Simultaneous inhibition of IGF1R and EGFR enhances the efficacy of standard treatment for colorectal cancer by the impairment of DNA repair and the induction of cell death.

Overexpression and activation of receptor tyrosine kinases (RTKs), such as the insulin-like growth factor 1 receptor (IGF1R) and the epidermal growth factor receptor (EGFR), are frequent phenomena in colorectal cancer (CRC). Here, we evaluated the effect and the cellular mechanisms of the simultaneous inhibition of these two RTKs both in vitro and in vivo in addition to a 5-fluoruracil (5-FU)-based radiochemotherapy (RCT), which is a standard treatment scheme for CRC. Using the small molecule inhibitors AEW541 and erlotinib, specific against IGF1R and EGFR, respectively, different CRC cell lines exhibited a reduced survival fraction after RCT, with the highest effect after the simultaneous inhibition of IGF1R/EGFR. In vivo, xenograft mice simultaneously treated with low dose AEW541/erlotinib plus RCT revealed a significant reduction in tumour volume and weight compared with the tumours of mice treated with either AEW541 or erlotinib alone. In vitro, the combined inhibition of IGF1R/EGFR resulted in a stronger reduction of downstream signalling, an increase in DNA double strand breaks (DSBs), apoptosis and mitotic catastrophe after RCT depending on the cell line. Moreover, the existence of IGF1R/EGFR heterodimers in CRC cells and human rectal cancer samples was proven. The heterodimerisation of these RTKs was dependent on the presence of both ligands, IGF-1 and EGF, and functional receptors. In conclusion, these results demonstrate that the strategy of targeting both IGF1R and EGFR, in addition to basic RCT, could be of intriguing importance in CRC therapy.

Prophylactic Transcatheter Arterial Embolization After Successful Endoscopic Hemostasis in the Management of Bleeding Duodenal Ulcer.

GOALS: The aim of this study was to demonstrate the new strategy of prophylactic transcatheter arterial embolization (TAE) of the gastroduodenal artery after endoscopic hemostasis of bleeding duodenal ulcers. BACKGROUND: TAE is a well-established method for the treatment of recurrent or refractory ulcer bleeding resistant to endoscopic intervention, which increasingly replaces surgical procedures. A new approach for improving outcome and reducing rebleeding episodes is the supplemental and prophylactic TAE after successful endoscopic hemostasis. STUDY: This retrospective study included all patients (n=117) treated from 2008 to 2012 for duodenal ulcer bleeding. After initial endoscopic hemostasis, patients were assessed regarding their individual rebleeding risk. Patients with a low rebleeding risk (n=47) were conservatively treated, patients with a high risk for rebleeding (n=55) had prophylactic TAE of the gastroduodenal artery, and patients with endoscopically refractory ulcer bleeding received immediate TAE. RESULTS: The technical success of prophylactic TAE was 98% and the clinical success was 87% of cases. Rebleeding occurred in 11% of patients with prophylactic TAE and was successfully treated with repeated TAE or endoscopy. The major complication rate was 4%. Surgery was necessary in only 1 prophylactic TAE patient (0.9%) during the whole study period. Mortality associated with ulcer bleeding was 4% in patients with prophylactic TAE. CONCLUSIONS: Prophylactic TAE in patients with duodenal ulcers at high risk for rebleeding was feasible, effective at preventing the need for surgery, and had low major complication rates. Given these promising outcomes, prophylactic TAE should be further evaluated as a preventative therapy in high-risk patients.

Blockade of the PDGFR family together with SRC leads to diminished proliferation of colorectal cancer cells.

