The present study was conducted with the recommendations in the guide for the Care and Use of Laboratory Animals and was approved by the ethics committee of The Second Hospital of Tianjin Medical University, Tianjin, China (approval no. 20151105DGB). All animal protocols were conducted and maintained in accordance with the National Institutes of Health and approved by the ethics committee of Animal Experiments Defence Research of the Second Hospital of Tianjin Medical University (Tianjin, China; Approval validity: Oct 2015 to Oct 2017).

CAOV-3 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). All cells were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.). All cells were cultured in a 37°C humidified atmosphere containing 5% CO₂.

CAOV-3 cells (1×10³ cells/well) were incubated with PTX (5, 10 and 15 mg/ml) or PL-PTX (5, 10 and 15 mg/ml) in 96-well plates for 24, 48 and 72 h at 37°C in triplicate for each condition, with PBS serving as a control. Following incubation, 20 µl MTT (5 mg/ml) in PBS was added to each well and the plate was further incubated for 4 h. The majority of the medium was removed and 100 µl dimethyl sulfoxide was added into the wells to solubilize the crystals. Optical density was measured with an ELISA reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at a wavelength of 570 nm.

CAOV-3 cells (1×10⁵ cells/well) were cultured at 37°C in an environment containing 5% CO₂ until 90% confluence was attained. CAOV-3 cells were then incubated with PTX (10 mg/ml), PL-PTX (10 mg/ml) or extracellular signal-regulated kinase (ERK) inhibitor (cat. no. ab142271; Abcam, Cambridge, UK) treatment for 48 h at 37°C. For the invasion assay, 1×10⁵ CAOV-3 cells were suspended in 500 µl serum-free RPMI (Thermo Fisher Scientific, Inc.) and 500 µl RPMI with 5% FBS (Thermo Fisher Scientific, Inc.) was ploaced in the lower chamber for 48 h at 37°C. The cells were plated in the upper chambers of BD BioCoat Matrigel Invasion Chambers (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. Cotton swabs were used to remove cells and to plate cells. To investigate migration, CAOV-3 cells (1×10⁶) were cultured in RPMI medium in the upper chamber and RPMI medium with 5% FBS (Thermo Fisher Scientific, Inc.) in the lower chamber. Cells were then plated in a control insert chamber (BD Biosciences) for 48 h at 37°C. Cells were stained using 0.1% crystal violet dye (Sigma-Aldrich; Merck KGaA) for 30 min at 37°C. Tumor cell migration and invasion were observed in ≥3 randomly-selected stained-fields per membrane under a light microscope (BX51; Olympus Corporation, Tokyo, Japan).

CAOV-3 cells were then incubated with PTX (10 mg/ml), PL-PTX (10 mg/ml) or ERK inhibitor (cat. no. ab142271; Abcam) for 48 h at 37°C. Cells were collected and lysed in radioimmunoprecipitation assay buffer (M-PER reagent for cells and T-PER reagent for tissues; Thermo Fisher Scientific, Inc.) followed by homogenization at 4°C for 10 min. Protein concentration was measured with a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). A total of 20 µg protein was electrophoresed via 12.5% SDS-PAGE and subsequently transferred to nitrocellulose membranes. Prior to incubation with primary antibodies at 4°C overnight, membranes were incubated with blocking buffer (5% milk) for 2 h at 37°C. The primary antibodies used were against: Caspase-9 (1:1,200; ab32539), ERK (1:1,000; ab54230) and phosphorylayed (p)ERK 1/2 (phospho-Thr202/Tyr204; 1:1,000; ab214362), caspase-3 (1:1,200; ab2171), protein kinase B (AKT; 1:1,000; ab8805), p-AKT (phosphor-S473; 1:1,000; ab8932) and β-actin (1:500; ab8226; all Abcam) for 12 h at 4°C. Horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG; Bio-Rad Laboratories, Inc.) was applied to membranes at a 1:5,000 dilution for 2 h at 37°C and detected using an enhanced chemiluminescence substrate ECL Select™ (Roche Diagnostics, Basel, Switzerland). The density of the protein bands was analyzed by Quantity One software version 4.62 (Bio-Rad Laboratories, Inc.).

