Introduction
Pancreatic cancer is a relatively common type of cancer, with an incidence of 13.2/100,000 men and 10.2/100,000 women, accounting for approximately 227,000 deaths each year worldwide (1,2). Pancreatic cancer is usually clinically silent in its early stages, and usually becomes symptomatic at an advanced stage, or is an incidental finding on imaging performed for other or non-specific symptoms. Therefore, the prognosis of pancreatic cancer is poor, with a 3-year mortality rate of 80–98% (3). Pancreatic cancer occurs due to the malignant transformation of the pancreatic ductal epithelium. The etiology is uncertain, but may involve genetic alterations (4).
Due to the rapid growth of tumors, tissue hypoxia commonly occurs inside tumors, and adaptation to tissue hypoxia appears to be one of the most important characteristics of malignant cells (5). In addition, hypoxia induces resistance to chemotherapy and radiotherapy (5). Endothelial PAS domain protein 1 (EPAS1), also known as hypoxia-inducible factor-2 (HIF-2), is a member of the hypoxia-inducible transcription factor family. It contains a bHLH-PAS domain and is composed of homologous α subunit and a common β subunit (6,7). EPAS1 is a crucial transcription factor known to regulate tumor cell adaption to a hypoxic microenvironment (6,7). Cobalt(II)-chloride (CoCl2) binds to EPAS1 oxygen-dependent degradation domains (8), thereby inhibiting the degradation of EPAS1 and leading to the accumulation of EPAS1 protein. EPAS1 is an independent prognostic factor for cancer (9–12). EPAS1 has been suggested to play a role in metastatic spread (13). Therefore, EPAS1 may be a potential target for pancreatic cancer gene therapy, and has become the subject of considerable interest.
The use of nanoparticles to deliver drugs and therapeutic agents, such as siRNA to cancer cells is a promising novel approach in cancer therapy (14). A number of materials have been tested, but polyethylenimine-poly(lactide-coglycolide) (PLGA) nanoparticles have shown promising results for siRNA delivery due to their high cell penetration and uptake effectiveness, the protection of trapped siRNA against RNase and the high degree of adaptable, controllable and alterable degradation profile (15). PLGA nanoparticles also possess the ability to cross the blood-brain barrier, which is most promising for the treatment of brain metastases (16). Nanoparticles have been used successfully for the treatment of cancer in cells and animal models using different genes as targets, such as signal transducer and activator of transcription 3 (STAT3) (17), Kras (18), p53 (19) and vascular endothelial growth factor (VEGF) (20).
In the present study, PLGA/poloxamer nanoparticles were selected as the gene delivery vector and EPAS1 as the therapeutic target. The aim of the present study was to examine the effects of EPAS1 siRNA nanoparticles on pancreatic cancer cells and in a xenograft mouse model of pancreatic cancer. The data from this study may provide new insight into the selection of target genes and the optimization of gene vectors in pancreatic cancer gene therapy.
Materials and methods
Cell culture
The BxPC-3 pancreatic cancer cell line was purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The BxPC-3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Invitrogen Inc., Carlsbad, CA, USA) containing 10% heat-inactivated calf serum (Sijiqing, Hangzhou, China), 100 U/ml penicillin and 20 μg/ml streptomycin in a fully humidified incubator at 37°C and an atmosphere of 5% CO2 in air.
Animals
Male BALB/c nude mice at 5 weeks of age (n=120) were obtained from the Shanghai Institute of Zoology, Chinese Academy of Sciences (Shanghai, China) and kept under specific pathogen-free (SPF) conditions. The mice were allowed to adapt to their environment for 1 week, and had access to food and water ad libitum. All procedures and animal experiments were approved by the Animal Care and Use Committee of the Affiliated Hospital of the Medical College Qingdao University, Qingdao, China.
