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1.
Pancreas ; 48(5): 711-718, 2019.
Article in English | MEDLINE | ID: mdl-31091220

ABSTRACT

OBJECTIVE: Chronic pancreatitis is the consequence of multiple episodes of recurrent acute pancreatitis (RAP). We hypothesized that apigenin can minimize the sequelae of RAP by limiting acinar cells' proinflammatory signaling pathways. METHODS: AR42J acinar cells were treated in vitro with transforming growth factor ß (TGF-ß), apigenin, and other inhibitors. Dual luciferase reporter assay measured parathyroid hormone-related protein (PTHrP) promoter activity. MAPK/ERK pathway activity was assessed by immunoblotting and in vivo by immunohistochemistry with a cerulein-induced RAP mouse model. Nuclear factor κ B nuclear localization was analyzed in vitro in cells stimulated with tumor necrosis factor α. Primary acini were isolated and treated with cerulein; interleukin 6 messenger RNA was measured comparing PTHrP wild-type and knockout mice. RESULTS: Apigenin and PD98059 each downregulated TGF-ß stimulation of PTHrP P3 promoter activity. In a RAP mouse model, apigenin reduced pERK nuclear localization in acinar cells and preserved acinar cell architecture. Apigenin suppressed tumor necrosis factor α-mediated signaling by decreasing nuclear factor κ B nuclear localization and decreased interleukin 6 messenger RNA levels via a PTHrP-dependent mechanism. CONCLUSIONS: Apigenin reduced inflammatory responses in experimental models of RAP. The mechanisms mediating the actions of apigenin, in part, are owing to attenuation of PTHrP and TGF-ß proinflammatory signaling.


Subject(s)
Acinar Cells/drug effects , Apigenin/pharmacology , Pancreatitis, Chronic/metabolism , Parathyroid Hormone-Related Protein/metabolism , Transforming Growth Factor beta/pharmacology , Acinar Cells/metabolism , Acinar Cells/pathology , Acute Disease , Animals , Cell Line, Tumor , Cells, Cultured , Ceruletide , Interleukin-6/genetics , Interleukin-6/metabolism , Mice, Knockout , Mice, Transgenic , NF-kappa B/metabolism , Pancreatitis/chemically induced , Pancreatitis/genetics , Pancreatitis/metabolism , Pancreatitis, Chronic/genetics , Parathyroid Hormone-Related Protein/genetics , Signal Transduction/drug effects
2.
Pancreas ; 45(5): 659-70, 2016.
Article in English | MEDLINE | ID: mdl-26495794

ABSTRACT

OBJECTIVES: Transforming growth factor ß (TGF-ß) regulates immune and fibrotic responses of chronic pancreatitis. The bone morphogenetic protein 2 (BMP-2) antagonist gremlin is regulated by TGF-ß. Parathyroid hormone-related protein (PTHrP) levels are elevated in chronic pancreatitis. Here, we investigated the cross-talk between TGF-ß/BMP-2/gremlin and PTHrP signaling. METHODS: Reverse transcription/real-time polymerase chain reaction, chromatin immunoprecipitation, Western blotting, and transient transfection were used to investigate PTHrP regulation by TGF-ß and BMP-2 and gremlin regulation by PTHrP. The PTHrP antagonist PTHrP (7-34) and acinar cells with conditional Pthrp gene deletion (PTHrP) were used to assess PTHrP's role in the proinflammatory and profibrotic effects of TGF-ß and gremlin. RESULTS: Transforming growth factor ß increased PTHrP levels in acinar cells and pancreatic stellate cells (PSCs) through a Smad3-dependent pathway. Transforming growth factor ß's effects on levels of IL-6 and intercellular adhesion molecule 1 (ICAM-1) (acinar cells) and procollagen I and fibronectin (PSCs) were inhibited by PTHrP (7-34). PTHrP suppressed TGF-ß's effects on IL-6 and ICAM-1. Parathyroid hormone-related hormone increased gremlin in acinar cells, and inhibiting gremlin action suppressed TGF-ß's and PTHrP's effects on IL-6 and ICAM-1. Transforming growth factor ß-mediated gremlin up-regulation was suppressed in PTHrP cells. Bone morphogenetic protein 2 suppressed PTHrP levels in PSCs. CONCLUSIONS: Parathyroid hormone-related hormone functions as a novel mediator of the proinflammatory and profibrotic effects of TGF-ß. Transforming growth factor ß and BMP-2 regulate PTHrP expression, and PTHrP regulates gremlin levels.


