Susan A. Watson

Gastrin — active participant or bystander in gastric carcinogenesis?

Gastrin is a pro-proliferative, anti-apoptotic hormone with a central role in acid secretion in the gastric mucosa and a long-standing association with malignant progression in transgenic mouse models. However, its exact role in human gastric malignancy requires further validation. Gastrin expression is tightly regulated by two closely associated hormones, somatostatin and gastrin-releasing peptide, and aspects of their interaction may be deregulated during progression to gastric adenocarcinoma. Furthermore, agonists and antagonists of the receptors for all three hormones have shown modest clinical efficacy against gastric adenocarcinoma, which might provide useful information on the future combined use of these agents.

Gastrin: growth enhancing effects on human gastric and colonic tumour cells.

Two colorectal (HT29, LoVo) and one gastric (MKN45) human tumour cell lines were examined for their in vitro trophic response to human gastrin-17. MKN45 and HT29 responded by increased 75Se selenomethionine uptake to exogenous gastrin (139 +/- 5.5% and 123 +/- 3% of control values respectively) whereas LoVo showed no significant response to this hormone. When these same cell lines were grown as xenografts in nude mice, similar responses were seen to exogenously administered human gastrin-17 (10 micrograms mouse-1 day-1, subcutaneous injection). MKN45 xenografts showed a greater response to continuously administered gastrin (osmotic mini-pumps, (10 micrograms mouse-1 day-1) when compared to the same dose given via a subcutaneous bolus injection. The hormone-treated xenografts had a two-fold increase in tumour cross-sectional area and growth rate when compared to saline-treated controls. Dose-response studies revealed that 0.4 micrograms gastrin mouse-1 day-1 appeared to be the minimally effective dose. As gastric and colorectal tumour cells show a trophic response to gastrin, antagonists of the gastrin receptor may prevent this effect causing tumour stasis. The gastric tumour cell line, MKN45, is gastrin-responsive and would be an ideal model for screening potent receptor antagonists.

An antiapoptotic role for gastrin and the gastrin/CCK-2 receptor in Barrett's esophagus

Mechanisms by which premalignant Barrett’s metaplasia (BM) progresses to esophageal adenocarcinoma are currently being sought. This study investigated the role played by the polypeptide hormone gastrin, specifically its antiapoptotic effects through activation of protein kinase B/Akt (PKB/Akt). In esophageal cell lines with low basal levels of activated PKB/Akt, phosphorylation could be induced by exogenous amidated gastrin. High basal levels of activated PKB/Akt were linked to endogenous gastrin expression and were reduced by treatment with a cholecystokinin-type 2 receptor (CCK-2R) antagonist. Expression of a constitutively active splice variant of the CCK-2R additionally increased basal activation of PKB/Akt. It is proposed that gastrin acting in an autocrine and endocrine manner via a CCK-2R isoform may activate PKB/Akt and that with expression of gastrin and CCK-2R isoforms increasing in BM samples, gastrin may aid progression of BM through amplification of antiapoptotic pathways. Evidence for this proposal was provided through the observed specific up-regulation of PKB/Akt in BM samples.

Phase I/II trial of batimastat, a matrix metalloproteinase inhibitor, in patients with malignant ascites

Matrix metalloproteinases have been shown to be important in tumour invasion and metastasis, and the use of matrix metalloproteinase inhibitors in animal models has suggested that these agents may be useful in the control of malignant disease. This article reports the results of an early clinical trial of batimastat, one of the first generation of metalloproteinase inhibitors, in patients with malignant ascites. The drug was well absorbed via the intraperitoneal route and associated with few side-effects. Furthermore, a response to treatment was seen in about half the evaluable patients with advanced malignant disease. The results suggest that further research on the use of matrix metalloproteinase inhibitors in patients with malignant disease is worthwhile.

