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MACC1 wurde als prognostischer Biomarker für die Tumorprogression und das Metastasen-freie Überleben im KRK sowie in anderen soliden Tumoren beschrieben. Das Gen induziert Zellmotilität und Proliferation in Zellkultur sowie die Metastasierung im Mausmodell. Damit stellt MACC1 ein vielversprechendes Ziel für die Intervention bei Tumorprogression und –metastasierung und damit für die Behandlung von KRK-Patienten dar. Unser Ziel war es, die Transkription von MACC1 zu inhibieren. Hierfür identifizierten wir zunächst die Promoter-Region von MACC1 und untersuchten MACC1s transkriptionelles Regulationsnetzwerk. Durch ortsgerichtete Mutagenese, Chromatin Immunopräzipitation und Electrophoretic Mobility Shift Assay ermittelten wir, dass Transkriptionsfaktoren wie Ap-1, Sp1, C/EBPs und GIPC1 an den MACC1-Promoter binden und die Transkription des MACC1-Gens kontrollieren. Darüberhinaus konnten wir durch Hochdurchsatz-Screening die bisher ersten Inhibitoren gegen MACC1 identifizieren: Rottlerin und Lovastatin. Wir zeigten, dass diese spezifisch auf den endogenen MACC1-Promoter wirken, was eine zeit- und konzentrationsabhängige Reduktion der MACC1-Expression zur Folge hatte. Beide Inhibitoren begrenzten das Expressionsniveau von Sp1 und interferierten mit der Bindung von c-Jun mit dem MACC1-Promoter, was in einer Inhibition der MACC1-Transkription resultierte. Ferner führte die tägliche Behandlung von Xenograft-Mausmodellen mit Rottlerin zu einer Inhibition der MACC1-Expression im Primärtumor und einer damit einhergehenden Begrenzung des Tumorwachstums. Zusammenfassend lässt sich festhalten, dass in der vorliegenden Arbeit zum ersten Mal der MACC1-Promoter und seine transkriptionelle Regulation beleuchtet wurden. Die neuen Erkenntnisse wurden zur Identifizierung der ersten Inhibitoren gegen MACC1 genutzt. Zur Behandlung von KRK-Patienten mit einem hohen Risiko für MACC1-induzierte Metastasierung könnten diese Inhibitoren Potential für die klinische Anwendung beherbergen., MACC1 has been reported as a prognostic biomarker for tumor progression and metastasis-free survival in CRC along with other solid tumors. It induces cell motility and proliferation in cell culture and metastasis in mouse models. Consequently, targeting MACC1 to intervene in tumor progression and metastasis formation holds a promising approach to treat CRC patients. We designed a strategy to inhibit MACC1 via targeting its transcription. We first identified MACC1 gene promoter by creating various promoter-luciferase constructs. We then established that transcription factors such as Ap-1, Sp1, C/EBPs and GIPC1 bind to the MACC1 promoter and govern MACC1 transcription, expression and thus motility in vitro and in CRC patients. Using a high throughput screening targeting the MACC1 promoter, we identified small molecule MACC1 inhibitors, Rottlerin and Lovastatin. These inhibitors specifically restricted endogenous MACC1 promoter leading to reduced MACC1 expression in a time- and concentration-dependent manner. In vitro functional assays demonstrated the impact of the small molecule inhibitors on retarding cell proliferation and motility. Both inhibitors restricted Sp1 levels and interfered with the binding of c-Jun to the MACC1 promoter, thereby inhibiting MACC1 transcription. The study further described the effect of Rottlerin on a CRC-xenografted mouse model. Daily treatment of xenografted mice with Rottlerin resulted in the inhibition of MACC1 expression in the primary tumor accompanied with the restricted tumor growth. To summarize, this is the first study unraveling the MACC1 promoter, its transcriptional regulation and identification of newly identified MACC1 inhibitors. In clinical settings, inhibition of MACC1 expression using these inhibitors might provide immense potential for the treatment of CRC patients who are at high risk for MACC1-induced metastasis linked to shorter survival.

