withaferin-a and Osteosarcoma

Withaferin-A (WF-A) is a well-known dietary compound isolated from Withania somnifera. It has marked pharmacological potential and has been shown to exhibit antiproliferative activity against several types of cancerous cells. Currently, the main focus of anti-cancer therapeutic development is to identify apoptosis-inducing drug-like molecules. Osteosarcoma is a rare type of bone cancer affecting humans. The objective of the present study was therefore to evaluate the antitumor potential of WF-A against several osteosarcoma cell lines.. MTT assay was used to evaluate WF-A against osteosarcoma cell lines and to calculate the IC50. DAPI staining was used to confirm the apoptosis-inducing potential of WF-A. Mitochondrial membrane potential, reactive oxygen species (ROS) assay, and Western blotting were used to confirm the basis of apoptosis.. The results of the present study revealed that WF-A exhibited strong antiproliferative activity against all the cells lines, with IC50 ranging from 0.32 to 7.6 µM. The lowest IC50 (0.32 µM) was observed against U2OS cell line and, therefore, it was selected for further analysis. DAPI staining indicated that WF-A exhibited antiproliferative activity via induction of apoptosis. Moreover, WF-A induced a ROS-mediated reduction in mitochondrial membrane potential in a dose-dependent manner and activation of caspase-3 in osteosarcoma cells.. We suggest that WF-A may prove a potent therapeutic agent for inducing apoptosis in osteosarcoma cell lines via generation of ROS and disruption of mitochondrial membrane potential.

Topics: Apoptosis; Cell Line, Tumor; Cell Survival; Humans; Membrane Potential, Mitochondrial; Osteosarcoma; Reactive Oxygen Species; Withanolides

Lithium, a widely used drug for the treatment of mood disorders, has previously been shown to stimulate the release of fibroblast growth factor FGF23, a powerful regulator of 1,25(OH)2D3 formation and mineral metabolism. The cellular mechanisms involved have remained elusive. Lithium has been shown to modify Ca2+ signaling. In a wide variety of cells, Ca2+ entry is accomplished by the pore-forming Ca2+ channel subunit Orai1 and its regulator STIM, which stimulates Orai following Ca2+ depletion of intracellular stores. Transcription factors promoting Orai1 expression include NF-κB. The present study thus explored whether the effect of lithium on FGF23 involves and requires Ca2+ entry.. Experiments were performed in UMR106 osteoblastic cells and immortalized primary osteoblasts (IPO). FGF23 and Orai1 transcript levels were estimated from qRT-PCR, cytosolic Ca2+ concentration ([Ca2+]i) from Fura2 fluorescence and store-operated Ca2+ entry (SOCE) from an increase in [Ca2+]i following store depletion by inhibition of the sarcoendoplasmatic Ca2+ ATPase (SERCA) with thapsigargin (1 µM).. SOCE in UMR106 cells was enhanced by lithium treatment, an effect abrogated by Orai1 inhibitor 2-APB (50 µM). FGF23 transcript levels were increased by lithium and inhibited by Orai1 inhibitors 2-APB (50 µM) and YM58483 (100 nM) as well as NF-κB inhibitors wogonin (100 µM) and withaferin A (500 nM). Moreover, Orai1 transcript levels were up-regulated by lithium, an effect attenuated by wogonin and withaferin A.. Lithium stimulates FGF23 release at least in part by NF-κB dependent up-regulation of Orai1 transcription and store operated Ca2+ entry.

Topics: Analysis of Variance; Animals; Antimanic Agents; Calcium; Calcium-Transporting ATPases; Cell Line, Tumor; Enzyme Inhibitors; Fibroblast Growth Factors; Intracellular Fluid; Lithium; Osteosarcoma; Rats; Thapsigargin; Transcription Factor RelA; Withanolides