Zeitschriftenartikel: „Art. 9 (3) Rome I Regulation“

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Margottini, Claudio, Paolo Canuti und Kyoji Sassa. „The Second World Landslide Forum, Rome, 3–9 October 2011: state of art at May 2011“. Landslides 8, Nr. 2 (Juni 2011): 261–67. http://dx.doi.org/10.1007/s10346-011-0272-x.

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Ding, Zhongyang, Ying Li, Zhangfeng Tang, Xiaoyi Song, Fa Jing, Haotian Wu und Bei Lu. „Role of gambogenic acid in regulating PI3K/Akt/NF-kβ signaling pathways in rat model of acute hepatotoxicity“. Bioscience, Biotechnology, and Biochemistry 85, Nr. 3 (20.01.2021): 520–27. http://dx.doi.org/10.1093/bbb/zbaa039.

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ABSTRACT The purpose of this study is to investigate the protective effect of gambogenic acid (GA) in acetaminophen (APAP)-induced hepatotoxicity in rat models. GA (10 mg/kg) was administered intraperitoneal (i.p.) to rats for 7 consecutive days followed by APAP (500 mg/kg) single dose (i.p.) on the final day after GA administration. The levels of MDA, GSH, SOD, CAT, GPx, GST, ALP, AST, ALT, proinflammatory cytokines (TNF-α, IL-1β, IL-6), apoptosis markers (caspase-3 and -9, Bax, Bcl-2), 4-hydroxynonenal (4-HNE), and prostaglandin E2 (PGE2) were evaluated. Results exhibited protective effects of GA by inhibiting inflammation, preventing oxidative stress and apoptosis in APAP-induced liver. Histopathological changes caused by APAP were attenuated, protein expressions of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) were upregulated, and nuclear factor–kappa β (NF-kβ) was downregulated by GA. In summary, GA significantly exerted anti-inflammatory and antiapoptotic effects against APAP-induced hepatotoxicity potentially through regulation of PI3K/Akt and NF-kβ signaling pathways.

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Rammeloo, Stephan. „‘From Rome to Rome’ – Cross-border employment contract. European Private International Law: Intertemporal law and foreign overriding mandatory laws“. Maastricht Journal of European and Comparative Law 24, Nr. 2 (April 2017): 298–322. http://dx.doi.org/10.1177/1023263x17709754.

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To what extent are Greek saving laws, resulting in payment cuts in the public sector (that is employment conditions), capable of overriding the applicable (German) law? A dispute arising from an employment relationship between the Greek Republic and an employee habitually carrying out work in Germany, gave rise to preliminary questions having regard to the temporal scope of EU Regulation No. 593/2008 (the ‘Rome I Regulation’)1 and, closely related thereto, the functional reach of Article 9(3) of that Regulation in respect of ‘foreign’ mandatory laws, in light of the principle of sincere cooperation enshrined in Article 4(3) TEU. An analysis of the Advocate General’s Opinion and the Court of Justice of the European Union’s (CJEU) ruling is followed by critical commentary and suggestions for future EU legislative amendments to the Rome I regime.

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Bator-Bryła, Monika Patrycja. „Prohibition of Discrimination on Grounds of Nationality in the Freedom of Movement of Persons within the EU in the Light of Case Law of the Court of Justice of the European Union“. Review of European and Comparative Law 46, Nr. 3 (21.08.2021): 189–218. http://dx.doi.org/10.31743/recl.12340.

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The subject of this article is to analyze the meaning of the prohibition of discrimination on grounds of nationality in the light of the provisions of primary and secondary European Union law and the case law of the Court of Justice of the European Union, which is inherent to the functioning of the internal market and EU citizenship. The prohibition of discrimination on grounds of nationality is undoubtedly one of the main goals of the European Union[1] in the social and economic context, which was reflected in the localization of the matter in question in the primary law of the European Union[2], in secondary law and in the jurisprudence of the Court of Justice of the European Union (CJEU). The Treaty on European Union (TEU)[3] and the Treaty on the Functioning of the European Union (TFEU)[4] indicate equality as one of the EU values (Article 2 TEU), require it to be promoted and combat all discrimination (Articles 8 and 10 TFEU) and prohibit discrimination due to the criteria indicated therein (Articles 18 and 19 TFEU). In secondary law, this principle was expressed primarily in the Regulation of the European Parliament and of the Council No. 492/2011 on the free movement of workers within the Union and in art. 24 of Directive 2004/38/EC 2004 on the right of citizens of the Union and their relatives to move freely[5]. A special role in this area is played by the case law of the Court of Justice of the European Union (CJEU), which stated that all authorities of the Member States are obliged to refuse to apply a provision of national law that is contrary to the prohibition of discrimination on the grounds of citizenship (Article 18 TFEU)[6]. Moreover, national measures may be examined in the light of art. 18 TFEU, but only to the extent that they apply to situations not covered by specific non-discrimination provisions included in the Treaty[7]. The author puts forward the thesis that the analysis of CJEU jurisprudence reveals a visible dissonance between the application of national regulations of the Member States and the provisions of EU law in this matter, which significantly hinders the implementation of the principle of non-discrimination in practice. Discrepancies mainly occur in domestic legal acts due to the improper drafting of national legal provisions and / or their misinterpretation by national judicial or administrative authorities. It should be emphasized that the Member States are obliged to comply with EU law, which is not tantamount only to the obligation of state authorities to respect directly applicable acts, or to implement required regulations into internal law, but also the obligation to interpret and apply internal law in a manner that does not violate the requirement resulting from EU law[8]. Judicial and administrative authorities of the Member States should therefore interpret national law as far as possible, in line with EU law, because the limits of the pro-EU interpretation will be determined by the powers conferred by domestic law[9]. The study uses the legal-comparative method, consisting in a comparative analysis of the legal systems of the Member States and the European Union in the field of non-discrimination on the basis of nationality, rights and restrictions on the freedom of movement of authorized entities. Comparative verification of EU acts with the internal standards of individual EU Member States allows to reveal the degree of advancement of the implementation process of EU law provisions under the free movement of EU citizens and their family members in the discussed area in the legal systems of European Union Member States. The purpose of this analysis is to, inter alia, diagnose areas in which these countries have not implemented or improperly implemented EU regulations, or have misinterpreted them. The second method used is the method of analyzing the jurisprudence of the Court of Justice of the European Union - the rulings of the CJEU constitute a significant part of the study. The case law in question covers the period from the establishment of the Treaties of Rome to the present day. The use of the latter obligated the author to apply the comparative method of judgments based on same or similar legal bases in similar circumstances from different stages of the evolution of the free movement of citizens of the European Union and their family members under the prohibition of discrimination on the basis of nationality. [1] Cf. Olivier De Schutter, Links between migration and discrimination. A legal analysis of the situation in EU Member States (Brussels: European Commission, 2016), 102 and next; See also Brita Sundberg-Weitman, Discrimination on Grounds of Nationality. Free Movement of Workers and Freedom of Establishment under the EEC Treaty (Amsterdam, New York, Oxford: North-Holland Publishing Co., 1977). [2] Erica Szyszczak, “Antidiscrimination Law in the European Union,” Fordham International Law Journal, no. 32 (2008): 635. [3] The Treaty on European Union (consolidated version) OJ of the EU 2012, No. C 326/01. [4] The Treaty on the Functioning of the European Union (consolidated version) OJ of the EU 2012, No. C 326/01. [5] Directive of the European Parliament and of the Council No. 2004/38 on the right of citizens of the Union and their family members to move and reside freely within the territory of the Member States amending Regulation (EEC) No. 1612/68 and repealing Directives 64/221/EEC, 68/360/EEC, 72/194/EEC, 73/148/EEC, 75/34/EEC, 75/35/EEC, 90/364/EEC, 90/365/EEC and 93/96/EEC (O.J.E.C. L 158, 30 April 2004). [6] CJEU Judgement of 7 May 1998, Clean Car Autoservice GmbH p. Landeshauptmann von Wien, Case C-350/96, ECLI:EU:C:1998:205. [7] CJEU Judgement of 18 June 2019, Republic of Austria v Federal Republic of Germany, Case C-591/17, ECLI:EU:C:2019:504, pt 41. [8] Marek Górski, “Wpływ orzecznictwa Europejskiego Trybunału Sprawiedliwości na interpretację i stosowanie przepisów o ochronie środowiska,” in Wspólnotowe prawo ochrony środowiska i jego implementacja w Polsce trzy lata po akcesji, ed. Jerzy Jendrośka and Magdalena Bar (Wrocław: Centrum Prawa Ekologicznego Press, 2008), 31. [9] Monika Niedźwiedź, “Stosowanie prawa wspólnotowego przez organy administracyjne,” Casus, no. 32 (October 2004): 6.

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Zhao, Xiaomin, Xiangjun Song, Xiaoyuan Bai, Naijiao Fei, Yong Huang, Zhimin Zhao, Qian Du, Hongling Zhang, Liang Zhang und Dewen Tong. „miR-27b attenuates apoptosis induced by transmissible gastroenteritis virus (TGEV) infection via targeting runt-related transcription factor 1 (RUNX1)“. PeerJ 4 (04.02.2016): e1635. http://dx.doi.org/10.7717/peerj.1635.

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Transmissible gastroenteritis virus (TGEV), belonging to the coronaviridae family, is the key cause of the fatal diarrhea of piglets and results in many pathological processes. microRNAs (miRNAs) play a key role in the regulation of virus-induced apoptosis. During the process of apoptosis induced by TGEV infection in PK-15 cells, the miR-27b is notably down-regulated. Thus, we speculate that miR-27b is involved in regulating the process of apoptosis in PK-15 cells. In this study we demonstrated that the over-expression of miR-27b led to the inhibition of TGEV-induced apoptosis, reduction of Bax protein level, and decrease of caspase-3 and −9 activities. Conversely, silencing of miR-27b by miR-27b inhibitors enhanced apoptosis via up-regulating Bax expression and promoting the activities of caspase-3 and −9 in TGEV-infected cells. Subsequently, the runt-related transcription factor 1 (RUNX1) is a candidate target of miR-27b predicted by bioinformatics search. We further identified that the miR-27b directly bound to the 3′ UTR of RUNX1 mRNA and suppressed RUNX1 expression, which indicates RUNX1 is the direct target gene of miR-27b. The over-expression of RUNX1 increased apoptosis and knockdown RUNX1blocked apoptosis of viral-infected cells via regulating Bax expression and the activities of caspase-3 and −9. Our data reveal that miR-27b may repress the mitochondrial pathway of apoptosis by targeting RUNX1, indicating that TGEV may induce apoptosis via down-regulating miR-27b and that miR-27b may act as a target for therapeutic intervention.

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Toufaily, Chirine, Cyndia Charfi, Bayader Annabi und Borhane Annabi. „A Role for the Cavin-3/Matrix Metalloproteinase-9 Signaling Axis in the Regulation of PMA-Activated Human HT1080 Fibrosarcoma Cell Neoplastic Phenotype“. Cancer Growth and Metastasis 7 (Januar 2014): CGM.S18581. http://dx.doi.org/10.4137/cgm.s18581.

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Caveolae are specialized cell membrane invaginations known to regulate several cancer cell functions and oncogenic signaling pathways. Among other caveolar proteins, they are characterized by the presence of proteins of the cavin family. In this study, we assessed the impact of cavin-1, cavin-2, and cavin-3 on cell migration in a human HT-1080 fibrosarcoma model. We found that all cavin-1, -2 and -3 transcripts were expressed and that treatment with phorbol 12-myristate 13-acetate (PMA), which is known to prime cell migration and proliferation, specifically upregulated cavin-3 gene and protein expression. PMA also triggered matrix metalloproteinase (MMP)-9 secretion, but reduced the global cell migration index. Overexpression of recombinant forms of the three cavins demonstrated that only cavin-3 was able to reduce basal cell migration, and this anti-migratory effect was potentiated by PMA. Interestingly, cavin-3 overexpression inhibited PMA-induced MMP-9, while cavin-3 gene silencing led to an increase in MMP-9 gene expression and secretion. Furthermore, recombinant cavin-3 significantly prevented PMA-mediated dephosphorylation of AKT, a crucial regulator in MMP-9 transcription. In conclusion, our results demonstrate that cellular cavin-3 expression may repress MMP-9 transcriptional regulation in part through AKT. We suggest that the balance in cavin-3-to-MMP-9 expression regulates the extent of extracellular matrix degradation, confirming the tumor-suppressive role of cavin-3 in controlling the invasive potential of human fibrosarcoma cells.

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Kotelevets, Larissa, und Eric Chastre. „A New Story of the Three Magi: Scaffolding Proteins and lncRNA Suppressors of Cancer“. Cancers 13, Nr. 17 (24.08.2021): 4264. http://dx.doi.org/10.3390/cancers13174264.

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Scaffolding molecules exert a critical role in orchestrating cellular response through the spatiotemporal assembly of effector proteins as signalosomes. By increasing the efficiency and selectivity of intracellular signaling, these molecules can exert (anti/pro)oncogenic activities. As an archetype of scaffolding proteins with tumor suppressor property, the present review focuses on MAGI1, 2, and 3 (membrane-associated guanylate kinase inverted), a subgroup of the MAGUK protein family, that mediate networks involving receptors, junctional complexes, signaling molecules, and the cytoskeleton. MAGI1, 2, and 3 are comprised of 6 PDZ domains, 2 WW domains, and 1 GUK domain. These 9 protein binding modules allow selective interactions with a wide range of effectors, including the PTEN tumor suppressor, the β-catenin and YAP1 proto-oncogenes, and the regulation of the PI3K/AKT, the Wnt, and the Hippo signaling pathways. The frequent downmodulation of MAGIs in various human malignancies makes these scaffolding molecules and their ligands putative therapeutic targets. Interestingly, MAGI1 and MAGI2 genetic loci generate a series of long non-coding RNAs that act as a tumor promoter or suppressor in a tissue-dependent manner, by selectively sponging some miRNAs or by regulating epigenetic processes. Here, we discuss the different paths followed by the three MAGIs to control carcinogenesis.

