Promoting Effects Of Vitamin D Persistent Deficiency From Early Life On Prostatic Hyperplasia And Prostate Cancer Progression In Middle-aged Mice

Vitamin D deficiency is especially prevalent in pregnant women and children,and is increasingly recognized as a global public health problem.Maternal vitamin D deficiency is associated with adverse pregnant outcomes.Moreover,vitamin D deficiency in gestational period increases the risk of asthma and multiple sclerosis in offspring.Recently,our results showed that vitamin D deficiency in early life disturbed testicular development and spermatogenesis in mice.However,the effects and mechanisms of vitamin D persistent deficiency from early life on prostatic hyperplasia and prostate cancer progression in middle-aged mice are still unclear.The current study explored the effects and part of the mechanism of vitamin D persistent deficiency from early life on prostatic hyperplasia and prostate cancer progression in middle-aged mice.The objectives of present study are through to establish an ICR mouse model of vitamin D deficiency to investigate the effects of vitamin D persistent deficiency from early life on chronic prostatitis and prostatic hyperplasia in middle-aged mice and explore the roles of prostatic NF-kB and STAT3 signal in it,through to establish an TRAMP mouse model of vitamin D deficiency to observe the effects of vitamin D persistent deficiency from early life on prostate cancer growth and metastasis in middle-aged mice and explore the roles of prostatic NF-kB and STAT3 signal in it.To verify the roles of NF-kB and STAT3 signaling in the progression of prostate cancer and the protective effect of vitamin D,we observed the effects of active vitamin D supplementation on LPS-evoked proliferation,migration and invasion in PC-3 cell and explored the inhibitory effect of active vitamin D on LPS-induced activation of NF-kB and STAT3 signaling.This study will preliminary clarify the impact of vitamin D persistent deficiency from early life on prostatic hyperplasia and prostate cancer progression in middle-aged mice and part of the mechanism that may help to find the cause of prostate hyperplasia and prostate cancer and provide a theoretical basis for prevention and treatment.Study 1: Effects of vitamin D persistent deficiency from early life on prostatic hyperplasia in middle-aged mice.Objective This study aims to investigated the effects of vitamin D persistent deficiency from early life on prostatic hyperplasia in middle-aged mice and its mechanism.Methods Pregnant mice were divided randomly into two groups.In the control group,dams were fed with standard diets(Standard AIN93 G Rodent diet with 1000 IU vitamin D3/kg)throughout pregnancy and lactation.After weaning,male pups were fed with standard diets for 37 weeks.In the VDD group,dams were fed with VDD diets(with 0IU vitamin D3/kg)throughout pregnancy and lactation.After weaning,male pups were divided randomly into two groups.In the VDD group,male pups were fed with VDD diets for 37 weeks.In the VDS group,male pups were fed with VDD diets for 17 weeks and then replaced with standard diets for 20 weeks.Fortieth weeks after birth,mice were sacrificed.Serum was collected for 25(OH)D level.Prostate tissue was weighted and collected for immunoblot and real-time RT-PCR or fixed in 4% paraformaldehyde for histological examination or immunohistochemistry.Results Prostate weight was elevated and prostatic hyperplasia was observed in VDD-fed mice.The number of prostatic Ki67-positive epithelial cells,a proliferation marker,was increased in VDD-fed mice.Further analysis found that vitamin D deficiency promoted inflammatory infiltration and stromal fibrosis in prostate of middle-aged mice.Moreover,vitamin D deficiency activated NF-?B and up-regulated Il-6 m RNA in prostate of middle-aged mice.In addition,vitamin D deficiency activated prostatic STAT3,a proliferation pathway in middle-aged mice.Of interest,VDD-induced prostatic inflammation and hyperplasia were partially reversed when VDD diets was replaced with standard diets.Conclusion These results provide evidence that vitamin D persistent deficiency from early life promotes prostatic hyperplasia in middle-aged mice through exacerbating local inflammation.Study 2: Effects of vitamin D persistent deficiency from early life on prostate cancer progression in middle-aged mice.Objective This study aims to investigate the effects of vitamin D persistent deficiency from early life on growth and metastasis of prostate cancer in middle-aged mice.Methods To observe the effects of vitamin D persistent deficiency from early life on prostate cancer growth and metastasis in middle-aged mice in vivo,a vitamin D-deficient TRAMP mouse model was established.Male TRAMP mice(three week-old)were randomly divided into two groups.In the control group and the VDD group,mice were fed with standard diets and VDD diets respectively.Mice were sacrificed at twenty-eight weeks of age.Serum was collected for 25(OH)D level.Prostate tissue was weighted and collected for immunoblot or fixed in 4% paraformaldehyde for histological examination.Tumor metastasis of lymph nodes,lung,liver and kidney tissues were analyzed by histopathological method.Results