P53 Upregulated Modulator Of Apoptosis Research Articles

Sildenafil Reverses the Neuropathological Alzheimer's Disease Phenotype in Cholinergic-Like Neurons Carrying the Presenilin 1 E280A Mutation.

Familial Alzheimer's disease (FAD) presenilin 1 E280A (PSEN 1 E280A) is characterized by functional impairment and the death of cholinergic neurons as a consequence of amyloid-β (Aβ) accumulation and abnormal phosphorylation of the tau protein. Currently, there are no available therapies that can cure FAD. Therefore, new therapies are urgently needed for treating this disease. To assess the effect of sildenafil (SIL) on cholinergic-like neurons (ChLNs) harboring the PSEN 1 E280A mutation. Wild-type (WT) and PSEN 1 E280A ChLNs were cultured in the presence of SIL (25μM) for 24 h. Afterward, proteinopathy, cell signaling, and apoptosis markers were evaluated via flow cytometry and fluorescence microscopy. We found that SIL was innocuous toward WT PSEN 1 ChLNs but reduced the accumulation of intracellular Aβ fragments by 87%, decreased the non-physiological phosphorylation of the protein tau at residue Ser202/Thr205 by 35%, reduced the phosphorylation of the proapoptotic transcription factor c-JUN at residue Ser63/Ser73 by 63%, decreased oxidized DJ-1 at Cys106-SO3 by 32%, and downregulated transcription factor TP53 (tumor protein p53), BH-3-only protein PUMA (p53 upregulated modulator of apoptosis), and cleaved caspase 3 (CC3) expression by 20%, 32%, and 22%, respectively, compared with untreated mutant ChLNs. Interestingly, SIL also ameliorated the dysregulation of acetylcholine-induced calcium ion (Ca2+) influx in PSEN 1 E280A ChLNs. Although SIL showed no antioxidant capacity in the oxygen radical absorbance capacity and ferric ion reducing antioxidant power assays, it might function as an anti-amyloid and antiapoptotic agent and functional neuronal enhancer in PSEN 1 E280A ChLNs. Therefore, the SIL has therapeutic potential for treating FAD.

Induction of breast cancer cell apoptosis by novel thiouracil-fused heterocyclic compounds through boosting of Bax/Bcl-2 ratio and DFT study

Breast cancer is a common public health disease causing mortality worldwide. Thus, providing novel chemotherapies that tackle breast cancer is of great interest. In this investigation, novel pyrido[2,3-d]pyrimidine derivatives 3,4,(6a-c),(8a,b),9–20 were synthesized and characterized using a variety of spectrum analyses. The geometric and thermal parameters of the novel thiouracil derivatives 3,4,6a,(8a,b),11,12,17,18, 19 were measured using density functional theory (DFT) via DFT/B3LYP/6–31 + G(d,p) basis set. All synthesized compounds were evaluated by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) method using MCF-7 and MDA-MB-231 breast cancerous cells, compound 17 had the maximum anticancer activity against both breast cancerous cells, recording the lowest half-maximal inhibitory concentration (IC50) values (56.712 μg/mL for MCF-7 cells and 48.743 μg/mL for MDA-MB-231 cells). The results were confirmed in terms of the intrinsic mechanism of apoptosis, where compound 17 had the highest percentage in the case of both cancer cells and recorded Bax (Bcl-2 associated X)/Bcl-2 (B-cell lymphoma 2) ratio 17.5 and 96.667 for MCF-7 and MDA-MB-231 cells, while compound 19 came after 17 in the ability for induction of apoptosis, where the Bax/Bcl-2 ratio was 15.789 and 44.273 for both cancerous cells, respectively. Also, compound 11 recorded a high Bax/Bcl-2 ratio for both cells. The safety of the synthesized compounds was applied on normal WI-38 cells, showing minimum cytotoxic effect with undetectable IC50. Compounds 17, 11, and 19 recorded a significant increase of p53 upregulated modulator of apoptosis (PUMA) expression levels in the cancerous cells. The DFT method was also used to establish a connection between the experimentally determined values of the present investigated compounds and their predicted quantum chemical parameters. It was concluded that Compounds 17, 11, and 19 had anti-breast cancer potential through the induction of apoptotic Bax/Bcl-2 and PUMA expression levels.