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Wu, Meng, Jiaqiang Xiong, Lingwei Ma, Zhiyong Lu, Xian Qin, Aiyue Luo, Jinjin Zhang, Huan Xie, Wei Shen und Shixuan Wang. „Enrichment of Female Germline Stem Cells from Mouse Ovaries Using the Differential Adhesion Method“. Cellular Physiology and Biochemistry 46, Nr. 5 (2018): 2114–26. http://dx.doi.org/10.1159/000489452.

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Background/Aims: The isolation and establishment of female germline stem cells (FGSCs) is controversial because of questions regarding the reliability and stability of the isolation method using antibody targeting mouse vasa homologue (MVH), and the molecular mechanism of FGSCs self-renewal remains unclear. Thus, there needs to be a simple and reliable method for sorting FGSCs to study them. Methods: We applied the differential adhesion method to enrich FGSCs (DA-FGSCs) from mouse ovaries. Through four rounds of purification and 7-9 subsequent passages, DA-FGSC lines were established. In addition, we assessed the role of the phosphoinositide-3 kinase (PI3K)-AKT pathway in regulating FGSC self-renewal. Results: The obtained DA-FGSCs spontaneously differentiated into oocyte-like cells in vitro and formed functional eggs in vivo that were fertilized and produced healthy offspring. AKT was rapidly phosphorylated when the proliferation rate of FGSCs increased after 10 passages, and the addition of a chemical PI3K inhibitor prevented FGSCs self-renewal. Furthermore, over-expression of AKT-induced proliferation and differentiation of FGSCs, c-Myc, Oct-4 and Gdf-9 levels were increased. Conclusions: The differential adhesion method provides a more feasible approach and is an easier procedure to establish FGSC lines than traditional methods. The AKT pathway plays an important role in regulation of the proliferation and maintenance of FGSCs. These findings could help promote stem cell studies and provide a better understanding of causes of ovarian infertility, thereby providing potential treatments for infertility.

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Qiu, Q., M. Yang, B. K. Tsang und A. Gruslin. „EGF-induced trophoblast secretion of MMP-9 and TIMP-1 involves activation of both PI3K and MAPK signalling pathways“. Reproduction 128, Nr. 3 (September 2004): 355–63. http://dx.doi.org/10.1530/rep.1.00234.

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Epidermal growth factor (EGF) is present in the maternal-fetal environment and has an important role in placental development. Matrix metalloproteinase-9 (MMP-9) expression/activation is a pre-requisite in extravillous trophoblast invasion. Whereas EGF up-regulates MMP-9 activity in a variety of cell types, there is no direct evidence for the stimulation of MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) secretion by EGF in extravillous trophoblasts. In addition, the signalling pathways involved in this regulation are not clear. In the present study, we have examined the possible involvement of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways in the regulation of the MMP-9/TIMP-1 system by EGFin vitro. We used a well-established invasive extravillous trophoblast cell line (HTR8/Svneo) and measured gene and protein expression by semi-quantitative RT-PCR and western analysis respectively. MMP activity was determined by zymography. We showed for the first time that EGF activated both PI3K/Akt and MAPK/extracellular-signal regulated kinase (ERK) signalling in HTR8/SVneo, and increased both MMP-9 and TIMP-1 mRNAs and protein concentrations. Interfering with either signalling pathway via PI3K inhibitor LY294002 or MEK inhibitor U0126 in EGF-stimulated HTR8/SVneo cells blocked the induction of MMP-9 and TIMP-1. LY294002 inhibited Akt phosphorylation, but had no effect on ERK phosphorylation; U0126 suppressed ERK phosphorylation without interfering with the phosphorylation of Akt. In addition, expression of constitutively active Akt (Myr-Akt1, Myr-Akt2, Myr-Akt3) was not sufficient to induce proMMP-9 and TIMP-1 secretion. Our results suggest that the activation of both PI3K and MAPK pathways in extravillous trophoblasts is necessary for the up-regulation of MMP-9 and TIMP-1 expression by EGF.

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El Sheikh, Marwa, Ayman Mesalam, Ahmed Atef Mesalam, Muhammad Idrees, Kyeong-Lim Lee und Il-Keun Kong. „Melatonin Abrogates the Anti-Developmental Effect of the AKT Inhibitor SH6 in Bovine Oocytes and Embryos“. International Journal of Molecular Sciences 20, Nr. 12 (17.06.2019): 2956. http://dx.doi.org/10.3390/ijms20122956.

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Melatonin, a nighttime-secreted antioxidant hormone produced by the pineal gland, and AKT, a serine/threonine-specific protein kinase, have been identified as regulators for several cellular processes essential for reproduction. The current study aimed to investigate the potential interplay between melatonin and AKT in bovine oocytes in the context of embryo development. Results showed that the inclusion of SH6, a specific AKT inhibitor, during in vitro maturation (IVM) significantly reduced oocyte maturation, cumulus cell expansion, cleavage, and blastocyst development that were rescued upon addition of melatonin. Oocytes treated with SH6 in the presence of melatonin showed lower levels of reactive oxygen species (ROS) and blastocysts developed exhibited low apoptosis while the mitochondrial profile was significantly improved compared to the SH6-treated group. The RT-qPCR results showed up-regulation of the mRNA of maturation-, mitochondrial-, and cumulus expansion-related genes including GDF-9, BMP-15, MARF1, ATPase, ATP5F1E, POLG2, HAS2, TNFAIP6, and PTGS2 and down-regulation of Bcl-2 associated X apoptosis regulator (BAX), caspase 3, and p21 involved in apoptosis and cell cycle arrest in melatonin-SH6 co-treated group compared to SH6 sole treatment. The immunofluorescence showed high levels of caspase 3 and caspase 9, and low AKT phosphorylation in the SH6-treated group compared to the control and melatonin-SH6 co-treatment. Taken together, our results showed the importance of both melatonin and AKT for overall embryonic developmental processes and, for the first time, we report that melatonin could neutralize the deleterious consequences of AKT inhibition, suggesting a potential role in regulation of AKT signaling in bovine oocytes.

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Kronenberg, Alexander. „Foreign overriding mandatory provisions under the regulation (EC) No 593/2008 (Rome I Regulation). Judgment of the European Court of Justice of 18 october 2016, case c-135/15 = Leyes de policía de terceros estados en el ámbito del reglamento (CE) No 593/2008 (Reglamento Roma I). Comentario a la STJUE de 18 de octubre de 2016, asunto c-135/15“. CUADERNOS DE DERECHO TRANSNACIONAL 10, Nr. 2 (05.10.2018): 873. http://dx.doi.org/10.20318/cdt.2018.4409.

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Abstract: The role and treatment of foreign overriding mandatory provisions in international contract law have been subject to academic discussions for a long time. This has not changed with the introduction of Article 9 of the Rome I Regulation. In the judgment discussed in this case note, the European Court of Justice addressed some of the contentious issues in relation to Article 9(3) of the Rome I Regulation. This note examines and evaluates the solutions found by the ECJ and puts them into context. It also points out some questions the ECJ did not discuss; these questions remain open for now but will need to be addressed in the future.Keywords: Article 9(3) Rome I Regulation, foreign overriding mandatory provisions, conflict-of-law level consideration, substantive law level consideration, principle of sincere cooperation.Resumen: El tratamiento de las leyes de policía de terceros estados en derecho de contratos internacionales ha sido objeto de la polémica desde hace tiempo. Esto no ha cambiado con la entrada en vigor del artículo 9 del Reglamento Roma I. Con la sentencia comentada el Tribunal de Justicia de la Unión Europea ha tratado algunas de la cuestiones debatidas respecto al artículo 9.3 del Reglamento Roma I. Este comentario analiza, evalúa y pone en contexto las soluciones encontradas por el TJUE. También aborda las cuestiones que no han sido comentadas por el TJUE; estas cuestiones permanecen abiertas por el momento pero deberán ser examinadas en el futuro.Palabras clave: leyes de policía de terceros estados, consideración en nivel conflictual, consideración en nivel sustantivo, principio de cooperación leal.

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Liu, Feng, Ning Jiang, Zhi-yong Xiao, Jun-ping Cheng, Yi-zhou Mei, Pan Zheng, Li Wang et al. „Effects of poly (ADP-ribose) polymerase-1 (PARP-1) inhibition on sulfur mustard-induced cutaneous injuriesin vitroandin vivo“. PeerJ 4 (04.04.2016): e1890. http://dx.doi.org/10.7717/peerj.1890.

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Early studies with first-generation poly (ADP-ribose) polymerase (PARP) inhibitors have already indicated some therapeutic potential for sulfur mustard (SM) injuries. The available novel and more potential PARP inhibitors, which are undergoing clinical trials as drugs for cancer treatment, bring it back to the centre of interest. However, the role of PARP-1 in SM-induced injury is not fully understood. In this study, we selected a high potent specific PARP inhibitor ABT-888 as an example to investigate the effect of PARP inhibitor in SM injury. The results showed that in both the mouse ear vesicant model (MEVM) and HaCaT cell model, PARP inhibitor ABT-888 can reduce cell damage induced by severe SM injury. ABT-888 significantly reduced SM induced edema and epidermal necrosis in MEVM. In the HaCaT cell model, ABT-888 can reduce SM-induced NAD+/ATP depletion and apoptosis/necrosis. Then, we studied the mechanism of PARP-1 in SM injury by knockdown of PARP-1 in HaCaT cells. Knockdown of PARP-1 protected cell viability and downregulated the apoptosis checkpoints, including p-JNK, p-p53, Caspase 9, Caspase 8, c-PARP and Caspase 3 following SM-induced injury. Furthermore, the activation of AKT can inhibit autophagy via the regulation of mTOR. Our results showed that SM exposure could significantly inhibit the activation of Akt/mTOR pathway. Knockdown of PARP-1 reversed the SM-induced suppression of the Akt/mTOR pathway. In summary, the results of our study indicated that the protective effects of downregulation of PARP-1 in SM injury may be due to the regulation of apoptosis, necrosis, energy crisis and autophagy. However, it should be noticed that PARP inhibitor ABT-888 further enhanced the phosphorylation of H2AX (S139) after SM exposure, which indicated that we should be very careful in the application of PARP inhibitors in SM injury treatment because of the enhancement of DNA damage.

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Kurowski, Witold. „Glosa do wyroku Trybunału Sprawiedliwości Unii Europejskiej z dnia 12 lutego 2015 r. w sprawie Sähköalojen ammattiliitto ry c/a Elektrobudowa Spółka Akcyjna (C‑396/13)“. Problemy Prawa Prywatnego Międzynarodowego 24 (30.06.2019): 191–209. http://dx.doi.org/10.31261/pppm.2019.24.08.

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This paper aims to comment an important ruling concerning the Posted Workers Directive (Directive 96/71/EC). In the judgement C-396/13 (Sähköalojen ammattiliitto ry v. Elektrobudowa Spółka Akcyjna), the European Court of Justice providedits pro-worker’s interpretation of Art 3 of Directive 96/71/EC concerning the scope of the "minimum pay rate". The second issue raised by the European Court of Justice was the assignability of pay claims governed by Polish law based on Art 14 (2) of Rome I Regulation and prohibited under that law. In commented judgement, the Court admitted the assignment of claims arising from employment relationships in light of article 47 of the Charter of Fundamental Rights of the European Union and accepted the trade union’s right to represent the posted workers.

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Redondo-Muñoz, Javier, Elizabeth Escobar-Díaz, Rafael Samaniego, María José Terol, José A. García-Marco und Ángeles García-Pardo. „MMP-9 in B-cell chronic lymphocytic leukemia is up-regulated by α4β1 integrin or CXCR4 engagement via distinct signaling pathways, localizes to podosomes, and is involved in cell invasion and migration“. Blood 108, Nr. 9 (01.11.2006): 3143–51. http://dx.doi.org/10.1182/blood-2006-03-007294.

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Abstract B-cell chronic lymphocytic leukemia (B-CLL) progression is determined by malignant cell extravasation and lymphoid tissue infiltration. We have studied the role and regulation of matrix metalloproteinase-9 (MMP-9) in B-CLL cell migration and invasion. Adhesion of B-CLL cells to the fibronectin fragment FN-H89, VCAM-1, or TNF-α–activated human umbilical vein endothelial cells (HUVECs) up-regulated MMP-9 production, measured by gelatin zymography. This effect was mediated by α4β1 integrin and required PI3-K/Akt signaling. The chemokine CXCL12 also up-regulated MMP-9, independently of α4β1 and involving ERK1/2 but not Akt activity. Accordingly, α4β1 engagement activated the PI3-K/Akt/NF-κB pathway, while CXCL12/CXCR4 interaction activated ERK1/2/c-Fos signaling. Anti–MMP-9 antibodies, the MMP-9 inhibitor TIMP-1, or transfection with 3 different MMP-9 siRNAs significantly blocked migration through Matrigel or HUVECs. Cell-associated MMP-9 was mainly at the membrane and contained the proactive and mature forms. Moreover, B-CLL cells formed podosomes upon adhesion to FN-H89, VCAM-1, or fibronectin; MMP-9 localized to podosomes in a PI3-K–dependent manner and degraded a fibronectin/gelatin matrix. Our results are the first to show that MMP-9 is physiologically regulated by α4β1 integrin and CXCL12 and plays a key role in cell invasion and transendothelial migration, thus contributing to B-CLL progression. MMP-9 could therefore constitute a target for treatment of this malignancy.

