P53 Apoptotic Pathway Research Articles

Effective seed cake extract of O. sanctum inducing the p53 dependent apoptotic pathway in oral cancer cells

Objective Present study was to observe the therapeutic aspects of seed cake extracts of Ocimum sanctum against the oral cancer cell line with the activation of p53 apoptotic pathway. Method Seed cake extracts were characterized using GC-MS analysis. Cytotoxic activity was observed on KB cells and L929 cell through MTT assay and scratch assay. Antioxidant activity on KB cells were determined using enzymatic and non enzyme content in the treated cells. Chick chorioallantoic membrane (CAM) was established to check the presence of blood vessel formation and neuvasculature pattern in the treated fertilized eggs. DNA fragmentation and gene expression studies were also determined in the treated cells to check the upregulation of apoptotic pathways. Results GC-MS analysis confirmed alkaloids, phenols, and many. The cytotoxic activity showed maximum antiproliferative potential with aqueous extract, whereas no cytotoxic effect was observed on L929 cells. The ethanolic and aqueous extract has shown a greater SI value. Scratch assay has signified that aqueous extract has a lower migration rate of KB cells. Aqueous extract showed maximum enzymatic activity and lower malondialdehyde content in cells treated with ethanolic extract. CAM model confirmed that eggs treated with aqueous extract has shown inhibition of vasculature pattern and dissolutions of blood vessels. DNA Fragmentation and Gene expression studies confirmed maximum fold in the KB cell treated with an aqueous extract of seed cake leading to activation of p53 dependent apoptotic pathway. Conclusion The potent therapeutic properties of seed cake extracts have been proven, and they can be used as herbal treatments to prevent oral cancer.

Identification of characteristic genes and construction of regulatory network in gallbladder carcinoma

BackgroundGallbladder carcinoma (GBC) is a highly malignant tumor with a poor overall prognosis. This study aimed to identify the characteristic microRNAs (miRNAs) of GBC and the competing endogenous RNA (ceRNA) regulatory mechanisms.MethodsThe microarray data of GBC tissue samples and normal gallbladder (NGB) tissue samples from the Gene Expression Omnibus (GEO) database was downloaded. GBC-related differentially expressed miRNAs (DE-miRNAs) were identified by inter-group differential expression analysis and weighted gene co-expression network analysis (WGCNA). Machine learning algorithms were used to screen the characteristic miRNA based on the intersect between least absolute shrinkage and selection operator (LASSO) and Support vector machine-recursive feature elimination (SVM-RFE). Based on the differential expression analysis of GEO database, the ceRNA network of characteristic miRNA was predicted and constructed. The biological functions of the ceRNA network were revealed by carrying out the gene enrichment analysis was implemented. We further screened the key genes of ceRNA network and constructed a protein-protein interaction (PPI) network, and predicted and generated the transcription factors (TFs) network of signature miRNAs. The expression of characteristic miRNA in clinical samples was verified by quantitative real-time polymerase chain reaction (qRT-PCR).ResultsA total of 131 GBC-related DE-miRNAs were obtained. The hsa-miR-4770 was defined as characteristic miRNA for GBC. The ceRNA network containing 211 mRNAs, one miRNA, two lncRNAs, and 48 circRNAs was created. Gene enrichment analysis suggested that the downstream genes were mainly involved in actin filament organization, cell-substrate adhesion, cell-matrix adhesion, reactive oxygen species metabolic process, glutamine metabolic process and extracellular matrix (ECM)-receptor interaction pathway. 10 key genes in the network were found to be most correlated with disease, and involved in cell cycle-related processes, p53, and extrinsic apoptotic signaling pathways. The qRT-PCR result demonstrated that hsa-miR-4770 is down-regulated in GBC, and the expression trend is consistent with the public database.ConclusionsWe identified hsa-miR-4770 as the characteristic miRNA for GBC. The ceRNA network of hsa-miR-4770 may play key roles in GBC. This study provided some basis for potential pathogenesis of GBC.

Danhong Injection Up-regulates miR-125b in Endothelial Exosomes and Attenuates Apoptosis in Post-Infarction Myocardium.

