P53 Inhibitor Research Articles

In silico screening and identification of potential drug against p300 acetyltransferase activity in breast cancer via drug repurposing approach

Emerging evidence portray the involvement of epigenomic reprogramming in the onset and progression of several malignancies, including breast cancer. Histone acetyltransferase (HAT) p300 is a critical epigenetic regulator that acts as a transcription co-activator and regulates various cellular processes. p300 is overexpressed in breast cancer and promotes cellular invasion and survival, making it a promising druggable target. In this study, the relevance of p300 in different cancer pathways was established. Virtual screening of the FDA-approved drug library was carried out using molecular docking, and the top 10 potential repurposed drugs were identified. Further, recalculation of binding free energy of drug-p300 complexes was carried out using molecular mechanics Poisson-Boltzmann and surface area (MM-PBSA) method after molecular dynamic simulation. Based on molecular dynamic simulation parameters and binding free energy analysis, two drugs, namely Netarsudil (−305.068 kJ/mol) and Imatinib (−260.457 kJ/mol), were identified as potential repurposed drugs to inhibit the activity of p300. In conclusion, these findings suggest, Netarsudil and Imatinib might be a potential repurposed drug to combat breast cancer via p300 inhibition. Communicated by Ramaswamy H. Sarma

Mdm2 Targeting Reassembly Peptide (Trap) Nanoparticles For P53 Based Cancer Therapy.

Gene mutations and functional inhibition are the major obstacles for p53-mediated oncotherapy. For p53-wild type tumors, the underlying mechanisms of functional inhibition of p53 during oncogenesis are unknown. Our results revealed that the expression of the MDM2 inhibitor ARF was inhibited in p53-wild type tumors, indicating that the restoration of ARF could be a potential oncotherapy strategy for p53-wild type tumors. Therefore, we developed ARF-mimetic MDM2-targeting reassembly peptide nanoparticles (MtrapNPs) for p53-based tumor therapy. Our results elucidated that the MtrapNPs responded to and formed a nanofiber structure with MDM2. By trapping MDM2, the MtrapNPs stabilized and activated p53 for the inhibition of p53-wild type tumors. In most cases, reactivated mutant p53 is inhibited and degraded by MDM2. In the present study, MtrapNPs were used to load and deliver arsenic trioxide, a p53 mutation rescuer, for p53-mutated tumor treatment in both orthotopic and metastatic models, and they exhibited significant therapeutic effects. Therefore, our study provides evidence supporting a link between decreased ARF expression and tumor development in patients with p53-wild type tumors. Thus, the novel MDM2-trap strategy, which addresses both the inhibition and mutations of p53, is an efficient strategy for the treatment of p53-mutated tumors. This article is protected by copyright. All rights reserved.

Involvement of transcriptional co-activator p300 in upregulated expression of HSP70 by aquareovirus non-structural protein NS31

Members of Aquareovirus genus, including grass carp reovirus (GCRV), contribute to a serious threat to aquaculture animals accompanied by stress response. Our previous reports revealed that GCRV nonstructural protein NS31 serves as a potent contributor for virus selectively up-regulating specific heat shock protein 70-kd gene（HSP70），however，the mechanism by which inducing HSP70 gene expression is unknown. In this study, we further found that either the N- or C-terminal domain of GCRV NS31 is responsible for enhancing fish HSP70 promoter transcription, and recombinant NS31 protein purified from baculovirus expression system seems to not directly bind HSP70 basic promoter in vitro by an electrophoretic mobility shift assay. However, the transcriptional co-activator p300 was identified as a potential interacting partner for NS31 by pull-down assay. Moreover, knock-down of p300 or addition of p300 inhibitor resulted in obviously reduced HSP70 expression by NS31 or GCRV infection suggesting that the well-characterized heat-shock-responsive HSF1/p300 transcriptional complex might involve in the induction of HSP70. These results collectively reveal this aquareovirus generates cell stress response through its nonstructural protein NS31 recruiting transcriptional co-activator p300.

Cigarette smoke extract induces the senescence of endothelial progenitor cells by upregulating p300.

