Targeting MDM2-p53 interaction in Glioblastoma: Transcriptomic analysis and Peptide-Based inhibition strategy.

MDM2 is a gene that encodes a protein involved in cell survival, growth, and DNA repair. It has been implicated in the development and progression of glioblastoma (GBM). Inhibition of the MDM2-p53 interaction has emerged as a promising strategy for treating GBM. In this study, we performed comprehensive transcriptomic expression analysis from diverse datasets and observed MDM2 overexpression in a subset of GBM cases. MDM2 negatively regulates the major onco-suppressor p53. The interaction between MDM2 and p53 is a promising target for cancer therapy, as it can trigger p53-mediated cell death in response to different stress conditions, such as oncogene activation or DNA damage. In this study, we have identified a peptide-based inhibition of MDM2 as a therapeutic strategy for GBM. We have further validated the stability of the MDM2-peptide interaction using a molecular structural dynamics approach. The major trajectories, including root mean square of deviation (RMSD), root mean square of fluctuation (RMSF), and radius of gyration (RoG), indicate that the candidate peptides have a more stable binding compared to the native ligand and control drug. The stability of the binding interaction was further estimated by MMGBSA analysis, which also suggests that MDM2 has a stable binding with both peptide molecules. Based on these results, peptides P-1843 and P-3837 could be tested further for experimental validation to confirm their targeted inhibition of MDM-2. This approach could provide a highly selective and efficient inhibitor with potentially fewer side effects and less toxicity compared to small drug-based molecules.

Discovery of Novel p53-MDM2 Inhibitor (RG7388)-Conjugated PlatinumIV Complexes as Potent Antitumor Agents.

While a number of p53-MDM2 inhibitors have progressed into clinical trials for the treatment of cancer, their progression has been hampered by a variety of problems, including acquired drug resistance, dose-dependent toxicity, and limited clinical efficiency. To make more progress, we integrated the advantages of MDM2 inhibitors and platinum drugs to construct novel PtIV-RG7388 (a selective MDM2 inhibitor) complexes. Most complexes, especially 5a and 5b, displayed greatly improved antiproliferative activity against both wild-type and mutated p53 cancer cells. Remarkably, 5a exhibited potent in vivo tumor growth inhibition in the A549 xenograft model (66.5%) without apparent toxicity. It arrested the cell cycle at both the S phase and the G2/M phase and efficiently induced apoptosis via the synergistic effects of RG7388 and cisplatin. Altogether, PtIV-RG7388 complex 5a exhibited excellent in vitro and in vivo antitumor activities, highlighting the therapeutic potential of PtIV-RG7388 complexes as antitumor agents.

An overview of PROTACs targeting MDM2 as a novel approach for cancer therapy.

MDM2 genes amplification or altered expression is commonly observed in various cancers bearing wild-type TP53. Directly targeting the p53-binding pocket of MDM2 to activate the p53 pathway represents a promising therapeutic approach. Despite the development of numerous potent MDM2 inhibitors that have advanced into clinical trials, their utility is frequently hampered by drug resistance and hematologic toxicity such as neutropenia and thrombocytopenia. The emergence of PROTAC technology has revolutionized drug discovery and development, with applications in both preclinical and clinical research. Harnessing the power of PROTAC molecules to achieve MDM2 targeted degradation and p53 reactivation holds significant promise for cancer therapy. In this review, we summarize representative MDM2 PROTAC degraders and provide insights for researchers investigating MDM2 proteins and the p53 pathway.

Design, Synthesis, and In vitro Anti-cervical Cancer Activity of a Novel MDM2-p53 Inhibitor Based on a Chalcone Scaffold.

