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.

Corrigendum to "Targeting HIF-1α to Prevent Renal Ischemia-Reperfusion Injury: Does It Work?"

[This corrects the article DOI: 10.1155/2018/9852791.].

Metformin may offer no protective effect in men undergoing external beam radiation therapy for prostate cancer.

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.

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.

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.

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.

Distribution and characterisation of CCK containing enteroendocrine cells of the mouse small and large intestine.

There is general consensus that enteroendocrine cells, EEC, containing the enteric hormone cholecystokinin (CCK) are confined to the small intestine and predominate in the duodenum and jejunum. Contrary to this, EEC that express the gene for CCK have been isolated from the large intestine of the mouse and there is evidence for EEC that contain CCK-like immunoreactivity in the mouse colon. However, the human and rat colons do not contain CCK cells. In the current study, we use immunohistochemistry to investigate CCK peptide presence in endocrine cells, PCR to identify cck transcripts and chromatography to identify CCK peptide forms in the mouse small and large intestine. The colocalisation of CCK and 5-HT, hormones that have been hypothesised to derive from cells of different lineages, was also investigated. CCK immunoreactivity was found in EEC throughout the mouse small and large intestine but positive cells were rare in the rectum. Immunoreactive EEC were as common in the caecum and proximal colon as they were in the duodenum and jejunum. CCK gene transcripts were found in the mucosa throughout the intestine but mRNA for gastrin, a hormone that can bind some anti-CCK antibodies, was only found in the stomach and duodenum. Characterisation of CCK peptides of the colon by extraction, chromatographic separation and radioimmunoassay revealed bioactive amidated and sulphated forms, including CCK-8 and CCK-33. Moreover, CCK-containing EEC in the large intestine bound antibodies that target the biologically active sulfated form. Colocalisation of CCK and 5-HT occurred in a proportion of EEC throughout the small intestine and in the caecum but these hormones were not colocalised in the colon, where there was CCK and PYY colocalisation. It is concluded that authentic, biologically active, CCK occurs in EEC of the mouse large intestine.

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.

Up-regulation of stem cell markers by P21-activated kinase 1 contributes to 5-fluorouracil resistance of colorectal cancer.

Cancer stem cells (CSC) are tumorigenic and resistant to chemotherapy. In colorectal cancer (CRC), CSCs have been identified by the expression of specific markers, including CD44, Bmi1 and Nanog. Although p21-activated kinase 1 (PAK1), acting downstream of Ras, stimulates Wnt/ß-catenin signaling and is known to play an important role in CRC development and progression, the role of PAK1 in the expression of CSC markers has not previously been investigated. The effect of PAK1 over-expression, knockdown or inhibition on the expression or alteration (in the case of CD44) of CSC markers in human CRC cell lines was measured by immunofluorescence and Western blotting. The effect of PAK1 modulation on tumorigenesis, and on resistance to treatment with 5-fluorouracil (5-FU), was measured by sphere formation in vitro and by growth of xenografted tumors in vivo. The results show that PAK1 activity correlated with the expression of CSC markers and the CD44 isoform profile, and with tumor growth both in vitro and in vivo. Furthermore PAK overexpression partially overcame the inhibition of CRC growth by 5-FU, and PAK inhibition was synergistic with 5-FU treatment. Our findings lay the foundation for a combination therapy in which PAK1 inhibitors targeting CSCs may be combined with conventional 5-FU-based chemotherapy for the treatment of CRC.

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.

Glaucarubinone inhibits colorectal cancer growth by suppression of hypoxia-inducible factor 1α and ß-catenin via a p-21 activated kinase 1-dependent pathway.

p-21-Activated kinase 1 (PAK1) enhances colorectal cancer (CRC) progression by stimulating Wnt/ß-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 [(3)H]-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 ß-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 ß-catenin via a PAK1-dependent pathway.

HIF1α expression under normoxia in prostate cancer--which pathways to target?

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Increased gastrin gene expression provides a physiological advantage to mice under hypoxic conditions.

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.

CCK2R identifies and regulates gastric antral stem cell states and carcinogenesis.

OBJECTIVE: Progastrin is the incompletely cleaved precursor of gastrin that is secreted by G-cells in the gastric antrum. Both gastrin and progastrin bind to the CCK2 receptor (Cckbr or CCK2R) expressed on a subset of gastric epithelial cells. Little is known about how gastrin peptides and CCK2R regulate gastric stem cells and carcinogenesis. Interconversion among progenitors in the intestine is documented, but the mechanisms by which this occurs are poorly defined. DESIGN: We generated CCK2R-CreERT mice and performed inducible lineage tracing experiments. CCK2R+ antral cells and Lgr5+ antral stem cells were cultured in a three-dimensional in vitro system. We crossed progastrin-overexpressing mice with Lgr5-GFP-CreERT mice and examined the role of progastrin and CCK2R in Lgr5+ stem cells during MNU-induced carcinogenesis. RESULTS: Through lineage tracing experiments, we found that CCK2R defines antral stem cells at position +4, which overlapped with an Lgr5(neg or low) cell population but was distinct from typical antral Lgr5(high) stem cells. Treatment with progastrin interconverts Lgr5(neg or low) CCK2R+ cells into Lgr5(high) cells, increases CCK2R+ cell numbers and promotes gland fission and carcinogenesis in response to the chemical carcinogen MNU. Pharmacological inhibition or genetic ablation of CCK2R attenuated progastrin-dependent stem cell expansion and carcinogenesis. CONCLUSIONS: CCK2R labels +4 antral stem cells that can be activated and expanded by progastrin, thus identifying one hormonal trigger for gastric stem cell interconversion and a potential target for gastric cancer chemoprevention and therapy.

Identification of binding sites for C-terminal pro-gastrin-releasing peptide (GRP)-derived peptides in renal cell carcinoma: a potential target for future therapy.

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.

Demonstration and biological significance of a gastrin-P21-activated kinase 1 feedback loop in colorectal cancer cells.

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 (3)H-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 ß-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.

Expression and function of gastrin-releasing peptide (GRP) in normal and cancerous urological tissues.

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.