- Präklinische Studien zur Behandlung des Nierenzellkarzinoms durch den Histondeacetylase-Inhibitor Valproat und Interferon-alpha (2009)
- Das Nierenzellkarzinom (NZK) ist der häufigste maligne Tumor der Niere. In vielen Fällen sind bereits bei der Erstdiagnose Metastasen vorhanden oder entstehen im Verlauf der Therapie. Die Behandlungsmöglichkeiten für diese NZK-Patienten sind äußerst limitiert. Nahezu 2/3 der Betroffenen versterben an ihrer Erkrankung. Die Etablierung neuer Therapieansätze zur Behandlung des NZK ist dringend gefordert. Es wird postuliert, dass ein Therapiekonzept basierend auf dem Histondeacetylase (HDAC)-Inhibitor Valproat (VPA) kombiniert mit niedrig dosiertem Interferon (IFN)-alpha eine innovative und effiziente Behandlungsoption für austherapierte NZK-Patienten eröffnen könnte. In der vorliegenden Studie wurde der Einfluss einer VPA-Mono- versus VPA/IFN-alpha-Kombinationstherapie auf die malignen Eigenschaften verschiedener NZK-Zelllinien evaluiert. Mittels funktioneller Untersuchungen wurden Proliferations- und Adhäsionsphänomene unter den entsprechenden Therapien näher betrachtet. Fluorimetrische und molekularbiologische Studien dienten der detaillierten Aufklärung der den Veränderungen zugrunde liegenden Wirkmechanismen. Zur translationalen Gestaltung wurden zusätzlich tierexperimentelle Untersuchungen durchgeführt. VPA induzierte eine signifikante Reduktion der Proliferation von NZK-Zellen, die durch die additive Gabe von IFN-alpha weiter verstärkt wurde. Die anti-proliferativen Effekte korrelierten mit einer Zunahme der Zellen in der G0/G1-Phase und einer damit einhergehenden verminderten Anzahl an Zellen in der S-Phase. Die Verschiebung der Zellzyklusphasen war mit einer deutlichen Modulation relevanter regulatorischer Zellzyklusproteine assoziiert. Des Weiteren resultierte VPA und die korrespondierende Kombination mit IFN-alpha in einer signifikanten Inhibition der Adhäsion an Endothel und die extrazellulären Matrixproteine. VPA und die VPA/IFN-alpha-Kombination übten ihren Einfluss dabei offensichtlich über eine Modulation von Adhäsionsrezeptoren, implizit Integrine und CD44-Varianten, aus. Die antiproliferative und –adhäsive Wirkung war in der Regel nach längerer Inkubationszeit von 5 Tagen deutlich stärker als nach 3 Tagen. In analog behandelten normalen Nierenzellen zeigten sich im Vergleich keine solchen Effekte. Die Behandlung mit VPA und IFN-alpha scheint somit spezifisch maligne Zellen zu beeinflussen. VPA induzierte in den NZK-Zellen ferner die Reduktion von Protoonkogenen und MAP-Kinasen sowie die Zunahme von Tumorsuppressoren. Die zusätzliche Gabe von IFN-alpha resultierte in einer weiteren Wirkungsverstärkung gegenüber VPA allein. VPA und die Kombination mit IFN-alpha inhibierten zudem signifikant die HDAC6-Aktivität und -Proteinexpression der NZK-Zellen. Diese Hemmung ging mit einer Hyperacetylierung der Histone einher. Die epigenetische Modulation führte zur veränderten Genregulation und Transkription. So nahmen VPA und die korrespondierende Kombination neben den genannten funktionellen und molekularbiologischen Veränderungen maßgeblich Einfluss auf das Genexpressionsprofil der Tumorzellen. Die Expression negativer Regulatoren der Proliferation, Migration und Adhäsion sowie von Genen involviert in Differenzierung und Immunantwort wurden erhöht, wohingegen die Anzahl der Transkripte von Genen mitverantwortlich für die Ausbildung von Resistenzen und die Nährstoffversorgung der Tumoren reduziert wurde. Translationale tierexperimentelle Studien bestätigten die klinische Relevanz der VPA- und VPA/IFN-alpha-Behandlung, die in einer signifikanten Hemmung des Tumorwachstums resultierten. Die Wachstums-Inhibition war mit einer starken Modulation regulatorischer Proteine des Zellzyklus, der Apoptose und des HDAC-Systems assoziiert. Die vorliegenden Ergebnisse demonstrieren das viel versprechende Wirkungspotential von VPA und der korrespondierenden Kombination mit niedrig dosiertem IFN-alpha. VPAs anti-proliferative und -adhäsive Effekte in vitro und in vivo eröffnen die Perspektive für eine innovative Strategie in der Behandlung des NZK. Aufgrund der präsentierten Daten lässt sich postulieren, dass VPA und IFN-alpha die Grundlage für ein neues, effizientes Therapiekonzept bei austherapierten NZK-Patienten darstellen könnte.
