Oxymatrine and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA were obtained from Gibco-BRL (Invitrogen Life Technologies, Grand Island, NY, USA). Antibodies were obtained from the following commercial sources: rabbit NF-κB p65 and VEGF antibody (Abcam, Cambridge, UK); rabbit vascular endothelial growth factor receptor-2 (VEGFR-2) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit β-tubulin antibody (Cell Signaling Technology, Inc., Beverly, MA, USA); and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Beyotime Biotechnology, Haimen, China). The ultrasound contrast agent SonoVue was obtained from Bracco (Switzerland). The contrast agent was dissolved in 5 ml 0.9% sodium chloride solution and the final concentration was 45 μg/ml.

The human pancreatic cancer cell line PANC-1 was purchased from the Shanghai Cell Bank (Shanghai, China). Cells were cultured in DMEM with 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin at 37ºC under a humidified 5% CO₂ atmosphere. The medium was replaced every 2–3 days, and the cells were subcultured when confluency reached 70–80% by 0.25% trypsin-EDTA at 37ºC.

The Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Kunamoto, Japan) was used to assess the viability of cells after oxymatrine treatment. PANC-1 cells were seeded into 96-well plates at a density of ~5×10³ cells/well and grown for 24 h. Cells were treated with 0.25, 0.5, 1, 2, 4 and 6 mg/ml oxymatrine or DMSO (control) for 12, 24 or 36 h, and then 10 μl CCK-8 reagent was added to 100 μl of media in each well, and the incubation was continued for a further 3 h. The absorbance (A) of each well was determined with an enzyme-linked immunosorbant assay (ELISA) reader (ELx808; Bio-Tek Instruments, Inc., Winooski, VT, USA) at a wavelength of 450 nm. The percentage of cell viability was calculated using the following equation: Cell viability (%)= (A_sample − A_blank)/(A_control − A_blank) ×100%.

Approximately 5×10⁵ PANC-1 cells/well were seeded into 6-well plates, allowed to adhere overnight, and treated with 0.5, 1, 2 mg/ml oxymatrine or DMSO (control) respectively for 24 h. Treated cells were collected and total proteins were extracted using cell lysis buffer (20 mmol/l Tris-HCl pH 7.5, 150 mmol/l NaCl, 1 mmol/l Na₂EDTA, 1 mmol/l EGTA, 1% Triton, 2.5 mmol/l sodium pyrophosphate, 1 mmol/l β-glycerophosphate, 1 mmol/l Na₃VO₄, 1 μg/ml leupeptin, 1 mmol/l PMSF; Cell Signaling Technology, Inc.). After centrifugation at 14,000 × g for 15 min at 4ºC, the supernatant was collected and the protein concentration was detected using the BCA Protein Assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA), according to the manufacturer’s instructions. Equal amounts of protein were separated on 8–12% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk in TBST washing buffer, the membrane was incubated with the specific primary antibodies at 4ºC overnight. After being washed at room temperature with washing buffer, the blots were labeled with peroxidase-conjugated secondary antibodies. The formed immunocomplex was visualized using an enhanced chemiluminescence kit (Pierce Biotechnology, Inc.) according to the manufacturer’s instructions and exposed to X-ray film.

PANC-1 cells were seeded into 6-well plates at a density of ~5×10⁵ cells/well and grown for 24 h. Cells were treated with 0.5, 1, 2 mg/ml oxymatrine or DMSO (control) respectively for 24 h. Total RNA was isolated from the treated cells using the TRIzol reagent (Invitrogen Life Technologies). For reverse transcription (RT) analysis, 1 μg of total RNA was reverse transcribed in a 20-μl volume, using the RevertAid™ First Strand cDNA Synthesis kit (Fermentas, St. Leon-Rot, Germany). One microliter of the RT reaction mixture was then PCR-amplified in a PCR machine (Eppendorf, Hamburg, Germany). The PCR profile was as follows: 5 min at 94ºC followed by 35 cycles of 30 sec at 94ºC, 30 sec at 54ºC, 30 sec at 72ºC; the final cycle was modified to allow for a 5-min extension at 72ºC. The PCR primers were as follows: NF-κB (300 bp) sense, 5′-AGCACAGATACCACCAAGACCC-3′ and antisense, 5′-CCCACGCTGCTCTTCTATAGGAAC-3′; VEGF (336 bp) sense, 5′-TGCCCACTGAGGAGTCCAAC-3′ and antisense, 5′-TGGTTCCCGAAACGCTGAG-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (533 bp) sense, 5′-CGGAGTCAACGGATTTGGCC-3′ and antisense, 5′-GTGCAGAGATGGCATGGAC-3′. Sequences of the primers were designed using the software Primer Premier 5. GAPDH was used for quantification of the samples. PCR products were electrophoresed at 120 V for 45 min on a 1.2% agarose gel and imaged. The grey value was analyzed using Quantity One version 4.5 software (Bio-Rad, Hercules, CA, USA).

