Vitamin D3 has been widely used clinically, and the proper dosage and side effects have been well-documented [28
]. In the present study, we showed, for the first time, that gastric cancer and cholangiocarcinoma cells are sensitive to vitamin D3, and that vitamin D3 has synergism with other cancer drugs. Moreover, we also showed that the actions of vitamin D3 are possibly mediated through inhibition of Hh signaling.
The expression of Hh signaling target genes was regulated by vitamin D in the present study (). Hh signaling is one of the most important signaling pathways regulating proliferation, differentiation, embryogenesis, and cancer [29
]. In mammals, Hh consists of three different members, including Sonic hedgehog, Indian hedgehog, and Desert hedgehog [30
]. The trigger for signaling is the Hh ligand binding to a 12-pass transmembrane protein patched (Ptch1), which resembles the Niemann-Pick disease type C1 (NPC1) protein. The NPC1 protein is involved in cholesterol trafficking and has a pump function [26
]. After Hh binds to Ptch1, Ptch1 no longer blocks the 7-pass transmembrane protein, smoothened (Smo), and subsequently the intracellular signaling cascade is activated, including Fused, protein kinase A, GSK3, CKI, and Gli. Finally, the Gli protein is activated and translocated to the nucleus where it turns on target genes. However, in the absence of Hh, Smo is continuously repressed by Ptch1. Finally, the Hh target genes are turned off.
The association between vitamin D and Hh signaling was first reported by Bijlsma et al. [26
]. Bijlsma et al. [26
] reported that Ptch1 can secrete 3β-hydroxysteroids, which inhibit Smo on other cells. Vitamin D3 directly binds to Smo with high affinity in a cyclopamine-sensitive manner. Moreover, Bijlsma et al. [26
] showed that treating zebrafish embryos with vitamin D3 mimics the Smo-/-
phenotype. Their report suggested that vitamin D3 can be used as an anti-cancer drug. In contrast, Brüggemann et al. [25
] reported that vitamin D3 inhibits pancreatic cancer cell viability in the presence of 0.5% serum; however, it does not inhibit the pancreatic cancer cell viability in the presence of 10% serum. Moreover, vitamin D3 did not show any synergism with other anti-cancer drugs for inhibiting cell viability. Brüggemann et al. [25
] suggested that vitamin D3 is not an efficient anti-cancer drug. However, in the present study, vitamin D3 had the ability to inhibit cell viability and showed a synergistic effect with other anti-cancer drugs in 10% serum, as shown in . Hence, our data indicate that vitamin D3 could be used as an anti-cancer drug, and its effect may depend on cell type.
Vitamin D3 is related to cancer via various signaling pathways [2
]. However, in the present study, we focused on Hh signaling because cyclopamine, a specific inhibitor of Hh signaling, almost completely suppressed cancer cell viability, which was consistent with previous reports [24
]. Although it is unclear how the Hh signaling pathway is activated in gastric cancer and cholangiocarcinoma, it is clear that Hh signaling activity is critical for viability [24
]. We showed that vitamin D3 reduced the expression of Hh signaling target genes (), suggesting the possibility that Hh signaling may play a critical role in vitamin D3-induced inhibition of viability. Future studies such as Gli1 overexpression are required to confirm this possibility.