Subject(s)
Acinar Cells/drug effects , Bone Morphogenetic Protein 2/pharmacology , Intercellular Signaling Peptides and Proteins/metabolism , Pancreatic Stellate Cells/drug effects , Parathyroid Hormone-Related Protein/metabolism , Transforming Growth Factor beta/pharmacology , Acinar Cells/metabolism , Animals , Blotting, Western , Cell Line , Cells, Cultured , Fibrosis/metabolism , Gene Expression/drug effects , Inflammation Mediators/metabolism , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Intercellular Signaling Peptides and Proteins/genetics , Interleukin-6/genetics , Interleukin-6/metabolism , Mice, Knockout , Mice, Transgenic , Pancreas/metabolism , Pancreas/pathology , Pancreatic Stellate Cells/metabolism , Parathyroid Hormone-Related Protein/genetics , Protein Binding , Rats , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Smad3 Protein/metabolism
3.
Am J Physiol Gastrointest Liver Physiol ; 309(7): G554-65, 2015 Oct 01.
Article in English | MEDLINE | ID: mdl-26229008

ABSTRACT

Chronic pancreatitis (CP) is a devastating disease with no treatments. Experimental models have been developed to reproduce the parenchyma and inflammatory responses typical of human CP. For the present study, one objective was to assess and compare the effects of pancreatic duct ligation (PDL) to those of repetitive cerulein (Cer)-induced CP in mice on pancreatic production of bone morphogenetic protein-2 (BMP2), apelin, and parathyroid hormone-related protein (PTHrP). A second objective was to determine the extent of cross talk among pancreatic BMP2, apelin, and PTHrP signaling systems. We focused on BMP2, apelin, and PTHrP since these factors regulate the inflammation-fibrosis cascade during pancreatitis. Findings showed that PDL- and Cer-induced CP resulted in significant elevations in expression and peptide/protein levels of pancreatic BMP2, apelin, and PTHrP. In vivo mouse and in vitro pancreatic cell culture experiments demonstrated that BMP2 stimulated pancreatic apelin expression whereas apelin expression was inhibited by PTHrP exposure. Apelin or BMP2 exposure inhibited PTHrP expression, and PTHrP stimulated upregulation of gremlin, an endogenous inhibitor of BMP2 activity. Transforming growth factor-ß (TGF-ß) stimulated PTHrP expression. Together, findings demonstrated that PDL- and Cer-induced CP resulted in increased production of the pancreatic BMP2, apelin, and PTHrP signaling systems and that significant cross talk occurred among pancreatic BMP2, apelin, and PTHrP. These results together with previous findings imply that these factors interact via a pancreatic network to regulate the inflammation-fibrosis cascade during CP. More importantly, this network communicated with TGF-ß, a key effector of pancreatic pathophysiology. This novel network may be amenable to pharmacologic manipulations during CP in humans.


Subject(s)
Adipokines/metabolism , Bone Morphogenetic Protein 2/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Pancreatic Ducts/surgery , Pancreatitis, Chronic/metabolism , Parathyroid Hormone-Related Protein/metabolism , Animals , Apelin , Blotting, Western , Cell Culture Techniques , Ceruletide/pharmacology , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Humans , Ligation , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Real-Time Polymerase Chain Reaction , Signal Transduction
4.
Cancers (Basel) ; 7(2): 1091-108, 2015 Jun 18.
Article in English | MEDLINE | ID: mdl-26095761

ABSTRACT

Chronic pancreatitis (CP), a progressive inflammatory disease where acini are destroyed and replaced by fibrous tissue, increases the risk for pancreatic cancer. Risk factors include alcohol, smoking, and obesity. The effects of these risk factors are exacerbated in patients with mutations in genes that predispose to CP. The different environmental and genetic factors produce the same clinical phenotype; once CP develops, disease course is the same regardless of etiology. Critical questions still need to be answered to understand what modifies predisposition to develop CP in persons exposed to risk factors. We postulate that risk factors modulate endogenous pathways, with parathyroid hormone-related protein (PTHrP) signaling being one such pathway. In support, PTHrP levels are elevated in mice treated with alcohol, and in mouse models of cerulein- and pancreatic duct ligation-induced CP. Disrupting the Pthrp gene in acinar cells exerts protective effects (decreased edema, histological damage, amylase and cytokine release, and fibrosis) in these CP models. PTHrP levels are elevated in human CP. Currently, CP care lacks specific pharmacological interventions. Targeting PTHrP signaling may present a novel therapeutic strategy that inhibits pancreatic inflammation and fibrosis, especially since the risk of developing pancreatic cancer is strongly associated with duration of chronic inflammation.

5.
Cancer Lett ; 362(2): 199-207, 2015 Jul 01.
Article in English | MEDLINE | ID: mdl-25846868

ABSTRACT

Colorectal carcinoma (CRC) is the third most common cancer in developed countries. A large fraction of cases are linked to chronic intestinal inflammation, with concomitant increased TNF-α release and elevated Snail1/Snail2 levels. These transcription factors in turn suppress vitamin D receptor (VDR) expression, resulting in loss of responsiveness to the protective anti-proliferative and anti-migratory effects of 1,25-dihydroxyvitamin D (1,25D). Experimental and epidemiologic evidence support the use of natural products to target CRC. Here we show that the flavonolignan silibinin reverses the TNF-α-induced upregulation of Snail1 and Snail2 in the 1,25D-resistant human colon carcinoma cells HT-29. These silibinin effects are accompanied by an increase in VDR levels; Snail1 overexpression reverses these silibinin effects. Silibinin also restores promoter activity from a vitamin D-response element (VDRE) reporter construct. While 1,25D had no significant effect on HT-29 and SW480-R cell proliferation and migration, co-treatment with silibinin restored 1,25D responsiveness. In addition, co-treatment with silibinin plus 1,25D decreased proliferation and migration at doses where silibinin alone had no effect. These findings demonstrate that this combination may present a novel approach to target CRC in conditions of chronic colonic inflammation.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Colonic Neoplasms/drug therapy , Silymarin/pharmacology , Vitamin D/analogs & derivatives , Cell Movement/drug effects , Cell Proliferation/drug effects , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Drug Synergism , HT29 Cells , Humans , Receptors, Calcitriol/metabolism , Retinoid X Receptor alpha/metabolism , Silybin , Silymarin/administration & dosage , Snail Family Transcription Factors , Transcription Factors/metabolism , Tumor Necrosis Factor-alpha/metabolism , Vitamin D/administration & dosage , Vitamin D/pharmacology
6.
J Surg Res ; 196(1): 8-16, 2015 Jun 01.
Article in English | MEDLINE | ID: mdl-25799526

