Upregulation Of P53 Upregulated Modulator Of Apoptosis Research Articles

Platycodin D induces apoptosis through JNK1/AP-1/PUMA pathway in non-small cell lung cancer cells: A new mechanism for an old compound.

Platycodin D, a triterpenoid monomer, has been shown to possess an anti-tumor effect on various types of cancer. Although Platycodin D has been reported to suppress tumorigenesis, the detailed underlying mechanism remains elusive. Platycodin D treatment significantly reduced the cell viability, decreased the number of colonies, impaired the mitochondrial function, and induced apoptosis in non-small cell lung cancer (NSCLC) cells. To understand the mechanism by which platycodin D induces apoptosis, the expression levels of apoptosis-related proteins were examined, and we found that the expression of PUMA (p53 upregulated modulator of apoptosis) was upregulated upon platycodin D treatment. Knockdown of PUMA resulted in attenuation of platycodin D-induced apoptosis, indicating that PUMA up-regulation is essential for platycodin D to induce apoptosis. The induction of PUMA expression by platycodin D treatment was through activation of AP-1 since mutation of AP-1 binding site in the PUMA promoter abolished the PUMA promoter activity. In addition, the chromatin immunoprecipitation further demonstrated that platycodin D promoted AP-1 binding to PUMA promoter. Moreover, knockdown of JNK1, but not JNK2, significantly abolished the phosphorylation of c-Jun at Ser63 (a component of AP-1), decreased the platycodin D-induced expression of PUMA and cleaved caspase 3, indicating that platycodin D inhibits JNK1/AP-1 signaling pathway. Furthermore, immunohistochemical staining studies showed that tumors from the mice treated with platycodin D activated JNK by translocation of JNK into nuclei, increased phosphorylation of JNK and c-Jun at Ser63 in nuclei, and boosted the PUMA expression. Taken together, our in vitro and in vivo data revealed a novel mechanism by which platycodin D up-regulates PUMA to induce apoptosis through JNK1/AP-1 axis in NSCLC.

Abstract 5490: Comparative activation of cellular ISR and cell death induction by p53 pathway restoring small molecules

Abstract Restoration of the p53 pathway has been a long-term goal of the field of cancer research as an approach to treat tumors with mutated p53 and aggressive clinical behavior. In general, p53- pathway-restoring can be achieved by small molecules through p53-dependent or p53-independent ways. For short, hereafter p53-independent restoration of p53-pathway-signaling is also referred as ‘restoration of p53 pathway’. To perform a comparative activation of p53 target genes, novel compounds PG3 and PG3-Oc that activate a subset of p53-target genes in a p53-independent manner, and four known mutant p53-activating compounds were included (ZMC1, APR-246, CP-31398 and Ellipticine). Nutlin-3a was used as a negative control. PG3 and PG3-Oc induced significant upregulation of p21, PUMA (p53 upregulated modulator of apoptosis), and DR5 consistently in five tested cancer cell lines with various p53 status through ATF4 (Activating transcriptional factor 4) via the ISR in a p53-independent manner. However, mutant p53-targeting compounds induced the expression of the three p53 target genes and ATF4 in a highly variable and cell type-dependent manner. PG3 treatment activated ATF4 through the ISR via kinase HRI (Heme-regulated inhibitor). ATF4 mediated the upregulation of PUMA, p21 and NAG-1 (Nonsteroidal anti-inflammatory drug-activated gene 1). We identified PUMA-mediated cell apoptosis through activation of caspase-8 in HT29 cells and possibly caspase-10 in SW480 cells. In summary, we provide a new mechanism that PG3 induced cell death via an HRI/ATF4/PUMA axis that may be helpful in understanding drug efficacy. Citation Format: Xiaobing Tian, Wafik S. El-Deiry. Comparative activation of cellular ISR and cell death induction by p53 pathway restoring small molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5490.

