Parthenolide induces proliferation inhibition and apoptosis of pancreatic cancer cells in vitro

Human pancreatic cancer cell line BxPC-3 was purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). It was cultured in dulbecco's modified eagle's medium (DMEM, HyClone, Logan, Utah, USA) containing 10% fetal bovine serum (JRH Biosciences, Lenexa, Kansas, USA), peniciline streptomycin mixture at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Parthenolide (Sigma, St. Louis, MO, USA) supplied as a crystalline solid was dissolved in dimethylsulfoxide (100 mM stock) and stored at -20°C. Antibodies used in this study were obtained from Santa Cruz (CA, USA) and Cell Signaling Technology (CA, USA) respectively.

BxPC-3 cells were plated at a density of 1.0 × 10⁴ cells per well in 96 well plates. 24 hours after incubation, cells were treated by PTL at indicated concentrations for 48 hours; then the medium was removed and 200 μl of fresh medium plus 20 μl of 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT, 2.5 mg dissolved in 50 μl of dimethylsulfoxide, Sigma, St. Louis, MO, USA) were added to each well. After incubation for 4 hours at 37°C, the culture medium containing MTT was withdrawn and 200 μl of dimethylsulfoxide(DMSO) was added, followed by shaking for 10 minutes until the crystals were dissolved. Viable cells were detected by measuring absorbance at 570 nm using MRX II absorbance reader (DYNEX Technologies, Chantilly, Virginia, USA). The cell growth was expressed as a percentage of absorbance in cells with PTL treatment to that in cells without PTL treatment (100%). The inhibition rate (IR) was calculated as follows: IR = (1-A value of PTL well/A value of control well) × 100%

1× 10⁵ cells suspended in 2 ml fresh media were plated in each well of a 6-well flat-bottomed microtiter plate and incubated overnight. Then PTL with indicated concentrations were added. After 48 hours cells were harvested and washed twice with pre-cold PBS and then resuspended in 1× binding buffer at a concentration of 1 × 10⁶ cells/ml. 100 μl of such solution (1 × 10⁵ cells) was mixed with 5 μl of annexin V-FITC and 5 μl of Propidium Iodide (PI) (BD Biosciences, San Jose, CA, USA) according to the manufacturer's introduction. The mixed solution was incubated at room temperature (25°C) away from light for 15 minutes. Then 400 μl of 1× dilution buffer was added to each tube. Analysis was performed by Beckman Coulter FC500 Flow Cytometry System with CXP Software (Beckman Coulter, Fullerton, CA, USA) within 1 hour.

BxPC-3 cells (1 × 10⁶ cells) were seeded in 6-well microtiter plate. Then the cells were treated with the indicated concentrations of PTL for 48 hours. For analysis of genomic DNA, attached and nonattached cells in the supernatant were harvested and collected together. DNA was extracted by the DNA extraction kit (QIAGEN, German) according to the manufacturer's instruction. 5 μg of DNA was separated on a 2% agarose gel. DNA in the gel was stained with ethidium bromide, visualized under UV light, and photographed.

Cells were plated in 6-well-plates. When the cells grew into full confluency, a wound was created on the monolayer cells by scraping a gap using a micropipette tip and then PTL with indicated concentrations were added immediately after wound creation. The speed of wound closure was compared between PTL treated groups and the control group (PTL untreated cells). Photographs were taken under 100× magnifications using phase-contrast microscopy (OLYMPUS IX70, Olympus, Tokyo, Japan) immediately after wound incision and at later time points as showed.

A Transwell cell culture chamber (Millipore, Bedford, MA, USA) with a 6.5-mm-diameter polycarbonate filters (8-μm pore size) was coated with Matrigel, dried and reconstituted at 37°C with culture medium. Cells were divided into three groups: the control group, 7.5 μM group and 15 μM PTL group. We placed culture medium containing 20% FBS in the lower chamber (24-well-plates). Then the cells at 1 × 10⁵ cells per chamber were added to the upper chamber in DMEM containing 10% FBS. After 48 hours incubation at 37°C the suspended media in the lower chamber were removed. The cells that had invaded to the lower side of the filter were fixed in methanol, stained with GIMSA solution. The number of cells that passed through the pores into the lower chamber was counted under a phase-contrast microscope (Leica DMLB2, Leica Microsystems AG, Wetzlar, Germany) (five fields per chamber).

