P53 Mitochondrial Translocation Research Articles

BRD7 regulates cellular senescence and apoptosis in ALS by modulating p21 expression and p53 mitochondrial translocation respectively

Cellular senescence is involved in the progression of neurodegenerative diseases. Motor neurons exhibit senescence-like alterations in ALS. BRD7, identified as a regulatory factor associated with cellular senescence, its function in ALS remains unclear. This study aims to investigate the potential role and mechanisms of BRD7 in ALS. We analyzed RNA levels using qRT-PCR, protein levels through immunofluorescence and western blot, and apoptosis via TUNEL staining. Cell transfection was conducted for in vitro experiments. The level of β-galactosidase was measured by β-galactosidase activity detection kit. ALS motor neurons exhibited senescence-like alterations, characterized by increased activity of p53, p21, and β-galactosidase, as well as reduced lamin B1 staining. Additionally, the expression of BRD7 was upregulated and induced cellular senescence and apoptosis. Downregulation of BRD7 alleviates the cellular senescence by inhibiting p21 rather than p53. Knockdown of BRD7 inhibited p53 mitochondrial translocation, leading to reduced apoptosis. Our results suggest that BRD7 plays an important role in the survival of ALS motor neurons. BRD7 knockdown can reduce cellular senescence and apoptosis by inhibiting p21 and p53 mitochondrial translocation.

The cell fate regulator DACH1 modulates ferroptosis through affecting P53/SLC25A37 signaling in fibrotic disease.

Dachshund homolog 1 (DACH1) is widely acknowledged for its involvement in regulating diverse cell fates, but its precise regulatory mechanism in ferroptosis remains elusive. In this study, we investigated whether DACH1 modulates ferroptosis through affecting P53/solute carrier family 25 member 37 (SLC25A37) signaling in hepatic fibrogenesis. CRISPR-Cas9 system was used to knockout DACH1 in HSC to determine the effect of DACH1 on ferroptosis. Immunoprecipitation, pulldown, and mouse model of hepatic fibrogenesis were used to analyze the potential molecular mechanism of ferroptosis regulation by DACH1. We found that ferroptosis inducers increased the protein expression of DACH1 by suppressing the ubiquitin-proteasome signaling. DACH1 knockout can resist ferroptosis, whereas DACH1 knockin can enhance it. Interestingly, the upregulation of DACH1 resulted in the mitochondrial translocation of p53 by inducing phosphorylation at serine 392. The mutation of serine 392 can prevent the combination of DACH1 and p53, the mitochondrial translocation of p53, and DACH1-mediated ferroptosis. Moreover, SLC25A37 was identified as a candidate target for mitochondrial p53. The binding of p53 to SLC25A37 can enhance the iron uptake capacity of SLC25A37, which may cause an overload of iron in the mitochondria and hyperactive mitochondrial electron transport chain. Knockdown of SLC25A37 can impair p53-mediated mitochondrial iron overload and ferroptosis. Furthermore, treatment with erastin can induce HSC ferroptosis and relieve fibrotic lesion damage in the mouse model of hepatic fibrogenesis. HSC-specific knockdown of DACH1, p53, and SLC25A37 can abolish the induction of HSC ferroptosis and reversal of hepatic fibrogenesis by erastin treatment. Our findings suggest that the DACH1/P53/SLC25A37 signaling pathway is a promising target for fibrotic disorders and reveals new regulatory mechanisms of ferroptosis.

O101 Mitochondrial P53 inhibition ameliorates acute pancreatitis through reduction of mitochondrial injury and cell necrosis

