The human ovarian cancer cell line OVCAR-3 was purchased from the American Type Culture Collection (Manassas, VA, USA) and was grown as a monolayer in RPMI 1640 medium supplemented with 1% (w/v) penicillin/streptomycin and 10% (v/v) fetal bovine serum (FBS) at 37°C in a 5% CO₂ humidified atmosphere. The human ovarian cancer cell line A2780 and the cisplatin-resistant ovarian cancer cell line A2780cis were obtained from the European Collection of Animal Cell Cultures (Salisbury, UK) and maintained as described previously (11). PTX and cisplatin were purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). FBS, penicillin/streptomycin solution, trypsin and RPMI 1640 medium were purchased from HyClone (GE Healthcare Life Sciences, Logan, UT, USA).

A mixture of 2,4-dihydroxyacetophenone (2.0 g, 13.1 mmol), 2-bromopropane (2.47 ml, 26.3 mmol), and aluminum chloride (3.5 g, 26.3 mmol) in CH₂Cl₂ was stirred at 50°C for 12 h in a reflux condenser under argon. 2-Bromopropane (2.47 ml, 26.3 mmol) was added to the reaction mixture three times every 6 h. The mixture was neutralized with 10% NaOH to pH 5.0 and then extracted with ethyl acetate. The organic layer was washed with water three times, dried over Na₂SO₄, concentrated under reduced pressure and purified by medium pressure liquid chromatography (MPLC) to obtain compound 1 with a 46% yield. The ratio of the distance traveled by compound and the distance moved by solvent on the thin-layer chromatography plate (R_f)=0.33 (1:4 ethyl acetate:hexane). ¹H nuclear magnetic resonance (NMR) (400 MHz, CDCl₃): δ 12.61 (s, 1H), 7.52 (s, 1H), 6.35 (s, 1H), 6.31 (s, 1H), 3.22–3.12 (m, 1H), 2.61 (s, 3H), 1.27 (d, J=6.8 Hz, 6H). Electrospray ionization (ESI) mass spectrometry (MS) (me)=195 [M+1]⁺.

A mixture of compound 1 (0.1 g, 0.52 mmol), allyl bromide (1.4 g, 1.3 mmol) and potassium carbonate (0.19 g, 1.3 mmol) in dimethylformamide was stirred for 18 h. The solvent was removed under reduced pressure, and the remaining residue was dissolved in ethyl acetate. The organic layer was washed with saturated NaHCO₃ solution, dried over Na₂SO₄, concentrated under reduced pressure and purified by MPLC to obtain compound 2 with a 99% yield. R_f=0.45 (1:9 ethyl acetate:hexane). ¹H NMR (400 MHz, CDCl₃): δ 7.73 (s, 1H), 6.39 (s, 1H), 6.12–6.01 (m, 2H), 5.46 (s, 1H), 5.42 (s, 1H), 5.34–5.30 (m, 2H), 4.62–4.58 (m, 4H), 3.27–3.18 (m, 1H), 2.60 (s, 3H), 1.20 (d, J=7.2 Hz, 6H). ESI MS (me)=275 [M+1]⁺.

A mixture of compound 2 (0.21 g, 0.75 mmol), benzaldehyde (0.094 g, 0.83 mmol) and KOH (4.0 g) in 2 ml water and 10 ml ethanol was stirred for 1 h. The mixture was neutralized with 6 N HCl to pH 6.0 and then extracted with ethyl acetate. The organic layer was washed with saturated NaHCO₃ solution three times, dried over Na₂SO_4, concentrated under reduced pressure and purified by MPLC (SNAP HP-Sil column; Biotage, Uppsala, Sweden) to obtain compound 3. ¹H NMR (400 MHz, CDCl₃): δ 7.71 (d, J=15.8 Hz, 1H), 7.70 (s, 1H), 7.65 (d, J=15.8 Hz, 1H), 7.60–7.58 (m, 2H), 7.39–7.36 (m, 3H), 6.45 (s, 1H), 6.12–6.02 (m, 2H), 5.48–5.42 (m, 2H), 5.34–5.26 (m, 2H), 4.63–4.60 (m, 4H), 3.12–3.25 (m, 1H), 1.24 (d, J=7.3 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃): δ 190.7, 160.4, 157. 9, 141.5, 135.7, 132.9, 132.9, 130.3, 130.0, 129.3, 129.0, 128.5, 127.7, 121.7, 118.1, 117.7, 97.5, 70.0, 69.1, 26.8, 22.7. ESI MS (me)=363 [M+1]⁺.

