Molecular Mechanisms Of Cell Death Research Articles

The protumorigenic enzyme GPAT2 inhibits arachidonic acid-triggered apoptosis in breast cancer

BackgroundCancer is a significant health challenge and the leading cause of mortality globally. Tumor cells use multiple mechanisms to acquire their distinctive capacity for uncontrolled proliferation, one of which is the evasion of apoptosis. It has been shown that in breast, colon, and liver cancer, evasion of apoptosis is associated with the overexpression of enzymes that metabolize arachidonic acid (AA) because free AA is a strong inducer of apoptosis. Glycerol−3-phosphate acyltransferase 2 (GPAT2) is a key enzyme in AA metabolism and is highly expressed in breast and colon cancer, where it promotes the development of essential tumor features.MethodsIn this work, a model of GPAT2 silencing in the human breast cancer-derived cell line MDA-MB-231 was used, and the cells were exposed to exogenous AA. The role of GPAT2 in AA-induced cell death was studied using MTT and TUNEL assays and measurements of caspase activity. The underlying molecular mechanism of cell death was assessed by qRT‒PCR.ResultsThe results showed that AA reduced cell viability only in GPAT2-silenced cells, and that this cell death was a consequence of an apoptotic process involving BNIP3 overexpression. Additionally, it was demonstrated that GPAT2 silencing triggered a compensatory mechanism by overexpressing other genes involved in AA utilization for eicosanoid biosynthesis.ConclusionsWe concluded that GPAT2 expression is necessary to prevent AA-induced apoptotic cell death in MDA-MB-231 cells and that the overexpression of other AA-metabolizing genes is not sufficient to compensate for the lack of GPAT2 and prevent apoptosis.

Role of Exogenous Pyruvate in Maintaining Adenosine Triphosphate Production under High-Glucose Conditions through PARP-Dependent Glycolysis and PARP-Independent Tricarboxylic Acid Cycle.

Pyruvate serves as a key metabolite in energy production and as an anti-oxidant. In our previous study, exogenous pyruvate starvation under high-glucose conditions induced IMS32 Schwann cell death because of the reduced glycolysis-tricarboxylic acid (TCA) cycle flux and adenosine triphosphate (ATP) production. Thus, this study focused on poly-(ADP-ribose) polymerase (PARP) to investigate the detailed molecular mechanism of cell death. Rucaparib, a PARP inhibitor, protected Schwann cells against cell death and decreased glycolysis but not against an impaired TCA cycle under high-glucose conditions in the absence of pyruvate. Under such conditions, reduced pyruvate dehydrogenase (PDH) activity and glycolytic and mitochondrial ATP production were observed but not oxidative phosphorylation or the electric transfer chain. In addition, rucaparib supplementation restored glycolytic ATP production but not PDH activity and mitochondrial ATP production. No differences in the increased activity of caspase 3/7 and the localization of apoptosis-inducing factor were found among the experimental conditions. These results indicate that Schwann cells undergo necrosis rather than apoptosis or parthanatos under the aforementioned conditions. Exogenous pyruvate plays a pivotal role in maintaining the flux in PARP-dependent glycolysis and the PARP-independent TCA cycle in Schwann cells under high-glucose conditions.

Transferrin Receptor-Mediated Cellular Uptake of Fluorinated Chlorido[N,N'-bis(salicylidene)-1,2-phenylenediamine]iron(III) Complexes.