Preparation of nanoparticles loaded with EPAS1 siRNA
PLGA/poloxamer nanoparticles loaded with EPAS1 siRNA plasmids (Sangon Biotech, Shanghai, China) were prepared using the emulsion/solvent evaporation method as previously described (21,22). Briefly, PLGA and poloxamer were synthesized by Daigang Biotechnology Co., Ltd. (Jinan, China) and were dissolved in 2 ml of dichloromethane. Thereafter, 200 μl of siRNA plasmid solution in deinoized water containing 500 μg of EPAS1 siRNA or scramble siRNA plasmids were added (plasmids: PLGA/poloxamer, 2%, w/w), and the resulting mixture was stirred. The obtained emulsion was quickly poured into 25 ml of absolute ethyl alcohol and then the mixture was added to 25 ml of deionized water and stirred for 10 min, followed by evaporation using a rotary vacuum to remove the organic solvent at 30°C. The nanoparticles were collected by centrifugation at 7,104 × g for 1 h at 15°C. The diameters and ζ-potentials of the nanoparticles were measured by dynamic light scattering (DLS) using a Zeta Potential/Particle Sizer Nicomp™ 380 ZLS [Particle Sizing Systems (PSS), Santa Barbara, CA, USA]. Morphological examination was performed using a Model JEM-200CX transmission electron microscope (TEM; Jeol, Tokyo, Japan), and the in vitro release of the EPAS1 siRNA from the nanoparticles was also determined.
Cell grouping and exposure to hypoxia
The BxPC-3 cells were seeded in 96-well plates at a density of 2×105 cells/well and incubated for 24 h. The medium was then replaced with 200 μl of DMEM without serum and further incubated for 2 h. The BxPC-3 cells were assigned to 6 groups: i) the control group (BxPC-3 cells only); ii) the liposome/plasmid vector group; iii) the liposome/EPAS1 siRNA group (transfected with EPAS1 siRNA using Lipofectamine 2000; Invitrogen Inc.); iv) the EPAS1 siRNA nanoparticle group; v) the nanoparticle group; and vi) the scramble siRNA nanoparticle group. The cells were then cultured under normoxic or hypoxic conditions. CoCl2 solution was added to the medium to simulate hypoxic conditions at a final concentration of 125 μmol/l, as previously described (8).
Cell proliferation assay
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate the proliferation of the BxPC-3 cells. Briefly, the BxPC-3 cells were seeded in 96-well plates (2×105 cells/well), treated as described above and incubated at 37°C for 24, 48, 72 and 96 h, separately. MTT solution (100 μg; Invitrogen Inc.) was added to each well and incubated for 4 h. The medium was then removed, and 150 μl of dimethyl sulfoxide (DMSO) were added to dissolve the formazan crystals. After 10 min, the absorption (OD value) was measured at a wavelength of 570 nm.
In vitro cell apoptosis assay
After 48 h of incubation, the cells in each group were collected and lysed with trypsin. Binding buffer, Annexin V/FITC and propidium iodide (PI) were then added according to the instructions provided with the Annexin V/FITC assay kit (Invitrogen Inc.), and the cell apoptotic rate was analyzed using a flow cytometer (FACSCalibur™; BD Biosciences, San Jose, CA, USA) following incubation for 15 min at room temperature in the dark. Annexin V was set as the horizontal axis and PI was set as the vertical axis. Mechanically-damaged cells were located in the upper left quadrant, apoptotic or necrotic cells in the upper right quadrant, double-negative and normal cells in the lower left quadrant and early apoptotic cells in the lower right quadrant of the flow cytometric dot plot.
BxPC-3 tumor-bearing mouse model
Male BALB/c nude mice at 5 weeks of age (n=120) were randomly assigned to 6 groups: i) the control group (BxPC-3 cells only); ii) the plasmid vector liposome group; iii) the EPAS1 siRNA liposome group; iv) the EPAS1 siRNA nanoparticle group; v) the nanoparticle group and vi) the scramble siRNA nanoparticle group (n=20 in each group). Each mouse was injected subcutaneously into the armpit with BxPC-3 cells (1×107 cells/mouse) to establish the nude mouse model of pancreatic cancer. Three weeks after implantation, the tumors had grown to a volume of 0.3–0.5 cm3, and the mice in each group were then administered an injection of 1.5 μg of the plasmids or nanoparticles/Lipofectamine 2000 containing 1.5 μg of the plasmids into the tumors. This process was conducted once a day and repeated 5 times. The tumor volumes were measured with external calipers using the following formula: tumor volume (mm3) = 1/2(axb2), where ‘a’ is the longest longitudinal diameter and ‘b’ is the longest transverse diameter. The mice were sacrificed by cervical dislocation 6 weeks after implantation, and the tumors were excised for histological examination.
