Tan IIA was isolated from extracts of the roots of Salvia miltiorrhiza Bunge (Danshen) using methanol. Briefly, 2 kg Danshen root was extracted with 4 liters methanol for five days. The supernatant was subsequently filtered through Whatman Grade 4 filter paper (Sigma-Aldrich, St. Louis, MO, USA). The filtrate was concentrated under reduced pressure and the residue was dissolved in ethyl acetate. The ethyl acetate soluble fractions were concentrated under a vacuum and the obtained residue (125 g) was subjected to column chromatography with silica gel 60 (Sigma-Aldrich). The column was packed with methylene chloride and the sample was loaded in methylene chloride. The sample was subsequently eluted with hexane, which was followed by consecutive elution with hexane, containing 2, 5, 8 and 10% ethyl acetate. Tan IIA was purified by recrystallization in hexane and ethyl acetate. The structure of the compound was determined using nuclear magnetic resonance (¹H and ¹³C NMR; JEOL Ltd., Tokyo, Japan) and by comparison with authentic samples.

The HEI-193 cells, an immortalized VS cell line, and Nf2-/-mouse Schwann (SC4) cells were obtained from the House Research Institute (Los Angeles, CA, USA) and were maintained in Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin. To prevent changes in cell morphology, the schwannoma cells were used at passage six. The cell number was calculated using a hemocytometer (Marienfeld-Superior, Lauda-Königshofen, Germany). Hypoxic conditions were induced in an incubator chamber (Billups-Rothenberg, Inc., San Diego, CA, USA), which was flushed with a mixture of 1% O₂, 5% CO₂ and 94% N₂. For normoxic incubation, the gas contained 20% O₂, 5% CO₂ and 75% N₂.

HEI-193 cells and SC4 cells were seeded into 96-well plates at 5×10³ cells per well. Following incubation for 24 h, the culture medium was replaced with medium containing Tan IIA at 1, 3, 5, 7 or 10 µg/ml, with four wells per concentration. Following 24 h treatment, one of the plates was removed and 40 µl fresh 3-[4,5-Dimethylthiazol]-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) in 5 g/l phosphate-buffered saline (PBS) was added to each well. Following incubation for 4 h at 37°C, the culture medium was discarded and 100 µl dimethyl sulfoxide was added to each well. The plates were agitated at room temperature for 30 min to dissolve the formazan crystals and the optical density was measured at 595 nm using a micro-plate reader (VersaMax Microplate Reader with SoftMax Pro Software; Molecular Devices, LLC, Sunnyvale, CA, USA).

Apoptosis was analyzed using an Annexin V assay kit (BD Pharmingen, San Diego, CA, USA). Briefly, the HEI-193 cells were plated at 7×10³ cells per well into six-well plates. Following overnight attachment, the cells were treated with 2 or 4 µg/ml Tan IIA for 48 h. The floating cells in the medium were combined with the attached cells, which had been harvested by trypsinization. The cells were washed with cold PBS and resuspended in 1X binding buffer, containing 10 mM HEPES/NaOH (pH 7.4; BD Pharmingen), 140 mM NaCl and 2.5 mM CaCl₂, at a density of 1×10⁶ cells/ml. Next, the cell suspension was stained with Annexin V-fluorescein isothiocyanate (FITC) and PI, provided with the kit, at room temperature for at least 10 min in the dark. The cells were analyzed using a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) within 1 h of staining. The data were analyzed using WinList 5.0 software (Verity Software House, Topsham, ME, USA).

The enzymatic activity of capase-3 was assessed using a caspase colorimetric assay kit (BioVision Inc., Milpitas, CA, USA), according to the manufacturer's instructions. Briefly, the cells were rinsed twice with ice-cold PBS and lysed in lysis buffer (50 mM Tris-HCl, pH 8.0; 150 MM sodium citrate; 1% NP-40; 0.5% sodium deoxycholate; 0.1% sodium dodecyl sulfate) for 10 min on ice. The lysed cells were centrifuged at 10,000 × g for 5 min and an equal quantity of total protein in each lysate was quantified using a Lowry protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cell lysates were incubated with 50 µl 2X reaction buffer, containing 10 mM dithiothreitol and 200 µM DEVD-pNA substrate at 37°C for 1.5 h. The absorbance was measured at a wavelength of 405 nm on a spectrophotometer.

To assess the mRNA expression levels, total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. Total RNAs (3 µg) were reverse-transcribed at 55°C for 30 min and the cDNAs were amplified by 35 cycles in a PCR mixture (Qiagen Inc., Valencia, CA, USA). The oligo-nucleotide primers used in the PCR reactions were determined from the human HIF-1α sequence and β-actin, and were as follows: Forward, 5′-CCCCAGATTCAGGATCAGACA-3′ and reverse, 5′-CCATCATGTTCCATTTTTCG-3′ for HIF-1α, (701 bp fragment). The mRNA expression levels were normalized against β-actin. The PCR products were electro-phoresed on 1% agarose gels containing ethidium bromide and scanned using a PCR system (2720 Thermal Cycler; Applied Biosystems Life Technologies, Foster City, CA, USA).

