The kallikrein-Kinin system modulates the progression of colorectal liver metastases in a mouse model
The Kallikrein-Kinin System (KKS) has been found to play a role in tumor progression in several cancers. The KKS metabolic cascade depends on signalling through two cross talking receptors; bradykinin receptor 1 (B1R) and bradykinin receptor 2 (B2R). Activation of the Kinin receptor is responsible for multiple pathophysiologic functions including increase of vascular permeability and induction of host inflammatory responses that exert diverse effects on tumor growth.
B1R and B2R expression on mouse and human CRC cell lines was investigated. Changes in tumor growth and progression was assessed in male CBA mice bearing colorectal liver metastases (CRLM) following treatment with B1R or B2R blockers. In vitro cultures of human SW-480 and mouse colorectal cancer (MoCR) cell lines were examined for changes in their proliferation and migration properties following treatment with B1R or B2R blockers.
Both colorectal cancer cell lines tested strongly positive for B1R and B2R expression. Inhibition of both receptors retarded tumor growth but only B1R blockade significantly reduced tumor load and increased tumor apoptosis. Blockade of either receptor reduced tumor vascularization in vivo and significantly inhibited proliferation and migration of colorectal cancer cells in vitro.
Taken together, the present study demonstrated that kinin receptor blockade inhibited tumor growth and reduced its invading properties suggesting that KKS manipulation could be a novel target in colorectal cancer therapy.
KeywordsColorectal liver metastases Kallikrein-Kinin system Bradykinin receptor Kinin or bradykinin
colorectal liver metastasis
Mouse colorectal cancer
Colorectal cancer (CRC) is the third most common cancer worldwide . The majority of CRC related deaths are associated with liver metastasis [2, 3]. Surgical removal of colorectal liver metastases (CRLM) supported by systemic chemotherapy provides the best possibility for cure in a percentage of patients and even among these, 30% - 60% will develop tumor recurrence in the liver or in other organs [4, 5]. Recent studies have demonstrated involvement of the renin angiotensin system (RAS) in cancer progression, including CRLM [6, 7]. There is also evidence of crosstalk between the Kallikrein Kinin System KKS and RAS pathways. The angiotensin-converting enzyme (ACE) catalyses both the production of angiotensin II and the degradation of bradykinin, suggesting a cross-regulation between the two systems , however the effects of the Kallikrein Kinin System (KKS) on CRLM have not been as well studied.
The role of kinin receptors in cancer has been investigated [9, 10, 11]. Many cancer tissues display higher expression of B1R and/or B2R compared to their normal counterparts ; ‘kinin’ in humans and most mammal contexts refers to bradykinin (BK). Signalling by BK through either B1R, B2R or both can promote angiogenesis in different experimental models by promoting vascular cell proliferation and survival, and by increasing vascular permeability [12, 13]. Selective antagonists of B1R or B2R have shown anti-proliferative, anti-inflammatory, anti-angiogenic and anti-migratory properties [9, 12, 14]. Kinin receptors have been reported in mouse and human colon carcinoma cell lines [15, 16]. BK treatment of the CRC cell line SW-480 results in mitogenic activation . Zelawski et al. found a higher B1R expression in human tubular adenomas, a benign tumor that can become colonic carcinoma, suggesting that kinins may contribute to cellular transformation .
While the literature support the role of the Kallikrein Kinin System (KKS) signalling in tumor aggressiveness and progression, additional studies are needed in relevant animal models that closely represent the clinical tumor progression. We have developed and characterised a mouse model of CRLM in a fully immunocompetent host which we used in the present study to investigate the effects of kinin receptor inhibition on the CRLM tumors. Additionally we investigated the direct effects of KKS inhibitors on cultured CRC cell proliferation and migration.
CBA strain mice (Laboratory Animal services, University of Adelaide, South Australia) were used. Mice were kept in standard cages (2–5 animals per cage) in rooms with 12 h light/dark cycle, constant temperature and humidity. Food and water were provided ad libitum. Environmental enrichment was provided in the form of dry tissue ribbons and cardboard tunnels. All animal experiments were approved by the Austin Health Animal Ethics Committee.