OBJECTIVE: Several novel fluorinated chalcone derivatives were synthesized, and their in vitro anticervical cancer activity and mechanism of action were investigated using the parent nucleus of licorice chalcone as the lead compound backbone and MDM2-p53 as the target. METHODS: In this study, 16 novel chalcone derivatives (3a-3r) were designed and synthesized by molecular docking technology based on the licorice chalcone parent nucleus as the lead compound scaffold and the cancer apoptosis regulatory target MDM2-p53. The structures of these compounds were confirmed by 1H-NMR, 13C-NMR, and HR-ESI-MS. The inhibitory effects of the compounds on the proliferation of three human cervical cancer cell lines (SiHa, HeLa, and C-33A) and two normal cell lines (H8 and HaCaT) were determined by MTT assay, and the initialstructure-activity relationship was analyzed. Transwell and flow cytometry were used to evaluate the effects of target compounds on the inhibition of cancer cell migration and invasion, apoptosis induction, and cell cycle arrest. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) were used to detect the effects of candidate compounds on mRNA, p53, and Murine double minute 2 (MDM2) protein expression. The binding characteristics of the target compounds to the MDM2 protein target in the p53-MDM2 pathway were evaluated by molecular docking technology. RESULTS: The target compounds had considerable inhibitory activity on the proliferation of three cervical cancer cell lines. Among them, compound 3k (E)-3-(4-(dimethylamino)phenyl)-2-methyl-1-(3-(trifluoromethyl)phenyl) prop-2-en-1-one) showed the highest activity against HeLa cells (IC50=1.08 µmol/L), which was better than that of the lead compound Licochalcone B, and 3k showed lower toxicity to both normal cells. Compound 3k strongly inhibited the migration and invasion of HeLa cells and induced apoptosis and cell cycle arrest at the G0/G1 phase. Furthermore, compound 3k upregulated the expression of p53 and BAX and downregulated the expression of MDM2, MDMX, and BCL2. Moreover, molecular docking results showed that compound 3k could effectively bind to the MDM2 protein (binding energy: -9.0 kcal/mol). These results suggest that the compounds may activate the p53 signaling pathway by inhibiting MDM2 protein, which prevents cancer cell proliferation, migration, and invasion and induces apoptosis and cell cycle arrest in cancer cells. CONCLUSION: This study provides a new effective and low-toxicity drug candidate from licochalcone derivatives for treating cervical cancer.

Augmentation of bone morphogenetic protein signaling in cranial neural crest cells in mice deforms skull base due to premature fusion of intersphenoidal synchondrosis.

Craniofacial anomalies (CFAs) are a diverse group of disorders affecting the shapes of the face and the head. Malformation of the cranial base in humans leads CFAs, such as midfacial hypoplasia and craniosynostosis. These patients have significant burdens associated with breathing, speaking, and chewing. Invasive surgical intervention is the current primary option to correct these structural deficiencies. Understanding molecular cellular mechanism for craniofacial development would provide novel therapeutic options for CFAs. In this study, we found that enhanced bone morphogenetic protein (BMP) signaling in cranial neural crest cells (NCCs) (P0-Cre;caBmpr1a mice) causes premature fusion of intersphenoid synchondrosis (ISS) resulting in leading to short snouts and hypertelorism. Histological analyses revealed reduction of proliferation and higher cell death in ISS at postnatal day 3. We demonstrated to prevent the premature fusion of ISS in P0-Cre;caBmpr1a mice by injecting a p53 inhibitor Pifithrin-α to the pregnant mother from E15.5 to E18.5, resulting in rescue from short snouts and hypertelorism. We further demonstrated to prevent premature fusion of cranial sutures in P0-Cre;caBmpr1a mice by injecting Pifithrin-α through E8.5 to E18.5. These results suggested that enhanced BMP-p53-induced cell death in cranial NCCs causes premature fusion of ISS and sutures in time-dependent manner.

p53 inhibition attenuates cisplatin-induced acute kidney injury through microRNA-142-5p regulating SIRT7/NF-κB.