Abstract Introduction Persistent opening of the mitochondrial permeability transition pore (MPTP) is central to acute pancreatitis (AP). Mitochondrial translocation of P53 promotes MPTP opening, which induces necrotic cell death. We sought to determine whether and how mitochondrial P53 inhibition is protective in experimental AP. Methods Mitochondrial translocation of P53 was inhibited by pifithrin-mu (PFT-µ, mitochondrial specific P53 inhibitor) or by Trp53 KO. Mitochondrial membrane potential (ΔΨm) and necrosis of murine pancreatic acinar cells (PACs) were evaluated by confocal microscopy with TMRM and propidium iodide, respectively, in the presence of TLCS. Fluo4 was used for calcium imaging. Caerulein AP (Cer-AP) was induced in male (WT, P53−/+, P53−/−; 10-12 week old) mice by 7 hourly i.p. injections of 50 μg/kg caerulein. PFT-µ (5 mg/kg) was injected after the third injection of caerulein; sacrifice was made 12h after the first injection of caerulein. AP was assessed by standard biomarkers and blinded histopathology. Results TLCS-induced collapse of ΔΨm and cell necrosis of wild type PACs were significantly reduced in the presence of PFTµ or in PACs isolated from Trp53 KO mice; calcium overload was also reduced, indicative of improved calcium clearance, and ATP was maintained. Pharmacological or genetic P53 inhibition resulted in significant reductions of biochemical, immunological, and histopathological indices of severity in the Cer-AP model. Conclusion Inhibition of P53 maintained mitochondrial integrity, reduced PACs necrosis, and ameliorated the severity of Cer-AP. These data suggest that inhibition of mitochondrial translocation of P53 could be an effective strategy for the treatment of AP.

Elephant TP53-RETROGENE 9 induces transcription-independent apoptosis at the mitochondria

Approximately 20 TP53 retrogenes exist in the African and Asian elephant genomes (Loxodonta Africana, Elephas Maximus) in addition to a conserved TP53 gene that encodes a full-length protein. Elephant TP53-RETROGENE 9 (TP53-R9) encodes a p53 protein (p53-R9) that is truncated in the middle of the canonical DNA binding domain. This C-terminally truncated p53 retrogene protein lacks the nuclear localization signals and oligomerization domain of its full-length counterpart. When expressed in human osteosarcoma cells (U2OS), p53-R9 binds to Tid1, the chaperone protein responsible for mitochondrial translocation of human p53 in response to cellular stress. Tid1 expression is required for p53-R9-induced apoptosis. At the mitochondria, p53-R9 binds to the pro-apoptotic BCL-2 family member Bax, which leads to caspase activation, cytochrome c release, and cell death. Our data show, for the first time, that expression of this truncated elephant p53 retrogene protein induces apoptosis in human cancer cells. Understanding the molecular mechanism by which the additional elephant TP53 retrogenes function may provide evolutionary insight that can be utilized for the development of therapeutics to treat human cancers.

C60 Fullerene Reduces 3-Nitropropionic Acid-Induced Oxidative Stress Disorders and Mitochondrial Dysfunction in Rats by Modulation of p53, Bcl-2 and Nrf2 Targeted Proteins.

C60 fullerene as a potent free radical scavenger and antioxidant could be a beneficial means for neurodegenerative disease prevention or cure. The aim of the study was to define the effects of C60 administration on mitochondrial dysfunction and oxidative stress disorders in a 3-nitropropionic acid (3-NPA)-induced rat model of Huntington’s disease. Animals received 3-NPA (30 mg/kg i.p.) once a day for 3 consecutive days. C60 was applied at a dose of 0.5 mg/kg of body weight, i.p. daily over 5 days before (C60 pre-treatment) and after 3-NPA exposure (C60 post-treatment). Oxidative stress biomarkers, the activity of respiratory chain enzymes, the level of antioxidant defense, and pro- and antiapoptotic markers were analyzed in the brain and skeletal muscle mitochondria. The nuclear and cytosol Nrf2 protein expression, protein level of MnSOD, γ-glutamate-cysteine ligase (γ-GCLC), and glutathione-S-transferase (GSTP) as Nrf2 targets were evaluated. Our results indicated that C60 can prevent 3-NPA-induced mitochondrial dysfunction through the restoring of mitochondrial complexes’ enzyme activity, ROS scavenging, modulating of pro/antioxidant balance and GSH/GSSG ratio, as well as inhibition of mitochondria-dependent apoptosis through the limitation of p53 mitochondrial translocation and increase in Bcl-2 protein expression. C60 improved mitochondrial protection by strengthening the endogenous glutathione system via glutathione biosynthesis by up-regulating Nrf2 nuclear accumulation as well as GCLC and GSTP protein level.

