphoma; Recurrent Adult Lymphoblastic Lymphoma; Recurrent Adult T-cell Leukemia/Lymphoma; Recurrent Cutaneous T-cell Non-Hodgkin Lymphoma; Recurrent Grade 1 Follicular Lymphoma; Recurrent Grade 2 Follicular Lymphoma; Recurrent Grade 3 Follicular Lymphoma; Recurrent Mantle Cell Lymphoma; Recurrent Marginal Zone Lymphoma; Recurrent Mycosis Fungoides/Sezary Syndrome; Recurrent Small Lymphocytic Lymphoma; Refractory Hairy Cell Leukemia; Small Intestine Lymphoma; Splenic Marginal Zone Lymphoma; T-cell Adult Acute Lymphoblastic Leukemia; T-cell Large Granular Lymphocyte Leukemia; Testicular Lymphoma; Waldenström Macroglobulinemia

Harnessing the PI3K/Akt/mTOR pathway in T-cell acute lymphoblastic leukemia: Eliminating activity by targeting at different levels

PubMed Central

Ricci, Francesca; Tabellini, Giovanna; Chiarini, Francesca; Tazzari, Pier Luigi; Melchionda, Fraia; Buontempo, Francesca; Pagliaro, Pasqualepaolo; Pession, Andrea; McCubrey, James A.; Martelli, Alberto M.

2012-01-01

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant hematological disorder arising in the thymus from T-cell progenitors. T-ALL mainly affects children and young adults, and remains fatal in 20% of adolescents and 50% of adults, despite progress in polychemotherapy protocols. Therefore, innovative targeted therapies are desperately needed for patients with a dismal prognosis. Aberrant activation of PI3K/Akt/mTOR signaling is a common event in T-ALL patients and portends a poor prognosis. Preclinical studies have highlighted that modulators of PI3K/Akt/mTOR signaling could have a therapeutic relevance in T-ALL. However, the best strategy for inhibiting this highly complex signal transduction pathway is still unclear, as the pharmaceutical companies have disclosed an impressive array of small molecules targeting this signaling network at different levels. Here, we demonstrate that a dual PI3K/PDK1 inhibitor, NVP-BAG956, displayed the most powerful cytotoxic effects against T-ALL cell lines and primary patients samples, when compared with a pan class I PI3K inhibitor (GDC-0941), an allosteric Akt inhibitor (MK-2206), an mTORC1 allosteric inhibitor (RAD-001), or an ATP-competitive mTORC1/mTORC2 inhibitor (KU-63794). Moreover, we also document that combinations of some of the aforementioned drugs strongly synergized against T-ALL cells at concentrations well below their respective IC50. This observation indicates that vertical inhibition at different levels of the PI3K/Akt/mTOR network could be considered as a future innovative strategy for treating T-ALL patients. PMID:22885370

Harnessing the PI3K/Akt/mTOR pathway in T-cell acute lymphoblastic leukemia: eliminating activity by targeting at different levels.

PubMed

Bressanin, Daniela; Evangelisti, Camilla; Ricci, Francesca; Tabellini, Giovanna; Chiarini, Francesca; Tazzari, Pier Luigi; Melchionda, Fraia; Buontempo, Francesca; Pagliaro, Pasqualepaolo; Pession, Andrea; McCubrey, James A; Martelli, Alberto M

2012-08-01

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant hematological disorder arising in the thymus from T-cell progenitors. T-ALL mainly affects children and young adults, and remains fatal in 20% of adolescents and 50% of adults, despite progress in polychemotherapy protocols. Therefore, innovative targeted therapies are desperately needed for patients with a dismal prognosis. Aberrant activation of PI3K/Akt/mTOR signaling is a common event in T-ALL patients and portends a poor prognosis. Preclinical studies have highlighted that modulators of PI3K/Akt/mTOR signaling could have a therapeutic relevance in T-ALL. However, the best strategy for inhibiting this highly complex signal transduction pathway is still unclear, as the pharmaceutical companies have disclosed an impressive array of small molecules targeting this signaling network at different levels. Here, we demonstrate that a dual PI3K/PDK1 inhibitor, NVP-BAG956, displayed the most powerful cytotoxic affects against T-ALL cell lines and primary patients samples, when compared with a pan class I PI3K inhibitor (GDC-0941), an allosteric Akt inhibitor (MK-2206), an mTORC1 allosteric inhibitor (RAD-001), or an ATP-competitive mTORC1/mTORC2 inhibitor (KU63794). Moreover, we also document that combinations of some of the aforementioned drugs strongly synergized against T-ALL cells at concentrations well below their respective IC50. This observation indicates that vertical inhibition at different levels of the PI3K/Akt/mTOR network could be considered as a future innovative strategy for treating T-ALL patients.

oxLDL induces endothelial cell proliferation via Rho/ROCK/Akt/p27kip1 signaling: opposite effects of oxLDL and cholesterol loading.

PubMed

Zhang, Chongxu; Adamos, Crystal; Oh, Myung-Jin; Baruah, Jugajyoti; Ayee, Manuela A A; Mehta, Dolly; Wary, Kishore K; Levitan, Irena

2017-09-01

Oxidized modifications of LDL (oxLDL) play a key role in the development of endothelial dysfunction and atherosclerosis. However, the underlying mechanisms of oxLDL-mediated cellular behavior are not completely understood. Here, we compared the effects of two major types of oxLDL, copper-oxidized LDL (Cu 2+ -oxLDL) and lipoxygenase-oxidized LDL (LPO-oxLDL), on proliferation of human aortic endothelial cells (HAECs). Cu 2+ -oxLDL enhanced HAECs' proliferation in a dose- and degree of oxidation-dependent manner. Similarly, LPO-oxLDL also enhanced HAEC proliferation. Mechanistically, both Cu 2+ -oxLDL and LPO-oxLDL enhance HAEC proliferation via activation of Rho, Akt phosphorylation, and a decrease in the expression of cyclin-dependent kinase inhibitor 1B (p27 kip1 ). Both Cu 2+ -oxLDL or LPO-oxLDL significantly increased Akt phosphorylation, whereas an Akt inhibitor, MK2206, blocked oxLDL-induced increase in HAEC proliferation. Blocking Rho with C3 or its downstream target ROCK with Y27632 significantly inhibited oxLDL-induced Akt phosphorylation and proliferation mediated by both Cu 2+ - and LPO-oxLDL. Activation of RhoA was blocked by Rho-GDI-1, which also abrogated oxLDL-induced Akt phosphorylation and HAEC proliferation. In contrast, blocking Rac1 in these cells had no effect on oxLDL-induced Akt phosphorylation or cell proliferation. Moreover, oxLDL-induced Rho/Akt signaling downregulated cell cycle inhibitor p27 kip1 Preloading these cells with cholesterol, however, prevented oxLDL-induced Akt phosphorylation and HAEC proliferation. These findings provide a new understanding of the effects of oxLDL on endothelial proliferation, which is essential for developing new treatments against neovascularization and progression of atherosclerosis. Copyright © 2017 the American Physiological Society.

Vorinostat Enhances Cytotoxicity of SN-38 and Temozolomide in Ewing Sarcoma Cells and Activates STAT3/AKT/MAPK Pathways

PubMed Central

Sampson, Valerie B.; Vetter, Nancy S.; Kamara, Davida F.; Collier, Anderson B.; Gresh, Renee C.; Kolb, E. Anders

2015-01-01

Histone deacetylase inhibitors (HDACi) have been evaluated in patients with Ewing sarcoma (EWS) but demonstrated limited activity. To better understand the potential for HDACi in EWS, we evaluated the combination of the HDACi vorinostat, with DNA damaging agents SN-38 (the active metabolite of irinotecan and topoisomerase 1 inhibitor) plus the alkylating agent temozolomide (ST). Drugs were evaluated in sequential and simultaneous combinations in two EWS cell lines. Results demonstrate that cell viability, DNA damage and reactive oxygen species (ROS) production are dependent on the sequence of drug administration. Enhanced cytotoxicity is exhibited in vitro in EWS cell lines treated with ST administered before vorinostat, which was modestly higher than concomitant treatment and superior to vorinostat administered before ST. Drug combinations downregulate cyclin D1 to induce G0/G1 arrest and promote apoptosis by cleavage of caspase-3 and PARP. When ST is administered before or concomitantly with vorinostat there is activation of STAT3, MAPK and the p53 pathway. In contrast, when vorinostat is administered before ST, there is DNA repair, increased AKT phosphorylation and reduced H2B acetylation. Inhibition of AKT using the small molecule inhibitor MK-2206 did not restore H2B acetylation. Combining ST with the dual ALK and IGF-1R inhibitor, AZD3463 simultaneously inhibited STAT3 and AKT to enhance the cytotoxic effects of ST and further reduce cell growth suggesting that STAT3 and AKT activation were in part mediated by ALK and IGF-1R signaling. In summary, potent antiproliferative and proapoptotic activity were demonstrated for ST induced DNA damage before or simultaneous with HDAC inhibition and cell death was mediated through the p53 pathway. These observations may aid in designing new protocols for treating pediatric patients with high-risk EWS. PMID:26571493

CCNG2 Overexpression Mediated by AKT Inhibits Tumor Cell Proliferation in Human Astrocytoma Cells.

PubMed

Zhang, Danfeng; Wang, Chunhui; Li, Zhenxing; Li, Yiming; Dai, Dawei; Han, Kaiwei; Lv, Liquan; Lu, Yicheng; Hou, Lijun; Wang, Junyu

2018-01-01

The cyclin family protein CCNG2 has an important inhibitory role in cancer initiation and progression, but the exact mechanism is still unknown. In this study, we examined the relationship between CCNG2 and the malignancy of astrocytomas and whether the AKT pathway, which is upregulated in astrocytomas, may inhibit CCNG2 expression. CCNG2 expression was found to be negatively associated with the pathological grade and proliferative activity of astrocytomas, as the highest expression was found in control brain tissue ( N  = 31), whereas the lowest expression was in high-grade glioma tissue ( N  = 31). Additionally, CCNG2 overexpression in glioma cell lines, T98G and U251 inhibited proliferation and arrested cells in the G0/G1 phase. Moreover, CCNG2 overexpression could increase glioma cells apoptosis. In contrast, AKT activity increased in glioma cells that had low CCNG2 expression. Expression of CCNG2 was higher in cells treated with the AKT kinase inhibitor MK-2206 indicating that the presence of phosphorylated AKT may inhibit the expression of CCNG2. Inhibition of AKT also led to decreased colony formation in T98G and U251 cells and knocked down of CCNG2 reversed the result. Finally, overexpression of CCNG2 in glioma cells reduced tumor volume in a murine model. To conclude, low expression of CCNG2 correlated with the severity astrocytoma and CCNG2 overexpression could induce apoptosis and inhibit proliferation. Inhibition of AKT activity increased the expression of CCNG2. The present study highlights the regulatory consequences of CCNG2 expression and AKT activity in astrocytoma tumorigenesis and the potential use of CCNG2 in anticancer treatment.

Group II Metabotropic Glutamate Receptor Agonist Ameliorates MK801-Induced Dysfunction of NMDA Receptors via the Akt/GSK-3β Pathway in Adult Rat Prefrontal Cortex

PubMed Central

Xi, Dong; Li, Yan-Chun; Snyder, Melissa A; Gao, Ruby Y; Adelman, Alicia E; Zhang, Wentong; Shumsky, Jed S; Gao, Wen-Jun

2011-01-01

Pharmacological intervention targeting mGluRs has emerged as a potential treatment for schizophrenia, whereas the mechanisms involved remain elusive. We explored the antipsychotic effects of an mGluR2/3 agonist in the MK-801 model of schizophrenia in the rat prefrontal cortex. We found that the mGluR2/3 agonist LY379268 effectively recovered the disrupted expression of NMDA receptors induced by MK-801 administration. This effect was attributable to the direct regulatory action of LY379268 on NMDA receptors via activation of the Akt/GSK-3β signaling pathway. As occurs with the antipsychotic drug clozapine, acute treatment with LY379268 significantly increased the expression and phosphorylation of NMDA receptors, as well as Akt and GSK-3β. Physiologically, LY379268 significantly enhanced NMDA-induced current in prefrontal neurons and a GSK-3β inhibitor occluded this effect. In contrast to the widely proposed mechanism of modulating presynaptic glutamate release, our results strongly argue that mGluR2/3 agonists modulate the function of NMDA receptors through postsynaptic actions and reverse the MK-801-induced NMDA dysfunction via the Akt/GSK-3β pathway. This study provides novel evidence for postsynaptic mechanisms of mGluR2/3 in regulation of NMDA receptors and presents useful insights into the mechanistic actions of mGluR2/3 agonists as potential antipsychotic agents for treating schizophrenia. PMID:21326193

Airborne nitro-PAHs induce Nrf2/ARE defense system against oxidative stress and promote inflammatory process by activating PI3K/Akt pathway in A549 cells.

PubMed

Shang, Yu; Zhou, Qian; Wang, Tiantian; Jiang, Yuting; Zhong, Yufang; Qian, Guangren; Zhu, Tong; Qiu, Xinghua; An, Jing

2017-10-01

Ambient particulate matter (PM) is a worldwide health issue of concern. However, limited information is available regarding the toxic contributions of the nitro-derivatives of polycyclic aromatic hydrocarbons (nitro-PAHs). This study intend to examine whether 1-nitropyrene (1-NP) and 3-nitrofluoranthene (3-NF) could activate the nuclear factor-erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) antioxidant defense system, and whether the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway participates in regulating pro-inflammatory responses in A549 cells. Firstly, 1-NP and 3-NF concentration-dependently induced cellular apoptosis, reactive oxygen species (ROS) generation, DNA damage, S phase cell cycle arrest and differential expression of related cytokine genes. Secondly, 1-NP and 3-NF activated the Nrf2/ARE defense system, as evidenced by increased protein expression levels and nuclear translocation of transcription factor Nrf2, elevated Nrf2/ARE binding activity, up-regulated expression of the target gene heme oxygenase-1 (HO-1). Significantly increased protein expression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and phosphorylation level of Akt indicated that the PI3K/Akt pathway was activated during pro-inflammatory process. Further, both PI3K inhibitor (LY294002) and Akt inhibitor (MK-2206) reversed the elevated TNF-α expression to control level. Our results suggested that Nrf2/ARE pathway activation might cause an initiation step in cellular protection against oxidative stress caused by nitro-PAHs, and the PI3K/Akt pathway participated in regulating inflammatory responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

PI3K/Akt Signaling Pathway Activates the WNK-OSR1/SPAK-NCC Phosphorylation Cascade in Hyperinsulinemic db/db Mice

PubMed Central

Nishida, Hidenori; Sohara, Eisei; Nomura, Naohiro; Chiga, Motoko; Alessi, Dario R; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi

2013-01-01

Metabolic syndrome patients have insulin resistance, which causes hyperinsulinemia, which in turn causes aberrant increased renal sodium reabsorption. The precise mechanisms underlying this greater salt-sensitivity of hyperinsulinemic patients remain unclear. Abnormal activation of the recently-identified WNK kinase-OSR1/SPAK kinases-NCC transporter phosphorylation cascade results in the salt-sensitive hypertension of pseudohypoaldosteronism type II. Here, we report a study of renal WNK-OSR1/SPAK-NCC cascade activation in the db/db mouse model of hyperinsulinemic metabolic syndrome. Thiazide sensitivity was increased, suggesting greater activity of NCC in db/db mice. In fact, increased phosphorylation of OSR1/SPAK and NCC was observed. In both SpakT243A/+ and Osr1T185A/+ knock-in db/db mice, which carry mutations that disrupt the signal from WNK kinases, increased phosphorylation of NCC and elevated blood pressure were completely corrected, indicating that phosphorylation of SPAK and OSR1 by WNK kinases is required for the increased activation and phosphorylation of NCC in this model. Renal phosphorylated Akt was increased in db/db mice, suggesting that increased NCC phosphorylation is regulated by the PI3K/Akt signaling cascade in the kidney in response to hyperinsulinemia. A PI3K inhibitor (NVP-BEZ235) corrected the increased OSR1/SPAK-NCC phosphorylation. Another more specific PI3K inhibitor (GDC-0941) and an Akt inhibitor (MK-2206) also inhibited increased NCC phosphorylation. These results indicate that the PI3K/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in db/db mice. This mechanism may play a role in the pathogenesis of salt-sensitive hypertension in human hyperinsulinemic conditions such as the metabolic syndrome. PMID:22949526

Phosphatidylinositol 3-kinase/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in hyperinsulinemic db/db mice.

PubMed

Nishida, Hidenori; Sohara, Eisei; Nomura, Naohiro; Chiga, Motoko; Alessi, Dario R; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi

2012-10-01

Metabolic syndrome patients have insulin resistance, which causes hyperinsulinemia, which in turn causes aberrant increased renal sodium reabsorption. The precise mechanisms underlying this greater salt sensitivity of hyperinsulinemic patients remain unclear. Abnormal activation of the recently identified with-no-lysine kinase (WNK)-oxidative stress-responsive kinase 1 (OSR1)/STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NaCl cotransporter (NCC) phosphorylation cascade results in the salt-sensitive hypertension of pseudohypoaldosteronism type II. Here, we report a study of renal WNK-OSR1/SPAK-NCC cascade activation in the db/db mouse model of hyperinsulinemic metabolic syndrome. Thiazide sensitivity was increased, suggesting greater activity of NCC in db/db mice. In fact, increased phosphorylation of OSR1/SPAK and NCC was observed. In both SpakT243A/+ and Osr1T185A/+ knock-in db/db mice, which carry mutations that disrupt the signal from WNK kinases, increased phosphorylation of NCC and elevated blood pressure were completely corrected, indicating that phosphorylation of SPAK and OSR1 by WNK kinases is required for the increased activation and phosphorylation of NCC in this model. Renal phosphorylated Akt was increased in db/db mice, suggesting that increased NCC phosphorylation is regulated by the phosphatidylinositol 3-kinase/Akt signaling cascade in the kidney in response to hyperinsulinemia. A phosphatidylinositol 3-kinase inhibitor (NVP-BEZ235) corrected the increased OSR1/SPAK-NCC phosphorylation. Another more specific phosphatidylinositol 3-kinase inhibitor (GDC-0941) and an Akt inhibitor (MK-2206) also inhibited increased NCC phosphorylation. These results indicate that the phosphatidylinositol 3-kinase/Akt signaling pathway activates the WNK-OSR1/SPAK-NCC phosphorylation cascade in db/db mice. This mechanism may play a role in the pathogenesis of salt-sensitive hypertension in human hyperinsulinemic conditions, such as the metabolic syndrome.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Yong; Fang, Shi-ji; Zhu, Li-juan

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 wasmore » 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.« less

Benzothiophene inhibitors of MK2. Part 2: Improvements in kinase selectivity and cell potency

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, David R.; Meyers, Marvin J.; Kurumbail, Ravi G.

2010-10-01

Optimization of kinase selectivity for a set of benzothiophene MK2 inhibitors provided analogs with potencies of less than 500 nM in a cell based assay. The selectivity of the inhibitors can be rationalized by examination of X-ray crystal structures of inhibitors bound to MK2.

Dual Regulation of Glycogen Synthase Kinase 3 (GSK3)α/β by Protein Kinase C (PKC)α and Akt Promotes Thrombin-mediated Integrin αIIbβ3 Activation and Granule Secretion in Platelets*

PubMed Central

Moore, Samantha F.; van den Bosch, Marion T. J.; Hunter, Roger W.; Sakamoto, Kei; Poole, Alastair W.; Hers, Ingeborg

2013-01-01

Glycogen synthase kinase-3 is a Ser/Thr kinase, tonically active in resting cells but inhibited by phosphorylation of an N-terminal Ser residue (Ser21 in GSK3α and Ser9 in GSK3β) in response to varied external stimuli. Recent work suggests that GSK3 functions as a negative regulator of platelet function, but how GSK3 is regulated in platelets has not been examined in detail. Here, we show that early thrombin-mediated GSK3 phosphorylation (0–30 s) was blocked by PKC inhibitors and largely absent in platelets from PKCα knock-out mice. In contrast, late (2–5 min) GSK3 phosphorylation was dependent on the PI3K/Akt pathway. Similarly, early thrombin-mediated inhibition of GSK3 activity was blocked in PKCα knock-out platelets, whereas the Akt inhibitor MK2206 reduced late thrombin-mediated GSK3 inhibition and largely prevented GSK3 inhibition in PKCα knock-out platelets. More importantly, GSK3 phosphorylation contributes to platelet function as knock-in mice where GSK3α Ser21 and GSK3β Ser9 were mutated to Ala showed a significant reduction in PAR4-mediated platelet aggregation, fibrinogen binding, and P-selectin expression, whereas the GSK3 inhibitor CHIR99021 enhanced these responses. Together, these results demonstrate that PKCα and Akt modulate platelet function by phosphorylating and inhibiting GSK3α/β, thereby relieving the negative effect of GSK3α/β on thrombin-mediated platelet activation. PMID:23239877

Calcium oxalate crystals induces tight junction disruption in distal renal tubular epithelial cells by activating ROS/Akt/p38 MAPK signaling pathway.

PubMed

Yu, Lei; Gan, Xiuguo; Liu, Xukun; An, Ruihua

2017-11-01

Tight junction plays important roles in regulating paracellular transports and maintaining cell polarity. Calcium oxalate monohydrate (COM) crystals, the major crystalline composition of kidney stones, have been demonstrated to be able to cause tight junction disruption to accelerate renal cell injury. However, the cellular signaling involved in COM crystal-induced tight junction disruption remains largely to be investigated. In the present study, we proved that COM crystals induced tight junction disruption by activating ROS/Akt/p38 MAPK pathway. Treating Madin-Darby canine kidney (MDCK) cells with COM crystals induced a substantial increasing of ROS generation and activation of Akt that triggered subsequential activation of ASK1 and p38 mitogen-activated protein kinase (MAPK). Western blot revealed a significantly decreased expression of ZO-1 and occludin, two important structural proteins of tight junction. Besides, redistribution and dissociation of ZO-1 were observed by COM crystals treatment. Inhibition of ROS by N-acetyl-l-cysteine (NAC) attenuated the activation of Akt, ASK1, p38 MAPK, and down-regulation of ZO-1 and occludin. The redistribution and dissociation of ZO-1 were also alleviated by NAC treatment. These results indicated that ROS were involved in the regulation of tight junction disruption induced by COM crystals. In addition, the down-regulation of ZO-1 and occludin, the phosphorylation of ASK1 and p38 MAPK were also attenuated by MK-2206, an inhibitor of Akt kinase, implying Akt was involved in the disruption of tight junction upstream of p38 MAPK. Thus, these results suggested that ROS-Akt-p38 MAPK signaling pathway was activated in COM crystal-induced disruption of tight junction in MDCK cells.

Autophagy protects chondrocytes from glucocorticoids-induced apoptosis via ROS/Akt/FOXO3 signaling.

PubMed

Shen, C; Cai, G-Q; Peng, J-P; Chen, X-D

2015-12-01

Glucocorticoids (GCs) have been widely used in the management of osteoarthritis (OA) and rheumatoid arthritis (RA). Nevertheless, there has been some concern about their ability of increasing reactive oxygen species (ROS) in the cartilage. Forkhead-box class O (FOXO) transcription factors have been proved to have a protective role in chondrocytes through regulation of autophagy and defending oxidative stress. The objective of this study was to investigate the role of FOXO3 in Dex-induce up-regulation of ROS. Healthy cartilages debris from six patients were used for chondrocytes culture. After the treatment of dexamethasone (Dex), the ROS levels, autophagic flux, the expression of FOXO3 in chondrocytes were measured. RNA interference technique was also used to determine the role of FOXO3 in Dex-induced autophagy. The metabolism of the extra-cellular matrix was also investigated. Dex increased intracellular ROS level, the expression of Akt, FOXO3 as well as autophagy flux in human chondrocytes. The expression of aggrecanases also increased after the treatment of Dex. Catalase, the ROS scavenger, suppressed Dex-induced up-regulation of autophagy flux and expression of aggrecanases and Akt. MK-2206 and LY294002, the PI3K/Akt inhibitors, repressed Dex-induced up-regulation of FOXO3. Silencing FOXO3 resulted in down-regulation of Dex-induced autophagy. Moreover, knockdown of FOXO3 increased Dex-induced apoptosis as well as ROS levels in chondrocytes. In addition, up-regulation of autophagy by Rapamycin resulted in decreasing ROS level in chondrocytes. Dex could advance the degenerative process in cartilage. Autophagy was induced in response to Dex-induced up-regulation of ROS via ROS/Akt/FOXO3 signal pathway. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Pharmacokinetic and pharmacodynamic interaction between the lipoxygenase inhibitor MK-0591 and the cyclooxygenase inhibitor ibuprofen in man.

PubMed

Depré, M; Van Hecken, A; Verbesselt, R; De Lepeleire, I; Schwartz, J; Porras, A; Larson, P; Lin, C; De Schepper, P J

1998-01-01

Twelve healthy male subjects participated in a double-blind, placebo-controlled, randomized, three-period, crossover study to investigate the safety, tolerability, biochemical activity and pharmacokinetics of ibuprofen, a cyclooxygenase inhibitor and MK-0591, a 5-lipoxygenase inhibitor, given as single entities and in combination. Each subject received for three consecutive 8-day periods, separated by 1 week washout, each of the following treatments: ibuprofen 600 mg three times a day with 125 mg MK-0591 twice a day, ibuprofen 600 mg three times a day with placebo for MK-0591 and MK-0591 125 mg twice a day with placebo for ibuprofen. Cyclooxygenase inhibition was measured by platelet thromboxane (TxB2) generation test, and 5-lipoxygenase inhibition was measured by urinary leukotriene E4 excretion and ex vivo LTB4 generation in calcium-ionophore-stimulated blood. TxB2 suppression on day 8 by ibuprofen was not affected by concomitant treatment with MK-0591. MK-0591 alone had no effect on TxB2 generation. Leukotriene biosynthesis was inhibited by more than 90% by MK-0591 alone and by combined treatment, while ibuprofen alone had no effect. Coadministration appears to affect the pharmacokinetics of MK-0591 (decrease of area under the plasma concentration-vs-time curve [AUC] and maximum plasma concentrations [Cmax]) and of ibuprofen (increase of AUC and half-lives of elimination (t1/2) of the (S)-enantiomer, increase of t1/2 the (R)-enantiomer). Combined treatment had no effect on creatinine clearance nor on the number and intensity of the reported adverse experiences.

MK2 inhibitor reduces alkali burn-induced inflammation in rat cornea

PubMed Central

Chen, Yanfeng; Yang, Wenzhao; Zhang, Xiaobo; Yang, Shu; Peng, Gao; Wu, Ting; Zhou, Yueping; Huang, Caihong; Reinach, Peter S.; Li, Wei; Liu, Zuguo

2016-01-01

MK2 activation by p38 MAPK selectively induces inflammation in various diseases. We determined if a MK2 inhibitor (MK2i), improves cornea wound healing by inhibiting inflammation caused by burning rat corneas with alkali. Our study, for the first time, demonstrated that MK2i inhibited alkali burn-induced MK2 activation as well as rises in inflammation based on: a) blunting rises in inflammatory index, inflammatory cell infiltration, ED1+ macrophage and PMN+ neutrophil infiltration; b) suppressing IL-6 and IL-1β gene expression along with those of macrophage inflammatory protein-1α (MIP-1α), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1); c) reducing angiogenic gene expression levels and neovascularization (NV) whereas anti-angiogenic PEDF levels increased. In addition, this study found that MK2i did not affect human corneal epithelial cell (HCEC) proliferation and migration and had no detectable side effects on ocular surface integrity. Taken together, MK2i selectively inhibited alkali burn-induced corneal inflammation by blocking MK2 activation, these effects have clinical relevance in the treatment of inflammation related ocular surface diseases. PMID:27329698

Reactivation of AKT signaling following treatment of cancer cells with PI3K inhibitors attenuates their antitumor effects

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dufour, Marc; Dormond-Meuwly, Anne; Pythoud, Catherine

2013-08-16

Highlights: •PI3K inhibitors inhibit AKT only transiently. •Re-activation of AKT limits the anti-cancer effect of PI3K inhibitors. •The results suggest to combine PI3K and AKT inhibitors in cancer therapy. -- Abstract: Targeting the phosphatidylinositol-3-kinase (PI3K) is a promising approach in cancer therapy. In particular, PI3K blockade leads to the inhibition of AKT, a major downstream effector responsible for the oncogenic activity of PI3K. However, we report here that small molecule inhibitors of PI3K only transiently block AKT signaling. Indeed, treatment of cancer cells with PI3K inhibitors results in a rapid inhibition of AKT phosphorylation and signaling which is followed bymore » the reactivation of AKT signaling after 48 h as observed by Western blot. Reactivation of AKT signaling occurs despite effective inhibition of PI3K activity by PI3K inhibitors. In addition, wortmannin, a broad range PI3K inhibitor, did not block AKT reactivation suggesting that AKT signals independently of PI3K. In a therapeutical perspective, combining AKT and PI3K inhibitors exhibit stronger anti-proliferative and pro-apoptotic effects compared to AKT or PI3K inhibitors alone. Similarly, in a tumor xenograft mouse model, concomitant PI3K and AKT blockade results in stronger anti-cancer activity compared with either blockade alone. This study shows that PI3K inhibitors only transiently inhibit AKT which limits their antitumor activities. It also provides the proof of concept to combine PI3K inhibitors with AKT inhibitors in cancer therapy.« less

An allosteric Akt inhibitor effectively blocks Akt signaling and tumor growth with only transient effects on glucose and insulin levels in vivo

PubMed Central

Cherrin, Craig; Haskell, Kathleen; Howell, Bonnie; Jones, Raymond; Leander, Karen; Robinson, Ronald; Watkins, Aubrey; Bilodeau, Mark; Hoffman, Jacob; Sanderson, Philip; Hartman, George; Mahan, Elizabeth; Prueksaritanont, Thomayant; Jiang, Guoqiang; She, Qing-Bai; Rosen, Neal; Sepp-Lorenzino, Laura; Defeo-Jones, Deborah; Huber, Hans E.

2010-01-01

The PI3K-Akt pathway is dysregulated in the majority of solid tumors. Pharmacological inhibition of Akt is a promising strategy for treating tumors resistant to growth factor receptor antagonists due to mutations in PI3K or PTEN. We have developed allosteric, isozyme-specific inhibitors of Akt activity and activation, as well as ex vivo kinase assays to measure inhibition of individual Akt isozymes in tissues. Here we describe the relationship between PK, Akt inhibition, hyperglycemia and tumor efficacy for a selective inhibitor of Akt1 and Akt2 (AKTi). In nude mice, AKTi treatment caused transient insulin resistance and reversible, dose-dependent hyperglycemia and hyperinsulinemia. Akt1 and Akt2 phosphorylation was inhibited in mouse lung with EC50 values of 1.6 and 7 μM, respectively, and with similar potency in other tissues and xenograft tumors. Weekly subcutaneous dosing of AKTi resulted in dose-dependent inhibition of LNCaP prostate cancer xenografts, an AR-dependent tumor with PTEN deletion and constitutively activated Akt. Complete tumor growth inhibition was achieved at 200 mpk, a dose that maintained inhibition of Akt1 and Akt2 of greater than 80% and 50%, respectively, for at least 12 hours in xenograft tumor and mouse lung. Hyperglycemia could be controlled by reducing Cmax, while maintaining efficacy in the LNCaP model, but not by insulin administration. AKTi treatment was well tolerated, without weight loss or gross toxicities. These studies supported the rationale for clinical development of allosteric Akt inhibitors and provide the basis for further refining of pharmacokinetic properties and dosing regimens of this class of inhibitors. PMID:20139722

Akt Inhibitor MK2206 in Treating Patients With Progressive, Recurrent, or Metastatic Adenoid Cyst Carcinoma

ClinicalTrials.gov

2018-03-21

Recurrent Oral Cavity Adenoid Cystic Carcinoma; Recurrent Salivary Gland Carcinoma; Salivary Gland Adenoid Cystic Carcinoma; Stage IVA Major Salivary Gland Cancer AJCC v7; Stage IVA Oral Cavity Adenoid Cystic Carcinoma AJCC v6 and v7; Stage IVB Major Salivary Gland Cancer AJCC v7; Stage IVB Oral Cavity Adenoid Cystic Carcinoma AJCC v6 and v7; Stage IVC Major Salivary Gland Cancer AJCC v7; Stage IVC Oral Cavity Adenoid Cystic Carcinoma AJCC v6 and v7

Targeting activated Akt with GDC-0068, a novel selective Akt inhibitor that is efficacious in multiple tumor models.

PubMed

Lin, Jie; Sampath, Deepak; Nannini, Michelle A; Lee, Brian B; Degtyarev, Michael; Oeh, Jason; Savage, Heidi; Guan, Zhengyu; Hong, Rebecca; Kassees, Robert; Lee, Leslie B; Risom, Tyler; Gross, Stefan; Liederer, Bianca M; Koeppen, Hartmut; Skelton, Nicholas J; Wallin, Jeffrey J; Belvin, Marcia; Punnoose, Elizabeth; Friedman, Lori S; Lin, Kui

2013-04-01

We describe the preclinical pharmacology and antitumor activity of GDC-0068, a novel highly selective ATP-competitive pan-Akt inhibitor currently in clinical trials for the treatment of human cancers. The effect of GDC-0068 on Akt signaling was characterized using specific biomarkers of the Akt pathway, and response to GDC-0068 was evaluated in human cancer cell lines and xenograft models with various genetic backgrounds, either as a single agent or in combination with chemotherapeutic agents. GDC-0068 blocked Akt signaling both in cultured human cancer cell lines and in tumor xenograft models as evidenced by dose-dependent decrease in phosphorylation of downstream targets. Inhibition of Akt activity by GDC-0068 resulted in blockade of cell-cycle progression and reduced viability of cancer cell lines. Markers of Akt activation, including high-basal phospho-Akt levels, PTEN loss, and PIK3CA kinase domain mutations, correlate with sensitivity to GDC-0068. Isogenic PTEN knockout also sensitized MCF10A cells to GDC-0068. In multiple tumor xenograft models, oral administration of GDC-0068 resulted in antitumor activity ranging from tumor growth delay to regression. Consistent with the role of Akt in a survival pathway, GDC-0068 also enhanced antitumor activity of classic chemotherapeutic agents. GDC-0068 is a highly selective, orally bioavailable Akt kinase inhibitor that shows pharmacodynamic inhibition of Akt signaling and robust antitumor activity in human cancer cells in vitro and in vivo. Our preclinical data provide a strong mechanistic rationale to evaluate GDC-0068 in cancers with activated Akt signaling. ©2012 AACR.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meng, Zhen; Department of Oral & Maxillofacial Surgery, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081; Gan, Ye-Hua, E-mail: kqyehuagan@bjmu.edu.cn

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 translocationmore » 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.« less

Crystal Structure of Human AKT1 with an Allosteric Inhibitor Reveals a New Mode of Kinase Inhibition

PubMed Central

Wu, Wen-I; Voegtli, Walter C.; Sturgis, Hillary L.; Dizon, Faith P.; Vigers, Guy P. A.; Brandhuber, Barbara J.

2010-01-01

AKT1 (NP_005154.2) is a member of the serine/threonine AGC protein kinase family involved in cellular metabolism, growth, proliferation and survival. The three human AKT isozymes are highly homologous multi-domain proteins with both overlapping and distinct cellular functions. Dysregulation of the AKT pathway has been identified in multiple human cancers. Several clinical trials are in progress to test the efficacy of AKT pathway inhibitors in treating cancer. Recently, a series of AKT isozyme-selective allosteric inhibitors have been reported. They require the presence of both the pleckstrin-homology (PH) and kinase domains of AKT, but their binding mode has not yet been elucidated. We present here a 2.7 Å resolution co-crystal structure of human AKT1 containing both the PH and kinase domains with a selective allosteric inhibitor bound in the interface. The structure reveals the interactions between the PH and kinase domains, as well as the critical amino residues that mediate binding of the inhibitor to AKT1. Our work also reveals an intricate balance in the enzymatic regulation of AKT, where the PH domain appears to lock the kinase in an inactive conformation and the kinase domain disrupts the phospholipid binding site of the PH domain. This information advances our knowledge in AKT1 structure and regulation, thereby providing a structural foundation for interpreting the effects of different classes of AKT inhibitors and designing selective ones. PMID:20886116

miR-489 inhibits proliferation, cell cycle progression and induces apoptosis of glioma cells via targeting SPIN1-mediated PI3K/AKT pathway.

PubMed

Li, Yan; Ma, Xiaolin; Wang, Yanpeng; Li, Guohua

2017-09-01

microRNA-489 (miR-489), a newly identified tumor-related miRNA, functions as an oncogene or tumor suppressor via regulating growth and metastasis of human cancers. But, the clinical significance, biological function and underlying mechanisms of miR-489 in glioma remain rarely known. Here, we showed that the levels of miR-489 in glioma tissues were notably underexpressed compared to corresponding non-tumor tissues. In accordance, the relative levels of miR-489 were decreased in glioma cell lines compared with NHA cells. Kaplan-Meier plots indicated that miR-489 low expressing glioma patients showed a prominent shorter overall survival. In addition, miR-489 overexpression prohibited proliferation and cell cycle progression, and promoted apoptosis in U251 cells. While, miR-489 knockdown showed opposite effects on these cellular processes of U87 cells. In vivo experiments demonstrated that miR-489 restoration reduced the tumor volume and weight of subcutaneous glioma xenografts in nude mice. Notably, Spindlin 1 (SPIN1) was inversely and directly regulated by miR-489 in glioma cells. A negative correlation between the expression of miR-489 and SPIN1 mRNA was confirmed in glioma tissues. Interestingly, miR-489 inversely modulated activation of PI3K/AKT pathway and expression of downstream targets including p-mTOR, Cyclin D1 and BCL-XL. SPIN1 re-expression abolished the effects of miR-489 on U251 cells with enhanced activation of PI3K/AKT pathway and malignant phenotype. Meanwhile, AKT inhibitor MK-2206 blocked activation of PI3K/AKT pathway and resulted in reduced proliferation, cell cycle arrest and increased apoptosis in miR-489 down-regulating U87 cells. Altogether, our data support that miR-489 loss facilitates malignant phenotype of glioma cells probably via SPIN1-mediated PI3K/AKT pathway. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Targeting Mitogen-activated Protein Kinase-activated Protein Kinase 2 (MAPKAPK2, MK2): Medicinal Chemistry Efforts to Lead Small Molecule Inhibitors to Clinical Trials

PubMed Central

Fiore, Mario; Forli, Stefano; Manetti, Fabrizio

2015-01-01

The p38/MAPK-activated kinase 2 (MK2) pathway is involved in a series of pathological conditions (inflammation diseases and metastasis) and in the resistance mechanism to antitumor agents. None of the p38 inhibitors entered advanced clinical trials because of their unwanted systemic side effects. For this reason, MK2 was identified as an alternative target to block the pathway, but avoiding the side effects of p38 inhibition. However, ATP-competitive MK2 inhibitors suffered from low solubility, poor cell permeability, and scarce kinase selectivity. Fortunately, non-ATP-competitive inhibitors of MK2 have been already discovered that allowed circumventing the selectivity issue. These compounds showed the additional advantage to be effective at lower concentrations in comparison to the ATP-competitive inhibitors. Therefore, although the significant difficulties encountered during the development of these inhibitors, MK2 is still considered as an attractive target to treat inflammation and related diseases, to prevent tumor metastasis, and to increase tumor sensitivity to chemotherapeutics. PMID:26502061

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

2017-09-01

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. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Discovery of MK-7655, a β-lactamase inhibitor for combination with Primaxin®

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blizzard, Timothy A.; Chen, Helen; Kim, Seongkon

2014-02-01

β-Lactamase inhibitors with a bicyclic urea core and a variety of heterocyclic side chains were prepared and evaluated as potential partners for combination with imipenem to overcome class A and C β-lactamase mediated antibiotic resistance. The piperidine analog 3 (MK-7655) inhibited both class A and C β-lactamases in vitro. It effectively restored imipenem’s activity against imipenem-resistant Pseudomonas and Klebsiella strains at clinically achievable concentrations. A combination of MK-7655 and Primaxin® is currently in phase II clinical trials for the treatment of Gram-negative bacterial infections.

32 CFR 220.6 - Certain payers excluded.

Code of Federal Regulations, 2012 CFR

2012-07-01

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32 CFR 220.6 - Certain payers excluded.

Code of Federal Regulations, 2011 CFR

2011-07-01

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Code of Federal Regulations, 2010 CFR

2010-07-01

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2014-07-01

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32 CFR 220.6 - Certain payers excluded.

Code of Federal Regulations, 2013 CFR

2013-07-01

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PI3Kδ inhibitor idelalisib in combination with BTK inhibitor ONO/GS-4059 in diffuse large B cell lymphoma with acquired resistance to PI3Kδ and BTK inhibitors.

PubMed

Yahiaoui, Anella; Meadows, Sarah A; Sorensen, Rick A; Cui, Zhi-Hua; Keegan, Kathleen S; Brockett, Robert; Chen, Guang; Quéva, Christophe; Li, Li; Tannheimer, Stacey L

2017-01-01

Activated B-cell-like diffuse large B-cell lymphoma relies on B-cell receptor signaling to drive proliferation and survival. Downstream of the B-cell receptor, the key signaling kinases Bruton's tyrosine kinase and phosphoinositide 3-kinase δ offer opportunities for therapeutic intervention by agents such as ibrutinib, ONO/GS-4059, and idelalisib. Combination therapy with such targeted agents could provide enhanced efficacy due to complimentary mechanisms of action. In this study, we describe both the additive interaction of and resistance mechanisms to idelalisib and ONO/GS-4059 in a model of activated B-cell-like diffuse large B-cell lymphoma. Significant tumor regression was observed with a combination of PI3Kδ and Bruton's tyrosine kinase inhibitors in the mouse TMD8 xenograft. Acquired resistance to idelalisib in the TMD8 cell line occurred by loss of phosphatase and tensin homolog and phosphoinositide 3-kinase pathway upregulation, but not by mutation of PIK3CD. Sensitivity to idelalisib could be restored by combining idelalisib and ONO/GS-4059. Further evaluation of targeted inhibitors revealed that the combination of idelalisib and the phosphoinositide-dependent kinase-1 inhibitor GSK2334470 or the AKT inhibitor MK-2206 could partially overcome resistance. Characterization of acquired Bruton's tyrosine kinase inhibitor resistance revealed a novel tumor necrosis factor alpha induced protein 3 mutation (TNFAIP3 Q143*), which led to a loss of A20 protein, and increased p-IκBα. The combination of idelalisib and ONO/GS-4059 partially restored sensitivity in this resistant line. Additionally, a mutation in Bruton's tyrosine kinase at C481F was identified as a mechanism of resistance. The combination activity observed with idelalisib and ONO/GS-4059, taken together with the ability to overcome resistance, could lead to a new therapeutic option in activated B-cell-like diffuse large B-cell lymphoma. A clinical trial is currently underway to evaluate the

12 CFR 220.6 - Good faith account.

Code of Federal Regulations, 2011 CFR

2011-01-01

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12 CFR 220.6 - Good faith account.

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2012-01-01

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12 CFR 220.6 - Good faith account.

Code of Federal Regulations, 2013 CFR

2013-01-01

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12 CFR 220.6 - Good faith account.

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2014-01-01

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Synthesis and Biological Evaluation of Analogues of AKT (Protein Kinase B) Inhibitor-IV

PubMed Central

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

2011-01-01

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 ZrCl4-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. PMID:21319800

Reversing Melanoma Cross-Resistance to BRAF and MEK Inhibitors by Co-Targeting the AKT/mTOR Pathway

PubMed Central

Attar, Narsis; Ng, Charles; Chu, Connie; Guo, Deliang; Nazarian, Ramin; Chmielowski, Bartosz; Glaspy, John A.; Comin-Anduix, Begonya; Mischel, Paul S.; Lo, Roger S.; Ribas, Antoni

2011-01-01

Background The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAFV600 mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression. Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors. However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway. Methodology/Principal Findings The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically. There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS. In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance. Conclusions/Significance Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation. Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs. This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor. These combinations should be available for clinical testing in patients progressing on BRAF inhibitors. PMID:22194965

Pharmacodynamics of Imipenem in Combination with β-Lactamase Inhibitor MK7655 in a Murine Thigh Model

PubMed Central

Mavridou, Eleftheria; Melchers, Ria J. B.; van Mil, Anita C. H. A. M.; Mangin, E.; Motyl, Mary R.

2014-01-01

MK7655 is a newly developed beta-lactamase inhibitor of class A and class C carbapenemases. Pharmacokinetics (PK) of imipenem-cilastatin (IMP/C) and MK7655 were determined for intraperitoneal doses of 4 mg/kg to 128 mg/kg of body weight. MIC and pharmacodynamics (PD) studies of MK7655 were performed against several beta-lactamase producing Pseudomonas aeruginosa and Klebsiella pneumoniae strains to determine its effect in vitro and in vivo. Neutropenic mice were infected in each thigh 2 h before treatment with an inoculum of approximately 5 × 106 CFU. They were treated with IMP/C alone (every 2 hours [q2h], various doses) or in combination with MK7655 in either a dose fractionation study or q2h for 24 h and sacrificed for CFU determinations. IMP/MK7655 decreased MICs regarding IMP MIC. The PK profiles of IMP/C and MK7655 were linear over the dosing range studied and comparable with volumes of distribution (V) of 0.434 and 0.544 liter/kg and half-lives (t1/2) of 0.24 and 0.25 h, respectively. Protein binding of MK7655 was 20%. A sigmoidal maximum effect (Emax) model was fit to the PK/PD index responses. The effect of the inhibitor was not related to the maximum concentration of drug in serum (Cmax)/MIC, and model fits for T>MIC and area under the concentration-time curve (AUC)/MIC were comparable (R2 of 0.7 and 0.75), but there appeared to be no significant relationship of effect with dose frequency. Escalating doses of MK7655 and IMP/C showed that the AUC of MK7655 required for a static effect was dependent on the dose of IMP/C and the MIC of the strain, with a mean area under the concentration-time curve for the free, unbound fraction of the drug (fAUC) of 26.0 mg · h/liter. MK7655 shows significant activity in vivo and results in efficacy of IMP/C in otherwise resistant strains. The exposure-response relationships found can serve as a basis for establishing dosing regimens in humans. PMID:25403667

[The role of FOXO3a-Bim signaling in triptolide induced bladder cancer T24 cells apoptosis].

PubMed

Yang, L L; Wang, X Y; Zheng, L Y; Fang, S J; Xu, M; Zhao, Z W; Ji, J S

2017-04-18

Objective: To investigate the role of FOXO3a-Bim signaling in triptolide induced bladder cancer T24 cells apoptosis. Methods: T24 cells were used and divided into control group, triptolide group(50 nmol/L), MK2206 group(50 nmol/L triptolide+ 5 μmol/L MK2206), FOXO3a-siRNA group(50 nmol/L triptolide+ 100 nmol/L FOXO3a-siRNA), Bim-siRNA group (50 nmol/L triptolide+ 100 nmol/L Bim-siRNA). MTT assay was used to analyze the cells growth inhibition.Annexin V/PI staining was implemented to detect cell apoptosis rate, the expression of p-Akt, Akt, p-FOXO3a, FOXO3a, Bim, Bax.Cleaved-caspase 3 was analyzed by Western blot. Results: After treatment with triptolide 25, 50, 100, 250 nmol/L, the cell growth inhibition rates at 24 hours(17%±9%, 24%±5%, 43%±8%, 61%±8%), 48 hours (20%±7%, 34%±6%, 56%±7%, 74%±5%) and 72 hours(32%±8%, 41%±7%, 69%±7%, 84%±3%) were significantly higher than control group respectively.The IC(50) at 24, 48, 72 hours were (113±10), (91±8), (68±5) nmol/L; the cell apoptosis rates at 24 hours (10%±4%, 15%±5%, 29%±8%, 46%±8%), 48 hours (16%±5%, 24%±6%, 40%±7%, 55%±9%) and 72 hours (27%±4%, 38%±5%, 50%±9%, 65%±8%) were significantly increased ( P <0.05). Western blot showed that triptolide reduced the expression of p-Akt, p-FOXO3a and increased the expression of Bim, Bax, cleaved-caspase 3.The cell inhibition rate in Triptolide group (30%±8%) was significantly higher than that in the control group ( P <0.05) and the rates in MK2206 group (54% ±6%), FOXO3a-siRNA group (18%±7%) and Bim-siRNA group (11%±6%) were also higher than the control group.Compared with the triptolide group, the inhibition rate in MK2206 group was significantly increased, but decreased in FOXO3a-siRNA group and Bim-siRNA group( P <0.05). Conclusion: Triptolide induces T24 cells apoptosis through FOXO3a-Bim signaling pathway.

Discovery of MK-8718, an HIV Protease Inhibitor Containing a Novel Morpholine Aspartate Binding Group

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bungard, Christopher J.; Williams, Peter D.; Ballard, Jeanine E.

A novel HIV protease inhibitor was designed using a morpholine core as the aspartate binding group. Analysis of the crystal structure of the initial lead bound to HIV protease enabled optimization of enzyme potency and antiviral activity. This afforded a series of potent orally bioavailable inhibitors of which MK-8718 was identified as a compound with a favorable overall profile.

Discovery of chiral dihydropyridopyrimidinones as potent, selective and orally bioavailable inhibitors of AKT.

PubMed

Parthasarathy, Saravanan; Henry, Kenneth; Pei, Huaxing; Clayton, Josh; Rempala, Mark; Johns, Deidre; De Frutos, Oscar; Garcia, Pablo; Mateos, Carlos; Pleite, Sehila; Wang, Yong; Stout, Stephanie; Condon, Bradley; Ashok, Sheela; Lu, Zhohai; Ehlhardt, William; Raub, Tom; Lai, Mei; Geeganage, Sandaruwan; Burkholder, Timothy P

2018-06-01

During the course of our research efforts to develop potent and selective AKT inhibitors, we discovered enatiomerically pure substituted dihydropyridopyrimidinones (DHP) as potent inhibitors of protein kinase B/AKT with excellent selectivity against ROCK 2 . A key challenge in this program was the poor physicochemical properties of the initial lead compound 5. Integration of structure-based drug design and physical properties-based design resulted in replacement of a highly hydrophobic poly fluorinated aryl ring by a simple trifluoromethyl that led to identification of compound 6 with much improved physicochemical properties. Subsequent SAR studies led to the synthesis of new pyran analog 7 with improved cell potency. Further optimization of pharmacokintetics properties by increasing permeability with appropriate fluorinated alkyl led to compound 8 as a potent, selective AKT inhibitors that blocks the phosphorylation of GSK3β in vivo and had robust, dose and concentration dependent efficacy in the U87MG tumor xenograft model. Copyright © 2018 Elsevier Ltd. All rights reserved.

In Vitro Characterization of MK-1439, a Novel HIV-1 Nonnucleoside Reverse Transcriptase Inhibitor

PubMed Central

Feng, Meizhen; Falgueyret, Jean-Pierre; Tawa, Paul; Witmer, Marc; DiStefano, Daniel; Li, Yuan; Burch, Jason; Sachs, Nancy; Lu, Meiqing; Cauchon, Elizabeth; Campeau, Louis-Charles; Grobler, Jay; Yan, Youwei; Ducharme, Yves; Côté, Bernard; Asante-Appiah, Ernest; Hazuda, Daria J.; Miller, Michael D.

2014-01-01

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a mainstay of therapy for treating human immunodeficiency type 1 virus (HIV-1)-infected patients. MK-1439 is a novel NNRTI with a 50% inhibitory concentration (IC50) of 12, 9.7, and 9.7 nM against the wild type (WT) and K103N and Y181C reverse transcriptase (RT) mutants, respectively, in a biochemical assay. Selectivity and cytotoxicity studies confirmed that MK-1439 is a highly specific NNRTI with minimum off-target activities. In the presence of 50% normal human serum (NHS), MK-1439 showed excellent potency in suppressing the replication of WT virus, with a 95% effective concentration (EC95) of 20 nM, as well as K103N, Y181C, and K103N/Y181C mutant viruses with EC95 of 43, 27, and 55 nM, respectively. MK-1439 exhibited similar antiviral activities against 10 different HIV-1 subtype viruses (a total of 93 viruses). In addition, the susceptibility of a broader array of clinical NNRTI-associated mutant viruses (a total of 96 viruses) to MK-1439 and other benchmark NNRTIs was investigated. The results showed that the mutant profile of MK-1439 was superior overall to that of efavirenz (EFV) and comparable to that of etravirine (ETR) and rilpivirine (RPV). Furthermore, E138K, Y181C, and K101E mutant viruses that are associated with ETR and RPV were susceptible to MK-1439 with a fold change (FC) of <3. A two-drug in vitro combination study indicated that MK-1439 acts nonantagonistically in the antiviral activity with each of 18 FDA-licensed drugs for HIV infection. Taken together, these in vitro data suggest that MK-1439 possesses the desired properties for further development as a new antiviral agent. PMID:24379202

Selective Akt Inhibitors Synergize with Tyrosine Kinase Inhibitors and Effectively Override Stroma-Associated Cytoprotection of Mutant FLT3-Positive AML Cells

PubMed Central

Zhang, Xin; Nelson, Erik; Sattler, Martin; Liu, Feiyang; Nicolais, Maria; Zhang, Jianming; Mitsiades, Constantine; Smith, Robert W.; Stone, Richard; Galinsky, Ilene; Nonami, Atsushi; Griffin, James D.; Gray, Nathanael

2013-01-01

Objectives Tyrosine kinase inhibitor (TKI)-treated acute myeloid leukemia (AML) patients commonly show rapid and significant peripheral blood blast cell reduction, however a marginal decrease in bone marrow blasts. This suggests a protective environment and highlights the demand for a better understanding of stromal:leukemia cell communication. As a strategy to improve clinical efficacy, we searched for novel agents capable of potentiating the stroma-diminished effects of TKI treatment of mutant FLT3-expressing cells. Methods We designed a combinatorial high throughput drug screen using well-characterized kinase inhibitor-focused libraries to identify novel kinase inhibitors capable of overriding stromal-mediated resistance to TKIs, such as PKC412 and AC220. Standard liquid culture proliferation assays, cell cycle and apoptosis analysis, and immunoblotting were carried out with cell lines or primary AML to validate putative candidates from the screen and characterize the mechanism(s) underlying observed synergy. Results and Conclusions Our study led to the observation of synergy between selective Akt inhibitors and FLT3 inhibitors against mutant FLT3-positive AML in either the absence or presence of stroma. Our findings are consistent with evidence that Akt activation is characteristic of mutant FLT3-transformed cells, as well as observed residual Akt activity following FLT3 inhibitor treatment. In conclusion, our study highlights the potential importance of Akt as a signaling factor in leukemia survival, and supports the use of the co-culture chemical screen to identify agents able to potentiate TKI anti-leukemia activity in a cytoprotective microenvironment. PMID:23437141

Sequential combination therapy of ovarian cancer with cisplatin and γ-secretase inhibitor MK-0752.

PubMed

Chen, XiuXiu; Gong, LiHua; Ou, RongYing; Zheng, ZhenZhen; Chen, JinYan; Xie, FengFeng; Huang, XiaoXiu; Qiu, JianGe; Zhang, WenJi; Jiang, QiWei; Yang, Yang; Zhu, Hua; Shi, Zhi; Yan, XiaoJian

2016-03-01

Ovarian cancer is one of the most lethal of women cancers and lack potent therapeutic options. There have many evidences demonstrate the Notch signaling has deregulation in variety of human malignancies.MK-0752 is a novel potent γ-secretase inhibitor and now assessed in clinical trial for treatment of several types of cancer, our objective was to investigate the anticancer effects and mechanisms of MK-0752 alone or combined with cisplatin in ovarian cancer. Cell lines used: A2780, OVCAR3, SKOV3, HO8910PM, the effects of MK-0752 and cisplatin on cell proliferation were measured by MTT assay. The effect of combination treatment was examined by isobologram analysis. The distribution of cell cycle and cell apoptosis were analyzed using PI and Annexin V-FITC/PI staining by flow cytometric analysis. The mechanism in biochemistry was analyzed by using Western blot. Mouse xenograft model of A2780 was established to observe the anti-ovarian cancer effects in vivo setting, nude mice were randomized into four groups (n=6 per group) and treated every 4 days with control (solvent) group, MK-0752(25mg/kg) group, cisplatin (2mg/kg)group, combination group (both of MK-0752 and cisplatin). MK-0752 alone actively induced cell growth inhibition, G2/M phase cell cycle arrest and apoptosis with down-regulation of Notch1 and its downstream effectors including Hes1, XIAP, c-Myc and MDM2 in a dose- and time-dependent manner. Moreover, sequential combination of cisplatin prior to MK-0752 significantly promoted cell apoptosis and inhibited the subcutaneous xenograft growth of ovarian cancer in nude mice. Our data supports the sequential combination of cisplatin prior to MK-0752 is a highly promising novel experimental therapeutic strategy against ovarian cancer. Copyright © 2015 Elsevier Inc. All rights reserved.

Structure of the β-form of human MK2 in complex with the non-selective kinase inhibitor TEI-L03090

PubMed Central

Fujino, Aiko; Fukushima, Kei; Kubota, Takaharu; Matsumoto, Yoshiyuki; Takimoto-Kamimura, Midori

2013-01-01

Mitogen-activated protein kinase-activated protein kinase 2 (MK2 or MAPKAP-K2), a serine/threonine kinase from the p38 mitogen-activated protein kinase signalling pathway, plays an important role in the production of TNF-α and other cytokines. In a previous report, it was shown that MK2 in complex with the selective inhibitor TEI-I01800 adopts an α-helical glycine-rich loop that is induced by the stable nonplanar conformer of TEI-I01800. To understand the mechanism of the structural change, the structure of MK2 bound to TEI-L03090, which lacks the key substituent found in TEI-I01800, was determined. MK2–TEI-L03090 has a β-sheet glycine-rich loop in common with other kinases, as predicted. This result suggests that a small compound can induce a drastic conformational change in the target protein structure and can be used to design potent and selective inhibitors. PMID:24316826

40 CFR 85.2205-85.2206 - [Reserved

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 18 2010-07-01 2010-07-01 false [Reserved] 85.2205-85.2206 Section 85.2205-85.2206 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) CONTROL OF AIR POLLUTION FROM MOBILE SOURCES Emission Control System Performance Warranty Short...

Effect of the Angiotensin I Converting Enzyme Inhibitor, MK-421, on Experimentally Induced Drinking

NASA Technical Reports Server (NTRS)

Fregley, Melvin J.; Fater, Dennis C.; Greenleaf, John E.

1982-01-01

MK-421, the ethyl ester maleate salt of N-(S)-1-(ethoxycarbonyl)-3-phenyl-propyl- Ala-L-Pro, is an angiotensin I converting enzyme inhibitor. An initial objective was to determine whether MK-421, administered at 0, 2.5, 5.0, 10.0, 20.0 and 40.0 mg/kg, ip to 96 female rats 15 min prior to administration of the beta-adrenergic agonist, isoproterenol (25 microgram/kg, ip), would inhibit the drinking induced by isoproterenol during 2 h after its administration. The water intake induced by isoproterenol was inhibited significantly by 2.5 mg MK-421/kg. When a similar experiment was performed using Angiotensin I (AI) (200 microgram/kg, ip) as the dipsogenic agent, MK-421 (5 mg/kg, ip), administered 15 min prior to AI, inhibited significantly both the dipsogenic and the diuretic effect of AI. However, administration of angiotensin II (AII, 200 microgram/kg, ip) 15 min after MK-421 (5mg/kg) was accompanied by a water intake that did not differ from AII alone. The drink induced by ip administration of 1.0 m NaCl solution (1% of body wt, ip) was not inhibited by administration of MK-421 (5 mg/kg) 15 min prior to allowing access to water while the drink induced by a 24 h dehydration was partially inhibited. Thus, the drinks induced by administraition of either isoproterenol or AI are dependent on formation of AII. That induced by dehydration is partially dependent, while that induced by hypertonic siilinc is independent of the formation of AII.

