Relationship between IGF-1 and body weight in inflammatory bowel diseases: Cellular and molecular mechanisms involved.

Inflammatory bowel diseases (IBD), represented by ulcerative colitis (UC) and Crohn's disease (CD), are characterized by chronic inflammation of the gastrointestinal tract, what leads to diarrhea, malnutrition, and weight loss. Depression of the growth hormone-insulin-like growth factor-1 axis (GH-IGF-1 axis) could be responsible of these symptoms. We demonstrate that long-term treatment (54 weeks) of adult CD patients with adalimumab (ADA) results in a decrease in serum IGF-1 without changes in serum IGF-1 binding protein (IGF1BP4). These results prompted us to conduct a preclinical study to test the efficiency of IGF-1 in the medication for experimental colitis. IGF-1 treatment of rats with DSS-induced colitis has a beneficial effect on the following circulating biochemical parameters: glucose, albumin, and total protein levels. In this experimental group we also observed healthy maintenance of colon size, body weight, and lean mass in comparison with the DSS-only group. Histological analysis revealed restoration of the mucosal barrier with the IGF-1 treatment, which was characterized by healthy quantities of mucin production, structural maintenance of adherers junctions (AJs), recuperation of E-cadherin and ß-catenin levels and decrease in infiltrating immune cells and in metalloproteinase-2 levels. The experimentally induced colitis caused activation of apoptosis markers, including cleaved caspase 3, caspase 8, and PARP and decreases cell-cycle checkpoint activators including phosphorylated Rb, cyclin E, and E2F1. The IGF-1 treatment inhibited cyclin E depletion and partially protects PARP levels. The beneficial effects of IGF-1 in experimental colitis could be explained by a re-sensitization of the IGF-1/IRS-1/AKT cascade to exogenous IGF-1. Given these results, we postulate that IGF-1 treatment of IBD patients could prove to be successful in reducing disease pathology.

Actinomycin D Arrests Cell Cycle of Hepatocellular Carcinoma Cell Lines and Induces p53-Dependent Cell Death: A Study of the Molecular Mechanism Involved in the Protective Effect of IRS-4.

Actinomycin D (ActD) is an FDA-approved NCI oncology drug that specifically targets and downregulates stem cell transcription factors, which leads to a depletion of stem cells within the tumor bulk. Recently, our research group demonstrated the importance of IRS-4 in the development of liver cancer. In this study, we evaluated the protective effects of IRS-4 against ActD. For this study, three hepatocellular carcinoma cell lines (HepG2, Huh7, and Chang cells) were used to study the mechanism of actinomycin D. Most assays were carried out in the Hep G2 cell line, due to the high expression of stem cell biomarkers. We found that ActD caused HepG2 cell necroptosis characterized by DNA fragmentation, decreased mitochondrial membrane potential, cytochrome c depletion, and decreased the levels of reduced glutathione. However, we did not observe a clear increase in apoptosis markers such as annexin V presence, caspase 3 activation, or PARP fragmentation. ActD produced an activation of MAP kinases (ERK, p38, and JNK) and AKT. ActD-induced activation of AKT and MAP kinases produced an activation of the Rb-E2F cascade together with a blockage of cell cycle transitions, due to c-jun depletion. ActD led to the inhibition of pCdK1 and pH3 along with DNA fragmentation resulting in cell cycle arrest and the subsequent activation of p53-dependent cell death in the HepG2 cell line. Only JNK and AKT inhibitors were protective against the effects of ActD. N-Acetyl-L-cysteine also had a protective effect as it restored GSH levels. A likely mechanism for this is IRS-4 stimulating GCL-GSH and inhibiting the Brk-CHK1-p53 pathway. The assessment of the IRS-4 in cancer biopsies could be of interest to carry out a personalized treatment with ActD.

Possible Role of IRS-4 in the Origin of Multifocal Hepatocellular Carcinoma.

New evidence suggests that insulin receptor substrate 4 (IRS-4) may play an important role in the promotion of tumoral growth. In this investigation, we have evaluated the role of IRS-4 in a pilot study performed on patients with liver cancer. We used immunohistochemistry to examine IRS-4 expression in biopsies of tumoral tissue from a cohort of 31 patient suffering of hepatocellular carcinoma (HCC). We simultaneously analyzed the expression of the cancer biomarkers PCNA, Ki-67, and pH3 in the same tissue samples. The in vitro analysis was conducted by studying the behavior of HepG2 cells following IRS-4 overexpression/silencing. IRS-4 was expressed mainly in the nuclei of tumoral cells from HCC patients. In contrast, in healthy cells involved in portal triads, canaliculi, and parenchymal tissue, IRS-4 was observed in the cytosol and the membrane. Nuclear IRS-4 in the tumoral region was found in 69.9 ± 3.2%, whereas in the surrounding healthy hepatocytes, nuclear IRS-4 was rarely observed. The percentage of tumoral cells that exhibited nuclear PCNA and Ki-67 were 52.1 ± 7%, 6.1 ± 1.1% and 1.3 ± 0.2%, respectively. Furthermore, we observed a significant positive linear correlation between nuclear IRS-4 and PCNA (r = 0.989; p < 0.001). However, when we correlated the nuclear expression of IRS-4 and Ki-67, we observed a significant positive curvilinear correlation (r = 0.758; p < 0.010). This allowed us to define two populations, (IRS-4 + Ki-67 ≤ 69%) and (IRS-4 + Ki-67 > 70%). The population with lower levels of IRS-4 and Ki-67 had a higher risk of suffering from multifocal liver cancer (OR = 16.66; CI = 1.68-164.8 (95%); p < 0.05). Immunoblot analyses showed that IRS-4 in normal human liver biopsies was lower than in HepG2, Huh7, and Chang cells. Treatment of HepG2 with IGF-1 and EGF induced IRS-4 translocation to the nucleus. Regulation of IRS-4 levels via HepG2 transfection experiments revealed the protein's role in proliferation, cell migration, and cell-collagen adhesion. Nuclear IRS-4 is increased in the tumoral region of HCC. IRS-4 and Ki-67 levels are significantly correlated with the presence of multifocal HCC. Moreover, upregulation of IRS-4 in HepG2 cells induced proliferation by a ß-catenin/Rb/cyclin D mechanism, whereas downregulation of IRS-4 caused a loss in cellular polarity and in its adherence to collagen as well as a gain in migratory and invasive capacities, probably via an integrin α2 and focal adhesion cascade (FAK) mechanism.