Endothelial progenitor cells (EPCs) are the main source of endothelial cells. The senescence of EPCs is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke extract (CSE) can directly induce the dysfunction and increased expression of senescence-related markers in EPCs cultured in vitro. Histone acetyltransferase p300 is a transcriptional activator, and its changes can lead to cell senescence. The present study investigated whether CSE can induce the senescence of EPCs by upregulating p300. EPCs were isolated from bone marrow of C57BL/6J mice by density gradient centrifugation. The p300 inhibitor C646 and agonist CTPB were used to interfere with EPCs, cell cycle and apoptosis were detected by flow cytometry, the proportion of senile cells was counted by β-galactosidase staining, the protein expression of p300, H4K12, Cyclin D1, TERT and Ki67 were detected by western blot. Compared with the control group, the cell cycle of CSE group and CTPB group were blocked, the apoptosis rate and early apoptosis rate were increased, the proportion of senile cells counted by β-galactosidase staining was increased, the expression of p300 and H4K12 protein were increased, the expression of Cyclin D1, TERT and Ki67 protein were decreased. C646 could partly alleviate the damages caused by CSE. CSE may promote the apoptosis and senescence of EPCs by upregulating the expression of p300 and H4K12 protein, thus preventing the transition of EPCs from G1 phase to S phase, affecting telomerase synthesis, and reducing EPCs proliferation.

The Role of JNK and p53 in the Regulation of Secretory Function of Neuroglial Cells of Various Types in β-Amyloid-Induced Neurodegeneration.

We studied the effect of JNK and p53 inhibitors on the production of neurotrophic factors stimulating the realization of the growth potential of neural stem cells by neuroglial cells of various types under conditions of simulation of induced β-amyloid neurodegeneration in vitro. It was shown that β-amyloid stimulates the production of neurotrophins by astrocytes and microglial cells, but does not affect the functioning of oligodendrocytes. JNK and p53 were not involved in the secretion of neurotrophins by intact astrocytes. The stimulating role of p53 on the implementation of their secretory activity under the influence of a neurotoxic agent was revealed. At the same time, the inhibitory role of JNK and p53 in the production of neurotrophic growth factors by oligodendrocytes and microglial cells was revealed both under conditions of their optimal vital activity and when β-amyloid was added to the cell culture.

PFT-α protects the blood-brain barrier through the Wnt/β-catenin pathway after acute ischemic stroke.

The dysfunction of blood-brain barrier (BBB) plays a pivotal role in brain injury and subsequent neurological deficits of ischemic stroke. The current study aimed to examine the potential correlation between p53 inhibition and the neuroprotective effect of on the BBB. Rat middle cerebral artery occlusion and reperfusion model (MCAO/R) and oxygen-glucose deprivation/re-oxygenation model (OGD/R) were employed to simulate cerebral ischemia-reperfusion (CI/R) injury occurrence in vivo and in vitro. mNSS and TTC staining were applied to evaluate neurological deficits and brain infarct volumes. Evans blue (EB) staining was carried out to examine the permeability of BBB. RT-qPCR and Western blot to examine the mRNA and protein levels. Cell viabilities were detected by CCK-8. Flow cytometry and ELISA assaywere employed to examine apoptosis and neuroinflammation levels. TEER value and sodium fluorescein were carried out to explore the permeability of HBMEC cells. PFT-α inhibited P53 and promoted the expression of β-catenin and cyclin D1, which were reversed by DKK1. PFT-α inhibited neurological deficits, brain infarct volume, neuroinflammation, apoptosis, and BBB integrity than the MCAO/R rats; however, this inhibition was reversed by DKK1. PFT-α promoted OGD/R-induced cell viability in NSCs, and suppressed inflammation and apoptosis, but DKK1 weakened the effect of PFT-α. PFT-α increased OGD/R-induced TEER values in cerebrovascular endothelial cells, inhibited sodium fluorescein permeability, and increased the mRNA levels of tight junction protein, but they were all attenuated by DKK1. PFT-α protects the BBB after acute ischemic stroke via the Wnt/β-catenin pathway, which in turn improves neurological function.