Fluorinated chlorido[salophene]iron(III) complexes (salophene = N,N'-bis(salicylidene)-1,2-phenylenediamine) are promising anticancer agents. Apoptosis and necrosis induction have already been described as part of their mode of action. However, the involvement of ferroptosis in cell death induction, as confirmed for other chlorido[salophene]iron(III) complexes, has not yet been investigated. Furthermore, the mechanism of cellular uptake of these compounds is unknown. Therefore, the biological activity of the fluorescent chlorido[salophene]iron(III) complexes with a fluorine substituent at positions 3, 4, 5, or 6 at the salicylidene moieties (C1-C4) was evaluated in malignant and nonmalignant cell lines with focus on the involvement of the transferrin receptor-1 (TfR-1) in cellular uptake, the influence of the complexes on mitochondrial function, and the analysis of the molecular mechanism of cell death. All complexes significantly decreased the metabolic activity in the tested ovarian cancer (A2780, A2780cis), breast cancer (MDA-MB 231), and leukemia (HL-60) cell lines, while the nonmalignant human stroma cell line HS-5 at a concentration of 0.5 μM, which represents the IC50 of the complexes in most of the used tumorigenic cell lines, was not affected. The mitochondrial function was impaired, as evidenced by a reduced mitochondrial membrane potential ΔΨm and decreased mitochondrial activity. Besides apoptosis and necroptosis, ferroptosis was identified as part of the mode of action. It was further demonstrated for the first time that fluorinated chlorido[salophene]iron(III) complexes downregulate TfR-1 expression, comparable to ferristatin II, an iron transport inhibitor that acts via TfR-1 degradation. FerroOrange staining further indicated that the complexes strongly increased the intracellular iron(II) level as a driving force to induce ferroptosis. In conclusion, these fluorinated chlorido[salophene]iron(III) complexes are potent, tumor cell-specific chemotherapeutic agents, with the potential to treat various types of cancers.

Preliminary study on cytotoxicity of selegiline on different cancer cell lines: exploration of the induction of ROS-independent apoptosis in breast cancer cells.

The concept of drug repurposing is now widely utilized by biomedical scientists for drug discovery. An example of this is the use of selegiline (SEL), a monoamine oxidase inhibitor that was initially used for the management of depression but is now being considered for another purpose. This study compares the cytotoxic effects of SEL on different cancer cells. Further, the study explores the molecular mechanism of cell death, validating the possibility of its repurposing for cancer. Preliminary analysis of network pharmacological data was conducted in silico, followed by in vitro cytotoxicity tests on PC12, G361, MDA-MB231, MCF7, THP-1, and Hela cells under normoxic and hypoxic conditions, using the MTT assay. The mechanism of cell death was then confirmed by performing DAPI and FITC-conjugated Annexin V and Propidium Iodide (PI) staining assays. Additionally, ROS levels and PKC phosphorylation were also evaluated. In silico analysis has revealed that SEL is associated with ten genes linked to different cancer types. Specifically, SEL was most cytotoxic to neuronal pheochromocytoma, triple-negative human epithelial breast cancer cells, and ER+ and PR+ breast cancer cells. Furthermore, it was observed that this cell death occurred through ROS-independent apoptosis pathways. In addition, SEL was found to inhibit the phosphorylation of PKC, which may contribute to cell death. SEL induces apoptosis in breast cancer cells independently of reactive oxygen species and inhibits the phosphorylation of protein kinase C, which merits further exploration.

Antheraea proylei J. Sericin Induces Apoptosis in a Caspase-dependent Manner in A549 and HeLa Cells.

In spite of much progress in cancer, the global cancer burden is still significant and increasing. Sericin, an adhesive protein of silk cocoons, has been shown to be a potential protein in various biomedical applications, including cancer therapeutics. The present study evaluates the anticancer property of sericin from cocoons of Antheraea proylei J (SAP) against human lung cancer (A549) and cervical cancer (HeLa) cell lines. This is the first report of anti-cancer activity of the non-mulberry silkworm A. proylei J. Establish the antiproliferative potential of SAP. 2. Identify the molecular mechanism of cell death induced by SAP on two different cell lines. To investigate the anticancer activity of sericin preparation from cocoons of A. proylei. SAP was prepared from cocoons of A. proylei J. by the process of the degumming method. Cytotoxic activity was assessed by MTT assay, and genotoxicity was assessed by comet assay. Cleavage of caspase and PARP proteins and phosphorylation of MAPK pathway members were analysed by Western blotting. Cell cycle analysis was done by flow cytometer. SAP causes cytotoxicity to A549 and HeLa cell lines with the IC50 values 3.8 and 3.9 μg/μl respectively. SAP induces apoptosis in a dose-dependent manner through caspase-3 and p38, MAPK pathways in A549 and HeLa cells. Moreover, in A549 and HeLa cells, SAP induces cell cycle arrest at the S phase in a dose-dependent manner. The difference in the molecular mechanisms of apoptosis induced by SAP in A549 and HeLa cell lines may be due to the difference in the genotypes of the cancer cell lines. However, further investigation is warranted. The overall results of the present study envisage the possibility of using SAP as an anti-tumorigenic agent.

