Department of Orthopaedics, the First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, China.
Chin Med J (Engl). 2010 Jul;123(13):1768-73.
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.
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.
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.
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.
骨肉瘤是一种常见的原发性恶性骨肿瘤，由于其易发生转移，预后较差。患者的预后高度依赖于是否存在肺转移以及对其的治疗效果。据报道，人骨肉瘤细胞中 Fas 蛋白的低表达与肺转移密切相关。大量研究表明，三氧化二砷（ATO）能抑制多种癌细胞系的增殖并诱导其凋亡；然而，其对人骨肉瘤细胞（Saos-2 细胞系）的影响尚不清楚。本研究旨在探讨 ATO 对 Saos-2 细胞的作用，并探讨其 Fas 表达的机制。
一组 Saos-2 细胞分别用或不用 0.5、1、2、4 和 8 微摩尔/升的 ATO 处理 3 天，并用 3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四唑溴盐（MTT）测定 ATO 的细胞毒性。用吖啶橙/溴化乙锭（AO/EB）双重染色研究细胞形态变化。用流式细胞术（FCM）检测细胞 DNA 分布。另一组细胞先用 10 纳摩尔/升基质金属蛋白酶 7（MMP-7）预处理 3 小时，然后再用或不用 2 微摩尔/升的 ATO 孵育 24、48 和 72 小时。用 MTT、Western blot 和实时 PCR 分别系统研究细胞毒性、Fas 蛋白和 mRNA 水平。本研究检测了细胞增殖、细胞周期进程和细胞凋亡。
ATO 呈剂量和时间依赖性抑制 Saos-2 细胞的增殖。24、48 和 72 小时的 IC（50）值分别为 9.30、5.54 和 3.49 微摩尔/升。在 MMP-7 和 ATO 共同处理组中，Saos-2 细胞的存活率明显高于 ATO 组，但低于对照组。ATO 诱导 Saos-2 细胞 G（1）期细胞周期阻滞，并通过流式细胞术检测到亚 G（1）DNA 含量和 AO/EB 染色，有效地刺激细胞凋亡。Western blot 结果表明，ATO 处理后骨肉瘤细胞 Fas（FasL）蛋白表达呈时间依赖性显著增加。与对照组相比，用 2 微摩尔/升和 4 微摩尔/升的 ATO 处理 48 小时，Fas 基因表达分别增加到 28.31%和 56.74%。我们的结果表明，ATO 诱导的 Saos-2 细胞凋亡可能是通过 Fas 途径介导的。
ATO 呈剂量和时间依赖性抑制 Saos-2 细胞的增殖，并增加 Fas 蛋白表达。然而，MMP-7 不完全阻断 Fas 介导的凋亡，这表明其他分子机制可能介导这一过程。