- The histone deacetylase inhibitor valproic acid alters growth properties of renal cell carcinoma in vitro and in vivo (2009)
- Histone deacetylase (HDAC) inhibitors represent a promising class of antineoplastic agents which affect tumour growth, differentiation and invasion. The effects of the HDAC inhibitor valproic acid (VPA) were tested in vitro and in vivo on pre-clinical renal cell carcinoma (RCC) models. Caki-1, KTC-26 or A498 cells were treated with various concentrations of VPA during in vitro cell proliferation 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and to evaluate cell cycle manipulation. In vivo tumour growth was conducted in subcutaneous xenograft mouse models. The anti-tumoural potential of VPA combined with low-dosed interferon-α (IFN-α) was also investigated. VPA significantly and dose-dependently up-regulated histones H3 and H4 acetylation and caused growth arrest in RCC cells. VPA altered cell cycle regulating proteins, in particular CDK2, cyclin B, cyclin D3, p21 and Rb. In vivo, VPA significantly inhibited the growth of Caki-1 in subcutaneous xenografts, accompanied by a strong accumulation of p21 and bax in tissue specimens of VPA-treated animals. VPA–IFN-α combination markedly enhanced the effects of VPA monotherapy on RCC proliferation in vitro, but did not further enhance the anti-tumoural potential of VPA in vivo. VPA was found to have profound effects on RCC cell growth, lending support to the initiation of clinical testing of VPA for treating advanced RCC.
- Resistance after chronic application of the HDAC-inhibitor valproic acid is associated with elevated Akt activation in renal cell carcinoma in vivo (2013)
- Targeted drugs have significantly improved the therapeutic options for advanced renal cell carcinoma (RCC). However, resistance often develops, negating the benefit of these agents. In the present study, the molecular mechanisms of acquired resistance towards the histone deacetylase (HDAC) inhibitor valproic acid (VPA) in a RCC in vivo model were investigated. NMRI:nu/nu mice were transplanted with Caki-1 RCC cells and then treated with VPA (200 mg/kg/day). Controls remained untreated. Based on tumor growth dynamics, the mice were divided into “responders” and “non-responders” to VPA. Histone H3 and H4 acetylation and expression of cell signaling and cell cycle regulating proteins in the RCC mouse tumors were evaluated by Western blotting. Tumor growth of VPA responders was significantly diminished, whereas that of VPA non-responders even exceeded control values. Cdk1, 2 and 4 proteins were strongly enhanced in the non-responders. Importantly, Akt expression and activity were massively up-regulated in the tumors of the VPA non-responders. Chronic application (12 weeks) of VPA to Caki-1 cells in vitro evoked a distinct elevation of Akt activity and cancer cells no longer responded with cell growth reduction, compared to the short 2 week treatment. We assume that chronic use of an HDAC-inhibitor is associated with (re)-activation of Akt, which may be involved in resistance development. Consequently, combined blockade of both HDAC and Akt may delay or prevent drug resistance in RCC.