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Kaushal, Gur P., Ling Liu, Varsha Kaushal, Xiaoman Hong, Oksana Melnyk, Rohit Seth, Robert Safirstein und Sudhir V. Shah. „Regulation of caspase-3 and -9 activation in oxidant stress to RTE by forkhead transcription factors, Bcl-2 proteins, and MAP kinases“. American Journal of Physiology-Renal Physiology 287, Nr. 6 (Dezember 2004): F1258—F1268. http://dx.doi.org/10.1152/ajprenal.00391.2003.

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Cytotoxicity to renal tubular epithelial cells (RTE) is dependent on the relative response of cell survival and cell death signals triggered by the injury. Forkhead transcription factors, Bcl-2 family member Bad, and mitogen-activated protein kinases are regulated by phosphorylation that plays crucial roles in determining cell fate. We examined the role of phosphorylation of these proteins in regulation of H2O2-induced caspase activation in RTE. The phosphorylation of FKHR, FKHRL, and Bcl-2 family member Bad was markedly increased in response to oxidant injury, and this increase was associated with elevated levels of basal phosphorylation of Akt/protein kinase B. Phosphoinositol (PI) 3-kinase inhibitors abolished this phosphorylation and also decreased expression of antiapoptotic proteins Bcl-2 and BclxL. Inhibition of phosphorylation of forkhead proteins resulted in a marked increase in the proapoptotic protein Bim. These downstream effects of PI 3-kinase inhibition promoted the oxidant-induced activation of caspase-3 and -9, but not caspase-8 and -1. The impact of enhanced activation of caspases by PI 3-kinase inhibition was reflected on accelerated oxidant-induced cell death. Oxidant stress also induced marked phosphorylation of ERK1/2, P38, and JNK kinases. Inhibition of ERK1/2 phosphorylation but not P38 and JNK kinase increased caspase-3 and -9 activation; however, this activation was far less than induced by inhibition of Akt phosphorylation. Thus the Akt-mediated phosphorylation pathway, ERK signaling, and the antiapoptotic Bcl-2 proteins distinctly regulate caspase activation during oxidant injury to RTE. These studies suggest that enhancing renal-specific survival signals may lead to preservation of renal function during oxidant injury.

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Koltai, Tomas. „Fenofibrate in cancer: mechanisms involved in anticancer activity“. F1000Research 4 (26.02.2015): 55. http://dx.doi.org/10.12688/f1000research.6153.1.

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Objective: To review the mechanisms of anti-cancer activity of fenofibrate (FF) and other Peroxisome Proliferator Activator Receptor α (PPARα) agonists based on evidences reported in the published literature.Methods: We extensively reviewed the literature concerning FF as an off target anti-cancer drug. Controversies regarding conflicting findings were also addressed.Results: The main mechanism involved in anti-cancer activity is anti-angiogenesis through down-regulation of Vascular Endothelial Growth Factor (VEGF), Vascular Endothelial Growth Factor Receptor (VEGFR) and Hypoxia Inducible factor-1 α (HIF-1α), inhibition of endothelial cell migration, up-regulation of endostatin and thrombospondin-1, but there are many other contributing mechanisms like apoptosis and cell cycle arrest, down-regulation of Nuclear Factor Kappa B (NF-kB) and Protein kinase B (Akt) and decrease of cellular energy by impairing mitochondrial function. Growth impairment is related to down-regulation of Phospho-Inositol 3 Kinase (PI3K)/Akt axis and down-regulation of the p38 map kinase (MAPK) cascade. A possible role should be assigned to FF stimulated over-expression of Tribbles Homolog-3 (TRIB3) which inhibits Akt phosphorylation. Important anti-cancer and anti-metastatic activities are due to down-regulation of MCP-1 (monocyte chemotactic protein-1), decreased Metalloprotease-9 (MMP-9) production, weak down-regulation of adhesion molecules like E selectin, intercellular adhesion molecules (ICAM) and Vascular Endothelial Adhesion Molecules (VCAM), and decreased secretion of chemokines like Interleukin-6 (IL-6), and down-regulation of cyclin D-1. There is no direct link between FF activity in lipid metabolism and anticancer activity, except for the fact that many anticancer actions are dependent from PPARα agonism. FF exhibits also PPARα independent anti-cancer activities.Conclusions: There are strong evidences indicating that FF can disrupt growth-related activities in many different cancers, due to anti-angiogenesis and anti-inflammatory effects. Therefore FF may be useful as a complementary adjunct treatment of cancer, particularly included in anti-angiogenic protocols like those currently increasingly used in glioblastoma. There are sound reasons to initiate well planned phase II clinical trials for FF as a complementary adjunct treatment of cancer.

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Koltai, Tomas. „Fenofibrate in cancer: mechanisms involved in anticancer activity“. F1000Research 4 (07.07.2015): 55. http://dx.doi.org/10.12688/f1000research.6153.2.

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Objective: To review the mechanisms of anti-cancer activity of fenofibrate (FF) and other Peroxisome Proliferator Activator Receptor α (PPARα) agonists based on evidences reported in the published literature.Methods: We extensively reviewed the literature concerning FF as an off target anti-cancer drug. Controversies regarding conflicting findings were also addressed.Results: The main mechanism involved in anti-cancer activity is anti-angiogenesis through down-regulation of Vascular Endothelial Growth Factor (VEGF), Vascular Endothelial Growth Factor Receptor (VEGFR) and Hypoxia Inducible factor-1 α (HIF-1α), inhibition of endothelial cell migration, up-regulation of endostatin and thrombospondin-1, but there are many other contributing mechanisms like apoptosis and cell cycle arrest, down-regulation of Nuclear Factor Kappa B (NF-kB) and Protein kinase B (Akt) and decrease of cellular energy by impairing mitochondrial function. Growth impairment is related to down-regulation of Phospho-Inositol 3 Kinase (PI3K)/Akt axis and down-regulation of the p38 map kinase (MAPK) cascade. A possible role should be assigned to FF stimulated over-expression of Tribbles Homolog-3 (TRIB3) which inhibits Akt phosphorylation. Important anti-cancer and anti-metastatic activities are due to down-regulation of MCP-1 (monocyte chemotactic protein-1), decreased Metalloprotease-9 (MMP-9) production, weak down-regulation of adhesion molecules like E selectin, intercellular adhesion molecules (ICAM) and Vascular Endothelial Adhesion Molecules (VCAM), and decreased secretion of chemokines like Interleukin-6 (IL-6), and down-regulation of cyclin D-1. There is no direct link between FF activity in lipid metabolism and anticancer activity, except for the fact that many anticancer actions are dependent from PPARα agonism. FF exhibits also PPARα independent anti-cancer activities.Conclusions: There are strong evidences indicating that FF can disrupt growth-related activities in many different cancers, due to anti-angiogenesis and anti-inflammatory effects. Therefore FF may be useful as a complementary adjunct treatment of cancer, particularly included in anti-angiogenic protocols like those currently increasingly used in glioblastoma. There are sound reasons to initiate well planned phase II clinical trials for FF as a complementary adjunct treatment of cancer.

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Wang, Shujuan, Wenju Liu, Aiyou Wen, Bing Yang und Xunsheng Pang. „Luzindole and 4P-PDOT block the effect of melatonin on bovine granulosa cell apoptosis and cell cycle depending on its concentration“. PeerJ 9 (08.03.2021): e10627. http://dx.doi.org/10.7717/peerj.10627.

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Granulosa cells play an essential physiological role in mediating the follicle development and survival or apoptosis of granulosa cells dictate the follicle development or atresia. The aim of this study was to investigate the role of high dose (10−5 M) and low dose (10−9 M) melatonin in bovine granulosa cells, and assess whether MT1 and MT2 inhibiter affect granulosa cells response to melatonin. We found that the high dose (10−5 M) and low dose (10−9 M) both could act as an essential role in modulating granulosa cells apoptosis, cell cycle and antioxidant. The beneficial effect could be related to that melatonin promoted the expression of Bcl2, Bcl-xl, SOD1 and GPX4, and inhibited Bax, caspase-3 and p53 expression. Moreover P21 expression was decreased in granulosa cells treated with the high dose (10−5 M) melatonin and increased in that treated with the low dose (10−9 M) melatonin. To further reveal the role of MT1 and MT2 in mediating the effect of melatonin on granulosa cells apoptosis, cell cycle and antioxidant, we found that the luzindole and 4P-PDOT did not affect the effect of high dose (10−5 M) melatonin on regulating Bcl2, Bax, caspase-3, SOD1, GPX4 and p53 expression, while blocked its effect on modulating Bcl-xl and P21expression. However, luzindole and 4P-PDOT disturbed the effect of low dose (10−9 M) melatonin on regulating Bcl2, Bax, caspase-3, Bcl-xl, SOD1, GPX4, and p53 expression. In conclusion, these results reveal that the effect of low dose (10−9 M) melatonin on granulosa cells apoptosis are mediated by MT1 and MT2, and the high dose (10−5 M) melatonin affect the granulosa cells apoptosis by other pathway, besides MT1 and MT2. Moreover MT1 and MT2 may work in concert to modulate bovine granulosa cells function by regulating cellular progression and apoptosis.

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Liu, Shuang, Lin Wang, Yongwei Li, Yuanshan Cui, Yongqiang Wang und Chu Liu. „Long non-coding RNA CHRF promotes proliferation and mesenchymal transition (EMT) in prostate cancer cell line PC3 requiring up-regulating microRNA-10b“. Biological Chemistry 400, Nr. 8 (26.07.2019): 1035–45. http://dx.doi.org/10.1515/hsz-2018-0380.

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Abstract Despite the advance of diagnosis and treatment for prostate cancer, the prognosis of metastatic prostate cancer is poor. We aimed to explore the functional role of long non-coding RNA cardiac hypertrophy-related factor (lncRNA CHRF) in prostate cancer cells (PC3) as well as the molecular mechanisms. LncRNA CHRF silence repressed cell number (%), down-regulated expression of cyclinD1, CDK4 and CDK6, and promoted apoptosis along with activation of the casapse-3 and caspase-9. LncRNA CHRF promoted mesenchymal transition (EMT), showing down-regulation of E-cadherin and up-regulation of N-cadherin, vimentin and ZEB1. Afterwards, we found miR-10b expression was positively correlated with lncRNA CHRF expression, and miR-10b inhibition could reverse the effects of lncRNA CHRF on PC3 and LNCaP cell proliferation and EMT. Finally, lncRNA CHRF was found to activate the GSK3β/AKT and NF-κB pathways via up-regulation of miR-10b. LncRNA CHRF silence repressed proliferation and EMT while promoted apoptosis in PC3 cells via positive regulation of miR-10b. The GSK3β/AKT and NF-κB pathways were activated by lncRNA CHRF, possibly through up-regulation of miR-10b.

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Won, Yeong-Seon, und Kwon-Il Seo. „Sanggenol L Induces Apoptosis and Cell Cycle Arrest via Activation of p53 and Suppression of PI3K/Akt/mTOR Signaling in Human Prostate Cancer Cells“. Nutrients 12, Nr. 2 (14.02.2020): 488. http://dx.doi.org/10.3390/nu12020488.

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Prostate cancer is the most common cancer in Western countries. Recently, Asian countries are being affected by Western habits, which have had an important role in the rapid increase in cancer incidence. Sanggenol L (San L) is a natural flavonoid present in the root barks of Morus alba, which induces anti-cancer activities in ovarian cancer cells. However, the molecular and cellular mechanisms of the effects of sanggenol L on human prostate cancer cells have not been elucidated. In this study, we investigated whether sanggenol L exerts anti-cancer activity in human prostate cancer cells via apoptosis and cell cycle arrest. Sanggenol L induced caspase-dependent apoptosis (up-regulation of PARP and Bax or down-regulation of procaspase-3, -8, -9, Bid, and Bcl-2), induction of caspase-independent apoptosis (up-regulation of AIF and Endo G on cytosol), suppression of cell cycle (down-regulation of CDK1/2, CDK4, CDK6, cyclin D1, cyclin E, cyclin A, and cyclin B1 or up-regulation of p53 and p21), and inhibition of PI3K/Akt/mTOR signaling (down-regulation of PI3K, p-Akt, and p-mTOR) in prostate cancer cells. These results suggest the induction of apoptosis via suppression of PI3K/Akt/mTOR signaling and cell cycle arrest via activation of p53 in response to sanggenol L in prostate cancer cells.

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Ravingerová, Táňa, Slávka Čarnická, Martina Nemčeková, Veronika Ledvényiová, Adriana Adameová, Tara Kelly, Eleftheria Barlaka, Eleftheria Galatou, Vinoth Kumar Megraj Khandelwal und Antigone Lazou. „PPAR-alpha activation as a preconditioning-like intervention in rats in vivo confers myocardial protection against acute ischaemia–reperfusion injury: involvement of PI3K–Akt“. Canadian Journal of Physiology and Pharmacology 90, Nr. 8 (August 2012): 1135–44. http://dx.doi.org/10.1139/y2012-052.