Critical role of PI3-kinase/Akt activation in the PARP inhibitor induced heart function recovery during ischemia-reperfusion.

PubMed

Kovacs, Krisztina; Toth, Ambrus; Deres, Peter; Kalai, Tamas; Hideg, Kalman; Gallyas, Ferenc; Sumegi, Balazs

2006-02-14

Poly(ADP-ribose) polymerase (PARP) inhibitors protect hearts from ischemia-reperfusion (IR)-induced damages by limiting nicotinamide adenine dinucleotide (NAD+) and ATP depletion, and by other, not yet elucidated mechanisms. Our preliminary data suggested that PARP catalyzed ADP-ribosylations may affect signaling pathways in cardiomyocytes. To clarify this possibility, we studied the effect of a well-characterized (4-hydroxyquinazoline) and a novel (carboxaminobenzimidazol-derivative) PARP inhibitor on the activation of phosphatidylinositol-3-kinase (PI3-kinase)/Akt pathway in Langendorff-perfused hearts. PARP inhibitors promoted the restoration of myocardial energy metabolism (assessed by 31P nuclear magnetic resonance spectroscopy) and cardiac function compared to untreated hearts. PARP inhibitors also attenuated the infarct size and reduced the IR-induced lipid peroxidation, protein oxidation and total peroxide concentration. Moreover, PARP inhibitors facilitated Akt phosphorylation and activation, as well as the phosphorylation of its downstream target glycogen synthase kinase-3beta (GSK-3beta) in normoxia and, more robustly, during IR. Blocking PI3-kinase by wortmannin or LY294002 reduced the PARP inhibitor-elicited robust Akt and GSK-3beta phosphorylation upon ischemia-reperfusion, and significantly diminished the recovery of ATP and creatine phosphate showing the importance of Akt activation in the recovery of energy metabolism. In addition, inhibition of PI3-kinase/Akt pathway decreased the protective effect of PARP inhibitors on infarct size and the recovery of heart functions. All these data suggest that contrary to the original view, which considered preservation of NAD+ and consequently ATP pools as the exclusive underlying mechanism for the cytoprotective effect of PARP inhibitors, the activation of PI3-kinase/Akt pathway and related processes are at least equally important in the cardioprotective effects of PARP inhibitors during ischemia-reperfusion.

28 CFR 2.206 - Travel approval and transfers of supervision.

Code of Federal Regulations, 2010 CFR

2010-07-01

... supervision. 2.206 Section 2.206 Judicial Administration DEPARTMENT OF JUSTICE PAROLE, RELEASE, SUPERVISION... Supervised Releasees § 2.206 Travel approval and transfers of supervision. (a) A releasee's supervision officer may approve travel outside the district of supervision without approval of the Commission in the...

28 CFR 2.206 - Travel approval and transfers of supervision.

Code of Federal Regulations, 2011 CFR

2011-07-01

... supervision. 2.206 Section 2.206 Judicial Administration DEPARTMENT OF JUSTICE PAROLE, RELEASE, SUPERVISION... Supervised Releasees § 2.206 Travel approval and transfers of supervision. (a) A releasee's supervision officer may approve travel outside the district of supervision without approval of the Commission in the...

28 CFR 2.206 - Travel approval and transfers of supervision.

Code of Federal Regulations, 2013 CFR

2013-07-01

... supervision. 2.206 Section 2.206 Judicial Administration DEPARTMENT OF JUSTICE PAROLE, RELEASE, SUPERVISION... Supervised Releasees § 2.206 Travel approval and transfers of supervision. (a) A releasee's supervision officer may approve travel outside the district of supervision without approval of the Commission in the...

26 CFR 20.2206-1 - Liability of life insurance beneficiaries.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 14 2010-04-01 2010-04-01 false Liability of life insurance beneficiaries. 20.2206-1 Section 20.2206-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY... § 20.2206-1 Liability of life insurance beneficiaries. With respect to the right of the district...

26 CFR 20.2206-1 - Liability of life insurance beneficiaries.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 14 2011-04-01 2010-04-01 true Liability of life insurance beneficiaries. 20.2206-1 Section 20.2206-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY... § 20.2206-1 Liability of life insurance beneficiaries. With respect to the right of the district...

26 CFR 20.2206-1 - Liability of life insurance beneficiaries.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 26 Internal Revenue 14 2014-04-01 2013-04-01 true Liability of life insurance beneficiaries. 20.2206-1 Section 20.2206-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY... § 20.2206-1 Liability of life insurance beneficiaries. With respect to the right of the district...

26 CFR 20.2206-1 - Liability of life insurance beneficiaries.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 26 Internal Revenue 14 2013-04-01 2013-04-01 false Liability of life insurance beneficiaries. 20.2206-1 Section 20.2206-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY... § 20.2206-1 Liability of life insurance beneficiaries. With respect to the right of the district...

26 CFR 20.2206-1 - Liability of life insurance beneficiaries.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 26 Internal Revenue 14 2012-04-01 2012-04-01 false Liability of life insurance beneficiaries. 20.2206-1 Section 20.2206-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY... § 20.2206-1 Liability of life insurance beneficiaries. With respect to the right of the district...

Targeting Nuclear EGFR: Strategies for Improving Cetuximab Therapy in Lung Cancer

DTIC Science & Technology

2015-12-01

xenograft models to determine if, in vivo, SFK or AKT inhibition can block EGFR translocation to the nucleus leading to increased membrane expression...resistant NSCLC tumors using mouse xenografts . Mice bearing resistant tumors will be treated with 1) vehicle, 2) cetuximab, 3) dasatinib, 4) MK2206...phosphorylation of Y1101 and EGFR dimerization with HER3 leading to nuclear translocation. Key Research Accomplishments for Aim 2: • In vivo xenograft tumors

Oxidative stress reduces trophoblast FOXO1 and integrin β3 expression that inhibits cell motility.

PubMed

Chen, Chie-Pein; Chen, Cheng-Yi; Wu, Yi-Hsin; Chen, Chia-Yu

2018-06-08

Preeclampsia is a serious pregnancy complication associated with placental oxidative stress and impaired trophoblast migration. The mechanism of defective trophoblast migration remains unknown. Forkhead box O1 (FOXO1) is a transcription factor. Integrin β3 is involved in cell motility. We hypothesized that FOXO1 mediates expression of trophoblast integrin β3, which could be impaired by oxidative stress and have implications in preeclampsia. The expressions of FOXO1 and integrin β3 were significantly reduced in preeclamptic placentas (n = 15) compared to that of controls (n = 15; p < 0.01). HTR-8/SVneo and JEG-3 trophoblasts were transfected to express wild-type FOXO1-WT or constitutively-expressed nuclear mutant form, FOXO1-AAA. The FOXO1 in HTR-8/SVneo and 3A-Sub-E trophoblasts was silenced by small interfering RNA. AKT-mediated phosphorylation inactivated FOXO1, but FOXO1-AAA was not phosphorylated. The expression of trophoblast integrin β3 was significantly elevated by FOXO1 overexpression and inhibited by FOXO1 knockdown. FOXO1 regulates integrin β3 at the transcriptional level via binding to the putative FOXO1 response element site between position -1154 to -1139 (TGAGATGTTTTGAAAG) in HTR-8/SVneo trophoblasts. The level of phosphorylated FOXO1 was decreased, and the FOXO1 level was increased in trophoblasts treated with AKT inhibitor MK2206, leading to upregulation of integrin β3. The capabilities of trophoblast adhesion and migration were enhanced by FOXO1-overexpression or MK2206, and inhibited by silencing FOXO1 or oxidative stress with H 2 O 2 . These results suggest that FOXO1 enhances trophoblast integrin β3 expression, and mediates cell adhesion and migration. By affecting the expression of FOXO1 and cell motility in trophoblasts, oxidative stress plays a role in the development of preeclampsia. Copyright © 2018 Elsevier Inc. All rights reserved.

Psychotomimetic effects of different doses of MK-801 and the underlying mechanisms in a selective memory impairment model.

PubMed

Liu, Weiqing; Wang, Dong; Hong, Wenjuan; Yu, Yi; Tang, Jinsong; Wang, Jicai; Liu, Fang; Xu, Xiufeng; Tan, Liwen; Chen, Xiaogang

2017-03-01

Although N-methyl-d-aspartate receptor antagonists-induced hypoglutamate rodent models are the most well-established models for preclinical studies of schizophrenia-related deficits, they also evoke a wide spectrum of psychotomimetic side effects. It is significant to increase the specificity of hypoglutamate rodent models. In this study, the recognition memory was evaluated in rats by object recognition test (ORT), sensorimotor gating was evaluated by prepulse inhibition of the startle reflex (PPI), and locomotor activity was measured using open field test. High-performance liquid chromatography was used to measure neurotransmitters content in the medial prefrontal cortex (mPFC) and thalamus (THA). Total Akt and phospho-Akt protein was measured by Western blots. Results showed that 0.3mg/kg of MK-801 was most effective in inducing locomotion. 0.3mg/kg of MK-801 was most effective in decreasing PPI. 0.03mg/kg of MK-801 was most effective in decreasing object memory while not affecting exploration manners in the training session. 0.03mg/kg of MK-801 significantly increased HVA and Glu content in the mPFC. 0.1mg/kg of MK-801 significantly decreased GABA content in the THA. 0.03mg/kg of MK-801 significantly decreased Akt phosphorylation in the mPFC, which was related to the ORT index. In conclusion, a dose of 0.03mg/kg MK-801 can establish a "pure" memory impairment model without contaminations of sensorimotor gating and locomotor activity. MK-801-induced cognitive deficits is associated with increased DA metabolites and glutamate content in the mPFC and decreased GABA content in the THA as well as decrease in Akt phosphorylation in the mPFC. Copyright © 2016. Published by Elsevier B.V.

Akt Inhibitor MK2206 and Hydroxychloroquine in Treating Patients With Advanced Solid Tumors, Melanoma, Prostate or Kidney Cancer

ClinicalTrials.gov

2018-05-15

Adult Solid Neoplasm; Hormone-Resistant Prostate Carcinoma; Recurrent Melanoma; Recurrent Prostate Carcinoma; Recurrent Renal Cell Carcinoma; Stage IIIA Cutaneous Melanoma AJCC v7; Stage IIIB Cutaneous Melanoma AJCC v7; Stage IIIC Cutaneous Melanoma AJCC v7; Stage IV Cutaneous Melanoma AJCC v6 and v7; Stage IV Prostate Cancer AJCC v7; Stage IV Renal Cell Cancer AJCC v7

Evaluation of Biomarkers Predictive of Benefit From PD-1 Inhibitor MK-3475 in Patients with Non-Small Cell Lung Cancer and Brain Metastases

DTIC Science & Technology

2016-07-01

AWARD NUMBER: W81XWH-15-1-0203 TITLE: Evaluation of Biomarkers Predictive of Benefit From PD-1 Inhibitor MK-3475 in Patients with Non-Small...AND SUBTITLE 5a. CONTRACT NUMBER Evaluation of Biomarkers Predictive of Benefit From PD-1 Inhibitor MK-3475 in Patients with Non-Small Cell Lung...axis can result in dramatic responses and durable benefit in patients with non- small cell lung cancer (NSCLC). However, the overall response rate is

CysLTR1 Blockage Ameliorates Liver Injury Caused by Aluminum-Overload via PI3K/AKT/mTOR-Mediated Autophagy Activation in Vivo and in Vitro.

PubMed

Hu, Congli; Yang, Junqing; He, Qin; Luo, Ying; Chen, Zhihao; Yang, Lu; Yi, Honggang; Li, Huan; Xia, Hui; Ran, Dongzhi; Yang, Yang; Zhang, Jiahua; Li, Yuke; Wang, Hong

2018-05-07

Aluminum (Al) is a trivalent cation that can accumulate in animal organs, especially in the liver. We previously demonstrated that Al-overload could induce liver morphologic aberrations and dysfunction. However, the molecular mechanism underlying liver injury caused by Al-overload still remains unknown. In the present study, we investigated the relationship between leukotrienes receptors and the PI3K/AKT/mTOR pathway in Al-induced liver injury in vivo and in vitro. We demonstrated that Al-overload significantly increased the protein expression levels of CysLTR1, PI3K, AKT, mTOR, and p62, while significantly decreasing the LC3BII protein levels in rat liver; thus, suggesting that the autophagy process was inhibited in Al-overloaded rat liver. In addition, MK-571, an inhibitor of CysLTR1, effectively protected the human hepatocyte L02 cells against injury caused by Al exposure. Moreover, CysLTR1 blockage could significantly down-regulate the PI3K/AKT/mTOR pathway and activate autophagy. The effect of MK-571 on cell viability was abolished by the treatment with the autophagy inhibitor (wortmannin) but not with the autophagy agonist (rapamycin). Taken together, our results indicated that the blockage of the leukotriene receptor of CysLTR1 promotes autophagy and further reduces hepatocyte death through the PI3K/AKT/mTOR pathway inhibition. CysLTR1 thus could represent a potential target for the new drug development for chronic noninfective liver injury.

Omarigliptin (MK-3102): A Novel Long-Acting DPP-4 Inhibitor for Once-Weekly Treatment of Type 2 Diabetes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biftu, Tesfaye; Sinha-Roy, Ranabir; Chen, Ping

In our effort to discover DPP-4 inhibitors with added benefits over currently commercially available DPP-4 inhibitors, MK-3102 (omarigliptin), was identified as a potent and selective dipeptidyl peptidase 4 (DPP-4) inhibitor with an excellent pharmacokinetic profile amenable for once-weekly human dosing and selected as a clinical development candidate. This manuscript summarizes the mechanism of action, scientific rationale, medicinal chemistry, pharmacokinetic properties, and human efficacy data for omarigliptin, which is currently in phase 3 clinical development.

Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kizhakkayil, Jaleel; Thayyullathil, Faisal; Chathoth, Shahanas

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 glycogenmore » 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.« less

Microwave-Assisted Synthesis of a MK2 Inhibitor by Suzuki-Miyaura Coupling for Study in Werner Syndrome Cells

PubMed Central

Bagley, Mark C.; Baashen, Mohammed; Chuckowree, Irina; Dwyer, Jessica E.; Kipling, David; Davis, Terence

2015-01-01

Microwave-assisted Suzuki-Miyaura cross-coupling reactions have been employed towards the synthesis of three different MAPKAPK2 (MK2) inhibitors to study accelerated aging in Werner syndrome (WS) cells, including the cross-coupling of a 2-chloroquinoline with a 3-pyridinylboronic acid, the coupling of an aryl bromide with an indolylboronic acid and the reaction of a 3-amino-4-bromopyrazole with 4-carbamoylphenylboronic acid. In all of these processes, the Suzuki-Miyaura reaction was fast and relatively efficient using a palladium catalyst under microwave irradiation. The process was incorporated into a rapid 3-step microwave-assisted method for the synthesis of a MK2 inhibitor involving 3-aminopyrazole formation, pyrazole C-4 bromination using N-bromosuccinimide (NBS), and Suzuki-Miyaura cross-coupling of the pyrazolyl bromide with 4-carbamoylphenylboronic acid to give the target 4-arylpyrazole in 35% overall yield, suitable for study in WS cells. PMID:26046488

Microwave-Assisted Synthesis of a MK2 Inhibitor by Suzuki-Miyaura Coupling for Study in Werner Syndrome Cells.

PubMed

Bagley, Mark C; Baashen, Mohammed; Chuckowree, Irina; Dwyer, Jessica E; Kipling, David; Davis, Terence

2015-06-03

Microwave-assisted Suzuki-Miyaura cross-coupling reactions have been employed towards the synthesis of three different MAPKAPK2 (MK2) inhibitors to study accelerated aging in Werner syndrome (WS) cells, including the cross-coupling of a 2-chloroquinoline with a 3-pyridinylboronic acid, the coupling of an aryl bromide with an indolylboronic acid and the reaction of a 3-amino-4-bromopyrazole with 4-carbamoylphenylboronic acid. In all of these processes, the Suzuki-Miyaura reaction was fast and relatively efficient using a palladium catalyst under microwave irradiation. The process was incorporated into a rapid 3-step microwave-assisted method for the synthesis of a MK2 inhibitor involving 3-aminopyrazole formation, pyrazole C-4 bromination using N-bromosuccinimide (NBS), and Suzuki-Miyaura cross-coupling of the pyrazolyl bromide with 4-carbamoylphenylboronic acid to give the target 4-arylpyrazole in 35% overall yield, suitable for study in WS cells.

Metabolic biomarkers for response to PI3K inhibition in basal-like breast cancer

PubMed Central

2013-01-01

Introduction The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in cancer cells through numerous mutations and epigenetic changes. The recent development of inhibitors targeting different components of the PI3K pathway may represent a valuable treatment alternative. However, predicting efficacy of these drugs is challenging, and methods for therapy monitoring are needed. Basal-like breast cancer (BLBC) is an aggressive breast cancer subtype, frequently associated with PI3K pathway activation. The objectives of this study were to quantify the PI3K pathway activity in tissue sections from xenografts representing basal-like and luminal-like breast cancer before and immediately after treatment with PI3K inhibitors, and to identify metabolic biomarkers for treatment response. Methods Tumor-bearing animals (n = 8 per treatment group) received MK-2206 (120 mg/kg/day) or BEZ235 (50 mg/kg/day) for 3 days. Activity in the PI3K/Akt/mammalian target of rapamycin pathway in xenografts and human biopsies was evaluated using a novel method for semiquantitative assessment of Aktser473 phosphorylation. Metabolic changes were assessed by ex vivo high-resolution magic angle spinning magnetic resonance spectroscopy. Results Using a novel dual near-infrared immunofluorescent imaging method, basal-like xenografts had a 4.5-fold higher baseline level of pAktser473 than luminal-like xenografts. Following treatment, basal-like xenografts demonstrated reduced levels of pAktser473 and decreased proliferation. This correlated with metabolic changes, as both MK-2206 and BEZ235 reduced lactate concentration and increased phosphocholine concentration in the basal-like tumors. BEZ235 also caused increased glucose and glycerophosphocholine concentrations. No response to treatment or change in metabolic profile was seen in luminal-like xenografts. Analyzing tumor sections from five patients with BLBC demonstrated that two of these patients had an elevated pAktser473 level

Clinical Response of Carcinomas Harboring the BRD4-NUT Oncoprotein to the Targeted Bromodomain Inhibitor OTX015/MK-8628

PubMed Central

Stathis, Anastasios; Zucca, Emanuele; Bekradda, Mohamed; Gomez-Roca, Carlos; Delord, Jean-Pierre; de La Motte Rouge, Thibault; Uro-Coste, Emmanuelle; de Braud, Filippo; Pelosi, Giuseppe; French, Christopher A.

2016-01-01

The anti-neoplastic, pro-differentiative effects of bromodomain and extra-terminal (BET) bromodomain (BRD) inhibitors were initially discovered in NUT midline carcinoma (NMC), an aggressive subtype of squamous cancer driven by the BRD4-NUT fusion oncoprotein. BRD4-NUT blocks differentiation and maintains tumor growth through a potent chromatin modifying mechanism. OTX015/MK-8628, a novel oral BET inhibitor, targets BRD2/3/4/T with preclinical activity in NMC and several other tumor types, and is currently in clinical development. Antitumor activity was evaluated in four advanced stage NMC patients with confirmed BRD4-NUT fusions who were treated with 80 mg OTX015/MK-8628 once daily in a compassionate-use context. Two patients responded rapidly with tumor regression and symptomatic relief, and a third had meaningful disease stabilization with a minor metabolic response. The main side effects were mild to moderate gastrointestinal toxicity and fatigue, and reversible grade 3 thrombocytopenia. This is the first proof-of-concept evidence of clinical activity of a bromodomain inhibitor in targeting BRD4-NUT. PMID:26976114

Discovery and preclinical pharmacology of a selective ATP-competitive Akt inhibitor (GDC-0068) for the treatment of human tumors.

PubMed

Blake, James F; Xu, Rui; Bencsik, Josef R; Xiao, Dengming; Kallan, Nicholas C; Schlachter, Stephen; Mitchell, Ian S; Spencer, Keith L; Banka, Anna L; Wallace, Eli M; Gloor, Susan L; Martinson, Matthew; Woessner, Richard D; Vigers, Guy P A; Brandhuber, Barbara J; Liang, Jun; Safina, Brian S; Li, Jun; Zhang, Birong; Chabot, Christine; Do, Steven; Lee, Leslie; Oeh, Jason; Sampath, Deepak; Lee, Brian B; Lin, Kui; Liederer, Bianca M; Skelton, Nicholas J

2012-09-27

The discovery and optimization of a series of 6,7-dihydro-5H-cyclopenta[d]pyrimidine compounds that are ATP-competitive, selective inhibitors of protein kinase B/Akt is reported. The initial design and optimization was guided by the use of X-ray structures of inhibitors in complex with Akt1 and the closely related protein kinase A. The resulting compounds demonstrate potent inhibition of all three Akt isoforms in biochemical assays and poor inhibition of other members of the cAMP-dependent protein kinase/protein kinase G/protein kinase C extended family and block the phosphorylation of multiple downstream targets of Akt in human cancer cell lines. Biological studies with one such compound, 28 (GDC-0068), demonstrate good oral exposure resulting in dose-dependent pharmacodynamic effects on downstream biomarkers and a robust antitumor response in xenograft models in which the phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin pathway is activated. 28 is currently being evaluated in human clinical trials for the treatment of cancer.

Swertisin ameliorates pre-pulse inhibition deficits and cognitive impairment induced by MK-801 in mice.

PubMed

Oh, Hee Kyong; Jeon, Se Jin; Lee, Sunhee; Lee, Hyung Eun; Kim, Eunji; Park, Se Jin; Kim, Ha Neul; Jung, Won Yong; Cheong, Jae Hoon; Jang, Dae Sik; Ryu, Jong Hoon

2017-02-01

Swertisin, a plant-derived C-glucosylflavone, is known to have antidiabetic, anti-inflammatory and antioxidant effects. In the present study, we investigated in mice the effects of swertisin on glutamatergic dysfunction induced by dizocilpine (MK-801), a non-competitive N-methyl-D-aspartate receptor antagonist. In the Acoustic Startle Response test, their MK-801-induced (given 0.2 mg/kg i.p.) pre-pulse inhibition deficit was significantly attenuated by the administration of swertisin (30 mg/kg p.o.). In the Novel Object Recognition Test, the recognition memory impairments that were induced by MK-801 (0.2 mg/kg, given i.p.) were also reversed by administration of swertisin (30 mg/kg p.o.). In addition, swertisin normalized the MK-801-induced elevation of phosphorylation levels of Akt and GSK-3β signaling molecules in the prefrontal cortex. These results indicated that swertisin may be useful in managing the symptoms of schizophrenia, including sensorimotor gating disruption and cognitive impairment, and that these behavioral outcomes may be related to Akt-GSK-3β signaling in the prefrontal cortex.

The Akt-inhibitor Erufosine induces apoptotic cell death in prostate cancer cells and increases the short term effects of ionizing radiation.

PubMed

Rudner, Justine; Ruiner, Carola-Ellen; Handrick, René; Eibl, Hans-Jörg; Belka, Claus; Jendrossek, Verena

2010-11-16

The phosphatidylinositol-3-kinase (PI3K)/Akt pathway is frequently deregulated in prostate cancer and associated with neoplastic transformation, malignant progression, and enhanced resistance to classical chemotherapy and radiotherapy. Thus, it is a promising target for therapeutic intervention. In the present study, the cytotoxic action of the Akt inhibitor Erufosine (ErPC3) was analyzed in prostate cancer cells and compared to the cytotoxicity of the PI3K inhibitor LY294002. Moreover, the efficacy of combined treatment with Akt inhibitors and ionizing radiation in prostate cancer cells was examined. Prostate cancer cell lines PC3, DU145, and LNCaP were treated with ErPC3 (1-100 µM), LY294002 (25-100 µM), irradiated (0-10 Gy), or subjected to combined treatments. Cell viability was determined by the WST-1 assay. Apoptosis induction was analyzed by flow cytometry after staining with propidium iodide in a hypotonic citrate buffer, and by Western blotting using antibodies against caspase-3 and its substrate PARP. Akt activity and regulation of the expression of Bcl-2 family members and key downstream effectors involved in apoptosis regulation were examined by Western blot analysis. The Akt inhibitor ErPC3 exerted anti-neoplastic effects in prostate cancer cells, however with different potency. The anti-neoplastic action of ErPC3 was associated with reduced phosphoserine 473-Akt levels and induction of apoptosis. PC3 and LNCaP prostate cancer cells were also sensitive to treatment with the PI3K inhibitor LY294002. However, the ErPC3-sensitive PC3-cells were less susceptible to LY294002 than the ErPC3-refractory LNCaP cells. Although both cell lines were largely resistant to radiation-induced apoptosis, both cell lines showed higher levels of apoptotic cell death when ErPC3 was combined with radiotherapy. Our data suggest that constitutive Akt activation and survival are controlled by different different molecular mechanisms in the two prostate cancer cell lines

Sensitivity to PI3K and AKT inhibitors is mediated by divergent molecular mechanisms in subtypes of DLBCL.

PubMed

Erdmann, Tabea; Klener, Pavel; Lynch, James T; Grau, Michael; Vočková, Petra; Molinsky, Jan; Tuskova, Diana; Hudson, Kevin; Polanska, Urszula M; Grondine, Michael; Mayo, Michele; Dai, Beiying; Pfeifer, Matthias; Erdmann, Kristian; Schwammbach, Daniela; Zapukhlyak, Myroslav; Staiger, Annette M; Ott, German; Berdel, Wolfgang E; Davies, Barry R; Cruzalegui, Francisco; Trneny, Marek; Lenz, Peter; Barry, Simon T; Lenz, Georg

2017-07-20

Activated B-cell-like (ABC) and germinal center B-cell-like diffuse large B-cell lymphoma (DLBCL) represent the 2 major molecular DLBCL subtypes. They are characterized by differences in clinical course and by divergent addiction to oncogenic pathways. To determine activity of novel compounds in these 2 subtypes, we conducted an unbiased pharmacologic in vitro screen. The phosphatidylinositol-3-kinase (PI3K) α/δ (PI3Kα/δ) inhibitor AZD8835 showed marked potency in ABC DLBCL models, whereas the protein kinase B (AKT) inhibitor AZD5363 induced apoptosis in PTEN-deficient DLBCLs irrespective of their molecular subtype. These in vitro results were confirmed in various cell line xenograft and patient-derived xenograft mouse models in vivo. Treatment with AZD8835 induced inhibition of nuclear factor κB signaling, prompting us to combine AZD8835 with the Bruton's tyrosine kinase inhibitor ibrutinib. This combination was synergistic and effective both in vitro and in vivo. In contrast, the AKT inhibitor AZD5363 was effective in PTEN-deficient DLBCLs through downregulation of the oncogenic transcription factor MYC. Collectively, our data suggest that patients should be stratified according to their oncogenic dependencies when treated with PI3K and AKT inhibitors. © 2017 by The American Society of Hematology.

21 CFR 74.2206 - D&C Green No. 6.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 1 2011-04-01 2011-04-01 false D&C Green No. 6. 74.2206 Section 74.2206 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR....1206 (a) and (b). (b) Uses and restrictions. D&C Green No. 6 may be safely used for coloring externally...

Sorafenib induces cathepsin B-mediated apoptosis of bladder cancer cells by regulating the Akt/PTEN pathway. The Akt inhibitor, perifosine, enhances the sorafenib-induced cytotoxicity against bladder cancer cells

PubMed Central

Amantini, Consuelo; Morelli, Maria Beatrice; Santoni, Matteo; Soriani, Alessandra; Cardinali, Claudio; Farfariello, Valerio; Eleuteri, Anna Maria; Bonfili, Laura; Mozzicafreddo, Matteo; Nabissi, Massimo; Cascinu, Stefano; Santoni, Giorgio

2015-01-01

Sorafenib, a tyrosine kinase inhibitor, has been demonstrated to exert anti-tumor effects. However, the molecular mechanisms underlying its effects on bladder cancer remain unknown. Here, we evaluated the mechanisms responsible for the sorafenib-induced anti-tumor effects on 5637 and T24 bladder cancer cells. We demonstrated that sorafenib reduces cell viability, stimulates lysosome permeabilization and induces apoptosis of bladder cancer cells. These effects are dependent by the activation of cathepsin B released from lysosomes. The sorafenib-increased cathepsin B activity induced the proteolysis of Bid into tBid that stimulates the intrinsic pathway of apoptosis characterized by mitochondrial membrane depolarization, oxygen radical generation and cytochrome c release. Moreover, we found that cathepsin B enzymatic activity, induced by sorafenib, is dependent on its dephosphorylation via PTEN activation and Akt inactivation. Pretreatment with orthovanadate rescued bladder cancer cells from apoptosis. In addition, the Akt inhibitor perifosine increased the sensitivity of bladder cancer cells to sorafenib-induced cytotoxicity. Overall, our results show that apoptotic cell death induced by sorafenib in bladder cancer cells is dependent on cathepsin B activity and involved PTEN and Akt signaling pathways. The Akt inhibitor perifosine increased the cytotoxic effects of sorafenib in bladder cancer cells. PMID:26097873

Preclinical Characterization of the Phosphodiesterase 10A PET Tracer [(11)C]MK-8193.

PubMed

Hostetler, Eric D; Fan, Hong; Joshi, Aniket D; Zeng, Zhizhen; Eng, Waisi; Gantert, Liza; Holahan, Marie; Meng, Xianjun; Miller, Patricia; O'Malley, Stacey; Purcell, Mona; Riffel, Kerry; Salinas, Cristian; Williams, Mangay; Ma, Bennett; Buist, Nicole; Smith, Sean M; Coleman, Paul J; Cox, Christopher D; Flores, Brock A; Raheem, Izzat T; Cook, Jacquelynn J; Evelhoch, Jeffrey L

2016-08-01

A positron emission tomography (PET) tracer for the enzyme phosphodiesterase 10A (PDE10A) is desirable to guide the discovery and development of PDE10A inhibitors as potential therapeutics. The preclinical characterization of the PDE10A PET tracer [(11)C]MK-8193 is described. In vitro binding studies with [(3)H]MK-8193 were conducted in rat, monkey, and human brain tissue. PET studies with [(11)C]MK-8193 were conducted in rats and rhesus monkeys at baseline and following administration of a PDE10A inhibitor. [(3)H]MK-8193 is a high-affinity, selective PDE10A radioligand in rat, monkey, and human brain tissue. In vivo, [(11)C]MK-8193 displays rapid kinetics, low test-retest variability, and a large specific signal that is displaced by a structurally diverse PDE10A inhibitor, enabling the determination of pharmacokinetic/enzyme occupancy relationships. [(11)C]MK-8193 is a useful PET tracer for the preclinical characterization of PDE10A therapeutic candidates in rat and monkey. Further evaluation of [(11)C]MK-8193 in humans is warranted.

Specific gene expression signatures induced by the multiple oncogenic alterations that occur within the PTEN/PI3K/AKT pathway in lung cancer.

PubMed

De Marco, Carmela; Laudanna, Carmelo; Rinaldo, Nicola; Oliveira, Duarte Mendes; Ravo, Maria; Weisz, Alessandro; Ceccarelli, Michele; Caira, Elvira; Rizzuto, Antonia; Zoppoli, Pietro; Malanga, Donatella; Viglietto, Giuseppe

2017-01-01

Hyperactivation of the phosphatydil-inositol-3' phosphate kinase (PI3K)/AKT pathway is observed in most NSCLCs, promoting proliferation, migration, invasion and resistance to therapy. AKT can be activated through several mechanisms that include loss of the negative regulator PTEN, activating mutations of the catalytic subunit of PI3K (PIK3CA) and/or mutations of AKT1 itself. However, number and identity of downstream targets of activated PI3K/AKT pathway are poorly defined. To identify the genes that are targets of constitutive PI3K/AKT signalling in lung cancer cells, we performed a comparative transcriptomic analysis of human lung epithelial cells (BEAS-2B) expressing active mutant AKT1 (AKT1-E17K), active mutant PIK3CA (PIK3CA-E545K) or that are silenced for PTEN. We found that, altogether, aberrant PI3K/AKT signalling in lung epithelial cells regulated the expression of 1,960/20,436 genes (9%), though only 30 differentially expressed genes (DEGs) (15 up-regulated, 12 down-regulated and 3 discordant) out of 20,436 that were common among BEAS-AKT1-E17K, BEAS-PIK3CA-E545K and BEAS-shPTEN cells (0.1%). Conversely, DEGs specific for mutant AKT1 were 133 (85 up-regulated; 48 down-regulated), DEGs specific for mutant PIK3CA were 502 (280 up-regulated; 222 down-regulated) and DEGs specific for PTEN loss were 1549 (799 up-regulated, 750 down-regulated). The results obtained from array analysis were confirmed by quantitative RT-PCR on selected up- and down-regulated genes (n = 10). Treatment of BEAS-C cells and the corresponding derivatives with pharmacological inhibitors of AKT (MK2206) or PI3K (LY294002) further validated the significance of our findings. Moreover, mRNA expression of selected DEGs (SGK1, IGFBP3, PEG10, GDF15, PTGES, S100P, respectively) correlated with the activation status of the PI3K/AKT pathway assessed by S473 phosphorylation in NSCLC cell lines (n = 6). Finally, we made use of Ingenuity Pathway Analysis (IPA) to investigate the relevant Bio

Inhibition of the protein kinase MK-2 protects podocytes from nephrotic syndrome-related injury

PubMed Central

Pengal, Ruma; Guess, Adam J.; Agrawal, Shipra; Manley, Joshua; Ransom, Richard F.; Mourey, Robert J.; Smoyer, William E.

2011-01-01

While mitogen-activated protein kinase (MAPK) activation has been implicated in the pathogenesis of various glomerular diseases, including nephrotic syndrome (NS), its specific role in podocyte injury is not known. We hypothesized that MK-2, a downstream substrate of p38 MAPK, mediates the adverse effects of this pathway and that inhibition of MK-2 would protect podocytes from NS-related injury. Using cultured podocytes, we analyzed 1) the roles of MK-2 and p38 MAPK in puromycin aminonucleoside (PAN)-induced podocyte injury; 2) the ability of specific MK-2 and p38 MAPK inhibitors to protect podocytes against injury; 3) the role of serum albumin, known to induce podocyte injury, in activating p38 MAPK/MK-2 signaling; and 4) the role of p38 MAPK/MK-2 signaling in the expression of Cox-2, an enzyme associated with podocyte injury. Treatment with protein kinase inhibitors specific for both MK-2 (C23, a pyrrolopyridine-type compound) or p38 MAPK (SB203580) reduced PAN-induced podocyte injury and actin cytoskeletal disruption. Both inhibitors reduced baseline podocyte p38 MAPK/MK-2 signaling, as measured by the degree of phosphorylation of HSPB1, a downstream substrate of MK-2, but exhibited disparate effects on upstream signaling. Serum albumin activated p38 MAPK/MK-2 signaling and induced Cox-2 expression, and these responses were blocked by both inhibitors. Given the critical importance of podocyte injury to both NS and other progressive glomerular diseases, these data suggest an important role for p38 MAPK/MK-2 signaling in podocyte injury and identify MK-2 inhibition as a promising potential therapeutic strategy to protect podocytes in various glomerular diseases. PMID:21613416

Heightening energetic stress selectively targets LKB1-deficient non-small cell lung cancers

PubMed Central

Momcilovic, Milica; McMickle, Robert; Abt, Evan; Seki, Atsuko; Simko, Sarah A.; Magyar, Clara; Stout, David B.; Fishbein, Michael C.; Walser, Tonya C.; Dubinett, Steven M.; Shackelford, David B.

2015-01-01

Inactivation of the LKB1 tumor suppressor is a frequent event in non-small cell lung carcinoma (NSCLC) leading to the activation of mammalian target of rapamycin complex 1 (mTORC1) and sensitivity to the metabolic stress inducer phenformin. In this study, we explored the combinatorial use of phenformin with the mTOR catalytic kinase inhibitor MLN0128 as a treatment strategy for NSCLC bearing co-mutations in the LKB1 and KRAS genes. NSCLC is a genetically and pathologically heterogeneous disease, giving rise to lung tumors of varying histologies that include adenocarcinomas (ADCs) and squamous cell carcinomas (SCCs). We demonstrate that phenformin in combination with MLN0128 induced a significant therapeutic response in KRAS/LKB1 mutant human cell lines and genetically engineered mouse models of NSCLC that develop both ADCs and SCCs. Specifically, we found that KRAS/LKB1 mutant lung ADCs responded strongly to phenformin + MLN0128 treatment, but the response of SCCs to single or combined treatment with MLN0128 was more attenuated due to acquired resistance to mTOR inhibition through modulation of the AKT-GSK signaling axis. Combinatorial use of the mTOR inhibitor and AKT inhibitor MK2206 robustly inhibited the growth and viability of squamous lung tumors thus providing an effective strategy to overcome resistance. Taken together, our findings define new personalized therapeutic strategies that may be rapidly translated into clinical use for the treatment of KRAS/LKB1 mutant adenocarcinomas and squamous cell tumors. PMID:26574479

Heightening Energetic Stress Selectively Targets LKB1-Deficient Non-Small Cell Lung Cancers.

PubMed

Momcilovic, Milica; McMickle, Robert; Abt, Evan; Seki, Atsuko; Simko, Sarah A; Magyar, Clara; Stout, David B; Fishbein, Michael C; Walser, Tonya C; Dubinett, Steven M; Shackelford, David B

2015-11-15

Inactivation of the LKB1 tumor suppressor is a frequent event in non-small cell lung carcinoma (NSCLC) leading to the activation of mTOR complex 1 (mTORC1) and sensitivity to the metabolic stress inducer phenformin. In this study, we explored the combinatorial use of phenformin with the mTOR catalytic kinase inhibitor MLN0128 as a treatment strategy for NSCLC bearing comutations in the LKB1 and KRAS genes. NSCLC is a genetically and pathologically heterogeneous disease, giving rise to lung tumors of varying histologies that include adenocarcinomas and squamous cell carcinomas (SCC). We demonstrate that phenformin in combination with MLN0128 induced a significant therapeutic response in KRAS/LKB1-mutant human cell lines and genetically engineered mouse models of NSCLC that develop both adenocarcinomas and SCCs. Specifically, we found that KRAS/LKB1-mutant lung adenocarcinomas responded strongly to phenformin + MLN0128 treatment, but the response of SCCs to single or combined treatment with MLN0128 was more attenuated due to acquired resistance to mTOR inhibition through modulation of the AKT-GSK signaling axis. Combinatorial use of the mTOR inhibitor and AKT inhibitor MK2206 robustly inhibited the growth and viability of squamous lung tumors, thus providing an effective strategy to overcome resistance. Taken together, our findings define new personalized therapeutic strategies that may be rapidly translated into clinical use for the treatment of KRAS/LKB1-mutant adenocarcinomas and squamous cell tumors. ©2015 American Association for Cancer Research.

Pan-Pim Kinase Inhibitor AZD1208 Suppresses Tumor Growth and Synergistically Interacts with Akt Inhibition in Gastric Cancer Cells.

PubMed

Lee, Miso; Lee, Kyung-Hun; Min, Ahrum; Kim, Jeongeun; Kim, Seongyeong; Jang, Hyemin; Lim, Jee Min; Kim, So Hyeon; Ha, Dong-Hyeon; Jeong, Won Jae; Suh, Koung Jin; Yang, Yae-Won; Kim, Tae Yong; Oh, Do-Youn; Bang, Yung-Jue; Im, Seock-Ah

2018-06-06

Pim kinases are highly conserved serine/threonine kinases, and different expression patterns of each isoform (Pim-1, Pim-2, and Pim-3) have been observed in various types of human cancers, including gastric cancer. AZD1208 is a potent and selective inhibitor that affects all three isoforms of Pim. We investigated the effects of AZD1208 as a single agent and in combination with an Akt inhibitor in gastric cancer cells. The antitumor activity of AZD1208 with/without an Akt inhibitor was evaluated in a large panel of gastric cancer cell lines through growth inhibition assays. The underlying mechanism was also examined by western blotting, immunofluorescence assay, and cell cycle analysis. AZD1208 treatment decreased gastric cancer cell proliferation rates and induced autophagy only in long-term culture systems. Light chain 3B (LC3B), a marker of autophagy, was increased in sensitive cells in a dose-dependent manner with AZD1208 treatment, which suggested that the growth inhibition effect of AZD1208 was achieved through autophagy, not apoptosis. Moreover, we found that cells damaged by Pim inhibition were repaired by activation of the DNA damage repair pathway, which promoted cell survival and led the cells to become resistant to AZD1208. We also confirmed that the combination of an Akt inhibitor with AZD1208 produced a highly synergistic effect in gastric cancer cell lines. Treatment with AZD1208 alone induced considerable cell death through autophagy in gastric cancer cells. Moreover, the combination of AZD1208 with an Akt inhibitor showed synergistic antitumor effects through regulation of the DNA damage repair pathway.

Akt Inhibitor MK-2206 and Anastrozole With or Without Goserelin Acetate in Treating Patients With Stage II-III Breast Cancer

ClinicalTrials.gov

2018-04-06

Estrogen Receptor Positive; HER2/Neu Negative; Recurrent Breast Carcinoma; Stage IIA Breast Cancer; Stage IIB Breast Cancer; Stage IIIA Breast Cancer; Stage IIIB Breast Cancer; Stage IIIC Breast Cancer

Estradiol up-regulates L-type Ca2+ channels via membrane-bound estrogen receptor/phosphoinositide-3-kinase/Akt/cAMP response element-binding protein signaling pathway.

PubMed

Yang, Xiaoyan; Mao, Xiaofang; Xu, Gao; Xing, Shasha; Chattopadhyay, Ansuman; Jin, Si; Salama, Guy

2018-05-01

In long QT syndrome type 2, women are more prone than men to the lethal arrhythmia torsades de pointes. We previously reported that 17β-estradiol (E2) up-regulates L-type Ca 2+ channels and current (I Ca,L ) (∼30%) in rabbit ventricular myocytes by a classic genomic mechanism mediated by estrogen receptor-α (ERα). In long QT syndrome type 2 (I Kr blockade or bradycardia), the higher Ca 2+ influx via I Ca,L causes Ca 2+ overload, spontaneous sarcoplasmic reticulum Ca 2+ release, and reactivation of I Ca,L that triggers early afterdepolarizations and torsades de pointes. The purpose of this study was to investigate the molecular mechanisms whereby E2 up-regulates I Ca,L , which are poorly understood. H9C2 and rat myocytes were incubated with E2 ± ER antagonist, or inhibitors of downstream transcription factors, for 24 hours, followed by western blots of Cav1.2α1C and voltage-clamp measurements of I Ca,L . Incubation of H9C2 cells with E2 (10-100 nM) increased I Ca,L density and Cav1.2α1C expression, which were suppressed by the ER antagonist ICI182,780 (1 μM). Enhanced I Ca,L and Cav1.2α1C expression by E2 was suppressed by inhibitors of phosphoinositide-3-kinase (Pi3K) (30 μM LY294002; P <.05) and Akt (5 μM MK2206) but not of mitogen-activated protein kinase (5 μM U0126) or protein kinase A (1 μM KT5720). E2 incubation increased p-CREB via the Pi3K/Akt pathway, reached a peak in 20 minutes (3-fold), and leveled off to 1.5-fold 24 hours later. Furthermore, a CREB decoy oligonucleotide inhibited E2-induced Cav1.2α1C expression, whereas membrane-impermeable E2 (E2-bovine serum albumin) was equally effective at Cav1.2α1C up-regulation as E2. Estradiol up-regulates Cav1.2α1C and I Ca,L via plasma membrane ER and by activating Pi3K, Akt, and CREB signaling. The promoter regions of the CACNA1C gene (human-rabbit-rat) contain adjacent/overlapping binding sites for p-CREB and ERα, which suggests a synergistic regulation by these pathways. Copyright © 2018

Targeting of X-linked inhibitor of apoptosis protein and PI3-kinase/AKT signaling by embelin suppresses growth of leukemic cells

PubMed Central

Prabhu, Kirti S.; Siveen, Kodappully S.; Kuttikrishnan, Shilpa; Iskandarani, Ahmad; Tsakou, Magdalini; Achkar, Iman W.; Therachiyil, Lubna; Krishnankutty, Roopesh; Parray, Aijaz; Kulinski, Michal; Merhi, Maysaloun; Dermime, Said; Mohammad, Ramzi M.

2017-01-01

The X-linked inhibitor of apoptosis (XIAP) is a viable molecular target for anticancer drugs that overcome apoptosis-resistance of malignant cells. XIAP is an inhibitor of apoptosis, mediating through its association with BIR3 domain of caspase 9. Embelin, a quinone derivative isolated from the Embelia ribes plant, has been shown to exhibit chemopreventive, anti-inflammatory, and apoptotic activities via inhibiting XIAP activity. In this study, we found that embelin causes a dose-dependent suppression of proliferation in leukemic cell lines K562 and U937. Embelin mediated inhibition of proliferation correlates with induction of apoptosis. Furthermore, embelin treatment causes loss of mitochondrial membrane potential and release of cytochrome c, resulting in subsequent activation of caspase-3 followed by polyadenosin-5’-diphosphate-ribose polymerase (PARP) cleavage. In addition, embelin treatment of leukemic cells results in a decrease of constitutive phosphorylations/activation level of AKT and downregulation of XIAP. Gene silencing of XIAP and AKT expression showed a link between XIAP expression and activated AKT in leukemic cells. Interestingly, targeting of XIAP and PI3-kinase/AKT signaling augmented inhibition of proliferation and induction of apoptosis in leukemic cells. Altogether these findings raise the possibility that embelin alone or in combination with inhibitors of PI3-kinase/AKT pathway may have therapeutic usage in leukemia and possibly other malignancies with up-regulated XIAP pathway. PMID:28704451

In vivo analysis of insulin-like growth factor type 1 receptor humanized monoclonal antibody MK-0646 and small molecule kinase inhibitor OSI-906 in colorectal cancer

PubMed Central

LEIPHRAKPAM, PREMILA D.; AGARWAL, EKTA; MATHIESEN, MICHELLE; HAFERBIER, KATIE L.; BRATTAIN, MICHAEL G.; CHOWDHURY, SANJIB

2014-01-01

The development and characterization of effective anticancer drugs against colorectal cancer (CRC) is of urgent need since it is the second most common cause of cancer death. The study was designed to evaluate the effects of two IGF-1R antagonists, MK-0646, a recombinant fully humanized monoclonal antibody and OSI-906, a small molecule tyrosine kinase inhibitor on CRC cells. Xenograft study was performed on IGF-1R-dependent CRC cell lines for analyzing the antitumor activity of MK-0646 and OSI-906. Tumor proliferation and apoptosis were assessed using Ki67 and TUNEL assays, respectively. We also performed in vitro characterization of MK-0646 and OSI-906 treatment on CRC cells to identify mechanisms associated with drug-induced cell death. Exposure of the GEO and CBS tumor xenografts to MK-0646 or OSI-906 led to a decrease in tumor growth. TUNEL analysis showed an increase of approximately 45–55% in apoptotic cells in both MK-0646 and OSI-906 treated tumor samples. We report the novel finding that treatment with IGF-1R antagonists led to downregulation of X-linked inhibitor of apoptosis (XIAP) protein involved in cell survival and inhibition of cell death. In conclusion, IGF-1R antagonists (MK-0646 and OSI-906) demonstrated single agent inhibition of subcutaneous CRC xenograft growth. This was coupled to pro-apoptotic effects resulting in downregulation of XIAP and inhibition of cell survival. We report a novel mechanism by which MK-0646 and OSI-906 elicits cell death in vivo and in vitro. Moreover, these results indicate that MK-0646 and OSI-906 may be potential anticancer candidates for the treatment of patients with IGF-1R-dependent CRC. PMID:24173770

In vivo analysis of insulin-like growth factor type 1 receptor humanized monoclonal antibody MK-0646 and small molecule kinase inhibitor OSI-906 in colorectal cancer.

PubMed

Leiphrakpam, Premila D; Agarwal, Ekta; Mathiesen, Michelle; Haferbier, Katie L; Brattain, Michael G; Chowdhury, Sanjib

2014-01-01

The development and characterization of effective anticancer drugs against colorectal cancer (CRC) is of urgent need since it is the second most common cause of cancer death. The study was designed to evaluate the effects of two IGF-1R antagonists, MK-0646, a recombinant fully humanized monoclonal antibody and OSI-906, a small molecule tyrosine kinase inhibitor on CRC cells. Xenograft study was performed on IGF-1R-dependent CRC cell lines for analyzing the antitumor activity of MK-0646 and OSI-906. Tumor proliferation and apoptosis were assessed using Ki67 and TUNEL assays, respectively. We also performed in vitro characterization of MK-0646 and OSI-906 treatment on CRC cells to identify mechanisms associated with drug-induced cell death. Exposure of the GEO and CBS tumor xenografts to MK-0646 or OSI-906 led to a decrease in tumor growth. TUNEL analysis showed an increase of approximately 45-55% in apoptotic cells in both MK-0646 and OSI-906 treated tumor samples. We report the novel finding that treatment with IGF-1R antagonists led to downregulation of X-linked inhibitor of apoptosis (XIAP) protein involved in cell survival and inhibition of cell death. In conclusion, IGF-1R antagonists (MK-0646 and OSI-906) demonstrated single agent inhibition of subcutaneous CRC xenograft growth. This was coupled to pro-apoptotic effects resulting in downregulation of XIAP and inhibition of cell survival. We report a novel mechanism by which MK-0646 and OSI-906 elicits cell death in vivo and in vitro. Moreover, these results indicate that MK-0646 and OSI-906 may be potential anticancer candidates for the treatment of patients with IGF-1R-dependent CRC.

Anticonvulsant and adverse effects of MK-801, LY 235959, and GYKI 52466 in combination with Ca2+ channel inhibitors in mice.

PubMed

Gasior, M; Borowicz, K; Kleinrok, Z; Starownik, R; Czuczwar, S J

1997-04-01

This study was designed to investigate the influence of the calcium (Ca2+) channel inhibitors nicardipine, nifedipine, and flunarizine on the protective action of MK-801, LY 235959 [N-methyl-D-aspartate (NMDA) receptor antagonists], and GYKI 52466 (a non-NMDA receptor antagonist) against electroconvulsions in mice. Unlike nicardipine (15 mg/kg) or flunarizine (10 mg/kg) nifedipine (7.5 and 15 mg/kg) potentiated the protective potency of MK-801 (0.05 mg/kg), as reflected by significant elevation of the convulsive threshold (a CS50 value of the current strength in mA producing tonic hind limb extension in 50% of the animals). The protective activity of LY 235959 and GYKI 52466 was reflected by their ED50 values in mg/kg, at which the drugs were expected to protect 50% of mice against maximal electroshock-induced tonic extension of the hind limbs. Nicardipine (3.75 15 mg/kg), nifedipine (0.94-15 mg/kg), and flunarizine (2.5-10 mg/kg) in a dose-dependent manner markedly potentiated the antiseizure efficacy of LY 235959. Flunarizine (5 and 10 mg/kg) was the only Ca2+ channel inhibitor to enhance the protective action of GYKI 52466 against electroconvulsions. Except with MK-801 + flunarizine (motor performance) or GYKI 52466 + flunarizine (long-term memory), combination of NMDA or non-NMDA receptor antagonists with Ca2+ channel inhibitors produced an impairment of motor performance (evaluated in the chimney test) and long-term memory acquisition (measured in the passive avoidance task) as compared with vehicle treatment.

Preclinical rationale for PI3K/Akt/mTOR pathway inhibitors as therapy for epidermal growth factor receptor inhibitor-resistant non-small-cell lung cancer.

PubMed

Gadgeel, Shirish M; Wozniak, Antoinette

2013-07-01

Mutations in the epidermal growth factor receptor gene (EGFR) are frequently observed in non-small-cell lung cancer (NSCLC), occurring in about 40% to 60% of never-smokers and in about 17% of patients with adenocarcinomas. EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, have transformed therapy for patients with EGFR-mutant NSCLC and have proved superior to chemotherapy as first-line treatment for this patient group. Despite these benefits, there are currently 2 key challenges associated with EGFR inhibitor therapy for patients with NSCLC. First, only 85% to 90% of patients with the EGFR mutation derive clinical benefit from EGFR TKIs, with the remainder demonstrating innate resistance to therapy. Second, acquired resistance to EGFR TKIs inevitably occurs in patients who initially respond to therapy, with a median duration of response of about 10 months. Mutant EGFR activates various subcellular signaling cascades, including the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, which demonstrates maintained activity in a variety of TKI-resistant cancers. Given the fundamental role of the PI3K/Akt/mTOR pathway in tumor oncogenesis, proliferation, and survival, PI3K pathway inhibitors have emerged as a possible solution to the problem of EGFR TKI resistance. However resistance to EGFR TKIs is associated with considerable heterogeneity and complexity. Preclinical experiments investigating these phenomena suggest that in some patients, PI3K inhibitors will have to be paired with other targeted agents if they are to be effective. This review discusses the preclinical data supporting PI3K/Akt/mTOR pathway inhibitor combinations in EGFR TKI-resistant NSCLC from the perspective of the various agents currently being investigated in clinical trials. Copyright © 2013 Elsevier Inc. All rights reserved.

Gamma-glutamylcyclotransferase promotes the growth of human glioma cells by activating Notch-Akt signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen, Shang-Hang; Yu, Ning; Liu, Xi-Yao

Glioma as an aggressive type tumor is rapidly growing and has become one of the leading cause of cancer-related death worldwide. γ-Glutamylcyclotransferase (GGCT) has been shown as a diagnostic marker in various cancers. To reveal whether there is a correlation between GGCT and human glioma, GGCT expression in human glioma tissues and cell lines was first determined. We found that GGCT expression was up-regulated in human glioma tissues and cell lines. Further, we demonstrate that GGCT knockdown inhibits glioma cell T98G and U251 proliferation and colony formation, whereas GGCT overexpression leads to oppose effects. GGCT overexpression promotes the expression ofmore » Notch receptors and activates Akt signaling in glioma cells, and Notch-Akt signaling is activated in glioma tissues with high expression of GGCT. Finally, we show that inhibition of Notch-Akt signaling with Notch inhibitor MK-0752 blocks the effects of GGCT on glioma proliferation and colony formation. In conclusion, GGCT plays a critical role in glioma cell proliferation and may be a potential cancer therapeutic target. - Highlights: • GGCT expression is up-regulated in human glioma tissues and cell lines. • GGCT promotes glioma cell growth and colony formation. • GGCT promotes the activation of Notch-Akt signaling in glioma cells and tissues. • Notch inhibition blocks the role of GGCT in human glioma cells.« less

Antagonism of EGFR and HER3 Enhances the Response to Inhibitors of the PI3K-Akt Pathway in Triple-Negative Breast Cancer

PubMed Central

Tao, Jessica J.; Castel, Pau; Radosevic-Robin, Nina; Elkabets, Moshe; Auricchio, Neil; Aceto, Nicola; Weitsman, Gregory; Barber, Paul; Vojnovic, Borivoj; Ellis, Haley; Morse, Natasha; Viola-Villegas, Nerissa Therese; Bosch, Ana; Juric, Dejan; Hazra, Saswati; Singh, Sharat; Kim, Phillip; Bergamaschi, Anna; Maheswaran, Shyamala; Ng, Tony; Penault-Llorca, Frédérique; Lewis, Jason S.; Carey, Lisa A.; Perou, Charles M.; Baselga, José; Scaltriti, Maurizio

2014-01-01

Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidyl-inositol 3-kinase (PI3K)–Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting. PMID:24667376

Antagonism of EGFR and HER3 enhances the response to inhibitors of the PI3K-Akt pathway in triple-negative breast cancer.

PubMed

Tao, Jessica J; Castel, Pau; Radosevic-Robin, Nina; Elkabets, Moshe; Auricchio, Neil; Aceto, Nicola; Weitsman, Gregory; Barber, Paul; Vojnovic, Borivoj; Ellis, Haley; Morse, Natasha; Viola-Villegas, Nerissa Therese; Bosch, Ana; Juric, Dejan; Hazra, Saswati; Singh, Sharat; Kim, Phillip; Bergamaschi, Anna; Maheswaran, Shyamala; Ng, Tony; Penault-Llorca, Frédérique; Lewis, Jason S; Carey, Lisa A; Perou, Charles M; Baselga, José; Scaltriti, Maurizio

2014-03-25

Both abundant epidermal growth factor receptor (EGFR or ErbB1) and high activity of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway are common and therapeutically targeted in triple-negative breast cancer (TNBC). However, activation of another EGFR family member [human epidermal growth factor receptor 3 (HER3) (or ErbB3)] may limit the antitumor effects of these drugs. We found that TNBC cell lines cultured with the EGFR or HER3 ligand EGF or heregulin, respectively, and treated with either an Akt inhibitor (GDC-0068) or a PI3K inhibitor (GDC-0941) had increased abundance and phosphorylation of HER3. The phosphorylation of HER3 and EGFR in response to these treatments was reduced by the addition of a dual EGFR and HER3 inhibitor (MEHD7945A). MEHD7945A also decreased the phosphorylation (and activation) of EGFR and HER3 and the phosphorylation of downstream targets that occurred in response to the combination of EGFR ligands and PI3K-Akt pathway inhibitors. In culture, inhibition of the PI3K-Akt pathway combined with either MEHD7945A or knockdown of HER3 decreased cell proliferation compared with inhibition of the PI3K-Akt pathway alone. Combining either GDC-0068 or GDC-0941 with MEHD7945A inhibited the growth of xenografts derived from TNBC cell lines or from TNBC patient tumors, and this combination treatment was also more effective than combining either GDC-0068 or GDC-0941 with cetuximab, an EGFR-targeted antibody. After therapy with EGFR-targeted antibodies, some patients had residual tumors with increased HER3 abundance and EGFR/HER3 dimerization (an activating interaction). Thus, we propose that concomitant blockade of EGFR, HER3, and the PI3K-Akt pathway in TNBC should be investigated in the clinical setting.

Antiviral Activity of MK-4965, a Novel Nonnucleoside Reverse Transcriptase Inhibitor▿

PubMed Central

Lai, Ming-Tain; Munshi, Vandna; Touch, Sinoeun; Tynebor, Robert M.; Tucker, Thomas J.; McKenna, Philip M.; Williams, Theresa M.; DiStefano, Daniel J.; Hazuda, Daria J.; Miller, Michael D.

2009-01-01

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are the mainstays of therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infections. However, the effectiveness of NNRTIs can be hampered by the development of resistance mutations which confer cross-resistance to drugs in the same class. Extensive efforts have been made to identify new NNRTIs that can suppress the replication of the prevalent NNRTI-resistant viruses. MK-4965 is a novel NNRTI that possesses both diaryl ether and indazole moieties. The compound displays potency at subnanomolar concentrations against wild-type (WT), K103N, and Y181C reverse transcriptase (RT) in biochemical assays. MK-4965 is also highly potent against the WT virus and two most prevalent NNRTI-resistant viruses (viruses that harbor the K103N or the Y181C mutation), against which it had 95% effective concentrations (EC95s) of <30 nM in the presence of 10% fetal bovine serum. The antiviral EC95 of MK-4965 was reduced approximately four- to sixfold when it was tested in 50% human serum. Moreover, MK-4965 was evaluated with a panel of 15 viruses with NNRTI resistance-associated mutations and showed a superior mutant profile to that of efavirenz but not to that of etravirine. MK-4965 was similarly effective against various HIV-1 subtypes and viruses containing nucleoside reverse transcriptase inhibitor or protease inhibitor resistance-conferring mutations. A two-drug combination study showed that the antiviral activity of MK-4965 was nonantagonistic with each of the 18 FDA-licensed drugs tested vice versa in the present study. Taken together, these in vitro data show that MK-4965 possesses the desired properties for further development as a new NNRTI for the treatment of HIV-1 infection. PMID:19289522

Role of Akt/PKB and PFKFB isoenzymes in the control of glycolysis, cell proliferation and protein synthesis in mitogen-stimulated thymocytes.

