JNK1 Protects against Glucolipotoxicity-Mediated Beta-Cell Apoptosis

Pancreatic β-cell dysfunction is central to type 2 diabetes pathogenesis. Prolonged elevated levels of circulating free-fatty acids and hyperglycemia, also termed glucolipotoxicity, mediate β-cell dysfunction and apoptosis associated with increased c-Jun N-terminal Kinase (JNK) activity. Endoplasmic reticulum (ER) and oxidative stress are elicited by palmitate and high glucose concentrations further potentiating JNK activity. Our aim was to determine the role of the JNK subtypes JNK1, JNK2 and JNK3 in palmitate and high glucose-induced β-cell apoptosis. We established insulin-producing INS1 cell lines stably expressing JNK subtype specific shRNAs to understand the differential roles of the individual JNK isoforms. JNK activity was increased after 3 h of palmitate and high glucose exposure associated with increased expression of ER and mitochondrial stress markers. JNK1 shRNA expressing INS1 cells showed increased apoptosis and cleaved caspase 9 and 3 compared to non-sense shRNA expressing control INS1 cells when exposed to palmitate and high glucose associated with increased CHOP expression, ROS formation and Puma mRNA expression. JNK2 shRNA expressing INS1 cells did not affect palmitate and high glucose induced apoptosis or ER stress markers, but increased Puma mRNA expression compared to non-sense shRNA expressing INS1 cells. Finally, JNK3 shRNA expressing INS1 cells did not induce apoptosis compared to non-sense shRNA expressing INS1 cells when exposed to palmitate and high glucose but showed increased caspase 9 and 3 cleavage associated with increased DP5 and Puma mRNA expression. These data suggest that JNK1 protects against palmitate and high glucose-induced β-cell apoptosis associated with reduced ER and mitochondrial stress.     

NDRG2 is a new HIF-1 target gene necessary for hypoxia-induced apoptosis in A549 cells.

The NDRG2 gene belongs to a family of N-Myc downstream-regulated genes (NDRGs) and is expressed in many normal tissues. NDRG2 gene expression has been shown to be regulated in the stress response of certain cells. However, its function is not yet fully understood. Many studies have demonstrated that hypoxia, one of the stress responses, induced apoptosis in several cell types. In the current study, we investigated NDRG2 involvement in hypoxia response and found that NDRG2 expression was markedly up-regulated in several tumor cell lines exposed to hypoxic conditions or similar stresses at the mRNA and protein level. We also observed that the expression of NDRG2 was regulated by Hypoxia-inducible factor 1 (HIF-1) in tumor cells under hypoxia. Three hypoxia-responsive elements (HREs) in the NDRG2 promoter were identified. HRE1 could directly bind Hif-1 in vivo. Importantly, we found that silencing or enforcing the expression of NDRG2 could strongly inhibit or increase apoptosis. In addition, our data also showed that Ndrg2 was able to be translocated from the cytoplasm to the nucleus, and the segment from 101 to 178 amino acids of Ndrg2 is responsible for its translocation. Taken together, this study suggests that NDRG2 is a Hif-1 target gene and closely related with hypoxia-induced apoptosis in A549 cells.     

A retroviral vector for siRNA expression in mammalian cells

Utilization of RNA interference (RNAi) for knockdown of gene expression has become a standard tool for the study of gene function. Short hairpin RNAs (shRNAs) expressed from RNA polymerase III promoters are widely used to achieve stable knockdown of gene expression by RNAi. We have constructed a retroviral-based shRNA expression vector, pSiRPG, as a tool for shRNA-based functional genomic studies. This vector is based on a widely used shRNA expression system and was modified to harbor an enhanced green fluorescent protein (EGFP) and a puromycin selection marker. The functionality of the elements in the pSiRPG vector was validated. The H1(TetO₂) promoter in the vector facilitates doxycycline-inducible shRNA expression, which was demonstrated in cells expressing the Tet repressor (TetR). However, we also demonstrated limited efficiency of the inhibition of shRNA expression in an uninduced TetR-expressing cell line. This observation strongly indicates that the H1(TetO₂) promoter, which is used in a wide range of vectors, is not optimal for tightly regulated shRNA expression. Stable repression of the NDRG1 protein level was observed when introducing pSiRPG constructs expressing shRNAs targeting NDRG1 into two mammary epithelial cell lines by retroviral delivery. This vector should therefore facilitate functional studies in breast cell lines that are hard to transfect with conventional plasmid-based methods.     

