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OBJECTIVES: To assess whether metformin reduces radio-resistance and increases survival in men undergoing external beam radiation therapy (EBRT) for prostate cancer (PCa), and to determine its effect on hypoxia inducible factor 1-α (HIF1α). PATIENTS AND METHODS: All patients treated with curative intent with EBRT for PCa at a major cancer centre between 2000 and 2007 were included in this study. The outcome measures of time to biochemical failure (BF), metastasis, PCa-specific mortality and overall survival (OS) were analysed in those taking metformin vs those not, using competing risk and Cox regression models. To determine metformin's effect on HIF1α expression and survival in vitro, PC3 cells with high basal HIF1α levels were subjected to increasing doses of metformin after H2 O2 -induced oxidative stress. RESULTS: A total of 2055 eligible cases, including 113 who were on metformin, were identified, with a median follow-up of 95.7 months. There were no differences in age, initial prostate-specific antigen level, Gleason score, T-stage, D'Amico risk class or duration of androgen deprivation therapy (ADT) between patients who were or were not on metformin. Treatment with metformin did not result in any apparent improvement in time to BF, time to metastasis detection or OS, but there was a 1.5-fold increase in PCa-specific deaths (P = 0.045) in patients on metformin and ADT when adjusted for cancer risk and comorbidities. When comparing patients on high-dose metformin (>1 g/d) with those on low-dose metformin (≤1 g), there was no difference in either time to metastases or time to BF. In vitro metformin at a high concentration of 100 μM did not reduce HIF1α expression, nor did metformin affect the PC3 cell survival when exposed to oxidative stress (H2 O2 ). CONCLUSIONS: No association was found between the use of metformin and time to metastasis detection, time to BF or OS in patients undergoing radiation therapy with or without ADT for PCa. In vitro, low therapeutic concentrations of metformin had no effect on HIF1α, and this observation could explain the conflicting evidence for the effectiveness of metformin in men undergoing EBRT for PCa. Higher, more toxic doses of metformin may be required to inhibit the mammalian target of rapamycin-HIF1α pathway in this patient group.

Introduction & Objectives. Contrast media (CM) causes renal injury through both direct cellular injury (cytotoxicity) and regional vascular changes (renal hypoxia) mediated by reactive oxygen species (ROS). Zinc may be able to provide protection against CM-induced cytotoxicity due to its indirect antioxidant properties and subsequent effect on ROS. We aimed to determine the protective role of zinc preconditioning against contrast-induced renal injury in vitro. Methods. Normal human proximal renal kidney cells (HK-2) were preconditioned with either increasing doses of ZnCl2 or control. Following this preconditioning, cells were exposed to increasing concentrations of Iohexol 300 mg I2/ml for four hours. Key outcome measures included cell survival (MTT colorimetric assay) and ROS generation (H2DCFDA fluorescence assay). Results. Contrast media induced a dose-dependent reduction in survival of HK-2 cells. Compared to control, contrast media at 150, 225, and 300 mg I2/ml resulted in 69.5% (SD 8.8%), 37.3% (SD 4.8%), and 4.8% (SD 6.6%) cell survival, respectively ( p < 0.001 ). Preconditioning with 37.5 μM and 50 μM ZnCl2 increased cell survival by 173% (SD 27.8%) ( p < 0.001 ) and 219% (SD 32.2%) ( p < 0.001 ), respectively, compared to control preconditioning. Zinc preconditioning resulted in a reduction of ROS generation. Zinc pre-conditioning with 37.5 μM μM ZnCl2 reduced ROS generation by 46% ( p < 0.001 ) compared to control pre-conditioning. Conclusions. Zinc preconditioning reduces oxidative stress following exposure to radiographic contrast media which in turn results in increased survival of renal cells. Translation of this in vitro finding in animal models will lay the foundation for future use of zinc preconditioning against contrast induced nephropathy.

Acute kidney injury (AKI) as a result of ischaemia-reperfusion represents a major healthcare burden worldwide. Mortality rates from AKI in hospitalized patients are extremely high and have changed little despite decades of research and medical advances. In 1986, Murry et al. demonstrated for the first time the phenomenon of ischaemic preconditioning to protect against ischaemia-reperfusion injury (IRI). This seminal finding paved the way for a broad body of research, which attempted to understand and ultimately harness this phenomenon for human application. The ability of preconditioning to limit renal IRI has now been demonstrated in multiple different animal models. However, more than 30 years later, a safe and consistent method of protecting human organs, including the kidneys, against IRI is still not available. This review highlights agents which, despite strong preclinical data, have recently failed to reduce AKI in human trials. The multiple reasons which may have contributed to the failure to translate some of the promising findings to clinical therapies are discussed. Agents which hold promise in the clinic because of their recent efficacy in preclinical large animal models are also reviewed.

Pancreatic cancer is one of the most aggressive and lethal malignancies worldwide, with a very poor prognosis and a five-year survival rate less than 8%. This dismal outcome is largely due to delayed diagnosis, early distant dissemination and resistance to conventional chemo-therapies. Kras mutation is a well-defined hallmark of pancreatic cancer, with over 95% of cases harbouring Kras mutations that give rise to constitutively active forms of Kras. As important down-stream effectors of Kras, p21-activated kinases (PAKs) are involved in regulating cell proliferation, apoptosis, invasion/migration and chemo-resistance. Immunotherapy is now emerging as a promising treatment modality in the era of personalized anti-cancer therapeutics. In this review, basic knowledge of PAK structure and regulation is briefly summarised and the pivotal role of PAKs in Kras-driven pancreatic cancer is highlighted in terms of tumour biology and chemo-resistance. Finally, the involvement of PAKs in immune modulation in the tumour microenvironment is discussed and the potential advantages of targeting PAKs are explored.

Immature forms of the peptide hormone gastrin have been implicated in the development of colorectal cancer (CRC). The biological activity of glycine-extended gastrin (Ggly) is dependent on the binding of Fe3+ ions in vitro and in vivo. The aim of the present study was to determine the effect of blocking Fe3+ ion binding to Ggly, using Bi3+, In3+ or Ru3+ ions, on the development of intestinal tumors in APCΔ14/+ mice. APCΔ14/+ mice were treated orally with Bi3+, In3+ or Ru3+ ions for up to 60 days, serum trace metals were analyzed by inductively coupled plasma mass spectrometry, and the incidence and size of intestinal tumors were assessed. Bi3+ treatment significantly decreased the number of tumors larger than 3 mm in male mice. In3+ or Ru3+ treatment significantly increased the tumor burden in all animals and In3+ increased the number of tumors larger than 3 mm or 5 mm in male mice alone. The fact that binding of In3+ or Ru3+ ions to Ggly was orders of magnitude stronger than the binding of Bi3+ ions implies that the inhibitory effect of Bi3+ ions is not a consequence of a reduction in Ggly activity. However, further testing of higher doses of Bi3+ ions for longer periods as an oral treatment for intestinal tumors is warranted.