ABSTRACT

BACKGROUND: Chronic pancreatitis (CP) is characterized by recurrent pancreatic injury, resulting in inflammation, necrosis, and fibrosis. There are currently no drugs limiting pancreatic fibrosis associated with CP, and there is a definite need to fill this void in patient care. MATERIALS AND METHODS: Pancreatitis was induced in C57/BL6 mice using supraphysiologic doses of cerulein, and apigenin treatment (once daily, 50 µg per mouse by oral gavage) was initiated 1 wk into the recurrent acute pancreatitis (RAP) protocol. Pancreata were harvested after 4 wk of RAP. Immunostaining with fibronectin antibody was used to quantify the extent of pancreatic fibrosis. To assess how apigenin may decrease organ fibrosis, we evaluated the effect of apigenin on the proliferation and apoptosis of human pancreatic stellate cells (PSCs) in vitro. Finally, we assessed apigenin's effect on the gene expression in PSCs stimulated with parathyroid hormone-related protein, a profibrotic and proinflammatory mediator of pancreatitis, using reverse transcription-polymerase chain reaction. RESULTS: After 4 wk of RAP, apigenin significantly reduced the fibrotic response to injury while preserving acinar units. Apigenin inhibited viability and induced apoptosis of PSCs in a time- and dose-dependent manner. Finally, apigenin reduced parathyroid hormone-related protein-stimulated increases in the PSC messenger RNA expression levels of extracellular matrix proteins collagen 1A1 and fibronectin, proliferating cell nuclear antigen, transforming growth factor-beta, and interleukin-6. CONCLUSIONS: These in vivo and in vitro studies provide novel insights regarding apigenin's mechanism(s) of action in reducing the severity of RAP. Additional preclinical testing of apigenin analogs is warranted to develop a therapeutic agent for patients at risk for CP.


Subject(s)
Apigenin/therapeutic use , Pancreatic Stellate Cells/drug effects , Pancreatitis, Chronic/drug therapy , Animals , Apigenin/pharmacology , Cell Survival/drug effects , Cells, Cultured , Disease Models, Animal , Female , Humans , Male , Mice , Mice, Inbred C57BL , Pancreatic Stellate Cells/pathology , Pancreatitis, Chronic/metabolism , Pancreatitis, Chronic/pathology , Parathyroid Hormone-Related Protein/pharmacology
7.
Am J Physiol Gastrointest Liver Physiol ; 307(5): G533-49, 2014 Sep 01.
Article in English | MEDLINE | ID: mdl-25035110

ABSTRACT

Pancreatitis is a necroinflammatory disease with acute and chronic manifestations. Accumulated damage incurred during repeated bouts of acute pancreatitis (AP) can lead to chronic pancreatitis (CP). Pancreatic parathyroid hormone-related protein (PTHrP) levels are elevated in a mouse model of cerulein-induced AP. Here, we show elevated PTHrP levels in mouse models of pancreatitis induced by chronic cerulein administration and pancreatic duct ligation. Because acinar cells play a major role in the pathophysiology of pancreatitis, mice with acinar cell-specific targeted disruption of the Pthrp gene (PTHrP(Δacinar)) were generated to assess the role of acinar cell-secreted PTHrP in pancreatitis. These mice were generated using Cre-LoxP technology and the acinar cell-specific elastase promoter. PTHrP(Δacinar) exerted protective effects in cerulein and pancreatic duct ligation models, evident as decreased edema, histological damage, amylase secretion, pancreatic stellate cell (PSC) activation, and extracellular matrix deposition. Treating acinar cells in vitro with cerulein increased IL-6 expression and NF-κB activity; these effects were attenuated in PTHrP(Δacinar) cells, as were the cerulein- and carbachol-induced elevations in amylase secretion. The cerulein-induced upregulation of procollagen I expression was lost in PSCs from PTHrP(Δacinar) mice. PTHrP immunostaining was elevated in human CP sections. The cerulein-induced upregulation of IL-6 and ICAM-1 (human acinar cells) and procollagen I (human PSCs) was suppressed by pretreatment with the PTH1R antagonist, PTHrP (7-34). These findings establish PTHrP as a novel mediator of inflammation and fibrosis associated with CP. Acinar cell-secreted PTHrP modulates acinar cell function via its effects on proinflammatory cytokine release and functions via a paracrine pathway to activate PSCs.


