Inhibitory Effect of Aspirin on Cholangiocarcinoma Cells

Abstract

Aspirin and other non-steroidal anti-inflammatory drugs reduce the risk of cancer due to their anti-proliferative and apoptotic effects, which are the important mechanisms for their anti-tumor activity. Here, the effect of aspirin on human cholangiocarcinoma cells (KKU-214) and the underlying mechanisms of its action were explored. Cell proliferation was measured by sulforhodamine B (SRB) assay, while cell cycle distribution and apoptosis were determined by flow cytometry. Western blotting was used to explore protein expression underlying molecular mechanisms of anti-cancer treatment of aspirin. Aspirin reduced cell proliferation in a dose- and time-dependent manner, and altered the cell cycle phase distribution of KKU-214 cells by increasing the proportion of cells in the G0/G1 phase and reducing the proportion in the S and G2/M phases. Consistent with its effect on the cell cycle, aspirin also reduced the expression of cyclin D1 and cyclin‑dependent kinase 4 (Cdk-4), which are important for G0/G1 cell cycle progression. Treatment with aspirin led to increased induction of apoptosis in a dose-dependent manner. Further analysis of the mechanism underlying the effect of this drug showed that aspirin induced the expression of the tumor-suppressor protein p53 while inhibiting the anti-apoptotic protein B‑cell lymphoma-2 (Bcl-2). Correspondingly, the activation of caspase-9 and -3 was also increased. These findings suggest that aspirin causes cell cycle arrest and apoptosis, both of which could contribute to its anti-proliferative effect.

Keywords: Aspirin; cholangiocarcinoma; cell cycle analysis; apoptosis.

Figure 1

Dose-Dependent Effect of Aspirin on Growth of KKU-214 Cells. KKU-214 cells were plated into a 96-well plate for 24 h, then treated with different concentrations of aspirin (0, 1, 2, 4, 8, 16, 32 and 64 µM) for 24, 48 and 72 h. The percentage of cell viability was determined by SRB assay. Each value represents the mean ± SD of three independent experiments; *p<0.05, **p<0.01, and ***p<0.001, vs. the control group.

Figure 2

Effect of Aspirin on Cell Migration of KKU-214 Cells. Cells were seeded in 6-well plates in completed medium. After 24 h of incubation, the cells monolayers were wounded by scratched with a 1 mL plastic pipette tip and treated with the indicated concentration of aspirin (0, 6, 8, 16, 32 µM) for 24 h. (a) The wound migration areas were determined by a phase-contrast microscope at 10× magnification. (b) The gap distance was measured from photographs of three random fields. Each value represents the mean ± SD of three independent experiments; *p<0.05 and ***p<0.001, vs. the control group.

Figure 3

Change in Cell Cycle Distribution and Down-Regulation of G0/G1 Phase-Associated Proteins in KKU-214 Cells. (a) KKU-214 cells were seeded into 6-well plates in complete medium. After incubation, cells were treated with various concentrations of aspirin (0, 6, 8 and 10 µM) and flow cytometry was performed. (b) The total cell lysates were extracted and separated on 12% SDS-PAGE. Blotted proteins on a nitrocellulose membrane were probed with primary antibody specific to cyclin D1 and Cdk-4. (c) The band intensity of cyclin D1 and Cdk-4 was quantitated by densitometry and normalized to β-actin. Statistical analysis of control and treated cells was performed using Student’s t-test; *p<0.05, **p<0.01, ***p<0.001 indicate a significant difference.

Figure 4

Apoptosis Induction was Increased in KKU-214 Cells Compared with Control Cells. KKU-214 cells were exposed to various concentrations of aspirin for 24 h. (a) Cells were stained with annexin V-FITC/PI and flow cytometry was performed. Flow cytometric analysis was visualized for living cells (Q3), early apoptotic cells (Q4), late apoptotic cells (Q2) and necrotic cells (Q1). (b) The percentage of both early- and late-stage (Q2+Q4) apoptotic KKU-214 cells was analyzed. (c) Results of western blot analysis of apoptosis-regulating protein expression, including p53, Bcl-2, Bax, caspase-9 and caspase-3, and subsequent densitometry. (d) The density of protein expression after normalization to β-actin was measured in three independent experiments. Statistical analysis of cells was performed using Student’s t-test; *p<0.05, **p<0.01, ***p<0.001 indicate a significant difference.