Among the family of receptor tyrosine kinases (RTKs), platelet-derived growth factor receptor (PDGFR) has attracted increasing attention as a potential target of anti-tumor therapy in colorectal cancer (CRC). To study the function of PDGFRß in CRC cell lines, SW480, DLD-1 and Caco-2 cells showing high PDGFRß expression were used for receptor down-regulation by small interfering RNA (siRNA) and using the pharmacological inhibitor of PDGFRß Ki11502. Blockade of PDGFRß using both approaches led to moderate inhibition of proliferation and diminished activation of the downstream PI3K-signaling pathway in all three cell lines. Surprisingly, incubation with Ki11502 resulted in an arrest of SW480 cells in the G2 phase of the cell cycle, whereas the siRNA approach did not result in this effect. To address this difference, we analyzed the involvement of the PDGFRß family member c-KIT in Ki11502 effectiveness, but siRNA and proliferation studies in SW480 and DLD-1 cells could not prove the involvement of c-KIT inactivation during Ki11502 treatment. Hence, an RTK activation antibody array on SW480 cells led us to the identification of the non-receptor tyrosine kinase SRC, which is inactivated after Ki11502 treatment but not after the siRNA approach. Further studies using the SRC-specific inhibitor PP2 showed that SRC inhibition upon treatment with the inhibitor Ki11502 is responsible for the observed effects of Ki11502 in SW480 and DLD-1 CRC cells. In summary, our results demonstrate that the inhibition of PDGFRß alone using siRNA has only moderate cellular effects in CRC cell lines; however, the multi-target inhibition of PDGFRß, c-KIT and SRC, e.g., using Ki11502, represents a promising therapeutic intervention for the treatment of CRC.

High chromosomal instability in adenocarcinoma of the ileum arising from multifocal gastric heterotopia with gastritis cystica profunda.

Adenocarcinoma of the small intestine arising from heterotopic gastric mucosa is extremely rare. In this report, we present the case of a 68-year-old woman who complained of abdominal pain, weight loss and subileus. Gross examination of resected small bowel revealed multiple flat polypous lesions with cysts in the ileal submucosa, one of which containing an ulcerated, stenosing tumour. On microscopic examination, an adenocarcinoma of the ileum arising from multifocal gastric heterotopia with secondary gastritis cystica profunda was diagnosed. Comparative genomic hybridization of the adenocarcinoma revealed chromosomal gains at 1q, 3q, 5p, 8q, 11p, 12p, 13q and losses at Xp, 4q, 8p, 10p, 14q, 17p, 20p, compatible with a high degree of genomic instability.

A gene expression signature for chemoradiosensitivity of colorectal cancer cells.

PURPOSE: The standard treatment of patients with locally advanced rectal cancers comprises preoperative 5-fluorouracil-based chemoradiotherapy followed by standardized surgery. However, tumor response to multimodal treatment has varied greatly, ranging from complete resistance to complete pathologic regression. The prediction of the response is, therefore, an important clinical need. METHODS AND MATERIALS: To establish in vitro models for studying the molecular basis of this heterogeneous tumor response, we exposed 12 colorectal cancer cell lines to 3 µM of 5-fluorouracil and 2 Gy of radiation. The differences in treatment sensitivity were then correlated with the pretherapeutic gene expression profiles of these cell lines. RESULTS: We observed a heterogeneous response, with surviving fractions ranging from 0.28 to 0.81, closely recapitulating clinical reality. Using a linear model analysis, we identified 4,796 features whose expression levels correlated significantly with the sensitivity to chemoradiotherapy (Q <.05), including many genes involved in the mitogen-activated protein kinase signaling pathway or cell cycle genes. These data have suggested a potential relevance of the insulin and Wnt signaling pathways for treatment response, and we identified STAT3, RASSF1, DOK3, and ERBB2 as potential therapeutic targets. The microarray measurements were independently validated for a subset of these genes using real-time polymerase chain reactions. CONCLUSION: We are the first to report a gene expression signature for the in vitro chemoradiosensitivity of colorectal cancer cells. We anticipate that this analysis will unveil molecular biomarkers predictive of the response of rectal cancers to chemoradiotherapy and enable the identification of genes that could serve as targets to sensitize a priori resistant primary tumors.

Response of the primary tumor in symptomatic and asymptomatic stage IV colorectal cancer to combined interventional endoscopy and palliative chemotherapy.

