CYP24 inhibition preserves 1α,25-dihydroxyvitamin D(3) anti-proliferative signaling in lung cancer cells

Abstract

Human lung tumors aberrantly express the 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))-catabolizing enzyme, CYP24. We hypothesized that CYP24 reduces 1,25(OH)(2)D(3)-mediated transcription and allows lung cancer cells to escape its growth-inhibitory action. To test this, H292 lung cancer cells and the CYP24-selective inhibitor CTA091 were utilized. In H292 cells, CTA091 reduces 1,25(OH)(2)D(3) catabolism, significantly increases 1,25(OH)(2)D(3)-mediated growth inhibition, and increases 1,25(OH)(2)D(3) effects on induced and repressed genes in gene expression profiling studies. Pathway mapping of repressed genes uncovered cell cycle as a predominant 1,25(OH)(2)D(3) target. In H292 cells, 1,25(OH)(2)D(3) significantly decreases cyclin E2 levels and induces G(0)/G(1) arrest. A broader set of cyclins is down-regulated when 1,25(OH)(2)D(3) is combined with CTA091, and cell cycle arrest further increases. Effects of CTA091 on 1,25(OH)(2)D(3) signaling are vitamin D receptor-dependent. These data provide evidence that CYP24 limits 1,25(OH)(2)D(3) anti-proliferative signaling in cancer cells, and suggest that CTA091 may be beneficial in preserving 1,25(OH)(2)D(3) action in lung cancer.

Figure 1. CYP24 catabolizes 1,25(OH)₂D₃ and reduces its anti-proliferative activity in lung cancer cells

(A) H292 cells were treated with 100 nM 1,25(OH)₂D₃ ± 50 nM CTA091. At the times indicated, whole cell extracts were prepared. Equivalent amounts of protein from each extract were analyzed by immunoblot for CYP24 expression. (B) H292 cells were exposed to 100 nM 1,25(OH)₂D₃ in the absence or presence of CTA091 (0–100 nM). After 0h or 24h of treatment, cell homogenates were prepared and 1,25(OH)₂D₃ concentrations determined by LC-MS/MS, as described in Methods. Each data point represents a separate 1,25(OH)₂D₃ determination. Horizontal bars represent the mean of 3 determinations (**, P< 0.001 treatment vs. control). The lower limit of detection for 1,25(OH)₂D₃ using this assay is 1 ng/mL. The ability of 50 nM CTA091 to inhibit 1,25(OH)₂D₃ elimination from H292 cultures was confirmed in 3 additional experiments. (C) H292 cells were treated with vehicle (control) or the indicated concentrations of CTA091. Treatments were repeated every 3 days. Crystal violet staining was used to assess colony formation after 7 days of treatment. Bars represent means ± SD for triplicate determinations within a single experiment. No significant differences in growth were observed. (D) Cells were treated with vehicle (control) or the indicated concentrations of 1,25(OH)₂D₃ in the absence or presence of CTA091 (50 nM). As above, crystal violet staining was used to assess colony formation after 7 days of treatment. Bars represent means± SD for triplicate determinations within a single experiment. *, P<0.05, treatment vs. vehicle (control). Horizontal lines are used to indicate significant differences in mean % colonies remaining between 1,25(OH)₂D₃ treatment dose levels, and between 1,25(OH)₂D₃ and 1,25(OH)₂D₃ + CTA091 treatment groups. Clonogenic assays were conducted twice, and similar results were obtained.

Figure 2. CYP24 impairs transcriptional regulation by 1,25(OH)₂D₃

H292 cells were treated with vehicle (control), CTA091 alone (50 nM), 10 nM 1,25(OH)₂D₃ ± CTA091 (50 nM) or 100 nM1,25(OH)₂D₃. RNA was extracted 24 h post-treatment, and expression of CD14, LL37, CDH5, and GAPDHwas analyzed by RT-PCR. PCR products were resolved on 1.2% agarose gels, visualized with ethidium bromide, and photographed. Gel images from three separate experiments are presented in the left panels. Densitometry results for these three experiments are summarized in the right panels. Band intensities of the RT-PCR products were quantified by densitometry and normalized to the intensity of the band for GAPDH. Bars represent the mean normalized intensity ± SD. *, P<0.05, treatment vs. vehicle (control). No significant differences between 10 nM 1,25(OH)₂D₃ and 100 nM 1,25(OH)₂D₃ were observed. Brackets are used to indicate significant differences in mean normalized intensity between 10 nM 1,25(OH)₂D₃ and10 nM1,25(OH)₂D₃ + CTA091 treatment groups.

Figure 3. CYP24 restricts 1,25(OH)₂D₃–mediated repression of cyclin expression in lung cancer cells

H292 cells were treated with vehicle or the indicated concentrations of 1,25(OH)₂D₃ in the absence or presence of CTA091 (50 nM). Whole cell extracts were prepared after 24 h and analyzed by immunoblot for cyclin expression. Blots were reprobed for actin as a control for protein quantitation and loading. Densitometry was used to quantify the expression of each protein, and composite actin-normalized expression data from 4 independent experiments are presented. Bars represent the mean actin-normalized expression ± SD. *, P<0.05, treatment vs. vehicle (control). Horizontal lines are used to indicate significant differences in mean actin-normalized expression between 1,25(OH)₂D₃ treatment dose levels, and between 1,25(OH)₂D₃ and 1,25(OH)₂D₃ + CTA091 treatment groups.

Figure 4. The VDR is required for 1, 25(OH)₂D₃-mediated down-modulation of cyclin expression in lung cancer cells

H292 cells were cultured in complete medium for 24 h followed by transfection with human VDR siRNAor control siRNA. After 5 h, cells were treated with vehicle, CTA091 (50nM) 1,25(OH)₂D₃ (10 nM or 100 nM), or 1,25(OH)₂D₃ + CTA091. Cell extracts were prepared 3 d post-treatment and subjected to western blot analysis to evaluate protein expression. Blots were re-probed for actin as a control. Densitometry was used to quantify the expression of each protein, and protein expression was normalized to the intensity of the band for actin. Bars represent the mean normalized intensity ± SD from 3 independent experiments. Vehicle (control) was compared to each treatment.* P< 0.05, treatment vs. control.

Figure 5. CTA091 increases 1,25(OH)₂D₃-mediated G0/G1 arrest in H292 cells

H292 cells were seeded into 6-well dishes. After attachment, cells were treated with the indicated agents for 48 h. The cells were harvested and processed for cell cycle analysis. The cell cycle distribution was determined by flow cytometric analysis of PI-stained cells, as described in the Methods. Results, shown by phase, from 3 independent experiments are summarized. Bars represent mean % cells in that particular phase ± SD. *, P<0.05, treatment vs vehicle (control); ^, P<0.05, treatment vs 10 nM and 100 nM 1,25(OH)₂D₃ + CTA091. ^, P<0.05, treatment vs 100 nM 1,25(OH)₂D₃ + CTA091.