WITHDRAWN: The cell fate determinant NUMB regulates ferroptosis through interacting with P53/MFRN1 signaling

Abstract Ferroptosis is a kind of programmed cell necrosis accompanied by iron-dependent lipid peroxidation, but its regulatory mechanisms are largely unknown. In the present study, we showed that the cell fate determinant NUMB may regulate ferroptosis through interacting with P53/MFRN1 signaling in hepatic stellate cells (HSCs). Upon exposure to ferroptosis-inducing compounds, the expression of NUMB protein was evidently elevated through the inhibition of the ubiquitin-proteasome pathway. The knockout of NUMB led to ferroptosis resistance, whereas the overexpression of NUMB promoted HSC ferroptosis. Intriguingly, increased NUMB appeared to trigger the mitochondrial translocation of p53 protein via binding to its N-terminal transactivation domain. Site-directed mutations of serine 392 evidently impaired the binding of NUMB to p53, blocked the mitochondrial translocation of p53 protein, and prevented NUMB-enhanced HSC ferroptosis. Importantly, mitochondrial p53 could interact with mitoferrin-1 (MFRN1) to increase the activity of MFRN1, which may result in the overload of mitochondrial iron and the hyperfunction of electron transfer chain (ETC). Attractively, the knockdown of MFRN1 could damage NUMB- or p53-mediated HSC ferroptosis. In mice, treatment with erastin alleviated murine liver fibrosis by inducing HSC ferroptosis. HSC-specific blockade of NUMB/P53/MFRN1 signaling abrogated erastin-enhanced HSC ferroptosis. Noteworthy, we determined the impact of sorafenib on HSC ferroptosis in advanced fibrotic patients with hepatocellular carcinoma receiving sorafenib monotherapy. As expected, the upregulation of NUMB protein, the mitochondrial translocation of p53, the bind of p53 to MFRN1, mitochondrial iron accumulation, ETC hyperfunction, lipid peroxidation, and ferroptosis induction occurred in human HSCs. Overall, these results revealed novel molecular mechanisms and signaling pathways of ferroptosis, and further suggest NUMB/P53/MFRN1 signaling as potential targets for fibrotic diseases.

Rational design, synthesis and biological evaluation of triphenylphosphonium-ginsenoside conjugates as mitochondria-targeting anti-cancer agents

In anti-cancer therapy, targeting a single gene or a single metabolic pathway usually cannot effectively cure cancer, while targeting cellular mitochondria might be effective based on the role of mitochondria in the occurrence and development of cancer. Anti-cancer study on ginsenosides AD-1, AD-2 and PD have proved that they have broad spectrum anti-cancer activities in vitro and in vivo. However, they are not active at sufficiently low concentration, and their lower selectivity and cell permeability hindered therapeutic applications. In the present study, AD-1, AD-2 and PD are incorporated with triphenylphosphonium at the OH group in C-3 position through different length of alkyl chains, with the aim of targeting mitochondria and improving the efficacy and selectivity of parent compounds. Biological studies suggested that most of the conjugates had enhanced anti-proliferative activity, in particular, conjugate 1f had an IC50 value of 0.76 μM against MCF-7 cells while showed a high degree of selectivity to MCF-7 cells. In addition, 1f was obviously increased accumulation in the mitochondria, and induced apoptosis, elevated reactive oxygen species (ROS) level and caused mitochondrial membrane potential collapse in MCF-7 cells. Further study revealed that ROS-related mitochondrial translocation of p53 was also involved in 1f-induced mitochondrial apoptotic pathway. The results demonstrated that 1f could be a promising lead for the development of mitocans. These findings also provide a reference for the development of ginsenoside for mitochondria-targeted anti-cancer drugs.

MDM2 Antagonists Induce a Paradoxical Activation of Erk1/2 through a P53-Dependent Mechanism in Dedifferentiated Liposarcomas: Implications for Combinatorial Strategies.

The MDM2 gene is amplified in dedifferentiated liposarcoma (DDLPS). Treatment with MDM2 antagonists is a promising strategy to treat DDLPS; however, drug resistance is a major limitation when these drugs are used as a single agent. This study examined the impact of MDM2 antagonists on the mitogen-activated protein kinase (MAPK) pathway in DDLPS and investigated the potential synergistic activity of a MAPK kinase (MEK) inhibitor in combination with MDM2 antagonists. We identified a synergistic effect and identified the mechanism behind it. Combination effects of MDM2 antagonists and a MEK inhibitor were analyzed in a patient-derived xenograft mouse model and in DDLPS and leiomyosarcoma cell lines using different cell proliferation assays and immunoblot analysis. MDM2 antagonist (RG7388)-resistant IB115 [P4] cells and p53-silenced DDLPS cells were also established to understand the importance of functional p53. We found that MDM2 antagonists induced an upregulation of phosphorylated extracellular signal-regulated kinase (p-ERK) in DDLPS cells. The upregulation of p-ERK occurred due to mitochondrial translocation of p53, which resulted in increased production of reactive oxygen species, causing the activation of receptor tyrosine kinases (RTKs). Activated RTKs led to the activation of the downstream MEK/ERK signaling pathway. Treatment with a MEK inhibitor resulted in decreased expression of p-ERK, causing significant anti-tumor synergy when combined with MDM2 antagonists. Our results provide a framework for designing clinical studies of combination therapies in DDLPS patients.