Subject(s)
Acinar Cells/metabolism , Pancreatitis/metabolism , Parathyroid Hormone-Related Protein/metabolism , Acinar Cells/drug effects , Amylases/metabolism , Animals , Carbachol/pharmacology , Cells, Cultured , Ceruletide/toxicity , Fibrosis/metabolism , Humans , Inflammation/metabolism , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Mice , Mice, Inbred BALB C , Mice, Knockout , NF-kappa B/metabolism , Pancreatic Ducts/metabolism , Pancreatic Ducts/pathology , Parathyroid Hormone-Related Protein/blood , Parathyroid Hormone-Related Protein/genetics , Procollagen/genetics , Procollagen/metabolism
8.
Exp Cell Res ; 319(10): 1419-30, 2013 Jun 10.
Article in English | MEDLINE | ID: mdl-23499737

ABSTRACT

Parathyroid hormone-related protein (PTHrP) enhances prostate cancer (CaP) growth and metastasis in vivo. PTHrP also increases cell survival and migration, and upregulates pro-invasive integrin α6ß4 expression. We used the human CaP cell lines C4-2 and PC-3 as model systems to study the mechanisms via which PTHrP regulates α6ß4 levels. We report that PTHrP regulates α6 and ß4 levels via a transcriptional pathway; ß4 regulation involves the NF-κB pathway. PTHrP also regulates ß4 levels at the post-translational level. PTHrP inhibits caspase-3 and -7 activities. Post-translational regulation of ß4 by PTHrP is mediated via attenuation of its proteolytic cleavage by these caspases. Since α6 dimerizes with ß4, increased ß4 levels result in elevated α6 levels. Suppressing ß4 using siRNA attenuates the effect of caspase inhibition on apoptosis and cell migration. These results provide evidence of a link between PTHrP, integrin α6ß4 levels as a function of caspase activity, and cell survival and migration. Targeting PTHrP in CaP cancer, thereby reversing the effect on caspase activity and α6ß4 levels, may thus prove therapeutically beneficial.


Subject(s)
Integrin alpha6/metabolism , Integrin beta4/metabolism , Parathyroid Hormone-Related Protein/metabolism , Protein Processing, Post-Translational , Transcription, Genetic , Apoptosis , Caspase 3/genetics , Caspase 3/metabolism , Caspase 7/genetics , Caspase 7/metabolism , Cell Line, Tumor , Cell Movement , Cell Survival , Gene Silencing , Half-Life , Humans , Integrin alpha6/genetics , Integrin beta4/genetics , Mutagenesis, Site-Directed , NF-kappa B/metabolism , Parathyroid Hormone-Related Protein/genetics , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Promoter Regions, Genetic , Protein Multimerization , Proteolysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Transfection
9.
Regul Pept ; 175(1-3): 49-60, 2012 Apr 10.
Article in English | MEDLINE | ID: mdl-22280800

ABSTRACT

Pancreatitis is a common and potentially lethal necro-inflammatory disease with both acute and chronic manifestations. Current evidence suggests that the accumulated damage incurred during repeated bouts of acute pancreatitis (AP) can lead to chronic disease, which is associated with an increased risk of pancreatic cancer. While parathyroid hormone-related protein (PTHrP) exerts multiple effects in normal physiology and disease states, its function in pancreatitis has not been previously addressed. Here we show that PTHrP levels are transiently elevated in a mouse model of cerulein-induced AP. Treatment with alcohol, a risk factor for both AP and chronic pancreatitis (CP), also increases PTHrP levels. These effects of cerulein and ethanol are evident in isolated primary acinar and stellate cells, as well as in the immortalized acinar and stellate cell lines AR42J and irPSCc3, respectively. Ethanol sensitizes acinar and stellate cells to the PTHrP-modulating effects of cerulein. Treatment of acinar cells with PTHrP (1-36) increases expression of the inflammatory mediators interleukin-6 (IL-6) and intracellular adhesion protein (ICAM-1), suggesting a potential autocrine loop. PTHrP also increases apoptosis in AR42J cells. Stellate cells mediate the fibrogenic response associated with pancreatitis; PTHrP (1-36) increases procollagen I and fibronectin mRNA levels in both primary and immortalized stellate cells. The effects of cerulein and ethanol on levels of IL-6 and procollagen I are suppressed by the PTH1R antagonist, PTHrP (7-34). Together these studies identify PTHrP as a potential mediator of the inflammatory and fibrogenic responses associated with alcoholic pancreatitis.


Subject(s)
Acinar Cells/drug effects , Ceruletide/adverse effects , Ethanol/adverse effects , Inflammation/metabolism , Pancreatic Stellate Cells/drug effects , Pancreatitis/metabolism , Parathyroid Hormone-Related Protein/metabolism , Acinar Cells/cytology , Acinar Cells/metabolism , Animals , Apoptosis/drug effects , Blotting, Western , Bone Neoplasms/drug therapy , Bone Neoplasms/immunology , Bone Neoplasms/metabolism , Cells, Cultured , Central Nervous System Depressants/adverse effects , Collagen Type I/genetics , Collagen Type I/metabolism , Fibronectins/genetics , Fibronectins/metabolism , Fluorescent Antibody Technique , Humans , Immunoenzyme Techniques , Inflammation/chemically induced , Inflammation/immunology , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Mice , Mice, Inbred C57BL , Osteosarcoma/drug therapy , Osteosarcoma/immunology , Osteosarcoma/metabolism , Pancreatic Stellate Cells/cytology , Pancreatic Stellate Cells/metabolism , Pancreatitis/chemically induced , Pancreatitis/immunology , Parathyroid Hormone-Related Protein/genetics , RNA, Messenger/genetics , Rats , Real-Time Polymerase Chain Reaction
10.
Mol Cell Endocrinol ; 342(1-2): 32-40, 2011 Aug 06.
Article in English | MEDLINE | ID: mdl-21664243

