D,L-sulforaphane-induced apoptosis in human breast cancer cells is regulated by the adapter protein p66Shc

Cancer chemopreventive response to D,L-sulforaphane (SFN), a synthetic racemic analogue of broccoli constituent L-sulforaphane, is partly attributable to apoptosis induction, but the mechanism of cell death is not fully understood. The present study demonstrates a critical role for adapter protein p66(Shc) in SFN-induced apoptosis. Immortalized mouse embryonic fibroblasts (MEF) derived from p66(shc) knockout mice were significantly more resistant to SFN-induced apoptosis, collapse of mitochondrial membrane potential, and reactive oxygen species (ROS) production compared with MEF obtained from the wild-type mice. Notably, a spontaneously immortalized and non-tumorigenic human mammary epithelial cell line (MCF-10A) was resistant to SFN-induced ROS production and apoptosis. Stable overexpression of manganese superoxide dismutase in MCF-7 and MDA-MB-231 human breast cancer cells conferred near complete protection against SFN-induced apoptosis and mitochondrial membrane potential collapse. SFN treatment resulted in increased S36 phosphorylation and mitochondrial translocation of p66(shc) in MDA-MB-231 and MCF-7 cells, and SFN-induced apoptosis was significantly attenuated by RNA interference of p66(shc) in both cells. SFN-treated MDA-MB-231 and MCF-7 cells also exhibited a marked decrease in protein level of peptidyl prolyl isomerase (Pin1), which is implicated in mitochondrial translocation of p66(shc) . However, stable overexpression of Pin1 failed to alter proapoptotic response to SFN at least in MCF-7 cells. Finally, SFN-induced S36 phosphorylation of p66(Shc) was mediated by protein kinase Cβ (PKCβ), and pharmacological inhibition of PKCβ significantly inhibited apoptotic cell death resulting from SFN exposure. In conclusion, the present study provides new insight into the mechanism of SFN-induced apoptosis involving PKCβ -mediated S36 phosphorylation of p66(shc).     

Rac1 leads to phosphorylation-dependent increase in stability of the p66shc adaptor protein: role in Rac1-induced oxidative stress

The rac1 GTPase and the p66shc adaptor protein regulate intracellular levels of reactive oxygen species (ROS). We examined the relationship between rac1 and p66shc. Expression of constitutively active rac1 (rac1V12) increased phosphorylation, reduced ubiquitination, and increased stability of p66shc protein. Rac1V12-induced phosphorylation and up-regulation of p66shc was suppressed by inhibiting p38MAPK and was dependent on serine 54 and threonine 386 in p66shc. Phosphorylation of recombinant p66shc by p38MAPK in vitro was also partly dependent on serine 54 and threonine 386. Reconstitution of p66shc in p66shc-null fibroblasts increased intracellular ROS generated by rac1V12, which was significantly dependent on the integrity of residues 54 and 386. Overexpression of p66shc increased rac1V12-induced apoptosis, an effect that was also partly dependent on serine 54 and threonine 386. Finally, RNA interference-mediated down-regulation of endogenous p66shc suppressed rac1V12-induced cell death. These findings identify p66shc as a mediator of rac1-induced oxidative stress. In addition, they suggest that serine 54 and threonine 386 are novel phosphorylatable residues in p66shc that govern rac1-induced increase in its expression, through a decrease in its ubiquitination and degradation, and thereby mediate rac1-stimulated cellular oxidative stress and death.     

The mammalian longevity-associated gene product p66shc regulates mitochondrial metabolism

Previous studies have determined that mice with a homozygous deletion in the adapter protein p66(shc) have an extended life span and that cells derived from these mice exhibit lower levels of reactive oxygen species. Here we demonstrate that a fraction of p66(shc) localizes to the mitochondria and that p66(shc-/-) fibroblasts have altered mitochondrial energetics. In particular, despite similar cytochrome content, under basal conditions, the oxygen consumption of spontaneously immortalized p66(shc-/-) mouse embryonic fibroblasts were lower than similarly maintained wild type cells. Differences in oxygen consumption were particularly evident under chemically uncoupled conditions, demonstrating that p66(shc-/-) cells have a reduction in both their resting and maximal oxidative capacity. We further demonstrate that reconstitution of p66(shc) expression in p66(shc-/-) cells increases oxygen consumption. The observed defect in oxidative capacity seen in p66(shc-/-) cells is partially offset by augmented levels of aerobic glycolysis. This metabolic switch is manifested by p66(shc-/-) cells exhibiting an increase in lactate production and a stricter requirement for extracellular glucose in order to maintain intracellular ATP levels. In addition, using an in vivo NADH photobleaching technique, we demonstrate that mitochondrial NADH metabolism is reduced in p66(shc-/-) cells. These results demonstrate that p66(shc) regulates mitochondrial oxidative capacity and suggest that p66(shc) may extend life span by repartitioning metabolic energy conversion away from oxidative and toward glycolytic pathways.     

The p66Shc Adaptor Protein Controls Oxidative Stress Response in Early Bovine Embryos

The in vitro production of mammalian embryos suffers from high frequencies of developmental failure due to excessive levels of permanent embryo arrest and apoptosis caused by oxidative stress. The p66Shc stress adaptor protein controls oxidative stress response of somatic cells by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidant gene expression. We have previously demonstrated a strong relationship with elevated p66Shc levels, reduced antioxidant levels and greater intracellular ROS generation with the high incidence of permanent cell cycle arrest of 2–4 cell embryos cultured under high oxygen tensions or after oxidant treatment. The main objective of this study was to establish a functional role for p66Shc in regulating the oxidative stress response during early embryo development. Using RNA interference in bovine zygotes we show that p66Shc knockdown embryos exhibited increased MnSOD levels, reduced intracellular ROS and DNA damage that resulted in a greater propensity for development to the blastocyst stage. P66Shc knockdown embryos were stress resistant exhibiting significantly reduced intracellular ROS levels, DNA damage, permanent 2–4 cell embryo arrest and diminished apoptosis frequencies after oxidant treatment. The results of this study demonstrate that p66Shc controls the oxidative stress response in early mammalian embryos. Small molecule inhibition of p66Shc may be a viable clinical therapy to increase the developmental potential of in vitro produced mammalian embryos.     

Expression profiles of p53 and p66shc during oxidative stress-induced senescence in fetal bovine fibroblasts

Somatic cells undergo a permanent cell cycle arrest, called cellular senescence, after a limited number of cell divisions in vitro. Both the tumor suppressor protein p53 and the stress-response protein p66(shc) are suggested to regulate the molecular events associated with senescence. This study was undertaken to investigate the effect of different oxygen tensions and oxidative stress on cell longevity and to establish the role of p53 and p66(shc) in cells undergoing senescence. As a model of cellular senescence, primary fetal bovine fibroblasts were cultured in either 20% O(2) or 5% O(2) atmospheres until senescence was reached. Fibroblasts cultured under 20% O(2) tension underwent senescence after 30 population doublings (PD), whereas fibroblasts cultured under 5% O(2) tension did not exhibit signs of senescence. Oxidative stress, as measured by protein carbonyl content, was significantly elevated in senescent cells compared to their younger counterparts and to fibroblasts cultured under 5% O(2) at the same PD. p53 mRNA gradually decreased in 20% O(2) cultured fibroblasts until senescence was reached, whereas p53 protein levels were significantly increased as well as p53 phosphorylation on serine 20, suggesting that p53 might be stabilized by posttranslational modifications during senescence. Senescence was also associated with high levels of p66(shc) mRNA and protein levels, while the levels remained low and stable in dividing fibroblasts under 5% O(2) atmosphere. Taken together, our results show an effect of oxidative stress on the replicative life span of fetal bovine fibroblasts as well as an involvement of p53, serine 20-p53 phosphorylation and p66(shc) in senescence.     

Phosphorylation of p66shc mediates 6-hydroxydopamine cytotoxicity

6-Hydroxydopamine (6-OHDA) is a neurotoxin that has been widely used to generate Parkinson's disease (PD) models. Increased oxidative stress is suggested to play an important role in 6-OHDA-induced cell death. Given the lessened susceptibility to oxidative stress exhibited by mice lacking p66shc, this study investigated the role of p66shc in the cytotoxicity of 6-OHDA. 6-OHDA induced cell death and p66shc phosphorylation at Ser36 in SH-SY5Y cells. Pre-treatment with the protein kinase C β (PKCβ) inhibitor hispidin suppressed 6-OHDA-induced p66shc phosphorylation. Elimination of H(2)O(2) by catalase reduced cell death and p66shc phosphorylation induced by 6-OHDA. Cells deficient in p66shc were more resistant to 6-OHDA-induced cell death than wild-type cells. Furthermore, reconstitution of wild-type p66shc, but not the S36A mutant, in p66shc-deficient cells increased susceptibility to 6-OHDA. These results indicate that H(2)O(2) derived from 6-OHDA is an important mediator of cell death and p66shc phosphorylation induced by 6-OHDA and that p66shc phosphorylation at Ser36 is indispensable for the cytotoxicity of 6-OHDA.     

