ALDH enzyme activity is regulated by Nodal and histamine in the A549 cell line

Abstract

The present study aimed to examine whether the enzyme activity of aldehyde dehydrogenase (ALDH) was regulated by Nodal and histamine in the human alveolar adenocarcinoma A549 cell line. The regulated enzyme activity of ALDH was analyzed by flow cytometry in the A549 cell line. ALDH1 and Nodal expression was investigated by immunohistochemistry in28 cases of lung mixed adenocarcinoma. The enzyme activity of ALDH was upregulated by histamine and agonists of histamine H1 receptor (H1R) and histamine H2 receptor (H2R). ALDH activity was also downregulated by recombinant human Nodal and antagonists of H1R and H2R in the A549 cell line. In addition, expression of Nodal and ALDH1 were inversely correlated in lung mixed adenocarcinoma. ALDH enzyme activity was regulated by Nodal and histamine in lung adenocarcinoma.

Keywords: A549; aldehyde dehydrogenase; histamine; lung mixed adenocarcinoma; nodal.

Figure 1.

Expression of ALDH1 and Nodal in lung mixed adenocarcinoma. (A) Representative immunohistochemical staining of ALDH1 and Nodal in two representative lung mixed adenocarcinoma tissues. Magnification, ×100. (B) Assessment of the association between ALDH1 and Nodal expression (P<0.05; r=−0.253) indicated that the expression of ALDH1 and Nodal is inversely correlated. ALDH1, aldehyde dehydrogenase 1.

Figure 2.

Various concentrations of rhNodal downregulated the activity of ALDH in A549 cells, as determined by flow cytometry. (A) Representative flow cytometry results. The activity of ALDH was inhibited by DEAB; thus, A549 cells were used as a control for background fluorescence subsequent to receiving pretreatment with DEAB (DEAB⁺). (B) Quantitative analysis of the percentage of ALDH-hi cells after treatment with various concentrations of rhNodal. (C) Quantitative analysis of the mRNA level of ALDH1A1 in A549 cells after receiving histamine treatment for various durations, as determined by reverse transcription-quantitative polymerase chain reaction. *P<0.05, **P<0.01 vs. control; DEAB, diethylaminobenzaldehyde; ALDH-hi cells, ALDH-positive cells.

Figure 3.

Various concentrations and treatment times of histamine upregulated the activity of ALDH in A549 cells, as determined by flow cytometry. (A) Representative flow cytometry results of A549 cells treated with 10, 100 or 500 µg/ml histamine for 2 h. (B) Quantitative analysis of flow cytometry results of A549 cells treated with 10, 100 or 500 µg/ml histamine for 2 h.*P<0.05, **P<0.01 vs. control. (C) Representative flow cytometry results of A549 cells treated for 2,4 or 18 h with 10 µg/ml histamine. (D) Quantitative analysis of A549 cells treated with 10 µg/ml histamine for 2,4 or 18 h. *P<0.05 vs. control. ALDH-hi cells, ALDH-positive cells.

Figure 4.

Expression of human histamine receptors in A549 cells. (A) Reverse transcription-polymerase chain reaction analysis of histamine receptor mRNA expression. Lane H1RA, H1R in A549 cells (402 bp); lane H2RA, H2R in A549 cells (495 bp); lane H3RA, H3R in A549 cells; lane H4R, H4R in A549 cells. (B) Ratio of mRNA expression of H1R, H2R, H3R and H4R to the expression of GAPDH. H1R, H1 receptor; H2R, H2 receptor; H3R, H3 receptor; H4R, H4 receptor.

Figure 5.

Effect of agonists and antagonists of H1R and H2R on A549 cells for the activity of ALDH. (A) Representative flow cytometry results of A549 cells treated with the agonists of H1R and H2R. Histamine (10 µg/ml) was used as a quantity control. (B) Quantitative analysis of flow cytometry results of A549 cells treated with histamine and agonists of H1R and H2R. *P<0.05 vs. control. (C) Representative flow cytometry results of A549 cells treated with the antagonists of H1R and H2R. (D) Quantitative analysis of flow cytometry results of A549 cells treated with histamine and agonists of H1R and H2R. *P<0.05 vs. control. DEAB, diethylaminobenzaldehyde; ALDH-hi cells, ALDH-positive cells; H1R, H1 receptor; H2R, H2 receptor; HTM, trifluoromethyltoluididedimaleate; DIM, dimapritdihydrochloride; CIM, cimetidine; PYR, pyrilamine maleate salt.