Cancer Res Treat.  2005 Oct;37(5):313-317.

Pattern of Apoptosis by NS398, a Selective COX-2 Inhibitor, in Hepatocellular Carcinoma Cell Lines

• Park MK
• Kim MK
• Kim JC
• Sung YK

Abstract

PURPOSE
NS398, a selective COX-2 inhibitor, is known to inhibit the growth of COX-2 expressing hepatocellular carcinoma cells. The present study investigated whether the cytotoxic effect of NS398 was COX-2 dependent and whether caspases were involved in NS398-induced apoptosis in hepatocellular carcinoma cells. MATERIALS AND METHODS: The expressions of COX-2 in SNU 423 and SNU 449 hepatocellular carcinoma cell lines were examined using RT-PCR and Western blot. The cytotoxic effect of NS398 was measured using MTT in the presence or absence of caspase inhibitors. The distribution of the cell cycle and extent of apoptosis were analyzed using flow cytometry and a Cell Death Elisa kit, respectively. RESULTS: The expression of COX-2 was observed in SNU423 cells, but not in SNU 449 cells. NS398 treatment resulted in both dose-and time-dependent growth inhibitions, with increases in apoptotic cells in both cell lines. Treatment with the pan-caspase inhibitor, z-VAD- fmk, or the caspase-3 inhibitor, Ac-DMQD-CHO, showed no attenuation of the cytotoxic effect of NS398 in either cell line. CONCLUSIONS: This study demonstrated that the cytotoxic effect of NS398 was independent of COX-2 expression. Caspases were also shown not to be involved in NS398-induced apoptosis in either SNU 423 or SNU 449 Korean HCC cell lines. Our data suggests the feasibility of preventing hepatocellular carcinoma with the use of COX-2 inhibitors needs to be carefully evaluated.

Keyword

Apoptosis; Cyclooxygenase 2; Hepatocellular carcinoma; NS398

MeSH Terms

Apoptosis*
Blotting, Western
Carcinoma, Hepatocellular*
Caspase 3
Caspase Inhibitors
Caspases
Cell Cycle
Cell Death
Cell Line*
Cyclooxygenase 2
Cyclooxygenase 2 Inhibitors
Enzyme-Linked Immunosorbent Assay
Flow Cytometry
Caspase 3
Caspase Inhibitors
Caspases
Cyclooxygenase 2
Cyclooxygenase 2 Inhibitors

Figure

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  Fig. 1 COX-2 expression in SNU 423 and SNU 449 hepatocellular carcinoma cell lines. (A) RT-PCR and (B) Western blot analysis.

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  Fig. 2 Effect of NS398 on the growth of the SNU 423 and SNU 449 cell lines. (A) Dose-dependent growth inhibition. Cells were treated with NS398 for 72 h. (B) Time-dependent growth inhibition with 100 µM NS398. The MTT assay was employed to measure the cell viability in both cases. Data are the means±standard errors of six determinations per experiment from three independent experiments (*p<0.05).

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  Fig. 3 Cell cycle analysis using flow cytometry. A representative DNA histogram is shown. Cells were exposed to either DMSO or 100 µM NS398 for 72 h.

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  Fig. 4 Cell death Elisa assay. Cells were treated with 100 µM NS398 for 72 h. The results are presented as the fold increase in apoptosis relative to the controls that received DMSO only. Data are the means±standard errors of two determinations per experiment from 2 independent experiments.

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  Fig. 5 Demonstration of caspase-independence. Cells were treated with 100 µM NS398 for 48 h, in the presence (40 µM) or absence of the pan-caspase inhibitor, z-VAD-fmk, or the caspase-3 inhibitor, Ac-DMQD-CHO. The MTT assay was employed to check the cell viability. Data are the means±standard errors of six determinations per experiment from 3 independent experiments. (*p<0.05).

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