Susanne R. Meyer

Corazonin gene expression in the waxmoth Galleria mellonella

We cloned and sequenced a full length cDNA coding for [Arg7]‐corazonin in the greater wax moth Galleria mellonella. The deduced corazonin preprohormone consists of a nineteen amino acid signal peptide, the actual eleven amino acid corazonin sequence, followed by a Gly serving for amidation, a Lys‐Arg processing site and an eighty amino acid corazonin precursor‐related peptide. The data confirm the phylogenetic conservation of the actual corazonin sequence. The signal peptide and the precursor‐related peptide exhibit a similar spacing of a few amino acids as detected in the corazonin preprohormone of Drosophila melanogaster. Northern blots and in situ hybridization experiments revealed that the G. mellonella corazonin gene is tissue‐specifically expressed in four pairs of lateral neurosecretory cells in the brains of penultimate and last instar larvae, as well as of pupae and adults. No corazonin mRNA was detected in other cells of the nervous system, fat body, gut, and several other organs.

Cancer immunotherapy based on recombinant Salmonella enterica serovar Typhimurium aroA strains secreting prostate-specific antigen and cholera toxin subunit B

Prostate cancer is the most common malignant tumor in men and is normally associated with increased serum levels of prostate-specific antigen (PSA). Therefore, PSA is one potential target for a prostate cancer vaccine. In this study we analyzed the functionality of new bacterial PSA vaccines, expressed and secreted via the hemolysin (HlyA) secretion system of Escherichia coli, the prototype of Type I secretion systems (T1SS) using an attenuated Salmonella enterica serovar Typhimurium aroA strain as carrier. The data demonstrate that a bacterial live vaccine encompassing T1SS in combination with cholera toxin subunit B can be successfully used for delivery of PSA to induce cytotoxic CD8+ T-cell responses resulting in an efficient prevention of tumor growth in mice.

Effects of lobaplatin as a single agent and in combination with TRAIL on the growth of triple-negative p53-mutated breast cancers in vitro.

Lobaplatin as a single agent and in combination with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is investigated in in-vitro models of p53-negative triple-negative breast cancers (TNBCs) and compared with a model of oestrogen receptor-positive p53-positive breast cancer. In addition, the induction of programmed cell death by lobaplatin is further explored. By using cell viability assays and western blotting, the cytotoxic effects of lobaplatin alone and in combination with TRAIL are compared with cisplatin in HCC 1806, HCC 1937, and MCF 7 cells. The multicaspase inhibitor z-VAD-fmk and necrostatin, an inhibitor of necroptosis, are used to demonstrate the mechanism of cell death caused by lobaplatin. Lobaplatin displayed antitumour activity in all three cell lines, which increased time dependently. Cotreatment of lobaplatin and TRAIL induced an increase in cytotoxicity by 30–50% in the different cell lines. The pan-caspase inhibitor z-VAD-fmk as well as necrostatin could weaken but not abolish the cytotoxic effect of lobaplatin and cisplatin. Lobaplatin showed substantial cytotoxic effects in two in-vitro models of p53-mutated TNBC. Cotreatment with TRAIL and platinum agents resulted in increased antitumour activity in the TNBC cell lines investigated. Cell death subsequent to treatment with cisplatin and lobaplatin occurred because of apoptosis. However, caspase-independent mechanisms of programmed cell death were also involved. It was also demonstrated that platinum compounds could induce necroptosis, although to a minor extent.

Downregulation of AKT reverses platinum resistance of human ovarian cancers in vitro

Platinum resistance is the most crucial problem for treatment of ovarian cancer. Increasing evidence points towards AKT overexpression as a mechanistic reason for this clinical condition. The present study evaluates the effect of overexpression and downregulation of AKT on the sensitivity to cisplatin in a platinum-resistant human ovarian cancer cell line and the corresponding platinum-sensitive parental cell line. A2780 and A2780cis ovarian cancer cell lines were stably transfected with an AKT-sense and AKT-antisense plasmid. Successful transfection was evaluated by western blot analysis. Cytotoxic effects of cisplatin were evaluated by metabolic (MTT) and clonogenicity assays as well as by FACS analysis. AKT overexpression (confirmed by western blotting) converted platinum-sensitive A2780 into platinum-resistant cells as shown by MTT assay. Importantly, platinum resistance of A2780cis cells could be reversed by downregulation of AKT, as demonstrated by MTT and clonogenicity assays and FACS analysis. Our data provide strong evidence that cisplatin resistance in ovarian cancer is mediated by AKT overexpression and can be overcome by AKT downregulation, thus, providing a rationale for clinical phase II/III studies combining AKT inhibitors with cisplatin.