Up-regulation of PUMA caused the activation of p53 phosphorylation and acetylation, enhancing the interaction between PUMA and Bcl-X and mediating arsenic-induced apoptosis

Arsenic is a toxic metalloid vastly dispersed all over the occupational environments, manifesting multiple adverse health issues related to apoptosis. PUMA (p53 up-regulated modulator of apoptosis) is a crucial member of the Bcl-2 protein family and plays a key role in pro-apoptosis. The purpose of this work was to determine whether inorganic arsenic (NaAsO2) and its metabolites influenced the expression of PUMA in vivo and vitro, followed by investigating the mechanisms. RNA was extracted from serum and used to determine the expression of PUMA in vivo. The urine samples performed arsenic speciation analysis. This trial tested three-dose proportions in two cell lines (A549: 20, 40, 60 μM/L; 16HBE: 1.5, 3.0, 4.5 μM/L), respectively. According to the results of qRT-PCR and western blotting, NaAsO2 caused the overexpression of PUMA, not its metabolites. Furthermore, NaAsO2 induced phosphorylation of p53 at Ser315, 376, 392, and Thr55, and acetylation of p53 at K370, 382 with a dose-response relationship, suggesting the contribution of PUMA up-regulation to p53 phosphorylation and acetylation. CCK-8, JC-1 (5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetramethylbenzimi-dazolylcarbocyanine iodide), Hoechst33342/PI and the caspase3 and PARP1 blots were utilized to reveal apoptosis responding to NaAsO2 exposure. The co-immunoprecipitation assay showed that the interaction between PUMA and Bcl-X enhanced in intensity responding to NaAsO2 exposure, disrupting the complexes of Bcl-X with other pro-survival Bcl-2-related proteins. To our knowledge, we first reported that NaAsO2 activated phosphorylation of p53 at Ser315, 376, and Thr55, as well as acetylation of p53 at K370.

Forkhead Box O3a requires BAF57, a subunit of chromatin remodeler SWI/SNF complex for induction of p53 up-regulated modulator of apoptosis(Puma) in a model of Parkinson's disease.

Parkinson's disease (PD) results from the selective loss of dopaminergic neurons of substantia nigra pars compacta region of the midbrain. It has been reported that the transcription factor forkhead Box O3a (FoxO3a) is activated and induces pro-apoptotic protein such as Bcl-2-interacting mediator of cell death (BIM) and p53 up-regulated modulator of apoptosis (PUMA) in variety of neuron death paradigms. Activity of FoxO3a is governed by its post-translational modifications which control its subcellular localization. Aim of this study was to determine whether FoxO3a is activated and up-regulates its pro-apoptotic genes to induce neuron death in PD. We exposed neuronal PC12 cells or primary cultures of dopaminergic neurons to 6-hydroxy dopamine (6-OHDA) and infused 6-OHDA in rat brain to develop PD models. We found that FoxO3a undergoes multiple post-translational modifications which render its nuclear localization in dopaminergic neuronal cells in response to 6-OHDA. The nuclear redistribution of FoxO3a is significantly increased in dopaminergic neurons of 6-OHDA infused rat brains as well. Moreover, FoxO3a is required for dopaminergic neurodegeneration in response to 6-OHDA as RNAi-mediated silencing of FoxO3a protects these cells from 6-OHDA toxicity. In a search of the downstream targets we identified PUMA as a direct target of FoxO3a. By knocking down FoxO3a we could successfully block the up-regulation of the pro-apoptotic protein PUMA in this model. Recently, it has been reported that chromatin remodeler SWItch/sucrose non-fermentable binds to FOXO and activates transcription. We found that Brg-associated factor 57 (BAF57), a subclass of SWItch/sucrose non-fermentable is up-regulated and play a necessary role in neuron death induced by 6-OHDA. Moreover, it is required for induction of PUMA by FoxO3a in this cellular model of PD. Taken together, our study suggest that FoxO3a is activated, translocates to nucleus, induces its pro-apoptotic target PUMA in the presence of chromatin remodeler BAF57 to execute neuron death in cellular models of PD.

