Prohibitin protects against oxidative stress-induced cell injury in cultured neonatal cardiomyocyte

Oxidative stress is one of the main causes of myocardial injury, which is associated with cardiomyocyte death. Mitochondria play a key role in triggering the necrosis and apoptosis pathway of cardiomyocytes under oxidative stress. Although prohibitin (PHB) has been acknowledged as a mitochondrial chaperone, its functions in cardiomyocytes are poorly characterized. The present research was designed to investigate the cardioprotective role of PHB in mitochondria. Oxidative stress can increase the PHB content in mitochondria in a time-dependent manner. Overexpression of PHB in cultured cardiomyocytes by transfection of recombinant adenovirus vector containing PHB sense cDNA resulted in an increase of PHB in mitochondria. Compared with the non-transfection cardiomyocytes, PHB overexpression could protect the mitochondria from oxidative stress-induced injury. The mitochondria-mediated apoptosis pathway was consistently suppressed in PHB-overexpressed cardiomyocytes after hydrogen peroxide (H₂O₂) treatment, including a reduced change in mitochondrial membrane permeability transition and an inhibited release of cytochrome c from mitochondria to cytoplasma. As a result, the oxidative stress-induced cardiomyocyte apoptosis was suppressed. These data indicated that PHB protected the cardiomyocytes from oxidative stress-induced damage, and that increasing PHB content in mitochondria constituted a new therapeutic target for myocardium injury. XiaoHua Liu and Zhe Ren contributed equally to this work. ● Prohibitin is an evolutionarily conserved and ubiquitously expressed protein involved in mitochondrial structure, function, and inheritance whose function in cardiomyocyte is not known. In this study, we found oxidative stress could induce increased expression in cardiomyocytes and mitochondrial translocation of PHB, and PHB can protect against oxidative stress in cultured neonatal cardiomyocyte.     

Oleate prevents palmitate-induced cytotoxic stress in cardiac myocytes

The cytotoxicity of saturated fatty acids has been implicated in the pathophysiology of cardiovascular disease, though their effects on cardiac myocytes are incompletely understood. We examined the effects of palmitate and the mono-unsaturated fatty acid oleate on neonatal rat ventricular myocyte cell biology. Palmitate (0.5mM) increased oxidative stress, as well as activation of the stress-associated protein kinases (SAPK) p38, Erk1/2, and JNK, following 18h and induced apoptosis in approximately 20% of cells after 24h. Neither antioxidants nor SAPK inhibitors prevented palmitate-induced apoptosis. Low concentrations of oleate (0.1mM) completely inhibited palmitate-induced oxidative stress, SAPK activation, and apoptosis. Increasing mitochondrial uptake of palmitate with l-carnitine decreased apoptosis, while decreasing uptake with the carnitine palmitoyl transferase-1 inhibitor perhexiline nearly doubled palmitate-induced apoptosis. These results support a model for palmitate-induced apoptosis, activation of SAPKs, and protein oxidative stress in myocytes that involves cytosolic accumulation of saturated fatty acids.     

Regulation of prohibitin expression during follicular development and atresia in the mammalian ovary

Prohibitin is a ubiquitous and highly conserved protein implicated as an important regulator in cell survival. Prohibitin content is inversely associated with cell proliferation, but it increases during granulosa cell differentiation as well as in earlier events of apoptosis in a temperature-sensitive granulosa cell line. In the present study, we have characterized the spatial expression patterns for prohibitin using established in vivo models for the induction of follicular development and atresia in the mammalian ovary. Comparative Western blot analyses of granulosa cell lysates from control ovaries and from ovaries primed with eCG or treated with eCG plus anti-eCG (gonadotropin withdrawal) were conducted. Prohibitin was immunolocalized in rat ovarian sections probed with antibodies against either proliferating cell nuclear antigen (PCNA) or cholesterol side-chain cleavage cytochrome P450 (P450(scc)) or in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeled sections. Additionally, porcine oocytes, zygotes, and blastocyts were also immunolocalized with prohibitin antibody. Immunolocalization revealed the presence of prohibitin in granulosa cells, theca-interstitial cells, and the oocyte. The results indicate that prohibitin protein expression in the gonadotropin-treated cells was upregulated. Immunoreactivity of prohibitin was inversely related to PCNA expression during follicular maturation and colocalized with P450(scc). Prohibitin appeared to be translocated from the cytoplasm to the nucleus in atretic follicles, germinal vesicle-stage oocytes, zygotes, and blastocysts. These results suggest that prohibitin has several functional regulatory roles in granulosa and theca-interstitial cells and in the ovum during follicular maturation and atresia. It is likely that prohibitin may play an important role in determining the fate of these cells and eventual follicular destiny.     

