Brain and spinal cord trauma:what we know about the therapeutic potential of insulin growth factor 1 gene therapy

Although little attention has been paid to cognitive and emotional dysfunctions observed in patients after spinal co rd injury,several reports have described impairments in cognitive abilities.Our group also has contributed significantly to the study of cognitive impairments in a rat model of spinal co rd injury.These findings are very significant because they demonstrate that cognitive and mood deficits are not induced by lifestyle changes,drugs of abuse,and combined medication.They are related to changes in brain structures involved in cognition and emotion,such as the hippocampus.Chronic spinal cord injury decreases neurogenesis,enhances glial reactivity leading to hippocampal neuroinflammation,and trigge rs cognitive deficits.These brain distal abnormalities are recently called te rtiary damage.Given that there is no treatment for Tertiary Damage,insulin growth factor 1 gene therapy emerges as a good candidate.Insulin growth factor 1 gene thera py recove rs neurogenesis and induces the polarization from pro-inflammato ry towards anti-inflammatory microglial phenotypes,which represents a potential strategy to treat the neuroinflammation that supports te rtiary damage.Insulin growth factor 1 gene therapy can be extended to other central nervous system pathologies such as traumatic brain injury where the neuroinflammatory component is crucial.Insulin growth factor 1 gene therapy could emerge as a new therapeutic strategy for treating traumatic brain injury and spinal cord injury.

Overexpression of insulin-like growth factor-Ⅰ receptor as a pertinent biomarker for hepatocytes malignant transformation

AIM:To investigate the dynamic features of insulinlike growth factor-Ⅰreceptor(IGF-ⅠR)expression in rat hepatocarcinogenesis,and the relationship between IGF-ⅠR and hepatocytes malignant transformation at mRNA or protein level.METHODS:Hepatoma models were made by inducing with 2-fluorenylacetamide(2-FAA)on male SpragueDawley rats.Morphological changes of hepatocytes were observed by pathological Hematoxylin and eosin staining,the dynamic expressions of liver and serum IGF-ⅠR were quantitatively analyzed by an enzymelinked immunosorbent assay.The distribution of hepatic IGF-ⅠR was located by immunohistochemistry.The fragments of IGF-ⅠR gene were amplified by reverse transcription-polymerase chain reaction,and confirmed by sequencing.RESULTS:Rat hepatocytes after induced by 2-FAA were changed dynamically from granule-like degeneration,precancerous to hepatoma formation with the progressing increasing of hepatic mRNA or IGF-ⅠR expression.The incidences of liver IGF-ⅠR,IGF-ⅠR mRNA,specific IGF-ⅠR concentration(ng/mg wet liver),and serum IGF-ⅠR level(ng/mL)were 0.0%,0.0%,0.63±0.17,and 1.33±0.47 in the control;50.0%,61.1%,0.65±0.2,and 1.51±0.46 in the degeneration;88.9%,100%,0.66±0.14,and 1.92±0.29 in the precancerosis;and 100%,100%,0.96±0.09,and2.43±0.57 in the cancerous group,respectively.IGF-ⅠR expression in the cancerous group was significantly higher(P<0.01)than that in any of other groups at mRNA or protein level.The closely positive IGF-ⅠR relationship was found between livers and sera(r=0.91,t=14.222,P<0.01),respectively.CONCLUSION:IGF-ⅠR expression may participate in rat hepatocarcinogenesis and its abnormality should be an early marker for hepatocytes malignant transformation.

A candidate targeting molecule of insulin-like growth factor-Ⅰ receptor for gastrointestinal cancers