Functional dissection of the hexamerin receptor and its ligand arylphorin in the blowfly Calliphora vicina.

The process of receptor‐mediated uptake of hexamerin storage proteins from insect haemolymph by fat body cells is a unique feature of the class Insecta. We identified the binding domains of the hexamerin receptor and the hexamerin ligand arylphorin in the blowfly, by means of the yeast‐two‐hybrid‐system. The receptor‐binding domain of arylphorin was located within domain 3 of the arylphorin monomer. The ligand‐binding domain of the hexamerin receptor was mapped to the extreme N‐terminus of the receptor. The binding domains identified exhibit no similarity to any functional protein domains known to date. Additionally, we identified two previously unknown protein‐interactors of the hexamerin receptor. The results of this study provide further insights regarding the mechanism of the receptor‐mediated endocytosis of storage proteins in insects.

Improvement of the live vaccine strain Salmonella enterica serovar Typhi Ty21a for antigen delivery via the hemolysin secretion system of Escherichia coli

The attenuated Salmonella enterica serovar Typhi strain Ty21a (Ty21a) is the only attenuated live oral vaccine against typhoid fever. Ty21a is also an attractive carrier for the delivery of heterologous antigens. We have used Ty21a for antigen delivery via the hemolysin (HlyA) secretion system of Escherichia coli, the prototype of the type I secretion system (T1SS). In this study, we identified by genetic complementation that the specific mutation of rpoS correlated with the hemolysin production of strain Ty21a. We furthermore showed that complementation with a plasmid encoding rfaH, which is described to be a downstream target of rpoS, led to increased expression and secretion of hemolysin. Finally, we demonstrated a significant enhancement of antibody responses against the heterologous HlyA antigen of Ty21a after immunization of mice with rfaH complemented S. typhi strain secreting HlyA compared with the same strain without rfaH plasmid.

Immune escape of AKT overexpressing ovarian cancer cells

Platinum-resistance is the most crucial problem for treatment of ovarian cancer. There is a clinical need for new treatment strategies which overcome platinum resistance. As survival is strongly influenced by immunological parameters, immunotherapeutic strategies appear promising. Therefore a better understanding of the interaction between ovarian tumour cells and cells of the immune system is a necessary prerequisite. In the present study we aimed to enlighten the interactions between platinum resistant and platinum sensitive ovarian cancer cells and natural-killer (NK)-cells. Modified FATAL assay was used for determining the killing efficiency of NK-cells for the parental A2780 cells and the cis-platinum resistant A2780cis human ovarian cancer cells. Expression of pro- and anti-apoptotic genes as well as ligands involved in NK-cell receptor recognition were analysed by RT-PCR and flow cytometric analysis. The efficiency of NK mediated cell lysis differs between A2780 cells and the cis-platinum-resistant A2780cis cells. A2780cis cells are less accessible for NK-cell mediated killing. Based on this observation we characterized the molecular basis for resistance mechanisms. Besides an increase in anti-apoptotic genes (especially CIAP-1 and -2) that probably render A2780cis cells more resistant against apoptosis an increased amount of soluble MICA/B seems to be responsible for the lower killing rate of platinum-resistant A2780cis cells compared to their parental A2780 cells.

Anti-tumour activity of phosphoinositide-3-kinase antagonist AEZS-126 in models of ovarian cancer.

AbstractPurposePlatinum resistance is the most crucial problem for treatment of ovarian cancer. There is a clinical need for new treatment strategies which overcome platinum resistance. Recently high level of AKT was shown to be involved in platinum resistance and furthermore in resistance against Natural-killer (NK)-cell mediated killing in ovarian cancer.MethodsHere, we investigate the ability of the PI3K/AKT inhibitor AEZS-126 alone and in combination with rapamycin to selectively target ovarian cancer cell proliferation and survival in vitro by MTT-assays and FACS based analysis. Furthermore the mechanism of cytotoxicity is analysed by FACS based assays. The NK-killing efficiency of ovarian cancer cells with and without pre-treatment with AEZS-126 was analysed.ResultsAEZS-126 showed good anti-tumour activity in in vitro models of ovarian cancer. Main mechanism of cytotoxicity seems to be necroptosis which could be abrogated by co-incubation with necrostatin-1. Furthermore pre-treatment of platinum resistant cells with AEZS-126 resulted in an increased accessibility of these tumour cells for killing by NK-cells.ConclusionWe demonstrated the highly efficient anti-tumour activity of AEZS-126 in in vitro models of ovarian cancer. Due to the good anti-tumour activity and the expected increase in NK-cell mediated killing even of platinum resistant tumour cells, AEZS-126 seems to be a promising candidate for clinical testing in ovarian cancer.

