Insulin-secreting beta-cell dysfunction induced by human lipoproteins.

Diabetes is associated with significant changes in plasma concentrations of lipoproteins. We tested the hypothesis that lipoproteins modulate the function and survival of insulin-secreting cells. We first detected the presence of several receptors that participate in the binding and processing of plasma lipoproteins and confirmed the internalization of fluorescent low density lipoprotein (LDL) and high density lipoprotein (HDL) particles in insulin-secreting beta-cells. Purified human very low density lipoprotein (VLDL) and LDL particles reduced insulin mRNA levels and beta-cell proliferation and induced a dose-dependent increase in the rate of apoptosis. In mice lacking the LDL receptor, islets showed a dramatic decrease in LDL uptake and were partially resistant to apoptosis caused by LDL. VLDL-induced apoptosis of beta-cells involved caspase-3 cleavage and reduction in the levels of the c-Jun N-terminal kinase-interacting protein-1. In contrast, the proapoptotic signaling of lipoproteins was antagonized by HDL particles or by a small peptide inhibitor of c-Jun N-terminal kinase. The protective effects of HDL were mediated, in part, by inhibition of caspase-3 cleavage and activation of Akt/protein kinase B. In conclusion, human lipoproteins are critical regulators of beta-cell survival and may therefore contribute to the beta-cell dysfunction observed during the development of type 2 diabetes.

Analysis of the Cytoprotective Role of α-Crystallins in Cell Survival and Implication of the αA-Crystallin C-Terminal Extension Domain in Preventing Bax-Induced Apoptosis.

α-Crystallins, initially described as the major structural proteins of the lens, belong to the small heat shock protein family. Apart from their function as chaperones, α-crystallins are involved in the regulation of intracellular apoptotic signals. αA- and αB-crystallins have been shown to interfere with the mitochondrial apoptotic pathway triggering Bax pro-apoptotic activity and downstream activation of effector caspases. Differential regulation of α-crystallins has been observed in several eye diseases such as age-related macular degeneration and stress-induced and inherited retinal degenerations. Although the function of α-crystallins in healthy and diseased retina remains poorly understood, their altered expression in pathological conditions argue in favor of a role in cellular defensive response. In the Rpe65(-/-) mouse model of Leber's congenital amaurosis, we previously observed decreased expression of αA- and αB-crystallins during disease progression, which was correlated with Bax pro-death activity and photoreceptor apoptosis. In the present study, we demonstrated that α-crystallins interacted with pro-apoptotic Bax and displayed cytoprotective action against Bax-triggered apoptosis, as assessed by TUNEL and caspase assays. We further observed in staurosporine-treated photoreceptor-like 661W cells stably overexpressing αA- or αB-crystallin that Bax-dependent apoptosis and caspase activation were inhibited. Finally, we reported that the C-terminal extension domain of αA-crystallin was sufficient to provide protection against Bax-triggered apoptosis. Altogether, these data suggest that α-crystallins interfere with Bax-induced apoptosis in several cell types, including the cone-derived 661W cells. They further suggest that αA-crystallin-derived peptides might be sufficient to promote cytoprotective action in response to apoptotic cell death.

Role of JNK in neurodegenerative diseases

The c-Jun N-terminal kinases (JNK) are members of the MAPK family and can be activated by different stimuli such as cellular stress, heat shock and ultra-violet irradiation. JNKs have different physiological functions and they have been linked to apoptosis in different cell types. Therefore, the JNK signalling pathway is an important target to prevent cell death. In the present chapter, the role of JNKs in neurodegenerative diseases will be discussed, as well as the pharmacological compounds that inhibit this signalling pathway as therapeutic intervention to prevent neuronal death.

Role of JNK in neurodegenerative diseases

The c-Jun N-terminal kinases (JNK) are members of the MAPK family and can be activated by different stimuli such as cellular stress, heat shock and ultra-violet irradiation. JNKs have different physiological functions and they have been linked to apoptosis in different cell types. Therefore, the JNK signalling pathway is an important target to prevent cell death. In the present chapter, the role of JNKs in neurodegenerative diseases will be discussed, as well as the pharmacological compounds that inhibit this signalling pathway as therapeutic intervention to prevent neuronal death.

