ABSTRACT
In multiple myeloma, the presence of highly resistant cancer stem cells (CSC) that are responsible for tumor metastasis and relapse has been proven. Evidently, for achieving complete response, new therapeutic paradigms that effectively eradicate both, CSCs and bulk cancer populations, need to be developed. For achieving that goal, an innovative two-step treatment combining targeting of thymidine de novo synthesis pathway and a nanoirradiation by the Auger electron emitting thymidine analogue (123/125)I-5-iodo-4'-thio-2'-deoxyuridine ((123/125)I-ITdU) could be a promising approach. The pretreatment with thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine (FdUrd, 1 µmol/L for 1 hour) efficiently induced proliferation and terminal differentiation of isolated myeloma stem-like cells. Moreover, FdUrd stimulation led to a decreased activity of a functional CSC marker, aldehyde dehydrogenase (ALDH). The metabolic conditioning by FdUrd emerged to be essential for enhanced incorporation of (125)I-ITdU (incubation with 50 kBq/2 × 10(4) cells for 4 days) and, consequently, for the induction of irreparable DNA damage. (125)I-ITdU showed a pronounced antimyeloma effect on isolated tumor stem-like cells. More than 85% of the treated cells were apoptotic, despite activation of DNA repair mechanisms. Most important, exposure of metabolically conditioned cells to (125)I-ITdU resulted in a complete inhibition of clonogenic recovery. This is the first report showing that pretreatment with FdUrd sensitizes the stem-like cell compartment in multiple myeloma to apoptosis induced by (125)I-ITdU-mediated nanoirradiation of DNA.
Subject(s)
Multiple Myeloma/drug therapy , Neoplasm Recurrence, Local/drug therapy , Neoplastic Stem Cells/drug effects , Thymidylate Synthase/genetics , Apoptosis/drug effects , Cell Line, Tumor , Cell Lineage/drug effects , DNA Damage/drug effects , Deoxyuridine/administration & dosage , Deoxyuridine/analogs & derivatives , Enzyme Inhibitors/administration & dosage , Humans , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Neoplasm Recurrence, Local/pathology , Thymidylate Synthase/antagonists & inhibitorsABSTRACT
BACKGROUND: Recently, we identified hepatocytes as the major cellular source of profibrogenic connective tissue growth factor (CTGF/CCN2) in the liver. Based on reports of a hepatoprotective effect of coffee consumption, we were the first to provide evidence that caffeine suppresses transforming growth factor (TGF)-beta dependent and -independent CTGF expression in hepatocytes in vitro and in vivo, thus suggesting this xanthine-alkaloid as a potential therapeutic agent. AIM: This study aims at comparing the inhibitory capacities of caffeine and its three demethylated derivates paraxanthine, theophylline and theobromine on CTGF expression in hepatocytes and hepatic stellate cells (HSC). RESULTS: Our data suggest paraxanthine as the most important pharmacological repressor of hepatocellular CTGF expression among the caffeine-derived metabolic methylxanthines with an inhibitory dosage (ID)50 of 1.15 mM, i.e. 3.84-fold lower than what is observed for caffeine. In addition, paraxanthine displayed the least cell toxicity as proven by the water-soluble tetrazolium-1 cell vitality assay. However, caffeine or any of the metabolites did not inhibit CTGF expression in HSC. At the toxicological threshold concentration of 1 mM for paraxanthine, we observed an inhibition of hepatocellular CTGF synthesis by 44%, which was strongly reverted in the presence of the specific competitive cyclic adenosine monophosphate inhibitor Rp-adenosine 3',5-cyclic monophosphorothioate triethylammonium salt. Furthermore, CTGF protein expression induced by various concentrations of TGF-beta (0.13-1 ng/ml) is still reduced by, on average, 27%/45% in the presence of paraxanthine (1.25 mM/2.5 mM). CONCLUSION: Our data provide an evidence-based suggestion of the caffeine-derived primary metabolite paraxanthine as a potentially powerful antifibrotic drug by its inhibitory effect on (hepatocellular) CTGF synthesis.
