Proteinase-activated receptors (PARs) are 7 transmembrane-spanning domain G protein-coupled receptors comprising 4 receptors, PAR-1, PAR-2, PAR-3 and PAR-4,1 that are activated by proteolytic cleavage of their N-terminal domains.2 The newly released N-terminal sequence acts as a tethered ligand that binds to the core of the receptor and initiates signal transduction.3 Thrombin is a physiological activator of PAR-1, PAR-3 and PAR-4, whereas PAR-2 is activated by multiple trypsin-like enzymes, including trypsin and mast cell tryptase, but not thrombin.1, 2, 4, 5 The gene encoding human PAR-2 was cloned by Bohm et al.4 and Nystedt et al.,5 and PAR-2 is expressed widely and at high levels in gastrointestinal tract tissues such as small intestine, colon, liver, pancreas and stomach.4, 5, 6 In addition, it was recently reported that PAR-2 is expressed in colon cancer,7 gastric cancer,8 pancreatic cancer9 and gallbladder cancer.10 PAR-2 is also expressed by some human cell lines.5, 9, 11, 12
Trypsin, a pancreatic enzyme, was shown to induce cell proliferation via PAR-2.11, 12 Signaling through PAR-2 is stimulated preferentially by trypsin and tryptase and can be activated independently by peptide SLIGKV as ligand, which is known to be related to cell activation and intracellular signaling pathways following ligand binding.13, 14 A PAR-2 agonist causes cytosolic Ca2+ mobilization and activates p44/42 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) and p38 MAPK as well as the nuclear factor kappa B (NF-κB) pathway,14, 15, 16 resulting in proliferation and upregulation of COX-2. In our previous study, trypsin and a PAR-2 agonist induced proliferation and enhanced cyclooxygenase-2 (COX-2) mRNA expression in 2 pancreatic cancer cell lines, T3M4 and BxPC3.13 We also found that anti-PAR-2 antibody suppressed proliferation of activated cells.13 Recently, it was reported that downregulation of trypsin17 or serine protease inhibitors suppressed carcinogenesis in many different systems, both in vivo and in vitro.18 However, there have been no studies of PAR-2 signaling pathways in vivo.
The phenomenon of RNA interference (RNAi) was first discovered in the nematode Caenorhabditis elegans as a response to double-stranded RNA (dsRNA) that resulted in sequence-specific gene silencing.19 The dsRNAs are processed by Dicer, a cellular RNase III, to generate duplexes of ∼21 nucleotides (nt) with 3′-overhangs (small interfering RNA [siRNA]), which mediate sequence-specific mRNA degradation.19, 20 Several RNAi methodologies were rapidly established and showed promise to inhibit expression of specific genes in mammals.20 RNAi induced by siRNA has recently emerged as a powerful technique for highly specific suppression of expression of individual genes.21 Furthermore, there is an intense research effort aimed at developing siRNAs as therapies against various diseases, including viral infections, neurodegenerative disorders and cancers.22 Several researchers have investigated siRNAs in animal models.23, 24 In several studies, atelocollagen was used as a carrier of siRNAs.25, 26 Atelocollagen, which was first used as a plasmid DNA delivery system,27 is a liquid at 4°C and a gel at 37°C. Atelocollagen complexed with siRNA is resistant to nucleases and is efficiently taken up by cells, thereby allowing long-term gene silencing.25 Radiolabeled siRNA mixed with atelocollagen remains intact tumors for at least a week.26
The aim of the present study was to determine whether downregulation of PAR-2 by RNAi influences pancreatic tumor growth in vivo, and we discuss the possibility of RNAi-based treatment for pancreatic cancer.
Material and methods
Cell lines and reagents
Human Panc1 pancreatic cancer cells were obtained from the Japanese Cancer Research Bank (Tokyo, Japan). Panc1 cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) and 1% antibiotics. Each culture was maintained at 37°C in a humidified atmosphere of 5% CO2/air. Human PAR-2-activating peptide SLIGKV and the reverse sequence peptide VKGILS were obtained from Sigma-Genosys (Ishikari, Japan).