To investigate the involvement of endothelial cells (ECs)-derived exosomes in the anti-apoptotic effect of Danhong Injection (DHI) and the mechanism of DHI-induced exosomal protection against postinfarction myocardial apoptosis. A mouse permanent myocardial infarction (MI) model was established, followed by a 14-day daily treatment with DHI, DHI plus GW4869 (an exosomal inhibitor), or saline. Phosphate-buffered saline (PBS)-induced ECs-derived exosomes were isolated, analyzed by miRNA microarray and validated by droplet digital polymerase chain reaction (ddPCR). The exosomes induced by DHI (DHI-exo), PBS (PBS-exo), or DHI+GW4869 (GW-exo) were isolated and injected into the peri-infarct zone following MI. The protective effects of DHI and DHI-exo on MI hearts were measured by echocardiography, Masson's trichrome staining, and TUNEL apoptosis assay. The Western blotting and quantitative reverse transcription PCR (qRT-PCR) were used to evaluate the expression levels of miR-125b/p53-mediated pathway components, including miR-125b, p53, Bak, Bax, and caspase-3 activities. DHI significantly improved cardiac function and reduced infarct size in MI mice (P<0.01), which was abolished by the GW4869 intervention. DHI promoted the exosomal secretion in ECs (P<0.01). According to the results of exosomal miRNA microarray assay, 30 differentially expressed miRNAs in the DHI-exo were identified (28 up-regulated miRNAs and 2 down-regulated miRNAs). Among them, DHI significantly elevated miR-125b level in DHI-exo and DHI-treated ECs, a recognized apoptotic inhibitor impeding p53 signaling (P<0.05). Remarkably, treatment with DHI and DHI-exo attenuated apoptosis, elevated miR-125b expression level, inhibited capsase-3 activity, and down-regulated the expression levels of proapoptotic effectors (p53, Bak, and Bax) in post-MI hearts, whereas these effects were blocked by GW4869 (P<0.05 or P<0.01). DHI and DHI-induced exosomes inhibited apoptosis, promoted the miR-125b expression level, and regulated the p53 apoptotic pathway in post-infarction myocardium.

Developmental toxicity and inflammatory response induced by Botrytis cinerea in zebrafish (Danio rerio) larvae

Botrytis cinerea can reduce the yield of fruits and vegetables by infecting plants. The conidia produced by Botrytis cinerea can be transmitted to the aquatic environment via air and water, but the effects of Botrytis cinerea on aquatic animals is unknown. In this research, the influence of Botrytis cinerea on the development, inflammation, and apoptosis of zebrafish larvae and the underlying mechanism was evaluated. Results indicated that, compared with the control group, the larvae exposed to 101–103 CFU/mL of Botrytis cinerea spore suspension had a delayed hatching rate, lower head and eye area, shorter body length, and larger yolk sac at 72 h post-fertilization. In addition, the quantitative fluorescence intensity of treated larvae displayed a dose-dependent increase in apoptosis sign, revealing that Botrytis cinerea could generate apoptosis. Subsequently, zebrafish larvae were inflamed after exposure to Botrytis cinerea spore suspension, which was characterized as inflammatory infiltration and macrophage aggregation in the intestine. The enrichment of the pro-inflammatory factor TNF-α activated the NF-κB signaling pathway, generating the increase of the transcription level of target genes (jak3, pi3k, pdk1, akt, and ikk2) and the high expression of major proteins NF-κB (P65) in this pathway. Likewise, elevated content of TNF-α could activate JNK, which turned on the P53 apoptotic pathway, leading to a significant increase in the bax, caspase3, and caspase9 transcript abundances. This study demonstrated that Botrytis cinerea could cause developmental toxicity, morphological malformation, inflammation, and cell apoptosis in zebrafish larvae, which provided data support and a theoretical basis for ecological health risk assessment and filled the gap in biological research of Botrytis cinerea.

Erlotinib and Garcinia Cambogia Prevent Adenine Induced Nephrotoxicity in Adult Male Albino Rats Through Modulating EKR1/2, STAT3, and p53 Apoptotic Pathways

Erlotinib and Garcinia Cambogia Prevent Adenine Induced Nephrotoxicity in Adult Male Albino Rats Through Modulating EKR1/2, STAT3, and p53 Apoptotic Pathways

Condensed tannins from Ulmus pumila L. leaves induce G2/M phase arrest and apoptosis via caspase-cascade activation in TFK-1 cholangiocarcinoma cells.