Targeting survivin sensitizes cervical cancer cells to radiation treatment

ABSTRACT Survivin is an inhibitor of apoptosis protein that functions to inhibit apoptosis, promote proliferation, and enhance invasion. It is selectively up-regulated in many human tumors and implicated in cellular radiation response through its role in apoptosis, cell division, and DNA damage response. This study aimed to investigate the effect and mechanisms of targeting survivin radiosensitivity in cervical cancer C33A cells. Here, the authors designed a small interfering RNA (siRNA) or plasmid-based small hairpin RNA (shRNA) targeting survivin and tested its effects on radiosensitivity to ionizing radiation (IR) treatment of C33A cells in vitro, as well as on the tumorigenicity of C33A cells in nude mice in vivo. Transient transfection of survivin siRNA into C33A cells suppressed survivin expression, induced cell apoptosis and G2/M arrest and reduced cell proliferation, clone formation ability after IR, followed by p53 upregulated modulator of apoptosis (PUMA) upregulation. But, transient transfection of survivin siRNA alone has no significant effect on cell growth and apoptosis. To confirm that PUMA upregulation is necessary for survivin silencing -induced radiosensitivity to IR treatment, the effect of targeting PUMA in survivin sliencing cells was observed. The results showed that targeting PUMA in survivin sliencing cells rescued C33A cells’ radioresistance. Furthermore, knocking down survivin expression combined with IR treatment significantly slowed tumor growth and promoted tumor cell apoptosis in C33A xenografted tumors. It was concluded that survivin played a role in radiotherapy resistance. Targeting survivin increased the radiosensitivity of C33A cells through induction of PUMA expression.

Capsaicin Induces ATF4 Translation with Upregulation of CHOP, GADD34 and PUMA.

Primary Effusion Lymphoma (PEL) is a rare and aggressive B-lymphoma caused by Kaposi's sarcoma-associated herpes virus (KSHV) infection that occurs in immunocompromised patients. PEL patients have a poor prognosis. KSHV modulates various cellular signaling pathways to maintain latent infection, and causes malignant conversion of host cells. We previously reported that capsaicin suppressed extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling and induced apoptosis in PEL. Generally, cellular stress such as nutrient starvation, oxidation and virus infection induce CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) expression by activating transcription factor 4 (ATF4), however endoplasmic reticulum (ER) stress induces CHOP expression by both ATF4 and ATF6. CHOP is associated with apoptosis induction and upregulates growth arrest and DNA damage-inducible protein 34 (GADD34) and p53 up-regulated modulator of apoptosis (PUMA) mRNA expression. In this study, we found a new mechanism in which capsaicin induces apoptosis via ATF4-CHOP-PUMA. Capsaicin promoted transcriptional activation of CHOP, which increased mRNA expression of GADD34 and PUMA, resulting in PEL apoptosis. Furthermore, capsaicin increased ATF4 protein levels by promoting ATF4 translation, not transcription, and had no effect on ATF6-dependent transcriptional activation. In sum, capsaicin promotes ATF4 translation and transcriptional induction of CHOP, which results in PUMA expression and apoptosis in PEL cells.

Vitamin D receptor activation protects against lipopolysaccharide-induced acute kidney injury through suppression of tubular cell apoptosis.

Acute kidney injury (AKI) is a common complication of sepsis characterized by a rapid degradation of renal function. The effect of vitamin D on AKI remains poorly understood. Here, we showed that vitamin D receptor (VDR) activation protects against lipopolysaccharide (LPS)-induced AKI by blocking renal tubular epithelial cell apoptosis. Mice lacking VDR developed more severe AKI than wild-type (WT) control mice after LPS treatment, which was manifested by marked increases in body weight loss and accumulation of serum blood urea nitrogen and creatinine as well as the magnitude of apoptosis of tubular epithelial cells. In the renal cortex, LPS treatment led to more dramatic downregulation of Bcl-2, more robust induction of p53-upregulated modulator of apoptosis (PUMA) and miR-155, and more severe caspase-3 activation in VDR knockout mice compared with WT control mice. Conversely, paricalcitol pretreatment markedly prevented LPS-induced AKI. Paricalcitol ameliorated body weight loss, attenuated serum blood urea nitrogen and creatinine accumulation, blocked tubular cell apoptosis, prevented the suppression of Bcl-2, and reversed PUMA and miR-155 induction and caspase-3 activation in LPS-treated WT mice. In HK2 cells, LPS induced PUMA and miR-155 by activating NF-κB, whereas 1,25(OH)2D3 blocked PUMA and miR-155 induction by repressing NF-κB activation. Both PUMA and miR-155 target Bcl-2 to promote apoptosis; namely, PUMA inhibits Bcl-2 activity, whereas miR-155 promotes Bcl-2 mRNA degradation and inhibits Bcl-2 protein translation. Collectively, these data provide strong evidence that LPS induces tubular cell apoptosis via upregulating PUMA and miR-155, whereas vitamin D/VDR signaling protects against AKI by blocking NF-κB-mediated PUMA and miR-155 upregulation.

