Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter β1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells

Crocin suppresses multidrug resistance in MRP overexpressing ovarian cancer cell line

Background Crocin, one of the main constituents of saffron extract, has numerous biological effects such as anti-cancer effects. Multidrug resistance- associated proteins 1 and 2 (MRP1 and MRP2) are important elements in the failure of cancer chemotherapy. In this study we aimed to evaluate the effects of crocin on MRP1 and MRP2 expression and function in human ovarian cancer cell line A2780 and its cisplatin-resistant derivative A2780/RCIS cells. Methods The cytotoxicity of crocin was assessed by the MTT assay. The effects of crocin on the MRP1 and MRP2 mRNA expression and function were assessed by real-time RT-PCR and MTT assays, respectively. Results Our study indicated that crocin reduced cell proliferation in a dose-dependent manner in which the reduction in proliferation rate was more noticeable in the A2780 cell line compared to A2780/RCIS. Crocin reduced MRP1 and MRP2 gene expression at the mRNA level in A2780/RCIS cells. It increased doxorubicin cytotoxicity on the resistant A2780/RCIS cells in comparison with the drug-sensitive A2780 cells. Conclusion Totally, these results indicated that crocin could suppress drug resistance via down regulation of MRP transporters in the human ovarian cancer resistant cell line

The cell cycle arrest and the anti-invasive effects of nitrogen-containing bisphosphonates are not mediated by DBF4 in breast cancer cells.

Recent work has shown that a DBF4 analog in yeast may be a target of nitrogen-containing bisphosphonates. DBF4 is an essential protein kinase required for DNA replication from primary eukaryotes to humans and appears to play a critical role in the S-phase checkpoint. It is also required for cell migration and cell surface adhesion. The effects of Pamidronate, risedronate, or zoledronate on cell viability and DBF4 expression were measured via MTT assays and western blotting. In addition, FACS cell cycle analyses and invasion assays were conducted in cells in the presence of nitrogen-containing bisphosphonates to identify any correlations between DBF4 expression and S-phase arrest or anti-invasive effects of the bisphosphonates. Zoledronate transiently down-regulated DBF4 expression in all three cell lines in the first 24 h of the experiment, but after 72 h, DBF4 expression returned to the control levels in all treated cells. Following treatment of the tumor cells with the bisphosphonates, the number of cells in S-phase was increased. Pamidronate and zoledronate showed anti-invasive effects in BT20 cells. The anti-invasive effects of pamidronate, risedronate and zoledronate appeared after 48 h of exposure. In MDA-MB231 cells a reduction of invasiveness was only observed after 72 h of the pamidronate exposure. We finally concluded that the anti-invasive and cell cycle arrest-inducing effects of nitrogen-containing bisphosphonates are not DBF4 mediated, and other mediators are therefore needed to explain the observed complex behaviors

YB-1 dependent oncolytic adenovirus efficiently inhibits tumor growth of glioma cancer stem like cells

Background: The brain cancer stem cell (CSC) model describes a small subset of glioma cells as being responsible for tumor initiation, conferring therapy resistance and tumor recurrence. In brain CSC, the PI3-K/AKT and the RAS/mitogen activated protein kinase (MAPK) pathways are found to be activated. In consequence, the human transcription factor YB-1, knowing to be responsible for the emergence of drug resistance and driving adenoviral replication, is phosphorylated and activated. With this knowledge, YB-1 was established in the past as a biomarker for disease progression and prognosis. This study determines the expression of YB-1 in glioblastoma (GBM) specimen in vivo and in brain CSC lines. In addition, the capacity of Ad-Delo3-RGD, an YB-1 dependent oncolytic adenovirus, to eradicate CSC was evaluated both in vitro and in vivo. Methods: YB-1 expression was investigated by immunoblot and immuno-histochemistry. In vitro, viral replication as well as the capacity of Ad-Delo3-RGD to replicate in and, in consequence, to kill CSC was determined by real-time PCR and clonogenic dilution assays. In vivo, Ad-Delo3-RGD-mediated tumor growth inhibition was evaluated in an orthotopic mouse GBM model. Safety and specificity of Ad-Delo3-RGD were investigated in immortalized human astrocytes and by siRNA-mediated downregulation of YB-1. Results: YB-1 is highly expressed in brain CSC lines and in GBM specimen. Efficient viral replication in and virus-mediated lysis of CSC was observed in vitro. Experiments addressing safety aspects of Ad-Delo3-RGD showed that (i) virus production in human astrocytes was significantly reduced compared to wild type adenovirus (Ad-WT) and (ii) knockdown of YB-1 significantly reduced virus replication. Mice harboring othotopic GBM developed from a temozolomide (TMZ)-resistant GBM derived CSC line which was intratumorally injected with Ad-Delo3-RGD survived significantly longer than mice receiving PBS-injections or TMZ treatment. Conclusion: The results of this study supported YB-1 based virotherapy as an attractive therapeutic strategy for GBM treatment which will be exploited further in multimodal treatment concepts

