P53 Mitochondrial Translocation Research Articles

Heat stress induced apoptosis is triggered by transcription-independent p53, Ca(2+) dyshomeostasis and the subsequent Bax mitochondrial translocation.

In this study, We demonstrated that Bax mitochondrial translocation plays a vital role in the initiation of the mitochondrial signaling pathway upon activation by heat stress. In addition, both p53 mitochondrial translocation and Ca2+ signal mediated MPTP opening activate Bax mitochondrial translocation. Employing pifithrin-α (a p53 mitochondrial translocation inhibitor) and CsA (a permeability transition pore (MPTP) inhibitor), we found that heat stress induced Bax mitochondrial translocation was significantly inhibited in cells pretreated with both PFT and CsA. Furthermore, we demonstrated that generation of reactive oxygen species (ROS) is a critical mediator in heat stress induced apoptosis and that the antioxidant MnTBAP significantly decreased heat stress induced p53 mitochondrial translocation and Ca2+ signal mediated MPTP opening, as well as the subsequent Bax mitochondrial translocation and activation of the caspase cascade. Taken together, our results indicate that heat stress induces apoptosis through the mitochondrial pathway with ROS dependent mitochondrial p53 translocation and Ca2+ dyshomeostasis, and the ensuing intro Bax mitochondrial translocation as the upstream events involved in triggering the apoptotic process observed upon cellular exposure to heat stress.

ROS-p53-cyclophilin-D signaling mediates salinomycin-induced glioma cell necrosis.

BackgroundThe primary glioblastoma multiforme (GBM) is the most malignant form of astrocytic tumor with an average survival of approximately 12–14 months. The search for novel and more efficient chemo-agents against this disease is urgent. Salinomycin induces broad anti-cancer effects; however, its role in GBM and the underlying mechanism are not clear.ResultsHere we found that salinomycin induced both apoptosis and necrosis in cultured glioma cells, and necrosis played a major role in contributing salinomycin’s cytotoxicity. Salinomycin induced p53 translocation to mitochondria, where it formed a complex with cyclophilin-D (CyPD). This complexation was required for mitochondrial permeability transition pore (mPTP) opening and subsequent programmed necrosis. Blockade of Cyp-D by siRNA-mediated depletion or pharmacological inhibitors (cyclosporin A and sanglifehrin A) significantly suppressed salinomycin-induced glioma cell necrosis. Meanwhile, p53 stable knockdown alleviated salinomycin-induced necrosis in glioma cells. Reactive oxygen species (ROS) production was required for salinomycin-induced p53 mitochondrial translocation, mPTP opening and necrosis, and anti-oxidants n-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) inhibited p53 translocation, mPTP opening and glioma cell death.ConclusionsThus, salinomycin mainly induces programmed necrosis in cultured glioma cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0174-1) contains supplementary material, which is available to authorized users.

Mitochondrial p53 phosphorylation induces Bak-mediated and caspase-independent cell death

Chemoresistance in cancer has previously been attributed to gene mutations or deficiency. Caspase mutations or Bax deficiency can lead to resistance to cancer drugs. We recently demonstrated that Bak initiates a caspase/Bax-independent cell death pathway. We show that Plumbagin (PL) (5-hydroxy-2-methyl-1,4-napthoquinone), a medicinal plant-derived naphthoquinone that is known to have anti-tumor activity in a variety of models, induces caspase-independent cell death in HCT116 Bax knockout (KO) or MCF-7 Bax knockdown (KD) cells that express wild-type (WT) Bak. The re-expression of Bax in HCT116 Bax KO cells fails to enhance the PL-induced cell death. Additionally, Bak knockdown by shRNA efficiently attenuates PL-induced cell death. These results suggest that PL-induced cell death depends primarily on Bak, not Bax, in these cells. Further experimentation demonstrated that p53 Ser15 phosphorylation and mitochondrial translocation mediated Bak activation and subsequent cell death. Knockdown of p53 or a p53 Ser15 mutant significantly inhibited p53 mitochondrial translocation and cell death. Furthermore, we found that Akt mediated p53 phosphorylation and the subsequent mitochondrial accumulation. Taken together, our data elaborate the role of Bak in caspase/Bax-independent cell death and suggest that PL may be an effective agent for overcoming chemoresistance in cancer cells with dysfunctional caspases.

