The Effects And Mechanisms Of LY294002on Human Osteosarcoma Cancer Stem-like Cells

Objective1. To isolate and culture human osteosarcoma cancer stem-like cells with serum-free cell culture method and magnetic activated cell sorting method, and to explore the feasibility of both as sorting method.2. To investigate the expression and significance of Nanog in human osteosarcoma and its cancer stem-like cells.3. To investigate the effect of LY294002on the proliferation and cell cycle of human osteosarcoma cancer stem-like cells, and to explore the possible mechanism in the inhibitory effect on proliferation.4. To investigate the effect of LY294002on the apoptosis of human osteosarcoma cancer stem-like cells, and to explore the possible mechanism to enhance apoptosis.Methods1. Human osteosarcoma cancer stem-like cells were isolated with serum-free media. CD133+CD44+human osteosarcoma cancer stem-like cells were isolated with magnetic activated cell sorting method. The expression levels of CD44and CD133in human osteosarcoma cancer stem-like cells were detected by using immunohistochemistry. The expression of Nanog in human osteosarcoma and its cancer stem-like cells were detected by western blot and analyzed by semi-quantitative method.2. Experimental groups:the concentration of LY294002in culture medium was Oumol/L (control group),5μmol/L,15μmol/L and45μmol/L respectively.3. The proliferation of human osteosarcoma cancer stem-like cells under different LY294002concentrations was detected by CCK-8method. The cell cycle disturbutions of human osteosarcoma cancer stem-like cells under different LY294002concentrations was detected by Propyl iodide staining andflow cytometry. Western blotting was employed to detect the expression levels of total Akt, p-Akt and β-actin in human osteosarcoma cancer stem-like cells in different LY294002concentrations.4. Annexin V/PI double staining and flow cytometry was employed to detect the apoptosis of human osteosarcoma cancer stem-like cells under different LY294002concentrations. Western blotting was employed to detect the expression levels of Caspase-3cleaved Caspase-3、Caspase-9、cleaved poly ADP-ribose polymerase.Results1. Human osteosarcoma cancer stem-like cells coule be isolated from human osteosarcoma MG63cells with serum-free media method, and coule grow as cell spheres. The CD133+CD44+human osteosarcoma cells could be isolated from human osteosarcoma MG63cells with magnetic activated cell sorting method, and cells could form cell spheres in serum-free media contained EGF and bFGF. The human osteosarcoma cancer stem-like cells sphere as previously described could be passaged in serum-free media and form similar human osteosarcoma stem-like cells spheres. The osteosarcoma cancer stem-like cells show positive expressions of CD44and CD133. The Nanog was higher expressed in osteosarcoma cancer stem-like cells than human osteosarcoma MG63cells (P<0.05).2. Compared with the control group, the proliferation of human osteosarcoma cancer stem-like cells could be significantly inhibited by LY294002regardless of concentration, and the inhibitory effect of LY294002on osteosarcoma cancer stem-like cells maybe dose-dependent. Compared with the control group, LY294002increase the number of osteosarcoma cancer stem-like cells in G0/G1phase (P<0.01) and a corresponding decrease in proliferation index (P<0.01) after treatment with LY294002. LY294002led to a decrease phosphorylation of AKT, and the expressions of phosphorylated AKT was corresponding decreased as the increasing LY294002concentration.3. The decrease of viable cells was corresponding to a increase in apoptotic cells with different concentrations of LY294002. The effect of apoptotic enhancement also maybe dose-dependent, and in45μmol/L group was significant (P<0.01). Caspase-3expression could be detected in both control group and the intervention groups, but cleaved caspases-3expression could only be obviously observed in the intervention groups, and cleaved caspases-3and poly ADP-ribose polymerase expression increased with concentration of LY294002.Conclusion1. Human osteosarcoma cancer stem-like cells could be isolated and cultured in serum-free medium; Nanog, a key protein in the maintenance of cell pluripotency, may serve as a marker for prostate cancer stem cells for isolation and identification of human osteosarcoma cancer stem-like cells.2. LY294002could inhibit the proliferation of human osteosarcoma cancer stem-like cells, and this effect may be relative to by inhibition of PI3K and its downstream AKT activity, and induction of G0/G1cell cycle arrest.3. LY294002could induce the apoptosis of human osteosarcoma cancer stem-like cells, and the effect may be related to by AKT activity reduction caused by inhibition of PI3K, and activation of Caspase-9, Caspaser3and poly ADP-ribose polymerase.