Semi-quantitative RT-PCR
RT-PCR assay was performed to measure the changes in EPAS1 mRNA expression levels in the pancreatic cancer tissues. The tumor tissues were harvested and grinded in liquid nitrogen, and then the total RNA was extracted from each sample using TRIzol reagent (Takara, Tokyo, Japan). RNA purity was determined by the absorbance at 260 and 280 nm (A260/280). Complementary DNA (cNDA) was reverse transcribed from total RNA using an RT-PCR kit (Takara). PCR was performed to amplify EPAS1 using GAPDH as an endogenous control. The gene-specific primers used were as follows: EPAS1 forward, 5′-AGCCTTGGAGGGTTTCAT-3′ and reverse, 5′-AGCCTTGGAGGGTTTCAT-3′; GAPDH forward, 5′-AGAAGGCTGGGGCTCATTTG-3′ and reverse, 5′-AGGGGCCATCCACAGTCTTC-3′. The PCR reaction was performed as follows: 94°C initial denaturation for 5 min, 94°C denaturation for 30 sec, 58°C annealing for 35 sec, 72°C extension for 50 sec, 30 cycles, and 72°C extension for 10 min. The amplified PCR products were resolved on 2% agarose gels, visualized and photographed using a gel-imaging system (Tanon Science & Technology Co., Ltd., Shanghai, Technology, Shanghai, China), and analyzed using Image Analysis software (National Institutes of Health, Bethesda, MD, USA).
Western blot analysis
Western blot analysis was performed to measure the changes in the EPAS1 protein expression levels in the pancreatic cancer tissues. Briefly, the tumor tissues were weighed, cut into small sections and placed in a homogenizer. After adding cold protein extraction reagent containing pre-cooling inhibitor (Beyotime, Jiangsu, China), the tissues were homogenized at low speed. The homogenates were then centrifuged (12,000 × g, 4°C, 5 min) and the supernatants were collected to extract the proteins. Protein concentrations were measured using the bicinchoninic acid (BCA) method. Equal amounts of protein (50 μg) were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes using standard procedures. The membranes were blocked with 5% non-fat dried milk and then incubated with primary antibodies against EPAS1 (Cat. no. sc-8712) and β-actin (Cat. no. sc-130656; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). β-actin was used as an internal reference. Following incubation at room temperature for 2 h, the protein blots were then washed with Tris-buffered saline and Tween-20 (TBST) and blocked with horseradish peroxidase-labeled IgG (1:5,000 dilution; Origene, Beijing, China) at 37°C for 1.5 h. The protein blots were washed 3 times with TBST, and then developed with electrogenerated chemiluminescence (ECL; Millipore, Billerica, MA, USA) using a gel-imaging system and analyzed using Image Analysis software.
Microvessel density in the pancreatic cancer tissues
Fresh pancreatic cancer tissues were fixed with 10% formaldehyde and embedded in paraffin. The tissues were then cut into slices and the detection of VEGF and CD34 was carried out according to the instructions provided with the relevant kits (Wuhan Boster Biological Technology, Ltd., Wuhan, China). The cells stained brown were counted as positive. Each slice was observed in 20 fields at an original magnifications of ×200 using an inverted phase contrast microscope (Nikon, Tokyo, Japan). The microvessel density indicated by CD34 in the pancreatic cancer tissues was detected.
Statistical analysis
All data are expressed as the means ± standard deviation (SD) from 3 independent experiments. Statistical analyses were performed using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). Continuous data were analyzed using one-way ANOVA with the Student-Newman-Keuls (SNK) test for post hoc analysis. The Student’s t-test was used for two-group comparisons. P-values <0.05 were considered to indicate statistically significant differences.