Molecular mechanisms of cell death by parthanatos: More questions than answers.

Regulated cell death by a non-apoptotic pathway known as parthanatos is increasingly recognised as a central player in pathological processes, including ischaemic tissue damage and neurodegenerative diseases. Parthanatos is activated under conditions that induce high levels of DNA damage, leading to hyperactivation of the DNA damage sensor PARP1. While this strict dependence on PARP1 activation is a defining feature of parthanatos that distinguishes it from other forms of cell death, the molecular events downstream of PARP1 activation remain poorly understood. In this mini-review, we highlight a number of important questions that remain to be answered about this enigmatic form of cell death.

Cell death of Acanthamoeba castellanii following exposure to antimicrobial agents commonly included in contact lens disinfecting solutions.

Several antimicrobial agents are commonly included in contact lens disinfectant solutions including chlorhexidine diacetate (CHX), polyhexamethylene biguanide (PHMB) or myristamidopropyl dimethylamine (MAPD); however, their mode of action, i.e. necrosis versus apoptosis is incompletely understood. Here, we determined whether a mechanism of cell death resembling that of apoptosis was present in Acanthamoeba castellanii of the T4 genotype (NEFF) following exposure to the aforementioned antimicrobials using the anticoagulant annexin V that undergoes rapid high affinity binding to phosphatidylserine in the presence of calcium, making it a sensitive probe for phosphatidylserine exposure. The results revealed that under the conditions employed in this study, an apoptotic pathway of cell death in this organism at the tested conditions does not occur. Our findings suggest that necrosis is the likely mode of action; however, future mechanistic studies should be accomplished in additional experimental conditions to further comprehend the molecular mechanisms of cell death in Acanthamoeba.

Protein stability governed by α1-2 helices in Pvr4 is essential for localization and cell death.

Plant nucleotide-binding domain leucine-rich-repeat receptor (NLR) confers disease resistance to various pathogens by recognizing effectors derived from the pathogen. Previous studies have shown that overexpression of the CC domain in several NLRs triggers cell death, implying that the CC domain plays an important role as a signaling module. However, how CC domain transduces immune signals remains largely unknown. A Potyvirus-resistant NLR protein, Pvr4, possesses a CC domain (CCPvr4 ) that induces cell death upon transient overexpression in Nicotiana benthamiana. In this study, loss-of-function mutants were generated by error-prone PCR-based random mutagenesis to understand the molecular mechanisms underlying CCPvr4 -mediated cell death. Cell biology and biochemical studies revealed that M16 and Q52 in the α1 and α2 helices, respectively, are crucial for protein stability, and mutation of these residues disrupts localization to the plasma membrane and oligomerization activity. The increase of the protein stability of these mutants by tagging a green fluorescent protein (GFP) variant led to restoration of cell death-inducing activity and plasma membrane localization. Another mutant, I7E in the very N-terminal region, lost cell death-inducing activity by weakening the interaction with plasma membrane H+ -ATPase compared to CCPvr4 , although the protein remained in the plasma membrane. Moreover, most of the mutated residues are on the outer surface of the funnel shape in the predicted pentameric CCPvr4 , implying that the disordered N-terminal region plays a crucial role in association with PMA as well as targeting to the plasma membrane. This work could provide insights into the molecular mechanisms of cell death induced by NLR immune receptors.

Identification of cell death-related biomarkers and immune infiltration in ischemic stroke between male and female patients.