Statement of Retraction: Dexmedetomidine attenuates ischemia and reperfusion-induced cardiomyocyte injury through p53 and forkhead box O3a (FOXO3a)/p53-upregulated modulator of apoptosis (PUMA) signaling signaling

Statement of Retraction: Dexmedetomidine attenuates ischemia and reperfusion-induced cardiomyocyte injury through p53 and forkhead box O3a (FOXO3a)/p53-upregulated modulator of apoptosis (PUMA) signaling signaling

Olverembatinib (HQP1351) Enhances Antitumor Effects of Chemotherapy in Philadelphia Chromosome-Positive (Ph⁺) Acute Lymphoblastic Leukemia (ALL)

Introduction Ph⁺ ALL is the most common subtype of B-cell- (B-) ALL in adults. BCR-ABL1 tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML) cells. However, treatment with TKIs fails to maintain disease remission in Ph⁺ B-ALL. Although the complete hematologic remission rate is very high, cytogenetic and molecular remission rates are low, so that TKIs are still considered as complements to chemotherapy and bridges to allogeneic stem cell transplantation. Olverembatinib is a novel, third-generation TKI with more potent activity against BCR-ABL1 and overcomes various resistant mutations, including the “gatekeeper” T315I, which confers resistance against all first- and second-generation TKIs. The aim of this study was to assess whether olverembatinib combined with chemotherapeutic agents vincristine or doxorubicin can promote anti-cancer effects in Ph⁺ ALL cells and explore the potential mechanism of action of the combinations. Methods Cell-based antiproliferation assays to evaluate the activities of olverembatinib alone or in combination with vincristine or doxorubicin were conducted using the CellTiter-Glo® Luminescent Cell Viability assay kits. Drug induced cell apoptosis was measured by flow cytometry. Potential mechanism of action analysis was conducted using western blot to evaluate the protein expression involved in BCR-ABL1 and apoptosis signaling pathways. Results Cell-based antiproliferation studies showed synergistic activity of olverembatinib in combination with vincristine or doxorubicin in Ph⁺ ALL SUP-B15 cells compared with single agents after 72 hours of treatment. The percentage of apoptotic cells induced by 24-hour treatment of olverembatinib (100 nM), vincristine (300 nM), and the combination were 27.7%, 27.2%, and 40.7%, respectively, while the percentage of apoptotic cells induced by 24-hour treatment of doxorubicin (100 nM) alone and in combination with olverembatinib (100 nM) were 35.3 and 74.1, respectively (apoptotic cells % in DMSO control group is 6.74). The combination significantly increased cell apoptosis compared with single agents ( p < 0.0001). Mechanistically, the combination of olverembatinib with vincristine showed synergistic effects in inhibiting protein expression and phosphorylation of BCR-ABL1, and downstream signal transducer and activator of transcription 5 (STAT5). The synergistic effects were also seen in decreased phosphorylation of AKT and ERK1/2. In addition, the combination significantly decreased protein levels of antiapoptotic MCL-1, BCL-2, and BCL-xL and increased protein levels of proapoptotic BAX and p53 upregulated modulator of apoptosis (PUMA) in a dose-dependent manner, which led to marked augmentation of cleavage of caspase-3 and poly (ADP-ribose) polymerase 1 (PARP). A similar pattern was also observed when olverembatinib was combined with doxorubicin. Conclusions Olverembatinib, a novel, third-generation TKI, in combination with chemotherapeutic agents vincristine or doxorubicin decreased MCL-1, BCL-2, and BCL-xL and increased BAX and PUMA in a dose-dependent manner, the combinations have demonstrated synergistic antitumor effects by enhancing apoptosis and antiproliferation in Ph⁺ ALL cells, which may provide an alternative approach for treatment of patients with Ph⁺ ALL.

PUMA/RIP3 Mediates Chemotherapy Response via Necroptosis and Local Immune Activation in Colorectal Cancer.

