Katja Evert

Tissue Tolerable Plasma (TTP) induces apoptosis in pancreatic cancer cells in vitro and in vivo

BackgroundThe rate of microscopic incomplete resections of gastrointestinal cancers including pancreatic cancer has not changed considerably over the past years. Future intra-operative applications of tissue tolerable plasmas (TTP) could help to address this problem. Plasma is generated by feeding energy, like electrical discharges, to gases. The development of non-thermal atmospheric plasmas displaying spectra of temperature within or just above physiological ranges allows biological or medical applications of plasmas.MethodsWe have investigated the effects of tissue tolerable plasmas (TTP) on the human pancreatic cancer cell line Colo-357 and PaTu8988T and the murine cell line 6606PDA in vitro (Annexin-V-FITC/DAPI-Assay and propidium iodide DNA staining assay) as well as in the in vivo tumour chorio-allantoic membrane (TUM-CAM) assay using Colo-357.ResultsTTP of 20 seconds (s) induced a mild elevation of an experimental surface temperature of 23.7 degree Celsius up to 26.63+/−0.40 degree Celsius. In vitro TTP significantly (p=0.0003) decreased cell viability showing the strongest effects after 20s TTP. Also, TTP effects increased over time levelling off after 72 hours (30.1+/−4.4% of dead cells (untreated control) versus 78.0+/−9.6% (20s TTP)). However, analyzing these cells for apoptosis 10s TTP revealed the largest proportion of apoptotic cells (34.8+/−7.2%, p=0.0009 versus 12.3+/−6.6%, 20s TTP) suggesting non-apoptotic cell death in the majority of cells after 20s TTP. Using solid Colo-357 tumours in the TUM-CAM model TUNEL-staining showed TTP-induced apoptosis up to a depth of tissue penetration (DETiP) of 48.8+/−12.3μm (20s TTP, p<0.0001). This was mirrored by a significant (p<0.0001) reduction of Ki-67+ proliferating cells (80.9+/−13.2% versus 37.7+/−14.6%, p<0.0001) in the top cell layers as well as typical changes on HE specimens. The bottom cell layers were not affected by TTP.ConclusionsOur data suggest possible future intra-operative applications of TTP to reduce microscopic residual disease in pancreatic cancer resections. Further promising applications include other malignancies (central liver/lung tumours) as well as synergistic effects combining TTP with chemotherapies. Yet, adaptations of plasma sources as well as of the composition of effective components of TTP are required to optimize their synergistic apoptotic actions.

V-AKT murine thymoma viral oncogene homolog/mammalian target of rapamycin activation induces a module of metabolic changes contributing to growth in insulin-induced hepatocarcinogenesis†‡

Mounting epidemiological evidence supports a role for insulin‐signaling deregulation and diabetes mellitus in human hepatocarcinogenesis. However, the underlying molecular mechanisms remain unknown. To study the oncogenic effect of chronically elevated insulin on hepatocytes in the presence of mild hyperglycemia, we developed a model of pancreatic islet transplantation into the liver. In this model, islets of a donor rat are transplanted into the liver of a recipient diabetic rat, with resulting local hyperinsulinism that leads to the development of preneoplastic lesions and hepatocellular carcinoma (HCC). Here, we investigated the metabolic and growth properties of the v‐akt murine thymoma viral oncogene homolog/mammalian target of rapamycin (AKT/mTOR) pathway, a major downstream effector of insulin signaling, in this model of insulin‐induced hepatocarcinogenesis. We found that activation of insulin signaling triggers a strong induction of the AKT/mTOR cascade that is paralleled by increased synthesis of fatty acids, cholesterol, and triglycerides, induction of glycolysis, and decrease of fatty acid oxidation and gluconeogenesis in rat preneoplastic and neoplastic liver lesions, when compared with the healthy liver. AKT/mTOR metabolic effects on hepatocytes, after insulin stimulation, were found to be mTORC1 dependent and independent in human HCC cell lines. In these cells, suppression of lipogenesis, glycolysis, and the pentose phosphate pathway triggered a strong growth restraint, despite insulin administration. Noticeably, metabolic abnormalities and proliferation driven by insulin were effectively reverted using the dual PI3K/mTOR inhibitor, NVP‐BEZ235, both in vitro and in vivo. Conclusions: The present results indicate that activation of the AKT/mTOR cascade by unconstrained insulin signaling induces a defined module of metabolic alterations in hepatocytes contributing to aberrant cell growth. Thus, inhibition of AKT/mTOR and related metabolic changes might represent a novel preventive and therapeutic approach to effectively inhibit insulin‐induced hepatocarcinogenesis. (Hepatology 2012;)

