Hh‐inhibitory effects of calcitriol are specifically enhanced by azoles

ASZ001 cells were treated with calcitriol in combination with ITZ or KTZ using normal culture medium containing 2 % FCS. As expected from our recent studies, single calcitriol treatment significantly inhibited Gli1 expression (Fig. 10 A and D), induced Cyp24a1 expression (Fig. 10 B and E) and reduced proliferation of ASZ001 cells (Fig. 10 C and F). Single KTZ or ITZ treatments had no effects on Gli1 or Cyp24a1 expression or proliferation, but the combinations calcitriol/KTZ and calcitriol/ITZ also significantly inhibited Gli1 and induced Cyp24a1 expression and reduced proliferation. Only the combination calcitriol/KTZ resulted in significantly reduced Gli1 expression compared to both single treatments. Only Cyp24a1 expression was significantly lower after calcitriol/ITZ treatment compared to calcitriol alone (Fig. 10 E).

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Figure 10: Combined calcitriol/azole treatment of ASZ001 cells using FCS-containing medium. Relative quantification of (A and D) Gli1 or (B and E) Cyp24a1 expression levels and (C and F) BrdU-incorporation assays of ASZ001 cells after treatment with 10 nM calcitriol (cal), (A to C) 1 µM ketoconazole (KTZ), (D to F) 1 µM itraconazole (ITZ) or a combination of both. Gene expression levels were normalized to 18S rRNA expression. The respective solvent-treated controls (solvent) for each experiment were set to 1. All experiments were performed in 154-CF supplemented with 2 % heat-inactivated, chelexed FCS, 0.05 mM CaCl2 and 1 % PS as described in the material and method section. The treatments for gene expression were conducted for 24 h, BrdU-incorporation-assays for 48 h. All data represent at least 3 independent experiments measured in triplicates, represented as mean +/-SEM; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

Together these results show that ITZ and KTZ do not have an impact on Hh signaling activity or cellular proliferation. They also do not enhance the calcitriol-mediated anti-tumoral effects if used in FCS-containing medium.

Because the effectiveness of ITZ-mediated Hh signaling inhibition can be quenched by FCS concentration higher than 2 % in the culture medium (Kim et al. 2010), we next performed a similar experiment using FCS-free medium. To decrease the colloid-osmotic pressure on the cells 1.5 % (w/v) BSA was added to the medium. Using these settings we found that single treatments with the azoles significantly inhibited Hh signaling activity as measured by reduced Gli1 expression (Fig. 11 A and D). Moreover, the combination of the

Results

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azoles with calcitriol significantly intensified Hh signaling inhibition compared to either of the single treatments. Again Cyp24a1 expression was strongly induced by single calcitriol and calcitriol/azole treatments (Fig. 11 B and E). In addition, calcitriol/ITZ, but not calcitriol/KTZ-treatment, significantly induced Cyp24a1 expression compared to the respective single calcitriol treatments (Fig. 11 E). The combination of calcitriol with the azoles also significantly reduced cellular proliferation compared to solvent (ITZ, Fig. 11 F) or single-treatments (KTZ, Fig. 11 C).

Taken together, these experiments reveal that under serum-starved conditions azoles significantly intensify the Hh-inhibitory potential of calcitriol. Moreover the data suggest that azoles also cooperate with calcitriol in inhibition of cellular proliferation.