Induction of programmed cell death (PCD) is a key cytotoxic effect of anticancer therapies. PCD is not confined to caspase-dependent apoptosis, but includes necroptosis, a regulated form of necrotic cell death controlled by receptor-interacting protein (RIP) kinases 1 and 3, and mixed lineage kinase domain-like (MLKL) pseudokinase. Necroptosis functions as a defense mechanism against oncogenic mutations and pathogens and can be induced by a variety of anticancer agents. However, the functional role and regulatory mechanisms of necroptosis in anticancer therapy are poorly understood. In this study, we found that RIP3-dependent but RIP1-independent necroptosis is engaged by 5-fluorouracil (5-FU) and other widely used antimetabolite drugs, and functions as a major mode of cell death in a subset of colorectal cancer cells that express RIP3. We identified a novel 5-FU-induced necroptosis pathway involving p53-mediated induction of the BH3-only Bcl-2 family protein, p53 upregulated modulator of apoptosis (PUMA), which promotes cytosolic release of mitochondrial DNA and stimulates its sensor z-DNA-binding protein 1 (ZBP1) to activate RIP3. PUMA/RIP3-dependent necroptosis mediates the in vitro and in vivo antitumor effects of 5-FU and promotes a robust antitumor immune response. Our findings provide a rationale for stimulating necroptosis to enhance tumor cell killing and antitumor immune response leading to improved colorectal cancer treatments.

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.

Cardioprotective effect of tetra(aniline) containing terpolymers through miR-15a-5p and MFN-2 regulation against hypertrophic responses

ObjectiveCardiac hypertrophy is a condition of abnormal cardiomyocyte enlargement accompanied by ventricular wall thickening. The study aims to investigate the role of miR-15a-5p in the regulation of mitofusin-2 (MFN-2) and to explore the cardioprotective effect of terpolymers ES-37 and L-37. MethodsIn this study, the Sprague Dawley rats’ cardiac hypertrophic model was established by administering 5 mg/kg Isoproterenol subcutaneously every other day for 14 days. As treatment rats received NAC (50 mg/kg), NAC treatment (50 mg/kg NAC + 5 mg/kg ISO), ES-37 (1 mg/kg) and ES-37 treatment (1 mg/kg ES-37+5 mg/kg ISO), L-37 (1 mg/kg) and L-37 treatment (1 mg/kg L-37+5 mg/kg ISO). subcutaneously every other day for 14 days. NAC, ES 37 and L-37 were given after 1 h of Isoproterenol administration in treatment groups. Cardiac hypertrophy was confirmed through morphological and histological analysis. For estimation of oxidative stress profiling, ROS and TBARS and antioxidative profiling superoxide dismutase (SOD), Catalase, and Glutathione (GSH) levels were checked. Triglyceride, cholesterol, alanine transaminase (ALT), and aspartate transaminase (AST) were performed to evaluate levels of lipid profiling and liver profiling. Molecular expression analysis was checked through real-time PCR, and western blotting both at the transcriptional and translational levels. Molecular docking studies were performed to study the interactions and modes of binding between the synthetic polymers with three proteins (Mitofusin-2, DRP-1 and PUMA). All the studies were carried out using the AutoDock Vina software and the protein-ligand complexes were visualized in Biovia Discovery Studio.Cardiac hypertrophy was confirmed by the relative changes in the cellular structure of the heart by histopathological examination and physiological changes by estimating organ weights. Biochemical profiling results depict elevated oxidative and lipid profiles signify myocardial damage. N-acetyl cysteine (NAC), ES-37, and L-37 overcome the cardiac hypertrophic responses through attenuating oxidative stress and enhancing the antioxidative signaling mechanism. miR-15a-5p was identified as hypertrophic microRNA directly regulating the expression of Mitofusin-2 (MFN-2). Significantly increased expression of miR-15a-5p, Dynamin related protein 1 (Drp1), and P53 upregulated modulator of apoptosis (PUMA), was observed in the disease group, whereas MFN-2 expression was observed downregulated. N-acetyl cysteine (NAC), ES-37, and L-37 showed increased expression of antiapoptotic maker MFN-2 and decreased expression of miR-15a-5p, Drp1, and PUMA in treatment groups suggesting their cardioprotective role in attenuation of cardiac hypertrophy. An analysis of the docking results shows that ES-37 has greater binding affinity with the target proteins compared to L-37, with the highest binding values reported for MFN-2. ConclusionThe physiochemical properties of ES-37 and L-37 predicted it as a good drug-like molecule and its mechanism of action is predictably through inhibition of ROS. Molecular docking results shows that the polymer ES-37 has greater binding affinity with the target proteins compared to L-37, with the highest binding values reported for MFN-2. Thus, the study validates the role and targeting of miR-15a-5p and MFN-2 in cardiac hypertrophy as well as the therapeutic potential of NAC, ES-37, and L-37 in overcoming oxidative stress and myocardial damage.

