Zinc Preconditioning Provides Cytoprotection following Iodinated Contrast Media Exposure in In Vitro Models.

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.

Inhibition of PAK1 suppresses pancreatic cancer by stimulation of anti-tumour immunity through down-regulation of PD-L1.

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.

PAK inhibition by PF-3758309 enhanced the sensitivity of multiple chemotherapeutic reagents in patient-derived pancreatic cancer cell lines.

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.

Antitumor effects of all-trans retinoic acid and its synergism with gemcitabine are associated with downregulation of p21-activated kinases in pancreatic cancer.

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.

Preconditioning against renal ischaemia reperfusion injury: the failure to translate to the clinic.

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.

p21-activated kinase signalling in pancreatic cancer: New insights into tumour biology and immune modulation.

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.

Zinc preconditioning protects against renal ischaemia reperfusion injury in a preclinical sheep large animal model.

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.

Zinc ion dyshomeostasis increases resistance of prostate cancer cells to oxidative stress via upregulation of HIF1α.

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.

Oral trivalent bismuth ions decrease, and trivalent indium or ruthenium ions increase, intestinal tumor burden in ApcΔ14/+ mice.

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.

Protective effect of zinc preconditioning against renal ischemia reperfusion injury is dose dependent.

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.

Complexes of gastrin with In3+, Ru3+ or Ga3+ ions are not recognised by the cholecystokinin 2 receptor.

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.

Depletion of p21-activated kinase 1 up-regulates the immune system of APC∆14/+ mice and inhibits intestinal tumorigenesis.

BACKGROUND: P21-activated kinase 1 (PAK1) stimulates growth and metastasis of colorectal cancer (CRC) through activation of multiple signalling pathways. Up-regulation of CRC stem cell markers by PAK1 also contributes to the resistance of CRC to 5-fluorouracil. The aim of this study was to investigate the effect of PAK1 depletion and inhibition on the immune system and on intestinal tumour formation in APC∆14/+ mice. METHODS: The PAK1 KO APC∆14/+ mice were generated by cross-breeding of PAK1 KO mice with APC∆14/+ mice. Splenic lymphocytes were analysed by flow cytometry, and immunohistochemical staining. The numbers of intestinal tumours were counted. Blood cells were also counted. RESULTS: Compared to APC+/+ mice, the numbers of both T- and B- lymphocytes were reduced in the spleen of APC∆14/+ mice. Depletion of PAK1 in APC∆14/+ mice increased the numbers of splenic T- and B- lymphocytes and decreased the numbers of intestinal tumours. Treatment of APC∆14/+ mice with PF-3758309, a PAK inhibitor reduced the numbers of intestinal tumours and increased the numbers of blood lymphocytes. CONCLUSION: Depletion of active PAK1 up-regulates the immune system of APC∆14/+ mice and suppresses intestinal tumour development. These observations suggest an important role for PAK1 in the immune response to tumours.

Progastrin: a potential predictive marker of liver metastasis in colorectal cancer.

BACKGROUND AND AIMS: 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. METHODS: 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. RESULTS: 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 < 0.001). CONCLUSIONS: This is the first study to show that progastrin expression may be predictive of aggressive tumour behaviour in patients with colorectal cancer and supports its clinical relevance and potential use as a biomarker.

Inhibition of group 1 p21-activated kinases suppresses pancreatic stellate cell activation and increases survival of mice with pancreatic cancer.

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.

Zinc Ions Mediate Gastrin Expression, Proliferation, and Migration Downstream of the Cholecystokinin-2 Receptor.

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.

Glaucarubinone Combined with Gemcitabine Improves Pancreatic Cancer Survival in an Immunocompetent Orthotopic Murine Model.

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 3H-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.

FRAX597, a PAK1 inhibitor, synergistically reduces pancreatic cancer growth when combined with gemcitabine.

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.

High Affinity Binding of Indium and Ruthenium Ions by Gastrins.

The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10-7 and 1.1 x 10-6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10-15 and 1.7 x 10-7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10-13 and 1.2 x 10-5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0-3.3 Å, the Ru complex clearly demonstrated a short range Ru-Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.

Retro-inverso forms of gastrin5-12 are as biologically active as glycine-extended gastrin in vitro but not in vivo.

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.

Activation by zinc of the human gastrin gene promoter in colon cancer cells in vitro and in vivo.

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.