The Dose-Response of Vitamin D on Cell Proliferation, Differentiation and Apoptosis in Human Osteosarcoma Cells

Abstract

Osteosarcoma is a malignant bone tumor predominantly affecting children and adolescents. Osteosarcoma has a 60-70% survival rate with current treatments; hence there is a need to identify novel adjuncts to chemotherapeutic regimens. The active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25D), is being increasingly recognized for its anti-cancer properties. The dose-response of 1,25D and 25-hydroxyvitamin D3 (25D) was examined on human osteosarcoma cell lines, SaOS-2 (tumorigenic, p53 null, metastatic) and 143B (tumorigenic, ras gene transformed and highly metastatic). It was hypothesized that both forms of vitamin D would inhibit proliferation, stimulate differentiation and induce apoptosis of these cells in a dose-dependent manner. Osteosarcoma cell lines, SaOS-2 and 143B, were treated with 1,25D, 25D or an ethanol control respectively at concentrations ranging from 1-1000nM. Cellular proliferation was measured after 1,25D or 25D exposure using a cell viability assay (MTS), Ki67 immunocytochemistry and cell cycle analysis by flow cytometry. Osteoblastic differentiation was measured by alkaline phosphatase (ALP) activity, osteocalcin secretion and in vitro osteoblastic mineralization. Apoptosis was determined by Terminal Deoxynucleotide Transferase dUTP Nick End Labeling (TUNEL). Neither 25D nor 1,25D inhibited proliferation or affected cell cycle in SaOS-2 or 143B cells, although in 143B cells, proliferation was increased significantly in cells exposed to 25D at 1000nM versus control. Markers of osteoblastic differentiation were upregulated. In particular, a significant increase in ALP in 143B cells and mineralization in SaOS-2 and 143B cells was observed. The effect of 1,25D on apoptosis in SaOS-2 and 143B cells was not significant; 25D at high concentration (1000nM) increased numbers of apoptotic cells significantly (p<0.05). Biological differences between SaOS-2 and 143B control cells were observed. Cell cycle analysis revealed significantly more SaOS-2 control cells in G0/G1 than 143B, and significantly fewer SaOS-2 cells in synthesis phase than 143B (p<0.05). SaOS-2 control cells had ALP levels significantly higher than 143B cells (p<0.05). Both forms of vitamin D (25D and 1,25D) did not inhibit proliferation but acted as differentiation agents in SaOS-2 and 143B cells through activation or upregulation of markers of osteoblastic differentiation, including ALP and/or osteoblastic mineralization. At high concentration (1000nM), 25D increases apoptosis. There are also inherent differences in the biology of SaOS-2 and 143B osteosarcoma cells which modulate 1,25D response.