BACKGROUND: The treatment of the primary tumor in advanced metastatic colorectal cancer (CRC) is still a matter of discussion. Little attention has thus far been paid to the endoscopically observable changes of the primary in non-curatively resectable stage IV disease. METHODS: 20 patients [14 men, 6 women, median age 67 (39-82) years] were observed after initial diagnosis of non-curatively resectable metastasized symptomatic (83%) or asymptomatic (17%) CRC, from June 2002 to April 2009. If necessary, endoscopic tumor debulking was performed. 5-FU based chemotherapy was given immediately thereafter. In 10 patients, chemotherapy was combined with antibody therapy. RESULTS: Response of the primary was observed in all patients. Local symptoms were treated endoscopically whenever necessary (obstruction or bleeding), and further improved after chemotherapy was started: Four patients showed initial complete endoscopic disappearance of the primary. In an additional 6 patients, only adenomatous tissue was histologically detected. In both these groups, two patients revealed local tumor relapse after interruption of therapy. Local tumor regression or stable disease was achieved in the remaining 10 patients. 15 patients died during the observation time. In 13 cases, death was related to metastatic disease progression. The mean overall survival time was 19.6 (3-71) months. No complications due to the primary were observed. CONCLUSION: This study shows that modern anti-cancer drugs combined with endoscopic therapy are an effective and safe treatment of the symptomatic primary and ameliorate local complaints without the need for surgical intervention in advanced UICC stage IV CRC.

Dual silencing of insulin-like growth factor-I receptor and epidermal growth factor receptor in colorectal cancer cells is associated with decreased proliferation and enhanced apoptosis.

Overexpression and activation of tyrosine kinase receptors are common features of colorectal cancer. Using the human colorectal cancer cell lines DLD-1 and Caco-2, we evaluated the role of the insulin-like growth factor-I (IGF-I) receptor (IGF-IR) and epidermal growth factor receptor (EGFR) in cellular functions of these cells. We used the small interfering RNA (siRNA) technology to specifically down-regulate IGF-IR and EGFR expression. Knockdown of IGF-IR and EGFR resulted in inhibition of cell proliferation of DLD-1 and Caco-2 cells. An increased rate of apoptosis was associated with siRNA-mediated silencing of IGF-IR and EGFR as assessed by activation of caspase-3/caspase-7. The combined knockdown of both EGFR and IGF-IR decreased cell proliferation and induced cell apoptosis more effectively than did silencing of either receptor alone. Comparable effects on cell proliferation and apoptosis were observed after single and combinational treatment of cells by the IGF-IR tyrosine kinase inhibitor NVP-AEW541 and/or the EGFR tyrosine kinase inhibitor erlotinib. Combined IGF-IR and EGFR silencing by either siRNAs or tyrosine kinase inhibitors diminished the phosphorylation of downstream signaling pathways AKT and extracellular signal-regulated kinase (ERK)-1/2 more effectively than did the single receptor knockdown. Single IGF-IR knockdown inhibited IGF-I-dependent phosphorylation of AKT but had no effect on IGF-I- or EGF-dependent phosphorylation of ERK1/2, indicating a role of EGFR in ligand-dependent ERK1/2 phosphorylation. The present data show that inhibition of the IGF-IR transduction cascade augments the antipoliferative and proapoptotic effects of EGFR inhibition in colorectal cancer cells. A clinical application of combination therapy targeting both EGFR and IGF-IR could be a promising therapeutic strategy.

Functional analysis of NKX3.1 in LNCaP prostate cancer cells by RNA interference.

The function of the androgen-regulated homeobox protein NKX3.1 in prostate cancer is controversial. NKX3.1 is necessary for correct prostate development and undergoes frequent allelic loss in prostate cancer. However, no mutations occur in the coding region and some particularly aggressive cancers over-express the protein. Nevertheless NKX3.1 is often referred to as candidate tumor suppressor gene. Recent findings suggest a function in protection against oxidative damage involved in prostate carcinogenesis. Thus NKX3.1 may act differently at various stages of prostate cancer. Unlike a classical tumor suppressor NKX3.1 is up-regulated by androgens and down-regulated by phytoestrogens. In this study we performed RNAi based functional analysis by knocking down NKX3.1 expression in LNCaP prostate cancer cells and analyzing the impact of NKX3.1 on gene expression and cell proliferation. Knock-down of NKX3.1 evoked a massive down-regulation of NKX3.1 expression, followed by reduction in mRNA expression of the androdrogen receptor (AR) and the insulin-like growth factor receptor (IGF-1R). Western blot analysis showed strong decreases of NKX3.1, AR, and IGF-1R protein expression. Concomitantly, cell proliferation decreased and expression of prostate-specific antigen (PSA) mRNA and its secretion were diminished, whereas expression of IGF binding protein 3 (IGFBP-3) and MMP tissue inhibitor 3 (TIMP-3) was up-regulated. In tumor cells not deprived of NKX3.1 expression this gene still has a function which might differ from its role in prostate development and carcinogenesis. NKX3.1 knock-down altered the expression of genes highly relevant in prostate cancer cell proliferation and apoptosis. In LNCaP NKX3.1 most probably plays the role of an androgen-regulated transcription factor whose down-regulation is paralleled by anti-proliferative and pro-apoptotic effects. Since NKX3.1 can regulate AR expression it may become a target for interference in hormone refractory prostate carcinoma.

