Sarah Bhargava

Epithelial to Mesenchymal Transition: Expression of the Regulators Snail, Slug, and Twist in Pancreatic Cancer

Purpose: Epithelial to mesenchymal transitions are vital for tumor growth and metastasis. Several inducers of epithelial to mesenchymal transition are transcription factors that repress E-cadherin expression, such as Snail, Slug, and Twist. In this study, we aimed to examine the expression of these transcription factors in pancreatic cancer. Experimental Design: The expression of Snail, Slug, and Twist was detected by immunohistochemistry in tissue samples from patients with pancreatic ductal adenocarcinoma. Five human pancreatic cancer cell lines (AsPC-1, Capan-1, HPAF-2, MiaPaCa-2, and Panc-1) were analyzed by reverse transcription-PCR, real-time PCR, and Western blotting. An orthotopic nude mouse model of pancreatic cancer was applied for in vivo experiments. Results: Seventy-eight percent of human pancreatic cancer tissues showed an expression of Snail, and 50% of the patients displayed positive expression of Slug. Twist showed no or only weak expression. Snail expression was higher in undifferentiated cancer cell lines (MiaPaCa-2 and Panc-1) than in more differentiated cell lines (Capan-1, HPAF-2, AsPC-1). Expression of Slug was detected in all cell lines with different intensities. Twist was not expressed. After exposure to hypoxia, the Twist gene was activated in all five pancreatic cancer cell lines. Conclusions: The transcription factors Snail and Slug are expressed in pancreatic cancer but not in normal tissue, suggesting a role in the progression of human pancreatic tumors. Twist, activated by hypoxia, may play an important role in the invasive behavior of pancreatic tumors.

Cyanine Dye Labeled Vasoactive Intestinal Peptide and Somatostatin Analog for Optical Detection of Gastroenteropancreatic Tumors

Many gastroenteropancreatic (GEP) tumors express vasoactive intestinal peptide (VIP) and/or somatostatin (SST) receptors in high densities. 1 These receptors can, therefore, be used as targets for the tumor directed delivery of contrast agents for detection of primary tumors as well as metastases. Radiolabeled SST analogues are used in daily clinical routine for receptor scintigraphy of neuroendocrine GEP tumors, whereas radiolabeled VIP has recently been introduced for the detection of GEP adenocarcinomas (for a review see Ref. 2). In the last few years optical (nearinfrared) imaging, a method that does not expose patients to ionizing radiation, has emerged as a new diagnostic modality for oncological applications. 3 The method is based on the detection of differences in the absorption and/or fluorescence of normal and tumor tissue. Due to the limited penetration depth of the excitation light, optical imaging is mainly applied to the detection of superficial lesions. A potential application is the detection of gastrointestinal tumors by fluorescence guided endoscopy after administration of a tumor specific, fluorescent contrast agent. We previously reported the synthesis and characterization of cyanine dyes as fluorescent contrast agents for optical imaging. 4 Conjugates consisting of a cyanine dye and a peptide that binds with high affinity to receptors on tumor cells may provide new tumor specific contrast agents for the optical detection of superficial GEP tumors.

Suramin inhibits not only tumor growth and metastasis but also angiogenesis in experimental pancreatic cancer.

Suramin inhibits the proliferation of several human tumors in vivo and in vitro. In this study, the effects of Suramin on proliferation and angiogenesis were investigated in human pancreatic cancer cell lines and in an orthotopic nude mouse model of human pancreatic cancer. The effects of Suramin on proliferation, viability, cell cycle, and apoptosis were studied in five human pancreatic cancer cell lines. Suramin inhibited the proliferation of pancreatic cancer cells in a dose-dependent manner and reduced viability at high concentrations. Cell cycle analysis revealed a decreased S-phase fraction in most cell lines, whereas the apoptotic fraction was not notably different. In vivo treatment with Suramin significantly reduced pancreatic tumor size (MiaPaCa-2, −74%; AsPC-1, −41%; and Capan-1, −49%) and metastatic spread (MiaPaCa-2, −79%; AsPC-1, −34%; and Capan, −38%). As a parameter for angiogenic activity, vascular endothelial growth factor (VEGF) secretion was measured, revealing reduced VEGF concentrations under Suramin treatment in both cell culture medium and ascites. Also, microvessel density quantified in primary tumors was reduced in animals treated with Suramin. Therefore, Suramin inhibits the proliferation of human pancreatic cancer in vitro and in vivo. The therapeutic effects seem to involve cell cycle kinetics and may be in part related to the antiangiogenic action of the drug.

