Cell Death Signaling Pathways Research Articles

Biosynthesis and Characterization of Silver Nanoparticles Using Cleistanthus collinus; Investigation on Molecular Mechanism of Apoptosis Cell Death Against Lung Cancer Cells

Background: The discovery of novel molecular and cellular signaling pathways for cancer medicines would enhance the efficacy of cancer therapy. In the current study, we made an effort to explore the mitochondrial-mediated apoptosis cell death signaling pathway in A549 lung cancer cells by silver nanoparticles (AgNPs) synthesized from leaf extract of Cleistanthus collinus. Methods: In-depth, A549 lung cancer cells were treated with AgNPs, and further studies such as HOECHST 33342, AO/EB, Rhodamine-123 staining, flowcytometry, RT-PCR, and Western blotting techniques (24, 48, and 72 h) evidenced apoptosis pathway in cancer cells. Results: Indeed, the microscopic studies proved that AgNPs-treated lung cancer cells appeared with cell shrinkage, membrane swelling, and apoptotic body formation whereas the untreated cells (control) failed to show any morphological consequences. The flow cytometry analysis revealed the G2/M phase of cell cycle arrest by cells were accumulated in large numbers. The RT-PCR data confirmed the expression (Bax and p53) and suppression (Bcl 2) apoptosis-responsive genes in cancer cells. Finally, immunoblotting proved the increased expression of cytochrome c, initiator caspase 9, and executioner caspase 3, which are effective proteins of intrinsic apoptosis activation. Conclusion: Ultimately, the overall present investigations confirmed the mitochondrial signaling pathway played a key role in inducing apoptosis in A549 cancer cell death and demonstrated that AgNPs are novel therapeutic agents for cancer nanomedicine

Single-cell RNA sequencing analysis identifies acute changes in the tumor microenvironment induced by interferon α gene therapy in a murine bladder cancer model.

Nadofaragene firadenovec (Ad-IFNα/Syn3) is now approved for BCG-unresponsive bladder cancer (BLCA). IFNα is a pleiotropic cytokine that causes direct tumor cell killing via TRAIL-mediated apoptosis, angiogenesis inhibition, and activation of the innate and adaptive immune system. We established an immunocompetent murine BLCA model to study the effects of murine adenoviral IFNα (muAd-Ifnα) gene therapy on cancer cells and the tumor microenvironment using a novel murine equivalent of Nadofaragene firadenovec (muAd-Ifnα). Tumors were induced by instilling MB49 cells into the bladders of mice; luciferase imaging confirmed tumor development. Mice were treated with adenovirus control (Ad-Ctrl; empty vector), or muAd-Ifnα (3x1011 VP/mL), and survival analysis was performed. For single-cell sequencing (scRNAseq) analysis (72h), bladders were harvested and treated with collagenase/hyaluronidase and TrypLE for cell dissociation. Single cells were suspended in PBS/1% FBS buffer; viability was assessed with Vicell cell counter. scRNAseq analysis was performed using 10X genomics 3' sequencing. Raw RNAseq data were pre-processed using Cell Ranger single-cell software. Seurat (R package) was used to normalize and cluster the scRNA data. Pooled differential gene expression analysis in specific cell clusters was performed with DESeq2. We identified 16 cell clusters based on marker expression which were grouped into epithelial (tumor), uroplakin-enriched, endothelial, T-cells, neutrophils, and macrophage clusters. Top differentially expressed genes between muAd-Ifnα and Ad-Ctrl were identified. Within the specific cell clusters, IPA analysis revealed significant differences between muAd-Ifnα and control. IFNα signaling and hypercytokinemia/chemokinemia were upregulated in all clusters. Cell death pathways were upregulated in tumor and endothelial clusters. T-cells demonstrated upregulation of the immunogenic cell death signaling pathway and a decrease in the Th2 pathway genes. Macrophages showed upregulation of PD1/PD-L1 pathways along with downregulation of macrophage activation pathways (alternate and classical). Multiplex immunofluorescence confirmed increased infiltration with macrophages in muAd-Ifnα treated tumors compared to controls. PD1/PD-L1 expression was reduced at 72h. This single-cell analysis builds upon our understanding of the impact of Ad-IFNα on tumor cells and other compartments of the microenvironment. These data will help identify mechanisms to improve patient selection and therapeutic efficacy of Nadofaragene firadenovec.

