Timo Weiland

Resveratrol as a Pan-HDAC Inhibitor Alters the Acetylation Status of Jistone Proteins in Human-Derived Hepatoblastoma Cells

The polyphenolic alcohol resveratrol has demonstrated promising activities for the prevention and treatment of cancer. Different modes of action have been described for resveratrol including the activation of sirtuins, which represent the class III histone deacetylases (HDACs). However, little is known about the activity of resveratrol on the classical HDACs of class I, II and IV, although these classes are involved in cancer development or progression and inhibitors of HDACs (HDACi) are currently under investigation as promising novel anticancer drugs. We could show by in silico docking studies that resveratrol has the chemical structure to inhibit the activity of different human HDAC enzymes. In vitro analyses of overall HDAC inhibition and a detailed HDAC profiling showed that resveratrol inhibited all eleven human HDACs of class I, II and IV in a dose-dependent manner. Transferring this molecular mechanism into cancer therapy strategies, resveratrol treatment was analyzed on solid tumor cell lines. Despite the fact that hepatocellular carcinoma (HCC) is known to be particularly resistant against conventional chemotherapeutics, treatment of HCC with established HDACi already has shown promising results. Testing of resveratrol on hepatoma cell lines HepG2, Hep3B and HuH7 revealed a dose-dependent antiproliferative effect on all cell lines. Interestingly, only for HepG2 cells a specific inhibition of HDACs and in turn a histone hyperacetylation caused by resveratrol was detected. Additional testing of human blood samples demonstrated a HDACi activity by resveratrol ex vivo. Concluding toxicity studies showed that primary human hepatocytes tolerated resveratrol, whereas in vivo chicken embryotoxicity assays demonstrated severe toxicity at high concentrations. Taken together, this novel pan-HDACi activity opens up a new perspective of resveratrol for cancer therapy alone or in combination with other chemotherapeutics. Moreover, resveratrol may serve as a lead structure for chemical optimization of bioavailability, pharmacology or HDAC inhibition.

Kaempferol, a new nutrition-derived pan-inhibitor of human histone deacetylases

Kaempferol is a natural polyphenol belonging to the group of flavonoids. Different biological functions like inhibition of oxidative stress in plants or animal cells and apoptosis induction have been directly associated with kaempferol. The underlying mechanisms are only partially understood. Here we report for the first time that kaempferol has a distinct epigenetic activity by inhibition of histone deacetylases (HDACs). In silico docking analysis revealed that it fits into the binding pocket of HDAC2, 4, 7 or 8 and thereby binds to the zinc ion of the catalytic center. Further in vitro profiling of all conserved human HDACs of class I, II and IV showed that kaempferol inhibited all tested HDACs. In clinical oncology, HDAC inhibitors are currently under investigation as new anticancer compounds. Therefore, we studied the effect of kaempferol on human-derived hepatoma cell lines HepG2 and Hep3B as well as on HCT-116 colon cancer cells and found that it induces hyperacetylation of histone complex H3. Furthermore, kaempferol mediated a prominent reduction of cell viability and proliferation rate. Interestingly, toxicity assays revealed signs of relevant cellular toxicity in primary human hepatocytes only starting at 50 μM as well as in an in vivo chicken embryotoxicity assay at 200 μM. In conclusion, the identification of a novel broad inhibitory capacity of the natural compound kaempferol for human-derived HDAC enzymes opens up the perspective for clinical application in both tumor prevention and therapy. Moreover, kaempferol may serve as a novel lead structure for chemical optimization of pharmacokinetics, pharmacology or inhibitory activities.

