Levels Of P53 Upregulated Modulator Of Apoptosis Research Articles

Abstract 17456: Inositol 1,4,5-Trisphosphate Receptors (ITP3Rs) and Activation of Cardiac Fibroblasts in Ischemia/Reperfusion Injury

Introduction: Inositol 1,4,5-trisphosphate receptors (ITP3Rs) are Ca 2+ release channels on the endoplasmic reticulum (ER). The role of ITP3Rs in cardiomyocytes has been previously investigated for hypertrophy and arrhythmias, however, the exact function in the activation of cardiac fibroblasts (FBs) is yet to be determined. The main goal of this project is to investigate the mechanisms linking FB ITP3Rs to post-ischemia/reperfusion (I/R) cardiac fibrosis. Hypothesis: We hypothesize that ITP3Rs activate FB via a direct transcriptional regulation of αSMA and other genes involved in myoFB activation. Indeed, myoFB differentiation and persistence is regulated by a balance between pro-and anti-apoptotic signaling pathways. Methods: We generated myoFB-specific ITP3Rs KO mice (ITP3R KO ) and isolated FBs as well as cardiomyocytes via collagenase digestion and performed immunoprecipitation (IP) assays showing that ITP3Rs bind p53 upregulated modulator of apoptosis (PUMA). Cytosolic levels of Cytochrome C (Cyt C) were assessed in myoFB isolated from areas remote from injury and scar areas 7 d after I/R. After having immunoprecipitated for ITP3R1 we blotted the isolated FBs for PUMA and, vice versa. PUMA levels were measured in cytosol, ER, and mitochondria of FBs isolated from regions remote from injury and scar areas 7 d post I/R. Results: Our data show reduced levels of cytosolic Cyt C following I/R, which was prevented by IP3Rs ablation, providing an explanation for the persistence of myoFBs in remote areas. We found that the mitochondrial expression of PUMA was significantly decreased - while ER levels were markedly increased - in myoFBs isolated from areas remote from I/R injury, indicating a sequestration of PUMA by ITP3Rs; this phenotype was rescued by ITP3Rs ablation. Instead, in myoFBs isolated from the scar areas, mitochondrial PUMA levels were increased both in ITP3R fl/fl and ITP3R KO mice, and no sequestration was observed, consistent with the absence of ITP3R upregulation. Conclusions: Using a specific Cre/lox KO model, we provide the first assessment of functional contribution of ITP3R to post-I/R activation of cardiac FBs and understanding the molecular mechanisms underlying excessive reactive fibrosis in the heart.

FBXL20 promotes breast cancer malignancy by inhibiting apoptosis through degradation of PUMA and BAX

Apoptosis is a programmed cell death that efficiently removes damaged cells to maintain tissue homeostasis. Defect in apoptotic machinery can lead to tumor development, progression, and resistance to chemotherapy. PUMA (p53 upregulated modulator of apoptosis) and BAX (BCL2-associated X protein) are among the most well-known inducers of apoptosis. It has been reported that expression levels of BAX and PUMA are controlled at the posttranslational level by phosphorylation. However, the posttranslational regulation of these proapoptotic proteins remains largely unexplored. In this study, using biochemical, molecular biology, flow cytometric, and immunohistochemistry techniques, we show that PUMA and BAX are the direct target of the F-box protein FBXL20, which restricts their cellular levels. FBXL20 directs the proteasomal degradation of PUMA and BAX in a protein kinase AKT1-dependent manner to promote cancer cell proliferation and tumor growth. Interestingly, inactivation of AKT1 results in activation of another protein kinase GSK3α/β, which facilitates the proteasomal degradation of FBXL20 by another F-box protein, FBXO31. Thus, a switch between two signaling kinases AKT1 and GSK3α/β modulates the functional activity of these proapoptotic regulators, thereby determining cell survival or death. RNAi-mediated ablation of FBXL20 results in increased levels of PUMA as well as BAX, which further enhances the sensitivity of cancer cells to chemotherapeutic drugs. We showed that high level expression of FBXL20 in cancer cells reduces therapeutic drug-induced apoptosis and promotes chemoresistance. Overall, this study highlights the importance of targeting FBXL20 in cancers in conjunction with chemotherapy and may represent a promising anticancer strategy to overcome chemoresistance.