The peptide hormone gastrin (Gamide) binds trivalent metal ions, including indium (In), ruthenium (Ru) and gallium (Ga), with high affinity. Complexes of gastrin with chelated isotopes of In and Ga have previously been used for the location of tumours expressing the cholecystokinin 2 receptor (CCK2R). The aim of the present study was to purify the complexes of Gamide with radioactive isotopes of In, Ru or Ga and to investigate their ability to bind to the CCK2R. The radioactive Gamide complexes were purified on Sep-Pak C18 cartridges or by anion exchange HPLC. Binding to the CCK2R was assessed with a stably transfected clone of the gastric carcinoma cell line AGS. The 106Ru-Gamide complex could be eluted from the C18 cartridge; the 111In-Gamide and 68Ga-Gamide complexes bound irreversibly. All three complexes were successfully purified by anion exchange HPLC. The failure to detect binding of the 111In-Gamide, 106Ru-Gamide and 68Ga-Gamide complexes to the CCK2R suggests that formation of these complexes will not be useful for the detection of tumours expressing this receptor, but may instead provide alternative ways to block the actions of Gamide as a growth factor or a stimulant of gastric acid secretion. The complexes between the hormone gastrin and radioactive 111In, 106Ru or 68Ga ions were purified by anion exchange HPLC using a NaCl gradient. The failure to detect binding of the complexes to the cholecystokinin 2 receptor suggests that metal ion treatment may provide novel approaches to block the biological actions of gastrin.

Staging of colorectal cancer often fails to discriminate outcomes of patients with morphologically similar tumours that exhibit different clinical behaviours. Data from several studies suggest that the gastrin family of growth factors potentiates colorectal cancer tumourigenesis. The aim of this study was to investigate whether progastrin expression may predict clinical outcome in colorectal cancer. Patients with colorectal adenocarcinoma of identical depth of invasion who had not received neoadjuvant therapy were included. The patients either had stage IIa disease with greater than 3-year disease-free survival without adjuvant therapy or stage IV disease with liver metastases on staging CT. Progastrin expression in tumour sections was scored with reference to the intensity and area of immunohistochemical staining. Progastrin expression by stage IV tumours was significantly greater than stage IIa tumours with mean progastrin immunopositivity scores of 2.1 ± 0.2 versus 0.5 ± 0.2, respectively (P

Immunotherapies have not yielded significant clinical benefits for pancreatic ductal adenocarcinoma (PDA) because of the existence of an immunosuppressive tumour microenvironment (TME) characterized by a desmoplastic stroma containing infiltrated immune cells and activated pancreatic stellate cells (PSCs). This study aims to investigate the involvement of PAK1 in anti-tumour immunity. In PDA patients, low PAK1 expression, low activation of PSC and high CD8+ T cell/PAK1 ratios correlated with longer overall survival. In a murine PDA model, PAK1 knockout increased intra-tumoral CD4+ and CD8+ T cells, inhibited PSCs activation and extended survival. Inhibition of PAK1 reduced PSC-stimulated PDA cell proliferation and migration, blocked PSC-mediated protection of PDA cells from killing by cytotoxic lymphocytes and decreased intrinsic and PSC-stimulated PD-L1 expression in PDA cells, which further sensitized PDA cells to cytotoxic lymphocytes. Inhibition of PAK1 stimulates anti-tumour immunity by increasing intra-tumoral CD4+ and CD8+ T cells, and by sensitizing PDA cells to killing by cytotoxic lymphocytes via down-regulation of intrinsic and PSC-stimulated PD-L1 expression. PAK1 inhibitors, especially in combination with immune checkpoint inhibitors may result in improved efficacy of immunotherapy of PDA.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies worldwide. All-trans retinoic acid (ATRA) has been used as an antistromal agent in PDA, and its antitumor effect has also been reported in various kinds of cancer, including PDA. Inhibition of p21-activated kinases (PAKs) is associated with decreased tumor growth and increased gemcitabine sensitivity. The aim of this study was to evaluate the inhibitory effects of ATRA alone and in combination with gemcitabine on cell growth and migration of wild-type and gemcitabine-resistant PDA cells and the potential mechanism(s) involved. Human (MiaPaCa-2) and murine (TB33117) PDA cell lines were incubated in increasing concentrations of gemcitabine to establish resistant clones. Cell growth, clonogenicity, and migration/invasion were determined using a sulforhodamine B assay, a colony formation assay, and a Boyden chamber assay, respectively. Protein expression was measured by Western blotting. ATRA reduced cell proliferation, colony formation, and migration/invasion in both wild-type and gemcitabine-resistant cell lines. PAK1 expression was significantly increased in resistant cells. Cells treated with ATRA showed decreased expression of PAK1, PAK2, PAK4, and α-smooth muscle actin. The combination of ATRA and gemcitabine synergistically reduced cell growth in both wild-type and gemcitabine-resistant cell lines. Depletion of PAK1 enhanced ATRA sensitivity in MiaPaCa-2 cells. In conclusion, the antitumor effects of ATRA and its synergism with gemcitabine are associated with downregulation of PAKs.NEW & NOTEWORTHY The inhibitory effect of all-trans retinoic acid (ATRA) on cell proliferation, colony formation, and migration/invasion was associated with downregulation of p21-activated kinases (PAKs), and depletion of PAK1 enhanced ATRA sensitivity in MiaPaCa-2 cells. The combination of ATRA and gemcitabine synergistically reduced cell growth in both wild-type and gemcitabine-resistant pancreatic ductal adenocarcinoma cells. As an important prognostic marker, α-smooth muscle actin also can be downregulated by ATRA in pancreatic ductal adenocarcinoma cells.

Pancreatic cancer remains one of the most lethal of all solid tumors. Pancreatic stellate cells (PSCs) are primarily responsible for the fibrosis that constitutes the stroma and p21-activated kinase 1 (PAK1) may have a role in signalling pathways involving PSCs. This study aimed to examine the role of PAK1 in PSCs and in the interaction of PSCs with pancreatic cancer cells. Human PSCs were isolated using the modified outgrowth method. The effect of inhibiting PAK1 with group 1 PAK inhibitor, FRAX597, on cell proliferation and apoptosis in vitro was measured by thymidine incorporation and annexin V assays, respectively. The effect of depleting host PAK1 on the survival of mice with pancreatic Pan02 cell tumors was evaluated using PAK1 knockout (KO) mice. PAK1 was expressed in isolated PSCs. FRAX597 reduced the activation of PSCs, inhibited PSC proliferation, and increased PSC apoptosis at least in partial by inhibiting PAK1 activity. The decreased expression and activity of PAK1 in PAK1 KO mice tumors was associated with an increased mouse survival. These results implicate PAK1 as a regulator of PSC activation, proliferation and apoptosis. Targeting stromal PAK1 could increase therapeutic response and survival of patients with pancreatic cancer.

