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.

Infliximab therapy reverses the increase of allograft inflammatory factor-1 in serum and colonic mucosa of rats with inflammatory bowel disease.

OBJECTIVE: Our purpose was to study the molecular basis of infliximab (IFX) effect on colon mucosa in a colitis model and to identify new biomarkers of mucosal healing. METHODS: Healthy rats and rats which were subjected to experimental colitis induced by dextran sulfate sodium, with or without IFX treatment (in the short- and long-term), were studied along with forty-seven IBD patients. Colon mucosal integrity by periodic acid Schiff (PAS) staining, intestinal damage by immunohistochemistry (proliferating cell nuclear antigen, ß-catenin, E-cadherin, phosphotyrosine, p-p38, allograft inflammatory factor-1 (AIF-1) and colonic mucosal apoptosis by TUNEL staining were evaluated in rats while serum and colon AIF-1 levels were determined in IBD patients. RESULTS: In rats with colitis, IFX reestablished the epithelial barrier integrity, recovered mucus production and decreased colon inflammation, as verified by reduced serum and colon AIF-1 levels; colon and serum AIF-1 levels were also lower in inactive IBD patients compare to active ones. P38 activation after IFX treatment tended to induce differentiation/proliferation of epithelial cells along the colonic crypt-villous axis. CONCLUSIONS: These findings support AIF-1 as a new biomarker of mucosal healing in experimental colitis and suggest that p38 activation is involved in the mucosal healing intracellular mechanism induced by IFX treatment.

Effect of Infliximab in oxidised serum albumin levels during experimental colitis.

Infliximab (IFX) is widely used in ulcerative colitis and in Crohn's disease treatment. Both diseases are characterised by increased oxidative stress, which may affect albumin oxidation. In order to test this hypothesis, the effect of IFX on colitis induced by dextran sulphate sodium (DSS) in rats was evaluated by measuring the Disease Activity Index, biochemical parameters, serum albumin oxidation and colonic mucosa oxidation. Rats with colitis showed an increase in oxidised serum albumin levels and in the oxidation of colon mucous cells. Both decreased after IFX treatment. This suggests that oxidised albumin could be a useful biomarker for monitoring inflammatory bowel disease.

Azathioprine desensitizes liver cancer cells to insulin-like growth factor 1 and causes apoptosis when it is combined with bafilomycin A1.

Hepatoblastoma is a primary liver cancer that affects children, due to the sensitivity of this tumor to insulin-like growth factor 1 (IGF-1). In this paper we show that azathioprine (AZA) is capable of inhibiting IGF1-mediated signaling cascade in HepG2 cells. The efficiency of AZA on inhibition of proliferation differs in the evaluated cell lines as follows: HepG2 (an experimental model of hepatoblastoma)>Hep3B (derived from a hepatocellular carcinoma)>HuH6 (derived from a hepatoblastoma)>>HuH7 (derived from a hepatocellular carcinoma)=Chang Liver cells (a non-malignant cellular model). The effect of AZA in HepG2 cells has been proven to derive from activation of Ras/ERK/TSC2, leading to activation of mTOR/p70S6K in a sustained manner. p70S6K phosphorylates IRS-1 in serine 307 which leads to the uncoupling between IRS-1 and p85 (the regulatory subunit of PI3K) and therefore causing the lack of response of HepG2 to IGF-1. As a consequence, proliferation induced by IGF-1 is inhibited by AZA and autophagy increases leading to senescence of HepG2 cells. Our results suggest that AZA induces the autophagic process in HepG2 activating senescence, and driving to deceleration of cell cycle but not to apoptosis. However, when simultaneous to AZA treatment the autophagy was inhibited by bafilomycin A1 and the degradation of regulatory proteins of cell cycle (e.g. Rb, E2F, and cyclin D1) provoked apoptosis. In conclusion, AZA induces resistance in hepatoblastoma cells to IGF-1, which leads to autophagy activation, and causes apoptosis when it is combined with bafilomycin A1. We are presenting here a novel mechanism of action of azathioprine, which could be useful in treatment of IGF-1 dependent tumors, especially in its combination with other drugs.

Is the autophagy induced by thiopurines beneficial or deleterious?

