Roles of insulin-like growth factor binding protein-3 in gastrointestinal stromal tumors

Imatinib mesylate, a selective inhibitor of KIT, PDGFR, and Abl kinases, has shown significant success as a therapy for patients with advanced gastrointestinal stromal tumors (GISTs). However, the underlying mechanisms of imatinib-induced cytotoxicity are not well understood. Using gene expression profiling and real-time PCR for target validation, we identified insulin-like growth factor binding protein-3 (IGFBP3) to be to be up-regulated after imatinib treatment in imatinib-sensitive GISTs. IGFBP3 is a multifunctional protein that regulates cell proliferation and survival and mediates the effects of a variety of anti-cancer agents through IGF-dependent and IGF-independent mechanisms. Therefore, we hypothesized that IGFBP3 mediates GIST cell response to imatinib. To test this hypothesis, we manipulated IGFBP3 protein levels in two KIT mutant, imatinib-sensitive GIST cell lines and assessed the resultant changes in cell viability, survival, and imatinib sensitivity. In GIST882 cells, endogenous IGFBP3 was required for cell viability. However, inhibiting imatinib-induced IGFBP3 up-regulation by RNA interference or neutralization resulted in reduced drug sensitivity, suggesting that IGFBP3 sensitizes GIST882 cells to imatinib. GIST-T1 cells, on the other hand, had no detectable levels of endogenous IGFBP3, nor did imatinib induce IGFBP3 up-regulation, in contrast to our previous findings. IGFBP3 overexpression in GIST-T1 cells reduced viability but did not induce cell death; rather, the cells became polyploid through a mechanism that may involve attenuated Cdc20 expression and securin degradation. Moreover, IGFBP3 overexpression resulted in a loss of KIT activation and decreased levels of mature KIT. Consistent with this, GIST-T1 cells overexpressing IGFBP3 were less sensitive to imatinib. Furthermore, as neither GIST882 cells nor GIST-T1 cells expressed detectable levels of IGF-1R, IGFBP3 is likely not exerting its effects by modulating IGF signaling through IGF-1R or IR/IGF-1R hybrid receptors in these cell lines. Collectively, these findings demonstrate that IGFBP3 has cell-dependent effects and would, therefore, not be an ideal marker for identifying imatinib response in GISTs. Nevertheless, our results provide preliminary evidence that IGFBP3 may have some therapeutic benefits in GISTs. ^

Is depression associated with insulin resistance? A systematic review

Studies suggest that depression affects glucose metabolism, and therefore is a risk factor for insulin resistance. The association between depression and insulin resistance has been investigated in a number of studies, but there is no agreement on the results. The objective of this study is to survey the epidemiological studies, identify the ones that measured the association of depression (as exposure) with insulin resistance (as outcome), and perform a systematic review to assess the reliability and strength of the association. For high quality reporting, and assessment, this systematic review used the outlined procedures, guidelines and recommendations for reviews in health care, suggested by the Centre for Reviews and Dissemination, along with recommendations from the STROBE group (Strengthening the Reporting of Observational Studies in Epidemiology). Ovid MEDLINE 1996 to April Week 1 2010, was used to identify the relevant epidemiological studies. To identify the most relevant set of articles for this systematic review, a set of inclusion and exclusion criteria were applied. Six studies that met the specific criteria were selected. Key information from identified studies was tabulated, and the methodological quality, internal and external validity, and the strength of the evidence of the selected studies were assessed. The result from the tabulated data of the reviewed studies indicates that the studies either did not apply a case definition for insulin resistance in their investigation, or did not state a specific value for the index used to define insulin resistance. The quality assessment of the reviewed studies indicates that to assess the association between insulin resistance and depression, specifying a case definition for insulin resistance is important. The case definition for insulin resistance is defined by the World Health Organization and the European Group for the Study of Insulin Resistance as the insulin sensitivity index of the lowest quartile or lowest decile of a general population, respectively. Three studies defined the percentile cut-off point for insulin resistance, but did not give the insulin sensitivity index value. In these cases, it is not possible to compare the results. Three other studies did not define the cut-off point for insulin resistance. In these cases, it is hard to confirm the existence of insulin resistance. In conclusion, to convincingly answer our question, future studies need to adopt a clear case definition, define a percentile cut-off point and reference population, and give value of the insulin resistance measure at the specified percentile.^

Macular edema in Hispanics with non-insulin dependent diabetes mellitus in Starr County, Texas: Prevalence, incidence and risk factors