Results
Preparation and characterization of PLGA/poloxamer nanoparticles loaded with EPAS1 siRNA
The PLGA/poloxamer nanoparticles had spherical and uniform characteristics, as shown under a scanning electron microscope (Fig. 1A). The particle size was 160–220 nm, and the ζ-potential was -0.41±1.23 mV. The encapsulation efficiency was 40%. The in vitro release of the EPAS1 siRNA from the nanoparticles was investigated, and there was a rapid initial release (>30% release during the first day), and then a slower, continuous and almost linear release in the following 9 days (approximately 90% total release; Fig. 1B). As time further progressed, the release of EPAS1 siRNA was still relatively slow, indicating that the in vitro release of the EPAS1 siRNA from the nanoparticles presented the sustained-release effect. We then used MTT assay to evaluate the cytotoxicity of the PLGA/poloxamer nanoparticles; they did not appear to have any significant effect on the growth of the BxPC-3 pancreatic cancer cells (Fig. 1C).
Effect of EPAS1 siRNA nanoparticles on BxPC-3 cell proliferation under normoxic and hypoxic conditions
The results of BxPC-3 cell proliferation are shown in Table I. Under normoxic conditions, no differences were observed among the treatment groups. Under hypoxic conditions, the EPAS1 siRNA nanoparticle and liposome/EPAS1 siRNA-transfected groups showed a decreased cell proliferation at 48, 72 and 96 h following treatment compared with the other 4 groups (all P<0.05). Importantly, the EPAS1 siRNA nanoparticles significantly inhibited BxPC-3 cell proliferation compared with the EPAS1 siRNA liposome-transfected group. These results suggest that nanoparticles loaded with EPAS1 siRNA facilitate the inhibition of cell proliferation under hypoxic conditions in vitro.
Effect of EPAS1 siRNA nanoparticles on BxPC-3 cell apoptosis under normoxic and hypoxic conditions
The pancreatic cancer cell apoptotic rates were analyzed by flow cytometry 48 h after treatment. Under normoxic conditions, the cell apoptotic rates of the control, liposome/plasmid vector, liposome/EPAS1 siRNA, EPAS1 siRNA nanoparticle, nanoparticle and scramble siRNA nanoparticle groups were 1.17±0.31, 1.25±0.33, 1.48±0.29, 1.31±0.32, 1.50±0.35 and 1.38±0.29%, respectively (all P>0.05; Fig. 2). Under hypoxic conditions, the cell apoptotic rates of the control, liposome/plasmid vector, liposome/EPAS1 siRNA, EPAS1 siRNA nanoparticle, nanoparticle and scramble siRNA nanoparticle groups were 2.38±0.0.29, 2.34±0.32, 9.37±2.35, 17.57±4.25, 2.89±0.30 and 3.31±0.30%, respectively. Compared with the other 4 groups, the cell apoptotic rates of the EPAS1 siRNA nanoparticle and the liposome/EPAS1 siRNA-transfected group were significantly higher (all P<0.05). Furthermore, the EPAS1 siRNA nanoparticle group demonstrated the highest cell apoptotic rates compared with the other groups.
EPAS1 siRNA nanoparticles downregulate EPAS1 expression in pancreatic cancer tissues from the xenograft mouse model of ectopic human pancreatic cancer
At 6 weeks after implantation, the mice were sacrificed by cervical dislocation and the tumors were excised. Semi-quantitative RT-PCR was conducted to measure the EPAS1 mRNA expression levels. The results revealed that compared with the other 4 groups, the EPAS1 siRNA nanoparticle or liposome/EPAS1 siRNA-transfected groups demonstrated a markedly lower mRNA expression of EPAS1. More importantly, the EPAS1 siRNA nanoparticle group showed the lowest expression (all P<0.05), suggesting that the nanoparticles loaded with EPAS1 siRNA downregulated the mRNA expression of EPAS1 in the pancreatic tumors (Fig. 3A).
Western blot analysis revaled that compared with the other 4 groups, the EPAS1 siRNA nanoparticle or liposome/EPAS1 siRNA-transfected groups demonstrated a notably lower protein expression of EPAS1 in the pancreatic tumors. Furthermore, the EPAS1 siRNA nanoparticle group showed the lowest expression (all P<0.05), which was in agreement with the results from semi-quantitative RT-PCR. Taken together, these results indicate that nanoparticles loaded with EPAS1 siRNA downregulated the protein expression of EPAS1 in the pancreatic tumors (Fig. 3B).