MACC1 wurde als prognostischer Biomarker für die Tumorprogression und das Metastasen-freie Überleben im KRK sowie in anderen soliden Tumoren beschrieben. Das Gen induziert Zellmotilität und Proliferation in Zellkultur sowie die Metastasierung im Mausmodell. Damit stellt MACC1 ein vielversprechendes Ziel für die Intervention bei Tumorprogression und –metastasierung und damit für die Behandlung von KRK-Patienten dar. Unser Ziel war es, die Transkription von MACC1 zu inhibieren. Hierfür identifizierten wir zunächst die Promoter-Region von MACC1 und untersuchten MACC1s transkriptionelles Regulationsnetzwerk. Durch ortsgerichtete Mutagenese, Chromatin Immunopräzipitation und Electrophoretic Mobility Shift Assay ermittelten wir, dass Transkriptionsfaktoren wie Ap-1, Sp1, C/EBPs und GIPC1 an den MACC1-Promoter binden und die Transkription des MACC1-Gens kontrollieren. Darüberhinaus konnten wir durch Hochdurchsatz-Screening die bisher ersten Inhibitoren gegen MACC1 identifizieren: Rottlerin und Lovastatin. Wir zeigten, dass diese spezifisch auf den endogenen MACC1-Promoter wirken, was eine zeit- und konzentrationsabhängige Reduktion der MACC1-Expression zur Folge hatte. Beide Inhibitoren begrenzten das Expressionsniveau von Sp1 und interferierten mit der Bindung von c-Jun mit dem MACC1-Promoter, was in einer Inhibition der MACC1-Transkription resultierte. Ferner führte die tägliche Behandlung von Xenograft-Mausmodellen mit Rottlerin zu einer Inhibition der MACC1-Expression im Primärtumor und einer damit einhergehenden Begrenzung des Tumorwachstums. Zusammenfassend lässt sich festhalten, dass in der vorliegenden Arbeit zum ersten Mal der MACC1-Promoter und seine transkriptionelle Regulation beleuchtet wurden. Die neuen Erkenntnisse wurden zur Identifizierung der ersten Inhibitoren gegen MACC1 genutzt. Zur Behandlung von KRK-Patienten mit einem hohen Risiko für MACC1-induzierte Metastasierung könnten diese Inhibitoren Potential für die klinische Anwendung beherbergen., MACC1 has been reported as a prognostic biomarker for tumor progression and metastasis-free survival in CRC along with other solid tumors. It induces cell motility and proliferation in cell culture and metastasis in mouse models. Consequently, targeting MACC1 to intervene in tumor progression and metastasis formation holds a promising approach to treat CRC patients. We designed a strategy to inhibit MACC1 via targeting its transcription. We first identified MACC1 gene promoter by creating various promoter-luciferase constructs. We then established that transcription factors such as Ap-1, Sp1, C/EBPs and GIPC1 bind to the MACC1 promoter and govern MACC1 transcription, expression and thus motility in vitro and in CRC patients. Using a high throughput screening targeting the MACC1 promoter, we identified small molecule MACC1 inhibitors, Rottlerin and Lovastatin. These inhibitors specifically restricted endogenous MACC1 promoter leading to reduced MACC1 expression in a time- and concentration-dependent manner. In vitro functional assays demonstrated the impact of the small molecule inhibitors on retarding cell proliferation and motility. Both inhibitors restricted Sp1 levels and interfered with the binding of c-Jun to the MACC1 promoter, thereby inhibiting MACC1 transcription. The study further described the effect of Rottlerin on a CRC-xenografted mouse model. Daily treatment of xenografted mice with Rottlerin resulted in the inhibition of MACC1 expression in the primary tumor accompanied with the restricted tumor growth. To summarize, this is the first study unraveling the MACC1 promoter, its transcriptional regulation and identification of newly identified MACC1 inhibitors. In clinical settings, inhibition of MACC1 expression using these inhibitors might provide immense potential for the treatment of CRC patients who are at high risk for MACC1-induced metastasis linked to shorter survival.

Rottlerin is a natural polyphenolic ketone isolated from the pericarps of Mallotus phillippinensis. In previous studies we showed that parenteral administration of rottlerin reduced tumor growth in murine xenograft models of pancreatic cancer. The aim of this study was to develop a simple and validated method for the quantitative determination of rottlerin in plasma and tumor tissues of mice fed a rottlerin diet. A xenograft model of pancreatic cancer was prepared by injection of 2×106 HPAF-II cells subcutaneously into nude mice. One week before tumor implantation, mice were randomly allocated to standard diet (AIN76A) and standard diet supplement with 0.012% rottlerin (n=6 per group). Mice were sacrificed after 6 weeks on diets. Rottlerin was extracted from the plasma and tissues using protein precipitation-extraction and analyzed by reverse-phase HPLC-DAD method. The same HPLC method was also applied to determine rottlerin levels in conditioned culture media and in cell lysates from HPAF-II cells exposed to 25 µM concentration of rottlerin. A substantial amount of rottlerin was detected in tumor (2.11 ± 0.25 nmol/g tissue) and plasma (2.88 ± 0.41 µM) in mice fed rottlerin diet. In addition, significant levels of rottlerin (57.4 ± 5.4 nmol/mg protein) were detected in cell lysates from rottlerin-treated HPAF-II cells. These data indicate that rottlerin is efficiently absorbed in cells and tissues both in vivo and in vitro and suggest a strong potential for rottlerin as a preventive or adjuvant supplement for pancreatic cancer.