Implantation of a colorectal stent as a therapeutic approach in the treatment of esophageal leakage.

BACKGROUND: While the mortality of esophageal surgery has decreased due to technological advancements, there is still a complication rate of about 30%. One of the main complications is the anastomotic leakage associated with a significant rate of morbidity and mortality. To close the leakage the efficacy of self-expanding stents (SES) has been shown in different studies. However, the high rate of stent migration limits the use of commercial available stents. In our case we were faced with the problem that the diameter of all available stents was too small to attach tightly to the mucosal wall of the esophagogastric anastomosis. CASE PRESENTATION: We used, for the first time to our knowledge, a metal stent designed for colorectal application in an extensive anastomotic leak after esophageal resection in a patient with an esophageal cancer. After primary surgery with subtotal esohagectomy the anastomotic leak was stented endoscopically with a Polyflex self-expanding covered plastic stent after no response to intensive conventional management. Even though the stent was placed correctly, the diameter of the Polyflex stent was too small to attach onto the wall of the esophagogastric anastomosis. Again surgery was performed with a thoracal resection of the esophageal remnant and a hand made anastomosis. Unfortunately, again an anastomotic leak was detected soon after. To close the leak we decided to use a covered colorectal stent (Hanarostent) with an inner diameter of 30 mm. Sixteen weeks later the stent was extracted and complete mucosal healing of the esophageal leak was observed. CONCLUSION: The stent implantation with a large wide diameter offers a good chance to close more extensive leaks and prevent stent migration.

Alteration of the insulin-like growth factor axis during in vitro differentiation of the human osteosarcoma cell line HOS 58.

The insulin-like growth factors I and II (IGF-I, IGF-II), their receptors, and high affinity binding proteins (IGFBPs) represent a family of cellular modulators that play essential roles in the development and differentiation of cells and tissues including the skeleton. Recently, the human osteosarcoma cell line HOS 58 cells were used as an in vitro model of osteoblast differentiation characterized by (i) a rapid proliferation rate in low-density cells that decreased continuously with time of culture and (ii) an increasing secretion of matrix proteins during their in vitro differentiation. In the present paper, HOS 58 cells with low cell density at early time points of the in vitro differentiation (i) displayed a low expression of IGF-I and -II; (ii) synthesized low levels of IGFBP-2, -3, -4, and -5, but (iii) showed high expression levels of both the type I and II IGF receptors. During the in vitro differentiation of HOS 58 cells, IGF-I and -II expressions increased continuously in parallel with an upregulation of IGFBP-2, -3, -4, and -5 whereas the IGF-I receptor and IGF-II/M6P receptor mRNA were downregulated. In conclusion, the high proliferative activity in low cell density HOS 58 cells was associated with high mRNA levels of the IGF-IR, but low concentrations of IGFBP-2. The rate of proliferation of HOS 58 cells continuously decreased during cultivation in parallel with a decline in IGF-IR expression, but increase of mitoinhibitory IGFBP-2. These data are indicative for a role of the IGF axis during the in vitro differentiation of HOS 58 cells.

Crosstalk between PDGF and IGF-I receptors in rat liver myofibroblasts: implication for liver fibrogenesis.

