S. Rosewicz

Activated signal transducer and activator of transcription 3 (STAT3) supports the malignant phenotype of human pancreatic cancer

BACKGROUND & AIMS Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been implicated in regulation of growth and malignant transformation. We therefore analyzed the expression and biologic significance of STAT3 in human pancreatic cancer cells. METHODS Expression and activation of STAT3 were investigated by immunohistochemistry and immunoblotting. Functional inactivation of STAT3 was achieved by stable transfection of dominant-negative STAT3 constructs in 2 pancreatic cancer cell lines and confirmed by electrophoretic mobility shift assay and immunoblotting. Cell proliferation and tumorigenicity were evaluated by cell counting, colony formation in soft agar, and xenotransplantation in nude mice. STAT3-dependent cell cycle distribution was monitored by flow cytometry, immunoprecipitation, immunoblotting, and histone H1 and GST-Rb kinase assays. RESULTS Compared with nontransformed human pancreas, activated STAT3 is overexpressed in ductal carcinoma cells but not in ducts from chronic pancreatitis. Constitutive activation was also observed in all human pancreatic cancer cell lines examined. Functional inactivation of STAT3 resulted in significant inhibition of anchorage-dependent and -independent proliferation in vitro and reduced tumor growth in vivo. Cell cycle analysis showed a delay of G(1)/S-phase progression due to inhibition of cyclin-dependent kinase 2 activity based on increased expression of p21(WAF1) in vitro and in vivo. Blocking of the STAT3 upstream activator Janus kinase 2 by tyrphostin also resulted in growth arrest because of delayed G(1)/S-phase progression and increased expression of p21(WAF1). CONCLUSIONS On malignant transformation, activated STAT3 promotes cellular proliferation by acceleration of G(1)/S-phase progression and thereby contributes to the malignant phenotype of human pancreatic cancer.

Dual mechanism of vascular endothelial growth factor upregulation by hypoxia in human hepatocellular carcinoma

BACKGROUND/AIMS Vascular endothelial growth factor (VEGF) plays a key role in regulation of tumour associated angiogenesis. In the current study we analysed expression of VEGF and its receptors in human hepatocellular carcinoma (HCC) and investigated the molecular mechanisms of VEGF regulation by hypoxia. METHODS VEGF, kinase domain region (KDR)/fetal liver kinase 1 (flk-1), and flt-1 expression were examined by immunohistochemistry and in situ hybridisation in 15 human HCC tissues. Expression of VEGF and regulation by hypoxia were assessed in three human HCC cell lines using a quantitative competitive reverse transcription-polymerase chain reaction, ELISA, and a series of 5′ deletion reporter gene constructs of the human VEGF promoter in transient transfection assays. RESULTS We observed over expression of VEGF mRNA and protein in HCC compared with cirrhosis or normal liver. Expression of VEGF in tumour cells was strongly increased in areas directly adjacent to necrotic/hypoxic regions. Both VEGF receptors were detected in vascular endothelia of HCC while only KDR/flk-1 receptors were detected in endothelial cells of cirrhotic livers. Expression of VEGF was observed in all human HCC cell lines examined. Hypoxia (1% oxygen) resulted in profound upregulation of VEGF mRNA and protein levels. Furthermore, hypoxia treatment resulted in a doubling of VEGF mRNA stability. Deletion analysis of the human VEGF 5′ flanking region −2018 and +50 demonstrated induction of VEGF promoter activity under hypoxic conditions which was significantly decreased following deletion of the region −1286 and −789 suggesting a substantial contribution of the −975 putative hypoxia inducible factor 1 binding site to hypoxia mediated transcriptional activation of the VEGF gene. CONCLUSION These data suggest hypoxia as a central stimulus of angiogenesis in human HCC through upregulation of VEGF gene expression by at least two distinct molecular mechanisms: activation of VEGF gene transcription and an increase in VEGF mRNA stability.

Molecular mechanism of interferon alfa-mediated growth inhibition in human neuroendocrine tumor cells.

BACKGROUND & AIMS Although human neuroendocrine tumors respond to interferon (IFN)-alpha treatment in vivo, the underlying mechanisms of growth inhibition are poorly understood. To characterize the antiproliferative effects at a molecular level, we explored the growth-regulatory action of IFN-alpha in the human neuroendocrine tumor cell lines BON and QGP1. METHODS IFN-alpha receptor expression and signal transduction were examined by reverse-transcription polymerase chain reaction, immunoblotting, subcellular fractionation, and transactivation assays. Growth regulation was evaluated by cell numbers, soft agar assays, and cell cycle analysis using flow cytometry. Expression and activity of cell cycle-regulatory molecules were determined by immunoblotting and histone H1-kinase assays. RESULTS Both cell lines expressed IFN-alpha receptor mRNA transcripts. Ligand binding initiated phosphorylation of Jak kinases and Stat transcription factors, resulting in Stat activation, nuclear translocation, and transcription from an ISRE-reporter construct. Prolonged IFN-alpha treatment dose-dependently inhibited both anchorage-dependent and -independent growth. Cell cycle analysis of IFN-alpha-treated, unsynchronized cultures revealed an increased S-phase population, which was further substantiated in G(1) synchronized QGP1 cells. IFN-alpha-treated cells entered S phase in parallel to control cultures, but their progress into G(2)/M phase was delayed. Both cellular cyclin B levels and CDC 2 activity were substantially reduced. The extent and time course of this reduction corresponded to the observed S-phase accumulation. CONCLUSIONS IFN-alpha directly inhibits growth of human neuroendocrine tumor cells by specifically delaying progression through S phase and into G(2)/M. These cell cycle changes are associated with inhibition of cyclin B expression, resulting in reduced CDC2 activity.