The tumor suppressor gene ARF as a sensor of oxidative stress

Oxidative stress as a result of either exogenous stimuli or cellular metabolism affects several cellular processes such as proliferation, apoptosis, cell death and senescence. Consequently, it is implicated in the pathogenesis of various human diseases like cancer, diabetes mellitus, atherosclerosis, neurodegenerative diseases and aging. Oxidative stress is implicated in carcinogenesis either by directly provoking DNA damage or through the regulation of intracellular signaling cascades. In both cases the cellular response to oxidative stress is determined by the cellular context. ARF, the alternative protein product of the CDKN2A locus has been recently recognized as a novel sensor of oxidative stress, in a β-catenin and Hsp70-mediated manner. Since, improved understanding of cellular responses to oxidative stress may facilitate the design of novel antineoplastic regimens, we herein review the mechanisms by which oxidative stress promotes carcinogenesis, focusing on the role of ARF as a sensor of oxidative stress.     

Daxx is required for stress-induced cell death and JNK activation

Daxx has been implicated in the modulation of apoptosis in response to various stimuli. In the nucleus, Daxx interacts and colocalizes with the promyelocytic leukemia protein (PML) into the PML-nuclear body. Moreover, overexpressed Daxx positively modulates FAS-ligand and TGFbeta-induced apoptosis. However, recent reports indicate that Daxx can also act as an antiapoptotic factor. As most studies on the role of Daxx in cell death have been conducted using tumour cell lines, we analysed the function of Daxx in physiological settings. We found that Daxx is induced upon exposure to ultraviolet (UV) irradiation and hydrogen peroxide treatment. We employed RNA interference to downregulate Daxx in primary fibroblasts. Remarkably, Daxx-depleted cells are resistant to cell death induced by both UV irradiation and oxidative stress. Furthermore, the downregulation of Daxx results in impaired MKK/c-Jun-N-terminal kinase (JNK) activation. This is the first evidence that Daxx promotes cell death and JNK activation in physiological conditions.     

Low doses of reactive oxygen species protect endothelial cells from apoptosis by increasing thioredoxin-1 expression

The redox regulator thioredoxin-1 (Trx-1) is required for the redox potential of the cell and exerts important functions in cell growth and apoptosis. Severe oxidative stress has been implicated in the oxidation of proteins and cell death. However, the role of low doses of reactive oxygen species (ROS) is poorly understood. Here, we show that 10 and 50 microM H2O2 and short-term exposure to shear stress significantly increased Trx-1 mRNA and protein levels in endothelial cells. Since it is known that Trx-1 exerts anti-apoptotic functions, we next investigated whether low doses of ROS can inhibit basal and serum-depletion induced endothelial cell apoptosis. Indeed, treatment of endothelial cells with 10 and 50 microM H2O2 significantly reduced apoptosis induction. Reduction of Trx-1 expression using an antisense oligonucleotide approach resulted in the induction of apoptosis and abolished the inhibitory effect of low doses of H2O2. Taken together, our results demonstrate that low doses of ROS act as signaling molecules and exert anti-apoptotic functions in endothelial cells via upregulation of the redox-regulator Trx-1.     

Tat-glyoxalase protein inhibits against ischemic neuronal cell damage and ameliorates ischemic injury

Methylglyoxal (MG), a metabolite of glucose, is the major precursor of protein glycation and induces apoptosis. MG is associated with neurodegeneration, including oxidative stress and impaired glucose metabolism, and is efficiently metabolized to S-D-lactoylglutathione by glyoxalase (GLO). Although GLO has been implicated as being crucial in various diseases including ischemia, its detailed functions remain unclear. Therefore, we investigated the protective effect of GLO (GLO1 and GLO2) in neuronal cells and an animal ischemia model using Tat-GLO proteins. Purified Tat-GLO protein efficiently transduced into HT-22 neuronal cells and protected cells against MG- and H₂O₂-induced cell death, DNA fragmentation, and activation of caspase-3 and mitogen-activated protein kinase. In addition, transduced Tat-GLO protein increased D-lactate in MG- and H₂O₂-treated cells whereas glycation end products (AGE) and MG levels were significantly reduced in the same cells. Gerbils treated with Tat-GLO proteins displayed delayed neuronal cell death in the CA1 region of the hippocampus compared with a control. Furthermore, the combined neuroprotective effects of Tat-GLO1 and Tat-GLO2 proteins against ischemic damage were significantly higher than those of each individual protein. Those results demonstrate that transduced Tat-GLO protein protects neuronal cells by inhibiting MG- and H₂O₂-mediated cytotoxicity in vitro and in vivo. Therefore, we suggest that Tat-GLO proteins could be useful as a therapeutic agent for various human diseases related to oxidative stress including brain diseases.     