Rottlerin is a natural polyphenolic ketone isolated from the pericarps of Mallotus phillippinensis. In previous studies we showed that parenteral administration of rottlerin reduced tumor growth in murine xenograft models of pancreatic cancer. The aim of this study was to develop a simple and validated method for the quantitative determination of rottlerin in plasma and tumor tissues of mice fed a rottlerin diet. A xenograft model of pancreatic cancer was prepared by injection of 2×106 HPAF-II cells subcutaneously into nude mice. One week before tumor implantation, mice were randomly allocated to standard diet (AIN76A) and standard diet supplement with 0.012% rottlerin (n=6 per group). Mice were sacrificed after 6 weeks on diets. Rottlerin was extracted from the plasma and tissues using protein precipitation-extraction and analyzed by reverse-phase HPLC-DAD method. The same HPLC method was also applied to determine rottlerin levels in conditioned culture media and in cell lysates from HPAF-II cells exposed to 25 µM concentration of rottlerin. A substantial amount of rottlerin was detected in tumor (2.11 ± 0.25 nmol/g tissue) and plasma (2.88 ± 0.41 µM) in mice fed rottlerin diet. In addition, significant levels of rottlerin (57.4 ± 5.4 nmol/mg protein) were detected in cell lysates from rottlerin-treated HPAF-II cells. These data indicate that rottlerin is efficiently absorbed in cells and tissues both in vivo and in vitro and suggest a strong potential for rottlerin as a preventive or adjuvant supplement for pancreatic cancer.

The naturally occurring toxin rottlerin has been used by other laboratories as a specific inhibitor of protein kinase C-delta (PKC-d) to obtain evidence that the activitydependent distribution of glutamate transporter GLAST is regulated by PKC-d mediated phosphorylation. Using immunofluorescence labelling for GLAST and deconvolution microscopy we have observed that D-aspartateinduced redistribution of GLAST towards the plasma membranes of cultured astrocytes was abolished by rottlerin. In brain tissue in vitro, rottlerin reduced apparent activity of (Na?, K?)-dependent ATPase (Na?, K?-ATPase) and increased oxygen consumption in accordance with its known activity as an uncoupler of oxidative phosphorylation (``metabolic poison¿¿). Rottlerin also inhibited Na?, K?-ATPase in cultured astrocytes. As the glutamate transport critically depends on energy metabolism and on the activity of Na?, K?-ATPase in particular, we suggest that the metabolic toxicity of rottlerin and/or the decreased activity of the Na?, K?-ATPase could explain both the glutamate transport inhibition and altered GLAST distribution caused by rottlerin even without any involvement of PKC-d-catalysed phosphorylation in the process.

The naturally occurring toxin rottlerin has been used by other laboratories as a specific inhibitor of protein kinase C-delta (PKC-d) to obtain evidence that the activitydependent distribution of glutamate transporter GLAST is regulated by PKC-d mediated phosphorylation. Using immunofluorescence labelling for GLAST and deconvolution microscopy we have observed that D-aspartateinduced redistribution of GLAST towards the plasma membranes of cultured astrocytes was abolished by rottlerin. In brain tissue in vitro, rottlerin reduced apparent activity of (Na?, K?)-dependent ATPase (Na?, K?-ATPase) and increased oxygen consumption in accordance with its known activity as an uncoupler of oxidative phosphorylation (``metabolic poison¿¿). Rottlerin also inhibited Na?, K?-ATPase in cultured astrocytes. As the glutamate transport critically depends on energy metabolism and on the activity of Na?, K?-ATPase in particular, we suggest that the metabolic toxicity of rottlerin and/or the decreased activity of the Na?, K?-ATPase could explain both the glutamate transport inhibition and altered GLAST distribution caused by rottlerin even without any involvement of PKC-d-catalysed phosphorylation in the process.