ABSTRACT

Parathyroid hormone-related protein (PTHrP) increases the growth and osteolytic potential of prostate cancer cells, making it important to control PTHrP expression. PTHrP expression is suppressed by 1,25-dihydroxyvitamin D(3) (1,25D). The aim of this study was to identify the pathways via which 1,25D exerts these effects. Our main findings are that 1,25D regulates PTHrP levels via multiple pathways in PC-3 and C4-2 (human prostate cancer) cell lines, and regulation is dependent on VDR expression. The human PTHrP gene has three promoters (P); PC-3 cells preferentially utilize P2 and P3, while C4-2 cells preferentially utilize P1. 1,25D regulates PTHrP transcriptional activity from both P1 and P3. The 1,25D-mediated decrease in PTHrP mRNA levels also involves a post-transcriptional pathway since 1,25D decreases PTHrP mRNA stability. 1,25D also suppresses PTHrP expression directly at the protein level by increasing its degradation. Regulation of PTHrP levels is dependent on VDR expression, as using siRNAs to deplete VDR expression negates the 1,25D-mediated downregulation of PTHrP expression. These results indicate the importance of maintaining adequate 1,25D levels and VDR status to control PTHrP levels.


Subject(s)
Calcitriol/metabolism , Parathyroid Hormone-Related Protein/metabolism , Protein Processing, Post-Translational , RNA Processing, Post-Transcriptional , Transcription, Genetic , Cell Line, Tumor , Cell Proliferation , Humans , Male , Parathyroid Hormone-Related Protein/genetics , Promoter Regions, Genetic , Prostatic Neoplasms/metabolism , RNA Interference , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Receptors, Calcitriol/metabolism
11.
Cancer Lett ; 298(1): 119-27, 2010 Dec 01.
Article in English | MEDLINE | ID: mdl-20637541

ABSTRACT

Parathyroid hormone-related protein (PTHrP) is expressed by human colon cancer tissue and cell lines. Rac1 GTPase enhances colon cancer cell migration and invasion. Here we report a positive correlation between PTHrP expression and Rac1 activity in LoVo (human colon cancer) cells. The positive effects of PTHrP on Rac1 activity and on cell migration and invasion are mediated via the guanine nucleotide exchange factor Tiam1. Knockdown of integrin α6ß4, which is upregulated by PTHrP, negates the PTHrP-mediated increase in Rac1 activation. Integrin α6ß4 signals synergistically with growth factor receptors to activate the phosphatidylinositol 3-kinase (PI3-K) pathway. Chemical inhibition of PI3-K negates the PTHrP-mediated effects on Tiam1 and Rac1 activity. Tumors from PTHrP-overexpressing LoVo cells also show increased expression of Tiam1. Taken together, these observations provide evidence of a link between PTHrP and Rac1 activity through integrin α6ß4, resulting in enhanced cell migration and invasion. Targeting PTHrP production in colon cancer may thus prove therapeutically beneficial.


Subject(s)
Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Integrin alpha6beta4/metabolism , Parathyroid Hormone-Related Protein/metabolism , rac1 GTP-Binding Protein/metabolism , Cell Line, Tumor , Cell Movement/physiology , Colonic Neoplasms/genetics , Guanine Nucleotide Exchange Factors/metabolism , Humans , Neoplasm Invasiveness , Parathyroid Hormone-Related Protein/biosynthesis , Parathyroid Hormone-Related Protein/genetics , T-Lymphoma Invasion and Metastasis-inducing Protein 1 , Transfection
12.
Free Radic Biol Med ; 47(10): 1414-21, 2009 Nov 15.
Article in English | MEDLINE | ID: mdl-19686837

ABSTRACT

In this study, we investigated the role of Trypanosoma cruzi invasion and inflammatory processes in reactive oxygen species (ROS) production in a mouse atrial cardiomyocyte line (HL-1) and primary adult rat ventricular cardiomyocytes. Cardiomyocytes were incubated with T. cruzi (Tc) trypomastigotes, Tc lysate (TcTL), or Tc secreted proteins (TcSP) for 0-72 h, and ROS were measured by amplex red assay. Cardiomyocytes infected by T. cruzi (but not those incubated with TcTL or TcSP) exhibited a linear increase in ROS production for 2-48 h postinfection (max 18-fold increase), which was further enhanced by recombinant cytokines (IL-1beta, TNF-alpha, and IFN-gamma). We observed no increase in NADPH oxidase, xanthine oxidase, or myeloperoxidase activity, and specific inhibitors of these enzymes did not block the increased rate of ROS production in infected cardiomyocytes. Instead, the mitochondrial membrane potential was perturbed and resulted in inefficient electron transport chain (ETC) activity and enhanced electron leakage and ROS formation in infected cardiomyocytes. HL-1 rho (rho) cardiomyocytes lacked a functional ETC and exhibited no increase in ROS formation in response to T. cruzi. Together, these results demonstrate that invasion by T. cruzi and an inflammatory milieu affect mitochondrial integrity and contribute to electron transport chain inefficiency and ROS production in cardiomyocytes.