Pivotal roles of p53 transcription-dependent and -independent pathways in manganese-induced mitochondrial dysfunction and neuronal apoptosis

Chronic exposure to excessive manganese (Mn) has been known to lead to neuronal loss and a clinical syndrome resembling idiopathic Parkinson's disease (IPD). p53 plays an integral role in the development of various human diseases, including neurodegenerative disorders. However, the role of p53 in Mn-induced neuronal apoptosis and neurological deficits remains obscure. In the present study, we showed that p53 was critically involved in Mn-induced neuronal apoptosis in rat striatum through both transcription-dependent and -independent mechanisms. Western blot and immunohistochemistrical analyses revealed that p53 was remarkably upregulated in the striatum of rats following Mn exposure. Coincidentally, increased level of cleaved PARP, a hallmark of apoptosis, was observed. Furthermore, using nerve growth factor (NGF)-differentiated PC12 cells as a neuronal cell model, we showed that Mn exposure decreased cell viability and induced apparent apoptosis. Importantly, p53 was progressively upregulated, and accumulated in both the nucleus and the cytoplasm. The cytoplasmic p53 had a remarkable distribution in mitochondria, suggesting an involvement of p53 mitochondrial translocation in Mn-induced neuronal apoptosis. In addition, Mn-induced impairment of mitochondrial membrane potential (ΔΨm) could be partially rescued by pretreatment with inhibitors of p53 transcriptional activity and p53 mitochondrial translocation, Pifithrin-α (PFT-α) and Pifithrin-μ (PFT-μ), respectively. Moreover, blockage of p53 activities with PFT-α and PFT-μ significantly attenuated Mn-induced reactive oxidative stress (ROS) generation and mitochondrial H2O2 production. Finally, we observed that pretreatment with PFT-α and PFT-μ ameliorated Mn-induced apoptosis in PC12 cells. Collectively, these findings implicate that p53 transcription-dependent and -independent pathways may play crucial roles in the regulation of Mn-induced neuronal death.

Salidroside inhibits oxygen glucose deprivation (OGD)/re-oxygenation-induced H9c2 cell necrosis through activating of Akt–Nrf2 signaling

Oxygen glucose deprivation (OGD)/re-oxygenation has been applied to cultured cardiomyocytes to create a cellular model of ischemic heart damage. In the current study, we explored the potential role of salidroside against OGD/re-oxygenation-induced damage in H9c2 cardiomyocytes, and studied the underlying mechanisms. We found that OGD/re-oxygenation primarily induced necrosis in H9c2 cells, which was inhibited by salidroside. Salidroside suppressed OGD/re-oxygenation-induced reactive oxygen species (ROS) production, p53 mitochondrial translocation and cyclophilin D (Cyp-D) association as well as mitochondrial membrane potential (MMP) decrease in H9c2 cells. Meanwhile, salidroside activated Akt and promoted transcription of NF-E2-related factor 2 (Nrf2)-regulated genes (heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO-1)). Significantly, Nrf2 shRNA knockdown or Akt inhibitors (LY 294002 and wortmannin) not only prevented salidroside-induced HO-1/NQO-1 transcription, but also alleviated salidroside-mediated cytoprotective effect against OGD/re-oxygenation in H9c2 cells. These observations suggest that salidroside activates Nrf2-regulated anti-oxidant signaling, and protects against OGD/re-oxygenation-induced H9c2 cell necrosis via activation of Akt signaling.

AS-2, a novel inhibitor of p53-dependent apoptosis, prevents apoptotic mitochondrial dysfunction in a transcription-independent manner and protects mice from a lethal dose of ionizing radiation

In a previous study, we reported that some tetradentate zinc(II) chelators inhibit p53 through the denaturation of its zinc-requiring structure but a chelator, Bispicen, a potent inhibitor of in vitro apoptosis, failed to show any efficient radioprotective effect against irradiated mice because the toxicity of the chelator to mice. The unsuitability of using tetradentate chelators as radioprotectors prompted us to undertake a more extensive search for p53-inhibiting agents that are weaker zinc(II) chelators and therefore less toxic. Here, we show that an 8-hydroxyquinoline (8HQ) derivative, AS-2, suppresses p53-dependent apoptosis through a transcription-independent mechanism. A mechanistic study using cells with different p53 characteristics revealed that the suppressive effect of AS-2 on apoptosis is specifically mediated through p53. In addition, AS-2 was less effective in preventing p53-mediated transcription-dependent events than pifithrin-μ (PFTμ), an inhibitor of transcription-independent apoptosis by p53. Fluorescence visualization of the extranuclear distribution of AS-2 also supports that it is ineffective on the transcription-dependent pathway. Further investigations revealed that AS-2 suppressed mitochondrial apoptotic events, such as the mitochondrial release of intermembrane proteins and the loss of mitochondrial membrane potential, although AS-2 resulted in an increase in the mitochondrial translocation of p53 as opposed to the decrease of cytosolic p53, and did not affect the apoptotic interaction of p53 with Bcl-2. AS-2 also protected mice that had been exposed to a lethal dose of ionizing radiation. Our findings indicate that some types of bidentate 8HQ chelators could serve as radioprotectors with no substantial toxicity in vivo.