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Peroxisome proliferator-activated receptors (PPAR) regulate the expression of genes involved in lipid metabolism, energy production, and inflammation. Their role in ischaemia–reperfusion (I/R) is less clear, although research indicates involvement of PPARs in some forms of preconditioning. This study aimed to explore the effects of PPAR-α activation on the I/R injury and potential cardioprotective downstream mechanisms involved. Langendorff-perfused hearts of rats pretreated with the selective PPAR-α agonist WY-14643 (WY, pirinixic acid; 3 mg·(kg body mass)·day–1; 5 days) were subjected to 30 min ischaemia – 2 h reperfusion with or without the phosphatidylinositol 3-kinase (PI3K)–Akt inhibitor wortmannin for the evaluation of functional (left ventricular developed pressure, LVDP) recovery, infarct size (IS), and reperfusion-induced arrhythmias. A 2-fold increase in baseline PPAR-α mRNA levels (qPCR) in the WY-treated group and higher post-I/R PPAR-α levels compared with those in untreated controls were accompanied by similar changes in the expression of PPAR-α target genes PDK4 and mCPT-1, regulating glucose and fatty acid metabolism, and by enhanced Akt phosphorylation. Post-ischaemic LVDP restoration in WY-treated hearts reached 60% ± 9% of the pre-ischaemic values compared with 24% ± 3% in the control hearts (P < 0.05), coupled with reduced IS and incidence of ventricular fibrillation that was blunted by wortmannin. Results indicate that PPAR-α up-regulation may confer preconditioning-like protection via metabolic effects. Downstream mechanisms of PPAR-α-mediated cardioprotection may involve PI3K–Akt activation.

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Van Themsche, Céline, Lyne Lafontaine und Eric Asselin. „X-Linked Inhibitor of Apoptosis Protein Levels and Protein Kinase C Activity Regulate the Sensitivity of Human Endometrial Carcinoma Cells to Tumor Necrosis Factorα-Induced Apoptosis“. Endocrinology 149, Nr. 8 (08.05.2008): 3789–98. http://dx.doi.org/10.1210/en.2008-0275.

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Endometrial carcinomas are often chemoresistant. TNFα shows potent antitumor activity against various cancers, and if it demonstrates good antitumor activity against endometrial cancer, the cytokine could represent a valuable alternative therapeutic approach. We have tested the ability of TNFα to induce apoptosis in endometrial carcinoma cells, and examined a putative role for X-linked inhibitor of apoptosis protein (XIAP) in regulating cellular sensitivity to the cytokine. Exposure to TNFα triggered TNF-R1-dependent activation of caspases-8, -9, and -3, down-regulated Akt and XIAP proteins and induced dose-dependent and time-dependent apoptosis in Ishikawa cells. On the opposite, TNFα up-regulated XIAP in Hec-1A cells; in these cells, the cytokine induced delayed TNF-R1-dependent activation of caspase-8, and failed to activate caspases -9 and -3 and to induce apoptosis. However, XIAP small interfering RNA restored TNFα-induced caspase signaling and apoptosis in Hec-1A cells; XIAP small interfering RNA also increased TNFα-induced apoptosis in Ishikawa cells. In addition, inhibition of protein kinase C activity enhanced TNFα-induced down-regulation of XIAP and potentiated apoptosis induction, in both Ishikawa and Hec-1A cells. Finally, we found XIAP immunoreactivity in epithelial cells from a large number of human endometrial tumor tissue samples, indicating that XIAP is produced by endometrial tumor cells in vivo. This could allow XIAP to play a putative in vivo role in counteracting TNFα-induced apoptosis in endometrial tumor cells; in this case, direct or indirect targeting of XIAP should potentiate the antitumor effect of TNFα.

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DÜMMLER, Katrin, Stefan MÜLLER und Hans J. SEITZ. „Regulation of adenine nucleotide translocase and glycerol 3-phosphate dehydrogenase expression by thyroid hormones in different rat tissues“. Biochemical Journal 317, Nr. 3 (01.08.1996): 913–18. http://dx.doi.org/10.1042/bj3170913.

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Thyroid hormone (T3)-dependent gene expression of the adenine nucleotide translocase (ANT) and the FAD-linked glycerol 3-phosphate dehydrogenase (mGPDH) was investigated in several rat tissues. Both proteins provide an important link between cytosolic and mitochondrial metabolic pathways and seem to be involved in the stimulation of mitochondrial oxygen consumption in response to T3. Here we show that two ANT isoforms are expressed in rat, the muscle-specific ANT1 form and the ubiquitous ANT2 form. The expression of ANT1 mRNA is not sensitive to T3 whereas the amount of ANT2 mRNA is increased 7–9-fold in liver and heart within 12–48 h after T3 application. Little or no effect of T3 on ANT2 mRNA was observed in kidney and brain. The mRNA changes are paralleled by an increase in ANT protein, thus explaining the accelerated ADP/ATP exchange observed in mitochondria isolated from hyperthyroid rats. The key role of ANT2 in the control of hyperthyroid metabolism is evident because the expression of the mersalyl-sensitive phosphate carrier and the mitochondrial creatine kinase mRNA, which are functionally linked to ANT, did not respond to T3. Similarly to the ADP/ATP exchange, the transfer of cytosolic NADH to the respiratory chain via the glycerophosphate shuttle is very sensitive to T3. Recently we demonstrated the 10–15-fold induction of mGPDH mRNA in rat liver after administration of T3 [Müller and Seitz (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10581–10585]. Here we show that, in contrast with ANT2, the time course of induction is fast (4–6 h). Furthermore, mGPDH mRNA is induced 6-fold by T3 in heart and 4-fold in kidney. From these results we conclude that the T3-mediated transcriptional induction leading to increased activity of ANT2 and mGPDH contributes considerably to the increase in mitochondrial oxygen consumption in rat tissues.

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Lee, Chyan-Jang, Ching-Len Liao und Yi-Ling Lin. „Flavivirus Activates Phosphatidylinositol 3-Kinase Signaling To Block Caspase-Dependent Apoptotic Cell Death at the Early Stage of Virus Infection“. Journal of Virology 79, Nr. 13 (01.07.2005): 8388–99. http://dx.doi.org/10.1128/jvi.79.13.8388-8399.2005.

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ABSTRACT Flaviviruses such as dengue virus (DEN) and Japanese encephalitis virus (JEV) are medically important in humans. The lipid kinase, phosphatidylinositol 3-kinase (PI3K) and its downstream target Akt have been implicated in the regulation of diverse cellular functions such as proliferation, and apoptosis. Since JEV and DEN appear to trigger apoptosis in cultured cells at a rather late stage of infection, we evaluated the possible roles of the PI3K/Akt signaling pathway in flavivirus-infected cells. We found that Akt phosphorylation was noticeable in the JEV- and DEN serotype 2 (DEN-2)-infected neuronal N18 cells in an early, transient, PI3K- and lipid raft-dependent manner. Blocking of PI3K activation by its specific inhibitor LY294002 or wortmannin greatly enhanced virus-induced cytopathic effects (CPEs), even at an early stage of infection, but had no effect on virus production. This severe CPE was characterized as apoptotic cell death as evidenced by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining and cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Mechanically, the initiator and effector caspases involved are mainly caspase-9 and caspase-6, since only a pan-caspase inhibitor and the inhibitors preferentially target caspase-9 and -6, but not the ones antagonizing caspase-8, -3, or -7 alleviated the levels of PARP cleavage after virus infection and PI3K blockage. Furthermore, Bcl-2 appears to be a crucial mediator downstream of PI3K/Akt signaling, since overexpression of Bcl-2 reduced virus-induced apoptosis even when PI3K activation was repressed. Collectively, our results suggest an antiapoptotic role for the PI3K/Akt pathway triggered by JEV and DEN-2 to protect infected cells from early apoptotic cell death.

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Liu, Aihua, Zhongfu Zuo, Linlin Liu und Lihua Liu. „Down-regulation of NTSR3 inhibits cell growth and metastasis, as well as the PI3K–AKT and MAPK signaling pathways in colorectal cancer“. Biochemistry and Cell Biology 98, Nr. 5 (Oktober 2020): 548–55. http://dx.doi.org/10.1139/bcb-2019-0351.

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Colorectal cancer is a common malignancy. NTS receptor 3 (NTSR3) is known to play an important role in several cancers. This study examined the effects of NTSR3 on cell growth and metastasis in colorectal cancer. Western blot analysis, real-time PCR, immunofluorescence staining, MTT, cell cycle assay, cell apoptosis assay, Hoechst staining, caspase-3 and caspase-9 activity assays, cell adhesion assay, wound healing assay, and a Transwell assay were used in this study. We found that NTSR3 was expressed at relatively high levels in the colorectal cancer cell lines SW620 and SW480. NTSR3 knockdown suppressed cell growth and promoted cell apoptosis. Meanwhile, the protein expression levels of cyclinD1, cyclinE1, CDK4, and p-RB were reduced, and the levels of p-P27, P15, P21, cleaved caspase-3, and cleaved caspase-9 protein were increased. Cell invasiveness and cell migration were reduced with knockdown of NTSR3. In addition, our rescue experiments demonstrated that overexpression of the siRNA-resistant alleles of NTSR3 abrogated the NTSR3-siRNA-mediated effects on cell function. Further, down-regulation of NTSR3 inactivated the PI3K–AKT and MAPK signaling pathways. Collectively, these data demonstrate that knockdown of NTSR3 inhibits cell growth and metastasis, as well as the PI3K–AKT and MAPK signaling pathways in colorectal cancer. Thus, our results indicate that NTSR3 is a potential therapeutic target for treating colorectal cancer.

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Kubiatowski, Tomas, Taichang Jang, Mahesh B. Lachyankar, Rebecca Salmonsen, Roya R. Nabi, Peter J. Quesenberry, N. Scott Litofsky, Alonzo H. Ross und Lawrence D. Recht. „Association of increased phosphatidylinositol 3-kinase signaling with increased invasiveness and gelatinase activity in malignant gliomas“. Journal of Neurosurgery 95, Nr. 3 (September 2001): 480–88. http://dx.doi.org/10.3171/jns.2001.95.3.0480.

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Object. Glioblastoma multiforme is the most malignant of the primary brain tumors and aggressively infiltrates surrounding brain tissue, resulting in distant foci within the central nervous system, thereby rendering this tumor surgically incurable. The recent findings that both phosphatidylinositol 3-kinase (PI 3-K) and the phosphatase and tensin homolog (PTEN) regulate tumor cell invasiveness have led the authors to surmise that these lipid signaling molecules might play a role in regulating matrix metalloproteinases (MMPs), which are essential for tumor cell invasion. Methods. Using the C6 glioma cell line, which does not express measurable amounts of PTEN protein and in which in vitro invasiveness is MMP dependent, the authors determined that in vitro glioma cell invasiveness was significantly reduced when cells were preincubated overnight with LY294002 or wortmannin, two specific inhibitors of PI 3-K signaling. Next, using gelatin zymography, it was noted that these compounds significantly inhibited MMP-2 and MMP-9 activities. Moreover, the decrease in MMP activity correlated with the decrease in PI 3-K activity, as assessed by Akt phosphorylation. Finally, using semiquantitative reverse transcriptase—polymerase chain reaction, the authors demonstrated that LY294002 decreased messenger (m)RNA levels for both MMPs. Thus, these in vitro data indicate that PI 3-K signaling modulates gelatinase activity at the level of mRNA. Using immunostaining of phosphorylated Akt (p-Akt) as a measure of PI 3-K activity, the authors next assessed rat brains implanted with C6 cells. Compared with surrounding brain, there was marked p-Akt staining in C6 glioma cells and in neurons immediately adjacent to the tumor, but not in normal brain. The p-Akt staining in tumors was especially intense in perivascular areas. Using double-labeling techniques, colocalization of p-Akt with MMP-2 and MMP-9 was also noted in perivascular tumor areas. Conclusions. The increase in p-Akt staining within these PTEN-deficient gliomas is consistent with what would be predicted from unchecked PI 3-K signaling. Furthermore, the immunohistochemically detected colocalization of p-Akt and MMP-2 and MMP-9 supports the authors' in vitro studies and the proposed linkage between PI 3-K signaling and MMP activity in gliomas.

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Taha, Tarek A., Walid Osta, Lina Kozhaya, Jacek Bielawski, Korey R. Johnson, William E. Gillanders, Ghassan S. Dbaibo, Yusuf A. Hannun und Lina M. Obeid. „Down-regulation of Sphingosine Kinase-1 by DNA Damage“. Journal of Biological Chemistry 279, Nr. 19 (26.02.2004): 20546–54. http://dx.doi.org/10.1074/jbc.m401259200.