Advances in molecular research in cancer have brought new therapeutic strategies into clinical usage.One new group of targets is tyrosine kinase receptors,which can be treated by several strategies,including small molecule tyrosine kinase inhibitors(TKIs) and monoclonal antibodies(mAbs).Aberrant activation of growth factors/receptors and their signal pathways are required for malignant transformation and progression in gastrointestinal(GI) carcinomas.The concept of targeting specif ic carcinogenic receptors has been validated by successful clinical application of many new drugs.Type I insulin-like growth factor(IGF) receptor(IGF-IR) signaling potently stimulates tumor progression and cellular differentiation,and is a promising new molecular target in human malignancies.In this review,we focus on this promising therapeutic target,IGF-IR.The IGF/IGF-IR axis is an important modifier of tumor cell proliferation,survival,growth,and treatment sensitivity in many malignant diseases,including human GI cancers.Preclinical studies demonstrated that downregulation of IGF-IR signals reversed the neoplastic phenotype and sensitized cells to anticancer treatments.These results were mainly obtained through our strategy of adenoviruses expressing dominant negative IGF-IR(IGF-IR/dn) against gastrointestinal cancers,including esophagus,stomach,colon,and pancreas.We also summarize a variety of strategies to interrupt the IGFs/IGF-IR axis and their preclinical experiences.Several mAbs and TKIs targeting IGF-IR have entered clinical trials,and early results have suggested that these agents have generally acceptable safety profiles as single agents.We summarize the advantages and disadvantages of each strategy and discuss the merits/demerits of dual targeting of IGF-IR and other growth factor receptors,including Her2 and the insulin receptor,as well as other alternatives and possible drug combinations.Thus,IGF-IR might be a candidate for a molecular therapeutic target in human GI carcinomas.

Expression of insulin-like growth factor Ⅱ and its receptor in liver cells of chronic liver diseases

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Insulin-like growth factor-I receptor in proliferation and motility of pancreatic cancer

AIM:To develop a molecular therapy for pancreatic cancer, the insulin-like growth factor-I (IGF-I) signaling pathway was analyzed.METHODS: Pancreatic cancer cell lines (MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4) were cultured in media with 10 mL/L fetal bovine serum. Western blotting analysis was performed to clarify the expression of IGF-I receptor (IGF-IR). Picropodophyllin (PPP), a specific inhibitor of IGF-IR, LY294002, a specific inhibitor of phosphatidylinositol3 kinase (PI3K), and PD98059, a specific inhibitor of mitogen-activated protein kinase, were added to the media. After 72 h, a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay was performed to analyze cell proliferation. A wound assay was performed to analyze cell motility with hematoxylin and eosin (HE) staining 48 h after addition of each inhibitor. RESULTS: All cell lines clearly expressed not only IGF-IR but also phosphorylated IGF-IR. PPP significantly suppressed proliferation of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 36.9% ± 2.4% (mean ± SD), 30.9% ± 5.5%, 23.8% ± 3.9%, 37.1% ± 5.3%, 10.4% ± 4.5%, 52.5% ± 4.5% and 22.6% ± 0.4%, at 2 μmol/L, respectively (P < 0.05). LY294002 significantly suppressed proliferation of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 44.4% ± 7.6%, 32.9% ± 8.2%, 53.9% ± 8.0%, 52.8% ± 4.0%, 32.3% ± 4.2%, 51.8% ± 4.5%, and 30.6% ± 9.4%, at 50 μmol/L, respectively (P < 0.05). PD98059 did not significantly suppress cell proliferation. PPP at 2 μmol/L suppressed motility of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 3.0% ± 0.2%, 0%, 0%, 2.0% ± 0.1%, 5.0% ± 0.2%, 3.0% ± 0.1%, and 5.0% ± 0.2%, respectively (P < 0.05). LY294002 at 50 μmol/L suppressed motility of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 to 3.0% ± 0.2%, 0%, 3.0% ± 0.2%, 0%, 0%, 0% and 3% ± 0.1%, respectively (P < 0.05). PD980509 at 20 μmol/L did not suppress motility. Cells were observed by microscopy to analyze the morphological changes induced by the inhibitors. Cells in medium treated with 2 μmol/L PPP or 50 μmol/L LY294002 had pyknotic nuclei, whereas those in medium with 20 μmol/L PD98059 did not show apoptosis.CONCLUSION: IGF-IR and PI3K are good candidates for molecular therapy of pancreatic cancer.

Targeting the insulin-like growth factor pathway in hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is the third leading cause of cancer-related deaths worldwide. Only 30%-40% of the patients with HCC are eligible for curative treatments, which include surgical resection as the first option, liver transplantation and percutaneous ablation. Unfortunately, there is a high frequency of tumor recurrence after surgical resection and most HCC seem resistant to conventional chemotherapy and radiotherapy. Sorafenib, a multi-tyrosine kinase inhibitor, is the only chemotherapeutic option for patients with advanced hepatocellular carcinoma. Patients treated with Sorafenib have a significant increase in overall survival of about three months. Therefore, there is an urgent need to develop alternative treatments. Due to its role in cell growth and development, the insulin-like growth factor system is commonly deregulated in many cancers. Indeed, the insulin-like growth factor(IGF) axis has recently emerged as a potential target for hepatocellular carcinoma treatment. To this aim, several inhibitors of the pathway have been developed suchas monoclonal antibodies, small molecules, antisense oligonucleotides or small interfering RNAs. However recent studies suggest that, unlike most tumors, HCC development requires increased signaling through insulin growth factor Ⅱ rather than insulin growth factor Ⅰ. This may have great implications in the future treatment of HCC. This review summarizes the role of the IGF axis in liver carcinogenesis and the current status of the strategies designed to target the IGF-Ⅰ signaling pathway for hepatocellular carcinoma treatment.