Scribble promotes fibrosis-dependent mechanisms of hepatocarcinogenesis by p53/PUMA-mediated glycolysis

Backgrounds and aimsLiver cancer is the sixth most common type of cancer and the fifth leading cause of cancer mortality worldwide. Scribble has been shown to function as a neoplastic tumor suppressor gene in most tumors. Our previous studies reported that down-regulation or mislocalization of Scribble was sufficient to initiate mammary tumorigenesis and NSCLC. Recently, it was reported that Scribble was highly expressed in hepatocellular carcinoma (HCC). We aim to study how it was up-regulated and the contradictory role of Scribble in HCC. Methods and resultsUsing a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis system, we showed that Scribble was over-expressed and which may protect the mice against hepatic fibrosis. Unexpectedly, we found out the potential for Scribble to act as a tumor driver at the advanced stage of N-nitrosodiethylamine (DEN) plus CCl4 induced HCC mice model in vivo. In addition, we observed even higher expression of Scribble in HCC tumors harboring elevated levels of wild-type p53. Most importantly, nuclear translocated Scribble could interact with p53, which lead to enhanced stability and transcriptional activity of p53. Mechanistically, our data suggested that Scribble might drive HCC progression by promoting metabolic regulation of p53 through p53-upregulated modulator of apoptosis (PUMA)-mediated Warburg effect. ConclusionsOur data identified the molecular basis of hepatic fibrosis-specific gene expression of polarity gene, such as Scribble. Interestingly, with the progression from fibrosis to cirrhosis to HCC, its nuclear translocation promoted a wild-type p53-mediated cancer metabolic switch and tumor progression in HCC. Taken together, we demonstrated that Scribble was up-regulated and served a protective role in liver fibrosis, while also apparently acting as a tumor driver in fibrosis-dependent hepatocarcinogenesis.

In vivo hematopoietic stem cell modification by mRNA delivery.

Hematopoietic stem cells (HSCs) are the source of all blood cells over an individual's lifetime. Diseased HSCs can be replaced with gene-engineered or healthy HSCs through HSC transplantation (HSCT). However, current protocols carry major side effects and have limited access. We developed CD117/LNP-messenger RNA (mRNA), a lipid nanoparticle (LNP) that encapsulates mRNA and is targeted to the stem cell factor receptor (CD117) on HSCs. Delivery of the anti-human CD117/LNP-based editing system yielded near-complete correction of hematopoietic sickle cells. Furthermore, in vivo delivery of pro-apoptotic PUMA (p53 up-regulated modulator of apoptosis) mRNA with CD117/LNP affected HSC function and permitted nongenotoxic conditioning for HSCT. The ability to target HSCs in vivo offers a nongenotoxic conditioning regimen for HSCT, and this platform could be the basis of in vivo genome editing to cure genetic disorders, which would abrogate the need for HSCT.

The effect of Oxaliplatin in leading to apoptosis of intestinal cancer cells byinducing PUMA protein expression through reactive oxygen species

Previous studies demonstrated Oxaliplatin, a drug used in treating intestinal cancers by inducing cancer cell apoptosisregulated by p53 upregulated modulator of apoptosis (PUMA) protein gene expression. Increasing the reactiveoxygen species (ROS) level is possible to speed up the expression of PUMA protein in Oxaliplatin. This study aimsto investigate the effect of Oxaliplatin in intestinal cancer cells, leading to its apoptosis by inducing PUMA proteinexpression through ROS, compared to Cisplatin and PBS. The result of the study will provide important insight into thepreclinical effectiveness of Oxaliplatin in intestinal cancer cells, and it also testifies to the underlying mechanism of thisdrug. Future studies should focus on investigating drug combinations with Oxaliplatin that provides synergism towardthe disease and decreases its toxicity.