BACKGROUND: Pancreatic cancer continues to have a poor survival rate with an urgent need for improved treatments. Glaucarubinone, a natural product first isolated from the seeds of the tree Simarouba glauca, has recently been recognized as having anti-cancer properties that may be particularly applicable to pancreatic cancer. METHODS: The effect of glaucarubinone on the growth and migration of murine pancreatic cancer cells was assessed by (3)H-thymidine incorporation assay. The survival impact of glaucarubinone alone and in combination with gemcitabine chemotherapy was assessed using an immunocompetent orthotopic murine model of pancreatic cancer. RESULTS: Glaucarubinone inhibited the growth of the murine pancreatic cancer cell lines LM-P and PAN02. Treatment with either glaucarubinone or gemcitabine reduced proliferation in vitro and the combination was synergistic. The combination treatment improved survival two-fold compared to gemcitabine treatment alone (p = 0.046) in PAN02 cells. CONCLUSIONS: The synergistic inhibition by glaucarubinone and gemcitabine observed in vitro and the improved survival in vivo suggest that glaucarubinone may be a useful adjunct to current chemotherapy regimens.

Background/objective: Pancreatic ductal adenocarcinoma (PDA) remains the most lethal malignancy due to lack of an effective treatment. P21-activated kinases (PAKs) play a key role not only in cell proliferation and migration, but also in mediating chemo-resistance in PDA. The aim of this study was to investigate the combined effect of a PAK inhibitor PF-3758309 with multiple chemotherapeutic reagents on a panel of patient-derived PDA cell lines, and potential mechanisms involved. Methods: Cells were treated with PF-3758309 plus or minus gemcitabine, 5-fluorouracil (5-FU) or abraxane, and cell growth was determined using a cell proliferation assay kit. Protein expression profiles were measured by Western blot. PDA cells were subcutaneously injected into the flanks of SCID mice which were then treated with saline, gemcitabine, PF-3758309, gemcitabine plus PF-3758309 or abraxane. Tumour growth was measured by volume and weight. Results: PAK1 was correlated with CK19 expression, and PAK4 with α-SMA and palladin expression. Combination of PF-3758309 with 5-FU, gemcitabine or abraxane further suppressed cell growth of patient-derived PDA cell lines in vitro. The combination of PF-3758309 with gemcitabine maximally inhibited tumour growth in vivo by suppressing cell proliferation. PF-3758309 inhibited the expression of HIF-1α, palladin and α-SMA both in vitro and in vivo. Conclusions: PAK inhibitor PF-3758309 can enhance anti-tumour effects of multiple chemotherapeutic reagents on a panel of patient-derived PDA cell lines. Combination of PF-3758309 with gemcitabine achieves comparable efficacy to combination of gemcitabine with abraxane, and thus provides a potential targeted therapy in the management of PDA.

Pancreatic ductal adenocarcinoma (PDA) remains the most lethal malignancy due to lack of an effective treatment. P21-activated kinases (PAKs) play key roles in PDA growth, and the PAK inhibitor PF-3758309 synergistically reduced PDA growth with gemcitabine. The aim of this study was to determine the effect of PF-3758309 with multiple chemotherapeutic reagents on a panel of patient-derived PDA cell lines. Cells were treated with PF-3758309 plus or minus gemcitabine, 5-fluorouracil (5-FU) or abraxane, and cell proliferation was determined. Protein expression profiles were measured by Western blot. PDA cells were subcutaneously injected into the flanks of SCID mice which were then treated with PF-3758309, gemcitabine, PF-3758309 plus gemcitabine, or gemcitabine plus abraxane. Tumour growth was measured by volume and weight. PF-3758309 enhanced the inhibitory effects of 5-FU, gemcitabine and abraxane on a panel of patient-derived PDA cells, inhibited HIF-1 protein expression and reduced the protein levels of palladin and -SMA in these cells. The combination of PF-3758309 with gemcitabine maximally inhibited PDA growth in vivo, which was comparable to the combination of gemcitabine with abraxane. PF-3758309 enhanced the suppressive effects of multiple chemotherapeutic reagents on the growth of a panel of patient-derived PDA cell lines. The combination of PF-3758309 with gemcitabine provides a potential treatment option with less toxicity than gemcitabine plus abraxane.

Ischaemia-reperfusion injury (IRI) during various surgical procedures, including partial nephrectomy for kidney cancer or renal transplantation, is a major cause of acute kidney injury and chronic kidney disease. Currently there are no drugs or methods for protecting human organs, including the kidneys, against the peril of IRI. The aim of this study was therefore to investigate the reno-protective effect of Zn2+ preconditioning in a clinically relevant large animal sheep model of IRI. Further the reno-protective effectiveness of Zn2+ preconditioning was tested on normal human kidney cell lines HK-2 and HEK293. Anaesthetised sheep were subjected to uninephrectomy and 60 min of renal ischaemia followed by reperfusion. Sheep were preconditioned with intravenous injection of zinc chloride prior to occlusion. Serum creatinine and urea were measured before ischaemia and for 7 days after reperfusion. HK-2 and HEK293 cells were subjected to in vitro IRI using the oxygen- and glucose-deprivation model. Zn2+ preconditioning reduced ischaemic burden determined by creatinine and urea rise over time by ~ 70% in sheep. Zn2+ preconditioning also increased the survival of normal human kidney cells subjected to cellular stress such as hypoxia, hydrogen peroxide injury, and serum starvation. Overall, our protocol incorporating specific Zn2+ dosage, number of dosages (two), time of injection (24 and 4 h prior), mode of Zn2+ delivery (IV) and testing of efficacy in a rat model, a large preclinical sheep model of IRI and cells of human origin has laid the foundation for assessment of the benefit of Zn2+ preconditioning for human applications.

Non-amidated gastrin peptides such as glycine-extended gastrin (Ggly) are biologically active in vitro and in vivo and have been implicated in the development of gastric and colonic cancers. Previous studies have shown that the truncated form of Ggly, the octapeptide LE5AY, was still biologically active in vitro, and that activity was dependent on ferric ion binding but independent of binding to the cholecystokinin 2 (CCK2) receptor. The present work was aimed at creating more stable gastrin-derived 'super agonists' using retro-inverso technology. The truncated LE5AY peptide was synthesized using end protecting groups in three forms with l-amino acids (GL), d-amino acids (GD) or retro-inverso (reverse order with d-amino acids; GRI). All of these peptides bound ferric ions with a 2:1 (Fe: peptide) ratio. As predicted, Ggly, GL and GRI were biologically active in vitro and increased cell proliferation in mouse gastric epithelial (IMGE-5) and human colorectal cancer (DLD-1) cell lines, and increased cell migration in DLD-1 cells. These activities were likely via the same mechanism as Ggly since no CCK1 or CCK2 binding was identified, and GD remained inactive in all assays. Surprisingly, unlike Ggly, GL and GRI were not active in vivo. While Ggly stimulated colonic crypt height and proliferation rates in gastrin knockout mice, GL and GRI did not. The apparent lack of activity may be due to rapid clearance of these smaller peptides. Nevertheless further work designing and testing retro-inverso gastrins is warranted, as it may lead to the generation of super agonists that could potentially be used to treat patients with gastrointestinal disorders with reduced mucosal function.