The BRD7-P53-SLC25A28 axis regulates ferroptosis in hepatic stellate cells

Ferroptosis is a recently discovered form of programmed cell death, but its regulatory mechanisms are not fully understood. In the current study, we reported that the BRD7-P53-SLC25A28 axis played a crucial role in regulating ferroptosis in hepatic stellate cells (HSCs). Upon exposure to ferroptosis inducers, bromodomain-containing protein 7 (BRD7) protein expression was remarkably increased through the inhibition of the ubiquitin-proteasome pathway. CRISPR/Cas9-mediated BRD7 knockout conferred resistance to HSC ferroptosis, whereas specific BRD7 plasmid-mediated BRD7 overexpression facilitated HSC ferroptosis. Interestingly, the elevated BRD7 expression exhibited to promote p53 mitochondrial translocation via direct binding with p53 N-terminal transactivation domain (TAD), which may be the underlying mechanisms for BRD7-enhanced HSC ferroptosis. Site-directed mutations of serine 392 completely blocked the binding of BRD7 to p53, and, in turn, prevented p53 mitochondrial translocation and HSC ferroptosis. Importantly, mitochondrial p53 interacted with solute carrier family 25 member 28 (SLC25A28) to form complex and enhanced the activity of SLC25A28, which could lead to the abnormal accumulation of redox-active iron and hyperfunction of electron transfer chain (ETC). SLC25A28 knockdown impaired BRD7-or p53-mediated ferroptotic events. In mice, erastin treatment ameliorated pathological damage of liver fibrosis through inducing HSC ferroptosis. HSC-specific blockade of BRD7-P53-SLC25A28 axis could abrogate erastin-induced HSC ferroptosis. Of note, we analyzed the effect of sorafenib on HSC ferroptosis in advanced fibrotic patients with hepatocellular carcinoma receiving sorafenib monotherapy. Attractively, BRD7 upregulation, p53 mitochondrial translocation, combination of SLC25A28 and p53, and ferroptosis induction occurred in primary human HSCs. Overall, these findings reveal novel signal transduction and regulatory mechanism of ferroptosis, and also suggest BRD7-P53-SLC25A28 axis as potential targets for liver fibrosis.

Remote Ischemic Postconditioning Inhibits Hippocampal Neuronal Apoptosis and Mitophagy After Cardiopulmonary Resuscitation in Rats.

Studies have shown that remote ischemic post-conditioning can improve brain damage caused by ischemia and hypoxia. However, the specific mechanism underlying this phenomenon is still unclear. The purpose of this study was to investigate the effects of remote ischemic post-conditioning on neuronal apoptosis and mitophagy after cardiopulmonary resuscitation (CPR) in rats. Male Sprague-Dawley rats were used to establish an asphyxia cardiac arrest model by clamping the tracheal duct. First, the expression levels of P53, Cytochrome c (Cytc), and Parkin in the cytoplasm and mitochondria were observed at 3, 6, 24, and 72 h after the restoration of spontaneous circulation (ROSC). Then neurological deficit scores, hippocampal neuron apoptosis, mitochondrial P53 and Parkin, cytoplasmic Cytc, and neuron ultrastructure were evaluated 24 h after ROSC. P53 and Parkin can translocate from the cytoplasm to the mitochondria, promoting the translocation of cytoplasmic Cytc to mitochondria after CPR, reaching a peak at 24 h after the ROSC. The P53 inhibitor Pifithrin-μ reduced apoptosis induced by P53 mitochondrial translocation. Apoptosis was induced after cardiac arrest and attenuated by remote ischemic postconditioning via inhibiting P53 mitochondrial translocation and the release of Cytc to the cytoplasm. In addition, remote ischemic postconditioning could inhibit Parkin-mediated mitophagy. Taken together, our results show that remote ischemic post-conditioning improves neural function after CPR by inhibiting P53 mitochondrial translocation-induced apoptosis and Parkin-mediated mitophagy.