PubMed

Houddane, Amina; Bultot, Laurent; Novellasdemunt, Laura; Johanns, Manuel; Gueuning, Marie-Agnès; Vertommen, Didier; Coulie, Pierre G; Bartrons, Ramon; Hue, Louis; Rider, Mark H

2017-06-01

Proliferating cells depend on glycolysis mainly to supply precursors for macromolecular synthesis. Fructose 2,6-bisphosphate (Fru-2,6-P 2 ) is the most potent positive allosteric effector of 6-phosphofructo-1-kinase (PFK-1), and hence of glycolysis. Mitogen stimulation of rat thymocytes with concanavalin A (ConA) led to time-dependent increases in lactate accumulation (6-fold), Fru-2,6-P 2 content (4-fold), 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase isoenzyme 3 and 4 (PFKFB3 and PFKFB4) protein levels (~2-fold and ~15-fold, respectively) and rates of cell proliferation (~40-fold) and protein synthesis (10-fold) after 68h of incubation compared with resting cells. After 54h of ConA stimulation, PFKFB3 mRNA levels were 45-fold higher than those of PFKFB4 mRNA. Although PFKFB3 could be phosphorylated at Ser461 by protein kinase B (PKB) in vitro leading to PFK-2 activation, PFKFB3 Ser461 phosphorylation was barely detectable in resting cells and only increased slightly in ConA-stimulated cells. On the other hand, PFKFB3 and PFKFB4 mRNA levels were decreased (90% and 70%, respectively) by exposure of ConA-stimulated cells to low doses of PKB inhibitor (MK-2206), suggesting control of expression of the two PFKFB isoenzymes by PKB. Incubation of thymocytes with ConA resulted in increased expression and phosphorylation of the translation factors eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) and ribosomal protein S6 (rpS6). Treatment of ConA-stimulated thymocytes with PFK-2 inhibitor (3PO) or MK-2206 led to significant decreases in Fru-2,6-P 2 content, medium lactate accumulation and rates of cell proliferation and protein synthesis. These data were confirmed by using siRNA knockdown of PFKFB3, PFKFB4 and PKB α/β in the more easily transfectable Jurkat E6-1 cell line. The findings suggest that increased PFKFB3 and PFKFB4 expression, but not increased PFKFB3 Ser461 phosphorylation, plays a role in increasing glycolysis in mitogen

Restoration of Circulating MFGE8 (Milk Fat Globule-EGF Factor 8) Attenuates Cardiac Hypertrophy Through Inhibition of Akt Pathway.

PubMed

Deng, Ke-Qiong; Li, Jing; She, Zhi-Gang; Gong, Jun; Cheng, Wen-Lin; Gong, Fu-Han; Zhu, Xue-Yong; Zhang, Yan; Wang, Zhihua; Li, Hongliang

2017-10-01

Cardiac hypertrophy occurs in response to numerous stimuli like neurohumoral stress, pressure overload, infection, and injury, and leads to heart failure. Mfge8 (milk fat globule-EGF factor 8) is a secreted protein involved in various human diseases, but its regulation and function during cardiac hypertrophy remain unexplored. Here, we found that circulating MFGE8 levels declined significantly in failing hearts from patients with dilated cardiomyopathy. Correlation analyses revealed that circulating MFGE8 levels were negatively correlated with the severity of cardiac dysfunction and remodeling in affected patients. Deleting Mfge8 in mice maintained normal heart function at basal level but substantially exacerbated the hypertrophic enlargement of cardiomyocytes, reprogramming of pathological genes, contractile dysfunction, and myocardial fibrosis after aortic banding surgery. In contrast, cardiac-specific Mfge8 overexpression in transgenic mice significantly blunted aortic banding-induced cardiac hypertrophy. Whereas MAPK (mitogen-activated protein kinase) pathways were unaffected in either Mfge8 -knockout or Mfge8 -overexpressing mice, the activated Akt/PKB (protein kinase B)-Gsk-3β (glycogen synthase kinase-3β)/mTOR (mammalian target of rapamycin) pathway after aortic banding was significantly potentiated by Mfge8 deficiency but suppressed by Mfge8 overexpression. Inhibition of Akt with MK-2206 blocked the prohypertrophic effects of Mfge8 deficiency in angiotensin II-treated neonatal rat cardiomyocytes. Finally, administering a recombinant human MFGE8 in mice in vivo alleviated cardiac hypertrophy induced by aortic banding. Our findings indicate that Mfge8 is an endogenous negative regulator of pathological cardiac hypertrophy and may, thus, have potential both as a novel biomarker and as a therapeutic target for treatment of cardiac hypertrophy. © 2017 American Heart Association, Inc.

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

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.

Effects of MK-886, a 5-lipoxygenase activating protein (FLAP) inhibitor, and 5-lipoxygenase deficiency on the forced swimming behavior of mice

PubMed Central

Uz, Tolga; Dimitrijevic, Nikola; Imbesi, Marta; Manev, Hari; Manev, Radmila

2008-01-01

A common biological pathway may contribute to the comorbidity of atherosclerosis and depression. Increased activity of the enzymatic 5-lipoxygenase (5-LOX; 5LO) pathway is a contributing factor in atherosclerosis and a 5-LOX inhibitor, MK-886, is beneficial in animal models of atherosclerosis. In the brain, MK-886 increases phosphorylation of the glutamate receptor subunit GluR1, and the increased phosphorylation of this receptor has been associated with antidepressant treatment. In this work, we evaluated the behavioral effects of MK-886 in an automated assay of mouse forced swimming, which identifies antidepressant activity as increased climbing behavior and/or decreased rest time. Whereas a single injection of MK-886 (3 and 10 mg/kg) did not affect forced swimming behaviors assayed 30 min later, 6 daily injections of 3 mg/kg MK-886 slightly increased climbing and significantly reduced rest time in wild-type mice but not in 5-LOX-deficient mice. A diet delivery of MK-886, 4 μg per 100 mg body-weight per day, required three weeks to affect forced swimming; it increased climbing behavior. Climbing behavior was also increased in naive 5-LOX-deficient mice compared to naive wild-type controls. These results suggest that 5-LOX inhibition and deficiency may be associated with antidepressant activity. Increased climbing in a forced swimming assay is a typical outcome of antidepressants that increase noradrenergic and dopaminergic activity. Interestingly, 5-LOX deficiency and MK-886 treatment have been shown to be capable of increasing the behavioral effects of a noradrenaline/dopamine-potentiating drug, cocaine. Future research is needed to evaluate the clinical relevance of our findings. PMID:18403121

The Efficacy of the Wee1 Inhibitor MK-1775 Combined with Temozolomide Is Limited by Heterogeneous Distribution across the Blood-Brain Barrier in Glioblastoma.

PubMed

Pokorny, Jenny L; Calligaris, David; Gupta, Shiv K; Iyekegbe, Dennis O; Mueller, Dustin; Bakken, Katrina K; Carlson, Brett L; Schroeder, Mark A; Evans, Debra L; Lou, Zhenkun; Decker, Paul A; Eckel-Passow, Jeanette E; Pucci, Vincenzo; Ma, Bennett; Shumway, Stuart D; Elmquist, William F; Agar, Nathalie Y R; Sarkaria, Jann N

2015-04-15

Wee1 regulates key DNA damage checkpoints, and in this study, the efficacy of the Wee1 inhibitor MK-1775 was evaluated in glioblastoma multiforme (GBM) xenograft models alone and in combination with radiation and/or temozolomide. In vitro MK-1775 efficacy alone and in combination with temozolomide, and the impact on DNA damage, was analyzed by Western blotting and γH2AX foci formation. In vivo efficacy was evaluated in orthotopic and heterotopic xenografts. Drug distribution was assessed by conventional mass spectrometry (MS) and matrix-assisted laser desorption/ionization (MALDI)-MS imaging. GBM22 (IC50 = 68 nmol/L) was significantly more sensitive to MK-1775 compared with five other GBM xenograft lines, including GBM6 (IC50 >300 nmol/L), and this was associated with a significant difference in pan-nuclear γH2AX staining between treated GBM22 (81% cells positive) and GBM6 (20% cells positive) cells. However, there was no sensitizing effect of MK-1775 when combined with temozolomide in vitro. In an orthotopic GBM22 model, MK-1775 was ineffective when combined with temozolomide, whereas in a flank model of GBM22, MK-1775 exhibited both single-agent and combinatorial activity with temozolomide. Consistent with limited drug delivery into orthotopic tumors, the normal brain to whole blood ratio following a single MK-1775 dose was 5%, and MALDI-MS imaging demonstrated heterogeneous and markedly lower MK-1775 distribution in orthotopic as compared with heterotopic GBM22 tumors. Limited distribution to brain tumors may limit the efficacy of MK-1775 in GBM. ©2015 American Association for Cancer Research.

The efficacy of the Wee1 inhibitor MK-1775 combined with temozolomide is limited by heterogeneous distribution across the blood-brain barrier in glioblastoma

PubMed Central

Pokorny, Jenny L.; Calligaris, David; Gupta, Shiv K.; Iyekegbe, Dennis O.; Mueller, Dustin; Bakken, Katrina K.; Carlson, Brett L.; Schroeder, Mark A.; Evans, Debra L.; Lou, Zhenkun; Decker, Paul A.; Eckel-Passow, Jeanette E.; Pucci, Vincenzo; Ma, Bennett; Shumway, Stuart D.; Elmquist, William; Agar, Nathalie Y.; Sarkaria, Jann N.

2015-01-01

Purpose Wee1 regulates key DNA damage checkpoints, and in this study, the efficacy of the Wee1 inhibitor MK-1775 was evaluated in GBM xenograft models alone and in combination with radiation and/or temozolomide (TMZ). Experimental design In vitro MK-1775 efficacy alone and in combination with TMZ, and the impact on DNA damage was analyzed by western blotting and γH2AX foci formation. In vivo efficacy was evaluated in orthotopic and heterotopic xenografts. Drug distribution was assessed by conventional mass spectrometry (MS) and matrix-assisted laser desorption/ionization (MALDI) -MS imaging. Results GBM22 (IC50 = 68 nM) was significantly more sensitive to MK-1775 compared to 5 other GBM xenograft lines including GBM6 (IC50 >300 nM), and this was associated with a significant difference in pan-nuclear γH2AX staining between treated GBM22 (81% cells positive) and GBM6 (20% cells positive) cells. However, there was no sensitizing effect of MK-1775 when combined with TMZ in vitro. In an orthotopic GBM22 model, MK-1775 was ineffective when combined with TMZ, while in a flank model of GBM22, MK-1775 exhibited both single agent and combinatorial activity with TMZ. Consistent with limited drug delivery into orthotopic tumors, the normal brain to whole blood ratio following a single MK-1775 dose was 5%, and MALDI-MS imaging demonstrated heterogeneous and markedly lower MK-1775 distribution in orthotopic as compared to heterotopic GBM22 tumors. Conclusions Limited distribution to brain tumors may limit the efficacy of MK-1775 in GBM. PMID:25609063

Overexpression of miR-223 Tips the Balance of Pro- and Anti-hypertrophic Signaling Cascades toward Physiologic Cardiac Hypertrophy*

PubMed Central

Yang, Liwang; Li, Yutian; Wang, Xiaohong; Mu, Xingjiang; Qin, Dongze; Huang, Wei; Alshahrani, Saeed; Nieman, Michelle; Peng, Jiangtong; Essandoh, Kobina; Peng, Tianqing; Wang, Yigang; Lorenz, John; Soleimani, Manoocher; Zhao, Zhi-Qing; Fan, Guo-Chang

2016-01-01

MicroRNAs (miRNAs) have been extensively examined in pathological cardiac hypertrophy. However, few studies focused on profiling the miRNA alterations in physiological hypertrophic hearts. In this study we generated a transgenic mouse model with cardiac-specific overexpression of miR-223. Our results showed that elevation of miR-223 caused physiological cardiac hypertrophy with enhanced cardiac function but no fibrosis. Using the next generation RNA sequencing, we observed that most of dys-regulated genes (e.g. Atf3/5, Egr1/3, Sfrp2, Itgb1, Ndrg4, Akip1, Postn, Rxfp1, and Egln3) in miR-223-transgenic hearts were associated with cell growth, but they were not directly targeted by miR-223. Interestingly, these dys-regulated genes are known to regulate the Akt signaling pathway. We further identified that miR-223 directly interacted with 3′-UTRs of FBXW7 and Acvr2a, two negative regulators of the Akt signaling. However, we also validated that miR-223 directly inhibited the expression of IGF-1R and β1-integrin, two positive regulators of the Akt signaling. Lastly, Western blotting did reveal that Akt was activated in miR-223-overexpressing hearts. Adenovirus-mediated overexpression of miR-223 in neonatal rat cardiomyocytes induced cell hypertrophy, which was blocked by the addition of MK2206, a specific inhibitor of Akt. Taken together, these data represent the first piece of work showing that miR-223 tips the balance of promotion and inactivation of Akt signaling cascades toward activation of Akt, a key regulator of physiological cardiac hypertrophy. Thus, our study suggests that the ultimate phenotype outcome of a miRNA may be decided by the secondary net effects of the whole target network rather than by several primary direct targets in an organ/tissue. PMID:27226563

A Novel Oncolytic Herpes Simplex Virus that Synergizes with Phosphatidylinositol 3-Kinase/Akt Pathway Inhibitors to Target Glioblastoma Stem Cells

PubMed Central

Kanai, Ryuichi; Wakimoto, Hiroaki; Martuza, Robert L.; Rabkin, Samuel D.

2011-01-01

Purpose To develop a new oncolytic herpes simplex virus (oHSV) for glioblastoma therapy that will be effective in glioblastoma stem cells (GSCs), an important and untargeted component of glioblastoma. One approach to enhance oHSV efficacy is by combination with other therapeutic modalities. Experimental design MG18L, containing a US3 deletion and an inactivating LacZ insertion in UL39, was constructed for the treatment of brain tumors. Safety was evaluated after intracerebral injection in HSV-susceptible mice. The efficacy of MG18L in human GSCs and glioma cell lines in vitro was compared to other oHSVs, alone or in combination with PI3K/Akt inhibitors (LY294002, triciribine, GDC-0941, BEZ235). Cytotoxic interactions between MG18L and PI3K/Akt inhibitors were determined using Chou-Talalay analysis. In vivo efficacy studies were performed using a clinically relevant mouse model of GSC-derived glioblastoma. Results MG18L was severely neuroattenuated in mice, replicated well in GSCs, and had anti-glioblastoma activity in vivo. PI3K/Akt inhibitors displayed significant but variable anti-proliferative activities in GSCs, while their combination with MG18L synergized in killing GSCs and glioma lines, but not human astrocytes, through enhanced induction of apoptosis. Importantly, synergy was independent of inhibitor sensitivity. In vivo, the combination of MG18L and LY294002 significantly prolonged survival of mice, as compared to either agent alone, achieving 50% long-term survival in glioblastoma-bearing mice. Conclusions This study establishes a novel therapeutic strategy: oHSV manipulation of critical oncogenic pathways to sensitize cancer cells to molecularly-targeted drugs. MG18L is a promising agent for the treatment of glioblastoma, being especially effective when combined with PI3K/Akt pathway-targeted agents. PMID:21505062

Sirt2 Deacetylase Is a Novel AKT Binding Partner Critical for AKT Activation by Insulin*

PubMed Central

Ramakrishnan, Gopalakrishnan; Davaakhuu, Gantulga; Kaplun, Ludmila; Chung, Wen-Cheng; Rana, Ajay; Atfi, Azeddine; Miele, Lucio; Tzivion, Guri

2014-01-01

AKT/PKB kinases transmit insulin and growth factor signals downstream of phosphatidylinositol 3-kinase (PI3K). AKT activation involves phosphorylation at two residues, Thr308 and Ser473, mediated by PDK1 and the mammalian target of rapamycin complex 2 (mTORC2), respectively. Impaired AKT activation is a key factor in metabolic disorders involving insulin resistance, whereas hyperactivation of AKT is linked to cancer pathogenesis. Here, we identify the cytoplasmic NAD+-dependent deacetylase, Sirt2, as a novel AKT interactor, required for optimal AKT activation. Pharmacological inhibition or genetic down-regulation of Sirt2 diminished AKT activation in insulin and growth factor-responsive cells, whereas Sirt2 overexpression enhanced the activation of AKT and its downstream targets. AKT was prebound with Sirt2 in serum or glucose-deprived cells, and the complex dissociated following insulin treatment. The binding was mediated by the pleckstrin homology and the kinase domains of AKT and was dependent on AMP-activated kinase. This regulation involved a novel AMP-activated kinase-dependent Sirt2 phosphorylation at Thr101. In cells with constitutive PI3K activation, we found that AKT also associated with a nuclear sirtuin, Sirt1; however, inhibition of PI3K resulted in dissociation from Sirt1 and increased association with Sirt2. Sirt1 and Sirt2 inhibitors additively inhibited the constitutive AKT activity in these cells. Our results suggest potential usefulness of Sirt1 and Sirt2 inhibitors in the treatment of cancer cells with up-regulated PI3K activity and of Sirt2 activators in the treatment of insulin-resistant metabolic disorders. PMID:24446434

Platelets, neutrophils, and vasoconstriction after arterial injury by angioplasty in pigs: effects of MK-886, a leukotriene biosynthesis inhibitor

PubMed Central

Provost, Patrick; Borgeat, Pierre; Merhi, Yahye

1998-01-01

Leukotrienes constitute a class of potent bioactive mediators known to play a pivotal role in inflammation. Since their biosynthesis has been shown to be enhanced by platelet-neutrophil interactions, leukotrienes may be involved in these interactions and the arterial response to injury. Therefore, we investigated the effects of the selective leukotriene biosynthesis inhibitor 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid (MK-886) on the acute thrombotic and vasomotor responses after arterial injury by angioplasty.Carotid arterial injury was produced by balloon dilatation in control (molecusol vehicle; n=10) and treated (MK-886, 10 mg kg−1, i.v.; n=9) pigs. The acute thrombotic reaction following deep arterial wall injury was quantified with 51Cr labelled platelets and 111In labelled neutrophils, and the vasomotor response was determined angiographically.Platelet deposition at the site of deep arterial wall injury averaged 56.4±11.0×106 platelets cm−2 in the control group, and was significantly reduced to 18.2±3.8×106 platelets cm−2 (P<0.005) by treatment with MK-886. Neutrophil deposition was also decreased by MK-886, from 242.8±36.8 to 120.9±20.3×103 neutrophils cm−2 (P<0.01). MK-886-treated animals had a significant decrease in the postangioplasty vasoconstrictive response at the site of endothelial injury distally, from 37.5±3.1% in the control group to 13.5±2.5% (P<0.001).The effects of MK-886 were associated with a profound inhibition of ex vivo leukotriene B4 (LTB4) synthesis in blood stimulated by the calcium ionophore A23187 and a significant reduction of neutrophil aggregation in whole blood (P<0.01), whereas neutrophil superoxide anion production, serum thromboxane B2 and platelet aggregation in whole blood were not influenced.The relevant effects of MK-886 are primarily related to inhibition of neutrophil function and suggest an important modulatory role for leukotrienes in the

40 CFR 35.2206 - Operation and maintenance.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Section 35.2206 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY GRANTS AND OTHER FEDERAL... operation and maintenance manual. (2) In projects where a component is placed in operation before completion... project until a final operation and maintenance manual for the operating component is furnished and...

Microsecond molecular dynamics simulations provide insight into the ATP-competitive inhibitor-induced allosteric protection of Akt kinase phosphorylation.

PubMed

Mou, Linkai; Cui, Tongwei; Liu, Weiguang; Zhang, Hong; Cai, Zhanxiu; Lu, Shaoyong; Gao, Guojun

2017-05-01

Akt is a serine/threonine protein kinase, a critical mediator of growth factor-induced survival in key cellular pathways. Allosteric signaling between protein intramolecular domains requires long-range communication mediated by hotspot residues, often triggered by ligand binding. Here, based on extensive 3 μs explicit solvent molecular dynamics (MD) simulations of Akt1 kinase domain in the unbound (apo) and ATP-competitive inhibitor, GDC-0068-bound states, we propose a molecular mechanism for allosteric regulation of Akt1 kinase phosphorylation by GDC-0068 binding to the ATP-binding site. MD simulations revealed that the apo Akt1 is flexible with two disengaged N- and C-lobes, equilibrated between the open and closed conformations. GDC-0068 occupancy of the ATP-binding site shifts the conformational equilibrium of Akt1 from the open conformation toward the closed conformation and stabilizes the closed state. This effect enables allosteric signal propagation from the GDC-0068 to the phosphorylated T308 (pT308) in the activation loop and restrains phosphatase access to pT308, thereby protecting the pT308 in the GDC-0068-bound Akt1. Importantly, functional hotspots involved in the allosteric communication from the GDC-0068 to the pT308 are identified. Our analysis of GDC-0068-induced allosteric protection of Akt kinase phosphorylation yields important new insights into the molecular mechanism of allosteric regulation of Akt kinase activity. © 2016 John Wiley & Sons A/S.

D-Serine and a glycine transporter inhibitor improve MK-801-induced cognitive deficits in a novel object recognition test in rats.

PubMed

Karasawa, Jun-Ichi; Hashimoto, Kenji; Chaki, Shigeyuki

2008-01-10

Compounds enhancing N-methyl-d-aspartate (NMDA) glutamate receptor function have been reported to improve cognitive deficits. Since cognitive deficits are considered to be the core symptom of schizophrenia, enhancing NMDA receptor function represents a promising approach to treating schizophrenia. In the present study, we investigated whether d-serine or a glycine transporter inhibitor N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine (NFPS), both of which enhance NMDA receptor function, could improve MK-801-induced cognitive deficits in rats, and compared their effects with those of the atypical antipsychotic clozapine and of the typical antipsychotic haloperidol. To assess cognitive function, we used a novel object recognition test in rats that measured spontaneous exploratory activity of a novel object when paired with a familiar object. We then evaluated the effects of the compounds on cognitive deficits induced by treatment with MK-801, the NMDA receptor antagonist. Pretreatment with clozapine (1, 5 mg/kg, i.p.) but not haloperidol (0.03, 0.1 mg/kg, i.p.) significantly improved MK-801-induced cognitive deficits. Pretreatment with D-serine at 800 mg/kg (i.p.) or NFPS (0.3, 1 mg/kg, i.p.) significantly improved MK-801-induced cognitive deficits under this test paradigm. These findings suggest that impaired preference for novel objects induced by MK-801 in the novel object recognition test could be a useful animal model for evaluating the efficacy of compounds targeting the cognitive deficits observed in schizophrenic patients. The results also suggest that enhancing NMDA receptor function is an effective way for treating the cognitive deficits associated with schizophrenia.

Targeting protein kinase-b3 (akt3) signaling in melanoma.

PubMed

Madhunapantula, SubbaRao V; Robertson, Gavin P

2017-03-01

Deregulated Akt activity leading to apoptosis inhibition, enhanced proliferation and drug resistance has been shown to be responsible for 35-70% of advanced metastatic melanomas. Of the three isoforms, the majority of melanomas have elevated Akt3 expression and activity. Hence, potent inhibitors targeting Akt are urgently required, which is possible only if (a) the factors responsible for the failure of Akt inhibitors in clinical trials is known; and (b) the information pertaining to synergistically acting targeted therapeutics is available. Areas covered: This review provides a brief introduction of the PI3K-Akt signaling pathway and its role in melanoma development. In addition, the functional role of key Akt pathway members such as PRAS40, GSK3 kinases, WEE1 kinase in melanoma development are discussed together with strategies to modulate these targets. Efficacy and safety of Akt inhibitors is also discussed. Finally, the mechanism(s) through which Akt leads to drug resistance is discussed in this expert opinion review. Expert opinion: Even though Akt play key roles in melanoma tumor progression, cell survival and drug resistance, many gaps still exist that require further understanding of Akt functions, especially in the (a) metastatic spread; (b) circulating melanoma cells survival; and (c) melanoma stem cells growth.

7 CFR 220.6 - Use of funds.

Code of Federal Regulations, 2011 CFR

2011-01-01

... CHILD NUTRITION PROGRAMS SCHOOL BREAKFAST PROGRAM § 220.6 Use of funds. (a) Federal funds made available under the School Breakfast Program shall be used by State agencies, or FNSROs where applicable, to reimburse or make advance payments to School Food Authorities in connection with breakfasts served in...

7 CFR 220.6 - Use of funds.

Code of Federal Regulations, 2010 CFR

2010-01-01

... CHILD NUTRITION PROGRAMS SCHOOL BREAKFAST PROGRAM § 220.6 Use of funds. (a) Federal funds made available under the School Breakfast Program shall be used by State agencies, or FNSROs where applicable, to reimburse or make advance payments to School Food Authorities in connection with breakfasts served in...

Efficacy of AKT Inhibitor ARQ 092 Compared with Sorafenib in a Cirrhotic Rat Model with Hepatocellular Carcinoma.

PubMed

Roth, Gaël S; Macek Jilkova, Zuzana; Zeybek Kuyucu, Ayca; Kurma, Keerthi; Ahmad Pour, Séyédéh Tayébéh; Abbadessa, Giovanni; Yu, Yi; Busser, Benoit; Marche, Patrice N; Leroy, Vincent; Decaens, Thomas

2017-10-01

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related mortality worldwide. The AKT pathway has been found activated in 50% of HCC cases, making it a promising target. Therefore, we assess efficacy of the allosteric AKT inhibitor ARQ 092 compared with untreated control and standard treatment, sorafenib, in vitro and in vivo ARQ 092 blocked phosphorylation of AKT in vitro and strongly inhibited cell growth with significantly higher potency than sorafenib. Similarly, apoptosis and cell migration were strongly reduced by ARQ 092 in vitro To mimic human advanced HCC, we used a diethylnitrosamine-induced cirrhotic rat model with fully developed HCC. MRI analyses showed that ARQ 092 significantly reduced overall tumor size. Furthermore, number of tumors was decreased by ARQ 092, which was associated with increased apoptosis and decreased proliferation. Tumor contrast enhancement was significantly decreased in the ARQ 092 group. Moreover, on tumor tissue sections, we observed a vascular normalization and a significant decrease in fibrosis in the surrounding liver of animals treated with ARQ 092. Finally, pAKT/AKT levels in ARQ 092-treated tumors were reduced, followed by downregulation of actors of AKT downstream signaling pathway: pmTOR, pPRAS40, pPLCγ1, and pS6K1. In conclusion, we demonstrated that ARQ 092 blocks AKT phosphorylation in vitro and in vivo In the HCC-rat model, ARQ 092 was well tolerated, showed antifibrotic effect, and had stronger antitumor effect than sorafenib. Our results confirm the importance of targeting AKT in HCC. Mol Cancer Ther; 16(10); 2157-65. ©2017 AACR . ©2017 American Association for Cancer Research.

In Vitro Activity of MK-7655, a Novel β-Lactamase Inhibitor, in Combination with Imipenem against Carbapenem-Resistant Gram-Negative Bacteria

PubMed Central

Hirsch, Elizabeth B.; Ledesma, Kimberly R.; Chang, Kai-Tai; Schwartz, Michael S.; Motyl, Mary R.

2012-01-01

Carbapenem-resistant bacteria represent a significant treatment challenge due to the lack of active antimicrobials available. MK-7655 is a novel β-lactamase inhibitor under clinical development. We investigated the combined killing activity of imipenem and MK-7655 against four imipenem-resistant bacterial strains, using a mathematical model previously evaluated in our laboratory. Time-kill studies (TKS) were conducted with imipenem and MK-7655 against a KPC-2-producing Klebsiella pneumoniae isolate (KP6339) as well as 3 Pseudomonas aeruginosa isolates (PA24226, PA24227, and PA24228) with OprD porin deletions and overexpression of AmpC. TKS were performed using 25 clinically achievable concentration combinations in a 5-by-5 array. Bacterial burden at 24 h was determined in triplicate by quantitative culture and mathematically modeled using a three-dimensional response surface. Mathematical model assessments were evaluated experimentally using clinically relevant dosing regimens of imipenem, with or without MK-7655, in a hollow-fiber infection model (HFIM). The combination of imipenem and MK-7655 was synergistic for all strains. Interaction indices were as follows: for KP6339, 0.50 (95% confidence interval [CI], 0.42 to 0.58); for PA24226, 0.60 (95% CI, 0.58 to 0.62); for PA24227, 0.70 (95% CI, 0.66 to 0.74); and for PA24228, 0.55 (95% CI, 0.49 to 0.61). In the HFIM, imipenem plus MK-7655 considerably reduced the bacterial burden at 24 h, while failure with imipenem alone was seen against all isolates. Sustained suppression of bacterial growth at 72 h was achieved with simulated doses of 500 mg imipenem plus 500 mg MK-7655 in 2 (KP6339 and PA24227) strains, and it was achieved in an additional strain (PA24228) when the imipenem dose was increased to 1,000 mg. Additional studies are being conducted to determine the optimal dose and combinations to be used in clinical investigations. PMID:22526311

Inhibitor of G protein-coupled receptor kinase 2 normalizes vascular endothelial function in type 2 diabetic mice by improving β-arrestin 2 translocation and ameliorating Akt/eNOS signal dysfunction.

PubMed

Taguchi, Kumiko; Matsumoto, Takayuki; Kamata, Katsuo; Kobayashi, Tsuneo

2012-07-01

In type 2 diabetes, although Akt/endothelial NO synthase (eNOS) activation is known to be negatively regulated by G protein-coupled receptor kinase 2 (GRK2), it is unclear whether the GRK2 inhibitor would have therapeutic effects. Here we examined the hypotensive effect of the GRK2 inhibitor and its efficacy agonist both vascular (aortic) endothelial dysfunction (focusing especially on the Akt/eNOS pathway) and glucose intolerance in two type 2 diabetic models (ob/ob mice and nicotinamide+streptozotocin-induced diabetic mice). Mice were treated with a single injection of the GRK2 inhibitor or vehicle, and the therapeutic effects were compared by examining vascular function and by Western blotting. The GRK2 inhibitor lowered blood pressure in both diabetic models but not in their age-matched controls. The GRK2 inhibitor significantly improved clonidine-induced relaxation only in diabetic (ob/ob and DM) mice, with accompanying attenuations of GRK2 activity and translocation to the plasma membrane. These protective effects of the GRK2 inhibitor may be attributable to the augmented Akt/eNOS pathway activation (as evidenced by increases in Akt phosphorylation at Ser(473) and at Thr(308), and eNOS phosphorylation at Ser(1177)) and to the prevention of the GRK2 translocation and promotion of β-arrestin 2 translocation to the membrane under clonidine stimulation. Moreover, the GRK2 inhibitor significantly improved the glucose intolerance seen in the ob/ob mice. Our work provides the first evidence that in diabetes, the GRK2 inhibitor ameliorates vascular endothelial dysfunction via the Akt/eNOS pathway by inhibiting GRK2 activity and enhancing β-arrestin 2 translocation under clonidine stimulation, thereby contributing to a blood pressure-lowering effect. We propose that the GRK2 inhibitor may be a promising therapeutic agent for cardiovascular complications in type 2 diabetes.

MANF attenuates neuronal apoptosis and promotes behavioral recovery via Akt/MDM-2/p53 pathway after traumatic spinal cord injury in rats.

PubMed

Gao, Liansheng; Xu, Weilin; Fan, Shuangbo; Li, Tao; Zhao, Tengfei; Ying, Guangyu; Zheng, Jingwei; Li, Jianru; Zhang, Zhongyuan; Yan, Feng; Zhu, Yongjian; Chen, Gao

2018-05-24

The aim of this study was to investigate the potential effect and mechanism of action of MANF in attenuating neuronal apoptosis following t-SCI. A clip compressive model was used to induce a crush injury of the spinal cord in a total of 230 rats. The Basso, Beattie, and Bresnahan (BBB) score, spinal cord water content, and blood spinal cord barrier (BSCB) permeability were evaluated. The expression levels of MANF and its downstream proteins were examined by western blotting. Immunofluorescence staining of MANF, NeuN, GFAP, Iba-1, cleaved caspase-3, and TUNEL staining were also performed. Cells were counted in six randomly selected fields in the gray matter regions of the sections from two spinal cord sites (2 mm rostral and caudal to the epicenter of the injury) per sample. A cell-based mechanical injury model was also conducted using SH-SY5Y cells. Cell apoptosis and viability were assessed by flow cytometry, an MTT assay, and trypan blue staining. Subcellular structures were observed by transmission electron microscopy. MANF was mainly expressed in neurons. The expression levels of MANF, and its downstream target, p-Akt, were gradually increased and after t-SCI. Treatment with MANF increased Bcl-2 and decreased Bax and CC-3 levels; these effects were reversed on treatment with MK2206. The BBB score, spinal cord water content, and BSCB destruction were also ameliorated by MANF treatment. MANF decreases neuronal apoptosis and improves neurological function through Akt/MDM-2/p53 pathway after t-SCI. Therefore, MANF might be a potential treatment for patients with t-SCI.© 2018 BioFactors, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

Intrinsic BET inhibitor resistance in SPOP-mutated prostate cancer is mediated by BET protein stabilization and AKT-mTORC1 activation.

PubMed

Zhang, Pingzhao; Wang, Dejie; Zhao, Yu; Ren, Shancheng; Gao, Kun; Ye, Zhenqing; Wang, Shangqian; Pan, Chun-Wu; Zhu, Yasheng; Yan, Yuqian; Yang, Yinhui; Wu, Di; He, Yundong; Zhang, Jun; Lu, Daru; Liu, Xiuping; Yu, Long; Zhao, Shimin; Li, Yao; Lin, Dong; Wang, Yuzhuo; Wang, Liguo; Chen, Yu; Sun, Yinghao; Wang, Chenji; Huang, Haojie

2017-09-01

Bromodomain and extraterminal domain (BET) protein inhibitors are emerging as promising anticancer therapies. The gene encoding the E3 ubiquitin ligase substrate-binding adaptor speckle-type POZ protein (SPOP) is the most frequently mutated in primary prostate cancer. Here we demonstrate that wild-type SPOP binds to and induces ubiquitination and proteasomal degradation of BET proteins (BRD2, BRD3 and BRD4) by recognizing a degron motif common among them. In contrast, prostate cancer-associated SPOP mutants show impaired binding to BET proteins, resulting in decreased proteasomal degradation and accumulation of these proteins in prostate cancer cell lines and patient specimens and causing resistance to BET inhibitors. Transcriptome and BRD4 cistrome analyses reveal enhanced expression of the GTPase RAC1 and cholesterol-biosynthesis-associated genes together with activation of AKT-mTORC1 signaling as a consequence of BRD4 stabilization. Our data show that resistance to BET inhibitors in SPOP-mutant prostate cancer can be overcome by combination with AKT inhibitors and further support the evaluation of SPOP mutations as biomarkers to guide BET-inhibitor-oriented therapy in patients with prostate cancer.

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.

HSP27 phosphorylation modulates TRAIL-induced activation of Src-Akt/ERK signaling through interaction with β-arrestin2.

PubMed

Qi, Shimei; Xin, Yinqiang; Qi, Zhilin; Xu, Yimiao; Diao, Ying; Lan, Lei; Luo, Lan; Yin, Zhimin

2014-03-01

Heat shock protein 27 (HSP27) regulates critical cellular functions such as development, differentiation, cell growth and apoptosis. A variety of stimuli induce the phosphorylation of HSP27, which affects its cellular functions. However, most previous studies focused on the role of HSP27 protein itself in apoptosis, the particular role of its phosphorylation state in signaling transduction remains largely unclear. In the present study, we reported that HSP27 phosphorylation modulated TRAIL-triggered pro-survival signaling transduction. In HeLa cells, suppression of HSP27 phosphorylation by specific inhibitor KRIBB3 or MAPKAPK2 (MK2) knockdown and by overexpression of non-phosphorylatable HSP27(3A) mutant demonstrated that hindered HSP27 phosphorylation enhanced the TRAIL-induced apoptosis. In addition, reduced HSP27 phosphorylation by KRIBB3 treatment or MK2 knockdown attenuated the TRAIL-induced activation of Akt and ERK survival signaling through suppressing the phosphorylation of Src. By overexpression of HSP27(15A) or HSP27(78/82A) phosphorylation mutant, we further showed that phosphorylation of HSP27 at serine 78/82 residues was essential to TRAIL-triggered Src-Akt/ERK signaling transduction. Co-immunoprecipitation and confocal microscopy showed that HSP27 interacted with Src and scaffolding protein β-arrestin2 in response of TRAIL stimulation and suppression of HSP27 phosphorylation apparently disrupted the TRAIL-induced interaction of HSP27 and Src or interaction of HSP27 and β-arrestin2. We further demonstrated that β-arrestin2 mediated HSP27 action on TRAIL-induced Src activation, which was achieved by recruiting signaling complex of HSP27/β-arrestin2/Src in response to TRAIL. Taken together, our study revealed that HSP27 phosphorylation modulates TRAIL-triggered activation of Src-Akt/ERK pro-survival signaling via interacting with β-arrestin2 in HeLa cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Development of a New Structural Class of Broadly Acting HCV Non-Nucleoside Inhibitors Leading to the Discovery of MK-8876

DOE Office of Scientific and Technical Information (OSTI.GOV)

McComas, Casey C.; Palani, Anandan; Chang, Wei

Studies directed at developing a broadly acting non-nucleoside inhibitor of HCV NS5B led to the discovery of a novel structural class of 5-aryl benzofurans that simultaneously interact with both the palm I and palm II binding regions. An initial candidate was potent in vitro against HCV GT1a and GT1b replicons, and induced multi-log reductions in HCV viral load when orally dosed to chronic GT1 infected chimpanzees. However, in vitro potency losses against clinically relevant GT1a variants prompted a further effort to develop compounds with sustained potency across a broader array of HCV genotypes and mutants. Ultimately, a biology and medicinalmore » chemistry collaboration led to the discovery of the development candidate MK-8876. MK-8876 demonstrated a pan-genotypic potency profile and maintained potency against clinically relevant mutants. It demonstrated moderate bioavailability in rats and dogs, but showed low plasma clearance characteristics consistent with once-daily dosing. Herein we describe the efforts which led to the discovery of MK-8876, which advanced into Phase 1 monotherapy studies for evaluation and characterization as a component of an all-oral direct-acting drug regimen for the treatment of chronic HCV infection.« less

Discovery of 3-(3-(4-(1-Aminocyclobutyl)phenyl)-5-phenyl-3 H -imidazo[4,5- b ]pyridin-2-yl)pyridin-2-amine (ARQ 092): An Orally Bioavailable, Selective, and Potent Allosteric AKT Inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lapierre, Jean-Marc; Eathiraj, Sudharshan; Vensel, David

The work in this paper describes the optimization of the 3-(3-phenyl-3H-imidazo[4,5-b]pyridin-2-yl)pyridin-2-amine chemical series as potent, selective allosteric inhibitors of AKT kinases, leading to the discovery of ARQ 092 (21a). The cocrystal structure of compound 21a bound to full-length AKT1 confirmed the allosteric mode of inhibition of this chemical class and the role of the cyclobutylamine moiety. Compound 21a demonstrated high enzymatic potency against AKT1, AKT2, and AKT3, as well as potent cellular inhibition of AKT activation and the phosphorylation of the downstream target PRAS40. Compound 21a also served as a potent inhibitor of the AKT1-E17K mutant protein and inhibited tumormore » growth in a human xenograft mouse model of endometrial adenocarcinoma.« less

Novel PI3K/Akt Inhibitors Screened by the Cytoprotective Function of Human Immunodeficiency Virus Type 1 Tat

PubMed Central

Kim, Dong-Hyun; Kim, Baek

2011-01-01

The PI3K/Akt pathway regulates various stress-related cellular responses such as cell survival, cell proliferation, metabolism and protein synthesis. Many cancer cell types display the activation of this pathway, and compounds inhibiting this cell survival pathway have been extensively evaluated as anti-cancer agents. In addition to cancers, several human viruses, such as HTLV, HPV, HCV and HIV-1, also modulate this pathway, presumably in order to extend the life span of the infected target cells for productive viral replication. The expression of HIV-1 Tat protein exhibited the cytoprotective effect in macrophages and a human microglial cell line by inhibiting the negative regulator of this pathway, PTEN. This cytoprotective effect of HIV-1 appears to contribute to the long-term survival and persistent HIV-1 production in human macrophage reservoirs. In this study we exploited the PI3K/Akt dependent cytoprotective effect of Tat-expressing CHME5 cells. We screened a collection of compounds known to modulate inflammation, and identified three novel compounds: Lancemaside A, Compound K and Arctigenin that abolished the cytoprotective phenotype of Tat-expressing CHME5 cells. All three compounds antagonized the kinase activity of Akt. Further detailed signaling studies revealed that each of these three compounds targeted different steps of the PI3K/Akt pathway. Arctigenin regulates the upstream PI3K enzyme from converting PIP2 to PIP3. Lancemaside A1 inhibited the movement of Akt to the plasma membrane, a critical step for Akt activation. Compound K inhibited Akt phosphorylation. This study supports that Tat-expressing CHME5 cells are an effective model system for screening novel PI3K/Akt inhibitors. PMID:21765914

17β-Estradiol Promotes Schwann Cell Proliferation and Differentiation, Accelerating Early Remyelination in a Mouse Peripheral Nerve Injury Model

PubMed Central

Chen, Yan; Guo, Wenjie; Li, Wenjuan; Cheng, Meng; Hu, Ying; Xu, Wenming

2016-01-01

Estrogen induces oligodendrocyte remyelination in response to demyelination in the central nervous system. Our objective was to determine the effects of 17β-estradiol (E2) on Schwann cell function and peripheral nerve remyelination after injury. Adult male C57BL/6J mice were used to prepare the sciatic nerve transection injury model and were randomly categorized into control and E2 groups. To study myelination in vitro, dorsal root ganglion (DRG) explant culture was prepared using 13.5-day-old mouse embryos. Primary Schwann cells were isolated from the sciatic nerves of 1- to 3-day-old Sprague–Dawley rats. Immunostaining for myelin basic protein (MBP) expression and toluidine blue staining for myelin sheaths demonstrated that E2 treatment accelerates early remyelination in the “nerve bridge” region between the proximal and distal stumps of the transection injury site in the mouse sciatic nerve. The 5-bromo-2′-deoxyuridine incorporation assay revealed that E2 promotes Schwann cell proliferation in the bridge region and in the primary culture, which is blocked using AKT inhibitor MK2206. The in vitro myelination in the DRG explant culture determined showed that the MBP expression in the E2-treated group is higher than that in the control group. These results show that E2 promotes Schwann cell proliferation and myelination depending on AKT activation. PMID:27872858

FABP4 reversed the regulation of leptin on mitochondrial fatty acid oxidation in mice adipocytes

PubMed Central

Gan, Lu; Liu, Zhenjiang; Cao, Weina; Zhang, Zhenzhen; Sun, Chao

2015-01-01

Fatty acid binding protein 4 (FABP4), plays key role in fatty acid transportation and oxidation, and increases with leptin synergistically during adipose inflammation process. However, the regulation mechanism between FABP4 and leptin on mitochondrial fatty acid oxidation remains unclear. In this study, we found that FABP4 reduced the expression of leptin, CPT-1 and AOX1 in mice adipocytes. Conversely, FABP4 was down-regulated in a time-dependent manner by leptin treatment. Additionally, forced expression of FABP4 attenuated the expression of PGC1-α, UCP2, CPT-1, AOX1 and COX2 compared with leptin incubation. Moreover, mitochondrial membrane potential, fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase (MCAD), long-chain acyl-CoA dehydrogenase (LCAD) and Cyt C levels were reduced in response to the overexpression of FABP4. These reductions correspond well with the reduced release of free fatty acid and the inactivation of mitochondrial complexes I and III by FABP4 overexpression. Furthermore, addition of the Akt/mTOR pathway-specific inhibitor (MK2206) blocked the mitochondrial fatty acid oxidation and respiration factors, whereas interference of FABP4 overcame these effects. Taken together, FABP4 could reverse the activation of the leptin-induced mitochondrial fatty acid oxidation, and the inhibition of Akt/mTOR signal pathway played a key role in this process. PMID:26310911

FABP4 reversed the regulation of leptin on mitochondrial fatty acid oxidation in mice adipocytes.

PubMed

Gan, Lu; Liu, Zhenjiang; Cao, Weina; Zhang, Zhenzhen; Sun, Chao

2015-08-27

Fatty acid binding protein 4 (FABP4), plays key role in fatty acid transportation and oxidation, and increases with leptin synergistically during adipose inflammation process. However, the regulation mechanism between FABP4 and leptin on mitochondrial fatty acid oxidation remains unclear. In this study, we found that FABP4 reduced the expression of leptin, CPT-1 and AOX1 in mice adipocytes. Conversely, FABP4 was down-regulated in a time-dependent manner by leptin treatment. Additionally, forced expression of FABP4 attenuated the expression of PGC1-α, UCP2, CPT-1, AOX1 and COX2 compared with leptin incubation. Moreover, mitochondrial membrane potential, fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase (MCAD), long-chain acyl-CoA dehydrogenase (LCAD) and Cyt C levels were reduced in response to the overexpression of FABP4. These reductions correspond well with the reduced release of free fatty acid and the inactivation of mitochondrial complexes I and III by FABP4 overexpression. Furthermore, addition of the Akt/mTOR pathway-specific inhibitor (MK2206) blocked the mitochondrial fatty acid oxidation and respiration factors, whereas interference of FABP4 overcame these effects. Taken together, FABP4 could reverse the activation of the leptin-induced mitochondrial fatty acid oxidation, and the inhibition of Akt/mTOR signal pathway played a key role in this process.

Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity.

PubMed

Chan, Tung O; Zhang, Jin; Rodeck, Ulrich; Pascal, John M; Armen, Roger S; Spring, Maureen; Dumitru, Calin D; Myers, Valerie; Li, Xue; Cheung, Joseph Y; Feldman, Arthur M

2011-11-15

Phosphorylation of a threonine residue (T308 in Akt1) in the activation loop of Akt kinases is a prerequisite for deregulated Akt activity frequently observed in neoplasia. Akt phosphorylation in vivo is balanced by the opposite activities of kinases and phosphatases. Here we describe that targeting Akt kinase to the cell membrane markedly reduced sensitivity of phosphorylated Akt to dephosphorylation by protein phosphatase 2A. This effect was amplified by occupancy of the ATP binding pocket by either ATP or ATP-competitive inhibitors. Mutational analysis revealed that R273 in Akt1 and the corresponding R274 in Akt2 are essential for shielding T308 in the activation loop against dephosphorylation. Thus, occupancy of the nucleotide binding pocket of Akt kinases enables intramolecular interactions that restrict phosphatase access and sustain Akt phosphorylation. This mechanism provides an explanation for the "paradoxical" Akt hyperphosphorylation induced by ATP-competitive inhibitor, A-443654. The lack of phosphatase resistance further contributes insight into the mechanism by which the human Akt2 R274H missense mutation may cause autosomal-dominant diabetes mellitus.

Assay development and case history of a 32K-biased library high-content MK2-EGFP translocation screen to identify p38 mitogen-activated protein kinase inhibitors on the ArrayScan 3.1 imaging platform.

PubMed

Trask, Oscar J; Baker, Audrey; Williams, Rhonda Gates; Nickischer, Debra; Kandasamy, Ramani; Laethem, Carmen; Johnston, Patricia A; Johnston, Paul A

2006-01-01

This chapter describes the conversion and assay development of a 96-well MK2-EGFP translocation assay into a higher density 384-well format high-content assay to be screened on the ArrayScan 3.1 imaging platform. The assay takes advantage of the well-substantiated hypothesis that mitogen-activated protein kinase-activating protein kinase-2 (MK2) is a substrate of p38 MAPK kinase and that p38-induced phosphorylation of MK-2 induces a nucleus-to-cytoplasm translocation. This chapter also presents a case history of the performance of the MK2-EGFP translocation assay, run as a "high-content" screen of a 32K kinase-biased library to identify p38 inhibitors. The assay performed very well and a number of putative p38 inhibitor hits were identified. Through the use of multiparameter data provided by the nuclear translocation algorithm and by checking images, a number of compounds were identified that were potential artifacts due to interference with the imaging format. These included fluorescent compounds, or compounds that dramatically reduced cell numbers due to cytotoxicity or by disrupting cell adherence. A total of 145 compounds produced IC(50) values <50.0 muM in the MK2-EGFP translocation assay, and a cross target query of the Lilly-RTP HTS database confirmed their inhibitory activity against in vitro kinase targets, including p38a. Compounds were confirmed structurally by LCMS analysis and profiled in cell-based imaging assays for MAPK signaling pathway selectivity. Three of the hit scaffolds identified in the MK2-EGFP translocation HCS run on the ArrayScan were selected for a p38a inhibitor hit-to-lead structure activity relationship (SAR) chemistry effort.

10 CFR 20.2206 - Reports of individual monitoring.

Code of Federal Regulations, 2010 CFR

2010-01-01

... Energy NUCLEAR REGULATORY COMMISSION STANDARDS FOR PROTECTION AGAINST RADIATION Reports § 20.2206 Reports...) Operate a nuclear reactor designed to produce electrical or heat energy pursuant to § 50.21(b) or § 50.22... nuclear material in a quantity exceeding 5,000 grams of contained uranium-235, uranium-233, or plutonium...

10 CFR 20.2206 - Reports of individual monitoring.

Code of Federal Regulations, 2012 CFR

2012-01-01

... Energy NUCLEAR REGULATORY COMMISSION STANDARDS FOR PROTECTION AGAINST RADIATION Reports § 20.2206 Reports...) Operate a nuclear reactor designed to produce electrical or heat energy pursuant to § 50.21(b) or § 50.22... nuclear material in a quantity exceeding 5,000 grams of contained uranium-235, uranium-233, or plutonium...

10 CFR 20.2206 - Reports of individual monitoring.

Code of Federal Regulations, 2014 CFR

2014-01-01

... Energy NUCLEAR REGULATORY COMMISSION STANDARDS FOR PROTECTION AGAINST RADIATION Reports § 20.2206 Reports...) Operate a nuclear reactor designed to produce electrical or heat energy pursuant to § 50.21(b) or § 50.22... nuclear material in a quantity exceeding 5,000 grams of contained uranium-235, uranium-233, or plutonium...

Dimethylarginine dimethylaminohydrolase 1 modulates endothelial cell growth through NO and Akt

PubMed Central

Zhang, Ping; Hu, Xinli; Xu, Xin; Chen, Yingjie; Bache, Robert J.

2011-01-01

Objective Dimethylarginine dimethylaminohydrolase 1 (DDAH1) modulates NO production by degrading the endogenous NO synthase (NOS) inhibitors ADMA and L-NMMA. This study examined whether, in addition to degrading ADMA, DDAH1 exerts ADMA independent effects that influence endothelial function. Methods and Results Using selective gene silencing of DDAH1 with small interfering RNA and overexpression of DDAH1 in HUVEC, we found that DDAH1 acts to promote endothelial cell proliferation, migration and tube formation both by Akt phosphorylation as well as through the traditional role of degrading ADMA. Incubation of HUVEC with the NOS inhibitors L-NAME or ADMA, the soluble guanylyl cyclase inhibitor ODQ, or the cGMP analog 8-pCPT-cGMP had no effect on p-AktSer473, indicating that the increase of p-AktSer473 produced by DDAH1 was independent of the NO-cGMP signaling pathway. DDAH1 formed a protein complex with Ras, and DDAH1 overexpression increased Ras activity. The Ras inhibitor manumycin-A or dominant-negative Ras significantly attenuated the DDAH1-induced increase of p-AktSer473. Furthermore, DDAH1 knockout impaired endothelial sprouting from cultured aortic rings, and overexpression of constitutively active Akt or DDAH1 rescued endothelial sprouting in the aortic rings from these mice. Conclusions DDAH1 exerts a unique role in activating Akt that affects endothelial function independent of degrading endogenous NOS inhibitors. PMID:21212404

Akt-mediated regulation of NFkappaB and the essentialness of NFkappaB for the oncogenicity of PI3K and Akt.

PubMed

Bai, Dong; Ueno, Lynn; Vogt, Peter K

2009-12-15

The serine/threonine kinase Akt (cellular homolog of murine thymoma virus akt8 oncogene), also known as PKB (protein kinase B), is activated by lipid products of phosphatidylinositol 3-kinase (PI3K). Akt phosphorylates numerous protein targets that control cell survival, proliferation and motility. Previous studies suggest that Akt regulates transcriptional activity of the nuclear factor-kappaB (NFkappaB) by inducing phosphorylation and subsequent degradation of inhibitor of kappaB (IkappaB). We show here that NFkappaB-driven transcription increases in chicken embryonic fibroblasts (CEF) transformed by myristylated Akt (myrAkt). Accordingly, both a dominant negative mutant of Akt and Akt inhibitors repress NFkappaB-dependent transcription. The degradation of the IkappaB protein is strongly enhanced in Akt-transformed cells, and the loss of NFkappaB activity by introduction of a super-repressor of NFkappaB, IkappaBSR, interferes with PI3K- and Akt-induced oncogenic transformation of CEF. The phosphorylation of the p65 subunit of NFkappaB at serine 534 is also upregulated in Akt-transformed cells. Our data suggest that the stimulation of NFkappaB by Akt is dependent on the phosphorylation of p65 at S534, mediated by IKK (IkappaB kinase) alpha and beta. Akt phosphorylates IKKalpha on T23, and this phosphorylation event is a prerequisite for the phosphorylation of p65 at S534 by IKKalpha and beta. Our results demonstrate two separate functions of the IKK complex in NFkappaB activation in cells with constitutive Akt activity: the phosphorylation and consequent degradation of IkappaB and the phosphorylation of p65. The data further support the conclusion that NFkappaB activity is essential for PI3K- and Akt-induced oncogenic transformation. Copyright (c) 2009 UICC.

Effects of a glycine transporter-1 inhibitor and D-serine on MK-801-induced immobility in the forced swimming test in rats.

PubMed

Kawaura, Kazuaki; Koike, Hiroyuki; Kinoshita, Kohnosuke; Kambe, Daiji; Kaku, Ayaka; Karasawa, Jun-ichi; Chaki, Shigeyuki; Hikichi, Hirohiko

2015-02-01

Glutamatergic dysfunction, particularly the hypofunction of N-methyl-D-aspartate (NMDA) receptors, is involved in the pathophysiology of schizophrenia. The positive modulation of the glycine site on the NMDA receptor has been proposed as a novel therapeutic approach for schizophrenia. However, its efficacy against negative symptoms, which are poorly managed by current medications, has not been fully addressed. In the present study, the effects of the positive modulation of the glycine site on the NMDA receptor were investigated in an animal model of negative symptoms of schizophrenia. The subchronic administration of MK-801 increased immobility in the forced swimming test in rats without affecting spontaneous locomotor activity. The increased immobility induced by MK-801 was attenuated by the atypical antipsychotic clozapine but not by either the typical antipsychotic haloperidol or the antidepressant imipramine, indicating that the increased immobility induced by subchronic treatment with MK-801 in the forced swimming test may represent a negative symptom of schizophrenia. Likewise, positive modulation of the glycine sites on the NMDA receptor using an agonist for the glycine site, D-serine, and a glycine transporter-1 inhibitor, N-[(3R)-3-([1,1'-biphenyl]-4-yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine hydrochloride (NFPS), significantly reversed the increase in immobility in MK-801-treated rats without reducing the immobility time in vehicle-treated rats. The present results show that the stimulation of the NMDA receptor through the glycine site on the receptor either directly with D-serine or by blocking glycine transporter-1 attenuates the immobility elicited by the subchronic administration of MK-801 and may be potentially useful for the treatment of negative symptoms of schizophrenia. Copyright © 2014 Elsevier B.V. All rights reserved.

BRAFV600E Negatively Regulates the AKT Pathway in Melanoma Cell Lines

PubMed Central

Chen, Brenden; Tardell, Christine; Higgins, Brian; Packman, Kathryn; Boylan, John F.; Niu, Huifeng

2012-01-01

Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways. PMID:22880048

BRAFV600E negatively regulates the AKT pathway in melanoma cell lines.

PubMed

Chen, Brenden; Tardell, Christine; Higgins, Brian; Packman, Kathryn; Boylan, John F; Niu, Huifeng

2012-01-01

Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways.

21 CFR 74.2206 - D&C Green No. 6.

Code of Federal Regulations, 2010 CFR

2010-04-01

... ADDITIVES SUBJECT TO CERTIFICATION Cosmetics § 74.2206 D&C Green No. 6. (a) Identity and specifications. The color additive D&C Green No. 6 shall conform in identity and specifications to the requirements of § 74... applied cosmetics in amounts consistent with good manufacturing practice. (c) Labeling requirements. The...

21 CFR 74.2206 - D&C Green No. 6.

Code of Federal Regulations, 2012 CFR

2012-04-01

... ADDITIVES SUBJECT TO CERTIFICATION Cosmetics § 74.2206 D&C Green No. 6. (a) Identity and specifications. The color additive D&C Green No. 6 shall conform in identity and specifications to the requirements of § 74... applied cosmetics in amounts consistent with good manufacturing practice. (c) Labeling requirements. The...

21 CFR 74.2206 - D&C Green No. 6.

Code of Federal Regulations, 2013 CFR

2013-04-01

... ADDITIVES SUBJECT TO CERTIFICATION Cosmetics § 74.2206 D&C Green No. 6. (a) Identity and specifications. The color additive D&C Green No. 6 shall conform in identity and specifications to the requirements of § 74... applied cosmetics in amounts consistent with good manufacturing practice. (c) Labeling requirements. The...

21 CFR 74.2206 - D&C Green No. 6.

Code of Federal Regulations, 2014 CFR

2014-04-01

... ADDITIVES SUBJECT TO CERTIFICATION Cosmetics § 74.2206 D&C Green No. 6. (a) Identity and specifications. The color additive D&C Green No. 6 shall conform in identity and specifications to the requirements of § 74... applied cosmetics in amounts consistent with good manufacturing practice. (c) Labeling requirements. The...

The erythropoietin-derived peptide MK-X and erythropoietin have neuroprotective effects against ischemic brain damage

PubMed Central

Yoo, Seung-Jun; Cho, Bongki; Lee, Deokho; Son, Gowoon; Lee, Yeong-Bae; Soo Han, Hyung; Kim, Eunjoo; Moon, Chanil; Moon, Cheil

2017-01-01

Erythropoietin (EPO) has been well known as a hematopoietic cytokine over the past decades. However, recent reports have demonstrated that EPO plays a neuroprotective role in the central nervous system, and EPO has been considered as a therapeutic target in neurodegenerative diseases such as ischemic stroke. Despite the neuroprotective effect of EPO, clinical trials have shown its unexpected side effects, including undesirable proliferative effects such as erythropoiesis and tumor growth. Therefore, the development of EPO analogs that would confer neuroprotection without adverse effects has been attempted. In this study, we examined the potential of a novel EPO-based short peptide, MK-X, as a novel drug for stroke treatment in comparison with EPO. We found that MK-X administration with reperfusion dramatically reduced brain injury in an in vivo mouse model of ischemic stroke induced by middle cerebral artery occlusion, whereas EPO had little effect. Similar to EPO, MK-X efficiently ameliorated mitochondrial dysfunction followed by neuronal death caused by glutamate-induced oxidative stress in cultured neurons. Consistent with this effect, MK-X significantly decreased caspase-3 cleavage and nuclear translocation of apoptosis-inducing factor induced by glutamate. MK-X completely mimicked the effect of EPO on multiple activation of JAK2 and its downstream PI3K/AKT and ERK1/2 signaling pathways, and this signaling process was involved in the neuroprotective effect of MK-X. Furthermore, MK-X and EPO induced similar changes in the gene expression patterns under glutamate-induced excitotoxicity. Interestingly, the most significant difference between MK-X and EPO was that MK-X better penetrated into the brain across the brain–blood barrier than did EPO. In conclusion, we suggest that MK-X might be used as a novel drug for protection from brain injury caused by ischemic stroke, which penetrates into the brain faster in comparison with EPO, even though MK-X and EPO have

Dimethylarginine dimethylaminohydrolase 1 modulates endothelial cell growth through nitric oxide and Akt.