Pancreatic cancer is an aggressive cancer with a poor prognosis and an overall 5-year survival rate of less than 5%. Management has not improved significantly over the last 30 years, and a better understanding of the genetic and molecular changes that occur is urgently required. Many of these changes appear to involve the p21-activated kinases (PAKs). The PAK family consists of 6 isoforms, 2 of which, PAK1 and PAK4, are up-regulated and/or hyperactivated in pancreatic cancer. p21-Activated kinases can mediate many different cellular processes especially those contributing to cancer development and progression. These processes include the regulation of cytoskeletal dynamics and cell adhesion, the evasion of apoptosis, and the promotion of cell survival, proliferation, migration, and invasion. p21-Activated kinases may also be involved in characteristics unique to pancreatic tumors, such as interplay with the pancreatic stroma, the re-emergence of embryonic pathways, and the involvement of a subset of microRNAs and heat shock proteins. This review highlights the potential role of PAKs in pancreatic cancer and provides a foundation for more effective therapeutics to improve our current treatment of pancreatic cancer.

Immature forms of the peptide hormone gastrin have been implicated in the development of colorectal cancer (CRC). The biological activity of glycine-extended gastrin (Ggly) is dependent on the binding of Fe

OBJECTIVE: To determine the expression and biology of the neuroendocrine growth factor gastrin-releasing peptide (GRP) and other proGRP-derived peptides in renal cancer. MATERIALS AND METHODS: Receptor binding studies, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay, were used to quantitate the presence of proGRP-derived peptide receptors and their ligands in renal cancer cell lines and human renal cancers. Biological activity of proGRP peptides was confirmed with proliferation, migration, and extracellular-signal-regulated kinases 1 and 2 (ERK1/2) activation assays in vitro. In vivo, ACHN renal cancer xenografts were treated with proGRP-derived peptides to assess tumour size and necrosis. hypoxia-inducible factor 1α (HIF1α) and vascular endothelial growth factor (VEGF) expression were investigated with Western blotting and ELISA respectively, to determine the possible contribution of the proGRP peptides to tumour viability. RESULTS: In ACHN cells that expressed both proGRP- and GRP-receptors, the expression of proGRP binding sites was 80-fold greater than the GRP-receptor (GRPR). C-terminal proGRP-derived peptides stimulated the activation of ERK1/2, but with a different time course to GRP, consistent with the suggestion that these peptides may have unique cellular functions. Both GRP and proGRP47-68 stimulated proliferation and migration of ACHN cells in vitro, but only GRP reduced the extent of tumour necrosis in ACHN xenografts. GRP, but not proGRP47-68, was able to induce HIF1α and VEGF expression in ACHN cells. This may account in part for the reduction in necrosis after GRP treatment. C-terminal proGRP-derived peptides were present in all three renal cancer cell lines and a panel of human renal cancers, but mature amidated GRP was absent. CONCLUSION: C-terminal proGRP peptides are more abundant in renal cancers and their cell lines than the more extensively studied amidated peptide, GRP. These results suggest that C-terminal proGRP-derived peptides may be a better target for novel renal cancer treatments.

Hypoxia, or a low concentration of O2, is encountered in humans undertaking activities such as mountain climbing and scuba diving and is important pathophysiologically as a limiting factor in tumor growth. Although data on the interplay between hypoxia and gastrins are limited, gastrin expression is upregulated by hypoxia in gastrointestinal cancer cell lines, and gastrins counterbalance hypoxia by stimulating angiogenesis in vitro and in vivo. The aim of this study was to determine if higher concentrations of the gastrin precursor progastrin are protective against hypoxia in vivo. hGAS mice, which overexpress progastrin in the liver, and mice of the corresponding wild-type FVB/N strain were exposed to normoxia or hypoxia. Iron status was assessed by measurement of serum iron parameters, real-time PCR for mRNAs encoding critical iron regulatory proteins, and Perls' stain and atomic absorption spectrometry for tissue iron concentrations. FVB/N mice lost weight at a faster rate and had higher sickness scores than hGAS mice exposed to hypoxia. Serum iron levels were lower in hGAS than FVB/N mice and decreased further when the animals were exposed to hypoxia. The concentration of iron in the liver was strikingly lower in hGAS than FVB/N mice. We conclude that increased circulating concentrations of progastrin provide a physiological advantage against systemic hypoxia in mice, possibly by increasing the availability of iron stores. This is the first report of an association between progastrin overexpression, hypoxia, and iron homeostasis.

Over-expression of growth factors can contribute to the development and progression of cancer, and gastrins in particular have been implicated in accelerating the development of gastrointestinal cancers. Previously our group showed that hypoxia, cobalt chloride (a hypoxia mimetic) and zinc chloride could activate the expression of the gastrin gene in vitro. To characterise activation of the gastrin promoter by zinc ions further in vivo, TALEN technology was used to engineer a luciferase reporter construct into the endogenous human gastrin gene promoter in SW480 colon cancer cells. Gastrin promoter activity in the resultant Gast(luc) SW480 colon cancer cells was then measured by bioluminescence in cell culture and in tumour xenografts in SCID mice. Activation of intracellular signalling pathways was assessed by Western blotting. Activation of the gastrin promoter by zinc ions was concentration dependent in vitro and in vivo. Zinc ions significantly stimulated phosphorylation of ERK1/2 (MAPK pathway) but not of Akt (PI3K pathway). We conclude that the endogenous gastrin promoter is responsive to zinc ions, likely via activation of the MAPK pathway.

Zinc ions (Zn2+) are known to influence cell survival and proliferation. However the homeostatic regulation of Zn2+ and their role in prostate cancer (PC) progression is poorly understood. Therefore the subcellular distribution and uptake of Zn2+ in PC cells were investigated. Inductively coupled plasma mass spectroscopy and fluorescent microscopy with the Zn2+-specific fluorescent probe FluoZin-3 were used to quantify total and free Zn2+, respectively, in the normal prostate epithelial cell line (PNT1A) and three human PC cell lines (PC3, DU145 and LNCaP). The effects of Zn2+ treatment on proliferation and survival were measured in vitro using MTT assays and in vivo using mouse xenografts. The ability of Zn2+ to protect against oxidative stress via a HIF1α-dependent mechanism was investigated using a HIF1α knock-down PC3 model. Our results demonstrate that the total Zn2+ concentration in normal PNT1A and PC cells is similar, but PC3 cells contain significantly higher free Zn2+ than PNT1A cells (p < 0.01). PNT1A cells can survive better in the presence of high concentrations of Zn2+ than PC3 cells. Exposure to 10 µM Zn2+ over 72 hours significantly reduces PC3 cell proliferation in vitro but not in vivo. Zn2+ increases PC3 cell survival up to 2.3-fold under oxidative stress, and this protective effect is not seen in PNT1A cells or in a HIF1α-KD PC3 cell model. A state of Zn2+ dyshomeostasis exists in PC. HIF1α is an integral component of a Zn2+-dependent protective mechanism present in PC3 cells. This pathway may be clinically significant through its contribution to castrate-resistant PC survival.