The resulting compound 3 was stirred under microwave irradiation (Initiator; Biotage) for 30 min at 120°C in the presence of PdCl₂ (PPh₃)₂ (10 mg) and ammonium formate (150 mg) in 4 ml tetrahydrofuran. The reaction mixture was diluted with ethyl acetate. The organic layer was washed with water, dried over Na₂SO₄, concentrated under reduced pressure and purified by MPLC to obtain compound SY-016 with a 32% yield in two steps. ¹H NMR (400 MHz, CDCl₃): δ 13.29 (bs, 1H), 7.89 (d, J=15.4 Hz, 1H), 7.69–7.65 (m, 3H), 7.59 (d, J=15.4 Hz, 1H), 7.46–7.43 (m, 3H), 6.39 (s, 1H), 6.20 (bs, 1H), 3.22–3.16 (m, 1H), 1.30 (d, J=6.8 Hz, 6H). ¹³C NMR (100 MHz, CDCl₃): δ 192.1, 164.3, 160.8, 144.4, 134.8, 130.7, 129.0, 128.6, 128.0, 127.0, 120.4, 114.2, 103.6, 26.9, 22.6. ESI MS (me)=283 [M+1]⁺ (Fig. 1).

The PTX-resistant ovarian cancer cell line OVCAR-3PTX was induced by continuous exposure of OVCAR-3 cells to PTX at low concentrations, which were gradually increased. When the cells were in logarithmic growth phase, PTX was added to the medium to a final concentration of 20 nM. After 48 h of incubation, the old medium was discarded, and fresh medium was added. Cells were passed when they were 80% confluent, and PTX (20 nM) was then added. The concentration of PTX was gradually increased once the cells had grown stably. Finally, a cell line resistant to 100 nM PTX was derived from the OVCAR-3 cell line, and was maintained in complete medium containing 10 nM PTX.

The number of viable cells exposed to Hsp90 inhibitor were evaluated by a colorimetric MTT assay. The intensity of the purple color formed by this assay is proportional to the number of viable cells. A total of 5×10⁴ OVCAR-3 cells were seeded in 24-well plates and treated for 48 h with PTX or SY-016, and then with 0.5 mg/ml MTT. The optical density (OD) was then measured at 540 nm. The mean value and standard deviation (SD) were calculated from triplicate experiments.

To determine the distribution of cells in the different phases of the cell cycle, profile fluorescence-activated cell sorting analysis was performed. OVCAR-3PTX cells (4–5×10⁵) were seeded in 6-well plates and treated with 10 nM PTX and/or 5 µM SY-016 for 48 h. Next, cells were harvested by trypsinization and centrifugation (500 × g at room temperature for 5 min), washed with cold PBS, and fixed in ice-cold 70% ethanol at 4°C for 24 h. Ethanol-fixed cells were washed and treated with RNase A for 30 min at 37°C. Subsequently, cells were stained with propidium iodide and incubated for 30 min at room temperature. DNA fluorescence was measured by flow cytometry (FACSCalibur™; BD Biosciences, Franklin Lakes, NJ, USA).

Hsp90 inhibitor-treated cells were harvested in 1X radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors with EDTA (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Protein concentrations were measured using Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA) following the manufacturer's protocol. The cell lysates were mixed with 4X sample buffer, boiled for 10 min and then separated by electrophoresis on 10–12% SDS polyacrylamide gels. After electrophoresis, the proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% skimmed milk powder in Tris-buffered saline with Tween-20 at room temperature for 1.5 h and then incubated overnight at 4°C with the following primary antibodies: Anti-β-actin (1:10,000; Sigma-Aldrich; Merck Millipore), anti-Hsp70 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-Hsp90 (1:1,000; Cell Signaling Technology, Inc.), anti-Akt (1:1,000; Cell Signaling Technology, Inc.), anti-nuclear factor (NF)-κB (1:1,000; Cell Signaling Technology, Inc.), anti-B-cell lymphoma (Bcl)-2 (1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-Bcl-2-associated X protein (Bax) (1:1,000; BD Pharmingen, San Diego, CA, USA), anti-X-linked inhibitor of apoptosis protein (XIAP) (1:1,000; BD Pharmingen), anti-cyclin-dependent kinase (CDK)4 (1:1,000; Santa Cruz Biotechnology, Inc.) and anti-survivin (1:1,000; Santa Cruz Biotechnology, Inc.). The blots were next incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc.) at room temperature for 2 h, and signals were detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.). The densitometry of the bands was determined using a ChemiDoc XRS+ imaging system (Bio-Rad Laboratories, Inc.) and normalized to β-actin, which served as the loading control.