The activity of the anti-apoptotic fragment generated by the caspase-3/p120 RasGAP stress-sensing module displays strict Akt isoform specificity.

The caspase-3/p120 RasGAP module acts as a stress sensor that promotes pro-survival or pro-death signaling depending on the intensity and the duration of the stressful stimuli. Partial cleavage of p120 RasGAP generates a fragment, called fragment N, which protects stressed cells by activating Akt signaling. Akt family members regulate many cellular processes including proliferation, inhibition of apoptosis and metabolism. These cellular processes are regulated by three distinct Akt isoforms: Akt1, Akt2 and Akt3. However, which of these isoforms are required for fragment N mediated protection have not been defined. In this study, we investigated the individual contribution of each isoform in fragment N-mediated cell protection against Fas ligand induced cell death. To this end, DLD1 and HCT116 isogenic cell lines lacking specific Akt isoforms were used. It was found that fragment N could activate Akt1 and Akt2 but that only the former could mediate the protective activity of the RasGAP-derived fragment. Even overexpression of Akt2 or Akt3 could not rescue the inability of fragment N to protect cells lacking Akt1. These results demonstrate a strict Akt isoform requirement for the anti-apoptotic activity of fragment N.

Cannabinoid 1 receptor promotes cardiac dysfunction, oxidative stress, inflammation, and fibrosis in diabetic cardiomyopathy.

Endocannabinoids and cannabinoid 1 (CB(1)) receptors have been implicated in cardiac dysfunction, inflammation, and cell death associated with various forms of shock, heart failure, and atherosclerosis, in addition to their recognized role in the development of various cardiovascular risk factors in obesity/metabolic syndrome and diabetes. In this study, we explored the role of CB(1) receptors in myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type 1 diabetic cardiomyopathy. Diabetic cardiomyopathy was characterized by increased myocardial endocannabinoid anandamide levels, oxidative/nitrative stress, activation of p38/Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs), enhanced inflammation (tumor necrosis factor-α, interleukin-1β, cyclooxygenase 2, intracellular adhesion molecule 1, and vascular cell adhesion molecule 1), increased expression of CB(1), advanced glycation end product (AGE) and angiotensin II type 1 receptors (receptor for advanced glycation end product [RAGE], angiotensin II receptor type 1 [AT(1)R]), p47(phox) NADPH oxidase subunit, β-myosin heavy chain isozyme switch, accumulation of AGE, fibrosis, and decreased expression of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a). Pharmacological inhibition or genetic deletion of CB(1) receptors attenuated the diabetes-induced cardiac dysfunction and the above-mentioned pathological alterations. Activation of CB(1) receptors by endocannabinoids may play an important role in the pathogenesis of diabetic cardiomyopathy by facilitating MAPK activation, AT(1)R expression/signaling, AGE accumulation, oxidative/nitrative stress, inflammation, and fibrosis. Conversely, CB(1) receptor inhibition may be beneficial in the treatment of diabetic cardiovascular complications.

Role of JNK in neurodegenerative diseases

The c-Jun N-terminal kinases (JNK) are members of the MAPK family and can be activated by different stimuli such as cellular stress, heat shock and ultra-violet irradiation. JNKs have different physiological functions and they have been linked to apoptosis in different cell types. Therefore, the JNK signalling pathway is an important target to prevent cell death. In the present chapter, the role of JNKs in neurodegenerative diseases will be discussed, as well as the pharmacological compounds that inhibit this signalling pathway as therapeutic intervention to prevent neuronal death.

Role of JNK in neurodegenerative diseases

The c-Jun N-terminal kinases (JNK) are members of the MAPK family and can be activated by different stimuli such as cellular stress, heat shock and ultra-violet irradiation. JNKs have different physiological functions and they have been linked to apoptosis in different cell types. Therefore, the JNK signalling pathway is an important target to prevent cell death. In the present chapter, the role of JNKs in neurodegenerative diseases will be discussed, as well as the pharmacological compounds that inhibit this signalling pathway as therapeutic intervention to prevent neuronal death.

Specific targeting of pro-death NMDA receptor signals with differing reliance on the NR2B PDZ ligand.

NMDA receptors (NMDARs) mediate ischemic brain damage, for which interactions between the C termini of NR2 subunits and PDZ domain proteins within the NMDAR signaling complex (NSC) are emerging therapeutic targets. However, expression of NMDARs in a non-neuronal context, lacking many NSC components, can still induce cell death. Moreover, it is unclear whether targeting the NSC will impair NMDAR-dependent prosurvival and plasticity signaling. We show that the NMDAR can promote death signaling independently of the NR2 PDZ ligand, when expressed in non-neuronal cells lacking PSD-95 and neuronal nitric oxide synthase (nNOS), key PDZ proteins that mediate neuronal NMDAR excitotoxicity. However, in a non-neuronal context, the NMDAR promotes cell death solely via c-Jun N-terminal protein kinase (JNK), whereas NMDAR-dependent cortical neuronal death is promoted by both JNK and p38. NMDAR-dependent pro-death signaling via p38 relies on neuronal context, although death signaling by JNK, triggered by mitochondrial reactive oxygen species production, does not. NMDAR-dependent p38 activation in neurons is triggered by submembranous Ca(2+), and is disrupted by NOS inhibitors and also a peptide mimicking the NR2B PDZ ligand (TAT-NR2B9c). TAT-NR2B9c reduced excitotoxic neuronal death and p38-mediated ischemic damage, without impairing an NMDAR-dependent plasticity model or prosurvival signaling to CREB or Akt. TAT-NR2B9c did not inhibit JNK activation, and synergized with JNK inhibitors to ameliorate severe excitotoxic neuronal loss in vitro and ischemic cortical damage in vivo. Thus, NMDAR-activated signals comprise pro-death pathways with differing requirements for PDZ protein interactions. These signals are amenable to selective inhibition, while sparing synaptic plasticity and prosurvival signaling.

Combined loss of the BH3-only proteins Bim and Bmf restores B-cell development and function in TACI-Ig transgenic mice.

Terminal differentiation of B cells depends on two interconnected survival pathways, elicited by the B-cell receptor (BCR) and the BAFF receptor (BAFF-R), respectively. Loss of either signaling pathway arrests B-cell development. Although BCR-dependent survival depends mainly on the activation of the v-AKT murine thymoma viral oncogene homolog 1 (AKT)/PI3-kinase network, BAFF/BAFF-R-mediated survival engages non-canonical NF-κB signaling as well as MAPK/extracellular-signal regulated kinase and AKT/PI3-kinase modules to allow proper B-cell development. Plasma cell survival, however, is independent of BAFF-R and regulated by APRIL that signals NF-κB activation via alternative receptors, that is, transmembrane activator and CAML interactor (TACI) or B-cell maturation (BCMA). All these complex signaling events are believed to secure survival by increased expression of anti-apoptotic B-cell lymphoma 2 (Bcl2) family proteins in developing and mature B cells. Curiously, how lack of BAFF- or APRIL-mediated signaling triggers B-cell apoptosis remains largely unexplored. Here, we show that two pro-apoptotic members of the 'Bcl2 homology domain 3-only' subgroup of the Bcl2 family, Bcl2 interacting mediator of cell death (Bim) and Bcl2 modifying factor (Bmf), mediate apoptosis in the context of TACI-Ig overexpression that effectively neutralizes BAFF as well as APRIL. Surprisingly, although Bcl2 overexpression triggers B-cell hyperplasia exceeding the one observed in Bim(-/-)Bmf(-/-) mice, Bcl2 transgenic B cells remain susceptible to the effects of TACI-Ig expression in vivo, leading to ameliorated pathology in Vav-Bcl2 transgenic mice. Together, our findings shed new light on the molecular machinery restricting B-cell survival during development, normal homeostasis and under pathological conditions. Our data further suggest that Bcl2 antagonists might improve the potency of BAFF/APRIL-depletion strategies in B-cell-driven pathologies.

The activity of the anti-apoptotic fragment generated by the caspase-3/p120 RasGAP stress-sensing module displays strict Akt isoform specificity.