Downregulation of p21waf/cip-1 mediates apoptosis of human hepatocellular carcinoma cells in response to interferon-γ

There is no effective treatment for advanced hepatocellular carcinoma (HCC). We therefore explored the molecular mechanisms of interferon-gamma (IFN-gamma)-mediated growth regulation in human HCC cell lines. IFN-gamma receptor expression, signal transduction, and regulation of effectors were examined by RT-PCR, immunoprecipitation, immunoblotting, and reporter gene assays. Growth and apoptosis were determined based on cell numbers, cell cycle analyses, kinase assays, DNA fragmentation, and PARP cleavage. HCC cell lines express functionally intact IFN-gamma receptors and downstream effectors. IFN-gamma profoundly inhibited growth of HCC cells via two different mechanisms: inhibition of G1 cell cycle progression and induction of apoptosis. Analyses in SK-Hep-1 cells revealed a deficient cyclin D induction in IFN-gamma-treated cells, resulting in reduced activity of CDK4 and CDK2 kinases and pRB hypophosphorylation. In contrast, apoptosis prevailed in IFN-gamma-treated HepG2 cultures. A survey of apoptosis relevant IFN-gamma effectors including IRF-1, caspase-1, caspase-3, and p21(waf/cip-1) documented a dramatic transcriptional downregulation of p21(waf/cip-1) exclusively in apoptosis-susceptible HepG2 cells. Reconstitution of p21(waf/cip-1) rescued HepG2 cells from IFN-gamma-induced apoptosis, indicating that p21(waf/cip-1) reduction was required for apoptosis execution. Inversely, downregulation of p21(waf/cip-1) sensitized SK-Hep-1 cells to IFN-gamma-induced apoptosis. Thus, downregulation of p21(waf/cip-1) in HCC cells functions as a novel, critical determinant of alternative growth inhibitory pathways in response to IFN-gamma.

Stromelysin 3 is overexpressed in human pancreatic carcinoma and regulated by retinoic acid in pancreatic carcinoma cell lines

Background—Matrix metalloproteinases play an important role in the control of local tumour growth and metastasis of human pancreatic cancer. Aims—To examine expression of recently discovered stromelysin 3 (STR-3) in human pancreatic cancer and pancreatic carcinoma cell lines and to investigate their regulation by retinoids. Methods—STR-3 expression was examined by immunohistochemistry in 21 human pancreatic carcinomas. Expression of STR-3 and regulation by retinoids was assessed in five human pancreatic carcinoma cell lines using western and northern blotting as well as nuclear run on assays. Results—There was pronounced overexpression of STR-3 in 17 of 21 (80.9%) pancreatic carcinoma specimens. STR-3 expression was predominantly located in peritumourous stromal cells. Six of 21 (28.5%) carcinomas also revealed STR-3 expression in epithelial tumour cells whereas no STR-3 expression was observed in non-transformed pancreas. All five pancreatic carcinoma cell lines expressed STR-3 mRNA and protein. Furthermore, retinoid treatment results in a time and dose dependent inhibition of STR-3 protein expression. This inhibition seems to be post-transcriptional as neither STR-3 gene transcription nor mRNA steady state concentrations were affected by retinoids. Conclusions—STR-3 overexpression in stromal as well as epithelial elements during pancreatic carcinogenesis might contribute to the aggressive local growth and metastasis of pancreatic cancer and can be therapeutically targeted by retinoids.

Induction of matrix metalloprotease-1 gene expression by retinoic acid in the human pancreatic tumour cell line Dan-G.

SummaryWe have investigated the effects of retinoic acid (RA) on matrix metalloprotease-1 (MMP-1) gene expression in the human pancreatic tumour cell line Dan-G. 13-cis RA results in a time- and dose-dependent increase of MMP-1 protein concentration. These stimulatory effects were paralleled by a time- and dose-dependent increase of MMP-1 mRNA steady-state concentrations. Nuclear run-on analysis revealed that the increase of MMP-1 mRNA was partially due to an increase of MMP-1 gene transcription. In addition, 13-cis RA treatment results in an increase of MMP-1 mRNA stability. These data demonstrate that RA stimulates MMP-1 gene expression in human pancreatic carcinoma cells by transcriptional and post-transcriptional mechanisms.