Condensed tannins the polyphenolic compounds that are widespread in plants have been proved to have antitumor potential. Here, we purified the bioactive condensed tannins from leaves of Ulmus pumila L. and explored their structural characteristics, antitumor effect on TFK-1 cholangiocarcinoma cells as well as the related potential mechanism. The UV-Vis, FT-IR spectroscopy, ESI-Full-MS, and thiolysis-HPLC-ESI-MS demonstrated that U. pumila condensed tannins (UCTs) consisted essentially of procyanidins with epicatechin as the main flavan-3-ol extension unit. The UCTs could significantly reduce the survival rate of human cholangiocarcinoma TFK-1, SK-CHA-1, and MZ-CHA-1 cells with the better inhibitory effect on TFK-1 cell proliferation. Flow cytometric assay showed that UCTs affected TFK-1 survival by G2/M phase arrest and inducing apoptosis in a dose-dependent manner. In addition, a total of 6592 differentially expressed genes (DEGs), consisting of 94 upregulated and 6498 downregulated DEGs, were identified between untreated and UCTs-treated TFK-1 cells using RNA-seq technology. Enrichment analysis based on the KEGG database revealed that these DEGs were closely associated with cell cycle and p53 apoptotic signaling pathways. Furthermore, qRT-PCR confirmed that treatment of UCTs to TFK-1 cells caused significant changes in the expression of cyclin E, cdc25 A, cytochrome c, caspase-3, and caspase-8. These results indicated that UCTs exhibited the growth inhibition effect on TFK-1 cells possibly via G2/M cell cycle arrest and activation of caspase-cascade to induce apoptosis, and had potential asan anti-cholangiocarcinoma drug for further development. PRACTICAL APPLICATIONS: Ulmus pumila L. as a valuable tree species has been widely used in fields of medicine and food. Condensed tannins, the polyphenolic compounds widespread in plants, have been proved to have antitumor potential and be safe to normal cells.In this study, the condensed tannins from leaves of U. pumila (UCTs) remarkably suppressed cholangiocarcinoma (CCA) cell viability possibly via G2/M cell cycle arrest and activation of caspase-cascade to induce apoptosis. The results provided evidence for the application of UCTs as a potential therapeutic drug for CCA tumor.

Investigation and experimental validation of curcumin-related mechanisms against hepatocellular carcinoma based on network pharmacology.

To determine the potential molecular mechanisms underlying the therapeutic effect of curcumin on hepatocellular carcinoma (HCC) by network pharmacology and experimental in vitro validation. The predictive targets of curcumin or HCC were collected from several databases. the identified overlapping targets were crossed with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) platform. Two of the candidate pathways were selected to conduct an experimental verification. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) assay was used to determine the effect of curcumin on the viability of HepG2 and LO2 cells. The apoptosis and autophagy of HepG2 cells were respectively detected by flow cytometry and transmission electron microscopy. Besides, western blot and real-time polymerase chain reaction (PCR) were employed to verify the p53 apoptotic pathway and adenosine 5'-monophosphate (AMP)‍-activated protein kinase (AMPK) autophagy pathway. HepG2 cells were pretreated with pifithrin-‍α (PFT-‍α) and GSK690693 for further investigation. The 167 pathways analyzed by KEGG included apoptosis, autophagy, p53, and AMPK pathways. The GO enrichment analysis demonstrated that curcumin was involved in cellular response to drug, regulation of apoptotic pathway, and so on. The in vitro experiments also confirmed that curcumin can inhibit the growth of HepG2 cells by promoting the apoptosis of p53 pathway and autophagy through the AMPK pathway. Furthermore, the protein and messenger RNA (mRNA) of the two pathways were downregulated in the inhibitor-pretreated group compared with the experimental group. The damage-regulated autophagy modulator (DRAM) in the PFT-‍α-pretreated group was downregulated, and p62 in the GSK690693-pretreated group was upregulated. Curcumin can treat HCC through the p53 apoptotic pathway and the AMPK/Unc-51-like kinase 1 (ULK1) autophagy pathway, in which the mutual transformation of autophagy and apoptosis may occur through DRAM and p62.

Biosafety and differentially expressed genes analysis of melanoma cells treated with cold atmospheric plasma.

Cold atmospheric plasma (CAP) has attracted increasing attention due to its anti-bacterial and anti-tumor effects. Melanoma is an aggressive malignancy with increasing incidence rate and poor prognosis. Evaluating cell viability, apoptosis rate and reactive species injection efficiency of melanoma cells and human keratinocyte cells (HaCaT) treated with CAP to analyze biological safety of CAP. RNA-sequencing (RNA-seq) of A875 cells before and after treatment was performed to further explore the anti-tumor mechanism of CAP. CAP had a more significant biological effect on melanoma cells than HaCaT cells by inhibiting proliferation and promoting apoptosis. RNA-sequencing analysis showed that besides MAPK and p53 apoptotic signaling pathways, necroptosis and autophagy also played important roles in CAP-induced melanoma cells death. CAP can selectively kill melanoma cells and has good biosafety cytologically. Besides apoptosis, CAP can induce cell death via autophagy and necroptosis.