Delayed treatment with NSC23766 in streptozotocin-induced diabetic rats ameliorates post-ischemic neuronal apoptosis through suppression of mitochondrial p53 translocation

NSC23766, a specific inhibitor of Rac1, has recently been shown to protect against cerebral ischemic injury, although the effects of NSC23766 in a diabetic model have not been examined. Therefore, the aim of our study was to investigate if NSC23766 provided neuroprotection in streptozotocin-induced diabetic rats and to determine the potential mechanism through which NSC23766 works. Diabetic Sprague–Dawley rats were subjected to right middle cerebral artery occlusion (MCAO) for 90 min. NSC23766 (10 or 30 mg kg−1) or isotonic saline were administered intraperitoneally twice daily starting 24 h after cerebral ischemia, for three consecutive days. Cerebral infarct volume, neurological deficit scores, neuronal apoptosis, and the release of cytochrome c, as well as the generation of ROS and mitochondrial integrity, were evaluated 96 h after reperfusion. In addition, the mitochondrial translocation of p53 and the expression of p53-upregulated modulator of apoptosis (PUMA) in the mitochondria of the cerebral ischemic cortex were determined by western blotting. NSC23766 not only ameliorated post-ischemic neuronal apoptosis but also decreased cerebral ischemia-induced mitochondrial p53 translocation and the expression of PUMA in mitochondria in diabetic rats. Thus, our data indicate that NSC23766 has therapeutic potential against cerebral ischemic reperfusion injury and that NSC23766 significantly ameliorates neuronal apoptosis by suppressing mitochondrial p53 translocation in streptozotocin-induced diabetic rats.

Mitochondrial protein cyclophilin-D-mediated programmed necrosis attributes to berberine-induced cytotoxicity in cultured prostate cancer cells

The prostate cancer is one of the leading causes of men’s cancer mortality. The development of alternative chemotherapeutic strategies is urgent. Berberine has displayed significant anti-prostate cancer activities. The underlying mechanisms are not fully understood. In the current study, we found that berberine induced apoptosis and programmed necrosis in cultured prostate cancer cells (LNCaP and PC-82 lines), and necrosis weighted more than apoptosis in contributing berberine’s cytotoxicity. We demonstrated that mitochondrial protein cyclophilin-D (Cyp-D) is required for berberine-induced programmed necrosis. Inhibition of Cyp-D by its inhibitors cyclosporin A (CSA) or sanglifehrin A (SFA), and by Cyp-D shRNA depletion alleviated berberine-induced prostate cancer cell necrosis (but not apoptosis). Our data found that in prostate cancer cells, berberine induced reactive oxygen species (ROS) production, which dictated P53 translocation to mitochondria, where it physically interacted with Cyp-D to open mitochondrial permeability transition pore (mPTP). The anti-oxidant N-acetylcysteine (NAC), the P53 inhibitor pifithrin-α (PFTα) as well as P53 siRNA knockdown suppressed berberine-induced P53 mitochondrial translocation and Cyp-D association, thus inhibiting mitochondrial membrane potential (MMP) decrease and prostate cancer cell necrosis. In summary, the results of the present study provide mechanistic evidence that both apoptosis and programmed necrosis attribute to berberine’s cytotoxicity in prostate cancer cells.

Nutlin-3 induces BCL2A1 expression by activating ELK1 through the mitochondrial p53-ROS-ERK1/2 pathway.