Stroke is the second leading cause of death and the third leading cause of disability worldwide, with ischemic stroke (IS) being the most prevalent. A substantial number of irreversible brain cell death occur in the short term, leading to impairment or death in IS. Limiting the loss of brain cells is the primary therapy target and a significant clinical issue for IS therapy. Our study aims to establish the gender specificity pattern from immune cell infiltration and four kinds of cell-death perspectives to improve IS diagnosis and therapy. Combining and standardizing two IS datasets (GSE16561 and GSE22255) from the GEO database, we used the CIBERSORT algorithm to investigate and compare the immune cell infiltration in different groups and genders. Then, ferroptosis-related differently expressed genes (FRDEGs), pyroptosis-related DEGs (PRDEGs), anoikis-related DEGs (ARDEGs), and cuproptosis-related DEGs (CRDEGs) between the IS patient group and the healthy control group were identified in men and women, respectively. Machine learning (ML) was finally used to generate the disease prediction model for cell death-related DEGs (CDRDEGs) and to screen biomarkers related to cell death involved in IS. Significant changes were observed in 4 types of immune cells in male IS patients and 10 types in female IS patients compared with healthy controls. In total, 10 FRDEGs, 11 PRDEGs, 3 ARDEGs, and 1 CRDEG were present in male IS patients, while 6 FRDEGs, 16 PRDEGs, 4 ARDEGs, and 1 CRDEG existed in female IS patients. ML techniques indicated that the best diagnostic model for both male and female patients was the support vector machine (SVM) for CDRDEG genes. SVM's feature importance analysis demonstrated that SLC2A3, MMP9, C5AR1, ACSL1, and NLRP3 were the top five feature-important CDRDEGs in male IS patients. Meanwhile, the PDK4, SCL40A1, FAR1, CD163, and CD96 displayed their overwhelming influence on female IS patients. These findings contribute to a better knowledge of immune cell infiltration and their corresponding molecular mechanisms of cell death and offer distinct clinically relevant biological targets for IS patients of different genders.

Abstract 2674: Synergistic combinations of lurbinectedin with irinotecan and ONC212 in pancreatic cancer

Abstract Pancreatic cancer is a devastating disease with a five-year survival rate below 10% according to the American Cancer Society. Novel chemotherapeutics and targeted therapies in pancreatic cancer have shown limited success, illustrating the urgent need for new treatments. Lurbinectedin is a chemotherapeutic synthetic tetrahydroisoquinoline alkaloid that inhibits active transcription by binding to guanine rich sequences in the minor groove of DNA. Lurbinectedin has been shown to reduce oncogenic transcription by stalling and degrading RNA Polymerase II while also inducing single- and double-stranded breaks to DNA, causing subsequent apoptosis. Lurbinectedin received accelerated FDA approval in 2020 for metastatic small cell lung cancer on or after platinum-based chemotherapy and is currently undergoing clinical trials in a variety of tumor types. We recently described a synergistic interaction between lurbinectedin and ONC201/TIC10, a compound that induces the TRAIL pathway, in killing SCLC cell lines associated with activation of p-Chk1 and the integrated stress response. We now demonstrate lurbinectedin’s efficient killing of pancreatic tumor cells as a single agent in PANC-1, BxPC-3, and HPAF-II cell lines, with IC-50s corresponding to sub-nanomolar concentrations. We also demonstrate that a combination of lurbinectedin and irinotecan, a topoisomerase I inhibitor with FDA approval for advanced pancreatic cancer, results in synergistic killing of pancreatic tumor cells in vitro. We further demonstrate a combination of lurbinectedin and ONC212, an imipridone, the same class of compounds as ONC201, also results in synergistic killing of pancreatic tumor cells. Cell viability was measured using the CellTiterGlo assay at varying drug concentrations. We hypothesize a combination therapy of lurbinectedin and irinotecan or ONC212 can enhance immune cell killing of pancreatic tumor cells. This is being explored by co-culturing CD8+ T lymphoblast cells with pancreatic tumor cells treated with lurbinectedin, irinotecan, ONC212, and combination, along with molecular mechanisms of cell death and effects on cytokines in the tumor microenvironment. Our results are developing insights regarding molecular mechanisms underlying therapeutic efficacy of a novel combination drug treatment for pancreatic cancer. Citation Format: Tej Tummala, Arielle De La Cruz, Ash Uruchurtu, Nicholas R. Liguori, Abbas E. Abbas, Leiqing Zhang, Christopher G. Azzoli, Lanlan Zhou, Wafik S. El-Deiry. Synergistic combinations of lurbinectedin with irinotecan and ONC212 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2674.