Expression and inhibitory effects of p53-upregulated modulator of apoptosis in gallbladder carcinoma.

The p53-upregulated modulator of apoptosis (PUMA) has been reported to be involved in various types of cancer. However, its potential biological role in gallbladder carcinoma (GBC) has not been fully elucidated. The present study aimed to determine the expression levels of PUMA and its biological effects on GBC. The mRNA and protein expression levels of PUMA in GBC tissues and cell lines were measured using reverse transcription-quantitative PCR and western blotting, respectively. The effects of PUMA overexpression on cell viability, proliferation and invasive ability were determined in vitro using the MTT, colony formation and Transwell invasion assays, respectively. The apoptotic rates were detected using the Annexin V-FITC apoptosis detection kit. Furthermore, follow-up of patients with GBC was performed to identify the association between PUMA expression levels and GBC prognosis. The results of the present study demonstrated that the expression levels of PUMA were significantly lower in the GBC tissues and cell lines compared with those in adjacent normal gallbladder tissues and normal gallbladder cells, respectively. Further experiments indicated that overexpression of PUMA inhibited the viability, proliferation and invasive ability of GBC cells compared with those in the control-transfected GBC cells. In addition, overexpression of PUMA significantly promoted apoptosis in GBC cells. Furthermore, overexpression of PUMA inhibited epithelial-mesenchymal transition, and promoted Bax upregulation and Bcl-2 downregulation compared with those in the control group. Low PUMA expression levels were associated with a short overall survival time in patients with GBC. In conclusions, PUMA may act as a tumor suppressor in GBC and may serve as a potential novel treatment target for human GBC.

Conditioned medium from adipose-derived stem cells attenuates ischemia/reperfusion-induced cardiac injury through the microRNA-221/222/PUMA/ETS-1 pathway.

Rationale: Cardiovascular diseases, such as myocardial infarction (MI), are the leading causes of death worldwide. Reperfusion therapy is the common standard treatment for MI. However, myocardial ischemia/reperfusion (I/R) causes cardiomyocyte injury, including apoptosis and fibrosis. We aimed to investigate the effects of conditioned medium from adipose-derived stem cells (ADSC-CM) on apoptosis and fibrosis in I/R-treated hearts and hypoxia/reoxygenation (H/R)-treated cardiomyocytes and the underlying mechanisms.Methods: ADSC-CM was collected from ADSCs. The effects of intramuscular injection of ADSC-CM on cardiac function, cardiac apoptosis, and fibrosis examined by echocardiography, Evans blue/TTC staining, TUNEL assay, and Masson's trichrome staining in I/R-treated mice. We also examined the effects of ADSC-CM on apoptosis and fibrosis in H/R-treated H9c2 cells by annexin V/PI flow cytometry, TUNEL assay, and immunocytochemistry.Results: ADSC-CM treatment significantly reduced heart damage and fibrosis of I/R-treated mice and H/R-treated cardiomyocytes. In addition, the expression of apoptosis-related proteins, such as p53 upregulated modulator of apoptosis (PUMA), p-p53 and B-cell lymphoma 2 (BCL2), as well as the fibrosis-related proteins ETS-1, fibronectin and collagen 3, were significantly reduced by ADSC-CM treatment. Moreover, we demonstrated that ADSC-CM contains a large amount of miR-221/222, which can target and regulate PUMA or ETS-1 protein levels. Furthermore, the knockdown of PUMA and ETS-1 decreased the induction of apoptosis and fibrosis, respectively. MiR-221/222 overexpression achieved similar results. We also observed that cardiac I/R markedly increased apoptosis and fibrosis in miR-221/222 knockout (KO) mice, while ADSC-CM decreased these effects. The increased phosphorylation of p38 and NF‐κB not only mediated myocardial apoptosis through the PUMA/p53/BCL2 pathway but also regulated fibrosis through the ETS-1/fibronectin/collagen 3 pathway.Conclusions: Overall, our results show that ADSC-CM attenuates cardiac apoptosis and fibrosis by reducing PUMA and ETS-1 expression, respectively. The protective effect is mediated via the miR-221/222/p38/NF-κB pathway.