Therapie des Pankreasadenokarzinoms

BACKGROUND Despite significant advances in the areas of epidemiology, risk factors, molecular genetics and diagnosis pancreatic carcinoma is characterized by a dismal prognosis and ranks 5th among malignancy-associated deaths. This article attempts to critically review the current literature and analyze therapeutic recommendations based on published evidence. Therapeutic options are based on the stage of the disease. SURGICAL TREATMENT Surgical resection with curative intention is feasible only in a minority of patients presenting with locally confined tumor disease. RADIO- AND CHEMOTHERAPY: Adjuvant combined radiochemotherapy might potentially improve survival and can also be considered in unresectable, locally advanced disease. The role of chemotherapy in advanced disease is exclusively palliative. Up to now, no chemotherapeutic regimen has demonstrated convincing impact on survival. Newer substances, such as gemcitabine, appear to be of some value in respect to quality of life. Best supportive care oriented at clinical symptoms remains a cornerstone in the therapeutic concept of patients with pancreatic carcinoma. CONCLUSION Development of innovative therapeutic strategies is therefore mandatory.Zusammenfassung□ HintergrundTrotz erheblicher Fortschritte im Bereich der Epidemiologie, Molekulargenetik und Diagnostik ist das Adenokarzinom des Pankreas unverändert durch eine extrem schlechte Prognose gekennzeichnet. In der Häufigkeit tumorbedingter Todesfälle liegt es an fünfter Stelle Die vorliegende Arbeit gibt einen kritischen Überblick über die umfangreiche Datenlage und analysiert Therapieempfehlungen, die auf gesicherten Erkenntnissen beruhen.□ Chirurgische TherapieBei einer Minderheit von Patienten wird ein lokal begrenztes Krankheitsstadium diagnostiziert. Hier ist eine chirurgische Resektion unter kurativer Zielsetzung möglich und aufgrund der verbesserten Operationstechnik in erfahrenen Zentren gerechtfertigt.□ Radio-und ChemotherapieMöglicherweise führt eine adjuvante Radiochemotherapie zu einer Überlebensverbesserung. Eine kombinierte Radiochemotherapie kann ferner bei irresektabler, lokal fortgeschrittener Erkrankung indiziert sein. Der systemischen Chemotherapie kommt in fortgeschrittenem Krankheitsstadium eine rein palliative Rolle zu. Kein Behandlungsprotokoll hat bislang reproduzierbar eine eindeutige Lebensverlängerung demonstrieren können. Möglicherweise profitiert jedoch ein Teil der Patienten klinisch von einer Chemotherapie. Grundpfeiler in jedem Stadium stellt eine an Symptomen orientierte supportive Behandlung dar.□ SclußfolgerungAngesichts der aktuellen Datenlage ist die Entwicklung innovativer therapeutischer Strategien von herausragender Bedeutung.Abstract□ BackgroundDespite significant advances in the areas of epidemiology, risk factors, molecular genetics and diagnosis pancreatic carcinoma is characterized by a dismal prognosis and ranks 5th among malignancy-associated deaths. This article attempts to critically review the current literature and analyze therapeutic recommendations based on published evidence. Therapeutic options are based on the stage of the disease.□ Surgical TreatmentSurgical resection with curative intention is feasible only in a minority of patients presenting with locally confined tumor disease.□ Radio- and ChemotherapyAdjuvant combined radiochemotherapy might potentially improve survival and can also be considered in unresectable, locally advanced disease. The role of chemotherapy in advanced disease is exclusively palliative. Up to now, no chemotherapeutic regimen has demonstrated convincing impact on survival. Newer substances, such as gemcitabine, appear to be of some value in respect to quality of life. Best supportive care oriented at clinical symptoms remains a cornerstone in the therapeutic concept of patients with pancreatic carcinoma.□ ConclusionDevelopment of innovative therapeutic strategies is therefore mandatory.

TGF beta-1 stimuliert die VEGF-Gentranskription im humanen cholangiozellulären Karzinom

The expression pattern and functional interaction of proangiogenic factors in human cholangiocellular carcinoma (CCC) have not been fully defined. We therefore investigated the expression of VEGF and TGFs-1 as well as their respective receptors in human CCC tumor samples and further analysed their functional interaction in vitro. Expression of VEGF, TGFs-1 and their receptors was examined by immunohistochemistry, quantitative competitive (QC) RT-PCR and ELISA. VEGF promoter analysis and identification of transcription factors involved in promoter regulation was investigated using transient transfection and electrophoretic mobility shift assays. Coexpression of VEGF and TGFs and its receptors in tumor cells suggests a possible functional interaction between both cytokines. In vitro studies confirmed a paracrine/autocrine stimulation of VEGF by TGFs-1 at a transcriptional level. Further molecular studies using 5′-deletion and mutational analysis of the human VEGF promoter revealed that TGFs stimulates VEGF through Sp1-dependent transcriptional activation. These data suggest that overexpression and functional interaction of TGFs and VEGF might contribute to the „angiogenic switch“ and the malignant phenotype in human CCC.