Transient Receptor Potential Ankyrin 1 (TRPA1) Channel Mediates Acrolein Cytotoxicity in Human Lung Cancer Cells

Abstract

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective ion channel implicated in thermosensation and inflammatory pain. It has been reported that expression of the TRPA1 channel is induced by cigarette smoke extract. Acrolein found in cigarette smoke is highly toxic and known as an agonist of the TRPA1 channel. However, the role of TRPA1 in the cytotoxicity of acrolein remains unclear. Here, we investigated whether the TRPA1 channel is involved in the cytotoxicity of acrolein in human lung cancer A549 cells. The IC₅₀ of acrolein in A549 cells was 25 μM, and acrolein toxicity increased in a concentration- and time-dependent manner. When the effect of acrolein on TRPA1 expression was examined, the expression of TRPA1 in A549 cells was increased by treatment with 50 μM acrolein for 24 h or 500 μM acrolein for 30 min. AP-1, a transcription factor, was activated in the cells treated with 50 μM acrolein for 24 h, while induction of NF-κB and HIF-1α was observed in the cells treated with 500 μM acrolein for 30 min. These results suggest that acrolein induces TRPA1 expression by activating these transcription factors. Overexpression of TRPA1 in A549 cells increased acrolein sensitivity and the level of protein-conjugated acrolein (PC-Acro), while knockdown of TRPA1 in A549 cells or treatment with a TRPA1 antagonist caused tolerance to acrolein. These findings suggest that acrolein induces the TRPA1 channel and that an increase in TRPA1 expression promotes the cytotoxicity of acrolein.

Keywords: acrolein; cell damage; cytotoxicity; transient receptor potential ankyrin 1 (TRPA1).

Figure 1

Effect of acrolein on viability of A549 cells. (A,B) A549 cells were treated with various concentrations of acrolein (ACR) for 72 h (A) or different times (B). The living cells were then detected using a CCK-8 kit. The cell viability of the control group was set at 100%.

Figure 2

Levels of protein and mRNA of TRPA1. (A) TRPA1 in membrane fraction in A549 cells was measured using western blot analysis after incubation of the cells with various concentrations of acrolein (ACR) for 24 h or 30 min. (B) The mRNA level of TRPA1 in A549 cells cultured with or without acrolein was determined using qRT-PCR, as described in Materials and Methods. ** p < 0.01.

Figure 3

Levels of transcription factors in A549 cells. (A) Locations of active sites of transcription factors [AP-1 (c-Jun and c-Fos; green), NF-κB (blue), and HIF-1α (red)] in TRPA1 gene. (B) Levels of the nuclear transcription factors in A549 cells were measured using western blot analysis after incubation of the cells with 50 μM acrolein (ACR) for 24 h or 500 μM acrolein for 30 min. Nuclear histone H3 in A549 cells was used as a control.

Figure 4

Effect of TRPA1 on acrolein toxicity. (A) A549 cells were first preincubated with the TRPA1 antagonist HC-030031 (HC, 100 μM) or vehicle (dimethyl sulfoxide, DMSO) for 30 min. The cells were then exposed to 500 μM acrolein (ACR) for 30 min. Cell viability was measured as described in Materials and Methods. (B) Plasmids for knockdown or overexpression of TRPA1 were transfected into A549 cells. Protein levels were analyzed using western blot analysis. (C) The cells were treated with 500 μM acrolein for 30 min. Cell viability was measured as described in Materials and Methods. ** p < 0.01; *** p < 0.001.

Figure 5

Effect of TRPA1 on the level of PC-Acro in A549 cells. (A,B) Plasmids for control vector or overexpression of TRPA1 were transfected into A549 cells. These cells were treated with 500 μM acrolein for 30 min. CBB (Coomassie Brilliant Blue) staining and measurement of PC-Acro and polyamines were performed as described in Materials and Methods. The degree of protein polymerization through crosslinking by acrolein (ACR) (shown by square brackets) is indicated as relative amount (%). PUT, putrescine; SPD, spermidine; SPM, spermine.