Romiplostim administration shows reduced megakaryocyte response-capacity and increased myelofibrosis in a mouse model of MYH9-RD

Macrothrombocytopenia in MYH9-related disease (MYH9-RD) results from defects in nonmuscular myosin-IIA function. Thrombopoietin receptor agonists (eltrombopag; romiplostim) seem to improve hemostasis, but little is known about their biologic effects in MYH9-RD. We administered romiplostim to Myh9(-/-) mice (100 μg/kg, every 3 days, during 1 month). MKs increased to similar numbers in Myh9(-/-) and wild-type (WT) mice (with an increase in immature MKs), but Myh9(-/-) platelet count response was much less (2.5-fold vs 8-fold increase). A strong increase in MK nuclei emboli in the lung, in WT and Myh9(-/-) mice, indicates increased transmigration of MKs from the BM. Prolonged (but not acute) treatment with romiplostim decreased expression of GPIb-IX-V complex and GPVI, but not of GPIIbIIIa, and bleeding time increased in WT mice. Microcirculation was not altered by the increased number of large platelets in any of the assessed organs, but in Myh9(-/-) mice a much stronger increase in BM reticulin fibers was present after 4 weeks of romiplostim treatment vs WT mice. These data further encourage short-term use of thrombopoietic agents in patients with MYH9-RDs; however, myelofibrosis has to be considered as a potential severe adverse effect during longer treatment. Reduction of GPIbIX/GPVI expression by romiplostim requires further studies.

Cardiolipin composition correlates with prostate cancer cell proliferation

Prostate cancer (PC) is the second most diagnosed cancer in men. It has been recognized that diet can play a crucial role in PC genesis and progression. In this context, free fatty acids are considered as modulators of cell proliferation. Recently, a relationship between the composition of the mitochondrial phospholipid cardiolipin (CL) and cell proliferation has been discussed. The aim of this study was to analyse the interrelationship between CL composition and the proliferation of prostate cells by exposing PC-3 tumour cells to different fatty acids and by analysing the CL composition in prostate tissue from PC patients after prostatectomy. Among the applied fatty acids, palmitic acid was found to stimulate proliferation of PC-3 cells, whereas oleic acid (OA) had an inhibiting effect. The lipidomic analysis of CL revealed that fatty acids supplied to PC-3 cells were incorporated into CL molecules. Further, the CL content of palmitoleic acid (C16:1) exclusively correlated with the proliferation of PC-3 cells. The CL composition significantly differed between tumour and normal prostate tissue from PC patients. In five out of six patients, the CL content of palmitoleic acid was higher in tumour prostate tissue in comparison to normal prostate tissue. Our data illustrate that the composition of CL can be easily modified by the fatty acid environment of cells. OA was most effective in decreasing the amount of palmitoleic acid within the CL molecules and deceleration of PC-3 cell proliferation. In conclusion, a diet rich in OA might be beneficial in protecting from rapid proliferation of PC cells.

Rho kinase inhibition mitigates sunitinib-induced rise in arterial pressure and renal vascular resistance but not increased renal sodium reabsorption.

Objectives: The therapeutic use of the vascular endothelial growth factor (VEGF) antagonist sunitinib is limited by sunitinib-induced hypertension. The hypotheses were tested that sunitinib increases renal vascular resistance (RVR) and renal Na+ reabsorption, and that Rho kinase (ROCK) inhibition blunts sunitinib-induced hypertension. Methods: Sunitinib actions on human and rat resistance arteries were investigated by myography. The effects of sunitinib alone or in combination with a ROCK inhibitor on arterial pressure and renal function were investigated in rats by radiotelemetry, renal function and metabolism studies accompanied by biochemical, molecular and histological analyses. Results: Sunitinib blunted agonist-induced vasoconstriction and facilitated endothelium-dependent vasodilation. Within 4 days, sunitinib treatment caused arterial pressure and RVR to rise by 30 mmHg and 5 mmHg × ml–1 × min × g kidney weight, respectively, accompanied by reduced glomerular filtration rate and fractional Na+ excretion with unaffected fractional Li+ excretion. ROCK inhibition blunted sunitinib-induced hypertension and prevented the early rise in RVR, but not the decrease in fractional Na+ excretion, which may explain its modest effect on sunitinib-induced hypertension. Conclusion: Our data indicate that early sunitinib-induced hypertension is associated with modest alterations in renal vascular function, but markedly increased renal sodium reabsorption, probably due to direct actions of the VEGF antagonist on the collecting duct, suggesting that VEGF receptors regulate renal Na+ absorption.

