Cisplatin modulates B-cell translocation gene 2 to attenuate cell proliferation of prostate carcinoma cells in both p53-dependent and p53-independent pathways

Abstract

Cisplatin is a widely used anti-cancer drug. The B-cell translocation gene 2 (BTG2) is involved in the cell cycle transition regulation. We evaluated the cisplatin effects on prostate cancer cell proliferation and the expressions of BTG2, p53, androgen receptor (AR) and prostate specific antigen (PSA) in prostate carcinoma, p53 wild-type LNCaP or p53-null PC-3, cells. Cisplatin treatments attenuated cell prostate cancer cell growth through inducing Go/G1 cell cycle arrest in lower concentration and apoptosis at higher dosage. Cisplatin treatments enhanced p53 and BTG2 expression, repressed AR and PSA expression, and blocked the activation of androgen on the PSA secretion in LNCaP cells. BTG2 knockdown in LNCaP cells attenuated cisplatin-mediated growth inhibition. Cisplatin enhanced BTG2 gene expression dependent on the DNA fragment located within -173 to -82 upstream of BTG2 translation initiation site in prostate cancer cells. Mutation of the p53 response element from GGGCAGAGCCC to GGGCACC or mutation of the NFκB response element from GGAAAGTCC to GGAAAGGAA by site-directed mutagenesis abolished the stimulation of cisplatin on the BTG2 promoter activity in LNCaP or PC-3 cells, respectively. Our results indicated that cisplatin attenuates prostate cancer cell proliferation partly mediated by upregulation of BTG2 through the p53-dependent pathway or p53-independent NFκB pathway.

Figure 1. Cisplatin regulates cell proliferation and cell cycle progression in LNCaP cells.

(a) LNCaP cells were treated with various concentrations of cisplatin, as indicated, for 24 (black circle) and 48 (white circle) hours and the cell proliferation was determined by the H³-thymidine incorporation assay. (b) LNCaP cells were serum starved for 24 hours and then were treated with 0–80 μM of cisplatin as indicated for 24 hours. The cells were stained with PI, and the cell cycle distribution was analyzed by flow cytometry. Each box represents the mean ± SE (n = 6). (c) LNCaP cells were treated with indicated concentrations of cisplatin for 24 hours. Cells were lysed and expressions of PARP, cleaved PARP (c-PARP) were investigated with β-actin serving as an internal control.(Cropped gel) (*P < 0.05, **P < 0.01).

Figure 2. Cisplatin modulates p53 and BTG2 expression in LNCaP cells and BTG2 mediates partial cisplatin-induced growth inhibition in LNCaP cells.

(a) LNCaP cells were treated with various concentrations of cisplatin, as indicated, for 24 hours. Cells were lysed and expressions of BTG2 and p53 were determined by immunoblotting assay.(Cropped gel) (b). Data of quantitative immunoblot analysis are expressed as relative density (mean ± SE; n = 3). (c) LNCaP cells were treated with various concentrations of cisplatin, as indicated, for 24 hours. BTG2 mRNA levels were determined by RT-qPCR. (d) LNCaP cells were pretreated with or without MG132 for 2 hours and then treated with 80 μM cisplatin for 24 hours. Cells were lysed and expression of BTG2 was determined by immnuoblotting assay.(Cropped gel) (e) Luciferase activity of BTG2 reporter vectors-transfected LNCaP cells after treated with various concentrations of cisplatin for 24 hours. (f) LN-BTGsi cells (BTG2 knockdown LNCaP cells) and LN-COLsi cells (mock-knockdown LNCaP cells) were treated with indicated concentrations of cisplatin for 48 hours. Cell proliferation was measured by the CyQUANT cell proliferation assay. Each value is presented as the % in relation to the control (solvent-treated) group. Data are shown as the mean percentage ± SE (n = 6) (*P < 0.05, **P < 0.01).

Figure 3. Cisplatin regulates cell proliferation, cell cycle progression and BTG2 expression in PC-3 cells.

(a) PC-3 cells were treated with various concentrations of cisplatin for 24 (black circle) or 48 (white circle) hours. Cell proliferation was determined by the H³-thymidine incorporation assay. (b) PC-3 cells were serum starved for 24 hours and then were treated with 0–80 μM of cisplatin as indicated for 24 hours. The cells were stained with PI, and the cell cycle distribution was analyzed by flow cytometry. Each box represents the mean ± SE (n = 6). PC-3 cells were treated with various concentrations of cisplatin as indicated for 24 hours. Expressions of BTG2 were determined by immunoblotting assays (c) (Cropped gel) and RT-qPCR (d). (e) Luciferase activity of BTG2 promoter vectors-transfected PC-3 cells treated with various concentrations of cisplatin. Each value is the % in relation to the control (solvent-treated) group. Data are presented as the mean percentage ± SE (n = 6). (*P < 0.05, **P < 0.01).

Figure 4. Cisplatin modulates androgen receptor and prostate-specific antigen expression in LNCaP cells.

(a) LNCaP cells were treated with indicated concentrations cisplatin for 24 hours. Cells were lysed and expressions of androgen receptor (AR) ad prostate-specific antigen (PSA) were determined by immunoblotting assay. (Cropped gel) (b) Data of quantitative immunoblot analysis are expressed as relative density (mean ± SE; n = 3). (c) LNCaP cells were treated with 40 μM of cisplatin with or without R1881 (1 nM) for 24 hours. The conditional media were analyzed by ELISA for PSA concentrations. Each value is presented as % in relation to the control (solvent-treated) group. Data are shown as the mean percentage ± SE (n = 6). (*P < 0.05, **P < 0.01).

Figure 5. Cisplatin modulates BTG2 expression depending on DNA fragment 173 to −82 bp upstream of BTG2 gene p53-dependently or p53-independently in prostate carcinoma cells.

Luciferase activity of nested deletion or mutation constructs BTG2 reporter vectors-transfected LNCaP (a) or PC-3 (b) cells after treatment of 40 μM of cispalatin (black bars) or control-solvent (white bars). X represented the mutation of p53 response element. Each value is the % in relation to the control solvent-treated group. Data are presented as the mean percentage ± SE (n = 6). (**P < 0.01).

Figure 6. Cisplatin modulates BTG2 expression via the dwonregulation of NFκB activity in PC-3 cells.

(a) Luciferase activity of NFκB reporter vectors-transfected PC-3 cells treated with indicated concentrations of cisplatin. (b) Luciferase activity of PC-3 cells cotrasfected with BTG2 reporter vectors along with IκB or MAP3K14 expression vectors. (c) Luciferase activity of wild-type BTG2 reporter vectors (pGL3-BTG2) - or BTG2 reporter vector with mutated NFκB response element (pGL3-NFκBm) -transfected PC-3 cells cotransfected with pcDNA3 (white bars) or IκB expression vector (black bars). (d). Luciferase activity of wild-type BTG2 reporter vectors (pGL3-BTG2)- or BTG2 reporter vector with mutated NFκB response element (pGL3-NFκBm)-transfected PC-3 cells treated with (black bars) or without (white bars) 20 μM cisplatin. Each value is the % in relation to the control group. Data are presented as the mean percentage ± SE (n = 6). (*P < 0.05, **P < 0.01).