Targeting XIST induced apoptosis of human osteosarcoma cells by activation of NF-kB/PUMA signal

ABSTRACT The long noncoding RNA X-inactive specific transcript (XIST) plays vital roles in tumor progression. However, the underlying mechanisms remain unclear. This study investigated the effects and mechanisms of targeting XIST on osteosarcoma (OS) cells in vitro and in vivo. We used shRNA to knockdown XIST to evaluate cell growth and apoptosis in U2OS cells in vitro and xenograft formation in vivo. An observed relationship between XIST and the p53 upregulated modulator of apoptosis (PUMA) and nuclear factor-kappa B (NF-kB) pathway was further explored by using small interfering RNA (siRNA). Our results showed that suppression of XIST by short hairpin RNA (shRNA) impeded U2OS cell growth, induced apoptosis and lessened OS xenograft tumor growth. Targeting XIST increased NF-kB-dependent PUMA upregulation in U2OS cells. Upregulation of PUMA is correlated with suppression of XIST-induced apoptosis in U2OS cells. Therefore, inhibition of XIST could promote U2OS cell death via activation of NF-kB/PUMA pathways.

Sorafenib induces renal cell carcinoma apoptosis via upregulating activating transcription factor 4.

Previous studies have shown sorafenib to function as a multitargeted tyrosine kinase inhibitor in different tumors. However, whether sorafenib improves renal cell carcinoma (RCC) through activating transcription factor 4 (ATF4) has never been explored. In the current study, we showed that sorafenib could suppress RCC cell viability in a time- and dose-dependent manner. Furthermore, sorafenib is demonstrated to enhance the mRNA and protein levels of ATF4. Meanwhile, overexpression of ATF4 was demonstrated to induce ACHN cell cycle arrest and cell apoptosis. Moreover, treatment with sorafenib could enhance the expression of CCAAT/enhancer-binding protein-homologous protein (CHOP) and p53 upregulated modulator of apoptosis (PUMA), thereby leading to ACHN cell apoptosis. More importantly, silencing of ATF4 could largely abolish sorafenib-induced upregulation of CHOP and PUMA in ACHN cells. Meanwhile, sorafenib-induced cell apoptosis may be dependent on the activation of ATF4 since knockdown of ATF4 partially reversed sorafenib-induced ACHN cell apoptosis. In summary, the present study demonstrates that sorafenib activates ATF4-CHOP-PUMA pathway in RCC cells, resulting in enhanced ER stress-related cell apoptosis.

Effect of taurine on cell proliferation and apoptosis human lung cancer A549 cells.

To investigate the effects of taurine on cell proliferation and apoptosis, the human lung cancer A549 cell line and xenograft tumors in nude mice were used. The effects of taurine on cell proliferation and apoptosis were observed at time points of 24, 48 and 72 h after treatment using an MTT assay to detect the survival rate, and flow cytometry to detect the apoptotic rate. Western blot analysis was performed to examine the levels of p53 upregulated modulator of apoptosis (PUMA), BCL2, apoptosis regulator (Bcl-2) and BCL2-associated X, apoptosis regulator (Bax) in A549 cells. The level of PUMA, Bax and Bcl-2 proteins in the mouse xenograft tumors treated with taurine and/or exogenous PUMA were assessed by immunohistochemistry, with taurine suppressing the proliferation of the human lung cancer A549 cell line in a concentration-dependent manner, and it significantly enhanced the apoptosis rate at all concentrations. Taurine induced the significant upregulation of PUMA and Bax, but led to downregulation of Bcl-2. In comparison to the control group, taurine treatment markedly reduced the volume and weight of A549-derived xenograft tumors in nude mice. Expression of PUMA and Bax were upregulated in the xenograft tumors following taurine treatment, whereas Bcl-2 was downregulated. In addition, the inhibitory effect of taurine and exogenous PUMA on tumor growth was significantly higher than that of a single treatment of taurine or exogenous PUMA. It can therefore be concluded that taurine can inhibit cell proliferation of the human lung cancer A549 cell line and the growth of the xenograft tumors, whereas PUMA serves an important role in taurine-induced growth suppression.

P53-derived hybrid peptides induce apoptosis of synovial fibroblasts in the rheumatoid joint.

