Arsenic trioxide up-regulates Fas expression in human osteosarcoma cells.
BACKGROUND
Osteosarcoma is a common primary malignant tumor of bone with a poor prognosis due to its propensity for metastasis. The prognosis of patients is highly dependent on the presence or absence of lung metastasis and on the effectiveness of treatment against it. It has been reported that low level expression of Fas protein in human osteosarcoma cell is closely associated with lung metastasis. A large number of studies have shown that arsenic trioxide (ATO) can inhibit proliferation and induce apoptosis of many cancer cell lines; however, its effects on human osteosarcoma cells (Saos-2 cell line) remains unknown. The aim of this study was to investigate the effects of ATO on Saos-2 cells and to characterize its mechanism of Fas-expressing.


METHODS
A group of Saos-2 cells was treated with or without 0.5, 1, 2, 4 and 8 micromol/L ATO for three successive days, and the cytotoxicity of ATO was determined by an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Morphological changes in cells were studied by acridine orange/ethidium bromide (AO/EB) double staining. Flow cytometry (FCM) was used to assay cell DNA distribution. Another group of cells was pretreated with 10 nmol/L matrix metalloproteinase 7 (MMP-7) for 3 hours. They were then incubated with or without 2 micromol/L ATO for 24, 48 and 72 hours. Cytotoxicity, Fas protein and mRNA levels were systematically studied using MTT, Western blotting and real-time PCR, respectively. Cell proliferation, cell cycle progression and apoptosis were examined in this study.


RESULTS
Proliferation of Saos-2 cells was inhibited by ATO in both a dose- and time-dependent manner. The IC(50) values at 24, 48 and 72 hours were 9.30, 5.54 and 3.49 micromol/L, respectively. The survival rate of Saos-2 cells in the MMP-7 and ATO co-treated group was significantly higher than the ATO group, but it was lower than the control group. ATO induced G(1) phase arrest of the cell cycle and very efficiently stimulated apoptosis in Saos-2 cells, as evidenced by flow cytometric detection of sub-G(1) DNA content and AO/EB staining. Western blotting results indicated that Fas (FasL) protein expression in osteosarcoma cultures markedly increases in a time dependent manner after exposure to ATO. Compared with control, treatment with ATO 2 micromol/L and 4 micromol/L for 48 hours, resulted in increase of Fas gene expression to 28.31% and 56.74%, respectively. Our results indicated that ATO induced-apoptosis of Saos-2 cells may be mediated through the Fas pathway.


CONCLUSIONS
ATO suppressed cell proliferation of Saos-2 cell in a dose- and time-dependent manner and increased Fas protein expression. However, Fas-mediated apoptosis was incompletely interrupted by MMP-7, which suggested that other molecular mechanisms may mediate this process.
Yang
Guo-Fu
GF
Department of Orthopaedics, the First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, China.
Li
Xiang-Hui
XH
Zhao
Zhe
Z
Wang
Wen-Bo
WB
D000970:Antineoplastic Agents / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000627:therapeutic use*; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D012516:Osteosarcoma / Q000188:drug therapy* / Q000378:metabolism*; D010087:Oxides / Q000627:therapeutic use*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism*
D000970:Antineoplastic Agents; D001152:Arsenicals; C491901:FAS protein, human; D010087:Oxides; D019014:fas Receptor; D000077237:Arsenic Trioxide