p66shc inhibits pro-survival epidermal growth factor receptor/ERK signaling during severe oxidative stress in mouse renal proximal tubule cells

The fully executed epidermal growth factor receptor (EGFR)/Ras/MEK/ERK pathway serves a pro-survival role in renal epithelia under moderate oxidative stress. We and others have demonstrated that during severe oxidative stress, however, the activated EGFR is disconnected from ERK activation in cultured renal proximal tubule cells and also in renal proximal tubules after ischemia/reperfusion injury, resulting in necrotic death. Studies have shown that the tyrosine-phosphorylated p46/52 isoforms of the ShcA family of adaptor proteins connect the activated EGFR to activation of Ras and ERK, whereas the p66(shc) isoform can inhibit this p46/52(shc) function. Here, we determined that severe oxidative stress (after a brief period of activation) terminates activation of the Ras/MEK/ERK pathway, which coincides with ERK/JNK-dependent Ser(36) phosphorylation of p66(shc). Isoform-specific knockdown of p66(shc) or mutation of Ser(36) to Ala, but not to Asp, attenuated severe oxidative stress-mediated ERK inhibition and cell death in vitro. Also, severe oxidative stress (unlike ligand stimulation and moderate oxidative stress, both of which support survival) increased binding of p66(shc) to the activated EGFR and Grb2. This binding dissociated the SOS1 adaptor protein from the EGFR-recruited signaling complex, leading to termination of Ras/MEK/ERK activation. Notably, Ser(36) phosphorylation of p66(shc) and its increased binding to the EGFR also occurred in the kidney after ischemia/reperfusion injury in vivo. At the same time, SOS1 binding to the EGFR declined, similar to the in vitro findings. Thus, the mechanism we propose in vitro offers a means to ameliorate oxidative stress-induced cell injury by either inhibiting Ser(36) phosphorylation of p66(shc) or knocking down p66(shc) expression in vivo.     

High levels of p66shc and intracellular ROS in permanently arrested early embryos

A high incidence of permanent embryo arrest occurs during the first week of in vitro development. We hypothesize that this developmental arrest event is regulated by the stress adaptor protein p66shc, a genetic determinant of life span in mammals, which regulates ROS metabolism, apoptosis, and cellular senescence. The aim of this study was to assess the relationship between intracellular oxidative stress levels with the incidence of embryo arrest and the expression of senescent-associated genes in embryos produced under different oxygen tensions. Embryos cultured under 20% oxygen conditions showed approximately 10-fold increase in oxidative stress, 2-fold increase in the percentage of 2- to 4-cell arrest, and significantly lower developmental capabilities compared to embryos cultured under a 5% oxygen environment. Quantification by real-time PCR and by semiquantitative immunofluorescence showed significantly higher p66shc mRNA and protein levels, respectively, in embryos cultured in 20% versus those cultured in 5% oxygen atmosphere. No significant changes in p53 mRNA and protein levels were detected among embryos derived from both oxygen tensions. Taken together, these results demonstrate that p66shc, but not p53, is significantly more abundant in an embryo population that exhibits higher frequencies of embryo arrest and quantities of intracellular ROS. These results further substantiate that p66shc and oxidative stress are associated with a p53-independent embryonic arrest event for in vitro-produced embryos.     

PARK2-mediated mitophagy is involved in regulation of HBEC senescence in COPD pathogenesis

