Graham S Baldwin

University of Melbourne, Melbourne, Victoria, Australia

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Publications (199)882.87 Total impact

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    ABSTRACT: 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.
    Full-text · Article · Dec 2016 · BMC Cancer
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    Graham S. Baldwin · Ioulia Sims
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    ABSTRACT: The peptide hormone gastrin 17 , 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 gastrin 17 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 gastrin 17 , gastrin 17 SO 4 and gastrin 17 PO 4 on addition of Fe 3+ ions were monitored. Binding of gastrin 17 , gastrin 17 SO 4 and gastrin 17 PO 4 to the human CCK1 and CCK2 receptors was assessed by competition with [ 125 I]-Bolton and Hunter-labelled cholecystokinin 8 in transiently transfected COS cells. Tyrosine sulphation or phosphorylation increased the affinity of gastrin 17 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 gastrin 17 for the second ferric ion bound was reduced from 94 pM to 7.32 µM and 671 nM, respectively. While sulphation of gastrin 17 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.
    Preview · Article · Dec 2015 · SpringerPlus
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    Full-text · Conference Paper · Oct 2015
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    ABSTRACT: 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.
    No preview · Article · Oct 2015 · Peptides
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    Graham S Baldwin · Graham N George · M Jake Pushie
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    ABSTRACT: 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.
    Full-text · Article · Oct 2015 · PLoS ONE
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    ABSTRACT: 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.
    No preview · Article · Sep 2015 · Metallomics
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    Dannel Yeo · Hong He · Graham Baldwin · Mehrdad Nikfarjam

    Full-text · Article · Jun 2015 · Pancreatology
  • D. Wetherell · J. Ischia · G. Baldwin · A. Shulkes · O. Patel · D. Bolton