Potential Anticancer Effects of Isoflavone Prunetin and Prunetin Glycoside on Apoptosis Mechanisms.

Cancer is a deadly disease caused by cells that deviate from the normal differentiation and proliferation behaviors and continue to multiply. There is still no definitive cure, and many side effects occur even after treatment. However, apoptosis, one of the programs imprinted on cells, is becoming an important concept in controlling cancer. Flavonoids are polyphenolic compounds found in plants, are naturally bioactive compounds, have been studied for their anticancer effects, and have fewer side effects than chemical treatments. Isoflavones are phytoestrogens belonging to the flavonoid family, and this review discusses in depth the potential anticancer effects of prunetin, one of the many flavonoid families, via the apoptotic mechanism. In addition, a glycoside called prunetin glucoside has been investigated for its anticancer effects through apoptotic mechanisms. The primary intention of this review is to identify the effects of prunetin and its glycoside, prunetin glucoside, on cell death signaling pathways in various cancers to enhance the potential anticancer effects of these natural compounds.

Piperine, a black pepper compound, induces autophagy and cellular senescence mediated by NF-κB and IL-6 in acute leukemia

Acute leukemia is characterized by abnormal white blood cell proliferation with rapid onset and severe complications. Natural compounds, which are alternative treatments, are widely used in cancer treatment. Piperine, an alkaloid compound from black pepper, exerts anticancer effects through the cell death signaling pathway. Autophagy and senescence signaling pathways are considered target signaling pathways for cancer treatment. In this study, we investigated the effects of piperine via autophagy and senescence signaling pathways in NB4 and MOLT-4 cells. The MTT assay results demonstrated that piperine significantly decreased the viability of NB4 and MOLT-4 cells. Piperine induced autophagy by increasing LC3, Beclin-1 and ULK1 and decreasing mTOR and NF-κB1 expression in NB4 and MOLT-4 cells. In addition, piperine increased senescence-associated beta-galactosidase fluorescence intensity by increasing p21 and IL-6 expression while decreasing CDK2 expression in NB4 and MOLT-4 cells. In conclusion, our study provides additional information about the induction of autophagy and senescence by piperine in acute leukemia.

Advancements in Stevens–Johnson Syndrome/Toxic Epidermal Necrolysis Treatment: Utilizing Fas–FasL Inhibition to Target Cell Death Signaling Pathways for Practical Human Application

Advancements in Stevens–Johnson Syndrome/Toxic Epidermal Necrolysis Treatment: Utilizing Fas–FasL Inhibition to Target Cell Death Signaling Pathways for Practical Human Application

Digital spatial profiling identifies the tumor center as a topological niche in prostate cancer characterized by an upregulation of BAD

Prostate cancer is characterized by a high degree of intratumoral heterogeneity. However, little is known about the spatial distribution of cancer cells with respect to specific functional characteristics and the formation of spatial niches. Here, we used digital spatial profiling (DSP) to investigate differences in protein expression in the tumor center versus the tumor periphery. Thirty-seven regions of interest were analyzed for the expression of 47 proteins, which included components of the PI3K-AKT, MAPK, and cell death signaling pathways as well as immune cell markers. A total of 1739 data points were collected from five patients. DSP identified the BCL-2 associated agonist of cell death (BAD) protein as the most significantly upregulated protein in the tumor center. BAD upregulation was confirmed by conventional immunohistochemistry, which furthermore showed a phosphorylation of BAD at serine 112 indicating its inactivation. Knockdown of BAD in prostate cancer cells in vitro led to decreased cell viability and colony growth. Clinically, high BAD expression was associated with a shorter time to biochemical recurrence in 158 mostly high-risk prostate cancer patients. Collectively, our results suggest that the tumor center is a topological niche with high BAD expression that may drive prostate cancer progression.