Reactivation of the insulin-like growth factor-Ⅱsignaling pathway in human hepatocellular carcinoma

Constitutive activation of the insulin-like growth factor (IGF)-signaling axis is frequently observed in human hepatocellular carcinoma(HCC).Especially the over- expression of the fetal growth factor IGF-Ⅱ,IGF-Ⅰ receptor(IGF-IR),and cytoplasmic downstream effectors such as insulin-receptor substrates(IRS)contribute to proliferation,anti-apoptosis,and invasive behavior. This review focuses on the relevant alterations in this signaling pathway and independent in vivo models that support the central role IGF-Ⅱsignaling during HCC development and progression.Since this pathway has become the center of interest as a target for potential anti-cancer therapy in many types of malignancies,various experimental strategies have been developed,including neutralizing antibodies and selective receptor kinase inhibitors,with respect to the specific and efficient reduction of oncogenic IGF-Ⅱ/IGF-IR-signaling.

Modified insulin-like growth factor 1 containing collagen-binding domain for nerve regeneration

Insulin-like growth factor 1 （IGF-I） is a potential nutrient for nerve repair. However, it is impractical as a therapy because of its limited half- life, rapid clearance, and limited target specificity. To achieve targeted and long-lasting treatment, we investigated the addition of a binding structure by fusing a collagen-binding domain to IGF- 1. After confirming its affinity for collagen, the biological activity of this construct was examined by measuring cell proliferation after transfection into PC12 and Schwann cells using a 3-（4,5-dimethyl-2-thiazolyl）-2,5-di- phenyl-2-H-tetrazolium bromide assay. Immunofluorescence staining was conducted to detect neurofilament and microtubule-associated protein 2 expression, while real time-polymerase chain reaction was utilized to determine IGF-1 receptor and nerve growth/actor mRNA expression. Our results demonstrate a significant increase in collagen-binding activity of the recombinant protein compared with IGF-1. Moreover, the recombinant protein promoted proliferation of PC12 and Schwann cells, and increased the expression of neurofilament and microtubule-associated protein 2. Importantly, the recombinant protein also stimulated sustained expression of IGF-1 receptor and nerve growth factor mRNA for days. These results show that the recombinant protein achieved the goal of targeting and long-lasting treatment, and thus could become a clinically used factor for promoting nerve regeneration with a prolonged therapeutic effect.

Insulin-like Growth Factor-Ⅰ Gene Cloning and Protein Expression in Bovine Trabecular Meshwork Tissue and Cells

Whether cultured bovine trabecular meshwork cells and trabecular tissue ex vivo express insulin like growth factor I (IGF Ⅰ) messenger RNA (mRNA) and protein was investigated. Total RNA of cultured bovine trabecular meshwork cells as well as trabecular meshwork tissue freshly excised from bovine eyes was extracted, and reverse transcriptase polymerase chain reaction (RT PCR) was used to detect IGF Ⅰ mRNA. RT PCR product was verified by sequencing. Immunohistochemical stain was used to detect IGF Ⅰ protein. The results showed that a single PCR amplified product was obtained, and the sequence was homologous to the known sequence.. IGF Ⅰ immunostain was positive in the cytoplasm of trabecular meshwork cells. It was concluded that trabecular meshwork cells produce IGF Ⅰ and contribute to the presence of IGF Ⅰ in trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by IGF Ⅰ not only through paracrine, but also autocrine action. Whether abnormal down regulations in IGF Ⅰ production may contribute to the pathogenesis of primary open angle glaucoma and the possibility of promoting the autocrine action of IGF Ⅰ by trabecular meshwork cells to treat the diesease is worth further investigation.