This project is based on secondary analyses of data collected in Starr County, Texas from 1981 till 1991 to determine the prevalence, incidence and risk factors for macular edema in Hispanics with non-insulin-dependent diabetes in Starr County, Texas. Two studies were conducted. The first study examined the prevalence of macular edema in this population. Of the 310 diabetics that were included in the study 22 had macular edema. Of these 22 individuals 9 had clinically significant macular edema. Fasting blood glucose was found to be significantly associated with macular edema. For each 10 mg/dl increase in fasting blood glucose there was a 1.07 probability of an increase in the risk of having macular edema. Individuals with fasting blood glucose $\ge$200 mg/dl were found to be more than three times at risk of having macular edema compared to those with fasting blood glucose $<$200 mg/dl.^ In the second study the incidence and the risk factors that could cause macular edema in this Hispanic population were examined. 240 Hispanics with non-insulin-dependent diabetes mellitus and without macular edema were followed for 1223 person-years. During the follow-up period 27 individuals developed macular edema (2.21/100 person-years). High fasting blood glucose and glycosylated hemoglobin were found to be strong and independent risk factors for macular edema. Participants taking insulin were 3.9 times more at risk of developing macular edema compared to those not taking insulin. Systolic blood pressure was significantly related to macular edema, where each 10 mmHg increase in systolic blood pressure was associated with a 1.3 increase in the risk of macular edema.^ In summary, this study suggests that hyperglycemia is the main underlying factor for retinal pathological changes in this diabetic population, and that macular edema probably is not the result of sudden change in the blood glucose level. It also determined that changes in blood pressure, particularly systolic blood pressure, could trigger the development of macular edema.^ Based on the prevalence reported in this study, it is estimated that 35,500 Hispanic diabetics in the US have macular edema. This imposes a major public health challenge particularly in areas with high concentration of Mexican Americans. It also highlights the importance of public health measures directed to Mexican Americans such as health education, improved access to medical care, and periodic and careful ophthalmologic examination by ophthalmologists knowledgeable and experienced in the management of diabetic macular edema. ^

A GENETIC MARKER STUDY OF NON - INSULIN DEPENDENT DIABETICS IN THE MEXICAN-AMERICAN COMMUNITY FROM STARR COUNTY, TEXAS (PLASMA GLUCOSE, HEMOGLOBIN ANALYSIS)

Diabetes mellitus occurs in two forms, insulin-dependent (IDDM, formerly called juvenile type) and non-insulin dependent (NIDDM, formerly called adult type). Prevalence figures from around the world for NIDDM, show that all societies and all races are affected; although uncommon in some populations (.4%), it is common (10%) or very common (40%) in others (Tables 1 and 2).^ In Mexican-Americans in particular, the prevalence rates (7-10%) are intermediate to those in Caucasians (1-2%) and Amerindians (35%). Information about the distribution of the disease and identification of high risk groups for developing glucose intolerance or its vascular manifestations by the study of genetic markers will help to clarify and solve some of the problems from the public health and the genetic point of view.^ This research was designed to examine two general areas in relation to NIDDM. The first aims to determine the prevalence of polymorphic genetic markers in two groups distinguished by the presence or absence of diabetes and to observe if there are any genetic marker-disease association (univariate analysis using two by two tables and logistic regression to study the individual and joint effects of the different variables). The second deals with the effect of genetic differences on the variation in fasting plasma glucose and percent glycosylated hemoglobin (HbAl) (analysis of Covariance for each marker, using age and sex as covariates).^ The results from the first analysis were not statistically significant at the corrected p value of 0.003 given the number of tests that were performed. From the analysis of covariance of all the markers studied, only Duffy and Phosphoglucomutase were statistically significant but poor predictors, given that the amount they explain in terms of variation in glycosylated hemoglobin is very small.^ Trying to determine the polygenic component of chronic disease is not an easy task. This study confirms the fact that a larger and random or representative sample is needed to be able to detect differences in the prevalence of a marker for association studies and in the genetic contribution to the variation in glucose and glycosylated hemoglobin. The importance that ethnic homogeneity in the groups studied and standardization in the methodology will have on the results has been stressed. ^