Liver metastases were established by intrasplenic injection. as described previously [6, 18] Briefly, 2.5 × 104 MoCR cells were injected into the spleen of 6 to 8 week old male CBA mice (body weight 24–28 g), under anaesthesia (isoflurane) following an abdominal incision to exteriorize the spleen. Mice were given long-acting analgesic (carprofen, 5 mg/kg) just before surgery, hydrating jelly post surgery and were monitored closely for the next 24 h. In this model tumors are well established by day 21 post-induction, when liver samples were collected and fixed in fresh 10% formalin.
Drugs/agents and treatments for the in vivo studies
Three groups of six animals were used, one control and two experimental groups. Animals were assigned randomly to each group. The B1R inhibitor (SSR240612) was given at 15 mg/kg/day. The B2R inhibitor (FR173657) was given at15 mg/kg twice daily (early morning and late afternoon), totalling 30 mg/kg/day. Both drugs were dissolved prior to administration in 10% DMSO diluted in distilled H2O. Control animals were administered 10% DMSO in distilled H2O. Each treatment was administered via oral gavage (volume of 0.25 ml) starting from day 1 post tumor induction.
Stereological assessment of tumor growth
Tumor volume and burden were assessed using quantitative stereology as described previously . Briefly the fixed tissues were sliced into 1.5 mm sections and imaged using a Lumenera Infinity4 digital CCD camera. The imaging software Image-ProPlus 6.0 was used to collect the data. The researcher collecting the data was blinded in regards to the group origin of the tissue.
Immunohistochemistry and data collection were performed as described previously  Changes in tumor angiogenesis and apoptosis were determined by immunostaining formalin fixed liver tissues with anti-CD34 (rat anti-mouse monoclonal, Serotec MCA 18256 at 1 μg/ml and anti-Caspase 3 (rabbit polyclonal, R&D system AF835 at 2 μg/ml) antibodies, respectively. Antigen retrieval was performed by heat in citrate buffer.
Human and mouse cell lines
SW480 (ATCC® CCL-228™) cell line is a human colorectal cancer cell line, derived from grade 3–4 colon adenocarcinoma and grown adherent in cell culture. SW480 cells were cultured in DMEM/10% FBS at 37 °C and 5%CO2/95% air. The mouse MoCR cell line used in the current studies was originally derived in our laboratory from a dimethyl hydrazine-induced CBA mouse colon carcinoma as described previously  MoCR cells were cultured in RPMI/10% FBS at 37°Cand 5%CO2/95% air. Both cell lines were mycoplasma free upon testing. Culture passages 4–15 were used for the experiments described in these studies.
Drugs/agents and treatments for the in vitro studies
SSR240612 (Sanofi Aventis) was used at 10 μM to block B1R and FR173657 (Astellas Pharma) at 10 μM to block B2R. The agonist desArg9-Bradykinin (DABK) (Sigma-Aldrich) at 0.1 μM was used to activate B1R. Bradykinin fragment 1–8 (BK) (Sigma-Aldrich) at 0.1 μM was used to activate B2R.
Cell proliferation was determined by counting the incorporation of [H3]-thymidine. Cells were seeded at 6 × 104 cells/well in RPMI/10%FBS (MoCR cells) or DMEM/10%FBS (SW480) in 96-well plates and allowed to adhere overnight before being starved in 0% FBS RPMI or DMEM for 18 h. Following starving, fresh RPMI/DMEM and 10 μCi/ml 3H-thymidine, +/− agonists (DABK or BK) and/or antagonists (SSR240612 or FR173657) were added and cultured for 48 h. The cell cultures were then collected using the NUNC cell harvester. The 3H-thymidine incorporation into the dividing cells was measured using a β-counter (Packard, Meriden, CT).