PubMed

Zhang, Ping; Hu, Xinli; Xu, Xin; Chen, Yingjie; Bache, Robert J

2011-04-01

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) modulates NO production by degrading the endogenous nitric oxide (NO) synthase (NOS) inhibitors asymmetrical dimethylarginine (ADMA) and L-NG-monomethyl arginine (L-NMMA). This study examined whether, in addition to degrading ADMA, DDAH1 exerts ADMA-independent effects that influence endothelial function. Using selective gene silencing of DDAH1 with small interfering RNA and overexpression of DDAH1 in human umbilical vein endothelial cells, we found that DDAH1 acts to promote endothelial cell proliferation, migration, and tube formation by Akt phosphorylation, as well as through the traditional role of degrading ADMA. Incubation of human umbilical vein endothelial cells with the NOS inhibitors l-NG-nitro-arginine methyl ester (L-NAME) or ADMA, the soluble guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo-(4,3-2)quinoxalin-1-one, or the cGMP analog 8-(4-Chlorophenylthio)-cGMP had no effect on phosphorylated (p)-Akt(Ser473), indicating that the increase in p-Akt(Ser473) produced by DDAH1 was independent of the NO-cGMP signaling pathway. DDAH1 formed a protein complex with Ras, and DDAH1 overexpression increased Ras activity. The Ras inhibitor manumycin-A or dominant-negative Ras significantly attenuated the DDAH1-induced increase in p-Akt(Ser473). Furthermore, DDAH1 knockout impaired endothelial sprouting from cultured aortic rings, and overexpression of constitutively active Akt or DDAH1 rescued endothelial sprouting in the aortic rings from these mice. DDAH1 exerts a unique role in activating Akt that affects endothelial function independently of degrading endogenous NOS inhibitors.

Expression of SLP-2 was associated with invasion of esophageal squamous cell carcinoma.

PubMed

Cao, Wenfeng; Zhang, Bin; Ding, Fang; Zhang, Weiran; Sun, Baocun; Liu, Zhihua

2013-01-01

Stomatin-like protein 2 (SLP-2), a member of the Stomatin superfamily, has been identified as an oncogenic-related protein and found to be up-regulated in multi-cancers. Nonetheless, the expression pattern and regulation of SLP-2 in human esophageal squamous cell carcinoma (ESCC) remain unexplored. Immunohistochemistry and immunofluorescence staining analysis were performed to show SLP-2 expression and location. RNAi method was used to inhibit specific protein expression. Transwell assay was done to investigate cells invasive capability. RT-PCR and Western blot analysis were used to detect mRNA and protein expression levels. Immunohistochemical analysis showed that up-regulation of SLP-2 was found in invasive front compared with cancer central tissue in ESCC. Inhibition of SLP-2 by SLP-2 siRNA can decrease ESCC cells invasive capability through MMP-2 dependent manner. Up-regulation of SLP-2 was effectively abrogated by the ERK1/2 inhibitors either PD98059 or U0126, but no effect was showed by the treatment of AKT inhibitors either LY294002 or MK-2206. So the regulation of SLP-2 was involved in activation of the MAPK/ERK pathway. We found that PMA/EGF could induce the up-regulated expression of SLP-2 probably through activating ERK signalling. The current study suggests that SLP-2 may represent an important molecular hallmark that is clinically relevant to the invasion of ESCC.

20 CFR 416.2206 - Basic qualifications for alternate participants.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Provisions § 416.2206 Basic qualifications for alternate participants. (a) General. We may arrange for VR... (that is, any entity whether for-profit or not-for-profit), other than a State VR agency. (1) An... provide VR services in the State in which it provides services; and (ii) Under the terms of the written...

20 CFR 416.2206 - Basic qualifications for alternate participants.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Provisions § 416.2206 Basic qualifications for alternate participants. (a) General. We may arrange for VR... (that is, any entity whether for-profit or not-for-profit), other than a State VR agency. (1) An... provide VR services in the State in which it provides services; and (ii) Under the terms of the written...

20 CFR 416.2206 - Basic qualifications for alternate participants.

Code of Federal Regulations, 2013 CFR

2013-04-01

... Provisions § 416.2206 Basic qualifications for alternate participants. (a) General. We may arrange for VR... (that is, any entity whether for-profit or not-for-profit), other than a State VR agency. (1) An... provide VR services in the State in which it provides services; and (ii) Under the terms of the written...

20 CFR 416.2206 - Basic qualifications for alternate participants.

Code of Federal Regulations, 2014 CFR

2014-04-01

... Provisions § 416.2206 Basic qualifications for alternate participants. (a) General. We may arrange for VR... (that is, any entity whether for-profit or not-for-profit), other than a State VR agency. (1) An... provide VR services in the State in which it provides services; and (ii) Under the terms of the written...

20 CFR 416.2206 - Basic qualifications for alternate participants.

Code of Federal Regulations, 2012 CFR

2012-04-01

... Provisions § 416.2206 Basic qualifications for alternate participants. (a) General. We may arrange for VR... (that is, any entity whether for-profit or not-for-profit), other than a State VR agency. (1) An... provide VR services in the State in which it provides services; and (ii) Under the terms of the written...

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

Selumetinib and Akt Inhibitor MK-2206 in Treating Patients With Refractory or Advanced Gallbladder or Bile Duct Cancer That Cannot Be Removed By Surgery

ClinicalTrials.gov

2014-09-08

Adenocarcinoma of the Gallbladder; Adenocarcinoma With Squamous Metaplasia of the Gallbladder; Adult Primary Cholangiocellular Carcinoma; Advanced Adult Primary Liver Cancer; Cholangiocarcinoma of the Extrahepatic Bile Duct; Localized Unresectable Adult Primary Liver Cancer; Metastatic Extrahepatic Bile Duct Cancer; Recurrent Adult Primary Liver Cancer; Recurrent Extrahepatic Bile Duct Cancer; Stage II Gallbladder Cancer; Stage IIIA Gallbladder Cancer; Stage IIIB Gallbladder Cancer; Stage IVA Gallbladder Cancer; Stage IVB Gallbladder Cancer; Unresectable Extrahepatic Bile Duct Cancer

Complications of hyperglycaemia with PI3K-AKT-mTOR inhibitors in patients with advanced solid tumours on Phase I clinical trials.

PubMed

Geuna, E; Roda, D; Rafii, S; Jimenez, B; Capelan, M; Rihawi, K; Montemurro, F; Yap, T A; Kaye, S B; De Bono, J S; Molife, L R; Banerji, U

2015-12-01

PI3K-AKT-mTOR inhibitors (PAMi) are promising anticancer treatments. Hyperglycaemia is a mechanism-based toxicity of these agents and is becoming increasingly important with their use in larger numbers of patients. Retrospective case-control study comparing incidence and severity of hyperglycaemia (all grades) between a case group of 387 patients treated on 18 phase I clinical trials with PAMi (78 patients with PI3Ki, 138 with mTORi, 144 with AKTi and 27 with PI3K/mTORi) and a control group of 109 patients treated on 10 phase I clinical trials with agents not directly targeting the PAM pathway. Diabetic patients were excluded in both groups. The incidence of hyperglycaemia was not significantly different between cases and controls (86.6% vs 80.7%, respectively, P=0.129). However, high grade (grade 3-4) hyperglycaemia was more frequent in the PAMi group than in controls (6.7% vs 0%, respectively, P=0.005). The incidence of grade 3-4 hyperglycaemia was greater with AKT and multikinase inhibitors compared with other PAMi (P<0.001). All patients with high-grade hyperglycaemia received antihyperglycemic treatment and none developed severe metabolic complications (diabetic ketoacidosis or hyperosmolar hyperglycemic nonketotic state). High-grade hyperglycaemia was the cause of permanent PAMi discontinuation in nine patients. PI3K-AKT-mTOR inhibitors are associated with small (6.7%) but statistically significant increased risk of high-grade hyperglycaemia compared with non-PAM targeting agents. However, PAMi-induced hyperglycaemia was not found to be associated with severe metabolic complications in this non-diabetic population of patients with advanced cancers.

Celecoxib promotes c-FLIP degradation through Akt-independent inhibition of GSK3

PubMed Central

Chen, Shuzhen; Cao, Wei; Yue, Ping; Hao, Chunhai; Khuri, Fadlo R.; Sun, Shi-Yong

2011-01-01

Celecoxib is a COX2 inhibitor that reduces the risk of colon cancer. However, the basis for its cancer chemopreventive activity is not fully understood. In this study, we defined a mechanism of celecoxib action based on degradation of c-FLIP, a major regulator of the death receptor pathway of apoptosis. c-FLIP protein levels are regulated by ubiquitination and proteasome-mediated degradation. We found that celecoxib controlled c-FLIP ubiquitination through Akt-independent inhibition of GSK3 kinase, itself a candidate therapeutic target of interest in colon cancer. Celecoxib increased the levels of phosphorylated GSK3 (p-GSK3), including the α and β forms, even in cell lines where p-Akt levels were not increased. PI3K inhibitors abrogated Akt phosphorylation as expected but had no effect on celecoxib-induced GSK3 phosphorylation. In contrast, PKC inhibitors abolished celecoxib-induced GSK3 phosphorylation, implying that celecoxib influenced GSK3 phosphorylation through a mechanism relied upon PKC but not Akt. GSK3 blockade either by siRNA or kinase inhibitors was sufficient to attenuate c-FLIP levels. Combining celecoxib with GSK3 inhibition enhanced attenuation of c-FLIP and increased apoptosis. Proteasome inhibitor MG132 reversed the effects of GSK3 inhibition and increased c-FLIP ubiquitination, confirming that c-FLIP attenuation was mediated by proteasomal turnover as expected. Our findings reveal a novel mechanism through which the regulatory effects of c-FLIP on death receptor signaling are controlled by GSK3, which celecoxib acts at an upstream level to control independently of Akt. PMID:21868755

Inhibition of Akt with small molecules and biologics: historical perspective and current status of the patent landscape

PubMed Central

Mattmann, Margrith E; Stoops, Sydney L; Lindsley, Craig W

2014-01-01

Introduction Akt plays a pivotal role in cell survival and proliferation through a number of downstream effectors; unregulated activation of the PI3K/PTEN/Akt pathway is a prominent feature of many human cancers. Akt is considered an attractive target for cancer therapy by the inhibition of Akt alone or in combination with standard cancer chemotherapeutics. Both preclinical animal studies and clinical trials in humans have validated Akt as an important target of cancer drug discovery. Area covered A historical perspective of Akt inhibitors, including PI analogs, ATP-competitive and allosteric Akt inhibitors, along with other inhibitory mechanisms are reviewed in this paper with a focus on issued patents, patent applications and a summary of clinical trial updates since the last review in 2007. Expert opinion A vast diversity of inhibitors of Akt, both small molecule and biologic, have been developed in the past 5 years, with over a dozen in various phases of clinical development, and several displaying efficacy in humans. While it is not yet clear which mechanism of Akt inhibition will be optimal in humans, or which Akt isoforms to inhibit, or whether a small molecule or biologic agent will be best, data to all of these points will be available in the near future. PMID:21635152

Molecular and functional interactions between AKT and SOX2 in breast carcinoma

PubMed Central

Mir, Perihan; Konantz, Martina; Pereboom, Tamara C.; Paczulla, Anna M.; Merz, Britta; Fehm, Tanja; Perner, Sven; Rothfuss, Oliver C.; Kanz, Lothar; Schulze-Osthoff, Klaus; Lengerke, Claudia

2015-01-01

The transcription factor SOX2 is a key regulator of pluripotency in embryonic stem cells and plays important roles in early organogenesis. Recently, SOX2 expression was documented in various cancers and suggested as a cancer stem cell (CSC) marker. Here we identify the Ser/Thr-kinase AKT as an upstream regulator of SOX2 protein turnover in breast carcinoma (BC). SOX2 and pAKT are co-expressed and co-regulated in breast CSCs and depletion of either reduces clonogenicity. Ectopic SOX2 expression restores clonogenicity and in vivo tumorigenicity of AKT-inhibited cells, suggesting that SOX2 acts as a functional downstream AKT target. Mechanistically, we show that AKT physically interacts with the SOX2 protein to modulate its subcellular distribution. AKT kinase inhibition results in enhanced cytoplasmic retention of SOX2, presumably via impaired nuclear import, and in successive cytoplasmic proteasomal degradation of the protein. In line, blockade of either nuclear transport or proteasomal degradation rescues SOX2 expression in AKT-inhibited BC cells. Finally, AKT inhibitors efficiently suppress the growth of SOX2-expressing putative cancer stem cells, whereas conventional chemotherapeutics select for this population. Together, our results suggest the AKT/SOX2 molecular axis as a regulator of BC clonogenicity and AKT inhibitors as promising drugs for the treatment of SOX2-positive BC. PMID:26498353

3D-QSAR and Molecular Docking Studies on Derivatives of MK-0457, GSK1070916 and SNS-314 as Inhibitors against Aurora B Kinase

PubMed Central

Zhang, Baidong; Li, Yan; Zhang, Huixiao; Ai, Chunzhi

2010-01-01

Development of anticancer drugs targeting Aurora B, an important member of the serine/threonine kinases family, has been extensively focused on in recent years. In this work, by applying an integrated computational method, including comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), homology modeling and molecular docking, we investigated the structural determinants of Aurora B inhibitors based on three different series of derivatives of 108 molecules. The resultant optimum 3D-QSAR models exhibited (q2 = 0.605, r2pred = 0.826), (q2 = 0.52, r2pred = 0.798) and (q2 = 0.582, r2pred = 0.971) for MK-0457, GSK1070916 and SNS-314 classes, respectively, and the 3D contour maps generated from these models were analyzed individually. The contour map analysis for the MK-0457 model revealed the relative importance of steric and electrostatic effects for Aurora B inhibition, whereas, the electronegative groups with hydrogen bond donating capacity showed a great impact on the inhibitory activity for the derivatives of GSK1070916. Additionally, the predictive model of the SNS-314 class revealed the great importance of hydrophobic favorable contour, since hydrophobic favorable substituents added to this region bind to a deep and narrow hydrophobic pocket composed of residues that are hydrophobic in nature and thus enhanced the inhibitory activity. Moreover, based on the docking study, a further comparison of the binding modes was accomplished to identify a set of critical residues that play a key role in stabilizing the drug-target interactions. Overall, the high level of consistency between the 3D contour maps and the topographical features of binding sites led to our identification of several key structural requirements for more potency inhibitors. Taken together, the results will serve as a basis for future drug development of inhibitors against Aurora B kinase for various tumors. PMID:21151441

3D-QSAR and molecular docking studies on derivatives of MK-0457, GSK1070916 and SNS-314 as inhibitors against Aurora B kinase.

PubMed

Zhang, Baidong; Li, Yan; Zhang, Huixiao; Ai, Chunzhi

2010-11-02

Development of anticancer drugs targeting Aurora B, an important member of the serine/threonine kinases family, has been extensively focused on in recent years. In this work, by applying an integrated computational method, including comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), homology modeling and molecular docking, we investigated the structural determinants of Aurora B inhibitors based on three different series of derivatives of 108 molecules. The resultant optimum 3D-QSAR models exhibited (q(2) = 0.605, r(2) (pred) = 0.826), (q(2) = 0.52, r(2) (pred) = 0.798) and (q(2) = 0.582, r(2) (pred) = 0.971) for MK-0457, GSK1070916 and SNS-314 classes, respectively, and the 3D contour maps generated from these models were analyzed individually. The contour map analysis for the MK-0457 model revealed the relative importance of steric and electrostatic effects for Aurora B inhibition, whereas, the electronegative groups with hydrogen bond donating capacity showed a great impact on the inhibitory activity for the derivatives of GSK1070916. Additionally, the predictive model of the SNS-314 class revealed the great importance of hydrophobic favorable contour, since hydrophobic favorable substituents added to this region bind to a deep and narrow hydrophobic pocket composed of residues that are hydrophobic in nature and thus enhanced the inhibitory activity. Moreover, based on the docking study, a further comparison of the binding modes was accomplished to identify a set of critical residues that play a key role in stabilizing the drug-target interactions. Overall, the high level of consistency between the 3D contour maps and the topographical features of binding sites led to our identification of several key structural requirements for more potency inhibitors. Taken together, the results will serve as a basis for future drug development of inhibitors against Aurora B kinase for various tumors.

Recent Development of Anticancer Therapeutics Targeting Akt

PubMed Central

Morrow, John K.; Du-Cuny, Lei; Chen, Lu; Meuillet, Emmanuelle J.; Mash, Eugene A.; Powis, Garth; Zhang, Shuxing

2013-01-01

The serine/threonine kinase Akt has proven to be a significant signaling target, involved in various biological functions. Because of its cardinal role in numerous cellular responses, Akt has been implicated in many human diseases, particularly cancer. It has been established that Akt is a viable and feasible target for anticancer therapeutics. Analysis of all Akt kinases reveals conserved homology for an N-terminal regulatory domain, which contains a pleckstrin-homology (PH) domain for cellular translocation, a kinase domain with serine/threonine specificity, and a C-terminal extension domain. These well defined regions have been targeted, and various approaches, including in silico methods, have been implemented to develop Akt inhibitors. In spite of unique techniques and a prolific body of knowledge surrounding Akt, no targeted Akt therapeutics have reached the market yet. Here we will highlight successes and challenges to date on the development of anticancer agents modulating the Akt pathway in recent patents as well as discuss the methods employed for this task. Special attention will be given to patents with focus on those discoveries using computer-aided drug design approaches. PMID:21110830

Protein kinase C negatively regulates Akt activity and modifies UVC-induced apoptosis in mouse keratinocytes.

PubMed

Li, Luowei; Sampat, Keeran; Hu, Nancy; Zakari, Julia; Yuspa, Stuart H

2006-02-10

Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both the PKC and the Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phospho-Ser-473-Akt. IGF-1 also selectively induced translocation of PKCdelta and PKCepsilon from soluble to particulate fractions in mouse keratinocytes. Furthermore, the PKC-specific inhibitor, GF109203X, increased IGF-1-induced phospho-Ser-473-Akt and Akt kinase activity and enhanced IGF-1 protection from UVC-induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced phospho-Ser-473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuated IGF-1 and epidermal growth factor-induced phospho-Ser-473-Akt, reduced Akt kinase activity, and blocked IGF-1 protection from UVC-induced apoptosis. The inhibition of Akt activity by TPA was reduced by inhibitors of protein phosphatase 2A, and TPA stimulated the association of phosphatase 2A with Akt. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors or inhibited with PKC-selective inhibitors. These studies indicated that PKCdelta and PKCepsilon were selectively potent at causing dephosphorylation of Akt and modifying cell survival, whereas PKCalpha enhanced phosphorylation of Akt on Ser-473. Our results suggested that activation of PKCdelta and PKCepsilon provide a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes and serve as modulators of cell

17β-estradiol activates mTOR in chondrocytes by AKT-dependent and AKT-independent signaling pathways

PubMed Central

Tao, Yulei; Sun, Haibiao; Sun, Hongyan; Qiu, Xianxing; Xu, Changbo; Shi, Changxiu; Du, Jiahui

2015-01-01

To confirm whether 17β-estradiol (E2) activates mammalian target of rapamycin (mTOR) signaling pathway in chondrocytes and in what way activates mTOR. Human immortalized chondrocytes cell lines TC28a2 and C28/I2 were subjected to incubate with or without E2, LY294002 (the inhibitor of PI3K), rapamycin (the inhibitor of mTOR), or E2 in combination with LY294002 or rapamycin. Thereafter, protein levels of S6K1, p-S6K1, protein kinase B (AKT), and p-AKT were determined by Western blot analysis. Matrix metallopeptidase (MMP) 3 or MMP13 mRNA levels were evaluated by quantitative real-time PCR (qRT-PCR). Co-immunoprecipitation and Western blot analysis were performed to verify the interaction between ERα and mTOR. Both p-S6K1 and p-AKT protein levels in TC28a2 and C28/I2E2 cells were significantly increased by incubation with E2 (0.5 h and 1 h) (P < 0.05). Rapamycin did not affect the levels of p-AKT, but were significantly reduced by LY294002 or E2 in combination with LY294002. The levels of p-S6K1 were significantly decreased by incubation with LY294002, but the effect could be reversed by E2 in combination with LY294002. Rabbit anti-mTOR antibody was able to immunoprecipitate ERα after incubation with E2. Moreover, E2 inhibited the mRNA levels of MMP3 and MMP13 by mTOR pathway. E2 actives mTOR in chondrocytes through AKT-dependent and independent ways. PMID:26884863

Celecoxib promotes c-FLIP degradation through Akt-independent inhibition of GSK3.

PubMed

Chen, Shuzhen; Cao, Wei; Yue, Ping; Hao, Chunhai; Khuri, Fadlo R; Sun, Shi-Yong

2011-10-01

Celecoxib is a COX-2 inhibitor that reduces the risk of colon cancer. However, the basis for its cancer chemopreventive activity is not fully understood. In this study, we defined a mechanism of celecoxib action based on degradation of cellular FLICE-inhibitory protein (c-FLIP), a major regulator of the death receptor pathway of apoptosis. c-FLIP protein levels are regulated by ubiquitination and proteasome-mediated degradation. We found that celecoxib controlled c-FLIP ubiquitination through Akt-independent inhibition of glycogen synthase kinase-3 (GSK3), itself a candidate therapeutic target of interest in colon cancer. Celecoxib increased the levels of phosphorylated GSK3, including the α and β forms, even in cell lines, where phosphorylated Akt levels were not increased. Phosphoinositide 3-kinase inhibitors abrogated Akt phosphorylation as expected but had no effect on celecoxib-induced GSK3 phosphorylation. In contrast, protein kinase C (PKC) inhibitors abolished celecoxib-induced GSK3 phosphorylation, implying that celecoxib influenced GSK3 phosphorylation through a mechanism that relied upon PKC and not Akt. GSK3 blockade either by siRNA or kinase inhibitors was sufficient to attenuate c-FLIP levels. Combining celecoxib with GSK3 inhibition enhanced attenuation of c-FLIP and increased apoptosis. Proteasome inhibitor MG132 reversed the effects of GSK3 inhibition and increased c-FLIP ubiquitination, confirming that c-FLIP attenuation was mediated by proteasomal turnover as expected. Our findings reveal a novel mechanism through which the regulatory effects of c-FLIP on death receptor signaling are controlled by GSK3, which celecoxib acts at an upstream level to control independently of Akt.

Delayed administration of parecoxib, a specific COX-2 inhibitor, attenuated postischemic neuronal apoptosis by phosphorylation Akt and GSK-3β.

PubMed

Ye, Zhi; Wang, Na; Xia, Pingping; Wang, E; Yuan, Yajing; Guo, Qulian

2012-02-01

Parecoxib is a recently described novel COX-2 inhibitor whose functional significance and neuroprotective mechanisms remain elusive. Therefore, in this study, we aimed to investigate whether delayed administration of parecoxib inhibited mitochondria-mediated neuronal apoptosis induced by ischemic reperfusion injury via phosphorylating Akt and its downstream target protein, glycogen synthase kinase 3β (GSK-3β). Adult male Sprague-Dawley rats were administered parecoxib (10 or 30 mg kg(-1), IP) or isotonic saline twice a day starting 24 h after middle cerebral artery occlusion (MCAO) for three consecutive days. Cerebral infarct volume, apoptotic neuron, caspase-3 immunoreactivity and the protein expression of p-Akt, p-GSK-3β and Cytochrome C in cerebral ischemic cortex were evaluated at 96 h after reperfusion. Parecoxib significantly diminished infarct volume and attenuated neuron apoptosis in a dose-independent manner, compared with MCAO group alone. Increased p-Akt and p-GSK-3β was observed in the ischemic penumbra of parecoxib group after stroke. Moreover, parecoxib also reduced the release of Cytochrome C from mitochondrial into cytosol and attenuated the caspase-3 immunoreactivity in the penumbra. Taken together, these results suggested that parecoxib ameliorated postischemic mitochondria-mediated neuronal apoptosis induced by focal cerebral ischemia in rats and this neuroprotective potential is involved in phosphorylation of Akt and GSK-3β.

Antidepressant Effects of (+)-MK-801 and (-)-MK-801 in the Social Defeat Stress Model.

PubMed

Yang, Bangkun; Ren, Qian; Ma, Min; Chen, Qian-Xue; Hashimoto, Kenji

2016-12-01

Current data on antidepressant action of the N-methyl-D-aspartate receptor antagonist, (+)-MK-801, is inconsistent. This study was conducted to examine the effects of (+)-MK-801 and its less potent stereoisomer, (-)-MK-801, in the social defeat stress model of depression. The antidepressant effects of (+)-MK-801 (0.1mg/kg) and (-)-MK-801 (0.1mg/kg) in the social defeat stress model were examined. In the tail suspension and forced swimming tests, both stereoisomers significantly attenuated increased immobility time in susceptible mice. In the sucrose preference test, (+)-MK-801, but not (-)-MK-801, significantly enhanced reduced sucrose consumption 2 or 4 days after a single dose. However, no antianhedonia effects were detected 7 days after a single dose of either stereoisomer. Both stereoisomers of MK-801 induced rapid antidepressant effects in the social defeat stress model, although neither produced a long-lasting effect (7 days). © The Author 2016. Published by Oxford University Press on behalf of CINP.

Antidepressant Effects of (+)-MK-801 and (-)-MK-801 in the Social Defeat Stress Model

PubMed Central

Yang, Bangkun; Ren, Qian; Ma, Min; Chen, Qian-Xue

2016-01-01

Background: Current data on antidepressant action of the N-methyl-D-aspartate receptor antagonist, (+)-MK-801, is inconsistent. This study was conducted to examine the effects of (+)-MK-801 and its less potent stereoisomer, (-)-MK-801, in the social defeat stress model of depression. Methods: The antidepressant effects of (+)-MK-801 (0.1mg/kg) and (-)-MK-801 (0.1mg/kg) in the social defeat stress model were examined. Results: In the tail suspension and forced swimming tests, both stereoisomers significantly attenuated increased immobility time in susceptible mice. In the sucrose preference test, (+)-MK-801, but not (-)-MK-801, significantly enhanced reduced sucrose consumption 2 or 4 days after a single dose. However, no antianhedonia effects were detected 7 days after a single dose of either stereoisomer. Conclusions: Both stereoisomers of MK-801 induced rapid antidepressant effects in the social defeat stress model, although neither produced a long-lasting effect (7 days). PMID:27608811

Insulin induces drug resistance in melanoma through activation of the PI3K/Akt pathway

PubMed Central

Chi, Mengna; Ye, Yan; Zhang, Xu Dong; Chen, Jiezhong

2014-01-01

Introduction There is currently no curative treatment for melanoma once the disease spreads beyond the original site. Although activation of the PI3K/Akt pathway resulting from genetic mutations and epigenetic deregulation of its major regulators is known to cause resistance of melanoma to therapeutic agents, including the conventional chemotherapeutic drug dacarbazine and the Food and Drug Administration-approved mutant BRAF inhibitors vemurafenib and dabrafenib, the role of extracellular stimuli of the pathway, such as insulin, in drug resistance of melanoma remains less understood. Objective To investigate the effect of insulin on the response of melanoma cells to dacarbazine, and in particular, the effect of insulin on the response of melanoma cells carrying the BRAFV600E mutation to mutant BRAF inhibitors. An additional aim was to define the role of the PI3K/Akt pathway in the insulin-triggered drug resistance. Methods The effect of insulin on cytotoxicity induced by dacarbazine or the mutant BRAF inhibitor PLX4720 was tested by pre-incubation of melanoma cells with insulin. Cytotoxicity was determined by the MTS assay. The role of the PI3K/Akt pathway in the insulin-triggered drug resistance was examined using the PI3K inhibitor LY294002 and the PI3K and mammalian target of rapamycin dual inhibitor BEZ-235. Activation of the PI3K/Akt pathway was monitored by Western blot analysis of phosphorylated levels of Akt. Results Recombinant insulin attenuated dacarbazine-induced cytotoxicity in both wild-type BRAF and BRAFV600E melanoma cells, whereas it also reduced killing of BRAFV600E melanoma cells by PLX4720. Nevertheless, the protective effect of insulin was abolished by the PI3K and mTOR dual inhibitor BEZ-235 or the PI3K inhibitor LY294002. Conclusion Insulin attenuates the therapeutic efficacy of dacarbazine and PLX4720 in melanoma cells, which is mediated by activation of the PI3K/Akt pathway and can be overcome by PI3K inhibitors. PMID:24600206

Activation of Akt by Advanced Glycation End Products (AGEs): Involvement of IGF-1 Receptor and Caveolin-1

PubMed Central

Yang, Su-Jung; Chen, Chen-Yu; Chang, Geen-Dong; Wen, Hui-Chin; Chen, Ching-Yu; Chang, Shi-Chuan; Liao, Jyh-Fei; Chang, Chung-Ho

2013-01-01

Diabetes is characterized by chronic hyperglycemia, which in turn facilitates the formation of advanced glycation end products (AGEs). AGEs activate signaling proteins such as Src, Akt and ERK1/2. However, the mechanisms by which AGEs activate these kinases remain unclear. We examined the effect of AGEs on Akt activation in 3T3-L1 preadipocytes. Addition of AGEs to 3T3-L1 cells activated Akt in a dose- and time-dependent manner. The AGEs-stimulated Akt activation was blocked by a PI3-kinase inhibitor LY 294002, Src inhibitor PP2, an antioxidant NAC, superoxide scavenger Tiron, or nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase inhibitor DPI, suggesting the involvement of Src and NAD(P)H oxidase in the activation of PI3-kinase-Akt pathway by AGEs. AGEs-stimulated Src tyrosine phosphorylation was inhibited by NAC, suggesting that Src is downstream of NAD(P)H oxidase. The AGEs-stimulated Akt activity was sensitive to Insulin-like growth factor 1 receptor (IGF-1R) kinase inhibitor AG1024. Furthermore, AGEs induced phosphorylation of IGF-1 receptorβsubunit (IGF-1Rβ) on Tyr1135/1136, which was sensitive to PP2, indicating that AGEs stimulate Akt activity by transactivating IGF-1 receptor. In addition, the AGEs-stimulated Akt activation was attenuated by β-methylcyclodextrin that abolishes the structure of caveolae, and by lowering caveolin-1 (Cav-1) levels with siRNAs. Furthermore, addition of AGEs enhanced the interaction of phospho-Cav-1 with IGF-1Rβ and transfection of 3T3-L1 cells with Cav-1 Y14F mutants inhibited the activation of Akt by AGEs. These results suggest that AGEs activate NAD(P)H oxidase and Src which in turn phosphorylates IGF-1 receptor and Cav-1 leading to activation of IGF-1 receptor and the downstream Akt in 3T3-L1 cells. AGEs treatment promoted the differentiation of 3T3-L1 preadipocytes and addition of AG1024, LY 294002 or Akt inhibitor attenuated the promoting effect of AGEs on adipogenesis, suggesting that IGF-1 receptor, PI3

Possible involvement of nitric oxide (NO) signaling pathway in the antidepressant-like effect of MK-801(dizocilpine), a NMDA receptor antagonist in mouse forced swim test.

PubMed

Dhir, Ashish; Kulkarni, S K

2008-03-01

L-arginine-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) is an important signaling pathway involved in depression. With this information, the present study aimed to study the involvement of this signaling pathway in the antidepressant-like action of MK-801 (dizocilpine; N-methyl-d-aspartate receptor antagonist) in the mouse forced-swim test. Total immobility period was recorded in mouse forced swim test for 6 min. MK-801 (5-25 microg/kg., ip) produced a U-shaped curve in reducing the immobility period. The antidepressant-like effect of MK-801 (10 microg/kg, ip) was prevented by pretreatment with L-arginine (750 mg/kg, ip) [substrate for nitric oxide synthase (NOS)]. Pretreatment of mice with 7-nitroindazole (7-NI) (25 mg/kg, ip) [a specific neuronal nitric oxide synthase inhibitor] produced potentiation of the action of subeffective dose of MK-801 (5 microg/kg, ip). In addition, treatment of mice with methylene blue (10 mg/kg, ip) [direct inhibitor of both nitric oxide synthase and soluble guanylate cyclase] potentiated the effect of MK-801 (5 microg/kg, ip) in the forced-swim test. Further, the reduction in the immobility period elicited by MK-801 (10 microg/kg, ip) was also inhibited by pretreatment with sildenafil (5 mg/kg, ip) [phosphodiesterase 5 inhibitor]. The various modulators used in the study and their combination did not produce any changes in locomotor activity per se and in combination with MK-801. MK-801 however, at higher doses (25 microg/kg, ip) produced hyperlocomotion. The results demonstrated the involvement of nitric oxide signaling pathway in the antidepressant-like effect of MK-801 in mouse forced-swim test.

Inhibition of PTEN and activation of Akt by menadione.

PubMed

Yoshikawa, Kyoko; Nigorikawa, Kiyomi; Tsukamoto, Mariko; Tamura, Namiko; Hazeki, Kaoru; Hazeki, Osamu

2007-04-01

Menadione (vitamin K(3)) has been shown to activate Erk in several cell lines. This effect has been shown to be due to the activation of EGF receptors (EGFR) as a result of inhibition of some protein tyrosine phosphatases. In the present study, we examined the effects of menadione on Akt in Chinese hamster ovary cells. The phosphorylation of Akt by menadione was not inhibited by AG1478, an inhibitor of EGFR. Menadione inhibited the lipid phosphatase activity of PTEN in a cell-free system. In an intact cell system, menadione inhibited the effect of transfected PTEN on Akt. Thus, one mechanism of its action was considered the accelerated activation of Akt through inhibition of PTEN. This was not the sole mechanism responsible for the EGFR-independent activation of Akt, because menadione attenuated the rate of Akt dephosphorylation even in PTEN-null PC3 cells. The decelerated inactivation of Akt, probably through inhibition of some tyrosine phosphatases, was considered another mechanism of its action.

Expression of SLP-2 Was Associated with Invasion of Esophageal Squamous Cell Carcinoma

PubMed Central

Cao, Wenfeng; Zhang, Bin; Ding, Fang; Zhang, Weiran; Sun, Baocun; Liu, Zhihua

2013-01-01

Introduction Stomatin-like protein 2 (SLP-2), a member of the Stomatin superfamily, has been identified as an oncogenic-related protein and found to be up-regulated in multi-cancers. Nonetheless, the expression pattern and regulation of SLP-2 in human esophageal squamous cell carcinoma (ESCC) remain unexplored. Methods Immunohistochemistry and immunofluorescence staining analysis were performed to show SLP-2 expression and location. RNAi method was used to inhibit specific protein expression. Transwell assay was done to investigate cells invasive capability. RT-PCR and Western blot analysis were used to detect mRNA and protein expression levels. Results Immunohistochemical analysis showed that up-regulation of SLP-2 was found in invasive front compared with cancer central tissue in ESCC. Inhibition of SLP-2 by SLP-2 siRNA can decrease ESCC cells invasive capability through MMP-2 dependent manner. Up-regulation of SLP-2 was effectively abrogated by the ERK1/2 inhibitors either PD98059 or U0126, but no effect was showed by the treatment of AKT inhibitors either LY294002 or MK-2206. So the regulation of SLP-2 was involved in activation of the MAPK/ERK pathway. Conclusions We found that PMA/EGF could induce the up-regulated expression of SLP-2 probably through activating ERK signalling. The current study suggests that SLP-2 may represent an important molecular hallmark that is clinically relevant to the invasion of ESCC. PMID:23667687

Activation of Akt, not connexin 43 protein ubiquitination, regulates gap junction stability.

PubMed

Dunn, Clarence A; Su, Vivian; Lau, Alan F; Lampe, Paul D

2012-01-20

The pore-forming gap junctional protein connexin 43 (Cx43) has a short (1-3 h) half-life in cells in tissue culture and in whole tissues. Although critical for cellular function in all tissues, the process of gap junction turnover is not well understood because treatment of cells with a proteasomal inhibitor results in larger gap junctions but little change in total Cx43 protein whereas lysosomal inhibitors increase total, mostly nonjunctional Cx43. To better understand turnover and identify potential sites of Cx43 ubiquitination, we prepared constructs of Cx43 with different lysines converted to arginines. However, when transfected into cells, a mutant version of Cx43 with all lysines converted to arginines behaved similarly to wild type in the presence of proteasomal and lysosomal inhibitors, indicating that ubiquitination of Cx43 did not appear to be playing a role in gap junction stability. Through the use of inhibitors and dominant negative constructs, we found that Akt (protein kinase B) activity controlled gap junction stability and was necessary to form larger stable gap junctions. Akt activation was increased upon proteasomal inhibition and resulted in phosphorylation of Cx43 at Akt phosphorylation consensus sites. Thus, we conclude that Cx43 ubiquitination is not necessary for the regulation of Cx43 turnover; rather, Akt activity, probably through direct phosphorylation of Cx43, controls gap junction stability. This linkage of a kinase involved in controlling cell survival and growth to gap junction stability may mechanistically explain how gap junctions and Akt play similar regulatory roles.

Akt1 binds focal adhesion kinase via the Akt1 kinase domain independently of the pleckstrin homology domain.

PubMed

Basson, M D; Zeng, B; Wang, S

2015-10-01

for developing novel inhibitors that target this specific Akt1/FAK interaction.

Brain Cancer Stem Cells Display Preferential Sensitivity to Akt Inhibition

PubMed Central

Eyler, Christine E.; Foo, Wen-Chi; LaFiura, Katherine M.; McLendon, Roger E.; Hjelmeland, Anita B.; Rich, Jeremy N.

2009-01-01

Malignant brain tumors are among the most lethal cancers, and conventional therapies are largely limited to palliation. Novel therapies targeted against specific molecular pathways may offer improved efficacy and reduced toxicity compared to conventional therapies, but initial clinical trials of molecular targeted agents in brain cancer therapy have been frequently disappointing. In brain tumors and other cancers, subpopulations of tumor cells have recently been characterized by their ability to self-renew and initiate tumors. Although these cancer stem cells, or tumor initiating cells, are often only present in small numbers in human tumors, mounting evidence suggests that cancer stem cells contribute to tumor maintenance and therapeutic resistance. Thus, the development of therapies that target cancer stem cell signal transduction and biologies may improve brain tumor patient survival. We now demonstrate that populations enriched for cancer stem cells are preferentially sensitive to an inhibitor of Akt, a prominent cell survival and invasion signaling node. Treatment with an Akt inhibitor more potently reduced the numbers of viable brain cancer stem cells relative to matched non-stem cancer cells associated with a preferential induction of apoptosis and a suppression of neurosphere formation. Akt inhibition also reduced the motility and invasiveness of all tumor cells but had a greater impact on cancer stem cell behaviors. Furthermore, inhibition of Akt activity in cancer stem cells increased survival of immunocompromised mice bearing human glioma xenografts in vivo. Together, these results suggest that Akt inhibitors may function as effective anti-cancer stem cell therapies. PMID:18802038

PDK1 inhibitor GSK2334470 synergizes with proteasome inhibitor MG‑132 in multiple myeloma cells by inhibiting full AKT activity and increasing nuclear accumulation of the PTEN protein.

PubMed

Zhang, Jin; Yang, Chunmei; Zhou, Fengping; Chen, Xiaohui

2018-06-01

Phosphoinositide‑dependent kinase 1 (PDK1) is generally active in multiple myeloma (MM) and higher expression than other hematopoietic cells, which is associated with the drug resistance and the disease progression. Previous studies have demonstrated that PDK1 can be targeted therapeutically in MM. In the present study, we examined the combination effect of GSK2334470 (GSK‑470), a novel and highly specific inhibitor of PDK1, with proteasome inhibitor MG‑132 in MM cell lines. GSK‑470 monotherapy significantly inhibited growth of MM cell lines and induced apoptosis that was associated with the activation of both the intrinsic mitochondrial pathway and the extrinsic death receptor pathway. Moreover, GSK‑470 demonstrated synergistic growth inhibitory effects with MG‑132. Notably, treatment with these inhibitors resulted in an almost complete inhibition of phosphorylation of mammalian target of rapamycin on Ser2448 and Ser2481 and full activation of AKT. The combination therapy also caused an upregulation of PTEN and an increased nuclear accumulation of PTEN protein. Collectively, our results provide the rationale for novel combination treatment with PDK1 inhibitor and proteasome inhibitors to improve outcomes in patients with MM.

Cross regulation between cGMP-dependent protein kinase and Akt in vasodilatation of porcine pulmonary artery.

PubMed

Liu, Juan; Liu, Huixia; Li, Yanjing; Xu, Xiaojian; Chen, Zhengju; Liu, Limei; Yu, Xiaoxing; Gao, Yuansheng; Dou, Dou

2014-11-01

cGMP-dependent protein kinase (PKG) plays a crucial role in vasodilatation induced by cGMP-elevating agents. Akt has been demonstrated to be involved in modulating vasoreactivity. The present study was to determine the interaction between PKG and Akt and their influences on nitric oxide (NO)-induced vasodilatation. Isolated fourth-generation porcine pulmonary arteries were dissected from the lung and cut into rings in ice-cold modified Krebs-Ringer bicarbonate buffer. The relaxant responses of vessels were determined by organ chamber technique, cGMP was assayed by using enzyme-linked immunosorbent assay kit, the protein levels of phosphorylated Akt were examined by Western blotting, and the activity of phosphodiesterase type 5 (PDE5) was assayed by measuring the rate of cGMP degradation. Incubation with DETA NONOate (a stable NO donor) and 8-Br-cGMP (a cell membrane permeable analog of cGMP) attenuated Akt phosphorylation at Ser-473, which was prevented by Rp-8-Br-PET-cGMPS (a specific inhibitor of PKG) and calyculin A (an inhibitor of protein phosphatase 1 and 2A) but not by okadaic acid (a selective inhibitor of protein phosphatase 2A). Inhibition of Akt enhanced the relaxation and cGMP elevation of porcine pulmonary arteries induced by DETA NONOate or sodium nitroprusside, which was prevented by zaprinast, a specific inhibitor of PDE5. Incubation with LY294002 or Akt inhibitor reduced PDE5 activity in porcine pulmonary arteries. The present study indicates that PKG may attenuate Akt phosphorylation through protein phosphatase 1, which leads to an augmented cGMP elevation by inhibition of PDE5. The increased cGMP in turn activates PKG. Such a positive feedback may play an important role in NO-induced pulmonary vasodilatation.

miR-21 inhibitor suppresses cell proliferation and colony formation through regulating the PTEN/AKT pathway and improves paclitaxel sensitivity in cervical cancer cells.

PubMed

Du, Guohui; Cao, Dongmei; Meng, Lingzheng

2017-05-01

The present study aimed to investigate the role and the molecular mechanisms underlying the effects of microRNA-21 (miR-21) on the proliferation, apoptosis and colony formation of cervical cancer cells, and to examine the role of miR-21 in mediating the sensitivity of cervical cancer cells to paclitaxel (PTX). Reverse transcription‑quantitative polymerase chain reaction was employed to determine the level of miR‑21 in various cervical cancer and normal cervical cells. The results revealed that the expression levels of miR-21 in cervical cancer cells were markedly higher when compared with normal cervical cells. Subsequently, a miR‑21 inhibitor or negative control (NC) was transfected into cervical cancer cells. Cell viability, colony formation and apoptosis were then analyzed using an MTT assay, crystal violet and Annexin V-fluorescein isothiocyanate/propidium iodide staining, respectively. The protein expression level of B-cell lymphoma‑2 (Bcl‑2), Bcl‑2‑associated X (Bax), programmed cell death 4 (PDCD4), survivin, c‑myc, phosphatase and tensin homolog (PTEN) and phosphorylated (p)‑AKT were determined by western blot analysis. The sensitivity of cervical cancer cells to PTX (25, 50 and 100 µg/ml) was characterized using an MTT assay. The results demonstrated that the miR-21 inhibitor promoted apoptosis of cervical cancer cells and suppressed their proliferation and colony formation when compared with the NC. In addition, the expression levels of Bcl‑2, survivin, c‑myc and p‑AKT were significantly downregulated in cells transfected with the miR‑21 inhibitor, whilst the expression levels of Bax, PDCD4 and PTEN were significantly upregulated. Furthermore, the miR‑21 inhibitor significantly enhanced the inhibition efficacy of PTX at a range of concentrations in cervical cancer cells. It was concluded that inhibition of miR‑21 suppressed cell proliferation and colony formation through regulating the PTEN/AKT pathway, and improved PTX

Isoginkgetin inhibits tumor cell invasion by regulating phosphatidylinositol 3-kinase/Akt-dependent matrix metalloproteinase-9 expression.

PubMed

Yoon, Sang-Oh; Shin, Sejeong; Lee, Ho-Jae; Chun, Hyo-Kon; Chung, An-Sik

2006-11-01

Matrix metalloproteinase (MMP)-9 plays a key role in tumor invasion. Inhibitors of MMP-9 were screened from Metasequoia glyptostroboides (Dawn redwood) and one potent inhibitor, isoginkgetin, a biflavonoid, was identified. Noncytotoxic levels of isoginkgetin decreased MMP-9 production profoundly, but up-regulated the level of tissue inhibitor of metalloproteinase (TIMP)-1, an inhibitor of MMP-9, in HT1080 human fibrosarcoma cells. The major mechanism of Ras-dependent MMP-9 production in HT1080 cells was phosphatidylinositol 3-kinase (PI3K)/Akt/nuclear factor-kappaB (NF-kappaB) activation. Expression of dominant-active H-Ras and p85 (a subunit of PI3K) increased MMP-9 activity, whereas dominant-negative forms of these molecules decreased the level of MMP-9. H-Ras did not increase MMP-9 in the presence of a PI3K inhibitor, LY294002, and a NF-kappaB inhibitor, SN50. Further studies showed that isoginkgetin regulated MMP-9 production via PI3K/Akt/NF-kappaB pathway, as evidenced by the findings that isoginkgetin inhibited activities of both Akt and NF-kappaB. PI3K/Akt is a well-known key pathway for cell invasion, and isoginkgetin inhibited HT1080 tumor cell invasion substantially. Isoginkgetin was also quite effective in inhibiting the activities of Akt and MMP-9 in MDA-MB-231 breast carcinomas and B16F10 melanoma. Moreover, isoginkgetin treatment resulted in marked decrease in invasion of these cells. In summary, PI3K/Akt is a major pathway for MMP-9 expression and isoginkgetin markedly decreased MMP-9 expression and invasion through inhibition of this pathway. This suggests that isoginkgetin could be a potential candidate as a therapeutic agent against tumor invasion.

The AKT-mTOR signalling pathway in kidney cancer tissues

NASA Astrophysics Data System (ADS)

Spirina, L. V.; Usynin, Y. A.; Kondakova, I. V.; Yurmazov, Z. A.; Slonimskaya, E. M.; Kolegova, E. S.

2015-11-01

An increased expression of phospho-AKT, m-TOR, glycogen regulator GSK-3-beta and transcription inhibitor 4E-BP1 was observed in kidney cancer tissues. Tumor size growth was associated with a high level of c-Raf and low content of phospho-m-TOR. Cancer metastasis development led to a decreased PTEN and phospho-AKT expression.

Targeting the PI3K/Akt/mTOR pathway: effective combinations and clinical considerations

PubMed Central

LoPiccolo, Jaclyn; Blumenthal, Gideon M.; Bernstein, Wendy B.; Dennis, Phillip A.

2008-01-01

The PI3K/Akt/mTOR pathway is a prototypic survival pathway that is constitutively activated in many types of cancer. Mechanisms for pathway activation include loss of tumor suppressor PTEN function, amplification or mutation of PI3K, amplification or mutation of Akt, activation of growth factor receptors, and exposure to carcinogens. Once activated, signaling through Akt can be propagated to a diverse array of substrates, including mTOR, a key regulator of protein translation. This pathway is an attractive therapeutic target in cancer because it serves as a convergence point for many growth stimuli, and through its downstream substrates, controls cellular processes that contribute to the initiation and maintenance of cancer. Moreover, activation of the Akt/mTOR pathway confers resistance to many types of cancer therapy, and is a poor prognostic factor for many types of cancers. This review will provide an update on the clinical progress of various agents that target the pathway, such as the Akt inhibitors perifosine and PX-866 and mTOR inhibitors (rapamycin, CCI-779, RAD-001) and discuss strategies to combine these pathway inhibitors with conventional chemotherapy, radiotherapy, as well as newer targeted agents. We will also discuss how the complex regulation of the PI3K/Akt/mTOR pathway poses practical issues concerning the design of clinical trials, potential toxicities and criteria for patient selection. PMID:18166498

Design and synthesis of 2-oxindole based multi-targeted inhibitors of PDK1/Akt signaling pathway for the treatment of glioblastoma multiforme.

PubMed

Sestito, Simona; Nesi, Giulia; Daniele, Simona; Martelli, Alma; Digiacomo, Maria; Borghini, Alice; Pietra, Daniele; Calderone, Vincenzo; Lapucci, Annalina; Falasca, Marco; Parrella, Paola; Notarangelo, Angelantonio; Breschi, Maria C; Macchia, Marco; Martini, Claudia; Rapposelli, Simona

2015-11-13

Aggressive behavior and diffuse infiltrative growth are the main features of Glioblastoma multiforme (GBM), together with the high degree of resistance and recurrence. Evidence indicate that GBM-derived stem cells (GSCs), endowed with unlimited proliferative potential, play a critical role in tumor development and maintenance. Among the many signaling pathways involved in maintaining GSC stemness, tumorigenic potential, and anti-apoptotic properties, the PDK1/Akt pathway is a challenging target to develop new potential agents able to affect GBM resistance to chemotherapy. In an effort to find new PDK1/Akt inhibitors, we rationally designed and synthesized a small family of 2-oxindole derivatives. Among them, compound 3 inhibited PDK1 kinase and downstream effectors such as CHK1, GS3Kα and GS3Kβ, which contribute to GCS survival. Compound 3 appeared to be a good tool for studying the role of the PDK1/Akt pathway in GCS self-renewal and tumorigenicity, and might represent the starting point for the development of more potent and focused multi-target therapies for GBM. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

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.

Synemin promotes AKT-dependent glioblastoma cell proliferation by antagonizing PP2A.

PubMed

Pitre, Aaron; Davis, Nathan; Paul, Madhumita; Orr, A Wayne; Skalli, Omar

2012-04-01

The intermediate filament protein synemin is present in astrocyte progenitors and glioblastoma cells but not in mature astrocytes. Here we demonstrate a role for synemin in enhancing glioblastoma cell proliferation and clonogenic survival, as synemin RNA interference decreased both behaviors by inducing G1 arrest along with Rb hypophosphorylation and increased protein levels of the G1/S inhibitors p21(Cip1) and p27(Kip1). Akt involvement was demonstrated by decreased phosphorylation of its substrate, p21(Cip1), and reduced Akt catalytic activity and phosphorylation at essential activation sites. Synemin silencing, however, did not affect the activities of PDPK1 and mTOR complex 2, which directly phosphorylate Akt activation sites, but instead enhanced the activity of the major regulator of Akt dephosphorylation, protein phosphatase type 2A (PP2A). This was accompanied by changes in PP2A subcellular distribution resulting in increased physical interactions between PP2A and Akt, as shown by proximity ligation assays (PLAs). PLAs and immunoprecipitation experiments further revealed that synemin and PP2A form a protein complex. In addition, treatment of synemin-silenced cells with the PP2A inhibitor cantharidic acid resulted in proliferation and pAkt and pRb levels similar to those of controls. Collectively these results indicate that synemin positively regulates glioblastoma cell proliferation by helping sequester PP2A away from Akt, thereby favoring Akt activation.

Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines.

PubMed

Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

2016-07-01

Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application.

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

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Atorvastatin enhances neurite outgrowth in cortical neurons in vitro via up-regulating the Akt/mTOR and Akt/GSK-3β signaling pathways

PubMed Central

Jin, Ying; Sui, Hai-juan; Dong, Yan; Ding, Qi; Qu, Wen-hui; Yu, Sheng-xue; Jin, Ying-xin

2012-01-01

Aim: To investigate whether atorvastatin can promote formation of neurites in cultured cortical neurons and the signaling mechanisms responsible for this effect. Methods: Cultured rat cerebral cortical neurons were incubated with atorvastatin (0.05–10 μmol/L) for various lengths of time. For pharmacological experiments, inhibitors were added 30 min prior to addition of atorvastatin. Control cultures received a similar amount of DMSO. Following the treatment period, phase-contrast digital images were taken. Digital images of neurons were analyzed for total neurite branch length (TNBL), neurite number, terminal branch number, and soma area by SPOT Advanced Imaging software. After incubation with atorvastatin for 48 h, the levels of phosphorylated 3-phosphoinoside-dependent protein kinase-1 (PDK1), phospho-Akt, phosphorylated mammalian target of rapamycin (mTOR), phosphorylated 4E-binding protein 1 (4E-BP1), p70S6 kinase (p70S6K), and glycogen synthase kinase-3β (GSK-3β) in the cortical neurons were evaluated using Western blotting analyses. Results: Atorvastatin (0.05–10 μmol/L) resulted in dose-dependent increase in neurite number and length in these neurons. Pretreatment of the cortical neurons with phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 (30 μmol/L) and wortmannin (5 μmol/L), Akt inhibitor tricribine (1 μmol/L) or mTOR inhibitor rapamycin (100 nmol/L) blocked the atorvastatin-induced increase in neurite outgrowth, suggesting that atorvastatin promoted neurite outgrowth via activating the PI3K/Akt/mTOR signaling pathway. Atorvastatin (10 μmol/L) significantly increased the levels of phosphorylated PDK1, Akt and mTOR in the cortical neurons, which were prevented by LY294002 (30 μmol/L). Moreover, atorvastatin (10 μmol/L) stimulated the phosphorylation of 4E-BP1 and p70S6K, the substrates of mTOR, in the cortical neurons. In addition, atorvastatin (10 μmol/L) significantly increased the phosphorylated GSK-3β level in the cortical

Substance P induces cardioprotection in ischemia-reperfusion via activation of AKT.

PubMed

Jubair, Shaiban; Li, Jianping; Dehlin, Heather M; Manteufel, Edward J; Goldspink, Paul H; Levick, Scott P; Janicki, Joseph S

2015-08-15

Accumulating evidence indicates that substance P is cardioprotective following ischemia-reperfusion primarily due to its potent coronary vasodilator actions. However, an anti-apoptotic effect of substance P has been observed in tenocytes following ischemia, which involved activation of the AKT pathway. This suggests the possibility that substance P also provides cardioprotection via direct actions to activate AKT in myocardial cells. The purpose of this study was to test the hypothesis that substance P attenuates ischemia-related cell death via direct effects on myocardial cells by activating cell survival pathways. Seven-week-old male Sprague-Dawley rats, anesthetized with intraperitoneal pentobarbital sodium (100 mg/kg), were used. The ability of substance P to prevent cellular damage was assessed following ischemia-reperfusion in an isolated heart preparation and in short-term hypoxia without reperfusion using a left ventricular tissue slice culture preparation. In addition, the NK-1 receptor and AKT involvement was assessed using the NK-1 receptor antagonist L732138 and the AKT inhibitor LY294002. The results indicate that substance P reduced the ischemia-related release of lactate dehydrogenase in both preparations and the degree of apoptosis and necrosis in the hypoxic left ventricular slices, indicating its ability to attenuate cell damage; and induced AKT phosphorylation, with both the AKT inhibitor and NK-1 receptor antagonist preventing the increased phosphorylation of AKT and the ability of substance P to attenuate hypoxic cellular damage. It is concluded that substance P reduces ischemia/hypoxia-induced myocardial cell death by acting directly on cardiac cells to initiate cell survival pathways via the NK-1 receptor and AKT. Copyright © 2015 the American Physiological Society.

Substance P induces cardioprotection in ischemia-reperfusion via activation of AKT

PubMed Central

Jubair, Shaiban; Li, Jianping; Dehlin, Heather M.; Manteufel, Edward J.; Goldspink, Paul H.; Levick, Scott P.

2015-01-01

Accumulating evidence indicates that substance P is cardioprotective following ischemia-reperfusion primarily due to its potent coronary vasodilator actions. However, an anti-apoptotic effect of substance P has been observed in tenocytes following ischemia, which involved activation of the AKT pathway. This suggests the possibility that substance P also provides cardioprotection via direct actions to activate AKT in myocardial cells. The purpose of this study was to test the hypothesis that substance P attenuates ischemia-related cell death via direct effects on myocardial cells by activating cell survival pathways. Seven-week-old male Sprague-Dawley rats, anesthetized with intraperitoneal pentobarbital sodium (100 mg/kg), were used. The ability of substance P to prevent cellular damage was assessed following ischemia-reperfusion in an isolated heart preparation and in short-term hypoxia without reperfusion using a left ventricular tissue slice culture preparation. In addition, the NK-1 receptor and AKT involvement was assessed using the NK-1 receptor antagonist L732138 and the AKT inhibitor LY294002. The results indicate that substance P reduced the ischemia-related release of lactate dehydrogenase in both preparations and the degree of apoptosis and necrosis in the hypoxic left ventricular slices, indicating its ability to attenuate cell damage; and induced AKT phosphorylation, with both the AKT inhibitor and NK-1 receptor antagonist preventing the increased phosphorylation of AKT and the ability of substance P to attenuate hypoxic cellular damage. It is concluded that substance P reduces ischemia/hypoxia-induced myocardial cell death by acting directly on cardiac cells to initiate cell survival pathways via the NK-1 receptor and AKT. PMID:26071541

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

The role of purinergic and dopaminergic systems on MK-801-induced antidepressant effects in zebrafish.

PubMed

da Silva, Raquel Bohrer; Siebel, Anna Maria; Bonan, Carla Denise

2015-12-01

Depression is a serious disease characterized by low mood, anhedonia, loss of interest in daily activities, appetite and sleep disturbances, reduced concentration, and psychomotor agitation. There is a growing interest in NMDA antagonists as a promising target for the development of new antidepressants. Considering that purinergic and dopaminergic systems are involved in depression and anxiety states, we characterized the role of these signaling pathways on MK-801-induced antidepressant effects in zebrafish. Animals treated with MK-801 at the doses of 5, 10, 15, or 20μM during 15, 30, or 60min spent longer time in the top area of aquariums in comparison to control group, indicating an anxiolytic/antidepressant effect induced by this drug. Animals treated with MK-801 spent longer time period at top area until 2 (5μM MK-801) and 4 (20μM MK-801) hours after treatment, returning to basal levels from 24h to 7days after exposure. Repeated MK-801 treatment did not induce cumulative effects, since animals treated daily during 7days had the same behavioral response pattern observed since the first until the 7th day. In order to investigate the effects of adenosine A1 and A2A receptor antagonist and agonist and the influence of modulation of adenosine levels on MK-801 effects, we treated zebrafish with caffeine, DPCPX, CPA, ZM 241385, CGS 21680, AMPCP, EHNA, dipyridamole, and NBTI during 30min before MK-801 exposure. The non-specific adenosine receptor antagonist caffeine (50mg/kg) and the selective A1 receptor antagonist DPCPX (15mg/kg) prevented the behavioral changes induced by MK-801. The non-specific nucleoside transporter (NT) inhibitor dipyridamole (10mg/kg) exacerbated the behavioral changes induced by MK-801. Dopamine receptor antagonists (sulpiride and SCH 23390) did not change the behavioral alterations induced by MK-801. Our findings demonstrated that antidepressant-like effects of MK-801 in zebrafish are mediated through adenosine A1 receptor activation

The CYP2E1 inhibitor DDC up-regulates MMP-1 expression in hepatic stellate cells via an ERK1/2- and Akt-dependent mechanism.