Involvement of Bcl-2 and Bax in photodynamic therapy-mediated apoptosis. Antisense Bcl-2 oligonucleotide sensitizes RIF 1 cells to photodynamic therapy apoptosis

Photodynamic therapy (PDT), a promising treatment modality, is an oxidative stress that induces apoptosis in many cancer cells in vitro and tumors in vivo. Understanding the mechanism(s) involved in PDT-mediated apoptosis may improve its therapeutic efficacy. Although studies suggest the involvement of multiple pathways, the triggering event(s) responsible for PDT-mediated apoptotic response is(are) not clear. To investigate the role of Bcl-2 in PDT-mediated apoptosis, we employed Bcl-2-antisense and -overexpression approaches in two cell types differing in their responses toward PDT apoptosis. In the first approach, we treated radiation-induced fibrosarcoma (RIF 1) cells, which are resistant to silicon phthalocyanine (Pc 4)-PDT apoptosis, with Bcl-2-antisense oligonucleotide. This treatment resulted in sensitization of RIF 1 cells to PDT-mediated apoptosis as demonstrated by i) cleavage of poly(ADP-ribose) polymerase, ii) DNA ladder formation, iii) terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells, and iv) DEVDase activity. This treatment also resulted in oligonucleotide concentration-dependent decrease in cell viability and down-regulation of Bcl-2 protein with a concomitant increase in apoptosis. However, the level of Bax, a pro-apoptotic member of Bcl-2 family, remained unaltered. In the second approach, an overexpression of Bcl-2 in PDT apoptosis-sensitive human epidermoid carcinoma (A431) cells resulted in enhanced apoptosis and up-regulation of Bax following PDT. In both the approaches, the increased Bax/Bcl-2 ratio was associated with an increased apoptotic response of PDT. Our data also demonstrated that PDT results in modulation of other Bcl-2 family members in a way that the overall ratio of pro-apoptotic and anti-apoptotic member proteins favors apoptosis.     

Evaluation of Lovastatin Effects on Expression of Anti-apoptotic Nrf2 and PGC-1α Genes in Neural Stem Cells Treated with Hydrogen Peroxide

Reactive oxygen species and oxidative stress are associated with various cell processes, including cell survival and apoptosis. Oxidative stress has been implicated in the pathogenesis of several neurological disorders including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and multiple sclerosis (MS). In the present study, we evaluated the effects of lovastatin chemoprotection against hydrogen peroxide-induced oxidative stress in bone marrow stromal cell-derived neural stem cells (BMSC-derived NSCs) and whether it has protective effects. BMSC-derived NSCs were pretreated with different doses of lovastatin for 48 h and then exposed to 125 μM H₂O₂ for 30 min. Using MTT, TUNEL assay, and real-time RT-PCR, we evaluated the effects of lovastatin on cell survival, apoptosis, and PGC-1α and Nrf2 expression rates in pretreated BMSC-derived NSCs compared to control groups. Results showed that apoptosis rate in the lovastatin-pretreated BMSC-derived NSCs was significantly decreased compared to the control group. Our findings suggest that lovastatin protects NSCs against oxidative stress-induced cell death, and therefore, it may be used to promote the survival rate of NSCs and can be a candidate for treatment of oxidative stress-mediated neurological diseases.     

