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Aug 22, 2009 - Abstract Given that arsenic trioxide (As2O3) has been successfully used as a chemotherapeutic agent for refrac- tory malignant tumors, this ...
Mol Biol Rep (2010) 37:2509–2515 DOI 10.1007/s11033-009-9765-2

Arsenic trioxide inhibits the growth of Adriamycin resistant osteosarcoma cells through inducing apoptosis Hui Zhao Æ Wei Guo Æ Changliang Peng Æ Tao Ji Æ Xinchang Lu

Received: 8 March 2009 / Accepted: 14 August 2009 / Published online: 22 August 2009 Ó Springer Science+Business Media B.V. 2009

Abstract Given that arsenic trioxide (As2O3) has been successfully used as a chemotherapeutic agent for refractory malignant tumors, this study is aimed at investigating the effect of As2O3 on human Adriamycin resistant osteosarcoma cell line Saos-2. The mechanism underlying multi drug resistance (MDR) in osteosarcoma cells and the anti-tumor effect of As2O3 on Adriamycin resistant osteosarcoma cells were analyzed. In our experiment, we first selected Adriamycin resistant osteosarcoma cell line by growing the classic osteosarcoma cell line Saos-2 in the medium with increasing drug concentrations. Then, we compared the IC50s of the osteosarcoma cells treated with different anticancer drugs by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay. Subsequently, we assessed the expression of classic MDR related molecules, Pgp, multidrug resistance-associated protein (MRP) and glutathione (GSH) activity in the wild type and Adriamycin resistant Saos-2 cells. Furthermore, the apoptosis was assessed by concerning DNA fragment and flow cytometry with Annexin-V staining. To elucidate the underlying mechanism of the apoptosis, related proteins Bcl-2, Bcl-xL, Bax, Bak, cleaved Caspase-3 and cleaved Caspase-9 were analyzed by western blotting. The data showed that the resistance to Adriamycin affected the sensitivity of osteosarcoma cell to other chemotherapeutic agents. The IC50s of Saos-2/ADM cells for methotrexate (1.74-fold), Each author certifies that his or her institution has approved the experimental protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research. H. Zhao  W. Guo (&)  C. Peng  T. Ji  X. Lu Musculoskeletal Tumor Center, Peking University People’s Hospital, #11 South Xizhimen Street, 100044 Beijing, People’s Republic of China e-mail: [email protected]

Cisplatin (1.43-fold) and As2O3 (1.21-fold) were increased compared with Saos-2 control cells. The expression of Pgp was upregulated comparing with the control cells. No significant difference was detected about the MRP and the glutathione-S-transferase activity and intracellular GSH concentration among different treated osteosarcoma cells. Apoptosis was observed and proved. The western blotting showed that the expression of Bcl-2 and Bcl-xL was downregulated. Meanwhile, the level of Bax, Bak, cleaved Caspase-3 and cleaved Caspase-9 was upregulated after treated with As2O3. The study suggests that Adriamycin resistant osteosarcoma cells have good response to As2O3based chemotherapy in vitro, probably via the pathway of inducing apoptosis. And As2O3 might serve as an excellent alternative candidate for adjuvant chemotherapeutic agent on this incurable pediatric sarcoma. Keywords MDR

Arsenic trioxide  Osteosarcoma  Apoptosis 

Introduction Arsenic trioxide (As2O3), an effective anti-cancer agent for acute promyelocytic leukemia (APL) found in 1970s, has been recognized as a potential alterative agent for chemotherapy [1]. During the last decade, numerous studies had demonstrated that the As2O3 were effective in cytotoxicity, such as gastric cancer, lung cancer, breast cancer, hepatocellular carcinoma, esophageal carcinoma, cervical cancer and ovarian cancer. And the past studies have shown that As2O3 could induce cytotoxic effects on these tumors in a dose and time dependent manner [2]. The detailed mechanisms of As2O3 cytotoxicity have not been completely elucidated, but many preclinical studies have