Radio-miRs: a comprehensive view of radioresistance-related microRNAs.

Radiotherapy is a key treatment option for a wide variety of human tumors, employed either alone or alongside with other therapeutic interventions. Radiotherapy uses high-energy particles to destroy tumor cells, blocking their ability to divide and proliferate. The effectiveness of radiotherapy is due to genetic and epigenetic factors that determine how tumor cells respond to ionizing radiation. These factors contribute to the establishment of resistance to radiotherapy, which increases the risk of poor clinical prognosis of patients. Although the mechanisms by which tumor cells induce radioresistance are unclear, evidence points out several contributing factors including the overexpression of DNA repair systems, increased levels of reactive oxygen species, alterations in the tumor microenvironment, and enrichment of cancer stem cell populations. In this context, dysregulation of microRNAs or miRNAs, critical regulators of gene expression, may influence how tumors respond to radiation. There is increasing evidence that miRNAs may act as sensitizers or enhancers of radioresistance, regulating key processes such as the DNA damage response and the cell death signaling pathway. Furthermore, expression and activity of miRNAs have shown informative value in overcoming radiotherapy and long-term radiotoxicity, revealing their potential as biomarkers. In this review, we will discuss the molecular mechanisms associated with the response to radiotherapy and highlight the central role of miRNAs in regulating the molecular mechanisms responsible for cellular radioresistance. We will also review radio-miRs, radiotherapy-related miRNAs, either as sensitizers or enhancers of radioresistance that hold promise as biomarkers or pharmacological targets to sensitize radioresistant cells.

N-Myc and STAT Interactor is an Endometriosis Suppressor.

In patients with endometriosis, refluxed endometrial fragments evade host immunosurveillance, developing into endometriotic lesions. However, the mechanisms underlying this evasion have not been fully elucidated. N-Myc and STAT Interactor (NMI) have been identified as key players in host immunosurveillance, including interferon (IFN)-induced cell death signaling pathways. NMI levels are markedly reduced in the stromal cells of human endometriotic lesions due to modulation by the Estrogen Receptor beta/Histone Deacetylase 8 axis. Knocking down NMI in immortalized human endometrial stromal cells (IHESCs) led to elevated RNA levels of genes involved in cell-to-cell adhesion and extracellular matrix signaling following IFNA treatment. Furthermore, NMI knockdown inhibited IFN-regulated canonical signaling pathways, such as apoptosis mediated by Interferon Stimulated Gene Factor 3 and necroptosis upon IFNA treatment. In contrast, NMI knockdown with IFNA treatment activated non-canonical IFN-regulated signaling pathways that promote proliferation, including β-Catenin and AKT signaling. Moreover, NMI knockdown in IHESCs stimulated ectopic lesions' growth in mouse endometriosis models. Therefore, NMI is a novel endometriosis suppressor, enhancing apoptosis and inhibiting proliferation and cell adhesion of endometrial cells upon IFN exposure.

Protective Effects of Changbudodam-tang on Cell Death Signals on the Bone Marrow-Derived Human Mesenchymal Stem Cells via Regulation of MKK7/JNK/c-Jun Signaling Pathway.

Polycystic ovary syndrome (PCOS) is one of the most common disorders and it shows up to 20% prevalence in reproductive-aged women populations, but no cures are available to date. We aimed to investigate the protective effects of Changbudodam-tang (CBD) on cell death signaling pathways, inflammation, and oxidative stress observed in Bone-Marrow derived human mesenchymal stem cell (BM-hMSC) by means of PCOS therapeutics in the future. BM-hMSCs were applied with cell deaths and injuries. Apoptosis and pyroptosis signals were quenched with their related signaling pathways using quantitative PCR, Western blot, and fluorescence image analysis. Our data clearly displayed hydrogen peroxide- and nigericin-treated cell death signaling pathways via regulations of mitochondrial integrity and interleukin (IL)-1β at the cellular levels (p < 0.01 or 0.001). We further observed that pre-treatment with CBD showed protective effects against oxidative stress by enhancement of antioxidant components at the cellular level, with respect to both protein and mRNA expression levels (p < 0.05, 0.01 or 0.001). The mechanisms of CBD were examined by Western blot analysis, and it showed anti-cell death, anti-inflammatory, and antioxidant effects via normalizations of the Jun N-terminal kinase/mitogen-activated protein kinase kinase 7/c-Jun signaling pathways. This study confirmed the pharmacological properties of CBD by regulation of cellular oxidation and the inflammation-provoked cell death condition of BM-hMSCs, which is mediated by the MKK7/JNK/c-Jun signaling pathway.