Cigarette smoke (CS)-induced mitochondrial damage with increased reactive oxygen species (ROS) production has been implicated in COPD pathogenesis by accelerating senescence. Mitophagy may play a pivotal role for removal of CS-induced damaged mitochondria, and the PINK1 (PTEN-induced putative kinase 1)-PARK2 pathway has been proposed as a crucial mechanism for mitophagic degradation. Therefore, we sought to investigate to determine if PINK1-PARK2-mediated mitophagy is involved in the regulation of CS extract (CSE)-induced cell senescence and in COPD pathogenesis. Mitochondrial damage, ROS production, and cell senescence were evaluated in primary human bronchial epithelial cells (HBEC). Mitophagy was assessed in BEAS-2B cells stably expressing EGFP-LC3B, using confocal microscopy to measure colocalization between TOMM20-stained mitochondria and EGFP-LC3B dots as a representation of autophagosome formation. To elucidate the involvement of PINK1 and PARK2 in mitophagy, knockdown and overexpression experiments were performed. PINK1 and PARK2 protein levels in lungs from patients were evaluated by means of lung homogenate and immunohistochemistry. We demonstrated that CSE-induced mitochondrial damage was accompanied by increased ROS production and HBEC senescence. CSE-induced mitophagy was inhibited by PINK1 and PARK2 knockdown, resulting in enhanced mitochondrial ROS production and cellular senescence in HBEC. Evaluation of protein levels demonstrated decreased PARK2 in COPD lungs compared with non-COPD lungs. These results suggest that PINK1-PARK2 pathway-mediated mitophagy plays a key regulatory role in CSE-induced mitochondrial ROS production and cellular senescence in HBEC. Reduced PARK2 expression levels in COPD lung suggest that insufficient mitophagy is a part of the pathogenic sequence of COPD.     

Replicative senescence induced by Romo1-derived reactive oxygen species

Persistent accumulation of DNA damage induced by reactive oxygen species (ROS) is proposed to be a major contributor toward the aging process. Furthermore, an increase in age-associated ROS is strongly correlated with aging in various species, including humans. Here we showed that the enforced expression of the ROS modulator 1 (Romo1) triggered premature senescence by ROS production, and this also contributed toward induction of DNA damage. Romo1-derived ROS was found to originate in the mitochondrial electron transport chain. Romo1 expression gradually increased in proportion to population doublings of IMR-90 human fibroblasts. An increase in ROS production in these cells with high population doubling was blocked by the Romo1 knockdown using Romo1 small interfering RNA. Romo1 knockdown also inhibited the progression of replicative senescence. Based on these results, we suggest that age-related ROS levels increase, and this contributes to replicative senescence, which is directly associated with Romo1 expression.     

Postmitotic neurons develop a p21‐dependent senescence‐like phenotype driven by a DNA damage response

In senescent cells, a DNA damage response drives not only irreversible loss of replicative capacity but also production and secretion of reactive oxygen species (ROS) and bioactive peptides including pro‐inflammatory cytokines. This makes senescent cells a potential cause of tissue functional decline in aging. To our knowledge, we show here for the first time evidence suggesting that DNA damage induces a senescence‐like state in mature postmitotic neurons in vivo. About 40–80% of Purkinje neurons and 20–40% of cortical, hippocampal and peripheral neurons in the myenteric plexus from old C57Bl/6 mice showed severe DNA damage, activated p38MAPkinase, high ROS production and oxidative damage, interleukin IL‐6 production, heterochromatinization and senescence‐associated β‐galactosidase activity. Frequencies of these senescence‐like neurons increased with age. Short‐term caloric restriction tended to decrease frequencies of positive cells. The phenotype was aggravated in brains of late‐generation TERC?/? mice with dysfunctional telomeres. It was fully rescued by loss of p21(CDKN1A) function in late‐generation TERC?/?CDKN1A?/? mice, indicating p21 as the necessary signal transducer between DNA damage response and senescence‐like phenotype in neurons, as in senescing fibroblasts and other proliferation‐competent cells. We conclude that a senescence‐like phenotype is possibly not restricted to proliferation‐competent cells. Rather, dysfunctional telomeres and/or accumulated DNA damage can induce a DNA damage response leading to a phenotype in postmitotic neurons that resembles cell senescence in multiple features. Senescence‐like neurons might be a source of oxidative and inflammatory stress and a contributor to brain aging.     

Mitochondrial longevity pathways

Production of reactive oxygen species (ROS) is a tightly regulated process, and increased levels of ROS within mitochondria are the principal trigger not only for mitochondrial dysfunctions but, more in general, for the diseases associated with aging, thus representing a powerful signaling molecules. One of the key regulators of ROS production, mitochondrial dysfunction, and aging is the 66-kDa isoform of the growth factor adapter shc (p66^shc) that is activated by stress and generates ROS within the mitochondria, driving cells to apoptosis. Accordingly, p66^shc knockout animals are one of the best characterized genetic model of longevity.On the other hand, caloric restriction is the only non-genetic mechanism that is shown to increase life span. Several studies have revealed a complex network of signaling pathways modulated by nutrients, such as IGF-1, TOR, sirtuins, AMP kinase, and PGC-1α that are connected and converge to inhibit oxidative stresses within the mitochondria. Animal models in which components of these signaling pathways are induced or silenced present a general phenotype characterized by the deceleration of the aging process. This review will summarize the main findings in the process that link mitochondria to longevity and the connections between the different signaling molecules involved in this intriguing relationship.     

