Cell Counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). RPMI-1640 media was purchased from Thermo Fisher Scientific (Beijing, China), and fetal bovine serum (FBS) was obtained from Gibco Life Technologies (Carlsbad, CA, USA). Propidium iodide (PI) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Zedoray Turmeric Oil Injection, containing 0.1 g/10 ml CZEO, was purchased from Xuzhou Lai’en Pharmaceutical Co., Ltd (Xuzhou, China). The main components of CZEO are neocurdione, curdione, germacrone, curzerene, furanodiene, γ-elemene and 8,9-dehydro-9-formyl-cycloisolongifolene (15).

The SKOV3 human ovarian cancer cells were purchased from the Cell Bank of the Cinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco Life Technologies), at 37°C in a humidified atmosphere containing 5% CO₂.

The SKOV3 cells were dispersed in culture medium supplemented with 10% FBS and were seeded in a 96-well plate at a density of 2×10⁴ cells/ml. The cells were then treated with PTX (10 nM; Xi’an Sanjiang Bio-Engineering Co. Ltd, Xi’an, China), CZEO (62.5 μg/ml) or a combination of the two for 48 h, following 24 h growth. Cell in the control group were treated with 0.2 ml phosphate-buffered saline (PBS). A total of 10 μl CCK-8 was added to each well, and the cells were then cultured in an incubator for a further 3 h. The optical density (OD) of the cells was measured at 490 nm using a microplate spectrophotometer (Spectramax 190; Molecular Devices Corp., Sunnyvale, CA, USA). Each concentration corresponds to three parallel wells for detection. The cell viability was calculated as follows: Cell viability=(O_{Dtreated group}-OD_blank)/(OD_control-OD_blank)×100. The interaction between PTK and CZEO was analyzed using CalcuSyn 2.0 software (Biosoft, Cambridge, UK), using the Chou and Talalay method (16). The combination index (CI) was determined on the basis of the isobologram analysis: CI<1, synergistic effect; CI=1, additive effect; and CI>1, antagonistic effect.

The SKOV3 cells (5×10⁵ cells/ml) were seeded in six-well plates, and cultured overnight. Following treatment with PTX (10 nM), CZEO (62.5 μg/ml) or a combination of the two for 24–48 h in a 37°C incubator containing 5% CO₂, the cells were incubated with Hoechst 33342 (5 μl; Sigma-Aldrich) for 30 min. Hoechst 33342-stained cell nuclei were observed using an inverted fluorescence microscope (BX60; Olympus Optical Co., Tokyo, Japan), and images were captured with a confocal microscope (LSM510; Carl Zeiss AG, Oberkochen, Germany).

The SKOV3 cells (5×10⁵ cells/ml) were seeded in six-well plates and cultured for 12 h. Following treatment of the cells with PTX, CZEO, or a combination of the two for 48 h in a 37°C incubator containing 5% CO₂, the cells were collected and washed twice with cold PBS in order to remove the medium. The cells were then resuspended in 100 μl of 1X binding buffer (Invitrogen Life Technologies, Carlsbad, CA, USA), prior to the addition of 5 μl Annexin-V (Invitrogen Life Technologies) and 1 μl propidium iodide (PI; Sigma-Aldrich) and incubation in the dark on ice. Finally, 400 μl of 1X binding buffer was added to the cells and they were analyzed by flow cytometry using a BD FACSAria cell sorter (Becton-Dickinson, San Jose, CA, USA).

The SKOV3 cells were seeded in six-well plates and treated as mentioned in the previous section. The cells were then harvested, washed twice with PBS and resuspended in 0.3 ml PBS. RNase A (Roche, Indianapolis, IN, USA) was added, in order to digest the cells for 30 min at 37°C, after which the cells were collected and washed twice with PBS. The reaction was terminated by placing the mixture on ice. PI was added to the cells in the dark, in order to prepare the samples for flow cytometry.

The SKOV3 cells (5×10⁵ cells/ml) were seeded in 60 mm culture dishes in the presence of PTX, CZEO, or a combination of the two for 48 h, and cultured in a 37°C incubator containing 5% CO₂. The cells were collected according to the standard western blotting procedure. The protein samples were used straight after protein concentration determination, or were stored at −20°C until further use.