Thirty-six male BALB/c nude mice, aged 7–8 weeks and weighing 20–24 g, were used in this study. All animals were kept under standard laboratory conditions (temperature, 20–24°C; relative humidity, 50–60%; 12 hr light/dark cycles) and given a standard laboratory diet with free access to water and food. This study was approved by the Animal Committee of the Oita University Faculty of Medicine and conformed to the Guidelines for Animal Experimentation of the Oita University Faculty of Medicine.
Preparation of siRNAs
Three siRNAs targeting human PAR-2 and one scrambled siRNA (as negative control) with the following sense and antisense sequences were used: PAR-2 siRNA no.#1 (bases 578–602), 5′-GGGAAGCUCUUUGUAAUGUGCUUAU-3′ (sense) and 5′-AUAAGCACAUUACAAAGAGCUUCCC-3′ (antisense); PAR-2 siRNA no.#2 (bases 579–603), 5′-GGAAGCUCUUUGUAAUGUGCUUAUU-3′ (sense) and 5′-AAUAAGCACAUUACAAAGAGCUUCC-3′ (antisense); PAR-2 siRNA no.#3 (bases 1203–1227), 5′-GGAUCAUGCAAAGAACGCUCUCCUU-3′ (sense) and 5′-AAGGAGAGCGUUCUUUGCAUGAUCC-3′ (antisense); PAR-2 siRNA no.#2-scrambled, 5′-GGACUCUUUAUGGUAC GUUUAGAUU-3′ (sense) and 5′-AAUCUAAACGUACCAUA AAGAGUCC-3′ (antisense). All siRNAs were designed and synthesized by Invitrogen as a modified type that does not cause interferon responses. Invitrogen refers to such siRNAs as “Stealth™RNAis.” All PAR-2 siRNAs scored 5 stars, which indicate the highest probability of success, with by “Star Scoring System” in BLOCK-iT™ RNAi Designer (https://rnaidesigner.invitrogen.com/rnaiexpress/). Stealth™ RNAi Negative Control Duplexes was also used as an insensitive siRNA. Each freeze-dried siRNA was reconstituted with RNase-free water to prepare a 20 μM stock solution.
Cell culture conditions and transfection of siRNAs
Cells were plated at a density of 5 × 105 Panc1 cells/6-well dish. When cells reached 70–80% confluence (approximately 24 hr of culture) the medium was removed, and the attached cells were rinsed twice and incubated with serum-free medium for 12 hr. The cells were transfected with siRNAs in serum-free medium with LipofectAMINE 2000 (Invitrogen). Each siRNA stock solution (8–24 μl; final concentration, 40–120 nM) and LipofectAMINE 2000 reagent were mixed (1:1) in Opti-MEM (Invitrogen) and adjusted to a total volume of 400 μl in a small sterile tube. After immediate mixing and incubation at room temperature for 20 min, 1.6 ml of Opti-MEM was added to make the siRNA-lipid complex. This siRNA-lipid complex (2.0 ml total volume) was added to the cultured cells after the regular medium was removed. The cultures were incubated 4–24 hr at 37°C. The medium was then replaced with fresh RPMI 1640 containing 10% FBS and 1% antibiotics. The treated cells were cultured for 24 hr and then collected for analysis.
Total RNA isolation
Total RNAs from cultured cancer cells and tumor samples were isolated with the BioRobot EZ1 RNA system with EZ1 RNA Cell Mini Kit (Qiagen, Tokyo, Japan) or EZ1 RNA Tissue Mini Kit (Qiagen) according to the manufacturer's instructions. The complementary DNA (cDNA) was synthesized as described previously.13 Total RNA (1.0 μg) was reverse transcribed in a 25-μl reaction containing 80 pmol random primer (Takara, Shiga, Japan) and 200 U Moloney murine leukemia virus reverse transcriptase (M-MLV Reverse Transcriptase; Invitrogen) according to the manufacturer's instructions. The cDNA was used as a template for subsequent real-time polymerase chain reaction (PCR).