DIABETIC RETINOPATHY IN NON-INSULIN-DEPENDENT DIABETES MELLITUS IN BLACK WOMEN: A DESCRIPTIVE STUDY OF RISK FACTORS

This cross-sectional study examines the prevalence of selected potential risk factors by stage of diabetic retinopathy (DR) among Black American women with non-insulin-dependent diabetes mellitus (NIDDM) followed at a university diabetes clinic. DR was assessed by ophthalmoscopy and five-field retinography, and graded on counts of microaneurysms, hemorrhages and/or exudates, and presence of proliferative DR. Prevalence of other vascular diseases was assessed from medical records. Potential risk factors included age, known duration of diabetes, type of hypoglycemic treatment, concentrations of random capillary blood glucose, glycosylated hemoglobin, urine protein and fibrinogen, body mass index, and blood pressure. Prevalence of these risk factors is reported for three categories: No DR, mild background DR, severe background or proliferative DR (including surgically treated DR). Duration, age at diagnosis and treatment of diabetes, concentration of urine protein and average blood glucose, hypertension and cardiovascular disease were significantly associated with DR in univariate analysis. The covariance analysis employed stratification on duration, age at diagnosis and therapy of diabetes. The highest DR scores were calculated for those diagnosed before age 45, regardless of duration, therapy, or average blood glucose. Only individuals diagnosed before age 45 had high blood glucose concentrations in all categories of duration. These findings suggest that in this clinic population of Black women, those diagnosed with NIDDm before age 45 who eventually required insulin treatment were at the greatest risk of developing DR and that longterm poor glucose control is a contributing factor. These results suggest that greater emphasis be placed on this subgroup in allocating the limited resources available to improve the quality of glucose regulation, particularly through measures affecting compliance behavior.^ Findings concerning the association of DR with concentration of blood glucose and urine protein, blood pressure/hypertension and weight were compared with those reported from American Indian and Mexican American populations of the Southwestern United States where prevalence of NIDDM, hypertension and obesity is also high. Additional comparative analyses are outlined to substantiate the preliminary finding that there are systematic differences between these ethnic populations. ^

The Role of Type I Insulin-Like Growth Factor Receptor Signaling in Breast Cancer Brain Metastasis

Brain metastasis is a common cause of mortality in cancer patients. Approximately 20-30% of breast cancer patients acquire brain metastasis, yet potential therapeutic targets remain largely unknown. The type I insulin-like growth factor receptor (IGF- IR) is known to play a role in the progression of breast cancer and is currently being investigated in the clinical setting for various types of cancer. The present study demonstrates that the IGF-IR signaling axis is constitutively active in brain-seeking sublines of breast cancer cells, driving an increase in in vitro metastatic properties. We demonstrate that IGF-IR signaling is activated in an autocrine manner as a result of IGFBP3 overexpression in brain-seeking cells. Transient and stable knockdown of IGF-IR results in a downregulation of IGF-IR downstream signaling through phospho-AKT, as well as decreased in vitro migration and invasion of MDA- MB-231Br brain-seeking cells. Using an in vivo experimental brain metastasis model, we show that IGF-IR ablation attenuates the establishment of brain metastases and prolongs survival. Finally, we demonstrate that the malignancy of brain-seeking cells is attenuated by pharmacological inhibition with picropodophyllin, an IGF-IR-specific tyrosine kinase inhibitor. Together, our data suggest that the IGF-IR is an important mediator of brain metastasis and its ablation delays the onset of brain metastases in our model system.

Characterization of anti -sulfatide autoantibodies in demyelinating diseases

Multiple sclerosis (MS) is the most common autoimmune disease of the central nerve system and Guillain Barré Syndrome (GBS) is an inflammatory neuropathy involving the peripheral nerves. Anti-myelin immunoglobins may play a role in the demyelination processes of the both diseases. Sulfatide is an abundant glycolipid on myelin and is a candidate target antigen for disease related autoantibodies. The objective of this study was to characterize anti-sulfatide antibodies and compare antibodies from GBS and MS patients with fetal antibodies. Our hypothesis is that some B cells producing disease-associated autoantibodies are derived from or related to B cells of the fetal repertoire. Here we report that reactivity of plasma IgM against sulfatide was elevated in twelve MS patients compared with twelve normal subjects. This result implies that anti-sulfatide antibodies are disease-related. A total of sixteen human B lymphocyte clones producing anti-sulfatide autoantibodies were isolated from MS patients, GBS patients and a human fetus. Seven of the clones were from three MS patients, four of the clones were from three GBS patients and five were from the spleen of a twenty-week human fetus. Sequences have been obtained for the heavy and light chain variable regions (VDJ and VJ regions) of all of the anti-sulfatide immunoglobulins. Seven of the sixteen antibodies used VH3 for the variable region gene of the heavy chain consistent with the rate of VH3 usage in randomly selected B cells. Somatic mutations were significantly more frequent in the patient antibodies than in the fetus and somatic mutations in CDR's (Complementarity Determining Region) were significantly more frequent than in framework regions. No significant difference was found between patients and fetus in length of VH CDRIII. However, it is reported that antibodies from randomly selected normal adult B cells have longer CDRIII lengths than those of the fetus (Sanz I, 1991 Journal of Immunology Sep 1;147(5):1720-9). Our results are consistent with derivation of the precursors of B cells producing these autoantibodies from B cells related to those of the fetal repertoire. These findings are consistent with a model in which quiescent B cells from clones produced early in development undergo proliferation in dysregulated disease states, accumulating somatic mutations and increasing in reactivity toward self-antigens. ^ Epitope mapping and molecular modeling were done to elucidate the relationships between antibody structure and binding characteristics. The autoantibodies were tested for binding activity to three different antigens: sulfatide, galactoceramide and ceramide. Molecular modeling suggests that antibodies with positive charge surrounded by or adjacent to hydrophobic groups in the binding pocket bind to the head of sulfatide via the sulfate group through electrostatic interactions. However, the antibodies with hydrophobic groups separated from positive charges appear to bind to the hydrophobic tail of sulfatide. This observation was supported by a study of the effect of NaCl concentration on antigen binding. The result suggested that electrostatic interactions played a major role in sulfate group binding and that hydrophobic interactions were of greater importance for binding to the ceramide group. Our three-dimensional structure data indicated that epitope specificity of these antibodies is more predictable at the level of tertiary than primary structure and suggested positive selection based on structure occurred in the. formation of those autoantibodies. ^