Cardioprotective effect of Ginger in a rat model of myocardial damage and its possible intervention in PERK-ATF4-CHOP-PUMA apoptotic pathway

For today the exact mechanisms of myocardial infarction and ischemia/reperfusion injury are still not fully understood. ER stress and integrated stress response pathways are thought to play an essential role in myocardial damage. This includes activation of endoplasmic reticulum kinase PERK, induction of activating transcription factor ATF4, expression of pro-apoptotic transcription factor CHOP and P53 up-regulated modulator of apoptosis (PUMA) involved in apoptosis control. We used a rat model of isoproterenol-induced myocardial damage to elucidate the possible cardioprotective effect of Ginger through the influence on ER stress-induced apoptotic pathway. We also compared its effect with the ACE inhibitor Captopril. Male albino Wistar rats received 1.0 or 2.0 ml of Zingiber officinale (Ginger) powder suspension (200 mg/ml) daily by intra-gastric intubation for 28 days. Isoproterenol at a dose of 85 mg/kg was IP injected on the 27th and 28th days. Serum aspartate transaminase (AST) level was measured using kinetic kit. Heart tissue was used for RNA extraction, evaluation of gene expression by Q-RT-PCR, immuno-histochemical determination of caspase-3 expression and histopathological studies. Our results showed that Isoproterenol administration increased CHOP-mRNA expression 4 folds in cardiac muscle tissue compared to normal control. Ginger pretreatment significantly decreased both CHOP and ATF4, and PUMA mRNA expression compared to Isoproterenol-treated groups. A significant reduction in ATF4 mRNA expression in a group pretreated with Captopril and Ginger compared to normal control group was observed. The results showed that Ginger reduced AST serum levels which correlated with results of histopathological studies of heart tissue. Our findings suggest that the protective effects of Ginger against myocardium damage induced by Isoproterenol may be mediated by reducing­ the endoplasmic reticulum stress by affecting the ATF4-CHOP-PUMA pathway. Keywords: AST, ATF4, Captopril, CHOP, Ginger, ischemia-reperfusion, Isoproterenol, myocardial damage, PUMA

Abstract 3929: MDMX overexpression in cancer cells confers significant resistance to MDMX inhibitor XI-006 and may modulate chemosensitivity through suppressing p53 activation of pro-apoptotic factors

Abstract MDM2 inhibition has gained popularity in recent years as a therapeutic strategy in targeting both solid tumors and hematological malignancies, and several small molecule inhibitors of the p53-MDM2 binding interface have progressed into clinical trials in recent years. Alongside direct stabilization of p53, MDM2 has further been identified to have immune implications in terms of CD8+ T-cell mediated anti-tumor immunity and checkpoint-blockade therapies. However, little work has been done to explore the therapeutic targeting potential of related protein MDMX (MDM4). Pan-cancer analyses conducted by our lab (in part presented at the ASCO 2022 Annual Meeting) identify an important role of MDMX in several tumor types. Particularly, MDMX amplification is associated with a higher risk of brain and liver metastasis, and MDMX amplification itself also presents with significant frequency (10%) in glioblastoma multiforme (GBM). Notably, MDMX amplification also served as a better predictor of reduced overall survival in NSCLC following checkpoint-blockade. We have also previously reported on associations between MDMX amplification and CDKN2A deep deletions in melanoma, a predictor of favorable response to checkpoint blockade. Such findings establish the need for further exploration to be done and the role of MDMX both in cell phenotype and potential immune regulation to be better characterized. Preliminary data acquired from transient overexpression experiments using lipofection of MDMX cDNA and MDMX inhibitor XI-006 (NSC207895) alone and in combination with chemotherapy further supports the viability of MDMX-inhibition as a therapeutic strategy in several cell lines. Overexpression of MDMX in HCT116 p53WT cells demonstrably confers remarkable resistance to MDMX inhibitor XI-006 at a 72-hour timepoint (IC50 of 104.75 uM in overexpressing cells versus 29.03 uM in WT), and treatment with oxaliplatin reveals both reduced basal and post-treatment expression of p53-upregulated modulator of apoptosis (PUMA). Further mechanistic workup of this phenomenon is underway, particularly with an emphasis on immuno-oncological therapeutic translations. Altogether, initial findings ultimately establish the need for further investigation into the mechanistic basis of MDMX-correlated clinical outcomes and the therapeutic potential of MDMX inhibition. Citation Format: Andrew George, Arielle De La Cruz, Shengliang Zhang, Ilyas Sahin, Praveen Srinivasan, Maximilian Schwermann, Morgan Turcotte, Taylor Arnoff, Wafik S. El-Deiry. MDMX overexpression in cancer cells confers significant resistance to MDMX inhibitor XI-006 and may modulate chemosensitivity through suppressing p53 activation of pro-apoptotic factors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3929.

