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Trondl R.,University of Vienna | Trondl R.,Research Platform Translational Cancer Therapy Research | Flocke L.S.,University of Vienna | Kowol C.R.,University of Vienna | And 18 more authors.
Molecular Pharmacology | Year: 2014

Triapine (3-AP; 3-aminopyridine-2-carboxaldehyde thiosemicarbazone), a ribonucleotide reductase inhibitor, has been extensively evaluated in clinical trials in the last decade. This study addresses the role of endoplasmic reticulum (ER) stress in the anticancer activity of 3-AP and the derivative N4,N4-dimethyltriapine (3-AP-Me), differing from 3-AP only by dimethylation of the terminal nitrogen. Treatment of colon cancer cells with 3-AP or 3-AP-Me activated all three ER stress pathways (PERK, IRE1a, ATF6) by phosphorylation of eIF2a and upregulation of gene expression of activating transcription factors ATF4 and ATF6. In particular, 3-AP-Me led to an upregulation of the alternatively spliced mRNA variant XBP1 (16-fold). Moreover, 3-AP and 3-AP-Me activated the cellular stress kinases c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases, and inhibition of JNK activity antagonized the cytotoxic effect of both compounds. Subsequent to induction of the unfolded protein response, a significant upregulation of proapoptotic proteins was detected, including the transcription factor CHOP and Bim, an essential factor for ER stress-related apoptosis. In correlation with the higher degree of ER stress after 3-AP-Me treatment, also a more potent depolarization of mitochondrial membranes was found. These data suggest that 3-AP and 3-AP-Me induce apoptosis via ER stress. This was further corroborated by showing that inhibition of protein biosynthesis with cycloheximide prior to 3-AP and 3-AP-Me treatment leads to a significant reduction of the antiproliferative properties of both compounds. Taken together, this study demonstrates that induction of ER stress contributes to the mode of action of 3-AP and that terminal dimethylation leads to an even more pronounced manifestation of this effect. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Rodriguez-Carrasco Y.,University of Valencia | Heilos D.,University of Vienna | Heilos D.,Medical University of Vienna | Richter L.,TU Berlin | And 11 more authors.
Toxicology Letters | Year: 2016

The fusariotoxins Enniatin B (Enn B) and Beauvericin (Bea) have recently aroused interest as food contaminants and as potential anticancer drugs. However, limited data are available about their toxic profile. Aim of this study was to investigate their pharmacological behavior in vivo and their persistence in mice. Therefore, liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to analyze the distribution of Enn B and Bea in selected tissue samples and biological fluids originating from mice treated intraperitoneally with these cyclohexadepsipeptides. Overall, no toxicological signs during life time or pathological changes were observed. Moreover, both fusariotoxins were found in all tissues and serum but not in urine. Highest amounts were measured in liver and fat demonstrating the moleculeś tendency to bioaccumulate in lipophilic tissues. While for Bea no metabolites could be detected, for Enn B three phase I metabolites (dioxygenated-Enn B, mono- and di-demethylated-Enn B) were found in liver and colon, with dioxygenated-Enn B being most prominent. Consequently, contribution of hepatic as well as intestinal metabolism seems to be involved in the overall metabolism of Enn B. Thus, despite their structural similarity, the metabolism of Enn B and Bea shows distinct discrepancies which might affect long-term effects and tolerability in humans. © 2016 Elsevier Ireland Ltd.

Groschel C.,Medical University of Vienna | Aggarwal A.,Medical University of Vienna | Tennakoon S.,Medical University of Vienna | Hobaus J.,Medical University of Vienna | And 7 more authors.
Journal of Steroid Biochemistry and Molecular Biology | Year: 2016

Epidemiological studies suggest a correlation between vitamin D deficiency and colorectal cancer (CRC) incidence. The majority of sporadic tumors develop from premalignant lesions with aberrant activation of the Wnt/β-catenin signaling pathway. The adenoma cell line LT97 harbors an adenomatous polyposis coli (APC) mutation leading to constitutively active Wnt signaling. In these cells, expression of Wnt target genes leads to increased survival capacity. We hypothesized that 1,25-dihydroyvitamin D3 (1,25-D3), the active form of vitamin D3, promotes differentiation by modulating β-catenin/T-cell factor (TCF) 4-mediated gene transcription. The effect of dietary vitamin D on colonic Wnt signaling was investigated in mice fed either with 100 IU or 2500 IU vitamin D/kg diet. We examined the effect of 1,25-D3 on differentiation by measuring alkaline phosphatase activity. We analyzed mRNA expression of Wnt target genes by real time qRT-PCR. The impact of 1,25-D3 on β-catenin and TCF4 protein expression was assessed by western blot and immunohistochemistry. In LT97 cells, 1,25-D3 increased cellular differentiation and reduced nuclear β-catenin levels. Further, 1,25-D3 decreased mRNA expression of the Wnt target genes BCL-2, Cyclin D1, Snail1, CD44 and LGR5. In healthy colon of mice fed with high vitamin D diet, the mRNA levels of Wnt5a and ROR2, that promote degradation of β-catenin, were upregulated whereas β-catenin and TCF4 protein expression were decreased. In conclusion, 1,25-D3 inhibits Wnt signaling even in nonmalignant cells underlining its importance in protection against colorectal tumorigenesis and early tumor progression. This article is part of a Special Issue entitled '17th Vitamin D Workshop'. © 2015 Elsevier Ltd. All rights reserved.