Helicobacter pylori potentiates epithelial:mesenchymal transition in gastric cancer: links to soluble HB-EGF, gastrin and matrix metalloproteinase-7

Background and aims Helicobacter pylori (H pylori) infection is a major risk factor in the development of distal gastric adenocarcinoma. Development of the invasive phenotype is associated with the phenomenon of epithelial:mesenchymal transition (EMT). Soluble heparin-binding epidermal growth factor (HB-EGF) has been implicated in this process. A study was undertaken to investigate the possibility that matrix metalloproteinase (MMP)-7 is upregulated in H pylori infection as a result of hypergastrinaemia, which may enhance shedding of HB-EGF and contribute towards EMT in gastric adenocarcinoma cell lines. Methods Three gastric epithelial cell lines (AGS, MGLVA1 and ST16) were co-cultured with the pathogenic H pylori strain 60190 and non-pathogenic strain Tx30a in an in vitro infection model. Gene expression was quantified by real-time PCR, HB-EGF shedding by ELISA and protein expression by immunofluorescence or immunohistochemistry. The INS-GAS mouse, a transgenic mouse model of gastric carcinogenesis which overexpresses amidated gastrin, was used to investigate the in vivo relationship between HB-EGF, MMP-7, gastrin and EMT. Results The pathogenic strain of H pylori significantly upregulated EMT-associated genes Snail, Slug and vimentin in all three gastric cell lines to a greater degree than the non-pathogenic strain. Pathogenic H pylori also upregulated HB-EGF shedding, a factor implicated in EMT, which was partially dependent on both gastrin and MMP-7 expression. Gastrin and MMP-7 siRNAs and MMP-7 neutralising antibody significantly reduced upregulation of HB-EGF shedding in H pylori infected gastric cell lines and reduced EMT gene expression. The effect of H pylori on EMT was also reversed by gastrin siRNA. Neutralisation of gastrin in the INS-GAS mouse model reduced expression of MMP-7, HB-EGF and key EMT proteins. Conclusion The upregulation of MMP-7 by pathogenic H pylori is partially dependent on gastrin and may have a role in the development of gastric cancer, potentially through EMT, by indirectly increasing levels of soluble HB-EGF.

Matrix metalloproteinase expression and activity in human airway smooth muscle cells

Airway remodelling is a feature of chronic asthma comprising smooth muscle hypertrophy and deposition of extracellular matrix (ECM) proteins. Matrix metalloproteinases (MMPs) breakdown ECM, are involved in tissue remodelling and have been implicated in airway remodelling. Although mesenchymal cells are an important source of MMPs, little data are available on airway smooth muscle (ASM) derived MMPs. We therefore investigated MMP and tissue inhibitor of metalloproteinase (TIMP) production and activity in human ASM cells. MMPs and TIMPs were examined using quantitative real‐time RT–PCR, Western blotting, zymography and a quench fluorescence (QF) assay of total MMP activity. The most abundant MMPs were pro‐MMP‐2, pro‐ MMP‐3, active MMP‐3 and MT1‐MMP. TIMP‐1 and TIMP‐2 expression was low in cell lysates but high in conditioned medium. High TIMP secretion was confirmed by the ability of ASM‐conditioned medium to inhibit recombinant MMP‐2 in a QF assay. Thrombin increased MMP activity by activation of pro‐MMP‐2 independent of the conventional smooth muscle thrombin receptors PAR 1 and 4. In conclusion, ASM cells express pro‐MMP‐2, pro and active MMP‐3, MMP‐9 and MT1‐MMP. Unstimulated cells secrete excess TIMP 1 and 2, preventing proteolytic activity. MMP‐2 can be activated by thrombin which may contribute to airway remodelling.

Selection of Radiolabeled Gastrin Analogs for Peptide Receptor–Targeted Radionuclide Therapy

The gastrin/cholecystokinin-2 (CCK-2) receptor has been identified as a possible target for peptide receptor radionuclide imaging and therapy. Several radiolabeled peptides binding to this receptor have been explored in animal models and clinical trials but either low tumor uptake or high renal retention has been found. The aim of this study was to identify a peptide with improved tumor-to-kidney pharmacodynamics when compared with current candidates. Methods: A small peptide-chelator library of 34 compounds based on the C-terminal sequences of CCK-8 or minigastrin was constructed. The peptides were radiolabeled with 111In with high labeling efficiency (>90%), as determined by high-performance liquid chromatographic analysis. The labeled peptides were screened by assessing tumor and kidney uptake in pancreatic xenograft nude mouse models, including AR42J. An extensive biodistribution analysis was performed on the lead candidate from the library. Results: Minigastrin analogs containing a pentaglutamate sequence showed the highest tumor uptake but very high renal retention. CCK analogs showed the lowest tumor and renal uptake. Deletion of the pentaglutamate sequence in the gastrin analogs lowered the tumor uptake by a factor of 3 but decreased the kidney uptake by a factor of 20. Insertion of histidine residues in the sequence reduced kidney uptake by a further factor of almost 2-fold. In AR42J tumor-bearing mice, the peptide with the sequence DOTA-HHEAYGWMDF-NH2 (DOTA is tetraazacyclododecane tetraacetic acid) showed the highest tumor-to-kidney ratio of all peptides studied, with saturable uptake in target organs and low uptake by nontarget tissues other than the kidney. Conclusion: This peptide is a worthwhile candidate for clinical studies to determine whether it is suitable for use in peptide receptor–targeted radionuclide therapy.