Insulin-like growth factor I (IGF-I) and platelet-derived growth factor (PDGF) have been identified as significant mitogens for liver myofibroblasts (LMFs), one of the cell populations playing a role in liver fibrogenesis. In the present work, we aimed to elucidate a possible interaction between PDGF receptor (PDGFR) and IGF-I receptor (IGF-IR) signaling in LMFs. Among different rat liver cells, PDGFR alpha- and beta-subunits were mainly expressed in hepatic stellate cells and LMFs, and were upregulated during their in vitro cultivation. In LMFs, PDGF-BB (10 ng/ml) stimulated DNA synthesis approximately two-fold and this effect was similar to that of IGF-I. IGF-I and PDGF-BB differentially affected IGF-IR and PDGFR signaling. High concentrations of IGF-I decreased levels of IGF-IR and IRS-1 and inhibited the expression and activation of PDGFRalpha. PDGF-BB prevented IGF-I-induced downregulation of the IGF-IR, but did not affect expression of its cognate receptor subunits. Transphosphorylation of PDGFR and IGF-IR was not observed. PDGF effectively activated terminal MAP kinases, PI3 kinase and Akt kinase, whereas IGF-I demonstrated weaker effects. PLCgamma(1) was phosphorylated only in response to PDGF, but not to IGF-I. In rat LMFs, blockade of the IGF-IR via inhibition of the IGF-IR kinase completely abrogated IGF- and PDGF-induced mitogenesis and the ability of PDGF to phosphorylate PLCgamma(1). In conclusion, the presented data demonstrate that the PDGFR signaling requires a functional IGF-IR and that PDGF-BB stabilizes the IGF-IR function through preventing the IGF-I-induced downregulation of the IGF-IR. These interactions might be relevant in vivo for the fibroproliferative response during liver injury.

Oxygen-dependent modulation of insulin-like growth factor binding protein biosynthesis in primary cultures of rat hepatocytes.

Higher levels of IGF-binding protein 1 (IGFBP-1) mRNA are expressed in the less aerobic perivenous zone of the liver. Because gradients in oxygen tension (pO(2)) may contribute to zonated gene expression, the influence of arterial and venous pO(2) on IGFBP-1 biosynthesis was studied in primary cultures of rat hepatocytes. Maximal IGFBP-1 mRNA and protein levels were observed under venous pO(2), whereas less than 30% of maximal levels were observed under arterial pO(2). In contrast, the expression of IGFBP-4 was greatest under arterial pO(2), indicating that this effect of hypoxia on IGFBP-1 gene expression is specific. The response to hypoxia appears to involve reactive oxygen species, because treatment with H(2)O(2) results in a dose-dependent decrease of IGFBP-1 mRNA levels under venous pO(2), whereas IGFBP-1 mRNA expression under arterial pO(2) was not affected. Inhibition of the hypoxia-dependent IGFBP-1 mRNA induction by actinomycin D indicates that this effect is mediated at the level of gene transcription, and inhibition of IGFBP-1 mRNA by the iron chelator desferrioxamine under both venous and arterial pO(2) suggested the involvement of hypoxia-inducible transcription factors (HIF). Transfection experiments demonstrated that especially HIF-3alpha and HIF-2alpha, and to a lesser extent HIF-1alpha, contribute to the induction of IGFBP-1 mRNA expression in isolated hepatocytes, whereas experiments with vectors for the HIF prolyl hydroxylases (PHD) indicated a major role of PHD-2 in destabilization of HIFs, attenuating the induction of IGFBP-1 under venous pO(2). Reporter gene studies indicate that hypoxia stimulates IGFBP-1 expression through a putative HIF response element located approximately 250 bp upstream from the transcription initiation site. Together, these results support the concept that iron, radical oxygen species, and the HIF-2 and -3 as well as the PHD pathways play important roles in mediating effects of hypoxia on IGFBP-1 gene expression in the liver.

Tectorigenin and other phytochemicals extracted from leopard lily Belamcanda chinensis affect new and established targets for therapies in prostate cancer.

Isoflavones have been shown to exert antiproliferative effects on cancer cells by steroid receptor signaling. In this study, we demonstrate the potential of plant constituents extracted from Belamcanda chinensis as anticancer drugs, which regulate the aberrant expression of genes relevant in proliferation, invasion, immortalization and apoptosis. LNCaP cells were treated with B.chinensis extract, tectorigenin or other isoflavones and mRNA expression was quantified by using real time RT-PCR. In addition, ELISA, TRAP assays and western blots were used to measure protein expression or activity. Male nude mice (n=18) were injected subcutaneously with LNCaP cells and were fed with extracts from B.chinensis, and tumor development was monitored versus a control animal group (n=18). Tectorigenin and several other phytochemicals downregulated PDEF, PSA and IGF-1 receptor mRNA expression in vitro. Furthermore, PSA secretion and IGF-1 receptor protein expression were diminished, and hTERT mRNA expression and telomerase activity decreased after tectorigenin treatments. However, TIMP-3 mRNA was upregulated on tectorigenin treatment. Growth of subcutaneous tumors in nude mice was delayed and diminished in animals fed with extracts from B.chinensis. The downregulation of PDEF, PSA, hTERT and IGF-1 receptor gene expression by tectorigenin demonstrates the antiproliferative potential of these agents. The upregulation of TIMP-3 gene expression indicates a pro-apoptotic function of the drug and a reduction of the invasiveness of tumors. The animal experiments demonstrate that B.chinensis markedly inhibited the development of tumors in vivo. Thus, these compounds may be useful for the prevention or treatment of human prostate cancer.