Increased hepatic expression of insulin-like growth factor-Ⅰreceptor in chronic hepatitis C

瞄准：尽管增加像胰岛素的生长因素 -- 我受体(IGF 红外) 基因表示在肝细胞癌被报导了，在长期的丙肝(CHC ) 估计 IGF 红外的研究和肝硬化是少见的。我们因此试图与 CHC 从病人在肝评估 IGF 红外和 IGF-I mRNA 表示。方法：IGF 红外和 IGF-I mRNA 内容被半量的 RT-PCR 决定， IGF 红外蛋白质表示由在以前与 CHC 从病人获得的肝的织物(34 个病人) 并且在以后的 immunohisto 化学是坚定的(10 个病人) 有 interferon-alpha 和 ribavirin 的治疗。结果：IGF 红外 mRNA 内容的增加在从所有 CHC 病人以及从与正常的肝相比跟随 orthopic 移植(评估正常的肝的一次尝试) 的 6 个尸体肝施主获得的肝的织物被观察，当没有相关修正在 IGF-I mRNA 内容被检测时。组织化学的结果显示出的免疫在 IGF 红外 mRNA 的加薪满足，这与 ductular 反应并且到在 hepatocytes 的增加的 IGF 红外表示相关。在 IGF 红外 mRNA 内容的减少在在治疗以后完成了持续 virological 反应的病人被观察，建议处于肝的损坏的改进。结论：在有 CHC 的病人的 hepatocytes 的 IGF 红外表示的起来规定能组成一次尝试刺激 hepatocyte 新生。考虑到那个肝是有 IGF-I 的高水平的机关，我们在尖锐、长期的肝的损坏以后发现增加的 IGF 红外表示加亮需要让另外的研究阐明在肝的 IGF-I 的角色新生。

Insulin-like growth factor-1 induces lymphangiogenesis and facilitates lymphatic metastasis in colorectal cancer

AIM:To investigate the expression of insulin-like growth factor-1(IGF-1)/insulin-like growth factor-1 receptor(IGF-1R)in colorectal cancer(CRC)tissues and to analyze their correlation with lymphangiogenesis and lymphatic metastasis.METHODS:Immunohistochemistry was used to evaluate IGF-1 and IGF-1R expression and lymphatic vessel density(LVD)in 40 CRC specimens.The correlation between IGF-1/IGF-1R and LVD was investigated.Effects of IGF-1 on migration and invasion of CRC cells were examined using transwell chamber assays.A LoVo cell xenograft model was established to further detect the role of IGF-1 in CRC lymphangiogenesis in vivo. RESULTS:Elevated IGF-1 and IGF-1R expression in CRC tissues was correlated with lymph node metastasis(r=0.715 and 0.569,respectively,P<0.05)and tumor TNM stage(r=0.731 and 0.609,P<0.05).A higher LVD was also found in CRC tissues and was correlated with lymphatic metastasis(r=0.405,P<0.05).A positive correlation was found between LVD and IGF-1R expression(r=0.437,P<0.05).Transwell assays revealed that IGF-1 increased the migration and invasion of CRC cells.In vivo mouse studies showed that IGF-1 also increased LVD in LoVo cell xenografts.CONCLUSION:IGF-1/IGF-1R signaling induces tumorassociated lymphangiogenesis and contributes to lymphatic metastasis of CRC.

Integrating insulin-like growth factor 1 and sex hormones into neuroprotection:Implications for diabetes

Brain integrity and cognitive aptitude are often impaired in patients with diabetes mellitus, presumably a result of the metabolic complications inherent to the disease. However, an increasing body of evidence has demonstrated the central role of insulin-like growth factor 1(IGF1) and its relation to sex hormones in many neuroprotective processes. Both male and female patients with diabetes display abnormal IGF1 and sexhormone levels but the comparison of these fluctuations is seldom a topic of interest. It is interesting to note that both IGF1 and sex hormones have the ability to regulate phosphoinositide 3-kinase-Akt and mitogen-activated protein kinases-extracellular signal-related kinasesignaling cascades in animal and cell culture models of neuroprotection. Additionally, there is considerable evidence demonstrating the neuroprotective coupling of IGF1 and estrogen. Androgens have also been implicated in many neuroprotective processes that operate on similar signaling cascades as the estrogen-IGF1 relation. Yet, androgens have not been directly linked to the brain IGF1 system and neuroprotection. Despite the sex-specific variations in brain integrity and hormone levels observed in diabetic patients, the IGF1-sex hormone relation in neuroprotection has yet to be fully substantiated in experimental models of diabetes. Taken together, there is a clear need for the comprehensive analysis of sex differences on brain integrity of diabetic patients and the relationship between IGF1 and sex hormones that may influence brain-health outcomes. As such, this review will briefly outline the basic relation of diabetes and IGF1 and its role in neuroprotection. We will also consider the findings on sex hormones and diabetes as a basis for separately analyzing males and females to identify possible hormone-induced brain abnormalities. Finally, we will introduce the neuroprotective interplay of IGF1 and estrogen and how androgen-derived neuroprotection operates through similar signaling cascades. Future research on both neuroprotection and diabetes should include androgens into the interplay of IGF1 and sex hormones.