Pancreatic ductal adenocarcinoma (PDA) is one of the major types of cancer that exhibit high mortality worldwide because of the late diagnosis and the lack of effective treatment. Immunotherapy appears to be ineffective in PDA treatment due to the existence of a unique immune-suppressive microenvironment in PDA. Gemcitabine-based therapy is still the most commonly used chemotherapy to treat PDA patients with only marginal increased survival rates. This prompted us to continue the search for more effective therapy for PDA treatment. The effects of p21 activated kinases (PAKs) on tumour immune response and gemcitabine response were examined in PDA. An orthotopic murine PDA model, in which pancreatic cancer cells were injected to the tail of pancreas, was used. The mice were treated with PAK inhibitor, PF‑3758309, plus or minus gemcitabine. Tumour growth was measured by volume and weight. Tumour immune response was determined by flow cytometry analysis of splenic cells and immunohistochemical staining of intratumoural lymphocytes. Inhibition of PAKs by PF‑3758309, not only suppressed tumour growth, but also stimulated tumour immune response by increasing the numbers of splenic and intratumoural T lymphocytes. Furthermore, inhibition of PAKs decreased PDA cell growth synergistically with gemcitabine in vitro and in vivo. The dual effects of inhibition of PAKs make PAK-targeted therapy more potent for the treatment of PDA. The combination of PAK inhibitors with gemcitabine may be a more effective therapeutic approach in PDA treatment.

The peptide hormone gastrin (Gamide) binds trivalent metal ions, including indium (In), ruthenium (Ru) and gallium (Ga), with high affinity. Complexes of gastrin with chelated isotopes of In and Ga have previously been used for the location of tumours expressing the cholecystokinin 2 receptor (CCK2R). The aim of the present study was to purify the complexes of Gamide with radioactive isotopes of In, Ru or Ga and to investigate their ability to bind to the CCK2R. The radioactive Gamide complexes were purified on Sep-Pak C18 cartridges or by anion exchange HPLC. Binding to the CCK2R was assessed with a stably transfected clone of the gastric carcinoma cell line AGS. The

Pancreatic cancer is one of the most lethal of human malignancies. Nearly 100% cases of pancreatic cancer carry mutations in KRas. P-21-activated kinases (PAKs) are activated by and act downstream of KRas. Gla- ucarubinone, a natural product first isolated from the seeds of the tree Simarouba glauca, was originally developed as an antimalarial drug, and has more recently been recognised as an anticancer agent. The aims of this study were to determine whether glaucarubinone, alone or in combination with the front- line chemotherapeutic agent gemcitabine, would inhibit the growth of pancreatic cancer cells in vitro or in vivo and the mechanism involved. Growth of the human pancreatic cancer cell lines PANC-1 and MiaPaCa-2 was measured by 3 H-thymidine incorporation in vitro, and by volume as xenografts in SCID mice. The expression and activities of the two serine/threonine kinases PAK1 and PAK4, which are key regulators of cancer progression, were measured by Western blotting. Here we report that glaucarubi- none decreased proliferation and migration of pancreatic cancer cells in vitro, and reduced their growth as xenografts in vivo. Treatment with glaucarubinone and gemcitabine reduced proliferation in vitro and tumor growth in vivo more than treatment with either glaucarubinone or gemcitabine alone. Treatment with glaucarubinone reduced PAK1 and PAK4 activities, which were further decreased by the combina- tion of glaucarubinone and gemcitabine. These results indicate that glaucarubinone reduced pancreatic cancer cell growth at least in part via inhibition of pathways involving PAK1 and PAK4. The synergistic inhibition by glaucarubinone and gemcitabine observed both in vitro and in vivo suggests that glaucaru- binone may be a useful adjunct to current regimes of chemotherapy.

The Src-family tyrosine kinases (SFKs) are oncogenic enzymes that contribute to the initiation and progression of many types of cancer. In normal cells, SFKs are kept in an inactive state mainly by phosphorylation of a consensus regulatory tyrosine near the C-terminus (Tyr(530) in the SFK c-Src). As recent data indicate that tyrosine modification enhances binding of metal ions, the hypothesis that SFKs might be regulated by metal ions was investigated. The c-Src C-terminal peptide bound two Fe(3+) ions with affinities at pH4.0 of 33 and 252μM, and phosphorylation increased the affinities at least 10-fold to 1.4 and 23μM, as measured by absorbance spectroscopy. The corresponding phosphorylated peptide from the SFK Lyn bound two Fe(3+) ions with much higher affinities (1.2pM and 160nM) than the Src C-terminal peptide. Furthermore, when Lyn or Hck kinases, which had been stabilised in the inactive state by phosphorylation of the C-terminal regulatory tyrosine, were incubated with Fe(3+) ions, a significant enhancement of kinase activity was observed. In contrast Lyn or Hck kinases in the unphosphorylated active state were significantly inhibited by Fe(3+) ions. These results suggest that Fe(3+) ions can regulate SFK activity by binding to the phosphorylated C-terminal regulatory tyrosine.

Gastrin-releasing peptide (GRP) acts as an important regulatory peptide in several normal physiological processes and as a growth factor in certain cancers. In this review we provide a comprehensive overview of the current state of knowledge of GRP in urological tissues under both normal and cancerous conditions. GRP and its receptor, GRP-R, are expressed in the normal kidney and renal cancers. GRP can stimulate the growth of renal cancer cells. GRP and GRP-R are expressed in prostate cancer and GRP can stimulate the growth of prostate cancer cell lines. Importantly, GRP is a key neuroendocrine peptide, which may be involved in the progression of advanced prostate cancer and in the neuroendocrine differentiation of prostate cancer. Recent animal studies have shown that GRP and GRP-R are an integral part of male sexual function and play a crucial role in spinal control of erections and ejaculation.

Background and Aim An increase in circulating concentrations of gastrin or gastrin precursors such as progastrin and glycine-extended gastrin has been proposed to promote the development of colorectal carcinomas (CRC). The aim of this study was to investigate whether or not circulating gastrin concentrations were increased in patients with an increased risk of developing CRC. Method Patients were divided according to their risk into the five following groups: familial adenomatous polyposis (n = 20), hereditary non-polyposis colorectal cancer (n = 53), cluster of common colorectal cancers (n = 13), personal history and/or family history of adenomatous polyps or CRC (n = 150) and controls (n = 42). Radioimmunoassay with four region-specific gastrin antisera was used to measure progastrin, glycine-extended gastrin (gastrin-gly), amidated gastrin (gastrin-amide), and total gastrin in peripheral blood taken at the time of colonoscopy. Results Compared with the control group, familial adenomatous polyposis patients had significantly higher median values of total gastrin (29.8 pM vs 16.9 pM, P = 0.003) and gastrin-amide (17.1 pM vs 12.0 pM, P = 0.015). Patients with a personal or family history of adenomatous polyps or CRC also had higher circulating concentrations of total gastrin (21.8 pM) compared with controls (P