Evaluation of Microarray Preprocessing Algorithms Based on Concordance with RT-PCR in Clinical Samples

BACKGROUND Several preprocessing algorithms for Affymetrix gene expression microarrays have been developed, and their performance on spike-in data sets has been evaluated previously. However, a comprehensive comparison of preprocessing algorithms on samples taken under research conditions has not been performed. METHODOLOGY/PRINCIPAL FINDINGS We used TaqMan RT-PCR arrays as a reference to evaluate the accuracy of expression values from Affymetrix microarrays in two experimental data sets: one comprising 84 genes in 36 colon biopsies, and the other comprising 75 genes in 29 cancer cell lines. We evaluated consistency using the Pearson correlation between measurements obtained on the two platforms. Also, we introduce the log-ratio discrepancy as a more relevant measure of discordance between gene expression platforms. Of nine preprocessing algorithms tested, PLIER+16 produced expression values that were most consistent with RT-PCR measurements, although the difference in performance between most of the algorithms was not statistically significant. CONCLUSIONS/SIGNIFICANCE Our results support the choice of PLIER+16 for the preprocessing of clinical Affymetrix microarray data. However, other algorithms performed similarly and are probably also good choices

Nanolipoparticles-mediated MDR1 siRNA delivery reduces doxorubicin resistance in breast cancer cells and silences MDR1 expression in xenograft model of human breast cancer

Objective(s): P-glycoprotein (P-gp) is an efflux protein, the overexpression of which has been associated with multidrug resistance in various cancers. Although siRNA delivery to reverse P-gp expression may be promising for sensitizing of tumor cells to cytotoxic drugs, the therapeutic use of siRNA requires effective carriers that can deliver siRNA intracellularly with minimal toxicity on target cells. We investigated a special class of PEGylated lipid-based nanoparticles (NP), named nanolipoparticles (NLPs), for siRNA- mediated P-gp downregulation. Materials and Methods: NLPs were prepared based on low detergent dialysis method. After characterization, we evaluated the effect of NLPs on siRNA delivery, and P-gp downregulation compared to oligofectamineTM (OFA) in vitro and in vivo. Results: Our results showed a significant decrease in P-gp expression and subsequent enhancement of chemosensitivity to doxorubicin in vitro. Although the effectiveness of NLPs for in vitro siRNA delivery compared to OFA was limited, the results of in vivo studies showed noticeable effectiveness of NLPs for systemic siRNA delivery. siRNA delivery using NLPs could downregulate MDR1 in tumor cells more than 80%, while OFA had a reverse effect on MDR1 expression in vivo. Conclusion: The results indicated that the prepared NLPs could be suitable siRNA delivery systems for tumor therapy

Estrogen Receptor Alpha Expression in Ovarian Cancer Predicts Longer Overall Survival

Estrogen as a potential factor of ovarian carcinogenesis, acts via two nuclear receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), but the cellular signal pathways involved are not completely clear so far. In this study we have described the expression of ERα, detected by immunocytochemistry in 11 ovarian carcinoma cell lines and by immunohistochemistry in 43 Federation Internationale des Gyneacologistes et Obstetristes stage III ovarian carcinoma specimens prepared before and after treatment with cisplatin-based schemes. For cisplatin resistance is a major obstacle in the treatment of ovarian carcinoma, analysis of cisplatin sensitivity in 11 ovarian carcinoma cell line was also performed. The strong nuclear ERα expression was only shown in the single A2780P cell line. Expression of ERα in tissue specimens did not reveal any correlations between histopathological parameters (histologic type and grading). We demonstrated a significant association with ERα expression in specimens from primary laparotomies (PL) and cause–specific survival. In the cases terminated by death of the patient, overall immunoreactivity score of ERα expression at PL was significantly lower than in surviving patients. In addition, Kaplan-Meier analysis revealed significantly shorter overall survival time and progression-free time in cases with lower immunoreactivity score of ERα expression at PL. Our findings support the hypothesis that aberrant hormone activity, by way of altered receptor expression, might be an important factor in the malignant transformation of ovarian cancer