The naturally occurring toxin rottlerin has been used by other laboratories as a specific inhibitor of protein kinase C-delta (PKC-d) to obtain evidence that the activitydependent distribution of glutamate transporter GLAST is regulated by PKC-d mediated phosphorylation. Using immunofluorescence labelling for GLAST and deconvolution microscopy we have observed that D-aspartateinduced redistribution of GLAST towards the plasma membranes of cultured astrocytes was abolished by rottlerin. In brain tissue in vitro, rottlerin reduced apparent activity of (Na?, K?)-dependent ATPase (Na?, K?-ATPase) and increased oxygen consumption in accordance with its known activity as an uncoupler of oxidative phosphorylation (``metabolic poison¿¿). Rottlerin also inhibited Na?, K?-ATPase in cultured astrocytes. As the glutamate transport critically depends on energy metabolism and on the activity of Na?, K?-ATPase in particular, we suggest that the metabolic toxicity of rottlerin and/or the decreased activity of the Na?, K?-ATPase could explain both the glutamate transport inhibition and altered GLAST distribution caused by rottlerin even without any involvement of PKC-d-catalysed phosphorylation in the process.

The naturally occurring toxin rottlerin has been used by other laboratories as a specific inhibitor of protein kinase C-delta (PKC-d) to obtain evidence that the activitydependent distribution of glutamate transporter GLAST is regulated by PKC-d mediated phosphorylation. Using immunofluorescence labelling for GLAST and deconvolution microscopy we have observed that D-aspartateinduced redistribution of GLAST towards the plasma membranes of cultured astrocytes was abolished by rottlerin. In brain tissue in vitro, rottlerin reduced apparent activity of (Na?, K?)-dependent ATPase (Na?, K?-ATPase) and increased oxygen consumption in accordance with its known activity as an uncoupler of oxidative phosphorylation (``metabolic poison¿¿). Rottlerin also inhibited Na?, K?-ATPase in cultured astrocytes. As the glutamate transport critically depends on energy metabolism and on the activity of Na?, K?-ATPase in particular, we suggest that the metabolic toxicity of rottlerin and/or the decreased activity of the Na?, K?-ATPase could explain both the glutamate transport inhibition and altered GLAST distribution caused by rottlerin even without any involvement of PKC-d-catalysed phosphorylation in the process.

The naturally occurring toxin rottlerin has been used by other laboratories as a specific inhibitor of protein kinase C-delta (PKC-d) to obtain evidence that the activitydependent distribution of glutamate transporter GLAST is regulated by PKC-d mediated phosphorylation. Using immunofluorescence labelling for GLAST and deconvolution microscopy we have observed that D-aspartateinduced redistribution of GLAST towards the plasma membranes of cultured astrocytes was abolished by rottlerin. In brain tissue in vitro, rottlerin reduced apparent activity of (Na?, K?)-dependent ATPase (Na?, K?-ATPase) and increased oxygen consumption in accordance with its known activity as an uncoupler of oxidative phosphorylation (``metabolic poison¿¿). Rottlerin also inhibited Na?, K?-ATPase in cultured astrocytes. As the glutamate transport critically depends on energy metabolism and on the activity of Na?, K?-ATPase in particular, we suggest that the metabolic toxicity of rottlerin and/or the decreased activity of the Na?, K?-ATPase could explain both the glutamate transport inhibition and altered GLAST distribution caused by rottlerin even without any involvement of PKC-d-catalysed phosphorylation in the process.

Rottlerin is a pharmacological inhibitor of protein kinase C (PKC) theta, a novel PKC selectively expressed in T lymphocytes. PKC theta is known to regulate T cell receptor (TCR)/CD28 signalling pathways in T lymphocytes, but the impact of PKC theta inhibition on human T cell responses remains undefined. In this work, we describe the effects of rottlerin on the responses of CD4+ and CD8+ human T lymphocytes upon polyclonal activation. We observed a dose-dependent inhibition of CD4+ and CD8+ T cell proliferation in response to anti-CD3/anti-CD28 antibodies stimulation in the presence of rottlerin. This inhibition was associated with impaired CD25 expression and decreased interleukin (IL)-2 production in activated T cells. In contrast, rottlerin did not alter IL-2-induced T cell proliferation. Furthermore, we demonstrated that rottlerin blocked interferon (IFN) gamma, IL-10 and IL-13 mRNA expression in TCR/CD28 activated CD4+ T cells. These findings place rottlerin as a potent immunosuppressive agent for the development of novel therapies in T cell mediated immune disorders., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published