The Effects of Ludartin on Cell Proliferation, Cell Migration, Cell Cycle Arrest and Apoptosis Are Associated with Upregulation of p21WAF1 in Saos-2 Osteosarcoma Cells In Vitro

Abstract

BACKGROUND The aim of this study was investigate the effects of the sesquiterpene lactone, ludartin, on cell proliferation, cell migration, apoptosis, and the cell cycle in osteosarcoma cell lines, compared with a normal osteoblast cell line. MATERIAL AND METHODS Osteosarcoma cell lines, MG-63 Saos-2 U-2OS, T1-73 143B, and HOS, and normal hFOB 1.19 osteoblasts, were cultured and treated with increasing doses of ludartin, The MTT colorimetric assay was used to measure cell metabolic activity and viability. Apoptosis was studied by fluorescence-activated cell sorting (FACS) using 4',6-diamidino-2-phenylindole (DAPI) nuclear staining and Annexin-V/propidium iodide (PI) staining. Cell cycle was studied using flow cytometry. Cell migration and invasion were studied using wound healing and Boyden chamber assays. Protein expression was measured by Western blotting. RESULTS Ludartin inhibited cell viability, cell migration, cell proliferation, and increased cell apoptosis, in all osteosarcoma cell lines, with an IC50 dose ranging from 15-30 µM. The greatest effects were on the Saso-2 osteosarcoma cells, with an IC50 of 15 µM. However, ludartin showed minor cytotoxic effects of the normal hFOB 1.19 osteoblasts (IC50 >100 µM). Ludartin exerted its anti-proliferative effects on Saos-2 cells via induction of apoptosis and cell cycle arrest at the G2/M checkpoint, associated with reduced expression of Cdc25c (Ser216), Cdc25c, pCdc2 (Tyr15), and Cdc2 and increased expression of p21WAF1. Ludartin inhibited cell migration and invasion of the Saos-2 cells. CONCLUSIONS The dose-dependent effects of ludartin on cell proliferation, migration, apoptosis, cell cycle arrest at the G2/M checkpoint involved p21WAFI in Saos-2 osteosarcoma cells.

Figure 1

Effect of ludartin on the viability of Saos-2 osteosarcoma cells. (A) The effect of ludartin on osteosarcoma Saos-2 cells in the MTT assay. (B) The effect of ludartin of the morphology of Saos-2 cells. The experiments were performed in triplicate and the values presented as mean ±SD (* P<0.05).

Figure 2

Ludartin triggered apoptosis in Saso-2 osteosarcoma cells. Apoptosis was studied by fluorescence-activated cell sorting (FACS) using 4′, 6-diamidino-2-phenylindole (DAPI) nuclear staining. The experiments were performed in triplicate and the values presented as mean ±SD (* P<0.05).

Figure 3

Estimation of apoptotic of Saos-2 osteosarcoma cell populations at indicated concentrations of ludartin. Annexin-V/propidium iodide (PI) staining. The experiments were performed in triplicate.

Figure 4

Ludartin treatment and G2/M cell cycle arrest in Saso-2 osteosarcoma cells as determined by flow cytometry. The experiments were performed in triplicate.

Figure 5

Western blots showing the effect of ludartin on cell cycle proteins in Saso-2 osteosarcoma cells. (A) Cdc25c, pCdc25c, Cdc2, pCdc2, p21WAFI, and cyclin B1, and (B) p21, p53, and p27. Ludartin treatment increased the expression of cyclin-dependent kinase inhibitor p21 (WAF1) or p21WAF1 in Saso-2 osteosarcoma cells but did not alter the expression of p27 or p53 tumor suppressor proteins. The experiments were performed in triplicate and the values presented as mean ±SD (* P<0.05).

Figure 6

Effect of ludartin at the IC₅₀ of the Saso-2 osteosarcoma cells. (A) Cell migration of Saso-2 osteosarcoma cells. (B) Cell invasion of Saso-2 osteosarcoma cells. (C) Expression of MMP-2 and 9 by Saso-2 osteosarcoma cells. The experiments were performed in triplicate and the values presented as mean ±SD (* P<0.05).