Antiproliferative activity of various fused heterocyclic derivatives of benzoxazoles and benzimidazoles in different tumour cell lines with resistance against DNA-topoisomerase II-inhibiting agents

Promotion mit DeckblattResistenzen von Tumorzellen gegenüber einer chemotherapeutischen Behandlung stellen ein zentrales Problem in der Onkologie dar. Chemoresistenzen gegenüber Zytostatika, die mit DNA Topoisomerase II (Topo II) interagieren, können über Modifikationen der Topo II Aktivität vermittelt werden. Eine Option zur Überwindung von Topo II vermittelter Chemoresistenz stellt der Einsatz alternativer Topo II Inhibitoren dar. Es wurden daher 20 neu synthetisierte Benzoxazol- und Benzimidazol-Derivate sowie davon abgeleiteter fusionierte heterozyklischer Verbindungen, die im zellfreien System Topo II inhibierende Eigenschaften zeigten, hinsichtlich einer möglichen antiproliferativen Aktivität in 18 unterschiedlichen Tumorzellinien untersucht. Die Zellmodelle leiten sich von unterschiedlichen Tumorentitäten ab und bestehen aus chemosensiblen und chemoresistenten Varianten, die hinsichtlich der vorhandenen Resistenzmechanismen, insbesondere der Bedeutung von Topo II, näher charakterisiert wurden. In chemosensitiven Zellen zeigten lediglich zwei Substanzen, BD3 und G35, eine ähnliche Effektivität wie der klassische Topo II Inhibitor Etoposid. Die meisten heterozyklischen Verbindungen zeigten eine höhere antiproliferative Aktivität in chemoresistenten Zellen als in chemosensitiven Zellinien. Dabei waren die Substanzen BD13, BD14 und BD16 hoch wirksam in mehreren chemoresistenten Zellmodellen. Substanz D23 war hoch effizient in zwei resistenten Linien und Substanz D24, mit der höchsten Effiktivität überhaupt, zeigte in einer chemoresistenten Zellinie antiproliferative Aktivität. Zusammenfassend konnten die Substanzen BD13, BD14, BD16, D23 und D24 als wirksam in der Behandlung von unterschiedlichen Tumorzellmodellen identifiziert werden. Sie können daher potentiell als Ausgangssubstanzen für die Synthese neuer, verbesserter Topo II-Inhibitoren genutzt werden.Resistance of tumour cells against treatment with chemotherapeutic agents is a major obstacle in oncology. Drug resistance against cytotoxic agents that interact with DNA-topoisomerase II (Topo II) can be caused by modulation of Topo II activity. One promising strategy to overcome Topo II-mediated drug resistance is the application of alternative Topo II inhibitors. Thus, 20 new synthesized fused heterocyclic derivatives of benzoxazoles and benzimidazoles with Topo II-inhibiting activity in a cell-free system were investigated in 18 different tumour cell lines. The cell models were derived from different tumour entities and consisted of drug-sensitive and drug-resistant variants. All cell models were characterized concerning the relevance of known drug resistance mechanisms including modulation of Topo II expression. In drug- sensitive cells merely the compounds BD3 and G35 showed efficacies, in terms of µM, that were similar to that of the classical Topo II inhibitor etoposide. On the other hand, most of the tested heterocyclic compounds were found more effective in drug-resistant cells than in drug-sensitive ones, and some of the compounds showed high antineoplastic efficacy in several drug-resistant cell models. Compounds BD13, BD14 and BD16 exhibited the most pronounced antineoplastic activity in various drug-resistant cell variants. Compound D23 was found to be highly efficient in two drug-resistant cell lines and compound D24 exhibited the highest antineoplastic activity among the tested compounds in a single drug-resistant cell variant. In conclusion, compounds BD 13, BD 14, BD 16, D 23, and D 24, may be useful for the treatment of different multidrug-resistant cancer cells with cross resistance against classical Topo II-targeting drugs. Thus, they may be useful for the synthesis of new, improved heterocyclic compounds with Topo II-inhibiting activity