Loss of p53-mediated suppression by its dominant-negative counterpart is commonly observed in human cancers, and activating p73 is a therapeutic strategy in p53-mutated oncological patients. In synovial fibroblasts (SFs) from rheumatoid arthritis (RA), mutant p53 can lead to the transformation-like features with resistance to the apoptosis induction. We examined whether intra-articular (i.a.) administration of p53-derived hybrid peptides to activate p73 can induce apoptosis of SFs by using adenoviral vectors encoding 37 amino acid (Ad37AA), a p53-derived hybrid peptide capable of activating p73, to transduce SFs in vitro and inject collagen-induced arthritis (CIA) joints in vivo. Increased p73 expression was found in synovial lining layers and SFs of RA patients and CIA rats. Higher expression of p53 up-regulated modulator of apoptosis (PUMA) and Bax with enhanced apoptosis were found in Ad37AA-transduced SFs, and silencing p73 abrogated the up-regulation of PUMA and Bax. Articular indexes and histologic scores were reduced in Ad37AA-injected joints with decreased SF densities, increased apoptotic cell numbers, and higher PUMA expression levels. We demonstrate that i.a. administration of p53-derived hybrid peptides can activate p73 to induce apoptosis of SFs and ameliorate the rheumatoid joint, implicating an enhancement of the p73-dependent apoptotic mechanism as a pharmacological strategy in the RA therapy.

Proapoptotic PUMA targets stem-like breast cancer cells to suppress metastasis.

Breast cancer cells with stem cell properties are key contributors to metastatic disease, and there remains a need to better understand and target these cells in human cancers. Here, we identified rare stem-like cells in patients' tumors characterized by low levels of the proapoptotic molecule p53-upregulated modulator of apoptosis (PUMA) and showed that these cells play a critical role in tumor progression that is independent of clinical subtype. A signaling axis consisting of the integrin αvβ3, Src kinase, and the transcription factor Slug suppresses PUMA in these cells, promoting tumor stemness. We showed that genetic or pharmacological disruption of αvβ3/Src signaling drives PUMA expression, specifically depleting these stem-like tumor cells; increases their sensitivity to apoptosis; and reduces pulmonary metastasis, with no effect on primary tumor growth. Taken together, these findings point to PUMA as a key vulnerability of stem-like cells and suggest that pharmacological upregulation of PUMA via Src inhibition may represent a strategy to selectively target these cells in a wide spectrum of aggressive breast cancers.

Small interfering RNA (siRNA) against Slug induces apoptosis and sensitizes human anaplastic thyroid carcinoma cells to doxorubicin.

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human cancers and often shows resistance to multimodal therapeutic approaches. It has been shown that the transcriptional repressor Slug inhibits the chemotherapeutic agent-induced apoptosis of cancer cells. We evaluated whether targeting of Slug could augment doxorubicin (DOX)-induced apoptosis of ATC cells. We also determined changes in PUMA (p53-upregulated modulator of apoptosis) expression levels to identify possible mechanisms of their combined actions. SW1736 cells were transfected with Slug siRNA or/and PUMA siRNA and then exposed to DOX (0.1, 1, and 5 μ M) for selected times. Scrambled siRNA was used as a control. The effects on cell viability were determined via MTT assay. Apoptosis was assessed using TUNEL assays and annexin V staining, and was confirmed by flow cytometry analyses. Slug and PUMA levels were determined using western blotting, RT-PCR and immunofluorescence analyses. We used a subcutaneous implanted tumor model of SW1736 cells in nude mice to assess the effects of Slug silencing in combination with DOX on tumor development. Apoptosis was assessed via TUNEL assay. Targeting of Slug using siRNA inhibits growth of SW1736 cells and sensitizes SW1736 cells to DOX in vitro and vivo. Targeting of Slug combined with DOX led to lower cell viability than treatment with DOX alone in SW1736 cells. TUNEL and flow cytometry analyses showed that targeting of Slug enhanced DOX-induced apoptosis of SW1736 cells. In addition, targeting of Slug increased PUMA expression, and targeting of PUMA restored the chemoresistance of SW1736/Slug siRNA cells to DOX. Knockdown of Slug enhanced the antitumor activity of DOX in SW1736 cells via induction of PUMA upregulation. Our results suggest that targeting of Slug has good potential for the development of new therapeutic strategies for ATC.

Cisplatin induced apoptosis of ovarian cancer A2780s cells by activation of ERK/p53/PUMA signals.