Thiopurines (azathioprine, 6-mercaptopurine and 6-thioguanine), are drugs useful in the treatment of leukemia, autoimmune diseases, as well as in organ transplantation. After many years of use is still not well understood their mode of action. Recently, several groups have found that thiopurines can activate autophagy by different mechanisms. Autophagy is a process of auto-digestion. After an infection, radiation, injury, oxidative stress, or after drug treatment, the cellular organelles may be damaged. In those cases the damaged structures are recognized by the cell, isolated in a double-membrane vacuole and finally degraded in autolysosomes. The digestion gives rise to biosynthetic precursors needed to regenerate partially destroyed structures, so as to produce the energy essential in the anabolic process. During fasting, the protein aggregates, lipid droplets and glycogen deposits are degraded by this pathway for releasing nutrients to the blood. Therefore this process is of vital importance in the maintaining of cellular functions and in the systemic homeostasis of whole organism. The therapy with thiopurines leads to adverse effects such as myelosuppression and hepatotoxicity whose mechanism is not well understood today. Autophagy is also involved in liver degeneration induced by drugs, alcohol or viruses. Therefore, seems to be very attractive know whether the autophagy induced by thiopurines is the cause of the hepatotoxicity associated with these drugs, or rather, autophagy is a compensatory response that protects the liver against the deleterious effects of the thiopurines. Our results and previous data suggest that autophagy is beneficial for the liver because protects it against the deleterious effects of thiopurines.

Preclinical evaluation of azathioprine plus buthionine sulfoximine in the treatment of human hepatocarcinoma and colon carcinoma.

AIM: To evaluate the efficacy and the safety of azathioprine (AZA) and buthionine sulfoximine (BSO) by localized application into HepG2 tumor in vivo. METHODS: Different hepatoma and colon carcinoma cell lines (HepG2, HuH7, Chang liver, LoVo, RKO, SW-48, SW-480) were grown in minimal essencial medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution and maintained in a humidified 37 °C incubator with 5% CO2. These cells were pretreated with BSO for 24 h and then with AZA for different times. We examined the effects of this combination on some proteins and on cellular death. We also studied the efficacy and the safety of AZA (6 mg/kg per day) and BSO (90 mg/kg per day) in HepG2 tumor growth in vivo using athymic mice. We measured safety by serological markers such as aminotransferases and creatine kinase. RESULTS: The in vitro studies revealed a new mechanism of action for the AZA plus BSO combination in the cancer cells compared with other thiopurines (6-mercaptopurine, 6-methylmercaptopurine, 6-thioguanine and 6-methylthioguanine) in combination with BSO. The cytotoxic effect of AZA plus BSO in HepG2 cells resulted from necroptosis induction in a mitochondrial-dependent manner. From kinetic studies we suggest that glutathione (GSH) depletion stimulates c-Jun amino-terminal kinase and Bax translocation in HepG2 cells with subsequent deregulation of mitochondria (cytochrome c release, loss of membrane potential), and proteolysis activation leading to loss of membrane integrity, release of lactate dehydrogenase and DNA degradation. Some of this biochemical and cellular changes could be reversed by N-acetylcysteine (a GSH replenisher). In vivo studies showed that HepG2 tumor growth was inhibited when AZA was combined with BSO. CONCLUSION: Our studies suggest that a combination of AZA plus BSO could be useful for localized treatment of hepatocellular carcinoma as in the currently used transarterial chemoembolization method.

RNAi-mediated silencing of insulin receptor substrate-4 enhances actinomycin D- and tumor necrosis factor-alpha-induced cell death in hepatocarcinoma cancer cell lines.

Insulin receptor substrate-4 (IRS-4) transmits signals from the insulin-like growth factor receptor (IGF-IR) and the insulin receptor (IR) to the PI3K/AKT and the ERK1/2 pathways. IRS-4 expression increases dramatically after partial hepatectomy and plays an important role in HepG2 hepatoblastoma cell line proliferation/differentiation. In human hepatocarcinoma, IRS-4 overexpression has been associated with tumor development. Herein, we describe the mechanism whereby IRS-4 depletion induced by RNA interference (siRNA) sensitizes HepG2 cells to treatment with actinomycin D (Act D) and combined treatment with Act D plus tumor necrosis factor-alpha (TNF-alpha). Similar results have been obtained in HuH 7 and Chang cell lines. Act D therapy drove the cells to a mitochondrial-dependent apoptotic program involving cytochrome c release, caspase 3 activation, PARP fragmentation and DNA laddering. TNF-alpha amplifies the effect of Act D on HepG2 cell apoptosis increasing c-jun N-terminal kinase (JNK) activity, IkappaB-alpha proteolysis and glutathione depletion. IRS-4 depleted cells that were treated with Act D showed an increase in cytochrome c release and procaspase 3 and PARP proteolysis with respect to control cells. The mechanism involved in IRS-4 action is independent of Akt, IkappaB kinase and JNK. IRS-4 down regulation, however, decreased gamma-glutamylcysteine synthetase content and cell glutathione level in the presence of Act D plus TNF-alpha. These results suggest that IRS-4 protects HepG2 cells from oxidative stress induced by drug treatment.