Role of the SIRT1/p53 regulatory axis in oxidative stress‑mediated granulosa cell apoptosis.

Oxidative stress has been suggested to induce granulosa cell apoptosis, which contributes to follicular atresia. However, the mechanism via which oxidative stress mediates granulosa cell apoptosis remains elusive. Therefore, the aim of this study was to elucidate the molecular mechanisms regulating oxidative stress‑induced granulosa cell apoptosis. The present study demonstrated that reactive oxygen species induced by H2O2 resulted in human granulosa COV434 cell apoptosis via the regulation of sirtuin1 (SIRT1)‑mediated p53 activity. Endogenous SIRT1 expression was alleviated by H2O2 treatment of COV434 cells in a time‑dependent manner. In addition, knockdown or inhibition of SIRT1 promoted H2O2‑induced poly(ADP‑ribose) polymerase (PARP) cleavage and p53 acetylation, which led to an increase in COV434 cell apoptosis. Treatment with H2O2 enhanced the expression levels of the p53‑dependent proteins, p53‑upregulated modulator of apoptosis (PUMA) and phorbol‑12‑myristate‑13‑acetate‑induced protein1 (PMAIP1), as well as those of p53; however, knockdown of p53 decreased cleaved PARP, PUMA and PMAIP1 expression levels induced by H2O2 treatment. Moreover, knockdown of PUMA or PMAIP1 attenuated the H2O2 induction of PARP cleavage and COV434 cell apoptosis. In conclusion, the present findings suggested that H2O2‑induced oxidative stress causes granulosa COV434 cell apoptosis via the upregulation of p53 activity by SIRT1 suppression, indicating a mechanistic role of the SIRT1/p53 axis in H2O2‑induced granulosa cell apoptosis.

Effect of electroacupuncture at the ST36 and GB39 acupoints on apoptosis by regulating the p53 signaling pathway in adjuvant arthritis rats.

p53 and mouse double minute 2 homolog (MDM2) serve key regulatory roles in the apoptosis of synovial cells. The present study aimed to investigate the effects of electroacupuncture (EA) at the ‘Zusanli’ (ST36) and ‘Xuanzhong’ (GB39) acupoints on apoptosis in an adjuvant arthritis (AA) rat model. A total of 40 male Sprague-Dawley rats were randomly divided into Control, AA, AA + EA and AA + sham EA groups (n=10 rats in each group). Rats in all the groups, with the exception of the control group, were injected with Complete™ Freund's adjuvant into the bilateral hindlimb footpad to establish the AA model. Rats in the AA + EA group were treated with EA at the ST36 and GB39 acupoints. Rats in the AA + sham EA group were treated with percutaneous electrical stimulation at a position of 5 mm away from the ST36 and GB39 acupoints. The arthritis index scores and hindlimb paw volumes of the rats in each group were recorded. Subsequently, pathological changes in the synovial tissue were evaluated by hematoxylin and eosin (H&E) staining, and the apoptotic rate of the synovial cells was detected by TUNEL staining. In addition, the expression levels of the apoptosis-associated proteins, Bax, phorbol-12-myristate-13-acetate-induced protein 1 (Noxa) and p53 upregulated modulator of apoptosis (PUMA), were determined by western blot analysis. The expression of both the gene and protein of p53 and MDM2 in synovial tissue was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. The results indicated that the arthritis index scores and hindlimb paw volumes upon EA stimulation were significantly decreased compared with those of the AA group (P<0.05). H&E staining revealed that the synovial inflammation of EA stimulation was significantly decreased compared with the AA group (P<0.05). The TUNEL assay results indicated that the apoptotic rate of synovial cells in the AA + EA group was significantly increased compared with that in the AA group (P<0.05). Furthermore, an increased expression of proapoptotic proteins was confirmed by the increased expression levels of Bax, Noxa and PUMA in the AA + EA group. The results of RT-qPCR and western blot analysis demonstrated that, compared with the AA group, EA stimulation led to a marked increase in p53 (P<0.05) and a significant decrease in MDM2 (P<0.05) gene and protein expression. Taken together, these results demonstrated that EA performed on the ST36 and GB39 acupoints led to a significant amelioration in AA injury of model rats, by regulating the p53 signaling pathway and inducing apoptosis.