Mitochondria and cell death.

Mitochondria are cellular factories for energy production, calcium homeostasis and iron metabolism, but they also have an unequivocal and central role in intrinsic apoptosis through the release of cytochrome c. While the subsequent activation of proteolytic caspases ensures that cell death proceeds in the absence of collateral inflammation, other phlogistic cell death pathways have been implicated in using, or engaging, mitochondria. Here we discuss the emerging complexities of intrinsic apoptosis controlled by the BCL-2 family of proteins. We highlight the emerging theory that non-lethal mitochondrial apoptotic signalling has diverse biological roles that impact cancer, innate immunity and ageing. Finally, we delineate the role of mitochondria in other forms of cell death, such as pyroptosis, ferroptosis and necroptosis, and discuss mitochondria as central hubs for the intersection and coordination of cell death signalling pathways, underscoring their potential for therapeutic manipulation.

Plumbagin induces G2/M arrest and apoptosis and ferroptosis via ROS/p38 MAPK pathway in human osteosarcoma cells

Plumbagin (PLB), a naphthoquinone compound, has been proven to have anti-cancer properties in different types of cancers. However, the anti-tumor actions as well as molecular processes in osteosarcoma (OS) remain unclear. This study investigates the underlying mechanisms of PLB in human OS cells in vitro. Our study showed that PLB significantly inhibited the proliferation of human OS cells in a time- and dosage-dependent manner. RNA sequencing data revealed that the efficacy of PLB is related to cell cycle, cell death, and p38 MAPK signaling pathway in KHOS-240S cells. Further studies indicated that PLB triggers G2/M phase arrest, mitochondrial-dependent apoptosis, as well as ferroptosis in human OS cells. Mechanistically, PLB treatment increased ROS generation that activates the p38 MAPK signaling pathway. BIRB 796, a p38 MAPK inhibitor, partly reversed PLB-induced phase arrest of G2/M and cell death. Furthermore, N-acetylcysteine (NAC), a ROS scavenger, significantly decreased G2/M phase arrest and cell death and reversed p38 MAPK activation caused by PLB. In conclusion, PLB induces arrest G2/M arrest, apoptosis as well as ferroptosis in human OS cells via ROS generation and p38 MAPK activation. There is potential for PLB to be an effective treatment for OS.

Potency and efficacy of pharmacological PIP4K2 inhibitors in acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL), a complex malignancy, displays varying expression profiles of PIP4K2-related genes in adult patients. While PIP4K2A expression is elevated in ALL bone marrow cells compared to healthy bone marrow cells, PIP4K2B is downregulated, and PIP4K2C remains relatively unchanged. Despite the correlation between increased PIP4K2A expression and increased percentage of peripheral blood blasts, clinical outcomes do not strongly correlate with the expression of these genes. Here we investigated the therapeutic potential of three PIP4K2 inhibitors (THZ-P1-2, a131, and CC260) in ALL cell models. THZ-P1-2 emerges as the most effective inhibitor, inducing cell death and mitochondrial damage while reducing cell viability and metabolism significantly. Comparative analyses highlight the superior efficacy of THZ-P1-2 over a131 and CC260. Notably, THZ-P1-2 uniquely disrupts autophagic flux and inhibits the PI3K/AKT/mTOR pathway, indicating a distinct molecular mechanism. In summary, our findings elucidate the differential expression of PIP4K2-related genes in ALL and underscore the potential role of PIP4K2A in disease pathogenesis. The therapeutic promise of THZ-P1-2 in ALL treatment, along with its distinct effects on cell death mechanisms and signaling pathways, enriches our understanding of PIP4K2's involvement in ALL development and offers targeted therapy prospects.