Nutlin-3 which occupies the p53 binding pocket in HDM2, has been reported to activate apoptosis through both the transcriptional activity-dependent and -independent programs of p53. Transcription-independent apoptosis by nutlin-3 is triggered by p53 which is translocated to mitochondria. However, we previously demonstrated that the nutlin-3-induced mitochondrial translocation of p53 stimulates ERK1/2 activation, an anti-apoptosis signal, via mitochondrial ROS generation. We report on how nutlin-3-stimulated ERK1/2 activity inhibits p53-induced apoptosis. Among the anti-apoptotic BCL2 family proteins, BCL2A1 expression was increased by nutlin-3 at both the mRNA and protein levels, and this increase was prevented by the inhibition of ERK1/2. TEMPO, a ROS scavenger, and PFT-μ , a blocker of the mitochondrial translocation of p53, also inhibited BCL2A1 expression as well as ERK1/2 phosphorylation. In addition, nutlin-3 stimulated phosphorylation of ELK1, which was prevented by all compounds that inhibited nutlin-3-induced ERK1/2 such as U0126, PFT-μ and TEMPO. Moreover, an increase in BCL2A1 expression was weakened by the knockdown of ELK1. Finally, nutlin-3-induced apoptosis was found to be potentiated by the knockdown of BCL2A1, as demonstrated by an increase of in hypo-diploidic cells and Annexin V-positive cells. Parallel to the increase in apoptotic cells, the knockdown of BCL2A1 augmented the cleavage of poly(ADP-ribose) polymerase-1. It is noteworthy that the augmented levels of apoptosis induced by the knockdown of BCL2A1 were comparable to those of apoptosis induced by U0126. Collectively, these results suggest that nutlin-3-activated ERK1/2 may stimulate the transcription of BCL2A1 via the activation of ELK1, and BCL2A1 expression may contribute to the inhibitory effect of ERK1/2 on nutlin-3-induced apoptosis, thereby constituting a negative feedback loop of p53-induced apoptosis.

Heat stress induces apoptosis through transcription-independent p53-mediated mitochondrial pathways in human umbilical vein endothelial cell

Cells apoptosis induced by intense heat stress is the prominent feature of heat-related illness. However, little is known about the biological effects of heat stress on cells apoptosis. Herein, we presented evidence that intense heat stress could induce early apoptosis of HUVEC cells through activating mitochondrial pathway with changes in mitochondrial membrane potential(ΔΨm), release of cytochrome c, and activation of caspase-9 and -3. We further revealed that p53 played a crucial role in heat stress-induced early apoptosis, with p53 protein rapidly translocated into mitochondria. Using pifithrin-α(PFT), a p53's mitochondrial translocation inhibitor, we found that pretreated with PFT, heat stress induced mitochondrial p53 translocation was significantly suppressed, accompanied by a significant alleviation in the loss of ΔΨm, cytochrome c release and caspase-9 activation. Furthermore, we also found that generation of reactive oxygen species (ROS) was a critical mediator in heat stress-induced apoptosis. In addition, the antioxidant MnTMPyP significantly decreased the heat stress-induced p53's mitochondrial translocation, followed by the loss of ΔΨm, cytochrome c release, caspase-9 activation and heat stress-mediated apoptosis. Conclusively, these findings indicate the contribution of the transcription-independent mitochondrial p53 pathway to early apoptosis in HUVEC cells induced by oxidative stress in response to intense heat stress.

Mitochondrial Translocation of p53 Modulates Neuronal Fate by Preventing Differentiation-Induced Mitochondrial Stress

Apoptosis regulatory proteins, such as p53, play a pivotal role in neural differentiation, through mechanisms independent of cell death. In addition, p53 has been identified as an important regulator of mitochondrial survival response, maintaining mitochondrial DNA (mtDNA) integrity and oxidative protection. The aim of this study was to determine the role of mitochondrial p53 in organelle damage and neural differentiation. Our results show that mitochondrial apoptotic events such as reactive oxygen species production, mitochondrial membrane permeabilization, and cytochrome c release are typical of early-stage mouse neural stem cell differentiation, which occurs 3-18 h after induction of differentiation, with no evidence of cell death. In addition, decreased mtDNA content, lipidated LC3 (LC3-II), colocalization of mitochondria and LC3-II puncta, and mitochondria-associated Parkin are consistent with activation of mitophagy. Importantly, at early stages of neural differentiation, p53 was actively translocated to mitochondria and attenuated mitochondrial oxidative stress, cytochrome c release, and mitophagy. Forced mitochondrial translocation of p53 increased neurogenic potential and neurite outgrowth. In conclusion, our results reveal a novel role for mitochondrial p53, which modulates mitochondrial damage and apoptosis-related events in the context of neural differentiation, thus enhancing neuronal fate.