Molecular targeting of breast and colon cancer cells by PAR1 mediated apoptosis through a novel pro-apoptotic peptide

A novel activating peptide was designed and synthesized from V. cholerae hemagglutinine protease (HAP) mediated cleavage site of mouse PAR1. The peptide “PFISED” interacts with PAR1 in a new site which is different from its thrombin mediated conventional activation site and induced a series of new downstream signaling pathways. The peptide showed apoptosis in human and mouse breast (MCF-7 and EAC) and colon (HT29 and CT26) cancer cells where as in the same peptide concentration in normal human breast epithelial cells (MCF-10A), normal human fibroblast cells (MRC-5), normal mouse peritoneal macrophage cells and normal mouse breast and colon tissues did not show any effect. Treatment with this peptide enhanced the survival kinetics of EAC induced mice. The peptide mediated apoptosis was inhibited in presence of PAR1 inhibitor and was significantly reduced in si-PAR1 treated cells that indicate the activating peptide “PFISED” induced PAR1 mediated apoptosis of colon and breast cancer cells. This peptide induced over expression and activation of PAR1 and its downstream MAP kinase and NFκB signaling pathways. These signaling pathways enhanced the cellular ROS level to kill malignant cells. We report a novel pro-apoptotic peptide which can selectively kill malignant cells via its specific target receptor PAR1 which is over expressed in the malignant cells and can be used as a molecular target therapy for cancer treatment.     

Nitric oxide suppresses tumor cell migration through N-Myc downstream-regulated gene-1 (NDRG1) expression: role of chelatable iron

N-Myc downstream-regulated gene 1 (NDRG1) is a ubiquitous cellular protein that is up-regulated under a multitude of stress and growth-regulatory conditions. Although the exact cellular functions of this protein have not been elucidated, mutations in this gene or aberrant expression of this protein have been linked to both tumor suppressive and oncogenic phenotypes. Previous reports have demonstrated that NDRG1 is strongly up-regulated by chemical iron chelators and hypoxia, yet its regulation by the free radical nitric oxide (^•NO) has never been demonstrated. Herein, we examine the chemical biology that confers NDRG1 responsiveness at the mRNA and protein levels to ^•NO. We demonstrate that the interaction of ^•NO with the chelatable iron pool (CIP) and the appearance of dinitrosyliron complexes (DNIC) are key determinants. Using HCC 1806 triple negative breast cancer cells, we find that NDRG1 is up-regulated by physiological ^•NO concentrations in a dose- and time-dependant manner. Tumor cell migration was suppressed by NDRG1 expression and we excluded the involvement of HIF-1α, sGC, N-Myc, and c-Myc as upstream regulatory targets of ^•NO. Augmenting the chelatable iron pool abolished ^•NO-mediated NDRG1 expression and the associated phenotypic effects. These data, in summary, reveal a link between ^•NO, chelatable iron, and regulation of NDRG1 expression and signaling in tumor cells.     

Protein S-glutathionylation induced by hypoxia increases hypoxia-inducible factor-1α in human colon cancer cells

Hypoxia is a common characteristic of many types of solid tumors. Intratumoral hypoxia selects for tumor cells that survive in a low oxygen environment, undergo epithelial–mesenchymal transition, are more motile and invasive, and show gene expression changes driven by hypoxia-inducible factor-1α (HIF-1α) activation. Therefore, targeting HIF-1α is an attractive strategy for disrupting multiple pathways crucial for tumor growth. In the present study, we demonstrated that hypoxia increases the S-glutathionylation of HIF-1α and its protein levels in colon cancer cells. This effect is significantly prevented by decreasing oxidized glutathione as well as glutathione depletion, indicating that S-glutathionylation and the formation of protein-glutathione mixed disulfides is related to HIF-1α protein levels. Moreover, colon cancer cells expressing glutaredoxin 1 are resistant to inducing HIF-1α and expressing hypoxia-responsive genes under hypoxic conditions. Therefore, S-glutathionylation of HIF-1α induced by tumor hypoxia may be a novel therapeutic target for the development of new drugs.     

Variation of NDRG2 and c-Myc expression in rat heart during the acute stage of ischemia/reperfusion injury

N-Myc downstream regulated gene 2 (NDRG2), a Myc-repressed gene, is highly expressed in heart tissue. NDRG2 increases in response to hypoxia-induced stress and is involved in hypoxia-induced radioresistance. However, little is known about the expression changes and possible roles of NDRG2 in the heart under hypoxia condition. Here, the authors show that NDRG2, mainly localized in cardiomyocyte cytoplasm, was significantly reduced in myocardial tissue after acute ischemia/reperfusion (I/R) injury. Meanwhile, c-Myc was up-regulated following acute I/R injury, and the expression of c-Myc was significantly inversely correlated with that of NDRG2. In addition, overexpression of c-Myc in primary cultured cardiomyocyte repressed NDRG2 expression. Furthermore, the increase of cardiomyocyte apoptosis was correlated with the decrease of NDRG2 protein during the acute phase of reperfusion. These data suggested for the first time that I/R injury-induced up-regulation of pro-apoptotic c-Myc expression may contribute to the down-regulation of anti-apoptotic NDRG2. This stress response might be involved in the novel mechanism of myocardial apoptosis induced by I/R injury in rat.     

