Celecoxib inhibits proliferation and induces apoptosis via prostaglandin E2 pathway in human cholangiocarcinoma cell lines

Abstract

AIM: To evaluate the roles and mechanisms of celecoxib in inducing proliferation inhibition and apoptosis of human cholangiocarcinoma cell lines.

METHODS: Cyclooxygenase-2-overexpressing human cholangiocarcinoma cell line QBC939 and cyclooxygenase-2-deficient human cholangiocarcinoma cell line SK-CHA-1 were used in the present study. The anti-proliferative effect was measured by methabenzthiazuron (MTT) assay; apoptosis was determined by transferase-mediated dUTP nick end labeling (TUNEL) detection and transmission electron microscopy (TEM). Cell cycle was analyzed by flow cytometry (FCM). The PGE₂ levels in the supernatant of cultured cholangiocarcinoma cells were quantitated by enzyme-linked immunoabsordent assay (ELISA).

RESULTS: Celecoxib suppressed the production of PGE₂ and inhibited the growth of QBC939 cells. Celecoxib at 10, 20, and 40 μmol/L inhibited PGE₂ production by 26%, 58%, and 74% in QBC939 cells. The PGE₂ level was much lower constitutively in SK-CHA-1 cells (18.6 ± 3.2) compared with that in QBC939 (121.9 ± 5.6) cells (P < 0.01) and celecoxib had no significant influence on PGE₂ level in the SK-CHA-1 cells. The PGE₂ concentration in SK-CHA-1 cells also reduced but not significantly after treatment with celecoxib. The PGE₂ concentration in SK-CHA-1 cells was (16.5 ± 2.9) ng/well, (14.8 ± 3.4) ng/well, (13.2 ± 2.0) ng/well and (12.6 ± 3.1) ng/well respectively, when pre-treated with 1 μmol/L, 10 μmol/L, 20 μmol/L and 40 μmol/L of celecoxib for 48 h (P > 0.05, vs control). The anti-proliferation effect of celecoxib (20 μmol/L) on QBC939 cells was time-dependent, it was noticeable on day 2 (OD490 = 0.23 ± 0.04) and became obvious on day 3 (OD₄₉₀ = 0.31 ± 0.07) to day 4 (OD490 = 0.25 ± 0.06), and the OD₄₉₀ in the control group (day 1) was 0.12 ± 0.03 (P < 0.01, vs control). The anti-proliferation effect of celecoxib could be abolished by the addition of 200 pg/mL PGE₂. The proliferation of SK-CHA-1 cells was inhibited slightly by celecoxib, the cell density OD490 in the presence of celecoxib and in control group was 0.31 ± 0.04 and 0.42 ± 0.03 respectively on day 2 (P > 0.05), 0.58 ± 0.07 and 0.67 ± 0.09 respectively on day 3 (P > 0.05), and 0.71 ± 0.08 and 0.78 ± 0.06 respectively on day 4 (P > 0.05). Celecoxib induced proliferation inhibition and apoptosis by G₁-S cell cycle arrest: the percentage of QBC939 cells in G₀-G₁phase after treatment with 40 μmol/L (74.6 ± 66.21) and 20 μmol/L (68.63 ± 4.36) celecoxib increased significantly compared with control cells (54.41 ± 5.12, P < 0.01). The percentage of SK-CHA-1 cells in G₀-G₁ phase after treatment with various concentrations of celecoxib didn't change significantly compared with control cells. The TUNEL index was much higher in QBC939 cells treated with 20 μmol/L celecoxib for 2 d (0.063 ± 0.018) and for 4 d (0.102 ± 0.037) compared with control cells (0.017 ± 0.004, P < 0.01).

CONCLUSION: The current in vitro study indicates that inhibition of proliferation and induction of apoptosis in human cholangiocarcinoma cells by cyclooxygenase-2 specific inhibitor celecoxib may involve in COX-dependent mechanisms and PGE₂pathway. Celecoxib as a chemopreventive and chemotherapeutic agent might be effective primarily on COX-2-expressing cholangiocarcinoma.

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