Expressions of p53 and PUMA in fibroblasts of systemic sclerosis patients are normal at transcription level.

Systemic sclerosis (SSc) fibroblasts show resistance apoptosis mechanisms, which enhances the fibrosis stage of the disease. Impaired function of p53 upregulated modulator of apoptosis (PUMA) has been related to deficits in p53-dependant apoptosis pathway. This study aimed to evaluate the transcriptional levels of p53 and PUMA mRNAs in fibroblasts from SSc patients and compare it with healthy individuals. In this case-control study, skin biopsy samples were obtained from 19 patients with diffuse cutaneous SSc (DcSSc) and 16 healthy controls. Afterward, dermal fibroblasts were isolated and cultured. After extraction of total RNA from cultured fibroblasts, complementary DNA (cDNA) was synthesized. mRNA quantification was carried out using real-time PCR, SYBR Green PCR master mix, and specific primers for p53 and PUMA. No significant alteration was observed in mRNA expression levels of p53 and PUMA (P=.99 and .23, respectively) in fibroblasts from SSc patients compared with controls. Apoptosis pathways are impaired in fibroblasts from patients with SSc, leading to chronic fibrosis. Nonetheless, PUMA/p53 pathway may not be involved in dysfunction of apoptosis mechanisms in fibroblasts of patients with SSc.

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.

Inhibitory effect and mechanism of exogenous mammalian sterile 20-like kinase 1 on the growth of human colorectal cancer.

The present study aimed to observe the inhibitory effect and preliminary mechanism of exogenous mammalian sterile 20-like kinase 1 (MST1) on the growth of colorectal cancer SW480 cells. The SW480 cells were randomly divided into the following groups: Control, empty enhanced green fluorescent protein (EGFP) plasmid (pEGFP-N1), MST1 EGFP plasmid (pEGFP-MST1), 20 µmol/l fluorouracil (5-FU) and pEGFP-MST1 + 5-FU. An MTS colorimetric assay was used to detect cell viability, Hoechst 33342 staining was used to observe cell apoptosis, and western blotting and immunohistochemistry were used to detect the levels of the proteins MST1, yes-associated protein (YAP), phospho-YAP1 (Ser127), p53 and p53 upregulated modulator of apoptosis (PUMA). In addition, nude mice were injected with SW480 cells to assess the tumor inhibition rates. Compared with the control group, the growth inhibition and apoptosis rates, the levels of MST1, p53 and PUMA, and the ratios of phospho-YAP1/YAP in the pEGFP-MST1 and pEGFP-MST1 + 5-FU groups were increased significantly (P<0.01). Additionally, relative to the control group, the tumor inhibition rates in the nude mice transplanted with SW480 cells of the pEGFP-MST1 and pEGFP-MST1 + 5-FU groups were 48.52±1.63 and 87.28±2.58%, respectively, and the positive rates of phospho-YAP1 (Ser127) protein in nuclei increased significantly (P<0.01). Overall, exogenous MST1 effectively inhibited the proliferation and growth of transplanted human colorectal cancer cells and promoted cancer cell apoptosis. The mechanism involved may be associated with the increase of intracellular phospho-YAP1 (Ser127) protein.

Clinical significance and expression of PUMA, MCL-1, and p53 in human renal cell carcinoma and para-carcinoma tissues.

