The cell lines used (presented in Table I) in this study were obtained from the American Type Culture Collection (Manassas, VA, USA) or Shanghai Cell Bank (Shanghai, China). DMEM, F12, RPMI-1640 and fetal bovine serum (FBS) were purchased from Gibco BRL (Gaithersburg, MD, USA). 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium-bromide (MTT) was obtained from Sigma-Aldrich (St. Louis, MO, USA). 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcar-bocyanine iodide (JC-1) was obtained from Molecular Probes Life Technologies (Carlsbad, CA, USA). The cytochrome c enzyme-linked immunosorbent assay kit was obtained from R&D systems (Minneapolis, MN, USA). Caspase-3, caspase-8 and caspase-9 activity assay kits were obtained from Kaiji Institute of Biological Engineering (Nanjing, China). The Cell Death Detection ELISA plus kit was from obtained Roche Applied Sciences (Basel, Switzerland). Primary monoclonal antibodies (rabbit anti-caspase-3, #9665, 1:2,000; rabbit anti-caspase-8, #4790, 1:2,000; mouse anti-caspase-9, #9508, 1:2,000; rabbit anti-Bcl-2, #2870, 1:3,000; and rabbit anti-Bax, #5023, 1:3,000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and secondary goat anti-rabbit (NEF812001EA, 1:5,000) and goat anti-mouse (NEF822001EA, 1:5,000) horseradish peroxidase-labeled IgG antibodies were purchased from PerkinElmer, Inc. (Boston, MA, USA). Taxol and cisplatin were obtained from Lukang Pharmaceutical Co., Ltd. (Jining, China). All solvents and other chemicals used in this study were of analytical grade and were obtained from Sinopharm chemical reagent Co., Ltd. (Shanghai, China). X609 was synthesized and identified by the institute of Materia Medica, Shangdong Academy of Medical Sciences (Jinan, China), and had a purity of 98%. For the in vitro experiments, treated chemicals were dissolved in DMSO (Sigma-Aldrich). For the animal experiments, taxol and cisplatin were prepared freshly using sterile saline. X609 was prepared in 0.5% carboxymethylated cellulose as a stock solution, and stored was at −20°C prior to use.

T47D cells were cotransfected with 0.01 μg pRL-CMV (Promega Corporation, Madison, WI, USA) and 0.2 μg pGL2-TK-HRE, with a firefly luciferase gene driven by 3 tandem repeats of HRE sequences (13), using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 24 h, cells were treated by test compounds for 30 min, moved to a hypoxia chamber (5% CO₂, 1% O₂, 94% N₂), and incubated for 16 h for the dual luciferase activity assays. VEGF protein expression was measured using an ELISA assay. T47D cells were treated as above, and conditioned media at 37°C were transferred to a new plate for 16 h. The quantity of VEGF was determined according to the manufacturer’s instructions (R&D Systems).

Cells were maintained in DMEM, DMEM/F12 or RPMI-1640 medium, supplemented with 10% FBS, 100 U/mL Penicillin and 100 μg/mL Streptomycin, at 37°C in humidified 95% air/5% CO₂. Cell viability following treatments with tested compounds (manas-santin A, 10 nM-10 μM; X609, 10 nM-10 μM; taxol, 1–100 nM) for 72 h were determined using an MTT assay, as described previously (14). Briefly, cells in the 96-well plate were incubated with 0.5 mg/ml MTT solution for 4 h. MTT was then dissolved in DMSO. The absorbance was measured at 570 nm, using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). IC₅₀ values were calculated using the software GraphPad Prism 5.0.

The MMP was measured using the fluorescent probe, JC-1. The ratio between aggregate (red) and monomer (green) fluorescence reflects the membrane potential of the mitochondria. Following treatment, MG-63 cells were washed twice with PBS and incubated with JC-1 for 15 min at 37°C in darkness. After rinsing twice, the red/green fluorescence intensity of the cells was determined using a fluorescence microplate reader (Tecan Polarion, Tecan, Reading, UK), at an excitation of 490 nm, and an emission of 530 nm (green fluorescent monomers) or 590 nm (red fluorescent aggregates). The change in MMP was expressed as a percentage of the negative control, which was set to 100%.

Cytosolic cytochrome c was measured using the assay kit. Following treatment, MG-63 cells were washed twice with PBS and fractionated. Cytochrome c was measured according to the manufacturer’s instructions. The optical density was measured using a microplate reader (3550; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 490 nm. The level of cytochrome c was expressed as a percentage of the negative control, which was set to 100%.

Quantification of DNA fragmentation was measured using the Cell Death Detection ELISA plus kit. Following treatment, MG-63 cells were washed twice and lysed for 30 min. Cell extracts were centrifuged at 50 × g for 10 min at 4°C. The supernatant (20 μl) was transferred to a streptavidin-coated microplate, and incubated with a mixture of anti-histone-biotin and anti-DNA-peroxidase (Roche Diagnostics Corporation, Indianapolis, IN, USA). The peroxidase in the immunocomplex was quantified by adding 2,2′-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (Sigma-Aldrich) as the substrate. The absorbance was measured using a microplate reader at 405 nm. The extent of DNA fragmentation was expressed as a percentage of the negative control, which was set to 100%.