Colorectal cancer in two pre-teenage siblings with familial adenomatous polyposis.

UNLABELLED: Familial adenomatous polyposis (FAP) is an autosomal dominant disorder that characteristically presents with colon cancer in early adult life. We describe a Pakistani FAP family in which two sons had an unusually early manifestation of colorectal cancer. The index patient presented at 11 years of age with abdominal pain, rectal bleeding and iron deficiency anaemia. Colonoscopy showed that the colon was carpeted with a myriad of polyps. Oesophago-gastric and duodenal endoscopy revealed that polyps had also developed in the duodenum. Multiple biopsies indicated neoplastic lesions. The patient underwent a proctocolectomy and endoscopic duodenal mucosectomy. The diagnosis of an adenocarcinoma of the colon and further adenomatous polyps with low-grade and high-grade dysplasia was confirmed by histology. Family screening including a blood test for anaemia and bowel examination revealed that his 12-year-old brother was also affected. CONCLUSION: Children with familial adenomatous polyposis are at risk for colon cancer and emphasise the need for early tumour recognition. Gastrointestinal symptoms in children should be thoroughly evaluated and standard screening for colonic polyposis should be performed in all individuals with a positive family history and/or known mutations in cancer-associated genes, particularly in children who are under 10 years of age.

Interferon type I gene expression in chronic hepatitis C.

Hepatitis C virus (HCV) frequently causes chronic liver disease. The cause of viral persistence might be an inappropriate type I interferon (IFN) induction. To analyze the host's IFN response in chronic hepatitis C, we measured the transcription level of type I IFN genes as well as type I IFN-regulated genes in liver tissue and corresponding blood samples from patients with chronic hepatitis C, nonviral liver diseases, and a suspected but later excluded liver disease. Competitive and real-time RT-PCR assays were used to quantify the messenger RNA (mRNA) levels of all known IFN-alpha, IFN-beta, and IFN-lambda genes and those of some IFN-regulated genes. We failed to detect any hepatic type I IFN mRNA induction, although liver tissue of chronic hepatitis C patients contained high numbers of some type I IFN-inducible effector mRNA molecules. Analysis of peripheral blood samples, however, showed a clear type I IFN induction. Parallel experiments employing HCV replicon cell lines revealed that replication of HCV RNA is not sufficient to induce any type I IFN nor to induce directly type I IFN-regulated genes such as MxA. In conclusion, our data provide evidence for the absence of an induction of type I IFN genes by HCV in the human liver and argue for a further development of type I IFN-based therapies.

Expression and regulation of the insulin-like growth factor axis components in rat liver myofibroblasts.

UNLABELLED: Apart from hepatic stellate cells (HSC), liver myofibroblasts (MF) represent a second mesenchymal cell population involved in hepatic fibrogenesis. The IGF system including the insulin-like growth factors I and II (IGF-I, -II), their receptors (IGF-I receptor, IGF-IR; IGF-II/mannose 6-phosphate receptor, IGF-II/M6-PR), and six high affinity IGF binding proteins (IGFBPs) participate in the regulation of growth and differentiation of cells of the fibroblast lineage, possibly contributing to the fibrogenic process. The aim of this work was to study the expression and regulation of the IGF axis components in rat liver MF. METHODS: Cultures of MF from passages 1 to 4 (P1-4) were studied. IGFBP secretion was analyzed by [(125)I]-IGF-I ligand and immunoblotting. IGF-I, IGF-IR, IGF-II/M6-PR, and IGFBP messenger RNA (mRNA) expression was assessed by Northern blot hybridization. DNA synthesis was evaluated by 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. RESULTS: MF from P1 to 4 constitutively expressed mRNA transcripts specific for IGF-I, IGF-IR, and IGF-II/M6-PR. In MF, biosynthesis of IGFBP-3 and -2 was observed that was stimulated by IGF-I, insulin, and transforming growth factor beta (TGF-beta), whereas platelet-derived growth factor (PDGF-BB) revealed inhibitory effects. IGF-I and to a lesser extent insulin increased DNA synthesis of MF. Simultaneous addition of recombinant human IGFBP-2 or -3 with IGF-I diminished the mitogenic effect of IGF-I on MF whereas preincubation of MF with IGFBP-2 or -3 further potentiated the IGF-I stimulated DNA synthesis. In conclusion, the present study demonstrates that the IGF axis may play a role in the regulation of MF proliferation in vitro which might be relevant in vivo for the process of fibrogenesis during acute and chronic liver injury.