Renal tubular epithelial cell apoptosis is the main mechanism of cisplatin-induced acute kidney injury. The role of microRNAs (miRNAs) in the apoptosis of renal tubular epithelial cells has been suggested, but the underlying mechanism has not been fully elucidated. We used microarray analysis to identify miR-142-5p involved in cisplatin-induced acute kidney injury. miR-142-5p was down-regulated in human renal tubular epithelial (HK-2) cells with cisplatin treatment. Notably, the overexpression of miR-142-5p attenuated the cisplatin-induced HK-2 cell apoptosis and inhibition of miR-142-5p aggravated cisplatin-induced HK-2 cell apoptosis. During cisplatin treatment, p53 was activated. The inhibition of p53 by pifithrin-α attenuated the cisplatin-induced kidney injury and up-regulated miR-142-5p expression. We also identified the Sirtuin7 (SIRT7) as a target of miR-142-5p. Furthermore, we demonstrated that the inhibition of SIRT7 prevented cisplatin-induced HK-2 cell apoptosis and decreased the expression of nuclear factor kappa B (NF-κB). Our data revealed that p53 inhibition could attenuate cisplatin-induced acute kidney injury by up-regulating miR-142-5p to repress SIRT7/NF-κB. These findings may provide a novel therapeutic target of cisplatin-induced acute kidney injury.

Urea transporter B downregulates polyamines levels in melanoma B16 cells via p53 activation.

Urea transporter B (UT-B, encoded by the SLC14A1 gene) is a membrane channel protein involved in urea transmembrane transport. Compared with normal tissues, UT-B expression is significantly decreased in most tumours, especially melanoma. However, the UT-B role in tumorigenesis and development is still unclear. Herein, we investigated the effects of UT-B overexpression on polyamine metabolism and the urea cycle in murine melanoma B16 cells, to explore the roles of mitochondrial dysfunction and p53 activation in cell growth and polyamines metabolism. UT-B overexpression in B16 cells decreased cell growth, increased apoptosis, and significantly altered metabolic pathways related to the urea cycle, which were characterized by reduced production of urea and polyamines and increased production of nitric oxide. Subsequently, we observed that activation of the p53 pathway may be the main cause of the above phenomena. The p53 inhibitor pifithrin-α partially restored the production of polyamines, but the mitochondrial morphology and function were still impaired. Further treatment of UT-B-overexpressing B16 cells with reactive oxygen species scavenging agent N-acetyl-l-cysteine and coenzyme Q10 restored cell viability and mitochondrial function and increased polyamine production. In conclusion, UT-B overexpression caused mitochondrial dysfunction and increased oxidative stress in B16 cells, and then activated p53 expression, which may be one of the mechanisms leading to the decrease in intracellular polyamines.

Phe19 modification of HDM2-p53 PPI inhibitors to alleviate CYP3A4 metabolism/mechanism-based inhibition liability.

The discovery of potent, bioavailable small molecule inhibitors of p53-HDM2 PPI led us to investigate subsequent modifications to address a CYP3A4 time-dependent inhibition liability. On the basis of the crystal structure of HDM2 in complex with 2, further functionalization of the solvent exposed area of the molecule that binds to Phe19 pocket were investigated as a strategy to modulate the molecule liphophilicity. Introduction of 2-oxo-nicotinic amide at Phe19 proved a viable strategy in obtaining inhibitors exempt from CYP3A4 time-dependent inhibition liability.

Therapeutic potential of p53 reactivation in prostate cancer: Strategies and opportunities.

Metastatic prostate cancer (mCaP) remains one of the leading causes of cancer-related death in men worldwide. Androgen receptor (AR) drives the progression of most of the mCaP, and hence the androgen deprivation therapy (ADT) is the first-line treatment of choice for mCaP. Although the responses of ADT and next-generation AR inhibitors initially improve the disease burden, the responses of this combinatorial drug therapy varied widely due to molecular alteration in mCaP patients. In addition to the altered AR signaling, loss of potent tumor-suppressor protein p53 exhibits poor outcomes. p53 influences cell plasticity and is frequently lost in more aggressive prostate cancer (CaP) with neuroendocrine differentiation. Loss of p53 antagonizes the effect of AR inhibitors and enhances the proliferation rate of CaP cells. Considering the important role of p53 inactivation in cancer development, restoration of wild-type p53 function by p53-reactivating compounds developed with different approaches, seems to be an attractive therapeutic strategy for prostate cancer therapy. In this review, we discuss the therapeutic potential of these compounds with a particular focus on the pharmacological rescue of p53 in mCaP. In addition, we also highlight the challenges and new opportunities of p53-targeted therapy for the future.

