Human lung cancer A549 and H460 cell lines were obtained from the ATCC and maintained in RPMI 1640 medium with 10% fetal bovine serum and 1% penicillin. As₂O₃ was purchased from Yida Pharmaceutical Co.(GMP, Ha'erbin, PR. China) and DDP was from Bristol-Myers Squibb Co.(Shanghai, PR. China).

Briefly, cells were seeded at a density of 2,000 to 5,000 cells/well in 96-well plates and incubated overnight. After treatment with As₂O₃, DDP, or their combination (described below), 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) was added (50 μL/well) for 4 hours. Solubilization of the converted purple formazan dye was accomplished by placing cells in 100 μL of 0.01 N HCl/10% SDS and incubating them overnight at 37°C. The reaction product was quantified by absorbance at 570 nm. All samples were repeated three times, and data were analyzed by Student's t test.

Human lung carcinoma cells were counted after trypsinization. Cells were serially diluted to appropriate concentrations and removed into 25-cm² flasks in 5-mL medium in triplicate per data point. After various treatments, cells were maintained for 8 days. Cells were then fixed for 15 minutes with a 3:1 ratio of methanol:acetic acid and stained for 15 minutes with 0.5% crystal violet (Sigma) in methanol. After staining, colonies were counted by the naked eye, with a cutoff of 50 viable cells. Error bars represent ± SE by pooling of the results of three independent experiments. Surviving fraction was calculated as (mean colony counts)/(cells inoculated)*(plating efficiency), where plating efficiency was defined as mean colony counts/cells inoculated for untreated controls.

Flow cytometry analysis of DNA content was performed to assess the cell cycle phase distribution as described previously6. Cells were harvested and stained for DNA content using propidium iodide fluorescence. The computer program Multicycle from Phoenix Flow System (San Diego, CA, USA) was used to generate histograms which were used to determine the cell cycle phase distribution and apoptosis. TUNEL staining was also used to detect apoptosis as described previously 7. The TUNEL stained apoptotic cells were separately numbered in four randomly selected microscopic fields (400*) and graphed.

After various treatments, cells were washed with ice-cold PBS twice before the addition of lysis buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium NaPPi, 1 mM phenylmethylsulfonyl fluoride, and leupeptin). Protein concentrations were quantified separately by the Bio-Rad Bradford assay. Equal amounts of protein were loaded into each well and separated by 10% SDS PAGE, followed by transfer onto nitrocellulose membranes. Membranes were blocked using 5% nonfat dry milk in PBS for 1 hour at room temperature. The blots were then incubated with anti-p21, anti-cyclin D1, anti-bax, anti-bcl-2, anti-clusterin, and anti-caspase-3 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. Blots were then incubated in secondary antibody conjugated with HRP (1:1000; Santa Cruz Biotechnology) for 1 hour at room temperature.

Immunoblots were developed using the enhanced chemiluminescence (ECL) detection system (Amersham, Piscataway, NJ) according to the manufacturer's protocol and autoradiography.

The MTT assay showed that 10^-2 μM to 10 μM inhibited the proliferation of A549 and H460 at 72 hours (Fig. 1). In vitro clonogenic assay proved 10^-1 μM to 12.5 μM As₂O₃ inhibited the proliferation of A549 and H460 cells (Fig. 2). MTT assay results showed that 2.5 μM As₂O₃ and 3 μg/ml DDP exerted synergistic inhibition effects on A549 and H460 cells at 48 hours. (Fig. 3A,B). To confirm the synergistic effects of As₂O₃ with DDP CalcuSyn™ program (Version 2.0, Biosoft, Inc., UK) was explored to make dose-effect curves and to determine the combination indices (CI) (Fig. 4A,B). The CI for A549 and H460 were 0.5 and 0.6, respectively which confirmed the synergism of As₂O₃ with DDP.

A549 cells were treated with 2.5 μM As₂O₃ and/or 3 μg/ml DDP for 48 hours. FCM cell cycle analysis showed that the treatment of As₂O₃ and/or DDP did not significantly alter G0/G1 fractions of A549 cells compared with those of the control. The G0/G1 fraction ranged from 57% to 62% for controlled A549 cells and cells treated with As₂O₃ and/or DDP; the G0/G1 fraction ranged from 37% to 42% for controlled H460 cells and cells treated with As₂O₃ and/or DDP (Fig. 5). Western blot analysis showed that As₂O₃ and/or DDP did not affect the expression of cell cycle related protein p21 and cyclin D1 (data not shown).

A549 cells were treated with 2.5 μM As₂O₃ and/or 3 μg/ml DDP for 48 hours. FCM analysis showed the apoptotic indices (AI) for the controlled A549 cells and cells treated with As₂O₃, DDP, or the combination were 0.25 ± 0.01%, 10.6 ± 0.53%, 15.85 ± 0.79%, and 20 ± 1%, respectively. The AI for the controlled H460 cells and cells treated with As₂O₃, DDP, or the combination were 1.95 ± 0.11%, 13.6 ± 0.65%, 7.53 ± 0.43%, and 35.6 ± 1.71%, respectively (Fig. 6). As₂O₃ and DDP significantly increased the AI compared with the control cells. TUNEL staining was performed to further confirm AI results from FCM analysis. With TUNEL staining, the AI for the control A549 cells, cells treated with As₂O₃, DDP, or the combination were 3.1 ± 0.16%, 15.41 ± 0.77%, 14 ± 0.7%, and 30 ± 1.5%, respectively. The AI for the control H460 cells, cells treated with As₂O₃, DDP, or the combination were 5.95 ± 0.25%, 18.6 ± 1.13%, 9.53 ± 0.49%, and 40.6 ± 2.11%, respectively (Fig. 7). Western blot analysis showed Bax expression increasing by 2-fold in the A549 cells treated with As₂O₃ and DDP over levels in control cells. In H460 cells treated with As₂O₃ and DDP, Bax expression was 3.7 times greater than in the control (Fig. 8). Bcl-2 expression was 72% less in the As₂O₃ and DDP treated A549 cells than in control cells, and 25% less in the As₂O₃ and DDP treated H460 cells than in control cells (Fig. 9). Expression of another tumor suppressed protein, clusterin, was 70% less in the As₂O₃ and DDP treated A549 cells than in control cells, and in H460 cells, clusterin expression was 90% less with treatment with the combination of As₂O₃ and DDP than in control cells (Fig. 10). For both A549 and H460, caspase-3 expression increased with the treatment of As₂O₃ and/or DDP over control levels, but caspase-3 expression was not different in cells treated with the combination of As₂O₃ and DDP and cells treated with each single agent (Fig. 11).