Following drug treatment, cells were centrifuged at 500 × g for 5 min and harvested. Cells (2×10⁶) were then lysed in 50 µl lysis buffer, which was from the Caspase Colorimetric Assay kit (Clontech Laboratories, Inc., Mountainview, CA, USA) on ice for 10 min and centrifuged at 10,000 × g for 5 min at 4°C, and the supernatant was then collected. The supernatant (50 µl) was added to an equal volume of 2X dithiothreitol solution supplemented with Asp-Glu-Val-Asp p-nitroanilide (pNA) (50 µM; Caspase Colorimetric Assay kit) and incubated at 37°C for 3 h. Caspase-3 activity was measured as the absorbance at 405 nm of the cleaved substrate pNA, according to the protocol of the Caspase Colorimetric Assay kit (Clontech Laboratories, Inc.).

Fragmented nucleosomal DNA was quantified with the Cell Death Detection ELISAPLUS kit (Roche Diagnostics GmbH, Mannheim, Germany) as described in the manufacturer's manual. Supernatant (20 µl) was used to detect apoptosis with a microplate reader (Tecan Austria GmbH, Grödig, Austria) at 405 nm. Background values were subtracted, and OD values representing nucleosomal DNA fragments in Hsp90 inhibitor-treated samples were compared with those values obtained from control cells and expressed as percentage of control.

The data are presented as the mean ± SD. Statistical analysis was conducted using one-way analysis of variance followed by Duncan's multiple range test for post hoc comparison with SPSS 17.0 (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The potential of SY-016 to inhibit the growth of ovarian cancer cells and chemoresistant ovarian cancer cells was determined by MTT assay. PTX (10 nM) alone inhibited the growth of OVCAR-3 and OVCAR-3PTX cells by 25 and 2%, respectively. The percentages of inhibition of growth of OVCAR-3 and OVCAR-3PTX cells by PTX combined with SY-016 (5 µM) were 68 and 59%, respectively (Fig. 2A). Based on these data, 10 nM PTX and 5 µM SY-016 were employed in all subsequent experiments. Cisplatin (10 µM) inhibited the growth of A2780 and A2780cis cells by 33 and 5%, respectively. The proliferation of A2780 and A2780cis cells was inhibited by 65 and 39%, respectively, when cisplatin was administered in combination with SY-016 (5 µM) (Fig. 2B).

To identify the mechanism of cell growth inhibition by combined treatment with PTX and SY-016, the effects of these drugs on the cell cycle distribution were examined by flow cytometry. As shown in Fig. 3, the percentage of G2/M-phase cells in the control group was 18.1%. In cells treated with PTX and SY-016, the percentage of G2/M-phase cells significantly increased to 31.6%. These results indicate that the cell cycle arrest at the G2/M checkpoint was due to the growth inhibitory effect of SY-016 on PTX-resistant ovarian cancer cells.

Hsp90 is involved in the folding, stabilization and activation of its substrate proteins, which are collectively referred to as ‘client’ proteins. The present study examined whether SY-016 induced the degradation of Hsp90 client proteins in OVCAR-3PTX cells. Combination treatment with PTX and SY-016 significantly reduced the protein levels of Akt, NF-κB and CDK4 (Fig. 4A). In addition, induction of Hsp70, a marker of Hsp90 inhibition (7), was also observed. Combination treatment with PTX and SY-016 effectively downregulated Bcl-2, XIAP and survivin. By contrast, Bax was upregulated following the above combination treatment (Fig. 4B). These results suggest that SY-016 induced cell growth inhibition and apoptotic cell death in part via the inhibition of survival factors and inactivation of the Akt/NF-κB pathway.

The activity of caspase-3, an executioner caspase of apoptosis (6), increased following combination treatment with PTX and SY-016 (Fig. 5A). The ELISA results revealed a significant increase in histone-associated DNA fragments after exposure to PTX and SY-016 for 48 h (Fig. 5B).