Cisplatin (CDDP) is one of the most effective anticancer agents widely used in the treatment of solid tumors, including ovarian cancer. It is generally considered as a cytotoxic drug which kills cancer cells by causing DNA damage, and subsequently inducing apoptosis in cancer cells. However, the underlying mechanisms leading to cell apoptosis remain obscure. In this study, the signaling pathways involved in CDDP-induced apoptosis were examined using CDDP-sensitive ovarian cancer A2780s cells. A2780s cells were treated with CDDP (1.5-3 μg/ml) for 6h, 12h and 24h. Using siRNA targeting P53 and PUMA, and a selective MEK inhibitor, PD98059 to examine the relation between ERK1/2 activation, p53 and PUMA expression after exposure to CDDP, and the effect on CDDP-induced apoptosis. The results shown that treatment of A2780s cells with CDDP (3 μg/ml) for 6-24h induced apoptosis, resulting in the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and accumulation of p53 and PUMA (p53 upregulated modulator of apoptosis) protein. Knockdown of P53 or PUMA by siRNA transfection blocked CDDP-induced apoptosis. Inhibition of ERK1/2 using PD98059, a selective MEK inhibitor, blocked the apoptotic cell death but prevented CDDP-induced accumulation of p53 and PUMA. Knockdown of P53 by siRNA transfection also blocked CDDP-induced accumulation of PUMA. We therefore concluded that CDDP activated ERK1/2 and induced-p53-dependent PUMA upregulation, resulting in triggering apoptosis in A2780s cells. Our study clearly demonstrates that the ERK1/2/p53/PUMA axis is related to CDDP-induced cell death in A2780s cells.

PUMA and NF-kB Are Cell Signaling Predictors of Reovirus Oncolysis of Breast Cancer.

Background and purposeReovirus is a ubiquitous RNA virus that exploits aberrant signaling pathways for its replication. The oncolytic potential of reovirus against numerous cancers under pre-clinical/clinical conditions has been documented by us and others. Despite its proven clinical activity, the underlying mechanisms of reovirus oncolysis is still not well elucidated. If reovirus therapy is to be optimized for cancer, including breast cancer patients, it is imperative to understand the mechanisms of reovirus oncolysis, especially in treatment of resistant tumour.Experimental approach and resultsIn the present study global gene expression profiling was utilized as a preliminary roadmap to tease-out pivotal molecules involved in reovirus induced apoptosis in breast cancer. Reovirus treated HTB133 and MCF7 breast cancer cells revealed transcriptional alteration of a defined subset of apoptotic genes and members of the nuclear factor-kappa B (NF-kB) family and p53 upregulated modulator of apoptosis (PUMA) were prominent. Since NF-kB can paradoxically suppress or promote apoptosis in cancer, the significance of NF-kB in reovirus oncolysis of breast cancer was investigated. Real time PCR analysis indicated a 2.9–4.3 fold increase in NF-kB p65 message levels following reovirus infection of MCF7 and HTB133, respectively. Nuclear translocation of NF-kB p65 protein was also dramatically augmented post reovirus treatment and correlated with enhanced DNA binding. Pharmacologic inhibition of NF-kB lead to oncolytic protection and significant down regulation of PUMA message levels. PUMA down regulation using siRNA suppressed reovirus oncolysis via significantly repressed apoptosis in p53 mutant HTB133 cells.ConclusionsThis study demonstrates for the first time that a prominent pathway of reovirus oncolysis of breast cancer is mediated through NF-kB and that PUMA upregulation is dependent on NF-kB activation. These findings represent potential therapeutic indicators of reovirus treatment in future clinical trials.

Post-traumatic administration of the p53 inactivator pifithrin-α oxygen analogue reduces hippocampal neuronal loss and improves cognitive deficits after experimental traumatic brain injury