Shen Shuai II Recipe Attenuates Apoptosis in 5/6 Renal Ablation/Infarction Rats by Inhibiting p53 and the Mitochondrial Pathway of Apoptosis.

Background. Chronic kidney disease (CKD) is a global health burden with high mortality and morbidity. Clinical efficacy has been demonstrated for Shen Shuai II Recipe (SSR), an approved and widely used Chinese herbal medicine for over 20 years in China, to attenuate CKD progression. In this study, we explored the underlying molecular mechanisms of SSR benefits and studied its effects on apoptosis, a critical process in CKD development and progression. CKD was induced in rats with 5/6 renal ablation and infarction (A/I). Eight weeks after SSR treatment, we mainly assessed the severity of renal injury and fibrosis, the translocation of apoptotic factors in the mitochondrial apoptosis pathway, the degree of mitochondrial dysfunction, and the nuclear and mitochondrial translocation of p53. Furthermore, we detected the interaction of p53 with antiapoptotic Bcl-xL and Bcl-2 proteins. Our results showed that SSR significantly attenuated renal injury and fibrosis and inhibited the mitochondrial accumulation of proapoptotic proteins Bax and Puma and release of cytochrome c from mitochondria to the cytosol in a rat CKD model. In addition, SSR also improved the mitochondrial function and inhibited the nuclear and mitochondrial translocation of p53. In addition, SSR suppressed the p53 transactivation and the interaction of p53 with Bcl-xL and Bcl-2. These results suggested that SSR could block apoptosis in CKD by inhibiting p53 transcriptional-dependent and transcriptional-independent proapoptotic function and the mitochondrial pathway of apoptosis.

TPP-related mitochondrial targeting copper (II) complex induces p53-dependent apoptosis in hepatoma cells through ROS-mediated activation of Drp1

BackgroundIn recent years, copper complexes have gradually become the focus of potential anticancer drugs due to their available redox properties and low toxicity. In this study, a novel mitochondrion-targeting copper (II) complex, [Cu (ttpy-tpp)Br2] Br (simplified as CTB), is first synthesized by our group. CTB with tri-phenyl-phosphine (TPP), a targeting and lipophilic group, can cross the cytoplasmic and mitochondrial membranes of tumor cells. The present study aims to investigate how CTB affects mitochondrial functions and exerts its anti-tumor activity in hepatoma cells.MethodsMultiple molecular experiments including Flow cytometry, Western blot, Immunofluorescence, Tracker staining, Transmission Electron Microscopy and Molecular docking simulation were used to elucidate the underlying mechanisms. Human hepatoma cells were subcutaneously injected into right armpit of male nude mice for evaluating the effects of CTB in vivo.ResultsCTB induced apoptosis via collapse of mitochondrial membrane potential (MMP), ROS production, Bax mitochondrial aggregation as well as cytochrome c release, indicating that CTB-induced apoptosis was associated with mitochondrial pathway in human hepatoma cells. Mechanistic study revealed that ROS-related mitochondrial translocation of p53 was involved in CTB-mediated apoptosis. Simultaneously, elevated mitochondrial Drp1 levels were also observed, and interruption of Drp1 activation played critical role in p53-dependent apoptosis. CTB also strongly suppressed the growth of liver cancer xenografts in vivo.ConclusionIn human hepatoma cells, CTB primarily induces mitochondrial dysfunction and promotes accumulation of ROS, leading to activation of Drp1. These stimulation signals accelerate mitochondrial accumulation of p53 and lead to the eventual apoptosis. Our research shows that CTB merits further evaluation as a chemotherapeutic agent for the treatment of Hepatocellular carcinoma (HCC).

Stretching magnitude-dependent inactivation of AKT by ROS led to enhanced p53 mitochondrial translocation and myoblast apoptosis.