Natural Small Molecules Enabled Efficient Immunotherapy through Supramolecular Self-Assembly in P53-Mutated Colorectal Cancer.

Nanomedicine, constructed from therapeutics, presents an advantage in drug delivery for cancer therapies. However, nanocarrier-based treatment systems have problems such as interbatch variability, multicomponent complexity, poor drug delivery, and carrier-related toxicity. To solve these issues, the natural molecule honokiol (HK), an anticancer agent in a phase I clinical trial (CTR20170822), was used to form a self-assembly nanoparticle (SA) through hydrogen bonding and hydrophobicity. The preparation of SA needs no molecular precursors or excipients in aqueous solution, and 100% drug-loaded SA exhibited superior tumor-targeting ability due to the enhanced permeability and retention (EPR) effect. Moreover, SA significantly enhanced the antitumor immunity relative to free HK, and the mechanism has notable selectivity to the p53 pathway. Furthermore, SA exhibited excellent physiological stability and inappreciable toxicity. Taken together, this supramolecular self-assembly strategy provides a safe and "molecular economy" model for rational design of clinical therapies and is expected to promote targeted therapy of HK, especially in colorectal cancer patients with obvious p53 status.

HIWI2 induces G2/M cell cycle arrest and apoptosis in human fibrosarcoma via the ROS/DNA damage/p53 axis.

AIMS: Piwi-like RNA-mediated gene silencing 4 (PIWIL4) or HIWI2, are seen deregulated in human cancers and possibly play critical roles in tumorigenesis. It is unknown what role HIWI2 plays in the regulation of fibrosarcoma, an early metastatic lethal type of soft tissue sarcoma (STS). The present study aimed to investigate the role of HIWI2 in the tumorigenesis of fibrosarcoma. MAIN METHODS: The expression of HIWI2 in HT1080 fibrosarcoma cells was determined by qRT-PCR and western blotting. The MTT assay, colony formation assay, cell cycle, and PE-AnnexinV/7AAD apoptosis assay using flow cytometry, DNA laddering assay, comet assay, and γH2AX accumulation assay were performed to study the effect of HIWI2 overexpression in HT1080 cells. Further, the effect of silencing of HIWI2 was determined by cell viability assay, transwell migration, and invasion assay. KEY FINDINGS: HIWI2 is under-expressed in STS cell lines and tissues, which is associated with poor disease-free survival, disease-specific survival, and progression-free survival of the patients. Overexpression of HIWI2 in HT1080 cells causes DNA damage by increasing intracellular ROS by inhibiting the expression of antioxidant genes (SOD1, SOD2, GPX1, GPX4, and CAT). Furthermore, an increase in H2AX phosphorylation was observed, which activates p53 that promotes p21 expression and caspase-3 activation, leading to G2/M phase cell cycle arrest and apoptosis. HIWI2 silencing, on the contrary, promotes cell growth, migration, and invasion by activating MMP2 and MMP9. SIGNIFICANCE: These results are the first to show that HIWI2 acts as a tumor suppressor in fibrosarcoma by modulating the ROS/DNA damage/p53 pathway.

A computer-aided drug design approach to discover tumour suppressor p53 protein activators for colorectal cancer therapy.

Colorectal cancer (CRC) is the third most detected cancer and the second foremost cause of cancer deaths in the world. Intervention targeting p53 provides potential therapeutic strategies, but thus far no p53-based therapy has been successfully translated into clinical cancer treatment. Here we developed a Quantitative Structure-Activity Relationships (QSAR) classification models using empirical molecular descriptors and fingerprints to predict the activity against the p53 protein, using the potency value with the active or inactive label, were developed. These models were built using in total 10,505 molecules that were extracted from the ChEMBL, ZINC and Reaxys® databases, and recent literature. Three machine learning (ML) techniques e.g., Random Forest, Support Vector Machine, Convolutional Neural Network were explored to build models for p53 inhibitor prediction. The performances of the models were successfully evaluated by internal and external validation. Moreover, based on the best in silico p53 model, a virtual screening campaign was carried out using 1443 FDA-approved drugs that were extracted from the ZINC database. A list of virtual screening hits was assented on base of some limits established in this approach, such as: (1) probability of being active against p53; (2) applicability domain; (3) prediction of the affinity between the p53, and ligands, through molecular docking. The most promising according to the limits established above was dihydroergocristine. This compound revealed cytotoxic activity against a p53-expressing CRC cell line with an IC50 of 56.8 µM. This study demonstrated that the computer-aided drug design approach can be used to identify previously unknown molecules for targeting p53 protein with anti-cancer activity and thus pave the way for the study of a therapeutic solution for CRC.