Hypoxia-inducible factors: details create a picture. Part I. HIF-1

The review presents a comparative analysis of scientific data on the structural and functional characteristics of subunits (HIF-1α and HIF-1β) of hypoxia-inducible transcription factor-1 (HIF-1). Differences between the main regulatory HIF-1α subunit and the constitutively expressed HIF-1β subunit, sensitivity to endo- and exogenous regulators of their stability, and intracellular content depending on the cell's oxygen supply state (normoxia and hypoxia conditions) are described. In normoxia, the intracellular content of HIF-1α is determined by oxygen-dependent and oxygen-independent mechanisms. Oxygen-dependent enzymatic degradation of HIF-1α occurs by PHD-dependent hydroxylation, VHL-dependent ubiquitination, and FIH-1-dependent hydroxylation. Oxygen-independent pathways of HIF-1α pool regulation include: 1) HIF-1α gene transcription (Notch and/or NF-KB-dependent, STAT3 and Sp1 cytokine-dependent), 2) mRNA translation (cap-dependent or IRES-dependent, as well as cytokine-dependent activation of the PI-3K/AKT pathway activation under the effect of growth factors and vasoactive hormones), 3) protein-protein interactions, 4) various mechanisms of post-translational modification. Changes in Krebs cycle enzyme activity and active oxygen forms confer HIF-1α stability through PHD activity inhibition and reduction of ubiquitin-proteasome degradation. PHD-independent post-translational stabilizers of HIF-1α are: cytosolic reductase NQO1, sirtuin-2, prostaglandin E2, activated protein kinase C1 receptor competing with heat shock protein 90, human Hdm2 (a natural inhibitor of p53), glycogen synthase kinase 3β, and negative modifiers are enzymes - methyltransferase SET7/9, lysine-specific demethylase-1, sex-like kinase 3, β-arrestin-2, casein kinase-1. Under hypoxic conditions, non-hydroxylated HIF-1α subunits migrate to the nucleus where they heterodimerize with HIF-1β, HIF-1α/β heterodimers bind the main 5'-(A/G)CGTG-3' consensus sequence within the hypoxia-reaction element (HRE) of the target genes, and recruit co-activators (p300, histone modifying enzymes, histone readers, chromatin remodeling proteins, and mediator proteins for target genes transcription enhancement with the aid of RNA polymerase II), resulting in the formation of HIF-1, acting as a transcription factor for the target genes providing metabolic reprogramming from oxidative phosphorylation to anaerobic glycolysis (genes encoding glucose transporters (GLUT1 and GLUT3), genes for glycolytic enzymes hexokinase 1 and 2 (HK1 and HK2), phosphoglycerate kinase 1), as well as genes for erythropoietin, vascular endothelial growth factor and its receptors FLT1 and FLK1, endothelin 1 and angiopoietin 1, resulting in adaptation to hypoxia.

Cellular metabolic pathways of aging in dogs: could p53 and SIRT1 be at play?

Aging and cancer seem to be closely associated, such that cancer is generally considered a disease of the elderly in both humans and dogs. Additionally, cancer is a metabolic shift in itself towards aerobic glycolysis. Larger dog breeds with shorter lifespans, and increased glycolytic cellular metabolic rates, die of cancer more often than smaller breeds. The tumor suppressor p53 factor is a key suppressor oncogene, and the p53 pathway arrests cellular proliferation and prevents DNA mutations from accumulating during cellular stress. The p53 pathway is also associated with the control of cellular metabolism to prevent cellular metabolic shifts common to cancerous phenotypes. SIRT1 deacetylates the p53 tumor suppressor protein, downregulating p53 via effects on stability and activity during stress. Here, we used primary fibroblast cells from small and large puppies and old dogs. Using UV radiation to upregulate the p53 system (100J/m2), control cells and UV-treated cells were used to measure aerobic and glycolytic metabolic rates using a Seahorse XFe96 oxygen flux analyzer. We also quantified p53 expression and SIRT1 concentration in canine primary fibroblasts before and after UV treatment. We demonstrate that, due to a higher p53 nuclear to cytoplasmic ratio in large breed dogs after UV treatment, p53 could have a more regulatory effect on large breed dogs' metabolism compared with smaller breeds. Thus, there may be a link between p53 upregulation and inhibition of glycolysis in large breed dogs during times of cellular stress compared with small breed dogs. However, SIRT1 concentrations decrease with age in domestic dogs of both size classes, suggesting a possible release of inhibition of p53 through the SIRT1 pathway with age. This may lead to increased incidences of cancer, especially due to the more pronounced upregulation of p53 with cellular stress.