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Sphingosine kinase 1 (SK1), a key enzyme in sphingosine 1-phosphate (S1P) synthesis, regulates various aspects of cell behavior, including cell survival and proliferation. DNA damaging anti-neoplastic agents have been shown to induce p53, ceramide levels, and apoptosis; however, the effects of anti-neoplastic agents on SK have not been assessed. In this study, we investigated the effects of a DNA damaging agent, actinomycin D (Act D), on the function of sphingosine kinase (SK1). Act D caused a reduction in the protein levels of SK1, as indicated by Western blot analysis, with a concomitant decrease in SK activity. The down-regulation was post-transcriptional, because the mRNA levels of SK1 remained unchanged. Similar decreases in SK1 protein were observed with other DNA damaging agents such as doxorubicin, etoposide, and γ-irradiation. ZVAD, the pancaspase inhibitor, and Bcl-2 annulled the effect of Act D on SK1, demonstrating a role for cysteine proteases downstream of Bcl-2 in the down-regulation of SK1. Inhibition of caspases 3, 6, 7, and 9 only partially reversed Act D-induced SK1 loss. Inhibition of cathepsin B, a lysosomal protease, produced a significant reversal of SK1 decline by Act D, suggesting that a multitude of ZVAD-sensitive cysteine proteases downstream of Bcl-2 mediated the SK1 decrease. When p53 up-regulation after Act D treatment was inhibited, SK1 down-regulation was rescued, demonstrating p53 dependence of SK1 modulation. Treatment of cells with S1P, the product of SK1, partially inhibited Act D-induced cell death, raising the possibility that a decrease in SK1 may be in part necessary for cell death to occur. Furthermore, the knockdown of SK1 by small interfering RNA in MCF-7 cells resulted in a significant reduction in cell viability. These studies demonstrate that SK1 is down-regulated by genotoxic stress, and that basal SK1 function may be necessary for the maintenance of tumor cell growth.

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Chae, Kwon-Seok, Miguel Martin-Caraballo, Marc Anderson und Stuart E. Dryer. „Akt Activation Is Necessary for Growth Factor–Induced Trafficking of Functional KCa Channels in Developing Parasympathetic Neurons“. Journal of Neurophysiology 93, Nr. 3 (März 2005): 1174–82. http://dx.doi.org/10.1152/jn.00796.2004.

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The protein kinase Akt is a crucial regulator of neuronal survival and apoptosis. Here we show that Akt activation is necessary for mobilization of large-conductance KCa channels in ciliary ganglion (CG) neurons evoked by β-neuregulin-1 (NRG1) and transforming growth factor-β1 (TGFβ1). Application of NRG1 to embryonic day 9 (E9) CG neurons increased Akt phosphorylation, as observed previously for TGFβ1. NRG1- and TGFβ1-evoked stimulation of KCa is blocked by inhibitors of PI3K by overexpression of a dominant-negative form of Akt, by overexpression of CTMP, an endogenous negative regulator of Akt, and by application of the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-( R)-2- O-methyl-3- O-octadecylcarbonate (HIMO). Conversely, overexpression of a constitutively-active form of Akt was sufficient by itself to increase mobilization of functional KCa channels. NRG1 and TGFβ1 evoked an Akt-dependent increase in cell-surface SLO α-subunits. These procedures have no effect on voltage-activated Ca2+ currents. Thus Akt plays an essential role in the developmental regulation of excitability in CG neurons.

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Iashchenko, A. „MEASURES OF CRIMINAL JUSTICE RESPONSE TO PROHIBITIONS IN THE FIELD OF TRAFFIC SAFETY AND VEHICLE OPERATION: SOME ASPECTS OF REGULATORY CERTAINTY AND PRACTICE OF USE“. Archives of Criminology and Forensic Sciences 2 (01.09.2020): 59–68. http://dx.doi.org/10.32353/acfs.2.2020.05.

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The article is devoted to the research of measures of criminal justice response to prohibitions in the field of traffic safety and vehicle operation. It is noted that the primary role in state response to violation of criminal justice prohibitions in the field of traffic safety and vehicle operation is given to punishment, but no less important role is paid to other alternative to prohibition measures of criminal justice nature based on the concussion (special confiscation) or the encouragement (exemption from criminal responsibility or serving a sentence). It is concluded that the normative regulations of threats of application of certain punitive measures of criminal justice nature in sanctions of the articles of this section of the Special part in which the legislator defines the threat of application of various types of punishment for committing the crimes stipulated in crimes’ dispositions, needs specification from the point of view of the system interconnection, along with the provisions of the General Part of the Criminal Code of Ukraine, whereas the practice of application of special confiscation its further distribution and development, considering the proposed recommendations of its delimitation with the so called criminal procedural confiscation as means of criminal procedural concussion. In particular, it is noted that such clarification may be implemented either by enforcing additional penalties specified in the sanctions of Part 1, 2, 3 of Article 286, part 1 of Article 287 of the Criminal Code of Ukraine, to the common list of types of punishments, with their separate meaningful definition in the corresponding articles of the section X of the General part of the Criminal Code of Ukraine, or by covering normative definition in sanctions of the specified articles of section XI of the Special part of threats of application of such additional types of punishments according to the existing parts of the Criminal Code of Ukraine. In this regard the sanctions of Article 286 and 287 of the Criminal Code of Ukraine propose to make appropriate changes. As for the practice of applying special confiscation for committing crimes in the field of traffic safety and vehicle operation, it is recommended that the question of its implementation should be based on the fact that the subject of special confiscation may be defined in paragraph 1 of Part 1 of Art. 96-2 of the Criminal Code - items 6, 6-1 part 9 of Art. 100 of the Criminal Procedure Code, paragraph 2, part 1 of Art. 96-2 of the Criminal Code - item 2 part 9 of Art. 100 of the CPC, paragraph 3, part 1 of Art. 96-2 of the Criminal Code - item 5 part 9 of Art. 100 of the CPC, paragraph 4, part 1 of Art. 96-2 of the Criminal Code - item 1 part 9 of Art. 100 of the CPC items of the material world that possess a certain property value, and are usually considered as physical evidence in criminal proceedings initiated on the fact of committing certain crimes in the field of traffic safety and vehicle operation.

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Civallero, Monica, Maria Cosenza und Stefano Sacchi. „New Targets and Potential Strategy to Enhance the Anticancer Efficacy BKM-120 and BEZ235 in Lymphoma Cell Lines“. Blood 124, Nr. 21 (06.12.2014): 1773. http://dx.doi.org/10.1182/blood.v124.21.1773.1773.

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Abstract Background. The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays an important role in many physiological functions, including cell cycle progression, differentiation, survival, motility, apoptosis, protein synthesis and metabolism modification. The PI3K/AKT/mTOR signaling pathway also inhibits cell autophagy, a catabolic process involving the degradation of a cell’s own components through the lysosomal machinery. BKM120 and BEZ235 are synthetic small molecules belonging to the class of imidazo-quinolones that show preclinical activity against a range of solid and hematological malignancies. BKM-120 inhibits the catalytic subunit of class I PI3K by competitive binding to its ATP binding site, while BEZ235 is a dual class I PI3K/mTOR inhibitor. In this study we investigated the effects of BKM120 and BEZ235 on survival rate, apoptosis, signaling pathways expression, autophagy, metabolism modification, cell cycle arrest and tubulin polymerization kinetic in lymphoma cell lines. Method. Lymphoma cell lines (WSU-NHL, Jeko-1 and Karpas-299) were treated with different concentrations of BKM120 and BEZ235 (Novartis) for 24 and 48h and the IC50 values were evaluated using MTT assay. To assess for apoptosis, we used annexin V/PI staining kit/ flow cytometer analysis and Western Blot to evaluate caspase 3, 8, 9 and PARP expression. The cell cycle was performed applying PI incorporation and flow cytometer analysis. A flow cytometry based technique for the analysis of tubulin polymerization using a-tubulin staining was done to test whether the two drugs could cause a mitotic block. Western blot was utilized for phosphorylation status of protein kinases and for monitoring autophagy and metabolism. Results and conclusions. BKM120 and BEZ235 induced significant increase of apoptosis evidenced by annexin IV/PI staining and confirmed by the cleavage of caspases -3, -9 -8 and PARP. BKM120 and BEZ235 induced an up regulation of pro apoptotic protein Bim, Bax and Bad. Treatment for 24h with BKM120 and BEZ235 resulted in different effects on cell cycle. BKM120 induced an increase of G2-phase with down regulation of Cyclin D and E, and an up-regulation of Cyclin A, p21 and p27. The increase in G2-M caused by BKM120 treatment occurred in a dose dependent manner. BEZ235 induced an increase of G0/G1-phase with up regulation of Cyclin A, D, E and p21 and p27. Quantitative analysis of a-tubulins polymerization of the cell lines revealed that treatment with BKM120 induced an accumulation of mitotic cells. BKM120 and BEZ235 are inhibitors of intracellular pathways in targeting p-Akt, p- mTOR, pS6K, 4EBP1, MYC and STAT. BKM120 and BEZ235 increased the levels of type II LC3 and p62, hallmarks of autophagy, in addition to increasing caspase 3 cleavage and annexin positive cells, suggesting that the two drugs induced both apoptosis and autophagy. The combination of BKM120 and BEZ235 with chloroquine which are in clinical use, with the lysosomotropic autophagy inhibitor chloroquine demonstrating marked cooperates with inhibition of autophagy to elicit apoptosis through the intrinsic mitochondrial pathway (BAX, BIM and BAD). Treatment with BKM120 and BEZ235 induced an increase of GLUT1 and HIF-1 protein expression. GLUT1 plays a role in regulation of ROS levels in particular after BEZ235 treatment. We concluded that BKM120 and BEZ235 inhibit PI3K/AKT and mTOR signaling, induce autophagy-apoptosis, affect lymphoid cell metabolism and promote ER stress via ROS increase. Moreover, we observed that BKM120 can act as a microtubule destabilizer inducing cell cycle arrest. In these study, we highlight new targets of BKM120 and BEZ235 in addition to the known PI3K/AKT signaling pathway; these results can help to identify new potential strategies to enhance the anticancer efficacy BKM-120 and BEZ235 in lymphoma cell lines. Disclosures No relevant conflicts of interest to declare.

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Van Themsche, Céline, Isabelle Mathieu, Sophie Parent und Eric Asselin. „Transforming Growth Factor-β3 Increases the Invasiveness of Endometrial Carcinoma Cells through Phosphatidylinositol 3-Kinase-dependent Up-regulation of X-linked Inhibitor of Apoptosis and Protein Kinase C-dependent Induction of Matrix Metalloproteinase-9“. Journal of Biological Chemistry 282, Nr. 7 (06.12.2006): 4794–802. http://dx.doi.org/10.1074/jbc.m608497200.

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Tumor cells often acquire intrinsic resistance to the growth inhibitory and pro-apoptotic effects of transforming growth factor-β (TGF-β); moreover, TGF-β can confer invasive properties to established tumor cells. In the present study, we show that TGF-β isoforms (TGF-β1, TGF-β2, and TGF-β3) trigger proper Smad signaling in human endometrial carcinoma cell lines and efficiently inhibit cellular proliferation. These cells, however, exhibit a high degree of resistance to TGF-β pro-apoptotic effects; we found that this resistant phenotype would be acquired through up-regulation of X-linked inhibitor of apoptosis protein (XIAP) levels. In addition, using RNA interference and pharmacological inhibitors, we show that TGF-β increases cellular invasiveness via two distinct signaling pathways in endometrial carcinoma cells: phosphatidylinositol 3-kinase/AKT-dependent up-regulation of XIAP and protein kinase C-dependent induction of matrix-metalloproteinase-9 (MMP-9) expression. Additionally, these findings were correlated with clinical observations showing abundant TGF-β immunoreactivity in human endometrial carcinoma tumors in vivo, extending from the epithelial compartment to the stroma upon acquisition of an invasive phenotype (gradually from grades I to III). Collectively our results describe for the first time a role for TGF-β3 in tumor invasiveness.

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Shaik, Zabeena P., E. Kim Fifer und Grażyna Nowak. „Protein kinase B/Akt modulates nephrotoxicant-induced necrosis in renal cells“. American Journal of Physiology-Renal Physiology 292, Nr. 1 (Januar 2007): F292—F303. http://dx.doi.org/10.1152/ajprenal.00082.2006.

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Protein kinase B (Akt) activation is well known for its protective effects against apoptosis. However, the role of Akt in regulation of necrosis is unknown. This study was designed to test whether Akt activation protects against nephrotoxicant-induced injury and death in renal proximal tubular cells (RPTC). Exposure of primary cultures of RPTC to the nephrotoxic cysteine conjugate, S-(1,2-dichlorovinyl)-l-cysteine (DCVC), resulted in 9% apoptosis and 30% necrosis at 24 h following the exposure. Akt was activated during 8 h but not at 24 h following toxicant exposure. No RPTC necrosis was observed during Akt activation. Blocking Akt activation using a phosphatidylinositol 3-kinase inhibitor, LY294002 (20 μM), or expressing dominant negative (inactive) Akt increased DCVC-induced RPTC necrosis to 42%. In contrast, Akt activation by expression of constitutively active Akt diminished necrosis to 15%. Modulation of Akt activity had no effect on DCVC-induced apoptosis. DCVC-induced RPTC injury was accompanied by decreases in respiration (51% of controls) and ATP levels (57% of controls). Akt inhibition exacerbated decreases in RPTC respiration and intracellular ATP content (both to 30% of controls). In contrast, Akt activation reduced DCVC-induced decreases in respiration (80% of controls) and prevented decline in ATP content. These data show that in RPTC, Akt activation reduces 1) toxicant-induced mitochondrial dysfunction, 2) decreases in ATP levels, and 3) necrosis. We conclude that Akt activation plays a protective role against necrosis caused by nephrotoxic insult in RPTC. Furthermore, we identified mitochondria as a subcellular target of protective actions of Akt against necrosis.

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Lhuillier, Loic, und Stuart E. Dryer. „Developmental Regulation of Neuronal KCa Channels by TGFβ1: An Essential Role for PI3 Kinase Signaling and Membrane Insertion“. Journal of Neurophysiology 88, Nr. 2 (01.08.2002): 954–64. http://dx.doi.org/10.1152/jn.2002.88.2.954.