Studies on the role of osteopontin-1 in endometrial cancer cell lines.

BackgroundOsteopontin-1 is a well characterized protein in many tumour entities. Multiple roles in the processes invasion, metastasis and angiogenesis of tumours are attributed to osteopontin-1. The putative role of osteopontin-1 has not been characterized for endometrial cancer.Material and methodsWe investigated multiple endometrial cancer cell lines for osteopontin-1 mRNA- and protein-expression. Osteopontin-1 dependent effects were analysed in vitro by siRNA inhibition.ResultsAll endometrial cell lines expressed osteopontin-1. Expression of osteopontin-1 was successfully inhibited by specific siRNA. Cells with reduced osteopontin-1 expression showed decreased migration in the Boyden chamber assay and invasion was reduced in the wound-healing assay. Osteopontin-1 seems to play a role in apoptotic processes of endometrial cancer cells. Inhibition of osteopontin-1 expression was associated with an increased susceptibility for radiation therapy.ConclusionOsteopontin-1 seems to play a role in endometrial cancer. Inhibition of osteopontin-1 expression leads to a higher susceptibility for radiation therapy. Our results suggest that a reduced expression of osteopontin-1 in endometrial cancer could inhibit the development of invasion and metastasis in these cells.ZusammenfassungHintergrundOsteopontin-1 ist in vielen Tumorarten ein gut untersuchtes Protein, dem eine vielfältige Rolle bei den diversen Prozessen der Tumorentwicklung wie Invasion, Metastasierung und Angiogenese zugeschrieben wird. Bisher fehlt jedoch eine breite funktionelle Untersuchung zur Rolle von Osteopontin-1 in Endometriumkarzinomen.Material und MethodeIn mehreren Endometriumzelllinien wurde untersucht, ob Osteopontin-1 auf RNA- und Proteinebene nachweisbar ist. Osteopontin-1-abhängige Effekte wurden in vitro durch spezifische Inhibition mittels siRNA („small interfering RNA“) analysiert.ErgebnisseAlle verwendeten Endometriumzelllinien exprimierten Osteopontin-1. Diese Expression konnte durch spezifische siRNA reprimiert werden. Zellen mit einer verminderten Osteopontin-1-Expression zeigten ein vermindertes Migrations- und Invasionsverhalten sowohl im Wund- als auch im Boyden-Chamber-Assay. Osteopontin-1 scheint beim Endometriumkarzinom an apoptotischen Vorgängen beteiligt zu sein. Die Hemmung von Osteopontin-1 erhöht die Suszeptibilität von Endometriumkarzinomzellen für eine Strahlentherapie.SchlussfolgerungOsteopontin-1 spielt auch in Endometriumkarzinomen eine bedeutende Rolle. Durch eine Inhibition von Osteopontin-1 werden Endometriumzellen für eine Strahlentherapie suszeptibler. Zudem deuten unsere Ergebnisse darauf hin, dass durch eine Inhibition von Osteopontin-1 diverse Prozesse der Tumorentwicklung, wie Invasion und Metastasierung bei Endometriumkarzinomen, gehemmt werden können.

The effect of Cordyceps extract and a mixture of Ganoderma lucidum/Agaricus Blazi Murill extract on human endometrial cancer cell lines in vitro

Endometrial carcinoma is the most common gynaecological malignancy. Nevertheless there is a lack of curative therapies, especially for patients diagnosed with late stage, recurrent or aggressive disease, who have a poor prognosis. Cordyceps Sinensis, Ganoderma lucidum and Agaricus Blazi Murill are three fungi widely used in traditional Chinese medicine, and effects as adjuvants in tumour therapy have been demonstrated. However, the function and effects of these fungi in regard to endometrial cancer are not known. Three endometrial cancer cell lines, Ishikawa, Hec-1A and AN3-CA (derived from endometrial cancers grade I, II and III, respectively), were used to determine the effect of the fungi extracts on endometrial cancer cell function and to analyze the molecular mechanism. All fungi extracts had an inhibitory effect on cell viability and proliferation most probably exerted through induction of autophagy. Our data suggest that these fungi extracts may be used as adjuvants in endometrial tumour therapy.