Quantitative real-time PCR was done with the Light-Cycler System (Roche, Lewes, East Sussex, UK). PAR-2-specific primers were 5′-G CCATCAAACT CATTGTCAC-3′ (forward) and 5′-GGCT ACAATGTACA GGGCATA-3′ (reverse) (Nihon Gene Research Laboratories, Sendai, Japan). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH primer set; Search-LC, Heidelberg, Germany) was amplified according to the manufacturer's protocol as an internal control to allow quantitation of the PAR-2 amplification product. A fresh standard dilution series was prepared. The PCR mix, which contained 9.4 μl PCR-grade water, 1 μl of each PAR-2-specific primers (10 μM), 1.6 μl of 25 mM MgCl2 and 2 μl LightCycler Faststart DNA Master SYBR Green I, was added to a 1.5-ml light-protected reaction tube on ice. This PCR mix was pipetted into the precooled LightCycler capillary, and 5 μl cDNA template (diluted 20×) was added. Fifteen microliters of the PCR mix was pipetted into 4 precooled LightCycler capillary tubes and 5 μl undiluted and 5 μl freshly diluted standard were then added to each capillary. Each capillary was sealed with a stopper and centrifuged at 700g for 15 sec. The capillaries were placed into the rotor of the LightCycler, and the samples were amplified. PCR cycles were monitored continuously with SYBR Green I dye. After amplification, melting curve analysis permitted accurate identification of the PCR amplicons. Data were analyzed with the LightCycler analysis software (Roche), and a standard curve that correlated cycle number with the amount of product formed was plotted for each sequence of interest. PAR-2 expression was then normalized to that of GAPDH.
Two days after siRNA treatment, cultured cells were washed twice with cold phosphate-buffered saline (PBS) and then lysed immediately with cold T-PER™ Tissue Protein Extraction Reagent (Pierce Chemical, Rockford, IL) containing protease inhibitors (0.5 mg/ml aprotinin, 0.5 mg/ml leupeptin, 0.5 mg/ml pepstatin). Lysates were homogenized and then cleared by centrifugation at 10,000g for 10 min at 4°C. Protein concentration in cell lysates was determined by the Quick Start™ Bradford protein assay (Bio-Rad Laboratories, Hercules, CA) with the supplied Quick Start™ bovine serum albumin standard set (Bio-Rad Laboratories).
Western blot analysis
Western blot analyses were done as described previously.28 Lysate aliquots were prepared for electrophoresis in Laemmli sample buffer (Bio-Rad) containing 5% 2-mercaptoethanol, and the samples were boiled for 5 min. Equal amounts of protein from each lysate sample, typically 10 μg, were separated on a 10% Tris-HCl Ready Gel (Bio-Rad) and then transferred electrophoretically to Sequi-Blot polyvinylidene difluoride membranes (Bio-Rad). Gel electrophoresis was performed at 50 mA for 90 min at room temperature. Transfer was performed at 40 V for 120 min at room temperature. Membrane blots were washed briefly in PBS containing 0.1% Tween-20 (PBS/Tw), blocked in PBS/Tw plus 2% nonfat dry milk and then incubated at room temperature for 60 min. Anti-human PAR-2 mouse monoclonal antibody (SAM-11, 1:100, Santa Cruz Biotechnology, CA) and Anti-β-Actin mouse monoclonal antibody (1:5,000, Sigma, MO) diluted in PBS/Tw plus 0.4% nonfat milk was used as the primary antibody. Primary antibody incubation was carried out at room temperature for 2 hr. The membrane was then washed and incubated with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G anibody (1:2,000, Santa Cruz Biotechnology) at room temperature for 45 min. Antibody-labeled proteins were detected with protocols and reagents contained in the Enhanced Chemiluminescence (ECL™) Western Blotting Detection Reagent and Analysis System (Amersham Biosciences UK Ltd., Buckinghamshire, UK) on Hyperfilm ECL (Amersham Biosciences, Piscataway, NJ).