Potential role for peroxisome proliferator activated receptor (PPAR) in preventing colon cancer.

Background: Peroxisome proliferator activated receptors (PPARs) are nuclear hormone receptors involved in genetic control of many cellular processes. PPAR and PPAR have been implicated in colonic malignancy. Here we provide three lines of evidence suggesting an inhibitory role for PPAR in colorectal cancer development. Methods: Levels of PPAR mRNA and protein in human colorectal cancers were compared with matched non-malignant mucosa using RNAse protection and western blotting. APCMin/+ mice were randomised to receive the PPAR activator methylclofenapate 25 mg/kg or vehicle for up to 16 weeks, and small and large intestinal polyps were quantified by image analysis. The effect of methylclofenapate on serum stimulated mitogenesis (thymidine incorporation), linear cell growth, and annexin V and propidium iodide staining were assessed in human colonic epithelial cells. Results: PPAR (mRNA and protein) expression levels were significantly depressed in colorectal cancer compared with matched non-malignant tissue. Methylclofenapate reduced polyp area in the small intestine from 18.7 mm2 (median (interquartile range 11.1, 26.8)) to 9.90 (4.88, 13.21) mm2 (p=0.003) and in the colon from 9.15 (6.31, 10.5) mm2 to 3.71 (2.71, 5.99) mm2 (p=0.009). Methylclofenapate significantly reduced thymidine incorporation and linear cell growth with no effect on annexin V or propidium iodide staining. Conclusions: PPAR may inhibit colorectal tumour progression, possibly via inhibition of proliferation, and may be an important therapeutic target.

Gastrin enhances the angiogenic potential of endothelial cells via modulation of heparin-binding epidermal-like growth factor

This study examined whether gastrin modulates endothelial cell activity via heparin-binding epidermal growth factor-like growth factor (HB-EGF) expression. Human umbilical vascular endothelial cells (HUVEC) were assessed for tubule formation in the presence of amidated gastrin-17 (G17) and glycine-extended gastrin-17 (GlyG17) peptides. HB-EGF gene and protein expressions were measured by quantitative reverse transcription-PCR, immunocytochemistry, and Western blotting, and HB-EGF shedding by ELISA. Matrix metalloproteinases MMP-2, MMP-3, and MMP-9 were assessed by Western blotting. Chick chorioallantoic membrane studies measured the in vivo angiogenic potential of gastrin and microvessel density (MVD) was assessed in large intestinal premalignant lesions of hypergastrinaemic APC(Min) mice. MVD was also examined in human colorectal tumor and resection margin normals and correlated with serum-amidated gastrin levels (via RIA) and HB-EGF protein expression (via immunohistochemistry). HUVEC cells showed increased tubule and node formation in response to G17 (186%, P < 0.0005) and GlyG17 (194%, P < 0.0005). This was blockaded by the cholecystokinin-2 receptor (CCK-2R) antagonists JB95008 and JMV1155 and by antiserum to gastrin and HB-EGF. Gastrin peptides increased HB-EGF gene expression/protein secretion in HUVEC and microvessel-derived endothelial cells and the levels of MMP-2, MMP-3, and MMP-9. G17 promoted angiogenesis in a chorioallantoic membrane assay, and MVD was significantly elevated in premalignant large intestinal tissue from hypergastrinaemic APC(Min) mice. In terms of the clinical situation, MVD in the normal mucosa surrounding colorectal adenocarcinomas correlated with patient serum gastrin levels and HB-EGF expression. Gastrin peptides, acting through the CCK-2R, enhance endothelial cell activity in models of angiogenesis. This may be mediated through enhanced expression and shedding of HB-EGF, possibly resulting from increased activity of matrix metalloproteinases. This proangiogenic effect translates to the in vivo and human situations and may add to the tumorigenic properties attributable to gastrin peptides in malignancy.

Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion

Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3–4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents.