Bisindolylmaleimide alkaloid BMA-155Cl induces autophagy and apoptosis in human hepatocarcinoma HepG-2 cells through the NF-κB p65 pathway.

Bisindolylmaleimides, a series of derivatives of a PKC inhibitor staurosporine, exhibit potential as anti-cancer drugs and have received considerable attention in clinical trials. This study aims to investigate the effects of a bisindolylmaleimide alkaloid 155Cl (BMA-155Cl) with a novel structure on autophagy and apoptosis in human hepatocarcinoma HepG-2 cells in vitro and in vivo. The cell poliferation was assessed with a MTT assay. Autophagy was evaluated by MDC staining and TEM analysis. Apoptosis was investigated using Annexin V-FITC/PI and DAPI staining. The antitumor effects were further evaluated in nude mice bearing HepG-2 xenografts, which received BMA-155Cl (10, 20 mg/kg, ip) for 18 days. Autophagy- and apoptosis-associated proteins and their mRNA levels were examined with Western blotting, immunohistochemistry, and RT-PCR. BMA-155Cl (2.5-20 µmol/L) inhibited the growth of HepG-2 cells with IC50 values of 16.62±1.34, 12.21±0.83, and 8.44±1.82 µmol/L at 24, 48, and 72 h, respectively. Furthermore, BMA-155Cl (5-20 µmol/L) dose-dependently induced autophagy and apoptosis in HepG-2 cells. The formation of autophagic vacuoles was induced by BMA-155Cl (10 µmol/L) at approximately 6 h and peaked at approximately 15 h. Pretreatment with 3-MA potentiated BMA-155Cl-mediated apoptotic cell death. This compound dose-dependently increased the mRNA and protein levels of Beclin-1, NF-κB p65, p53, and Bax, but decreased the expression of IκB and Bcl-2. Pretreatment with BAY 11-7082, a specific inhibitor of NF-κB p65, blocked BMA-155Cl-induced expression of autophagy- and apoptosis-associated proteins. BMA-155Cl administration effectively suppressed the growth of HepG-2 xenografts in vivo, and increased the protein expression levels of LC3B, Beclin-1, NF-κB p65, and Bax in vivo. We conclude that the NF-κB p65 pathway is involved in BMA-155Cl-triggered autophagy, followed by apoptosis in HepG-2 cells in vitro and in vivo. Hence, BMA-155Cl could be a promising pro-apoptotic candidate for developing as a novel anti-cancer drug.

Lithium attenuates lipopolysaccharide-induced hypothermia in rats.

BACKGROUND: Changes in body temperature are common features among patients with sepsis and septic shock. Similarly, systemic administration of bacterial endotoxin (lipopolysaccharide, LPS) to rats leads to an initial hypothermia followed by elevation in body temperature. These changes in body temperature are accompanied by increased levels of prostaglandin E2 (PGE2) in the hypothalamus. OBJECTIVE: This study examined the effects of lithium and SB216763 - two different glycogen synthase kinase (GSK)-3 inhibitors - on LPS-induced changes in body temperature and hypothalamic PGE2 levels in endotoxemic rats. MATERIALS AND METHODS: Endotoxemia was induced by intraperitoneal injection of LPS (10 mg/kg). Lithium (100 mg/kg) and SB216763 (5 mg/kg) were administered at 2 h before LPS. Body temperature and mortality were monitored during 48 h after LPS injection. In another protocol, rats were sacrificed at 2 h post LPS injection and then, blood, liver and hypothalamus were extracted for inflammatory mediators determination. RESULTS: Lithium but not SB216763 significantly reduced LPS-induced hypothermia, while both compounds did not alter the subsequent elevation in body temperature. Moreover, only lithium significantly reduced hypothalamic PGE2 levels. On the other hand, both compounds significantly reduced plasma, hepatic and hypothalamic tumor necrosis factor-α and decreased plasma PGE2 levels. Both compounds did not alter LPS-induced mortality. CONCLUSIONS: These results suggest that the attenuation of LPS-induced hypothermia by lithium may derive from its reduction of hypothalamic PGE2 levels.

Inhibiting glycogen synthase kinase-3 mitigates the hematopoietic acute radiation syndrome in mice.

Exposure to a nuclear accident or radiological attack can cause death from acute radiation syndrome (ARS), which results from radiation injury to vital organs such as the hematopoietic system. However, the U.S. Food and Drug Administration (FDA) has not approved any medical countermeasures for this specific purpose. With growing concern over nuclear terrorism, there is an urgent need to develop small molecule deliverables that mitigate mortality from ARS. One emerging modulator of hematopoietic stem/progenitor cell (HSPC) activity is glycogen synthase kinase-3 (GSK-3). The inhibition of GSK-3 has been shown to augment hematopoietic repopulation in mouse models of bone marrow transplantation. In this study, we performed an in vitro screen using irradiated bone marrow mononuclear cells (BM-MNCs) to test the effects of four GSK-3 inhibitors: CHIR99021; 6-Bromoindirubin-3'-oxime (BIO); SB415286; and SB216763. This screen showed that SB216763 significantly increased the frequency of c-Kit(+) Lin(-) Sca1(+) (KLS) cells and hematopoietic colony-forming cells in irradiated BM-MNCs. Importantly, administration of a single dose of SB216763 to C57BL/6J mice by subcutaneous injection 24 h after total-body irradiation significantly improved hematopoietic recovery and mitigated hematopoietic ARS. Collectively, our results demonstrate that the GSK-3 inhibitor SB216763 is an effective medical countermeasure against acute radiation injury of the hematopoietic system.

Impact of acellular hemoglobin-based oxygen carriers on brain apoptosis in rats.

BACKGROUND: Extracellular hemoglobin (Hb)-based oxygen carriers (HBOCs) are under extensive consideration as oxygen therapeutics. Their effects on cellular mechanisms related to apoptosis are of particular interest, because the onset of proapoptotic pathways may give rise to tissue damage. STUDY DESIGN AND METHODS: The objective was to assess whether the properties of the Hb that replaces blood during an isovolemic hemodilution would modulate apoptotic-response mechanisms in rat brain and whether such signaling favors cytoprotection or damage. We exposed rats to exchange transfusion (ET; 50% blood volume and isovolemic replacement with Hextend [negative colloid control], MP4OX [PEGylated HBOC with high oxygen affinity], and ααHb [αα-cross-linked HBOC with low oxygen affinity; n=4-6/group]). Sham rats acted as control. Animals were euthanized at 2, 6, and 12 hours after ET; brain tissue was harvested and processed for analysis. RESULTS: In MP4OX animals, the number of neurons that overexpressed the hypoxia-inducible factor (HIF)-1α was higher than in ααHb, particularly at the early time points. In addition, MP4OX was associated with greater phosphorylation of protein kinase B (Akt), a well-known cytoprotective factor. Indeed, the degree of apoptosis, measured as terminal deoxynucleotidyl transferase-positive neurons and caspase-3 cleavage, ranked in order of MP4OX < Hextend < ααHb. CONCLUSION: Even though both HBOCs showed increased levels of HIF-1α compared to shams or Hextend-treated animals, differences in signaling events resulted in very different outcomes for the two HBOCs. ααHb-treated brain tissue showed significant neuronal damage, measured as apoptosis. This was in stark contrast to the protection seen with MP4OX, apparently due to recruitment of Akt and neuronal specific HIF-1α pathways.

Curcumin protects against regional myocardial ischemia/reperfusion injury through activation of RISK/GSK-3ß and inhibition of p38 MAPK and JNK.

BACKGROUND: Curcumin, the active ingredient of turmeric (Curcuma longa), is known to have anti-inflammatory and antioxidative properties. The present study was aimed to determine the effect of curcumin in regional myocardial ischemia/reperfusion (I/R) injury and its underlying mechanisms involving the role of prosurvival kinases and apoptotic kinases. METHODS: Sprague-Dawley rats (n = 109) subjected to a 30-minute left anterior descending coronary artery (LAD) occlusion followed by reperfusion were assigned to receive saline (control), curcumin (100 mg/kg), wortmannin (inhibitor of phosphatidylinositol-3-OH kinase [PI3K]-Akt), wortmannin + curcumin, U0126 (inhibitor of extracellular signal-regulated kinase [ERK1/2]), U0126 + curcumin, SB216763 (inhibitor of glycogen synthase kinase [GSK-3ß]), and SB216763 + curcumin 20 minutes before LAD occlusion. Infarct size was measured after 2 hours of reperfusion by triphenyl tetrazolium chloride staining. The phosphorylation of Akt, ERK1/2, GSK-3ß, p38, and c-Jun N-terminal kinases (JNK) was determined by immunoblotting after 10 minutes of reperfusion. RESULTS: Curcumin significantly reduced the infarct size compared with the control (33.1% ± 6.2% vs 50.1% ± 3.9%; P < .05). Wortmannin or U0126 alone did not affect the infarct size but abolished the curcumin-induced cardioprotective effect. Curcumin significantly enhanced the phosphorylation of Akt, ERK1/2, and GSK-3ß, while it reduced that of p38 and JNK. Wortmannin or U0126 abolished enhanced phosphorylation of GSK-3ß induced by curcumin. SB216763 alone or combined with curcumin reduced the infarct size and enhanced phosphorylation of GSK-3ß compared with the control. CONCLUSIONS: Preconditioning by curcumin effectively protects against regional myocardial I/R injury through the activation of prosurvival kinases involving PI3K-Akt, ERK1/2, and GSK-3ß, and attenuation of p38 and JNK.

PKCß inhibition with ruboxistaurin reduces oxidative stress and attenuates left ventricular hypertrophy and dysfunction in rats with streptozotocin-induced diabetes.

Oxidative stress plays critical roles in the development of diabetic cardiovascular complications, including myocardial hypertrophy. The ß isoform of PKC (protein kinase C) is preferentially overexpressed in the myocardium of diabetic subjects accompanied with increased activation of the pro-oxidant enzyme NADPH oxidase, which may exacerbate oxidative stress. We hypothesized that myocardial PKCß is a major upstream mediator of oxidative stress in diabetes and that PKCß inhibition can attenuate myocardial hypertrophy and dysfunction. Control or streptozotocin-induced diabetic rats were treated with the selective PKCß inhibitor RBX (ruboxistaurin; 1 mg/kg of body weight per day) or the antioxidant NAC (N-acetylcysteine) for 4 weeks. LV (left ventricular) dimensions and functions were detected by echocardiography. 15-F2t-isoprostane (a specific index of oxidative stress) and myocardial activities of superoxide dismutase as well as protein levels of NADPH oxidase were assessed by immunoassay or Western blotting. Echocardiography revealed that the LV mass/body weight ratio was significantly increased in diabetic rats (P<0.01 compared with the control group) in parallel with the impaired LV relaxation. A significant increase in cardiomyocyte cross-sectional area was observed in diabetic rats accompanied by an increased production of O2- (superoxide anion) and 15-F2t-isoprostane (all P<0.05 compared with the control group). RBX normalized these changes with concomitant inhibition of PKCß2 activation and prevention of NADPH oxidase subunit p67phox membrane translocation and p22phox overexpression. The effects of RBX were comparable with that of NAC, except that NAC was inferior to RBX in attenuating cardiac dysfunction. It is concluded that RBX can ameliorate myocardial hypertrophy and dysfunction in diabetes, which may represent a novel therapy in the prevention of diabetic cardiovascular complications.

Antioxidants in the treatment of diabetes.

Diabetes is a chronic metabolic disorder that continues to present as a major health problem worldwide. It is characterized by absolute or relative deficiencies in insulin secretion and/or insulin action and is associated with chronic hyperglycemia and disturbances of carbohydrate, lipid, and protein metabolism. Many studies suggest a central role for oxidative stress in the pathogenesis of this multi-faceted metabolic disorder. This has prompted investigations in the use of antioxidants as a complementary therapeutic approach. In this review we briefly summarize oxidative mechanisms implicated in diabetic complications and then focus on the findings resulting from human clinical trials where antioxidants were studied as an adjuvant to standard diabetes treatment during the last ten years. A literature search using PubMed (last ten years) was performed using the following terms: vitamin E, vitamin C, coenzyme Q10, alpha lipoic acid, L-carnitine, ruboxistaurin or LY 333531 and diabetes. This search was limited to human clinical trials. We concluded there is not any established benefit for antioxidants use in the management of diabetic complications. Therefore, routine vitamin or mineral supplementation is not generally recommended.

Ruboxistaurin mesilate hydrate for diabetic retinopathy.

Diabetic retinopathy remains a major worldwide cause of preventable blindness. The beta isoform of protein kinase C (PKC) may play an important role in the pathogenesis of this disorder. Ruboxistaurin mesylate hydrate is an orally bioavailable, highly-specific inhibitor of PKC beta, which has shown some efficacy in several large, multicenter, randomized clinical trials. The U.S. Food and Drug Administration issued an approvable letter for ruboxistaurin in 2006, but at this time the medication is not available for routine clinical use.

Targeting the protein kinase C family in the diabetic kidney: lessons from analysis of mutant mice.

The protein kinase C (PKC) superfamily comprises proteins that are activated in response to various pathogenic stimuli in the diabetic state. Hyperglycaemia is the predominant stimulus that induces the activation of distinct PKC isoforms within a cell, each mediating specific functions, probably through differential subcellular localisation. The contribution of individual PKC isoforms can be directly addressed in vivo using innovative PKC-isoform-specific knockout (KO) mouse models, which are providing key insights into the physiological function of PKC isoform diversity in the development of diabetic nephropathy. Such studies can be a valuable complementary approach to more commonly used pharmacological analyses using agents such as ruboxistaurin mesylate (Arxxant, LY333531), which is claimed to specifically inhibit the PKC-beta-isoform. As expected given the multiple and specific properties of the isoforms in vitro, deletion of different PKC isoform signalling pathways leads to distinct phenotypes in mice. Notably, KOs of the individual PKCs assigned specific non-redundant biological functions to each isoform, which were not compensated for by the others. Thus, PKC isoform specificity and cellular diversity seem to be responsible for the divergent outcomes leading to albuminuria and/or renal fibrosis according to studies on the streptozotocin-induced mouse model of diabetes. This review discusses the role of individual PKC isoforms in diabetic nephropathy and their potential therapeutic implications. Defining and targeting mediators of increased intracellular activation in the diabetic microvasculature will have important clinical and therapeutic benefits and help in the design of novel effective therapies in the near future.

New potential agents in treating diabetic kidney disease: the fourth act.