WST-1 cell proliferation assay
Cellular proliferation was examined with cell proliferation reagent WST-1 (Roche Molecular Biochemicals, Mannheim, Germany). Panc1 cells treated with siRNAs were seeded sparsely (1 × 104 cells/well) in 96-well plates and allowed to attach for 24 hr at 37°C in a humidified atmosphere of 5% CO2/air. The medium was removed, and the attached cells were rinsed with PBS twice and incubated with serum-free medium for 12 hr. One hundred microliters of fresh medium with 0.5% FBS were added with or without SLIGKV or VKGILS at various concentrations. After cells were cultured for 48 hr, the absorbances of representative wells were measured at 450 nm with a microtiter plate reader (Model 3550, Bio-Rad Laboratories). No significant change in cell death was observed after treatment with either peptide regardless of the concentration.
PAR-2 siRNA treatment of a Panc1 xenograft model
A total of 5.0 × 106 Panc1 cells in 0.5 ml of PBS were inoculated subcutaneously through a 24-gauge needle into the lower flank of 8-week-old, male BALB/c nude mice as described previously.29 After 1 week, the tumors reached an average volume of 10–20 mm3, and the tumor-bearing nude mice were treated with PAR-2 siRNA with atelocollagen (Koken, Tokyo, Japan). Each siRNA was used after dilution with PBS and the final concentration of atelocollagen was 0.5%. Each therapeutic reagent was injected into the tumor once each week after the first injection. Tumor diameters were measured at 1-week interval with digital calipers, and the tumor volume was calculated according to the formula V = A × B2/2 (mm3), where A is the largest diameter (mm) and B is the smallest diameter (mm) as described previously.29
All data are presented as mean ± standard deviation (SD). One-way ANOVA followed by Bonferroni correction were performed for multiple comparisons. The significance of differences in the in vitro proliferation assay and the tumor therapy were analyzed by repeated ANOVA. A p value of less than 0.05 was considered statistically significant.
Testing PAR-2 siRNAs and concentrations
We made 3 PAR-2-specific siRNAs, and all 3 PAR-2 siRNAs (80 nM) suppressed expression of PAR-2 mRNA in a human pancreatic carcinoma cell line, Panc1, after a 24 hr transfection time (Fig. 1a). Differences in toxicity between the 3 siRNAs were not observed (Fig. 1b). When the concentration of each siRNA was changed to 40 nM and the transfection time was shifted to 4 hr, PAR-2 siRNA no. 2 showed the most significant suppression of expression of PAR-2 mRNA (p < 0.05, Fig. 1c). Scrambled (control) siRNA (PAR-2 siRNA no.2-SCR) had no effect on PAR-2 mRNA levels. These results suggested that PAR-2 siRNA no. 2 was useful for further study. And different concentration of PAR-2 siRNA no. 2 (24 hr) was compared (Fig. 1d). PAR-2 siRNA no. 2 (80 nM, 24 hr) suppressed the PAR-2 mRNA level to 8.9% ± 2.8% of that of the control cultures (120 nM, 24 hr). There were significant differences in suppression of expression of PAR-2 mRNA between 40 nM and 80 nM of PAR-2 siRNA no. 2 (p < 0.05). Therefore, we selected 80 nM of PAR-2 siRNA no. 2 for later experiments, and also used 80 nM of PAR-2 siRNA no. 3 as a multiplicity control.
To confirm the effects of the siRNA on expression at the protein level, we performed Western blot analyses of lysates of siRNA-treated Panc1 cells. PAR-2 siRNAs no. 2 and no. 3 significantly decreased PAR-2 protein expression when compared with the scrambled and insensitine siRNAs (Fig. 2).
Inhibition of pancreatic cancer cell proliferation induced by PAR-2 siRNA
After incubation with 10−6 M–10−4 M PAR-2 agonist peptide SLIGKV, Panc1 cells proliferated in a dose-dependent manner. Panc1 cells treated with the reverse sequence peptide, VKGILS did not show increased proliferation. Cells incubated with SLIGKV showed greater proliferation than those incubated with VKGILS (p < 0.05).