Protein concentration of the samples was determined using bicinchoninic acid protein reagent. (Pierce Biotechnology, Inc., Rockford, IL, USA). The protein samples were separated by SDS electrophoresis on polyacrylamide gels and transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA). The membranes were then blocked in 5% bovine serum albumin (Sigma-Aldrich) for 3 h at room temperature, followed by incubation for 2 h at room temperature with primary antibodies (rabbit polyclonal anti-PARP and anti-caspase-3; 1:1,000; Cell Signaling Technology, Inc., Beverly, MA, USA). The membranes were washed (3×10 min) in tris-buffered saline with Tween^® (TBST) and were then incubated with a horseradish peroxidase-conjugated sheep anti-rabbit secondary antibody (1:2,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature, followed by further washing (3×10 min) with TBST. Immunoblot signals were detected by Odyssey Infrared Imaging v1.2 system (LI-COR Biosciences, Lincoln, NE, USA).

All of the data are expressed as the mean ± standard error of the mean. Student’s t-test was used to determine the statistical significance between two observations and a one-way analysis of variance followed by the Bonferroni test were used for the multiple comparisons. Statistical analyses were performed using SPSS version 11.0 (SPSS Inc., Chicago, IL, USA). P≤0.05 was considered to indicate a statistically significant difference.

Treatment with CZEO or PTX significantly inhibited the viability of SKOV3 cells in a dose-dependent manner, at concentrations between 2.5 and 40 nM PTX, and between 15.625 and 250 μg/ml CZEO (Fig. 1A). Furthermore, treatment with a combination of CZEO and PTX enhanced the suppressive effect on the SKOV3 cells (Fig. 1B; confidence interval, 0.526 to 0.705).

Flow cytometry was used to detect apoptosis, following treatment of the SKOV3 cells with CZEO (62.5 μg/ml), PTX (10 nM), or a combination of the two for 48 h. The cells were collected for dual staining using Annexin V/PI and then underwent flow cytometry. The cells stained with Annexin V but not PI were considered to be in the early-apoptotic phase. Treatment with a combination of CZEO and PTX significantly enhanced the rate of apoptosis (Fig. 2A). The rate of apoptosis of the cells treated with CZEO (62.5 μg/ml), PTX (10 nM) and a combination of the two was 6.4%, 9.5% and 22.2%, respectively.

Flow cytometry was used to perform a cell cycle analysis, following treatment of the SKOV3 cells with CZEO (62.5 μg/ml), PTX (10 nM), or a combination of the two for 24 h. The number of cells arrested at G₂/M phase were significantly increased (Fig. 2B), which may be partially attributed to the anti-proliferative effects of CZEO and PTX.

The SKOV3 cells exhibited morphological changes characteristic of apoptosis following treatment with CZEO (62.5 μg/ml), PTX (10 nM), or a combination of the two. A microscopic observation demonstrated that the cells treated with CZEO (62.5 μg/ml), PTX (10 nM), or a combination of the two for 48 h had significantly reduced growth and exhibited morphological changes characteristic of apoptosis, including widespread chromatin condensation, exocytosis, and condensation and fragmentation of nuclei (Fig. 3).

Hoechst 33342 staining was also performed to detect apoptosis following treatment with CZEO (62.5 μg/ml), PTX (10 nM) or a combination of the two for 48 h. The untreated control cells exhibited normal chromatin without condensation or fragmentation, with no bright staining in the nuclei, thus indicating the absence of dying cells. Whereas, the treated cells exhibited significantly condensed and fragmented chromatin, disintegration of nuclei and formation of apoptotic bodies.

Following treatment with CZEO (62.5 μg/ml), PTX (10 nM), or a combination of the two for 24 h, the cells were harvested for western blotting, in order to detect the expression levels of proteins involved in the apoptotic pathway. Treatment with a combination of CZEO and PTX activated caspase-3, to form a cleaved product of the substrate PARP (Fig. 4).