Physicochemical characterization of a polysaccharide from Rosa roxburghii Tratt fruit and its antitumor activity by activating ROS mediated pathways

Rosa roxburghii Tratt fruit is a highly valued fruit that contains abundant functional and nutritional constituents. In this study, a novel polysaccharide, named RTFP-1, was isolated and purified from R. roxburghii Tratt fruit. Structural characterization indicated that RTFP-1 was a homogeneous heteropolysaccharide with the molecular weight (Mw) of 128.7 kDa and consisted of arabinose, galactose, glucose, mannose, xylose, and fucose with molar ratio percentages of 34.84, 40.59, 12.11, 5.06, 3.39, and 4.01%, respectively. A CCK-8 assay indicated that RTFP-1 inhibited the cell growth of HepG2 cells in a dose-dependent manner. Morphological analysis and flow cytometry experiment showed that RTFP-1 promoted the apoptosis of HepG2 cells and increased reactive oxygen species (ROS) level. The underlying molecular mechanisms indicated that RTFP-1 activated the apoptosis of HepG2 cells through ROS-mediated MAPK, STAT, and p53 apoptotic pathways. These results suggest that RTFP-1 might be a potential chemopreventive and antitumor agent.

Malignant glioma subset from actuate 1801: Phase I/II study of 9-ING-41, GSK-3β inhibitor, monotherapy or combined with chemotherapy for refractory malignancies.

BackgroundGSK3β serine/threonine kinase regulates metabolism and glycogen biosynthesis. GSK3β overexpression promotes progression and resistance through NF-κB and p53 apoptotic pathways. GSK3β inhibits immunomodulation by downregulating PD-L1 and LAG-3 checkpoints and increasing NK and T-cell tumor killing. 9-ING-41, a small-molecule, selective GSK3β inhibitor, showed preclinical activity in chemo-resistant PDX glioblastoma models, including enhanced lomustine antitumor effect.MethodsRefractory malignancies (n = 162) were treated with 9-ING-41 monotherapy (n = 65) or combined with 8 cytotoxic regimens after prior exposure (NCT03678883). Recurrent gliomas (n = 18) were treated with 9-ING-41 IV TIW q21day cycles at 3.3, 5, 9.3, 15 mg/kg, as monotherapy or combined with lomustine 30 mg/m² PO weekly q84day cycles. Primary objective was safety.ResultsRP2D of 15 mg/kg IV TIW was confirmed across all 9 regimens, no accentuated chemotherapy toxicity noted. Glioma subtypes included: 13 glioblastoma, 2 anaplastic astrocytomas, 1 anaplastic oligodendroglioma, 1 astrocytoma. Median age 52 (30–69) years; 6 female, 12 male; median ECOG 1 (0–2); median recurrences 3 (1–6). All received upfront radiation/temozolomide (18/18), plus salvage nitrosoureas (15/18), bevacizumab (8/18), TTFields (6/18), or immunotherapy (4/18). IDH/mutation(3/18); 1p19q/codeletion(1/18); MGMT/methylated(1/18). Four received 9-ING-41 monotherapy, 14 concurrent with lomustine. No severe toxicities were attributed to 9-ING-41, only mild vision changes (9/18, 50%), or infusion reactions (4/18, 22%). Lomustine-related toxicities: G3/4 thrombocytopenia (3/14, 21%), G1/2 fatigue (4/14, 28%). Median days on therapy was 55 (4–305); 1 partial response (>50%) was noted. Median OS was 5.5 (95% CI: 2.8–11.4) months and PFS-6 was 16.7%.Conclusion9-ING-41 plus/minus lomustine is safe and warrants further study in glioma patients.