To assess the protein expression levels, the cells at 80–90% confluence were starved by culturing in 0.2% FBS in DMEM for a further 20 h. The cells were stimulated with Tan IIA for 48 h and then rinsed twice in PBS (pH 7.4). The cells were lysed on ice for 30 min in radioim-munoprecipitation buffer [50 mM Tris (pH 7.5), 50 mM NaCl, 0.1% SDS, 1% Triton X-100 and 1% sodium deoxycholate], centrifuged at 11,700 x g for 30 min at 4°C and the insoluble materials were collected. The protein concentration was determined using the Lowry protein assay (Bio-Rad Laboratories, Inc.). The supernatant (30 µg) was separated on 6–12% acryl-amide gels (Bio-Rad Laboratories, Inc.) and the proteins were transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked for 30 min at room temperature in Tris-buffered saline, containing 5% non-fat milk and 0.1% Tween 20. The proteins were detected by immunoblotting using the following specific primary antibodies: Monoclonal mouse anti-human HIF-1α (1:500; cat. no. 610958; BD Biosciences), polyclonal rabbit anti-human caspase-3 (1:1,000; cat. no. 9661; Cell Signaling Technology, Inc., Danvers, MA, USA) polyclonal rabbit anti-human phosphorylated-AKT (1:1,000; cat. no. sc-7985R; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), polyclonal rabbit anti-human total AKT (1:1,000; cat. no. 9272; Cell Signaling Technology, Inc.), polyclonal rabbit anti-human phosphorylated-extracellular signal-regulated kinases (ERKs) (1:1,000; cat. no. 9101; Cell Signaling Technology, Inc.), polyclonal rabbit anti-human total ERK (1:1,000; cat. no. 9102; Cell Signaling Technology, Inc.), and monoclonal mouse anti-human α-tubulin (1:5,000; cat. no. CP06; EMD Millipore) overnight at 4°C. Following incubation with the primary antibodies, the membranes were washed three times with Tris-buffered saline containing 0.1% Tween 20, and incubated with horseradish peroxidase-conjugated goat anti-rabbit (1:3,000; cat. no. sc-2004) and goat anti-mouse (1:3,000; cat. no. sc-2005) secondary antibodies (Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. Detection was performed using an ECL-Plus western blotting detection system (GE Healthcare Life Sciences, Piscataway, NJ, USA).

To examine the transcription of HIF-1, the HEI-193 cells were transfected with the firefly lucif-erase reporter plasmid, pGL 4.42 [luc2P/HRE/Hygro]. The HEI-193 cells were plated into DMEM, containing 10% FBS, in a 12-well plate. The cells were transiently transfected with 1 µg luciferase reporter plasmid (Promega Corporation, Madison, WI, USA), along with the desired combination of expression plasmids, using Lipofectamine Plus (Invitrogen Life Technologies), according to the manufacturer's instructions. Following incubation for 6 h, the cells were rinsed and provided fresh 10% FBS/DMEM. Following stabilization, the cells were treated for 24 h with Tan IIA (1 or 3 µg/ml) and were subsequently washed with PBS, lysed with passive lysis buffer and the luciferase activity was measured. The experiments were performed a minimum of three times. The luciferase activity was measured using the dual-luciferase assay system (Promega Corporation) using a luminometer (PerkinElmer, Inc., Waltham, MA, USA).

All data are presented as the mean ± standard deviation from three independent experiments. Statistical analysis by Student's t-test was performed using SPSS 13.0 software for Windows (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

An MTT assay was used to detect the inhibitory effect of Tan IIA on the HEI-193 and SC4 cells. Cell proliferation of the HEI-193 and SC4 cells was inhibited as the concentration of Tan IIA increased from 1 to 10 µg/ml under normoxic and hypoxic conditions (Fig. 1). All experiments were repeated a minimum of three times and revealed similar results.

To demonstrate the induction of apoptosis by Tan IIA, western blotting and flow cytometric analysis with Annexin V/PI staining were performed. Caspase-3 has previously been associated with apoptotic changes in Tan IIA-induced cell death. Tan IIA activated the cleavage of caspase-3 under hypoxic conditions, which is a hallmark of apoptotic activation (Fig. 2A). Exposure to 1 or 3 µg/ml Tan IIA increased the activity of caspase-3 when compared with the untreated control (Fig. 2B). Following flow cytometric analysis, the PI fluorescence was plotted over the Annexin V-FITC fluorescence (Fig. 2C). Using this method, healthy cells exhibited low levels of FITC and PI fluorescence, whereas early apoptotic cells exhibited strong FITC fluorescence, but low PI fluorescence. By contrast, late apoptotic cells exhibited strong FITC and PI fluorescence, whereas dead cells exhibited low levels of FITC fluorescence and strong PI fluorescence. The proportions (%) of apoptotic cells (Q2 + Q4) were significantly higher in the HEI-193 cells that were exposed to 2 and 4 µg/ml Tan IIA (Fig. 2D).

The effect of Tan IIA on the expression of HIF-1α in the HEI-193 cells was assessed. The HIF-1α protein was not expressed in the HEI-193 cells under normoxic conditions and following a 4-h exposure to hypoxia, the expression of HIF-1α was markedly increased in the HEI-193 cells. Tan IIA decreased the hypoxia-induced protein expression of HIF-1α in the HEI-193 cells (Fig. 3A). However, Tan IIA treatment did not alter the mRNA expression of HIF-1α in the HEI-193 cells under normoxic and hypoxic conditions (Fig. 3B). AKT and ERK (upstream modulators of HIF-1α) signaling was not altered by treatment with Tan IIA (Fig. 3C).

The HEI-193 cells were treated with 1 or 3 µg/ml Tan IIA under hypoxic conditions for 4 h. The hypoxia response element (HRE) promoter activity in HEI-193 cells following a 24-h incubation period was assessed using a luciferase reporter assay with the luc2P/HRE/Hygro construct. The expression of HRE-regulated luciferase reporter gene was reduced following treatment with Tan IIA, suggesting that Tan IIA was able to inhibit the translational activity fo HIF-1α (Fig. 4).