CAOV-3 cells (5×10⁶) were then incubated with PTX (10 mg/ml), PL-PTX (10 mg/ml) or and or ERK inhibitor (ERKIR; IR7936) and/or anti-TNFα (1:1,000; cat. no. ab6671; 2 mg/ml; both Abcam) treatment for 48 h at 37°C. Following trypsinization, cells were washed in cold PBS and adjusted to 1×10⁶ cells/ml using PBS 3 times at room temperature; CAOV-3 cells were labeled with Annexin V-fluorescein isothiocyanate (V-FITC) and propidium iodide (Annexin V-FITC kit, BD Biosciences) for 2 h at 37°C, according to the manufacturer's protocol, and analyzed with a FACScan flow cytometer (BD Biosciences) using BD FACSChorus™ intuitively designed software version 1.2 (BD Biosciences).

Specific pathogen-free male Balb/c mice (n=90; 8 weeks old; 32–35 g body weight) were purchased from the Shanghai Laboratory Animal Center Co., Ltd. (Shanghai, China). All mice were housed separately and maintained in a 12 h light/dark cycle with 23±1°C with a relative and humidity of 50±5%. All mice had free access to food and water. Nude mice were subcutaneously injected with CAOV-3 cells (1×10⁵) into the right forelimb under aseptic conditions. All mice were housed in a temperature-controlled facility at 23±1°C with free access to food and water. Mice were randomly divided into three groups (n=30 per group) and received a treatment of 10 mg/kg PTX, 10 mg/kg PL-PTX or PBS by intravenous injection. Treatments were initiated on day 3 following tumor implantation (diameter: 5–6 mm) and continued 10 times daily for a total of 20 days. Tumor volumes were calculated once every 3 days according to the formula: V=0.5xa²xb, where a: Short diameter and b: Long diameter of tumor as measured with a Vernier caliper.

Tumors from xenograft mice, extracted as previously described (16), were fixed by using 10% formaldehyde for 4 h at 37°C and embedded in paraffin (4-µm thick sections). Antigen retrieval was performed using a microwave to heat the sections (standard microwave settings; 20s) and graded series of ethanol, followed by blocking of endogenous peroxidase activity with 3% hydrogen peroxide for 10 min at room temperature. Tumor sections were incubated with 5% BSA (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 2 h at 37°C and then incubated with rabbit anti-mouse primary antibodies caspase-9 (1:1,200; ab32539) or caspase-3 (1:1,200; ab2171; both Abcam) for 12 h at 4°C. Tumor tissues were washed with PBS three times and incubated with biotinylated secondary antibodies anti-rabbit IgG (1:2,000; Pierce; Thermo Fisher Scientific, Inc.) for 2 h at 37°C. Biotin-peroxidase signals were detected using 0.5 mg/ml 3′3′-diaminobenzidine (DAB)/0.003% H₂O₂ (Dako; Agilent Technologies, Inc. Santa Clara, CA, USA) as a substrate. Results were recorded using a laser confocal microscope (BX51; Olympus Corporation).

Apoptotic cells (5×10⁶) of tumor specimens were fixed using 4% formaldehyd for 30 min at 37°C and analyzed using a TUNEL assay (DeadEnd™ Colorimetric TUNEL System; Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. Tumor sections were incubated with the reaction mixture (terminal deoxynucleotidyl transferase, equilibration buffer and biotinylated nucleotide mix) for 1 h at 37°C. Subsequently, streptavidin- and DAB-bound biotin was quantified and counterstained with hemalum (1%; Merck KGaA, Darmstadt, Germany) and aquatex (Merck KGaA) for 1 h at 37°C. Tumor sections were washed with PBS three times for 5 min at room temperature. DNA fragmentation was analyzed in 3 randomly selected fields of 4-µm tumor sections using light microscope (magnification, ×40).