CTNI-13. MALIGNANT GLIOMA SUBSET FROM ACTUATE 1801 PHASE 1/2 STUDY OF 9-ING-41, A GLYCOGEN SYNTHASE KINASE 3 BETA (GSK-3β) INHIBITOR, AS A SINGLE AGENT AND COMBINED WITH CHEMOTHERAPY REFRACTORY

Abstract BACKGROUND GSK3β serine/threonine kinase regulates metabolism and glycogen biosynthesis. GSK3β overexpression promotes tumor progression and resistance through NF-κB and p53 apoptotic pathways. GSK3β inhibits immunomodulation by downregulating checkpoints, e.g. PD-L1 and LAG-3, and increasing NK and T-cell mediated tumor killing. 9-ING-41 is a small-molecule, potent, selective GSK3β inhibitor with preclinical activity. In chemoresistant PDX glioblastoma models, 9-ING-41 enhanced lomustine antitumor effect. METHODS Patients with refractory malignancies were treated with 9-ING-41 monotherapy (n=65) or combined with 8 cytotoxic regimens after prior exposure (n=162) in the first-in-human study (NCT03678883). The recurrent gliomas subset was treated with 9-ING-41 monotherapy IV TIW q21day cycles at 3.3, 5, 9.3, 15mg/kg, or combined with lomustine 30 mg/m² PO weekly q84day cycles. Primary objective was safety and tolerability. RESULTS An RP2D of 15mg/kg IV TIW was confirmed across all 9 regimens, no accentuation of chemotherapy toxicity noted. Of 18 glioma patients enrolled, 13 were glioblastoma, 2 anaplastic astrocytomas, 1 anaplastic oligodendroglioma, and 1 diffuse astrocytoma; 6 female, 12 male; median age 52 (30-69) years; median ECOG was 1 (0-2). All received initial radiation and temozolomide (18/18), prior salvage therapies included nitrosoureas (15/18), bevacizumab (8/18), TTFields (6/18), checkpoint inhibitor (4/18). Median recurrences 3 (1-6). NGS alterations included: IDH/wildtype (11), IDH/mutation(3); 1p19q/codeletion(10); MGMT/unmethylated(11), MGMT/methylated(1); EGFR/amplification(6), EGFR/v3mutation(3), TERT/mutation(6), PTEN/loss(3), NF1/rearrangement(2), ATRX/loss (2), TP53/mutation(4), CDKN2A/deletion(2), RB1/loss(1), PALB2/mutation(10). Four patients received 9-ING-41 monotherapy, 14 concurrently treated with lomustine. No SAEs or grade 3/4 AEs attributed to 9-ING-41 noted, only G1/2 vision changes (9/18, 50%), infusion reactions (4/18, 22%). Lomustine-related toxicities included G3/4 thrombocytopenia (3/14, 21%), and G1/2 fatigue (4/14, 28%). Median therapy duration was 55 (4-305); 1 partial response ( &amp;gt;50%) noted with 9-ING-41/lomustine. Median PFS and OS were 1.9 (0.3-11.1) and 6.0 (1.6-16.6) months, respectively. CONCLUSIONS 9-ING-41 plus/minus lomustine is safe and warrants further study in glioma patients.

Knock-down of LRP/LR influences signalling pathways in late-stage colorectal carcinoma cells

BackgroundThe 37 kDa/67 kDa laminin receptor (LRP/LR) is involved in several tumourigenic-promoting processes including cellular viability maintenance and apoptotic evasion. Thus, the aim of this study was to assess the molecular mechanism of LRP/LR on apoptotic pathways in late stage (DLD-1) colorectal cancer cells upon siRNA-mediated down-regulation of LRP/LR.MethodssiRNAs were used to down-regulate the expression of LRP/LR in DLD-1 cells which was assessed using western blotting and qPCR. To evaluate the mechanistic role of LRP/LR, proteomic analysis of pathways involved in proliferation and apoptosis were investigated. The data from the study was analysed using a one-way ANOVA, followed by a two-tailed student’s t-test with a confidence interval of 95%.ResultsHere we show that knock-down of LRP/LR led to significant changes in the proteome of DLD-1 cells, exposing new roles of the protein. Moreover, analysis showed that LRP/LR may alter components of the MAPK, p53-apoptotic and autophagic signalling pathways to aid colorectal cancer cells in continuous growth and survival. Knock-down of LRP/LR also resulted in significant decreases in telomerase activity and telomerase-related proteins in the DLD-1 cells.ConclusionsThese findings show that LRP/LR is critically implicated in apoptosis and cell viability maintenance and suggest that siRNA-mediated knock-down of LRP/LR may be a possible therapeutic strategy for the treatment of colorectal cancer.