Rhl deficient-Pseudomonas aeruginosa induces enhanced host necroptosis through upregulation pqs within quorum sensing

Abstract As the leading cause of nosocomial infections, the relationship between Pseudomoans aeruginosa (P. aeruginosa) and host innate immune responses still arouses the interest in innate immunity research. Quorum sensing (QS), a communication system evolved by P. aeruginosa to mediates its density, is involved in various activities during bacterial infection. Recent study has revealed the link between QS and host innate inflammatory responses, extending the role of QS in interaction with host defense. Although previous evidence suggests PCD exists during anti-P. aeruginosa responses, it remains unclear that what role of quorum sensing plays in connection with PCD during infection. In this study, we exhibited that the deficiency of QS subsystems, rhl, dramatically increased the cell death induced by P. aeruginosa in mouse bone marrow-derived macrophages (BMMs). However, the cell death was completely blocked by using the Casp1/11−/−Casp8−/−Ripk3−/− BMMs, indicating the association between QS and host PANoptosis. Intriguingly, we discovered the rhl deficiency upregulated downstream pqs signaling in P. aeruginosa, which significantly promoted the activation of necroptosis through elevated phosphorylation of MLKL. Deletion of pqsA gene in rhl deficiency strains of P. aeruginosa successfully rescued the necroptosis in BMMs. Taken together, our data highlight the crosstalk between QS and host PCD, ae well as a novel rhl – pqs – necroptosis axis in cell death signaling pathway.

The gene encoding flavonol synthase contributes to lesion mimic in wheat

Lesion mimic often exhibits leaf disease-like symptoms even in the absence of pathogen infection, and is characterized by a hypersensitive-response (HR) that closely linked to plant disease resistance. Despite this, only a few lesion mimic genes have been identified in wheat. In this investigation, a lesion mimic wheat mutant named je0297 was discovered, showing no alteration in yield components when compared to the wild type (WT). Segregation ratio analysis of the F2 individuals resulting from the cross between the WT and the mutant revealed that the lesion mimic was governed by a single recessive gene in je0297. Using Bulked segregant analysis (BSA) and exome capture sequencing, we mapped the lesion mimic gene designated as lm6 to chromosome 6BL. Further gene fine mapping using 3315 F2 individuals delimited the lm6 within a 1.18 Mb region. Within this region, we identified 16 high-confidence genes, with only two displaying mutations in je0297. Notably, one of the two genes, responsible for encoding flavonol synthase, exhibited altered expression levels. Subsequent phenotype analysis of TILLING mutants confirmed that the gene encoding flavonol synthase was indeed the causal gene for lm6. Transcriptome sequencing analysis revealed that the DEGs between the WT and mutant were significantly enriched in KEGG pathways related to flavonoid biosynthesis, including flavone and flavonol biosynthesis, isoflavonoid biosynthesis, and flavonoid biosynthesis pathways. Furthermore, more than 30 pathogen infection-related (PR) genes exhibited upregulation in the mutant. Corresponding to this expression pattern, the flavonoid content in je0297 showed a significant decrease in the 4th leaf, accompanied by a notable accumulation of reactive oxygen, which likely contributed to the development of lesion mimic in the mutant. This investigation enhances our comprehension of cell death signaling pathways and provides a valuable gene resource for the breeding of disease-resistant wheat.

