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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Ruminant Diseases and Immunology Research » Research » Publications at this Location » Publication #184929
Title: SIRNA AGAINST 25-OHD3 1ALPHA-HYDROXYLASE INHIBITS LOCAL PRODUCTION OF 1,25-(OH)2D3 AND PREVENTS 25-OHD3 INDUCED GROWTH INHIBITION OF CANCER CELLS IN VITRO AND IN VIVO

Author
item HUANG, D - MCGILL UNIVERSITY
item YANG, X - MCGILL UNIVERSITY
item RHIM, J - UNIF SRVCS UNIV HLTH SCI
item Horst, Ronald
item KREMER, R - MCGILL UNIVERSITY

Submitted to: American Society for Bone and Mineral Research
Publication Type: Abstract Only
Publication Acceptance Date: 9/23/2005
Publication Date: 9/23/2005
Citation: Huang, D.C., Yang, X.F., Rhim, J.S., Horst, R.L., Kremer, R. 2005. siRNA against 25-OHD3 1alpha-hydroxylase inhibits local production of 1,25-(OH)2D3 and prevents 25-OHD3 induced growth inhibition of cancer cells in vitro and in vivo [abstract]. American Society for Bone and Mineral Research. Available: http://www.asbmr.org/meeting/abstracts.cfm.

Interpretive Summary:

Technical Abstract: Extra-renal expression of the 1 alpha-hydroxylase enzyme which catalyzes the conversion of 25-hydroxyvitamin D3 to 1 alpha, 25-dihydroxyvitamine D3 may play an important autocrine/paracrine antiproliferative function. In the present study, we used a small interfering double-stranded RNA (siRNA) to knockdown 1 alpha-hydroxylase in two cancer cell lines previously shown to express this enzyme. A hairpin oligonucleotide sequence corresponding to the N-terminal region of the 1 alpha-hydroxylase gene was cloned into the siRNA vector. Transfection of this plasmid into the human amelanotic melanoma cell line A375 and the human prostate cancer cell lines FNC267B1-ras led to a dramatic decrease in the conversion of 25-OHD3 to 1,25-(OH)2D3. We then examined the effect of substrate 25-OHD3 on parameters of growth and differentiation in both 1 alpha-hydroxylase knockdown cell lines and compared them to the same parameters in the corresponding wild type control cell lines transfected with vector alone. Addition of 25-OHD3 (10**-6 M) to wild type cells inhibited growth by 28 +/- 5% in A375 cells (p < 0.001) and by 36 +/- 6% in the human transformed prostate cell line FNC267B1 (p < 0.001). In contrast, no significant growth inhibition was observed in both knockdown A375 and FNC267B1 cell lines in the presence of 25-OHD3 (10**-6 M). This growth inhibitory effect was further analyzed in vivo following subcutaneous injection of 1 alpha-hydroxylase knockdown or wild type cells in severely compromised immunodeficient (SCID) mice. Alzet osmotic mini-pump containing 25-OHD3 were implanted two days before tumor cell transplantation to deliver a constant infusion of 25-OHD3 (2000 pM/24 h) or vehicle. Circulating 25-OHD3 concentrations were at least 10 fold higher in 25-OHD3 treated animals as compared to vehicle treated animals without significant changes in circulating calcium levels. A significant inhibition of tumor growth was observed in mice transplanted with wild type A375 or FNC267B1 control cells treated with 25-OHD3 as compared to vehicle (p < 0.001). In contrast no tumor reduction was observed in mice transplanted with siRNA knockdown cells. In summary our data demonstrate that local conversion of 25-OHD3 to 1,25-(OH)2D3 inhibits tumor growth in both melanoma and prostate cancer cells. These data therefore suggest potential therapeutic cancer applications for the non-calcemic vitamin D precursor 25-OHD3.