Hexarelin alleviates apoptosis on ischemic acute kidney injury via MDM2/p53 pathway

IntroductionHexarelin exhibits significant protection against organ injury in models of ischemia/reperfusion (I/R)-induced injury (IRI). Nevertheless, the impact of Hexarelin on acute kidney injury (AKI) and its underlying mechanism remains unclear. In this study, we investigated the therapeutic potential of Hexarelin in I/R-induced AKI and elucidated its molecular mechanisms.MethodsWe assessed the protective effects of Hexarelin through both in vivo and in vitro experiments. In the I/R-induced AKI model, rats were pretreated with Hexarelin at 100 μg/kg/d for 7 days before being sacrificed 24 h post-IRI. Subsequently, kidney function, histology, and apoptosis were assessed. In vitro, hypoxia/reoxygenation (H/R)-induced HK-2 cell model was used to investigate the impact of Hexarelin on apoptosis in HK-2 cells. Then, we employed molecular docking using a pharmmapper server and autodock software to identify potential target proteins of Hexarelin.ResultsIn this study, rats subjected to I/R developed severe kidney injury characterized by tubular necrosis, tubular dilatation, increased serum creatinine levels, and cell apoptosis. However, pretreatment with Hexarelin exhibited a protective effect by mitigating post-ischemic kidney pathological changes, improving renal function, and inhibiting apoptosis. This was achieved through the downregulation of conventional apoptosis-related genes, such as Caspase-3, Bax and Bad, and the upregulation of the anti-apoptotic protein Bcl-2. Consistent with the in vivo results, Hexarelin also reduced cell apoptosis in post-H/R HK-2 cells. Furthermore, our analysis using GSEA confirmed the essential role of the apoptosis pathway in I/R-induced AKI. Molecular docking revealed a strong binding affinity between Hexarelin and MDM2, suggesting the potential mechanism of Hexarelin’s anti-apoptosis effect at least partially through its interaction with MDM2, a well-known negative regulator of apoptosis-related protein that of p53. To validate these findings, we evaluated the relative expression of MDM2 and p53 in I/R-induced AKI with or without Hexarelin pre-administration and observed a significant suppression of MDM2 and p53 by Hexarelin in both in vivo and in vitro experiments.ConclusionCollectively, Hexarelin was identified as a promising medication in protecting apoptosis against I/R-induced AKI.

Escherichia coli infection mediates pyroptosis via activating p53-p21 pathway-regulated apoptosis and cell cycle arrest in bovine mammary epithelial cells

Escherichia coli (E. coli) is a major environmental pathogen that causes mammary tissue damage and cell death, which results in substantial economic losses. Pyroptosis, a novel form of programmed cell death characterized by DNA fragmentation, chromatin condensation, cell swelling and leakage of cell contents, often occurs after inflammatory apoptotic pathways activation. Our objective was to investigate the intraction between E. coli infection and bovine mammary epithelial cells (bMECs) with pyroptosis and to explore the underlying regulatory mechanism. bMECs were infected with E. coli for 6 h. Lactic dehydrogenase activities, interleukin (IL)-10, IL-1β, IL-18 and tumor necrosis factor-α concentrations, total apoptosis indexes, and protein expressions of P-cdc25c, P-CDK1, cleaved caspase 9, cleaved caspase 3, cleaved PARP, P–NF-κB, NLRP3, ASC, caspase 1, gasdermin D N-terminal, IL-1β and IL-18 were significantly increased in E. coli infected bMECs. Whereas, cell membrane potential, protein levels of cdc25c, CDK1, cyclin B1, and Bcl-2/Bax level were markedly reduced. Furthermore, Ac-DEVD-CHO (specific inhibitor of apoptosis) dramatically suppressed pyroptosis in bMECs. Moreover, expressions of p53 and p21 promptly improved after E. coli infection, however, Pifithrin-α (specific inhibitor of p53) inhibited p53-p21 pathway, apoptosis, cell cycle arrest and pyroptosis. These results elaborated that E. coli infection of bMECs induced pyroptosis through activating the p53-p21 pathway-mediated apoptosis and cell cycle arrest. Taken together, inhibition of pyroptosis via suppressing of p53-p21 pathway may be an effective therapeutic approach for treating E. coli-induced mastitis, offering efficient theoretical support for the protection and treatment of bovine mastitis.

Benzo[b]fluoranthene induces male reproductive toxicity and apoptosis via Akt-Mdm2-p53 signaling axis in mouse Leydig cells: Integrating computational toxicology and experimental approaches.

This study aims to explore the male reproductive toxicity of Benzo[b]fluoranthene (BbF) and related mechanisms. The results of computational toxicology analysis indicated male reproductive toxicity of BbF was related to apoptosis of Leydig cells and that Akt/p53 pathway might play a key role. In experiments, BbF induced testosterone decline, decreased concentration and motility of sperm and aggravated testicular pathological injury in mice. Besides, BbF led to apoptosis in Leydig cells, and decreased expressions of p-Akt and Bcl2, while improving the expressions of p53, Bax and Cleaved Caspase-3 in vivo and in vitro. Further, compared with BbF group, Akt activator SC79 significantly reduced cell apoptosis rate, improved cell viability, promoted the expressions of p-Akt and p-Mdm2, and reversed the above molecular expressions. Similarly, p53 inhibitor Pifithrin-α also significantly enhanced the cell vitality, alleviated the apoptosis of TM3 cells induced by BbF, and decreased the expressions of Bax and Cleaved Caspase-3, with the up-regulation of Bcl2. To sum up, by inhibiting Akt-Mdm2 signaling, BbF activated the p53-mediated mitochondrial apoptosis pathway, further inducing the apoptosis of Leydig cells, therefore resulting in testosterone decline and male reproductive damage. Besides, this study provided a valid mode integrating computational toxicology and experimental approaches in toxicity testing.

MiR-214 could promote myocardial fibrosis and cardiac mesenchymal transition in VMC mice through regulation of the p53 or PTEN-PI3K-Akt signali pathway, promoting CF proliferation and inhibiting its ng pathway

IntroductionThis study aimed to investigate the role of miR-214 in the bidirectional regulation of p53 and PTEN and its influence on myocardial fibrosis and cardiac mesenchymal transformation in mice with viral myocarditis (VMC). MethodsThe study established a VMC model in BALB/c mice by injecting them with the CVB3 virus intraperitoneally. Techniques such as ELISA, H&E staining, Masson staining, immunohistochemical staining, RT-qPCR, western blot, and dual-luciferase reporter gene assay were used to detect the expression levels of relevant factors in tissues and cells. Isolation and culture of cardiac fibroblasts (CFs) were also conducted. ResultsThe study found that miR-214 bidirectional regulation of p53 and PTEN promotes myocardial fibrosis and cardiac mesenchymal transformation in mice with VMC. The expression levels of collagen-related peptides, inflammatory-related factors, miR-214, mesenchymal transformation-related factors, and fibrosis-related factors were significantly increased, while the expression levels of p53, PTEN, and epithelial/endothelial cell phenotype marker factors were significantly decreased. Downregulation of miR-214 or upregulation of p53 and PTEN expression inhibited inflammatory cell and fibroblast infiltration in VMC mouse myocardial tissue. It reduced the proliferation ability while increasing the apoptosis of cardiac fibroblasts. ConclusionmiR-214 plays a significant role in the bidirectional inhibition of p53 and PTEN, which leads to myocardial fibrosis and cardiac mesenchymal transformation in mice with VMC. Downregulation of miR-214 or upregulation of p53 and PTEN expression may provide potential therapeutic targets for treating VMC-induced cardiac fibrosis and mesenchymal transformation.

The Effects of Pifithrin-µ on Spermatogonial Stem Cell Viability and Pluripotency

Introduction: Spermatogonial stem cells (SSCs) offer remarkable competencies for animal reproduction and overcoming human disease as a result of their differentiation capability. We evaluated the effect of small molecule pifithrin-mu (PFT-µ), a well-known inhibitor of P53 on SSC biological processes such as viability, apoptosis, and gene expression pattern. Methods: The SSCs were isolated from the testes of adult NMRI mice and then cultured in DMEM/F12 medium containing 10% FBS. Then, they were characterized by the immunocytochemistry technique by high PLZF and low c-Kit expressions. SSC colony formation assay was carried out and their viability was estimated by methylthiazolyldiphenyl-tetrazolium bromide (MTT, or 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay upon exposure to PFT-µ (0, 0.6, 1.2, 2.5, and 5 µm). The apoptosis percentages were also measured using FACS analysis, and finally, Oct4 and Stra8 expression at mRNA levels was assessed using real-time quantitative PCR. Results: The 0.6 and 1.2 µm PFT-µ improved the viability of SSC based on MTT assay results; however, 2.5 and 5 µm PFT-µ reduced SSC viability compared with the control group. Moreover, PFT-µ at lower concentrations enhanced the colony size of SSCs and diminished their apoptosis. As well, exposure to PFT-µ upregulated Oct4 expression while downregulating the meiotic entry marker, Stra8. Conclusion: Based on findings, optimized concentrations of PFT-µ can decrease SSC apoptosis, and conversely potentiate their pluripotency and self-renewal capacities in vitro.

Inhibition of high level E2F in a RB1 proficient MYCN overexpressing chicken retinoblastoma model normalizes neoplastic behaviour

PurposeRetinoblastoma, a childhood cancer, is most frequently caused by bi-allelic inactivation of RB1 gene. However, other oncogenic mutations such as MYCN amplification can induce retinoblastoma with proficient RB1. Previously, we established RB1-proficient MYCN-overexpressing retinoblastoma models both in human organoids and chicken. Here, we investigate the regulatory events in MYCN-induced retinoblastoma carcinogenesis based on the model in chicken.MethodsMYCN transformed retinal cells in culture were obtained from in vivo MYCN electroporated chicken embryo retina. The expression profiles were analysed by RNA sequencing. Chemical treatments, qRT-PCR, flow cytometry, immunohisto- and immunocytochemistry and western blot were applied to study the properties and function of these cells.ResultsThe expression profile of MYCN-transformed retinal cells in culture showed cone photoreceptor progenitor signature and robustly increased levels of E2Fs. This expression profile was consistently observed in long-term culture. Chemical treatments confirmed RB1 proficiency in these cells. The cells were insensitive to p53 activation but inhibition of E2f efficiently induced cell cycle arrest followed by apoptosis.ConclusionIn conclusion, with proficient RB1, MYCN-induced high level of E2F expression dysregulates the cell cycle and contributes to retinoblastoma carcinogenesis. The increased level of E2f renders the cells to adopt a similar mechanistic phenotype to a RB1-deficient tumour.

Dynamic 3D genome reorganization during senescence: defining cell states through chromatin.

Cellular senescence, a cell state characterized by growth arrest and insensitivity to growth stimulatory hormones, is accompanied by a massive change in chromatin organization. Senescence can be induced by a range of physiological signals and pathological stresses and was originally thought to be an irreversible state, implicated in normal development, wound healing, tumor suppression and aging. Recently cellular senescence was shown to be reversible in some cases, with exit being triggered by the modulation of the cell's transcriptional program by the four Yamanaka factors, the suppression of p53 or H3K9me3, PDK1, and/or depletion of AP-1. Coincident with senescence reversal are changes in chromatin organization, most notably the loss of senescence-associated heterochromatin foci (SAHF) found in oncogene-induced senescence. In addition to fixed-cell imaging, chromatin conformation capture and multi-omics have been used to examine chromatin reorganization at different spatial resolutions during senescence. They identify determinants of SAHF formation and other key features that differentiate distinct types of senescence. Not surprisingly, multiple factors, including the time of induction, the type of stress experienced, and the type of cell involved, influence the global reorganization of chromatin in senescence. Here we discuss how changes in the three-dimensional organization of the genome contribute to the regulation of transcription at different stages of senescence. In particular, the distinct contributions of heterochromatin- and lamina-mediated interactions, changes in gene expression, and other cellular control mechanisms are discussed. We propose that high-resolution temporal and spatial analyses of the chromatin landscape during senescence will identify early markers of the different senescence states to help guide clinical diagnosis.

