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1.  A Novel Armed Oncolytic Measles Vaccine Virus for the Treatment of Cholangiocarcinoma 
Human Gene Therapy  2013;24(5):554-564.
Abstract
Cholangiocarcinoma (CC) is curable only in early stages by complete surgical resection. Thus, in advanced disease stages in which a complete removal of the tumor mass is no longer possible and palliative chemotherapy achieves only modest success, therapeutics employing new methods of action are desperately needed. Oncolytic viruses employed in clinical studies have been shown to spread preferentially in cancer cells. Beyond that, virotherapeutic cell killing can be enhanced by virus-based expression of suicide genes. We engineered a measles vaccine virus (MeV) vector expressing super cytosine deaminase (SCD), a fusion protein of yeast cytosine deaminase and uracil phosphoribosyltransferase, which converts the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) and subsequently to 5-fluorouridine-monophosphate. This novel vector was evaluated using three different human-derived CC cell lines. In vitro, all CC cell lines were found to be permissive to MeV infection. Partial blocking of MeV-mediated oncolysis could be overcome by employment of the SCD transgene together with administration of 5-FC. In vivo, intratumoral application of SCD-armed MeV together with a systemic 5-FC treatment showed a significant reduction in tumor size in a TFK-1 xenograft mouse model when compared with virus-only treatment. In a second animal experiment employing a HuCCT1 xenograft tumor model, an enhanced SCD-armed MeV vector, in which the SCD transgene was expressed from a different genomic position, led not only to reduced tumor volumes, but also to a significant survival benefit. On the basis of these encouraging preclinical data on employment of SCD-armed MeV for the virotherapeutic treatment of chemotherapy-resistant CC, a clinical virotherapy trial is set up currently.
Lange and colleagues engineer a measles vaccine virus (MeV) vector expressing an enzyme that converts 5-fluorocytosine (5-FC) prodrug into 5-fluorouracil-monophosphate. Partial blocking of MeV-mediated oncolysis could be overcome by vector and 5–FC co-administration in cell lines. In vivo, intratumoral vector injection along with systemic 5–FC treatment resulted in significant tumor reduction in a xenograft mouse model.
doi:10.1089/hum.2012.136
PMCID: PMC3655633  PMID: 23550539
2.  Innate Immune Defense Defines Susceptibility of Sarcoma Cells to Measles Vaccine Virus-Based Oncolysis 
Journal of Virology  2013;87(6):3484-3501.
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.
doi:10.1128/JVI.02106-12
PMCID: PMC3592150  PMID: 23302892
3.  Oncolytic Viruses 
Advances in Virology  2012;2012:320206.
doi:10.1155/2012/320206
PMCID: PMC3384932  PMID: 22754567
4.  Attenuated and Protease-Profile Modified Sendai Virus Vectors as a New Tool for Virotherapy of Solid Tumors 
PLoS ONE  2014;9(3):e90508.
Multiple types of oncolytic viruses are currently under investigation in clinical trials. To optimize therapeutic outcomes it is believed that the plethora of different tumor types will require a diversity of different virus types. Sendai virus (SeV), a murine parainfluenza virus, displays a broad host range, enters cells within minutes and already has been applied safely as a gene transfer vector in gene therapy patients. However, SeV spreading naturally is abrogated in human cells due to a lack of virus activating proteases. To enable oncolytic applications of SeV we here engineered a set of novel recombinant vectors by a two-step approach: (i) introduction of an ubiquitously recognized cleavage-motive into SeV fusion protein now enabling continuous spreading in human tissues, and (ii) profound attenuation of these rSeV by the knockout of viral immune modulating accessory proteins. When employing human hepatoma cell lines, newly generated SeV variants now reached high titers and induced a profound tumor cell lysis. In contrast, virus release from untransformed human fibroblasts or primary human hepatocytes was found to be reduced by about three log steps in a time course experiment which enables the cumulation of kinetic differences of the distinct phases of viral replication such as primary target cell infection, target cell replication, and progeny virus particle release. In a hepatoma xenograft animal model we found a tumor-specific spreading of our novel recombinant SeV vectors without evidence of biodistribution into non-malignant tissues. In conclusion, we successfully developed novel tumor-selective oncolytic rSeV vectors, constituting a new tool for virotherapy of solid tumors being ready for further preclinical and clinical development to address distinct tumor types.
doi:10.1371/journal.pone.0090508
PMCID: PMC3944018  PMID: 24598703
5.  Epigenetic Impacts of Ascorbate on Human Metastatic Melanoma Cells 
Frontiers in Oncology  2014;4:227.