P21 activated kinase 1 (PAK1) enhances colorectal cancer (CRC) progression by stimulating Wnt/β-catenin and Ras oncogene, which promote CRC survival via stimulation of hypoxia-inducible factor 1α (HIF-1α). The aim of this study was to assess the mechanism involved in the stimulation by PAK1 of CRC survival. PAK1 promoted CRC cell survival by up-regulation of HIF-1α. PAK1 was over-expressed and hyper-activated in tumors of ApcΔ(14/+) mice, which was correlated with over-expression of HIF-1α and β-catenin. Inhibition of PAK1 decreased tumor growth and the expression of HIF-1α and β-catenin in tumors of ApcΔ(14/+) mice, and suppressed xenograft tumor survival in SCID mice. These findings indicate that PAK1 stimulates CRC survival by up-regulation of HIF-1α.

p21-activated kinases (PAKs) were initially identified as effector proteins downstream from GTPases of the Rho family. To date, six members of the PAK family have been discovered in mammalian cells. PAKs play important roles in growth factor signalling, cytoskeletal remodelling, gene transcription, cell proliferation and oncogenic transformation. A large body of research has demonstrated that PAKs are up-regulated in several human cancers, and that their overexpression is linked to tumour progression and resistance to therapy. Structural and biochemical studies have revealed the mechanisms involved in PAK signalling, and opened the way to the development of PAK-targeted therapies for cancer treatment. Here we summarise recent findings from biological and clinical research on the role of PAKs in gastrointestinal cancer, and discuss the current status of PAK-targeted anticancer therapies.

Gastrin, acting via the cholecystokinin-2 receptor (CCK2R), activates its own promoter in a positive-feed-forward loop that may result in hypergastrinemia. Activity of the gastrin promoter is also stimulated by exogenous Zn2+ ions. Here, the role of intracellular zinc and calcium signaling in the gastrin positive-feed-forward loop was investigated. Gastrin promoter activity was measured in the human gastric carcinoma cell line AGS-CCK2R and in Jurkat cells transfected with various gastrin promoter-luciferase constructs after treatment with gastrin in the presence and absence of zinc- and calcium-chelating agents. The free intracellular zinc ion concentrations were measured in the same cells with the fluorescent indicator FluoZin-3. Cell proliferation and migration/invasion were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide cell proliferation assay and in Boyden chamber assays, respectively. The zinc chelator N,N,N,N-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) abolished gastrin-stimulated gastrin promoter activity, and the inhibition was completely reversed by exogenous Zn2+ ions. In contrast, the calcium chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) potentiated gastrin-stimulated gastrin promoter activity. Treatment with gastrin increased the intracellular concentration of free Zn2+ ions, and the increase was blocked by TPEN, but not by BAPTA-AM. TPEN also inhibited the stimulation of cell proliferation and migration/invasion by gastrin, but BAPTA-AM had no effect. These results, which are the first report of the existence of Zn2+ signaling downstream of CCK2R activation, suggest that zinc chelation therapies may be effective in counteracting gastrin-dependent tumor growth.

Objectives Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury and chronic kidney disease. Two promising preconditioning methods for the kidney, intermittent arterial clamping (IC) and treatment with the hypoxia mimetic cobalt chloride, have never been directly compared. Furthermore, the protective efficacy of the chemically related transition metal Zn2+ against renal IRI is unclear. Although Co2+ ions have been shown to protect the kidney via hypoxia inducible factor (HIF), the effect of Zn2+ ions on the induction of HIF1α, HIF2α and HIF3α has not been investigated previously. Materials and methods The efficacy of different preconditioning techniques was assessed using a Sprague-Dawley rat model of renal IRI. Induction of HIF proteins following Zn2+ treatment of the human kidney cell lines HK-2 (immortalized normal tubular cells) and ACHN (renal cancer) was measured using Western Blot. Results Following 40 minutes of renal ischemia in rats, cobalt preconditioning offered greater protection against renal IRI than IC as evidenced by lower peak serum creatinine and urea concentrations. ZnCl2 (10 mg/kg) significantly lowered the creatinine and urea concentrations compared to saline-treated control rats following a clinically relevant 60 minutes of ischemia. Zn2+ induced expression of HIF1α and HIF2α but not HIF3α in HK-2 and ACHN cells. Conclusion ZnCl2 preconditioning protects against renal IRI in a dose-dependent manner. Further studies are warranted to determine the possible mechanisms involved, and to assess the benefit of ZnCl2 preconditioning for clinical applications.

Pancreatic cancer remains one of the most lethal of all solid tumors. Pancreatic stellate cells (PSCs) are primarily responsible for the fibrosis that constitutes the stroma and p21-activated kinase 1 (PAK1) may have a role in signalling pathways involving PSCs. This study aimed to examine the role of PAK1 in PSCs and in the interaction of PSCs with pancreatic cancer cells. Human PSCs were isolated using the modified outgrowth method. The effect of inhibiting PAK1 with group 1 PAK inhibitor, FRAX597, on cell proliferation and apoptosis in vitro was measured by thymidine incorporation and annexin V assays, respectively. The effect of depleting host PAK1 on the survival of mice with pancreatic Pan02 cell tumors was evaluated using PAK1 knockout (KO) mice. PAK1 was expressed in isolated PSCs. FRAX597 reduced the activation of PSCs, inhibited PSC proliferation, and increased PSC apoptosis at least in partial by inhibiting PAK1 activity. The decreased expression and activity of PAK1 in PAK1 KO mice tumors was associated with an increased mouse survival. These results implicate PAK1 as a regulator of PSC activation, proliferation and apoptosis. Targeting stromal PAK1 could increase therapeutic response and survival of patients with pancreatic cancer.

Gastrin and its precursors have been shown to promote mitogenesis and angiogenesis in gastrointestinal tumors. Hypoxia stimulates tumor growth, but its effect on gastrin gene regulation has not been examined in detail. Here we have investigated the effect of hypoxia on the transcription of the gastrin gene in human gastric cancer (AGS) cells. Gastrin mRNA was measured by real-time PCR, gastrin peptides were measured by RIA, and gastrin promoter activity was measured by dual-luciferase reporter assay. Exposure to a low oxygen concentration (1%) increased gastrin mRNA concentrations in wild-type AGS cells (AGS) and in AGS cells overexpressing the gastrin receptor (AGS-cholecystokinin receptor 2) by 2.1 ± 0.4- and 4.1 ± 0.3-fold (P < 0.05), respectively. The hypoxia mimetic, cobalt chloride (300 μM), increased gastrin promoter activity in AGS cells by 2.4 ± 0.3-fold (P < 0.05), and in AGS-cholecystokinin receptor 2 cells by 4.0 ± 0.3-fold (P < 0.05), respectively. The observations that either deletion from the gastrin promoter of the putative binding sites for the transcription factor hypoxia-inducible factor 1 (HIF-1) or knockdown of either the HIF-1α or HIF-1β subunit did not affect gastrin promoter inducibility under hypoxia indicated that the hypoxic activation of the gastrin gene is likely HIF independent. Mutational analysis of previously identified Sp1 regulatory elements in the gastrin promoter also failed to abrogate the induction of promoter activity by hypoxia. The observations that hypoxia up-regulates the gastrin gene in AGS cells by HIF-independent mechanisms, and that this effect is enhanced by the presence of gastrin receptors, provide potential targets for gastrointestinal cancer therapy.