PubMed

Liu, Tianhui; Wang, Ping; Cong, Min; Xu, Youqing; Jia, Jidong; You, Hong

2013-06-05

DDC (diethyldithiocarbamate) could block collagen synthesis in HSC (hepatic stellate cells) through the inhibition of ROS (reactive oxygen species) derived from hepatocyte CYP2E1 (cytochrome P450 2E1). However, the effect of DDC on MMP-1 (matrix metalloproteinase-1), which is the main collagen degrading matrix metalloproteinase, has not been reported. In co-culture experiments, we found that DDC significantly enhanced MMP-1 expression in human HSC (LX-2) that were cultured with hepatocyte C3A cells either expressing or not expressing CYP2E1. The levels of both proenzyme and active MMP-1 enzyme were up-regulated in LX-2 cells, accompanied by elevated enzyme activity of MMP-1 and decreased collagen I, in both LX-2 cells and the culture medium. H2O2 treatment abrogated DDC-induced MMP-1 up-regulation and collagen I decrease, while catalase treatment slightly up-regulated MMP-1 expression. These data suggested that the decrease in ROS by DDC was partially responsible for the MMP-1 up-regulation. ERK1/2 (extracellular signal-regulated kinase 1/2), Akt (protein kinase B) and p38 were significantly activated by DDC. The ERK1/2 inhibitor (U0126) and Akt inhibitor (T3830) abrogated the DDC-induced MMP-1 up-regulation. In addition, a p38 inhibitor (SB203580) improved MMP-1 up-regulation through the stimulation of ERK1/2. Our data indicate that DDC significantly up-regulates the expression of MMP-1 in LX-2 cells which results in greater MMP-1 enzyme activity and decreased collagen I. The enhancement of MMP-1 expression by DDC was associated with H2O2 inhibition and coordinated regulation by the ERK1/2 and Akt pathways. These data provide some new insights into treatment strategies for hepatic fibrosis.

The CYP2E1 inhibitor DDC up-regulates MMP-1 expression in hepatic stellate cells via an ERK1/2- and Akt-dependent mechanism

PubMed Central

Liu, Tianhui; Wang, Ping; Cong, Min; Xu, Youqing; Jia, Jidong; You, Hong

2013-01-01

DDC (diethyldithiocarbamate) could block collagen synthesis in HSC (hepatic stellate cells) through the inhibition of ROS (reactive oxygen species) derived from hepatocyte CYP2E1 (cytochrome P450 2E1). However, the effect of DDC on MMP-1 (matrix metalloproteinase-1), which is the main collagen degrading matrix metalloproteinase, has not been reported. In co-culture experiments, we found that DDC significantly enhanced MMP-1 expression in human HSC (LX-2) that were cultured with hepatocyte C3A cells either expressing or not expressing CYP2E1. The levels of both proenzyme and active MMP-1 enzyme were up-regulated in LX-2 cells, accompanied by elevated enzyme activity of MMP-1 and decreased collagen I, in both LX-2 cells and the culture medium. H2O2 treatment abrogated DDC-induced MMP-1 up-regulation and collagen I decrease, while catalase treatment slightly up-regulated MMP-1 expression. These data suggested that the decrease in ROS by DDC was partially responsible for the MMP-1 up-regulation. ERK1/2 (extracellular signal-regulated kinase 1/2), Akt (protein kinase B) and p38 were significantly activated by DDC. The ERK1/2 inhibitor (U0126) and Akt inhibitor (T3830) abrogated the DDC-induced MMP-1 up-regulation. In addition, a p38 inhibitor (SB203580) improved MMP-1 up-regulation through the stimulation of ERK1/2. Our data indicate that DDC significantly up-regulates the expression of MMP-1 in LX-2 cells which results in greater MMP-1 enzyme activity and decreased collagen I. The enhancement of MMP-1 expression by DDC was associated with H2O2 inhibition and coordinated regulation by the ERK1/2 and Akt pathways. These data provide some new insights into treatment strategies for hepatic fibrosis. PMID:23577625

Regulation of D-cyclin translation inhibition in myeloma cells treated with mTOR inhibitors: Rationale for combined treatment with ERK inhibitors and rapamycin

PubMed Central

Frost, Patrick; Shi, Yijiang; Hoang, Bao; Gera, Joseph; Lichtenstein, Alan

2009-01-01

We have shown that heightened AKT activity sensitized multiple myeloma (MM) cells to the anti-tumor effects of the mTOR-inhibitor, CCI-779. To test the mechanism of AKT’s regulatory role, we stably transfected U266 MM cell lines with an activated AKT allele or empty vector. The AKT-transfected cells were more sensitive to cytostasis induced in vitro by rapamycin or in vivo by its analog, CCI-779, whereas cells with quiescent AKT were resistant. The ability of mTOR inhibitors to downregulate D-cyclin expression was significantly greater in AKT-transfected MM cells, due in part, to AKT’s ability to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. Similar AKT-dependent regulation of rapamycin responsiveness was demonstrated in a second myeloma model: the PTEN-null OPM-2 cell line transfected with wild type PTEN. As ERK/p38 activity facilitates IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of AKT-dependent effects on rapamycin sensitivity. AKT-transfected U266 cells demonstrated significantly decreased ERK and p38 activity. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant “low AKT” myeloma cells. Furthermore, the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down regulated D-cyclin protein expression and G1 arrest. However, ectopic over-expression of an activated MEK gene did not increase cap-independent translation of D-cyclin in “high AKT” myeloma cells indicating that MEK/ERK activity was required but not sufficient for activation of the IRES. These data support a scenario where heightened AKT activity down-regulates D-cyclin IRES function in MM cells and ERK facilitates activity. PMID:19139116

The role of the PTEN/AKT Pathway in NOTCH1-induced leukemia

PubMed Central

Palomero, Teresa; Dominguez, Maria; Ferrando, Adolfo A.

2008-01-01

Activating mutations in NOTCH1 are the most prominent genetic abnormality in T-cell acute Lymphoblastic Leukemia (T-ALL) and inhibition of NOTCH1 signaling with γ-secretase inhibitors (GSIs) has been proposed as targeted therapy in this disease. However, most T-ALL cell lines with mutations in NOTCH1 fail to respond to GSI therapy. Using gene expression profiling and mutation analysis we showed that mutational loss of PTEN is a common event in T-ALL and is associated with resistance to NOTCH inhibition. Furthermore, our studies revealed that NOTCH1 induces upregulation of the PI3K-AKT pathway via HES1, which negatively controls the expression of PTEN. This regulatory circuitry is evolutionary conserved from Drosophila to humans as demonstrated by the interaction of overexpression of Delta and Akt in a model of Notch-induced transformation in the fly eye. Loss of PTEN and constitutive activation of AKT in T-ALL induce increased glucose metabolism and bypass the requirement of NOTCH1 signaling to sustain cell growth. Importantly, PTEN-null/GSI resistant T-ALL cells switch their oncogene addiction from NOTCH1 to AKT and are highly sensitive to AKT inhibitors. These results should facilitate the development of molecular therapies targeting NOTCH1 and AKT for the treatment of T-ALL. PMID:18414037

Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines

PubMed Central

Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

2016-01-01

Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application. PMID:27408334

Dual PI3K/mTOR inhibitor BEZ235 as a promising therapeutic strategy against paclitaxel-resistant gastric cancer via targeting PI3K/Akt/mTOR pathway.

PubMed

Chen, Dongshao; Lin, Xiaoting; Zhang, Cheng; Liu, Zhentao; Chen, Zuhua; Li, Zhongwu; Wang, Jingyuan; Li, Beifang; Hu, Yanting; Dong, Bin; Shen, Lin; Ji, Jiafu; Gao, Jing; Zhang, Xiaotian

2018-01-26

Paclitaxel (PTX) is widely used in the front-line chemotherapy for gastric cancer (GC), but resistance limits its use. Due to the lack of proper models, mechanisms underlying PTX resistance in GC were not well studied. Using established PTX-resistant GC cell sublines HGC-27R, we for the first time integrated biological traits and molecular mechanisms of PTX resistance in GC. Data revealed that PTX-resistant GC cells were characterized by microtubular disorders, an EMT phenotype, reduced responses to antimitotic drugs, and resistance to apoptosis (marked by upregulated β-tubulin III, vimentin, attenuated changes in G 2 /M molecules or pro-apoptotic factors in response to antimitotic drugs or apoptotic inducers, respectively). Activation of the phosphoinositide 3-kinase, the serine/threonine kinase Akt and mammalian target of rapamycin (PI3K/Akt/mTOR) and mitogen-activated protein kinase (MAPK) pathways were also observed, which might be the reason for above phenotypic alternations. In vitro data suggested that targeting these pathways were sufficient to elicit antitumor responses in PTX-resistant GC, in which the dual PI3K/mTOR inhibitor BEZ235 displayed higher therapeutic efficiency than the mTOR inhibitor everolimus or the MEK inhibitor AZD6244. Antitumor effects of BEZ235 were also confirmed in mice bearing HGC-27R tumors. Thus, these data suggest that PI3K/Akt/mTOR and MAPK pathway inhibition, especially PI3K/mTOR dual blockade, might be a promising therapeutic strategy against PTX-resistant GC.

Targeting Aurora Kinase with MK-0457 Inhibits Ovarian Cancer Growth

PubMed Central

Lin, Yvonne G.; Immaneni, Anand; Merritt, William M.; Mangala, Lingegowda S.; Kim, SeungWook; Shahzad, Mian M.K.; Tsang, Yvonne T.M.; Armaiz-Pena, Guillermo N.; Lu, Chunhua; Kamat, Aparna A.; Han, Liz Y.; Spannuth, WhitneyA.; Nick, Alpa M.; Landen, Charles N.; Wong, Kwong K.; Gray, Michael J.; Coleman, Robert L.; Bodurka, Diane C.; Brinkley, William R.; Sood, Anil K.

2009-01-01

Purpose The Aurora kinase family plays pivotal roles in mitotic integrity and cell cycle.We sought to determine the effects of inhibiting Aurora kinase on ovarian cancer growth in an orthotopic mouse model using a small molecule pan-Aurora kinase inhibitor, MK-0457. Experimental Design We examined cell cycle regulatory effects and ascertained the therapeutic efficacy of Aurora kinase inhibition both alone and combined with docetaxel using both in vitro and in vivo ovarian cancer models. Results In vitro cytotoxicity assays with HeyA8 and SKOV3ip1 cells revealed >10-fold greater docetaxel cytotoxicity in combination with MK-0457. After in vivo dose kinetics were determined using phospho-histone H3 status, therapy experiments with the chemosensitive HeyA8 and SKOV3ip1as well as the chemoresistant HeyA8-MDR and A2780-CP20 models showed that Aurora kinase inhibition alone significantly reduced tumor burden compared with controls (P values < 0.01). Combination treatment with docetaxel resulted in significantly improved reduction in tumor growth beyond that afforded by docetaxel alone (P ≤ 0.03). Proliferating cell nuclear antigen immunohistochemistry revealed that MK-0457 alone and in combination with docetaxel significantly reduced cellular proliferation (P values < 0.001). Compared with controls, treatment with MK-0457 alone and in combination with docetaxel also significantly increased tumor cell apoptosis by ∼3-fold (P < 0.01). Remarkably, compared with docetaxel monotherapy, MK-0457 combined with docetaxel resulted in significantly increased tumor cell apoptosis. Conclusions Aurora kinase inhibition significantly reduces tumor burden and cell proliferation and increases tumor cell apoptosis in this preclinical orthotopic model of ovarian cancer. The role of Aurora kinase inhibition in ovarian cancer merits further investigation in clinical trials. PMID:18765535

Defibrotide Stimulates Angiogenesis and Protects Endothelial Cells from Calcineurin Inhibitor-Induced Apoptosis via Upregulation of AKT/Bcl-xL.

PubMed

Wang, Xiangmin; Pan, Bin; Hashimoto, Yuko; Ohkawara, Hiroshi; Xu, Kailin; Zeng, Lingyu; Ikezoe, Takayuki

2018-01-01

Sinusoidal obstruction syndrome is a life-threatening complication that can occur after haematopoietic stem cell transplantation. Defibrotide (DF) has been approved for the treatment of individuals with severe sinusoidal obstruction syndrome following haematopoietic stem cell transplantation in the European Union and the United States. However, the precise mechanisms by which DF protects endothelial cells remain to be elucidated. In this study, we found that DF stimulated angiogenesis in vitro and in vivo as assessed by vascular tube formation, scratch-wound repair and Matrigel plug assays. These effects were associated with an activation of pro-survival signalling pathways, including AKT (protein kinase B), ERK (extracellular signal-regulated kinases) and p38. More importantly, DF alleviated calcineurin inhibitor-induced growth inhibition and apoptosis of human umbilical vein endothelial cells and human hepatic sinusoidal endothelial cells in parallel with upregulation of anti-apoptotic protein B-cell lymphoma-extra-large (Bcl-xL), which was mediated by AKT (protein kinase B). Notably, these effects were abrogated when Bcl-xL was depleted by small interfering RNA (ribonucleic acid). In addition, DF counteracted calcineurin inhibitor-induced activation of nuclear factor-κB and Janus kinase 2 (JAK2)/Signal Transducer and Activator of Transcription 3 (STAT3) signalling and production of cytokines in vascular endothelial cell-derived EA.hy926 cells. Taken together, DF has pro-angiogenic, anti-apoptotic and anti-inflammatory effects on endothelial cells. DF is a potentially useful agent to prevent the development of, and treat individuals with, endothelial cell injury-related complications after haematopoietic stem cell transplantation. Schattauer GmbH Stuttgart.

Icotinib, a potent and specific EGFR tyrosine kinase inhibitor, inhibits growth of squamous cell carcinoma cell line A431 through negatively regulating AKT signaling.

PubMed

Gao, Zhenzhen; Chen, Wei; Zhang, Xiaohua; Cai, Peifen; Fang, Xianying; Xu, Qiang; Sun, Yang; Gu, Yanhong

2013-06-01

Icotinib is a potent and specific epidermal growth factor receptor tyrosine kinase inhibitor. In this study, we reported that icotinib had the antitumor activity on human squamous cell carcinoma cell line A431 in vitro. Meanwhile, adhesion to fibronectin and expression of integrin α3 and β1 were significantly reduced in a dose-dependent manner after the treatment of icotinib. Moreover, icotinib induced cell cycle arrested and affected expression of various cell cycle related proteins in squamous cancer cell line A431, whereas it did not cause apoptosis. Furthermore, icotinib remarkably down-regulated phosphorylation of protein kinase B (AKT) though blocking the interaction between 3-phosphoinositide-dependent protein kinase-1 (PDK1) and AKT in A431 cells. Taken together, it is shown that the small molecular compound, icotinib, has an anti-squamous cell carcinoma activity in vitro and its antitumor mechanism is associated with the blockage of the interaction between PDK1 and AKT. These results provide a novel strategy for anti-squamous cell carcinoma therapy. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Crosstalk between the IGF-1R/AKT/mTORC1 pathway and the tumor suppressors p53 and p27 determines cisplatin sensitivity and limits the effectiveness of an IGF-1R pathway inhibitor

PubMed Central

Davaadelger, Batzaya; Duan, Lei; Perez, Ricardo E.; Gitelis, Steven; Maki, Carl G.

2016-01-01

The insulin-like growth factor-1 receptor (IGF-1R) signaling pathway is aberrantly activated in multiple cancers and can promote proliferation and chemotherapy resistance. Multiple IGF-1R inhibitors have been developed as potential therapeutics. However, these inhibitors have failed to increase patient survival when given alone or in combination with chemotherapy agents. The reason(s) for the disappointing clinical effect of these inhibitors is not fully understood. Cisplatin (CP) activated the IGF-1R/AKT/mTORC1 pathway and stabilized p53 in osteosarcoma (OS) cells. p53 knockdown reduced IGF-1R/AKT/mTORC1 activation by CP, and IGF-1R inhibition reduced the accumulation of p53. These data demonstrate positive crosstalk between p53 and the IGF-1R/AKT/mTORC1 pathway in response to CP. Further studies showed the effect of IGF-1R inhibition on CP response is dependent on p53 status. In p53 wild-type cells treated with CP, IGF-1R inhibition increased p53s apoptotic function but reduced p53-dependent senescence, and had no effect on long term survival. In contrast, in p53-null/knockdown cells, IGF-1R inhibition reduced apoptosis in response to CP and increased long term survival. These effects were due to p27 since IGF-1R inhibition stabilized p27 in CP-treated cells, and p27 depletion restored apoptosis and reduced long term survival. Together, the results demonstrate 1) p53 expression determines the effect of IGF-1R inhibition on cancer cell CP response, and 2) crosstalk between the IGF-1R/AKT/mTORC1 pathway and p53 and p27 can reduce cancer cell responsiveness to chemotherapy and may ultimately limit the effectiveness of IGF-1R pathway inhibitors in the clinic. PMID:27050276

Lithium ions attenuate serum-deprivation-induced apoptosis in PC12 cells through regulation of the Akt/FoxO1 signaling pathways.

PubMed

Zeng, Zhiwen; Wang, Haitao; Shang, Fu; Zhou, Lihua; Little, Peter J; Quirion, Remi; Zheng, Wenhua

2016-03-01

Lithium is currently used in the treatment of mental illness. We have previously reported that lithium stimulated the protein kinase B/Forkhead box O1 (Akt/FoxO1) pathway in rats. However, little information is available regarding its neuroprotective role of this pathway and underlying mechanisms. PC12 cells treated with serum deprivation were used as a toxicity model to study the protective effect of lithium and its underlying mechanisms. Cell viability was determined by methyl thiazolyl tetrazolium assay and Hoechst staining. FoxO1 subcellular location and its overexpression were used to study the underlying mechanisms. Various pathway inhibitors were used to investigate the possible pathways, while the phosphorylation of Akt and FoxO1 was analyzed by Western blot. Lithium pretreatment dose-dependently reduced PC12 cell apoptosis induced by serum starvation. The protective effect of lithium was abolished by LY294002, a PI3K-specific inhibitor, and Akt inhibitor Akt inhibitor VIII, whereas mitogen-activated protein kinase kinase (MEK kinase) inhibitor U0126 had no effect. Lithium induced the phosphorylation of Akt and FoxO1 in a time- and concentration-dependent manner. Lithium-induced phosphorylation of Akt and FoxO1 is mediated by the PI3K/Akt pathway. Serum deprivation caused nuclear translocation of FoxO1 while application of lithium reversed the effect of serum deprivation. Moreover, overexpression of FoxO1 enhanced cell apoptosis induced by serum withdrawal. Finally, lithium was found to reduce the exogenous and endogenous FoxO1 protein levels in PC12 cells in a concentration-dependent fashion. The protective effect of lithium against serum starvation cell death is mediated by the PI3K/Akt/FoxO1 pathway.

Aberrant AKT activation drives well-differentiated liposarcoma

PubMed Central

Gutierrez, Alejandro; Snyder, Eric L.; Marino-Enriquez, Adrian; Zhang, Yi-Xiang; Sioletic, Stefano; Kozakewich, Elena; Grebliunaite, Ruta; Ou, Wen-bin; Sicinska, Ewa; Raut, Chandrajit P.; Demetri, George D.; Perez-Atayde, Antonio R.; Wagner, Andrew J.; Fletcher, Jonathan A.; Fletcher, Christopher D. M.; Look, A. Thomas

2011-01-01

Well-differentiated liposarcoma (WDLPS), one of the most common human sarcomas, is poorly responsive to radiation and chemotherapy, and the lack of animal models suitable for experimental analysis has seriously impeded functional investigation of its pathobiology and development of effective targeted therapies. Here, we show that zebrafish expressing constitutively active Akt2 in mesenchymal progenitors develop WDLPS that closely resembles the human disease. Tumor incidence rates were 8% in p53 wild-type zebrafish, 6% in p53 heterozygotes, and 29% in p53-homozygous mutant zebrafish (P = 0.013), indicating that aberrant Akt activation collaborates with p53 mutation in WDLPS pathogenesis. Analysis of primary clinical specimens of WDLPS, and of the closely related dedifferentiated liposarcoma (DDLPS) subtype, revealed immunohistochemical evidence of AKT activation in 27% of cases. Western blot analysis of a panel of cell lines derived from patients with WDLPS or DDLPS revealed robust AKT phosphorylation in all cell lines examined, even when these cells were cultured in serum-free media. Moreover, BEZ235, a small molecule inhibitor of PI3K and mammalian target of rapamycin that effectively inhibits AKT activation in these cells, impaired viability at nanomolar concentrations. Our findings are unique in providing an animal model to decipher the molecular pathogenesis of WDLPS, and implicate AKT as a previously unexplored therapeutic target in this chemoresistant sarcoma. PMID:21930930

Resveratrol Overcomes Cellular Resistance to Vemurafenib Through Dephosphorylation of AKT in BRAF-mutated Melanoma Cells.

PubMed

Luo, Hao; Umebayashi, Masayo; Doi, Keiko; Morisaki, Takashi; Shirasawa, Senji; Tsunoda, Toshiyuki

2016-07-01

The serine/threonine-protein kinase B-Raf (BRAF) V600E mutant (BRAF(V600E)) inhibitor vemurafenib, has improved clinical outcomes for patients with BRAF(V600E) melanoma, but acquired cellular resistance mediated by AKT serine/threonine kinase 1 (AKT) phosphorylation limits its efficacy. We examined the effect of resveratrol on vemurafenib-resistant melanoma cells. A vemurafenib-resistant human metastatic melanoma cell line positive for the BRAF V600E mutation was established. The anti-tumorigenic effects of vemurafenib and resveratrol, both alone and in combination, were examined through analysis of cell proliferation and protein expression. The level of phosphorylated AKT (p-AKT) was increased in the primary melanoma cells after treatment with vemurafenib, and the basal level of p-AKT was increased in vemurafenib-resistant melanoma cells. Notably, resveratrol both alone and in combination with vemurafenib effectively suppressed cell proliferation and AKT phosphorylation in both parental and vemurafenib-resistant melanoma cells. Vemurafenib resistance can be reversed by addition of resveratrol in patients undergoing treatment with BRAF inhibitors. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7.

PubMed

Gallardo Bolaños, Juan M; Balao da Silva, Carolina M; Martín Muñoz, Patricia; Morillo Rodríguez, Antolín; Plaza Dávila, María; Rodríguez-Martínez, Heriberto; Aparicio, Inés M; Tapia, José A; Ortega Ferrusola, Cristina; Peña, Fernando J

2014-08-01

AKT, also referred to as protein kinase B (PKB or RAC), plays a critical role in controlling cell survival and apoptosis. To gain insights into the mechanisms regulating sperm survival after ejaculation, the role of AKT was investigated in stallion spermatozoa using a specific inhibitor and a phosphoflow approach. Stallion spermatozoa were washed and incubated in Biggers-Whitten-Whittingham medium, supplemented with 1% polyvinyl alcohol (PVA) in the presence of 0 (vehicle), 10, 20 or 30 μM SH5, an AKT inhibitor. SH5 treatment reduced the percentage of sperm displaying AKT phosphorylation, with inhibition reaching a maximum after 1 h of incubation. This decrease in phosphorylation was attributable to either dephosphorylation or suppression of the active phosphorylation pathway. Stallion spermatozoa spontaneously dephosphorylated during in vitro incubation, resulting in a lack of a difference in AKT phosphorylation between the SH5-treated sperm and the control after 4 h of incubation. AKT inhibition decreased the proportion of motile spermatozoa (total and progressive) and the sperm velocity. Similarly, AKT inhibition reduced membrane integrity, leading to increased membrane permeability and reduced the mitochondrial membrane potential concomitantly with activation of caspases 3 and 7. However, the percentage of spermatozoa exhibiting oxidative stress, the production of mitochondrial superoxide radicals, DNA oxidation and DNA fragmentation were not affected by AKT inhibition. It is concluded that AKT maintains the membrane integrity of ejaculated stallion spermatozoa, presumably by inhibiting caspases 3 and 7, which prevents the progression of spermatozoa to an incomplete form of apoptosis. © 2014 Society for Reproduction and Fertility.

Activity of MK-7655 combined with imipenem against Enterobacteriaceae and Pseudomonas aeruginosa.

PubMed

Livermore, David M; Warner, Marina; Mushtaq, Shazad

2013-10-01

MK-7655 is a novel inhibitor of class A and C β-lactamases. We investigated its potential to protect imipenem. Chequerboard MICs were determined by CLSI agar dilution: (i) for Enterobacteriaceae with carbapenemases; (ii) for Enterobacteriaceae with carbapenem resistance contingent on combinations of impermeability together with an extended-spectrum β-lactamase or AmpC enzyme; and (iii) for Pseudomonas aeruginosa and other non-fermenters. At a concentration of 4 mg/L, MK-7655 reduced imipenem MICs for Enterobacteriaceae with KPC carbapenemases from 16-64 mg/L to 0.12-1 mg/L. Synergy also was seen for Enterobacteriaceae with impermeability-mediated carbapenem resistance, with weaker synergy seen for isolates with the OXA-48 enzyme. On the other hand, MK-7655 failed to potentiate imipenem against Enterobacteriaceae with metallo-carbapenemases. In the case of P. aeruginosa, where endogenous AmpC confers slight protection versus imipenem, 4 mg/L MK-7655 reduced the MIC of imipenem for all isolates, except those with metallo-carbapenemases: the MICs of imipenem fell from 1-2 mg/L to 0.25-0.5 mg/L for imipenem-susceptible P. aeruginosa and from 16-64 mg/L to 1-4 mg/L for OprD-deficient strains. No potentiation was seen for chryseobacteria or for Stenotrophomonas maltophilia. MK-7655 potentiated imipenem against Enterobacteriaceae with KPC carbapenemases or combinations of β-lactamase and impermeability, but not those with metallo-carbapenemases. It augmented the activity of imipenem against P. aeruginosa in general and OprD mutants in particular.

Efficacy of Histone Deacetylase and Estrogen Receptor Inhibition in Breast Cancer Cells Due to Concerted down Regulation of Akt

PubMed Central

Thomas, Scott; Thurn, K. Ted; Raha, Paromita; Chen, Stephanie; Munster, Pamela N.

2013-01-01

Hormonal therapy resistance remains a considerable barrier in the treatment of breast cancer. Activation of the Akt-PI3K-mTOR pathway plays an important role in hormonal therapy resistance. Our recent preclinical and clinical studies showed that the addition of a histone deacetylase inhibitor re-sensitized hormonal therapy resistant breast cancer to tamoxifen. As histone deacetylases are key regulators of Akt, we evaluated the effect of combined treatment with the histone deacetylase inhibitor PCI-24781 and tamoxifen on Akt in breast cancer cells. We demonstrate that while both histone deacetylase and estrogen receptor inhibition down regulate AKT mRNA and protein, their concerted effort results in down regulation of AKT activity with induction of cell death. Histone deacetylase inhibition exerts its effect on AKT mRNA through an estrogen receptor-dependent mechanism, primarily down regulating the most abundant isoform AKT1. Although siRNA depletion of AKT modestly induces cell death, when combined with an anti-estrogen, cytotoxicity is significantly enhanced. Thus, histone deacetylase regulation of AKT mRNA is a key mediator of this therapeutic combination and may represent a novel biomarker for predicting response to this regimen. PMID:23874830

Homocysteine enhances MMP-9 production in murine macrophages via ERK and Akt signaling pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Seung Jin; Lee, Yi Sle; Seo, Kyo Won

2012-04-01

Homocysteine (Hcy) at elevated levels is an independent risk factor of cardiovascular diseases, including atherosclerosis. In the present study, we investigated the effect of Hcy on the production of matrix metalloproteinases (MMP) in murine macrophages. Among the MMP known to regulate the activities of collagenase and gelatinase, Hcy exclusively increased the gelatinolytic activity of MMP-9 in J774A.1 cells as well as in mouse peritoneal macrophages. Furthermore, this activity was found to be correlated with Western blot findings in J774A.1 cells, which showed that MMP-9 expression was concentration- and time-dependently increased by Hcy. Inhibition of the ERK and Akt pathways ledmore » to a significant decrease in Hcy-induced MMP-9 expression, and combined treatment with inhibitors of the ERK and Akt pathways showed an additive effects. Activity assays for ERK and Akt showed that Hcy increased the phosphorylation of both, but these phosphorylation were not affected by inhibitors of the Akt and ERK pathways. In line with these findings, the molecular inhibition of ERK and Akt using siRNA did not affect the Hcy-induced phosphorylation of Akt and ERK, respectively. Taken together, these findings suggest that Hcy enhances MMP-9 production in murine macrophages by separately activating the ERK and Akt signaling pathways. -- Highlights: ► Homocysteine (Hcy) induced MMP-9 production in murine macrophages. ► Hcy induced MMP-9 production through ERK and Akt signaling pathways. ► ERK and Akt signaling pathways were activated by Hcy in murine macrophages. ► ERK and Akt pathways were additively act on Hcy-induced MMP-9 production. ► Hcy enhances MMP-9 production in macrophages via activation of ERK and Akt signaling pathways in an independent manner.« less

Calcineurin mediates AKT dephosphorylation in the ischemic rat retina.

PubMed

Park, Chang Hwan; Kim, Yoon Sook; Kim, Young Hee; Choi, Mee Young; Yoo, Ji Myong; Kang, Sang Soo; Choi, Wan Sung; Cho, Gyeong Jae

2008-10-09

Calcineurin (CaN) is a calcium/calmodulin-dependent protein phosphatase that has an important role in ischemia-induced apoptosis. The serine/threonine kinase, Akt, which is also known as protein kinase B, has an important role in the cell death/survival pathways. Akt is activated by its phosphorylation, which is positively regulated by phosphatidylinositol 3-kinase (PI3K) and negatively regulated by a class of protein phosphatases (PPs) in tissue. However, the relationship between CaN and Akt after transient ischemia remains unclear. In the present study, we investigated whether CaN is involved in neuronal cell apoptosis and Akt dephosphorylation that occur during ischemic injury. We examined the interdependence between CaN and Akt/protein kinase B (PKB) in the rat retina after transient ischemia. After ischemic damage, we detected changes in levels of CaN, Akt and Bad in rats in the presence or absence FK506, CaN inhibitor. Our results show that CaN cleavage reduced Akt phosphorylation at Thr308 and Ser473, and led to apoptosis via dephosphorylation of the proapoptotic Bcl-2 family member Bad. After treatment with FK506, Akt and Bad dephosphorylation was greatly reduced. The total number of TUNEL-positive neurons was reduced by intravitreal injection of FK506 after transient ischemia. These results indicate that CaN cleavage negatively regulates Akt phosphorylation and is involved in retinal cell apoptosis after transient ischemia.

Inhibition of Akt enhances the chemopreventive effects of topical rapamycin in mouse skin

USGS Publications Warehouse

Dickinson, Sally E; Janda, Jaroslav; Criswell, Jane; Blohm-Mangone, Karen; Olson, Erik R.; Liu, Zhonglin; Barber, Christie; Rusche, Jadrian J.; Petricoin, Emmanuel; Calvert, Valerie; Einspahr, Janine G.; Dickinson, Jesse; Stratton, Steven P.; Curiel-Lewandrowski, Clara; Saboda, Kathylynn; Hu, Chengcheng; Bode, Ann M.; Dong, Zigang; Alberts, David S.; Bowden, G. Timothy

2016-01-01

The PI3Kinase/Akt/mTOR pathway has important roles in cancer development for multiple tumor types, including UV-induced non-melanoma skin cancer. Immunosuppressed populations are at increased risk of aggressive cutaneous squamous cell carcinoma (SCC). Individuals who are treated with rapamycin, (sirolimus, a classical mTOR inhibitor) have significantly decreased rates of developing new cutaneous SCCs compared to those that receive traditional immunosuppression. However, systemic rapamycin use can lead to significant adverse events. Here we explored the use of topical rapamycin as a chemopreventive agent in the context of solar simulated light (SSL)-induced skin carcinogenesis. In SKH-1 mice, topical rapamycin treatment decreased tumor yields when applied after completion of 15 weeks of SSL exposure compared to controls. However, applying rapamycin during SSL exposure for 15 weeks, and continuing for 10 weeks after UV treatment, increased tumor yields. We also examined whether a combinatorial approach might result in more significant tumor suppression by rapamycin. We validated that rapamycin causes increased Akt (S473) phosphorylation in the epidermis after SSL, and show for the first time that this dysregulation can be inhibited in vivo by a selective PDK1/Akt inhibitor, PHT-427. Combining rapamycin with PHT-427 on tumor prone skin additively caused a significant reduction of tumor multiplicity compared to vehicle controls. Our findings indicate that patients taking rapamycin should avoid sun exposure, and that combining topical mTOR inhibitors and Akt inhibitors may be a viable chemoprevention option for individuals at high risk for cutaneous SCC.

Inhibition of Akt Enhances the Chemopreventive Effects of Topical Rapamycin in Mouse Skin

PubMed Central

Dickinson, Sally E.; Janda, Jaroslav; Criswell, Jane; Blohm-Mangone, Karen; Olson, Erik R.; Liu, Zhonglin; Barber, Christie; Petricoin, Emanuel F.; Calvert, Valerie S.; Einspahr, Janine; Dickinson, Jesse E.; Stratton, Steven P.; Curiel-Lewandrowski, Clara; Saboda, Kathylynn; Hu, Chengcheng; Bode, Ann M.; Dong, Zigang; Alberts, David S.; Bowden, G. Timothy

2016-01-01

The PI3Kinase/Akt/mTOR pathway has important roles in cancer development for multiple tumor types, including UV-induced non-melanoma skin cancer. Immunosuppressed populations are at increased risk of aggressive cutaneous squamous cell carcinoma (SCC). Individuals who are treated with rapamycin, (sirolimus, a classical mTOR inhibitor) have significantly decreased rates of developing new cutaneous SCCs compared to those that receive traditional immunosuppression. However, systemic rapamycin use can lead to significant adverse events. Here we explored the use of topical rapamycin as a chemopreventive agent in the context of solar simulated light (SSL)-induced skin carcinogenesis. In SKH-1 mice, topical rapamycin treatment decreased tumor yields when applied after completion of 15 weeks of SSL exposure compared to controls. However, applying rapamycin during SSL exposure for 15 weeks, and continuing for 10 weeks after UV treatment, increased tumor yields. We also examined whether a combinatorial approach might result in more significant tumor suppression by rapamycin. We validated that rapamycin causes increased Akt (S473) phosphorylation in the epidermis after SSL, and show for the first time that this dysregulation can be inhibited in vivo by a selective PDK1/Akt inhibitor, PHT-427. Combining rapamycin with PHT-427 on tumor prone skin additively caused a significant reduction of tumor multiplicity compared to vehicle controls. Our findings indicate that patients taking rapamycin should avoid sun exposure, and that combining topical mTOR inhibitors and Akt inhibitors may be a viable chemoprevention option for individuals at high risk for cutaneous SCC. PMID:26801880

Inhibition of Akt Enhances the Chemopreventive Effects of Topical Rapamycin in Mouse Skin.

PubMed

Dickinson, Sally E; Janda, Jaroslav; Criswell, Jane; Blohm-Mangone, Karen; Olson, Erik R; Liu, Zhonglin; Barber, Christy; Petricoin, Emanuel F; Calvert, Valerie S; Einspahr, Janine; Dickinson, Jesse E; Stratton, Steven P; Curiel-Lewandrowski, Clara; Saboda, Kathylynn; Hu, Chengcheng; Bode, Ann M; Dong, Zigang; Alberts, David S; Timothy Bowden, G

2016-03-01

The PI3Kinase/Akt/mTOR pathway has important roles in cancer development for multiple tumor types, including UV-induced nonmelanoma skin cancer. Immunosuppressed populations are at increased risk of aggressive cutaneous squamous cell carcinoma (SCC). Individuals who are treated with rapamycin (sirolimus, a classical mTOR inhibitor) have significantly decreased rates of developing new cutaneous SCCs compared with those that receive traditional immunosuppression. However, systemic rapamycin use can lead to significant adverse events. Here, we explored the use of topical rapamycin as a chemopreventive agent in the context of solar-simulated light (SSL)-induced skin carcinogenesis. In SKH-1 mice, topical rapamycin treatment decreased tumor yields when applied after completion of 15 weeks of SSL exposure compared with controls. However, applying rapamycin during SSL exposure for 15 weeks, and continuing for 10 weeks after UV treatment, increased tumor yields. We also examined whether a combinatorial approach might result in more significant tumor suppression by rapamycin. We validated that rapamycin causes increased Akt (S473) phosphorylation in the epidermis after SSL, and show for the first time that this dysregulation can be inhibited in vivo by a selective PDK1/Akt inhibitor, PHT-427. Combining rapamycin with PHT-427 on tumor prone skin additively caused a significant reduction of tumor multiplicity compared with vehicle controls. Our findings indicate that patients taking rapamycin should avoid sun exposure, and that combining topical mTOR inhibitors and Akt inhibitors may be a viable chemoprevention option for individuals at high risk for cutaneous SCC. ©2016 American Association for Cancer Research.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sang-pil; Ha, Jung Min; Yun, Sung Ji

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 amore » 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.« less

GLI inhibitor GANT-61 diminishes embryonal and alveolar rhabdomyosarcoma growth by inhibiting Shh/AKT-mTOR axis

PubMed Central

Srivastava, Ritesh K.; Kaylani, Samer Zaid; Edrees, Nayf; Li, Changzhao; Talwelkar, Sarang S.; Xu, Jianmin; Palle, Komaraiah; Pressey, Joseph G.; Athar, Mohammad

2014-01-01

Rhabdomyosarcoma (RMS) typically arises from skeletal muscle. Currently, RMS in patients with recurrent and metastatic disease have no successful treatment. The molecular pathogenesis of RMS varies based on cancer sub-types. Some embryonal RMS but not other sub-types are driven by sonic hedgehog (Shh) signaling pathway. However, Shh pathway inhibitors particularly smoothened inhibitors are not highly effective in animals. Here, we show that Shh pathway effectors GLI1 and/or GLI2 are over-expressed in the majority of RMS cells and that GANT-61, a specific GLI1/2 inhibitor dampens the proliferation of both embryonal and alveolar RMS cells-derived xenograft tumors thereby blocking their growth. As compared to vehicle-treated control, about 50% tumor growth inhibition occurs in mice receiving GANT-61 treatment. The proliferation inhibition was associated with slowing of cell cycle progression which was mediated by the reduced expression of cyclins D1/2/3 & E and the concomitant induction of p21. GANT-61 not only reduced expression of GLI1/2 in these RMS but also significantly diminished AKT/mTOR signaling. The therapeutic action of GANT-61 was significantly augmented when combined with chemotherapeutic agents employed for RMS therapy such as temsirolimus or vincristine. Finally, reduced expression of proteins driving epithelial mesenchymal transition (EMT) characterized the residual tumors. PMID:25432075

Establishment of a luciferase assay-based screening system: Fumitremorgin C selectively inhibits cellular proliferation of immortalized astrocytes expressing an active form of AKT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Lei; Sasai, Ken; Akagi, Tsuyoshi

2008-08-29

The AKT pathway is frequently activated in glioblastoma, and as such, inhibitors of this pathway could prove very useful as anti-glioblastoma therapies. Here we established immortalized astrocytes expressing Renilla luciferase as well as those expressing both an active form of AKT and firefly luciferase. Since both luciferase activities represent the numbers of corresponding cell lines, novel inhibitors of the AKT pathway can be identified by treating co-cultures containing the two types of luciferase-expressing cells with individual compounds. Indeed, such a screening system succeeded in identifying fumitremorgin C as an efficient inhibitor of the AKT pathway, which was further confirmed bymore » the ability of fumitremorgin C to selectively inhibit the growth of immortalized astrocytes expressing an active form of AKT. The present study proposes a broadly applicable approach for identifying therapeutic agents that target the pathways and/or molecules responsible for cancer development.« less

Resveratrol Inhibits the Epidermal Growth Factor-Induced Migration of Osteoblasts: the Suppression of SAPK/JNK and Akt.

PubMed

Kawabata, Tetsu; Tokuda, Haruhiko; Fujita, Kazuhiko; Kainuma, Shingo; Sakai, Go; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Otsuka, Takanobu

2017-01-01

Resveratrol is a polyphenol enriched in the skins of grapes and berries, that shows various beneficial effects for human health. In the present study, we investigated the mechanism behind the epidermal growth factor (EGF)-induced migration of osteoblast-like MC3T3-E1 cells, and the effect of resveratrol on this cell migration. The cell migration was examined using Boyden chamber, and phosphorylation of each kinase was analyzed by Western blotting. The EGF-induced migration was suppressed by PD98059, an inhibitor of MEK1/2, as well as SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of SAPK/JNK, and deguelin, an inhibitor of Akt. In contrast, rapamycin, an inhibitor of upstream kinase of p70 S6 kinase, and fasudil, an inhibitor of Rho-kinase, hardly affected the migration. Resveratrol significantly reduced the EGF-induced migration in a dose-dependent manner. SRT1720, an SIRT1 activator, suppressed the migration by EGF. In addition, resveratrol markedly attenuated the EGF-induced phosphorylation of SAPK/JNK and Akt without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. The phosphorylation of SAPK/JNK and Akt induced by EGF was down-regulated by SRT1720. Our results strongly suggest that resveratrol reduces the EGF-stimulated migration of osteoblasts via suppression of SAPK and Akt, and that the inhibitory effect of resveratrol is mediated in part via SIRT1. © 2017 The Author(s). Published by S. Karger AG, Basel.

Elongation Factor 1 alpha interacts with phospho-Akt in breast cancer cells and regulates their proliferation, survival and motility.

PubMed

Pecorari, Luisa; Marin, Oriano; Silvestri, Chiara; Candini, Olivia; Rossi, Elena; Guerzoni, Clara; Cattelani, Sara; Mariani, Samanta A; Corradini, Francesca; Ferrari-Amorotti, Giovanna; Cortesi, Laura; Bussolari, Rita; Raschellà, Giuseppe; Federico, Massimo R; Calabretta, Bruno

2009-08-03

Akt/PKB is a serine/threonine kinase that has attracted much attention because of its central role in regulating cell proliferation, survival, motility and angiogenesis. Activation of Akt in breast cancer portends aggressive tumour behaviour, resistance to hormone-, chemo-, and radiotherapy-induced apoptosis and it is correlated with decreased overall survival. Recent studies have identified novel tumor-specific substrates of Akt that may provide new diagnostic and prognostic markers and serve as therapeutic targets. This study was undertaken to identify pAkt-interacting proteins and to assess their biological roles in breast cancer cells. We confirmed that one of the pAkt interacting proteins is the Elongation Factor EF1alpha. EF1alpha contains a putative Akt phosphorylation site, but is not phosphorylated by pAkt1 or pAkt2, suggesting that it may function as a modulator of pAkt activity. Indeed, downregulation of EF1alpha expression by siRNAs led to markedly decreased expression of pAkt1 and to less extent of pAkt2 and was associated with reduced proliferation, survival and invasion of HCC1937 cells. Proliferation and survival was further reduced by combining EF1alpha siRNAs with specific pAkt inhibitors whereas EF1alpha downregulation slightly attenuated the decreased invasion induced by Akt inhibitors. We show here that EF1alpha is a pAkt-interacting protein which regulates pAkt levels. Since EF1alpha is often overexpressed in breast cancer, the consequences of EF1alpha increased levels for proliferation, survival and invasion will likely depend on the relative concentration of Akt1 and Akt2.

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

Cyclic mechanical strain maintains Nanog expression through PI3K/Akt signaling in mouse embryonic stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Horiuchi, Rie; Akimoto, Takayuki, E-mail: akimoto@m.u-tokyo.ac.jp; Institute for Biomedical Engineering, Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Waseda-tsurumaki, Shinjuku, Tokyo 162-0041

2012-08-15

Mechanical strain has been reported to affect the proliferation/differentiation of many cell types; however, the effects of mechanotransduction on self-renewal as well as pluripotency of embryonic stem (ES) cells remains unknown. To investigate the effects of mechanical strain on mouse ES cell fate, we examined the expression of Nanog, which is an essential regulator of self-renewal and pluripotency as well as Nanog-associated intracellular signaling during uniaxial cyclic mechanical strain. The mouse ES cell line, CCE was plated onto elastic membranes, and we applied 10% strain at 0.17 Hz. The expression of Nanog was reduced during ES cell differentiation in responsemore » to the withdrawal of leukemia inhibitory factor (LIF); however, two days of cyclic mechanical strain attenuated this reduction of Nanog expression. On the other hand, the cyclic mechanical strain promoted PI3K-Akt signaling, which is reported as an upstream of Nanog transcription. The cyclic mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor wortmannin. Furthermore, cytochalasin D, an inhibitor of actin polymerization, also inhibited the mechanical strain-induced increase in phospho-Akt. These findings imply that mechanical force plays a role in regulating Nanog expression in ES cells through the actin cytoskeleton-PI3K-Akt signaling. -- Highlights: Black-Right-Pointing-Pointer The expression of Nanog, which is an essential regulator of 'stemness' was reduced during embryonic stem (ES) cell differentiation. Black-Right-Pointing-Pointer Cyclic mechanical strain attenuated the reduction of Nanog expression. Black-Right-Pointing-Pointer Cyclic mechanical strain promoted PI3K-Akt signaling and mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor and an inhibitor of actin polymerization.« less

Hydrogen peroxide-induced Akt phosphorylation regulates Bax activation.

PubMed

Sadidi, Mahdieh; Lentz, Stephen I; Feldman, Eva L

2009-05-01

Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) are involved in many cellular processes that positively and negatively regulate cell fate. H(2)O(2), acting as an intracellular messenger, activates phosphatidylinositol-3 kinase (PI3K) and its downstream target Akt, and promotes cell survival. The aim of the current study was to understand the mechanism by which PI3K/Akt signaling promotes survival in SH-SY5Y neuroblastoma cells. We demonstrate that PI3K/Akt mediates phosphorylation of the pro-apoptotic Bcl-2 family member Bax. This phosphorylation suppresses apoptosis and promotes cell survival. Increased survival in the presence of H(2)O(2) was blocked by LY294002, an inhibitor of PI3K activation. LY294002 prevented Bax phosphorylation and resulted in Bax translocation to the mitochondria, cytochrome c release, caspase-3 activation, and cell death. Collectively, these findings reveal a mechanism by which H(2)O(2)-induced activation of PI3K/Akt influences post-translational modification of Bax and inactivates a key component of the cell death machinery.

A novel AKT1 mutant amplifies an adaptive melanoma response to BRAF inhibition

PubMed Central

Shi, Hubing; Hong, Aayoung; Kong, Xiangju; Koya, Richard C.; Song, Chunying; Moriceau, Gatien; Hugo, Willy; Yu, Clarissa C.; Ng, Charles; Chodon, Thinle; Scolyer, Richard A.; Kefford, Richard F.; Ribas, Antoni; Long, Georgina V.; Lo, Roger S.

2013-01-01

BRAF inhibitor (BRAFi) therapy leads to remarkable anti-melanoma responses, but the initial tumor shrinkage is commonly incomplete, providing a nidus for subsequent disease progression. Adaptive signaling may underlie early BRAFi resistance and influence the selection pattern for genetic variants causing late, acquired resistance. We show here that BRAFi (or BRAFi+MEKi) therapy in patients frequently led to rebound p-AKT levels in their melanomas early on treatment. In cell lines, BRAFi treatment led to rebound levels of RTKs (including PDGFRβ), PIP3, pleckstrin homology domain (PHD) recruitment, and p-AKT. PTEN expression limited this BRAFi-elicited PI3K-AKT signaling, which could be rescued by introduction of a mutant AKT1 (Q79K) kown to confer acquired BRAFi resistance. Functionally, AKT1 Q79K conferred BRAFi resistance via amplifying BRAFi-elicited PI3K-AKT signaling. Additionally, MAPK pathway inhibition enhanced clonogenic growth dependency on PI3K or AKT. Thus, adaptive or genetic upregulation of AKT critically participates in melanoma survival during BRAFi therapy. PMID:24265152

Glutaredoxin exerts an antiapoptotic effect by regulating the redox state of Akt.

PubMed

Murata, Hiroaki; Ihara, Yoshito; Nakamura, Hajime; Yodoi, Junji; Sumikawa, Koji; Kondo, Takahito

2003-12-12

Glutaredoxin (GRX) is a small dithiol protein involved in various cellular functions, including the redox regulation of certain enzyme activities. GRX functions via a disulfide exchange reaction by utilizing the active site Cys-Pro-Tyr-Cys. Here we demonstrated that overexpression of GRX protected cells from hydrogen peroxide (H2O2)-induced apoptosis by regulating the redox state of Akt. Akt was transiently phosphorylated, dephosphorylated, and then degraded in cardiac H9c2 cells undergoing H2O2-induced apoptosis. Under stress, Akt underwent disulfide bond formation between Cys-297 and Cys-311 and dephosphorylation in accordance with an increased association with protein phosphatase 2A. Overexpression of GRX protected Akt from H2O2-induced oxidation and suppressed recruitment of protein phosphatase 2A to Akt, resulting in a sustained phosphorylation of Akt and inhibition of apoptosis. This effect was reversed by cadmium, an inhibitor of GRX. Furthermore an in vitro assay revealed that GRX reduced oxidized Akt in concert with glutathione, NADPH, and glutathione-disulfide reductase. Thus, GRX plays an important role in protecting cells from apoptosis by regulating the redox state of Akt.

Novel small molecule inhibitors of 3-phosphoinositide-dependent kinase-1.

PubMed

Feldman, Richard I; Wu, James M; Polokoff, Mark A; Kochanny, Monica J; Dinter, Harald; Zhu, Daguang; Biroc, Sandra L; Alicke, Bruno; Bryant, Judi; Yuan, Shendong; Buckman, Brad O; Lentz, Dao; Ferrer, Mike; Whitlow, Marc; Adler, Marc; Finster, Silke; Chang, Zheng; Arnaiz, Damian O

2005-05-20

The phosphoinositide 3-kinase/3-phosphoinositide-dependent kinase 1 (PDK1)/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis and represents a promising target for anticancer drugs. Here, we describe three potent PDK1 inhibitors, BX-795, BX-912, and BX-320 (IC(50) = 11-30 nm) and their initial biological characterization. The inhibitors blocked PDK1/Akt signaling in tumor cells and inhibited the anchorage-dependent growth of a variety of tumor cell lines in culture or induced apoptosis. A number of cancer cell lines with elevated Akt activity were >30-fold more sensitive to growth inhibition by PDK1 inhibitors in soft agar than on tissue culture plastic, consistent with the cell survival function of the PDK1/Akt signaling pathway, which is particularly important for unattached cells. BX-320 inhibited the growth of LOX melanoma tumors in the lungs of nude mice after injection of tumor cells into the tail vein. The effect of BX-320 on cancer cell growth in vitro and in vivo indicates that PDK1 inhibitors may have clinical utility as anticancer agents.

The significance of PTEN and AKT aberrations in pediatric T-cell acute lymphoblastic leukemia

PubMed Central

Zuurbier, Linda; Petricoin, Emanuel F.; Vuerhard, Maartje J.; Calvert, Valerie; Kooi, Clarissa; Buijs-Gladdines, Jessica G.C.A.M.; Smits, Willem K.; Sonneveld, Edwin; Veerman, Anjo J.P.; Kamps, Willem A.; Horstmann, Martin; Pieters, Rob; Meijerink, Jules P.P.

2012-01-01

Background PI3K/AKT pathway mutations are found in T-cell acute lymphoblastic leukemia, but their overall impact and associations with other genetic aberrations is unknown. PTEN mutations have been proposed as secondary mutations that follow NOTCH1-activating mutations and cause cellular resistance to γ-secretase inhibitors. Design and Methods The impact of PTEN, PI3K and AKT aberrations was studied in a genetically well-characterized pediatric T-cell leukemia patient cohort (n=146) treated on DCOG or COALL protocols. Results PTEN and AKT E17K aberrations were detected in 13% and 2% of patients, respectively. Defective PTEN-splicing was identified in incidental cases. Patients without PTEN protein but lacking exon-, splice-, promoter mutations or promoter hypermethylation were present. PTEN/AKT mutations were especially abundant in TAL- or LMO-rearranged leukemia but nearly absent in TLX3-rearranged patients (P=0.03), the opposite to that observed for NOTCH1-activating mutations. Most PTEN/AKT mutant patients either lacked NOTCH1-activating mutations (P=0.006) or had weak NOTCH1-activating mutations (P=0.011), and consequently expressed low intracellular NOTCH1, cMYC and MUSASHI levels. T-cell leukemia patients without PTEN/AKT and NOTCH1-activating mutations fared well, with a cumulative incidence of relapse of only 8% versus 35% for PTEN/AKT and/or NOTCH1-activated patients (P=0.005). Conclusions PI3K/AKT pathway aberrations are present in 18% of pediatric T-cell acute lymphoblastic leukemia patients. Absence of strong NOTCH1-activating mutations in these cases may explain cellular insensitivity to γ-secretase inhibitors. PMID:22491738

Calmodulin-mediated activation of Akt regulates survival of c-Myc-overexpressing mouse mammary carcinoma cells.

PubMed

Deb, Tushar B; Coticchia, Christine M; Dickson, Robert B

2004-09-10

c-Myc-overexpressing mammary epithelial cells are proapoptotic; their survival is strongly promoted by epidermal growth factor (EGF). We now demonstrate that EGF-induced Akt activation and survival in transgenic mouse mammary tumor virus-c-Myc mouse mammary carcinoma cells are both calcium/calmodulin-dependent. Akt activation is abolished by the phospholipase C-gamma inhibitor U-73122, by the intracellular calcium chelator BAPTA-AM, and by the specific calmodulin antagonist W-7. These results implicate calcium/calmodulin in the activation of Akt in these cells. In addition, Akt activation by serum and insulin is also inhibited by W-7. EGF-induced and calcium/calmodulin-mediated Akt activation occurs in both tumorigenic and non-tumorigenic mouse and human mammary epithelial cells, independent of their overexpression of c-Myc. These results imply that calcium/calmodulin may be a common regulator of Akt activation, irrespective of upstream receptor activator, mammalian species, and transformation status in mammary epithelial cells. However, only c-Myc-overexpressing mouse mammary carcinoma cells (but not normal mouse mammary epithelial cells) undergo apoptosis in the presence of the calmodulin antagonist W-7, indicating the vital selective role of calmodulin for survival of these cells. Calcium/calmodulin-regulated Akt activation is mediated directly by neither calmodulin kinases nor phosphatidylinositol 3-kinase (PI-3 kinase). Pharmacological inhibitors of calmodulin kinase kinase and calmodulin kinases II and III do not inhibit EGF-induced Akt activation, and calmodulin antagonist W-7 does not inhibit phosphotyrosine-associated PI-3 kinase activation. Akt is, however, co-immunoprecipitated with calmodulin in an EGF-dependent manner, which is inhibited by calmodulin antagonist W-7. We conclude that calmodulin may serve a vital regulatory function to direct the localization of Akt to the plasma membrane for its activation by PI-3 kinase.

PPARα induced NOS1 phosphorylation via PI3K/Akt in guinea pig antral mucous cells: NO-enhancement in Ca(2+)-regulated exocytosis.

PubMed

Tanaka, Saori; Hosogi, Shigekuni; Sawabe, Yukinori; Shimamoto, Chikao; Matsumura, Hitoshi; Inui, Toshio; Marunaka, Yoshinori; Nakahari, Takashi

2016-01-01

A PPARα (peroxisome proliferation activation receptor α) agonist (GW7647) activates nitric oxide synthase 1 (NOS1) to produce NO leading to cGMP accumulation in antral mucous cells. In this study, we examined how PPARα activates NOS1. The NO production stimulated by GW7647 was suppressed by inhibitors of PI3K (wortmannin) and Akt (AKT 1/2 Kinase Inhibitor, AKT-inh), although it was also suppressed by the inhibitors of PPARα (GW6471) and NOS1 (N-PLA). GW7647 enhanced the ACh (acetylcholine)-stimulated exocytosis (Ca(2+)-regulated exocytosis) mediated via NO, which was abolished by GW6471, N-PLA, wortmannin, and AKT-inh. The Western blotting revealed that GW7647 phosphorylates NOS1 via phosphorylation of PI3K/Akt in antral mucous cells. The immunofluorescence examinations demonstrated that PPARα existing with NOS1 co-localizes with PI3K and Akt in the cytoplasm of antral mucous cells. ACh alone and AACOCF3, an analogue of arachidonic acid (AA), induced the NOS1 phosphorylation via PI3K/Akt to produce NO, which was inhibited by GW6471. Since AA is a natural ligand for PPARα, ACh stimulates PPARα probably via AA. In conclusion, PPARα activates NOS1 via PI3K/Akt phosphorylation to produce NO in antral mucous cells during ACh stimulation.

TRPC3- and ETB receptor-mediated PI3K/AKT activation induces vasogenic edema formation following status epilepticus.

PubMed

Kim, Ji-Eun; Kang, Tae-Cheon

2017-10-01

Status epilepticus (SE, a prolonged seizure activity) is a high risk factor of developing vasogenic edema, which leads to secondary complications following SE. In the present study, we investigated whether transient receptor potential canonical channel-3 (TRPC3) may link vascular endothelial growth factor (VEGF) pathway to NFκB/ET B receptor axis in the rat piriform cortex during vasogenic edema formation. Following SE, TRPC3 and ET B receptor independently activated phosphatidylinositol 3 kinase (PI3K)/AKT/eNOS signaling pathway. SN50 (a NFκB inhibitor) attenuated the up-regulations of eNOS, TRPC3 and ET B receptor expressions following SE, accompanied by reductions in PI3K/AKT phosphorylations. Inhibition of SE-induced VEGF over-expression by leptomycin B also abrogated PI3K and AKT phosphorylations, but not TRPC3 expression. Wortmannin (a PI3K inhibitor) and 3CAI (an AKT inhibitor) effectively inhibited up-regulation of eNOS expressions and vasogenic edema lesion following SE. These findings indicate that PI3K/AKT may be common down-stream molecules for TRPC3- and ET B receptor signaling pathways during vasogenic edema formation. In addition, the present data demonstrate for the first time that TRPC3 may integrate VEGF- and NFκB-mediated vasogenic edema formation following SE. Thus, we suggest that PI3K/AKT signaling pathway may be one of considerable therapeutic targets for vasogenic edema. Copyright © 2017 Elsevier B.V. All rights reserved.

Hyperforin inhibits Akt1 kinase activity and promotes caspase-mediated apoptosis involving Bad and Noxa activation in human myeloid tumor cells.

PubMed

Merhi, Faten; Tang, Ruoping; Piedfer, Marion; Mathieu, Julie; Bombarda, Isabelle; Zaher, Murhaf; Kolb, Jean-Pierre; Billard, Christian; Bauvois, Brigitte

2011-01-01

The natural phloroglucinol hyperforin HF displays anti-inflammatory and anti-tumoral properties of potential pharmacological interest. Acute myeloid leukemia (AML) cells abnormally proliferate and escape apoptosis. Herein, the effects and mechanisms of purified HF on AML cell dysfunction were investigated in AML cell lines defining distinct AML subfamilies and primary AML cells cultured ex vivo. HF inhibited in a time- and concentration-dependent manner the growth of AML cell lines (U937, OCI-AML3, NB4, HL-60) by inducing apoptosis as evidenced by accumulation of sub-G1 population, phosphatidylserine externalization and DNA fragmentation. HF also induced apoptosis in primary AML blasts, whereas normal blood cells were not affected. The apoptotic process in U937 cells was accompanied by downregulation of anti-apoptotic Bcl-2, upregulation of pro-apoptotic Noxa, mitochondrial membrane depolarization, activation of procaspases and cleavage of the caspase substrate PARP-1. The general caspase inhibitor Z-VAD-fmk and the caspase-9- and -3-specific inhibitors, but not caspase-8 inhibitor, significantly attenuated apoptosis. HF-mediated apoptosis was associated with dephosphorylation of active Akt1 (at Ser(473)) and Akt1 substrate Bad (at Ser(136)) which activates Bad pro-apoptotic function. HF supppressed the kinase activity of Akt1, and combined treatment with the allosteric Akt1 inhibitor Akt-I-VIII significantly enhanced apoptosis of U937 cells. Our data provide new evidence that HF's pro-apoptotic effect in AML cells involved inhibition of Akt1 signaling, mitochondria and Bcl-2 members dysfunctions, and activation of procaspases -9/-3. Combined interruption of mitochondrial and Akt1 pathways by HF may have implications for AML treatment.