Differential Induction of Apoptosis and Senescence by the DNA Methyltransferase Inhibitors 5-Azacytidine and 5-Aza-2′-Deoxycytidine in Solid Tumor Cells

Epigenetic alterations are a hallmark of cancer that govern the silencing of genes. Up to now, 5-azacytidine (5-aza-CR, Vidaza) and 5-aza-2′-deoxycytidine (5-aza-dC, Dacogen) are the only clinically approved DNA methyltransferase inhibitors (DNMTi). Current effort tries to exploit DNMTi application beyond acute leukemia or myelodysplastic syndrome, especially to solid tumors. Although both drugs only differ by a minimal structural difference, they trigger distinct molecular mechanisms that are highly relevant for a rational choice of new combination therapies. Therefore, we investigated cell death pathways in vitro in human hepatoma, colon, renal, and lung cancer cells and in vivo in chorioallantoic membrane and xenograft models. Real-time cancer cell monitoring and cytokine profiling revealed a profoundly distinct response pattern to both drugs. 5-aza-dC induced p53-dependent tumor cell senescence and a high number of DNA double-strand breaks. In contrast, 5-aza-CR downregulated p53, induced caspase activation and apoptosis. These individual response patterns of tumor cells could be verified in vivo in chorioallantoic membrane assays and in a hepatoma xenograft model. Although 5-aza-CR and 5-aza-dC are viewed as drugs with similar therapeutic activity, they induce a diverse molecular response in tumor cells. These findings together with other reported differences enable and facilitate a rational design of new combination strategies to further exploit the epigenetic mode of action of these two drugs in different areas of clinical oncology. Mol Cancer Ther; 12(10); 2226–36. ©2013 AACR.

Innate immune defense defines susceptibility of sarcoma cells to measles vaccine virus based oncolysis

ABSTRACT The oncolytic potential of measles vaccine virus (MeV) has been demonstrated in several tumor entities. Here, we investigated the susceptibility of eight sarcoma cell lines to MeV-mediated oncolysis and found five to be susceptible, whereas three proved to be resistant. In the MeV-resistant cell lines, we often observed an inhibition of viral replication along with a strong upregulation of the intracellular virus-sensing molecule RIG-I and of the interferon (IFN)-stimulated gene IFIT1. Not only expression of IFIT1 but also phosphorylation of IFN-stimulated Stat1 took place rapidly and were found to be persistent over time. In contrast, susceptible cell lines showed a much weaker, delayed, or completely missing expression of IFIT1 as well as a delayed or only transient phosphorylation of Stat1, whereas exogenic stimulation with beta interferon (IFN-β) resulted in a comparable profound activation of Stat1 and expression of IFIT1 in all cell lines. Pretreatment with IFN-β rendered three of the susceptible cell lines more resistant to MeV-mediated oncolysis. These data suggest that differences in the innate immune defense often account for different degrees of susceptibility of sarcoma cell lines to MeV-mediated oncolysis. From a therapeutic perspective, we were able to overcome resistance to MeV by increasing the multiplicity of infection (MOI) and by addition of the prodrug 5-fluorocytosine (FC), thereby exploiting the suicide gene function of virotherapeutic vector MeV-SCD armed with the SCD fusion protein, which consists of yeast cytosine deaminase and yeast uracil phosphoribosyltransferase.

An armed oncolytic measles vaccine virus eliminates human hepatoma cells independently of apoptosis.

Due to late diagnosis and a pronounced chemoresistance, most patients with hepatocellular carcinoma (HCC) have an overall poor prognosis. Measles vaccine viruses (MeV) have been shown to possess anti-tumor properties and their efficacy has been enhanced by arming with suicide genes. To test armed MeV for the treatment of HCC, we equipped it with the suicide gene Super-cytosine deaminase (SCD) and tested the efficacy in cell culture and in a mouse xenograft model of human HCC. Prodrug conversion was investigated in cell culture and quantified by high-performance liquid chromatography. We observed a strong oncolytic activity of MeV-SCD against human HCC in vitro and in vivo. The prodrug was efficiently converted in infected cells leading to a significant enhancement of the cytotoxic effect. Treatment of HCC xenografts with MeV caused long-term virus replication in tumor tissue. We show that the suicide gene therapy induces an apoptosis-like cell death but is not dependent on intact apoptosis pathways. These results demonstrate that MeV-based suicide gene therapy is a promising novel therapy regimen for HCC overcoming resistance towards conventional therapy. The independence from apoptosis raises hopes for the treatment of patients whose tumor cells exert defects in this cell death mechanism.

