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1.
Am J Physiol Gastrointest Liver Physiol ; 316(5): G632-G640, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30844294

RESUMO

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.


Assuntos
Carcinoma Ductal Pancreático , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas , Tretinoína/farmacologia , Animais , Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Camundongos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Quinases Ativadas por p21/metabolismo , Gencitabina
2.
Biometals ; 31(5): 821-834, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29974287

RESUMO

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.


Assuntos
Cloretos/farmacologia , Modelos Animais de Doenças , Substâncias Protetoras/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Ovinos , Compostos de Zinco/farmacologia , Animais , Cloretos/administração & dosagem , Cloretos/análise , Células HEK293 , Humanos , Peróxido de Hidrogênio , Espectrometria de Massas , Traumatismo por Reperfusão/induzido quimicamente , Traumatismo por Reperfusão/metabolismo , Compostos de Zinco/administração & dosagem , Compostos de Zinco/análise
3.
Int J Cancer ; 140(9): 2101-2111, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28109008

RESUMO

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.


Assuntos
Proliferação de Células/efeitos dos fármacos , Neoplasias Pancreáticas/genética , Células Estreladas do Pâncreas/metabolismo , Quinases Ativadas por p21/genética , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Células Estreladas do Pâncreas/patologia , Piridonas/administração & dosagem , Pirimidinas/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida
4.
J Biol Inorg Chem ; 22(7): 999-1006, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28702751

RESUMO

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.


Assuntos
Complexos de Coordenação/metabolismo , Gálio/metabolismo , Gastrinas/metabolismo , Índio/metabolismo , Receptor de Colecistocinina B/metabolismo , Rutênio/metabolismo , Linhagem Celular , Complexos de Coordenação/química , Gálio/química , Radioisótopos de Gálio/metabolismo , Gastrinas/química , Humanos , Índio/química , Ligação Proteica , Rutênio/química , Radioisótopos de Rutênio/metabolismo
5.
BMC Cancer ; 17(1): 431, 2017 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-28629331

RESUMO

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.


Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Genes APC , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Imunomodulação/genética , Quinases Ativadas por p21/genética , Animais , Biomarcadores , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Genótipo , Imuno-Histoquímica , Contagem de Leucócitos , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Pirazóis/farmacologia , Pirróis/farmacologia , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/metabolismo
6.
Int J Colorectal Dis ; 32(7): 1061-1064, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28432443

RESUMO

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.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/metabolismo , Gastrinas/metabolismo , Neoplasias Hepáticas/secundário , Precursores de Proteínas/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Idoso , Neoplasias Colorretais/patologia , Feminino , Humanos , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias
7.
Biochim Biophys Acta ; 1853(1): 157-65, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25409929

RESUMO

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.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Glaucarubina/análogos & derivados , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , beta Catenina/antagonistas & inibidores , Quinases Ativadas por p21/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Glaucarubina/farmacologia , Humanos
8.
BMC Cancer ; 16: 24, 2016 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-26774265

RESUMO

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.


Assuntos
Adenocarcinoma/tratamento farmacológico , Sinergismo Farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Piridonas/administração & dosagem , Pirimidinas/administração & dosagem , Quinases Ativadas por p21/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Humanos , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Ductos Pancreáticos/efeitos dos fármacos , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases Ativadas por p21/antagonistas & inibidores , Gencitabina
9.
Biochim Biophys Acta ; 1843(11): 2471-80, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24998603

RESUMO

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.

10.
Biochim Biophys Acta ; 1844(3): 487-96, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24334106

RESUMO

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


Assuntos
Compostos Férricos/metabolismo , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Cátions , Ativação Enzimática , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Ressonância de Plasmônio de Superfície , Quinases da Família src/química
11.
Am J Physiol Gastrointest Liver Physiol ; 308(2): G76-84, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25394662

RESUMO

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.


Assuntos
Colo/metabolismo , Gastrinas/metabolismo , Expressão Gênica/fisiologia , Hipóxia/metabolismo , Animais , Eritropoetina/sangue , Feminino , Hipóxia/genética , Ferro/metabolismo , Fígado/metabolismo , Masculino , Camundongos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos
12.
J Urol ; 193(3): 763-70, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25444956

RESUMO

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.


Assuntos
Biomarcadores Tumorais/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Oxigênio/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/terapia , Regulação para Cima , Humanos , Masculino
13.
BJU Int ; 115(5): 829-38, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25130393

RESUMO

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.