In recent years, increasing evidence has emerged demonstrating that high-dose ascorbate bears cytotoxic effects on cancer cells in vitro and in vivo, making ascorbate a pro-oxidative drug that catalyzes hydrogen peroxide production in tissues instead of acting as a radical scavenger. This anticancer effect of ascorbate is hypoxia-inducible factor-1α- and O2-dependent. However, whether the intracellular mechanisms governing this effect are modulated by epigenetic phenomena remains unknown. We treated human melanoma cells with physiological (200 μM) or pharmacological (8 mM) ascorbate for 1 h to record the impact on DNA methyltransferase (DNMT)-activity, histone deacetylases (HDACs), and microRNA (miRNA) expression after 12 h. The results were analyzed with the MIRUMIR online tool that estimates the power of miRNA to serve as potential biomarkers to predict survival of cancer patients. FACS cell-cycle analyses showed that 8 mM ascorbate shifted BLM melanoma cells toward the sub-G1 fraction starting at 12 h after an initial primary G2/M arrest, indicative for secondary apoptosis induction. In pharmacological doses, ascorbate inhibited the DNMT activity in nuclear extracts of MeWo and BLM melanoma cells, but did not inhibit human HDAC enzymes of classes I, II, and IV. The expression of 151 miRNAs was altered 12 h after ascorbate treatment of BLM cells in physiological or pharmacological doses. Pharmacological doses up-regulated 32 miRNAs (≥4-fold) mainly involved in tumor suppression and drug resistance in our preliminary miRNA screening array. The most prominently up-regulated miRNAs correlated with a significantly increased overall survival of breast cancer or nasopharyngeal carcinoma patients of the MIRUMIR database with high expression of the respective miRNA. Our results suggest a possible epigenetic signature of pharmacological doses of ascorbate in human melanoma cells and support further pre-clinical and possibly even clinical evaluation of ascorbate for melanoma therapy.
doi:10.3389/fonc.2014.00227
PMCID: PMC4142417  PMID: 25202679
ascorbate; vitamin C; cancer; melanoma; epigenetics; microRNA; HDAC; DNMT
6.  Feasibility of Telaprevir-Based Triple Therapy in Liver Transplant Patients with Hepatitis C Virus: SVR 24 Results  
PLoS ONE  2013;8(11):e80528.
Management of recurrent Hepatitis C virus (HCV) infection following liver transplantation remains a major challenge. In non-transplanted HCV genotype 1 patients, the introduction of protease inhibitor-based regimens has significantly increased the rate of sustained virological response. In this follow-up study, on the first published cohort of post-liver transplant patients treated with telaprevir-based triple therapy, we investigated both efficacy and safety data in follow-up to 24 weeks (SVR 24) after end of treatment (EOT). SVR 24 efficacy and safety data from 9 liver transplant HCV patients being treated with telaprevir, pegylated interferon, and ribavirin, showed 5 of the transplanted patients accomplished the full duration of the 48 week triple therapy.
Notable were the 4 patients found to be HCV RNA-negative at week 4, and 8 patients at week 12. Upon EOT, at week 48, 6 patients were HCV RNA-negative. Importantly, at follow-up (24 weeks after EOT), a favorable sustained virological response rate was observed in 5 of these patients with HCV RNA remaining negative, including in one patient who discontinued treatment prematurely. Due to side effects, 2 patients discontinued, 2 suffered from virological breakthrough after the telaprevir treatment phase, and 1 patient had a relapse after EOT. Two thirds of patients exhibited hematological side effects requiring ribavirin dose reductions, administration of erythropoetin, or even blood transfusions.