Improvement in erectile dysfunction after insulin-like growth factor-1 gene therapy in diabetic rats

Aim： To determine whether adenoviral gene transfer of insulin like growth factor-1 （IGF-1） to the penis of streptozotocin （STZ）-induced diabetic rats could improve erectile capacity. Methods： The STZ diabetic rats were transfected with AdCMV-βgal or AdCMV-IGF-1. These rats underwent cavernous nerve stimulation to assess erectile function and their responses were compared with those of age-matched control rats 1 to 2 days after transfection. In control and transfected STZ diabetic rats, IGF-1 expression were examined by reverse transcription polymerase chain reaction （RT-PCR）, Western blot and histology. The penis β-galactosidase activity and localization of the STZ diabetic rats were also determined. Results： One to two days after transfection, the β-galactosidase was found in the smooth muscle cells of the diabetic rat penis transfected with AdCMV-βgal. One to 2 days after administration of AdCMV- IGF-1, the cavernosal pressure, as determined by the ratio of maximal intracavernous pressure-to-mean arterial pressure （ICP/MAP） and total intracavernous pressure （ICP）, was increased in response to cavernous nerve stimulation. Transgene expression was confirmed by RT-PCR, Western blot and histology. Conclusion： Gene transfer of IGF-1 significantly increased erectile function in the STZ diabetic rats. These results suggest that in vivo gene transfer of IGF- 1 might be a new therapeutic intervention for the treatment of erectile dysfunction （ED） in the STZ diabetic rats.

Effect of endogenous insulin-like growth factor and stem cell factor on diabetic colonic dysmotility

AIM: To investigate whether the reduction of stem cell factor (SCF) is mediated by decreased endogenous insulin-like growth factor (IGF)-1 in diabetic rat colon smooth muscle. METHODS: Sixteen Sprague-Dawley rats were randomly divided into two groups: control group and streptozotocin-induced diabetic group. After 8 wk of streptozotocin administration, colonic motility function and contractility of circular muscle strips were measured. The expression of endogenous IGF-1 and SCF was tested in colonic tissues. Colonic smooth muscle cells were cultured from normal adult rats. IGF-1 siRNA transfection was used to investigate whether SCF expression was affected by endogenous IGF-1 expression in smooth muscle cells, and IGF-1 induced SCF expression effects were studied. The effect of high glucose on the expression of endogenous IGF-1 and SCF was also investigated. RESULTS: Diabetic rats showed prolonged colonic transit time (252 ± 16 min vs 168 ± 9 min, P < 0.01) and weakness of circular muscle contraction (0.81 ± 0.09 g vs 2.48 ± 0.23 g, P < 0.01) compared with the control group. Endogenous IGF-1 and SCF protein expression was significantly reduced in the diabetic colonic muscle tissues. IGF-1 and SCF mRNA expression also showed a paralleled reduction in diabetic rats. In the IGF-1 siRNA transfected smooth muscle cells, SCF mRNA and protein expression was significantly decreased. IGF-1 could induce SCF expression in a concentration and time-dependent manner, mainly through the extracellular-signal-regulated kinase 1/2 signal pathway. High glucose inhibited endogenous IGF-1 and SCF expression and the addition of IGF-1 to the medium reversed the SCF expression. CONCLUSION: Myopathy may resolve in colonic motility dysfunction in diabetic rats. Deficiency of endogenous IGF-1 in colonic smooth muscle cells leads to reduction of SCF expression.