    No preview · Conference Paper · Apr 2015

  • No preview · Conference Paper · Apr 2015
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    Dannel Yeo · Hong He · Graham S Baldwin · Mehrdad Nikfarjam
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    ABSTRACT: 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.
    Full-text · Article · Apr 2015 · Pancreas
  • Marie Laval · Graham S Baldwin · Arthur Shulkes · Kathryn M Marshall
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    ABSTRACT: Hypoxia, or a low concentration of oxygen, 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 over-express progastrin in the liver, and mice of the corresponding wild-type FVB/N strain, were exposed to normoxia or to hypoxia. Iron status was assessed by measurement of serum iron parameters, by real-time PCR for mRNAs encoding critical iron regulatory proteins, and by 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 under hypoxia. Serum iron levels were lower in hGAS mice than in FVB/N mice and decreased further when exposed to hypoxia. The concentration of iron in the livers of hGAS mice was strikingly lower than in 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.
    No preview · Article · Nov 2014 · AJP Gastrointestinal and Liver Physiology
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    Nhi Huynh · John A. Beutler · Arthur Shulkes · Graham S. Baldwin · Hong He
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    ABSTRACT: p-21-Activated kinase 1 (PAK1) enhances colorectal cancer (CRC) progression by stimulating Wnt/β-catenin, ERK and AKT pathways. PAK1 also promotes CRC survival via up-regulation of hypoxia-inducible factor 1α (HIF-1α), a key player in cancer survival. Glaucarubinone, a quassinoid natural product, inhibits pancreatic cancer growth by down-regulation of PAK1. The aim of this study was to investigate the effect of glaucarubinone on CRC growth and metastasis, and the mechanism involved. Cell proliferation was measured in vitro by [3H]-thymidine incorporation and in vivo by volume of tumor xenografts. Protein concentrations were measured by Western blotting of cell extracts. We report here that glaucarubinone inhibited CRC growth both in vitro and in vivo. The potency of glaucarubinone as an inhibitor of cell proliferation was negatively correlated to PAK1 expression in CRC cells. Glaucarubinone suppressed the expression of HIF-1α and β-catenin. Knockdown of PAK1 by shRNA enhanced inhibition by glaucarubinone while constitutively active PAK1 blocked the inhibitory effect. Our findings indicate that glaucarubinone inhibited CRC growth by down-regulation of HIF-1α and β-catenin via a PAK1-dependent pathway.
    Full-text · Article · Oct 2014 · Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
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    ABSTRACT: Hypoxia-inducible factor 1α (HIF1α) over-expression has been correlated with poor prognosis in a number of cancers. Although it is widely accepted that hypoxia-induced up-regulation of HIF1α expression occurs by reduction in oxygen-dependent degradation, up-regulation of HIF1α under normoxic conditions is being demonstrated with increasing frequency in many cancers. We review the current knowledge of mechanisms of normoxic and hypoxic up-regulation of HIF1α and its therapeutic implications, with a particular focus on its role as a potential biomarker in prostate cancer (PC). Although the literature regarding the role of HIFs in cancer development and progression has been reviewed extensively, publications' specifically considering the role of HIFs in prostate cancer is sparse. Therefore Pubmed and Google searches with the keywords prostate cancer, castration resistance, metastasis, hypoxia, HIF1α, HIF2α and regulation were performed. Relevant articles including original research papers and reviews were selected based on their contents and a synopsis was generated. Normoxic expression of HIF1α plays an important role in the development of chemo-resistance, radio-resistance and castrate resistance of PC and HIF1α could thus be used as a potential biomarker. Furthermore, agents that target HIF1α could be used in adjuvant therapies in order to reduce resistance to conventional therapeutic modalities. Over-expression of HIF1α in PC can be regulated at the three levels of transcription, translation and protein stability, via a number of different mechanisms including 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 upregulated in PC is still unanswered. Over-expression of HIF1α under normoxia could be used as a biomarker for development of chemo-resistance, radio-resistance and castrate resistance in PC. There is an urgent need to identify the cause of over-expression of HIF1α in castrate-resistant PC cells and tumors, in order to guide the choice of HIF inhibitors (transcription- or translation-based) best suited for the treatment of castrate-resistant PC. Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.
    No preview · Article · Oct 2014 · The Journal of Urology
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    ABSTRACT: 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 methodsReceptor binding studies, 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 ERK1/2 activation assays in vitro. In vivo, ACHN renal cancer xenografts were treated with proGRP-derived peptides to assess tumour size and necrosis. HIF1α and VEGF expression was investigated with western blotting and ELISA respectively to determine the possible contribution of the proGRP peptides to tumour viability.ResultsIn ACHN cells that express 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 tumor 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 following 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.ConclusionC-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.
    No preview · Article · Aug 2014 · BJU International
  • David A. Westwood · Oneel Patel · Graham S Baldwin
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    ABSTRACT: Aim: Gastrins act as growth factors for the normal and neoplastic colorectal mucosa. The aim of this study was to determine the role of gastrins in the response of human colorectal cancer (CRC) cells to hypoxia in vitro and in vivo. Methods: Expression of the gastrin gene in the human CRC cell line LoVo was examined under normoxia and hypoxia by quantitative PCR and by radioimmunoassay. Gastrin expression was knocked down with shRNA, and the effect on cell proliferation was measured by cell counting, on cell apoptosis by annexin V staining, and on cell migration by Boyden chamber assay. The effect of gastrin knockdown on tumourigenesis in mouse xenografts was analysed by measurement of tumour volumes and weights, and by immunohistochemistry. Results: Gastrin gene expression in LoVo cells was stimulated by hypoxia via binding of hypoxia-inducible factor-1α to the gastrin promoter. The viability of gastrin knockdown cells exposed to hypoxia (1% O2) in vitro was diminished because of loss of resistance against hypoxia-induced apoptosis, and the effect was partly reversed by treatment with non-amidated, but not amidated, gastrin. Conditioned medium from control LoVo cells under hypoxia simulated proliferation but not migration, and the effect was blocked by an inhibitor of non-amidated gastrins, but not by an inhibitor of amidated gastrins. In xenografts in mice exposed to hypoxia (10% O2) for 21days, tumour necrosis was significantly increased by knocking down gastrin expression. Conclusion: These results provide evidence that non-amidated gastrins are involved in the adaptation of CRCs to hypoxic microenvironments through increasing resistance to apoptosis.
    No preview · Article · Jul 2014 · Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