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TGFβ1 is a target-derived factor responsible for the developmental expression of large-conductance Ca2+-activated K+(KCa) channels in ciliary neurons of the chick ciliary ganglion. The acute effects of TGFβ1 on KCa channels are mediated by posttranslational events and require activation of the MAP kinase Erk. Here we show that TGFβ1 evokes robust phosphorylation of Akt/PKB, a protein kinase dependent on the products of phosphatidylinositol 3-OH kinase (PI3K). TGFβ1-evoked stimulation of KCa channels is blocked by the PI3K inhibitors wortmannin and LY294002. These drugs also inhibit TGFβ1 effects on Akt/PKB phosphorylation but have no effect on TGFβ1-evoked Erk activation. Application of the MEK1 inhibitor PD98059 blocked TGFβ1 effects on Erk but had no effect on Akt/PKB phosphorylation. These results indicate that PI3K and Erk represent parallel signaling cascades activated by TGFβ1 in ciliary neurons. The effects of TGFβ1 on functional expression of KCa are blocked by the microtubule inhibitors colchicine and nocodazole, by botulinum toxins A and E, and by brefeldin-A, an agent that disrupts the Golgi apparatus. These data indicate that translocation of a membrane protein, possibly Slowpoke (SLO), is required for the acute posttranslational effects of TGFβ1 on KCa channels. Confocal immunofluorescence studies with three different SLO antisera showed robust expression of SLO in multiple intracellular compartments of embryonic day 9–13 ciliary neurons, including the cell nucleus. These data suggest that TGFβ1 evokes insertion of SLO channels into the plasma membrane as a result of signaling cascades that entail activation of Erk and PI3K.

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Li, Dongjun, und Donna Woulfe. „Arrestin-2 Differentially Regulates PAR4 and P2Y12 Receptor Signaling in Platelets“. Blood 112, Nr. 11 (16.11.2008): 110. http://dx.doi.org/10.1182/blood.v112.11.110.110.

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Abstract Arrestins play important roles in the function of G Protein-Coupled Receptors (GPCRs) in many cells, but their roles in platelets remain uncharacterized. While the classical role of arrestins is considered to be the internalization and desensitization of GPCRs, more recent studies suggest that arrestins can serve as molecular scaffolds to recruit phosphatidyl inositol-3 kinases (PI3Ks) to GPCRs and promote PI3K-dependent signaling. Due to the multifunctional role of arrestins, we sought to determine whether arrestins regulate Akt activation in platelets and thrombosis in living animals. Co-immunoprecipitation experiments indicate that arrestin-2 associates with PAR4 in thrombin-treated platelets and P2Y12 in ADP-treated platelets, but neither receptor in resting cells. Interestingly, association of arrestin-2 with PAR4 was also stimulated by ADP and PAR4-induced association of arrestin with PAR4 was inhibited by P2Y12 antagonists or apyrase. To determine the functional role of arrestin-2 in platelets, ADP- and thrombin receptor-stimulated Akt phosphorylation was compared in platelets from arrestin-2 knock-out versus WT mice. Akt phosphorylation stimulated by 0.8 mM AYPGKF PAR4 agonist peptide was reduced by an average of 77% in arrestin-2 knock-out platelets compared to WT controls (significantly different, p=0.007, n=3 in each group), but ADP-stimulated Akt phosphorylation was unaffected (p=.38, n=3 each). PAR4-stimulated fibrinogen binding was also reduced in arrestin2−/− platelets (by 58.5% in 1 mM AYPGKF-stimulated platelets compared to WT controls), whereas ADP-stimulated fibrinogen binding was not. Finally, arrestin2 knock-out mice were less sensitive to ferric chloride-induced thrombosis than WT mice: 55% of WT mice (n-=9) formed occlusive thrombi after 2min15sec exposure of the carotid artery to 10% ferric chloride, whereas only 11% of WT mice (n=9) formed occlusive thrombi under the same conditions. In conclusion, arrestin-2 associates with both PAR4 and P2Y12 receptors, but differentially regulates their signaling to Akt and fibrinogen binding and appears to play a net positive role in regulating thrombosis in vivo.

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Care, A. S., M. J. Jasper, W. V. Ingman und S. A. Robertson. „146. MACROPHAGES ARE ESSENTIAL FOR MAINTENANCE OF EARLY PREGNANCY THROUGH REGULATION OF CORPUS LUTEUM FUNCTION“. Reproduction, Fertility and Development 21, Nr. 9 (2009): 64. http://dx.doi.org/10.1071/srb09abs146.

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Macrophages are abundant within the ovary, and have been identified in the corpus luteum (CL) of most species studied, including in rodents and human. Through their secretory products, macrophages are thought to be involved in ovarian tissue remodelling, including luteinization, and in regulating steroidogenesis [1, 2]. Macrophages co-cultured with granulosa or luteal cells act to stimulate progesterone secretion [3]. To determine the impact of macrophage ablation during early pregnancy, we utilised the macrophage specific CD11b-DTR diphtheria toxin receptor (DTR) transgenic mouse to cause transient systemic ablation of macrophages via the administration of DT (25 ng/g). The effects of macrophage ablation during the pre-implantation phase of pregnancy were evaluated and implantation sites were counted. Ablation of macrophages on day 1 pc (n=13 wild-type; n=9 CD11b-DTR) or day 4 pc (n=6 CD11b-DTR; n=9 wild-type) caused complete pregnancy loss in all DT-treated CD11b-DTR mice, while wild-type mice maintained viable pregnancies [mean ± SEM implantation sites = 5.0 ± 1.4 (day 1 treated), and 6.0 ± 1.9 (day 4 treated)]. Serum progesterone was analysed 24 h following macrophage ablation. A single DT injection on day 3 pc significantly reduced serum progesterone (P4) levels [n=7 wild-type P4 (ng/ml)= 29.6 ± 3.3; n=8 CD11b-DTR = 11.1 ± 2.1]. The administration of exogenous P4 (2 mg) on each of day 4-7 pc prevented fetal loss in DT-treated CD11b-DTR mice (n=6; implantation sites = 7.8 ± 1.6), while no pregnancies remained viable in DT-treated mice administered vehicle only (n=9). In conclusion, luteal insufficiency appears to be the cause of pregnancy failure following macrophage ablation. These data indicate a critical role for macrophages in corpus luteum function in early pregnancy.

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Wang, Jing, Zhichun Dong, Liyin Lou, Lijuan Yang und Jingying Qiu. „MiR-122 Participates in Oxidative Stress and Apoptosis in STZ-Induced Pancreatic β Cells by Regulating PI3K/AKT Signaling Pathway“. International Journal of Endocrinology 2021 (12.05.2021): 1–11. http://dx.doi.org/10.1155/2021/5525112.

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At present, there are few reports concerning the relationship between miR-122 and diabetes. In addition, the effect of miR-122 on streptozotocin- (STZ-) induced oxidative damage in INS-1 cells remains unclear. The present study aimed to investigate the role and modulatory mechanisms involving miR-122 in diabetes. STZ was used to induce INS-1 cell damage. Reverse transcription-quantitative PCR was used to investigate the expression of miR-122. A TUNEL cell apoptosis detection kit was used to detect apoptosis. Intracellular ROS levels were determined using dichlorofluorescein-diacetate. The activities of insulin secretion, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-px) were measured using ELISA kits. Western blotting was used to measure the expression levels of Bax, Bcl-2, PI3K, p-PI3K, caspase-3 and caspase-9, cleaved-caspase-3 and cleaved-caspase-9, AKT, and p-AKT. Then, LY294002 (LY, PI3K inhibitor) was used to treat INS-1 cells, and oxidative stress and apoptosis were measured. The results showed that STZ-induced inhibitory effects on insulin secretion were mitigated by miR-122 inhibitor, and the activities of SOD, CAT, and GSH-px were also increased. Furthermore, miR-122 inhibitor inhibited apoptosis and oxidative stress in STZ-induced INS-1 cells. Finally, the addition of LY increased insulin levels; reduced the activities of SOD, CAT, and GSH-px; and promoted apoptosis in STZ-induced INS-1 cells. In conclusion, interference with miR-122 can inhibit oxidative stress and apoptosis in STZ-induced INS-1 cells, involving a mechanism of action related to the PI3K/AKT pathway.

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Zhan, Haibing, Xingchang Sun, Xiaoxia Wang, Qing Gao, Mengmeng Yang, Han Liu, Jinfa Zheng et al. „LncRNA MEG3 Involved in NiO NPs-Induced Pulmonary Fibrosis via Regulating TGF-β1-Mediated PI3K/AKT Pathway“. Toxicological Sciences 182, Nr. 1 (24.04.2021): 120–31. http://dx.doi.org/10.1093/toxsci/kfab047.

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Abstract Long noncoding RNA maternally expressed gene 3 (MEG3) involves in fibrotic diseases, but its role in nickel oxide nanoparticles (NiO NPs)-induced pulmonary fibrosis remains unclear. The present study aimed to explore the relationships among MEG3, transforming growth factor-β1 (TGF-β1) and phosphoinositide 3-kinase (PI3K)/AKT pathway in NiO NPs-induced pulmonary fibrosis. Wistar rats were intratracheally instilled with NiO NPs twice a week for 9 weeks, and human lung adenocarcinoma epithelial cells (A549 cells) were exposed to NiO NPs for 24 h. The pathological alterations and increased hydroxyproline indicated that NiO NPs caused pulmonary fibrosis in rats. The up-regulated type I collagen (Col-I) suggested that NiO NPs-induced collagen deposition in A549 cells. Meanwhile, NiO NPs could significantly down-regulate MEG3, up-regulate TGF-β1 and activate PI3K/AKT signaling pathway both in vivo and in vitro. However, we found that the PI3K/AKT pathway activated by NiO NPs could be suppressed by 10 μM TGF-β1 inhibitor (SB431542) in A549 cells. The protein markers (Col-I, Fibronectin, and alpha-smooth muscle actin) of collagen deposition up-regulated by NiO NPs were reduced by 10 μM PI3K inhibitor (LY294002). Furthermore, we further found that overexpressed MEG3 inhibited the expression of TGF-β1, resulting in the inactivation of PI3K/AKT pathway and the reduction of collagen formation. In summary, our results validated that MEG3 could arrest NiO NPs-induced pulmonary fibrosis via inhibiting TGF-β1-mediated PI3K/AKT pathway.

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Liu, Yu, Liang Yang, Fan Liao, Wei Wang und Zhi-Fei Wang. „MiR-450a-5p strengthens the drug sensitivity of gefitinib in glioma chemotherapy via regulating autophagy by targeting EGFR“. Oncogene 39, Nr. 39 (20.08.2020): 6190–202. http://dx.doi.org/10.1038/s41388-020-01422-9.

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Abstract Glioma reported to be refractory to EGFR tyrosine kinase inhibitor is the most common malignant tumor in central nervous system. Our research showed the low expression of miR-450a-5p and high expression of EGFR in glioma tissues. MiR-450a-5p was also observed to synergize with gefitinib to inhibit the proliferation, migration and invasion and induce the apoptosis and autophagy of glioma cells. Furthermore, miR-450a-5p was demonstrated to target 3′UTR of EGFR, and regulated EGFR-induced PI3K/AKT/mTOR signaling pathway. Moreover, the above effects induced by miR-450a-5p in glioma cells were reversed by WIPI1 silencing. The inhibition role of miR-450a-5p on glioma growth was also confirmed in vivo by subcutaneous and intracranial tumor xenografts. Therefore, we conclude that miR-450a-5p synergizes with gefitinib to inhibit the glioma tumorigenesis through inducing autophagy by regulating the EGFR-induced PI3K/AKT/mTOR signaling pathway, thereby enhancing the drug sensitivity of gefitinib.

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Friedrich, Erik B., Emerson Liu, Sumita Sinha, Stuart Cook, David S. Milstone, Calum A. MacRae, Massimo Mariotti et al. „Integrin-Linked Kinase Regulates Endothelial Cell Survival and Vascular Development“. Molecular and Cellular Biology 24, Nr. 18 (15.09.2004): 8134–44. http://dx.doi.org/10.1128/mcb.24.18.8134-8144.2004.

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ABSTRACT Integrin-linked kinase (ILK) is a phosphoinositide 3-kinase-dependent serine/threonine kinase that interacts with β integrins. Here we show that endothelial cell (EC)-specific deletion of ILK in mice confers placental insufficiency with decreased labyrinthine vascularization, yielding no viable offspring. Deletion of ILK in zebra fish using antisense morpholino oligonucleotides results in marked patterning abnormalities of the vasculature and is similarly lethal. To dissect potential mechanisms responsible for these phenotypes, we performed ex vivo deletion of ILK from purified EC of adult mice. We observed downregulation of the active-conformation of β1 integrins with a striking increase in EC apoptosis associated with activation of caspase 9. There was also reduced phosphorylation of the ILK kinase substrate, Akt. However, phenotypic rescue of ILK-deficient EC by wild-type ILK, but not by a constitutively active mutant of Akt, suggests regulation of EC survival by ILK in an Akt-independent manner. Thus, endothelial ILK plays a critical role in vascular development through integrin-matrix interactions and EC survival. These data have important implications for both physiological and pathological angiogenesis.

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Nimmanapalli, Ramadevi, Erica O'Bryan, Deborah Kuhn, Hirohito Yamaguchi, Hong-Gang Wang und Kapil N. Bhalla. „Regulation of 17-AAG—induced apoptosis: role of Bcl-2, Bcl-xL, and Bax downstream of 17-AAG—mediated down-regulation of Akt, Raf-1, and Src kinases“. Blood 102, Nr. 1 (01.07.2003): 269–75. http://dx.doi.org/10.1182/blood-2002-12-3718.