Selective inhibition of endothelin receptor A as an anti-angiogenic and anti-proliferative strategy for human pancreatic cancer

Endothelin-1 (ET-1) plays a major role in tumor proliferation and angiogenesis of various types of cancer acting through endothelin receptors A and B (ETRA and ETRB). The aim of this study was to analyze theET-1/ETRsystem inhumanpancreatic cancer cell linesandto evaluate the effect of a selective endothelin A inhibitor in vitro and in vivo in an orthotopic mouse model. Three different human pancreatic cancer cell lines, MiaPaCa-2, AsPC-1, and Panc-1, were studied. We found that proliferation of human pancreatic carcinoma cells expressing ETRA was significantly reduced with a selective antagonist. Hypoxic conditions led to improved results compared to a normoxic environment (MiaPaCa-2: -53% vs. -18%; AsPC-1: -54% vs. -46%). Proliferation of ETRA negative Panc-1 cells was not decreased. In vivo, the selective ETRA inhibition resulted in reduced angiogenesis as measured by lower microvessel densities (MiaPaCa-2: -47%; AsPC-1: -55%). The blockade of ETRA decreased the volume (MiaPaCa-2: -87%; AsPC-1: -28%) and metastatic spread (MiaPaCa-2: -95.5%; AsPC-1: -27%) of receptorpositive tumors, thereby increasing survival in experimental pancreatic cancer. ETRA blockade did not show an effect on ETRA negative Panc-1 tumors. Therefore, targeting ETRA with a selective antagonist might provide a new approach to reducing proliferation and angiogenesis in human pancreatic cancer.

Spezifisches Endothelzell-Targeting mit einem Shiga-like-Toxin-VEGF Fusionsprotein als neue Therapiestrategie für das Pankreaskarzinom

SLT-VEGF Fusionsprotein reduziert Primartumorwachstum und Disseminierung des experimentellen Pankreaskarzinoms, was in einem verbesserten 14-Wochen-Uberleben der Versuchstiere resultiert. Die reduzierte mikrovaskulare Gefasdichte deutet darauf hin, das dieser Effekt in erster Linie durch einen toxischen Effekt von SLT-VEGF auf Tumorendothelzellen vermittelt wird. Die eingesetzten Therapien waren nicht mit systemischen Nebenwirkungen wie Gewichtsverlust assoziiert.

Der Angiogenese-Inhibitor IM862 verbessert das Überleben beim experimentellen Pankreaskarzinom auch nach verzögertem Therapiebeginn

IM862 is a dipeptide of L-glutamyl-L-tryptophan with antiangiogenic properties and potential antitumor activity. This study evaluated the effect of IM862 on human pancreatic cancer in vitro and in vivo. Proliferation of three pancreatic cancer cell lines (MIAPaCa-2, AsPC-1, HPAF-2) was significantly inhibited only at high concentrations of IM862 (> 100 µg/ml). In vivo, IM862 (100 mg/kg) reduced tumor size and metastasis in an orthotopic nude mouse model, thereby increasing survival, even after a delayed onset of therapy (6 weeks after tumor induction). In vitro data, reduced microvessel density in tumors, and diminished plasma concentration of the proangiogenic mediator VEGF indicate that IM862 inhibits tumor angiogenesis rather than proliferation of pancreatic cancer cells.

Gemcitabine steigert die Effektivität des spezifischen Endothelzell-Targetings im Nacktmausmodell des humanen Pankreaskarzinoms

Targeting of tumor endothelial cells by af usion protein where aV ascular Endothelial Growth Factor isoform is linked to a fragment of Shiga-like Toxin (SLT-VEGF) is effective in experimental pancreatic cancer. This study investigated whether a combination of SLT-VEGF with Gemcitabine increases the therapeutic potential. Human pancreatic cancer cells (AsPC-1) were incubated with SLT-VEGF or Gemcitabine. Cell proliferation was assessed after 72 hours. AsPC-1 tumors were orthotopically induced in 84 nude mice. Animals were randomized and application of SLT-VEGF, Gemcitabine, or the combination began 3 days (prophylaxis) or 6 weeks (therapy) after tumor induction. Treatment was continued for 14 weeks. Volume of the primary tumor, tumor dissemination, and microvessel density were determined at autopsy. SLT-VEGF did not influence proliferation of pancreatic cancer cells, whereas Gemcitabine treatment resulted in a dose-dependent reduction. The in vivo results of Gemcitabine alone were comparable with SLT-VEGF monotherapy. Combination therapy increased the therapeutic effects in comparison to the respective monotherapies. SLT-VEGF acts by an antiangiogenic mechanism, whereas Gemcitabine directly inhibits pancreatic cancer cell growth.

Etablierung eines Resektionsmodells des duktalen Pankreaskarzinoms in der Nacktmaus zur Evaluierung neuer adjuvanter Therapien

1. Eine makroskopisch und mikroskopisch kurative Resektion ist in diesem Tiermodell nach 2 Wochen moglich; spater liegt bereits eine Tumordisseminierung vor. 2. Scheinbar kurativ resezierte Tiere zeigen im weiteren Verlauf im Vergleich zu unbehandelten Kontrolltieren, wie auch zu den Tieren, denen nur die Milz entfernt wurde, keine signifikanten Unterschiede hinsichtlich Uberleben und Metastasierung. 3. Die Ergebnisse dieser Studie spiegeln den haufigen klinischen Verlauf nach Resektion des Primartumors beim Menschen wieder. Neue adjuvante Therapiestrategien konnten in diesem Modell erprobt werden.