This retrospective analysis provides evidence that - with respect to SVR 24 - liver transplant patients suffering from HCV genotype 1 recurrence may benefit from a telaprevir-based triple therapy as this new regimen showed acceptable antiviral efficacy in this small cohort of mostly pre-treated patients. Management of drug-drug interactions is challenging, but feasible. In part severe side effects are frequent during treatment and require therapeutic interventions.
doi:10.1371/journal.pone.0080528
PMCID: PMC3827217  PMID: 24265827
8.  Optical Detection and Virotherapy of Live Metastatic Tumor Cells in Body Fluids with Vaccinia Strains 
PLoS ONE  2013;8(9):e71105.
Metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. Here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (VACV). In contrast to the FDA-approved CellSearch system, VACV detects circulating tumor cells (CTCs) in a cancer biomarker-independent manner, thus, free of any bias related to the use of antibodies, and can be potentially a universal system for detection of live CTCs of any tumor type, not limited to CTCs of epithelial origin. Furthermore, we demonstrate for the first time that VACV was effective in preventing and reducing circulating tumor cells in mice bearing human tumor xenografts. Importantly, a single intra-peritoneal delivery of VACV resulted in a dramatic decline in the number of tumor cells in the ascitic fluid from a patient with gastric cancer. Taken together, these results suggest VACV to be a useful tool for quantitative detection of live tumor cells in liquid biopsies as well as a potentially effective treatment for reducing or eliminating live tumor cells in body fluids of patients with metastatic disease.
doi:10.1371/journal.pone.0071105
PMCID: PMC3760980  PMID: 24019862
9.  Resveratrol as a Pan-HDAC Inhibitor Alters the Acetylation Status of Jistone Proteins in Human-Derived Hepatoblastoma Cells 
PLoS ONE  2013;8(8):e73097.
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.
doi:10.1371/journal.pone.0073097
PMCID: PMC3758278  PMID: 24023672
10.  Selective Protection of Human Liver Tissue in TNF-Targeting of Cancers of the Liver by Transient Depletion of Adenosine Triphosphate 
PLoS ONE  2012;7(12):e52496.
Background
Tumor necrosis factor alpha (TNF) is able to kill cancer cells via receptor-mediated cell death requiring adenosine triphosphate (ATP). Clinical usage of TNF so far is largely limited by its profound hepatotoxicity. Recently, it was found in the murine system that specific protection of hepatocytes against TNF's detrimental effects can be achieved by fructose-mediated ATP depletion therein. Before employing this quite attractive selection principle in a first clinical trial, we here comprehensively investigated the interdependence between ATP depletion and TNF hepatotoxicity in both in vitro and ex vivo experiments based on usage of primary patient tissue materials.
Methods
Primary human hepatocytes, and both non-tumorous and tumorous patient-derived primary liver tissue slices were used to elucidate fructose-induced ATP depletion and TNF-induced cytotoxicity.
Results
PHH as well as tissue slices prepared from non-malignant human liver specimen undergoing a fructose-mediated ATP depletion were both demonstrated to be protected against TNF-induced cell death. In contrast, due to tumor-specific overexpression of hexokinase II, which imposes a profound bypass on hepatocytic-specific fructose catabolism, this was not the case for human tumorous liver tissues.
Conclusion
Normal human liver tissues can be protected transiently against TNF-induced cell death by systemic pretreatment with fructose used in non-toxic/physiologic concentrations. Selective TNF-targeting of primary and secondary tumors of the liver by transient and specific depletion of hepatocytic ATP opens up a new clinical avenue for the TNF-based treatment of liver cancers.
doi:10.1371/journal.pone.0052496
PMCID: PMC3525543  PMID: 23272249
11.  Efficient Colonization and Therapy of Human Hepatocellular Carcinoma (HCC) Using the Oncolytic Vaccinia Virus Strain GLV-1h68 
PLoS ONE  2011;6(7):e22069.
Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In this study, we analyzed for the first time the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 in two human hepatocellular carcinoma cell lines HuH7 and PLC/PRF/5 (PLC) in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 efficiently colonized, replicated in, and did lyse these cancer cells in culture. Experiments with HuH7 and PLC xenografts have revealed that a single intravenous injection (i.v.) of mice with GLV-1h68 resulted in a significant reduction of primary tumor sizes compared to uninjected controls. In addition, replication of GLV-1h68 in tumor cells led to strong inflammatory and oncolytic effects resulting in intense infiltration of MHC class II-positive cells like neutrophils, macrophages, B cells and dendritic cells and in up-regulation of 13 pro-inflammatory cytokines. Furthermore, GLV-1h68 infection of PLC tumors inhibited the formation of hemorrhagic structures which occur naturally in PLC tumors. Interestingly, we found a strongly reduced vascular density in infected PLC tumors only, but not in the non-hemorrhagic HuH7 tumor model. These data demonstrate that the GLV-1h68 vaccinia virus may have an enormous potential for treatment of human hepatocellular carcinoma in man.
doi:10.1371/journal.pone.0022069
PMCID: PMC3133637  PMID: 21779374
12.  Improved reproducibility in preparing precision-cut liver tissue slices 
Cytotechnology  2009;61(3):145-152.
Precision-cut liver tissue slices (PCLS) have been used for decades to study pharmacological metabolism as well as toxicology and efficacy of novel substances on primary material under standardized conditions. Slicing of primary liver tissue has been done using different slicing machines. Since there has been great variability in the results, we sought to compare the reproducibility of tissue slices generated using the newly developed Leica VT1200 S vibrating blade microtome with Vibrocheck (LV) and the Krumdieck tissue slicer (KD) which has been the standard apparatus for this application so far. Liver samples from five different species (human, pig, cattle, rat, mouse) were cut and the reproducibility of slice thickness was analyzed by cross sectioning the PCLS. The quality of the sliced tissue was determined via measurement of the ATP content. As a result, we found an improved accuracy and reproducibility of rat, mouse and human tissue slices using the new Leica vibrating blade microtome.
doi:10.1007/s10616-009-9246-4
PMCID: PMC2825296  PMID: 20091220
Primary culture; Precision-cut liver slices (PCLS); Leica VT1200 S vibrating blade microtome; Krumdieck tissue slicer
13.  Early MRI response monitoring of patients with advanced hepatocellular carcinoma under treatment with the multikinase inhibitor sorafenib 
BMC Cancer  2009;9:208.
Background
New therapeutic principles in clinical oncology require the adjustment of response criteria to govern therapy decisions. For advanced hepatocellular carcinoma (HCC) a new era has recently begun by the approval of the multikinase inhibitor sorafenib. As a unique feature, HCC usually develops in a diseased liver and current imaging technologies employing classical response criteria have not been prospectively evaluated for this new treatment.
Methods
MRI signal patterns were assessed in 21 advanced HCC patients receiving sorafenib. MRI was performed at baseline and in short-term intervals thereafter. Signal changes under therapy on T1WI, T2WI and post-gadolinium images including necrosis volume and its ratio to the entire tumor volume were compared to baseline imaging. To assess the association between the categorical variables, Fisher's exact tests were applied for a statistical analysis. Survey time ranged from 2–65 weeks, and a total of 39 target lesions were evaluated.
Results
Signal abnormalities during sorafenib therapy were disclosed by T1WI and T2WI in 15/21 patients. The predominant tumor signal change was hyperintensity on both T1WI and T2WI. Interestingly, most patients developed MRI signal changes within 4 weeks of therapy; in contrast, two non-responders did not show any signal alteration at follow-up. Under therapy, 16/21 patients presented with new or progressive necrosis, whereas 7 patients achieved temporarily >75% tumor necrosis under sorafenib. Significantly associated MRI variables were increase in T1WI signal and tumor necrosis (p = 0.017) as well as increase of tumor necrosis with an elevated ratio of necrotic to vital tumor areas (p = 0.002). Remarkably, some (3/13) of the patients developing necrotic tumor areas showed a relevant (>20%) increase in tumor volume, which should be considered in the assessment of imaging studies.
Conclusion
As sorafenib induces early intralesional necrosis with profound changes in T1WI/T2WI MRI signal intensities and measurable necrotic tumor areas in most HCC patients, early MRI-based evaluation could pave the way for its rationale and cost-effective application.
doi:10.1186/1471-2407-9-208
PMCID: PMC2714320  PMID: 19558720

Results 1-13 (13)