Proteins were extracted from cultured cells and were subjected to western blot analysis using specific antibodies for bcl-2, caspase-9 and pro-caspase-3 protein. The cells (~2 × 10⁸ cells) were harvested and rinsed twice with PBS after PTL treatment for 48 hours. Cell extracts were prepared with pre-cold lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 2% Cocktail) and cleared by centrifugation at 12000g for 30 minutes at 4°C. Total protein concentration was measured using the BCA assay kit (Sigma) according to the manufacturer's instruction. Cell extracts containing 30 μg of total protein were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were electrotransferred onto nitrocellulose membrane (Millipore, Bedford, MA, USA). The membrane was then blocked with TBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20) containing 5% w/v nonfat milk, and then incubated with primary antibody (dilution factor, 1:1000) in TBST with gentle agitation overnight at 4°C. The membrane was washed 3 times for 10 minutes incubation with TBST and hybridized with redish-peroxidase (HRP)-conjugated secondary antibody (1:2000 dilution, Dakocytomation corporation, Glostrup, Denmark) corresponding to each primary antibody with gentle agitation for 2 hours at room temperature. Protein bands specific for antibody were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

All the detection items in this study were repeated at least 3 times. Statistical analysis was done using SPSS software (Version 13.0, SPSS Inc, Chicago, IL, USA). The data was expressed as mean ± SD. Statistical significance of the differences between the control- and PTL-treated cells was determined by a two-tailed Student's t test with a 95% confidence interval.

The survival and inhibition rate of BxPC-3 cells following treatment with different PTL concentrations was measured. Cells treated with PTL for 48 hours were compared with PTL-untreated cells. The MTT assay (Fig. 1) demonstrated that treatment with 1 μM and 2 μM for 48 hours insignificantly triggered cell death (P > 0.05 VS control). However, concentrations from 5 μM to 30 μM could markedly inhibit tumor cells (P < 0.01 VS control). The bivariate correlation analysis confirmed the negative relationship between PTL concentrations and cell survival rates and the positive relationship between PTL concentrations and cell inhibition rates. In BxPC-3 cells, EC₅₀ was estimated to be 14.5 μM.

To investigate the effect of inducting apoptosis by PTL in BxPC-3 cells, the flow cytometry and DNA fragmentation analysis were preformed. Annexin-V/PI-FACS analysis (Fig. 2A) was applied to quantify the apoptotic phenotype. Annexin-V-positive cells (right quadrant in the density dot plot) were summarized, including early apoptotic and late apoptotic cell death. PTL-treated cells revealed morphologic events of apoptosis more significantly than cells treated with DMSO alone. The inductive effect of apoptosis presented as a concentration-dependent manner. The apoptosis induced was further confirmed using DNA fragmentation analysis (Fig. 2B). Disintegrated nuclei and nonrandom DNA fragmentation were found on gels. More apoptotic internucleosomal DNA fragmentation was observed after higher concentrations of PTL treatment. These results revealed that PTL effectively induced a dose-dependent apoptosis in human pancreatic cancer cell.

Increased migration rate is one of the characteristics in metastatic cancer cells [13]. Pancreatic cancer is a major health problem due to its high risk of metastasis. Accordingly the wound closure assay (Fig. 3) was used to investigate if PTL influenced migration ability of BxPC-3 cells. Wound gap of similar size was created in monolayer BxPC-3 cells at 0 hour. The gap was filled with migrating cells gradually and nearly closed in the control group after 24 hours. Oppositely, the wounds were still widely open at 24 hours after exposure to PTL at indicated concentrations. The results indicated that PTL treatment could inhibit migration of pancreatic cancer cell.

The effect of PTL on BxPC-3 cell invasion was detected by a reconsitituted Matrigel membrane. The number of cells that passed through the filter and into the lower chamber was counted and compared. As a result, PTL at different concentrations obviously inhibited invasive ability of pancreatic cancer cell. The cell numbers of 7.5 μM and 15 μM PTL groups were (94 ± 7)/HPF and (58 ± 8)/HPF respectively, which were less than (146 ± 10)/HPF of control group (P < 0.05) (Fig. 4).

The underlying mechanism of PTL was also explored in the study. The activation of several apoptosis-related proteins may contribute to PTL-induced apoptosis. In Bcl-2 family members, the expression of Bcl-2, Bax and Bad after PTL treatment for 48 hours were detected by Western blotting (Fig. 5A). PTL obviously decreased the protein expression of antiapoptotic Bcl-2 and increased the protein expression of proapoptotic Bax in the BxPC-3 cells after being treated with indicated concentrations. No change was found on Bad. Therefore, the susceptibility to PTL-induced apoptosis might be attributable to the imbalance of Bcl-2/Bax (Fig. 5B).

Data have showed many anticancer agents are capable of initiating the caspase activation and inducing apoptosis [14]. Hence the caspase cascade in PTL-induced effect was also analyzed. After PTL treatment at indicated concentrations for 48 hours, Caspase-9 and Pro-caspase-3 expressions of BxPC-3 cell were explored (Fig. 5C). The dose-dependent proteolytic cleavage of caspase-9 was detected. Following caspase-9 activation, Pro-caspase-3 expression was downregulated significantly. Therefore, PTL-induced apoptosis was confirmed to be caspase-dependent.