Characterization of histone deacetylase inhibitor-induced cell death and sensitization to chemotherapeutics in rhabdomyosarcoma

HDAC inhibitors (HDACI), a new class of anticancer agents, induce apoptosis in many cancer entities. JNJ-26481585 is a second generation class І HDACI that displays improved efficacy in preclinical studies compared to th
HDAC inhibitors (HDACI), a new class of anticancer agents, induce apoptosis in many cancer entities. JNJ-26481585 is a second generation class І HDACI that displays improved efficacy in preclinical studies compared to the established HDACI SAHA (Vorinostat). Therefore, this study aims at evaluating the effects of JNJ-26481585 on human rhabdomyosarcoma (RMS) and at identifying novel synergistic interactions of JNJ-26481585 or the more common HDACI SAHA with different anticancer drugs in RMS cells. Indeed, we show that JNJ-26481585 and SAHA significantly increase chemotherapeutic drug-induced apoptosis in embryonal and alveolar RMS cell lines, when used in combination with chemotherapeutic agents (i.e. doxorubicin, etoposide, vincristine, and cyclophosphamide) which are currently used in the clinic for the treatment of RMS.
We demonstrate that JNJ-26481585 as single agent and in combination with doxorubicin induces apoptosis, which is characterized by activation of the caspase cascade, PARP cleavage, and DNA fragmentation. Induction of caspase-dependent apoptotic cell death is confirmed by the use of the broad-range caspase inhibitor zVAD.fmk, which significantly decreases both JNJ-26481585-triggered and combination treatment-mediated DNA fragmentation, and in addition completely abrogates loss of cell viability. Importantly, JNJ-26481585 significantly inhibits tumor growth in vivo in two preclinical RMS models, i.e. the chicken chorioallantoic membrane (CAM) model and a xenograft mouse model, supporting the notion that JNJ-26481585 hampers tumor maintenance. Also, in combination with doxorubicin JNJ-26481585 significantly reduces tumor growth in in vivo experiments using the CAM model.
Mechanistically, we identify that JNJ-26481585-induced apoptosis is mediated via the intrinsic apoptotic pathway, since we observe increased loss of mitochondrial membrane potential and activation of the proapoptotic Bcl-2 family members Bax and Bak. Interestingly, we find that JNJ-26481585 triggers induction of Bim, Bmf, Puma, and Noxa on mRNA level as well as on protein level, pointing to an altered transcription of BH3-only proteins as important event for the Bax/Bak-mediated loss of mitochondrial membrane potential as well as mitochondrial apoptosis induction upon JNJ-26481585 treatment. JNJ-26481585-initiated activation of Bax and Bak is not prevented with the addition of zVAD.fmk, suggesting that JNJ-26481585 first disrupts the mitochondria and subsequently activates the caspase cascade. When JNJ-26481585 is used in combination with doxorubicin, we observe not only an increase of proapoptotic Bcl-2 proteins, but also a decrease in the level of the antiapoptotic mitochondrial proteins Bcl-2, Mcl-1, and Bcl-xL. This indicates that Bax, Bak, Bim, and Noxa are crucial for JNJ-26481585-induced as well as JNJ/Dox treatment-induced apoptosis, since RNAi mediated silencing of Bax, Bak, Bim, and Noxa significantly impedes DNA fragmentation upon those treatments.
Furthermore, ectopic overexpression of Bcl-2 profoundly impairs both JNJ-26481585 and combination treatment-mediated apoptosis, abrogates caspase cleavage, and reduces activation of Bax and Bak, underlining the hypothesis that JNJ-26481585 initially targets the mitochondria and then activates caspases.
With the more commonly used HDACI SAHA we confirm the results obtained with the HDACI JNJ-26481585, since combination treatment with SAHA and doxorubicin also induces intrinsic apoptosis, which can be significantly diminished by zVAD.fmk or ectopic overexpression of Bcl-2. Treatment with SAHA and doxorubicin also affects expression levels of pro- and antiapoptotic mitochondrial proteins, thus shifting the balance towards the proapoptotic mitochondrial machinery, resulting in Bax/Bak activation, caspase activation, and subsequently apoptosis.
Taken together, we provide evidence that the HDACIs JNJ-26481585 and SAHA are promising therapeutic agents for the treatment of RMS and that combination regimens with HDACIs represent an efficient strategy to prime RMS cells for chemotherapy-induced apoptosis. These findings have important implications for mitochondrial apoptosis-targeted therapies of RMS.
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In der Entwicklung vieler Organismen spielt der programmierte Zelltodmechanismus „Apoptose“ eine bedeutende Rolle. Er kann aufgrund von zellinternen Prozessen (z.B. durch Mutationen oder Fehler bei der Zellteilung) einge
In der Entwicklung vieler Organismen spielt der programmierte Zelltodmechanismus „Apoptose“ eine bedeutende Rolle. Er kann aufgrund von zellinternen Prozessen (z.B. durch Mutationen oder Fehler bei der Zellteilung) eingeleitet werden und unterliegt in normalen Zellen einer strengen Kontrolle. Bei der Apoptose handelt es sich um einen Stoffwechselprozess, bei dem die Zelle ohne Schädigung des Nachbargewebes abgebaut und verdaut wird. Proteolytische Enzyme (Caspasen) sind in diesem programmierten Zelltodmechanismus im Vergleich zu anderen Zelltod-Signalwegen von wesentlicher Bedeutung. Die Apoptose lässt sich in zwei gut bekannte Signalwege unterteilen, zum einen in den extrinsischen (Todesrezeptor-) und zum anderen in den intrinsischen (mitochondrialen) Signalweg.
Bei vielen Krankheiten, insbesondere vielen verschiedenen Krebsarten, unterliegt die Apoptose keiner strengen Regulation mehr. Krebszellen exprimieren z.B. im hohen Maße antiapoptotische Proteine wie Bcl-2, während proapoptotische Proteine wie z.B. Bim oder Noxa herunterreguliert sind. Die feine Balance zwischen Proliferation und Apoptose ist somit gestört, was bedeutet, dass sich fehlerhafte Zellen ungehindert weiter teilen können. Ein wichtiger Therapieansatz ist es daher, Apoptose in Krebszellen extrinsisch wieder zu initiieren und somit ein fortschreitendes Wachstum des Tumors zu verhindern. Histondeacetylasen (HDACs) werden in vielen Krebsarten verstärkt exprimiert, was eine erhöhte Expression von proliferationsfördernden und einer unterdrückten Expression von proapoptotischen Proteinen zur Folge hat, welche das Krebswachstum fördern...
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Metadaten
Author:Ulrike Elisabeth Brigitte Heinicke
URN:urn:nbn:de:hebis:30:3-416148
Referee:Volker Dötsch, Simone Fulda
Document Type:Doctoral Thesis
Language:English
Year of Completion:2016
Year of first Publication:2015
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Granting Institution:Johann Wolfgang Goethe-Universität
Date of final exam:2016/03/08
Release Date:2016/10/26
Pagenumber:164
Note:
Diese Dissertation steht außerhalb der Universitätsbibliothek leider (aus urheberrechtlichen Gründen) nicht im Volltext zur Verfügung, die CD-ROM kann (auch über Fernleihe) bei der UB Frankfurt am Main ausgeliehen werden.
HeBIS PPN:397073305
Institutes:Biochemie und Chemie
Pharmazie
Dewey Decimal Classification:570 Biowissenschaften; Biologie
610 Medizin und Gesundheit
Sammlungen:Universitätspublikationen
Licence (German):License LogoArchivex. zur Lesesaalplatznutzung § 52b UrhG

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