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Figure 11: Combined calcitriol/azole treatment of ASZ001 cells using FCS-free medium. Relative quantification of (A and D) Gli1 or (B and E) Cyp24a1 expression levels and (C and G) BrdU-incorporation assays of ASZ001 cells after treatment with 10 nM calcitriol (cal), (A to C) 1 µM ketoconazole (KTZ), (D to F) 1 µM itraconazole (ITZ) or a combination of both. Gene expression levels were normalized to 18S rRNA expression. The respective solvent-controls for each experiment were set to 1. All experiments were performed in 154-CF supplemented with 1.5 % BSA, 0.05 mM CaCl2 and 1 % PS as described in the material and method section. Prior to the treatment a 24 h starvation step was included. For gene expression analyses the cells were treated with the substances for 24 h, for BrdU-incorporation assays 48 h. All data represent at least 3 independent experiments measured in triplicates, represented as mean +/-SEM. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

To analyze if the enhancement of calcitriol-mediated inhibition of Hh signaling and proliferation is specific for KTZ and ITZ, we next replaced the azoles with the well-known Hh inhibitor CP. Since the effectiveness of CP is not altered by FCS (Uhmann et al. 2011a) these experiments were conducted in FCS-containing growth medium. As expected single treatments with calcitriol and CP significantly reduced Gli1 expression levels compared to solvent-treated controls (Fig. 12 A). However, the combination did not intensify this inhibition when compared to the single treatments. Cyp24a1 expression was strongly induced by calcitriol, both alone or in combination with CP (Fig. 12 B). Furthermore, the cellular proliferation was significantly reduced by calcitriol and the combination calcitriol/CP, but not

Results

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by CP alone (Fig. 12 C). Again, the anti-proliferative effect of the combined treatment was not enhanced compared to the single calcitriol treatment. These results suggest that the combination of calcitriol and CP, in contrast to azoles, does not enhance Hh inhibition or anti-proliferative effects in ASZ001 cells.

Figure 12: Combined calcitriol/CP treatment of ASZ001 cells does not result in combined antitumoral effects. Relative quantification of (A) Gli1 or (B) Cyp24a1 expression levels and (C) BrdU-incorporation assays of ASZ001 cells after treatment with 10 nM calcitriol (cal) and 5 µM cyclopamine (CP) alone or in combination.

Gene expression levels were normalized to 18S rRNA expression levels. The respective solvent-treated controls for each experiment were set to 1. Experiments were performed in 154-CF supplemented with 2 % chelexed FCS, 0.05 mM CaCl2 and 1 % PS. The treatments for gene expression were conducted for 24 h, the BrdU-incorporation assays for 48 h. All data represent at least 3 independent experiments measured in triplicates represented as mean +/-SEM. *, p<0.05; **, p<0.01; ***, p<0.001.

Finally, ASZ001 cells were treated with ITZ and CP. These experiments were conducted in FCS-free medium supplemented with 1.5 % BSA. Both compounds significantly reduced Gli1 expression (Fig. 13 A). However, no cooperative effect was observed. As expected none of the treatments altered Cyp24a1 expression (Fig. 13 B). Significant anti-proliferative effects were observed by the single ITZ and combined CP/ITZ treatment (Fig. 13 C).

Taken together these experiments reveal that azoles can significantly intensify the Hh-inhibitory potential of calcitriol. Moreover these data suggest a cooperative antiproliferative effect of the substances. In contrast, neither the combination of calcitriol and CP nor of CP and an azole are sufficient to enhance the anti-tumoral effects of the respective other drug.

Therefore, the observed cooperative effects must be a specific phenomenon for calcitriol and azoles.

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Figure 13: Combined treatment of ASZ001 with CP and ITZ does not result in combined antitumoral effects. Relative quantification of (A) Gli1 or (B) Cyp24a1 expression levels and (C) BrdU-incorporation-assays of ASZ001 cells after treatment with 5 µM cyclopamine (CP) and 1 µM itraconazole (ITZ) alone or in combination. Gene expression levels were normalized to 18S rRNA expression levels. The respective solvent-controls for each experiment were set to 1. Experiments were performed in 154-CF supplemented with 1.5 % BSA, 0.05 mM CaCl2 and 1 % PS. The treatments for gene expression were conducted for 24 h, the BrdU-incorporation-assays for 48 h. All data represent at least 3 independent experiments measured in triplicates represented as mean +/-SEM; *, p<0.05; **, p<0.01.