Dysregulation of Expression of the FOXO3 Transcription Factors Pathway in Helicobacter pylori Associated Gastric Cancer

The transcription factor FOXO3 acts as a tumor suppressor and is the major target of AKT kinases pathway. The protein encoded by FOXO3, prevents the activation of genes that are important for proliferation, differentiation and death cell. In addition, FOXO3 interacts with BCL-2 family proteins; and among them, BBC3 (PUMA) and BCL2L (BIM) are responsible for regulating apoptosis through the BAX and BAK chains. This study investigated the expression of FOXO3a, BBC3 and BCL2L in gastric cancer considering the presence of Helicobacter pylori and its involvement in the modulation of gene and protein expression. A total of 225 gastric biopsy samples were evaluated from individuals with normal gastric mucosa (n=54), gastritis (n=108), and gastric cancer (n=63), of both genders and over 18 years old. Samples were subclassified according to the presence or absence of Helicobacter pylori, detected by PCR. Real-time quantitative RT-PCR (qRT-PCR) and western blotting, were used to detect the expression. FOXO3 showed increased gene (p<0.0001) and protein (p=0.036) expression in patients with gastric cancer, who also exhibited a boost in mRNA expression through H. pylori infection. BCL2L had a subtle increase in its expression independent of the presence of H. pylori (p<0.0001), while the BBC3 gene showed no alterations. Our results suggested that increase FOXO3 expression may play an important role in carcinogenesis by performing positive feedback in an attempt to eliminate neoplastic cells. FOXO3 could be suggested a prognostic marker as well as a potential molecular therapy target for gastric cancer patients.

Inhibition of secretory leukocyte protease inhibitor (SLPI) promotes the PUMA-mediated apoptosis and chemosensitivity to cisplatin in colorectal cancer cells

BackgroundAberrant expression of Secretory Leukocyte Protease Inhibitor (SLPI) has been associated with human cancer growth and its suppression was identified as a potential target for anti-cancer drugs, particularly in colorectal cancer. However, the underlying mechanism by which SLPI affected the development of drug resistance in CRC remains unclear.ObjectiveThis study investigated the role of SLPI in the p53-up-regulated modulator of apoptosis (PUMA)-mediated CRC cells’ apoptosis and their chemosensitivity to Cisplatin.MethodsA series of qRT-PCR and western blot analyses were performed to characterize the expressions of SLPI, PUMA, and Akt in CRC lines. Tunel, transwell, and CCK-8 analyses were monitored to define the impacts of the siRNA-mediated knockdown of SLPI on CRC cell development. Furthermore, in vivo development of CRC was evaluated in nude mice infected with siSLPI or Cisplatin alone or both, and Ki67 and caspase-3 immunohistochemistry assay was monitored on multiple tissue microarray from the same cohort.ResultsOur results showed that SLPI inhibition strongly promoted the expressions of the pro-apoptotic protein PUMA, cleaved-caspase3 and Bax and reduced the cell viability of HT29 and HT116 cell lines in vitro. In addition, siSLPI knockdown effectively suppressed both Akt and FoxO3 proteins and improved the sensitivity to cisplatin chemotherapy. Xenograft tumor assay revealed a lowered growth in mice treated with Cisplatin, while combined treatment of siSLPI achieved more significant anticancer effects than Cisplatin alone.ConclusionsTaken together, these findings demonstrated that suppression of SLPI might repress the growth of human colorectal cancer cells both in vitro and in vivo. These results suggested SLPI as a novel resistance factor to Cisplatin, and a combination of Cisplatin and SLPI inhibitor be beneficial for colorectal cancer therapy.

Interdependent Nuclear Co-Trafficking of ASPP1 and p53 Aggravates Cardiac Ischemia/Reperfusion Injury.

