Tuberc Respir Dis.  2007 Jan;62(1):33-42. 10.4046/trd.2007.62.1.33.

The Effect of Inhibition of Heme Oxygenase-1 on Chemosensitivity of Cisplatin in Lung Cancer Cells

• Kim SY
• Kim EJ
• Jang HY
• Hwang KE
• Park JH
• Kim HJ
• Jo HJ
• Yang SH
• Jeong ET
• Kim HR

• KMID: 1518487
• DOI: http://doi.org/10.4046/trd.2007.62.1.33

Abstract

BACKGROUND: Heme oxygenase-1 (HO-1) is known to modulates the cellular functions, including cell proliferation and apoptosis. It is known that a high level of HO-1 expression is found in many tumors, and HO-1 plays an important role in rapid tumor growth on account of its antioxidant and antiapoptotic effects. Cisplatin is a widely used anti-cancer agent for the treatment of lung cancer. However, the development of resistance to cisplatin is a major obstacle to its use in clinical treatment. We previously demonstrated that inhibiting HO-1 expression through the transcriptional activation of Nrf2 induces apoptosis in A549 cells. The aim of this study was to determine of the inhibiting HO-1 enhance the chemosensitivity of A549 cells to cisplatin.
MATERIALS AND METHODS
The human lung cancer cell line, A549, was treated cisplatin, and the cell viability was measured by a MTT assay. The change in HO-1, Nrf2, and MAPK expression after the cisplatin treatment was examined by Western blotting. HO-1 inhibition was suppressed by ZnPP, which is a specific pharmacologic inhibitor of HO activity, and small interfering RNA (siRNA). Flow cytometry analysis and Western blot were performed in to determine the level of apoptosis. The level of hydrogen peroxide (H2O2) generation was monitored fluoimetrically using 2',7'-dichlorofluorescein diacetate.
RESULTS
The A549 cells showed more resistance to the cisplatin treatment than the other cell lines examined, whereas cisplatin increased the expression of HO-1 and Nrf2, as well as the phosphorylation of MAPK in a time-dependent fashion. Inhibitors of the MAPK pathway blocked the induction of HO-1 and Nrf2 by the cisplatin treatment in A549 cells. In addition, the cisplatin-treated A549 cells transfected with dither the HO-1 small interfering RNA (siRNA) or ZnPP, specific HO-1 inhibitor, showed in a more significantly decrease in viability than the cisplatin-only-treated group. The combination treatment of ZnPP and cisplatin caused in a marked increase in the ROS generation and a decrease in the HO-1 expression.
CONCLUSION
Cisplatin increases the expression of HO-1, probably through the MAPK-Nrf2 pathway, and the inhibition of HO-1 enhances the chemosensitivity of A549 cells to cisplatin.

Keyword

Heme oxygenase-1; Cisplatin; Lung Cancer

MeSH Terms

Apoptosis
Blotting, Western
Cell Line
Cell Proliferation
Cell Survival
Cisplatin*
Flow Cytometry
Heme Oxygenase-1*
Heme*
Humans
Hydrogen Peroxide
Lung Neoplasms*
Lung*
Phosphorylation
RNA, Small Interfering
Transcriptional Activation
Cisplatin
Heme
Heme Oxygenase-1
Hydrogen Peroxide
RNA, Small Interfering

Figure

• 

  Figure 1 Sensitivity to cisplatin in A549 cells. It was revealed that A549 cells had more significantly resistance to cisplatin treatment than other cell lines in a dose-dependent manner, especially 10 µM concentration. The data represent the mean±S.D. of triplicate. *, p<0.05.

• 

  Figure 2 Expression of HO-1, Nrf2, and MAPK after treatment with cisplatin. (A) Cisplatin resulted in a marked increase in the expression of HO-1 proteins in a time-dependent fashion with the same kinetics. It attained its peak at 12 h, and sustained in cells treated with cisplatin. Western blot was also performed to ascertain whether Nrf2 was expressed or not in A549 cells. Cisplatin resulted in a increase in the expression of Nrf2 proteins in nucleic fraction in a time-dependent fashion. (B) Activation of MAPK was evaluated by Western analysis using phospho-specific antibodies to ERK1/ERK2, p38, JNK. Cisplatin resulted in a increase in the expression of phosphorylated MAPKs proteins in a time-dependent fashion.

• 

  Figure 3 Effects of MEK1 inhibitor PD098059, p38 MAPK inhibitor SB203580, and JNK inhibitor SP600125 on cisplatin-induced HO-1 and Nrf2 expression. (A) Cisplatin resulted in an over twofold increase in HO-1 expression compared with the control, and MAPKs inhibitors decreased cisplatin-induced HO-1 expression from 50% to about 75%. (B) Under the same experimental conditions, cisplatin resulted in an 1.8 folds increase in HO-1 expression compared with the control, and MAPKs inhibitors decreased cisplatin-induced HO-1 expression of about 50%.

• 

  Figure 4 Sensitivity to ZnPP, a specific pharmacologic inhibitor of HO activity, in cisplatin-induced cytotoxicity. In contrast to ZnPP or various concentrations of cisplatin alone, combination of 10 µM ZnPP with varying doses of cisplatin from 5 to 20 µM resulted in a significant decrease of the viability of A549 cells in a dose-dependent fasion. The data represent the mean±S.D. of triplicate. *, p<0.05.

• 

  Figure 5 Sensitivity to HO-1 siRNA transfection in cisplatin-induced cytotoxicity. In contrast to cisplatin alone or combination with nonsense and cisplatin, combination with HO-1 siRNA and cisplatin resulted in a significant decrease in cell viability (62% of control cells). The data represent the mean±S.D. of triplicate. *, p<0.05.

• 

  Figure 6 Contribution of ROS generation and HO-1 expression to anticancer activity of cisplatin combined with ZnPP in A549 cells. (a) Combination treatment with ZnPP and cisplatin of blue line resulted in a significant increase in the generation of ROS. (b) The expression of HO-1 in endogenous state was marked increased in cells treated with cisplatin only, whereas combination treatment with ZnPP and cisplatin resulted in a decrease of about 50% of the expression of HO-1.

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