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Abstract
Purpose
Increasing evidences have indicated the critical role of HO-1 in cytoprotection and more diverse biological functions. HO-1 has been reported to stimulate tumor cell growth and proliferation. And several tumors, including renal cell carcinoma, prostate tumor, hepatoma, and sarcoma, express a high level of HO-1. Indeed, inhibition of HO-1 by using specific HO inhibitors demonstrated in vivo antitumor activity. However, the precise mechanism of HO-1 induction and signals in lung cancer is not clearly known yet. We aimed to analyze the role of HO-1, characterize the mechanism of HO-1 induction, the role of Nrf2 in the induction, and investigated whether inhibition of HO-1 may induce apoptosis in lung cancer cells.
Materials and Methods:
Western blot and immunostaining analyses were performed to ascertain whether HO-1, Nrf2, and NFkB were expressed or not in various lung cancer cell lines. Apoptosis by HO-1 inhibition through siRNA transfectiori was measured by flow cytometric analysis and Western blot. And the expression of HO-1 by siRNA of Nrf2 and NFkB was examined by ARE-driven luciferase activity and Western blot.
Results:
We demonstrated that HO-1 was expressed highly in A549 cells than other lung cancer cells. And A549 cells were transfected by HO-1 small interfering RNA (siRNA) induced apoptosis. Nrf2 siRNA, next, resulted in a decrease of HO-1 expression. However, NFkB siRNA had no influence on the expression of HO-1.
Conclusion:
Increasing HO-1 expression in A549 cells may be resulted from the transcriptional activation of Nrf2, and inhibition of Nrf2-HO-1 pathway induces apoptosis. Therefore it provides new important insights into the possible molecular mechanism of the antitumor therapy. (J Lung Cancer 2006;5(1):39-46)
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Fig. 1.
Expression of HO-1 in endogenous lung cancer cells. (A) Expression levels of HO-1 protein were measured by Western blotting using ant卜HO-1 antibody. Lysates of untreated lung cancer cells were subjected to 12.5% SDS-PAGE to measure the expression of HO-1 protein. (B) Untreated lung cancer cells were reacted with Hoechst and ariti-HO-1 antibody. Then, cells were visualized under fluorescent microscope.
Fig. 2.
Apoptosis in A549 celts after HO-1 siRNA transfection. Nonsense indicates the result for cells treated with Silencer Negative Control siRNA. Cells were transfected with 0.5 ᆻg siRNA of HO-1 and control plasmid. (A) Cellular DNA was stained with PI and celt cycle was analyzed by flow cytometry. The data represent the mean 士 S.D. of triplicate. ∗p<0.05. (B) Cells were transfected with 0.5 ᆻg siRNA of HO-1 and control plasmid. After 48 hrs, lysate was subjected to 12.5% SDS-PAGE to measure the expression of pro- caspase-3.
Fig. 3.
Expression of Nrf2 and NFkB in endogenous lung cancer cells. (A) Expression levels in both cytosolic and nuclear fractions of Nrf2 and NFkB protein were measured by Western blotting. Lysate was fractionated into cytosolic and nuclear portions and proteins were separated on 15% SDS-PAGE to immunoblot for Nrf2 and NFkB in nucleus and IkB in cytoplasm. (B) Untreated A549 cells were reacted with Hoechst and ant卜HO-1 antl· body. Then, cells were visualized under fluorescent microscope.
Fig. 4.
Expression of Nrf2 and NFkB siRNA transfection on HO-1 promotor activity. In A549 cells, Nrf2 and NFkB expression vectors were respectively cotransfected with ARE—luciferase reporter gene for 24 hrs, and then luciferase activity was measured at 18 h later. The data represent the mean 土 S.D. of triplicate. ∗p<0.05.
Fig. 5.
Expression of Nrf2 and NFkB siRNA transfectiori on HO-1 protein expression. Nonsense indicates the result for cells treated with Silencer Negative Control siRNA. (A) Cells were transfected with 0,5 μ g siRNA of Nrf2 and control plasmid. Suppression of Nrf2 levels after siRNA treatment after 48 hrs was respectively confirmed by Western blot. Simultaneously lysates were subjected to 12.5% SOS-PAGE to measure the expression of HO-1 protein, (B) Cells were transfected with 0.5μ g siRNA of NFkB and control plasmid Suppression of NFkB levels after siRNA treatment after 48 hrs was respectively confirmed by Western blot Simultaneously lysates were subjected to 12.5% SDS-PAGE to measure the expression of HO-1 protein.
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