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provided insight into the processes involved. The mechanism includes induction of apoptosis, cellular differentiation, anti-proliferation and inhibition of angiogenesis. Osteosarcoma is the most common primary malignant bone tumor, mainly occurring in children and adolescents. With current treatment strategies, including a combination of limb salvage surgery and neo-adjuvant chemotherapy, long-term disease-free survival rates of 60–76% would be resulted among localized disease patients, while for patients who are poorly responsive to chemotherapy; there is a higher risk of relapse and adverse outcome [3, 4]. The most challenging problem facing orthopedics oncologist is to counter the multidrug resistance (MDR) induced by the classic chemotherapeutic agents, such as Methotrexate, Adriamycin and Cisplatin [3–6]. Therefore, there is crying need for exploring mechanism of chemotherapy-resistance and developing new or additional treatment regimen on chemotherapy-resistant osteosarcoma. As far as the chemotherapeutic protocol is concerned, Adriamycin combined with high dose Methotrexate and Cisplatin were used in the preoperative chemotherapy to osteosarcoma. After surgery, the surgeons and pathologists together reviewed and evaluated chemotherapeutic responses. Responses were evaluated following the criteria previously reported. And graded ‘‘good’’ ([90% tumor necrosis) or ‘‘poor’’ (\90% tumor necrosis). With regard to the patients with graded ‘‘good’’, the postoperative chemotherapeutic protocol was the same as the preoperative protocol. While, the graded ‘‘poor’’ indicated that the tumor cells might be resistant to the preoperative agents. And alternated chemotherapeutic agents should be applied in the postoperative chemotherapy [7]. To get a better understanding of the intricate mechanism underlying anticancer agents’ resistance in the neo-adjuvant chemotherapy, we established a model of the tumor suffered from the preoperative chemotherapy and treated with alternative agent for the postoperative chemotherapy. In this study, human Adriamycin-resistant osteosarcoma cell line Saos-2/ADM was selected in vitro by growing them in progressively increasing drug concentrations in the medium. Then, the Saos-2/ADM cells were treated with other different agents and their IC50s were compared. Subsequently, expression of classic MDR related molecules was analyzed and the lurked network pathway of apoptosis was assessed to explore the mechanism of As2O3 induced antitumor effect.

Methods and materials Cell lines and culture The human osteosarcoma cell line Saos-2, was obtained from Memorial Sloan-Kettering Cancer Center. Human

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Adriamycin-resistant osteosarcoma cell line Saos-2/ADM was selected in vitro by growing them in progressively increasing drug concentrations in the medium as described previously [8]. The Saos-2/ADM cells were approximately 49.82-fold more resistant to Adriamycin than sensitive wild type Saos-2 cells. All the cells were routinely maintained in RPMI medium 1640 supplemented with 10% heat-inactivated fetal calf serum in 37°C humidified incubator with a mixture of 95% air and 5% CO2, fed every 3 days with complete medium, and subcultrued when confluence was reached. In vitro drug sensitivity assay Methotrexate (MTX), Adriamycin (ADM), Cisplatin (CDDP), and As2O3 were freshly prepared before each experiment. Drug sensitivity was assessed using 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [9]. Briefly, cells were diluted with the standard culture medium to the seeding density (1 9 104/ well), suspended in 96-well plates (200 ll/well), and incubated at 37°C for 24 h. Then, cells were incubated for 72 h in the absence or presence of various concentrations of the anticancer agents in 200 ll medium. Each assay was performed according to the manufacturer’s instructions. All samples and standards were run in triplicate. After cells being cultured for 72 h, 50 ll of 2 mg/ml MTT (Sigma Amresco) was added into each well, and cells were cultured for another 4 h. Then, supernatants were discarded and 150 ll DMSO was added into each well to dissolve crystals. Absorbance was determined by spectrophotometry (Biohit, BP800, Finland) using a wavelength of 490 nm. Cell survival rates were calculated according to the formula: survival rate = (mean A490 of treated wells/mean A490 of untreated wells) 9 100%. Finally, the IC50s of different anticancer drugs to the osteosarcoma cells were calculated. Western blotting Cells were plated at 5 9 105 per well in six well plates. The following day, cells were treated with different anticancer drugs. After treatment, for whole-cell extracts, cells were washed with PBS and lysed in the culture dishes using lysis buffer (150 mM NaCl, 50 mM Tris–HCl, pH 7.4, 2 mM EDTA, 1% NP-40) containing protease inhibitors. Then the total cellular proteins were quantified by Bradford method. Equal amounts of total protein (50– 100 lg per lane) were electrophoresed in 12% SDS–PAGE and blotted on a nitrocellulose membrane (0.45 lm, Millipore, USA) in 25 mM Tris-base, 190 mM glycine, and 20% methanol using a semi-dry blotter. Membranes were blocked with 8% fat-free milk powder at room temperature

