Elsevier

Gynecologic Oncology

Volume 88, Issue 3, March 2003, Pages 429-433
Gynecologic Oncology

Regular article
Inhibition of paclitaxel-induced apoptosis by the specific COX-2 inhibitor, NS398, in epithelial ovarian cancer cells

https://doi.org/10.1016/S0090-8258(03)00084-2Get rights and content

Abstract

Objective

In vitro studies have revealed that treatment of various human cancer cell lines with specific cyclo-oxygenase 2 (COX-2) inhibitors induces apoptotic cell death. It is currently proposed that the combination of COX-2 inhibitors with chemotherapeutic agents improves the efficacy of cancer treatment.

Materials and methods

In this study we sought to determine the effects of combining paclitaxel and the COX-2 inhibitor NS398 on apoptosis of epithelial ovarian cancer (EOC) cells. Two EOC cell lines, SKOV3 and MDAH2774, were exposed to increasing concentrations of paclitaxel (0.1, 10, and 100 μM) and NS398 (10, 100 μM) as well as a combination of both drugs. Apoptosis was evaluated by the Tunel assay. The fluorescein-labeled DNA was visualized directly by fluorescence microscopy and quantitated by flow cytometry.

Results

While NS398 did not significantly alter apoptosis of either EOC cell lines after 24 h of continuous exposure, treatment of both cell lines with paclitaxel resulted in a significant increase in the rate of apoptosis (60–70%). Concomitant treatment of both SKOV3 and MDAH2774 cells with paclitaxel and NS398 resulted in marked impairment of paclitaxel-induced apoptosis. Similarly, sequential treatment during which both cell lines were treated with NS398 for 4 h, triple-washed, and then exposed to paclitaxel for 24 h resulted in a significant inhibition of paclitaxel-induced apoptosis. Similar inhibition was seen when NS398 was replaced by aspirin.

Conclusion

Combining COX-2 inhibitors and paclitaxel does not have an additive or synergistic tumoricidal effect. On the contrary, NS398 treatment markedly inhibited the apoptotic effects of paclitaxel in each of these two EOC cell lines.

Introduction

Paclitaxel is one of the chemotherapeutic agents frequently used in the treatment of epithelial ovarian cancer. When compared to the combination of cyclophosphamide and platinum, treatment of patients with advanced stage ovarian cancer with paclitaxel and platinum has been shown to result in an increased tumor response rate and prolonged disease-free, as well as overall, survival [1]. Paclitaxel works by promoting assembly of microtubules and stabilizing them, preventing depolymerization. This inability to depolymerize microtubules prevents cellular replication. In addition, paclitaxel induces apoptotic cell death in cancer cell lines. Although the mechanism of apoptosis induction is not well delineated, it may be secondary to changes affecting the apoptotic proteins bcl2 and bax [2], [3], [4]

The enzyme prostaglandin endoperoxide synthase (PES), also referred to as cyclo-oxygenase (COX), is responsible for the conversion of arachidonic acid to prostaglandins. In mammalian cells, the COX enzyme consists of two isozymes encoded by separate genes. The COX-1 gene is constitutively expressed in most tissues and the protein levels do not fluctuate in response to stimuli such as cytokines or growth factors. Expression of the COX-2 gene is induced by a variety of agents, including growth factors [5], phorbol esters, and cytokines [6], [7]. The COX-2 gene has been characterized as an immediate–early gene associated with cellular growth and differentiation.

Evidence from in vitro and in vivo studies suggests an important role for prostaglandins and their synthesizing enzyme COX-2 in carcinogenesis [5], [8]. Prolonged intake of drugs which inhibit the COX enzymes activity reduces the incidence of a number of malignancies [9], [10], [11]. Laboratory data have also revealed that exposure of various human carcinoma cell lines to COX-2 inhibitors induce apoptotic cell death in vitro [12], [13], [14].

Currently, the combination of COX-2 inhibitors with chemotherapy agents is being proposed for cancer treatment trials. The objective of our study was to determine the effect of combining a COX-2 inhibitor with paclitaxel on apoptosis and proliferation of epithelial ovarian cancer cells.

Section snippets

Culture of ovarian cancer cell lines

The human epithelial ovarian cancer cell lines SKOV3 and MDAH-2774 were grown in T-150 flasks with RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U of penicillin, 0.1 μg of streptomycin, and 0.25 μg of amphotericin B. Cells were maintained at confluency at 37°C incubation with 5% CO2. The media were changed three times per week. For each experiment, cells were plated out in 0.3% FBS-containing medium in 100-cm2 culture dishes at a cell density of

Results

SKOV3 or MDAH 2774 cells, maintained at the control conditions, exhibited a very low level of apoptosis (1–1.5%). Treatment of both cell lines with paclitaxel resulted in a significant apoptotic death (60–70%) even at the smallest concentration tested (0.1 μM (Fig. 1). Treatment with NS398 at concentrations of 10 or 100 μM, and for extended periods of time (4 to 48 h) caused a slight increase in apoptosis (7–12%) that was not statistically significant.

When MDAH2774 cells were treated with

Discussion

The COX-2 enzyme seems to contribute to the process of carcinogenesis through a number of mechanisms. These include increased proliferation, reduced apoptosis, and stimulation of metastases and angiogenesis [16]. Treatment of various cancer cell lines with specific COX-2 inhibitors has been shown to inhibit cell growth and induce apoptotic death. A recent study has shown that treatment of established epithelial ovarian cancer cell lines (SKOV3, Caov-3, and OVCAR-3) with the specific COX-2

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