Tuberc Respir Dis.  2006 Mar;60(3):304-313.

The Role of Heme Oxygenase-1 in Lung Cancer Cells

• Jung JH
• Kim HR
• Kim EJ
• Hwang KE
• Kim SY
• Park JH
• Kim HJ
• Yang SH
• Jeong ET

Abstract

BACKGROUND: Heme oxygenase-1 (HO-1) is an inducible enzyme that catalyzes the oxidative degradation of heme to form biliverdin, carbon monoxide (CO), and free iron. The current evidence has indicated a critical role of HO-1 in cytoprotection and also in other, more diverse biological functions. It is known that the high expression of HO-1 occurs in various tumors, and that HO-1 has an important role in rapid tumor growth because of its antioxidative and antiapoptotic effects. Therefore, the role of HO-1 was analyzed in human lung cancer cell lines, and especially in the A549 cell line. MATERIAL AND METHODS: Human lung cancer cell lines, i.e., A549, NCI-H23, NCI-H157 and NCI-H460, were used for this study. The expression of HO-1 in the untreated state was defined by Western blotting. ZnPP, which is the specific HO inhibitor we used, and the viability of cells were tested for by conducting MTT assaysy. The HO enzymatic activity, as determined via the bilirubin level, was also indirectly measured. Moreover, the generation of intracellular hydrogen peroxide (H2O2) was monitored fluorimetrically with using a scopoletin-horse radish peroxidase (HRP) assay and 2',7'-dichlorofluorescein diacetate (DCFH-DA). We have also transfected small HO-1 interfering RNA (siRNA) into A549 cells, and the apoptotic effects were evaluated by flow cytometric analysis and Western blotting.
RESULTS
The A549 cells had a greater expression of HO-1 than the other cell lines, whereas ZnPP significantly decreased the viability of the A549 cells more than the viability of the other lung cancer cells in a dose-dependant fashion. Consistent with the viability, the HO enzymatic activity also was decreased. Moreover, intracellular H2O2 generation via ZnPP was induced in a dose-dependent manner. Apoptotic events were, then induced in the HO-1 siRNA transfected A549 cells.
CONCLUSION
HO-1 provides new important insights into the possible molecular mechanism of the antitumor therapy in lung cancer.

Keyword

Heme oxygenase-1; Lung cancer

MeSH Terms

Bilirubin
Biliverdine
Blotting, Western
Carbon Monoxide
Cell Line
Cytoprotection
Heme Oxygenase-1*
Heme*
Humans
Hydrogen Peroxide
Iron
Lung Neoplasms*
Lung*
Peroxidase
Raphanus
RNA
RNA, Small Interfering
Bilirubin
Biliverdine
Carbon Monoxide
Heme
Heme Oxygenase-1
Hydrogen Peroxide
Iron
Peroxidase
RNA
RNA, Small Interfering

Figure

• 

  Figure 1 (A) Under endogenous state, HO-1 was expressed highly in A549 cells than other lung cancer cells. Cell lysates of endogenous state were subjected to 12.5% SDS-PAGE to measure the expression of HO-1 protein. (B) ZnPP significantly decreased the viability of A549 cells than other lung cancer cells in a dose-dependent fashion. Cells were treated with different concentrations of ZnPP for 48 hrs, and viability was determined by MTT assay. The data represent the mean±S.D. of three independent experiments. *,p<0.05 compared to control.

• 

  Figure 2 ZnPP significantly decreased the enzymatic activity of HO-1 in A549 cells. Cells were treated wit h different concentrations of ZnPP for 48 hrs. Microsomal extracts were prepared and assayed for HO activity by measurement of bilirubin generation as described in Materials and Methods. HO activity was expressed as nmol·mg protein-1·h-1. The data represent the mean±S.D. of three independent experiments.

• 

  Figure 3 ZnPP induced the generation of intracellular H2O2 in A549 cells. (A) Cells were treated with different concentrations of ZnPP for 48 hrs. H2O2 generation was determined by monitoring the decrease in fluorescence of scopoletin (5 µM) during its oxidation catalyzed by HRP (1 U/ml). Fluorescence value of scopoletin is representative counts and relative fluorescence values are mean±S.D. of three independent experiments. *,p<0.05 compared to control. (B) Cells were treated with different concentrations of ZnPP for 48 hrs, and were harvested, washed with media, and then stained with oxidant-sensitive fluorescence dye DCFDA (50 µM) for 30 mins in the dark. After washing unbound dye out, the generation of H2O2 were visualized under fluorescent microscope.

• 

  Figure 4 A549 cells were transfected by HO-1 siRNA induced apoptosis. (A) Cells were transfected with siRNA for HO-1 mRNA. The nonsense HO-1 siRNA was used as a negative control. RT-PCR for the HO-1 mRNA shows expression of the cells sense or nonsense siRNA transfection. Total RNA of transfected cells was isolated and HO-1 gene was amplified by RT-PCR detailed in the materials and methods. (B) HO-1 siRNA increased sub-G0/G1 fraction in A549 cells. Cellular DNA were stained with PI staining solution and analyzed by flow cytometry. (C) HO-1 siRNA induced the activation of caspase-3. Cell lysate was subjected to 12.5% SDS-PAGE to measure the expression of pro-caspase- 3.

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