We investigated the expression level of p53 upregulated modulator of apoptosis (PUMA), myeloid cell leukemia-I (MCL-1), and p53 in renal cell carcinoma (RCC) and para-carcinoma tissues, as well as their clinical significance. The expression levels of PUMA, MCL-1, and p53 in RCC and para-carcinoma tissues were measured using immunohistochemical and quantitative real-time PCR methods. Correlations between protein expression and pathological characteristics were analyzed. Renal clear cell carcinoma showed elevated MCL-1 and p53 protein expression (P > 0.05) and reduced PUMA expression as compared to that in para-carcinoma tissues. Spearman ranking correlation analysis showed that expression of PUMA, MCL-1, and p53 in was negatively correlated with RCC (r = -0.504, P = 0.001; r = -0.413, P = 0.008). We also observed significant correlation between MCL-1 expression and tumor differentiation (P < 0.05), where MCL-1 expression was significantly higher in well-differentiated adenocarcinoma as compared to that in medium or lowly differentiated adenocarcinoma. In addition, p53 expression was highly correlated with TNM staging (P < 0.05). Single factor analysis on COX's proportional hazard model indicated that postoperative survival rate and prognosis of renal clear cell carcinoma was highly correlated with TNM staging (P < 0.05). Quantitative real-time PCR analysis indicated higher expression of PUMA, MCL-1, and p53 in cancer tissues as compared to that in para-carcinoma tissues (P < 0.05).The expression of PUMA, MCL-1, and p53 can reflect the biological behavior of renal cell carcinoma, and can be used to indicate tumor invasion, progression, and prognosis.

Lithocholic acid induces endoplasmic reticulum stress, autophagy and mitochondrial dysfunction in human prostate cancer cells.

Lithocholic acid (LCA) is a secondary bile acid that is selectively toxic to human neuroblastoma, breast and prostate cancer cells, whilst sparing normal cells. We previously reported that LCA inhibited cell viability and proliferation and induced apoptosis and necrosis of androgen-dependent LNCaP and androgen-independent PC-3 human prostate cancer cells. In the present study, we investigated the roles of endoplasmic reticulum (ER) stress, autophagy and mitochondrial dysfunction in the toxicity of LCA in PC-3 and autophagy deficient, androgen-independent DU-145 cells. LCA induced ER stress-related proteins, such as CCAAT-enhancer-binding protein homologous protein (CHOP), and the phosphorylation of eukaryotic initiation factor 2-alpha (p-eIF2α) and c-Jun N-terminal kinases (p-JNK) in both cancer cell-types. The p53 upregulated modulator of apoptosis (PUMA) and B cell lymphoma-like protein 11 (BIM) levels were decreased at overtly toxic LCA concentrations, although PUMA levels increased at lower LCA concentrations in both cell lines. LCA induced autophagy-related conversion of microtubule-associated proteins 1A/1B light chain 3B (LC3BI–LC3BII), and autophagy-related protein ATG5 in PC-3 cells, but not in autophagy-deficient DU-145 cells. LCA (>10 µM) increased levels of reactive oxygen species (ROS) concentration-dependently in PC-3 cells, whereas ROS levels were not affected in DU-145 cells. Salubrinal, an inhibitor of eIF2α dephosphorylation and ER stress, reduced LCA-induced CHOP levels slightly in PC-3, but not DU-145 cells. Salubrinal pre-treatment increased the cytotoxicity of LCA in PC-3 and DU-145 cells and resulted in a statistically significant loss of cell viability at normally non-toxic concentrations of LCA. The late-stage autophagy inhibitor bafilomycin A1 exacerbated LCA toxicity at subtoxic LCA concentrations in PC-3 cells. The antioxidant α-tocotrienol strongly inhibited the toxicity of LCA in PC-3 cells, but not in DU-145 cells. Collectively, although LCA induces autophagy and ER stress in PC-3 cells, these processes appear to be initially of protective nature and subsequently consequential to, but not critical for the ROS-mediated mitochondrial dysfunction and cytotoxicity of LCA. The full mechanism of LCA-induced mitochondrial dysfunction and cytotoxicity in the similarly sensitive DU-145 cells remains to be elucidated.

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.

Exogenous p53 upregulated modulator of apoptosis (PUMA) decreases growth of lung cancer A549 cells.