Subject(s)
Caffeine/chemistry , Connective Tissue Growth Factor/metabolism , Gene Expression Regulation/drug effects , Hepatocytes/metabolism , Theophylline/pharmacology , Animals , Blotting, Western , Cells, Cultured , DNA Primers/genetics , Electrophoresis, Polyacrylamide Gel , Inhibitory Concentration 50 , Male , Molecular Structure , Rats , Rats, Sprague-Dawley , Regression Analysis , Reverse Transcriptase Polymerase Chain Reaction , Theobromine/pharmacology , Theophylline/analysisABSTRACT
BACKGROUND AND AIM: Next to its role as a carrier protein for vitamin D and its plasma metabolites, the primary function of vitamin D-binding protein (DBP; Gc-globulin) is to serve and to bind and neutralize extracellular monomeric actin (G-actin) released from necrotic cells, which in its long filamentous form (F-actin) triggers coagulation. We, therefore, investigated the kinetics of serum concentrations of actin-free DBP during the pathogenetic sequence from liver fibrosis to hepatocellular carcinoma (HCC) to initiate a discussion on a hypothetical association of serum concentrations of actin-free DBP and, thus, altered actin clearance in the circulation, and an increased risk of thrombosis in patients with functional liver insufficiency. RESULTS: Our data show that serum levels of actin-free DBP are decreased in patients with liver fibrosis (n=288) and HCC (n=102) compared with healthy controls (n=120 and n=63) and nonliver disease sick (n=312) and that the level of decrease correlates with the severity of disease as determined according to the score by the French METAVIR Cooperative Study Group staging system for hepatic fibrosis or the Edmondson-Steiner's grading system for the assessment of HCC. CONCLUSION: Although further, in particular, longitudinal studies are still necessary to back up our data, the presented findings suggest that decreasing levels of the actin-scavenger DBP could potentially contribute to the thrombophilic predisposition frequently observed in patients with chronic fibrogenic liver disease and HCC.
Subject(s)
Carcinoma, Hepatocellular/pathology , Liver Cirrhosis/pathology , Liver Neoplasms/pathology , Vitamin D-Binding Protein/blood , Adolescent , Adult , Aged , Aged, 80 and over , Biomarkers/blood , Carcinoma, Hepatocellular/blood , Female , Germany , Humans , Liver Cirrhosis/blood , Liver Neoplasms/blood , Male , Middle Aged , Treatment Outcome , Vitamin D-Binding Protein/biosynthesis , Young AdultSubject(s)
Caffeine/pharmacology , Connective Tissue Growth Factor/metabolism , Galactosamine/pharmacology , Liver/metabolism , Phosphodiesterase Inhibitors/pharmacology , Animals , Caffeine/administration & dosage , Cyclic AMP/metabolism , Galactosamine/administration & dosage , Injections, Intraperitoneal , Liver/drug effects , Phosphodiesterase Inhibitors/administration & dosage , Rats , Rats, Sprague-DawleyABSTRACT
UNLABELLED: In vivo knockdown of connective tissue growth factor (CTGF/CCN2) was recently shown to attenuate the formation of experimental liver fibrosis. The secreted, cysteine-rich growth factor is proposed to adversely modulate the binding of profibrogenic transforming growth factor beta (TGF-beta) and its natural antagonist bone morphogenetic protein (BMP) to their cognate receptors in several cellular systems, but the functionality of CTGF in modulation of the TGF-beta/BMP signaling pathways is still unknown. This study aims at characterizing a potentially differential modulating role of CTGF on TGF-beta- and BMP7-dependent transactivation of reporter gene [Ad-(CAGA)(12)-MLP-luc, Ad-hCTGF-luc, and Ad-(BRE)(2)-luc reporter gene] expression in rat hepatocytes. In this context, emphasis is also placed on the differential roles of Smad2 and Smad3 in the TGF-beta-dependent transactivation of the endogenous CTGF gene and the CTGF gene reporter, as investigated following adenoviral infection of wild-type and dominant negative Smad2/3 or treatment with the specific inhibitor of Smad3 or ALK5-specific (SB-431542) inhibitor. In this analysis, we found (1) a selective transcriptional activation of the CTGF promoter by Smad2 (but not Smad3); (2) the failure of BMP7 to inhibit the transcriptional activation of the Smad3-selective (CAGA)(12)-luc reporter by TGF-beta, as well as the failure of TGF-beta to inhibit the transcriptional activation of the Smad5-selective (BRE)(2)-luc reporter by BMP7; and (3) the sensitization of hepatocytes toward TGF-beta type I receptor (ALK5)/Smad2 and Smad3-mediated TGF-beta signaling by CTGF, whereas BMP type I receptor (ALK1)/Smad5-mediated BMP7 signaling is not modulated. CONCLUSION: CTGF acts as a Smad2-dependent sensitizer of TGF-beta actions that does not influence BMP7 signaling in hepatocytes.