МОЛЕКУЛЯРНЫЕ И МОРФОЛОГИЧЕСКИЕ МАРКЕРЫ ГИБЕЛИ НЕЙРОНОВ ПРИ ОСТРЫХ НАРУШЕНИЯХ МОЗГОВОГО КРОВООБРАЩЕНИЯ

Acute cerebral circulatory disorders are characterised by various changes in brain cells, often leading to mass death. This review presents a list of markers associated with different types of cell death occurring in acute cerebral circulation disorders and identifies the importance of these markers in the diagnosis of haemorrhagic and ischaemic stroke.Acute cerebral circulation disorder is one of the most debated issues in modern resuscitation and medicine, asit is asevere condition leading to stroke and subsequent patient death, if not treated promptly. However, rapid treatment and diagnosis of stroke is difficult due to the lack of study of morphological signs and biomarkers to reliably determine the nature of the injury. Anin-depth analysis and systematization of the available information on this topic is needed.Purpose of the review:to reveal the correspondence between the molecular mechanisms of cell death in acute disorders ofcerebral circulation and their morphological manifestations.Material and Methods.A total of 50 most relevant sources of information were selected. The sources were selected from the databases of medical and biological publications PubMed, Scopus, Web of Science, RSCI, and fundamental works of scientificliterature on the considered topic were involved.Results.The main mechanisms of cell death in stroke were identified and analyzed, the morphological and histological features ofthe observed processes and their structural manifestations were reviewed. Besides, the most frequently detected molecular markers specific for each type of cell death were listed.Conclusion.The study of molecular pathways andcellular reorganization processes characteristic of different types of cell death as well as their corresponding biological markers is of important diagnostic value in the detection of cerebral circulatory disorders. Determination of morphological and molecular markers typical for this condition will allow a prompt diagnosis ofstroke and minimization of its negative consequences.

Molecular mechanisms of cell death in bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) in neonates is the most common pulmonary disease that causes neonatal mortality, has complex pathogenesis, and lacks effective treatment. It is associated with chronic obstructive pulmonary disease, pulmonary hypertension, and right ventricular hypertrophy. The occurrence and development of BPD involve various factors, of which premature birth is the most crucial reason for BPD. Under the premise of abnormal lung structure and functional product, newborns are susceptible to damage to oxides, free radicals, hypoxia, infections and so on. The most influential is oxidative stress, which induces cell death in different ways when the oxidative stress balance in the body is disrupted. Increasing evidence has shown that programmed cell death (PCD), including apoptosis, necrosis, autophagy, and ferroptosis, plays a significant role in the molecular and biological mechanisms of BPD and the further development of the disease. Understanding the mode of PCD and its signaling pathways can provide new therapeutic approaches and targets for the clinical treatment of BPD. This review elucidates the mechanism of BPD, focusing on the multiple types of PCD in BPD and their molecular mechanisms, which are mainly based on experimental results obtained in rodents.

Iron dyshomeostasis and ferroptosis in Alzheimer's disease: Molecular mechanisms of cell death and novel therapeutic drugs and targets for AD.

