Effect Of 1,25(OH)₂VitD₃ On Proliferation, Apoptosis And Bax/bcl-2 MRNA Expression Of Human Retinoblastoma Cells

Background 1,25(OH)₂VitD₃,the hormonal form of vitamin D is synthesized in response to sunlight.Experimental evidence suggests that 1,25(OH)₂VitD₃ may reduce the risk of cancer through regulation of cellular proliferation and differentiation as well as induction of apoptosis.It has been demonstrated that 1,25(OH)₂VitD₃ can induce apoptosis in several cancers(such as prostate,breast cancer and leukemia etc).However,it is not clear how 1,25(OH)₂VitD₃ induces apoptosis in human retinoblastoma and whether the apoptosis is related to apoptosis-regulated gene bcl-2 and bax.Objective This study aimed to investigated the effects and mechanisms of 1,25(OH)₂VitD₃ on the proliferation and apoptosis of human retinoblastoma cells,and to detect the relationship between cell apoptosis and expression of bax and bcl-2,so as to provide evidences for the clinical application of 1,25(OH)₂VitD₃.Methods Retinoblastoma cell strain(HXO-RB₄₁) were treated with 1,25(OH)₂VitD₃ in culture media with 10^-7,10^-8 and10^-9 mol/L concentration for 48 hours respectively,control group only treated with dehydrated alcohol(solvent of calcitriol).Survival and proliferation of the cells was evaluated by blue tetrazolium colorimetric(MTT) assay and trypan blue dye exclusion test.Morphological changes were observed by light microscope.Apoptosis of the cells was detected by flow cytometry (FCM) and dual staining of AnnexinV/PI assay,and the expression of bax /bcl-2 mRNA was determined by reverse transcriptional polymerase chain reaction(RT-PCR).The outputs were semiquantitative analysised.Results 1,25(OH)₂VitD₃ had obviorsly inhibitory effect on the growth of retinoblastoma cells in a dose-dependent manner.The inhibitory rate of trypan blue dye exclusion test is 57.8%in 1×10^-7 mol/L,34.7%in 1×10^-8mol/Land 21.8%in 1×10^-9mol/L(p＜0.01 vs control);The inhibitory rate of MTT test is 66.9%in 1×10^-7 mol/L,46.3%in 1×10^-8mol/L and 34.7% in 1×10^-9mol/L(p＜0.01 vs control).1,25(OH)₂VitD₃ could induce apoptosis of retinoblastoma cells.The induction rate of retinoblastoma cells detected by FCM is 15.033±1.537%in 1×10^-7 mol/L,12.866±0.907% in 1×10^-8 mol/L,10.600±1.082%in 1×10^-9 mol/L and 2.233±0.839%in control group(p＜0.01 vs control).The gray scale of bax/bcl-2 mRNA from RT-PCR show 1.508±0.174 in 1×10^-7 mol/L group,0.944±0.069 in 1×10^-8 mol/L,0.695±0.040 in 1×10^-9 mol/L and 0.504±0.053 in control(p＜0.05 vs control).1,25(OH)₂VitD₃ induced apoptosis of retinoblastoma cells is associated with reciprocal changes of bcl-2 and bax.Conclusions 1,25(OH)₂VitD₃ could inhibit the proliferation of human retinoblastoma cells and induce the apoptosis in human retinoblastoma cells which were proved by dual AnnexinV-FITC/PI stain and flow cytometry in vitro.This apoptosis may be mediated by down-espression of bcl-2 and up-expression of bax.Clinical application of 1,25(OH)₂VitD₃ and its analogue in treating human retinoblastoma is expected.