The caspase-3/p120 RasGAP module acts as a stress sensor that promotes pro-survival or pro-death signaling depending on the intensity and the duration of the stressful stimuli. Partial cleavage of p120 RasGAP generates a fragment, called fragment N, which protects stressed cells by activating Akt signaling. Akt family members regulate many cellular processes including proliferation, inhibition of apoptosis and metabolism. These cellular processes are regulated by three distinct Akt isoforms: Akt1, Akt2 and Akt3. However, which of these isoforms are required for fragment N mediated protection have not been defined. In this study, we investigated the individual contribution of each isoform in fragment N-mediated cell protection against Fas ligand induced cell death. To this end, DLD1 and HCT116 isogenic cell lines lacking specific Akt isoforms were used. It was found that fragment N could activate Akt1 and Akt2 but that only the former could mediate the protective activity of the RasGAP-derived fragment. Even overexpression of Akt2 or Akt3 could not rescue the inability of fragment N to protect cells lacking Akt1. These results demonstrate a strict Akt isoform requirement for the anti-apoptotic activity of fragment N.

Dysregulation of apoptosis in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a major health problem, being the sixth most common cancer world-wide. Dysregulation of the balance between proliferation and cell death represents a pro-tumorigenic principle in human hepatocarcinogenesis. This review updates the recent relevant contributions reporting molecular alterations for HCC that induce an imbalance in the regulation of apoptosis. Alterations in the expression and/or activation of p53 are frequent in HCC cells, which confer on them resistance to chemotherapeutic drugs. Many HCCs are also insensitive to apoptosis induced either by death receptor ligands, such as FasL or TRAIL, or by transforming growth factor-beta (TGF-beta). Although the expression of some pro-apoptotic genes is decreased, the balance between death and survival is dysregulated in HCC mainly due to overactivation of anti-apoptotic pathways. Indeed, some molecules involved in counteracting apoptosis, such as Bcl-XL, Mcl-1, c-IAP1, XIAP or survivin are over-expressed in HCC cells. Furthermore, some growth factors that mediate cell survival are up-regulated in HCC, as well as the molecules involved in the machinery responsible for cleavage of their pro-forms to an active peptide. The expression and/or activation of the JAK/STAT, PI3K/AKT and RAS/ERKs pathways are enhanced in many HCC cells, conferring on them resistance to apoptotic stimuli. Finally, recent evidence indicates that inflammatory processes, as well as the epithelial-mesenchymal transitions that occur in HCC cells to facilitate their dissemination, are related to cell survival. Therefore, therapeutic strategies to selectively inhibit anti-apoptotic signals in liver tumor cells have the potential to provide powerful tools to treat HCC.

Lethal weapons : novel approaches for receptor-targeted cancer cell elimination

The currently used forms of cancer therapy are associated with drug resistance and toxicity to healthy tissues. Thus, more efficient methods are needed for cancer-specific induction of growth arrest and programmed cell death, also known as apoptosis. Therapeutic forms of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are investigated in clinical trials due to the capability of TRAIL to trigger apoptosis specifically in cancer cells by activation of cell surface death receptors. Many tumors, however, have acquired resistance to TRAIL-induced apoptosis and sensitizing drugs for combinatorial treatments are, therefore, in high demand. This study demonstrates that lignans, natural polyphenols enriched in seeds and cereal, have a remarkable sensitizing effect on TRAIL-induced cell death at non-toxic lignan concentrations. In TRAIL-resistant and androgen-dependent prostate cancer cells we observe that lignans repress receptor tyrosine kinase (RTK) activity and downregulate cell survival signaling via the Akt pathway, which leads to increased TRAIL sensitivity. A structure-activity relationship analysis reveals that the γ-butyrolactone ring of the dibenzylbutyrolactone lignans is essential for the rapidly reversible TRAIL-sensitizing activity of these compounds. Furthermore, the lignan nortrachelogenin (NTG) is identified as the most efficient of the 27 tested lignans and norlignans in sensitization of androgen-deprived prostate cancer cells to TRAIL-induced apoptosis. While this combinatorial anticancer approach may leave normal cells unharmed, several efficient cancer drugs are too toxic, insoluble or unstable to be used in systemic therapy. To enable use of such drugs and to protect normal cells from cytotoxic effects, cancer-targeted drug delivery vehicles of nanometer scale have recently been generated. The newly developed nanoparticle system that we tested in vitro for cancer cell targeting combines the efficient drug-loading capacity of mesoporous silica to the versatile particle surface functionalization of hyperbranched poly(ethylene imine), PEI. The mesoporous hybrid silica nanoparticles (MSNs) were functionalized with folic acid to promote targeted internalization by folate receptor overexpressing cancer cells. The presented results demonstrate that the developed carrier system can be employed in vitro for cancer selective delivery of adsorbed or covalently conjugated molecules and furthermore, for selective induction of apoptotic cell death in folate receptor expressing cancer cells. The tested carrier system displays potential for simultaneous delivery of several anticancer agents specifically to cancer cells also in vivo.