An allomaltol derivative triggers distinct death pathways in luminal a and triple-negative breast cancer subtypes

Breast cancer is the most common cancer in women that shows a predisposition to metastasize to the distant organs. Kojic acid is a natural fungal metabolite exhibiting various biological activities. Compounds derived from kojic acid have been extensively studied and proved to demonstrate anti-neoplastic features on different cancer types. In the present study, allomaltol-structural analog of kojic acid and its seven derivatives including four novel compounds, have been synthesized, characterized and their possible impact on breast cancer cell viability was investigated. It was discovered that compound 5, bearing 3,4-dichlorobenzyl piperazine moiety, could decrease the viability of both MCF-7 and MDA-MB-231 cell lines distinctively. To ascertain the death mechanism, cells were subjected to different tests following the application of IC50 concentration of compound 5. Data obtained from lactate dehydrogenase activity and gene expression assays pointed out that necrosis had taken place predominantly in MDA-MB-231. On the other hand, in MCF-7 cells, the p53 apoptotic pathway was activated by overexpression of the pro-apoptotic TP53 and Bax genes and suppression of the anti-apoptotic Mdm-2 and Bcl-2 genes. Furthermore, Bax/Blc-2 ratio was escalated by 3.5 fold in the study group compared to the control. Compound 5 did not provoke drug resistance in MCF-7 cells since the Mdr-1 gene expression, drug efflux, and H2O2 content remained unaltered. As for MDA-MB-231 cells, only a 1.4 fold increase in the Mdr-1 gene expression was detected. These results indicate the advantage of the allomaltol derivative over the chemotherapeutic agents conventionally used for breast cancer treatment that can be highly toxic and mostly lead to drug resistance. Thus, this specific allomaltol derivative offers an alternative therapeutic approach for breast cancer which needs further investigation.

Dihydroartemisinin Induces Apoptosis of Human Acute T Lymphocytic Leukemia Cells by Activating Oxidative Stress

To investigate the effects of dihydroartemisinin (DHA) on the proliferation and apoptosis of human T-cell acute lymphoblastic leukemia (T-ALL) Jurkat cell. The effects of DHA on the proliferation of Jurkat cells and the recovery of DHA-inhibited cell viability by N-acetyl-L-cysteine (NAC) were examined by CCK-8 assay. Flow cytometry was performed to analyze the cell apoptosis and generation of reactive oxygen species (ROS). Western-blot was used to detected protein expression of DNA damage-related genes, as well as apoptosis-associated genes, respectively. DHA inhibited the proliferation of Jurkat cells, and shows a concentration-dependent manner(r =0.936), and NAC could partially restore the activity of DHA on cell proliferation inhibition. With the increase of drug concentration, the apoptosis rate (r =0.946) and ROS accumulation was increased (r =0.965). Western blot showed that the protein expressions of DNA damage-related gene γ-H2AX and apoptosis-related genes p53, c-Caspase3, BAX and cPARP were significantly increased, and BCL-2 protein expression was decreased. DHA can induce ROS production in Jurkat cells, which can cause DNA damage, activate the P53 apoptotic pathway, and promote apoptosis of cells.

A212 CHROMOFUNGIN PROTECTS AGAINST DSS-INDUCED COLITIS BY REGULATING P-53 APOPTITIC PATHWAY

Abstract Background Development of ulcerative colitis is associated with epithelial apoptosis mediated by p53-apoptotic pathway through the activation B-cell lymphoma 2 (Bcl-2), Bcl-2 associated-X protein (BAX) and Bcl-2 antagonist/killer-1 (BAK1) proteins. Chromofungin (CHR), a chromogranin-A derived peptide expressing a cell penetrating peptide motif, decreased the severity of colitis via the suppression of mucosal and pro-inflammatory macrophages-related p53-dependent apoptosis. Aims We aimed to investigate a) whether the gene profile expression of apoptosis could be extended to other p53-associated genes; b) whether the gene expression of some of the p53-apoptosis marker could be confirmed by protein analysis; and c) whether due to the cell penetrating peptide motif, CHR could enter into peritoneal macrophages. Methods UC-related colitis was induced in C57BL/6 mice (7 weeks) by administering dextran sulfate sodium (DSS) (5%, 5 days). Preventive CHR (2.5 mg/kg/day) or vehicle treatment started 1-day before colitis induction and lasted for 5-days. Profiler™ PCR Array was performed to screen a panel of 84 genes representative of the p53 signal pathway in colitic whole mucosa distal colonic samples treated or not with CHR. Western blot analysis was performed to confirm individual protein changes. Naïve macrophages were plated overnight and nonadherent cells were removed the next day. Cells were incubated with rhodamined CHR (4 ul) for 5, 10, 20, 30 min before washing and fixing them, detection was made via confocal microscopy. Results In colitic conditions, an up regulation of 26 genes associated to the p53-dependent apoptosis pathway were detected including Apaf1, Bax, Bbc3, Bcl2, Cradd, Fadd, Cul9, Pmaip1, Tnfrsf10b. In vivo CHR treatment decreased significantly the colitis and was associated with a significant downregulation of 19 genes including the 9 aforementioned when compared with biopsies from colitic groups. Compared to untreated groups, colitic mice treated with CHR demonstrated a significant decrease of BAX and BAK protein and the apoptotic ER stress inducer marker, X-Binding Protein 1. A large number of peritoneal macrophages displayed rhodamine within the all intracellular compartment. The presence of the peptide inside the cell can be visible as early as 5 min and the signal gradually increases. Conclusions CHR decreases the inflammatory process via the suppression of a large number of p53-related apoptotic proteins. CHR quickly enters the macrophage but the exact mechanism of entrance needs to be further defined. Targeting functional analysis of CHR may lead in the future to novel therapeutics for UC. Funding Agencies CCCNSERC