Hyperforin Inhibits Akt1 Kinase Activity and Promotes Caspase-Mediated Apoptosis Involving Bad and Noxa Activation in Human Myeloid Tumor Cells

PubMed Central

Merhi, Faten; Tang, Ruoping; Piedfer, Marion; Mathieu, Julie; Bombarda, Isabelle; Zaher, Murhaf; Kolb, Jean-Pierre; Billard, Christian; Bauvois, Brigitte

2011-01-01

Background The natural phloroglucinol hyperforin HF displays anti-inflammatory and anti-tumoral properties of potential pharmacological interest. Acute myeloid leukemia (AML) cells abnormally proliferate and escape apoptosis. Herein, the effects and mechanisms of purified HF on AML cell dysfunction were investigated in AML cell lines defining distinct AML subfamilies and primary AML cells cultured ex vivo. Methodology and Results HF inhibited in a time- and concentration-dependent manner the growth of AML cell lines (U937, OCI-AML3, NB4, HL-60) by inducing apoptosis as evidenced by accumulation of sub-G1 population, phosphatidylserine externalization and DNA fragmentation. HF also induced apoptosis in primary AML blasts, whereas normal blood cells were not affected. The apoptotic process in U937 cells was accompanied by downregulation of anti-apoptotic Bcl-2, upregulation of pro-apoptotic Noxa, mitochondrial membrane depolarization, activation of procaspases and cleavage of the caspase substrate PARP-1. The general caspase inhibitor Z-VAD-fmk and the caspase-9- and -3-specific inhibitors, but not caspase-8 inhibitor, significantly attenuated apoptosis. HF-mediated apoptosis was associated with dephosphorylation of active Akt1 (at Ser473) and Akt1 substrate Bad (at Ser136) which activates Bad pro-apoptotic function. HF supppressed the kinase activity of Akt1, and combined treatment with the allosteric Akt1 inhibitor Akt-I-VIII significantly enhanced apoptosis of U937 cells. Significance Our data provide new evidence that HF's pro-apoptotic effect in AML cells involved inhibition of Akt1 signaling, mitochondria and Bcl-2 members dysfunctions, and activation of procaspases -9/-3. Combined interruption of mitochondrial and Akt1 pathways by HF may have implications for AML treatment. PMID:21998731

Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A

NASA Technical Reports Server (NTRS)

Boo, Yong Chool; Sorescu, George; Boyd, Nolan; Shiojima, Ichiro; Walsh, Kenneth; Du, Jie; Jo, Hanjoong

2002-01-01

Recently, we have shown that shear stress stimulates NO(*) production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574-1581). Akt has been believed to regulate shear-dependent production of NO(*) by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser(1179) residue (eNOS-S(1179)), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt(AA) or Akt(AAA)) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S(1179). Here, we show that shear-dependent phosphorylation of eNOS-S(1179) is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt(AA) or Akt(AAA) in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S(1179) if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt(AA) inhibited shear-dependent NO(*) production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S(1179) and NO(*) production. In contrast, H89 did not inhibit phosphorylation of eNOS-S(1179) induced by expressing a constitutively active Akt mutant (Akt(Myr)) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S(1179) within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S(1179) in a PKA-dependent, but Aktindependent manner, whereas the NO(*) production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction

Cancer Associated E17K Mutation Causes Rapid Conformational Drift in AKT1 Pleckstrin Homology (PH) Domain

PubMed Central

Kumar, Ambuj; Purohit, Rituraj

2013-01-01

Background AKT1 (v-akt murine thymoma viral oncogene homologue 1) kinase is one of the most frequently activated proliferated and survival pathway of cancer. Recently it has been shown that E17K mutation in the Pleckstrin Homology (PH) domain of AKT1 protein leads to cancer by amplifying the phosphorylation and membrane localization of protein. The mutant has shown resistance to AKT1/2 inhibitor VIII drug molecule. In this study we have demonstrated the detailed structural and molecular consequences associated with the activity regulation of mutant protein. Methods The docking score exhibited significant loss in the interaction affinity to AKT1/2 inhibitor VIII drug molecule. Furthermore, the molecular dynamics simulation studies presented an evidence of rapid conformational drift observed in mutant structure. Results There was no stability loss in mutant as compared to native structure and the major cation–π interactions were also shown to be retained. Moreover, the active residues involved in membrane localization of protein exhibited significant rise in NHbonds formation in mutant. The rise in NHbond formation in active residues accounts for the 4-fold increase in the membrane localization potential of protein. Conclusion The overall result suggested that, although the mutation did not induce any stability loss in structure, the associated pathological consequences might have occurred due to the rapid conformational drifts observed in the mutant AKT1 PH domain. General Significance The methodology implemented and the results obtained in this work will facilitate in determining the core molecular mechanisms of cancer-associated mutations and in designing their potential drug inhibitors. PMID:23741320

Apatinib Inhibits Angiogenesis Via Suppressing Akt/GSK3β/ANG Signaling Pathway in Anaplastic Thyroid Cancer.

PubMed

Jin, Zhijian; Cheng, Xi; Feng, Haoran; Kuang, Jie; Yang, Weiping; Peng, Chenghong; Shen, Baiyong; Qiu, Weihua

2017-01-01

Anaplastic thyroid carcinoma (ATC) is one of the most lethal human malignancies, and there is no efficient method to slow its process. Apatinib, a novel tyrosine kinase inhibitor (TKI), has been confirmed for its efficacy and safety in the treatment of advanced gastric carcinoma patients. However, the effects of Apatinib in ATC are still unknown. In this study, we explored the effects and mechanisms of Apatinib on tumor growth and angiogenesis in vitro and in vitro in ATC cells. Angiogenesis antibodies array was utilized to detect the expression of angiogenesis-related genes after Apatinib treatment in ATC cells. In addition, we used Akt activator, Akt inhibitor and GSK3β inhibitor to further study the mechanism for how Apatinib suppressed angiogenesis. Apatinib treatment could suppress the growth of ATC cells in a dose- and time-dependent manner via inducing apoptosis and blocking cell cycle progression at G0/G1 phase. Moreover, Apatinib treatment decreased the expression of angiogenin (ANG) and inhibited angiogenesis of ATC cells in vitro and in vitro. We further confirmed that recombinant human ANG (rhANG) significantly abrogated Apatinib-mediated anti-angiogenic ability in ATC cells. Additionally, Apatinib treatment decreased the level of p-Akt and p-GSK3β. Moreover, the Apatinib-mediated decrease of ANG and anti-angiogenic ability were partly reversed when an Akt activator, SC79, was administered. Furthermore, the anti-angiogenic ability of Apatinib can be enhanced in the presence of Akt inhibitor, and the inhibition of GSK3β attenuated the anti-angiogenic ability of Apatinib. Our results demonstrated that Apatinib treatment inhibited tumor growth, and Apatinib-induced suppression of Akt/GSK3β/ANG signaling pathway may play an important role in the inhibition of angiogenesis in ATC, supporting a potential therapeutic approach for using Apatinib in the treatment of ATC. © 2017 The Author(s). Published by S. Karger AG, Basel.

Akt activation enhances ribosomal RNA synthesis through casein kinase II and TIF-IA.

PubMed

Nguyen, Le Xuan Truong; Mitchell, Beverly S

2013-12-17

Transcription initiation factor I (TIF-IA) plays an essential role in regulating ribosomal RNA (rRNA) synthesis by tethering RNA polymerase I (Pol I) to the rDNA promoter. We have found that activated Akt enhances rRNA synthesis through the phosphorylation of casein kinase IIα (CK2α) on a threonine residue near its N terminus. CK2 in turn phosphorylates TIF-IA, thereby increasing rDNA transcription. Activated Akt also stabilizes TIF-IA, induces its translocation to the nucleolus, and enhances its interaction with Pol I. Treatment with AZD8055, an inhibitor of both Akt and mammalian target of rapamycin phosphorylation, but not with rapamycin, disrupts Akt-mediated TIF-IA stability, translocation, and activity. These data support a model in which activated Akt enhances rRNA synthesis both by preventing TIF-IA degradation and phosphorylating CK2α, which in turn phosphorylates TIF-IA. This model provides an explanation for the ability of activated Akt to promote cell proliferation and, potentially, transformation.

A phase I trial of the human double minute 2 inhibitor (MK-8242) in patients with refractory/recurrent acute myelogenous leukemia (AML)

PubMed Central

Ravandi, Farhad; Gojo, Ivana; Patnaik, Mrinal M.; Minden, Mark D.; Kantarjian, Hagop; Johnson-Levonas, Amy O.; Fancourt, Craig; Lam, Raymond; Jones, Mary Beth; Knox, Clayton D.; Rose, Shelonitda; Patel, Payal Shah; Tibes, Raoul

2017-01-01

Objective Evaluate safety/tolerability/efficacy of MK-8242 in subjects with refractory/recurrent AML. Methods MK-8242 was dosed p.o. QD (30–250 mg) or BID (120–250 mg) for 7on/7off in 28-day cycle. Dosing was modified to 7on/14off, in 21-day cycle (210 or 300 mg BID). Results 26 subjects enrolled (24 evaluable for response); 5/26 discontinued due to AEs. There were 7 deaths; 1 (fungal pneumonia due to marrow aplasia) possibly drug-related. With the 7on/7off regimen, 2 subjects had DLTs in the 250 mg BID group (both bone marrow failure and prolonged cytopenia). With the 7on/14off, no DLTs were observed in 210 mg BID or 300 mg BID (doses >300 mg not tested). Best responses were: 1/24 PR (11 weeks;120 mg QD, 7on/7off); 1/24 CRi (2 weeks;210 mg BID, 7on/14off); 1/24 morphologic leukemia-free state (4 weeks; 250 mg BID, 7on/7off). PK on Day7 at 210 mg BID revealed AUC0-12hr 8.7 μM*hr, Cmax 1.5 μM (n=5, Tmax, 2–6 hr), T1/2 7.9 hr, CLss/F 28.8 L/hr, and Vss/F 317 L. Conclusions The 7on/14off regimen showed a more favorable safety profile; no MTD was established. Efficacy was seen using both regimens providing impetus for further study of HDM2 inhibitors in subjects with AML. PMID:27544076

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.

Inhibition of multidrug/xenobiotic resistance transporter by MK571 improves dye (Fura 2) accumulation in crustacean tissues from lobster, shrimp, and isopod.

PubMed

Lüders, Ann-Katrin; Saborowski, Reinhard; Bickmeyer, Ulf

2009-09-01

Multidrug/xenobiotic resistance transporters are present in living organisms as a first line defence system against small, potentially harmful molecules from the environment or from internal metabolic reactions. Multidrug resistance associated proteins (MRP) are one type of ATP-Binding-Cassette (ABC) transporters, which also transport dyes such as Fura 2, a calcium chelating fluorescence indicator. The specific MRP inhibitor MK571 was used to investigate the fluorescence intensity of cells in tissues of the brain and the midgut gland of the crustaceans Homarus gammarus (lobster), Crangon crangon (brown shrimp) and Idotea emarginata (isopod) during incubation with Fura 2AM (1 microM). In the presence of the inhibitor MK571 (50 microM), the fluorescence of brain tissue significantly increased in all of the three species. The midgut gland of H. gammarus showed a significant increase of fluorescence, whereas there was no effect in the midgut glands of C. crangon and I. baltica. The half maximal concentration of MK571 was 50 microM as measured in the midgut gland of H. gammarus. In conclusion, MRP transporters are present in the three investigated crustacean nervous systems. Using the midgut glands of the three species, only in H. gammarus MK571 inhibited dye extrusion, indicating species-specific differences of transporter systems, their specificity, or tissue specific expression.

Id-1 activation of PI3K/Akt/NFkappaB signaling pathway and its significance in promoting survival of esophageal cancer cells.

PubMed

Li, Bin; Cheung, Pak Yan; Wang, Xianghong; Tsao, Sai Wah; Ling, Ming Tat; Wong, Yong Chuan; Cheung, Annie L M

2007-11-01

Inhibitor of differentiation or DNA binding (Id-1) is a helix-loop-helix protein that is over-expressed in many types of cancer including esophageal cancer. This study aims to investigate its effects on the phosphatidylinositol-3-kinase (PI3K)/Akt/ nuclear factor kappa B (NFkappaB) signaling pathway and the significance in protecting esophageal cancer cells against apoptosis. We found elevated expression of phosphorylated forms of Akt, glycogen synthase kinase 3beta and inhibitor of kappa B, as well as increased nuclear translocation of NFkappaB subunit p65 and NFkappaB DNA-binding activity, in esophageal cancer cells with stable ectopic Id-1 expression. Transient transfection of Id-1 into HEK293 cells confirmed activation of PI3K/Akt/NFkappaB signaling and the effects were counteracted by the PI3K inhibitor LY294002. Treatment with tumor necrosis factor-alpha (TNF-alpha) elicited a significantly weaker apoptotic response, following a marked and sustained activation of Akt and NFkappaB in the Id-1-over-expressing cells, compared with the vector control. The effects of Id-1 on the PI3K/Akt/NFkappaB signaling pathway and apoptosis were reversed in esophageal cancer cells transfected with siRNA against Id-1. In addition, inhibition of PI3K or NFkappaB signaling using the PI3K inhibitor LY294002 or the NFkappaB inhibitor Bay11-7082 increased the sensitivity of Id-1-over-expressing esophageal cancer cells to TNF-alpha-induced apoptosis. Our results provide the first evidence that Id-1 induces the activation of PI3K/Akt/NFkappaB signaling pathway, and protects esophageal cancer cells from TNF-alpha-induced apoptosis in vitro. Inactivation of Id-1 may provide us with a novel strategy to improve the treatment and survival of patients with esophageal cancer.

Suppression of transforming growth factor-beta-induced apoptosis through a phosphatidylinositol 3-kinase/Akt-dependent pathway.

PubMed

Chen, R H; Su, Y H; Chuang, R L; Chang, T Y

1998-10-15

Insulin and insulin receptor substrate 1 (IRS-1) are capable of protecting liver cells from apoptosis induced by transforming growth factor-beta1 (TGF-beta). The Ras/mitogen-activated protein kinase (MAP kinase) and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways are both activated upon insulin stimulation and can protect against apoptosis under certain circumstances. We investigated which of these pathways is responsible for the protective effect of insulin on TGF-beta-induced apoptosis. An activated Ras, although elicited a strong mitogenic effect, could not protect Hep3B cells from TGF-beta-induced apoptosis. Furthermore, PD98059, a selective inhibitor of MEK, did not suppress the antiapoptotic effect of insulin. In contrast, the PI 3-kinase inhibitor, LY294002, efficiently blocked the effect of insulin. Protection against TGF-beta-induced apoptosis conferred by PI 3-kinase was further verified by stable transfection of an activated PI 3-kinase. Downstream targets of PI 3-kinase involved in this protection was further investigated. An activated Akt mimicked the antiapoptotic effect of insulin, whereas a dominant-negative Akt inhibited such effect. However, rapamycin, the p70S6 kinase inhibitor, had no effect on the protectivity of insulin against TGF-beta-induced apoptosis, suggesting that the antiapoptotic target of PI 3-kinase/Akt pathway is independent or lies upstream of the p70S6 kinase. The mechanism by which PI 3-kinase/Akt pathway interferes with the apoptotic signaling of TGF-beta was explored. Activation of PI 3-kinase did not lead to a suppression of Smad hetero-oligomerization or nuclear translocation but blocked TGF-beta-induced caspase-3-like activity. In summary, the PI 3-kinase/Akt pathway, but not the Ras/MAP kinase pathway, protects against TGF-beta-induced apoptosis by inhibiting a step downstream of Smad but upstream of caspase-3.

Disruption of PH–kinase domain interactions leads to oncogenic activation of AKT in human cancers

PubMed Central

Parikh, Chaitali; Janakiraman, Vasantharajan; Wu, Wen-I; Foo, Catherine K.; Kljavin, Noelyn M.; Chaudhuri, Subhra; Stawiski, Eric; Lee, Brian; Lin, Jie; Li, Hong; Lorenzo, Maria N.; Yuan, Wenlin; Guillory, Joseph; Jackson, Marlena; Rondon, Jesus; Franke, Yvonne; Bowman, Krista K.; Sagolla, Meredith; Stinson, Jeremy; Wu, Thomas D.; Wu, Jiansheng; Stokoe, David; Stern, Howard M.; Brandhuber, Barbara J.; Lin, Kui; Skelton, Nicholas J.; Seshagiri, Somasekar

2012-01-01

The protein kinase v-akt murine thymoma viral oncogene homolog (AKT), a key regulator of cell survival and proliferation, is frequently hyperactivated in human cancers. Intramolecular pleckstrin homology (PH) domain–kinase domain (KD) interactions are important in maintaining AKT in an inactive state. AKT activation proceeds after a conformational change that dislodges the PH from the KD. To understand these autoinhibitory interactions, we generated mutations at the PH–KD interface and found that most of them lead to constitutive activation of AKT. Such mutations are likely another mechanism by which activation may occur in human cancers and other diseases. In support of this likelihood, we found somatic mutations in AKT1 at the PH–KD interface that have not been previously described in human cancers. Furthermore, we show that the AKT1 somatic mutants are constitutively active, leading to oncogenic signaling. Additionally, our studies show that the AKT1 mutants are not effectively inhibited by allosteric AKT inhibitors, consistent with the requirement for an intact PH–KD interface for allosteric inhibition. These results have important implications for therapeutic intervention in patients with AKT mutations at the PH–KD interface. PMID:23134728

An essential role for the Id1/PI3K/Akt/NFkB/survivin signalling pathway in promoting the proliferation of endothelial progenitor cells in vitro.

PubMed

Li, Wei; Wang, Hang; Kuang, Chun-Yan; Zhu, Jin-Kun; Yu, Yang; Qin, Zhe-Xue; Liu, Jie; Huang, Lan

2012-04-01

The enhancement of re-endothelialisation is a critical therapeutic option for repairing injured blood vessels. Endothelial progenitor cells (EPCs) are the major source of cells that participate in endothelium repair and contribute to re-endothelialisation by reducing neointima formation after vascular injury. The over-expression of the inhibitor of differentiation or DNA binding 1 (Id1) significantly improved EPC proliferation. This study aimed to investigate the effects of Id1 on the phosphatidylinositol-3-kinase (PI3K)/Akt/nuclear factor kappa B (NFκB)/survivin signalling pathway and its significance in promoting EPC proliferation in vitro. Spleen-derived EPCs were cultured as previously described. Id1 was presented at low levels in EPCs, and was rapidly up-regulated by stimulation with vascular endothelial growth factor. We demonstrated that transient transfection of Id1 into EPCs activated the PI3K/Akt/NFκB/survivin signalling pathway and promoted EPC proliferation. The proliferation of EPCs was extensively inhibited by silencing of endogenous Id1, and knockdown of Id1 expression led to suppression of PI3K/Akt/NFκB/survivin signalling pathway in EPCs. In addition, blockade by the PI3K-specific inhibitor LY294002, Akt inhibitor, the NFκB inhibitor BAY 11-7082, the survivin inhibitor Curcumin, or the survivin inhibitor YM155 reduced the effects of Id1 transfection. These results suggest that the Id1/PI3K/Akt/NFκB/survivin signalling pathway plays a critical role in EPC proliferation. The Id1/PI3K/Akt/NFκB/survivin signalling pathway may represent a novel therapeutic target in the prevention of restenosis after vascular injury.

Development of highly sensitive cell-based AKT kinase ELISA for monitoring PI3K beta activity and compound efficacy.

PubMed

Yanamandra, Mahesh; Kole, Labanyamoy; Giri, Archana; Mitra, Sayan

2017-01-01

Phosphatidylinositol-3 kinase (PI3K) pathway regulates multiple cellular functions involving cell survival, growth, motility proliferation, apoptosis, and adhesion. These are deregulated in various diseases such as cancer, atherosclerosis, and inflammation. PI3Ks phosphorylate phosphatidylinositol 4,5-biphosphate (PIP2) yielding phosphatidylinositol 3, 4, 5 triphosphate (PIP3) which in turn activate AKT kinase (serine/threonine kinase), the central enzyme in regulation of metabolic functions. Due to their implications in disease pathophysiology, PI3K/AKT inhibitors became attractive targets for pharmaceutical industries. In order to assess the functional response generated by PI3K inhibitors, an appropriate cell-based screening system is essential in any screening cascade. Here we report the development of highly sensitive in-vitro cell-based kinase ELISA which quantifies the phosphorylated AKT kinase (serine 473) and total AKT kinase directly within the cells upon compound treatment. PI3Kβ overexpressing NIH3T3 cells stimulated by lysophosphatidic acid was used for PI3K/Akt pathway activation. Assay performance reliability and robustness were determined by percentage coefficient of variation (%CV) and Z factor which demonstrated an excellent agreement with assay guidelines. This 96-well plate medium throughput assay methodology was used to screen novel molecules and proved a commendable tool to study the mechanism of action property and target engagement of novel PI3K inhibitors in drug discovery.

The Atypical Antipsychotic Agent, Clozapine, Protects Against Corticosterone-Induced Death of PC12 Cells by Regulating the Akt/FoxO3a Signaling Pathway.

PubMed

Zeng, Zhiwen; Wang, Xue; Bhardwaj, Sanjeev K; Zhou, Xuanhe; Little, Peter J; Quirion, Remi; Srivastava, Lalit K; Zheng, Wenhua

2017-07-01

Schizophrenia is one of the most severe psychiatric disorders. Increasing evidence implicates that neurodegeneration is a component of schizophrenia pathology and some atypical antipsychotics are neuroprotective and successful in slowing the progressive morphological brain changes. As an antipsychotic agent, clozapine has superior and unique effects, but the intracellular signaling pathways that mediate clozapine action remain to be elucidated. The phosphatidylinositol-3-kinase/protein kinase B/Forkhead box O3 (PI3K/Akt/FoxO3a) pathway is crucial for neuronal survival. However, little information is available regarding this pathway with clozapine. In the present study, we investigated the protective effect of clozapine on the PC12 cells against corticosterone toxicity. Our results showed that corticosterone decreases the phosphorylation of Akt and FoxO3a, leading to the nuclear localization of FoxO3a and the apoptosis of PC12 cells, while clozapine concentration dependently protected PC12 cells against corticosterone insult. Pathway inhibitors studies displayed that the protective effect of clozapine was reversed by LY294002 and wortmannin, two PI3K inhibitors, or Akt inhibitor VIII although several other inhibitors had no effect. The shRNA knockdown results displayed that downregulated Akt1 or FoxO3a attenuated the protective effect of clozapine. Western blot analyses revealed that clozapine induced the phosphorylation of Akt and FoxO3a by the PI3K/Akt pathway and reversed the reduction of the phosphorylated Akt and FoxO3a and the nuclear translocation of FoxO3a evoked by corticosterone. Together, our data indicates that clozapine protects PC12 cells against corticosterone-induced cell death by modulating activity of the PI3K/Akt/FoxO3a pathway.

LPS Increases 5-LO Expression on Monocytes via an Activation of Akt-Sp1/NF-κB Pathways.

PubMed

Lee, Seung Jin; Seo, Kyo Won; Kim, Chi Dae

2015-05-01

5-Lipoxygenase (5-LO) plays a pivotal role in the progression of atherosclerosis. Therefore, this study investigated the molecular mechanisms involved in 5-LO expression on monocytes induced by LPS. Stimulation of THP-1 monocytes with LPS (0~3 µg/ml) increased 5-LO promoter activity and 5-LO protein expression in a concentration-dependent manner. LPS-induced 5-LO expression was blocked by pharmacological inhibition of the Akt pathway, but not by inhibitors of MAPK pathways including the ERK, JNK, and p38 MAPK pathways. In line with these results, LPS increased the phosphorylation of Akt, suggesting a role for the Akt pathway in LPS-induced 5-LO expression. In a promoter activity assay conducted to identify transcription factors, both Sp1 and NF-κB were found to play central roles in 5-LO expression in LPS-treated monocytes. The LPS-enhanced activities of Sp1 and NF-κB were attenuated by an Akt inhibitor. Moreover, the LPS-enhanced phosphorylation of Akt was significantly attenuated in cells pretreated with an anti-TLR4 antibody. Taken together, 5-LO expression in LPS-stimulated monocytes is regulated at the transcriptional level via TLR4/Akt-mediated activations of Sp1 and NF-κB pathways in monocytes.

Imatinib-mediated inactivation of Akt regulates ABCG2 function in head and neck squamous cell carcinoma.

PubMed

Chu, Theresa S; Chen, Jocelyn S; Lopez, Jay Patrick; Pardo, Francisco S; Aguilera, Joseph; Ongkeko, Weg M

2008-09-01

To investigate whether the mechanism for the reversal of ABCG2 (also known as ABCP, MXR, and BCRP)-mediated drug resistance by imatinib mesylate (Gleevec, STI571; Novartis Pharmaceuticals Corp, East Hanover, New Jersey) is caused by the downregulation of Akt kinase. The adenosine triphosphatase-binding cassette protein ABCG2 has been suggested to be involved in the resistance against various anticancer drugs. Recent studies show that imatinib reverses ABCG2-mediated drug resistance to topotecan hydrochloride and SN-38. In addition, we have previously reported that imatinib downregulates Akt kinase activity, which is elevated in head and neck squamous cell carcinoma. Flow cytometric analysis was used to determine the levels of drug or dye extrusion from the cells. We used Akt kinase inhibitors, transfection with short interfering RNA (siRNA) Akt, and the tyrosine kinase inhibitor imatinib to show that these treatments decreased the side population by 50% to 70% in Hoechst 33342 extrusion studies. Doxorubicin hydrochloride extrusion experiments also demonstrated 20% to 26% decrease in doxorubicin efflux on cells treated with imatinib, 1L6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, and transfection with siRNA Akt. With Western blot and immunofluorescence experiments, our data suggest that ABCG2 translocation is the mechanism by which imatinib and Akt regulate drug resistance. Clonogenic survival assays performed with imatinib-treated cells resulted in a dose-dependent decrease in cell survival compared with the control population. Our findings demonstrate that imatinib confers greater doxorubicin retention, presumably via inhibition of Akt, which regulates ABCG2 function.

Targeting the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway: an emerging treatment strategy for squamous cell lung carcinoma.

PubMed

Beck, Joseph Thaddeus; Ismail, Amen; Tolomeo, Christina

2014-09-01

Squamous cell lung carcinoma accounts for approximately 30% of all non-small cell lung cancers (NSCLCs). Despite progress in the understanding of the biology of cancer, cytotoxic chemotherapy remains the standard of care for patients with squamous cell lung carcinoma, but the prognosis is generally poor. The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is one of the most commonly activated signaling pathways in cancer, leading to cell proliferation, survival, and differentiation. It has therefore become a major focus of clinical research. Various alterations in the PI3K/AKT/mTOR pathway have been identified in squamous cell lung carcinoma and a number of agents targeting these alterations are in clinical development for use as single agents and in combination with other targeted and conventional treatments. These include pan-PI3K inhibitors, isoform-specific PI3K inhibitors, AKT inhibitors, mTOR inhibitors, and dual PI3K/mTOR inhibitors. These agents have demonstrated antitumor activity in preclinical models of NSCLC and preliminary clinical evidence is also available for some agents. This review will discuss the role of the PI3K/AKT/mTOR pathway in cancer and how the discovery of genetic alterations in this pathway in patients with squamous cell lung carcinoma can inform the development of targeted therapies for this disease. An overview of ongoing clinical trials investigating PI3K/AKT/mTOR pathway inhibitors in squamous cell lung carcinoma will also be included. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterization of the novel GlyT1 PET tracer [18F]MK-6577 in humans.

PubMed

Joshi, Aniket D; Sanabria-Bohórquez, Sandra M; Bormans, Guy; Koole, Michel; De Hoon, Jan; Van Hecken, Anne; Depre, Marleen; De Lepeleire, Inge; Van Laere, Koen; Sur, Cyrille; Hamill, Terence G

2015-01-01

Decreased glutamatergic neurotransmission is hypothesized to be involved in the pathophysiology of schizophrenia. Inhibition of glycine transporter Type-1 (GlyT1) reuptake is expected to increase the glutamatergic neurotransmission and may serve as treatment for cognitive and negative symptoms of schizophrenia. In this article, we present human data from a novel GlyT1 PET tracer, [(18) F]MK-6577. In the process of developing a GlyT1 inhibitor therapeutic, a PET tracer can assist in determining the dose with a high probability of sufficiently testing the mechanism of action. This article reports the human PET studies with [(18) F]MK-6577 for measuring GlyT1 receptor availability at baseline in normal human subjects and occupancy with a GlyT1 inhibitor, MK-2637. Studies were also performed to measure radiation burden and the baseline test-retest (T-RT) variability of the tracer. The effective dose from sequential whole-body dosimetry scans in three male subjects was estimated to be 24.5 ± 2.9 µSV/MBq (mean ± SD). The time-activity curves from T-RT scans modeled satisfactorily using a two tissue compartmental model. The tracer uptake was highest in the pons (VT  = 6.7 ± 0.9, BPND  = 4.1 ± 0.43) and lowest in the cortex (VT  = 2.1 ± 0.5, BPND  = 0.60 ± 0.23). VT T-RT variability measured in three subjects was <12% on average. The occupancy scans performed in a cohort of 15 subjects indicated absence of a reference region. The in vivo potency (Occ50 ) of MK-2637 was determined using two methods: A: Lassen plot with a population input function (Occ50  = 106 nM, SE = 20 nM) and B: pseudo reference tissue model using cortex as the pseudo reference region (Occ50  = 141 nM, SE = 21 nM). © 2014 Wiley Periodicals, Inc.

PI3K/Akt inhibitor LY294002 potentiates homoharringtonine antimyeloma activity in myeloma cells adhered to stromal cells and in SCID mouse xenograft.

PubMed

Chen, Ping; Wen, Xiaofang; Wang, Bin; Hou, Diyu; Zou, Hong; Yuan, Qin; Yang, Hui; Xie, Jieqiong; Huang, Huifang

2018-05-01

Homoharringtonine (HHT) is a known anti-leukemia drug that inhibits multiple myeloma (MM) cells both in vitro and in vivo. Our prior study demonstrated that the potency of HHT in MM cells was compromised significantly when myeloma cells were co-cultured with BM stromal cells. This study aimed to investigate whether PI3K/Akt inhibitor LY294002 could potentiate the antimyeloma activity of HHT against MM cells adhered to BM stromal cells and in vivo xenograft models. A co-culture system composed of MM cells and human stromal cells was employed to mimic MM cells in bone marrow niche. The inhibitory and pro-apoptotic effect of HHT and LY294002 was determined by CCK-8 assay or flow cytometry. Expression of PI3K/Akt signaling molecules and anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1) was assessed by western blot analysis and/or reverse transcription real-time quantitative PCR (RT-qPCR). MM xenografts were used to evaluate antitumor effect of combined therapy with HHT and LY294002. Adhesion to BM stromal cells rendered MM cells resistant to HHT whereas silencing Mcl-1 partly reversed the resistance. LY294002 induced apoptosis in MM cells and potentiated the antimyeloma effects of HHT by inhibiting the PI3K/Akt signal pathway which was abnormally activated during adhesion. LY294002 also enhanced the antimyeloma effect of HHT in in vivo xenograft models. These findings suggest that activation of PI3K/Akt signal pathway was responsible for the resistance to HHT in MM cells adhered to stromal cells. LY294002 can potentiate the antimyeloma activity of HHT both in vitro and in vivo, which may represent a new clinical treatment in MM.

Propofol mediates signal transducer and activator of transcription 3 activation and crosstalk with phosphoinositide 3-kinase/AKT.

PubMed

Shravah, Jayant; Wang, Baohua; Pavlovic, Marijana; Kumar, Ujendra; Chen, David Dy; Luo, Honglin; Ansley, David M

2014-01-01

We previously demonstrated that propofol, an intravenous anesthetic with anti-oxidative properties, activated the phosphoinositide 3-kinase (PI3K)/AKT pathway to increase the expression of B cell lymphoma (Bcl)-2 and, therefore the anti-apoptotic potential on cardiomyocytes. Here, we wanted to determine if propofol can also activate the Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3 pathway, another branch of cardioprotective signaling. The cellular response of nuclear factor kappa B (NFκB) and STAT3 was also evaluated. Cardiac H9c2 cells were treated by propofol alone or in combination with pretreatment by inhibitors for JAK2/STAT3 or PI3K/AKT pathway. STAT3 and AKT phosphorylation, and STAT3 translocation were measured by western blotting and immunofluorescence staining, respectively. Propofol treatment significantly increased STAT3 phosphorylation at both tyrosine 705 and serine 727 residues. Sustained early phosphorylation of STAT3 was observed with 25~75 μM propofol at 10 and 30 min. Nuclear translocation of STAT3 was seen at 4 h after treatment with 50 μM propofol. In cultured H9c2 cells, we further demonstrated that propofol-induced STAT3 phosphorylation was reduced by pretreatment with PI3K/AKT pathway inhibitors wortmannin or API-2. Conversely, pretreatment with JAK2/STAT3 pathway inhibitor AG490 or stattic inhibited propofol-induced AKT phosphorylation. In addition, propofol induced NFκB p65 subunit perinuclear translocation. Inhibition or knockdown of STAT3 was associated with increased levels of the NFκB p65 subunit. Our results suggest that propofol induces an adaptive response by dual activation and crosstalk of cytoprotective PI3K/AKT and JAK2/STAT3 pathways. Rationale to apply propofol clinically as a preemptive cardioprotectant during cardiac surgery is supported by our findings.

Acute Ethanol Inhibition of γ Oscillations Is Mediated by Akt and GSK3β

PubMed Central

Wang, JianGang; Zhao, JingXi; Liu, ZhiHua; Guo, FangLi; Wang, Yali; Wang, Xiaofang; Zhang, RuiLing; Vreugdenhil, Martin; Lu, Chengbiao

2016-01-01

Hippocampal network oscillations at gamma band frequency (γ, 30–80 Hz) are closely associated with higher brain functions such as learning and memory. Acute ethanol exposure at intoxicating concentrations (≥50 mM) impairs cognitive function. This study aimed to determine the effects and the mechanisms of acute ethanol exposure on γ oscillations in an in vitro model. Ethanol (25–100 mM) suppressed kainate-induced γ oscillations in CA3 area of the rat hippocampal slices, in a concentration-dependent, reversible manner. The ethanol-induced suppression was reduced by the D1R antagonist SCH23390 or the PKA inhibitor H89, was prevented by the Akt inhibitor triciribine or the GSk3β inhibitor SB415286, was enhanced by the NMDA receptor antagonist D-AP5, but was not affected by the MAPK inhibitor U0126 or PI3K inhibitor wortmanin. Our results indicate that the intracellular kinases Akt and GSk3β play a critical role in the ethanol-induced suppression of γ oscillations and reveal new cellular pathways involved in the ethanol-induced cognitive impairment. PMID:27582689

A phase 2 study of ofatumumab (Arzerra®) in combination with a pan-AKT inhibitor (afuresertib) in previously treated patients with chronic lymphocytic leukemia (CLL).

PubMed

Chen, Christine I; Paul, Harminder; Le, Lisa W; Wei, Ellen N; Snitzler, Susi; Wang, Trina; Levina, Olga; Kakar, Sumeet; Lau, Anthea; Queau, Michelle; Johnston, James B; Smith, Deborah A; Trudel, Suzanne

2018-06-19

AKT plays a centralized role in tumor proliferation and survival and is aberrantly activated in chronic lymphocytic leukemia (CLL). In this phase 2 trial, 30 relapsed/refractory CLL patients were treated with combination afuresertib, a novel oral AKT inhibitor, and ofatumumab for 6 months, followed by afuresertib maintenance for 12 months. We aimed to achieve deeper and more durable responses, without requiring long-term continuous treatment. Treatment was generally well tolerated but respiratory infections were common, with 18% severe requiring hospitalization. Hematologic toxicities were manageable (grade 3-4 neutropenia 39%). At a median follow-up of 13.4 months, overall responses were 50% (complete responses 3.6%). Median progression-free survival was 8.5 months and overall survival 34.8 months. Combination therapy with ofatumumab and afuresertib is active and well tolerated, but does not appear to lead to durable responses and may not provide additional benefit over single-agent ofatumumab in relapsed/refractory CLL. Novel agent combinations are currently undergoing intense investigation.

Halofuginone inhibits Smad3 phosphorylation via the PI3K/Akt and MAPK/ERK pathways in muscle cells: Effect on myotube fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roffe, Suzy; Hagai, Yosey; Institute of Animal Sciences, Volcani Center, Bet Dagan 50250

2010-04-01

Halofuginone, a novel inhibitor of Smad3 phosphorylation, has been shown to inhibit muscle fibrosis and to improve cardiac and skeletal muscle functions in the mdx mouse model of Duchenne muscular dystrophy. Here, we demonstrate that halofuginone promotes the phosphorylation of Akt and mitogen-activated protein kinase (MAPK) family members in a C2 muscle cell line and in primary myoblasts derived from wild-type and mdx mice diaphragms. Halofuginone enhanced the association of phosphorylated Akt and MAPK/extracellular signal-regulated protein kinase (ERK) with the non-phosphorylated form of Smad3, accompanied by a reduction in Smad3 phosphorylation levels. This reduction was reversed by inhibitors of themore » phosphoinositide 3'-kinase/Akt (PI3K/Akt) and MAPK/ERK pathways, suggesting their specific role in mediating halofuginone's inhibitory effect on Smad3 phosphorylation. Halofuginone enhanced Akt, MAPK/ERK and p38 MAPK phosphorylation and inhibited Smad3 phosphorylation in myotubes, all of which are crucial for myotube fusion. In addition, halofuginone increased the association Akt and MAPK/ERK with Smad3. As a consequence, halofuginone promoted myotube fusion, as reflected by an increased percentage of C2 and mdx myotubes containing high numbers of nuclei, and this was reversed by specific inhibitors of the PI3K and MAPK/ERK pathways. Together, the data suggest a role, either direct or via inhibition of Smad3 phosphorylation, for Akt or MAPK/ERK in halofuginone-enhanced myotube fusion, a feature which is crucial to improving muscle function in muscular dystrophies.« less

Effect of PPARG on AGEs-induced AKT/MTOR signaling-associated human chondrocytes autophagy.

PubMed

Wang, Zhao-Jun; Zhang, Hai-Bin; Chen, Cheng; Huang, Hao; Liang, Jian-Xia

2018-02-17

Accumulation of advanced glycation end products (AGEs) in articular cartilage is thought to represent a major risk factor for osteoarthritis development. In this study we aimed to probe the role of AGEs in human chondrocytes and to determine the impact of the peroxisome proliferator-activated receptor-γ (PPARG) on AGEs-induced cell autophagy. Cell viability was measured after human chondrocytes were treated with different concentrations of AGEs with or without the PPARG inhibitor, T0070907, or agonist, pioglitazone. Autophagy activation markers (MAP2LC3, BECN1 and SQSTM1/P62), expression of PPARG and the phosphorylation levels of Akt/MTOR were determined by Western blotting; autophagosome formation was analyzed by transmission electron microscopy (TEM); autophagic flux was detected with mRFP-GFP-LC3 tandem construct. Low doses of AGEs over a short amount of time stimulated chondrocyte proliferation and autophagy by limiting phosphorylation of Akt/MTOR signaling. The addition of PPARG inhibitor T0070907 lead to defective autophagy. High dose and long exposure to AGEs inhibited cell viability and autophagy by increasing phosphorylation levels of Akt/MTOR signaling. The agonist, pioglitazone, was shown to protect cell autophagy in a dose-dependent manner. Our findings suggest AGEs can downregulate PPARG and that PPARG maintains cell viability by activating the Akt/MTOR signaling pathway as well as inducing chondrocyte autophagy. © 2018 International Federation for Cell Biology.

The BATTLE-2 Study: A Biomarker-Integrated Targeted Therapy Study in Previously Treated Patients With Advanced Non–Small-Cell Lung Cancer

PubMed Central

Lee, J. Jack; Wistuba, Ignacio I.; Tsao, Anne S.; Fossella, Frank V.; Kalhor, Neda; Gupta, Sanjay; Byers, Lauren Averett; Izzo, Julie G.; Gettinger, Scott N.; Goldberg, Sarah B.; Tang, Ximing; Miller, Vincent A.; Skoulidis, Ferdinandos; Gibbons, Don L.; Shen, Li; Wei, Caimiao; Diao, Lixia; Peng, S. Andrew; Wang, Jing; Tam, Alda L.; Coombes, Kevin R.; Koo, Ja Seok; Mauro, David J.; Rubin, Eric H.; Heymach, John V.; Hong, Waun Ki; Herbst, Roy S.

2016-01-01

Purpose By applying the principles of real-time biopsy, biomarker-based, adaptively randomized studies in non–small-cell lung cancer (NSCLC) established by the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) trial, we conducted BATTLE-2 (BATTLE-2 Program: A Biomarker-Integrated Targeted Therapy Study in Previously Treated Patients With Advanced Non-Small Cell Lung Cancer), an umbrella study to evaluate the effects of targeted therapies focusing on KRAS-mutated cancers. Patients and Methods Patients with advanced NSCLC (excluding sensitizing EGFR mutations and ALK gene fusions) refractory to more than one prior therapy were randomly assigned, stratified by KRAS status, to four arms: (1) erlotinib, (2) erlotinib plus MK-2206, (3) MK-2206 plus AZD6244, or (4) sorafenib. Tumor gene expression profiling–targeted next-generation sequencing was performed to evaluate predictive and prognostic biomarkers. Results Two hundred patients, 27% with KRAS-mutated (KRAS mut+) tumors, were adaptively randomly assigned to erlotinib (n = 22), erlotinib plus MK-2206 (n = 42), MK-2206 plus AZD6244 (n = 75), or sorafenib (n = 61). In all, 186 patients were evaluable, and the primary end point of an 8-week disease control rate (DCR) was 48% (arm 1, 32%; arm 2, 50%; arm 3, 53%; and arm 4, 46%). For KRAS mut+ patients, DCR was 20%, 25%, 62%, and 44% whereas for KRAS wild-type patients, DCR was 36%, 57%, 49%, and 47% for arms 1, 2, 3, and 4, respectively. Median progression-free survival was 2.0 months, not different by KRAS status, 1.8 months for arm 1, and 2.5 months for arms 2 versus arms 3 and 4 in KRAS mut+ patients (P = .04). Median overall survival was 6.5 months, 9.0 and 5.1 months for arms 1 and 2 versus arms 3 and 4 in KRAS wild-type patients (P = .03). Median overall survival was 7.5 months in mesenchymal versus 5 months in epithelial tumors (P = .02). Conclusion Despite improved progression-free survival on therapy that did not contain

The BATTLE-2 Study: A Biomarker-Integrated Targeted Therapy Study in Previously Treated Patients With Advanced Non-Small-Cell Lung Cancer.

PubMed

Papadimitrakopoulou, Vassiliki; Lee, J Jack; Wistuba, Ignacio I; Tsao, Anne S; Fossella, Frank V; Kalhor, Neda; Gupta, Sanjay; Byers, Lauren Averett; Izzo, Julie G; Gettinger, Scott N; Goldberg, Sarah B; Tang, Ximing; Miller, Vincent A; Skoulidis, Ferdinandos; Gibbons, Don L; Shen, Li; Wei, Caimiao; Diao, Lixia; Peng, S Andrew; Wang, Jing; Tam, Alda L; Coombes, Kevin R; Koo, Ja Seok; Mauro, David J; Rubin, Eric H; Heymach, John V; Hong, Waun Ki; Herbst, Roy S

2016-08-01

By applying the principles of real-time biopsy, biomarker-based, adaptively randomized studies in non-small-cell lung cancer (NSCLC) established by the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) trial, we conducted BATTLE-2 (BATTLE-2 Program: A Biomarker-Integrated Targeted Therapy Study in Previously Treated Patients With Advanced Non-Small Cell Lung Cancer), an umbrella study to evaluate the effects of targeted therapies focusing on KRAS-mutated cancers. Patients with advanced NSCLC (excluding sensitizing EGFR mutations and ALK gene fusions) refractory to more than one prior therapy were randomly assigned, stratified by KRAS status, to four arms: (1) erlotinib, (2) erlotinib plus MK-2206, (3) MK-2206 plus AZD6244, or (4) sorafenib. Tumor gene expression profiling-targeted next-generation sequencing was performed to evaluate predictive and prognostic biomarkers. Two hundred patients, 27% with KRAS-mutated (KRAS mut+) tumors, were adaptively randomly assigned to erlotinib (n = 22), erlotinib plus MK-2206 (n = 42), MK-2206 plus AZD6244 (n = 75), or sorafenib (n = 61). In all, 186 patients were evaluable, and the primary end point of an 8-week disease control rate (DCR) was 48% (arm 1, 32%; arm 2, 50%; arm 3, 53%; and arm 4, 46%). For KRAS mut+ patients, DCR was 20%, 25%, 62%, and 44% whereas for KRAS wild-type patients, DCR was 36%, 57%, 49%, and 47% for arms 1, 2, 3, and 4, respectively. Median progression-free survival was 2.0 months, not different by KRAS status, 1.8 months for arm 1, and 2.5 months for arms 2 versus arms 3 and 4 in KRAS mut+ patients (P = .04). Median overall survival was 6.5 months, 9.0 and 5.1 months for arms 1 and 2 versus arms 3 and 4 in KRAS wild-type patients (P = .03). Median overall survival was 7.5 months in mesenchymal versus 5 months in epithelial tumors (P = .02). Despite improved progression-free survival on therapy that did not contain erlotinib for KRAS mut+ patients and improved

Phospholipase D2 Mediates Survival Signaling through Direct Regulation of Akt in Glioblastoma Cells*♦

PubMed Central

Bruntz, Ronald C.; Taylor, Harry E.; Lindsley, Craig W.; Brown, H. Alex

2014-01-01

The lack of innovative drug targets for glioblastoma multiforme (GBM) limits patient survival to approximately 1 year following diagnosis. The pro-survival kinase Akt provides an ideal target for the treatment of GBM as Akt signaling is frequently activated in this cancer type. However, the central role of Akt in physiological processes limits its potential as a therapeutic target. In this report, we show that the lipid-metabolizing enzyme phospholipase D (PLD) is a novel regulator of Akt in GBM. Studies using a combination of small molecule PLD inhibitors and siRNA knockdowns establish phosphatidic acid, the product of the PLD reaction, as an essential component for the membrane recruitment and activation of Akt. Inhibition of PLD enzymatic activity and subsequent Akt activation decreases GBM cell viability by specifically inhibiting autophagic flux. We propose a mechanism whereby phosphorylation of beclin1 by Akt prevents binding of Rubicon (RUN domain cysteine-rich domain containing beclin1-interacting protein), an interaction known to inhibit autophagic flux. These findings provide a novel framework through which Akt inhibition can be achieved without directly targeting the kinase. PMID:24257753

Cep55 regulates embryonic growth and development by promoting Akt stability in zebrafish.

PubMed

Jeffery, Jessie; Neyt, Christine; Moore, Wade; Paterson, Scott; Bower, Neil I; Chenevix-Trench, Georgia; Verkade, Heather; Hogan, Benjamin M; Khanna, Kum Kum

2015-05-01

CEP55 was initially described as a centrosome- and midbody-associated protein and a key mediator of cytokinesis. More recently, it has been implicated in PI3K/AKT pathway activation via an interaction with the catalytic subunit of PI3K. However, its role in embryonic development is unknown. Here we describe a cep55 nonsense mutant zebrafish with which we can study the in vivo physiologic role of Cep55. Homozygous mutants underwent extensive apoptosis by 24 hours postfertilization (hpf) concomitant with cell cycle defects, and heterozygous carriers were indistinguishable from their wild-type siblings. A similar phenotype was also observed in zebrafish injected with a cep55 morpholino, suggesting the mutant is a cep55 loss-of-function model. Further analysis revealed that Akt was destabilized in the homozygous mutants, which partially phenocopied Akt1 and Akt2 knockdown. Expression of either constitutively activated PIK3CA or AKT1 could partially rescue the homozygous mutants. Consistent with a role for Cep55 in regulation of Akt stability, treatment with proteasome inhibitor, MG132, partially rescued the homozygous mutants. Taken together, these results provide the first description of Cep55 in development and underline the importance of Cep55 in the regulation of Pi3k/Akt pathway and in particular Akt stability. © FASEB.

Ras inhibitors display an anti-metastatic effect by downregulation of lysyl oxidase through inhibition of the Ras-PI3K-Akt-HIF-1α pathway.

PubMed

Yoshikawa, Yoko; Takano, Osamu; Kato, Ichiro; Takahashi, Yoshihisa; Shima, Fumi; Kataoka, Tohru

2017-12-01

Metastasis stands as the major obstacle for the survival from cancers. Nonetheless most existing anti-cancer drugs inhibit only cell proliferation, and discovery of agents having both anti-proliferative and anti-metastatic properties would be more beneficial. We previously reported the discovery of small-molecule Ras inhibitors, represented by Kobe0065, that displayed anti-proliferative activity on xenografts of human colorectal cancer (CRC) cell line SW480 carrying the K-ras G12V gene. Here we show that treatment of cancer cells carrying the activated ras genes with Kobe0065 or a siRNA targeting Ras downregulates the expression of lysyl oxidase (LOX), which has been implicated in metastasis. LOX expression is enhanced by co-expression of Ras G12V through activation of phosphatidylinositol 3-kinase (PI3K)/Akt and concomitant accumulation of hypoxia-inducible factor (HIF)-1α. Furthermore, Kobe0065 effectively inhibits not only migration and invasion of cancer cells carrying the activated ras genes but also lung metastasis of human CRC cell line SW620 carrying the K-ras G12V gene. Collectively, these results indicate that Kobe0065 prevents metastasis through inhibition of the Ras-PI3K-Akt-HIF-1α-LOX signaling and suggest that Ras inhibitors in general might exhibit both anti-proliferative and anti-metastatic properties toward cancer cells carrying the activated ras genes. Copyright © 2017 Elsevier B.V. All rights reserved.

Agmatine Protects Against 6-OHDA-Induced Apoptosis, and ERK and Akt/GSK Disruption in SH-SY5Y Cells.

PubMed

Amiri, Esmat; Ghasemi, Rasoul; Moosavi, Maryam

2016-08-01

6-Hydroxydopamine (6-OHDA), a metabolite of dopamine is known to induce dopaminergic cell toxicity which makes that a suitable agent inducing an experimental model of Parkinson's disease (PD). Agmatine has been shown to protect against some cellular and animal PD models. This study was aimed to assess whether agmatine prevents 6-OHDA-induced SH-SY5Y cell death and if yes, then how it affects Akt/glycogen synthesis kinase-3β (GSK-3β) and extracellular signal-regulated kinases (ERK) signals. The cells were treated with different drugs, and their viability was examined via MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay and morphological observation. Western blot studies were done to assess cleaved caspase-3, Akt/GSK-3β, and ERK proteins. 6-OHDA-induced cell death and caspase-3 cleavage, while agmatine prevented those changes. 6-OHDA also decreased the amount of phosphorylated Akt (pAkt)/Akt while increased GSK-3β activity which was prevented by agmatine. Additionally, this toxin increased pERK/ERK ratio which was averted again by agmatine. The PI3/Akt inhibitor, LY294002, impeded the changes induced by agmatine, while ERK inhibitor (PD98059) did not disturb the effects of agmatine, and by itself, it preserved the cells against 6-OHDA toxicity. This study revealed that agmatine is protective in 6-OHDA model of PD and affects Akt/GSK-3β and ERK pathways.

Akt, mTOR and NF-κB pathway activation in Treponema pallidum stimulates M1 macrophages.

PubMed

Lin, Li-Rong; Gao, Zheng-Xiang; Lin, Yong; Zhu, Xiao-Zhen; Liu, Wei; Liu, Dan; Gao, Kun; Tong, Man-Li; Zhang, Hui-Lin; Liu, Li-Li; Xiao, Yao; Niu, Jian-Jun; Liu, Fan; Yang, Tian-Ci

2018-06-01

The polarization of macrophages and the molecular mechanism involved during the early process of syphilis infection remain unknown. This study was conducted to explore the influence of Treponema pallidum (T. pallidum) treatment on macrophage polarization and the Akt-mTOR-NFκB signaling pathway mechanism involved in this process. M0 macrophages derived from the phorbol-12-myristate-13-acetate-induced human acute monocytic leukemia cell line THP-1 were cultured with T. pallidum. T. pallidum induced inflammatory cytokine (IL-1β and TNF-α) expression in a dose- and time-dependent manner. However IL-10 cytokine expression decreased at the mRNA and protein levels. Additionally, the expression of the M1 surface marker iNOS was up-regulated with incubation time, and the expression of the M2 surface marker CD206 was low (vs. PBS treated macrophages, P < 0.001) and did not fluctuate over 12 h. Further studies revealed that Akt-mTOR-NFκB pathway proteins, including p-Akt, p-mTOR, p-S6, p-p65, and p-IκBα, were significantly higher in the T. pallidum-treated macrophages than in the PBS-treated macrophages (P < 0.05). In addition, inflammatory cytokine expression was suppressed in T. pallidum-induced M1 macrophages pretreated with LY294002 (an Akt-specific inhibitor) or PDTC (an NF-κB inhibitor), while inflammatory cytokine levels increased in T. pallidum-induced M1 macrophages pretreated with rapamycin (an mTOR inhibitor). These findings revealed that T. pallidum promotes the macrophage transition to pro-inflammatory M1 macrophages in vitro. The present study also provides evidence that Akt, mTOR and NF-κB pathway activation in T. pallidum stimulates M1 macrophages. This study provides novel insights into the innate immune response to T. pallidum infection. Copyright © 2018. Published by Elsevier B.V.

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

Synthesis and receptor binding studies of (+/-)1-iodo-MK-801

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, D.J.; Ciliax, B.J.; Van Dort, M.E.

1989-06-01

The glutamate analogue N-methyl-D-aspartate (NMDA) binds to a subset of glutamate receptors that are coupled to a voltage-sensitive cation channel. This NMDA-linked channel is the likely binding locus of the potent anticonvulsant MK-801. To develop single-photon emission computed tomography (SPECT) probes of this brain channel, we synthesized (+/)1-iodo-MK-801 and (+/-)1-({sup 125}I)iodo-MK-801. The effect of (+/-)1-iodo-MK-801 on ligand binding to the NMDA-linked glutamate receptor site was assessed using a rat brain homogenate assay. (+/-)1-Iodo-MK-801 displaced the dissociative anesthetic ligand ({sup 3}H)N-(1-(2-thienyl)cyclohexyl)piperidine (({sup 3}H)TCP) binding with an IC50 of 1 microM, which is a 10-fold lower binding affinity than that of (+/-)MK-801.more » In in vivo autoradiographic studies, (+/-)MK-801 failed to block selective uptake of (+/-)1-iodo-MK-801 in rat brain. These results suggest that (+/-)1-iodo-MK-801 may not be a suitable ligand for mapping NMDA-linked glutamate receptor channels.« less

Role for pAKT in rat urinary bladder with cyclophosphamide (CYP)-induced cystitis

PubMed Central

Arms, Lauren

2011-01-01

AKT phosphorylation following peripheral nerve injury or inflammation may play a role in somatic pain processes and visceral inflammation. To examine such a role in micturition reflexes with bladder inflammation, we induced bladder inflammation in adult female Wistar rats (200–300 g) by injecting cyclophosphamide (CYP) intraperitoneally at acute (150 mg/kg; 4 h), intermediate (150 mg/kg; 48 h), and chronic (75 mg/kg; every third day for 10 days) time points. Western blot analyses of whole urinary bladders showed significant increases (P ≤ 0.01) in phosphorylated (p) AKT at all time points; however, the magnitude of AKT phosphorylation varied with duration of CYP treatment. Immunohistochemical analyses of pAKT immunoreactivity (pAKT-IR) in cryostat bladder sections demonstrated duration-dependent, significant (P ≤ 0.01) increases in pAKT-IR in both the urothelium and detrusor smooth muscle of CYP-inflamed bladders. Additionally, a suburothelial population of pAKT-IR macrophages (CD68-, MAC2-, and F4/80-positive) was present in chronic CYP-treated bladders. The functional role of pAKT in micturition was evaluated using open, conscious cystometry with continuous instillation of saline in conjunction with administration of an inhibitor of AKT phosphorylation, deguelin (1.0 μg/10 μl), or vehicle (1% DMSO in saline) in control (no inflammation) and CYP (48 h)-treated rats. Bladder capacity, void volume, and intercontraction void interval increased significantly (P ≤ 0.05) following intravesical instillation of deguelin in CYP (48 h)-treated rats. These results demonstrate increased AKT phosphorylation in the urinary bladder with urinary bladder inflammation and that blockade of AKT phosphorylation in the urothelium improves overall bladder function. PMID:21632956

Methylglyoxal Mediates Adipocyte Proliferation by Increasing Phosphorylation of Akt1

PubMed Central

Jia, Xuming; Chang, Tuanjie; Wilson, Thomas W.; Wu, Lingyun

2012-01-01

Methylglyoxal (MG) is a highly reactive metabolite physiologically presented in all biological systems. The effects of MG on diabetes and hypertension have been long recognized. In the present study, we investigated the potential role of MG in obesity, one of the most important factors to cause metabolic syndrome. An increased MG accumulation was observed in the adipose tissue of obese Zucker rats. Cell proliferation assay showed that 5–20 µM of MG stimulated the proliferation of 3T3-L1 cells. Further study suggested that accumulated-MG stimulated the phosphorylation of Akt1 and its targets including p21 and p27. The activated Akt1 then increased the activity of CDK2 and accelerated the cell cycle progression of 3T3-L1 cells. The effects of MG were efficiently reversed by advanced glycation end product (AGE) breaker alagebrium and Akt inhibitor SH-6. In summary, our study revealed a previously unrecognized effect of MG in stimulating adipogenesis by up-regulation of Akt signaling pathway and this mechanism might offer a new approach to explain the development of obesity. PMID:22606274

PKI-587 and sorafenib targeting PI3K/AKT/mTOR and Ras/Raf/MAPK pathways synergistically inhibit HCC cell proliferation.

PubMed

Gedaly, Roberto; Angulo, Paul; Hundley, Jonathan; Daily, Michael F; Chen, Changguo; Evers, B Mark

2012-08-01

Deregulated Ras/Raf/MAPK and PI3K/AKT/mTOR signaling pathways are found in hepatocellular carcinoma (HCC). This study aimed to test the inhibitory effects of PKI-587 and sorafenib as single agents or in combination on HCC (Huh7 cell line) proliferation. (3)H-thymidine incorporation and MTT assay were used to assess Huh7 cell proliferation. Phosphorylation of the key enzymes in the Ras/Raf/MAPK and PI3K/AKT/mTOR pathways was detected by Western blot. We found that PKI-587 is a more potent PI3K/mTOR inhibitor than PI-103. Combination of PKI-587 and sorafenib was a more effective inhibitor of Huh7 proliferation than the combination of PI-103 and sorafenib. Combination of PKI-587 and sorafenib synergistically inhibited epidermal growth factor (EGF)-stimulated Huh7 proliferation compared with monodrug therapy. EGF increased phosphorylation of Ras/Raf downstream signaling proteins MEK and ERK; EGF-stimulated activation was inhibited by sorafenib. However, sorafenib, as a single agent, increased AKT (Ser473) phosphorylation. EGF-stimulated AKT (ser473) activation was inhibited by PKI-587. PKI-587 is a potent inhibitor of AKT (Ser473), mTOR (Ser2448), and S6K (Thr389) phosphorylation; in contrast, rapamycin stimulated mTOR complex 2 substrate AKT(Ser473) phosphorylation although it inhibited mTOR complex 1 substrate S6K phosphorylation. PKI-587, as a single agent, stimulated MEK and ERK phosphorylation. However, when PKI-587 and sorafenib were used in combination, they inhibited all the tested kinases in the Ras/Raf /MAPK and PI3K/AKT/mTOR pathways. The combination of PKI-587 and sorafenib has the advantage over monodrug therapy on inhibition of HCC cell proliferation by blocking both PI3K/AKT/mTOR and Ras/Raf/MAPK signaling pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

Exosomes promote cetuximab resistance via the PTEN/Akt pathway in colon cancer cells.