We examined the effects of PAR-2 siRNAs no. 2 and no. 3 and the scrambled and insensitive siRNAs, and the SILGKV and VKGILS peptides (Fig. 3). Cells treated with PAR-2 siRNA no. 2 and SLIGKV showed significantly lower cell proliferation than cells treated with scrambled or insensitive siRNA and VKGILS (p < 0.05). Cells treated with PAR-2 siRNA no. 3 and SLIGKV also showed significantly lower cell proliferation than the controls. There were no statistical differences in cell proliferation between the 6 groups except for 2 groups of SLIGKV and the control siRNAs. Thus, these results showed that these downregulation of PAR-2 by RNAis suppressed Panc1 cell proliferation in vitro.
Effect of PAR-2 siRNA on a xenograft model
By 1 week after injection of Panc1 cells, visible tumors had developed at the injection sites (mean tumor volume: 11.6 ± 5.1 mm3; n = 42). To evaluate the therapeutic effects of the PAR-2 siRNAs, intratumoral treatment with PAR-2 siRNAs or control siRNAs and atelocollagen was repeated every 7 days (total: 3 times). As shown in Figure 4a, PAR-2 siRNA no. 2 (both 5μM and 10 μM) significantly suppressed tumor growth when compared with the insensitive and scrambled siRNAs (10 μM) (p < 0.05). PAR-2 siRNA no. 3 (10 μM) also significantly suppressed tumor growth when compared with these control siRNAs (10 μM) (p < 0.05). The inhibitory effect might be dependent on the dose, but there were no significant differences in tumor growth between the doses of 5 μM and 10 μM.
Photos of Panc1 xenografts were showed in Figure 4b. Before and 3 weeks after the first injection of PAR-2 siRNAs no. 2 and no. 3 and the control siRNAs (concentration of siRNA: 10 μM), respectively, Panc1 tumors were photographed.
To confirm that expression of PAR-2 mRNA was inhibited in the tumors, expression of intratumoral PAR-2 mRNA was measured by quantitative real-time RT-PCR. Expression of PAR-2 mRNA was decreased significantly by PAR-2 siRNA no. 2 (10 μM) when compared with the control siRNAs (10 μM) (p < 0.05, Fig. 4c). Therefore, our results showed that Panc1 tumor growth in vivo was inhibited by blockade of PAR-2 using the RNAi phenomenon.
In this study, PAR-2 siRNA downregulated expression of PAR-2 mRNA and inhibited proliferation of a human pancreatic cancer cell line, Panc1. In addition, the PAR-2 siRNA markedly suppressed tumor growth in a xenograft model. These are the first demonstrations that a RNAi-based PAR-2 blockade system could have potential as a treatment for pancreatic cancer.
The pathophysiology of acute pancreatitis is strongly associated with autoactivation of trypsin.30 The biological activity of trypsin is attributed to activation of PAR-2.31 In pancreatic cancer, PAR-2 was expressed and signaling by activation of PAR-2 was also observed.13 Shimamoto et al. reported that xenograft tumor of pancreatic cancer cell including Panc1 significantly grew after treatment with PAR-2 agonist SLIGKV.32 The sequence of genetic events in the adenoma-carcinoma sequence of intraductal papillary mucinous neoplasms has been investigated,33 and PAR-2 may be related to proliferation or carcinogenesis of pancreatic neoplasms. In our previous study, an anti-PAR-2 monoclonal antibody suppressed proliferation of 4 pancreatic cancer cell lines in vitro13; however, there have been no studies as to whether blockade of the PAR-2 activation pathway could inhibit tumor growth in vivo. In our previous study, PAR-2-specific neutralizing monoclonal antibody (SAM11) did not inhibit tumor growth in a pancreatic cancer xenograft model (unpublished data), and therefore, we chose to use RNAi-based technology in the present study. siRNA may be better at blocking the signaling pathway than antibodies, because it is easily applicable to any therapeutic target. In the present study, PAR-2-specific siRNA inhibited growth of pancreatic cancer cells in vivo.