Dual antitumour effect of 5-azacytidine by inducing a breakdown of resistance-mediating factors and epigenetic modulation

Background The cytokine tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown promising anticancer activity in early clinical settings by selectively inducing apoptosis in different tumour types. However, some tumour entities such as hepatocellular carcinoma (HCC) display an inherent resistance to TRAIL. A huge effort has been made to unravel strategies for a clinically applicable sensitisation of resistant cancer cells to TRAIL. Reversible epigenetic alterations such as DNA methylation play a major role in development, maintenance and resistance phenomena of tumour cells. Currently, several clinical trials are exploiting the potential of epigenetic drugs, such as 5-azacytidine (5-aza-CR) or 5-aza-2′-deoxycytidine (5-aza-dC) to break primary or secondary resistance phenomena of cancer cells. Therefore, 5-aza-CR and 5-aza-dC were investigated in the context of TRAIL resistance. Methods Alterations in proliferation, apoptosis, regulatory proteins and toxicity were investigated in TRAIL-resistant hepatoma, and also in renal, colon and pancreatic cancer cells as well as non-transformed human-derived primary hepatocytes, tissue slices isolated from human liver and non-malignant colon cells, all of which had been exposed to demethylating drugs and/or TRAIL. Results Within hours, 5-aza-CR but not 5-aza-dC sensitised in vitro cultured tumour cells to TRAIL, first by activating caspases, followed by a subsequent induction of apoptosis. This surprisingly rapid sensitisation was confirmed in vivo employing a chorioallantoic membrane assay. As a major mechanism, a 5-aza-CR-induced inhibition of cellular protein synthesis was found which led to a breakdown of tumour-protecting factors such as the antiapoptotic factor FLICE inhibitory protein (FLIP). Importantly, TRAIL and 5-aza-CR did not induce relevant toxicity or apoptosis in primary hepatocytes, liver slices from different human donors and in normal colon cells. Conclusions Molecular evidence is provided for a novel 5-aza-CR-based translational approach enabling a twofold treatment of apoptosis-resistant tumour entities, not only by an epigenetic reversion of the malignancy-associated phenotype but also by an efficient resensitisation to apoptosis-inducing substances such as TRAIL.

Enhanced killing of therapy-induced senescent tumor cells by oncolytic measles vaccine viruses

Therapy‐induced senescence (TIS) as a permanent growth arrest can be induced by various stimuli, including anticancer compounds. TIS emerged as a promising strategy to overcome resistance phenomena. However, senescent cancer cells might regain proliferation activity in vivo or even secrete tumor‐promoting cytokines. Therefore, successful exploitation of TIS in cancer treatment simultaneously requires the development of effective strategies to eliminate senescent cancer cells. Virotherapy aims to selectively hit tumor cells, thus a combination with senescence‐inducing drugs was explored. As a model, we chose measles vaccine virus (MeV), which does not interfere with cellular senescence by itself. In different tumor cell types, such as hepatoma, pancreatic and mammary gland carcinoma, we demonstrate efficient viral replication and lysis after TIS by gemcitabine, doxorubicin or taxol. Applying real time imaging, we even found an accelerated lysis of senescent cancer cells, supporting an enhanced viral replication with an increase in cell‐associated and released infectious MeV particles. In summary, we show as a proof‐of‐concept that senescent tumor cells can be efficiently exploited as virus host cells by oncolytic MeV. These observations open up a new field for preclinical and clinical research to further investigate TIS and oncolytic viruses as an attractive combinatorial future treatment approach.