Modulation of replicative senescence of skeletal muscle satellite cells by insulin -like growth factor-I (IGF-I)

Growth and regeneration of postnatal skeletal muscle requires a population of mononuclear myogenic cells, called satellite cells to add/replace myonuclei, which are postmitotic. Wedged between the sarcolemma and the basal lamina of the skeletal muscle fiber, these cells function as the stem cells of mature muscle fibers. Like other normal diploid cells, satellite cells undergo cellular senescence. Investigations of aging in both rodents and humans have shown that satellite cell self-renewal capacity decreases with advanced age. As a consequence, this could be a potential reason for the characteristically observed age-associated loss in skeletal muscle mass (sarcopenia). This provided the rationale that any intervention that can further increase the proliferative capacity of these cells should potentially be able to either delay, or even prevent sarcopenia. ^ Using clonogenicity assays to determine a cell's proliferation potential, these studies have shown that IGF-I enhances the doubling potential of satellite cells from aged rodents. Using a transgenic model, where the mice express the IGF-I transgene specifically in their striated muscles, some of the underlying biochemical mechanisms for the observed increase in replicative life span were delineated. These studies have revealed that IGF-I activates the PI3/Akt pathway to mediate downregulation of p27KIP1, which consequently is associated with an increase in cyclin E-cdk2 kinase activity, phosphorylation of pRb, and upregulation of cyclin A protein. However, the beneficial effects of IGF-I on satellite cell proliferative potential appears to be limited as chronic overexpression of IGF-I in skeletal muscles did not protect against sarcopenia in 18-mo old mice, and was associated with an exhaustion of satellite cell replicative reserves. ^ These results have shown that replicative senescence can be modulated by environmental factors using skeletal muscle satellite cells as a model system. A better understanding of the molecular basis for enhancement of proliferative capacity by IGF-I will provide a rational basis for developing more effective counter-measures against physical frailty. However, the implications of these studies are that these beneficial effects of enhanced proliferative potential by IGF-I may only be over a short-term period, and other alternative approaches may need to be considered. ^

Estradiol and insulin cross -talk in breast cancer

Approximately 33% of clinical breast carcinomas require estrogens to proliferate. Epidemiological data show that insulin resistance and diabetes mellitus is 2–3 times more prevalent in women with breast cancer than those with benign breast lesions, suggesting a clinical link between insulin and estradiol. Insulin and estradiol have a synergistic effect on the growth of MCF7 breast cancer cells, and long-term estradiol treatment upregulates the expression of the key insulin signaling protein IRS-1. The goal of this study was to further define the mechanism(s) of cross-talk between insulin and estradiol in regulating the growth of breast cancer. Using MCF7 cells, acute treatment with insulin or estradiol alone was found to stimulate two activities associated with growth: Erk MAP kinase and PI 3-kinase. However, combined acute treatment had an antagonistic effect on both activities. Acute estradiol treatment inhibited the insulin-stimulated tyrosine phosphorylation of IRS-1 while increasing its serine phosphorylation; the serine phosphorylation was attenuated by the PI 3-kinase inhibitor wortmannin. The acute antagonism observed with combined estradiol and insulin are not consistent with the long-term synergistic effect on growth. In contrast, chronic estradiol treatment enhanced the insulin-sensitivity of breast cancer cells as measured by increases in total cellular insulin-stimulated tyrosine phosphorylation of IRS-1 and activation of PI 3-kinase. Estradiol stimulation of gene transcription was found to require PI 3-kinase activity but not MAP kinase activity. Insulin alone had no effect on ER transcriptional activity, but chronic treatment in combination with estradiol resulted in synergism of ER transcription. The synergistic effect of insulin and estradiol on MCF7 cell growth was also found to require PI 3-kinase but not MAP kinase activity. Therefore, chronic estradiol treatment increases insulin stimulation of PI 3-kinase, and PI 3-kinase is required for estradiol stimulation of gene transcription alone and in combined synergy with insulin. These data demonstrate that PI 3-kinase is the locus for the cross-talk between insulin and estradiol which results in enhanced breast cancer growth with long-term exposure to both hormones. This may have important clinical implications for women with high risk for breast cancer and/or diabetes mellitus. ^