RNAi technologies need suitable delivery methods for clinical application. Viral delivery systems are efficient but can have serious side effects.34 Cationic lipid complexes are another possible siRNA delivery agents,35 and recently, atelocollagen, which we used in the present study, was reported as an effective reagent for transfer of siRNAs in animal models.36 siRNAs, which are negatively charged, bind electrostatically to atelocollagen, which is positively charged, and the mixture easily penetrates tumor tissues. However, lipid-based delivery systems, such as atelocollagen, can induce immune responses in vivo.26 An important consideration for siRNA-mediated inhibition of gene expression is whether the observed effects are specific or nonspecific artifacts37 and whether the effects are free of potential interferon responses.38 To solve these problems, we used a Stealth™ RNAi modified by Invitrogen, which has higher specificity and can eliminate the sense strand “off-target” effects that can be problematic with traditional siRNA. In addition, Stealth™RNAi can eliminate the nonspecific stress response of the dsRNA-dependent protein kinase (PKR)/interferon pahtways that can be induced by traditional siRNA [http://www.invitrogen.co.jp/rnai/stealth.shtml]. Gene therapies, including RNAi technologies, still have problems. The RNAi technologies have high specificity at the gene level, but low specificity at the tissue level in vivo. If RNAi-based therapies are to be used, it will be necessary to inject the siRNA directly or administer it via a tissue-specific delivery system. Recently, there were reports of a tumor-targeted delivery system for gene therapies, including RNAi, in animal models.39, 40, 41 These new methods have utilized integrin ligand, sigma receptor ligand, transferrin or epidermal growth factor to bind targeted tumor cells. If PAR-2 siRNA is combined with these new tumor-targeted delivery systems, it may become a more effective and specific therapy for cancer.
Activation of the PAR-2 pathway accelerates cancer growth. Factors related to the PAR-2 pathway are closely associated to inflammatory reactions and include prostaglandins, cytokines, growth factors and proteases, including matrix metalloprotease (MMP) and mast cell tryptase.13, 14, 15, 42, 43, 44 Activation of PAR-2 in various human cell lines upregulates COX-2 expression.13, 14, 15 Nonsteroidal anti-inflammatory drugs, which can inhibit COX-2 activity, reduce the risks of several digestive diseases, including colon cancer.45 Yip-Schneider et al.46 reported that COX-2 inhibitors suppress pancreatic cancer cell growth and may be an effective treatment for pancreatic cancer. In addition, overexpression of COX-2 is related to poor prognosis of several cancers.47, 48 Other studies showed that activation of PAR-1 or PAR-2 in response to enhanced MMP-2 and MMP-9 activities is associated with metastases of cancer cells.43 MMPs, which are enzymes capable of degrading the extracellular matrix and basement membrane, have been shown to be strongly expressed in malignant tissues.49, 50
Trypsin, which is an activator of PAR-2, also activates a number of pro-collagenases, including MMP-1, MMP-2, MMP-7, MMP-8, MMP-9 and MMP-13.51, 52 Trypsin expression is increased in human ovary, prostate, lung, stomach and colon cancer cells.53 Trypsin overexpression in cancerous tissues, which is often referred to “tumor-associated trypsin,” is a poor prognostic factor.53 Conversely, PAR-2 activates trypsin, and this relation probably produces an autocrine-activating loop.54 Therefore, a unique triangle of PAR-2, trypsin and MMPs may regulate the growth and invasion of various tumors.
Tissue factor (TF), which is an activator of PAR-2, is a major initiator of blood coagulation and is related to the metastatic potential and poor prognosis of various malignancies.55 The subsequent formation of a TF-factor VIIa complex activates factors IX and X, which lead to thrombin generation and fibrin deposition. In addition to their roles in clotting, factors VIIa and Xa and thrombin activate various vascular cells.56 TF-factor VIIa and TF-factor VIIa-factor Xa complexes can activate PAR-2, and factor Xa alone can also activate PAR-2.56 This close relation between PAR-2 and TF may affect tumor growth.
The PAR-2 signaling pathway plays an important role in tumor growth, and further, PAR-2 signaling in tumors may promote cell proliferation and also produce a favorable environment for tumor growth and invasion. In this study, we have shown that PAR-2 siRNA has antitumor effects on pancreatic cancer cells. PAR-2-specific RNAi may hold promise as a novel strategy for treatment of pancreatic cancer.