Nuclear lamina component KAKU4 regulates chromatin states and transcriptional regulation in the Arabidopsis genome

BackgroundThe nuclear lamina links the nuclear membrane to chromosomes and plays a crucial role in regulating chromatin states and gene expression. However, current knowledge of nuclear lamina in plants is limited compared to animals and humans.ResultsThis study mainly focused on elucidating the mechanism through which the putative nuclear lamina component protein KAKU4 regulates chromatin states and gene expression in Arabidopsis leaves. Thus, we constructed a network using the association proteins of lamin-like proteins, revealing that KAKU4 is strongly associated with chromatin or epigenetic modifiers. Then, we conducted ChIP-seq technology to generate global epigenomic profiles of H3K4me3, H3K27me3, and H3K9me2 in Arabidopsis leaves for mutant (kaku4-2) and wild-type (WT) plants alongside RNA-seq method to generate gene expression profiles. The comprehensive chromatin state-based analyses indicate that the knockdown of KAKU4 has the strongest effect on H3K27me3, followed by H3K9me2, and the least impact on H3K4me3, leading to significant changes in chromatin states in the Arabidopsis genome. We discovered that the knockdown of the KAKU4 gene caused a transition between two types of repressive epigenetics marks, H3K9me2 and H3K27me3, in some specific PLAD regions. The combination analyses of epigenomic and transcriptomic data between the kaku4-2 mutant and WT suggested that KAKU4 may regulate key biological processes, such as programmed cell death and hormone signaling pathways, by affecting H3K27me3 modification in Arabidopsis leaves.ConclusionsIn summary, our results indicated that KAKU4 is directly and/or indirectly associated with chromatin/epigenetic modifiers and demonstrated the essential roles of KAKU4 in regulating chromatin states, transcriptional regulation, and diverse biological processes in Arabidopsis.

Targeting novel regulated cell death: Ferroptosis, pyroptosis and necroptosis in anti-PD-1/PD-L1 cancer immunotherapy.

Chemotherapy, radiotherapy, and immunotherapy represent key tumour treatment strategies. Notably, immune checkpoint inhibitors (ICIs), particularly anti-programmed cell death 1 (PD1) and anti-programmed cell death ligand 1 (PD-L1), have shown clinical efficacy in clinical tumour immunotherapy. However, the limited effectiveness of ICIs is evident due to many cancers exhibiting poor responses to this treatment. An emerging avenue involves triggering non-apoptotic regulated cell death (RCD), a significant mechanism driving cancer cell death in diverse cancer treatments. Recent research demonstrates that combining RCD inducers with ICIs significantly enhances their antitumor efficacy across various cancer types. The use of anti-PD-1/PD-L1 immunotherapy activates CD8+ T cells, prompting the initiation of novel RCD forms, such as ferroptosis, pyroptosis, and necroptosis. However, the functions and mechanisms of non-apoptotic RCD in anti-PD1/PD-L1 therapy remain insufficiently explored. This review summarises the emerging roles of ferroptosis, pyroptosis, and necroptosis in anti-PD1/PD-L1 immunotherapy. It emphasises the synergy between nanomaterials and PD-1/PD-L1 inhibitors to induce non-apoptotic RCD in different cancer types. Furthermore, targeting cell death signalling pathways in combination with anti-PD1/PD-L1 therapies holds promise as a prospective immunotherapy strategy for tumour treatment.

Impact of Complex Apoptotic Signaling Pathways on Cancer Cell Sensitivity to Therapy.

Anticancer drugs induce apoptotic and non-apoptotic cell death in various cancer types. The signaling pathways for anticancer drug-induced apoptotic cell death have been shown to differ between drug-sensitive and drug-resistant cells. In atypical multidrug-resistant leukemia cells, the c-Jun/activator protein 1 (AP-1)/p53 signaling pathway leading to apoptotic death is altered. Cancer cells treated with anticancer drugs undergo c-Jun/AP-1-mediated apoptotic death and are involved in c-Jun N-terminal kinase activation and growth arrest- and DNA damage-inducible gene 153 (Gadd153)/CCAAT/enhancer-binding protein homologous protein pathway induction, regardless of the p53 genotype. Gadd153 induction is associated with mitochondrial membrane permeabilization after anticancer drug treatment and involves a coupled endoplasmic reticulum stress response. The induction of apoptosis by anticancer drugs is mediated by the intrinsic pathway (cytochrome c, Cyt c) and subsequent activation of the caspase cascade via proapoptotic genes (e.g., Bax and Bcl-xS) and their interactions. Anticancer drug-induced apoptosis involves caspase-dependent and caspase-independent pathways and occurs via intrinsic and extrinsic pathways. The targeting of antiapoptotic genes such as Bcl-2 enhances anticancer drug efficacy. The modulation of apoptotic signaling by Bcl-xS transduction increases the sensitivity of multidrug resistance-related protein-overexpressing epidermoid carcinoma cells to anticancer drugs. The significance of autophagy in cancer therapy remains to be elucidated. In this review, we summarize current knowledge of cancer cell death-related signaling pathways and their alterations during anticancer drug treatment and discuss potential strategies to enhance treatment efficacy.