Mitochondria-targeted Ogg1 and Aconitase-2 Prevent Oxidant-induced Mitochondrial DNA Damage in Alveolar Epithelial Cells

Mitochondria-targeted human 8-oxoguanine DNA glycosylase (mt-hOgg1) and aconitase-2 (Aco-2) each reduce oxidant-induced alveolar epithelial cell (AEC) apoptosis, but it is unclear whether protection occurs by preventing AEC mitochondrial DNA (mtDNA) damage. Using quantitative PCR-based measurements of mitochondrial and nuclear DNA damage, mtDNA damage was preferentially noted in AEC after exposure to oxidative stress (e.g. amosite asbestos (5-25 μg/cm(2)) or H2O2 (100-250 μM)) for 24 h. Overexpression of wild-type mt-hOgg1 or mt-long α/β 317-323 hOgg1 mutant incapable of DNA repair (mt-hOgg1-Mut) each blocked A549 cell oxidant-induced mtDNA damage, mitochondrial p53 translocation, and intrinsic apoptosis as assessed by DNA fragmentation and cleaved caspase-9. In contrast, compared with controls, knockdown of Ogg1 (using Ogg1 shRNA in A549 cells or primary alveolar type 2 cells from ogg1(-/-) mice) augmented mtDNA lesions and intrinsic apoptosis at base line, and these effects were increased further after exposure to oxidative stress. Notably, overexpression of Aco-2 reduced oxidant-induced mtDNA lesions, mitochondrial p53 translocation, and apoptosis, whereas siRNA for Aco-2 (siAco-2) enhanced mtDNA damage, mitochondrial p53 translocation, and apoptosis. Finally, siAco-2 attenuated the protective effects of mt-hOgg1-Mut but not wild-type mt-hOgg1 against oxidant-induced mtDNA damage and apoptosis. Collectively, these data demonstrate a novel role for mt-hOgg1 and Aco-2 in preserving AEC mtDNA integrity, thereby preventing oxidant-induced mitochondrial dysfunction, p53 mitochondrial translocation, and intrinsic apoptosis. Furthermore, mt-hOgg1 chaperoning of Aco-2 in preventing oxidant-mediated mtDNA damage and apoptosis may afford an innovative target for the molecular events underlying oxidant-induced toxicity.

Abstract W MP47: Mitochondrial P53 Inhibition Abolishes Detrimental Effect of Post-Stroke Isolation on Ischemic Injury

Background: Social isolation is an important contributing risk factor for increased stroke injury and delayed functional recovery. Although previous studies strongly suggest that isolation prior to stroke increase injury in experimental models, little is known regarding the effects of SI after injury. Most patients do not seek medical attention until after the injury occurs; the ability to manipulate post-stroke housing environments has translational significance. In this study, we investigated the effects of post-stroke isolation. Methods: C57Bl6 mice were housed as pairs (PH), male with ovariectomized female for 2 weeks, then subjected to right middle cerebral artery occlusion and then assigned to single housing (SI) or continued PH. The effects of SI on infarct volumes and neurological deficit scores (NDS) at 72h were assessed. Brain lysates from a separate cohort at 6h post-stroke were used for mitochondrial p53, caspase3 and BCl2 protein levels by western blot. Using pifithrin-μ (PFT-μ), a mitochondrial p53inhibitor (2mg/kg given i.p. at 3h & 24h after stroke) we then explored the role of P53 on infarct in SI mice. Results: Post-stroke SI exacerbated infarct size compared to PH mice in males (Cortex: 61.6±4.8 vs 38.4±5.1; p<0.05); (Striatum: 79.2±6.1 vs 60.4±5.7; p<0.05); (Total: 58.1±4.6 vs 34.4±5.1; p<0.05). This effect is more pronounced in females (Cortex: 64.3±5.1 vs 37.1±3.1; p<0.01); (Striatum: 82.1±4.6 vs 56.5±4.6; p<0.01); (Total: 51.4±3.4 vs 30.3±3.8; p<0.01). PH mice displayed improved NDS compared to SI mice. The detrimental effects of SI are observed with an increase in mitochondrial p53 translocation (n=6/grp; p<0.05). Inhibition of mitochondrial p53 abolished the effects of SI and reduced TUNEL positive cells, suggesting that p53 plays a critical role in housing effects. Conclusions: We found that post-stroke SI leads to worse outcome in both males and females, along with increased mitochondrial p53 expression. P53 is a critical stress sensor activated in response to stress and ischemia. Inhibition of mitochondrial P53 by PFT-μ abolished the detrimental effects of SI on stroke volume and reduced apoptotic cell death. These findings suggest that mitochondrial P53 inhibition may minimize the detrimental effects of SI in stroke.

Nutlin-3 induces HO-1 expression by activating JNK in a transcription-independent manner of p53

A recent study reported that p53 can induce HO-1 by directly binding to the putative p53 responsive element in the HO-1 promoter. In this study, we report that nutlin-3, a small molecule antagonist of HDM2, induces the transcription of HO-1 in a transcription-independent manner of p53. Nutlin-3 induced HO-1 expression at the level of transcription in human cancer cells such as U2OS and RKO cells. This induction of HO-1 did not occur in SAOS cells in which p53 was mutated and was prevented by knocking down the p53 protein using p53 siRNA transfection, but not by PFT-α, an inhibitor of the transcriptional activity of p53. Accompanying HO-1 expression, nutlin-3 stimulated the accumulation of ROS and the phosphorylation of MAPKs such as JNK, p38 MAPK and ERK1/2. Nutlin-3-induced HO-1 expression was suppressed by TEMPO, a ROS scavenger, and chemical inhibitors of JNK and p38 MAPK but not ERK1/2. In addition, nutlin‑3-induced phosphorylation of JNK but not p38 MAPK was inhibited by TEMPO. Notably, the levels of nutlin-3-induced ROS were correlated with the mitochondrial translocation of p53 and this induction was prevented by PFT-μ, an inhibitor of the mitochondrial translocation of p53. Consistent with the effect of the ROS scavenger and MAPK inhibitors, PFT-μ reduced HO-1 expression and the phosphorylation of JNK induced by nutlin-3. In the experiments of analyzing cell death, the knockdown of HO-1 augmented nutlin-3-induced apoptosis. Collectively, these results suggest that nutlin-3 induces HO-1 expression via the activation of both JNK which is dependent on ROS generated by p53 translocated to the mitochondria and p38 MAPK which appears to be stimulated by a ROS-independent mechanism, and this HO-1 induction may inhibit nutlin-3-induced apoptosis, constituting a negative feedback loop of p53-induced apoptosis.

Capsaicin induces apoptosis in human osteosarcoma cells through AMPK-dependent and AMPK-independent signaling pathways

Recent studies have focused on the anti-tumor activity of capsaicin. However, the potential effects of capsaicin in osteosarcoma cells and the underlying mechanisms are not fully understood. In the current study, we observed that capsaicin-induced growth inhibition and apoptosis in cultured osteosarcoma cells (U2OS and MG63), which were associated with a significant AMP-activated protein kinase (AMPK) activation. AMPK inhibition by compound C or RNA interference suppressed capsaicin-induced cytotoxicity, while AMPK activators (AICAR and A769662) promoted osteosarcoma cell death. For the mechanism study, we found that AMPK activation was required for capsaicin-induced mTORC1 (mTOR complex 1) inhibition, B cell lymphoma 2 (Bcl-2) downregulation and Bax upregulation in MG63 cells. Capsaicin administration induced p53 activation, mitochondrial translocation and Bcl-2 killer association, such effects were dependent on AMPK activation. Interestingly, we observed a significant pro-apoptotic c-Jun NH2-terminal kinases activation by capsaicin in MG63 cells, which appeared to be AMPK independent. In conclusion, capsaicin possessed strong efficacy against human osteosarcoma cells. Molecular studies revealed that capsaicin activated AMPK-dependent and AMPK-independent signalings to mediate cell apoptosis. The results of this study should have significant translational relevance in managing this deadly malignancy.