PubMed

Zhang, S; Zhang, Y; Qu, J; Che, X; Fan, Y; Hou, K; Guo, T; Deng, G; Song, N; Li, C; Wan, X; Qu, X; Liu, Y

2017-11-13

Cetuximab is widely used in patients with metastatic colon cancer expressing wildtype KRAS. However, acquired drug resistance limits its clinical efficacy. Exosomes are nanosized vesicles secreted by various cell types. Tumor cell-derived exosomes participate in many biological processes, including tumor invasion, metastasis, and drug resistance. In this study, exosomes derived from cetuximab-resistant RKO colon cancer cells induced cetuximab resistance in cetuximab-sensitive Caco-2 cells. Meanwhile, exosomes from RKO and Caco-2 cells showed different levels of phosphatase and tensin homolog (PTEN) and phosphor-Akt. Furthermore, reduced PTEN and increased phosphorylated Akt levels were found in Caco-2 cells after exposure to RKO cell-derived exosomes. Moreover, an Akt inhibitor prevented RKO cell-derived exosome-induced drug resistance in Caco-2 cells. These findings provide novel evidence that exosomes derived from cetuximab-resistant cells could induce cetuximab resistance in cetuximab-sensitive cells, by downregulating PTEN and increasing phosphorylated Akt levels.

Inhibition of ERK1/2 or AKT Activity Equally Enhances Radiation Sensitization in B16F10 Cells

PubMed Central

Kalal, Bhuvanesh Sukhlal; Fathima, Faraz; Pai, Vinitha Ramanath; Sanjeev, Ganesh; Krishna, Chilakapati Murali; Upadhya, Dinesh

2018-01-01

Background The aim of the study was to evaluate the radiation sensitizing ability of ERK1/2, PI3K-AKT and JNK inhibitors in highly radiation resistant and metastatic B16F10 cells which carry wild-type Ras and Braf. Methods Mouse melanoma cell line B16F10 was exposed to 1.0, 2.0 and 3.0 Gy of electron beam radiation. Phosphorylated ERK1/2, AKT and JNK levels were estimated by ELISA. Cells were exposed to 2.0 and 3.0 Gy of radiation with or without prior pharmacological inhibition of ERK1/2, AKT as well as JNK pathways. Cell death induced by radiation as well as upon inhibition of these pathways was measured by TUNEL assay using flow cytometry. Results Exposure of B16F10 cells to 1.0, 2.0 and 3.0 Gy of electron beam irradiation triggered an increase in all the three phosphorylated proteins compared to sham-treated and control groups. B16F10 cells pre-treated with either ERK1/2 or AKT inhibitors equally enhanced radiation-induced cell death at 2.0 as well as 3.0 Gy (P < 0.001), while inhibition of JNK pathway increased radiation-induced cell death to a lesser extent. Interestingly combined inhibition of ERK1/2 or AKT pathways did not show additional cell death compared to individual ERK1/2 or AKT inhibition. This indicates that ERK1/2 or AKT mediates radiation resistance through common downstream molecules in B16F10 cells. Conclusions Even without activating mutations in Ras or Braf genes, ERK1/2 and AKT play a critical role in B16F10 cell survival upon radiation exposure and possibly act through common downstream effector/s. PMID:29581812

Inhibition of ERK1/2 or AKT Activity Equally Enhances Radiation Sensitization in B16F10 Cells.

PubMed

Kalal, Bhuvanesh Sukhlal; Fathima, Faraz; Pai, Vinitha Ramanath; Sanjeev, Ganesh; Krishna, Chilakapati Murali; Upadhya, Dinesh

2018-02-01

The aim of the study was to evaluate the radiation sensitizing ability of ERK1/2, PI3K-AKT and JNK inhibitors in highly radiation resistant and metastatic B16F10 cells which carry wild-type Ras and Braf . Mouse melanoma cell line B16F10 was exposed to 1.0, 2.0 and 3.0 Gy of electron beam radiation. Phosphorylated ERK1/2, AKT and JNK levels were estimated by ELISA. Cells were exposed to 2.0 and 3.0 Gy of radiation with or without prior pharmacological inhibition of ERK1/2, AKT as well as JNK pathways. Cell death induced by radiation as well as upon inhibition of these pathways was measured by TUNEL assay using flow cytometry. Exposure of B16F10 cells to 1.0, 2.0 and 3.0 Gy of electron beam irradiation triggered an increase in all the three phosphorylated proteins compared to sham-treated and control groups. B16F10 cells pre-treated with either ERK1/2 or AKT inhibitors equally enhanced radiation-induced cell death at 2.0 as well as 3.0 Gy (P < 0.001), while inhibition of JNK pathway increased radiation-induced cell death to a lesser extent. Interestingly combined inhibition of ERK1/2 or AKT pathways did not show additional cell death compared to individual ERK1/2 or AKT inhibition. This indicates that ERK1/2 or AKT mediates radiation resistance through common downstream molecules in B16F10 cells. Even without activating mutations in Ras or Braf genes, ERK1/2 and AKT play a critical role in B16F10 cell survival upon radiation exposure and possibly act through common downstream effector/s.

Comparative evaluation of two glycine transporter 1 radiotracers [11C]GSK931145 and [18F]MK-6577 in baboons.

PubMed

Zheng, Ming-Qiang; Lin, Shu-Fei; Holden, Daniel; Naganawa, Mika; Ropchan, Jim R; Najafzaden, Soheila; Kapinos, Michael; Tabriz, Mike; Carson, Richard E; Hamill, Terence G; Huang, Yiyun

2016-03-01

Glycine transporter type-1 (GlyT1) has been proposed as a target for drug development for schizophrenia. PET imaging with a GlyT1 specific radiotracer will allow for the measurement of target occupancy of GlyT1 inhibitors, and for in vivo investigation of GlyT1 alterations in schizophrenia. We conducted a comparative evaluation of two GlyT1 radiotracers, [(11) C]GSK931145, and [(18) F]MK-6577, in baboons. Two baboons were imaged with [(11) C]GSK931145 and [(18) F]MK-6577. Blocking studies with GSK931145 (0.3 or 0.2 mg/kg) were conducted to determine the level of tracer specific binding. [(11) C]GSK931145 and [(18) F]MK-6577 were synthesized in good yield and high specific activity. Moderately fast metabolism was observed for both tracers, with ∼ 30% of parent at 30 min post-injection. In the brain, both radiotracers showed good uptake and distribution profiles consistent with regional GlyT1 densities. [(18) F]MK-6577 displayed higher uptake and faster kinetics than [(11) C]GSK931145. Time activity curves were well described by the two-tissue compartment model. Regional volume of distribution (VT ) values were higher for [(18) F]MK-6577 than [(11) C]GSK931145. Pretreatment with GSK931145 reduced tracer uptake to a homogeneous level throughout the brain, indicating in vivo binding specificity and lack of a reference region for both radiotracers. Linear regression analysis of VT estimates between tracers indicated higher specific binding for [(18) F]MK-6577 than [(11) C]GSK931145, consistent with higher regional binding potential (BPND ) values of [(18) F]MK-6577 calculated using VT from the baseline scans and non-displaceable distribution volume (VND ) derived from blocking studies. [(18) F]MK-6577 appears to be a superior radiotracer with higher brain uptake, faster kinetics, and higher specific binding signals than [(11) C]GSK931145. © 2016 Wiley Periodicals, Inc.

Elastase inhibitors as potential therapies for ELANE-associated neutropenia.

PubMed

Makaryan, Vahagn; Kelley, Merideth L; Fletcher, Breanna; Bolyard, Audrey Anna; Aprikyan, A Andrew; Dale, David C

2017-10-01

Mutations in ELANE , the gene for neutrophil elastase (NE), a protease expressed early in neutrophil development, are the most frequent cause of cyclic (CyN) and severe congenital neutropenia (SCN). We hypothesized that inhibitors of NE, acting either by directly inhibiting enzymatic activity or as chaperones for the mutant protein, might be effective as therapy for CyN and SCN. We investigated β-lactam-based inhibitors of human NE (Merck Research Laboratories, Kenilworth, NJ, USA), focusing on 1 inhibitor called MK0339, a potent, orally absorbed agent that had been tested in clinical trials and shown to have a favorable safety profile. Because fresh, primary bone marrow cells are rarely available in sufficient quantities for research studies, we used 3 cellular models: patient-derived, induced pluripotent stem cells (iPSCs); HL60 cells transiently expressing mutant NE; and HL60 cells with regulated expression of the mutant enzyme. In all 3 models, the cells expressing the mutant enzyme had reduced survival as measured with annexin V and FACS. Coincubation with the inhibitors, particularly MK0339, promoted cell survival and increased formation of mature neutrophils. These studies suggest that cell-permeable inhibitors of neutrophil elastase show promise as novel therapies for ELANE -associated neutropenia. © Society for Leukocyte Biology.

[Signaling pathways mTOR and AKT in epilepsy].

PubMed

Romero-Leguizamon, C R; Ramirez-Latorre, J A; Mora-Munoz, L; Guerrero-Naranjo, A

2016-07-01

The signaling pathway AKT/mTOR is a central axis in regulating cellular processes, particularly in neurological diseases. In the case of epilepsy, it has been observed alteration in the pathophysiological process of the same. However, they have not described all the mechanisms of these signaling pathways that could open the opportunity to new research and therapeutic strategies. To review existing partnerships between intracellular signaling pathways AKT and mTOR in the pathophysiology of epilepsy. Epilepsy is a disease with a high epidemiological impact globally, so it is widely investigated regarding the pathophysiological components thereof. In that search they have been involved different intracellular signaling pathways in neurons, as determinants epileptogenic. Advances in this field have even allowed the successful implementation of new therapeutic strategies and to open the way to new research in the field. Improving knowledge about the pathophysiological role of the signaling pathway mTOR/AKT in epilepsy can raise new investigations regarding therapeutic alternatives. The use of mTOR inhibitors, has emerged in recent years as effective in treating this disease entity alternative however is clear the necessity of continue the research for new drug therapies.

Escin activates AKT-Nrf2 signaling to protect retinal pigment epithelium cells from oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Kaijun; Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou; Jiang, Yiqian

Here we explored the anti-oxidative and cytoprotective potentials of escin, a natural triterpene-saponin, against hydrogen peroxide (H{sub 2}O{sub 2}) in retinal pigment epithelium (RPE) cells. We showed that escin remarkably attenuated H{sub 2}O{sub 2}-induced death and apoptosis of established (ARPE-19) and primary murine RPE cells. Meanwhile, ROS production and lipid peroxidation by H{sub 2}O{sub 2} were remarkably inhibited by escin. Escin treatment in RPE cells resulted in NF-E2-related factor 2 (Nrf2) signaling activation, evidenced by transcription of anti-oxidant-responsive element (ARE)-regulated genes, including HO-1, NQO-1 and SRXN-1. Knockdown of Nrf2 through targeted shRNAs/siRNAs alleviated escin-mediated ARE gene transcription, and almost abolishedmore » escin-mediated anti-oxidant activity and RPE cytoprotection against H{sub 2}O{sub 2}. Reversely, escin was more potent against H{sub 2}O{sub 2} damages in Nrf2-over-expressed ARPE-19 cells. Further studies showed that escin-induced Nrf2 activation in RPE cells required AKT signaling. AKT inhibitors (LY294002 and perifosine) blocked escin-induced AKT activation, and dramatically inhibited Nrf2 phosphorylation, its cytosol accumulation and nuclear translocation in RPE cells. Escin-induced RPE cytoprotection against H{sub 2}O{sub 2} was also alleviated by the AKT inhibitors. Together, these results demonstrate that escin protects RPE cells from oxidative stress possibly through activating AKT-Nrf2 signaling.« less

Preclinical to Human Translational Pharmacology of the Novel M1 Positive Allosteric Modulator MK-7622.

PubMed

Uslaner, Jason M; Kuduk, Scott D; Wittmann, Marion; Lange, Henry S; Fox, Steve V; Min, Chris; Pajkovic, Natasa; Harris, Dawn; Cilissen, Caroline; Mahon, Chantal; Mostoller, Kate; Warrington, Steve; Beshore, Douglas C

2018-06-01

The current standard of care for treating Alzheimer's disease is acetylcholinesterase inhibitors, which nonselectively increase cholinergic signaling by indirectly enhancing activity of nicotinic and muscarinic receptors. These drugs improve cognitive function in patients, but also produce unwanted side effects that limit their efficacy. In an effort to selectively improve cognition and avoid the cholinergic side effects associated with the standard of care, various efforts have been aimed at developing selective M 1 muscarinic receptor activators. In this work, we describe the preclinical and clinical pharmacodynamic effects of the M 1 muscarinic receptor-positive allosteric modulator, MK-7622. MK-7622 attenuated the cognitive-impairing effects of the muscarinic receptor antagonist scopolamine and altered quantitative electroencephalography (qEEG) in both rhesus macaque and human. For both scopolamine reversal and qEEG, the effective exposures were similar between species. However, across species the minimum effective exposures to attenuate the scopolamine impairment were lower than for qEEG. Additionally, there were differences in the spectral power changes produced by MK-7622 in rhesus versus human. In sum, these results are the first to demonstrate translation of preclinical cognition and target modulation to clinical effects in humans for a selective M 1 muscarinic receptor-positive allosteric modulator. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

delta opioid receptors stimulate Akt-dependent phosphorylation of c-jun in T cells.

PubMed

Shahabi, Nahid A; McAllen, Kathy; Sharp, Burt M

2006-02-01

Activation of naive T cells markedly up-regulates the expression of delta opioid receptors (DORs). These receptors are bound by DOR peptides released by T cells, modulating T cell functions such as interleukin-2 production, cellular proliferation, and chemotaxis. Previous studies have shown that DOR agonists [e.g., [D-Ala(2)-D-Leu(5)]-enkephalin (DADLE)] modulate T cell antigen receptor signaling through mitogen-activated protein kinases (MAPKs; i.e., extracellular signal-regulated kinases 1 and 2) and that DORs directly induce phosphorylation of activating transcription factor-2 (implicated in cytokine gene transcription) and its association with the MAPK c-jun1 NH(2)-terminal kinase (JNK). Such observations suggest that DORs may induce the phosphorylation of c-jun. These experiments were performed to test this hypothesis and determine the potential roles of phosphoinositide 3-kinase (PI3K) and Akt (protein kinase B). DADLE (10(-10) to 10(-6) M) dose-dependently induced c-jun phosphorylation. This was blocked by pertussis toxin and the DOR-specific antagonist naltindole. Fluorescence flow cytometry showed that DADLE significantly stimulated c-jun phosphorylation by T cells. DADLE stimulated phosphorylation of membrane-associated Akt; wortmannin and LY294002 ([2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]), specific inhibitors of PI3K, abolished the DADLE-induced phosphorylation of c-jun. Finally, inhibitors of Akt and JNK blocked DADLE-induced phosphorylation of c-jun. Thus, activated DORs directly stimulate c-jun phosphorylation through a PI3K-dependent pathway in T cells, apparently involving Akt. This implies that DORs activate JNK through a novel pathway dependent on PI3K and Akt, thereby regulating the function of activator protein-1 transcription complexes containing c-jun and other transcription partners.

Wogonin induces cross-regulation between autophagy and apoptosis via a variety of Akt pathway in human nasopharyngeal carcinoma cells.

PubMed

Chow, Shu-Er; Chen, Yu-Wen; Liang, Chi-Ang; Huang, Yao-Kuan; Wang, Jong-Shyan

2012-11-01

Autophagy as well as apoptosis is an emerging target for cancer therapy. Wogonin, a flavonoid compound derived from the traditional Chinese medicine of Huang-Qin, has anticancer activity in many cancer cells including human nasopharyngeal carcinoma (NPC). However, the involvement of autophagy in the wogonin-induced apoptosis of NPC cells was still uninvestigated. In this study, we found wogonin-induced autophagy had interference on the process of apoptosis. Wogonin-induced autophagy formation evidenced by LC3 I/II cleavage, acridine orange (AO)-stained vacuoles and the autophagosome/autolysosome images of TEM analysis. Activation of autophagy with rapamycin resulted in increased wogonin-mediated autophagy via inhibition of mTOR/P70S6K pathway. The functional relevance of autophagy in the antitumor activity was investigated by annexin V-positive stained cells and PARP cleavage. Induction of autophagy by rapamycin ameliorated the wogonin-mediated apoptosis, whereas inhibition of autophagy by 3-methyladenine (3-MA) or bafilomycin A1 increased the apoptotic effect. Interestingly, this study also found, in addition the mTOR/P70S6K pathway, wogonin also inhibited Raf/ERK pathway, a variety of Akt pathways. Inactivation of PI(3) K/Akt by their inhibitors significantly induced apoptosis and markedly sensitized the NPC cells to wogonin-induced apoptosis. This anticancer effect of Akt was further confirmed by SH6, a specific inhibitor of Akt. Importantly, inactivation of its downstream molecule ERK by PD98059, a MEK inhibitor, also induced apoptosis. This study indicated wogonin-induced both autophagy and apoptosis through a variety of Akt pathways and suggested modulation of autophagy might provide profoundly the potential therapeutic effect. Copyright © 2012 Wiley Periodicals, Inc.

Neuregulin-1β promotes glucose uptake via PI3K/Akt in neonatal rat cardiomyocytes.

PubMed

Pentassuglia, Laura; Heim, Philippe; Lebboukh, Sonia; Morandi, Christian; Xu, Lifen; Brink, Marijke

2016-05-01

Nrg1β is critically involved in cardiac development and also maintains function of the adult heart. Studies conducted in animal models showed that it improves cardiac performance under a range of pathological conditions, which led to its introduction in clinical trials to treat heart failure. Recent work also implicated Nrg1β in the regenerative potential of neonatal and adult hearts. The molecular mechanisms whereby Nrg1β acts in cardiac cells are still poorly understood. In the present study, we analyzed the effects of Nrg1β on glucose uptake in neonatal rat ventricular myocytes and investigated to what extent mTOR/Akt signaling pathways are implicated. We show that Nrg1β enhances glucose uptake in cardiomyocytes as efficiently as IGF-I and insulin. Nrg1β causes phosphorylation of ErbB2 and ErbB4 and rapidly induces the phosphorylation of FAK (Tyr(861)), Akt (Thr(308) and Ser(473)), and its effector AS160 (Thr(642)). Knockdown of ErbB2 or ErbB4 reduces Akt phosphorylation and blocks the glucose uptake. The Akt inhibitor VIII and the PI3K inhibitors LY-294002 and Byl-719 abolish Nrg1β-induced phosphorylation and glucose uptake. Finally, specific mTORC2 inactivation after knockdown of rictor blocks the Nrg1β-induced increases in Akt-p-Ser(473) but does not modify AS160-p-Thr(642) or the glucose uptake responses to Nrg1β. In conclusion, our study demonstrates that Nrg1β enhances glucose uptake in cardiomyocytes via ErbB2/ErbB4 heterodimers, PI3Kα, and Akt. Furthermore, although Nrg1β activates mTORC2, the resulting Akt-Ser(473) phosphorylation is not essential for glucose uptake induction. These new insights into pathways whereby Nrg1β regulates glucose uptake in cardiomyocytes may contribute to the understanding of its regenerative capacity and protective function in heart failure. Copyright © 2016 the American Physiological Society.

Concurrent targeting Akt and sphingosine kinase 1 by A-674563 in acute myeloid leukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Lin; Shaoyang Central Hospital, Hunan Province; Zhang, Yanan

Akt signaling plays a pivotal role in acute myeloid leukemia (AML) development and progression. In the present study, we evaluated the potential anti-AML activity by a novel Akt kinase inhibitor A-674563. Our results showed that A-674563 dose-dependently inhibited survival and proliferation of U937 AML cells and six lines of human AML progenitor cells, yet sparing human peripheral blood mononuclear leukocytes (PBMCs). A-674563 activated caspase-3/9 and apoptosis in the AML cells. Reversely, the pan-caspase inhibitor z-VAD-CHO dramatically alleviated A-674563-induced AML cell apoptosis and cytotoxicity. For the molecular study, we showed that A-674563 blocked Akt activation in U937 cells and human AMLmore » progenitor cells. Further, A-674563 decreased sphingosine kinase 1 (SphK1) activity in above AML cells to deplete pro-survival sphingosine-1-phosphate (S1P) and boost pro-apoptotic ceramide production. Such an effect on SphK1 signaling by A-674563 appeared independent of Akt blockage. Significantly, K6PC-5, a novel SphK1 activator, or supplement with S1P attenuated A-674563-induced ceramide production, and subsequent U937 cell death and apoptosis. Importantly, intraperitoneal injection of A-674563 at well-tolerated doses suppressed U937 leukemic xenograft tumor growth in nude mice, whiling significantly improving the animal survival. The results of the current study demonstrate that A-674563 exerts potent anti-leukemic activity in vitro and in vivo, possibly via concurrent targeting Akt and SphK1 signalings. - Highlights: • A-674563 is cytotoxic and anti-proliferative in U937 and AML progenitor cells. • A-674563 activates caspase-3/9 and apoptosis in U937 and AML progenitor cells. • Whiling blocking Akt, A-674563 manipulates other signalings in AML cells. • A-674563 inhibits SphK1 activity in AML cells, independent of Akt blockage. • A-674563 injection inhibits U937 xenograft in vivo growth, and improves mice survival.« less

Design and Synthesis of Piperazine Sulfonamide Cores Leading to Highly Potent HIV-1 Protease Inhibitors.

PubMed

Bungard, Christopher J; Williams, Peter D; Schulz, Jurgen; Wiscount, Catherine M; Holloway, M Katharine; Loughran, H Marie; Manikowski, Jesse J; Su, Hua-Poo; Bennett, David J; Chang, Lehua; Chu, Xin-Jie; Crespo, Alejandro; Dwyer, Michael P; Keertikar, Kartik; Morriello, Gregori J; Stamford, Andrew W; Waddell, Sherman T; Zhong, Bin; Hu, Bin; Ji, Tao; Diamond, Tracy L; Bahnck-Teets, Carolyn; Carroll, Steven S; Fay, John F; Min, Xu; Morris, William; Ballard, Jeanine E; Miller, Michael D; McCauley, John A

2017-12-14

Using the HIV-1 protease binding mode of MK-8718 and PL-100 as inspiration, a novel aspartate binding bicyclic piperazine sulfonamide core was designed and synthesized. The resulting HIV-1 protease inhibitor containing this core showed an 60-fold increase in enzyme binding affinity and a 10-fold increase in antiviral activity relative to MK-8718 .

Hydrogen sulfide facilities production of nitric oxide via the Akt/endothelial nitric oxide synthases signaling pathway to protect human umbilical vein endothelial cells from injury by angiotensin II.

PubMed

Cui, Jiasen; Zhuang, Shunjiu; Qi, Shaohong; Li, Li; Zhou, Junwen; Zhang, Wan; Zhao, Yun; Qi, Ning; Yin, Yangjun; Huang, Lu

2017-11-01

Angiotensin II (Ang II) has been reported as key in inducing endothelial cell injury, and endothelial cells may produce nitric oxide (NO) to protect themselves. However, the underlying mechanism remains elusive. Human umbilical vein endothelial cells (HUVECs) were divided into five treatment groups as follows: Normal control, Ang II, Ang II + sodium hydrosulfide [NaHS; hydrogen sulfide (H2S) donor], Ang II + Akt inhibitors + NaHS, and Ang II + endothelial nitric oxide synthases (eNOS) inhibitors + NaHS. Subsequently, cell viability, apoptosis, migration, proliferation and adhesion ability were determined. In addition, tubular structure formation was observed, and the NO and phosphorylation levels of Akt and eNOS were evaluated. Compared with the normal control group, Ang II treatment reduced the viability of HUVECs and increased the level of cell apoptosis (P<0.05). Furthermore, Ang II treatment inhibited the phosphorylation level of eNOS and Akt, as well as the generation of NO (P<0.05). H2S reversed the above‑mentioned effects significantly and increased cell proliferation, adhesion ability and promoted tubular structure formation (P<0.05); however, H2S did not reverse the impact of eNOS and Akt phosphorylation levels after being processed with Akt and eNOS inhibitors, which indicates that H2S is capable of protecting HUVECs via the eNOS/Akt signaling pathway (P<0.05). Thus, H2S stimulates the production of NO and protects HUVECs via inducing the Akt/eNOS signaling pathway.

Akt2-Dependent Phosphorylation of Radixin in Regulation of Mrp-2 Trafficking in WIF-B Cells.

PubMed

Suda, Jo; Rockey, Don C; Karvar, Serhan

2016-02-01

The dominant ezrin/radixin/moesin protein in hepatocytes is radixin, which plays an important role in mediating the binding of F-actin to the plasma membrane after a conformational activation by phosphorylation at Thr564. Here we have investigated the importance of Akt-mediated radixin Thr564 phosphorylation on Mrp-2 distribution and function in WIF-B cells. Mrp-2 is an adenosine triphosphate (ATP)-binding cassette transporter that plays an important role in detoxification and chemoprotection by transporting a wide range of compounds, especially conjugates of lipophilic substances with glutathione, organic anions, and drug metabolites such as glucuronides. Akt1 and Akt2 expression were manipulated using dominant active and negative constructs as well as Akt1 and Akt2 siRNA. Cellular distribution of radixin and Mrp-2 was visualized by fluorescence microscopy. A 5-chloromethylfluorescein diacetate, which is a substrate of the Mrp-2 and is actively transported in canalicular lumina, was used to measure Mrp-2 function. Radixin phosphorylation was significantly increased in wild-type and dominant active Akt2 transfected cells. Furthermore, radixin and Mrp-2 were localized at the canalicular membrane, similar to control cells. In contrast, overexpression of dominant negative Akt2, siRNA knockdown of Akt2 and a specific Akt inhibitor prevented radixin phosphorylation and led to alteration of normal radixin and Mrp-2 localization; inhibition of Akt2, but not Akt1 function led to radixin localization to the cytoplasmic space. In addition, dominant negative and Akt2 knockdown led to a dramatically impaired hepatocyte secretory response, while wild-type and dominant active Akt2 transfected cells exhibited increased 5-chloromethylfluorescein diacetate excretion. In contrast to Akt2, Akt1 was not associated with radixin phosphorylation. These studies, therefore, identify Akt2 as a critical kinase that regulates radixin phosphorylation and leads to Mrp-2 translocation and

AKT Kinase Activity Is Required for Lithium to Modulate Mood-Related Behaviors in Mice

PubMed Central

Pan, Jen Q; Lewis, Michael C; Ketterman, Josh K; Clore, Elizabeth L; Riley, Misha; Richards, Keenan R; Berry-Scott, Erin; Liu, Xiulin; Wagner, Florence F; Holson, Edward B; Neve, Rachael L; Biechele, Travis L; Moon, Randall T; Scolnick, Edward M; Petryshen, Tracey L; Haggarty, Stephen J

2011-01-01

Bipolar disorder (BP) is a debilitating psychiatric disorder, affecting ∼2% of the worldwide population, for which the etiological basis, pathogenesis, and neurocircuitry remain poorly understood. Individuals with BP suffer from recurrent episodes of mania and depression, which are commonly treated with the mood stabilizer lithium. However, nearly half of BP patients do not respond adequately to lithium therapy and the clinically relevant mechanisms of lithium for mood stabilization remain elusive. Here, we modeled lithium responsiveness using cellular assays of glycogen synthase kinase 3 (GSK-3) signaling and mood-related behavioral assays in inbred strains of mice that differ in their response to lithium. We found that activating AKT through phosphosrylation of a key regulatory site (Thr308) was associated with lithium response—activation of signaling pathways downstream of GSK-3 in cells and attenuation of mood-related behaviors in mice—and this response was attenuated by selective and direct inhibition of AKT kinase activity. Conversely, the expression of constitutively active AKT1 in both the cellular and behavioral assays conferred lithium sensitivity. In contrast, selective and direct GSK-3 inhibition by the ATP-competitive inhibitor CHIR99021 bypassed the requirement for AKT activation and modulated behavior in both lithium-responsive and non-responsive mouse strains. These results distinguish the mechanism of action of lithium from direct GSK-3 inhibition both in vivo and in vitro, and highlight the therapeutic potential for selective GSK-3 inhibitors in BP treatment. PMID:21389981

Structural Basis of Wee Kinases Functionality and Inactivation by Diverse Small Molecule Inhibitors.

PubMed

Zhu, Jin-Yi; Cuellar, Rebecca A; Berndt, Norbert; Lee, Hee Eun; Olesen, Sanne H; Martin, Mathew P; Jensen, Jeffrey T; Georg, Gunda I; Schönbrunn, Ernst

2017-09-28

Members of the Wee family of kinases negatively regulate the cell cycle via phosphorylation of CDK1 and are considered potential drug targets. Herein, we investigated the structure-function relationship of human Wee1, Wee2, and Myt1 (PKMYT1). Purified recombinant full-length proteins and kinase domain constructs differed substantially in phosphorylation states and catalytic competency, suggesting complex mechanisms of activation. A series of crystal structures reveal unique features that distinguish Wee1 and Wee2 from Myt1 and establish the structural basis of differential inhibition by the widely used Wee1 inhibitor MK-1775. Kinome profiling and cellular studies demonstrate that, in addition to Wee1 and Wee2, MK-1775 is an equally potent inhibitor of the polo-like kinase PLK1. Several previously unrecognized inhibitors of Wee kinases were discovered and characterized. Combined, the data provide a comprehensive view on the catalytic and structural properties of Wee kinases and a framework for the rational design of novel inhibitors thereof.

Regulation of ENaC-mediated alveolar fluid clearance by insulin via PI3K/Akt pathway in LPS-induced acute lung injury.

PubMed

Deng, Wang; Li, Chang-Yi; Tong, Jin; Zhang, Wei; Wang, Dao-Xin

2012-03-30

Stimulation of epithelial sodium channel (ENaC) increases Na(+) transport, a driving force of alveolar fluid clearance (AFC) to keep alveolar spaces free of edema fluid that is beneficial for acute lung injury (ALI). It is well recognized that regulation of ENaC by insulin via PI3K pathway, but the mechanism of this signaling pathway to regulate AFC and ENaC in ALI remains unclear. The aim of this study was to investigate the effect of insulin on AFC in ALI and clarify the pathway in which insulin regulates the expression of ENaC in vitro and in vivo. A model of ALI (LPS at a dose of 5.0 mg/kg) with non-hyperglycemia was established in Sprague-Dawley rats receiving continuous exogenous insulin by micro-osmotic pumps and wortmannin. The lungs were isolated for measurement of bronchoalveolar lavage fluid(BALF), total lung water content(TLW), and AFC after ALI for 8 hours. Alveolar epithelial type II cells were pre-incubated with LY294002, Akt inhibitor and SGK1 inhibitor 30 minutes before insulin treatment for 2 hours. The expressions of α-,β-, and γ-ENaC were detected by immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting. In vivo, insulin decreased TLW, enchanced AFC, increased the expressions of α-,β-, and γ-ENaC and the level of phosphorylated Akt, attenuated lung injury and improved the survival rate in LPS-induced ALI, the effects of which were blocked by wortmannin. Amiloride, a sodium channel inhibitor, significantly reduced insulin-induced increase in AFC. In vitro, insulin increased the expressions of α-,β-, and γ-ENaC as well as the level of phosphorylated Akt but LY294002 and Akt inhibitor significantly prevented insulin-induced increase in the expression of ENaC and the level of phosphorylated Akt respectively. Immunoprecipitation studies showed that levels of Nedd4-2 binding to ENaC were decreased by insulin via PI3K/Akt pathway. Our study demonstrated that insulin alleviated pulmonary edema and

Normal cadmium uptake in microcytic anemia mk/mk mice suggests that DMT1 is not the only cadmium transporter in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki, Tomohito; Momoi, Kanae; Hosoyamada, Makoto

2008-03-15

Divalent metal transporter 1 (DMT1) is a mammalian iron (Fe) transporter and also transports Cadmium (Cd) in vitro. This study compared Cd absorption in DMT1-dysfunctional MK/Rej-{sup mk}/{sub mk} mice (mk/mk mice) and in DMT1-functional, Fe-deficient wild-type (WT) mice, to clarify the role of DMT1 in intestinal Cd absorption in vivo. Mice were given 1 ppm CdCl{sub 2} aq in drinking water for 2 weeks, and the concentrations of Cd and Fe in liver, kidney, and intestinal epithelium were subsequently determined. The Fe concentration in intestinal epithelia of WT mice was decreased in proportion to the level of dietary Fe limitation,more » while Cd accumulation under the same conditions was increased. DMT1 mRNA expression in the small intestine of Fe-deficient WT mice was clearly increased compared to that in Fe-sufficient WT mice. Iron deficiency resulted in up-regulation of Cd uptake in the intestine of Fe-deficient WT mice. The mk/mk mice have a mutation in DMT1 and loss of its function led to decreased intestinal Fe concentration. However, intestinal Cd accumulation was the same as in WT mice and it was also increased in Fe-deficient situation. There is the possibility that an unknown Cd pathway has taken a role on Cd intestinal absorption in vivo and that this pathway is regulated by food Fe concentrations. Therefore, DMT1 is not the sole transporter of intestinal cadmium absorption in vivo.« less

Down-regulation of Akt by methanol extracts of Impatiens balsamina L. promotes apoptosis in human oral squamous cell carcinoma cell lines.

PubMed

Shin, Ji-Ae; Ryu, Mi Heon; Kwon, Ki-Han; Choi, BuYoung; Cho, Sung-Dae

2015-07-01

The apoptotic activity of methanol extracts of Impatiens balsamina L. (MEIB) and related mechanisms in human oral squamous cell carcinoma (OSCC) cells have been systematically investigated. The effects of MEIB on human OSCC cell lines were investigated using trypan blue exclusion assay, MTS assay, Western blot, 4'-6-diamidino-2-phenylindole (DAPI) staining, Live/Dead assay, Immunohistochemistry, reverse transcription-polymerase chain reaction, and promoter assay. MEIB decreased cell viability and induced apoptosis in HSC-4 cells. Higher levels of p-Akt expression were observed in OSCC than in normal oral mucosa (NOM), and it correlated with poor survival of the patients. MEIB dephosphorylated p-Akt and decreased Akt expression through proteasome-dependent degradation. LY294002 (PI3K inhibitor) decreased p-Akt and Akt, resulting in enhancing MEIB-induced apoptosis. MEIB down-regulated the expression level of survivin protein at the transcriptional level and YM155 (survivin inhibitor) decreased survivin, which facilitated MEIB-induced apoptosis. MEIB and LY294002 significantly increased Bax, thereby inducing the conformational change, mitochondrial translocation, and oligomerization. In addition, MEIB-induced growth inhibition and apoptosis in OSC-20, another human OSCC cells were mediated by regulating Akt and it downstream targets, survivin and Bax. These results suggest that MEIB may serve as a potential drug candidate for the treatment of human OSCC. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun Yang; Sumi, Daigo; Kumagai, Yoshito

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, amore » 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.« less

Activation of Akt rescues endoplasmic reticulum stress-impaired murine cardiac contractile function via glycogen synthase kinase-3β-mediated suppression of mitochondrial permeation pore opening.

PubMed

Zhang, Yingmei; Xia, Zhi; La Cour, Karissa H; Ren, Jun

2011-11-01

The present study was designed to examine the impact of chronic Akt activation on endoplasmic reticulum (ER) stress-induced cardiac mechanical anomalies, if any, and the underlying mechanism involved. Wild-type and transgenic mice with cardiac-specific overexpression of the active mutant of Akt (Myr-Akt) were subjected to the ER stress inducer tunicamycin (1 or 3 mg/kg). ER stress led to compromised echocardiographic (elevated left ventricular end-systolic diameter and reduced fractional shortening) and cardiomyocyte contractile function, intracellular Ca(2+) mishandling, and cell survival in wild-type mice associated with mitochondrial damage. In vitro ER stress induction in murine cardiomyocytes upregulated the ER stress proteins Gadd153, GRP78, and phospho-eIF2α, and promoted reactive oxygen species production, carbonyl formation, apoptosis, mitochondrial membrane potential loss, and mitochondrial permeation pore (mPTP) opening associated with overtly impaired cardiomyocyte contractile and intracellular Ca(2+) properties. Interestingly, these anomalies were mitigated by chronic Akt activation or the ER chaperon tauroursodeoxycholic acid (TUDCA). Treatment with tunicamycin also dephosphorylated Akt and its downstream signal glycogen synthase kinase 3β (GSK3β) (leading to activation of GSK3β), the effect of which was abrogated by Akt activation and TUDCA. The ER stress-induced cardiomyocyte contractile and mitochondrial anomalies were obliterated by the mPTP inhibitor cyclosporin A, GSK3β inhibitor SB216763, and ER stress inhibitor TUDCA. This research reported the direct relationship between ER stress and cardiomyocyte contractile and mitochondrial anomalies for the first time. Taken together, these data suggest that ER stress may compromise cardiac contractile and intracellular Ca(2+) properties, possibly through the Akt/GSK3β-dependent impairment of mitochondrial integrity.

IL-1β-induced and p38MAPK-dependent activation of the mitogen-activated protein kinase-activated protein kinase 2 (MK2) in hepatocytes: Signal transduction with robust and concentration-independent signal amplification

PubMed Central

Kulawik, Andreas; Engesser, Raphael; Ehlting, Christian; Raue, Andreas; Albrecht, Ute; Hahn, Bettina; Lehmann, Wolf-Dieter; Gaestel, Matthias; Klingmüller, Ursula; Häussinger, Dieter; Timmer, Jens; Bode, Johannes G.

2017-01-01

The IL-1β induced activation of the p38MAPK/MAPK-activated protein kinase 2 (MK2) pathway in hepatocytes is important for control of the acute phase response and regulation of liver regeneration. Many aspects of the regulatory relevance of this pathway have been investigated in immune cells in the context of inflammation. However, very little is known about concentration-dependent activation kinetics and signal propagation in hepatocytes and the role of MK2. We established a mathematical model for IL-1β-induced activation of the p38MAPK/MK2 pathway in hepatocytes that was calibrated to quantitative data on time- and IL-1β concentration-dependent phosphorylation of p38MAPK and MK2 in primary mouse hepatocytes. This analysis showed that, in hepatocytes, signal transduction from IL-1β via p38MAPK to MK2 is characterized by strong signal amplification. Quantification of p38MAPK and MK2 revealed that, in hepatocytes, at maximum, 11.3% of p38MAPK molecules and 36.5% of MK2 molecules are activated in response to IL-1β. The mathematical model was experimentally validated by employing phosphatase inhibitors and the p38MAPK inhibitor SB203580. Model simulations predicted an IC50 of 1–1.2 μm for SB203580 in hepatocytes. In silico analyses and experimental validation demonstrated that the kinase activity of p38MAPK determines signal amplitude, whereas phosphatase activity affects both signal amplitude and duration. p38MAPK and MK2 concentrations and responsiveness toward IL-1β were quantitatively compared between hepatocytes and macrophages. In macrophages, the absolute p38MAPK and MK2 concentration was significantly higher. Finally, in line with experimental observations, the mathematical model predicted a significantly higher half-maximal effective concentration for IL-1β-induced pathway activation in macrophages compared with hepatocytes, underscoring the importance of cell type-specific differences in pathway regulation. PMID:28223354

[Exendin-4 promotes paracrine action of adipose-derived stem cells through PI3K/Akt signaling pathways].

PubMed

Zhou, Hao; Yang, Junjie; Wagn, Jing; Hu, Shunying; Chen, Guanghui; Chen, Yundai

2014-10-01

To investigate the mechanism by which exendin-4 promotes paracrine secretion of cytokines by adipose-derived stem cells (ADSCs). In vitro cultured SD rat ADSCs (fourth passage) with or without exendin-4 treatment underwent flow cytometry to characterize the surface markers. MTT assay was performed to assess the proliferation of the cells exposed to different concentrations (0-20 nm/L) of exendin-4, and the paracrine secretion of cytokines (bFGF, VEGF, HGF, and IGF-1) by the ADSCs was evaluated by qPCR. The changes in the expressions of p-Akt in the cells were analyzed by Western blotting and qPCR in response to exendin-4 (10 nm/L) with or without exposure to PI3K/Akt inhibitor LY-294002 (50 nm/L); bFGF, VEGF, HGF, and IGF-1 production in the cells were detected using ELISA kits. Treatment with exendin-4 for 12 h did not affect the surface marker profile of the ADSCs but promoted the cell proliferation (P<0.05). Exendin-4 significantly increased the mRNA expressions of VEGF, bFGF, HGF, and IGF-1 in a concentration-dependent manner, and 10 nm/L was the optimum concentration (P<0.05). Exendin-4 treatment resulted in significantly increased p-Akt expressions in the ADSCs, and PI3K/Akt inhibitor not only reversed such effects of exendin-4 on p-Akt but also diminished the exendin-4- mediated up-regulation of the paracrine cytokines. Exendin-4 can concentration-dependently promote the proliferative and paracrine capacities of ADSCs partially through the PI3K/Akt signaling pathway without affecting the surface marker profile of the cells.

Hepatic Warm Ischemia-Reperfusion-Induced Increase in Pulmonary Capillary Filtration Is Ameliorated by Administration of a Multidrug Resistance-Associated Protein 1 Inhibitor and Leukotriene D4 Antagonist (MK-571) Through Reducing Neutrophil Infiltration and Pulmonary Inflammation and Oxidative Stress in Rats.

PubMed

Yeh, D Y-W; Yang, Y-C; Wang, J-J

2015-05-01

Hepatopulmonary syndrome (HPS) is the major complication subsequent to liver ischemia and reperfusion (I/R) injury after resection or transplantation of liver. Hallmarks of HPS include increases in pulmonary leukotrienes and neutrophil recruitment and infiltrating across capillaries. We aimed to investigate the protective efficacy of MK-571, a multidrug resistance-associated protein 1 inhibitor and leukotriene D4 agonist, against hepatic I/R injury-associated change in capillary filtration. Eighteen Sprague-Dawley male rats were evenly divided into a sham-operated group, a hepatic I/R group, and an MK-571-treated I/R group. MK-571 was administered intraperitoneally 15 min before hepatic ischemia and every 12 hours during reperfusion. Ischemia was conducted by occluding the hepatic artery and portal vein for 30 min, followed by removing the clamps and closing the incision. Forty-eight hours after hepatic ischemia, we assessed the pulmonary capillary filtration coefficient (Kfc) through the use of in vitro-isolated, perfused rat lung preparation. We also measured the lung wet-to-dry weight ratio (W/D) and protein concentration in broncho-alveolar lavage fluid (PCBAL). Lung inflammation and oxidative stress were evaluated by use of tissue tumor necrosis factor (TNF)-α and malondialdehyde levels and lavage differential macrophage and neutrophil cell count. Hepatic I/R injury markedly increased Kfc, W/D, PCBAL, tissue TNF-α level, and differential neutrophil cell count (P < .05). MK-571 treatment reduced neutrophil infiltration and lung inflammation and improved pulmonary capillary filtration, collectively suggesting lung protection. Treatment with MK-571 before and during hepatic ischemia and reperfusion protects lung against pulmonary capillary barrier function impairment through decreasing pulmonary lung inflammation and lavage neutrophils. Copyright © 2015 Elsevier Inc. All rights reserved.

Differential roles of ERRFI1 in EGFR and AKT pathway regulation affect cancer proliferation.

PubMed

Cairns, Junmei; Fridley, Brooke L; Jenkins, Gregory D; Zhuang, Yongxian; Yu, Jia; Wang, Liewei

2018-03-01

AKT signaling is modulated by a complex network of regulatory proteins and is commonly deregulated in cancer. Here, we present a dual mechanism of AKT regulation by the ERBB receptor feedback inhibitor 1 (ERRFI1). We show that in cells expressing high levels of EGFR, ERRF1 inhibits growth and enhances responses to chemotherapy. This is mediated in part through the negative regulation of AKT signaling by direct ERRFI1-dependent inhibition of EGFR In cells expressing low levels of EGFR, ERRFI1 positively modulates AKT signaling by interfering with the interaction of the inactivating phosphatase PHLPP with AKT, thereby promoting cell growth and chemotherapy desensitization. These observations broaden our understanding of chemotherapy response and have important implications for the selection of targeted therapies in a cell context-dependent manner. EGFR inhibition can only sensitize EGFR-high cells for chemotherapy, while AKT inhibition increases chemosensitivity in EGFR-low cells. By understanding these mechanisms, we can take advantage of the cellular context to individualize antineoplastic therapy. Finally, our data also suggest targeting of EFFRI1 in EGFR-low cancer as a promising therapeutic approach. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

Epidermal growth factor–stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis

PubMed Central

Garay, Camilo; Judge, Gurjeet; Lucarelli, Stefanie; Bautista, Stephen; Pandey, Rohan; Singh, Tanveer; Antonescu, Costin N.

2015-01-01

Epidermal growth factor (EGF) binding to its receptor (EGFR) activates several signaling intermediates, including Akt, leading to control of cell survival and metabolism. Concomitantly, ligand-bound EGFR is incorporated into clathrin-coated pits—membrane structures containing clathrin and other proteins—eventually leading to receptor internalization. Whether clathrin might regulate EGFR signaling at the plasma membrane before vesicle scission is poorly understood. We compared the effect of clathrin perturbation (preventing formation of, or receptor recruitment to, clathrin structures) to that of dynamin2 (allowing formation of clathrin structures but preventing EGFR internalization) under conditions in which EGFR endocytosis is clathrin dependent. Clathrin perturbation by siRNA gene silencing, with the clathrin inhibitor pitstop2, or knocksideways silencing inhibited EGF-simulated Gab1 and Akt phosphorylation in ARPE-19 cells. In contrast, perturbation of dynamin2 with inhibitors or by siRNA gene silencing did not affect EGF-stimulated Gab1 or Akt phosphorylation. EGF stimulation enriched Gab1 and phospho-Gab1 within clathrin structures. ARPE-19 cells have low ErbB2 expression, and overexpression and knockdown experiments revealed that robust ErbB2 expression bypassed the requirement for clathrin for EGF-stimulated Akt phosphorylation. Thus clathrin scaffolds may represent unique plasma membrane signaling microdomains required for signaling by certain receptors, a function that can be separated from vesicle formation. PMID:26246598

Silencing of secretory clusterin sensitizes NSCLC cells to V-ATPase inhibitors by downregulating survivin.

PubMed

Kim, Young-Sun; Jin, Hyeon-Ok; Hong, Sung-Eun; Song, Jie-Young; Hwang, Chang-Sun; Park, In-Chul

2018-01-08

Secretory clusterin (sCLU) is a stress-associated protein that confers resistance to therapy when overexpressed. In this study, we observed that the V-ATPase inhibitors bafilomycin A1 and concanamycin A significantly stimulated sCLU protein expression. Knockdown of sCLU with siRNA sensitized non-small cell lung cancer (NSCLC) cells to bafilomycin A1, suggesting that sCLU expression renders cells resistant to V-ATPase inhibitors. The dual PI3K/AKT and mTOR inhibitor BEZ235 suppressed sCLU expression and enhanced cell sensitivity induced by bafilomycin A1. Notably, sCLU knockdown further decreased the expression of the survivin protein by bafilomycin A1, and the ectopic expression of survivin alleviated the cell sensitivity by bafilomycin A1 and sCLU depletion, suggesting that increased sensitivity to sCLU depletion in the cells with V-ATPase inhibitors is due, at least in part, to the down-regulation of survivin. Taken together, we demonstrated that the depletion of sCLU expression enhances the sensitivity of NSCLC cells to V-ATPase inhibitors by decreasing survivin expression. Inhibition of the PI3K/AKT/mTOR pathway enhances the sensitivity of NSCLC cells to V-ATPase inhibitors, leading to decreased sCLU and survivin expression. Thus, we suggest that a combination of PI3K/AKT/mTOR inhibitors with V-ATPase inhibitors might be an effective approach for NSCLC treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

The Bonn MK IV Correlator Project

NASA Astrophysics Data System (ADS)

Alef, W.; Graham, D. A.; Zensus, J. A.; Müskens, A.; Schlüter, W.

2000-05-01

We describe the present status of the VLBI correlator in Bonn. The old MK III correlator has been made Y2K compliant, but it is expected to be taken out of operation this year. We report our first experience with the new MK IV correlator, jointly operated by the MPIfR and the BKG, as well as our short-term and long-term plans, both with respect to astronomical and geodetic requirements.

Rhynchophylline Ameliorates Endothelial Dysfunction via Src-PI3K/Akt-eNOS Cascade in the Cultured Intrarenal Arteries of Spontaneous Hypertensive Rats

PubMed Central

Hao, Hui-Feng; Liu, Li-Mei; Pan, Chun-Shui; Wang, Chuan-She; Gao, Yuan-Sheng; Fan, Jing-Yu; Han, Jing-Yan

2017-01-01

Objectives: To examine the protective effect of Rhynchophylline (Rhy) on vascular endothelial function in spontaneous hypertensive rats (SHRs) and the underlying mechanism. Methods: Intrarenal arteries of SHRs and Wistar rats were suspended in myograph for force measurement. Expression and phosphorylation of endothelial nitric oxide (NO) synthase (eNOS), Akt, and Src kinase (Src) were examined by Western blotting. NO production was assayed by ELISA. Results: Rhy time- and concentration-dependently improved endothelium-dependent relaxation in the renal arteries from SHRs, but had no effect on endothelium-independent relaxation in SHR renal arteries. Wortmannin (an inhibitor of phosphatidylinositol 3-kinase) or PP2 (an inhibitor of Src) inhibited the improvement of relaxation in response to acetylcholine by 12 h-incubation with 300 μM Rhy. Western blot analysis revealed that Rhy elevated phosphorylations of eNOS, Akt, and Src in SHR renal arteries. Moreover, wortmannin reversed the increased phosphorylations of Akt and eNOS induced by Rhy, but did not affect the phosphorylation of Src. Furthermore, the enhanced phosphorylations of eNOS, Akt, and Src were blunted by PP2. Importantly, Rhy increased NO production and this effect was blocked by inhibition of Src or PI3K/Akt. Conclusion: The present study provides evidences for the first time that Rhy ameliorates endothelial dysfunction in SHRs through the activation of Src-PI3K/Akt-eNOS signaling pathway. PMID:29187825

The contribution of DNA replication stress marked by high-intensity, pan-nuclear γH2AX staining to chemosensitization by CHK1 and WEE1 inhibitors.

PubMed

Parsels, Leslie A; Parsels, Joshua D; Tanska, Daria M; Maybaum, Jonathan; Lawrence, Theodore S; Morgan, Meredith A

2018-06-12

Small molecule inhibitors of the checkpoint proteins CHK1 and WEE1 are currently in clinical development in combination with the antimetabolite gemcitabine. It is unclear, however, if there is a therapeutic advantage to CHK1 vs. WEE1 inhibition for chemosensitization. The goals of this study were to directly compare the relative efficacies of the CHK1 inhibitor MK8776 and the WEE1 inhibitor AZD1775 to sensitize pancreatic cancer cell lines to gemcitabine and to identify pharmacodynamic biomarkers predictive of chemosensitization. Cells treated with gemcitabine and either MK8776 or AZD1775 were first assessed for clonogenic survival. With the exception of the homologous recombination-defective Capan1 cells, which were relatively insensitive to MK8776, we found that these cell lines were similarly sensitized to gemcitabine by CHK1 or WEE1 inhibition. The abilities of either the CDK1/2 inhibitor roscovitine or exogenous nucleosides to prevent MK8776 or AZD1775-mediated chemosensitization, however, were both inhibitor-dependent and variable among cell lines. Given the importance of DNA replication stress to gemcitabine chemosensitization, we next assessed high-intensity, pan-nuclear γH2AX staining as a pharmacodynamic marker for sensitization. In contrast to total γH2AX, aberrant mitotic entry or sub-G1 DNA content, high-intensity γH2AX staining correlated with chemosensitization by either MK8776 or AZD1775 (R 2 0.83 - 0.53). In summary, we found that MK8776 and AZD1775 sensitize to gemcitabine with similar efficacy. Furthermore, our results suggest that the effects of CHK1 and WEE1 inhibition on gemcitabine-mediated replication stress best predict chemosensitization and support the use of high-intensity or pan-nuclear γH2AX staining as a marker for therapeutic response.

Critical role of endogenous Akt/IAPs and MEK1/ERK pathways in counteracting endoplasmic reticulum stress-induced cell death.

PubMed

Hu, Ping; Han, Zhang; Couvillon, Anthony D; Exton, John H

2004-11-19

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of many diseases and in cancer therapy. Although the unfolded protein response is known to alleviate ER stress by reducing the accumulation of misfolded proteins, the exact survival elements and their downstream signaling pathways that directly counteract ER stress-stimulated apoptotic signaling remain elusive. Here, we have shown that endogenous Akt and ERK are rapidly activated and act as downstream effectors of phosphatidylinositol 3-kinase in thapsigargin- or tunicamycin-induced ER stress. Introduction of either dominant-negative Akt or MEK1 or the inhibitors LY294002 and U0126 sensitized cells to ER stress-induced cell death in different cell types. Reverse transcription-PCR analysis of gene expression during ER stress revealed that cIAP-2 and XIAP, members of the IAP family of potent caspase suppressors, were strongly induced. Transcription of cIAP-2 and XIAP was up-regulated by the phosphatidylinositol 3-kinase/Akt pathway as shown by its reversal by dominant-negative Akt or LY294002. Ablation of these IAPs by RNA interference sensitized cells to ER stress-induced death, which was reversed by the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone. The protective role of IAPs in ER stress coincided with Smac release from mitochondria to the cytosol. Furthermore, it was shown that mTOR was not required for Akt-mediated survival. These results represent the first demonstration that activation of endogenous Akt/IAPs and MEK/ERK plays a critical role in controlling cell survival by resisting ER stress-induced cell death signaling.

I(2)(PP2A) regulates p53 and Akt correlatively and leads the neurons to abort apoptosis.

PubMed

Liu, Gong-Ping; Wei, Wei; Zhou, Xin; Zhang, Yao; Shi, Hai-Hong; Yin, Jun; Yao, Xiu-Qing; Peng, Cai-Xia; Hu, Juan; Wang, Qun; Li, Hong-Lian; Wang, Jian-Zhi

2012-02-01

A chronic neuron loss is the cardinal pathology in Alzheimer disease (AD), but it is still not understood why most neurons in AD brain do not accomplish apoptosis even though they are actually exposed to an environment with enriched proapoptotic factors. Protein phosphatase-2A inhibitor-2 (I(2)(PP2A)), an endogenous PP2A inhibitor, is significantly increased in AD brain, but the role of I(2)(PP2A) in AD-like neuron loss is elusive. Here, we show that I(2)(PP2A) regulates p53 and Akt correlatively. The mechanisms involve activated transcription and p38 MAPK activities. More importantly, we demonstrate that the simultaneous activation of Akt induced by I(2)(PP2A) counteracts the hyperactivated p53-induced cell apoptosis. Furthermore, I(2)(PP2A), p53 and Akt are all elevated in the brain of mouse model and AD patients. Our results suggest that the increased I(2)(PP2A) may trigger apoptosis by p53 upregulation, but due to simultaneous activation of Akt, the neurons are aborted from the apoptotic pathway. This finding contributes to the understanding of why most neurons in AD brain do not undergo apoptosis. Copyright © 2010. Published by Elsevier Inc.

Activation of AKT1/GSK-3β/β-Catenin-TRIM11/Survivin Pathway by Novel GSK-3β Inhibitor Promotes Neuron Cell Survival: Study in Differentiated SH-SY5Y Cells in OGD Model.

PubMed

Darshit, B S; Ramanathan, M

2016-12-01

The objective of this study is to elucidate the effect of a new glycogen synthase kinase-3β (GSK-3β) inhibitor in RA differentiated SH-SY5Y cells in oxygen and glucose deprivation (OGD) model. The pathway involved in GSK-3β signaling during OGD was measured to elucidate the mechanism of action. The differentiation of SH-SY5Y into mature neuronal cells was done with retinoic acid. During differentiation, upregulation of the growth-associated protein 43 (GAP43), neurogenin1 (NGN1), neuronal differentiation 2 (NeuroD2), and tripartite motif containing 11 (TRIM11) genes were observed. Twelve hours of optimal OGD exposure resulted in the alteration of GSK-3β functions of the neuron cells. Of the five molecules selected for this study, molecule G3 showed better effect in the initial phase of the study. Hence, G3 (0.5, 1, and 5 μM) was selected for further study in the OGD model. The standard GSK-3β inhibitor, AR-A014418 (1 μM), was used for comparison. Molecules were pretreated (30 min) and cotreated during OGD exposure. GSK-3β inhibitors showed antiapoptotic activity as evidenced by reduced caspase-3 enzyme activity and increased survivin transcription, as well as improved membrane integrity, evidenced by LDH assay. The inhibitor molecules also up-regulated survival AKT1/GSK-3β/β-catenin pathway and stabilized β-catenin. Inhibition of GSK-3β maintained neuronal survival by upregulating GAP43, Ngn1, and NeuroD2 gene transcription. Further GSK-3β inhibition reduced the TRIM11 gene transcription. In conclusion, both inhibitors have been found to control apoptosis and maintain neuronal functioning and this effect might have been mediated through AKT1/GSK-3β/β-catenin-TRIM11/survivin pathway.

Analysis of the activation status of Akt, NFkappaB, and Stat3 in human diffuse gliomas.

PubMed

Wang, Huamin; Wang, Hua; Zhang, Wei; Huang, Helen J; Liao, Warren S L; Fuller, Gregory N

2004-08-01

Loss of phosphatase and tensin homolog (PTEN) and amplification of the epidermal growth factor receptor (EGFR) gene contribute to the progression of gliomas. As downstream targets of the PTEN and EGFR signaling pathways, Akt, NFkappaB, and signal transducer and activator of transcription-3 (Stat3) have been shown to play important roles in the control of cell proliferation, apoptosis, and oncogenesis. We examined the activation status of Akt, NFkappaB, and Stat3 in 259 diffuse gliomas using tissue microarrays and immunohistochemistry, and evaluated their association with glioma grade. We observed significant positive correlations between the activation status of Akt and NFkappaB and glioma grade. In contrast, only focal immunoreactivity for phospho-Stat3 was observed in < 9% of high-grade gliomas. In addition, we observed a significant correlation between the activation of Akt and NFkappaB. Functional correlation between Akt activation and the activation of NFkappaB was confirmed in U251MG GBM cells in which inhibition of Akt activation either by stable expression of PTEN or by the PI3-kinase inhibitors, wortmannin and LY294002, led to a concomitant decrease in NFkappaB-binding activity. Thus, our results demonstrate that constitutive activation of Akt and NFkappaB, but not Stat3, contributes significantly to the progression of diffuse gliomas, and activation of Akt may lead to NFkappaB activation in high-grade gliomas.