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for 2 h and incubated overnight with primary antibody at 4°C. After three washes for 15 min in PBS-Tween 20, the membrane was incubated with the horseradish peroxidaseconjugated goat anti-mouse IgG antibody for 1 h at room temperature. The membrane was washed again in PBSTween 20; enhanced chemiluminescence (Amersham, NJ) was added and monitored for the development of color. The following antibodies were used: anti-Pgp, anti-MRP, anti-Bcl-2, anti-Bcl-xL, anti-Bax, anti-Bak, anti-cleaved Caspase-3, anti-cleaved Caspase-9, and anti-GAPDH. All these antibodies were from Santa Cruz (CA). The density of the bands was assessed using Multianalyst software. Glutathione-S-transferase activity and GSH measurements Total glutathione-S-transferase (GST) activity of osteosarcoma cells was evaluated by conjugation of reduced glutathione to 1-chloro-2,4-dinitrobenzene [10]. In brief, cells were resuspended in tetramethylsilane buffer and sonicated at 4°C for 30 s. After centrifuging, 100 lg cellular fractions were added to cuvettes containing 1.0 mM 1-chloro2,4-dinitrobenzene and 1.0 mM reduced glutathione at pH 6.5, and the final volume was adjusted to 1.0 ml by addition of PBS. Change in absorbance (340 nm) was measured over a span of 3 min to calculate the rate of conjugation of 1-chloro-2,4-dinitrobenzene. Intracellular GSH contents of osteosarcoma cells were measured using the GSH-400 assay kit (OXIS, Portland). Briefly, cells in log phase were harvested and resuspended in freshly prepared 5% metaphosphoric acid. The suspension was sonicated for 30 s and centrifuged at 3,000g for 10 min at 4°C. The supernatant (100 ll) was used in the assay for the detection at 400 nm of thione formed from the conjugation of reduced glutathione to 4-chloro-1-methyl-7-trifluoromethyl-quinolinium methylsulfate. DNA fragmentation assay After incubation with 0.8 mg/l As2O3 for 72 h, cells were collected and washed twice with ice-cold PBS [11]. Cell pellets were resuspended in lysis buffer and incubated

overnight at 50°C with continuously shaking. The nucleic acids were extracted with phenol–chloroform, precipitated with ethanol–sodium acetate, and redissolved in deionized water containing 100 mg/ml RNase A. After incubation in a water bath at 37°C for 30 min, the DNA samples were analyzed on 1.5% agarose gel containing 0.1 lg/ml ethidium bromide. Annexin V staining After incubation with 0.8 mg/l As2O3 for 72 h, cells were washed twice with cold PBS and resuspended in 100 ll binding buffer at a concentration of 1 9 106/ml. Then, 5 ll Annexin V-FITC (BD Biosciences, USA) and 10 ll of 20 lg/ml propidium iodide (50 lg/ml, Sigma) were added to these cells. After incubation at room temperature for 15 min, 400 ll Annexin-binding buffer was added to each sample, and the samples were kept on ice for counting the stained cells by flow cytometry. Annexin V binds to those cells that express phosphatidylserine on the outer layer of the cell membrane, and propidium iodide stains the cellular DNA of those cells with a compromised cell membrane. The fluorescence intensity of individual cells was measured by a flow cytometer (Becton–Dickinson, USA), at least 10,000 cells were counted and the data were analyzed using Cell fit Analysis Software. Viable cells were negative for both PI and Annexin V; apoptotic cells were positive for Annexin V and negative for PI, whereas late apoptotic dead cells displayed both high Annexin V and PI labeling Non-viable cells which underwent necrosis, were positive for PI and negative for Annexin V. The apoptotic percentage of 10,000 cells was determined. Statistical analysis The data were expressed as means ± SD. We determined the changes of IC50s, the GST activity and intracellular GSH content by ANOVA analysis of variance test. The statistical package SPSS was used to analyze the data (SPSS Inc., Chicago, IL). P \ 0.05 was considered significant.