To investigate the influence of exogenous p53 upregulated modulator of apoptosis (PUMA) expression on cell proliferation and apoptosis in human non-small cell lung cancer A549 cells and transplanted tumor cell growth in nude mice. A549 cells were divided into the following groups: control, non- carrier (NC), PUMA (transfected with pCEP4- (HA) 2-PUMA plasmid), DDP (10 μg/mL cisplatin treatment) and PUMA+DDP (transfected with pCEP4-(HA)2-PUMA plasmid and 10 μg/mL cisplatin treatment). The MTT method was used to detect the cell survival rate. Cell apoptosis rates were measured by flow cytometry, and PUMA, Bax and Bcl-2 protein expression levels were measured by Western blotting. Compared to the control group, the PUMA, DDP and PUMA+DDP groups all had significantly decreased A549 cell proliferation (p<0.01), with the largest reduction in the PUMA+DDP group. Conversely, the apoptosis rates of the three groups were significantly increased (P<0.01), and the PUMA and DDP treatments were synergistic. Moreover, Bax protein levels significantly increased (p<0.01), while Bcl-2 protein levels significantly decreased (p<0.01). Finally, both the volume and the weights of transplanted tumors were significantly reduced (p<0.01), and the inhibition ratio of the PUMA+DDP group was significantly higher than in the single DDP or PUMA groups. Exogenous PUMA effectively inhibited lung cancer A549 cell proliferation and transplanted tumor growth by increasing Bax protein levels and reducing Bcl-2 protein levels.

Apurinic/Apyrimidinic Endonuclease 1 Upregulation Reduces Oxidative DNA Damage and Protects Hippocampal Neurons from Ischemic Injury

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme that participates in base-excision repair of oxidative DNA damage and in the redox activation of transcription factors. We tested the hypothesis that APE1 upregulation protects neuronal structure and function against transient global cerebral ischemia (tGCI). Upregulation of APE1 by low-dose proton irradiation (PI) or by transgene overexpression protected hippocampal CA1 neurons against tGCI-induced cell loss and reduced apurinic/apyrimidinic sites and DNA fragmentation. Conversely, APE1 knockdown attenuated the protection afforded by PI and ischemic preconditioning. APE1 overexpression inhibited the DNA damage response, as evidenced by lower phospho-histone H2A and p53-upregulated modulator of apoptosis levels. APE1 overexpression also partially rescued dendritic spines and attenuated the decrease in field excitatory postsynaptic potentials in hippocampal CA1. Presynaptic and postsynaptic markers were reduced after tGCI, and this effect was blunted in APE1 transgenics. The Morris water maze test revealed that APE1 protected against learning and memory deficits for at least 27 days post-injury. Animals expressing DNA repair-disabled mutant APE1 (D210A) exhibited more DNA damage than wild-type controls and were not protected against tGCI-induced cell loss. This is the first study that thoroughly characterizes structural and functional protection against ischemia after APE1 upregulation by measuring synaptic markers, electrophysiological function, and long-term neurological deficits in vivo. Furthermore, disabling the DNA repair activity of APE1 was found to abrogate its protective impact. APE1 upregulation, either endogenously or through transgene overexpression, protects DNA, neuronal structures, synaptic function, and behavioral output from ischemic injury.

Over-Expression of PUMA Correlates with the Apoptosis of Spinal Cord Cells in Rat Neuropathic Intermittent Claudication Model

BackgroundNeuropathic intermittent claudication (NIC) is a typical clinical symptom of lumbar spinal stenosis and the apoptosis of neurons caused by cauda equina compression (CEC) has been proposed as an important reason. Whereas, the factors and the mechanism involved in the process of apoptosis induced by CEC remain unclear.Methodology and ResultsIn our modified rat model of NIC, a trapezoid-shaped silicon rubber was inserted into the epidural space under the L5 and L6 vertebral plate. Obvious apoptosis was observed in spinal cord cells after compression by TUNEL assay. Simultaneously, qRT-PCR and immunohistochemistry showed that the expression levels of PUMA (p53 up-regulated modulator of apoptosis) and p53 were upregulated significantly in spinal cord under compression, while the expression of p53 inhibitor MDM2 and SirT2 decreased in the same region. Furthermore, CEC also resulted in the upregulation of Bcl-2 pro-apoptotic genes expression and caspase-3 activation. With the protection of Methylprednisolone, the upregulation of PUMA and p53 expression as well as the decrease of MDM2 and SirT2 in spinal cord were partially rescued in western bolt analysis.ConclusionsThese results suggest that over-expression of PUMA correlates with CEC caused apoptosis of spinal cord cells, which is characterized by the increase of p53, Bax and Bad expression. PUMA upregulation might be crucial to induce apoptosis of spinal cord cells through p53-dependent pathway in CEC.