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Abstract 17-allylamino-demethoxy geldanamycin (17-AAG) inhibits the chaperone function of heat shock protein—90 (Hsp-90) and promotes the proteasomal degradation of its misfolded client proteins. Here, we demonstrate that treatment of the human acute myeloid leukemia HL-60 cells with 17-AAG attenuates the intracellular levels of a number of Hsp-90 client proteins, including Akt, c-Raf-1, and c-Src. Also, 17-AAG induced the mitochondrial release and cytosolic accumulation of cytochrome c (cyt c) and second mitochondria-derived activator of caspases (Smac)/DIABLO, resulting in the activation of caspase-9 and caspase-3 and apoptosis. Treatment with 17-AAG triggered the B-cell lymphoma—2 (Bcl-2)—associated X protein (Bax) conformational change associated with apoptosis, while Bax-deficient cells were resistant to 17-AAG—induced apoptosis. In addition, in HL-60/Bcl-2 and HL-60/Bcl-xL cells, which ectopically express Bcl-2 and Bcl-xL respectively, 17-AAG—induced Bax conformational change, cytosolic accumulation of cyt c and Smac/DIABLO, and apoptosis were markedly inhibited. Although the rate of 17-AAG—mediated decline in Akt, c-Raf-1, and c-Src levels was blunted, the total decline was not compromised in HL-60/Bcl-2 and HL-60/Bcl-xL cells. Cotreatment with HA14-1, a nonpeptidic ligand that can bind and inhibit the antiapoptotic activity of Bcl-2, significantly overcame the resistance to 17-AAG—induced apoptosis in HL-60/Bcl-2 cells. Together, these findings indicate that although 17-AAG treatment causes the levels of a number of survival-signaling protein kinases to decline, the downstream engagement of the mitochondrial pathway of apoptosis is regulated by the activity of the Bcl-2 family of proteins. Also, neutralizing the antiapoptotic effect of Bcl-2 would further enhance the antileukemia activity of 17-AAG. (Blood. 2003;102:269-275)

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Kuemmerle, John F., und Toni L. Bushman. „IGF-I stimulates intestinal muscle cell growth by activating distinct PI 3-kinase and MAP kinase pathways“. American Journal of Physiology-Gastrointestinal and Liver Physiology 275, Nr. 1 (01.07.1998): G151—G158. http://dx.doi.org/10.1152/ajpgi.1998.275.1.g151.

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Insulin-like growth factor I (IGF-I), acting via its cognate receptor, plays an autocrine role in the regulation of growth of intestinal muscle cells. In the present study the signaling pathways mediating the growth effects of IGF-I were characterized in cultured human intestinal smooth muscle cells. Growth induced by a maximally effective concentration of IGF-I (100 nM), measured as [3H]thymidine incorporation, was only partially inhibited by LY-294002 [phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor] or PD-98059 [mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor] (86 ± 7% and 35 ± 6% inhibition, respectively) alone but was abolished by the two combined (114 ± 18% inhibition), implying the participation of both pathways. IGF-I elicited time- and concentration-dependent increases in PI 3-kinase activity. This effect was inhibited only by LY-294002 (89 ± 12%). IGF-I elicited time- and concentration-dependent phosphorylation of p44/p42 MAP kinase and increased MAP kinase activity. These effects were inhibited only by PD-98059 (78 ± 9% and 98 ± 7%, respectively). We conclude that in human intestinal muscle cells IGF-I activates distinct PI 3-kinase and MAP kinase signaling pathways, which act in conjunction to mediate growth.

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Naik, Meghna Ulhas, Brendan Bachman, John C. Kostyak, Wei Dai und Ulhas P. Naik. „Polo-Like Kinase 3, a Novel Regulator of Thrombosis: Positive Regulation of TxA2 Generation, Granular Secretion and Clot Retraction, but Negative Regulation of Integrin Activation“. Blood 120, Nr. 21 (16.11.2012): 379. http://dx.doi.org/10.1182/blood.v120.21.379.379.

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Abstract Abstract 379 Polo-like kinase (Plk) family members are serine/threonine kinases involved in cell cycle regulation. Their expression and function in platelets are not known. We identified the presence of Plk3, a member of this family, in human and mouse platelets. We found that Plk3 is localized to the filopodia of activated platelets. Furthermore, it co-immunoprecipitates with integrin aIIbb3 in an aggregation-dependent manner. To understand the physiological function of Plk3 in thrombosis, we obtained Plk3−/− mice in C57Bl/6 background, examined the tail bleeding time of Plk3−/− mice, and compared it to the Plk3+/+ (WT) mice of the same genetic background. We found that the average tail bleeding time for WT mice was about 130 s, consistent with the values reported in the literature. Interestingly, the Plk3−/− mice had a significantly (P<0.05) delayed average tail bleeding time (325 s), suggesting that Plk3 deficiency results in a bleeding phenotype. These results suggest that there may be defects in the thrombotic process in these mice. To evaluate the in vivo thrombotic phenotype, we performed a 10% FeCl3-induced carotid artery injury and observed any differences in time of occlusion or unstable occlusions in Plk3−/− mice compared to WT mice. Consistent with our finding of extended tail bleeding time in Plk3−/− mice, our results showed that the WT mouse vessel occluded within 7–9 min, whereas Plk3−/− mouse took nearly twice that time (∼14 min) to initiate vessel occlusion (P<0.001). We also performed a collagen/epinephrine-induced pulmonary thromboembolism assay to investigate the role of platelet Plk3 in thrombosis. Our result suggests a marked protection from thromboembolism in Plk3−/− mice, since significantly more (P<0.0004) survived compared to WT mice. By assessing the ability of Evans blue dye to pass through the pulmonary circulation, we determined that this better survival rate in Plk3−/− mice is due to the failure of occlusion of pulmonary vessels in these mice. This was further supported by the histological examination of the lungs of these mice, which showed decreased size of the emboli and the reduced extent of arterial occlusion compared to WT. Ex vivo platelet functional studies suggested that thrombin-induced generation of TxA2, a potent activator of platelet function, was significantly attenuated (P<0.03) in Plk3−/− mice compared to WT. When tested for activation of cPLA2, a key enzyme in TxA2 generation, we found that the phosphorylation of cPLA2 is significantly attenuated (P<0.05) in Plk3 null platelets. Furthermore, thrombin-induced secretion of both a- and d- granules was significantly reduced (P<0.007) in Plk3−/− mouse platelets compared to WT, consistent with the observed anti-thrombotic phenotype in vivo. Surprisingly, however, platelet aggregation by low dose of thrombin or PAR4 peptide was significantly augmented (P<0.02) in Plk3 null platelets compared to WT. This was further supported by the significantly increased (P<0.05) fibrinogen receptor exposure on platelets. To determine the molecular mechanism of the observed hyperaggregation, we analyzed signaling events such as ERK1/2 and Akt, an upstream regulator of integrin aIIbb3 activation. Interestingly, we found that agonist-induced activation of ERK2 and Akt (both T308 and S473 phosphorylation) is significantly enhanced in the absence of Plk3. Furthermore, we found that levels of PTEN, a negative regulator of PI3-K/Akt pathway is reduced in the absence of Plk3. The severity of the anti-thrombotic phenotype in Plk3−/− mice may have been dampened due to the opposing role of Plk3. We next asked if the integrin outside-in signaling is also enhanced in these mice. As expected, platelet adhesion to immobilized fibrinogen was significantly increased (P<0.05) in the absence of Plk3. Surprisingly, when analyzed for fibrin clot retraction, we found that Plk3 null platelets failed to retract fibrin clot. These results suggest that Plk3, a mitotic kinase, plays a significant role in regulation of platelet function such as TxA2 generation, granular secretion, and clot retraction, thus affecting the process of thrombosis. Disclosures: No relevant conflicts of interest to declare.

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Li, Xiaojuan, Yunping Tang, Fangmiao Yu, Yu Sun, Fangfang Huang, Yan Chen, Zuisu Yang und Guofang Ding. „Inhibition of Prostate Cancer DU-145 Cells Proliferation by Anthopleura anjunae Oligopeptide (YVPGP) via PI3K/AKT/mTOR Signaling Pathway“. Marine Drugs 16, Nr. 9 (11.09.2018): 325. http://dx.doi.org/10.3390/md16090325.

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We investigated the antitumor mechanism of Anthopleura anjunae oligopeptide (AAP-H, YVPGP) in prostate cancer DU-145 cells in vitro and in vivo. Results indicated that AAP-H was nontoxic and exhibited antitumor activities. Cell cycle analysis indicated that AAP-H may arrest DU-145 cells in the S phase. The role of the phosphatidylinositol 3-kinase/protein kinase B/mammalian rapamycin target protein (PI3K/AKT/mTOR) signaling pathway in the antitumor mechanism of APP-H was investigated. Results showed that AAP-H treatment led to dose-dependent reduction in the levels of p-AKT (Ser473), p-PI3K (p85), and p-mTOR (Ser2448), whereas t-AKT and t-PI3K levels remained unaltered compared to the untreated DU-145 cells. Inhibition of PI3K/AKT/mTOR signaling pathway in the DU-145 cells by employing inhibitor LY294002 (10 μM) or rapamycin (20 nM) effectively attenuated AAP-H-induced phosphorylation of AKT and mTOR. At the same time, inhibitor addition further elevated AAP-H-induced cleaved-caspase-3 levels. Furthermore, the effect of AAP-H on tumor growth and the role of the PI3K/AKT/mTOR signaling pathway in nude mouse model were also investigated. Immunohistochemical analysis showed that activated AKT, PI3K, and mTOR levels were reduced in DU-145 xenografts. Western blotting showed that AAP-H treatment resulted in dose-dependent reduction in p-AKT (Ser473), p-PI3K (p85), and p-mTOR (Ser2448) levels, whereas t-AKT and t-PI3K levels remained unaltered. Similarly, Bcl-xL levels decreased, whereas that of Bax increased after AAP-H treatment. AAP-H also increased initiator (caspase 8 and 9) and executor caspase (caspase 3 and 7) levels. Therefore, the antitumor mechanism of APP-H on DU-145 cells may involve regulation of the PI3K/AKT/mTOR signaling pathway, which eventually promotes apoptosis via mitochondrial and death receptor pathways. Thus, the hydrophobic oligopeptide (YVPGP) can be developed as an adjuvant for the prevention or treatment of prostate cancer in the future.

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McGlynn, L. K., C. R. Mueller, M. Begbie, C. R. Notley und D. Lillicrap. „Role of the liver-enriched transcription factor hepatocyte nuclear factor 1 in transcriptional regulation of the factor V111 gene.“ Molecular and Cellular Biology 16, Nr. 5 (Mai 1996): 1936–45. http://dx.doi.org/10.1128/mcb.16.5.1936.

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Coagulation factor VIII is an essential cofactor required for normal hemostatic function. A deficiency in factor VIII results in the bleeding disorder hemophilia A. Despite the fact that the factor VIII gene was cloned a decade ago, the mechanisms which control its transcription remain unresolved. In our studies, we have characterized 12 protein binding sites within the factor VIII promoter by DNase I protection assays performed with rat liver nuclear extracts. Three of these elements (sites 1 to 3) are situated within the 5' untranslated region of the gene, while three other sites (sites 4 to 6) lie within the first 100 bp upstream of the transcriptional start site. We have identified an additional site (site 7) approximately 300 bp upstream from site 6, as well as a cluster of five sites in a 250-bp region which terminates approximately 1 kb from the transcriptional start site. Seven of these binding sites (sites 2, 3, 4, 6, 7, 9, and 10) bind members of the C/EBP family of transcription factors. DBP also binds to five of these sites (sites 3, 4, 6, 7, and 9). Utilizing transient transfection studies in HepG2 cells, we have shown that deletion of the factor VIII promoter sequences distal to nucleotide -44 results in a significant but small increase in promoter activity. The activity of each of the various 5' deletion constructs is significantly enhanced by cotransfection of C/EBPalpha and D-site-binding protein expression plasmids, while cotransfection of both C/EBPalpha and C/EBPbeta plasmids resulted in a further enhancement of transactivation. These studies also provide evidence of a repressor element located between nucleotides -740 and -1002. Since the minimal promoter sequence (-44 to +148) maintains the transcriptional activity of the full-length promoter sequence, we proceeded to identify additional factors binding to sites 1 to 4. Competition studies revealed that a ubiquitous transcription factor, NF-Y, binds to site 4, while the liver-enriched transcription factor hepatocyte nuclear factor I (HNF-1) binds to site 1. Mutation analysis of the minimal promoter demonstrated that HNF-1 is critical for activating transcription of the factor VIII gene in vitro. Our results also suggest that the multiple upstream elements that we have identified may act as a backup regulatory region in the event of disruption of the HNF-1 element in the 5' untranslated region.

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Balakrishnan, Ilango, Xiaodong Yang, Beverly Torok-Storb, Jay Hesselberth und Manoj M. Pillai. „High Throughput Sequencing Following Cross-Linked Immune Precipitation (HITS-CLIP) of Argonaute (AGO) Identifies Mir-9 As a Regulator of MMP2 in the Marrow Microenvironment (ME)“. Blood 118, Nr. 21 (18.11.2011): 2392. http://dx.doi.org/10.1182/blood.v118.21.2392.2392.