Oxygen Glucose Deprivation (OGD)/Re-Oxygenation-Induced In Vitro Neuronal Cell Death Involves Mitochondrial Cyclophilin-D/P53 Signaling Axis

Oxidative stress-induced neuronal cell death requires opening of the mitochondrial permeability transition pore. P53 mitochondrial translocation and association with Cyclophilin D (Cyp-D) is required for the pore opening. Here we tested this signaling axis in oxygen glucose deprivation (OGD)/re-oxygenation-induced in vitro neuronal death. Using mitochondrion immunoprecipitation, we found that p53 translocated to mitochondrion and associated with Cyp-D in SH-SY5Y cells exposed to (OGD)/re-oxygenation. Disruption of this complex by Cyp-D inhibitor Cyclosporine A (CsA), or by Cyp-D or p53 deficiency, significantly inhibited OGD/re-oxygenation-induced apoptosis-independent cell death. Conversely, over-expression of Cyp-D in SH-SY5Y cells caused spontaneous cell death, and these cells were more vulnerable to OGD/re-oxygenation. Finally, CsA or Cyp-D RNAi suppressed OGD/re-oxygenation-induced neuronal cell death in primary cultures. Together, our study suggests that OGD/re-oxygenation-induced in vitro cell death involves a mitochondrial Cyp-D/p53 signaling axis.

ERK1/2 activation mediated by the nutlin-3-induced mitochondrial translocation of p53

Nutlin-3 is a small-molecule antagonist of murine double minute 2 (MDM2) that blocks its binding to p53, leading to an increase in p53 protein levels. The tumor suppressor p53 induces growth arrest or apoptosis in response to genotoxic stress. Along with its growth-suppressive effect, it has been reported that p53 stimulates the mitogen-activated protein kinase (MAPK) pathway via the upregulation of heparin- binding epidermal growth factor-like growth factor (HB-EGF), an epidermal growth factor receptor (EGFR) ligand, and discoidin domain receptor 1 (DDR1), a tyrosine kinase receptor, at the transcription level. In this study, we propose a novel mechanism involved in the p53-induced MAPK activation. Nutlin-3 induced the phosphorylation of EGFR, MAPK/ERK kinase (MEK)1/2 and extracellular signal-regulated kinase (ERK)1/2 in U2OS human osteosarcoma cells harboring wild-type p53. This phosphorylation was completely inhibited by p53 siRNA, but not by pifithrin (PFT)-α, an inhibitor of the trans-criptional activity of p53. While the nutlin-3-induced EGFR phosphorylation was prevented by the inactivation of ERK1/2, the nutlin-3-induced MEK1/2-ERK1/2 phosphorylation was still observed in the cells in which EGFR phosphorylation was inhibited using EGFR siRNA and AG1478, an inhibitor of EGFR tyrosine kinase. Of note, nutlin-3 caused the accumulation of mitochondrial reactive oxygen species (ROS) and this correlated with the mitochondrial translocation of p53. 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO), a ROS scavenger, prevented the phosphorylation of ERK1/2. PFT-μ, which prevented the mitochondrial localization of p53, suppressed ERK1/2 phosphorylation, as well as ROS accumulation. Finally, we analyzed the effect of ERK1/2 activation on nutlin-3-induced apoptosis. The knockdown of MEK1/2 and ERK1/2 activity using U0126, a MEK inhibitor, or siRNAs, resulted in the enhancement of nutlin-3-induced apoptosis. In addition, TEMPO and PFT-μ also promoted nutlin-3-induced apoptosis. Taking the above findings into account, it can be concluded that nutlin-3 activates ERK1/2 prior to EGFR phosphorylation via ROS generation following the mitochondrial translocation of p53 and that nutlin-3-induced ERK1/2 activation may play a role in protecting U2OS cells from p53-dependent apoptosis, constituting a negative feedback loop for p53-induced apoptosis.

Pharmacological inhibition of GSK3 attenuates DNA damage-induced apoptosis via reduction of p53 mitochondrial translocation and Bax oligomerization in neuroblastoma SH-SY5Y CELLS