Cell migration/invasion assay
A modified Boyden Chamber assay  was used to investigate cell migration and invasion. Briefly membranes (8-μm pore size, BD353097, Becton Dickinson, NJ, USA) were coated with 30 μg/30 μl fibronectin (BD3534009, Becton Dickinson, USA) on their lower surface and placed into the wells of a 24-well plate containing 600 μl/well serum-free RPMI and 0.1% BSA. Tumor cells (2–5 × 104 cells/100 μl) were added to the upper chamber in the presence or absence of agonists (DABK or BK) and/or antagonists (SSR240612 or FR173657). The plates were placed in a humidifying incubator for 24 h at 37 °C and 5% CO2. The cells on the upper surface of the membranes were removed and the cells on the lower surface were fixed, stained with Quick-Dip (Fronine, Sydney, Australia) and counted using a NIKON Coolscope (Coherent Scientific, Adelaide, Australia).
Data was analysed using the statistical software package SPSS (SPSS Version 17.0; IBM Co., Armonk, NY, USA). Quantitative data are presented as mean values ± S.E.M. for each group. Parametric data was analysed using ANOVA with post hoc comparison (Tukey method) for parametric data and Mann-Whitney U test for non-parametric data. A p value ≤0.05 was considered statistically significant.
Mouse and human CRC cell lines express B1R and B2R.
We first wanted to establish whether our CRC cell lines (MoCR and SW480) express B1R and/or B2R. Immunohistochemical staining for these receptors confirmed their expression (Additional file 1), supporting previous published studies that CRC cells express bradykinin receptors .
B1R blockade reduced tumor burden in a mouse model.
B1R blockade increased tumor apoptosis in vivo
Kinin receptor blockade inhibited angiogenesis
Bradykinin receptor blockers inhibited in vitro tumor cell proliferation
Having established that blocking the B1R and B2R signalling slows CRC tumor progression and that CRC tumor cells express bradykinin receptors we wanted to know if receptor activation or blockade modulate tumor cell characteristics directly. The stimulation of the receptors on cell proliferation was also examined in MoCR cells by the addition of agonists 0.1 μM DABK (B1R) or BK (B2R). Exposure to 0.1 μM of DABK did not significantly change the proliferation of MoCR cells. In contrast 0.1 μM of BK increased MoCR proliferation by 30% at 48 h after treatment (*P < 0.05) (Additional file 2).
B1R and B2R blockade inhibited kinin mediated invasion in CRC
The KKS is a complex multifunctional signalling cascade and its role in cancer remains unclear. Early reports on the likely role of the KKS in cancer suggested enhanced vascular permeability and upregulation of nitric oxide synthase and prostaglandin [23, 24]. This led to the suggestion that KKS family members may be novel biomarkers for cancer [25, 26, 27, 28]. More recent studies further support the role of the KKS in cancer development [7, 29] and a number of KKS generated proteins confirmed to exhibit pro-inflammatory properties . Chronic inflammation is tightly associated with cancer development and progression .
In this study B1R or B2R blockade led to reduction in tumor progression compared to untreated controls, however this reduction did not reach significance for B2R blockade. Blockade of B1R resulted in significant viable tumor reduction and significant increase in apoptosis suggesting that the reduction in tumor progression is effected, at least in part, through tumor cell apoptosis. In contrast there was no reduction in the percentage of viable tumor or increase in apoptosis associated with B2R blockade. It has been shown that B1R and B2R can activate several intracellular signalling pathways including NF-κB [32, 33]. Indeed the p53 and NF-κB pathways are, arguably, the two major cellular stress response pathways leading to pro-apoptosis or anti-apoptosis pathways respectively. Webster et al. demonstrated that during injury or stress there is crosstalk between NF-κB and p53; they showed that these two transcription factors can modulate each other’s functions depending on their relative levels present. Following injury, the ability of NF-kB to supress p53 can determine the cell fate . Although both B1R and B2R have been shown to induce NF-kB activation; the patterns of signalling are different in terms of duration and intensity . It is possible that B1R activation of NF-kB leads to anti-apoptotic properties, therefore inhibition of B1R demonstrated a significant increase in tumor apoptosis. Another possible explanation for the reduced tumor burden following B1R blockade could be due to a reduction in chronic inflammation normally seen in cancer as a result of increased expression and signalling of the B1R. In contrast B2R is constitutively expressed and hence assumed to be mostly responsible for effecting normal physiological functions.