Alzheimer’s disease (AD) is a degenerative disease of the central nervous system that is the most common type of senile dementia. Ferroptosis is a new type of iron-dependent programmed cell death identified in recent years that is different from other cell death forms. Ferroptosis is induced by excessive accumulation of lipid peroxides and reactive oxygen species (ROS) in cells. In recent years, it has been found that ferroptosis plays an important role in the pathological process of AD. Iron dyshomeostasis contribute to senile plaques (SP) deposition and neurofibrillary tangles (NFTs). Iron metabolism imbalance in brain and the dysfunction of endogenous antioxidant systems including system Xc- and glutathione peroxidase (GPX) are closely related to the etiopathogenesis of AD. Dysfunction of nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy induced ferroptosis can accelerates the pathological process of AD. In addition, NRF2, through regulating the expression of a considerable number of genes related to ferroptosis, including genes related to iron and glutathione metabolism, plays an important role in the development of AD. Here, we review the potential interaction between AD and ferroptosis and the major pathways regulating ferroptosis in AD. We also review the active natural and synthetic compounds such as iron chelators, lipid peroxidation inhibitors and antioxidants available to treat AD by alleviating iron dyshomeostasis and preventing ferroptosis in mice and cell models to provide valuable information for the future treatment and prevention of AD.

Neuropeptide-Functionalized Gold Nanorod Enhanced Cellular Uptake and Improved In Vitro Photothermal Killing in LRP1-Positive Glioma Cells.

The therapeutic modalities for glioblastoma multiforme fail badly due to the limitations of poor penetration through the blood–brain barrier and the lack of tumor targeting. In this study, we synthesized a neuropeptide (ANGIOPEP-2)-functionalized gold nanorod (GNR-ANGI-2) and systemically evaluated the cellular uptake and photothermal effects enhanced by the neuropeptide functionalization of the gold nanorod under laser or sham exposure. The expression of LRP1, the specific ligand for ANGIOPEP-2, was the highest in C6 cells among five studied glioma cell lines. The cellular internalization studies showed higher uptake of gold nanorods functionalized with ANGIOPEP-2 than of those functionalized with scrambled ANGIOPEP-2. The in vitro photothermal studies of C6 cells treated with GNR-ANGI-2 and laser showed a higher rate of apoptosis at early and late stages than cells treated with GNR-ANGI-2 without laser. Correspondingly, in vitro ROS evaluation showed a higher intensity of ROS production in cells treated with GNR-ANGI-2 under laser irradiation. The Western blotting results indicated that GNR-ANGI-2 with laser exposure activated the caspase pathway of apoptosis, and GNR-ANGI-2 with sham exposure induced autophagy in C6 cells. The current study provides in-depth knowledge on the effective time point for maximum cellular uptake of GNR-ANGI-2 to achieve a better anti-glioma effect. Moreover, by exploring the molecular mechanism of cell death with GNR-ANGI-2-mediated photothermal therapy, we could modify the nanoshuttle with multimodal targets to achieve more efficient anti-glioma therapy in the future.

Mechanisms of cancer cell killing by metformin: a review on different cell death pathways.

Cancer resistance to anti-tumour agents has been one of the serious challenges in different types of cancer treatment. Usually, an increase in the cell death markers can predict a higher rate of survival among patients diagnosed with cancer. By increasing the regulation of survival genes, cancer cells can display a higher resistance to therapy through the suppression of anti-tumour immunity and inhibition of cell death signalling pathways. Administration of certain adjuvants may be useful in order to increase the therapeutic efficiency of anti-cancer therapy through the stimulation of different cell death pathways. Several studies have demonstrated that metformin, an antidiabetic drug with anti-cancer properties, amplifies cell death mechanisms, especially apoptosis in a broad-spectrum of cancer cells. Stimulation of the immune system by metformin has been shown to play a key role in the induction of cell death. It seems that the induction or suppression of different cell death mechanisms has a pivotal role in either sensitization or resistance of cancer cells to therapy. This review explains the cellular and molecular mechanisms of cell death following anticancer therapy. Then, we discuss the modulatory roles of metformin on different cancer cell death pathways including apoptosis, mitotic catastrophe, senescence, autophagy, ferroptosis and pyroptosis.

Sperm-Associated Antigen 9 Promotes Influenza A Virus-Induced Cell Death via the c-Jun N-Terminal Kinase Signaling Pathway.