Assuntos
Carcinoma de Células Renais/metabolismo , Peptídeo Liberador de Gastrina/metabolismo , Neoplasias Renais/metabolismo , Receptores da Bombesina/metabolismo , Sítios de Ligação , Carcinoma de Células Renais/tratamento farmacológico , Linhagem Celular Tumoral , Humanos , Neoplasias Renais/tratamento farmacológico , Peptídeos , Precursores de Proteínas
14.
Biochim Biophys Acta ; 1833(1): 33-9, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23092728

RESUMO

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


Assuntos
Carcinoma/genética , Neoplasias Gastrointestinais/genética , Quinases Ativadas por p21/fisiologia , Animais , Carcinoma/metabolismo , Neoplasias Gastrointestinais/metabolismo , Humanos , Modelos Biológicos , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , Quinases Ativadas por p21/química , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo
15.
BJU Int ; 113 Suppl 2: 40-7, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24894852

RESUMO

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.


Assuntos
Carcinoma de Células Renais/metabolismo , Peptídeo Liberador de Gastrina/metabolismo , Neoplasias Renais/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Receptores da Bombesina/metabolismo , Animais , Carcinoma de Células Renais/fisiopatologia , Linhagem Celular Tumoral , Coito , Progressão da Doença , Cães , Ejaculação , Peptídeo Liberador de Gastrina/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/fisiopatologia , Masculino , Neoplasias da Próstata/fisiopatologia , Ratos , Receptores da Bombesina/genética , Transmissão Sináptica
16.
J Gastroenterol Hepatol ; 29(3): 480-6, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24716212

RESUMO

BACKGROUND AND AIM: An increase in circulating concentrations of gastrin or gastrin precursors such as progastrin and glycine-extended gastrin has been proposed to promote the development of colorectal carcinomas (CRC). The aim of this study was to investigate whether or not circulating gastrin concentrations were increased in patients with an increased risk of developing CRC. METHOD: Patients were divided according to their risk into the five following groups: familial adenomatous polyposis (n = 20), hereditary non-polyposis colorectal cancer (n = 53), cluster of common colorectal cancers (n = 13), personal history and/or family history of adenomatous polyps or CRC (n = 150) and controls (n = 42). Radioimmunoassay with four region-specific gastrin antisera was used to measure progastrin, glycine-extended gastrin (gastrin-gly), amidated gastrin (gastrin-amide), and total gastrin in peripheral blood taken at the time of colonoscopy. RESULTS: Compared with the control group, familial adenomatous polyposis patients had significantly higher median values of total gastrin (29.8 pM vs 16.9 pM, P = 0.003) and gastrin-amide (17.1 pM vs 12.0 pM, P = 0.015). Patients with a personal or family history of adenomatous polyps or CRC also had higher circulating concentrations of total gastrin (21.8 pM) compared with controls (P < 0.05), while patients from all groups who presented with an adenomatous polyp on the day of colonoscopy had higher concentrations of total gastrin, progastrin, and gastrin-amide than patients without polyps. CONCLUSION: Concentrations of gastrin precursors are increased in particular groups with an increased risk of developing CRC.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/etiologia , Gastrinas/sangue , Polipose Adenomatosa do Colo/sangue , Polipose Adenomatosa do Colo/complicações , Polipose Adenomatosa do Colo/diagnóstico , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Precursores de Proteínas/sangue , Radioimunoensaio , Risco , Medição de Risco
17.
Am J Physiol Gastrointest Liver Physiol ; 304(6): G561-7, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23306081

RESUMO

Gastrins, including amidated (Gamide) and glycine-extended (Ggly) forms, function as growth factors for the gastrointestinal mucosa. The p-21-activated kinase 1 (PAK1) plays important roles in growth factor signaling networks that control cell motility, proliferation, differentiation, and transformation. PAK1, activated by both Gamide and Ggly, mediates gastrin-stimulated proliferation and migration, and activation of ß-catenin, in gastric epithelial cells. The aim of this study was to investigate the role of PAK1 in the regulation by gastrin of proliferation in the normal colorectal mucosa in vivo. Mucosal proliferation was measured in PAK1 knockout (PAK1 KO) mice by immunohistochemistry. The expression of phosphorylated and unphosphorylated forms of the signaling molecules PAK1, extracellular signal-regulated kinase (ERK), and protein kinase B (AKT), and the expression of ß-catenin and its downstream targets c-Myc and cyclin D1, were measured in gastrin knockout (Gas KO) and PAK1 KO mice by Western blotting. The expression and activation of PAK1 are decreased in Gas KO mice, and these decreases are associated with reduced activation of ERK, AKT, and ß-catenin. Proliferation in the colorectal mucosa of PAK1 KO mice is reduced, and the reduction is associated with reduced activation of ERK, AKT, and ß-catenin. In compensation, antral gastrin mRNA and serum gastrin concentrations are increased in PAK1 KO mice. These results indicate that PAK1 mediates the stimulation of colorectal proliferation by gastrins via multiple signaling pathways involving activation of ERK, AKT, and ß-catenin.