The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated gastrins in vitro and in vivo. Bi3+ ions also bind to glycine-extended gastrin17 (Ggly), but inhibit Ggly-induced cell proliferation and migration in gastrointestinal cell lines in vitro. The aims of the present study were firstly, to establish the mechanism by which Bi3+ ions inhibit the binding of Fe3+ ions to Ggly, and secondly, to test the effect of Bi3+ ions on the activity of non-amidated gastrins in vivo. The interaction between Bi3+ ions, Fe3+ ions and Ggly was investigated by ultraviolet spectroscopy. The effect of Bi3+ ions on colorectal mucosal proliferation was measured in three animal models. In vitro in the presence of Bi3+ ions the affinity of Fe3+ ions for Ggly was substantially reduced; the data was better fitted by a mixed, rather than a competitive, inhibition model. In rats treated with Ggly alone proliferation in the rectal mucosa was increased by 318%, but was reduced to control values (p < 0.001) in animals receiving oral bismuth plus Ggly. Proliferation in the colonic mucosa of mice overexpressing Ggly or progastrin was significantly greater than in wild-type mice, but was no greater than control (p < 0.01) in animals receiving oral bismuth. Thus a reduction in the binding of Fe3+ ions to Ggly and progastrin in the presence of Bi3+ ions is a likely explanation for the ability of oral bismuth to block the biological activity of non-amidated gastrins in vivo.

The gastrointestinal hormone gastrin is generated from an 80 amino acid precursor (progastrin) by cleavage after dibasic residues by prohormone convertase 1. Phosphorylation of Ser(75) has previously been suggested, on the basis of indirect evidence, to inhibit cleavage of progastrin after Arg(73)Arg(74). Gastrins bind two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated gastrins in vitro and in vivo. This study directly investigated the effect of iron binding and of serine phosphorylation on the cleavage of synthetic progastrin-derived peptides. The affinity of synthetic progastrin(55-80) for ferric ions, and the rate of cleavage by prohormone convertase 1, were not affected by phosphorylation of Ser(75). In contrast, in the presence of ferric ions the rate of cleavage of both progastrin(55-80) and phosphoSer(75)progastrin(55-80) by prohormone convertase 1 was significantly reduced. Hence iron binding to progastrin may regulate processing and secretion in vivo, and regulation may be particularly important in diseases with altered iron homeostasis.

The involvement of the gastrointestinal hormone gastrin in the development of gastrointestinal cancer is highly controversial. Here we demonstrate a positive-feedback loop whereby gastrin, acting via the CCK2 receptor, increases its own expression. Such an autocrine loop has not previously been reported for any other gastrointestinal hormone. Gastrin promoter activation was dependent on the MAP kinase pathway and did not involve Sp1 binding sites or epidermal growth factor receptor transactivation. As the treatment of gastrointestinal cancer cells with amidated gastrin led to increased expression of non-amidated gastrins, the positive-feedback loop may contribute to the sustained increase in circulating gastrins observed in colorectal cancer patients.

Precursors of the peptide hormone gastrin stimulate proliferation in the colorectal mucosa and promote the development of colorectal carcinoma. Gastrins bind two ferric ions selectively and with high affinity, and the biological activity of glycine-extended gastrin (Ggly) in vitro is dependent on the presence of ferric ions. The aim of the present study was to determine whether or not iron is required for biological activity of progastrin and Ggly in vivo. Rats that had undergone a colostomy were infused with Ggly, and proliferation was measured in the defunctioned rectal mucosa. Proliferation was also measured in the colonic mucosa of hGAS and MTI-Ggly mice, which, by definition, overexpress progastrin and Ggly, respectively. The requirement for iron was assessed by thrice-weekly injection of the chelating agent desferrioxamine (DFO). The proliferation index in the defunctioned rectal mucosa was significantly increased in the Ggly-infused rats, and the increase was significantly reduced after treatment with DFO. Treatment with DFO significantly reduced the crypt height and proliferation index in the colonic mucosa of hGAS and MTI-Ggly mice but had no effect on the same variables in wild-type mice. These observations are consistent with the hypothesis that the biological activity of progastrin and Ggly in vivo is dependent on the presence of ferric ions and further suggest that chelating agents may block the stimulatory effects of gastrin precursors in the development of colorectal carcinoma.

Transferrin is the main iron transport protein found in the circulation, and the level of transferrin saturation in the blood is an important indicator of iron status. The peptides amidated gastrin(17) (Gamide) and glycine-extended gastrin(17) (Ggly) are well known for their roles in controlling acid secretion and as growth factors in the gastrointestinal tract. Several lines of evidence, including the facts that transferrin binds gastrin, that gastrins bind ferric ions, and that the level of expression of gastrins positively correlates with transferrin saturation, suggest the possible involvement of the transferrin-gastrin interaction in iron homeostasis. In the present work, the interaction between gastrins and transferrin has been characterized by surface plasmon resonance and covalent crosslinking. First, an interaction between iron-free apo-transferrin and Gamide or Ggly was observed. The fact that no interaction was observed in the presence of the chelator EDTA suggested that the gastrin-ferric ion complex was the interacting species. Moreover, removal of ferric ions with EDTA reduced the stability of the complex between apo-transferrin and gastrins, and no interaction was observed between Gamide or Ggly and diferric transferrin. Second, some or all of glutamates at positions 8-10 of the Ggly molecule, together with the C-terminal domain, were necessary for the interaction with apo-transferrin. Third, monoferric transferrin mutants incapable of binding iron in either the N-terminal or C-terminal lobe still bound Ggly. These findings are consistent with the hypothesis that gastrin peptides bind to nonligand residues within the open cleft in each lobe of transferrin and are involved in iron loading of transferrin in vivo.

The cyclin-dependent kinase CDK2 is inactivated by phosphorylation on either of the two neighbouring residues Thr14 or Tyr15. The effect of phosphorylation on metal ion binding has been investigated with peptides incorporating residues 6-20 of CDK2. The stoichiometry of Ca(2+) binding increased from 1 in the un- and singly-phosphorylated peptides to 2 in the doubly phosphorylated peptide, without large changes in the affinity (75-250 microM). In contrast although binding of ferric ions to the un-phosphorylated peptide was not detected, both singly- and doubly-phosphorylated peptides bound two Fe(3+) ions. Binding of Ca(2+) or Zn(2+) ions to the doubly phosphorylated CDK2 peptide did not cause any change in absorbance, but increased the affinity of the peptide for Fe(3+) ions. These results demonstrate that double phosphorylation of CDK2 peptides increases the stoichiometry of metal ion binding, and hence may contribute to the previously observed regulation of CDK2 activity by metal ions.