Preclinical Evaluation of 4-Methylthiobutyl Isothiocyanate on Liver Cancer and Cancer Stem Cells with Different p53 Status

Isothiocyanates from plants of the order Brassicales are considered promising cancer chemotherapeutic phytochemicals. However, their selective cytotoxicity on liver cancer has been barely researched. Therefore, in the present study, we systematically studied the chemotherapeutic potency of 4-methylthiobutyl isothiocyanate (MTBITC). Selective toxicity was investigated by comparing its effect on liver cancer cells and their chemoresistant subpopulations to normal primary hepatocytes and liver tissue slices. Additionally, in a first assessment, the in vivo tolerability of MTBITC was investigated in mice. Growth arrest at G2/M and apoptosis induction was evident in all in vitro cancer models treated with MTBITC, including populations with cancer initiating characteristics. This was found independent from TP53; however cell death was delayed in p53 compromised cells as compared to wt-p53 cells which was probably due to differential BH3 only gene regulation i. e. Noxa and its antagonist A1. In normal hepatocytes, no apoptosis or necrosis could be detected after repeated administration of up to 50 µM MTBITC. In mice, orally applied MTBITC was well tolerated over 18 days of treatment for up to 50 mg/kg/day, the highest dose tested. In conclusion, we could show here that the killing effect of MTBITC has a definite selectivity for cancer cells over normal liver cells and its cytotoxicity even applies for chemoresistant cancer initiating cells. Our study could serve for a better understanding of the chemotherapeutic properties of isothiocyanates on human liver-derived cancer cells.

Kinetic Tracking of Therapy-Induced Senescence Using the Real-Time Cell Analyzer Single Plate System

In recent years, terminal growth arrest, that is, senescence, especially therapy-induced senescence (TIS), has become a major subject in cancer research and several fields of life sciences. Senescence is characterized by a specific set of morphological and biochemical changes. However, methods that evidence senescence induction are still very limited and show large variation between individual examiners. Most notably, these assays are classical endpoint assays, and, therefore, screening for senescence is time consuming and expensive. Here, we describe an efficient, simple, and objective method to screen for TIS over time by modifying the Real-Time Cell Analyzer SP system, thus enabling to pin point the induction of senescence. This method continuously detects the cells impedance in each well of a 96-microwell plate that allows to observe increment of cell size, a hallmark feature of cellular senescence. This technique is suitable for high-throughput TIS screening by measuring several compounds, small molecules, and/or cell lines simultaneously.

Sensitization by 5-Azacytidine toward Death Receptor-Induced Hepatic Apoptosis

5-Azacytidine (5-aza-CR) is a DNA-hypomethylating antineoplastic agent used because of its inhibitory activity on DNA methyltransferases. Today, it is approved as an epigenetically active drug therapy for treatment of myelodysplastic disorders, with a contraindication as to pre-existing liver diseases. Because the mechanism of its hepatotoxicity is still unknown, we investigated the pharmacodynamic properties of 5-aza-CR with regard to death receptor/ligand-induced apoptosis and the mode of execution of cell death. In a time- and concentration-dependent manner, primary murine, human hepatocytes and HepG2 cells exposed to 5-aza-CR became highly sensitive toward cell death induced by CD95L, tumor necrosis factor (TNF)-related apoptosis-inducing ligand, or TNF. Cell death was characterized as apoptotic by membrane blebbing, chromatin condensation, and exposure of phosphatidylserine on the outer membrane. Neither 5-aza-2′-deoxycytidine nor the common DNA methyltransferase inhibitors S-(5′-adenosyl)-l-homocysteine or RG 108 showed any significant effects under these conditions. Despite the complete protection of HepG2 by high concentrations of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (z-VAD-fmk), effector caspase-3/7 activity was completely abolished at approximately a 20-fold lower concentration of z-VAD-fmk. Under these conditions, the serine protease inhibitors N,α-tosyl-l-phenylalanine chloromethyl ketone, N,p-tosyl-l-lysine chloromethyl ketone, and 4-(2-aminoethyl)-benzenesulfonyl fluoride, respectively, conferred protection against death receptor ligands. We conclude that this caspase-independent apoptosis is executed by a yet-unidentified serine protease.