Subject(s)
Mitochondrial Membranes/immunology , Myocytes, Cardiac/immunology , Reactive Oxygen Species/immunology , Trypanosoma cruzi/isolation & purification , Animals , Cells, Cultured , Fluorescent Dyes/chemistry , Mice , Mitochondrial Membranes/drug effects , Myocytes, Cardiac/drug effects , Rats , Trypanocidal Agents/pharmacology , Trypanosoma cruzi/drug effects , Trypanosoma cruzi/immunology
13.
Mol Cancer Ther ; 8(7): 1787-98, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19584236

ABSTRACT

Parathyroid hormone-related protein (PTHrP) plays a major role in prostate carcinoma progression and bone metastasis. Once prostate cancers become androgen-independent, treatment options become limited. Vitamin D analogues represent a potentially valuable class of agents in this clinical context. Using the prostate cancer cell line C4-2 as a model, we studied the effects of PTHrP and the noncalcemic vitamin D analogue EB1089 on markers of prostate cancer cell progression in vitro and in vivo. C4-2 is a second-generation androgen-independent LNCaP subline that metastasizes to the lymph nodes and bone when injected into nude mice and produces mixed lytic/blastic lesions, mimicking the in vivo situation. We report that PTHrP increases cell migration and invasion, and that a pathway via which EB1089 inhibits these processes is through down-regulation of PTHrP expression. PTHrP also increases anchorage-independent cell growth in vitro and xenograft growth in vivo; EB1089 reverses these effects. The in vivo PTHrP effects are accompanied by increased tumor cell proliferation and survival. Treatment with EB1089 reverses the proliferative but not the antiapoptotic effects of PTHrP. PTHrP also increases intratumor vessel density and vascular endothelial growth factor expression; EB1089 reverses these effects. Intracardially injected C4-2 cells produce predominantly osteoblastic lesions; PTHrP overexpression decreases the latency, increases the severity and alters the bone lesion profile to predominantly osteolytic. EB1089 largely reverses these PTHrP effects. A direct correlation between PTHrP immunoreactivity and increasing tumor grade is observed in human prostate cancer specimens. Thus, decreasing PTHrP production by treatment with vitamin D analogues may prove therapeutically beneficial for prostate cancer.


Subject(s)
Adenocarcinoma/drug therapy , Antineoplastic Agents/therapeutic use , Bone Neoplasms/drug therapy , Calcitriol/analogs & derivatives , Parathyroid Hormone-Related Protein/metabolism , Prostatic Neoplasms/drug therapy , Adenocarcinoma/metabolism , Adenocarcinoma/secondary , Animals , Apoptosis/drug effects , Bone Neoplasms/metabolism , Bone Neoplasms/secondary , Calcitriol/therapeutic use , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Humans , Immunoenzyme Techniques , Male , Mice , Mice, Nude , Prostate/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Xenograft Model Antitumor Assays
14.
Mol Cancer Res ; 7(7): 1119-31, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19584267

ABSTRACT

Parathyroid hormone-related protein (PTHrP) is expressed by human prostatic tissues and cancer cell lines. PTHrP enhances tumor cell growth and metastasis in vivo and up-regulates proinvasive integrin alpha6beta4 expression in vitro. Hallmarks of malignant tumor cells include resistance to apoptosis and anchorage-independent cell growth. In this study, we used the human prostate cancer cell lines C4-2 and PC-3 as model systems to study the effects of PTHrP on these processes. We report that PTHrP protects these cells from doxorubicin-induced apoptosis and promotes anchorage-independent cell growth via an intracrine pathway. Conversely, autocrine/paracrine PTHrP action increases apoptosis in C4-2 cells and has no effect on apoptosis in PC-3 cells. The intracrine effects of PTHrP on apoptosis are mediated via activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. PTHrP also affects the phosphorylation state of Akt substrates implicated in apoptosis suppression, including glycogen synthase kinase-3 and Bad. The prosurvival effects of PTHrP are accompanied by increases in the ratio of antiapoptotic to proapoptotic members of the Bcl-2 family and in levels of c-myc. PTHrP also increases nuclear factor-kappaB activity via a PI3K-dependent pathway. Integrin alpha6beta4 is known to activate PI3K. Here, we also show that knockdown of integrin alpha6beta4 negates the PTHrP-mediated activation of the PI3K/Akt pathway. Taken together, these observations provide evidence of a link between PTHrP and the PI3K/Akt signaling pathway through integrin alpha6beta4, resulting in the activation of survival pathways. Targeting PTHrP production in prostate cancer may thus prove therapeutically beneficial.