Prostate weight and index were elevated and prostatic stroma thickening was observed in VDD-fed TRAMP mice.More importantly,the incidence of total and lung metastases was significantly increased in VDD-fed TRAMP mice.In addition,vitamin D deficiency up-regulates NF-?B and STAT3 signaling in prostate tissue of middle-aged TRAMP mice.Conclusion Vitamin D persistent deficiency from early life promotes growth and metastasis of prostate cancer in middle-aged TRAMP mice,and tumor microenvironment NF-kB and STAT3 signaling may play an important role in this process.Study 3: Effects of active vitamin D supplementation on inflammation-induced proliferation,migration,and invasion in PC-3 cell.Objective This study aims to further verify the roles of NF-kB and STAT3 signaling in the progression of prostate cancer and the protective effect of vitamin D.Methods PC-3 cells were divided into four groups: Control group,1,25(OH)2D3 alone group(VD3 group),LPS alone group(LPS group)and combined treatment group(VD3+LPS group).In the VD3 group and the VD3+LPS group,cells were pretreated with 1,25(OH)2D3(100 nmol/L)at 12 and 2 h before LPS(2.0 mg/ml).The culture supernatant and cells were collected at different time points after LPS treatment for subsequent experiments.This study consisted of five experiments.Experiment 1,to verify whether 1,25(OH)2D3 could activate VDR signal in PC-3 cells,the cells were harvested at 6 h after LPS treatment and the expression of Cyp24A1 m RNA,a VDR target gene,was detected by RT-PCR.Experiment 2,to investigate the effect of1,25(OH)2D3 on LPS-mediated proliferation of PC-3 cells,cells were harvested at 6 h after LPS treatment.(1)The harvested cells were fixed with ice cold 70% ethanol overnight at-20°C,and then analyzed cell cycle by Muse cell analyzer.(2)The harvested cells were extracted with nucleoprotein,and then detected the protein levels of Cyclin D1 and PCNA levels by Western blotting.Experiment 3,in order to investigate the effects of 1,25(OH)2D3 on LPS-induced migration and invasion of PC-3cells,migration and invasion activities of PC-3 cells were evaluated using Transwell filters at 12 and 48 h after LPS treatment respectively.In addition,matrix degradation related enzymes(MMP3,MMP9 and u PA)m RNA expression levels were detected by RT-PCR at 6 h after LPS incubation.Experiment 4,to investigate the effects of1,25(OH)2D3 on the LPS-induced expression of inflammatory cytokines,chemokines and adhesion molecules,the culture supernatant and cells were collected 6 h after LPS treatment.TNF-a,IL-1a,IL-1b,COX-2,IL-6,IL-8,MCP-1 and ICAM1 m RNAs were detected by RT-PCR.The levels of IL-6 and IL-8 in the culture supernatant were detected by ELISA.Experiment 5,in order to explore the mechanism of 1,25(OH)2D3inhibiting LPS-mediated proliferation,migration and invasion of PC-3 cells,the cells were harvested at 6 h after LPS treatment.NF-kB(cytoplasmic p I-kB/I-kB,nuclear NF-k B p50 and p65),AKT(cytoplasmic p-Akt/Akt),MAPK(cytoplasmic p-p38/p38)and STAT3(cytoplasmic p STAT3/STAT3 and nuclear p STAT3)signal-related protein levels were analyzed by western blotting.Results Experiment 1 showed that VDR signal in PC-3 cell was significantly activated after 1,25(OH)2D3 treatment,but LPS did not inhibit 1,25(OH)2D3-activated VDR signal.Experiment 2 showed that 1,25(OH)2D3 pretreatment significantly inhibited the percentage at S phase cells and the levels of nuclear Cyclin D1 and PCNA in LPS-stimulated PC-3 cells.Experiment 3 showed that 1,25(OH)2D3 pretreatment inhibited the cell migration and invasion ability,as well as the expression of MMP3,MMP9 and u PA m RNA in LPS-stimulated PC-3 cells.Experiment 4 showed that LPS induced the expressions of TNF-a,IL-1a,IL-1b,COX-2,IL-6,IL-8,MCP-1 and ICAM1 in PC-3 cells.However,1,25(OH)2D3 pretreatment significantly inhibited LPS-induced up-regulation of IL-6,COX-2,IL-8,MCP-1 and ICAM1.Correspondingly,the levels of IL-6 and IL-8 in the culture medium were significantly elevated in LPS-stimulated PC-3 cells.Although 1,25(OH)2D3 alone had no effect on IL-6 and IL-8,it significantly attenuated LPS-induced up-regulation of IL-6 and IL-8 in PC-3 cells.Experiment 5 demonstrated that LPS and 1,25(OH)2D3 had no effect on the AKT signal.Although 1,25(OH)2D3 treatment did not suppressed LPS-induced up-regulation of MAPK/p38,it significantly inhibited LPS-induced up-regulation of NF-kB and STAT3 signal in PC-3 cells.Conclusion These results suggest that 1,25(OH)2D3 inhibits proliferation,migration and invasion partially through suppressing inflammation-mediated activation of NF-?B and STAT3 in prostate cancer cells.Based on the results of the above three studies,this subject draws the following conclusion:(1)Vitamin D persistent deficiency from early life promotes prostatic hyperplasia in middle-aged ICR mice through exacerbating local inflammation.(2)Vitamin D persistent deficiency from early life promotes prostate cancer progression in middle-aged TRAMP mice,and tumor microenvironment NF-k B and STAT3 signaling may play an important role in this process.(3)Vitamin D inhibits proliferation,migration and invasion partially through suppressing inflammation-mediated activation of NF-?B and STAT3 in prostate cancer cells.