ABSTRACTUpon influenza A virus (IAV) infection, the IAV progeny ribonucleoprotein complex, with a defective viral genome, is sensed by DNA-dependent activator of interferon-regulatory factor (DAI). DAI initiates the recruitment of an array of proteins to form a multiprotein platform (PANoptosome), which triggers apoptosis, necroptosis, and pyroptosis during IAV infection. However, the mechanisms mediating the assembly of the PANoptosome are unclear. Here, we identified a scaffold protein, sperm-associated antigen 9 (SPAG9), which could interact with DAI to promote cell death during IAV infection. We further demonstrated that the cell death enhanced by SPAG9 was achieved through the DAI/SPAG9/c-Jun N-terminal kinase (JNK) axis, which could promote IAV-induced DAI-mediated cell death, including apoptosis, necroptosis, and pyroptosis. Our data further showed that the DAI/SPAG9/JNK signaling pathway enhanced the interactions among receptor-interacting serine/threonine kinase 1 (RIPK1), RIPK3, and DAI, thereby promoting IAV-induced PANoptosome formation. Overall, our study for the first time revealed a feed-forward circuit signaling pathway that enhanced IAV-induced DAI-mediated cell death, provided insights into the molecular mechanisms of cell death, and established therapeutic targets to address infectious and inflammatory diseases.

A Maize Necrotic Leaf Mutant Caused by Defect of Coproporphyrinogen III Oxidase in the Porphyrin Pathway.

Lesion mimic mutants provide ideal genetic materials for elucidating the molecular mechanism of cell death and disease resistance. The maize necrotic leaf mutant (nec-t) is a recessive mutant with necrotic spots and yellow-green leaves. In this study, we found that nec-t was a light and temperature-dependent mutant. Map-based cloning and the allelic test revealed that nec-t was a novel allelic mutant of the Necrotic4 gene. Necrotic4 encodes the coproporphyrinogen III oxidase (CPX1), a key enzyme in the tetrapyrrole pathway, catalyzing coproporphyrinogen III oxidate to protoporphyrinogen IX. Subcellular localization showed that the necrotic4 protein was localized in the chloroplast. Furthermore, RNA-seq analysis showed that the Necrotic4 mutation caused the enhanced chlorophyll degradation and reactive oxygen species (ROS) response. The mechanism of plant lesion formation induced by light and temperature is not clear. Our research provides a basis for understanding the molecular mechanism of necrosis initiation in maize.

Induction of Paraptotic Cell Death in Breast Cancer Cells by a Novel Pyrazolo[3,4-h]quinoline Derivative through ROS Production and Endoplasmic Reticulum Stress.

Chemotherapy has been a standard intervention for a variety of cancers to impede tumor growth, mainly by inducing apoptosis. However, development of resistance to this regimen has led to a growing interest and demand for drugs targeting alternative cell death modes, such as paraptosis. Here, we designed and synthesized a novel derivative of a pyrazolo[3,4-h]quinoline scaffold (YRL1091), evaluated its cytotoxic effect, and elucidated the underlying molecular mechanisms of cell death in MDA-MB-231 and MCF-7 breast cancer (BC) cells. We found that YRL1091 induced cytotoxicity in these cells with numerous cytoplasmic vacuoles, one of the distinct characteristics of paraptosis. YRL1091-treated BC cells displayed several other distinguishing features of paraptosis, excluding autophagy or apoptosis. Briefly, YRL1091-induced cell death was associated with upregulation of microtubule-associated protein 1 light chain 3B, downregulation of multifunctional adapter protein Alix, and activation of extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase. Furthermore, the production of reactive oxygen species (ROS) and newly synthesized proteins were also observed, subsequently causing ubiquitinated protein accumulation and endoplasmic reticulum (ER) stress. Collectively, these results indicate that YRL1091 induces paraptosis in BC cells through ROS generation and ER stress. Therefore, YRL1091 can serve as a potential candidate for the development of a novel anticancer drug triggering paraptosis, which may provide benefit for the treatment of cancers resistant to conventional chemotherapy.