Subject(s)
Integrin alpha6beta4/metabolism , Parathyroid Hormone-Related Protein/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Prostatic Neoplasms/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Analysis of Variance , Apoptosis/drug effects , Cell Adhesion/physiology , Cell Growth Processes/physiology , Cell Line, Tumor , Cell Survival/physiology , Doxorubicin/pharmacology , Enzyme Activation , Glycogen Synthase Kinase 3/metabolism , Humans , Male , NF-kappa B/metabolism , Phosphorylation , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Receptor, Parathyroid Hormone, Type 1/metabolism , Signal Transduction
15.
Clin Vaccine Immunol ; 15(8): 1158-64, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18550728

ABSTRACT

Trypanosoma cruzi is the etiologic agent of Chagas' disease, a major health problem in Latin America and an emerging infectious disease in the United States. Previously, we screened a T. cruzi sequence database by a computational-bioinformatic approach and identified antigens that exhibited the characteristics of good vaccine candidates. In this study, we tested the vaccine efficacy of three of the putative candidate antigens against T. cruzi infection and disease in a mouse model. C57BL/6 mice vaccinated with T. cruzi G1 (TcG1)-, TcG2-, or TcG4-encoding plasmids and cytokine (interleukin-12 and granulocyte-macrophage colony-stimulating factor) expression plasmids elicited a strong Th1-type antibody response dominated by immunoglobulin G2b (IgG2b)/IgG1 isotypes. The dominant IgG2b/IgG1 antibody response was maintained after a challenge infection and was associated with 50 to 90% control of the acute-phase tissue parasite burden and an almost undetectable level of tissue parasites during the chronic phase, as determined by a sensitive T. cruzi 18S rRNA gene-specific real-time PCR approach. Splenocytes from vaccinated-and-infected mice, compared to unvaccinated-and-infected mice, exhibited decreased (approximately 50% lower) proliferation and gamma interferon (IFN-gamma) production when stimulated in vitro with T. cruzi antigens, thus suggesting that protection from challenge infection was not provided by an active T-cell response. Subsequently, the serum and cardiac levels of IFN-gamma and tumor necrosis factor alpha and infiltration of inflammatory infiltrate in the heart were decreased in vaccinated mice during the course of infection and chronic disease development. Taken together, these results demonstrate the identification of novel vaccine candidates that provided protection from T. cruzi-induced immunopathology in experimental mice.


Subject(s)
Protozoan Vaccines , Trypanosoma cruzi/immunology , Animals , Antibodies, Protozoan/blood , Antigens, Protozoan/genetics , Antigens, Protozoan/immunology , Chagas Cardiomyopathy/immunology , Chagas Cardiomyopathy/parasitology , Chagas Cardiomyopathy/physiopathology , Chagas Cardiomyopathy/prevention & control , Chagas Disease/immunology , Chagas Disease/parasitology , Chagas Disease/physiopathology , Chagas Disease/prevention & control , Computational Biology , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Humans , Immunization , Immunoglobulin G/blood , Mice , Mice, Inbred C57BL , Plasmids , Protozoan Proteins/genetics , Protozoan Proteins/immunology , Protozoan Vaccines/administration & dosage , Protozoan Vaccines/genetics , Protozoan Vaccines/immunology , Th1 Cells/immunology , Trypanosoma cruzi/pathogenicity
16.
Am J Pathol ; 169(6): 1953-64, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17148660

ABSTRACT

In this study, we investigated the mechanism(s) of mitochondrial functional decline in acute Chagas' disease. Our data show a substantial decline in respiratory complex activities (39 to 58%) and ATP (38%) content in Trypanosoma cruzi-infected murine hearts compared with normal controls. These metabolic alterations were associated with an approximately fivefold increase in mitochondrial reactive oxygen species production rate, substantial oxidative insult of mitochondrial membranes and respiratory complex subunits, and >60% inhibition of mtDNA-encoded transcripts for respiratory complex subunits in infected myocardium. The antioxidant phenyl-alpha-tert-butyl nitrone (PBN) arrested the oxidative damage-mediated loss in mitochondrial membrane integrity, preserved redox potential-coupled mitochondrial gene expression, and improved respiratory complex activities (47 to 95% increase) and cardiac ATP level (>or=40% increase) in infected myocardium. Importantly, PBN resulted twofold decline in mitochondrial reactive oxygen species production rate in infected myocardium. Taken together, our data demonstrate the pathological significance of oxidative stress in metabolic decay and energy homeostasis in acute chagasic myocarditis and further suggest that oxidative injuries affecting mitochondrial integrity-dependent expression and activity of the respiratory complexes initiate a feedback cycle of electron transport chain inefficiency, increased reactive oxygen species production, and energy homeostasis in acute chagasic hearts. PBN and other mitochondria-targeted antioxidants may be useful in altering mitochondrial decay and oxidative pathology in Chagas' disease.


Subject(s)
Chagas Disease/drug therapy , Cyclic N-Oxides/pharmacology , Heart/parasitology , Mitochondria, Heart/drug effects , Oxidative Stress/drug effects , Animals , Antioxidants/metabolism , Cell Line , Electron Transport/drug effects , Male , Mice , Mice, Inbred C3H , Mitochondria, Heart/parasitology , Mitochondria, Heart/pathology
17.
Emerg Infect Dis ; 12(4): 624-30, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16704811

ABSTRACT

We used 5 diagnostic tests in a cross-sectional investigation of the prevalence of Trypanosoma cruzi in Tejupilco municipality, State of Mexico, Mexico. Our findings showed a substantial prevalence of immunoglobulin G (IgG) and IgM antibodies to T. cruzi in human (n = 293, IgG 2.05%, IgM 5.5%, both 7.1%) and dog (n = 114, IgG 15.8%, IgM 11.4%, both 21%) populations. We also found antibodies to T. cruzi (n = 80, IgG 10%, IgM 15%, both 17.5%) in dogs from Toluca, an area previously considered free of T. cruzi. Our data demonstrate the need for active epidemiologic surveillance programs in these regions. A direct correlation (r2 = 0.955) of seropositivity between humans and dogs suggests that seroanalysis in dogs may help identify the human prevalence of T. cruzi infection in these areas.