Azathioprine acts upon rat hepatocyte mitochondria and stress-activated protein kinases leading to necrosis: protective role of N-acetyl-L-cysteine.

Azathioprine is an immunosuppressant drug widely used. Our purpose was to 1) determine whether its associated hepatotoxicity could be attributable to the induction of a necrotic or apoptotic effect in hepatocytes, and 2) elucidate the mechanism involved. To evaluate cellular responses to azathioprine, we used primary culture of isolated rat hepatocytes. Cell metabolic activity, reduced glutathione, cell proliferation, and lactate dehydrogenase release were assessed. Mitochondria were isolated from rat livers, and swelling and oxygen consumption were measured. Mitogen-activated protein kinase pathways and proteins implicated in cell death were analyzed. Azathioprine decreased the viability of hepatocytes and induced the following events: intracellular reduced glutathione (GSH) depletion, metabolic activity reduction, and lactate dehydrogenase release. However, the cell death was not accompanied by DNA laddering, procaspase-3 cleavage, and cytochrome c release. The negative effects of azathioprine on the viability of hepatocytes were prevented by cotreatment with N-acetyl-L-cysteine. In contrast, 6-mercaptopurine showed no effects on GSH content and metabolic activity. Azathioprine effect on hepatocytes was associated with swelling and increased oxygen consumption of intact isolated rat liver mitochondria. Both effects were cyclosporine A-sensitive, suggesting an involvement of the mitochondrial permeability transition pore in the response to azathioprine. In addition, the drug's effects on hepatocyte viability were partially abrogated by c-Jun N-terminal kinase and p38 kinase inhibitors. In conclusion, our findings suggest that azathioprine effects correlate to mitochondrial dysfunction and activation of stress-activated protein kinase pathways leading to necrotic cell death. These negative effects of the drug could be prevented by coincubation with N-acetyl-L-cysteine.

Insulin receptor substrate-4 signaling in quiescent rat hepatocytes and in regenerating rat liver.

This study was designed to characterize insulin receptor substrate-4 (IRS-4) in isolated rat hepatocytes and to examine its role in liver regeneration. Subcellular fractionation revealed that 85% of IRS-4 is located at isolated hepatocyte plasma membranes. The distribution of IRS-4 among intracellular compartments remained unchanged in insulin-stimulated cells. Two bands corresponding to 145 and 138 kd were observed in immunoblotting experiments. Immunoprecipitation of hepatocyte lysates with a highly specific antibody against IRS-4 led to an insulin and insulin-like growth factor 1 (IGF-1)-dependent increase in phosphotyrosine residues of the 145-kd band. IRS-4 was found to be associated with Src homology 2 (SH2) domain-containing proteins (phosphatidylinositol 3-kinase [PI 3-kinase] and Src homology phosphatase [SHP-2]) and with protein kinase C zeta (PKC zeta). Insulin and IGF-1 elicited a rapid and dose-dependent binding of these 3 proteins to IRS-4. These data suggest that IRS-4 is insulin-/IGF-1-activated by phosphorylation and not by translocation, inducing the recruitment of SH2 domain-containing proteins and PKC zeta to the membrane. To evaluate the possible role of IRS-4 in liver regeneration, we also examined this system after partial hepatectomy (PH). One day after PH, IRS-1 expression increased, consistent with a stimulatory role in the regenerative process, whereas it decreased 7 days after liver resection. This drastic IRS-1 depletion occurred at the expense of increased IRS-2 and IRS-4 expression 7 days after PH. In addition, at this period of time after surgery, the in vivo insulin stimulation of remnant rat livers showed an increase in IRS-4/PI 3-kinase association. Given that 1 and 7 days after PH isolated hepatocytes responded similarly to insulin in terms of induced cell proliferation, a compensatory role is proposed for IRS-2/4 induction. In conclusion, IRS-4 is activated by insulin and IGF-1-like IRS-1 in rat hepatocytes, and the induced expression of IRS-4 is a compensatory mechanism that plays a role in conditions of liver regeneration.