EPAS siRNA nanoparticles inhibit tumor growth in the xenograft mouse model of ectopic human pancreatic cancer
At 7 days after implantation, subcutaneous tumor nodules of approximately 0.3–0.5 cm3 were observed, suggesting the successful establishment of the xenograft tumor model. The model success rate was 100%. The tumor volume in each group showed a progressive increase as time progressed. At 6 weeks after implantation, the mean tumor volumes in the control, liposome/plasmid vector, liposome/EPAS1 siRNA, EPAS1 siRNA nanoparticle, nanoparticle and scramble siRNA nanoparticles groups were 1786.36±24.20, 1821.30±22.35, 1329.25±19.25, 896.24±17.25, 1826.36±25.20 and 1821.25±21.25 mm3, respectively (Fig. 4). Compared with the other 4 groups, the tumor volumes in the EPAS1 siRNA nanoparticle or liposome/EPAS1 siRNA-transfected groups were significantly smaller (all P<0.05). The EPAS1 siRNA nanoparticle group demonstrated the smallest tumor volume compared with the other groups (all P<0.05). These results indicated that EPAS1 siRNA nanoparticles inhibited tumor growth in vivo.
EPAS1 siRNA nanoparticles inhibit the formation of microvessels in the xenograft mouse model of ectopic human pancreatic cancer
The results of immunohistochemical analysis revealed that the VEGF-positive and CD34-positive granules were mainly localized in the cytoplasm and stained brown (Fig. 5). The nanoparticles loaded with EPAS1 siRNA significantly downregulated the protein expression of VEGF in the pancreatic tumors (Fig. 5A).
The microvessel densities in the control, liposome/plasmid vector, liposome/EPAS1 siRNA, EPAS1 siRNA nanoparticle, nanoparticle and scramble siRNA nanoparticle groups were 19.5±1.7, 17.6±2.1, 9.7±1.4, 6.5±1.1, 18.5±1.6 and 17.9±2.3, respectively. Compared with the other 4 groups, the EPAS1 siRNA nanoparticle and the liposome/EPAS1 siRNA-transfected groups demonstrated lower microvessel densities (all P<0.05). The EPAS1 siRNA nanoparticle group showed the lowest microvessel density (all P<0.05; Fig. 5B). Taken together, our data indicate that nanoparticles loaded with EPAS1 siRNA inhibit the formation of microvessels in pancreatic tumors.
Discussion
The availability of a safe and efficient delivery vehicle for specifically transporting genes to target cells is an important requirement for gene therapy. siRNA delivery using nanoparticles is a novel, safe and efficient approach (23–25). The aim of the present study was to assess the effects of EPAS1 silencing using PLGA/poloxamer nanoparticles loaded with EPAS1 siRNA in pancreatic cancer. The results revealed that the nanoparticles loaded with EPAS1 siRNA decreased BxPC-3 cell proliferation under hypoxic conditions and increased apoptosis. The nanoparticles loaded with EPAS1 siRNA decreased EPAS1 expression. Pancreatic tumors from the mice injected with the nanoparticles loaded with EPAS1 siRNA were significantly smaller compared with those from the mice in the other groups. Importantly, pancreatic tumors from the mice injected with nanoparticles loaded with EPAS1 siRNA showed less microvessels. These results suggest that silencing EPAS1 using siRNA-loaded nanoparticles has beneficial effects against pancreatic cancer.
Currently, many types of polymers are used to condense DNA into nanoparticles to facilitate the crossing of therapeutic genes through the blood-pancreatic barrier and the blood-brain barrier, to achieve controlled release and to prolong gene expression, while avoiding the immunogenicity and potential tumorigenic defects caused by viral vectors (14–16). However, the efficiency of nanoparticles to deliver a specific siRNA depends upon the encalupsation process, cellular uptake and cytoplasmic release (26). The nanoparticles created in the present study showed a good siRNA incorporation efficiency, as well as a good siRNA release profile.