Previously, we had shown that high magnitude stretch (HMS), rather than low magnitude stretch (LMS), induced significant apoptosis of skeletal muscle C2C12 myoblasts. However, the molecular mechanism remains obscure. In this study, we found that p53 protein accumulated in the nucleus of LMS-loaded cells, whereas it translocated into mitochondria of HMS-loaded cells. Knocking down endogenous p53 by shRNA abrogated HMS-induced apoptosis. Furthermore, we demonstrated that overaccumulation of reactive oxygen species (ROS) during HMS-inactivated AKT that was activated in LMS-treated cells, which accounted for the distinct p53 subcellular localizations under HMS and LMS. Blocking ROS generation by N-acetylcysteine (NAC) or overexpressing constitutively active AKT vector (CA-AKT) inhibited HMS-incurred p53 mitochondrial translocation and promoted its nuclear targeting. Moreover, both NAC and CA-AKT significantly attenuated HMS-induced C2C12 apoptosis. Finally, we found that Ser389 phosphorylation of p53 was a downstream event of ROS-inactivated AKT pathway, which was critical to p53 mitochondrial trafficking during HMS stimuli. Transfecting p53-shRNA C2C12s with the mutant p53 (S389A) that was unable to target p53 to mitochondria underwent significantly lower apoptosis than transfection with wild-type p53. Altogether, our study uncovered that mitochondrial localization of p53, resulting from p53 Ser389 phosphorylation through ROS-inactivated AKT pathway, prompted C2C12 myoblast apoptosis during HMS stimulation.

Ser46 phosphorylation of p53 is an essential event in prolyl-isomerase Pin1-mediated p53-independent apoptosis in response to heat stress

Heat stroke has increased in frequency worldwide in recent years and continues to have a high morbidity and mortality. Identification of the mechanisms mediating heat stoke is important and necessary. Our preliminary study revealed heat stress (HS)-induced apoptosis of vascular endothelial cells was associated with reactive oxygen species (ROS)-induced p53 translocation into mitochondria. Previous studies have suggested the prolyl-isomerase Pin1 regulates p53 functioning through specific binding to p53 phosphorylation sites. Based on these studies, we presumed Pin1 is a key intermediate in regulation of mitochondrial p53 translocation through a HS-induced ROS-p53 transcription-independent apoptosis pathway. In this context, we revealed p53 had a crucial role in a HS-induced mitochondrial apoptotic pathway, where p53 protein rapidly translocated into mitochondria in endothelial cells both in vitro and in vivo. In particular, HS caused an increase in p53 phosphorylation at Ser46 that facilitated interactions with phosphorylation-dependent prolyl-isomerase Pin1, which has a key role in promoting HS-induced localization of p53 to mitochondria. Furthermore, we also found ROS production was a critical mediator in HS-induced Pin1/p53 signaling and was involved in regulating mitochondrial apoptosis pathway activation. Therefore, we have contributed to our profound understanding of the mechanism underlying HS-induced endothelial dysfunction in an effort to reduce the mortality and morbidity of heat stroke.

P53 knockout mice are protected from cocaine-induced kindling behaviors via inhibiting mitochondrial oxidative burdens, mitochondrial dysfunction, and proapoptotic changes

Previously we demonstrated that p53 mediates dopaminergic neurotoxicity via inducing mitochondrial burdens and proapoptotsis. However, little is known about the role of p53 in the excitotoxicity induced by psychostimulant, such as cocaine. Cocaine-induced kindling (convulsive) behaviors significantly increased p53 expression in the brain. Cocaine-induced p53 expression was more pronounced in hippocampus than in striatum or prefrontal cortex. Genetic depletion of p53 significantly attenuated cocaine-induced convulsive behaviors, followed by c-Fos immunoreactivity, and oxidative burdens in the hippocampus of mice. The antioxidant potentials mediated by genetic depletion of p53 were more pronounced in the mitochondrial-than cytosolic-fraction. Depletion of p53 significantly attenuated the changes in mitochondrial transmembrane potential, intramitochondrial Ca2+ level, and mitochondrial oxidative burdens induced by cocaine. Consistently, depletion of p53 significantly inhibited mitochondrial p53 translocation, and cleaved-PKCδ induced by cocaine. In addition, depletion of p53 protected from cytosolic cytochrome c release, and pro-apoptotic changes induced by cocaine. Importantly, the protective/anticonvulsant potentials by genetic depletion of p53 were comparable to those by pifithrin-μ (PFT), a p53 inhibitor. Our results suggest that depletion of p53 offers anticonvulsive and neuroprotective potentials mainly via attenuating mitochondrial oxidative burdens, mitochondrial dysfunction, and pro-apoptotic signalings against cocaine-induced convulsive neurotoxicity.