Effects of Halloysite Nanotubes and Multi-walled Carbon Nanotubes on Kruppel-like Factor 15-Mediated Downstream Events in Mouse Hearts After Intravenous Injection.

Halloysite nanotubes (HNTs) are nanomaterials (NMs) derived from natural clays and have been considered as biocompatible NMs for biomedical uses. However, the cardiovascular toxicity of HNTs has not been thoroughly investigated. In this study, we compared the cardiotoxicity of HNTs and multi-walled carbon nanotubes (MWCNTs), focusing on the changes in Kruppel-like factor (KLF)-mediated signaling pathways. Mice were intravenously injected with 50µg NMs, once a day, for 5 days, and then mouse hearts were removed for experiments. While HNTs or MWCNTs did not induce obvious pathological changes, RNA-sequencing data suggested the alterations of KLF gene expression. We further confirmed an increase of Klf15 positive cells, accompanied by changes in Klf15-related gene ontology (GO) terms. We noticed that most of the changed GO terms are related with the regulation of gene expression, and we confirmed that the NMs increased myoneurin (Mynn) but decreased snail family transcriptional repressor 1 (Snai1), two transcription factors (TFs) related with Klf15. Besides, the changed GO terms also include metal ion binding and positive regulation of glucose import, and we verified an increase of phosphoenolpyruvate carboxykinase 1 (Pck1) and insulin receptor (Insr). However, HNTs and MWCNTs only showed minimal impact on cell death signaling pathways, and no increase in apoptotic sites was observed after NM treatment. We concluded that intravenous administration of HNTs and MWCNTs activated a protective TF, namely Klf15 in mouse aortas, to alter gene expression and signaling pathways related with metal ion binding and glucose import.

A26 BIOLOGICAL CHARACTERIZATION OF THE INTERACTION BETWEEN SIRT1 AND HNF4Α2 IN INTESTINAL EPITHELIAL CELLS

Abstract Background Colorectal cancer (CRC) is the third most common cancer in Canada. HNF4A locus is amplified in CRC, while additional reports suggest that hepatocyte nuclear factor 4 alpha (HNF4α) is associated with increased proliferation and disease development. This dual role as a tumor suppressor or oncoprotein might be due to the production of 12 spliced isoforms with structural differences and variable tissue expression. It suggests distinct functions for each isoform according to their specific interaction complexes. However, little is known about the nature of these protein complexes and their biological functions during intestinal physiopathology. Recently, using a BioID2 and mass spectrometry approach, our laboratory showed a possible interaction between HNF4α2 and sirtuin 1 (SIRT1) in intestinal epithelial cells from a colon carcinoma (HCT116). HNF4α2 is one of the most potent isoforms in the control of transcriptional activity of multiple intestinal epithelial genes related to regulating cell death, angiogenesis, apoptosis, response to injury, and response to drugs, among others. Aims To characterize the possible interaction between HNF4α2 and SIRT1 in intestinal epithelial cells at the biological level. Methods We performed SIRT1 knockdown using shRNAs in HCT116 cells expressing the HNF4α2 isoform in an inducible manner by doxycycline (DOX). Subsequently, we conducted a proximity ligation assay to validate the interaction between HNF4α2 and SIRT1 in HCT116 cells. Additionally, protein purification was performed to validate their physical interaction through EMSA. A transcriptome analysis was carried out using RNA-seq to define the biological processes involved in the interaction between the two proteins. Results We observed an interaction signal between HNF4α2 and SIRT1 in HCT116 cells, similar to that observed for IRFBB2, whose interaction with HNF4α2 has been previously validated. This signal decreased when cells were treated with shRNA for SIRT1 and was not observed in cells not induced by DOX. EMSA analyses revealed a direct interaction signal between HNF4α2 and SIRT1. RNA-seq analyses showed a loss in the regulation of over 95% of genes targeted by HNF4α2 when cells were treated with shRNA for SIRT1. An enrichment analysis for GO annotations using ShyniGO v. 0.77 software showed an enrichment of genes for biological processes such as apoptosis, inflammation, cell migration, cell invasion, cell death, and various cancer-related signaling pathways. Conclusions SIRT1 displays physical interaction with HNF4α2 and significantly affects the transcriptional regulation of this isoform in the context of epithelial cells. Their interaction is involved in processes related to intestinal inflammation and cancer progression. Pharmacological targeting of SIRT1 could represent a viable strategy in treating CRC and other intestinal diseases Funding Agencies CIHRNSERC