  • No preview · Article · Jul 2014 · Nature Reviews Urology
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    Nhi Huynh · Kevin H. Liu · Mildred Yim · Arthur Shulkes · Graham S. Baldwin · Hong He
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    ABSTRACT: Gastrins, including amidated gastrin17 and glycine-extended gastrin17, are important growth factors in colorectal cancer (CRC). The p21-activated kinase 1 (PAK1) plays key roles in cellular processes including proliferation, survival, and motility, and in cell transformation and tumor progression. PAK1 expression increases with the progression of CRC, and knockdown of PAK1 blocks CRC cell growth and metastasis both in vitro and in vivo. The aim of this study was to determine the interaction between PAK1 and gastrins in CRC cells. PAK1 expression and activation were assayed by Western blots, and concentrations of gastrin mRNA and peptides by real-time PCR and radioimmunoassay, respectively. Proliferation of CRC cells was measured by 3H-thymidine incorporation, and vascular endothelial growth factor (VEGF) secretion was measured by ELISA. Gastrins activated PAK1 via PI3K-dependent pathways. Activated PAK1 in turn mediated gastrin-stimulated activation of β-catenin and VEGF secretion in CRC cells, as knockdown of PAK1 blocked stimulation of these cellular processes by gastrins. Downregulation of gastrin reduced the expression and activity of PAK1, but in contrast there was a compensatory increase in gastrins either when PAK1 was downregulated, or after treatment with a PAK inhibitor. Our results indicate that PAK1 is required for the stimulation of CRC cells by gastrins, and suggest the existence of an inhibitory feedback loop by which PAK1 downregulates gastrin production in CRC cells.
    Full-text · Article · Jun 2014

  • No preview · Article · Apr 2014 · The Journal of Urology
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    ABSTRACT: Expression of hypoxia-inducible factor (HIF)1α increases the risk of castrate-resistant prostate cancer (CRPC) and metastases in patients on androgen deprivation therapy (ADT) for prostate cancer (PC). We aimed to investigate the effects of nonspecific HIF1α inhibitors (Digoxin, metformin, and angiotensin-2 receptor blockers) on development of CRPC and metastases while on ADT. A retrospective review of prospectively collected medical records was conducted of all men who had continuous ADT as first-line therapy for CRPC at the Austin Hospital from 1983 to 2011. Association between HIF1α inhibitor medications and time to develop CRPC was investigated using actuarial statistics. Ninety-eight patients meeting the criteria were identified. Eighteen patients (21.4%) were treated with the nonspecific HIF1α inhibitors. Both groups had similar characteristics, apart from patients on HIF1α inhibitors being older (70 years vs. 63.9 years). The median CRPC-free survival was longer in men using HIF1α inhibitors compared to those not on inhibitors (6.7 years vs. 2.7 years, P = 0.01) and there was a 71% reduction in the risk of developing CRPC (HR 0.29 [95% CI 0.10-0.78] P = 0.02) after adjustment for Gleason score, age, and prostate-specific antigen (PSA). The median metastasis-free survival in men on HIF1α inhibitors was also significantly longer compared to those on no inhibitors (5.1 years vs. 2.6 years, P = 0.01) with an 81% reduction in the risk of developing metastases (HR 0.19 [CI 0.05-0.76] P = 0.02) after adjustment for Gleason score, age, and PSA. Nonspecific HIF1α inhibitors appear to increase the progression-free survival and reduce the risk of developing CRPC and metastases in patients on continuous ADT.
    Full-text · Article · Apr 2014 · Cancer Medicine
  • K. Rao · M. Yim · D. Bolton · L. Galea · G. Baldwin · A. Shulkes · O. Patel

    No preview · Article · Apr 2014 · European Urology Supplements

Publication Stats

3k Citations
882.87 Total Impact Points

Institutions

  • 1979-2016
    • University of Melbourne
      • Department of Surgery
      Melbourne, Victoria, Australia
  • 1995-2005
    • Austin Health
      Melbourne, Victoria, Australia
  • 1998-2001
    • Victoria University Melbourne
      Melbourne, Victoria, Australia
  • 2000
    • Diabetes Australia, Victoria
      Melbourne, Victoria, Australia
    • Université Montpellier
      • Faculté de Pharmacie
      Montpelhièr, Languedoc-Roussillon, France
    • University of Freiburg
      Freiburg, Baden-Württemberg, Germany
  • 1982-1998
    • Ludwig Institute for Cancer Research Australia
      Melbourne, Victoria, Australia
  • 1984-1996
    • Royal Melbourne Hospital
      • Department of Radiology
      Melbourne, Victoria, Australia
  • 1991
    • Ludwig Institute for Cancer Research
      La Jolla, California, United States
  • 1988
    • Ludwig Institute for Cancer Research Brazil
      San Paulo, São Paulo, Brazil