Figure 1.
Celastrol inhibits PC-3 cell growth through cell cycle arrest at G0/G1 and apoptosis induction.
(A) PC-3 cells were treated with indicated concentrations of celastrol for 48 hours and the cell proliferation was determined by the H3-thymidine incorporation. (B) PC-3 cells were treated with indicated concentrations of celastrol for48 hours. Cells were lysed and expressions of PARP, cleaved PARP (c-PARP) were determined by immunoblotting assay. (C) After one day of treatment, the apoptotic index of PC-3 cells treated with different concentrations of celastrol was calculated. Each value is a mean ± SE of 3 determinations. (D) PC-3 cells were serum starved for 24 hours and then were treated with 0 – 1 μM of celastrol as indicated for 48 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). (* p<0.05; **p<0.01).
Figure 2.
Knockdown of interleukin-6 attenuates the growth-inhibitorty effect ofcelastrol onPC-3 cells.
PC-COLsi cells (open circle) and PC-IL6si cells (close circle) were treated with various concentrations of celastrol, as indicated, for 48 hours. The cell proliferation was determined by the 3H-thymidine incorporation. Data are presented as mean percentage ± SE (n = 6). (* p<0.05; **p<0.01).
Figure 3.
Celastrol Modulates interleukin-6 secretion in PC-3 cells.
(A) PC-3 (black bars) and DU145 (white bars) cells were treated with various concentrations of celastrol, as indicated, for 24 hours. (B) PC-3 cells were treated with 1 μM celastrol, 40 nM PMA, and/or 10 ng/ml TNFα for 24 hours. IL-6 levels in the conditioned media were determined by ELISA. Data are expression as mean percentage stimulation ± SE of 6 preparation induced by different treatments relative to the control solvent treatment. (*p<0.05; **p<0.01).
Figure 4.
Celastrol downregulates interleukin-6 and NF-κB reporter activity in PC-3 cells.
(A) Luciferase activity of IL-6 reporter vector (pIL6-SX)-transfected PC-3 (black bars) and DU145 (white bars) cells treated with different concentrations of celastrol as indicated. (B) Luciferase activity of NFκB reporter vectors (black bars)- and MMTV reporter vector (white bars)-transfected PC-3 cells treated with different concentrations of celastrol as indicated. Data are presented as the mean percentage ± SE (n = 6) of the reporter activities induced by celastrol treatments in relation to the control solvent-treated group. (*p<0.05; **p<0.01).
Figure 5.
Celastrol blocks the activation of TNFα and PMA on interleukin-6 and NF-κB promoter activity.
Luciferase activity of IL-6 reporter vector- (A) and NF-κB reporter vectors- (B) transfected PC-3 cells treated with 1 μM celastrol (Cel), 40 nM PMA, or/and 10 ng/ml TNFα. Data are presented as the mean percentage ± SE (n = 6) of the reporter activities induce by different treatments in relation to the control solvent-treated group. (*p<0.05; **p<0.01).
Figure 6.
Celastrol represses PC-3 interleukin-6 expression through NF-κB signal pathway.
PC-3 cells were treated with various concentrations of celastrol as indicated for 24 hours. The expressions of IKKα, NIK, IκB, and β-actin were determined in the cytoplasmic fraction (A), and the NFκBp50, NFκBp65, and Lamin B were determined in nuclear fraction (B) by immunoblotting assay (top). The quantitative analysis was done by determining the intensity of each band from three independent experiments (bottom). Data are presented as the fold-induction (± SE; n = 3) of the relative density of the target gene/β-actin (for IKKα, IκB, and NIK) and target gene/Lamin B (for NFκBp50 and NFκBp65) of treatments in relation to the control solvent-treated group (*p<0.05, **p<0.01). (C) Luciferase activity of nested deletion or mutation constructs of IL-6 reporter vectors-transfected PC-3 cells after treatment with control solvent (white bars) or 1 μM of celastrol (black bars). Data are presented as the mean percentage ± SE (n = 6) of the IL-6 reporter activity induced by celastrol treatment in relation to the control solvent-treated group. (“X” represents the mutant NF-κB response element; **P<0.01).