Insight into the Protective Effect of Salidroside against H2O2-Induced Injury in H9C2 Cells.

Salidroside is the important active ingredient of Rhodiola species, which shows a wide range of pharmacological activities such as antioxidative stress, anti-inflammation, and antiliver fibrosis. In this paper, we aimed to study the protective effect and mechanism of salidroside against H2O2-induced oxidative damage in H9C2 cells by determining cell proliferation rate, intracellular reactive oxygen species (ROS) level, antioxidant enzyme activities, and the expression of apoptosis-related proteins. The results showed that salidroside significantly alleviated cell growth inhibition induced by H2O2 treatment in H9C2 cells, decreased the levels of intracellular ROS and malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD) and catalase (CAT); meanwhile, salidroside upregulated the expression of Bcl-2 while downregulated the expression of Bax, p53, and caspase-3 in H2O2-treated H9C2 cells. Furthermore, the antiapoptotic effect of salidroside was almost eliminated by the knockdown of Bcl-2. In the further exploration, the Bcl-2 expression was decreased by the p53 overexpression and increased by p53 knockdown in H2O2-treated H9C2 cells. Consequently, salidroside could protect H9C2 cells against H2O2-induced oxidative damage, and the underlying mechanism may be related to scavenging intracellular ROS, increasing the activities of intracellular antioxidant enzymes and inhibiting the expression of apoptosis-related proteins.

Resistance mechanisms to inhibitors of p53-MDM2 interactions in cancer therapy: can we overcome them?

Since the discovery of the first MDM2 inhibitors, we have gained deeper insights into the cellular roles of MDM2 and p53. In this review, we focus on MDM2 inhibitors that bind to the p53-binding domain of MDM2 and aim to disrupt the binding of MDM2 to p53. We describe the basic mechanism of action of these MDM2 inhibitors, such as nutlin-3a, summarise the determinants of sensitivity to MDM2 inhibition from p53-dependent and p53-independent points of view and discuss the problems with innate and acquired resistance to MDM2 inhibition. Despite progress in MDM2 inhibitor design and ongoing clinical trials, their broad use in cancer treatment is not fulfilling expectations in heterogenous human cancers. We assess the MDM2 inhibitor types in clinical trials and provide an overview of possible sources of resistance to MDM2 inhibition, underlining the need for patient stratification based on these aspects to gain better clinical responses, including the use of combination therapies for personalised medicine.

Colletofragarone A2 and Colletoins A-C from a Fungus Colletotrichum sp. Decrease Mutant p53 Levels in Cells.

p53 is frequently mutated in tumor cells. Mutant p53 (mut p53) accumulates in cells to promote cancer progression, invasion, and metastasis, and it is attracting attention as a target for cancer therapies. In this study, we used immunofluorescence staining of Saos-2 cells harboring doxycycline-inducible p53R175H [Saos-2 (p53R175H) cells] to search for compounds from natural sources that can target mut p53 and found an extract of Colletotrichum sp. (13S020) that was active. Bioassay-guided fractionation of the extract afforded a known polyketide, colletofragarone A2 (1), and three new analogues, colletoins A-C (2-4). The relative and absolute configurations of 1 were determined by the spectroscopic method and DFT calculation. Compounds 1 and 2 inhibited the growth of Saos-2 (p53R175H) cells and decreased mut p53 in the cells.

Lentiviral-Induced Spinal Cord Gliomas in Rat Model.