- Molecular targeting of prostate cancer cells by a triple drug combination down-regulates integrin driven adhesion processes, delays cell cycle progression and interferes with the cdk-cyclin axis (2011)
- Background: Single drug use has not achieved satisfactory results in the treatment of prostate cancer, despite application of increasingly widespread targeted therapeutics. In the present study, the combined impact of the mammalian target of rapamycin (mTOR)-inhibitor RAD001, the dual EGFr and VGEFr tyrosine kinase inhibitor AEE788 and the histone deacetylase (HDAC)-inhibitor valproic acid (VPA) on prostate cancer growth and adhesion in vitro was investigated. Methods: PC-3, DU-145 and LNCaP cells were treated with RAD001, AEE788 or VPA or with a RAD-AEE-VPA combination. Tumor cell growth, cell cycle progression and cell cycle regulating proteins were then investigated by MTT-assay, flow cytometry and western blotting, respectively. Furthermore, tumor cell adhesion to vascular endothelium or to immobilized extracellular matrix proteins as well as migratory properties of the cells was evaluated, and integrin alpha and beta subtypes were analyzed. Finally, effects of drug treatment on cell signaling pathways were determined. Results: All drugs, separately applied, reduced tumor cell adhesion, migration and growth. A much stronger anti-cancer effect was evoked by the triple drug combination. Particularly, cdk1, 2 and 4 and cyclin B were reduced, whereas p27 was elevated. In addition, simultaneous application of RAD001, AEE788 and VPA altered the membranous, cytoplasmic and gene expression pattern of various integrin alpha and beta subtypes, reduced integrin-linked kinase (ILK) and deactivated focal adhesion kinase (FAK). Signaling analysis revealed that EGFr and the downstream target Akt, as well as p70S6k was distinctly modified in the presence of the drug combination. Conclusions: Simultaneous targeting of several key proteins in prostate cancer cells provides an advantage over targeting a single pathway. Since strong anti-tumor properties became evident with respect to cell growth and adhesion dynamics, the triple drug combination might provide progress in the treatment of advanced prostate cancer.
- Combining the receptor tyrosine kinase inhibitor AEE788 and the mammalian target of rapamycin (mTOR) inhibitor RAD001 strongly inhibits adhesion and growth of renal cell carcinoma cells (2009)
- Background Treatment options for metastatic renal cell carcinoma (RCC) are limited due to resistance to chemo- and radiotherapy. The development of small-molecule multikinase inhibitors have now opened novel treatment options. The influence of the receptor tyrosine kinase inhibitor AEE788, applied alone or combined with the mammalian target of rapamycin (mTOR) inhibitor RAD001, on RCC cell adhesion and proliferation in vitro has been evaluated. Methods RCC cell lines Caki-1, KTC-26 or A498 were treated with various concentrations of RAD001 or AEE788 and tumor cell proliferation, tumor cell adhesion to vascular endothelial cells or to immobilized extracellular matrix proteins (laminin, collagen, fibronectin) evaluated. The anti-tumoral potential of RAD001 combined with AEE788 was also investigated. Both, asynchronous and synchronized cell cultures were used to subsequently analyze drug induced cell cycle manipulation. Analysis of cell cycle regulating proteins was done by western blotting. Results RAD001 or AEE788 reduced adhesion of RCC cell lines to vascular endothelium and diminished RCC cell binding to immobilized laminin or collagen. Both drugs blocked RCC cell growth, impaired cell cycle progression and altered the expression level of the cell cycle regulating proteins cdk2, cdk4, cyclin D1, cyclin E and p27. The combination of AEE788 and RAD001 resulted in more pronounced RCC growth inhibition, greater rates of G0/G1 cells and lower rates of S-phase cells than either agent alone. Cell cycle proteins were much more strongly altered when both drugs were used in combination than with single drug application. The synergistic effects were observed in an asynchronous cell culture model, but were more pronounced in synchronous RCC cell cultures. Conclusions Potent anti-tumoral activitites of the multikinase inhibitors AEE788 or RAD001 have been demonstrated. Most importantly, the simultaneous use of both AEE788 and RAD001 offered a distinct combinatorial benefit and thus may provide a therapeutic advantage over either agent employed as a monotherapy for RCC treatment.