Despite the worldwide epidemic of chronic kidney disease complicating diabetes mellitus, current therapies directed against nephroprogression are limited to angiotensin conversion or receptor blockade. Nonetheless, additional therapeutic possibilities are slowly emerging. The diversity of therapies currently in development reflects the pathogenic complexity of diabetic nephropathy. The three most important candidate drugs currently in development include a glycosaminoglycan, a protein kinase C (PKC) inhibitor and an inhibitor of advanced glycation. In targeting primary mechanisms by which hyperglycaemia contributes to diabetic complications, these drugs could provide risk reduction complementary to the partial reduction proven for ACE inhibitors and angiotensin II receptor antagonists (angiotensin receptor blockers). Glycosaminoglycans act to restore glycoproteins present in reduced amounts in the glomerular basement membrane and mesangium of diabetic animal models. Components of the drug sulodexide prevent pathological changes and proteinuria in diabetic rats. Reductions in albuminuria, a hallmark of early diabetic kidney disease, have been reported in initial human trials. In the US, a multicentre phase II study has been completed, with an interim analysis indicating reduction in urinary albumin losses. Pivotal phase II trials have begun in patients with type 2 diabetes. A second metabolic pathway of diabetic complications is overexpression of PKC. Several activators of this family of intracellular kinases have been identified and PKC activation may result in tissue damage through a variety of mechanisms. In animal models, the inhibitor ruboxistaurin reduces albuminuria, diabetic histological changes and kidney injury. Like sulodexide, drug development of ruboxistaurin has reached completion of a phase II evaluation with mixed results. The third metabolic target is the nonenzymatic formulation of advanced glycation end-products (AGEs) through well described biochemical pathways. Multiple pathways lead to AGE accumulation in tissues in diabetes and diverse AGE products are formed. AGE deposition has been implicated in animal models of diabetic nephropathy. The leading AGE inhibitor currently in development is pyridoxamine, which has multiple actions that inhibit glycation. Pyridoxamine is an efficient AGE inhibitor in experimental diabetes. A phase II study in diabetic patients with nephropathy reported mixed efficacy results and a favourable safety profile. Phase III evaluation of pyridoxamine has not begun. These three classes of potential therapies, if successfully developed, will confirm that diabetic kidney disease has entered the era of biochemical treatments.

Treatment of type 2 diabetes mellitus with agonists of the GLP-1 receptor or DPP-IV inhibitors.

Glucagon-like peptide-1 (GLP-1) is a peptide hormone from the gut that stimulates insulin secretion and protects beta-cells, inhibits glucagon secretion and gastric emptying, and reduces appetite and food intake. In agreement with these actions, it has been shown to be highly effective in the treatment of Type 2 diabetes, causing marked improvements in glycaemic profile, insulin sensitivity and beta-cell performance, as well as weight reduction. The hormone is metabolised rapidly by the enzyme dipeptidyl peptidase IV (DPP-IV) and, therefore, cannot be easily used clinically. Instead, resistant analogues of the hormone (or agonists of the GLP-1 receptor) are in development, along with DPP-IV inhibitors, which have been demonstrated to protect the endogenous hormone and enhance its activity. Agonists include both albumin-bound analogues of GLP-1 and exendin-4, a lizard peptide. Clinical studies with exendin have been carried out for > 6 months and have indicated efficacy in patients inadequately treated with oral antidiabetic agents. Orally active DPP-IV inhibitors, suitable for once-daily administration, have demonstrated similar efficacy. Diabetes therapy, based on GLP-1 receptor activation, therefore, appears very promising.

A novel potential therapy for diabetic nephropathy and vascular complications: protein kinase C beta inhibition.

Diabetic nephropathy is one of the most common microvascular complications of diabetes mellitus and the leading cause of end-stage renal disease in developed countries. Current treatment includes glycemic control, blood pressure control (with special emphasis on agents targeting the renin-angiotensin system), a low-protein (0.6 to 0.8 g/kg) diet, and the use of hypolipidemic agents. Although these therapeutic options may slow progression, the burden of disease remains large, and additional therapeutic agents are urgently needed. Ruboxistaurin (LY333531) mesylate is a bisindolylmaleimide that shows a high degree of specificity within the protein kinase C (PKC) gene family for inhibiting PKC beta isoforms. In animal models of diabetes, including the streptozotocin (STZ) rat, Lepr(db)/Lepr(db) mouse, and STZ-Ren 2 rat models, ruboxistaurin normalized glomerular hyperfiltration, decreased urinary albumin excretion, and reduced glomerular transforming growth factor-beta1 and extracellular matrix protein production. As a result, improvements were noted in mesangial expansion, glomerulosclerosis, tubulointerstitial fibrosis, and renal function. Other studies using less specific probes of PKC activity also have shown an important role for PKC in the development of diabetic nephropathy and a close relationship to pathways believed to be important in its pathogenesis. Inhibition of PKC beta, a common signaling molecule in diabetes-related renal and vascular injury, holds promise as a novel strategy to improve microvascular and macrovascular outcomes in diabetes. Such therapies are needed to reduce the occurrence of devastating diabetic complications.