Subject(s)
Bone Morphogenetic Protein 7/physiology , Connective Tissue Growth Factor/physiology , Hepatocytes/physiology , Smad2 Protein/physiology , Transforming Growth Factor beta/metabolism , Animals , Cells, Cultured , Male , Rats , Rats, Sprague-Dawley , Signal Transduction/physiologyABSTRACT
BACKGROUND/AIMS: Current knowledge confers a crucial role to connective tissue growth factor (CTGF/CCN2) in hepatic fibrogenesis. Hepatocytes are likely to be the major cellular source of CTGF in the liver in which CTGF is sensitively upregulated by TGF-beta. Recently, we demonstrated that the methylxanthine derivate caffeine leads to an upregulation of peroxisome proliferator activated receptor gamma (PPARgamma) expression in hepatocytes, thus sensitizing these cells to the well-known inhibitory effect of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15-d-PGJ(2)) on CTGF expression. However, upregulation of the receptor alone is not sufficient per se; its physiological ligand 15-d-PGJ(2) is required to exert an inhibitory effect on transforming growth factor-beta (TGF-beta) target genes such as CTGF. METHODS: This study compared serum concentrations of 15-d-PGJ(2) in Caucasian patients with fibrotic liver diseases (n=289), Caucasian controls (n=136) and Caucasian non-liver disease (NLD) sick (n=307), as well as of Chinese patients with hepatocellular carcinoma (HCC) (n=43) and Chinese healthy controls (n=63) in order to characterize their suitability for therapeutic approaches with PPARgamma-inducing (i.e. CTGF inhibitory) drugs such as caffeine. RESULTS: The presented data showed that Caucasian patients with ongoing hepatic fibrogenesis (mean 6.2+/-5.9 microg/L) displayed strikingly higher serum concentrations of 15-d-PGJ(2) than healthy probands (mean 2.3+/-1.0) and Caucasian patients with NLD (mean 2.7+/-1.4 microg/L). Similar results were found in Chinese patients with fully developed HCC (mean 1.3+/-0.7 microg/L) compared with Chinese healthy controls (mean 0.4+/-0.2 microg/L). CONCLUSIONS: In conclusion, our data thus proposed an increased suitability of these patient groups for therapeutic approaches with drugs inducing PPARgamma expression, such as methylxanthine derivates.
Subject(s)
Connective Tissue Growth Factor/antagonists & inhibitors , Liver Cirrhosis/blood , Liver Cirrhosis/metabolism , PPAR gamma/metabolism , Prostaglandin D2/analogs & derivatives , Adult , Aged , Asian People , Caffeine/pharmacology , Female , Hepatocytes/metabolism , Humans , Liver Cirrhosis/pathology , Male , Middle Aged , Prostaglandin D2/blood , Prostaglandin D2/metabolism , Up-Regulation/drug effects , White PeopleABSTRACT
BACKGROUND/AIMS: Epidemiological studies suggest that coffee drinking is inversely correlated with the risk of development of liver fibrosis but the molecular basis is unknown. METHODS: We investigated the pharmacological mechanisms involved in caffeine-dependent regulation of CTGF expression, an important modulator protein of fibrogenic TGF-beta, in rat hepatocytes using Western-blot, co-immunoprecipitations, reporter-gene-assays and ELISAs. RESULTS: It is demonstrated that caffeine, similar to 8-Br-cAMP, suppresses CTGF expression, decreases SMAD2 protein levels and inhibits SMAD1/3-phosphorylation. The SMAD2 level can be restored by a proteasome inhibitor. Additionally, caffeine leads to an up-regulation of PPARgamma expression, that enhances the inhibitory effect of the natural PPARgamma agonist 15-PGJ(2) on CTGF expression by inducing a dissociation of the SMAD2/3-CBP/p300-transcriptional complex. CONCLUSIONS: We show that caffeine strongly down-modulates TGF-beta-induced CTGF expression in hepatocytes by stimulation of degradation of the TGF-beta effector SMAD 2, inhibition of SMAD3 phosphorylation and up-regulation of the PPARgamma-receptor. Long-term caffeinization might be an option for anti-fibrotic trials in chronic liver diseases.
