Association on of 1α,25-(OH)₂D₃–mediated radiosensitivity of PC-3 cells and RelB suppression. A, luciferase reporter constructs shown were cotransfected into PC-3 cells with a β-galactosidase expression construct. Positions and orientations of four NF-κB elements are indicated by arrows . The transfected cells were treated with 1α,25-(OH)₂D₃ and IR as described in Fig. 1. After 24 h, activities of luciferase and β-galactosidase were measured. Transcription regulated by the NF-κB was estimated by β-galactosidase–normalized luciferase activity. *, significant difference compared with both untreated group and 1α,25-(OH)₂D₃–pretreated groups. B, mRNA and total proteins were extracted from the treated an and untreated treated PC-3 cells. Effect of IR alone or 1α,25-(OH)₂D₃ with IR on RelA or RelB expression was determine determined by RT-PCR (top) and by Western blots (bottom). β-actin serves as a control to normalize RelA and RelB signals. The relative sign signals in the treated groups were further normalized by untreated controls. Fold increases or decreases are indicated above corresponding bands. C, 24 h after treatment, nuclear clear extracts from the treated or untreated PC-3 cells were subjected to the NF-κB binding assay kit. Binding activity of each member of the NF-κB family was determined by ELISA analysis. *, significant differences between the untreated groups and the IR-treated groups; **, significant difference of the IR-treated group compared with both the untreated and the combined treatment of 1α,25-(OH)₂VD₃1α,25-(OH)₂D₃ and IR.

Radioresistance of PC-3 cells by overexpression of RelB. A, a RelB expression construct was transfected into 1α,25-(OH)₂D₃–pretreated or unpretreated PC-3 cells before the IR treatment. After 24 h, effect of the expressed RelB on cell survival was determined by trypan blue assay. B, ectopically expressed RelB was confirmed by Western blots normalized with β-actin.

1α,25-(OH)₂D₃ – mediated suppression of RelB target genes. A, chromatin from the treated and untreated PC-3 cells was precipitated using p50, transcription factor IIB (TFIIB), or IgG antibody. The NF-κB enhancer region of the human MnSOD gene was analyzed by PCR (top). Fragments of the exon 2 of the human MnSOD gene and the glyceraldehyde-3-phosphate dehydrogenase promoter region were amplified as an untargeted targeted control and an internal control, respectively. IgG-precipitated product served as a negative antibody control. RelB in the precipitated chromatin was measured by Western blots normalized with IgG (bottom). B, 24 h after treatment, mRNA and total proteins from the untreated or treated PC-3 cells were quantified for expression on levels of MnSOD and bcl-xl by RT-PCR (top), by Western blots (middle), and by SOD activity gel (bottom). The expression levels of the target genes shown in RT-PCR and Western blots were normalized by β-actin.MnSOD activity was compared with copper/zinc SOD (Cu-ZnSOD) activity. PCR products of the MnSOD enhancer region and RelB amounts in (A) and expression of MnSOD shown in the three panels of (B) were normalized by the relating controls, and fold increases or decreases were calculated by normalizing amounts in the treated groups with the untreated controls as described in Fig. 2B. C, a model showing the effect of IR on RelB and various steps where the expression of MnSOD SOD can be affected by 1α,25-(OH)₂D₃ in prostate cancer cells.

Radiosensitization of prostate cancer cells by 1α,25-(OH)₂D₃. A, three prostate cancer cell lines were treated with different concentrations of 1α,25-(OH)₂D₃ and then treated with different doses of IR as indicated. Effect of 1α,25-(OH)₂D₃ on IR-induced cell death was determined by colony survival analysis. Cellular extract from each cell line was used to determine level of VDR by Western blots normalized with β-actin. B, PC-3 (10⁵ cells per well) were treated with different concentrations of 1α,25-(OH)₂D₃ (10⁻⁸−10⁻⁶ mol/L) for 7 d. 1α,25-(OH)₂D₃ remaining in media was extracted and quantified using a 1α,25-(OH)₂D₃ ELISA kit (top). Concentrations of 1α,25-(OH)₂D₃ in the media were measured based on a standard curve and normalized with controls provided in the kit. Percentage of 1α,25-(OH)₂D₃ in the media was calculated by subtracting background levels in the untreated controls and then dividing by the amounts initially added into the media. CYP24 in the cells was detected as a 53-kDa protein by Western blots, which was normalized with β-actin(bottom).

Radioresistance of PC-3 cells by siRNA targeting VDR. A, VDR siRNA and siRNA control (Cont) were transfected into 1α,25-(OH)₂D₃–pretreated or unpretreated PC-3 cells before the IR treatment. After 24 h, effects of transfected siRNAs on cell survival were determined by trypan blue assay. B, knocked-down expression levels of VDR and resulting increases in RelB expression by the siRNA target were confirmed by Western blots normalized with β-actin.

Radiosensitization of PC-3 cells by siRNA targeting RelB. Control siRNA and RelB siRNA were transfected into 1α,25-(OH)₂D₃–pretreated and unpretreated PC-3 cells before the IR treatment. No siRNA transfection was added for control. After treatment for 24 h, effect of the knocked-down RelB level on cell survival was determined by trypan blue assay (A) and suppression of RelB by siRNA targeting was confirmed by Western blots normalized with β-actin (B). *, significant differences compared with non – IR-treated groups; **, significant differences compared red with the siRNA control groups in either non – IR-treated or IR-treated groups.