Intramedullary spinal cord tumors are a rare and understudied cancer with poor treatment options and prognosis. Our prior study used a combination of PDGF-B, HRAS, and p53 knockdown to induce the development of high-grade glioma in the spinal cords of minipigs. In this study, we evaluate the ability of each vector alone and combinations of vectors to produce high-grade spinal cord gliomas. Eight groups of rats (n = 8/group) underwent thoracolumbar laminectomy and injection of lentiviral vector in the lateral white matter of the spinal cord. Each group received a different combination of lentiviral vectors expressing PDGF-B, a constitutively active HRAS mutant, or shRNA targeting p53, or a control vector. All animals were monitored once per week for clinical deficits for 98 days. Tissues were harvested and analyzed using hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining. Rats injected with PDGF-B+HRAS+sh-p53 (triple cocktail) exhibited statistically significant declines in all behavioral measures (Basso Beattie Bresnahan scoring, Tarlov scoring, weight, and survival rate) over time when compared to the control. Histologically, all groups except the control and those injected with sh-p53 displayed the development of tumors at the injection site, although there were differences in the rate of tumor growth and the histopathological features of the lesions between groups. Examination of immunohistochemistry revealed rats receiving triple cocktail displayed the largest and most significant increase in the Ki67 proliferation index and GFAP positivity than any other group. PDGF-B+HRAS also displayed a significant increase in the Ki67 proliferation index. Rats receiving PDGF-B alone and PDGF-B+ sh-p53 displayed more a significant increase in SOX2-positive staining than in any other group. We found that different vector combinations produced differing high-grade glioma models in rodents. The combination of all three vectors produced a model of high-grade glioma more efficiently and aggressively with respect to behavioral, physiological, and histological characteristics than the rest of the vector combinations. Thus, the present rat model of spinal cord glioma may potentially be used to evaluate therapeutic strategies in the future.

p53 Inhibition Protects against Neuronal Ischemia/Reperfusion Injury by the p53/PRAS40/mTOR Pathway.

The underlying mechanisms of cerebral ischemia/reperfusion (I/R) injury are unclear. Within this study, we aimed to explore whether p53 inhibition exerts protective effects via the p53/PRAS40/mTOR pathway after stroke and its potential mechanism. Both an in vitro oxygen-glucose deprivation (OGD) model with a primary neuronal culture and in vivo stroke models (dMCAO or MCAO) were used. We found that the infarction size, neuronal apoptosis, and autophagy were less severe in p53 KO mice and p53 KO neurons after cerebral I/R or OGD/R injury. By activating the mTOR pathway, p53 knockdown alleviated cerebral I/R injury both in vitro and in vivo. When PRAS40 was knocked out, the regulatory effects of p53 overexpression or knockdown against stroke disappeared. PRAS40 knockdown could inhibit the activities of the mTOR pathway; moreover, neuronal autophagy and apoptosis were exacerbated by PRAS40 knockdown. To sum up, in this study, we showed p53 inhibition protects against neuronal I/R injury after stroke via the p53/PRAS40/mTOR pathway, which is a novel and pivotal cerebral ischemic injury signaling pathway. The induction of neuronal autophagy and apoptosis by the p53/PRAS40/mTOR pathway may be the potential mechanism of this protective effect.

Carcinomatosis under control by osimertinib in EGFR and TP53 mutated lung adenocarcinoma.

INTRODUCTION: Approximately 25%-30% of patients with non-small cell lung cancer (NSCLC) develop central nervous system (CNS) metastases during the course of the disease; this percentage is higher in patients with epidermal growth factor receptor (EGFR) mutations. Leptomeningeal metastases, infrequent in the advanced setting, have a particularly dismal prognosis. Osimertinib, a third-generation EGFR inhibitor, can provide effective and durable response in this setting. CASE DESCRIPTION: We present a 62-year-old man with progressive vomiting, headache, short-term memory impairment, and left lower limb hyposthenia. Computed tomography (CT) showed bilateral lung nodules, multiple lymphadenopathies, liver and bone metastases, and CNS and leptomeningeal dissemination, including multiple parenchymal nodules located at supra- and infratentorial brain. Bone needle biopsy documented TTF1+ lung adenocarcinoma. Whole brain radiotherapy (WBRT) and symptomatic treatments were started. Next-generation sequencing reported deletion of exon 19 of EGFR and mutation 8 of TP53. Osimertinib 80 mg was promptly started and WBRT interrupted. Some days after the patient experienced repetitive seizures and neurologic worsening, antiepileptic drugs and dexamethasone were implemented, with gradual improvement. Radiologic evaluation, including brain MRI and thorax-abdominal CT, showed partial response on CNS as well as extracranial sites, which was sustained. CONCLUSIONS: First-line treatment with osimertinib can be safe and effective in EGFR-mutated NSCLC even in presence of multiple negative predictive factors (poor Performance Status, diffuse leptomeningeal involvement, TP53 comutation), suggesting that deferring local treatments can be feasible in this setting, allowing the patient to maintain a good quality of life.

