Cigarette smoke induces nuclear translocation of heme oxygenase 1 (HO-1) in prostate cancer cells: nuclear HO-1 promotes vascular endothelial growth factor secretion

Abstract

Prostate cancer is the second leading cause of male-cancer related death in the United States. Despite a number of evidence-based studies which strongly suggest an association between cigarette smoking and prostate cancer, the underlying biological mechanism is largely unknown. Heme oxygenase 1 (HO-1) has been implicated in maintaining cellular homeostasis, but also in tumor angiogenesis. Nuclear HO-1 protein expression has been observed in various types of tumors including prostate cancer. These studies, however, were reported as clinical and pathological observations, and failed to investigate nuclear HO-1 at the molecular level in cancer. The present study explores the relationship between cigarette smoke and nuclear HO-1-modulated promotion of vascular endothelial growth factor (VEGF) secretion. We have demonstrated that cigarette smoke medium (SM)-induced HO-1 mRNA expression and upregulated HO-1 protein levels in the prostate cancer cell lines DU145 and PC3. We also observed that SM significantly induced nuclear expression of HO-1, and enhanced secretion of VEGF in cells. Nuclear-directed expression of HO-1 activated the transcriptional activity of VEGF and promoted VEGF secretion in prostate cancer cells. This study provides new insights into the molecular mechanism by which cigarette smoke-induced nuclear translocation of HO-1 promotes VEGF secretion in prostate cancer cells. Nuclear HO-1 may, therefore, constitute an attractive therapeutic target to inhibit angiogenesis and the progression of prostate cancer.

Figure 1

Effect of cigarette smoke on growth of prostate cancer cells. PC3 cells were plated onto 96-well plates and starved with culture medium containing 0.5% FBS. After 24 h, cell cultures were treated with serial dilutions of SM (2, 1 and 0.5%) as indicated. Concentrations of total particulate matter (TPM), tar, nicotine (Nico) and carbon monoxide (CO) were estimated based on information provided by the reference cigarette program. A CellTiter Non-Radioactive Cell Proliferation Assay was performed after 24 h, to determine effect of SM on cell growth. Growth rate was expressed as absorbance of the formazan product at 490 nm. Columns, mean; bars, SD; ^*p<0.05; ^**p<0.01.

Figure 2

Cigarette smoke induced expression of HO-1 in prostate cancer cells. (A and B) SM induced mRNA levels of HO-1 in prostate cancer cells. DU145 and PC3 were treated with SM for 0, 3 and 6 h. Total RNA was isolated and analyzed by semi-quantitative RT-PCR using primers for HO-1 and GAPDH (internal control). Relative HO-1 mRNA levels derived from three individual experiments were expressed in arbitrary units. Columns, mean; bars, SD; ^*p<0.05; ^**p<0.01. (C and D) SM increased HO-1 protein levels in prostate cancer cells. (C) DU145 and (D) PC3 cells were grown on 6-well plates and treated with SM for 0 and 24 h. Cell extracts were subjected to western blot analysis using anti-HO-1 and anti-GAPDH antibodies. GAPDH served as an internal control. Relative HO-1 protein levels derived from three individual experiments were expressed in arbitrary units. Columns, mean; bars, SD; ^*p<0.05; ^**p<0.01.

Figure 3

Cigarette smoke induced VEGF secretion in prostate cancer cells. (A) DU145 and (B) PC3 cells were grown in complete culture medium. Cells were refreshed with culture medium containing 0.5% FBS for 24 h, and then treated with SM. Culture supernatants were collected and cells were counted after 24 h. VEGF concentration was determined by ELISA and expressed as pg/10⁴ cells. Micrographs are representative of three individual experiments. Columns, mean; bars, SD; ^*p<0.05; ^**p<0.01.

Figure 4

Cigarette smoke induced nuclear translocation of HO-1 in prostate cancer cells. (A) DU145 and (B) PC3 cells were grown on 6-well-plates and treated with SM. After 24 h, cellular fractionation was performed, and the cytoplasmic and nuclear fractions were analyzed by western blotting using an anti-HO-1 antibody. The blots were re-probed with anti-GAPDH or anti-Lamin B1 antibodies. (C) DU145 and (D) PC3 cells were treated with SM. Nuclear extracts were probed with anti-HO1 and Lamin B1 antibodies. Nuclear expression of HO-1 was normalized to that of the nuclear marker Lamin B1, and relative expression of nuclear HO-1 was expressed in arbitrary units. Data shown in micrographs were derived from three individual experiments. Columns, mean; bars, SD; ^**p<0.01. CTL, control; CE, cytoplasmic extract; NE, nuclear extract.

Figure 5

Nuclear-directed expression of HO-1 in HEK293 cells. HO-1 and HO-1/NLS were constructed into pEGFP-N3 vector. Resultant constructs, (A) pEGFP-HO-1 and (B) pEGFP-HO-1/NLS, were transfected in HEK293 cells. After 24 h, cells were fixed and stained with F-actin phalloidin and DAPI. Images were taken by using Zeiss LSM 510 Meta confocal microscope. Bar, 50 μm.

Figure 6

Nuclear localization of HO-1 promoted transcriptional activity of VEGF. (A) Differential activation of VEGF transcriptional activity by cytoplasmic and nuclear HO-1. HEK293 cells were co-transfected with the VEGF promoter and HO-1 or HO-1/NLS in a dose-dependent fashion, as indicated. After 24 h, VEGF promoter activity (luciferase activity) was measured and normalized to β-Gal activity. Relative VEGF promoter activity derived from three experiments was expressed in arbitrary units. Cell extracts were also blotted with anti-HO-1 and anti-GAPDH antibodies. Columns, mean; bars, SD; ^*p<0.05; ^**p<0.01. pVEGF, VEGF promoter; HO-1, plasmid expressing HO-1 in cytosol; HO-1/NLS, plasmid expressing nuclear HO-1; NLS, nuclear localization signal. (B) Differential activation of VEGF transcriptional activity by heat shock proteins. HEK293 and COS7 cells were co-transfected with the VEGF promoter and pNuc-HO-1/NLS or EGFP-HSP72 in a dose-dependent manner as indicated. After 24 h, luciferase activity was measured and normalized to β-galactosidase (β-Gal) activity to quantify VEGF promoter activity. Relative VEGF promoter activity was expressed in arbitrary units. Micrograph is representative of three independent experiments. Cell extracts were also blotted with anti-HO-1, anti-GFP and anti-GAPDH antibodies. GAPDH served as internal control. Columns, mean; bars, SD; ^*p<0.05; ^**p<0.01. HO-1, also known as heat shock protein 32; and HSP72, heat shock protein 72; NLS, nuclear localization signal. Upper panel: HEK293; lower panel: COS7.

Figure 7

Ectopic expression of nuclear HO-1 promoted VEGF secretion in prostate cancer cells. (A) PC3 cells were transfected with mock, HO-1 or HO-1/NLS. After 24 h, cells were starved with cell culture medium containing 0.5% FBS. After 24 h, supernatants were collected, and VEGF secretions were measured by using ELISA. Columns, mean; bars, SD; ^*p<0.05; ^**p<0.01. (B) Cell culture, in (A), then were replenished with cell culture medium containing 0.5% FBS plus SM. After 24 h, supernatants were collected and VEGF secretions were measured by using ELISA. Columns, mean; bars, SD; ^*p<0.05; ^**p<0.01.