| 1,25(OH)2D3 is an active form of vitamin D.Besides its classic role in maintaining the extracellular calcium and phosphorus balance,recent studies have shown that 1,25(OH)2D3 is also associated with many aging related diseases including cancers,autoimmune diseases,cardiovascular disorders and infectious diseases.Epidemiological studies have shown that vitamin D deficiency is associated with high mortality;The length of telomere and life span are shorter in women with vitamin D deficiency.These results indicated that vitmin D plays an important role in preventing aging.Lots of epidemiological studies also demonstrated that Vitamin D deficiency is associated with cancers including breast cancer,prostate cancer,colon cancer.However,it is unclear whereby active vitamin D deficiency results in aging and cancers.To investigate whereby active vitamin D plays double actions in preventing aging and tumors,1?(OH)ase-/-and their wild-type littermates were fed on a normal diet or high calcium and phosphorus diet,or administrated with 1,25(OH)2D3 or fed a dietary antioxidant NAC and these treated mice were also subcutaneously transplanted TM40D mammary tumor cells.Phenotypes of tissues and xenograft tumors from those animals were analyzed and compared each other using histopathological,cellular and molecular techniches.Our results demonstrated that:1.Compared with wild-type mice,1?(OH)ase-/-mice on normal diet displayed a shorter life span(mean survival time only for 91 days),hypocalcemia,hypophosphatemia,no 1,25(OH)2D3 and a variety of ageing phenotypes,including weight loss,thinning skin,subcutaneous fat reduction,total-collagen reduction,lower bone mineral density and skeletal ossification,increased ROS levels in multi-tissues,down-regulated gene expression levels of the antioxidants including SOD2 and Prdx I,increased y-H2AX and senescence-associated P-galactosidase(SA-?-Gal)positive cells,up-regulated tumor suppressive gene expression including p53,p21and p16,down-regulated oncogene Bmi-1,reduced Ki67 positive cells.When 1?(OH)ase-/-mice were fed on a high calcium and phosphorus diet,serum calcium and phosphorus levels were normalized,mean survival time was prolonged to 252 days,aging associated phenotypes and above parameters were improved insignificantly.When 1?(OH)ase-/-mice were supplemented with exogenous 1,25(OH)2D3 or fed with the high calcium and phosphorus diet plus NAC,mean survival time was prolonged to 487 and 409 days,respectively,aging associated phenotypes and above parameters were rescued obviously.These results demonstrated that active vitamin D deficiency resulted in aging and growth retardation through increasing oxidative stress and DNA damage,inhibiting cell proliferation and inducing cell senescence;The supplementation of calcium and phosphorus prolonged the life span and stimulated the growth in active vitamin D deficient mice through normalized serum calcium and phosphorus;the supplementation of 1,25(OH)2D3 or combined calcium,phosphorus and NAC rescued aging and growth retardation caused by the deletion of 1?(OH)ase through normalizing serum calcium and phosphorus,inhibiting oxidative stress and DNA damage,increasing cell proliferation and inhibiting cell senescence.2.Spontaneous tumors were detected in 6 1?(OH)ase+-/-mice among over 1-year-old 52 1?(OH)ase+-/-mice fed on the normal diet and in 5 1?(OH)ase-/-mice among over 1-year-old 21 1?(OH)ase-/-mice fed on the rescue diet.The type of spontaneous tumors were included breast cancer,squamous-cell carcinoma,soft tissue sarcoma,hepatocellular carcinoma,granulocytic sarcoma,endometrial stromal tumor,pulmonary adenocarcinoma.However,no any Spontaneous tumor was detected in wild-type littermates and in 1?(OH)ase-/-mice supplemented with 1,25(OH)2D3 or with combined calcium,phosphorus and NAC.Compared with para-tumor tissues,the spontaneous tumor tissues displayed lower expression of the antioxidase SOD2,increased DNA damage,down-regulation of tumor suppressive genes including p27,p21 and p16,up-regulation of oncogenes including Bmi-1,RAS,c-Fos and increased cell proliferation.The serum protein expression differences were examined in 6-month-old wild-type and 1?(OH)ase-/-mice using serum protein array.The results showed that 7 proteins including HGF were up-regulated and 6 proteins were down-regulated in serum from 1?(OH)ase-/-mice compared those from wild-type mice.We confirmed that serum HGF levels were raised in 1?(OH)ase-/-mice by ELISA as detected by serum protein array.We also found that the expression level of c-Met,a receptor of HGF,were up-regulated dramatically in spontaneous tumor tissues compared para-tumor tissues.These results indicate that active vitamin D deficiency resulted in an increased spontaneous tumor development through increased oxidative stress and DNA damage,stimulating the proliferation of carcinomatous cells that also be enhanced by the molecules of senescence associated secreted phenotype caused by increased senescence cells in host tissues.3.Results from the xenograft tumors showed that the tumor formation rates and tumor volume were not altered significantly in the wild-type mice whatever they were fed on the normal diet or rescue diet or supplemented with 1,25(OH)2D3 or with combined calcium,phosphorus and NAC.The tumor formation rates and tumor volume were increased,ROS levels were increased,the gene expression levels of the antioxidants including SOD2 and Prdx I were up-regulated,8-OHdG-positive cells were increased,SA-?-Gal positive cells were reduced,the protein expression levels of p27,p21 and p16 were down-regulated,the protein expression levels of Bmi-1 and RAS were up-regulated and Ki67-positive cells were increased significantly in 1?(OH)ase-/-mice fed on the normal diet or rescue diet compared with wide-type mice on the same diet.The tumor formation rates and tumor volume were decreased significantly and all above parameters were rescued very well in 1?(OH)ase-/-mice supplemented with 1,25(OH)2D3 or with combined calcium,phosphorus and NAC compared with 1?(OH)ase-/-mice fed either the normal diet or the rescue diet These results demonstrated that active vitamin D deficiency accelerated the xenograft tumor formation and growth through increased oxidative stress and DNA damage,stimulated the proliferation of xenograft tumor cells and inhibited the senescence of xenograft tumor cells.The supplementation of calcium and phosphorus did not,but the supplementation of 1,25(OH)2D3 or combined calcium,phosphorus and NAC inhibited the xenograft tumor formation and growth through inhibiting oxidative stress,DNA damage and the proliferation of xenograft tumor cells and inducing the senescence of xenograft tumor cells.Results from this study indicated that 1,25(OH)2D3 deficiency accelerated anmal aging through increased oxidative stress,DNA damage and cell senescence,and accelerated tumor development and growth through disequilibrium between oncogenes and tumor repressive genes caused by oxidative stress induced DNA damage,and increased cell proliferation.Thus we demonstrated for the first time that active vitamin D plays double actions in preventing both aging and cancer.This study provided the experimental and theoretical basis for the application of active vitamin D to postpone ageing and to prevent and treat cancer. |
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