Subject(s)
Chagas Disease/epidemiology , Chagas Disease/veterinary , Dog Diseases/epidemiology , Dog Diseases/parasitology , Trypanosoma cruzi/isolation & purification , Animals , Antibodies, Protozoan/blood , Chagas Disease/immunology , Chagas Disease/parasitology , Dog Diseases/blood , Dog Diseases/immunology , Dogs , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Mexico/epidemiology , Seroepidemiologic Studies , Trypanosoma cruzi/immunology
18.
An Acad Bras Cienc ; 77(4): 695-715, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16341444

ABSTRACT

There is growing evidence to suggest that chagasic myocardia are exposed to sustained oxidative stress-induced injuries that may contribute to disease progression. Pathogen invasion- and replication-mediated cellular injuries and immune-mediated cytotoxic reactions are the common source of reactive oxygen species (ROS) in infectious etiologies. However, our understanding of the source and role of oxidative stress in chagasic cardiomyopathy (CCM) remains incomplete. In this review, we discuss the evidence for increased oxidative stress in chagasic disease, with emphasis on mitochondrial abnormalities, electron transport chain dysfunction and its role in sustaining oxidative stress in myocardium. We discuss the literature reporting the consequences of sustained oxidative stress in CCM pathogenesis.


Subject(s)
Chagas Cardiomyopathy/etiology , Mitochondria, Heart/metabolism , Oxidative Stress/physiology , Reactive Oxygen Species , Animals , Chagas Cardiomyopathy/immunology , Chagas Cardiomyopathy/metabolism , Electron Transport Chain Complex Proteins/physiology , Humans , Trypanosoma cruzi/immunology
19.
Expert Rev Vaccines ; 4(6): 867-80, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16372882

ABSTRACT

The clinically relevant pathognomonic consequences of human infection by Trypanosoma cruzi are dilation and hypertrophy of the left ventricle walls and thinning of the apex. The major complications and debilitating evolutionary outcomes of chronic infection include ventricular fibrillation, thromboembolism and congestive heart failure. American trypanosomiasis (Chagas disease) poses serious public healthcare and budgetary concerns. The currently available drugs, although effective against acute infection, are highly toxic and ineffective in arresting or attenuating clinical disease symptoms in chronic patients. The development of an efficacious prophylactic vaccine faces many challenges, and progress is slow, despite several years of effort. Studies in animal models and human patients have revealed the pathogenic mechanisms during disease progression, pathology of disease and features of protective immunity. Accordingly, several antigens, antigen-delivery vehicles and adjuvants have been tested in animal models, and some efforts have been successful in controlling infection and disease. This review will summarize the accumulated knowledge about the parasite and disease, as well as pathogenesis and protective immunity. The authors will discuss the efforts to date, and the challenges faced in achieving an efficient prophylactic vaccine against human American trypanosomiasis, and present the future perspectives.


Subject(s)
Antigens, Protozoan/immunology , Chagas Disease/prevention & control , Protozoan Vaccines , Trypanosoma cruzi/immunology , Vaccination , Animals , Drug Evaluation, Preclinical , Humans , Models, Animal
20.
An. acad. bras. ciênc ; 77(4): 695-715, Dec. 2005. ilus
Article in English | LILACS | ID: lil-418017

ABSTRACT

Há evidências que sugerem que as miocardites chagásicas são devidas aos danos induzidos pelo estresse oxidativo, podendo contribuir para a evolução da doença de Chagas. Em doenças infecciosas, a formação de espécies reativas do oxigênio (ROS) é, principalmente, derivada de danos celulares mediados pela invasão e replicação do patógeno e por reações citotóxicas mediadas pelo sistema imune. No entanto, como as ROS são formadas e sua função no estresse oxidativo na cardiomiopatia chagásica (CCM) não estão completamente elucidadas. Nesta revisão, nós discutimos as evidências para o aumento do estresse oxidativo na doença de Chagas, com ênfase nas anormalidades mitocondriais, na disfunção da cadeia de transporte de elétrons e seu papel na manutenção do estresse oxidativo no miocárdio. Discutimos ainda, os resultados da literatura que relatam as conseqüências da manutenção do estresse oxidativo na patogênese da CCM.


Subject(s)
Animals , Humans , Chagas Cardiomyopathy/etiology , Chagas Cardiomyopathy/physiopathology , Mitochondria, Heart , Oxidative Stress/physiology , Reactive Oxygen Species , Chagas Cardiomyopathy , Chagas Cardiomyopathy/immunology , Electron Transport Chain Complex Proteins/physiology , Trypanosoma cruzi , Trypanosoma cruzi/immunology
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