Akt Inhibitor MK2206, Lapatinib Ditosylate, and Trastuzumab in Treating Patients With Locally Advanced or Metastatic HER2-Positive Breast , Gastric, or Gastroesophageal Cancer That Cannot Be Removed By Surgery

ClinicalTrials.gov

2013-09-27

Adenocarcinoma of the Gastroesophageal Junction; HER2-positive Breast Cancer; Male Breast Cancer; Recurrent Breast Cancer; Recurrent Esophageal Cancer; Recurrent Gastric Cancer; Stage IIIC Breast Cancer; Stage IIIC Esophageal Cancer; Stage IIIC Gastric Cancer; Stage IV Breast Cancer; Stage IV Esophageal Cancer; Stage IV Gastric Cancer

Discovery of 1-(3-aryl-4-chlorophenyl)-3-(p-aryl)urea derivatives against breast cancer by inhibiting PI3K/Akt/mTOR and Hedgehog signalings.

PubMed

Li, Wenlu; Sun, Qinsheng; Song, Lu; Gao, Chunmei; Liu, Feng; Chen, Yuzong; Jiang, Yuyang

2017-12-01

PI3K/Akt/mTOR and hedgehog (Hh) signalings are two important pathways in breast cancer, which are usually connected with the drug resistance and cancer migration. Many studies indicated that PI3K/Akt/mTOR inhibitors and Hh inhibitors displayed synergistic effects, and the combination of the two signaling drugs could delay drug resistance and inhibit cancer migration in breast cancer. Therefore, the development of molecules simultaneously inhibiting these two pathways is urgent needed. Based on the structures of PI3K inhibitor buparlisib and Hh inhibitor vismodegib, a series of hybrid structures were designed and synthesized utilizing rational drug design and computer-based drug design. Several compounds displayed excellent antiproliferative activities against several breast cancer cell lines, including triple-negative breast cancer (TNBC) MDA-MB-231 cell. Further mechanistic studies demonstrated that the representative compound 9i could inhibit both PI3K/Akt/mTOR and hedgehog (Hh) signalings by inhibiting the phosphorylation of S6K and Akt as well as decreasing the SAG elevated expression of Gli1. Compound 9i could also induce apoptosis remarkably in T47D and MDA-MB-231 cells. In the transwell assay, 9i showed significant inhibition on the migration of MDA-MB-231. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Akt regulates the subcellular localization of the Rab27a-binding protein JFC1 by phosphorylation.

PubMed

Johnson, Jennifer L; Pacquelet, Sandrine; Lane, William S; Eam, Boreth; Catz, Sergio D

2005-08-01

Here, we show that the Rab27a-binding protein JFC1/Slp1 (synaptotagmin-like protein) is regulated by Akt-mediated phosphorylation. Using the phosphatase and tensin homolog-null LNCaP cells and the phosphatidylinositol 3-kinase inhibitor LY294002, we show that the phosphorylation of endogenous JFC1 is dependent on the phosphatidylinositol 3-kinase/Akt pathway. JFC1 was phosphorylated in cells expressing a constitutively active Akt, confirming that it is an Akt substrate in vivo. Direct phosphorylation of JFC1 by Akt was confirmed in vitro. Using microcapillary high-performance liquid chromatography tandem mass spectrometry, we identified five Akt-phosphorylation sites in JFC1. By mutagenesis analysis and subsequent immunoprecipitation (IP), we established that Akt phosphorylates JFC1 at serine 241. JFC1 and Rab27a colocalize in the proximity of the plasma membrane in LNCaP cells. The interaction was confirmed by IP analysis and was abolished by the point mutation W83S in JFC1. Phosphorylation did not alter the ability of JFC1 to bind to Rab27a. Instead, phosphorylation by Akt dramatically decreased when JFC1 was bound to Rab27a. Finally, we show that as a consequence of in vivo phosphorylation, JFC1 dissociates from the membrane, promoting JFC1 redistribution to the cytosol. Our results suggest that Akt regulates JFC1/Slp1 function by phosphorylation and may have implications on Rab27a-containing vesicle secretion.

Photoactivation of Akt1/GSK3β Isoform-Specific Signaling Axis Promotes Pancreatic β-Cell Regeneration.

PubMed

Huang, Lei; Jiang, Xiaoxiao; Gong, Longlong; Xing, Da

2015-08-01

Promotion of insulin-secreting β-cell regeneration in patients with diabetes is a promising approach for diabetes therapy, which can contribute to rescue the uncontrolled hyperglycemia. Low-power laser irradiation (LPLI) has been demonstrated to regulate multiple physiological processes both in vitro and in vivo through activation of various signaling pathways. In the present study, we showed that LPLI promoted β-cell replication and cell cycle progression through activation of Akt1/GSK3β isoform-specific signaling axis. Inhibition of PI3-K/Akt or GSK3 with specific inhibitors dramatically reduced or increased LPLI-induced β-cell replication, revealing Akt/GSK3 signaling axis was involved in β-cell replication and survival upon LPLI treatment. Furthermore, the results of shRNA-mediated knock down of Akt/GSK3 isoforms revealed that Akt1/GSK3β isoform-specific signaling axis regulated β-cell replication and survival in response to LPLI, but not Akt2/GSK3α. The mechanism by which LPLI promoted β-cell replication through Akt1/GSK3β signaling axis involved activation of β-catenin and down-regulation of p21. Taken together, these observations suggest that Akt1/GSK3β isoform signaling axis play a key role in β-cell replication and survival induced by LPLI. Moreover, our findings suggest that activation of Akt1/GSK3β isoform signaling axis by LPLI may provide guidance in practical applications for β-cell regenerative therapies. © 2015 Wiley Periodicals, Inc.

Pan-mTOR inhibitor MLN0128 is effective against intrahepatic cholangiocarcinoma in mice.

PubMed

Zhang, Shanshan; Song, Xinhua; Cao, Dan; Xu, Zhong; Fan, Biao; Che, Li; Hu, Junjie; Chen, Bin; Dong, Mingjie; Pilo, Maria G; Cigliano, Antonio; Evert, Katja; Ribback, Silvia; Dombrowski, Frank; Pascale, Rosa M; Cossu, Antonio; Vidili, Gianpaolo; Porcu, Alberto; Simile, Maria M; Pes, Giovanni M; Giannelli, Gianluigi; Gordan, John; Wei, Lixin; Evert, Matthias; Cong, Wenming; Calvisi, Diego F; Chen, Xin

2017-12-01

Intrahepatic cholangiocarcinoma (ICC) is a lethal malignancy without effective treatment options. MLN0128, a second generation pan-mTOR inhibitor, shows efficacy for multiple tumor types. We evaluated the therapeutic potential of MLN0128 vs. gemcitabine/oxaliplatin in a novel ICC mouse model. We established a novel ICC mouse model via hydrodynamic transfection of activated forms of AKT (myr-AKT) and Yap (YapS127A) protooncogenes (that will be referred to as AKT/YapS127A). Genetic approaches were applied to study the requirement of mTORC1 and mTORC2 in mediating AKT/YapS127A driven tumorigenesis. Gemcitabine/oxaliplatin and MLN0128 were administered in AKT/YapS127A tumor-bearing mice to study their anti-tumor efficacy in vivo. Multiple human ICC cell lines were used for in vitro experiments. Hematoxylin and eosin staining, immunohistochemistry and immunoblotting were applied for the characterization and mechanistic study. Co-expression of myr-AKT and YapS127A promoted ICC development in mice. Both mTORC1 and mTORC2 complexes were required for AKT/YapS127A ICC development. Gemcitabine/oxaliplatin had limited efficacy in treating late stage AKT/YapS127A ICC. In contrast, partial tumor regression was achieved when MLN0128 was applied in the late stage of AKT/YapS127A cholangiocarcinogenesis. Furthermore, when MLN0128 was administered in the early stage of AKT/YapS127A carcinogenesis, it led to disease stabilization. Mechanistically, MLN0128 efficiently inhibited AKT/mTOR signaling both in vivo and in vitro, inducing strong ICC cell apoptosis and only marginally affecting proliferation. This study suggests that mTOR kinase inhibitors may be beneficial for the treatment of ICC, even in tumors that are resistant to standard of care chemotherapeutics, such as gemcitabine/oxaliplatin-based regimens, especially in the subset of tumors exhibiting activated AKT/mTOR cascade. Lay summary: We established a novel mouse model of intrahepatic cholangiocarcinoma (ICC). Using this

Kaposi sarcoma associated herpesvirus (KSHV) induces AKT hyperphosphorylation, bortezomib-resistance and GLUT-1 plasma membrane exposure in THP-1 monocytic cell line.

PubMed

Gonnella, Roberta; Santarelli, Roberta; Farina, Antonella; Granato, Marisa; D'Orazi, Gabriella; Faggioni, Alberto; Cirone, Mara

2013-10-23

Phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway regulates multiple cellular processes such as cell proliferation, evasion from apoptosis, migration, glucose metabolism, protein synthesis and proper differentiation in immune cells. Kaposi sarcoma-associated herpesvirus (KSHV), an oncogenic virus associated with several human malignancies, expresses a variety of latent and lytic proteins able to activate PI3K/AKT pathway, promoting the growth of infected cells and a successful viral infection. We found that KSHV latent infection of THP-1 cells, a human monocytic cell line derived from an acute monocytic leukemia patient, resulted in an increase of AKT phoshorylation, not susceptible to bortezomib-induced dephosphorylation, compared to the mock-infected THP-1. Accordingly, THP-1-infected cells displayed increased resistance to the bortezomib cytotoxic effect in comparison to the uninfected cells, which was counteracted by pre-treatment with AKT-specific inhibitors. Finally, AKT hyperactivation by KSHV infection correlated with plasma membrane exposure of glucose transporter GLUT1, particularly evident during bortezomib treatment. GLUT1 membrane trafficking is a characteristic of malignant cells and underlies a change of glucose metabolism that ensures the survival to highly proliferating cells and render these cells highly dependent on glycolysis. GLUT1 membrane trafficking in KSHV-infected THP-1 cells indeed led to increased sensitivity to cell death induced by the glycolysis inhibitor 2-Deoxy-D-glucose (2DG), further potentiated by its combination with bortezomib. KSHV confers to the THP-1 infected cells an oncogenic potential by altering the phosphorylation, expression and localization of key molecules that control cell survival and metabolism such as AKT and GLUT1. Such modifications in one hand lead to resistance to cell death induced by some chemotherapeutic drugs such as bortezomib, but on the other hand, offer an

Involvement of PI3K/AKT and MAPK Pathways for TNF-α Production in SiHa Cervical Mucosal Epithelial Cells Infected with Trichomonas vaginalis.

PubMed

Yang, Jung-Bo; Quan, Juan-Hua; Kim, Ye-Eun; Rhee, Yun-Ee; Kang, Byung-Hyun; Choi, In-Wook; Cha, Guang-Ho; Yuk, Jae-Min; Lee, Young-Ha

2015-08-01

Trichomonas vaginalis; induces proinflammation in cervicovaginal mucosal epithelium. To investigate the signaling pathways in TNF-α production in cervical mucosal epithelium after T. vaginalis infection, the phosphorylation of PI3K/AKT and MAPK pathways were evaluated in T. vaginalis-infected SiHa cells in the presence and absence of specific inhibitors. T. vaginalis increased TNF-α production in SiHa cells, in a parasite burden-dependent and incubation time-dependent manner. In T. vaginalis-infected SiHa cells, AKT, ERK1/2, p38 MAPK, and JNK were phosphorylated from 1 hr after infection; however, the phosphorylation patterns were different from each other. After pretreatment with inhibitors of the PI3K/AKT and MAPK pathways, TNF-α production was significantly decreased compared to the control; however, TNF-α reduction patterns were different depending on the type of PI3K/MAPK inhibitors. TNF-α production was reduced in a dose-dependent manner by treatment with wortmannin and PD98059, whereas it was increased by SP600125. These data suggested that PI3K/AKT and MAPK signaling pathways are important in regulation of TNF-α production in cervical mucosal epithelial SiHa cells. However, activation patterns of each pathway were different from the types of PI3K/MAPK pathways.

Diallyl trisulfide exerts cardioprotection against myocardial ischemia-reperfusion injury in diabetic state, role of AMPK-mediated AKT/GSK-3β/HIF-1α activation

PubMed Central

Yu, Liming; Di, Wencheng; Dong, Xue; Li, Zhi; Xue, Xiaodong; Zhang, Jian; Wang, Qi; Xiao, Xiong; Han, Jinsong; Yang, Yang; Wang, Huishan

2017-01-01

Diallyl trisulfide (DATS), the major active ingredient in garlic, has been reported to confer cardioprotective effects. However, its effect on myocardial ischemia-reperfusion (MI/R) injury in diabetic state and the underlying mechanism are still unknown. We hypothesize that DATS reduces MI/R injury in diabetic state via AMPK-mediated AKT/GSK-3β/HIF-1α activation. Streptozotocin-induced diabetic rats received MI/R surgery with or without DATS (20mg/kg) treatment in the presence or absence of Compound C (Com.C, an AMPK inhibitor, 0.25mg/kg) or LY294002 (a PI3K inhibitor, 5mg/kg). We found that DATS significantly improved heart function and reduced myocardial apoptosis. Additionally, in cultured H9c2 cells, DATS (10μM) also attenuated simulated ischemia-reperfusion injury. We found that AMPK and AKT/GSK-3β/HIF-1α signaling were down-regulated under diabetic condition, while DATS markedly increased the phosphorylation of AMPK, ACC, AKT and GSK-3β as well as HIF-1α expression in MI/R-injured myocardium. However, these protective actions were all blunted by Com.C administration. Additionally, LY294002 abolished the stimulatory effect of DATS on AKT/GSK-3β/HIF-1α signaling without affecting AMPK signaling. While 2-methoxyestradiol (a HIF-1α inhibitor) reduced HIF-1α expression without affecting AKT/GSK-3β signaling. Taken together, these data showed that DATS protected against MI/R injury in diabetic state by attenuating cellular apoptosis via AMPK-mediated AKT/GSK-3β/HIF-1α signaling. Its cardioprotective effect deserves further study. PMID:29088824

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.

Vitamin E Facilitates the Inactivation of the Kinase Akt by the Phosphatase PHLPP1

PubMed Central

Huang, Po-Hsien; Chuang, Hsiao-Ching; Chou, Chih-Chien; Wang, Huiling; Lee, Su-Lin; Yang, Hsiao-Ching; Chiu, Hao-Chieh; Kapuriya, Naval; Wang, Dasheng; Kulp, Samuel K.; Chen, Ching-Shih

2014-01-01

rationale for the translational development of tocopherols into novel PH domain-targeted Akt inhibitors. PMID:23512990

Involvement of PI3K/Akt Signaling Pathway and Its Downstream Intracellular Targets in the Antidepressant-Like Effect of Creatine.

PubMed

Cunha, Mauricio P; Budni, Josiane; Ludka, Fabiana K; Pazini, Francis L; Rosa, Julia Macedo; Oliveira, Ágatha; Lopes, Mark W; Tasca, Carla I; Leal, Rodrigo B; Rodrigues, Ana Lúcia S

2016-07-01

Creatine has been proposed to exert beneficial effects in the management of depression, but the cell signaling pathways implicated in its antidepressant effects are not well established. This study investigated the involvement of PI3K/Akt signaling pathway and its downstream intracellular targets in the antidepressant-like effect of creatine. The acute treatment of mice with creatine (1 mg/kg, po) increased the Akt and P70S6K phosphorylation, and HO-1, GPx and PSD95 immunocontents. The pretreatment of mice with LY294002 (10 nmol/mouse, icv, PI3K inhibitor), wortmannin (0.1 μg/mouse, icv, PI3K inhibitor), ZnPP (10 μg/mouse, icv, HO-1 inhibitor), or rapamycin (0.2 nmol/mouse, icv, mTOR inhibitor) prevented the antidepressant-like effect of creatine (1 mg/kg, po) in the TST. In addition, the administration of subeffective dose of either the selective GSK3 inhibitor AR-A014418 (0.01 μg/mouse, icv), the nonselective GSK3 inhibitor lithium chloride (10 mg/kg, po), or the HO-1 inductor CoPP (0.01 μg/mouse, icv), in combination with a subeffective dose of creatine (0.01 mg/kg, po) reduced the immobility time in the TST as compared with either drug alone. No treatment caused significant changes in the locomotor activity of mice. These results indicate that the antidepressant-like effect of creatine in the TST depends on the activation of Akt, Nrf2/HO-1, GPx, and mTOR, and GSK3 inhibition.

Carbachol induces p70S6K1 activation through an ERK-dependent but Akt-independent pathway in human colonic epithelial cells.

PubMed

Jiang, Xiaohua; Sinnett-Smith, James; Rozengurt, Enrique

2009-09-25

Stimulation of human colonic epithelial T84 cells with the muscarinic receptor agonist carbachol, a stable analog of acetylcholine, induced Akt, p70S6K1 and ERK activation. Treatment of T84 cells with the selective inhibitor of EGF receptor (EGFR) tyrosine kinase AG1478 abrogated Akt phosphorylation on Ser(473) induced by either carbachol or EGF, indicating that carbachol-induced Akt activation is mediated through EGFR transactivation. Surprisingly, AG1478 did not suppress p70S6K1 phosphorylation on Thr(389) in response to carbachol, indicating the G protein-coupled receptor (GPCR) stimulation induces p70S6K1 activation, at least in part, via an Akt-independent pathway. In contrast, treatment with the selective MEK inhibitor U0126 (but not with the inactive analog U0124) inhibited carbachol-induced p70S6K1 activation, indicating that the MEK/ERK/RSK pathway plays a critical role in p70S6K1 activation in GPCR-stimulated T84 cells. These findings imply that GPCR activation induces p70S6K1 via ERK rather than through the canonical PI 3-kinase/Akt/TSC/mTORC1 pathway in T84 colon carcinoma cells.

Carbachol induces p70S6K1 activation through an ERK-dependent but Akt-independent pathway in human colonic epithelial cells

PubMed Central

Jiang, Xiaohua; Sinnett-Smith, James; Rozengurt, Enrique

2009-01-01

Stimulation of human colonic epithelial T84 cells with the muscarinic receptor agonist carbachol, a stable analog of acetylcholine, induced Akt, p70S6K1 and ERK activation. Treatment of T84 cells with the selective inhibitor of EGF receptor (EGFR) tyrosine kinase AG1478 abrogated Akt phosphorylation on Ser473 induced by either carbachol or EGF, indicating that carbachol-induced Akt activation is mediated through EGFR transactivation. Surprisingly, AG1478 did not suppress p70S6K1 phosphorylation on Thr389 in response to carbachol, indicating the G protein-coupled receptor (GPCR) stimulation induces p70S6K1 activation, at least in part, via an Akt-independent pathway. In contrast, treatment with the selective MEK inhibitor U0126 (but not with the inactive analog U0124) inhibited carbachol-induced p70S6K1 activation, indicating that the MEK/ERK/RSK pathway plays a critical role in p70S6K1 activation in GPCR-stimulated T84 cells. These findings imply that GPCR activation induces p70S6K1 via ERK rather than through the canonical PI 3-kinase/Akt/TSC/mTORC1 pathway in T84 colon carcinoma cells. PMID:19615971

Neuroprotective capabilities of TSA against cerebral ischemia/reperfusion injury via PI3K/Akt signaling pathway in rats.

PubMed

Ma, Xiao-Hui; Gao, Qiang; Jia, Zhen; Zhang, Ze-Wei

2015-02-01

Hundreds of previous studies demonstrated the cytoprotective effect of trichostatin-A (TSA), a kind of histone deacetylases inhibitors (HDACIs), against cerebral ischemia/reperfusion insult. Meanwhile, phosphatidylinositol-3 kinase/Akt (PI3K/Akt) is a well-known, important signaling pathway that mediates neuroprotection. However, it should be remains unclear whether the neuroprotective capabilities of TSA against cerebral ischemia/reperfusion is mediated by activation of the PI3K/Akt signaling pathway. Five groups rats (n = 12 each), with middle cerebral artery occlusion (MCAO) except sham group, were used to investigate the neuroprotective effect of certain concentration (0.05 mg/kg) of TSA, and whether the neuroprotective effect of TSA is associated with activation of the PI3K/Akt signaling pathway through using of wortmannin (0.25 mg/kg). TSA significantly increased the expression of p-Akt protein, reduced infarct volume, and attenuated neurological deficit in rats with transient MCAO, wortmannin weakened such effect of TSA dramatically. TSA could significantly decrease the neurological deficit scores and reduce the cerebral infarct volume during cerebral ischemia/reperfusion injury, which was achieved partly by activation of the PI3K/Akt signaling pathway via upgrading of p-Akt protein.

Chronic exposure of interleukin-13 suppress the induction of matrix metalloproteinase-1 by tumour necrosis factor α in normal and scleroderma dermal fibroblasts through protein kinase B/Akt.

PubMed

Brown Lobbins, M L; Shivakumar, B R; Postlethwaite, A E; Hasty, K A

2018-01-01

Peripheral blood mononuclear cells taken from patients with scleroderma express increased levels of interleukin (IL)-13. Moreover, the expression of matrix metalloproteinase-1 (MMP-1) from involved scleroderma skin fibroblasts is refractory to stimulation by tumour necrosis factor (TNF)-α. To elucidate the mechanism(s) involved, we examined the effect of IL-13 on TNF-α-induced MMP-1 expression in normal and scleroderma human dermal fibroblast lines and studied the involvement of serine/threonine kinase B/protein kinase B (Akt) in this response. Dermal fibroblast lines were stimulated with TNF-α in the presence of varying concentrations of IL-13. Total Akt and pAkt were quantitated using Western blot analyses. Fibroblasts were treated with or without Akt inhibitor VIII in the presence of IL-13 followed by TNF-α stimulation. MMP-1 expression was analysed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed using analysis of variance (anova) or Student's t-test. Upon TNF-α stimulation, normal dermal fibroblasts secrete more MMP-1 than systemic sclerosis (SSc) fibroblasts. This increase in MMP-1 is lost when fibroblasts are co-incubated with IL-13 and TNF-α. IL-13 induced a significant increase in levels of pAkt in dermal fibroblasts, while Akt inhibitor VIII reversed the suppressive effects of IL-13 on the response of cultured fibroblasts to TNF-α, increasing their expression of MMP-1. We show that IL-13 suppresses MMP-1 in TNF-α-stimulated normal and scleroderma dermal fibroblast. Akt inhibitor VIII is able to reverse the suppressive effect of IL-13 on MMP-1 expression and protein synthesis. Our data suggest that IL-13 regulates MMP-1 expression in response to TNF-α through an Akt-mediated pathway and may play a role in fibrotic diseases such as scleroderma. © 2017 British Society for Immunology.

Leptin-induced IL-6 production is mediated by leptin receptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase, Akt, NF-kappaB, and p300 pathway in microglia.

PubMed

Tang, Chih-Hsin; Lu, Da-Yuu; Yang, Rong-Sen; Tsai, Huei-Yann; Kao, Ming-Ching; Fu, Wen-Mei; Chen, Yuh-Fung

2007-07-15

Leptin, the adipocyte-secreted hormone that centrally regulates weight control, is known to function as an immunomodulatory regulator. We investigated the signaling pathway involved in IL-6 production caused by leptin in microglia. Microglia expressed the long (OBRl) and short (OBRs) isoforms of the leptin receptor. Leptin caused concentration- and time-dependent increases in IL-6 production. Leptin-mediated IL-6 production was attenuated by OBRl receptor antisense oligonucleotide, PI3K inhibitor (Ly294002 and wortmannin), Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate)), NF-kappaB inhibitor (pyrrolidine dithiocarbamate), IkappaB protease inhibitor (L-1-tosylamido-2-phenylenylethyl chloromethyl ketone), IkappaBalpha phosphorylation inhibitor (Bay 117082), or NF-kappaB inhibitor peptide. Transfection with insulin receptor substrate (IRS)-1 small-interference RNA or the dominant-negative mutant of p85 and Akt also inhibited the potentiating action of leptin. Stimulation of microglia with leptin activated IkappaB kinase alpha/IkappaB kinase beta, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser(276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. Leptin-mediated an increase of IkappaB kinase alpha/IkappaB kinase beta activity, kappaB-luciferase activity, and p65 and p50 binding to the NF-kappaB element was inhibited by wortmannin, Akt inhibitor, and IRS-1 small-interference RNA. The binding of p65 and p50 to the NF-kappaB elements, as well as the recruitment of p300 and the enhancement of histone H3 and H4 acetylation on the IL-6 promoter was enhanced by leptin. Our results suggest that leptin increased IL-6 production in microglia via the leptin receptor/IRS-1/PI3K/Akt/NF-kappaB and p300 signaling pathway.

M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells.

PubMed

Hara, Yasushi; Obata, Yuuki; Horikawa, Keita; Tasaki, Yasutaka; Suzuki, Kyohei; Murata, Takatsugu; Shiina, Isamu; Abe, Ryo

2017-01-01

Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to

M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells

PubMed Central

Hara, Yasushi; Obata, Yuuki; Horikawa, Keita; Tasaki, Yasutaka; Suzuki, Kyohei; Murata, Takatsugu; Shiina, Isamu; Abe, Ryo

2017-01-01

Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to

T-cell immunotherapy for human MK-1-expressing tumors using a fusion protein of the superantigen SEA and anti-MK-1 scFv antibody.

PubMed

Ueno, Aruto; Arakawa, Fumiko; Abe, Hironori; Matsumoto, Hisanobu; Kudo, Toshio; Asano, Ryutaro; Tsumoto, Kohei; Kumagai, Izumi; Kuroki, Motomu; Kuroki, Masahide

2002-01-01

The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on major histocompatibility complex (MHC) class II molecules. To develop a tumor-specific superantigen for cancer therapy, we constructed a recombinant fusion protein of SEA and the single-chain variable fragment (scFv) of the FU-MK-1 antibody, which recognizes a glycoprotein antigen (termed MK-1 antigen) present on most carcinomas. We employed recombinant DNA techniques to fuse recombinant mutant SEA to an scFv antibody derived from FU-MK-1 and the resulting fusion protein (SEA/FUscFv) was produced by a bacterial expression system, purified with a metal-affinity column, and characterized for its MK-1-binding specificity and its antitumor activity. The SEA/FUscFv fusion protein retained the reactivity with MK-1-expressing tumor cells, introduced a specific cytotoxicity of lymphokine-activated killer T-cells to the tumor cells, and consequently suppressed the tumor growth in a SCID mouse xenograft model. This genetically engineered SEA/FUscFv fusion protein may serve as a potentially useful immunotherapeutic reagent for human MK-1-expressing tumors.

Exercise activates the PI3K-AKT signal pathway by decreasing the expression of 5α-reductase type 1 in PCOS rats.

PubMed

Wu, Chuyan; Jiang, Feng; Wei, Ke; Jiang, Zhongli

2018-05-22

Hyperandrogenism and hyperinsulinemia are main clinical endocrine features of PCOS. Exercise can adjust the androgen level, as well as increase the sensitivity of insulin by activating PI3K-Akt insulin signaling pathways. 5αR1 has certain effects on insulin resistance and can synthesize dihydrotestosterone by metabolizing testosterone. So 5αR1 may be the target of androgen and insulin for exercise-induced regulation. To investigate the role of 5αR1 in the PI3K-Akt signaling pathway in skeletal muscle of PCOS rats activated by exercise, fifty-four female rats were randomly divided into the PCOS group (n = 42) and the control group(n = 12). After injection of testosterone propionate for 28 days, the remaining 36 rats in the PCOS group were randomly assigned to six groups: the sedentary group (PS, n = 6), sedentary and 5αRI (5α-reductase inhibitor) group (PS + RI, n = 6), sedentary and 5αR2I (5α-reductase type 2 selective inhibitor) group (PS + R2I, n = 6), exercise group (PE, n = 6), exercise and 5αRI group (PE + RI, n = 6), and exercise and 5αR2I group (PE + R2I, n = 6). The rats undergoing exercise were trained to swim for 14 days. Finasteride (5α-reductase type 2 selective inhibitor) and dutasteride (5α-reductase inhibitor) were administered once daily and were dosed based on weight. At the end, the expression of 5αR1 proteins, the phosphorylation level of PI3K and AKT, were determined by Western blot. The PCOS non-exercise group and the PE + RI group displayed significantly lower phosphorylation of Akt, PI3K p85 and GLUT4 expression, while in the PE + R2I group, the level of Akt phosphorylation and PI3K p85 expression was significantly higher than that of the PCOS non-exercise group and the PE + RI group. In summary, our study demonstrated that exercise can activate the PI3K/AKT signal pathway of PCOS rats by decreasing the expression of 5αR1.

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

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

PubMed

Penha, Alexandra Marcha; Burkhardt, Eva; Schaeffel, Frank; Feldkaemper, Marita P

2012-01-01

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. 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-Akt(Thr308), 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. 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 suppressed the effects of

MK-STYX, a Catalytically Inactive Phosphatase Regulating Mitochondrially Dependent Apoptosis ▿

PubMed Central

Niemi, Natalie M.; Lanning, Nathan J.; Klomp, Jeff A.; Tait, Stephen W.; Xu, Yong; Dykema, Karl J.; Murphy, Leon O.; Gaither, L. Alex; Xu, H. Eric; Furge, Kyle A.; Green, Douglas R.; MacKeigan, Jeffrey P.

2011-01-01

Evasion of apoptosis is a significant problem affecting an array of cancers. In order to identify novel regulators of apoptosis, we performed an RNA interference (RNAi) screen against all kinases and phosphatases in the human genome. We identified MK-STYX (STYXL1), a catalytically inactive phosphatase with homology to the mitogen-activated protein kinase (MAPK) phosphatases. Despite this homology, MK-STYX knockdown does not significantly regulate MAPK signaling in response to growth factors or apoptotic stimuli. Rather, RNAi-mediated knockdown of MK-STYX inhibits cells from undergoing apoptosis induced by cellular stressors activating mitochondrion-dependent apoptosis. This MK-STYX phenotype mimics the loss of Bax and Bak, two potent guardians of mitochondrial apoptotic potential. Similar to loss of both Bax and Bak, cells without MK-STYX expression are unable to release cytochrome c. Proapoptotic members of the BCL-2 family (Bax, Bid, and Bim) are unable to trigger cytochrome c release in MK-STYX-depleted cells, placing the apoptotic deficiency at the level of mitochondrial outer membrane permeabilization (MOMP). MK-STYX was found to localize to the mitochondria but is neither released from the mitochondria upon apoptotic stress nor proximal to the machinery currently known to control MOMP, indicating that MK-STYX regulates MOMP using a distinct mechanism. PMID:21262771

Evidence for involvement of nitric oxide and GABAB receptors in MK-801- stimulated release of glutamate in rat prefrontal cortex

PubMed Central

Roenker, Nicole L.; Gudelsky, Gary A.; Ahlbrand, Rebecca; Horn, Paul S.; Richtand, Neil M.

2012-01-01

Systemic administration of NMDA receptor antagonists elevates extracellular glutamate within prefrontal cortex. The cognitive and behavioral effects of NMDA receptor blockade have direct relevance to symptoms of schizophrenia, and recent studies demonstrate an important role for nitric oxide and GABAB receptors in mediating the effects of NMDA receptor blockade on these behaviors. We sought to extend those observations by directly measuring the effects of nitric oxide and GABAB receptor mechanisms on MK-801-induced glutamate release in the prefrontal cortex. Systemic MK-801 injection (0.3 mg/kg) to male Sprague-Dawley rats significantly increased extracellular glutamate levels in prefrontal cortex, as determined by microdialysis. This effect was blocked by pretreatment with the nitric oxide synthase inhibitor L-NAME (60 mg/kg). Reverse dialysis of the nitric oxide donor SNAP (0.5 – 5 mM) directly into prefrontal cortex mimicked the effect of systemic MK-801, dose-dependently elevating cortical extracellular glutamate. The effect of MK-801 was also blocked by systemic treatment with the GABAB receptor agonist baclofen (5 mg/kg). In combination, these data suggest increased nitric oxide formation is necessary for NMDA antagonist-induced elevations of extracellular glutamate in the prefrontal cortex. Additionally, the data suggest GABAB receptor activation can modulate the NMDA antagonist-induced increase in cortical glutamate release. PMID:22579658

Down-Regulation by Resveratrol of Basic Fibroblast Growth Factor-Stimulated Osteoprotegerin Synthesis through Suppression of Akt in Osteoblasts

PubMed Central

Kuroyanagi, Gen; Otsuka, Takanobu; Yamamoto, Naohiro; Matsushima-Nishiwaki, Rie; Nakakami, Akira; Mizutani, Jun; Kozawa, Osamu; Tokuda, Haruhiko

2014-01-01

It is firmly established that resveratrol, a natural food compound abundantly found in grape skins and red wine, has beneficial properties for human health. In the present study, we investigated the effect of basic fibroblast growth factor (FGF-2) on osteoprotegerin (OPG) synthesis in osteoblast-like MC3T3-E1 cells and whether resveratrol affects the OPG synthesis. FGF-2 stimulated both the OPG release and the expression of OPG mRNA. Resveratrol significantly suppressed the FGF-2-stimulated OPG release and the mRNA levels of OPG. SRT1720, an activator of SIRT1, reduced the FGF-2-induced OPG release and the OPG mRNA expression. PD98059, an inhibitor of upstream kinase activating p44/p42 mitogen-activated protein (MAP) kinase, had little effect on the FGF-2-stimulated OPG release. On the other hand, SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and Akt inhibitor suppressed the OPG release induced by FGF-2. Resveratrol failed to affect the FGF-2-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. The phosphorylation of Akt induced by FGF-2 was significantly suppressed by resveratrol or SRT1720. These findings strongly suggest that resveratrol down-regulates FGF-2-stimulated OPG synthesis through the suppression of the Akt pathway in osteoblasts and that the inhibitory effect of resveratrol is mediated at least in part by SIRT1 activation. PMID:25290095

PI3K/Akt/mTOR Intracellular Pathway and Breast Cancer: Factors, Mechanism and Regulation.

PubMed

Sharma, Var Ruchi; Gupta, Girish Kumar; Sharma, A K; Batra, Navneet; Sharma, Daljit K; Joshi, Amit; Sharma, Anil K

2017-01-01

The most recurrent and considered second most frequent cause of cancer-related deaths worldwide in women is the breast cancer. The key to diagnosis is early prediction and a curable stage but still treatment remains a great clinical challenge. Origin of the Problem: A number of studies have been carried out for the treatment of breast cancer which includes the targeted therapies and increased survival rates in women. Essential PI3K/mTOR signaling pathway activation has been observed in most breast cancers. The cell growth and tumor development in such cases involve phosphoinositide 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) complex intracellular pathway. Through preclinical and clinical trials, it has been observed that there are a number of other inhibitors of PI3K/Akt/mTOR pathway, which either alone or in combination with cytotoxic agents can be used for endocrine therapies. Structure and regulation/deregulation of mTOR provides a greater insight into the action mechanism. Also, through this review, one could easily scan first and second generation inhibitors for PI3K/Akt/mTOR pathway besides targeted therapies for breast cancer and the precise role of mTOR. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

PubMed Central

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

2010-01-01

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-triphosphate (PIP3) 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 PIP3 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 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. PMID:20875857

Inhibition of SRC-3 enhances sensitivity of human cancer cells to histone deacetylase inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zou, Zhengzhi, E-mail: zouzhengzhi@m.scnu.edu.cn; Luo, Xiaoyong; Nie, Peipei

SRC-3 is widely expressed in multiple tumor types and involved in cancer cell proliferation and apoptosis. Histone deacetylase (HDAC) inhibitors are promising antitumor drugs. However, the poor efficacy of HDAC inhibitors in solid tumors has restricted its further clinical application. Here, we reported the novel finding that depletion of SRC-3 enhanced sensitivity of breast and lung cancer cells to HDAC inhibitors (SAHA and romidepsin). In contrast, overexpression of SRC-3 decreased SAHA-induced cancer cell apoptosis. Furthermore, we found that SRC-3 inhibitor bufalin increased cancer cell apoptosis induced by HDAC inhibitors. The combination of bufalin and SAHA was particular efficient in attenuatingmore » AKT activation and reducing Bcl-2 levels. Taken together, these accumulating data might guide development of new breast and lung cancer therapies. - Highlights: • Depletion of SRC-3 enhanced sensitivity of breast and lung cancer cells to HDAC inhibitors. • Overexpression of SRC-3 enhanced cancer cell resistance to HDAC inhibitors. • SRC-3 inhibitor bufalin increased cancer cell apoptosis induced by HDAC inhibitors. • Bufalin synergized with HDAC inhibitor attenuated AKT activation and reduced Bcl-2 levels in human cancer cell.« less

Deficiency of Akt1, but not Akt2, attenuates the development of pulmonary hypertension

PubMed Central

Tang, Haiyang; Chen, Jiwang; Fraidenburg, Dustin R.; Song, Shanshan; Sysol, Justin R.; Drennan, Abigail R.; Offermanns, Stefan; Ye, Richard D.; Bonini, Marcelo G.; Minshall, Richard D.; Garcia, Joe G. N.; Machado, Roberto F.; Makino, Ayako

2014-01-01

Pulmonary vascular remodeling, mainly attributable to enhanced pulmonary arterial smooth muscle cell proliferation and migration, is a major cause for elevated pulmonary vascular resistance and pulmonary arterial pressure in patients with pulmonary hypertension. The signaling cascade through Akt, comprised of three isoforms (Akt1–3) with distinct but overlapping functions, is involved in regulating cell proliferation and migration. This study aims to investigate whether the Akt/mammalian target of rapamycin (mTOR) pathway, and particularly which Akt isoform, contributes to the development and progression of pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Compared with the wild-type littermates, Akt1−/− mice were protected against the development and progression of chronic HPH, whereas Akt2−/− mice did not demonstrate any significant protection against the development of HPH. Furthermore, pulmonary vascular remodeling was significantly attenuated in the Akt1−/− mice, with no significant effect noted in the Akt2−/− mice after chronic exposure to normobaric hypoxia (10% O2). Overexpression of the upstream repressor of Akt signaling, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and conditional and inducible knockout of mTOR in smooth muscle cells were also shown to attenuate the rise in right ventricular systolic pressure and the development of right ventricular hypertrophy. In conclusion, Akt isoforms appear to have a unique function within the pulmonary vasculature, with the Akt1 isoform having a dominant role in pulmonary vascular remodeling associated with HPH. The PTEN/Akt1/mTOR signaling pathway will continue to be a critical area of study in the pathogenesis of pulmonary hypertension, and specific Akt isoforms may help specify therapeutic targets for the treatment of pulmonary hypertension. PMID:25416384

Ghrelin promotes human non-small cell lung cancer A549 cell proliferation through PI3K/Akt/mTOR/P70S6K and ERK signaling pathways.

PubMed

Zhu, Jianhua; Yao, Jianfeng; Huang, Rongfu; Wang, Yueqin; Jia, Min; Huang, Yan

2018-04-06

Ghrelin is a gastric acyl-peptide that plays an important role in cell proliferation. In the present study, we explored the role of ghrelin in A549 cell proliferation and the possible molecular mechanisms. We found that ghrelin promotes A549 cell proliferation, knockdown of the growth hormone secretagogue receptor (GHSR) attenuated A549 cell proliferation caused by ghrelin. Ghrelin induced the rapid phosphorylation of phosphatidylinositol 3-kinase (PI3K), Akt, ERK, mammalian target of rapamycin (mTOR) and P70S6K. PI3K inhibitor (LY 294002), ERK inhibitor (PD98059) and mTOR inhibitor (Rapamycin) inhibited ghrelin-induced A549 cell proliferation. Moreover, GHSR siRNA inhibited phosphorylation of PI3K, Akt, ERK, mTOR and P70S6K induced by ghrelin. Akt and mTOR/P70S6K phosphorylation was inhibited by LY 294002 but not by PD98059. These results indicate that ghrelin promotes A549 cell proliferation via GHSR-dependent PI3K/Akt/mTOR/P70S6K and ERK signaling pathways. Copyright © 2018 Elsevier Inc. All rights reserved.

Akt phosphorylation and NFkappaB activation are counterregulated under conditions of oxidative stress.

PubMed

Taylor, Juliet M; Crack, Peter J; Gould, Jodee A; Ali, Uğur; Hertzog, Paul J; Iannello, Rocco C

2004-11-01

This study was designed to elucidate the mechanisms involved in elevated cell death arising from an altered endogenous oxidant state. Increased levels of cell death were detected in cells lacking Gpx1 following the addition of exogenous H2O2. This increased apoptosis correlated with a down-regulation in the activation of the PI(3)K-Akt survival pathway. The importance of this pathway in protecting against H2O2-induced cell death was highlighted by the increased susceptibility of wild-type cells to apoptosis when treated with the PI(3)K inhibitor, LY294002. Activation of the oxidative stress sensitive transcription factor, NFkappaB, was elevated in the Gpx1-/- cells. Significantly, NFkappaB activation could be increased in wild-type cells through the addition of dominant-negative Akt. Therefore, our results suggest that the increased susceptibility of Gpx1-/- cells to H2O2-induced apoptosis can be attributed in part to diminished activation of Akt despite an up-regulation in the activation of the prosurvival NFkappaB. Thus, the PI(3)K-Akt and NFkappaB pathways can act independently of each other in an endogenous model of oxidative stress.

Androgen-mediated sex bias impairs efficiency of leukotriene biosynthesis inhibitors in males

PubMed Central

Pace, Simona; Pergola, Carlo; Dehm, Friederike; Rossi, Antonietta; Gerstmeier, Jana; Troisi, Fabiana; Pein, Helmut; Schaible, Anja M.; Weinigel, Christina; Rummler, Silke; Northoff, Hinnak; Laufer, Stefan; Maier, Thorsten J.; Rådmark, Olof; Samuelsson, Bengt; Sautebin, Lidia

2017-01-01

Proinflammatory leukotrienes (LTs) are produced by 5-lipoxygenase (5-LO) aided by 5-LO–activating protein (FLAP). LT biosynthesis inhibitors are currently under clinical investigation as treatments for respiratory and cardiovascular diseases. Here, we have revealed a sex bias in the efficiency of clinically relevant LT biosynthesis inhibitors, showing that their effects are superior in females. We found that androgens cause these sex differences by impeding the LT-biosynthetic 5-LO/FLAP complex assembly. Lower doses of the FLAP inhibitor MK886 were required to reduce LTB4 levels in exudates of female versus male mice and rats. Following platelet-activating factor–induced shock, MK886 increased survival exclusively in female mice, and this effect was abolished by testosterone administration. FLAP inhibitors and the novel-type 5-LO inhibitors licofelone and sulindac sulfide exhibited higher potencies in human blood from females, and bioactive 5-LO/FLAP complexes were formed in female, but not male, human and murine leukocytes. Supplementation of female blood or leukocytes with 5α-dihydrotestosterone abolished the observed sex differences. Our data suggest that females may benefit from anti-LT therapy to a greater extent than males, prompting consideration of sex issues in LT modifier development. PMID:28737505

The inhibitory effect of alendronate, a nitrogen-containing bisphosphonate on the PI3K-Akt-NFkappaB pathway in osteosarcoma cells.

PubMed

Inoue, Ryosuke; Matsuki, Nori-aki; Jing, Gao; Kanematsu, Takashi; Abe, Kihachiro; Hirata, Masato

2005-11-01

1 Bisphosphonates are inhibitors of tumor cell growth as well as of bone resorption by inducing cell apoptosis. However, little is known regarding the mechanisms by which the drug induces cell apoptosis. The aim of the present study was to determine the effect of alendronate, one of the nitrogen-containing bisphosphonates on the phoshoinositide 3-kinase (PI3K)-Akt-NFkappaB pathway, the major cell survival pathway. 2 The PI3K-Akt-NFkappaB pathway was activated in the osteosarcoma cell line MG-63 treated with tumor necrosis factor-alpha or insulin. Saos-2 was also used in some experiments. This was assessed by the production of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), increased PI3K activity, phosphorylation of Akt at serine 473 and threonine 308, increase in activity of the inhibitor of nuclear factor kappaB (IkappaB) kinase (IKK) and finally phosphorylation of IkappaB and its subsequent degradation. 3 Pretreatment with alendronate at 100 microM for 24 h prior to the stimulation with tumor necrosis factor-alpha or insulin partially inhibited the IkappaB phosphorylation and degradation. These events were more clearly observed in the presence of inhibitors of proteasomes, which are responsible for the degradation of IkappaB. The drug also partially inhibited the activity of IKK, but almost fully inhibited the phosphorylation of Akt and the production of PtdIns(3,4,5)P(3). 4 The inhibitory effect of alendronate on IkappaB phosphorylation and degradation was not attenuated by the exogenous addition of geranylgeraniol to replenish the cytosolic isoprenyl lipid substrate. 5 The present findings demonstrate that alendronate inhibited the PI3K-Akt-NFkappaB cell survival pathway at the point of PI3K activation, thus indicating the presence of new targets of alendronate.

Clinical physiology and mechanism of dizocilpine (MK-801)

PubMed Central

Somanathan, Ratnasamy

2010-01-01

Dizocilpine (MK-801), an extensively investigated drug possessing secondary amine and benzenoid functions, displays a wide array of biological properties, including anticonvulsant and anesthetic. There is scant discussion of biomechanism. A relevant, important finding is formation of oxidative metabolites in the hydroxylamine and phenolic categories. Analogy to cocaine metabolites suggests participation of redox entities, such as, hydroxylamine, nitroxide and nitrosonium, which can lead to electron transfer and radical formation. There is also similarity to metabolism by 3,3′-iminodipropionitrile and phencyclidine. Alternatively, the phenolic metabolites are well-known precursors of ET quinones. The review documents various physiological effects, mainly involving the central nervous system. Also of interest are the pro- and anti-oxidant properties. Considerable attention has been paid to MK-801 as an antagonist of the N-methyl-D-aspartate receptor in the glutamate category. This aspect is often associated with effects on the central nervous system. The review also provides recent literature dealing with MK-801/NMDA receptor in various areas of bioactivity. Studies were made of MK-801 involvement in working memory processing. Deficits in behavior were noted after administration of the drug. Treatment of mice with dizocilpine induced learning impairment. The influence of MK-801 on fear has been investigated. The substance is known to exert an analgesic effect in pain control. A number of reports deal with anesthetic properties. PMID:20716924

Akt2/ZEB2 may be a biomarker for exfoliant cells in ascitic fluid in advanced grades of serous ovarian carcinoma.

PubMed

Liu, Changmei; Yang, Fangmei

2015-09-01

Ovarian cancers present a mild clinical course when diagnosed early but an aggressive pathway when diagnosed in the peri- and postmenopausal periods. However, the predictability of tumor progression is stochastic and is difficult to predict. In the present study, we hypothesized to examine the key pathways that are dysregulated to promote epithelial-mesenchymal transition in serous ovarian carcinoma. Examination of these steps would help to identify ascitic fluid with cells poised for metastasis or otherwise. We focused on examining the Akt2 expression, mainly because of its report as being overamplified in the aggressive variants of ovarian cancer, as well as TGFbeta-sensitivity of Akt2 that forms the key basis for metastasis initiation of most kinds of carcinoma. We obtained primary ovarian carcinoma samples as well as ascitic fluid and distantly metastatic ovarian carcinoma to examine the expression of Akt2. The results of the study demonstrated that in malignant exfoliated ovarian cancer cells, Smad4 expression was tremendously increased in the nuclei, suggesting nuclear translocation of Smad, which thereafter may have activated ZEB2, and thereafter genomically affected the expression of E-cadherin, myosin II, and vimentin, key components for initiating and sustaining metastasis. All of these may have been stimulated by increased cellular expression of Akt2 in metastatic variants of the serous ovarian carcinoma. The reliance on Akt2 and TGF beta signaling may also potentiate the case for Akt inhibitors or small molecule inhibitors of TGFbeta signaling like doxycycline as adjunct chemotherapy in serous ovarian carcinoma, especially the metastatic variants.

Anti-inflammatory effects of fimasartan via Akt, ERK, and NFκB pathways on astrocytes stimulated by hemolysate.

PubMed

Yang, Xiu-Li; Kim, Chi Kyung; Kim, Tae Jung; Sun, Jing; Rim, Doeun; Kim, Young-Ju; Ko, Sang-Bae; Jang, Hyunduk; Yoon, Byung-Woo

2016-02-01

The aim of this study was to investigate whether fimasartan, a novel angiotensin II receptor blocker, modulates hemolysate-induced inflammation in astrocytes. We stimulated astrocytes with hemolysate to induce hemorrhagic inflammation in vitro. Astrocytes were pretreated with fimasartan and then incubated with hemolysate at different durations. Anti-inflammatory cell signaling molecules including Akt, extracellular signal regulated kinase (ERK), NFκB and cyclooxygenase-2 (COX-2) were assessed by western blotting. Pro-inflammatory mediators were evaluated by real-time RT-PCR and ELISA. The stimulation by hemolysate generated a robust activation of inflammatory signaling pathways in astrocytes. Hemolysate increased the phosphorylation of Akt at 1 h, and ERK1/2 at 20 min compared with the control group and promoted the degradation of IκBα. Pretreated fimasartan significantly decreased hemolysate-induced phosphorylation of Akt and ERK1/2. In addition, fimasartan also suppressed NFκB-related inflammatory pathways induced by hemolysate, including reduction of the gene expression of NFκB, and decreased nuclear translocation of NFκB and degradation of IκB. This reduction of inflammatory upstream pathways decreased the expression of inflammatory end-products: COX-2 and interleukin-1 (IL-1β). Furthermore, the expression of COX-2 was attenuated by both Akt inhibitor (LY294002) and ERK inhibitor (U0126), and IκBα degradation was suppressed by LY294002. These results demonstrate that pretreatment with fimasartan to astrocytes suppresses the inflammatory responses induced by hemolysate. Akt, ERK and NFκB were associated with hemolysate-induced COX-2 and IL-1β expression. Based on these mechanisms, fimasartan could be a candidate anti-inflammatory regulator for the treatment of intracerebral hemorrhage.

Acute Alcohol Modulates Cardiac Function as PI3K/Akt Regulates Oxidative Stress

PubMed Central

Umoh, Nsini A.; Walker, Robin K.; Al-Rubaiee, Mustafa; Jeffress, Miara A.; Haddad, Georges E.

2015-01-01

Background Clinical manifestations of alcohol abuse on the cardiac muscle include defective contractility with the development of heart failure. Interestingly, low alcohol consumption has been associated with reduced risk of cardiovascular disease. Although several hypotheses have been postulated for alcoholic cardiomyopathy and for the low-dose beneficial cardiovascular effects, the precise mechanisms and mediators remain largely undefined. We hypothesize that modulation of oxidative stress by PI3K/Akt plays a key role in the cardiac functional outcome to acute alcohol exposure. Methods Thus, acutely exposed rat cardiac tissue and cardiocytes to low (LA: 5 mM), moderate (MA: 25 mM), and high (HA: 100 mM) alcohol were assessed for markers of oxidative stress in the presence and absence of PI3K/Akt activators (IGF-1 0.1 μM or constitutively active PI3K: Ad.BD110 transfection) or inhibitor (LY294002 1 μMor Akt-negative construct Ad.Akt(K179M) transfection). Results Acute LA reduced Akt, superoxide dismutase (SOD-3) and NFκB, ERK1, and p38 MAPK gene expression. Acute HA only increased that of SOD-3 and NFκB. These effects were generally inhibited by Ad.Akt(K179M) and enhanced with Ad.BD110 transfection. In parallel, LA reduced but HA enhanced Akt activity, which was reversed by IGF-1 and inhibited by Ad.Akt(K179M), respectively. Also, LA reduced caspase 3/7 activity and oxidative stress, while HA increased both. The former was blocked, while the latter effect was enhanced by Ad.Akt(K179M). The reverse was true with PI3K/Akt activation. This translated into reduced viability with HA, with no effect with LA. On the functional level, acute LA improved cardiac output and ejection fraction, mainly through increased stroke volume. This was accompanied with enhanced end-systolic pressure–volume relationship and preload recruitable stroke work. Opposite effect was recorded for HA. LA and HA in vivo functional effects were alleviated by LY and enhanced by IGF-1 treatment

The MK VI - A second generation attitude control system

NASA Astrophysics Data System (ADS)

Meredith, P. J.

1986-10-01

The MK VI, a new multipurpose attitude control system for the exoatmospheric attitude control of sounding rocket payloads, is described. The system employs reprogrammable microcomputer memory for storage of basic control logic and for specific mission event control data. The paper includes descriptions of MK VI specifications and configuration; sensor characteristics; the electronic, analog, and digital sections; the pneumatic system; ground equipment; the system operation; and software. A review of the MK VI performance for the Comet Halley flight is presented. Block diagrams are included.

Edaravone protects rats and human pulmonary alveolar epithelial cells against hyperoxia injury: heme oxygenase-1 and PI3K/Akt pathway may be involved.

PubMed

Cao, Huifang; Feng, Ying; Ning, Yunye; Zhang, Zinan; Li, Weihao; Li, Qiang

2015-01-01

Hyperoxic acute lung injury (HALI) is a clinical syndrome as a result of prolonged supplement of high concentrations of oxygen. As yet, no specific treatment is available for HALI. The present study aims to investigate the effects of edaravone on hyperoxia-induced oxidative injury and the underlying mechanism. We treated rats and human pulmonary alveolar epithelial cells with hyperoxia and different concentration of edaravone, then examined the effects of edaravone on cell viability, cell injury and two oxidative products. The roles of heme oxygenase-1 (HO-1) and PI3K/Akt pathway were explored using Western blot and corresponding inhibitors. The results showed that edaravone reduced lung biochemical alterations induced by hyperoxia and mortality of rats, dose-dependently alleviated cell mortality, cell injury, and peroxidation of cellular lipid and DNA oxidative damage. It upregulated cellular HO-1 expression and activity, which was reversed by PI3K/Akt pathway inhibition. The administration of zinc protoporphyrin-IX, a HO-1 inhibitor, and LY249002, a PI3K/Akt pathway inhibitor, abolished the protective effects of edaravone in cells. This study indicates that edaravone protects rats and human pulmonary alveolar epithelial cells against hyperoxia-induced injury and the antioxidant effect may be related to upregulation of HO-1, which is regulated by PI3K/Akt pathway.

Synergistic anti-tumor effect of 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 on human melanoma.

PubMed

Calero, R; Morchon, E; Martinez-Argudo, I; Serrano, R

2017-10-10

Drug resistance by MAPK signaling recovery or activation of alternative signaling pathways, such as PI3K/AKT/mTOR, is an important factor that limits the long-term efficacy of targeted therapies in melanoma patients. In the present study, we investigated the phospho-proteomic profile of RTKs and its correlation with downstream signaling pathways in human melanoma. We found that tyrosine kinase receptors expression correlated with the expression of pivotal downstream components of the RAS/RAF/MAPK and PI3K/AKT/mTOR pathways in melanoma cell lines and tumors. We also found high expression of HSP90 and the PI3K/AKT/mTOR pathway proteins, 4EBP1 and AKT compared with healthy tissue and this correlated with poor overall survival of melanoma patients. The combination of the HSP90 inhibitor 17AAG with the PI3K/mTOR inhibitor NVP-BEZ235 showed a synergistic activity decreasing melanoma cell growth, inducing apoptosis and targeting simultaneously the MAPK and PI3K/AKT/mTOR pathways. These results demonstrate that the combination of HSP90 and PI3K/mTOR inhibitors could be an effective therapeutic strategy that target the main survival pathways in melanoma and must be considered to overcome resistance to BRAF inhibitors in melanoma patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Phosphorylation of AKT induced by phosphorylated Hsp27 confers the apoptosis-resistance in t-AUCB-treated glioblastoma cells in vitro.

PubMed

Li, Rujun; Li, Junyang; Sang, Dongping; Lan, Qing

2015-01-01

The aim of this study is to determine whether phosphorylation of AKT could be effected by t-AUCB-induced p-Hsp27 and whether p-AKT inhibition sensitizes glioblastoma cells to t-AUCB, and to evaluate the effects of simultaneous inhibition of p-Hsp27 and p-AKT on t-AUCB treated glioblastoma cells. Cell growth was detected using CCK-8 assay; Caspase-3 activity assay kits and flow cytometry were used in apoptosis analysis; Western blot analysis was used to detect p-Hsp27 and p-AKT levels; RNA interference using the siRNA oligos of Hsp27 was performed to knockdown gene expression of Hsp27. All data were analyzed by the Student-Newman-Keul's test. We demonstrated that t-AUCB treatment induces AKT phosphorylation by activating Hsp27 in U251 and LN443 cell lines. Inhibition of AKT phosphorylation by AKT inhibitor IV sensitizes glioblastoma cells to t-AUCB, strengthens t-AUCB suppressing cell growth and inducing cell apoptosis. We also found inhibiting both p-Hsp27 and p-AKT synergistically strengthen t-AUCB suppressing cell growth. Thus, p-AKT induced by p-Hsp27 confers the apoptosis-resistance in t-AUCB-treated glioblastoma cells. Targeting p-Hsp27 and/or p-AKT may be a potential effective strategy for the treatment of glioblastoma.

Aspirin reduces the apoptotic effect of etoposide via Akt activation and up-regulation of p21(cip).

PubMed

Feng, Xiaocheng; Lu, Bin; Xu, Yingying; Li, Qin; Zhou, Wenbai; Yang, Zhihong; Yang, Zeng; Zhao, Weiwei; Shen, Zonghou; Hu, Renming

2011-10-01

Previous studies on the apoptotic effect of aspirin mainly focus on colorectal cancer and breast carcinoma. Few studies have been designed to explore the effect of aspirin on hepatocellular carcinoma. In the present study, we observed that aspirin caused G0/G1 phase cell cycle arrest and reduced etoposide induced caspase-3 activation in hepatocellular carcinoma G2 (HepG2) cells. Further investigation demonstrated that aspirin notably enhanced the activity of Akt and ERK1/2. Blocking the activation of Akt by the PI3-K-selective inhibitor wortmannin abrogated the anti-apoptotic effect of aspirin while the MEK inhibitor U0126 did not. p21(cip), an important substrate of Akt, is involved in the regulation of cell cycle arrest and apoptosis. Our data showed that the protein expression and ser146 phosphorylation levels of p21(cip) were significantly increased after treatment with aspirin, whereas p53 or p27 showed no change. The increase of p21(cip) protein levels was also scavenged by wortmannin but not by U0126. Moreover, reduction of caspase-3 activity induced by aspirin was attenuated by silencing p21(cip) expression. These results indicated that the anti-apoptotic effect of aspirin was dependent on activation of Akt which inhibited cell apoptosis by up-regulating p21(cip) and blocking caspase-3 activation. These findings could have clinical relevance in anticancer therapy and aspirin co-treatment of human malignancies.

Phosphatidic acid induces decidualization by stimulating Akt-PP2A binding in human endometrial stromal cells.

PubMed

Lee, So Young; Lee, Yun Young; Choi, Joong Sub; Yoon, Mee-Sup; Han, Joong-Soo

2016-11-01

Decidualization of human endometrial stromal cells (hESCs) is crucial for successful uterine implantation and maintaining pregnancy. We previously reported that phospholipase D1 (PLD1) is required for cAMP-induced decidualization of hESCs. However, the mechanism by which phosphatidic acid (PA), the product of PLD1 action, might regulate decidualization is not known. We confirmed that PA induced decidualization of hESCs by observing morphological changes and measuring increased levels of decidualization markers such as IGFBP1 and prolactin transcripts (P < 0.05). Treatment with PA reduced phosphorylation of Akt and consequently that of FoxO1, which led to the increased IGFBP1 and prolactin mRNA levels (P < 0.05). Conversely, PLD1 knockdown rescued Akt phosphorylation. Binding of PP2A and Akt increased in response to cAMP or PA, suggesting that their binding is directly responsible for the inactivation of Akt during decidualization. Consistent with this observation, treatment with okadaic acid, a PP2A inhibitor, also inhibited cAMP-induced decidualization by blocking Akt dephosphorylation. © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Jintao; Zhu, Dexiao; Zhang, Jing

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 pathwaymore » 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.« less

Allosteric modulation of Ras and the PI3K/AKT/mTOR pathway: emerging therapeutic opportunities

PubMed Central

Hubbard, Paul A.; Moody, Colleen L.; Murali, Ramachandran