Cellular responses to stress and kinase signaling activation : apoptosis and differentiation

Stressignaler avkänns många gånger av membranbundna proteiner som översätter signalerna till kemisk modifiering av molekyler, ofta proteinkinaser Dessa kinaser överför de avkodade budskapen till specifika transkriptionsfaktorer genom en kaskad av sekventiella fosforyleringshändelser, transkriptionsfaktorerna aktiverar i sin tur de gener som behövs för att reagera på stressen. En av de mest kända måltavlorna för stressignaler är transkriptionsfaktor AP-1 familjemedlemen c-Jun. I denna studie har jag identifierat den nukleolära proteinet AATF som en ny regulator av c-Jun-medierad transkriptionsaktivitet. Jag visar att stresstimuli inducerar omlokalisering av AATF vilket i sin tur leder till aktivering av c-Jun. Den AATF-medierad ökningen av c-Jun-aktiviteten leder till en betydande ökning av programmerad celldöd. Parallellt har jag vidarekarakteriserat Cdk5/p35 signaleringskomplexet som tidigare har identifierats i vårt laboratorium som en viktig faktor för myoblastdifferentiering. Jag identifierade den atypiska PKCξ som en uppströms regulator av Cdk5/p35-komplexet och visar att klyvning och aktivering av Cdk5 regulatorn p35 är av fysiologisk betydelse för differentieringsprocessen och beroende av PKCξ aktivitet. Jag visar att vid induktion av differentiering fosforylerar PKCξ p35 vilket leder till calpain-medierad klyvning av p35 och därmed ökning av Cdk5-aktiviteten. Denna avhandling ökar förståelsen för de regulatoriska mekanismer som styr c-Jun-transkriptionsaktiviteten och c-Jun beroende apoptos genom att identifiera AATF som en viktig faktor. Dessutom ger detta arbete nya insikter om funktionen av Cdk5/p35-komplexet under myoblastdifferentiering och identifierar PKCξ som en uppströms regulator av Cdk5 aktivitet och myoblast differentiering.

Caspase-8 and p38MAPK in DATS-induced apoptosis of human CNE2 cells

Nasopharyngeal carcinoma is a common malignancy in Southern China of uncertain etiologic origin. Diallyl trisulfide (DATS), one of the major components of garlic (Allium sativum), is highly bactericidal and fungicidal. In this study, we investigated the function of p38 mitogen-activated protein kinase (MAPK) and caspase-8 in DATS-induced apoptosis of human CNE2 cells using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], flow cytometry assay, and Western blotting. After CNE2 cells were treated with DATS (50, 100, or 150 μM) for 24 h, cell viability rates were 75.9, 63.4 and 39.6%, and apoptosis rates were 24.5, 36.9, and 62.4%, respectively. The data showed that DATS induced CNE2 cell death in a dose-dependent manner. After human CNE2 cells were treated with 100 μM DATS and inhibitors (10 μM SB203580 and Z-LETD-FMK for p38MAPK and caspase-8, respectively), changes in cell viability and apoptosis and in p38MAPK and caspase-8 activity were detected. Cell viability rates were 66.5 and 68.1% and decreased 9.9 and 11.5% compared with inhibitor treatment alone. Apoptosis rates were 31.53 and 29.98% and increased 9.1 and 10% compared with inhibitor treatment alone. The results indicated that DATS activates p38MAPK and caspase-8, but both inhibitors have an effect on P38MAPK and caspase-8 activity. In conclusion, our data indicate that p38MAPK and caspase-8 are involved in the process of DATS-induced apoptosis in human CNE2 cells and interact with each other.