Thromboxane A2 exerts promoting effects on cell proliferation through mediating cyclooxygenase-2 signal in lung adenocarcinoma cells

Abstract

Background: Lung cancer concerns a worldwide health problem and the efficacy of available treatments is unsatisfactory. Recently, thromboxane A2 (TXA2) synthase (TXAS) and receptor (TXA2R) have been documented to play a role in lung cancer development. Therefore, dual TXA2R modulator (i.e., the dual blocker of TXAS and TXA2R) may be more efficacious to kill lung tumor cells than single TXAS inhibitor or TXA2R antagonism. The close relationship between cyclooxygenase (COX)-2 and TXAS also raises whether or how TXA2 contributes to the oncogenic activity of COX-2. This study is therefore conducted to answer these questions.

Methods: Various inhibitors and siRNA were used to evaluate the roles of TXA2 and COX-2 in the proliferation and apoptosis of lung adenocarcinoma cells. Cell proliferation was detected using both MTS ELISA and BrdU labeling ELISA. Cell cycle distribution and apoptosis were examined by flow cytometric analysis. TXB2 level, reflecting the biosynthesis of TXA2, was detected by peroxidase-labeled TXB2 conjugates using an enzyme immunoassay kit. Western blotting was performed to evaluate many biomarkers for cell cycles, apoptosis and proliferation. The levels of COXs were screened by reverse transcriptase and real-time quantitative PCR.

Results: We found either single TXAS inhibitor/TXA2R antagonist or the dual TXA2 modulators offered a similar inhibition on cell proliferation. Moreover, inhibition of TXA2 arrested cells at the G2/M phase and induced apoptosis. It is further demonstrated that TXA2 was able to function as a critical mediator for tumor-promoting effects of COX-2 in lung adenocarcinoma cells.

Conclusion: The present study has for the first shown that dual TXA2 modulators and the single blocker of TXAS or TXA2R offer a similar inhibitory role in lung adenocarcinoma cell proliferation and that the tumor-promoting effects of COX-2 can largely be relayed by TXA2. Thus, TXA2 should be regarded as a critical molecule in COX-2-mediated tumor growth and a valuable target against lung cancer.

Fig. 1

Comparison of single TXAS inhibitor or TXA2R antagonist and dual TXA2 modulators. a–d BrdU cell proliferation assays were used to select the optimal concentration of chemicals. NCI-H23 cells were treated with graded concentrations of furegrelate, SQ29548, BM567, and PTXA2 for 24, 48, and 72 h, respectively. e–g The proliferation of NCI-H23 cells was further evaluated using MTS assays after treatment with furegrelate (1 mM), SQ29548 (5 μM), BM567 (10 μM) or PTXA2 (5 μM) for 24, 48 and 72 h. The results were presented as percentages of the control. Data are expressed as mean ± SD of three independent experiments done in quadruplicate. *p < 0.05, and **p < 0.01, ***p < 0.001 when compared to controls

Fig. 2

Effects of dual TXA2 modulators on cell cycle progression. a The number of NCI-H23 cells in G0/G1, G2/M, and S phases was determined by flow cytometry after staining the cells with PI. Values are expressed as mean ± SD of three independent experiments. *p < 0.05 and **p < 0.01 when compared to control. b The protein levels of cyclinB1 (58 kDa) and CDK1 (34 kDa) were measured by Western blot analysis, and actin was used as a loading control. Figure is the representative result selected from three independent experiments, and the densitometry for relative protein expression was shown in right panel. *p < 0.05 and **p < 0.01, as compared to control. In these experiments, NNK (10 μM) treatment was used as the additional controls

Fig. 3

Effects of dual TXA2 modulators on cell apoptosis. a flow cytometric analysis of cells treated with BM567 (10 μM) for 24 h. The percentage of cells in early or late apoptosis is provided in the lower right and upper right quadrants, respectively. b Protein expression of PARP (full length: 116 kDa; cleavage: 89 kDa) and Survivin (17 kDa) was analyzed by Western blotting. Actin was used as loading control. Figure is the representative result selected from three independent experiments, and densitometry for blots was shown in lower panel. *p < 0.05 and **p < 0.01, as compared to control. NNK (10 μM) treatment was used as the additional control in these experiments

Fig. 4

Relationship between COXs and TXA2 synthesis. a Differential expression of COX-1 and COX-2 in normal lung cell lines and lung tumor cells was detected by RT-PCR. Actin was used as a loading control. b Biosynthesis of TXA2 was determined by measuring the level of TXB2 in the culture medium. The results were presented as the percentage of the control. Data are expressed as mean ± SD of three independent experiments done in triplicate. *p < 0.05 and **p < 0.01, as compared to control

Fig. 5

Relationship between the effects of COX-2 and TXA2. a The upper panel, the proliferation of NCI-H23 cells was determined by MTT assays. The results were presented as the percentages of the control. Data are expressed as mean ± SD of three independent experiments done in triplicate. *p < 0.05 and **p < 0.01. Lower panel, Survivin (17 kDa) was evaluated by Western blot detection. Actin was used as a loading control. Figure is the representative result selected from three independent experiments and densitometry was shown below the blots image. *p < 0.05 and **p < 0.01. NNK treatment was used as the additional control. b The inhibitory effect of siRNA on expression of COX-2 mRNA was examined by RT-PCR after 24 h transfection. c The protein level of Survivin was detected using Western blot analysis. Actin was used as a loading control. Figure is the representative result selected from three independent experiments, and densitometry was shown. *p < 0.05 and **p < 0.01. NNK treatment was used as the additional control. d cell proliferation was determined by MTS assays after treatment of 1 μM U46619 in NCI-H23 cells transfected with COX-2 siRNA. Data were presented as percentages of the control and expressed as mean ± SD of three independent experiments done in triplicate. *p < 0.05 and **p < 0.01

Fig. 6

Schematic model. Lung cancer is a TXA2 abundant organ (Ref. 22). TXA2 is closely correlated with COX-2 and functions as a key mediator for tumor-promoting effects of COX-2 in lung tumor, as elucidated by the present study