Our results show that blockade of either the B1R or the B2R resulted in significant reduction in tumor vascular density and this may be one of the mechanisms by which tumor progression is retarded. B1R and B2R activation have been implicated in angiogenesis and neovascularization in other studies. Kinins induce EC proliferation in cell cultures through the B1-cAMP pathway. B1R stimulation has been shown to induce neovascularization in the rabbit cornea , while B2R stimulation can activate the mitogen-activated protein kinase pathways (MAPK) and P13K/AKT, contributing to proliferation and angiogenesis [37, 38, 39]. Other studies have shown tumor suppression and angiogenic inhibition following treatment with B2R inhibition  or in kininogen knock out mice . Moreover, Morbidelli et al. demonstrated the ability of BK to stimulate EC proliferation via activation of B1R or indirect activation of B2R, further supporting a role of kinin receptors in angiogenesis and tumor development .
In agreement with Wang et al.  we also detected B1R and B2R expression in both MoCR and SW480 colon cancer cells lines suggesting that receptor blockade may also have direct effects on the tumor cells. There are several studies which suggest that activation of B1R and B2R leads to stimulation of tumor cell proliferation and migration [37, 42, 43]. Unlike Barki-Harrington et al. who found increased proliferation following stimulation of B1R and B2R , our in vitro study failed to show increased proliferation with B1R agonist (desArg9-Bradykinin (DABK)), however, the involvement of B1R in proliferation cannot be completely ruled out, since treatment with a B1R antagonist (SSR240612) demonstrated significant reduction in tumor proliferation. McLean et al. found that the synthesis of DABK is significantly upregulated during inflammation ; it is possible that MoCR cells may have maximal DABK secretion therefore external addition of the DABK agonist does not further increase cell proliferation. Although DABK failed to increase tumor proliferation, our current study did find that BK treatment (B2R agonist) resulted in increased cell proliferation and the use of a B2R antagonist (FR173657) significantly decreased proliferation, further demonstrating that kinins do play a role in tumor proliferation. Further stimulation of kinin receptors was shown to increase tumor cell migration. Wang et al. reported that treatment with BK stimulated B2R and ERK1/2 leading to increased IL-6 production, ultimately increasing the invasiveness of colorectal cancer cells ., Ehrenfeld et al. found that activation of the B1R increased secretion of the metalloproteases (MMPs)-2 and − 9 by breast cancer cells . Previous studies have shown that MMP2 activates integrin αvβ3 resulting in cellular invasion ; similarly MMP-9 has been shown to enhance metastatic capacity through activation of αvβ3 in breast cancer cell lines  and αvβ6 integrin activation has been shown to promote invasion of squamous cell carcinoma cells . In agreement, using invasion assays, we observed significant decrease in tumor invasion following treatment with both B1R and B2R antagonists, further confirming a role of bradykinin receptors in CRLM migration.
Here we show that kinin receptors are involved in MoCR tumor progression In an in vivo CRLM mouse model tumor angiogenesis is inhibited and tumor progression retarded by B1R and B2R blockade. In addition, B1R inhibition led to significant reduction in the percentage of live tumor, possibly due to the increased tumor apoptosis. In an in vitro setting B1R and B2R antagonists decreased both tumor proliferation and migration. Taken together the results indicate that KKS manipulation could be a novel therapeutic target for treatment of colorectal cancer.
This study was supported by a grant from Cure Cancer Australia to Dr. Patricia Nunes da Costa. Cure Cancer Australia provided research support, they had no role in the study design, data collection, analysis and interpretation of results or in the preparation and submission of this manuscript.
Availability of data and materials
The datasets generated and analysed during this study are presented in the Figures and Additional Files. Raw data are available from the corresponding author on reasonable request.
PdaC and CC conceived, designed and supervised the study. DW and LN performed the experiments. PdaC, DW, MP and TF analysed and interpreted the data. PdaC and LN wrote the manuscript. TF and MP edited the manuscript. All authors read and approved the final manuscript.
All animal experiments were approved by the Austin Health Animal Ethics Committee in accordance with international guidelines for animal use. All efforts were made to minimize the number and suffering of animals
Consent for publication
The authors declare that they have no competing interests.
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