Assuntos
Proliferação de Células , Gastrinas/metabolismo , Mucosa Intestinal/metabolismo , Transdução de Sinais/fisiologia , Quinases Ativadas por p21/metabolismo , Animais , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Colo/patologia , Ciclina D1/metabolismo , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Imuno-Histoquímica , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reto/patologia , beta Catenina/metabolismo
18.
Biochim Biophys Acta ; 1813(5): 889-95, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21320535

RESUMO

The peptide hormone gastrin has been identified as a major regulator of acid secretion and a potent mitogen for normal and malignant gastrointestinal cells. The importance of gastric acid in the absorption of dietary iron first became evident 50 years ago when iron deficiency anemia was recognized as a long-term consequence of partial gastrectomy. This review summarizes the connections between circulating gastrins, iron status and colorectal cancer. Gastrins bind two ferric ions with micromolar affinity and, in the case of non-amidated forms of the hormone, iron binding is essential for biological activity in vitro and in vivo. The demonstration of an interaction between gastrin and transferrin by biochemical techniques led to the proposal that gastrins catalyze the loading of transferrin with iron. Several lines of evidence, including the facts that the concentrations of circulating gastrins are increased in mice and humans with the iron overload disease hemochromatosis and that transferrin saturation positively correlates with circulating gastrin concentration, suggest the potential involvement of gastrins in iron homeostasis. Conversely, recognition that ferric ions play an unexpected role in the biological activity of gastrins may assist in the development of useful therapies for colorectal carcinoma and other disorders of mucosal proliferation in the gastrointestinal tract. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.


Assuntos
Neoplasias Colorretais/metabolismo , Gastrinas/metabolismo , Homeostase , Ferro/metabolismo , Animais , Transporte Biológico , Humanos , Receptor de Colecistocinina B/metabolismo
19.
Biochim Biophys Acta ; 1803(9): 1106-13, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20595063

RESUMO

The p21-activated kinase (PAK) family of serine/threonine kinases plays an important role in cell proliferation, survival and motility, as well as in cell transformation and tumor progression. PAK1 promotes transformation through facilitating the ERK/MAPK pathway and enhances cell migration and survival by stimulating AKT. PAK1 expression increases with the progression of colorectal cancer (CRC). In this study, we have investigated the importance of PAK1 in the biology of colon cancer cells. Reduction of PAK1 expression decreased the activities of ERK and AKT leading to decreased cell proliferation, migration/invasion, and survival. Dual inhibition of ERK and AKT suppressed these cellular processes to levels comparable to those achieved by reduction of PAK1 expression, whereas inactivation of either the ERK or AKT pathway alone partially inhibited cell migration/invasion and survival and had no effect on proliferation. We conclude that PAK1 stimulates colon cancer cell proliferation, migration/invasion, and survival via ERK- and AKT-dependent pathways. These findings establish the central importance of PAK1 in CRC signal transduction and clarify the mechanism by which PAK1 regulates CRC growth and migration. Instead of simultaneously inhibiting both ERK and AKT, the PAK1 convergence point could be an alternative target for CRC therapy.


Assuntos
Carcinoma/patologia , Movimento Celular , Proliferação de Células , Neoplasias do Colo/patologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Proteína Oncogênica v-akt/fisiologia , Quinases Ativadas por p21/fisiologia , Carcinoma/genética , Carcinoma/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HCT116 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Modelos Biológicos , Invasividade Neoplásica , Proteína Oncogênica v-akt/metabolismo , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/genética
20.
Biochem Biophys Res Commun ; 404(4): 1083-7, 2011 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-21195058

RESUMO

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


Assuntos
Gastrinas/metabolismo , Ferro/metabolismo , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Gastrinas/química , Humanos , Ferro/química , Dados de Sequência Molecular , Fosforilação , Fosfosserina/metabolismo , Pró-Proteína Convertase 1/metabolismo , Precursores de Proteínas/química , Serina/metabolismo , Tripsina/metabolismo
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