Fenbendazole acts as a moderate microtubule destabilizing agent and causes cancer cell death by modulating multiple cellular pathways

Drugs that are already clinically approved or experimentally tested for conditions other than cancer, but are found to possess previously unrecognized cytotoxicity towards malignant cells, may serve as fitting anti-cancer candidates. Methyl N-(6-phenylsulfanyl-1H benzimidazol-2-yl) carbamate [Fenbendazole, FZ], a benzimidazole compound, is a safe and inexpensive anthelmintic drug possessing an efficient anti-proliferative activity. In our earlier work, we reported a potent growth-inhibitory activity of FZ caused partially by impairment of proteasomal function. Here, we show that FZ demonstrates moderate affinity for mammalian tubulin and exerts cytotoxicity to human cancer cells at micromolar concentrations. Simultaneously, it caused mitochondrial translocation of p53 and effectively inhibited glucose uptake, expression of GLUT transporters as well as hexokinase (HK II) - a key glycolytic enzyme that most cancer cells thrive on. It blocked the growth of human xenografts in nu/nu mice model when mice were fed with the drug orally. The results, in conjunction with our earlier data, suggest that FZ is a new microtubule interfering agent that displays anti-neoplastic activity and may be evaluated as a potential therapeutic agent because of its effect on multiple cellular pathways leading to effective elimination of cancer cells.

Role of cytoplasmic p53-mediated suppression of autophagy in heat stress-induced injury of cultured mouse aortic endothelial cells

To explore the association of cytoplasmic p53 with autophagy and apoptosis of primary aortic endothelial cells (MAECs) exposed to heat stress. Cultured mouse MAECs were exposed to heat stress induced by incubation at 43 ℃ for 2 h, with the cells in routine culture condition (37 ℃, 5% CO2) as the control group. All the cells were further incubated for 1, 3, 6 or 9 h at 37 ℃ before treatment with the autophagy inhibitor 3-MA (5 mmol/L), the autophagy inducer rapamycin (20 μmol/L), or the p53 inhibitor PFT (10 μmol/L) for 1 h. After the treatments, the cell viability was measured with CCK8 method, cell apoptosis analyzed by flow cytometry, and the mitochondrial membrane potential detected with flow cytometry with JC-1 staining; the subcellular localization of p53 and the autophagy- associated protein LC3-Ⅱ was detected with immunofluorescence staining, and their protein expressions were analyzed using Western blotting. Compared with the control cells, MAECs exposed to heat stress showed significantly decreased viability (P < 0.05). At 6 h after the exposure, the cells exhibited significantly decreased mitochondrial membrane potential with increased apoptotic rate (P < 0.05). The cytoplasmic fraction of p53 expression decreased and its mitochondrial fraction increased gradually with time within 6 h after heat stress. Treatment with 3- MA further decreased the mitochondrial membrane potential and significantly increased the apoptotic rate of the exposed cells (P < 0.05), while rapamycin obviously reversed these heat stress-induced cell injuries (P < 0.05). PFT significantly enhanced the expression of LC3-Ⅱ and also inhibited heat stress-induced mitochondrial membrane potential reduction and cell apoptosis (P < 0.05). Heat stress induces mitochondrial damage and apoptosis in MAECs possibly in relation with mitochondrial translocation of cytoplasmic p53 to result in autophagy inhibition.