Aggarwal A.,Medical University of Vienna | Hobaus J.,Medical University of Vienna | Tennakoon S.,Medical University of Vienna | Prinz-Wohlgenannt M.,Medical University of Vienna | And 8 more authors.
Journal of Steroid Biochemistry and Molecular Biology | Year: 2016

Epidemiological studies suggest an inverse correlation between dietary calcium (Ca2+) and vitamin D intake and the risk of colorectal cancer (CRC). It has been shown in vitro that the active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-D3) can upregulate expression of the calcium-sensing receptor (CaSR). In the colon, CaSR has been suggested to regulate proliferation of colonocytes. However, during tumorigenesis colonic CaSR expression is downregulated and we hypothesized that the loss of CaSR could influence the anti-tumorigenic effects of Ca2+ and vitamin D. Our aim was to assess the impact of CaSR expression and function on the anti-neoplastic effects of 1,25-D3 in colon cancer cell lines. We demonstrated that in the healthy colon of mice, high vitamin D diet (2500 IU/kg diet) increased expression of differentiation and apoptosis markers, decreased expression of proliferation markers and significantly upregulated CaSR mRNA expression, compared with low vitamin D diet (100 IU/kg diet). To determine the role of CaSR in this process, we transfected Caco2-15 and HT29 CRC cells with wild type CaSR (CaSR-WT) or a dominant negative CaSR mutant (CaSR-DN) and treated them with 1,25-D3 alone, or in combination with CaSR activators (Ca2+ and NPS R-568). 1,25-D3 enhanced the anti-proliferative effects of Ca2+ and induced differentiation and apoptosis only in cells with a functional CaSR, which were further enhanced in the presence of NPS R-568, a positive allosteric modulator of CaSR. The mutant CaSR inhibited the anti-tumorigenic effects of 1,25-D3 suggesting that the anti-neoplastic effects of 1,25-D3 are, at least in part, mediated by the CaSR. Taken together, our data provides molecular evidence to support the epidemiological observation that both, vitamin D and calcium are needed for protection against malignant transformation of the colon and that their effect is modulated by the presence of a functional CaSR. This article is part of a Special Issue entitled '17th Vitamin D Workshop'. © 2015 Z. Published by Elsevier Ltd.

Dornetshuber-Fleiss R.,University of Vienna | Dornetshuber-Fleiss R.,Medical University of Vienna | Heilos D.,University of Vienna | Heilos D.,Medical University of Vienna | And 13 more authors.
Biochemical Pharmacology | Year: 2015

During the last decades substantial progress has been made in developing systemic cancer therapy. However, tumors are frequently intrinsically resistant against structurally and mechanistically unrelated drugs. Thus, it is of predominant interest to overcome drug resistance and to encourage the research for novel chemotherapeutic approaches. Recently, we have introduced enniatins, naturally occurring cyclohexadepsipeptides produced by filamentous fungi of the genus Fusarium, as potential anticancer drugs. Here, we expend this approach by demonstrating antiangiogenic properties for enniatin B (Enn B) indicated by a strong inhibition of human endothelial cell migration and tube formation. Moreover, combination of Enn B with the clinically approved multi-kinase inhibitor sorafenib (Sora) displayed profound synergistic in vitro and in vivo anticancer effects against cervical cancer. Subsequent studies showed that this strong synergism is accompanied by a marked increase in mitochondrial injury and apoptosis induction reflected by mitochondrial membrane depolarization, caspase-7 activation, and subsequent cleavage of PARP. Additionally, cells were shown to stop DNA synthesis and accumulate in S and G2/M phase of the cell cycle. The multifaceted characteristics underlying this strong synergism were suggested to be based on interference with the p38 MAPK as well as the ERK signaling pathways. Finally, also in vivo studies revealed that the combination treatment is distinctly superior to single drug treatments against the KB-3-1 cervix carcinoma xenograft model. Taken together, our data confirm the anticancer benefits of the naturally occurring fusariotoxin Enn B and further present Enn B/Sora as a novel combination strategy especially for the treatment of cervical cancer. © 2014 Elsevier Inc.

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