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.
摘要：
维生素D的抗癌作用是最重要的。胆钙化醇依次内源性羟基化为骨化二醇和骨化三醇。这里,用胆钙化醇(10-2600nmol/L)处理SiHa表皮样宫颈癌细胞。使用结晶紫和台盼蓝测定细胞计数和活力，分别。使用流式细胞术评估早期和晚期生物标志物以及明场显微镜和透射电子显微镜。通过逆转录定量PCR和免疫印迹分析了自分泌维生素D的代谢，以激活酶：25-羟化酶（CYP2R1和CYP27A1）和1α-羟化酶（CYP27B1），分解代谢24-羟化酶（CYP24A1），和维生素D受体（VDR）。使用单向方差分析和Bonferroni事后检验分析数据，并且p<0.05被认为是显著的。在胆钙化醇之后，细胞计数（p=0.011）和活力（p<0.0001）降低，凋亡生物标志物呈阳性，线粒体膜电位降低（p=0.0145），和磷脂酰丝氨酸外化（p=0.0439），末端胱天蛋白酶活性（p=0.0025），核损伤(p=0.004)增加。显微镜检查显示细胞凋亡的经典特征。基因和蛋白质表达是一致的。免疫印迹显示CYP2R1增加（p=0.021），VDR（p=0.04），CYP24A1（p=0.0274）和CYP27B1（p=0.031）降低。作者得出结论，胆钙化醇自分泌激活为骨化二醇可能介导SiHa细胞中生长抑制和凋亡的VDR信号传导。