Hypoxia triggers endothelial endoplasmic reticulum stress and apoptosis via induction of VLDL receptor

Endothelial cells express very low density lipoprotein receptor (VLDLr). Beyond the function as peripheral lipoprotein receptor, other roles of VLDLr in endothelial cells have not been completely unraveled. In the present study, human umbilical vein endothelial cells were subjected to hypoxia, and VLDLr expression, endoplasmic reticulum (ER) stress, and apoptosis were assessed. Hypoxia triggered endothelial ER stress and apoptosis, and induced VLDLr expression. Silencing or stabilization of HIF-1α reduced and enhanced VLDLr expression, respectively. HIF-1α affected vldlr promoter activity by interacting with a hypoxia-responsive element (HRE). Knockdown or overexpression of VLDLr alleviated and exacerbated hypoxia-induced ER stress and apoptosis, respectively. Thus, hypoxia induces VLDLr expression through the interaction of HIF-1α with HRE at the vldlr promoter. VLDLr then mediates ER stress and apoptosis.     

Activation of CD95 (APO-1/Fas) signaling by ceramide mediates cancer therapy-induced apoptosis.

We report here that anticancer drugs such as doxorubicin lead to induction of the CD95 (APO-1/Fas) system of apoptosis and the cellular stress pathway which includes JNK/SAPKs. Ceramide, which accumulates in response to different types of cellular stress such as chemo- and radiotherapy, strongly induced expression of CD95-L, cleavage of caspases and apoptosis. Antisense CD95-L as well as dominant-negative FADD inhibited ceramide- and cellular stress-induced apoptosis. Fibroblasts from type A Niemann-Pick patients (NPA), genetically deficient in ceramide synthesis, failed to up-regulate CD95-L expression and to undergo apoptosis after gamma-irradiation or doxorubicin treatment. In contrast, JNK/SAPK activity was still inducible by doxorubicin in the NPA cells, suggesting that activation of JNK/SAPKs alone is not sufficient for induction of the CD95 system and apoptosis. CD95-L expression and apoptosis in NPA fibroblasts were restorable by exogenously added ceramide. In addition, NPA fibroblasts undergo apoptosis after triggering of CD95 with an agonistic antibody. These data demonstrate that ceramide links cellular stress responses induced by gamma-irradiation or anticancer drugs to the CD95 pathway of apoptosis.     

Salubrinal-Mediated Upregulation of eIF2α Phosphorylation Increases Doxorubicin Sensitivity in MCF-7/ADR Cells

Eukaryotic translation initiation factor 2 alpha (eIF2α), which is a component of the eukaryotic translation initiation complex, functions in cell death and survival under various stress conditions. In this study, we investigated the roles of eIF2α phosphorylation in cell death using the breast cancer cell lines MCF-7 and MCF-7/ADR. MCF-7/ADR cells are MCF-7-driven cells that have acquired resistance to doxorubicin (ADR). Treatment of doxorubicin reduced the viability and induced apoptosis in both cell lines, although susceptibility to the drug was very different. Treatment with doxorubicin induced phosphorylation of eIF2α in MCF-7 cells but not in MCF-7/ADR cells. Basal expression levels of Growth Arrest and DNA Damage 34 (GADD34), a regulator of eIF2α, were higher in MCF-7/ADR cells compared to MCF-7 cells. Indeed, treatment with salubrinal, an inhibitor of GADD34, resulted in the upregulation of eIF2α phosphorylation and enhanced doxorubicin-mediated apoptosis in MCF-7/ADR cells. However, MCF-7 cells did not show such synergic effects. These results suggest that dephosphorylation of eIF2α by GADD34 plays an important role in doxorubicin resistance in MCF-7/ADR cells.     

Up-regulation of NDRG2 in senescent lens epithelial cells contributes to age-related cataract in human

Background

Human N-Myc downstream regulated gene2 (NDRG2), a novel gene has been cloned and shown to be related to a number of cellular processes, including proliferation, differentiation, stress, and apoptosis. NDRG2 has also been linked to age-related Alzheimer''s disease. Since the role of this gene in senescence is limited, we have investigated the potential role of NDRG2 in human lens epithelial cells (HLECs), a paradigm implicated in age-related cataract.