CHIP has a protective role against oxidative stress-induced cell death through specific regulation of Endonuclease G

Oxidative stress is implicated in carcinogenesis, aging, and neurodegenerative diseases. The E3 ligase C terminus of Hsc-70 interacting protein (CHIP) has a protective role against various stresses by targeting damaged proteins for proteasomal degradation, and thus maintains protein quality control. However, the detailed mechanism by which CHIP protects cells from oxidative stress has not been demonstrated. Here, we show that depletion of CHIP led to elevated Endonuclease G (EndoG) levels and enhanced cell death upon oxidative stress. In contrast, CHIP overexpression reduced EndoG levels, and resulted in reduced or no oxidative stress-induced cell death in cancer cells and primary rat cortical neurons. Under normal conditions Hsp70 mediated the interaction between EndoG and CHIP, downregulating EndoG levels in a Hsp70/proteasome-dependent manner. However, under oxidative stress Hsp70 no longer interacted with EndoG, and the stabilized EndoG translocated to the nucleus and degraded chromosomal DNA. Our data suggest that regulation of the level of EndoG by CHIP in normal conditions may determine the sensitivity to cell death upon oxidative stress. Indeed, injection of H₂O₂ into the rat brain markedly increased cell death in aged mice compared with young mice, which correlated with elevated levels of EndoG and concurrent downregulation of CHIP in aged mice. Taken together, our findings demonstrate a novel protective mechanism of CHIP against oxidative stress through regulation of EndoG, and provide an opportunity to modulate oxidative stress-induced cell death in cancer and aging.     

Capsaicin binds to prohibitin 2 and displaces it from the mitochondria to the nucleus

Capsaicin is widely used as a food additive and as an analgesic agent. Besides its well-known role in nociception, which is mediated by vanilloid receptor 1 specifically expressed in dorsal root ganglion neurons, capsaicin has also been considered as a potential anticancer agent, as it inhibits cell proliferation and induces apoptosis in various types of cancer cells. Here we identified a new molecular target of capsaicin from human myeloid leukemia cells. We show that capsaicin binds to prohibitin (PHB) 2, which is normally localized to the inner mitochondrial membrane, and induces its translocation to the nucleus. PHB2 is implicated in the maintenance of mitochondrial morphology and the control of apoptosis. We also provide evidence suggesting that capsaicin causes apoptosis directly through the mitochondria and that PHB2 contributes to capsaicin-induced apoptosis at multiple levels. This work will serve as an important foundation for further understanding of anticancer activity of capsaicin.     

Aldose reductase in keratinocytes attenuates cellular apoptosis and senescence induced by UV radiation

Although aldose reductase (AR) has been implicated in the cellular response to oxidative stress, the role of AR in ultraviolet-B (UVB)-induced cellular injury has not been investigated. Here, we show that an increased expression of AR in human keratinocytes modulates UVB-induced apoptotic cell death and senescence. Overexpression of AR in HaCaT cells significantly attenuated UVB-induced cellular damage and apoptosis, with a decreased generation of reactive oxygen species (ROS) and aldehydes. Ablation of AR with small interfering RNA or inhibition of AR activity abolished these effects. We also show that increased AR activity suppressed UVB-induced activation of the p38 and c-Jun N-terminal kinases, but did not affect the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase pathways. Similarly, UVB-induced translocation of Bax and Bcl-2 to mitochondria and cytosol, respectively, was markedly attenuated in cells overexpressing AR. Knockdown or inhibition of AR activity in primary cultured keratinocytes enhanced UVB-induced cellular senescence and increased the level of a cell-cycle regulatory protein, p53. Finally, cellular apoptosis induced by UVB radiation was significantly reduced in the epidermis of transgenic mice overexpressing human AR. These findings suggest that AR plays an important role in the cellular response to oxidative stress by sequestering ROS and reactive aldehydes generated in keratinocytes.     

Polyol pathway-dependent osmotic and oxidative stresses in aldose reductase-mediated apoptosis in human lens epithelial cells: role of AOP2

Aldose reductase (AR) has been implicated as a major contributor to the pathogenesis of diabetic cataracts. AR activation generates osmotic and oxidative stresses via the polyol pathway and induces cell death signals. Antioxidant protein 2 (AOP2) protects cells from oxidative stress. We investigated the effect of AR overexpression on polyol accumulation and on hyperglycemic oxidative stress and osmotic stress, as well as the effects of these stresses on human lens epithelial cell (hLEC) survival. hLECs overexpressing the AR became apoptotic during hyperglycemia and showed elevated levels of intracellular polyols. Glutathione and AOP2 levels were significantly decreased in these cells. Interestingly, supply of AOP2 and/or the AR inhibitor fidarestat protected the cells against hyperglycemia-induced death. Overexpression of AR increased osmotic and oxidative stresses, resulting in increased apoptosis in hLECs. Because AOP2 protects hyperglycemia-induced hLEC apoptosis, this molecule may have the potential to prevent hyperglycemia-mediated complications in diabetes.