Table 1 IC50s (lg/l) of anticancer drugs for osteosarcoma cells ADM Saos-2 Saos-2/ADM

MTX

CDDP

As2O3

136.31 ± 24.80

23.93 ± 5.09

117.78 ± 18.01

894.77 ± 287.29

7,293.81 ± 1,263.72

41.72 ± 12.88

167.87 ± 47.93

1,082.42 ± 293.71

Survival rate of osteosarcoma cell to anticancer agents were evaluated by MTT assay as described in materials and methods. The IC50 were determined by the dose–effect curves of different anticancer agents. Data are means ± SD of different independent experiment P \ 0.01 versus Saos-2-MTX and Saos-2/ADM-MTX P \ 0.01 versus Saos-2-CDDP and Saos-2/ADM-CDDP P \ 0.01 versus Saos-2-As2O3 and Saos-2/ADM-As2O3

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Results

Mol Biol Rep (2010) 37:2509–2515 Table 2 GST activity (nmol/min/mg protein) and GSH (mg/mg protein) in osteosarcoma cells

In vitro drug sensitivity assay The wild type Saos-2 cells and Adriamycin resistant Saos2 cells were treated with different anticancer agents for 72 h. The IC50 of Saos-2 and Saos-2/ADM cells for MTX, CDDP and As2O3 were shown in Table 1. The IC50s of Saos-2/ADM cells for MTX (1.74-fold) and CDDP (1.43-fold) were increased compared with Saos-2 control cells. Moreover, differentiation was also found in the IC50 of cells treated with As2O3 (1.21-fold). Taken together, the resistance to Adriamycin would affect the sensitivity of osteosarcoma cell to different anticancer drugs. Effect of anti-cancer agents on classic MDR molecules To study the possible molecular mechanism involved in MDR of osteosarcoma, Pgp and MRP, two well characterized drug transporters, were examined in osteosarcoma cells as show in Fig. 1. The relative expression level of Pgp to GAPDH was markedly higher in Saos-2/ ADM cells (1.68-fold) and different anticancer drugs treated Saos-2/ADM cells [MTX (1.86-fold), CDDP (1.97-fold), As2O3 (2.12-fold)] compared with non resistant wild type Saos-2 control cells. Meanwhile, all these cell lines exhibited no obvious difference of MRP expression. GST activity and intracellular GSH content Glutathione-S-transferase-mediated drug-detoxifying systems were detected in all Saos-2 cells. Compared with control wild type Saos-2 cells, the resistant cells showed slightly altered GST activity and GSH concentration (Table 2). However, statistical analysis revealed that these changes between the As2O3 treated cells and other agents treated cells were not significant (P [ 0.05).

Fig. 1 Western blot analysis of Pgp and MRP in osteosarcoma cells. GAPDH was used as an internal control

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Saos-2

GST

GSH

16.22 ± 3.07

0.37 ± 0.13

Saos-2/ADM

18.53 ± 2.79

0.40 ± 0.11

Saos-2/ADM-MTX

17.31 ± 2.14

0.39 ± 0.12

Saos-2/ADM-CDDP

18.21 ± 1.70

0.43 ± 0.11

Saos-2/ADM-As2O3

18.21 ± 3.60

0.41 ± 0.24

P [ 0.05 among Saos-2, Saos-2/ADM, Saos-2/ADM-MTX, Saos-2/ ADM-CDDP and Saos-2/ADM-As2O3

Effect of As2O3 on apoptosis As induction of apoptosis is an important mechanism of anticancer drugs. We investigated the capacity of Saos-2 and Saos-2/ADM cells to undergo As2O3-induced apoptosis by DNA fragment and Annexin V staining. Treatment of Saos-2 cells with 0.8 mg/l As2O3 for 72 h resulted in internucleosomal DNA fragmentation, evidenced by the formation of DNA ladders on agarose gels (Fig. 2a), a hallmark of cells undergoing apoptosis. No DNA ladders were detected both in the As2O3 untreated osteosarcoma cells. Similarly, results of Annexin V staining suggested that As2O3 could induce both of the osteosarcoma cells apoptosis and the apoptotic rate of As2O3 treated osteosarcoma cells was significantly higher than that of untreated cells (Fig. 2b). Effect of As2O3 on proteins regulating apoptosis To gain insight into the molecular mechanisms involved in As2O3-mediated apoptosis, the expressions of Bcl-2, BclxL, Bax, Bak, Caspase-3, and Caspase-9 were assessed in Saos-2 cells treated with As2O3. As shown in Fig. 3, the expression of Bcl-2 protein and Bcl-xL was decreased in response to As2O3. However, the level of Bax and Bak was upregulated in osteosarcoma cells, and the expression of cleaved Caspase-3 and cleaved Caspase-9 were increased after As2O3 treatment.