Pan-mTOR inhibitor MLN0128 is effective against intrahepatic cholangiocarcinoma in mice.

BACKGROUND & AIMS Intrahepatic cholangiocarcinoma (ICC) is a lethal malignancy without effective treatment options. MLN0128, a second generation pan-mTOR inhibitor, shows efficacy for multiple tumor types. We evaluated the therapeutic potential of MLN0128 vs. gemcitabine/oxaliplatin in a novel ICC mouse model. METHODS We established a novel ICC mouse model via hydrodynamic transfection of activated forms of AKT (myr-AKT) and Yap (YapS127A) protooncogenes (that will be referred to as AKT/YapS127A). Genetic approaches were applied to study the requirement of mTORC1 and mTORC2 in mediating AKT/YapS127A driven tumorigenesis. Gemcitabine/oxaliplatin and MLN0128 were administered in AKT/YapS127A tumor-bearing mice to study their anti-tumor efficacy in vivo. Multiple human ICC cell lines were used for in vitro experiments. Hematoxylin and eosin staining, immunohistochemistry and immunoblotting were applied for the characterization and mechanistic study. RESULTS Co-expression of myr-AKT and YapS127A promoted ICC development in mice. Both mTORC1 and mTORC2 complexes were required for AKT/YapS127A ICC development. Gemcitabine/oxaliplatin had limited efficacy in treating late stage AKT/YapS127A ICC. In contrast, partial tumor regression was achieved when MLN0128 was applied in the late stage of AKT/YapS127A cholangiocarcinogenesis. Furthermore, when MLN0128 was administered in the early stage of AKT/YapS127A carcinogenesis, it led to disease stabilization. Mechanistically, MLN0128 efficiently inhibited AKT/mTOR signaling both in vivo and in vitro, inducing strong ICC cell apoptosis and only marginally affecting proliferation. CONCLUSIONS This study suggests that mTOR kinase inhibitors may be beneficial for the treatment of ICC, even in tumors that are resistant to standard of care chemotherapeutics, such as gemcitabine/oxaliplatin-based regimens, especially in the subset of tumors exhibiting activated AKT/mTOR cascade. Lay summary: We established a novel mouse model of intrahepatic cholangiocarcinoma (ICC). Using this new preclinical model, we evaluated the therapeutic potential of mTOR inhibitor MLN0128 vs. gemcitabine/oxaliplatin (the standard chemotherapy for ICC treatment). Our study shows the anti-neoplastic potential of MLN0128, suggesting that it may be superior to gemcitabine/oxaliplatin-based chemotherapy for the treatment of ICC, especially in the tumors exhibiting activated AKT/mTOR cascade.

Reassessment of endothelin receptor A expression in normal and neoplastic human tissues using the novel rabbit monoclonal antibody UMB-8.

The endothelins ET-1, ET-2 and ET-3 are a family of peptides, which exert their actions via two G protein-coupled receptor subtypes, ETA and ETB. ET-1 is a potent vasoconstrictor and is involved in the development of different cardiovascular and renal disorders. Additionally, ET-1 and the ETA receptor have been shown to be important mediators of cancer growth and metastasis. We have extensively characterized the novel monoclonal rabbit anti-ETA antibody UMB-8 using transfected cells as well as mouse, rat and human tissues. UMB-8 was then tested in a large series of formalin-fixed and paraffin-embedded human normal and neoplastic tissue specimens. Specificity of UMB-8 was demonstrated by detection of a broad band migrating at 70-80kDa in Western blot analyses of ETA-transfected HEK-293 cells and of different mouse tissues and by agonist-dependent translocation of the immunosignal from the surface of ETA-transfected cells into the cytosol. In tissue samples, UMB-8 yielded an efficient immunostaining of distinct cell populations with a predominance of plasma membrane staining, which was abolished after preadsorption of the antibody with its immunizing peptide. In normal tissue, ETA was present in the heart, blood vessels, gut and kidneys. ETA was also detected with a hitherto unappreciated high prevalence in all types of sarcomas and in glioblastomas, but also in various epithelial tumor entities and in tumor stroma. All in all, UMB-8 may prove of great value in the identification of ETA-expressing tumors during routine histopathological examinations.