ASPP1 (apoptosis stimulating of p53 protein 1) is critical in regulating cell apoptosis as a cofactor of p53 to promote its transcriptional activity in the nucleus. However, whether cytoplasmic ASPP1 affects p53 nuclear trafficking and its role in cardiac diseases remains unknown. This study aims to explore the mechanism by which ASPP1 modulates p53 nuclear trafficking and the subsequent contribution to cardiac ischemia/reperfusion (I/R) injury. The immunofluorescent staining showed that under normal condition ASPP1 and p53 colocalized in the cytoplasm of neonatal mouse ventricular cardiomyocytes, while they were both upregulated and translocated to the nuclei upon hypoxia/reoxygenation treatment. The nuclear translocation of ASPP1 and p53 was interdependent, as knockdown of either ASPP1 or p53 attenuated nuclear translocation of the other one. Inhibition of importin-β1 resulted in the cytoplasmic sequestration of both p53 and ASPP1 in neonatal mouse ventricular cardiomyocytes with hypoxia/reoxygenation stimulation. Overexpression of ASPP1 potentiated, whereas knockdown of ASPP1 inhibited the expression of Bax (Bcl2-associated X), PUMA (p53 upregulated modulator of apoptosis), and Noxa, direct apoptosis-associated targets of p53. ASPP1 was also increased in the I/R myocardium. Cardiomyocyte-specific transgenic overexpression of ASPP1 aggravated I/R injury as indicated by increased infarct size and impaired cardiac function. Conversely, knockout of ASPP1 mitigated cardiac I/R injury. The same qualitative data were observed in neonatal mouse ventricular cardiomyocytes exposed to hypoxia/reoxygenation injury. Furthermore, inhibition of p53 significantly blunted the proapoptotic activity and detrimental effects of ASPP1 both in vitro and in vivo. Binding of ASPP1 to p53 triggers their nuclear cotranslocation via importin-β1 that eventually exacerbates cardiac I/R injury. The findings imply that interfering the expression of ASPP1 or the interaction between ASPP1 and p53 to block their nuclear trafficking represents an important therapeutic strategy for cardiac I/R injury.

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.

Exosome-mediated delivery of Cas9 ribonucleoprotein complexes for tissue-specific gene therapy of liver diseases.

CRISPR-Cas9 gene editing has emerged as a powerful therapeutic technology, but the lack of safe and efficient in vivo delivery systems, especially for tissue-specific vectors, limits its broad clinical applications. Delivery of Cas9 ribonucleoprotein (RNP) owns competitive advantages over other options; however, the large size of RNPs exceeds the loading capacity of currently available delivery vectors. Here, we report a previously unidentified genome editing delivery system, named exosomeRNP, in which Cas9 RNPs were loaded into purified exosomes isolated from hepatic stellate cells through electroporation. ExosomeRNP facilitated effective cytosolic delivery of RNP in vitro while specifically accumulated in the liver tissue in vivo. ExosomeRNP showed vigorous therapeutic potential in acute liver injury, chronic liver fibrosis, and hepatocellular carcinoma mouse models via targeting p53 up-regulated modulator of apoptosis (PUMA), cyclin E1 (CcnE1), and K (lysine) acetyltransferase 5 (KAT5), respectively. The developed exosomeRNP provides a feasible platform for precise and tissue-specific gene therapies of liver diseases.

Effects of PUMA knockout on the apoptosis of H9C2 cardiomyocytes induced by high glucose

To investigate the effects of p53 upregulated modulator of apoptosis (PUMA) on the apoptosis of H9C2 cardiomyocytes induced by high glucose and its mechanisms. H9C2 cardiomyocytes were treated with 5.5mmol/L (control group) or 35 mmol/L glucose (HG group) for 6 h, 12 h, 24 h or 48 h respectively to induce apoptosis, each group sets 5 multiple wells. Apoptosis was tested by TUNEL assay. PUMA mRNA was measured by RT-PCR and protein expression was measured by Western blot assay. The mitochondrial membrane potential was detected by JC-1 method. The expressions of cleaved caspase-3 and cytochrome C (Cyt C) protein in mitochondria and cytoplasm were determined by Western blot assay. H9C2 cardiomyocytes were randomly divided into four groups, control group (5.5 mmol/L), HG (35 mmol/L) group, HG+si-scramble group(si-scramble treatment for 24 h, then 35 mmol/L high glucose treatment for 24 h) and HG-si-PUMA group (si-PUMA treatment for 24 h, then 35mmol/L high glucose treatment for 24 h). Si-PUMA was transfected into cardiomyocytes and the effects of PUMA on high glucose-induced apoptosis were studied. Compared with the control group, high glucose increased cardiomyocyte apoptosis and enhanced PUMA mRNA and protein expressions significantly (P＜0.05 or P＜0.01). Cell injury and increased PUMA expression were time-dependent and there was no significant difference between the high glucose 24 h group and the high glucose 48h group. The following experiment used high glucose 24 h as the stimulation time. The cardiomyocytes transfected with si-PUMA to inhibit PUMA expression had decreased apoptotic rate and cleaved caspase-3, increased mitochondria membrane potential and decreased Cyt C release (P＜0.05 or P＜0.01). There were no significant differences between the HG+si-scramble group and the high glucose group (P＞0.05). PUMA mediates high glucose-induced cardiomyocyte apoptosis suggesting PUMA may be an important target gene of diabetic cardiomyopathy.