Beta-catenin regulates cell-cell adhesion by binding to E-cadherin at the cell membrane and, when translocated into the nucleus, mediates signalling by activation of transcription factors such as TCF4. Mutations of the components of the Wnt/beta-catenin pathway are found in many gastrointestinal cancers. Gastrins, including amidated (Gamide) and glycine-extended (Ggly) gastrin(17), stimulate the proliferation of gastrointestinal cancer cells. Gastrins also regulate beta-catenin signalling through multiple pathways which seem to converge on p21-activated kinase 1 (PAK1). In this study, we have investigated the role of PAK1 in the regulation of beta-catenin signalling by gastrins. Here we report for the first time that PAK1 associated with beta-catenin. Both Gamide and Ggly stimulated the phosphorylation and activation of beta-catenin in a PAK1-dependent manner. A kinase-inactive mutant PAK1(K299A) blocked the gastrin-stimulated dissociation of beta-catenin from E-cadherin, translocation of beta-catenin from the cell membrane to the nucleus, and association of beta-catenin with the transcription factor TCF4. The PAK1(K299A) mutant also inhibited the stimulation of the expression of c-myc and cyclin D1, and of cell proliferation and migration, by gastrins. The results indicate that gastrins regulate beta-catenin signalling through a PAK1-dependent pathway. PAK1 seems to be the point of convergence of multiple signalling pathways activated by gastrins.

The observations that the peptide hormone gastrin interacts with transferrin in vitro and that circulating gastrin concentrations are increased in the iron-loading disorder hemochromatosis suggest a possible link between gastrin and iron homeostasis. This study tested the hypothesis that gastrin and iron status are interrelated by measurement of iron homeostasis in mice and humans with abnormal circulating gastrin concentrations. Intestinal iron absorption was determined by59Fe uptake following oral gavage, and concentrations of duodenal divalent metal transporter-1 (DMT-1) and hepatic hepcidin mRNAs were determined by quantitative real-time PCR in agastrinemic (GasKO), hypergastrinemic cholecystokinin 2 receptor-deficient (CCK2RKO), or wild-type mice. Iron status was measured by standard methods in the same mice and in hypergastrinemic humans with multiple endocrine neoplasia type 1 (MEN-1). Iron absorption was increased sixfold and DMT-1 mRNA concentration fourfold, and transferrin saturation was reduced 0.8-fold and hepcidin mRNA expression 0.5-fold in juvenile GasKO mice compared with age-matched wild-type mice. In mature mice, few differences were observed between the strains. Juvenile CCK2RKO mice were hypergastrinemic and had a 5.4-fold higher DMT-1 mRNA concentration than wild-type mice without any increase in iron absorption. In contrast to juvenile GasKO mice, juvenile CCK2RKO mice had a 1.5-fold greater transferrin saturation, which was reflected in a twofold increase in liver iron deposition at maturity compared with wild-type mice. The correlation between transferrin saturation and circulating gastrin concentration observed in mutant mice was also observed in human patients with MEN, in whom hypergastrinemia correlated positively ( P = 0.004) with an increased transferrin saturation. Our data indicate that, in juvenile animals when iron demand is high, circulating gastrin concentrations may alter iron status by a CCK2R-independent mechanism.

Gastrins, including amidated gastrin (Gamide) and glycine-extended gastrin (Ggly), accelerate the growth of gastrointestinal cancer cells by stimulation of proliferation and inhibition of apoptosis. Gamide and Ggly activate different G proteins of the Rho family of small GTPases. For example, Gamide signals Rac/Cdc42 to activate p21-activated kinase 1 while Ggly signals Rho to activate Rho-activated kinase. p21-activated kinase 1 and Rho-activated kinase induce changes in phosphorylation or expression, respectively, of proteins of the Bcl-2 family, which then affect the caspase cascade with consequent inhibition of apoptosis. In addition, interaction of p21-activated kinase 1 with beta-catenin results in phosphorylation of beta-catenin, which enhances its translocation in to the nucleus, activation of TCF4-dependent transcription, and proliferation and migration. The central role of the beta-catenin pathway in carcinogenesis suggests that specific inhibitors of p21-activated kinase 1 may in the future provide novel therapies for gastrointestinal malignancies.

Background Pancreatic ductal adenocarcinoma remains one of the most lethal of all solid tumours. Treatment options are limited and gemcitabine-based chemotherapy remains the standard of care. Although growing evidence shows that p21-activated kinase 1 (PAK1) plays a crucial role in pancreatic cancer, its role has not been fully elucidated. This study aimed to characterise the expression and functional relevance of PAK1 in pancreatic cancer. Methods PAK1 expression was measured in pancreatic cancer specimens by immunohistochemistry and in pancreatic cancer cell lines by western blotting. The effect of inhibition of PAK1 by either shRNA knock-down (KD), or by a selective inhibitor, FRAX597, alone or in combination with gemcitabine, on cell proliferation and migration/invasion was measured by thymidine uptake and Boyden chamber assays, respectively. The effect on tumour growth and survival was assessed in orthotopic murine models. Results PAK1 was expressed in all human pancreatic cancer samples tested, an7d was upregulated in all pancreatic cancer cell lines tested. PAK1 KD inhibited pancreatic cancer cell growth and survival, and increased sensitivity to gemcitabine treatment. AKT activity and HIF1α expression were also inhibited. FRAX597 inhibited pancreatic cancer cell proliferation, survival, and migration/invasion. When combined with gemcitabine, FRAX597 synergistically inhibited pancreatic cancer proliferation in vitro and inhibited tumour growth in vivo. Conclusions These results implicate PAK1 as a regulator of pancreatic cancer cell growth and survival. Combination of a PAK1 inhibitor such as FRAX597 with cytotoxic chemotherapy deserves further study as a novel therapeutic approach to pancreatic cancer treatment. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2057-z) contains supplementary material, which is available to authorized users.

The many biological activities of the hormone gastrin-releasing peptide (GRP), including stimulation of acid secretion and of tumour growth, are mediated by the gastrin-releasing peptide receptor (GRP-R). Here sequence comparisons are utilised to investigate the likely bioactive regions of the 125 amino acid GRP precursor and of GRP-R. Comparison of the sequences of the GRP precursor from 21 species revealed homology not only in the GRP region between amino acids 1 and 30, but also in C-terminal regions from amino acids 43 to 97. This observation is consistent with recent reports that peptides derived from the C-terminal region are biologically active. Comparison of the GRP-R sequence with the related receptors NMB-R and BRS-3 revealed that the family could be distinguished from other G-protein coupled receptors by the presence of the motif GVSVFTLTALS at the cytoplasmic end of transmembrane helix 3. Comparison of the sequences of the GRP-R from 21 species revealed that the most highly conserved regions occurred in transmembrane helices 2, 3, 5, 6 and 7, and in the third intracellular loop. These results will be important in guiding future structure-function studies of the GRP precursor and of GRP receptors.