Methodology/Principal Findings

Cultured HLECs (SRA01/04) were subjected to prolonged exposure to low dose of H₂O₂ to simulate senescence. After being exposed to 50 µM H₂O₂ for 2 weeks, HLECs senescent-morphological changes appeared, cell viability decreased dramatically, cell proliferation reduced from 37.4% to 16.1%, and senescence-associated β-galactosidase activity increased from 0 to 90.3%. Ndrg2 protein expression was also significantly increased in these senescent cells. To induce overexpression of NDRG2, SRA01/04 cells were infected with the adenoviral vector of NDRG2. In these cells, overexpression of NDRG2 resulted in a fibroblast-like appearance and the cell viability decreased about 20%. In addition, the NDRG2-overexpression cells demonstrated 20% lower viability when exposed to 50–200 µM H₂O₂ for acute oxidative stress. Furthermore, the expression of NDRG2 from age-related cataracts was up-regulated 2-fold at both mRNA and protein levels compared with the clear lenses.

Conclusions/Significance

NDRG2 is up regulated not only in the ageing process of HLECs in vitro but also in the cells from human age-related cortical cataract in vivo. Up-regulation of NDRG2 induces cell morphological changes, reduces cell viability, and especially lowers cellular resistance to oxidative stress. NDRG2-mediated affects in HLECs may associate with age-related cataract formation.     

HSP25 down-regulation enhanced p53 acetylation by dissociation of SIRT1 from p53 in doxorubicin-induced H9c2 cell apoptosis

Heat shock proteins (HSPs) play important roles in cellular stress resistance. Previous reports had already suggested that HSP27 played multiple roles in preventing doxorubicin-induced cardiotoxicity. Although HSP25 might have biological functions similar to its human homolog HSP27, the mechanism of HSP25 is still unclear in doxorubicin-induced cardiomyocyte apoptosis. To investigate HSP25 biological function on doxorubicin-induced apoptosis, flow cytometry was employed to analyze cell apoptosis in over-expressing HSP25 H9c2 cells in presence of doxorubicin. Unexpectedly, the H9c2 cells of over-expressing HSP25 have no protective effect on doxorubicin-induced apoptosis. Moreover, no detectable interactions were detected by coimmunoprecipitation between HSP25 and cytochrome c, and HSP25 over-expression failed in preventing cytochrome c release induced by doxorubicin. However, down-regulation of endogenous HSP25 by a specific small hairpin RNA aggravates apoptosis in H9c2 cells. Subsequent studies found that HSP25, but not HSP90, HSP70, and HSP20, interacted with SIRT1. Knockdown of HSP25 decreased the interaction between SIRT1 and p53, leading to increased p53 acetylation on K379, up-regulated pro-apoptotic Bax protein expression, induced cytochrome c release, and triggered caspase-3 and caspase-9 activation. These findings indicated a novel mechanism by which HSP25 regulated p53 acetylation through dissociation of SIRT1 from p53 in doxorubicin-induced H9c2 cell apoptosis.     

Regulation of epithelial cell turnover and macrophage phenotype by epithelial cell-derived transforming growth factor beta1 in the mammary gland

Transforming growth factor beta1 (TGFB1) is a multi-functional cytokine that regulates cell proliferation, apoptosis and immune system responses. In the breast, the mammary epithelium is the primary source of TGFB1 and increased expression is associated with increased breast cancer risk. This study was conducted to investigate the roles of epithelial cell-derived TGFB1 in regulation of epithelial cell activity and macrophage phenotype in the mammary gland. Tgfb1 null mutant and wildtype mammary epithelium was transplanted into contra-lateral sides of the cleared mammary gland of TGFB1 replete scid mice. Transplanted tissue was analysed for markers of proliferation and apoptosis to determine the effect of Tgfb1 null mutation on epithelial cell turnover, and was analysed by immunohistochemistry to investigate the location, abundance and phenotype of macrophages. The number of proliferating and dying ductal epithelial cells, determined by BrdU and TUNEL, was increased by 35% and 3.3-fold respectively in mammary gland transplanted with Tgfb1 null epithelium compared to wildtype epithelium (p < 0.05). Abundance of F4/80+ macrophages in between Tgfb1 null epithelial cells compared to wildtype epithelial cells was increased by 50%. The number of iNOS+ and CCR7+ cells in the stroma surrounding Tgfb1 null alveolar epithelium was increased by 78% and 2-fold respectively, and dendriform MHC class II+ cells within ductal epithelium were decreased by 30%. We conclude that epithelial cell-derived TGFB1 in the mammary gland has two functions: (1) regulation of cellular turnover of epithelial cells, and (2) regulation of local macrophage phenotype. These findings shed new light on the diversity of roles of TGFB1 in the mammary gland which are likely to impact on breast cancer risk.