Subject(s)
Caffeine/pharmacology , Connective Tissue Growth Factor/metabolism , Hepatocytes/drug effects , Hepatocytes/metabolism , PPAR gamma/metabolism , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Transforming Growth Factor beta/antagonists & inhibitors , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Animals , Base Sequence , Connective Tissue Growth Factor/genetics , DNA Primers/genetics , Gene Expression/drug effects , In Vitro Techniques , Male , PPAR gamma/genetics , Prostaglandin D2/analogs & derivatives , Prostaglandin D2/pharmacology , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Transforming Growth Factor beta/pharmacologyABSTRACT
BACKGROUND/AIMS: Up to now, the effect of activin A on the expression of the important transforming growth factor (TGF)-beta downstream modulator connective tissue growth factor (CTGF) is not known, but might be of relevance for the functional effects of this cytokine on several liver cell types. METHODS: In this study, activin A-dependent CTGF expression in hepatocytes (PC) primed by exogenous activin A and in PC maintained under complete activin-free culture conditions was analysed by Western blots, metabolic labelling, gene silencing, reverse transcriptase-polymerase chain reaction (RT-PCR) and CTGF reporter gene assays. This study was supplemented by immunocytochemical staining of activin A and CTGF in PC of injured liver. RESULTS: Using alkaline phosphatase alpha-alkaline phosphatase staining, it is demonstrated that activin A becomes increasingly detectable during the course of CCl(4)-liver damage. Addition of activin A to cultured PC induced CTGF protein expression via phosphorylation of Smad2 and Smad3. This induction can be inhibited by the antagonist follistatin and alpha-activin A antibody respectively. When PC were cultured under serum(i.e. activin A)-free culture conditions, a time-dependent increase of activin expression during the course of the culture was proven by RT-PCR. Silencing of inhibin beta(A) gene expression under serum-free conditions by small interfering RNAs greatly suppressed CTGF synthesis and the phosphorylations of Smad2 and Smad3. However, both the extracellularly acting follistatin and the alpha-activin A antibody could not inhibit spontaneous CTGF expression, which, however, was achieved by the cell-permeable TGF-beta Alk4/Alk5 receptor-kinase-inhibitor SB431542. CONCLUSIONS: In conclusion, the results point to activin A as an inducer of CTGF synthesis in PC. Intracellular activin A contributes to spontaneous CTGF expression in PC independent of exogenous activin A, which is proposed to occur via Alk4/Alk5-receptors. The findings might be important for many actions of activin A on the liver.
Subject(s)
Activins/metabolism , Connective Tissue Growth Factor/metabolism , Hepatocytes/metabolism , Paracrine Communication , Animals , Autocrine Communication , Carbon Tetrachloride/toxicity , Cells, Cultured , Chemical and Drug Induced Liver Injury/metabolism , Immunohistochemistry , Male , Rats , Rats, Sprague-Dawley , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Up-RegulationABSTRACT
Recently, synthesis and secretion of connective tissue growth factor (CTGF)/CYR61/CTGF/NOV-family member 2 (CCN2) in cultures of hepatocytes were shown, which are sensitively up-regulated by exogenous TGF-beta. In this study TGF-beta-dependent CTGF/CCN2 expression in hepatocytes cultured under completely TGF-beta-free conditions was analysed by Western-blots, metabolic labelling, and CTGF-reporter gene assays. In alkaline phosphatase monoclonal anti-alkaline phosphatase complex (APAAP)-staining of cultured hepatocytes it was demonstrated that latent TGF-beta within the hepatocytes becomes rapidly detectable during culture indicating an intracellular demasking of the mature TGF-beta antigen. Subsequent signaling to theCTGF/CCN2 promoter occurs via p-Smad2, whereas p-Smad3 does not seem to be involved. Cycloheximide did not abolish the rapid immunocytochemical appearance of mature TGF-beta, but calpain inhibitors partially suppressed intracellular TGF-beta activation and subsequently CTGF up-regulation. Calpain treatment had the reverse effect. None of the inhibitors of extracellular TGF-beta signalling was effective in the reduction of spontaneous CTGF synthesis, but intracellularly acting Alk 4-/Alk 5-specific inhibitor SB-431542 was able to diminish CTGF expression. The assumption that latent intracellular TGF-beta is activated by calpains during culture-induced stress or injurious conditions in the liver in vivo was further validated by a direct effect of calpains on the activation of recombinant latent TGF-beta. In conclusion, these data are the first to suggest the possibility of intracrine TGF-beta signalling due to calpain-dependent intracellular proteolytic activation leading to transcriptional activation of CTGF/CCN2 as a TGF-beta-sensitive reporter gene. This mechanism might be deleterious for keeping long-term hepatocyte cultures due to TGF-beta-induced apoptosis and, further, might be of relevance for induction of apoptosis or epithelial-mesenchymal transition of hepatocytes in injured liver.