Graham S. Baldwin

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 ( ). Preconditioning with 37.5 μM and 50 μM ZnCl₂ increased cell survival by 173% (SD 27.8%) ( ) and 219% (SD 32.2%) ( ), 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% ( ) 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.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies worldwide. All-trans retinoic acide (ATRA) has been used as an anti-stromal agent in PDA, and its anti-tumour effect has also been reported in various kinds of cancer, including PDA. Inhibition of p21-activated kinases (PAKs) is associated with decreased tumour 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. 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 was significantly increased in resistant cells. Cells treated with ATRA showed decreased expression of PAK1, PAK2, PAK4 and α-SMA. The combination of ATRA and gemcitabine synergistically reduced cell growth in both wild-type and gemcitabine-resistant cell lines. In conclusion, ATRA not only reduced cell growth and migration, but also synergistically enhanced gemcitabine sensitivity in both wild-type and gemcitabine-resistant cell lines via down-regulation of PAKs.

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.

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. 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. 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. 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.

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. 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. 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. 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.

The peptide hormone gastrin17, which occurs naturally in both tyrosine sulphated and unsulphated forms, binds two ferric ions with pM affinities. The aim of this study was to investigate the hypothesis that sulphation or phosphorylation of gastrin17 altered ferric ion binding, and/or affinity for the CCK1 or CCK2 receptor. To investigate the effect of tyrosine modification on ferric ion binding, the changes in absorbance of gastrin17, gastrin17SO4 and gastrin17PO4 on addition of Fe3+ ions were monitored. Binding of gastrin17, gastrin17SO4 and gastrin17PO4 to the human CCK1 and CCK2 receptors was assessed by competition with [125I]-Bolton and Hunter-labelled cholecystokinin8 in transiently transfected COS cells. Tyrosine sulphation or phosphorylation increased the affinity of gastrin17 for the first ferric ion bound from 267 to 83 pM and 14 pM, respectively, but had no effect on the stoichiometry of ferric ion binding. In contrast the affinity of gastrin17 for the second ferric ion bound was reduced from 94 pM to 7.32 µM and 671 nM, respectively. While sulphation of gastrin17 increased its affinity for the CCK2 receptor approximately 50 fold, phosphorylation had no effect on receptor binding. These results demonstrate that tyrosine modification may have profound effects on the interaction of gastrins with ferric ions and with the CCK2 receptor.

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 (K_d) 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 K_d 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 K_d 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.

HIF1α over expression correlates with poor prognosis in a number of cancers. Although it is widely accepted that hypoxia induces HIF1α expression up-regulation by a reduction in oxygen dependent degradation, HIF1α up-regulation under normoxic conditions is noted with increasing frequency in many cancers. We reviewed the current knowledge of mechanisms of normoxic and hypoxic HIF1α up-regulation, and its therapeutic implications with a particular focus on its role as a potential biomarker in prostate cancer. Although the literature on the role of HIFs in cancer development and progression has been reviewed extensively, few publications have specifically considered the role of HIFs in prostate cancer. Therefore, we searched PubMed® and Google® with the key words prostate cancer, castration resistance, metastasis, hypoxia, HIF1α, HIF2α and regulation. Relevant articles, including original research studies and reviews, were selected based on contents and a synopsis was generated. Normoxic expression of HIF1α has an important role in the development of prostate cancer chemoresistance, radioresistance and castrate resistance. Thus, HIF1α could serve as a potential biomarker. Furthermore, agents that target HIF1α could be used as adjuvant therapy to decrease resistance to conventional treatment modalities. HIF1α over expression in prostate cancer can be regulated at 3 levels, including transcription, translation and protein stability, by a number of mechanisms such as gene amplification, single nucleotide polymorphism, increased transcription of HIF1α mRNA, expression of truncated isoforms of HIF1α and stabilization of HIF1α. However, there is no definitive consensus and the intriguing question of how HIF1α is up-regulated in prostate cancer is still unanswered. HIF1α over expression under normoxia could serve as a biomarker for chemoresistance, radioresistance and castrate resistance in prostate cancer. There is an urgent need to identify the cause of HIF1α over expression in castrate resistant prostate cancer cells and tumors to guide the choice of HIF inhibitors (transcription or translation based) that are best suited for treating castrate resistant prostate cancer.

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.