Hesperetin Inhibits Bladder Cancer Cell Proliferation and Promotes Apoptosis and Cycle Arrest by PI3K/AKT/FoxO3a and ER Stress-mitochondria Pathways.

Hesperetin (HSE) is a natural flavonoid derived from the hydrolysis of Hesperidin, which is mainly found in traditional natural Chinese herbs, such as Chenpi and Hovenia caryophyllus. HSE displays anti-inflammatory and antioxidant activities. However, its potential mechanism of action on bladder cancer (BLCA) remains unknown. The aim of this study was to investigate the potential mechanism of action of HSE on BLCA cells. Network pharmacology analysis was used to construct a composite target network, combined with Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to identify HSE-induced cell death patterns and signaling pathway alterations. Cytotoxicity evaluation was determined by CCK-8 assay. A clone formation assay was performed to assess cell proliferative capacity. Scratch and Transwell assays were performed to evaluate cell migration and invasion ability. Hoechst 33342 staining was visualized to observe morphological features of apoptosis. Apoptosis, cycle distribution, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP) changes were examined by flow cytometry. Western blot analysis was performed to analyze the expression of key proteins associated with cell proliferation, apoptosis, cycle block, PI3K/AKT/FoxO3a and endoplasmic reticulum (ER) stress-mitochondrial pathways. Network pharmacology analysis was performed to identify 155 potential candidate targets of HSE-BLCA, and further topological analysis was performed to obtain 34 hub-gene. Enrichment analysis yielded patterns of death and key pathways, revealing that the anti-BLCA effect of HSE may be related to the positive regulation of PI3K/AKT/FoxO3a and ER stress-mitochondrial pathways. in vitro results showed that HSE blocked cell proliferation, migration, and invasion in a concentration-dependent manner and triggered apoptosis, G0/G1 phase blockade, ROS production, and MMP depolarization. In addition, Western blot results showed that HSE downregulated phosphorylated (p)-3-phosphoinositide-dependent kinase-1 (PI3K), phosphorylated (p)-AKT serine/threonine kinase 1 (AKT), phosphorylated (p)-Forkhead box O 3a (FoxO3a), anti-apoptotic proteins, proliferation-associated proteins, and cell cycle promoters, whereas the levels of proteins related to the expression of cell cycle regulators, pro-apoptotic proteins, and ER stress-mitochondrial pathway were up-regulated in BLCA cells by the intervention of HSE. PI3K agonist (YS-49) and ER stress inhibitor (4-PBA) partially or completely reversed HSE-mediated proliferation, apoptosis, and cycle blockade in BLCA cells. The anticancer effects of HSE in BLCA may be attributed to its coordination of actions, inhibiting cell proliferation, migration, and invasion, inducing apoptosis, G0/G1 phase arrest, generating reactive oxygen species, causing MMP loss, and engaging the caspase protein family. These actions are likely mediated through the PI3K/AKT/FoxO3a and ER stress-mitochondrial pathways. Thus, our findings suggest that HSE is a promising novel therapeutic candidate for the prevention and treatment of BLCA.