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Abstract Abstract 2392 There is increasing recognition of the role of small noncoding RNAs in post-transcriptional regulation of gene expression in diverse tissues of eukaryotic organisms including vertebrates. MicroRNAs (miRNAs) are the best studied amongst these small RNAs and are thought to act by binding to the 3' untranslated regions (3' UTRs) of mature mRNAs in a sequence-specific fashion and preventing the initiation of peptide translation and/ or initiating mRNA degradation. Recent evidence suggests that miRNA-based regulation might involve binding to regions other than 3' UTRs including coding regions. Current approaches to defining miRNA-mRNA interactions are mostly restricted to those based on bio-informatic prediction, protein down-regulation following in-vitro transfection of miRNA precursors and luciferase assays to determine binding to 3' UTRs. None of these methods however show direct interaction between a specific miRNA and its purported target RNA. Bio-informatics-based approaches are also prone to false positive and negative results given the short length of sequence matching, and reliance on heuristics and cross-species conservation. Newer genome-wide approaches like HITS-CLIP (High Throughput Sequencing following Cross Linked Immuno Precipitation, or CLIP-Seq) overcome some of these limitations by directly isolating the miRNA-mRNA interactome bound to argonaute (AGO), a critical component of the rna-induced silencing complex (RISC)1. HITS-CLIP utilizes the ability of ultraviolet (UV) light to cross-link RNAs to proteins in their close proximity. The crosslinked miRNA-mRNA-Ago complexes are then isolated and the RNA reverse transcribed to cDNA libraries and sequenced by next generation sequencing (NGS). Given the widespread role of miRNAs in several vertebrate tissues, we hypothesized that miRNA-regulation of gene expression is operant in the hematopoietic microenvironment (ME) and thus contributes to regulation of hematopoiesis. We hence used HITS-CLIP to analyze the miRNA-mRNA interactome of three key cellular components of the ME: stromal cells, endothelium and macrophages. We have previously reported on the use of the stromal cell lines Hs27a and Hs5 to define specific functional niches within the ME. Hs27a can functionally support primitive hematopoietic stem and progenitor cells (HSPC) in cobblestone areas (CSAs) and express high levels of factors known to support HSPC such as SDF1, Jagged1 and Angiopoietin1. In contrast, Hs5 drives HSPC to mature lineages and secretes high levels of cytokines like IL1, IL6 and GCSF. Human umbilical vein endothelial cells (HUVECs) and MCSF-treated CD14+ cells were utilized for the endothelial and macrophage cultures respectively. The HITS-CLIP datasets from each of these populations were enriched for a putative binding site for miR-9 in the coding region of Matrix Metalloproteinase 2 (MMP2) mRNA. MMP2 belongs to a family of endopeptidases critical in the remodeling of extracellular matrix in several tissues and in the egress/ homing of HSPC to their functional niches in the ME. Functional binding of miR-9 to MMP2 was validated by Western-blotting of stromal cells transfected with miR-9 which revealed > 50% reduction of protein levels when compared to control-transfected cells. This was also confirmed by gelatin zymography which showed significantly reduced MMP2 activity in stromal cells transfected with miR-9. Finally, to confirm direct binding of miR-9 to the putative binding region on the MMP2 transcript, we cloned this microRNA responsive region (MRE) downstream of the Renilla luciferase gene and assayed its activity by luciferase assays. MiR-9 transfection down-regulated luciferase activity > 50% confirming direct binding to the MRE. Our results show that genome-wide approaches such as HITS-CLIP can be used to define in vivo miRNA-mRNA interactions in the ME and should be considered in studies that define such interactions given the significant false-positive and false negative results associated with approaches based on bio-informatics alone. The approach can also define specific interactions between miRNAs and mRNAs such as MMP2, of relevance to regulation of the hematopoietic ME. Disclosures: No relevant conflicts of interest to declare.

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Youn, Byung-S., Young J. Kim, Charlie Mantel, Kang-Yeol Yu und Hal E. Broxmeyer. „Blocking of c-FLIPL–independent cycloheximide-induced apoptosis or Fas-mediated apoptosis by the CC chemokine receptor 9/TECK interaction“. Blood 98, Nr. 4 (15.08.2001): 925–33. http://dx.doi.org/10.1182/blood.v98.4.925.

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Chemokines play a pivotal role in regulating leukocyte migration as well as other biological functions. CC chemokine receptor 9 (CCR9) is a specific receptor for thymus-expressed CC chemokine (TECK). It is shown here that engagement of CCR9 with TECK leads to phosphorylation of Akt (protein kinase B), mitogen-activated protein kinases (MAPKs), glycogen synthase kinase–3β (GSK-3β), and a forkhead transcription factor, FKHR, in a human T-cell line, MOLT4, that naturally expresses CCR9. By means of chemical inhibitors, it is shown that phosphoinositide-3 kinase (PI-3 kinase), but not MAPK, is required for CCR9-mediated chemotaxis. Akt, GSK-3β, FKHR, and MAPK have been previously implicated in cell survival signals in response to an array of death stimuli. When MOLT4 cells, which expressed Fas as well as CXCR4, were stimulated with cycloheximide (CHX), an agonistic anti-Fas antibody, or a combination of these, the cells rapidly underwent apoptosis. However, costimulation of MOLT4 cells with TECK or stromal derived factor–1 significantly blocked CHX-mediated apoptosis, whereas stimulation only with TECK partially blocked Fas-mediated apoptosis. Concomitant with this blocking, cleavage of poly (adenosine 5′-diphosphate–ribose) polymerase and activation of caspase 3 were significantly attenuated, but the expression level of FLICE inhibitory protein c-FLIPL, which had been shown to be regulated by CHX, was unchanged. This demonstrates that activation of CCR9 leads to phosphorylation of GSK-3β and FKHR and provides a cell survival signal to the receptor expressing cells against CHX. It also suggests the existence of a novel pathway leading to CHX-induced apoptosis independently of c-FLIPL.

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Ou, Zhi-Jun, Jing Chen, Wei-Ping Dai, Xiang Liu, Yin-Ke Yang, Yan Li, Ze-Bang Lin et al. „25-Hydroxycholesterol impairs endothelial function and vasodilation by uncoupling and inhibiting endothelial nitric oxide synthase“. American Journal of Physiology-Endocrinology and Metabolism 311, Nr. 4 (01.10.2016): E781—E790. http://dx.doi.org/10.1152/ajpendo.00218.2016.

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Endothelial dysfunction is a key early step in atherosclerosis. 25-Hydroxycholesterol (25-OHC) is found in atherosclerotic lesions. However, whether 25-OHC promotes atherosclerosis is unclear. Here, we hypothesized that 25-OHC, a proinflammatory lipid, can impair endothelial function, which may play an important role in atherosclerosis. Bovine aortic endothelial cells were incubated with 25-OHC. Endothelial cell proliferation, migration, and tube formation were measured. Nitric oxide (NO) production and superoxide anion generation were determined. The expression and phosphorylation of endothelial NO synthase (eNOS) and Akt as well as the association of eNOS and heat shock protein (HSP)90 were detected by immunoblot analysis and immunoprecipitation. Endothelial cell apoptosis was monitored by TUNEL staining and caspase-3 activity, and expression of Bcl-2, Bax, cleaved caspase-9, and cleaved caspase-3 were detected by immunoblot analysis. Finally, aortic rings from Sprague-Dawley rats were isolated and treated with 25-OHC, and endothelium-dependent vasodilation was evaluated. 25-OHC significantly inhibited endothelial cell proliferation, migration, and tube formation. 25-OHC markedly decreased NO production and increased superoxide anion generation. 25-OHC reduced the phosphorylation of Akt and eNOS and the association of eNOS and HSP90. 25-OHC also enhanced endothelial cell apoptosis by decreasing Bcl-2 expression and increasing cleaved caspase-9 and cleaved caspase-3 expressions as well as caspase-3 activity. 25-OHC impaired endothelium-dependent vasodilation. These data demonstrated that 25-OHC could impair endothelial function by uncoupling and inhibiting eNOS activity as well as by inducing endothelial cell apoptosis. Our findings indicate that 25-OHC may play an important role in regulating atherosclerosis.

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Nassir, Fatiha, Justin J. Arndt, Sarah A. Johnson und Jamal A. Ibdah. „Regulation of mitochondrial trifunctional protein modulates nonalcoholic fatty liver disease in mice“. Journal of Lipid Research 59, Nr. 6 (26.03.2018): 967–73. http://dx.doi.org/10.1194/jlr.m080952.

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Mitochondrial trifunctional protein (MTP) plays a critical role in the oxidation of long-chain fatty acids. We previously reported that aging mice (>9 months old) heterozygous for an MTP defect (MTP+/−) develop nonalcoholic fatty liver disease (NAFLD). We tested whether a high-fat diet (HFD) accelerates NAFLD in young MTP+/−mice, and whether overexpression of the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase sirtuin 3 (SIRT3) deacetylates MTP and improves mitochondrial function and NAFLD. Three-month-old WT and MTP+/− mice were fed HFD (60% cal fat) for 16 weeks and livers were assessed for fatty acid oxidation (FAO) and NAFLD. Compared with WT, MTP+/− mice displayed reduced hepatic SIRT3 levels and reduced FAO, with increased hepatic steatosis and the inflammatory marker CD68. Hepatic overexpression of SIRT3 in HFD-fed MTP+/− mice increased hepatic MTP protein levels at the posttranscriptional level. Immunoprecipitation of MTP from liver mitochondria followed by Western blot with acetyl-lysine antibody showed higher acetylation of MTP in MTP+/− compared with WT mice. Overexpression of SIRT3 in MTP+/− mice significantly reduced the acetylation of MTP compared with β-galactosidase controls, increased mitochondrial FAO, and reduced hepatic steatosis, CD68, and serum ALT levels. Taken together, our data indicate that deacetylation of MTP by SIRT3 improves mitochondrial function and rescues NAFLD in MTP+/− mice.

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Loges, Sonja, Heike Tinnefeld, Anja Metzner, Manfred Jucker, Martin Butzal, Melanie Bruweleit, Uta Fischer et al. „Treatment of Patients with Refractory AML with SU5416 Downregulates VEGF-A, STAT5 and Akt Expression in Leukemic Blasts.“ Blood 106, Nr. 11 (16.11.2005): 2793. http://dx.doi.org/10.1182/blood.v106.11.2793.2793.

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Abstract Autocrine or paracrine activation of receptor tyrosine kinases are important pathogenetic factors in AML. Signal transduction via c-kit and FLT3 increase proliferation and apoptosis resistance of leukemic blasts. Additionally bone marrow angiogenesis plays a significant role in disease progression. This provided the rationale for a multicenter clinical trial in patients with refractory AML with SU5416, a small molecule kinase inhibitor which blocks phosphorylation of c-kit, FLT3, VEGFR-1, VEGFR-2 (KDR) and VEGFR-3. In the study presented here, we analysed expression levels of VEGF and the downstream signal transduction intermediates STAT5 and Akt in patients before and during treatment with SU5416. The levels of VEGF mRNA expression were investigated in peripheral blood leukemic blasts by means of quantitative Real-Time PCR. 74% (14 of 19) of analyzed patients had rapid and persistent down regulation of VEGF during therapy. Patients initially expressing high VEGF-levels had a stronger down regulation and a higher clinical response rate (mean 865-fold, n=9) than patients initially expressing low VEGF-levels (mean 4-fold, n=8). These results suggest that downregulation of high pretherapeutic VEGF expression by SU5416 treatment may serve as an early surrogate marker of therapeutic response to anti-angiogenetic therapy. Additionally, protein expression of STAT5 and Akt was assessed by Western blotting. Incubation of the leukemic cell line M-07e in-vitro with SU5416 using concentrations which are achievable in patients was used as a model system. In the AML patient samples parallel downregulation of both STAT5 and Akt was observed in several cases (STAT5 in 4 of 15, Akt in 3 of 6 examined patients). Decreased intracellular levels of STAT5 and Akt were also found in the line M-07e after incubation with SU5416 in vitro. In summary, our data provide evidence for suppression of expression of VEGF and of the key signal transduction intermediates STAT5 and Akt in AML blasts in-vivo during treatment with SU5416.

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Wowerka, Arkadiusz. „Obce przepisy wymuszające swoje zastosowanie. Glosa do wyroku Trybunału Sprawiedliwości Unii Europejskiej z dnia 18 października 2016 r. w sprawie C-135/15 Republika Grecji przeciwko Grigoriosowi Nikiforidisowi“. Problemy Prawa Prywatnego Międzynarodowego 25 (31.12.2019): 91–106. http://dx.doi.org/10.31261/pppm.2019.25.05.

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This commentary examines the judgement of the CJEU of 18 October 2016 in case C-135/15 Republik Griechenland v. Grigorios Nikiforidis. The judgement in question concerns the issue of treatment of foreign overriding mandatory provisions under the Article 9(3) of Regulation No 593/2008. This topic is the subject to a great deal of controversy and academic discussion. The ECJ concluded that the mentioned provision must be interpreted as precluding overriding mandatory provisions other than those of the State of the forum or of the State where the obligations arising out of the contract have to be or have been performed from being applied, as legal rules, by the court of the forum, but as not precluding it from taking such other overriding mandatory provisions into account as matters of fact in so far as this is provided for by the national law that is applicable to the contract pursuant to the Regulation. This interpretation is not affected by the principle of sincere cooperation laid down in Article 4(3) TEU. In this respect the judgement of CJEU brings significant clarification on the question, whether a court of the forum can have regard to foreign overriding mandatory provisions, which do not belong to the legal system of the country of performance of the contract on the level of the applicable substantive law. However, there are still questions arising under Article 9(3) of Rome I Regulation, which need to be clarified.

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