Purple sweet potato color protects against hepatocyte apoptosis through Sirt1 activation in high-fat-diet-treated mice.

BackgroundRecent evidence indicates that the inhibition of hepatocyte apoptosis is possible to develop a potential therapeutic strategy for nonalcoholic fatty liver disease (NAFLD). Our previous work suggested that purple sweet potato color (PSPC), a class of naturally occurring anthocyanins, effectively improved many features of high-fat diet (HFD)-induced NAFLD. However, whether PSPC ameliorates HFD-induced hepatocyte apoptosis has never been investigated.ObjectiveHere we investigated the effects of PSPC on HFD-induced hepatic apoptosis and the mechanisms underlying these effects.DesignMice were divided into four groups: Control group, HFD group, HFD + PSPC group and PSPC group. PSPC was administered by daily oral gavage at doses of 700 mg/kg/day for 20 weeks. EX-527 (a SirT1-selective inhibitor) and Sirt1 siRNA were used to demonstrate the Sirt1 dependence of PSPC-mediated effects on apoptotic and survival signaling pathways in vivo and in vitro.ResultsOur results showed that PSPC reduced body weights, hepatic triglyceride contents, histopathological lesions and serum ALT levels in a mouse model of NAFLD induced by HFD. Furthermore, PSPC attenuated HFD-induced hepatocyte apoptosis ratio from 7.27 ± 0.92% to 1.79 ± 0.27% in mouse livers, which is insignificant compared with that of controls. Moreover, PSPC activated Sirt1 by boosting NAD+ level in HFD-treated mouse livers. Furthermore, PSPC promoted Sirt1-dependent suppression of P53-mediated apoptotic signaling and activation of Akt survival signaling pathway in HFD-treated mouse livers, which was confirmed by EX527 treatment. Moreover, Sirt1 knockdown abolished these ameliorative effects of PSPC on apoptosis and P53 acetylation and protein expression in PA-treated L02 cells. Ultimately, PSPC reduced Caspase-3 activation and Bax level, and elevated the Bcl-2 level in HFD-treated mouse livers.ConclusionPSPC protected against HFD-induced hepatic apoptosis by promoting Sirt1- dependent inhibition of p53-apoptotic pathway and facilitation of Akt survival pathway. This study indicates that PSPC is a candidate for nutritional intervention of NAFLD.

Interactions among variants in P53 apoptotic pathway genes are associated with neurologic deterioration and functional outcome after acute ischemic stroke

ObjectiveNeurologic deterioration (ND) and functional outcome after ischemic stroke (IS) are not accurately predicted by clinical pictures on admission. The aim of present study was to investigate the association of variants in P53 apoptotic pathway genes with ND and functional outcome after IS.MethodsGenotypes of nine variants in apoptosis‐relevant genes were measured in patients with acute IS. Gene–gene interactions were analyzed by generalized multifactor dimensionality reduction (GMDR). The primary outcome was ND. ND was diagnosed in patients who worsened ≥2 points (National Institutes of Health Stroke Scale [NIHSS] score) within the first 10 days of stroke onset. The secondary outcome was functional status at 90 days after IS as measured by modified Rankin Scale (mRS) score.ResultsA total of 705 enrolled patients, ND occurred in 174 (24.7%) patients, and 184 (26.1%) patients were poor functional outcome (mRS score > 2). Although the nine variants were not significantly associated with ND and functional outcome by univariate analysis, there was a gene–gene interaction among P53rs1042522, MDM‐2rs2279744, and MMP‐9 rs3918242 using GMDR analysis. The high‐risk interaction among the three variants was independently associated with higher risk of ND (HR, 2.04, 95% CI: 1.22–5.64, p = .018) and poor functional outcome (OR, 2.68, 95% CI: 1.68–7.86, p = .004) after adjusting for the covariates.ConclusionThe interactions among P53 rs1042522, MDM‐2 rs2279744, and MMP‐9 rs3918242 may increase the risk of ND and poor functional outcome and may be considered as a genetic marker of predicting ND and poor functional outcome after stroke.