- Transient down-regulation of beta1 integrin subtypes on kidney carcinoma cells is induced by mechanical contact with endothelial cell membranes (2007)
- Adhesion molecules of the integrin beta1 family are thought to be involved in the malignant progression renal cell carcinoma (RCC). Still, it is not clear how they contribute to this process. Since the hematogenous phase of tumour dissemination is the rate-limiting step in the metastatic process, we explored beta1 integrin alterations on several RCC cell lines (A498, Caki1, KTC26) before and after contacting vascular endothelium in a tumour-endothelium (HUVEC) co-culture assay. Notably, alpha2, alpha3 and alpha5 integrins became down-regulated immediately after the tumour cells attached to HUVEC, followed by re-expression shortly thereafter. Integrin down-regulation on RCC cells was caused by direct contact with endothelial cells, since the isolated endothelial membrane fragments but not the cell culture supernatant contributed to the observed effects. Integrin loss was accompanied by a reduced focal adhesion kinase (FAK) expression, FAK activity and diminished binding of tumour cells to matrix proteins. Furthermore, intracellular signalling proteins RCC cells were altered in the presence of HUVEC membrane fragments, in particular 14-3-3 epsilon, ERK2, PKCdelta, PKCepsilon and RACK1, which are involved in regulating tumour cell motility. We, therefore, speculate that contact of RCC cells with the vascular endothelium converts integrin-dependent adhesion to integrin-independent cell movement. The process of dynamic integrin regulation may be an important part in tumour cell migration strategy, switching the cells from being adhesive to becoming motile and invasive.
- New histone deacetylase inhibitors as potential therapeutic tools for advanced prostate carcinoma (2008)
- The anti-epileptic drug valproic acid is also under trial as an anti-cancer agent due to its histone deacetylase (HDAC) inhibitory properties. However, the effects of valproic acid (VPA) are limited and concentrations required for exerting anti-neoplastic effects in vitro may not be reached in tumour patients. In this study, we tested in vitro and in vivo effects of two VPA-derivatives (ACS2, ACS33) on pre-clinical prostate cancer models. PC3 and DU-145 prostate tumour cell lines were treated with various concentrations of ACS2 or ACS33 to perform in vitro cell proliferation 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and to evaluate tumour cell adhesion to endothelial cell monolayers. Analysis of acetylated histones H3 and H4 protein expression was performed by western blotting. In vivo tumour growth was conducted in subcutaneous xenograft mouse models. Tumour sections were assessed by immunohistochemistry for histone H3 acetylation and proliferation. ACS2 and ACS33 significantly up-regulated histone H3 and H4 acetylation in prostate cancer cell lines. In micromolar concentrations both compounds exerted growth arrest in PC3 and DU-145 cells and prevented tumour cell attachment to endothelium. In vivo, ACS33 inhibited the growth of PC3 in subcutaneous xenografts. Immunohistochemistry and western blotting confirmed increased histone H3 acetylation and reduced proliferation. ACS2 and ACS33 represent novel VPA derivatives with superior anti-tumoural activities, compared to the mother compound. This investigation lends support to the clinical testing of ACS2 or ACS33 for the treatment of prostate cancer.
- Publication of original research in urologic journals - a neglected orphan? (2012)
- The pathophysiologic mechanisms behind urologic disease are increasingly being elucidated. The object of this investigation was to evaluate the publication policies of urologic journals during a period of progressively better understanding and management of urologic disease. Based on the ISI Web of Knowledge Journal Citation Reports and the PubMed database, the number and percentage of original experimental, original clinical, review or commentarial articles published between 2002–2010 in six leading urologic journals were analyzed. “British Journal of Urology International”, “European Urology”, “Urologic Oncology-Seminars and Original Investigations” (“Urologic Oncology”), “Urology”, “The Journal of Urology”, and “World Journal of Urology” were chosen, because these journals publish articles in all four categories. The publication policies of the six journals were very heterogeneous during the time period from 2002 to 2010. The percentage of original experimental and original clinical articles, related to all categories, remained the same in “British Journal of Urology International”, “Urologic Oncology”, “Urology” and “The Journal of Urology”. The percentage of experimental reports in “World Journal of Urology” between 2002–2010 significantly increased from 10 to 20%. A distinct elevation in the percentage of commentarial articles accompanied by a reduction of clinical articles became evident in “European Urology” which significantly correlated with a large increase in the journal’s impact factor. No clearly superior policy could be identified with regard to a general increase in the impact factors from all the journals. The publication policy of urologic journals does not expressly reflect the increase in scientific knowledge, which has occurred over the period 2002–2010. One way of increasing the exposure of urologists to research and expand the interface between experimental and clinical research, would be to enlarge the percentage of experimental articles published. There is no indication that such policy would be detrimental to a journal’s impact factor.