Each experiment was performed at least three times. All data are expressed as the mean ± standard deviation of triplicate dependent experiments and analyzed by using one-way analysis of variance followed by a Tukey test. All data were analyzed using SPSS software, version 19.0 (IBM Corp. Armonk, NY, USA), GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) and Microsoft Excel (Microsoft Corporation, Redmond, WA USA). P<0.05 and was considered to indicate a statistically significant difference.

The inhibitory effects of PL-PTX on the growth and aggressiveness of ovarian cancer cells were studied in vitro. PL-PTX and PTX treatment significantly inhibited ovarian cancer cell growth (Fig. 1A). In addition, PL-PTX and PTX inhibited growth of ovarian cancer cells in time-dependent manner (Fig. 1B). Treatments of 10 mg/ml PL-PTX or PTX significantly inhibited migration and invasiveness of ovarian cancer cells (Fig. 1C and D). The results of the present study indicated that PL-PTX significantly inhibited the growth and aggressiveness of ovarian cancer cells.

As presented in Fig. 2A, the apoptotic ability of CAOV-3 cells was promoted by PL-PTX. Significantly increased caspase-3 and caspase-9 activities were detected within PL-PTX-treated CAOV-3 cells (Fig. 2B). PL-PTX treatment increased caspase-3 and caspase-9 protein expression levels in CAOV-3 cells (Fig. 2C). These results demonstrated that PTX- and PL-PTX induced apoptosis of CAOV-3 cells (Fig. 2D). The results of the present study suggested that PL-PTX promoted ovarian cancer cell apoptosis via the caspase-dependent signaling pathway.

TNF has been reported to promote tumor cell apoptosis (17,18); the effects of TNF on PL-PTX-induced apoptosis were investigated in the present study. Employment of AntiTNF, an anti-TNF neutralizing antibody, partially reduced PTX- and PL-PTX-inhibited growth and aggressiveness of CAOV-3 cells (Fig. 3A-C). The results of the present study demonstrated that PL-PTX treatment led to a reduction in ERK and AKT expression and phosphorylation levels; however, anti-TNF inhibited the effects exhibited by PL-PTX (Fig. 3D and E). Additionally, inhibition of the TNF significantly reduced PTX- and PL-PTX-induced CAOV-3 cell apoptosis (Fig. 3F). These results suggested that PL-PTX may be associated with ovarian cancer metastasis, which is mediated by the TNF-induced inhibition of the ERK/AKT signaling pathway.

Associations between ERK/AKT and the caspase-3 cascade within CAOV-3 cells were analyzed in the present study. The inhibition of ERK/AKT activity using ERKIR decreased the expression levels of caspase-3 within CAOV-3 cells compared with in cells control group (Fig. 4A). ERKIR inhibited TNF-induced apoptosis of CAOV-3 cells compared with cells of the control group (Fig. 4B). PL-PTX-induced CAOV-3 cell apoptosis was also inhibited by an ERK inhibitor (Fig. 4C). The results of the present study suggested that the ERK/AKT signaling pathway may be involved in the activation of the TNF/caspase-3 cascade within ovarian cancer cells.

CAOV-3-bearing mouse model was established and received treatment of PL-PTX (10 mg/kg), PTX (10 mg/kg) or PBS once a day. In vivo analyses revealed that PL-PTX and PTX treatments significantly inhibited the growth of ovarian cancer cells compared with cells of the PBS groups in a 20 day observation (Fig. 5A). TUNEL analysis revealed that PL-PTX treatment significantly promoted tumor cell apoptosis compared with in cells of the PTX and PBS group (Fig. 5B). Caspase-3 and caspase-9 expression levels were upregulated in response to PL-PTX and PTX treatments (Fig. 5C). In addition, prolonged survival was observed within the PL-PTX treated group compared with in the PTX and PBS treated groups (Fig. 5D).