Insulin-like growth factor (IGF)-binding protein-1 is highly induced during acute carbon tetrachloride liver injury and potentiates the IGF-I-stimulated activation of rat hepatic stellate cells.

Hepatic stellate cells (HSC) play a pivotal role in hepatic tissue repair and fibrogenesis. IGF-I has been considered a mitogenic signal for activation and proliferation of HSC in vitro. In the present study IGF-I and IGF-binding protein (IGFBP) gene expression was studied in a model of acute liver injury induced by a single intragastric dose of carbon tetrachloride (CCl(4)) in adult rats. Northern blot analysis revealed a marked increase in IGFBP-1 mRNA levels, with a maximum between 3 and 9 h after CCl(4) application, whereas steady state mRNA levels of IGF-I were only moderately altered. In situ hybridization experiments demonstrated that this increase in IGFBP-1 mRNA was due to a strong expression of IGFBP-1 in the perivenous region 6-12 h after CCl(4) application, extending to the midzonal region of the acinus within 24-48 h. Consequently, a prominent immunostaining for IGFBP-1 was observed in perivenous areas, with a maximum 24-48 h after intoxication. Preincubation of early cultured HSC with a nonphosphorylated IGFBP-1 from human amniotic fluid resulted in a 3.4-fold increase in IGF-I-induced DNA synthesis. The mitogenic effect of IGF-I was also potentiated when HSC were cocultivated with IGFBP-1-overexpressing BHK-21 cells compared with nontransfected cells. These data suggest that IGFBP-1 released during the early steps of liver tissue damage and repair may interact with HSC and potentiate the sensitivity of IGF-I to mitogenic signals.

Isolation and characterization of circulating fragments of the insulin-like growth factor binding protein-3.

Proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3), the major carrier of IGFs in the circulation, is an essential mechanism to regulate IGF bioavailability. To analyze naturally occurring IGFBP-3 fragments a peptide library established from human hemofiltrate was screened. Three IGFBP-3 fragments were detected with apparent molecular masses of 34, 16, and 11 kDa. Mass spectrometric and sequence analysis identified the 16 and 11 kDa peptides as glycosylated and non-glycosylated N-terminal fragments spanning residues Gly1-Ala98 of IGFBP-3. Both the circulating forms and those secreted from IGFBP-3(1-98) overexpressing cells bound IGF. Additionally, two smaller fragments (IGFBP-3(139-157) and IGFBP-3(139-159)) were identified in the hemofiltrate. The data indicate that proteolysis of circulating IGFBP-3 occurs in the variable domain at residues alanine 98, phenylalanine 138, glutamine 157, and tyrosine 159.

Decreased intracellular degradation of insulin-like growth factor binding protein-3 in cathepsin L-deficient fibroblasts.

Proteolysis of insulin-like growth factor binding proteins (IGFBPs) is the major mechanism of releasing IGFs from their IGFBP complexes. Analysis of fibroblasts deficient for the lysosomal cysteine protease cathepsin L (CTSL) revealed an accumulation of IGFBP-3 in the medium which was due neither to alterations in IGFBP-3 mRNA expression nor to extracellular IGFBP-3 protease activity. Incubation of CTSL-deficient fibroblasts with radiolabeled IGFBP-3 followed by subcellular fractionation indicates that both intact and fragmented IGFBP-3 accumulate transiently in endosomal and lysosomal fractions of CTSL-deficient cells. This suggests the involvement of CTSL in the intracellular degradation of IGFBP-3 representing a new mechanism to regulate the extracellular concentration of IGFBP-3.