Epigenetic regulation of insulin-like growth factor axis in hepatocellular carcinoma

The insulin-like growth factor(IGF) signaling path-way is an important pathway in the process of hepa-tocarcinogenesis,and the IGF network is clearly dysregulated in many cancers and developmental abnormalities.In hepatocellular carcinoma(HCC),only a minority of patients are eligible for curative treatments,such as tumor resection or liver transplant.Unfortunately,there is a high recurrence of HCC after surgical tumor removal.Recent research efforts have focused on targeting IGF axis members in an attempt to find therapeutic options for many health problems.In this review,we shed lights on the regulation of members of the IGF axis,mainly by micro RNAs in HCC.Micro RNAs in HCC attempt to halt the aberrant expression of the IGF network,and a single micro RNA can have multiple downstream targets in one or more signaling pathways.Targeting micro RNAs is a relatively new approach for identifying an efficient radical cure for HCC.

Hepatogenic differentiation of mesenchymal stem cells induced by insulin like growth factor-Ⅰ

AIM:To improve hepatic differentiation of human mesenchymal stem cell(MSC)using insulin growth factor 1(IGF-Ⅰ),which has important role in liver development,hepatocyte differentiation and function.METHODS:Bone marrow of healthy donors was aspirated from the iliac crest.The adherent cells expanded rapidly and were maintained with periodic passages until a relatively homogeneous population was established.The identification of these cells was carried out by immunophenotype analysis and differentiation potential into osteocytes and adipocytes.To effectively induce hepatic differentiation,we designed a protocol based on a combination of IGF-Ⅰ and liver specificfactors(hepatocyte growth factor,oncostatin M and dexamethasone).Morphological features,hepatic functions and cytological staining were assessed to evaluate transdifferentiation of human marrow-derived MSCs.RESULTS:Flow cytometric analysis and the differentiation potential into osteoblasts and adipocytes showed that more than 90% of human MSCs which were isolated and expanded were positive by specif ic markers and functional tests.Morphological assessment and evaluation of glycogen storage,albumin and α-feto protein expression,as well as albumin and urea secretion revealed a statistically signif icant difference between the experimental groups and control.CONCLUSION:In vitro differentiated MSCs using IGF-Ⅰwere able to display advanced liver metabolic functions,supporting the possibility of developing them as potential alternatives to primary hepatocytes.

Insulin-like growth factor-1, IGF binding protein-3, and the risk of esophageal cancer in a nested case-control study

AIM To assess the relationship between serum levels of insulin-like growth factor-1(IGF1)/IGF-binding protein-3(IGFBP3)and the risk of esophageal carcinoma.METHODS We assessed the relationship between the serum levels of these molecules and the risk of esophageal cancer in a prospective,nested case-control study of participants from the Japan Collaborative Cohort Study.A baseline survey was conducted from 1988 to 1990.Of the 110585 enrolled participants,35%donated blood samples.Those who had been diagnosed with esophageal cancer were considered cases for nested case-control studies.A conditional logistic model was used to estimate odds ratios for the incidence of esophageal cancer associated with serum IGF1 and IGFBP3 levels.RESULTS Thirty-one cases and 86 controls were eligible for the present assessment.The molar ratio of IGF1/IGFBP3,which represents the free and active form of IGF1,was not correlated with the risk of esophageal carcinoma.A higher molar difference between IGFBP3and IGF1,which estimates the free form of IGFBP3,was associated with a decreased risk of esophageal carcinoma(P=0.0146),and people in the highest tertile had the lowest risk(OR=0.107,95%CI:0.017-0.669).After adjustment for body mass index,tobacco use,and alcohol intake,the molar difference of IGFBP3-IGF1 was inversely correlated with the risk of esophageal carcinoma(P=0.0150).CONCLUSION The free form of IGFBP3,which is estimated by this molar difference,may be inversely associated with esophageal cancer incidence.

Interplay between micro RNA-17-5p, insulin-like growth factor-Ⅱ through binding protein-3 in hepatocellular carcinoma