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Fig. 2 Apoptosis induced by As2O3. a Different osteosarcoma cells were incubated with 0.8 mg/l As2O3 for 72 h. Total DNA was extracted from cells and subjected to DNA fragmentation assay by electrophoresis on 1.5% agarose gel. b Annexin V/propidium iodide

Fig. 3 Detection of apoptosis related molecules in Saos-2 cells by western blot. Three different concentration of As2O3 were evaluated: 0.05, 0.2 and 0.8 mg/l. GAPDH was used as internal control

Discussion In the present study, we compared As2O3 with other anticancer agents on the Adriamycin-resistant osteosarcoma

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binding analyses of different osteosarcoma cells were presented. The apoptosis index of As2O3 treated osteosarcoma cells were significantly more than that of untreated osteosarcoma cells

cells, assessed the IC50 and the expression of classic MDR related molecules in the treated resistant Saos-2 cells. Then, the possible underlying anticancer mechanisms of As2O3 were further investigated. To obtain a better model in which cells of the same origin could be compared, we selected Adriamycin-resistant osteosarcoma cells by growing them in increasing drug concentration in the medium. MTT assay revealed that the Adriamycin-resistance would have influence on the sensitivity of other three kinds of anticancer drugs. Adriamycinresistant osteosarcoma cells showed a 53.6-fold increase of resistance to Adriamycin compared with control cells (P \ 0.01). It was interesting to find that Saos-2/ADM, selected by drug resistance to Adriamycin, not only showed resistance to Adriamycin in itself, but also changed sensitivity of Saos-2 to other anticancer drugs (Table 1). When Saos-2/ADM cells were treated with different anticancer agents, it showed a 1.74-fold increase of resistance to MTX (P \ 0.01), a 1.43-fold increase of resistance to CDDP (P \ 0.01) and a 1.21-fold increase of resistance to As2O3 (P \ 0.01). It should be noted that Adriamycin is the common substrates for Pgp and MRP. To clarify the association of Pgp and MRP with other anticancer drugs, we investigated their expression in the treated Adriamycinresistant osteosarcoma cells. The results showed that Pgp might mediate the Adriamycin-related MDR osteosarcoma. The Pgp, which is encoded by MDR1 gene and functions as an ATP-dependent drug efflux pump, discards the Adriamycin out of the cell membrane to decrease the intracellular concentrations [12]. It is a subject of future experiment to explore the exact

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mechanism by which Pgp influences the Adriamycin resistance of osteosarcoma. How Adriamycin resistant cells affected the sensitivity to MTX, CDDP and As2O3 might be explained by upregulation of Pgp via cross-resistance to Adriamycin [13–15]. Meanwhile, more studies have shown that the As2O3 based chemotherapy would also confer resistance in the application on the APL patients [16], and the cellular MRP1 and GSH levels were associated with sensitivity of this novel chemotherapeutic agent [17]. Thus, we further tested whether the GSTmediated drug detoxifying system was involved in the process of drugs treatment. GST catalyzed the conjugation of reduced glutathione to some anticancer drugs, which deprived these drugs of the possibility to reach their cellular targets. It has been reported that increased GST activity and reduced glutathione content in drug-resistant tumor cells were associated with resistance to nitrogen mustards, Memphian, and CDDP [18–20]. We detected total GST activity and intracellular GST content in all osteosarcoma cells. The result showed that GST mediated drug-detoxifying system was not found significantly involved in Adriamycin resistance. It has been reported that As2O3 would induce osteosarcoma cell line apoptosis in vitro and in vivo, but the intrinsic mechanism has not been explored [21–23]. The As2O3 has been used to APL for about three decades. And numerous studies had demonstrated that the As2O3 were effective in cytotoxicity for chemotherapy to tumors. But the metabolism of intra-cellular As2O3 is not completely defined. Furthermore, the pathway of apoptosis of As2O3 treated cells was explored for elucidating the mechanism of its anticancer effect. First, we examined the externalization of phosphatidylserine (PS) by flow cytometry with Annexin V-PI staining. PS externalization was observed in majority of treated cells. Meanwhile, to explore the mechanism proceeding to apoptosis induced by As2O3 on Saos-2 cells, several parameters were examined. Bcl-2 protein and Bcl-xL expression was down regulated after treatment of 0.8 mg/l As2O3 for 72 h. The level of Bax, Bak, cleaved Caspase-3 and cleaved Caspase-9 was upregulated in osteosarcoma cells after As2O3 treatment. During the past decade, extensive efforts have been made to explore the mechanisms by which As2O3 induces apoptosis and proliferation inhibition of tumor cells. Several pathways were involved in these events, for instance, the elevation of reactive oxygen species, loss of mitochondrial membrane potential, and release of cytochrome-c, etc., followed by activation of the Caspase cascade and programmed cell death [24, 25]. Moreover, the activation of JNK and the inhibition of the NF-kB also played an important role in these processes [26, 27]. Bcl-2 family, including bcl-2, bcl-xl, bax, bad, and bak, was a rapidly expanding family of proteins in mitochondrial-mediated apoptosis. These proteins were believed to modulate apoptosis by forming homodimers or