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Neuronal apoptosis in the hippocampus has been detected after TBI. The hippocampal dysfunction may result in cognitive deficits in learning, memory, and spatial information processing. Our previous studies demonstrated that a p53 inhibitor, pifithrin-α oxygen analogue (PFT-α (O)), significantly reduced cortical cell death, which is substantial following controlled cortical impact (CCI) TBI, and improved neurological functional outcomes via anti-apoptotic mechanisms. In the present study, we examined the effect of PFT-α (O) on CCI TBI-induced hippocampal cellular pathophysiology in light of this brain region's role in memory. To investigate whether p53-dependent apoptosis plays a role in hippocampal neuronal loss and associated cognitive deficits and to define underlying mechanisms, SD rats were subjected to experimental CCI TBI followed by the administration of PFT-α or PFT-α (O) (2mg/kg, i.v.) or vehicle at 5h after TBI. Magnetic resonance imaging (MRI) scans were acquired at 24h and 7days post-injury to assess evolving structural hippocampal damage. Fluoro-Jade C was used to stain hippocampal sub-regions, including CA1 and dentate gyrus (DG), for cellular degeneration. Neurological functions, including motor and recognition memory, were assessed by behavioral tests at 7days post injury. p53, p53 upregulated modulator of apoptosis (PUMA), 4-hydroxynonenal (4-HNE), cyclooxygenase-IV (COX IV), annexin V and NeuN were visualized by double immunofluorescence staining with cell-specific markers. Levels of mRNA encoding for caspase-3, p53, PUMA, Bcl-2, Bcl-2-associated X protein (BAX) and superoxide dismutase (SOD) were measured by RT-qPCR. Our results showed that post-injury administration of PFT-α and, particularly, PFT-α (O) at 5h dramatically reduced injury volumes in the ipsilateral hippocampus, improved motor outcomes, and ameliorated cognitive deficits at 7days after TBI, as evaluated by novel object recognition and open-field test. PFT-α and especially PFT-α (O) significantly reduced the number of FJC-positive cells in hippocampus CA1 and DG subregions, versus vehicle treatment, and significantly decreased caspase-3 and PUMA mRNA expression. PFT-α (O), but not PFT-α, treatment significantly lowered p53 and elevated SOD2 mRNA expression. Double immunofluorescence staining demonstrated that PFT-α (O) treatment decreased p53, annexin V and 4-HNE positive neurons in the hippocampal CA1 region. Furthermore, PUMA co-localization with the mitochondrial maker COX IV, and the upregulation of PUMA were inhibited by PFT-α (O) after TBI. Our data suggest that PFT-α and especially PFT-α (O) significantly reduce hippocampal neuronal degeneration, and ameliorate neurological and cognitive deficits in vivo via antiapoptotic and antioxidative properties.

Low-dose Actinomycin-D treatment re-establishes the tumoursuppressive function of P53 in RELA-positive ependymoma.

Ependymomas in children can arise throughout all compartments of the central nervous system (CNS). Highly malignant paediatric ependymoma subtypes are Group A tumours of the posterior fossa (PF-EPN-A) and RELA-fusion positive (ST-EPN-RELA) tumours in the supratentorial compartment. It was repeatedly reported in smaller series that accumulation of p53 is frequently observed in ependymomas and that immunohistochemical staining correlates with poor clinical outcome, while TP53 mutations are rare. Our TP53 mutation analysis of 130 primary ependymomas identified a mutation rate of only 3%. Immunohistochemical analysis of 398 ependymomas confirmed previous results correlating the accumulation of p53 with inferior outcome. Among the p53-positive ependymomas, the vast majority exhibited a RELA fusion leading to the hypothesis that p53 inactivation might be linked to RELA positivity.In order to assess the potential of p53 reactivation through MDM2 inhibition in ependymoma, we evaluated the effects of Actinomycin-D and Nutlin-3 treatment in two preclinical ependymoma models representing the high-risk subtypes PF-EPN-A and ST-EPN-RELA. The IC-50 of the agent as determined by metabolic activity assays was in the lower nano-molar range (0.2–0.7 nM). Transcriptome analyses of high-dose (100 nM), low-dose (5 nM) and non-treated cells revealed re-expression of p53 dependent genes including p53 upregulated modulator of apoptosis (PUMA) after low-dose treatment. At the protein level, we validated the Actinomycin-D induced upregulation of PUMA, and of p53 interaction partners MDM2 and p21. Proapoptotic effects of low-dose application of the agent were confirmed by flow cytometry. Thus, Actinomycin-D could constitute a promising therapeutic option for ST-EPN-RELA ependymoma patients, whose tumours frequently exhibit p53 inactivation.