AIM: To investigate the effect of microR NA on insulinlike growth factor binding protein-3(IGFBP-3) and hence on insulin-like growth factor-Ⅱ(IGF-Ⅱ) bioavailability in hepatocellular carcinoma(HCC).METHODS: Bioinformatic analysis was performed using microrna.org, DIANA lab and Segal lab softwares. Total RNA was extracted from 23 HCC and 10 healthy liver tissues using mir Vana mi RNA Isolation Kit. microR NA-17-5p(miR-17-5p) expression was mimicked and antagonized in Hu H-7 cell lines using Hi Per Fect Transfection Reagent, then total RNA was extracted using Biozol reagent then reverse transcribed into cD NA followed by quantification of mi R-17-5p and IGFBP-3 expression using Taq Man real-time quantitative PCR. Luciferase reporter assay was performed to validate the binding of miR-17-5p to the 3'UTR of IGFBP-3. Free IGF-Ⅱ protein was measured in transfected Hu H-7 cells using IGF-Ⅱ ELISA kit. RESULTS: Bioinformatic analysis revealed IGFBP-3 as a potential target for miR-17-5p. Screening of miR-17-5p and IGFBP-3 revealed a moderate negative correlation in HCC patients, where mi R-17-5p was extensively underexpressed in HCC tissues(P = 0.0012), while IGFBP-3 showed significant upregulation in the same set of patients(P = 0.0041) compared to healthy donors. Forcing mi R-17-5p expression in Hu H-7 cell lines showed a significant downregulation of IGFBP-3 mR NA expression(P = 0.0267) and a significant increase in free IGF-Ⅱ protein(P = 0.0339) compared to mock untransfected cells using unpaired t-test. Luciferase assay validated IGFBP-3 as a direct target of mi R-17-5p; luciferase activity was inhibited by 27.5% in cells co-transfected with miR-17-5p mimics and the construct harboring the wild-type binding region 2 of IGFBP-3 compared to cells transfected with this construct alone(P = 0.0474).CONCLUSION: These data suggest that regulating IGF-Ⅱ bioavailability and hence HCC progression can be achieved through targeting IGFBP-3 via manipulating the expression of miR NAs.

Mitochondrial protection by low doses of insulin-like growth factor-Ⅰin experimental cirrhosis

AIM: To characterize the mitochondrial dysfunction in experimental cirrhosis and to study whether insulin-like growth factor-Ⅰ(IGF-Ⅰ) therapy (4 wk) is able to induce beneficial effects on damaged mitochondria leading to cellular protection. METHODS: Wistar rats were divided into three groups: Control group, untreated cirrhotic rats and cirrhotic rats treated with IGF-Ⅰtreatment (2 μg/100 g bw/d). Mitochondrial function was analyzed by flow cytometry in isolated hepatic mitochondria, caspase 3 activation was assessed by Western blot and apoptosis by TUNEL in the three experimental groups. RESULTS: Untreated cirrhotic rats showed a mitochon- drial dysfunction characterized by a significant reduction of mitochondrial membrane potential (in status 4 and 3); an increase of intramitochondrial reactive oxigen species (ROS) generation and a significant reduction of ATPase activity. IGF-Ⅰtherapy normalized mitochondrial func-tion by increasing the membrane potential and ATPase activity and reducing the intramitochondrial free radical production. Activity of the electron transport complexes Ⅰand Ⅲ was increased in both cirrhotic groups. In addition, untreated cirrhotic rats showed an increase of caspase 3 activation and apoptosis. IGF-Ⅰtherapy reduced the expression of the active peptide of caspase 3 and resulted in reduced apoptosis.

Liver insulin-like growth factor 2 methylation in hepatitis C virus cirrhosis and further occurrence of hepatocellular carcinoma

AIM: To assess the predictive value of the insulinlike growth factor 2 (Igf2) methylation profile for the occurrence of Hepatocellular Carcinoma (HCC) in hepatitis C (HCV) cirrhosis. METHODS: Patients with: (1) biopsy-proven compensated HCV cirrhosis; (2) available baseline frozen liver sample; (3) absence of detectable HCC; (4) regular screening for HCC; (5) informed consent for genetic analysis were studied. After DNA extraction from liver samples and bisulfite treatment, unbiased PCR and DHPLC analysis were performed for methylation analysis at the Igf2 locus. The predictive value of the Igf2 methylation profile for HCC wasassessed by Kaplan-Meier and Cox methods. RESULTS: Among 94 included patients, 20 developed an HCC during follow-up (6.9 ± 3.2 years). The methylation profile was hypomethylated, intermediate and hypermethylated in 13, 64 and 17 cases, respectively. In univariate analysis, two baseline parameters were associated with the occurrence of HCC: age (P = 0.01) and prothrombin (P = 0.04). The test of linear tendency between the three ordered levels of Igf2 methylation and probability of HCC occurrence was significant (Log Rank, P = 0.043; Breslow, P = 0.037; Tarone-Ware, P = 0.039). CONCLUSION: These results suggest that hypomethylation at the Igf2 locus in the liver could be predictive for HCC occurrence in HCV cirrhosis.