ABSTRACT
A range of biological and molecular effects caused by nicotine are considered to effect bone metabolism. Vitamin C functions as a biological antioxidant. This study was to evaluate the in vitro effects of nicotine on human bone marrow stromal cells and whether Vitamin C supplementation show the antagonism action to high concentration nicotine. We used CCK-8, alkaline phosphatase (ALP) activity assay, Von Kossa staining, real-time polymerase chain reaction and Western Blot to evaluate the proliferation and osteogenic differentiation. The results indicated that the proliferation of BMSCs increased at the concentration of 50, 100 ng/ml, got inhibited at 1,000 ng/ml. When Vitamin C was added, the OD for proliferation increased. For ALP staining, we found that BMSCs treated with 50 and 100 ng/ml nicotine showed a higher activity compared with the control, and decreased at the 1,000 ng/ml. Bone morphogenetic protein-2 (BMP-2) expression and the calcium depositions decreased at 100 and 1,000 ng/ml nicotine, while the addition of Vitamin C reversed the down regulation. By real-time PCR, we detected that the mRNA expression of collagen type I (COL-I) and ALP were also increased in 50 and 100 ng/ml nicotine groups (P < 0.05), while reduced at 1,000 ng/ml (P < 0.05). When it came to osteocalcin (OCN), the changes were similar. Taken all together, it is found that nicotine has a two-phase effect on human BMSCs, showing that low level of nicotine could promote the proliferation and osteogenic differentiation while the high level display the opposite effect. Vitamin C could antagonize the inhibitory effect of higher concentration of nicotine partly.
Subject(s)
Antioxidants/pharmacology , Ascorbic Acid/pharmacology , Bone Marrow Cells/metabolism , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Ganglionic Stimulants/pharmacology , Nicotine/pharmacology , Osteogenesis/drug effects , Adult , Aged , Ascorbic Acid/antagonists & inhibitors , Bone Marrow Cells/cytology , Bone Morphogenetic Protein 2/biosynthesis , Cells, Cultured , Dose-Response Relationship, Drug , Drug Antagonism , Female , Ganglionic Stimulants/antagonists & inhibitors , Gene Expression Regulation/drug effects , Humans , Male , Middle Aged , Nicotine/antagonists & inhibitors , Stromal Cells/cytology , Stromal Cells/metabolismABSTRACT
Thymoquinone (TQ), the predominant bioactive constituent derived from the medicinal spice Nigella sativa (also known as black cumin), has been applied for medical purposes for more than 2,000 years. Recent studies reported that thymoquinone exhibited inhibitory effects on the cell proliferation of several cancer cell lines. This study was performed to investigate the antitumor and anti-angiogenic effects of thymoquinone on osteosarcoma in vitro and in vivo. Our results showed that thymoquinone induced a higher percentage of growth inhibition and apoptosis in the human osteosarcoma cell line SaOS-2 compared to that of control, and thymoquinone significantly blocked human umbilical vein endothelial cell (HUVEC) tube formation in a dose-dependent manner. To investigate the possible mechanisms involved in these events, we performed electrophoretic mobility shift assay (EMSA) and western blot analysis, and found that thymoquinone significantly downregulated NF-κB DNA-binding activity, XIAP, survivin and VEGF in SaOS-2 cells. Moreover, the expression of cleaved caspase-3 and Smac were upregulated in SaOS-2 cells after treatment with thymoquinone. In addition to these in vitro results, we also found that thymoquinone inhibits tumor angiogenesis and tumor growth through suppressing NF-κB and its regulated molecules. Collectively, our results demonstrate that thymoquinone effectively inhibits tumor growth and angiogenesis both in vitro and in vivo. Moreover, inhibition of NF-κB and downstream effector molecules is a possible underlying mechanism of the antitumor and anti-angiogenic activity of thymoquinone in osteosarcoma.