Caspase activity was measured using the assay kit, according to the manufacturer’s instructions. Following treatment, MG-63 cells were washed twice with PBS and lysed with a buffer, consisting of 1 mM EGTA-Na, 2 mM MgCl₂, 25 mM HEPES (pH 7.5), 0.1% CHAPS, 2 mM DTT, 100 mM PMSF and 1:100 broad spectrum protease inhibitor. Following centrifugation at 45,000 × g for 10 min at 4°C, aliquots of supernatants containing 25 mg protein were added to a reaction buffer that was supplemented with 0.1% CHAPS, 100 mM PMSF and 5 mM DTT. The reactions were initiated following addition of the following fluorescent substrates (50 mM final concentration): Ac-DEVD-Amc, for caspase-3 activity; Ac-IETD-Amc, for caspase-8 activity; and Ac-LEDH-Afc, for caspase-9 activity. After incubating for 2 h at 37°C, the cleavage of the substrates was measured (Amc: 390/475 nm; Afc: 400/505 nm) using a microplate reader. The activity of caspase was expressed as a percentage of the negative control, which was set to 100%.

Following treatment, MG-63 cells were harvested and washed twice with PBS. Total cellular protein was isolated using the protein extraction buffer and quantified using the protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). Cell lysates in 5X SDS sample buffer were boiled for 5 min, and equal amounts of protein (50 μg) from each sample were then subjected to electrophoresis on a 12% (v/v) SDS-polyacrylamide gel (Bio-Rad Laboratories, Inc.). Following electrophoresis, proteins were electroblotted onto a PVDF membrane (EMD Millipore, Temecula, CA, USA). After blocking with PBST (Sigma-Aldrich) containing 5% dried non-fat milk at room temperature, membranes were washed three times and incubated with the indicated primary antibodies at 4°C overnight, followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h. Following incubation, membranes were washed three times, and the antigen-antibody complexes were visualized using the enhanced chemiluminescence system (PerkinElmer). The relative protein expression, with the control defined as 100%, was measured using ImageJ software (National Institutes of Health, Bethseda, MA, USA).

The animal experiment was approved by the Ethics Committee of The Jinan Military General Hospital (Jinan, China). Female nude mice (BALB/c-nu/nu; 7 weeks old) were obtained from the Experimental Animal Center of Shandong (Jinan, China) and were housed in a sterile environment at 25°C on a 12-h light/dark cycle. MG-63 cells (1×10⁷) were injected subcutaneously into the right flank of five nude mice. After being allowed to grow for 3–4 weeks, nude mice were sacrificed by CO₂ inhalation, tumors were detached and weighed, cut into 2×2×2 mm³ pieces, and implanted subcutaneously into the right flank of 40 nude mice with a gauge trocar. When the tumor volume reached to 100–200 mm³, mice were randomized into four groups (8 mice per group: control, cisplatin, X609 50 mg/kg and X609 100 mg/kg groups), and treated with the tested compounds (X609, 50 mg/kg and 100 mg/kg; cisplatin, 5 mg/kg). Cisplatin (5 mg/kg) was injected twice weekly intraperitoneally for three weeks. X609 was administered orally for 6 days per week for three weeks. Tumor volume was calculated as described previously (15).

Data are presented as the mean ± standard deviation, and were analyzed using one-way analysis of variance, with the Sigma Stat statistical software, version 4.0 (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Manassantin A is an efficacious HIF-1 inhibitor. Based on its structure, a novel derivative, X609 (Fig. 1A), was designed and synthesized for use in the present study. Its HIF-1 inhibitory activity was determined using a cell-based HIF-1 reporter assay. Compared with the eight chiral centers of manassantin A, X609 has only two chiral centers, meaning it is easier to prepare and purify. Furthermore, this novel derivative inhibited HIF-1 activity more effectively than manassantin A (IC₅₀, 0.32 nM and 58 nM, respectively). In accordance with this HIF-1 inhibitory activity, X609 significantly downregulated the hypoxia-induced expression of VEGF, a known HIF-1 target gene (Fig. 1B).

In addition to the effect on HIF-1 activity, the antiproliferative activities of X609 on 12 different cancer cell lines was investigated using an MTT assay. Manassantin A and X609 each selectively inhibited the growth of osteosarcoma, and lung and pancreatic cancer cell lines. Notably, X609 appeared to exhibit a more potent antiproliferative activity than manassantin A, as the IC₅₀ values of this compound were generally 2–20-fold less than those of manassantin A (Table I). Furthermore, the IC₅₀ values under hypoxic conditions were lower than those under normoxic conditions, suggesting that X609 may inhibit cell growth through an HIF-1-dependent mechanism.

X609 significantly reduced cell viability, as demonstrated by the MTT assay. In order to explore the mechanisms underlying its antitumor activity, the expression of apoptosis-related markers was measured. Treatment with X609 led to an marked decrease in the MMP (Fig. 2A); significantly increased the quantity of cytochrome c released into the cytoplasm (Fig. 2B); increased the DNA fragmentation in MG-63 cells (Fig. 2C); and enhanced the activities of caspase-3, caspase-8 and caspase-9 (Fig. 2D), compared with the control group. Western blotting results confirmed that the expression of the proapoptotic proteins, caspases and bax, was increased, while that of the antiapopotic protein, bcl-2, was decreased, following treatment with X609 (Fig. 2E). Therefore, X609 appeared to exert its antitumor effect by inducing apoptosis.

In order to measure the antitumor activity of X609 in vivo, human osteosarcoma MG-63 cells were implanted into nude mice, and mice carrying established tumors were then treated with 50 or 100 mg/kg of X609. Cisplatin was used as a positive control. At a dose of 100 mg/kg, X609 significantly inhibited growth of MG-63 xenograft tumors (Fig. 3), in terms of tumor volume as well as tumor weight, at the time of mouse sacrifice. The relative tumor volume was reduced to 29.5% of that of the control, and the tumor weight was reduced to 65% of that of the control. At a dose of 50 mg/kg, X609 also reduced tumor volume and weight. Notably, X609 had no significant effect on mouse body weight during the experiments, suggesting that X609 is minimally toxic in mice. These results indicate that X609 has the potential for use in the treatment of osteosarcoma.