Pan-Caspase Inhibitor zVAD Induces Necroptotic and Autophagic Cell Death in TLR3/4-Stimulated Macrophages.

In addition to inducing apoptosis, caspase inhibition contributes to necroptosis and/or autophagy depending on the cell type and cellular context. In macrophages, necroptosis can be induced by co-treatment with Toll-like receptor (TLR) ligands (lipopolysaccharide [LPS] for TLR4 and polyinosinic-polycytidylic acid [poly I:C] for TLR3) and a cell-permeable pan-caspase inhibitor zVAD. Here, we elucidated the signaling pathways and molecular mechanisms of cell death. We showed that LPS/zVAD- and poly I:C/zVAD-induced cell death in bone marrow-derived macrophages (BMDMs) was inhibited by receptor-interacting protein kinase 1 (RIP1) inhibitor necrostatin-1 and autophagy inhibitor 3-methyladenine. Electron microscopic images displayed autophagosome/autolysosomes, and immunoblotting data revealed increased LC3II expression. Although zVAD did not affect LPS- or poly I:C-induced activation of IKK, JNK, and p38, it enhanced IRF3 and STAT1 activation as well as type I interferon (IFN) expression. In addition, zVAD inhibited ERK and Akt phosphorylation induced by LPS and poly I:C. Of note, zVAD-induced enhancement of the IRF3/IFN/STAT1 axis was abolished by necrostatin-1, while zVAD-induced inhibition of ERK and Akt was not. Our data further support the involvement of autocrine IFNs action in reactive oxygen species (ROS)-dependent necroptosis, LPS/zVAD-elicited ROS production was inhibited by necrostatin-1, neutralizing antibody of IFN receptor (IFNR) and JAK inhibitor AZD1480. Accordingly, both cell death and ROS production induced by TLR ligands plus zVAD were abrogated in STAT1 knockout macrophages. We conclude that enhanced TRIF-RIP1-dependent autocrine action of IFNβ, rather than inhibition of ERK or Akt, is involved in TLRs/zVAD-induced autophagic and necroptotic cell death via the JAK/STAT1/ROS pathway.

Investigation the Cytotoxicity of 5-AZA on Acute Lymphoblastic Leukemia Cell Line In Vitro and Characterization the Underlying Molecular Mechanisms of Cell Death and Motility.

DNA methylation is a reversible biochemical process determinant of gene expression that is frequently observed in acute lymphoblastic leukemia (ALL). This is believed to arise from aberrant DNA methyltransferase activity establishing abnormal levels of DNA methylation in tumor cells. DNA methyltransferase inhibitor, 5-azacytidine (5-AZA), is a clinically used epigenetic drug which induces promoter demethylation and gene re-expression in human cancers. In this study, we investigated the cytotoxicity of on MOLT4 and Jurkat leukemic cell line in vitro and characterized the underlying molecular mechanisms of cell death and motility. MOLT4 and Jurkat cells were treated with 5-AZA for 12, 24 and 48 hours. The effect of the 5-AZA treatment on cell viability (MTT assay), apoptosis (annexin V/PI staining), microRNA (miRNA) and mRNA expression (real-time PCR) was measured. The results showed that 5-AZA could induce MOLT4 and Jurkat apoptotic cell death in vitro in a time-dependent manner and probably via apoptotic mechanisms. We found that treatment with 5-AZA could increase the expression of epigenetically silenced miRNAs, miR-34a, miR-34b and miR-124-1 in treated cells. In addition, mRNA analyses demonstrated that MOLT4 and jurkat cells, expressed p53 gene more than 10-fold higher compared with untreated cells in three independent experiments while the cells suppressed the expression of a subset of functionally related genes including MYC, BCL2, APEX, SIRT1, SNAIL1 and vimentin to some extent, following 5-AZA treatment. We found that a miRNAs expression level in treated cell lines was closely correlated to the expression of their target genes. Together, these findings suggest that 5-AZA may affect the viability of MOLT4 and jurkat cells, at least in part, by regulating the transcription of genes that are associated with cellular apoptotic response.