Dopaminergic neuron-specific deletion of p53 gene is neuroprotective in an experimental Parkinson's disease model.

p53, a stress response gene, is involved in diverse cell death pathways and its activation has been implicated in the pathogenesis of Parkinson's disease (PD). However, whether the neuronal p53 protein plays a direct role in regulating dopaminergic (DA) neuronal cell death is unknown. In this study, in contrast to the global inhibition of p53 function by pharmacological inhibitors and in traditional p53 knock-out (KO) mice, we examined the effect of DA specific p53 gene deletion in DAT-p53KO mice. These DAT-p53KO mice did not exhibit apparent changes in the general structure and neuronal density of DA neurons during late development and in aging. However, in DA-p53KO mice treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we found that the induction of Bax and p53 up-regulated modulator of apoptosis (PUMA) mRNA and protein levels by MPTP were diminished in both striatum and substantia nigra of these mice. Notably, deletion of the p53 gene in DA neurons significantly reduced dopaminergic neuronal loss in substantia nigra, dopaminergic neuronal terminal loss at striatum and, additionally, decreased motor deficits in mice challenged with MPTP. In contrast, there was no difference in astrogliosis between WT and DAT-p53KO mice in response to MPTP treatment. These findings demonstrate a specific contribution of p53 activation in DA neuronal cell death by MPTP challenge. Our results further support the role of programmed cell death mediated by p53 in this animal model of PD and identify Bax, BAD and PUMA genes as downstream targets of p53 in modulating DA neuronal death in the invivo MPTP-induced PD model. We deleted p53 gene in dopaminergic neurons in late developmental stages and found that DA specific p53 deletion is protective in acute MPTP animal model possibly through blocking MPTP-induced BAX and PUMA up-regulation. Astrocyte activation measured by GFAP positive cells and GFAP gene up-regulation in the striatum shows no difference between wt and DA-p53 ko mice.

Evidence for caspase-dependent programmed cell death along with repair processes in affected skeletal muscle fibres in patients with mitochondrial disorders

Mitochondrial disorders are heterogeneous multisystemic disorders due to impaired oxidative phosphorylation causing defective mitochondrial energy production. Common histological hallmarks of mitochondrial disorders are RRFs (ragged red fibres), muscle fibres with abnormal focal accumulations of mitochondria. In contrast with the growing understanding of the genetic basis of mitochondrial disorders, the fate of phenotypically affected muscle fibres remains largely unknown. We investigated PCD (programmed cell death) in muscle of 17 patients with mitochondrial respiratory chain dysfunction. We documented that in affected muscle fibres, nuclear chromatin is condensed in lumpy irregular masses and cytochrome c is released into the cytosol to activate, along with Apaf-1 (apoptotic protease-activating factor 1), caspase 9 that, in turn, activates effector caspase 3, caspase 6, and caspase 7, suggesting the execution of the intrinsic apoptotic pathway. Whereas active caspase 3 underwent nuclear translocation, AIF (apoptosis-inducing factor) mainly stayed within mitochondria, into which an up-regulated Bax is relocated. The significant increase in caspase 2, caspase 3 and caspase 6 activity strongly suggest that the cell death programme is caspase-dependent and the activation of caspase 2 together with PUMA (p53 up-regulated modulator of apoptosis) up-regulation point to a role for oxidative stress in triggering the intrinsic pathway. Concurrently, in muscle of patients, the number of satellite cells was significantly increased and myonuclei were detected at different stages of myogenic differentiation, indicating that a reparative programme is ongoing in muscle of patients with mitochondrial disorders. Together, these data suggest that, in patients with mitochondrial disorders, affected muscle fibres are trapped in a mitochondria-regulated caspase-dependent PCD while repairing events take place.

Subcellular localization of PUMA regulates its pro-apoptotic activity in Burkitt's lymphoma B cells.

The BH3-only protein PUMA (p53-upregulated modulator of apoptosis) is a major regulator of apoptosis. It belongs to the Bcl-2 family of proteins responsible for maintaining mitochondrial outer membrane integrity by controlling the intrinsic (mitochondrial) apoptotic pathway. We describe here a new pathway regulating PUMA activation through the control of its subcellular distribution. Surprisingly, neither PUMA upregulation in normal activated human B lymphocytes nor high levels of PUMA in Burkitt's lymphoma (BL) were associated with cell death. We show that PUMA is localized to the cytosol in these cells. By contrast, various apoptosis-triggering signals were found to promote the translocation of PUMA to the mitochondria in these cells, leading to their death by apoptosis. This apoptosis was associated with the binding of mitochondrial PUMA to anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Mcl-1. This translocation was caspase-independent but was prevented by inhibiting or knocking down the expression of the MAPK kinase p38. Our data suggest that the accumulation of PUMA in the cytosol may be important for the participation of this protein in apoptosis without the need for prior transcription. This regulatory pathway may be an important feature of differentiation and tumorigenic processes.