Aus dem Institut für Pathologie der Medizinischen Fakultät Charité der Humboldt-Universität zu Berlin

Dissertation

Einfluß des Cyclooxygenase-2-Inhibitors NS-398 auf Proliferation und Apoptose von Ovarialkarzinomzellinien

Zur Erlangung des akademischen Grades
Doctor medicinae (Dr. med.)

vorgelegt der Medizinischen Fakultät Charité
der Humboldt-Universität zu Berlin

von Antje Fürstenberg
aus Rathenow

Dekan: Prof. Dr. Joachim W. Dudenhausen

Gutachter:
1. Prof. Dr. Steffen Hauptmann
2. Prof. Dr. Peter Dall
3. Prof. Dr. Sigurd Lax

Datum der Promotion:10.05.2004

Abstract

Several studies have provided evidence that the Cyclooxygenase-2 (COX-2) plays a key role in tumorigenesis and that this enzyme is linked to malignancy and prognosis of cancer. Therefore, COX-2-inhibitors are already evaluated in clinical trials as chemotherapeutics against diverse cancers, such as breast, prostate and colorectal cancer. COX-2 is the inducible isoform of cyclooxygenase - the enzyme catalyzing the synthesis of prostaglandins and other eicosanoids. Several studies suggest that COX-2 plays an important role in tumor development and progression. The usefulness of cyclooxygenase-inhibitors (Nonsteroidal Anti-Inflammatory Drugs, NSAIDs) as drugs against cancer has already been investigated in animal models and cell culture experiments. The results of these studies have shown, however, that it is not clear, whether the observed antitumor effects of NSAIDs are due to inhibition of the COX-enzyme or mediated via a COX-independent mechanism through a so-called non-COX-target.

In the present study we investigated effects of inhibition of COX isoforms by the NSAID

NS-398, a selective COX-2-inhibitor, as well as by COX-isoform specific RNA interference (RNAi) in the human ovarian carcinoma cell lines OVCAR-3 and SKOV-3. OVCAR-3 cells showed a constitutive expression of COX-1 and a highly inducible expression of COX-2 after stimulation with interleukin-1ß resulting in production of high levels of Prostaglandin E2 (PGE2). SKOV-3 cells were negative for both COX isoforms. While COX-2-inhibition with RNAi reduced PGE2-levels in the supernatants of OVCAR-3, COX-1-inhibition by RNAi had no influence on PGE2-production measured by PGE2-specific ELISA. Therefore COX-2 seems to be the main source of PGE2 in OVCAR-3 cells. In these cells PGE2-synthesis - and thus the COX-2-enzyme activity - was completely inhibited by NS-398 in concentrations of 1µM. Increasing amounts of NS-398 (>10µM) had an additional antiproliferative effect. This growth inhibition was also observed in the COX-negative cell line SKOV-3 and could not be reverted by exogenous addition of PGE2 (10µM). Flowcytometric analysis revealed, that this growth inhibition was based on a G0/G1-cell-cycle-arrest. There was no evidence for apoptosis induced by NS-398. In contrast, neither COX-1 nor COX-2-inhibition by RNAi led to similar effects on proliferation and cell cycle progression as observed with NS-398. COX-RNAi also did not induce apoptosis.

These results suggest that a COX-independent mechanism is responsible for the NS-398 induced G0/G1-arrest. Further investigations are needed to elucidate how NS-398 induces cell cycle arrest. Hopefully, this would make it possible to increase the specificity of NS-398 and other NSAIDs as chemotherapeutics.

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