BALB/C (nu/nu), 4 week-old, male mice were purchased from Shanghai Laboratory Animal Center (Shanghai, China) and maintained under specific pathogen-free conditions. Mice were allowed to acclimate for 1 week before the start of the experiments. All animal studies performed in this study were reviewed and approved by the Animal Research and Ethics Committee of Wenzhou Medical College. The orthotopic pancreatic cancer xenograft tumor model was established as described by us previously (20). The dose of oxymatrine was determined by balancing the antitumor and side effects on the basis of previous trials. Through numerous trials, we found that oxymatrine at a dose of 100 mg/kg body weight by intraperitoneal injection 3 days/week can effectively inhibit tumor growth without significant side effects, such as noticeable weight loss and decrease in overall activity. Therefore, oxymatrine treatment (100 mg/kg body weight) by intraperitoneal injection 3 days/week was administered in this study. Briefly, nude mice were anesthetized with pentobarbital sodium, a small left abdominal flank incision was made, and PANC-1 cells (5×10⁶) in 50 μl serum-free media were injected into the subcapsular region of the pancreas. All surgical procedures were conducted under sterilized conditions. One week after cell implantation, a total of 32 nude mice were randomized into 2 groups (control and oxymatrine group) with 16 mice/group. The control group mice were treated with 0.9% sodium chloride and the treatment was continued for 4 weeks.

After the first treatment, 8 mice in each group were used for a survival study which was carried out up to 60 days. When mice died during the period of the survival study, the number of living days were recorded. At the end of the survival study, the living mice were sacrificed. One week after the last treatment, the other 8 mice in each group were used for the study of tumor blood flow detected by contrast enhanced ultrasonography (CEUS). Then the mice were sacrificed, and the tumors were removed. The tumors were weighed with an electronic balance, and tumor volumes were calculated with a vernier caliper according to the following formula: Volume = (4π/3) × (width/2)² × (length/2).

One week after the last treatment, the mice were used for the study of tumor blood flow as detected by CEUS. Mice were anesthetized with sodium pentobarbital (50 mg/kg) and pronely positioned on an operating table. Mice were injected with 20 μl ultrasound SonoVue and 200 μl 0.9% sodium chloride solution per mouse via the tail vein and then imaged. A 1-min data collection was performed after the contrast agent injection. The images were analyzed with the ultrasound contrast analysis software that accompanies the ultrasonic imaging system (Siemens S2000, Germany). A parameter, peak intensity (PI), is in direct proportion to blood flow and was used here for quantification of the tumor blood perfusion in the pancreatic cancer xenografts.

Data are represented as means ± SD for the absolute values or percent of controls. SPSS13.0 software was used for statistical analysis. Differences between the oxymatrine-treated and DMSO-treated (control) groups were analyzed by the unpaired Student’s t-test or ANOVA analysis. A value of P<0.05 was considered to indicate a statistically significant result.

The viability of human pancreatic cancer PANC-1 cells were measured by CCK-8 assay. As shown in Fig. 1, oxymatrine inhibited the cell viability in a dose- and time-dependent manner. Notably, the cell viability was drastically decreased at the range of concentrations of 0.5–2 mg/ml of oxymatrine. However, at concentrations of 0.25–0.5 mg/ml oxymatrine, the cell viability was minimally altered, and higher concentrations of oxymatrine (>2 mg/ml) had a saturated inhibitory effect. Therefore, we chose the concentrations of 0.5, 1 and 2 mg/ml for the following in vitro studies. Meanwhile, we chose one time point (24 h) for the further studies.

We investigated whether NF-κB is involved in the antiproliferative effects of oxymatrine on pancreatic cancer PANC-1 cells. As shown in Fig. 2A, a dose-dependent decrease in NF-κB p65 expression as determined by western blot assay was observed after the cells were exposed to increasing concentrations of oxymatrine. The mRNA levels of NF-κB as detected by RT-PCR analysis was significantly decreased when compared with that in the control group (Fig. 2B, P<0.01).

Our data showed that the protein (Fig. 3A) and mRNA (Fig. 3B) expression levels of VEGF were significantly decreased after PANC-1 cells were treated with 1 or 2 mg/ml oxymatrine for 24 h. However, obvious inhibitory effects on VEGF by oxymatrine were not observed in the 0.5 mg/ml oxymatrine group. Moreover, we found that oxymatrine did not regulate the expression of VEGFR-2 (a receptor of VEGF) as determined by western blot assay in the PANC-1 cells (Fig. 3A).

To further confirm the antiproliferative effects of oxymatrine on pancreatic cancer, we established orthotopic pancreatic cancer xenograft tumors in nude mice. One week after the last oxymatrine treatment, the mice were sacrificed and tumors were removed (Fig. 4A). The weight of the tumors in the vehicle-treated mice was 1.54-fold higher than that of the oxymatrine-treated mice (P<0.01, Fig. 4B). Tumor volumes in the oxymatrine-treated mice and vehicle-treated mice were 438.23±123.40 and 702.73±101.00 mm³, respectively (P<0.01, Fig. 4C). The median survival time of mice in the oxymatrine group (58 days) was significantly longer than that in the control group (40 days) (Fig. 4D).

To further confirm the role of oxymatrine in the inhibition of tumor angiogenesis in pancreatic cancer, CEUS was employed to detect the tumor blood flow in pancreatic cancer xenograft mouse tumors 1 week after the last oxymatrine treatment (Fig. 5A). As shown in Fig. 5B, oxymatrine treatment markedly reduced the PI value, the data was 15.93±2.77 (oxymatrine group) to 22.87±2.56 (control group) (P<0.05).