Hereditary Hemorrhagic Telangiectasia

Hereditary hemorrhagic telangiectasia (HHT) describes the presenting manifestations of a disorder that is characterized by pathologic blood vessels. HHT is inherited as an autosomal dominant trait with variable penetrance. The abnormal vascular structures (dysplasias) can affect all the organs in the human body. The link between a physical stimulus and new lesion development has been established for mucosal trauma owing to nasal airflow turbulence, for ultraviolet exposure to the fingers, and for mechanical trauma to the dominant hand. The pressing question then is whether HHT treatment constitutes a stimulus that is sufficient to trigger new lesion development.

Efficacy of MEK inhibition in a K-Ras-driven cholangiocarcinoma preclinical model

Intrahepatic cholangiocarcinoma (iCCA) is a deadly malignancy with limited treatment options. Gain-of-function mutations in K-Ras is a very frequent alteration, occurring in ~15 to 25% of human iCCA patients. Here, we established a new iCCA model by expressing activated forms of Notch1 (NICD) and K-Ras (K-RasV12D) in the mouse liver (K-Ras/NICD mice). Furthermore, we investigated the therapeutic potential of MEK inhibitors in vitro and in vivo using human CCA cell lines and K-Ras/NICD mice, respectively. Treatment with U0126, PD901, and Selumetinib MEK inhibitors triggered growth restraint in all CCA cell lines tested, with the most pronounced growth suppressive effects being observed in K-Ras mutant cells. Growth inhibition was due to reduction in proliferation and massive apoptosis. Furthermore, treatment of K-Ras/NICD tumor-bearing mice with PD901 resulted in stable disease. At the molecular level, PD901 efficiently inhibited ERK activation in K-Ras/NICD tumor cells, mainly leading to increased apoptosis. Altogether, our study demonstrates that K-Ras/NICD mice represent a novel and useful preclinical model to study K-Ras-driven iCCA development and the effectiveness of MEK inhibitors in counteracting this process. Our data support the usefulness of MEK inhibitors for the treatment of human iCCA.

Chronische Pankreatitis als Risikofaktor für ein Pankreaskarzinom – diagnostische Herausforderungen

Patients with chronic pancreatitis are burdened with an increased risk of developing pancreatic cancer. Strategies or guidelines for the surveillance and early detection of pancreatic adenocarcinoma in patients with chronic pancreatitis are not established, because available clinical, serologic or imaging techniques are still of limited sensitivity and specificity. Despite these limitations do patients with chronic pancreatitis and suspected pancreatic cancer need a careful and sometimes repeated diagnostic work-up. A clear benefit of surveillance programs outside of clinical trials for the early detection of pancreatic cancer has not been demonstrated. A 51-year-old patient with chronic pancreatitis is reported who developed cancer of the pancreatic head while undergoing repeated imaging studies.ZusammenfassungDie chronische Pankreatitis geht mit einem erhöhten Risiko einher, ein Adenokarzinom des Pankreas zu entwickeln. Bis heute existieren keine etablierten Strategien zur Früherkennung des Pankreaskarzinoms bei Patienten mit chronischer Pankreatitis. Der Grund dafür liegt in der unzureichenden Sensitivität und Spezifität klinischer, bildgebender und serologischer Verfahren zur Diagnostik des Karzinoms in einer chronischen Pankreatitis. Ungeachtet dieser Schwierigkeiten sollten Patienten mit klinischem Verdacht auf ein Pankreaskarzinom sorgfältig und ggf. wiederholt untersucht werden. Der Stellenwert von Screeningmaßnahmen bei Patienten mit chronischer Pankreatitis außerhalb von kontrollierten Studien ist nicht gut belegt. Berichtet wird der Fall eines 51-jährigen Patienten mit einer chronischen Pankreatitis, bei dem sich unter engmaschigen bildgebenden Untersuchungen ein duktales Adenokarzinom des Pankreaskopfes entwickelt hat.AbstractPatients with chronic pancreatitis are burdened with an increased risk of developing pancreatic cancer. Strategies or guidelines for the surveillance and early detection of pancreatic adenocarcinoma in patients with chronic pancreatitis are not established, because available clinical, serologic or imaging techniques are still of limited sensitivity and specificity. Despite these limitations do patients with chronic pancreatitis and suspected pancreatic cancer need a careful and sometimes repeated diagnostic work-up. A clear benefit of surveillance programs outside of clinical trials for the early detection of pancreatic cancer has not been demonstrated. A 51-year-old patient with chronic pancreatitis is reported who developed cancer of the pancreatic head while undergoing repeated imaging studies.