Reactive oxygen species and mitochondrial sensitivity to oxidative stress determine induction of cancer cell death by p21

p21(Waf1/Cip1/Sdi1) is a cyclin-dependent kinase inhibitor that mediates cell cycle arrest. Prolonged p21 up-regulation induces a senescent phenotype in normal and cancer cells, accompanied by an increase in intracellular reactive oxygen species (ROS). However, it has been shown recently that p21 expression can also lead to cell death in certain models. The mechanisms involved in this process are not fully understood. Here, we describe an induction of apoptosis by p21 in sarcoma cell lines that is p53-independent and can be ameliorated with antioxidants. Similar levels of p21 and ROS caused senescence in the absence of significant death in other cancer cell lines, suggesting a cell-specific response. We also found that cells undergoing p21-dependent cell death had higher sensitivity to oxidants and a specific pattern of mitochondrial polarization changes. Consistent with this, apoptosis could be blocked with targeted expression of catalase in the mitochondria of these cells. We propose that the balance between cancer cell death and arrest after p21 up-regulation depends on the specific effects of p21-induced ROS on the mitochondria. This suggests that selective up-regulation of p21 in cancer cells could be a successful therapeutic intervention for sarcomas and tumors with lower resistance to mitochondrial oxidative damage, regardless of p53 status.     

The p53-p66shc-Manganese Superoxide Dismutase (MnSOD) network: a mitochondrial intrigue to generate reactive oxygen species

Once considered as a mere by-product of respiration, mitochondrial generation of reactive oxygen species (ROS) has recently emerged as a genetically controlled phenomenon, involved in complex intracellular signal transduction cascades that directly regulate cell survival and death in responses to environmental stressors. These cascades are involved in the pathogenesis of several major age-related diseases, such as cancer and neurodegeneration, and also appear to somehow regulate the "normal" ageing process. The present short review summarizes recent discoveries on mitochondrial reactive oxygen species regulation by p53, a tumor suppressor protein and p66shc, a protein implicated in the life-span determination. It also outlines the emerging network whereby these molecules cross-talk with each other and with the mitochondrial antioxidant system, namely MnSOD (SOD2), another life-span determining protein, to regulate oxidative stress in the organelle. This molecular circuit, which comprises two genetic determinants of longevity and a major tumor suppressor gene, also provides a theoretical framework connecting senescence and cancer.     

Reactive oxygen species mediated placental oxidative stress,mitochondrial content,and cell cycle progression through mitogen-activated protein kinases in intrauterine growth restricted pigs

Intrauterine growth restriction (IUGR) remains a significant obstacle in pig production; however, information regarding the relationship between reactive oxygen species (ROS)-induced placental dysfunction and IUGR is still unknown. This study aimed to explore the placental redox status, mitochondrial content, cellular progression, and mitogen-activated protein kinase (MAPK) pathways in IUGR. Placental tissues were collected from normal intrauterine gestation (NIUG) and IUGR fetuses at delivery. Compared with the NIUG, placental ROS production, lipid peroxidation, and DNA damage were increased in IUGR. Placental mitochondrial DNA (mtDNA) content and mtDNA-encoded gene expression decreased in IUGR. Moreover, p21 phosphorylation increased, cyclin E expression decreased in IUGR cases, which showed senescence characteristics. Analysis of signaling pathways showed that the ERK1/2 phosphorylation increased whereas the p38 and JNK phosphorylation decreased in IUGR. In cultured porcine trophectoderm (pTr) cells, exogenous H₂O₂ increased intracellular ROS production, decreased cell viability in a dose-dependent manner. Cell cycle distribution was found to arrest in S and G2/M phases. Our findings suggested that IUGR was associated with greater placental ROS and oxidative injury, which might be a factor that resulted in lower mitochondrial content, microvilli loss and senescence, and activation of MAPK pathways.