Cannabidiol-induced crosstalk of apoptosis and macroautophagy in colorectal cancer cells involves p53 and Hsp70

Although it has been established that cannabidiol (CBD), the major non-psychoactive constituent of cannabis, exerts antitumoral activities, the exact mechanism(s) via which tumor cells are killed by CBD are not well understood. This study provides new insights into the potential mechanisms of CBD-induced mutual antagonism of apoptosis and macroautophagy using wild type (HCT116 p53wt, LS174T p53wt), knockout (HCT116 p53−/−) and mutant (SW480 p53mut) human colorectal cancer cells (CRC). CBD causes a more pronounced loss in the viability of p53wt cells than p53−/− and p53mut cells, and a 5-week treatment with CBD reduced the volume of HCT116 p53wt xenografts in mice, but had no effect on the volume of HCT116 p53−/− tumors. Mechanistically, we demonstrate that CBD only significantly elevates ROS production in cells harboring wild-type p53 (HCT116, LS174T) and that this is associated with an accumulation of PARP1. CBD-induced elevated ROS levels trigger G0/G1 cell cycle arrest, a reduction in CDK2, a p53-dependent caspase-8/9/3 activation and macroautophagy in p53wt cells. The ROS-induced macroautophagy which promotes the activation of keap1/Nrf2 pathway might be positively regulated by p53wt, since inhibition of p53 by pifithrin-α further attenuates autophagy after CBD treatment. Interestingly, an inhibition of heat shock protein 70 (Hsp70) expression significantly enhances caspase-3 mediated programmed cell death in p53wt cells, whereas autophagy—which is associated with a nuclear translocation of Nrf2—was blocked. Taken together, our results demonstrate an intricate interplay between apoptosis and macroautophagy in CBD-treated colorectal cancer cells, which is regulated by the complex interactions of p53wt and Hsp70.

Involvement Value of FLT-3, c-Myc, STAT3, p27, and HOTAIR Gene Expression in Acute Myeloid Leukemia Patients: A Molecular Perspective to a Novel Leukemogenesis Mechanism.

Background: The identification of long non-coding RNAs (lncRNAs) in the pathogenesis of acute myeloid leukemia (AML) has marked a new era in the molecular understating of the disease. This study investigated the correlation between the changes in the expression of lncRNAs, including HOTAIR, PVT-1, and CRNDE, and the alteration in the expression profile of FLT-3, c-Myc, STAT3, STAT5, and p27 in AML patients. Materials and Methods: Blood samples were collected from forty-one newly diagnosed AML patients and ten healthy individuals to evaluate the expression levels of the study genes using qRT-PCR analysis. The probable correlation between the gene expressions was determined using Pearson's correlation test. Results: The results showed that while there was a significant elevation in the expression of FLT3, c-Myc, STAT3, and HOTAIR, p27 expression remarkably diminished in AML patients compared to the control group. Also, a correlation was found between the expression of FLT-3 and p27 and the expression of HOTAIR and STAT3. It was assumed that FLT-3 had a role in increasing the proliferative and survival capacity of AML cells, at least partly, through c-Myc-mediated suppression of p27. Moreover, lncRNA HOTAIR showed to be involved in leukemia proliferation assumably by enhancing the expression of STAT3. Conclusion: Overall, the results of gene profile analysis suggested that studying the expression of HOTAIR, FLT-3, c-Myc, STAT3, and p27 could be helpful to AML patients, and each of these genes could be a valuable target for pharmaceutic intervention.