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heterodimer with other Bcl-2 family members. Bcl-2 and BclxL act as negative regulars in mitochondrial-mediated apoptosis, which counteract the apoptosis stimulatory effect of Bax and prevent the release of cytochrome-c from mitochondria. Our studies showed that suppression of Bcl-2 and Bcl-xL protein expression by As2O3 directly or indirectly reduced the anti-apoptotic stimulus in osteosarcoma cells and in turn enhanced the induction of apoptosis. Some studies have demonstrated that downregulation of Bcl-2 involved in apoptosis induction and increased the sensitive of tumor cells to chemotherapeutic drugs. Meanwhile, the augmentation of Bax and Bak would facilitate the releasing of cytochrome-c from mitochondria [28, 29]. The subsequent directly or indirectly activation of Caspase-3 and Caspase-9 by the cytochrome-c and Apaf-1 contributes to the central trigger of the apoptotic process. This activation also appears to be responsible for many molecular and structural changes of apoptosis. In our studies, cleaved Caspase-3 and cleaved Caspase-9 antibodies were employed to detect the changes of the expression of Caspases [30]. The expression of cleaved Caspase-3 and cleaved Caspase-9 were increased after As2O3 treatment, thereby the apoptosis of osteosarcoma cells was induced. The current standard chemotherapy for patients with osteosarcoma consists of MTX, Adriamycin and CDDP plus limb salvage surgery. With these therapies, 50–65% of patients could achieve remission, and 50% of patients could be cured. However, due to the induced MDR by these classic chemotherapeutic agent, 20–30% of patients would experience relapse afterwards [3–6]. Because As2O3 has been used in clinical trials of APL for years and 0.4 mg/l of As2O3 was shown to be effective in inducing complete remission with no severe toxicity [31]. Moreover, our studies found that the IC50 concentration of As2O3 at a level of 0.894 mg/l with 72 h for osteosarcoma cells. These suggest that the clinical achieve concentration of 0.8 mg/l might exhibit its anti-cancer effect to patients with osteosarcoma through the induction of apoptosis. And As2O3 may be an alternative and applicable agent for chemotherapy on osteosarcoma. Since 2002, patients suffered from metastatic refractory osteosarcoma were treated with arsenic trioxide combined with other chemo agents after standard chemotherapy and surgical treatments in our previous clinical practices. About 15% patients would get complete response and 20% would get partial response and about 40% patients would get stable disease [21]. It must be emphasized that this study was performed using only Saos-2 cells, its relevance to osteosarcoma cells and its clinical implications need to be further investigated. In conclusion, our study demonstrated that the proliferation inhibition and apoptosis induction effects of As2O3 on osteosarcoma cell line Saos-2. These results suggested the use of As2O3 for the chemotherapy of human osteosarcoma.

Mol Biol Rep (2010) 37:2509–2515 Acknowledgments One or more of the authors (Guo Wei, MD, PhD) have received funding from the Capital Development Foundation of Beijing (Grant no. 2005-1009).

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