Glycogen synthase kinase-3 (GSK3) and p53 play crucial roles in the mitochondrial apoptotic pathway and are known to interact in the nucleus. However, it is not known if GSK3 has a regulatory role in the mitochondrial translocation of p53 that participates in apoptotic signaling following DNA damage. In this study, we demonstrated that lithium and SB216763, which are pharmacological inhibitors of GSK3, attenuated p53 accumulation and caspase-3 activation, as shown by PARP cleavage induced by the DNA-damaging agents doxorubicin, etoposide and camptothecin. Furthermore, each of these agents induced translocation of p53 to the mitochondria and activated the mitochondrial pathway of apoptosis, as evidenced by the release of cytochrome C from the mitochondria. Both mitochondrial translocation of p53 and mitochondrial release of cytochrome C were attenuated by inhibition of GSK3, indicating that GSK3 promotes the DNA damage-induced mitochondrial translocation of p53 and the mitochondrial apoptosis pathway. Interestingly, the regulation of p53 mitochondrial translocation by GSK3 was only evident with wild-type p53, not with mutated p53. GSK3 inhibition also reduced the phosphorylation of wild-type p53 at serine 33, which is induced by doxorubicin, etoposide and camptothecin in the mitochondria. Moreover, inhibition of GSK3 reduced etoposide-induced association of p53 with Bcl2 and Bax oligomerization. These findings show that GSK3 promotes the mitochondrial translocation of p53, enabling its interaction with Bcl2 to allow Bax oligomerization and the subsequent release of cytochrome C. This leads to caspase activation in the mitochondrial pathway of intrinsic apoptotic signaling.

Bax and B-cell-lymphoma 2 mediate proapoptotic signaling following chronic isolation stress in rat brain

Mitochondrial dysfunction has been implicated in several psychiatric disorders, including depression. Given that the B-cell-lymphoma 2 (Bcl-2) protein family plays a role in the regulation of mitochondrial apoptotic pathway, we hypothesized that ratio of proapoptotic to antiapoptotic proteins (e.g., Bcl-2-associated X protein (Bax)/Bcl-2) may determine prosurvival/proapoptotic intracellular signaling under stress. We tested this hypothesis by examining the effects of 2h of acute stress immobilization (IM) or cold (C), 21days of social isolation as chronic stress and combined stress (chronic stress followed by acute stress) on cytosolic/mitochondrial levels and ratios of Bax and Bcl-2 proteins in relation to cytosolic nitric oxide (NO) metabolites (nitrates and nitrites) and p53 protein redistribution between cytosolic and mitochondrial compartments in the prefrontal cortex (PFC) and hippocampus (HIPP) of male Wistar rats. The stress-induced changes in serum corticosterone (CORT) concentrations were also followed. Acute stressors resulting in an elevated CORT level did not change the Bax/Bcl-2 ratio in either brain region. However, chronic isolation, resulting in CORT levels similar to basal values, led to a translocation of mitochondrial Bcl-2 to the cytosol in the PFC. Furthermore, the Bax/Bcl-2 ratio in the PFC was significantly increased following chronic isolation and remained elevated after combined stressors. NO metabolites were increased by chronic isolation and the two combined stressors in the HIPP and following the combined stressors in the PFC. Translocation of p53 and proapoptotic Bax from the cytosol into mitochondria in response to NO overproduction following combined stressors was detected only in the PFC. These data indicate that chronic isolation stress exerts opposing actions on p53 and NO mechanisms in a tissue-specific manner (PFC vs. HIPP), triggering proapoptotic signaling via Bcl-2 translocation in the PFC.

Functional Study of Tumor Suppressor p53 Gene Variation: Effect on Cardiovascular Adaptation to Exercise Training

Aerobic exercise capacity is inversely related to CVD risk and all cause mortality. p53 plays a crucial role in mitochondrial DNA (mtDNA) maintenance, which is important for mitochondrial function and disease prevention. Here, we extend this concept and investigate whether the p53 codon 72 R/P polymorphism is associated with the adaptive response to aerobic exercise training (AEXT) with respect to enhancing aerobic capacity. 144 sedentary middle‐aged to older females underwent standardized AEXT for 6‐ month. We observed that R allele‐carriers (RR/RP, n = 117) had two‐fold greater improvement in VO2 max in response to the AEXT compared to the non‐carriers (PP, n = 27) (P < .05). To explore the likely molecular mechanisms of p53 being a potential regulator of cardiovascular adaptation to exercise, we further assessed 1) mitochondrial translocation of p53, 2) p53‐mtDNA binding complex, and 3) mtDNA contents in genotyped HUVEC (n = 10, from ten different donors) exposed to exercise‐simulated high flow shear stress (h‐flow, 20 dyne/cm2, 24 h) in vitro. We observed a dramatic increase in mitochondrial p53 level and p53‐mtDNA binding complex under h‐flow (n = 5). Also, RR/RP HUVEC (n = 7) showed significantly higher mtDNA contents compared to PP HUVEC (n = 3) (P < 0.01). Further molecular genetic mechanisms will be discussed. The findings in this study may provide new insights into physiological role of p53 in CV health.