Bcl-xL Reduces Chinese Giant Salamander Iridovirus-Induced Mitochondrial Apoptosis by Interacting with Bak and Inhibiting the p53 Pathway.

Chinese giant salamander iridovirus (GSIV) infection could lead to mitochondrial apoptosis in this animal, a process that involves B-cell lymphoma-2 (BCL-2) superfamily molecules. The mRNA expression level of Bcl-xL, a crucial antiapoptotic molecule in the BCL-2 family, was reduced in early infection and increased in late infection. However, the molecular mechanism remains unknown. In this study, the function and regulatory mechanisms of Chinese giant salamander (Andrias davidianus) Bcl-xL (AdBcl-xL) during GSIV infection were investigated. Western blotting assays revealed that the level of Bcl-xL protein was downregulated markedly as the infection progressed. Plasmids expressing AdBcl-xL or AdBcl-xL short interfering RNAs were separately constructed and transfected into Chinese giant salamander muscle cells. Confocal microscopy showed that overexpressed AdBcl-xL was translocated to the mitochondria after infection with GSIV. Additionally, flow cytometry analysis demonstrated that apoptotic progress was reduced in both AdBcl-xL-overexpressing cells compared with those in the control, while apoptotic progress was enhanced in cells silenced for AdBcl-xL. A lower number of copies of virus major capsid protein genes and a reduced protein synthesis were confirmed in AdBcl-xL-overexpressing cells. Moreover, AdBcl-xL could bind directly to the proapoptotic molecule AdBak with or without GSIV infection. In addition, the p53 level was inhibited and the mRNA expression levels of crucial regulatory molecules in the p53 pathway were regulated in AdBcl-xL-overexpressing cells during GSIV infection. These results suggest that AdBcl-xL plays negative roles in GSIV-induced mitochondrial apoptosis and virus replication by binding to AdBak and inhibiting p53 activation.

Transcriptome Analysis of Cells Exposed to Actinomycin D and Nutlin-3a Reveals New Candidate p53-Target Genes and Indicates That CHIR-98014 Is an Important Inhibitor of p53 Activity.

Co-treatment with actinomycin D and nutlin-3a (A + N) strongly activates p53. Previously we reported that CHIR-98014 (GSK-3 kinase inhibitor), acting in cells exposed to A + N, prevents activation of TREM2-an innate immunity and p53-regulated gene associated with Alzheimer's disease. In order to find novel candidate p53-target genes and genes regulated by CHIR-98014, we performed RNA-Seq of control A549 cells and the cells exposed to A + N, A + N with CHIR-98014 or to CHIR-98014. We validated the data for selected genes using RT-PCR and/or Western blotting. Using CRISPR/Cas9 technology we generated p53-deficient cells. These tools enabled us to identify dozens of candidate p53-regulated genes. We confirmed that p53 participates in upregulation of BLNK, APOE and IRF1. BLNK assists in activation of immune cells, APOE codes for apolipoprotein associated with Alzheimer's disease and IRF1 is activated by interferon gamma and regulates expression of antiviral genes. CHIR-98014 prevented or inhibited the upregulation of a fraction of genes stimulated by A + N. Downregulation of GSK-3 did not mimic the activity of CHIR-98014. Our data generate the hypothesis, that an unidentified kinase inhibited by CHIR-98014, participates in modification of p53 and enables it to activate a subset of its target genes, e.g., the ones associated with innate immunity.