Indomethacin-enhanced anticancer effect of arsenic trioxide in A549 cell line: involvement of apoptosis and phospho-ERK and p38 MAPK pathways

Abstract

Background: Focusing on novel drug combinations that target different pathways especially apoptosis and MAPK could be a rationale for combination therapy in successful treatment of lung cancer. Concurrent use of cyclooxygenase (COX) inhibitors with arsenic trioxide (ATO) might be a possible treatment option.

Methods: Cytotoxicity of ATO, dexamethasone (Dex), celecoxib (Cel), and Indomethacin (Indo) individually or in combination was determined at 24, 48, and 72 hrs in A549 lung cancer cells. The COX-2 gene and protein expression, MAPK pathway proteins, and caspase-3 activity were studied for the most cytotoxic combinations.

Results: The IC50s of ATO and Indo were 68.7 μmol/L and 396.5 μmol/L, respectively. Treatment of cells with combinations of clinically relevant concentrations of ATO and Indo resulted in greater growth inhibition and apoptosis induction than did either agent alone. Caspase-3 activity was considerably high in the presence of ATO and Indo but showed no difference in single or combination use. Phosphorylation of p38 and ERK1/2 was remarkable in the concurrent presence of both drugs.

Conclusions: Combination therapy with ATO and Indo exerted a very potent in vitro cytotoxic effect against A549 lung cancer cells. Activation of ERK and p38 pathways might be the mechanism of higher cytotoxic effect of ATO-Indo combination.

Figure 1

Effect of different incubation time on the cytotoxicity of ATO on the A549 cell line (Mean ± SE, n = 4; **P < 0.01 and ***P < 0.001; ^¶¶¶ P < 0.001 compared to ATO 2 μM).

Figure 2

Effect of different concentrations of ATO (a), Cel (b), Indo (c), and Dex (d) on the growth of A549 cell line (Mean ± SE, n = 4).

Figure 3

Inhibitory effects of single (a–c) and combination (d–f) of ATO and Indo, Cel and Dex on A549 lung cancer cell proliferation (Mean ± SE, n = 4; *P < 0.05, **P < 0.01 and ***P < 0.001).

Figure 4

The effects of ATO and Indo and their combinations on the expression of COX-2 mRNA. (a) RT-PCR reaction products were resolved on 1% agarose gel and stained with Ethidium bromide. (b) Densitometric analyses of COX-2 mRNA expression is presented as the band's density to control (β-actin) of three independent experiments (Mean ± SE, n = 3). (c) The effect of Indo alone (light columns) and combination with ATO 2 μM (dark columns) on COX-2 expression.

Figure 5

Western blot analysis of COX-2, Akt, ERK1/2, p38, JNK, and Bax proteins in A549 cells treated with ATO, Indo, Dex, ATO + Indo, and ATO + Dex combinations.

Figure 6

Phosphorylation of p38 and ERK in A549 cells treated with ATO, Indo, and ATO/Indo combination.

Figure 7

Activation of caspase-3 in A549 cells treated with ATO, Indo, and ATO/Indo combination.