Inhibition of p53 and ATRX increases telomeric recombination in primary fibroblasts.

Telomere length can be maintained either by the telomerase enzyme or by alternative lengthening of telomeres (ALT), which is based on telomeric recombination. However, both mechanisms are inactive in most human somatic cells. ATRX has been previously identified as an ALT repressor gene. Nonetheless, TP53 is also deficient in most ALT cell lines, and previous works showed that it is an inhibitor of homologous recombination (HR). Despite this, the role of p53 as an ALT repressor has not been previously examined. Therefore, we investigated the effects of p53 and ATRX inhibition on normal human fibroblasts (devoid of any mutation), in the presence or absence of X-ray-induced telomeric damage. Performing immunofluorescence with antibodies for RAD51, H2AX, and TRF1 (for studying HR-mediated DNA damage repair) and CO-FISH (for telomeric sister chromatid exchanges), we observed that HR is a normal mechanism for the repair of telomeric damage, present also in noncancer cells. Moreover, we discovered that telomeric HR, as for HR in general, is significantly inhibited by p53. Indeed, we observed that inhibition of p53 drastically increases telomeric sister chromatid exchanges. We also confirmed that ATRX inhibition increases telomeric recombination. In particular, we observed an increase in crossover products, but a much higher increase in noncrossover products.

MiR-147b is an oncomiR acting synergistically with HIPK2 to promote pancreatic carcinogenesis

MicroRNAs (miRs, miRNAs) are known players in the regulatory network of pancreatic tumorigenesis, but the downstream effectors remain poorly characterized. This study addressed this issue based on in silico prediction, in vitro experiments, and in vivo validation. The differentially expressed PCa-related miRNAs and bioinformatics tools predicted downstream regulators. The expression of miR-147b was examined in PCa cell lines. Putative targets of miR-147b were predicted by a publicly available database and confirmed by luciferase activity assay. Mimic/inhibitor, siRNA/overexpression plasmid, or pifithrin-α (p53 inhibitor) were delivered into PCa cells to assess the effect of miR-147b, HIPK2, and p53 on malignant phenotypes of PCa cells. AntagomiR-147b and shRNA targeting HIPK2 were introduced to xenograft-bearing nude mice for in vivo experiments. The expression of miR-147b was significantly increased in PCa cell lines. Ectopic expression of miR-147b promoted the malignant phenotypes of PCa cells and inhibited their apoptosis. HIPK2 was confirmed as a target gene of miR-147b. Inhibiting miR-147b could promote HIPK2 expression and potentially activate the p53 pathway, inhibiting PCa cell growth. In vivo experiments suggested that miR-147b inhibition suppressed the growth of xenograft tumors in nude mice, while HIPK2 knockdown counteracted its effect. Collectively, our work reveals a novel miR-147b-mediated carcinogenic regulatory network in PCa that may be a viable target for PCa treatment.

Molecular dynamics of genome editing with CRISPR-Cas9 and rAAV6 virus in human HSPCs to treat sickle cell disease

Exvivo gene correction with CRISPR-Cas9 and a recombinant adeno-associated virus serotype 6 (rAAV6) in autologous hematopoietic stem/progenitor cells (HSPCs) to treat sickle cell disease (SCD) has now entered early-phase clinical investigation. To facilitate the progress of CRISPR-Cas9/rAAV6 genome editing technology, we analyzed the molecular changes in key reagents and cellular responses during and after the genome editing procedure in human HSPCs. We demonstrated the high stability of rAAV6 to serve as the donor DNA template. We assessed the benefit of longer HSPC pre-stimulation in terms of increased numbers of edited cells. We observed that the p53 pathway was transiently activated, peaking at 6 h, and resolved over time. Notably, we revealed a strong correlation between p21 mRNA level and rAAV6 genome number in cells and beneficial effects of transient inhibition of p53 with siRNA on genome editing, cell proliferation, and cell survival. In terms of potential immunogenicity, we found that rAAV6 capsid protein was not detectable, while a trace amount of residual Cas9 protein was still detected at 48h post-genome editing. We believe this information will provide important insights for future improvements of gene correction protocols in HSPCs.