DNA damage and reticular stress in cytotoxicity and oncotic cell death of MCF-7 cells treated with fluopsin C

ABSTRACT Fluopsin C is an antibiotic compound derived from secondary metabolism of different microorganisms, which possesses antitumor, antibacterial, and antifungal activity. Related to fluopsin C antiproliferative activity, the aim of this study was to examine the following parameters: cytotoxicity, genotoxicity, cell cycle arrest, cell death induction (apoptosis), mitochondrial membrane potential (MMP), colony formation, and mRNA expression of genes involved in adaptive stress responses and cellular death utilizing a monolayer. In addition, a three-dimensional cell culture was used to evaluate the effects on growth of tumor spheroids. Fluopsin C was cytotoxic (1) producing cell division arrest in the G1 phase, (2) elevating expression of mRNA of the CDKN1A gene and (3) decrease in expression of mRNA H2AFX gene. Further, fluopsin C enhanced DNA damage as evidenced by increased expression of mRNA of GADD45A and GPX1 genes, indicating that reactive oxygen species (ROS) may be involved in the observed genotoxic response. Reticulum stress was also detected as noted from activation of the ribonuclease inositol-requiring protein 1 (IRE1) pathway, since a rise in mRNA expression of the ERN1 and TRAF2 genes was observed. During the cell death process, an increase in mRNA expression of the BBC3 gene was noted, indicating participation of this antibiotic in oncotic (ischemic) cell death. Data thus demonstrated for the first time that fluopsin C interferes with the volume of tumor spheroids, in order to attenuate their growth. Our findings show that fluopsin C modulates essential molecular processes in response to stress and cell death.

Tolfenamic acid inhibits ROS-generating oxidase Nox1-regulated p53 activity in intrastriatal injection of malonic acid rats

It has been reported that wild-type p53-induced gene 1 (Wig1), which is downstream of p53, regulates the expression of mutant huntingtin protein (mHtt) in Huntington’s disease (HD) patients and transgenic mouse brains. Intrastriatal injection of malonic acid in rats is often used as a model to study the pathological changes of Huntington’s disease, and this model has the advantages of a fast preparation and low cost. Therefore, in this study, we used intrastriatal injections of 6 μM malonic acid in rats to evaluate the effect of tolfenamic acid on motor and cognitive deficits and the effect of 6 mg/kg and 32 mg/kg tolfenamic acid on p53 and its downstream targets, such as Wig1. The results showed that 32 mg/kg tolfenamic acid attenuated motor and spatial memory dysfunction, prevented Nox1-mediated reactive oxygen species (ROS) production, and downregulated the activity of p53 by increasing the phosphorylation level at the Ser378 site and decreasing the acetylation level at the Lys382 site. Tolfenamic acid reduced mouse double minute 2 (Mdm2), phosphatase and tensin homologue (Pten), P53-upregulated modulator of apoptosis (Puma) and Bcl2-associated X (Bax) at the mRNA level to inhibit apoptosis and downregulated sestrin 2 (Sesn2) and hypoxia inducible factor 1, alpha subunit (Hif-1α) mRNA levels to exert antioxidative stress effects. In addition, 32 mg/kg tolfenamic acid played a role in neuroprotection by decreasing the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL)-positive cell numbers. However, there was no difference in the Wig mRNA level among all groups, and tolfenamic acid could not decrease the protein level of Wig1. In conclusion, tolfenamic acid inhibited the ROS-generating oxidase Nox1-regulated p53 activity and attenuated motor and spatial memory deficits in malonic acid-injected rats.