Article Figures & Data
Figures
- Fig. 1.
VES selectively increases VDR protein. LNCaP, PC3, and 22RV1 cells were treated with ethanol or VES (20 μmol/L) for 2 and 4 d. Protein samples of cell lysates were collected, and VDR, RXRα, ERβ, and β-actin protein levels were analyzed by Western blotting.
- Fig. 2.
The effect of combining calcitriol and VES is significantly superior to the sum effect of individual treatments in controlling prostate cancer cells. LNCaP (A) and PC3 (B) cells were used to test the growth inhibition effects. Cells were plated in 24-well plates (2-5 × 103/well) and pretreated with ethanol and VES (1, 5, and 10 μmol/L) for 24 h and then treated with calcitriol (1, 10, and 20 nmol/L) or in combination. MTT growth assay was done after 6 d of treatment. The results were reported as absorption at 595 nm, considering control cells as 100%. Data, mean ± SD (n = 3). *, statistically different (P < 0.05). C, CI values for combination treatment of calcitriol and VES in LNCaP and PC3 cells were calculated. Effect-CI plots were generated by CalcuSyn 2.0 software. 1, 2, and 3 represent 1, 10, and 20 nmol/L calcitriol combination, respectively.
- Fig. 3.
VES sensitizes calcitriol-induced expression of VDR target genes in prostate cancer cells. After treatment of VES (10 μmol/L) or ethanol for 24 h the cells were exposed to calcitriol (10 nmol/L) for an additional 24 h A, CYP24, IGFBP-3, and p21 mRNA levels in LNCaP cells were evaluated by real-time RT-PCR. B, CYP24, IGFBP-3, and p21 mRNA levels were analyzed in PC3 cells. Data, mean ± SD from three independent experiments; each sample was evaluated in triplicate in each experiment. *, statistically different (P < 0.05) versus VES and calcitriol single treatment. RT-PCR, reverse transcription-PCR.
- Fig. 4.
VDR is required for the VES-sensitized antiproliferation effect of calcitriol. A, VDR expression was decreased by retrovirus-based VDR siRNA in LNCaP and PC3 cells. Scramble and VDR siRNA stably transfected cells were generated as described in Materials and Methods. Protein lysates of scramble and VDR siRNA stable cells were collected for VDR Western blotting. Number under panel, percentage of VDR protein in comparison with the scramble control as measured by the Quantity One software. Ratio of VDR/β-actin in scramble siRNA stable cells as 1.00. B and C, LNCaP and PC3 VDR siRNA or scramble siRNA stable cells were treated with VES (10 μmol/L) for 24 h then exposed to calcitriol (10 nmol/L) for an additional 24 h. RNA was isolated, and CYP24, IGFBP-3, and p21 mRNA levels were analyzed by real-time RT-PCR. D, scramble and VDR siRNA stably transfected LNCaP and PC3 cells were treated with vehicle control, VES (10 μmol/L), calcitriol (10 nmol/L), or the combination therapy. MTT growth assay was done after 6 d of treatment. The results are reported as the percentage of inhibition, considering control cells as 0% inhibition. Point, mean ± SD (n = 3).
- Fig. 5.
VES enhances therapeutic effect of calcitriol on prostate cancer in the in vivo mouse cancer model. Animals were randomly divided into control and experimental groups after LNCaP or PC3 cells were injected in the dorsolateral flanks on both sides of each mouse. After tumors established to the size of 50 mm3 (1-fold), mice were i.p. injected with vehicle (DMSO), VES (100 mg/kg), calcitriol (0.0625 μg/mice), or in combination twice weekly. A, tumor volumes in different treatment groups. Changes in tumor volumes were measured once weekly. Relative changes in tumor volume were calculated by dividing tumor volumes measured during the course of therapy by the initial tumor volume (i.e., volume at day 0). Point, mean ± SE of 10 to 14 tumors. B, tumor weights in different treatment group. One day after the last treatment, tumors were harvested and weighed. Point, mean ± SE (n = 10-14 for each treatment group). C, no significant body weight changes in different treatment group. Body weights were monitored once a week. Point, mean ± SD (n = 7). D, no alteration of serum calcium in different treatment group. One day after the last treatment, mice were euthanized and sera were collected for calcium measurement. Point, mean ± SD of the 5 to 7 mice serum calcium value. *, P < 0.05 versus untreated controls. **, P < 0.05 versus single-agent VES or calcitriol. The animal experiments were repeated twice with similar results, and one set of results is presented.
- Fig. 6.
VES increased calcitriol-mediated antiproliferative activity and the expression of VDR target genes in in vivo prostate cancer model. A and B, LNCaP and PC3 control xenografts have poorly differentiated carcinoma cells without necrosis (original ×400). Xenografts from mice that received VES alone showed an increased proportion of noncellular stroma components and scattered necrotic lesions. Xenografts from mice that were treated with calcitriol alone have a similar histologic appearance as VES single-treatment xenografts (original ×400). Xenografts from mice treated with the combination of VES and calcitriol show less cell density and extensive tumor necrosis and fibrotic changes (original ×400). C, extracts from tumor homogenates were subjected to Western blotting for expression levels of VDR, PCNA, and β-actin. D, IGFBP-3 and p21 mRNA changes in different group xenografts were detected by real-time RT-PCR. *, statistically different (P < 0.05) versus VES tumor lysate. Lysate of each treatment group was a mixture of five individual tumors. PCNA, proliferating cell nuclear antigen.
Volume 15, Issue 1