This study demonstrated that using siRNA-loaded nanoparticles achieved a better inhibition of EPAS1 and tumor growth than transfecting the cells with a plasmid expressing the same siRNA. In addition, PLGA/poloxamer nanoparticles loaded with EPAS1 siRNA were successfully prepared and injected into nude mice with pancreatic tumor xenografts. The results demonstrated that compared with the other groups, PLGA/poloxamer nanoparticles loaded with EPAS1 siRNA significantly down-regulated EPAS1 expression and inhibited tumor growth. Albeit using different means of transport and different target genes, the results of the present study are similar to those of previous studies (14,17–20). Indeed, studies have demonstrated the efficiency of nanoparticle delivery systems in a number of cancer types, such as VEGF siRNA calcium phosphate/charge-conversional polymer hybrid nanoparticles in pancreatic cancer (20), STAT3 siRNA PEI-PLGA nanoparticles in lung cancer (17), Kras siRNA/arsenic PEG-PLL/PEG-PDLLA nanoparticles in pancreatic cancer (18), wt-p53/gemcitabine gelatin nanoparticles in pancreatic cancer (19), and HER2 siRNA nanosized immunoliposome-based delivery complex in pancreatic cancer (14).
One of the major hallmarks of solid tumors is tumor hypoxia. Sustained tumor hypoxia leads to resistance to chemotherapy and ionizing radiation, as well as to increased aggressiveness, thus improving tumor vascularity. Solid tumors are composed of both hypoxic and normoxic cells (27), and hypoxic cells allow for recurrence and metastases to occur even if the normoxic cells are destroyed by therapeutic agents since they represent a pool of cancer cells that may colonize other organs (28). Hypoxia induces changes in both DNA damage repair pathways and in cell survival pathways (28). HIFs such as HIF-1 or EPAS1 (also known as HIF-2) play a pivotal role in tumor cell survival (6,7,28,29). A number of approaches have been tested to overcome hypoxia-induced resistance, including hyperbaric oxygen chambers and targeting hypoxia proteins using drugs or siRNAs (28). The results of the present study demonstrated that treating normoxic cells with EPAS1 siRNA had no effect on cell proliferation and apoptosis, while the effects were observed under hypoxic conditions. In addition, considering the rapid growth of the tumors in our animal model, we hypothesized that areas of these tumors were under hypoxic conditions, and that these areas responded well to the EPAS1 siRNA therapy.
Angiogenesis is one of the responses taken by hypoxic tumors to improve their oxygen supply. It is well known that HIF-1 and EPAS1 activation leads to a more aggressive cancer phenotype (30), and that this upregulates VEGF expression (31–33). The present study demonstrated that inhibiting EPAS1 using siRNA decreased microvessel density in tumors, as revealed by VEGF and CD34 immunohistochemistry, and that the tumor size was smaller in the EPAS1 siRNA-treated tumors. In addition, the results of the present study support a role of EPAS1 in angiogenesis.
One of the main advantages of targeting hypoxia genes is that normal healthy tissues will be mainly untouched by the therapy. Even if human studies of siRNA-loaded nanoparticles are yet to be performed, it can be hypothesized that toxic side-effects should be reduced to a minimum. Therefore, using these delivery systems may be a promising treatment approach against cancer.
In conclusion, even if tumor regression was not observed in the present study, PLGA/poloxamer nanoparticles loaded with EPAS1 siRNA markedly inhibited the growth of pancreatic tumors and exhibited a significantly lower expression of VEGF and CD34. This suggests that EPAS1 and VEGF are closely related to angiogenesis, and that nanoparticles loaded with EPAS1 siRNA impede tumor growth by reducing angiogenesis through the inhibition of vascular endothelial cell proliferation. Nanoparticle technology and RNA interference were used to block the expression of EPAS1 in pancreatic cancer cells aiming at inhibiting the growth of pancreatic tumors, promoting cell apoptosis and downregulating the expression of EPAS1, VEGF and CD34, effectively inhibiting the growth of pancreatic cancer cells. This approach may lay a solid foundation for the clinical targeted therapy of pancreatic cancer.