Acylated Ghrelin Renders Chemosensitive Ovarian Cancer Cells Resistant to Cisplatin Chemotherapy via Activation of the PI3K/Akt/mTOR Survival Pathway

This study investigated the effect of acylated synthetic ghrelin (AG) on the survival and proliferation of human chemosensitive ovarian cancer cells (A2780) and explored some mechanisms of action with a focus on the p53 apoptotic pathway and PI3K/Akt and NF-κB survival pathways. Human A2780 ovarian cancer cells were cultured with or without AG treatment in the presence or absence of cisplatin. In some cases, cisplatin+AG-treated cells were pre-incubated either with [D-Lys3]-GHRP-6, a ghrelin receptor antagonist, or with LY294002, a PI3K inhibitor. mRNA of ghrelin receptors(GHS-R1a and GHS-R1b), as well as, protein levels of GHS-R1a, were expressed abundantly in A2780 cells. AG treatment did not affect the mRNA and protein levels of GHS-R1a and GHS-R1b in both control and Cis-treated cells. However, while AG treatment had no effect on control cell viability, it significantly increased cell viability and proliferation and inhibited cell death in Cis-treated cells. In both control and Cis-treated cells, AG treatment significantly increased PI3K/Akt/mTOR signaling and enhanced the nuclear accumulation of NF-κB. Concomitantly, in both control and Cis-treated cells, AG significantly lowered the protein levels of p53, p-p53 (Ser16), PUMA, cytochrome C, and cleaved caspase-3. Interestingly, pre-incubating the cells with either [D-Lys3]-GHRP-6 or LY294002 completely abolished the above-mentioned effect of AG in both control and Cis-treated cells. In conclusion, the findings of this study show that AG promotes cell survival of the OC cells and renders them resistat to Cis therapy, an effect that is mediated by the activation of PI3K/Akt/mTOR and activation of NF-κB, and requires GHS-R1a.

Life and Death: The Role of ADOR‐1 in Mediating Activation of the CEP‐1‐Apoptotic Pathway and IGF‐1 Signaling Pathway in Caenorhabditis elegans Exposed to Caffeine

Caffeine is the most widely used psychoactive drug in the world and forms a mild dependency in users who overuse the drug for an extended period of time. Other psychoactive drugs, including methamphetamine, have been shown to induce apoptosis in the brain's reward pathways, leading to lasting neuronal damage. Similarly, caffeine has been shown to activate the p53‐mediated apoptotic pathway in JB6 C141 cells derived from mice. However, other studies suggest that caffeine produces contradictory effects. Caffeine exposure has been reported to extend the lifespan of Caenorhabditis elegans through activation of the IGF‐1 signaling pathway. Additionally, caffeine has been shown to bind to ador‐1 receptors, the homolog of ADORA2A in humans, in C. elegans. We suspect that the seemingly contradictory effects of caffeine exposure are mediated by ador‐1, signifying that p53 and IGF‐1 activation occur downstream to adenosine receptors. Here we examine the role of ador‐1 in activation of the CEP‐1 apoptotic pathway, the C. elegans homolog of the p53‐apoptotic pathway in humans, and the IGF‐1 signaling pathway in C. elegans. Our current data clarifies the relationship between caffeine exposure and CEP‐1. We plan to further investigate the relationship of the CEP‐1 and IGF‐1 pathways in order to determine whether their activation is mediated by ador‐1.Support or Funding InformationThis work was supported by The Nueva School.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interactions Among Variants in P53 Apoptotic Pathway Genes are Associated with Neurologic Deterioration and Poor Functional Outcome after Ischemic Stroke

Interactions Among Variants in P53 Apoptotic Pathway Genes are Associated with Neurologic Deterioration and Poor Functional Outcome after Ischemic Stroke