Cholecalciferol induces apoptosis via autocrine metabolism in epidermoid cervical cancer cells

Abstract

The anti-cancer effects of vitamin D are of fundamental interest. Cholecalciferol is sequentially hydroxylated endogenously to calcidiol and calcitriol. Here, SiHa epidermoid cervical cancer cells were treated with cholecalciferol (10–2600 nmol/L). Cell count and viability were assayed using Crystal Violet and Trypan Blue, respectively. Apoptosis was assessed using flow cytometry for early and late biomarkers along with brightfield microscopy and transmission electron microscopy. Autocrine vitamin D metabolism was analysed by reverse transcription-quantitative PCR and immunoblotting for activating enzymes: 25-hydroxylases (CYP2R1 and CYP27A1) and 1α-hydroxylase (CYP27B1), the catabolic 24-hydroxylase (CYP24A1), and the vitamin D receptor (VDR). Data were analysed using one-way ANOVA and Bonferroni post-hoc test, and p < 0.05 was considered significant. After cholecalciferol, cell count ( p = 0.011) and viability ( p < 0.0001) decreased, apoptotic biomarkers were positive, mitochondrial membrane potential decreased ( p = 0.0145), and phosphatidylserine externalisation ( p = 0.0439), terminal caspase activity ( p = 0.0025), and nuclear damage ( p = 0.004) increased. Microscopy showed classical features of apoptosis. Gene and protein expression were concordant. Immunoblots revealed increased CYP2R1 ( p = 0.021), VDR ( p = 0.04), and CYP24A1 ( p = 0.0274) and decreased CYP27B1 ( p = 0.031). The authors conclude that autocrine activation of cholecalciferol to calcidiol may mediate VDR signalling of growth inhibition and apoptosis in SiHa cells.