Construction of gene targeting animal models for evaluating preventive and therapeutic anti-cancer effectiveness of active vitamin D analogues

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We have generated a line of animal model (1αOHase-/-) lacking the gene encoding vitamin D la-hydroxylase that is a key enzyme for activation of vitamin D. Phenotypic analysis revealed that similar to vitamin D. receptor gene knockout mice (VDR-/-), the animal exhibited growth retardation, hyperproliferation of cartilage, hypocalcemia, hyperparathyroidism, and impaired bone formation. As these abnormalities mainly derived from calcium malnutrition, the animals were almost completely rescued by feeding a high calcium/high lactose diet. We have been currently trying to generate (1αOHase-/-)/(VDR-/-) double knockout mice in order to clarify physiological function of vitamin D in terms of bone formation and growth of cartilage. In addition to the above mutant animals, we have generated a stable clone of human osteoblast MG-63 VDR knockdown cells and revealed that the cells tended to proliferate and differentiate much more rapidly and remarkably than their normal VDR expressing cells, sugges … More ting that VDR acts as a negative regulator in proliferation and differentiation of osteoblast. The VDR knockdown cells exhibited more rapid and stronger mineralization compared to their parent cells (normal MG-63 cells). We are currently conducting analysis of VDR knockdown effects on cancer metastasis and cancer induced angiogenesis using LLC-GFP lung carcinoma cells. We have developed a novel active vitamin D analogueα(2α-fluom-19-nor-22-oxa-lα, 25-dihydroxyvitamin D_3: 2αF-22-oxa-, 25-D_3) having a key structural motif related to non-calcemic action and it was found that 2αF-22-oxa-1,25-D_3αsuppressed tumorigenesis and angiogenesis of LLC-GFP cells more effectively and dose-dependently than the other active vitamin D3 analogues in a LLC-GFP induced cancer animal model. Furthermore, the anti-tumorigenetic and anti-angiogenetic effects of 2αF-22-oxa-1,25-D_3 were found in a VDR-/-mice fed a high calcium/high lactose diet, suggesting that 2αF-22-oxa-1,25-D_3 directly suppress tumorigenesis and angiogenesis of LLC-GFP cells without changing calcium metabolism.Through these studies, we could provide a useful animal model for evaluating preventive and therapeutic effectiveness of active vitamin D analogues on the tumorigenesis and angiogenesis of cancer cells, and provide a new approach for the development of anti-cancer drugs. Less