Supercritical Carbon Dioxide Extraction of Aromatic Turmerone from Curcuma longa Linn. Induces Apoptosis through Reactive Oxygen Species-Triggered Intrinsic and Extrinsic Pathways in Human Hepatocellular Carcinoma HepG2 Cells

The mechanisms underlying the antiproliferative and antitumor activities of aromatic turmerone (ar-turmerone), a volatile turmeric oil isolated from Curcuma longa Linn., have been largely unknown. In this study, 86% pure ar-turmerone was extracted by supercritical carbon dioxide and liquid-solid chromatography and its potential effects and molecular mechanisms on cell proliferation studied in human hepatocellular carcinoma cell lines. Ar-turmerone exhibited significant antiproliferative activity, with 50% inhibitory concentrations of 64.8 ± 7.1, 102.5 ± 11.5, and 122.2 ± 7.6 μg/mL against HepG2, Huh-7, and Hep3B cells, respectively. Ar-turmerone-induced apoptosis, confirmed by increased annexin V binding and DNA fragmentation, was accompanied by reactive oxygen species (ROS) production, mitochondrial membrane potential dissipation, increased Bax and p53 up-regulated modulator of apoptosis (PUMA) levels, Bax mitochondrial translocation, cytochrome c release, Fas and death receptor 4 (DR4) augmentation, and caspase-3, -8, and -9 activation. Exposure to caspase inhibitors, Fas-antagonistic antibody, DR4 antagonist, and furosemide (a blocker of Bax translocation) effectively abolished ar-turmerone-triggered apoptosis. Moreover, ar-turmerone stimulated c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) phosphorylation and activation; treatment with JNK and ERK inhibitors markedly reduced PUMA, Bax, Fas, and DR4 levels and reduced apoptosis but not ROS generation. Furthermore, antioxidants attenuated ar-turmerone-mediated ROS production; mitochondrial dysfunction; JNK and ERK activation; PUMA, Bax, Fas, and DR4 expression; and apoptosis. Taken together, these results suggest that ar-turmerone-induced apoptosis in HepG2 cells is through ROS-mediated activation of ERK and JNK kinases and triggers both intrinsic and extrinsic caspase activation, leading to apoptosis. On the basis of these observations, ar-turmerone deserves further investigation as a natural anticancer and cancer-preventive agent.

P53 and PUMA Independently Regulate Apoptosis of Intestinal Epithelial Cells in Patients and Mice With Colitis

Inflammatory bowel disease (IBD) is associated with increased apoptosis of intestinal epithelial cells (IECs). Mutations in the tumor suppressor p53 appear during early stages of progression from colitis to cancer. We investigated the role of p53 and its target, p53-upregulated modulator of apoptosis (PUMA), in inflammation-induced apoptosis of IECs. Apoptosis was induced in mouse models of mucosal inflammation. Responses of IECs to acute, T-cell activation were assessed in wild-type, p53⁻/⁻, Bid⁻/⁻, Bim⁻/⁻, Bax3⁻/⁻, Bak⁻/⁻, PUMA⁻/⁻, and Noxa⁻/⁻ mice. Responses of IECs to acute and chronic colitis were measured in mice following 1 or 3 cycles of dextran sulfate sodium (DSS), respectively. Apoptosis was assessed by TUNEL staining and measuring activity of caspases 3 and 9; levels of p53 and PUMA were assessed in colon tissue from patients with and without ulcerative colitis. Apoptosis of IECs occurred in the lower crypts of colitic tissue from humans and mice. Colitis induction with anti-CD3 or 3 cycles of DSS increased apoptosis and protein levels of p53 and PUMA in colonic crypt IECs. In p53⁻/⁻ and PUMA⁻/⁻ mice, apoptosis of IECs was significantly reduced but inflammation was not. Levels of p53 and PUMA were increased in inflamed mucosal tissues of mice with colitis and in patients with UC, compared with controls. Induction of PUMA in IECs of p53⁻/⁻ mice indicated that PUMA-mediated apoptosis was independent of p53. In mice and humans, colon inflammation induces apoptosis of IECs via p53-dependent and - independent mechanisms; PUMA also activates an intrinsic apoptosis pathway associated with colitis.