Abstract 552: PEPD is an essential regulator of p53 tumor suppressor

Abstract Peptidase D (PEPD), also known as prolidase among other names, is an enzyme that hydrolyzes dipeptides with proline or hydroxyproline at the carboxy terminus and is believed to be important for collagen metabolism, as proline and hydroxyproline are very abundant in collagen. Interestingly, we recently found that eliminating cellular PEPD causes cell death and tumor regression due to p53 activation. Here, we show that PEPD binds to and suppresses over half of nuclear and cytoplasmic p53 under normal conditions, independent of its enzymatic activity. PEPD binds to the proline-rich domain in p53, which inhibits phosphorylation of nuclear p53 and MDM2-mediated mitochondrial translocation of nuclear and cytoplasmic p53. Indeed, PEPD competes with MDM2 for p53 binding. However, the PEPD-p53 complex is critical for p53 response to stress, as stress signals doxorubicin (DOX) and hydrogen peroxide each must free p53 from PEPD in order to achieve robust p53 activation, which is mediated by reactive oxygen species. Thus, PEPD stores p53 for stress response, but this also renders cells dependent on PEPD for survival as it suppresses p53. Our results reveal a major p53 regulatory mechanism and a critical physiological function of PEPD. The p53-PEPD system likely operates in most if not all cells, since both p53 and PEPD are expressed ubiquitously. Disrupting PEPD suppression of p53 may be an important therapeutic strategy in cancer, as our data show that PEPD knockdown by RNA interference in tumors in mice causes p53 activation in the tumor tissues and tumor regression. Our study also reveals a previously unrecognized anticancer mechanism of DOX. We show that the key step in DOX-induced p53 activation is the disruption of p53 association with PEPD via reactive oxygen species. This finding also raises the intriguing question of whether other stress-inducing anticancer agents also disrupt the PEPD-p53 complex for p53 activation and suggests that antioxidants may inhibit the anticancer activity of DOX and other agents by inhibiting p53 separation from PEPD. This work is supported by NCI grants and Roswell Park Alliance Foundation Grants. Citation Format: Lu Yang, Yun Li, Arup Bhattacharya, Yuesheng Zhang. PEPD is an essential regulator of p53 tumor suppressor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 552.

PEDF regulates lipid metabolism and reduces apoptosis in hypoxic H9c2 cells by inducing autophagy related 5-mediated autophagy via PEDF-R.

Pigment epithelial-derived factor (PEDF) is a multifunctional secreted glycoprotein, which exerts a variety of physiological activities. PEDF may protect against hypoxia-induced cell death associated with its antioxidative effects and p53 mitochondrial translocation in cultured cardiomyocytes and H9c2 cells. Additionally, previous studies have suggested that autophagy is an important cell survival mechanism. However, the effect of PEDF on autophagy and the associated pathway in hypoxic H9c2 cells has not been fully established. Autophagy has been reported to regulate lipid metabolism; however, little is known about whether PEDF is able to regulate lipid metabolism by promoting autophagy. In the present study, western blotting results revealed that PEDF increased the level of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II. Transmission electron microscopy (TEM) and LC3 fluorescence demonstrated that PEDF increased the number of autophagosomes. PEDF also increased the viability of hypoxic H9c2 cells and decreased the level of cleaved caspase-3 protein, as evidenced by CCK-8 assays and western blotting, respectively. TEM and a triglyceride assay kit demonstrated that PEDF-induced autophagy may stimulate lipid degradation. Western blotting results revealed a novel mechanism underlying PEDF-induced H9c2 cell autophagy via the PEDF-R-mediated Atg5 pathway under hypoxic conditions. Furthermore, the results also suggest that PEDF-induced autophagy may stimulate lipid degradation. The survival function of autophagy suggests that modulation of PEDF-induced autophagy may be used as a therapeutic strategy to protect cells against lipid-associated metabolic diseases.

BFGF plays a neuroprotective role by suppressing excessive autophagy and apoptosis after transient global cerebral ischemia in rats

Transient global cerebral ischemia (tGCI) is a cerebrovascular disorder that can cause apoptotic neuronal damage and functional deficits. Basic fibroblast growth factor (bFGF) was reported to be highly expressed in the central nervous system (CNS) and to exert neuroprotective effects against different CNS diseases. However, the effects of bFGF on tGCI have not been studied intensively. This study was conducted to investigate the effect of bFGF and its underlying mechanism in an animal model of tGCI. After intracerebroventricular (i.c.v.) injection of bFGF, functional improvement was observed, and the number of viable neurons increased in the ischemia-vulnerable hippocampal CA1 region. Apoptosis was induced after tGCI and could be attenuated by bFGF treatment via inhibition of p53 mitochondrial translocation. In addition, autophagy was activated during this process, and bFGF could inhibit activation of autophagy through the mTOR pathway. Rapamycin, an activator of autophagy, was utilized to explore the relationship among bFGF, apoptosis, and autophagy. Apoptosis deteriorated after rapamycin treatment, which indicated that excessive autophagy could contribute to the apoptosis process. In conclusion, these results demonstrate that bFGF could exert neuroprotective effects in the hippocampal CA1 region by suppressing excessive autophagy via the mTOR pathway and inhibiting apoptosis by preventing p53 mitochondrial translocation. Furthermore, our results suggest that bFGF may be a promising therapeutic agent to for treating tGCI in response to major adverse events, including cardiac arrest, shock, extracorporeal circulation, traumatic hemorrhage, and asphyxiation.