Pifithrin-α reduces cerebral vasospasm by attenuating apoptosis of endothelial cells in a subarachnoid haemorrhage model of rat

The mechanism of cerebral vasospasm following subarachnoid haemorrhage (SAH) is not understood. Here, we hypothesized that apoptosis of endothelial cells induced by p53 and its target gene em dash p53 upregulated modulator of apoptosis (PUMA) played an important role in development of cerebral vasospasm. We also observed the effects of a p53 inhibitor, pifithrin-alpha (PFT-alpha), on reducing the expression of p53 and PUMA, consequently decreasing the apoptosis of endothelial cells and alleviating cerebral vasospasm. Male Sprague-Dawley rats weighing 300-350 g were randomly divided into five groups: a control group (sham surgery), a SAH group, a SAH+dimethyl sulfoxide (DMSO) group, a SAH + PFT-alpha (0.2 mg/kg) group and a SAH + PFT-alpha (2.0 mg/kg) group. PFT-alpha was injected intraperitoneally immediately after SAH. Rats were sacrificed 24 hours after SAH. Western blot and immunohistochemical staining were used to detect the levels of p53, PUMA and caspase-3 protein. In addition, mortality and neurological scores were assessed for each group. Statistical significance was assured by analysis of variance performed in one way ANOVA followed by the Tukey test. The neurological and mortality scores were analyzed by Dunn's method and Fisher exact test, respectively. After SAH, Western blot and immunohistochemical staining showed the levels of p53, PUMA and caspase-3 in the endothelial cells and the numbers of TdT mediated dUTP nick end labelling (TUNEL) positive endothelial cells were all significantly increased in the basilar arteries (P<0.05), but significantly reduced by PFT-alpha (P<0.05). These changes were accompanied by increasing diameters and declining wall thickness of basilar arteries (P<0.05), as well as reduced mortality and neurological deficits of the rats (P<0.05). PFT-alpha could protect cerebral vessels from development of vasospasm and improve neurological outcome as well as reduce the mortality via suppressing apoptosis induced by p53 in the endothelial cells of cerebral vessels.

Cyclin-dependent Kinase Inhibitors Uncouple Cell Cycle Progression from Mitochondrial Apoptotic Functions in DNA-damaged Cancer Cells

DNA damage results in transcriptional induction of p53 target genes, including the cyclin-dependent kinase (CDK) inhibitor p21(Cip1) (CDKN1A) and the proapoptotic Bcl-2 family member p53 up-regulated modulator of apoptosis (PUMA). Depending on the cellular context, p21(Cip1) and PUMA mediate cell cycle arrest and apoptosis, respectively. By imposing cell cycle arrest at the expense of apoptosis, p21(Cip1) can sharply reduce the effectiveness of DNA-damaging anticancer agents in colorectal cancer cells. We investigated the link between cell cycle progression and the onset of apoptosis in DNA-damaged cells by analyzing the activation of the apoptotic cascade in p21(Cip1)-deficient HCT116 colorectal cancer cells. DNA damage induced a similar level of p53 activation and PUMA induction in p21(Cip1)-deficient cells compared with wild-type isogenic counterparts. p21(Cip1) did not act as a direct blocker of PUMA. However, only p21(Cip1)-deficient cells showed extensive cytochrome c release, mitochondrial membrane depolarization, and caspase activation. An increase in caspase activation occurred as these cells reached M-phase and incurred polyploidy. When ectopically expressed in p21(Cip1)-deficient HCT116 cells, p21(Cip1), its family member p27(Kip1), and the structurally unrelated CDK inhibitor p16(Ink4a) were similarly effective at causing cell cycle arrest and inhibiting DNA damage-induced apoptotic events such as cytochrome c release, mitochondrial membrane depolarization, and activation of the caspase cascade. These observations suggest that by blocking dysregulated cell cycle progression, CDK inhibitors can influence the sensitivity of the mitochondria to proapoptotic signals in DNA damage-induced cancer cells.