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Extrachromosomal circular DNA (eccDNA) derived from linear chromosomes, are showed typical nucleosomal ladder pattern in agarose gel which as a known feature of apoptosis and demonstrated to be immunogenicity. In systemic lupus erythematosus (SLE) patients, elevated levels of cell-free DNA (cfDNA) can be found in either linear forms or circular forms, while circular ones are much less common and harder to detect. The molecular characteristics and function of circular forms in plasma SLE patients remains elusive. Herein, we characterized the hallmarks of plasma eccDNA in SLE patients, including the lower normalized number and GC content of eccDNA in SLE plasma than in the healthy, and SLE eccDNA number positively correlated with C3 and negatively with anti-dsDNA antibodies. The differential eccGenes (eccDNAs carrying the protein coding gene sequence) of SLE was significantly enriched in apoptosis-related pathways. The artificially synthesized eccDNA with sequences of the PRF1 exon region could promote transcriptional expression of PRF1, IFNA and IFIT3 and inhibit early-stage apoptosis. Plasma eccDNA can serve as a novel autoantigen in the pathogenesis of SLE., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objectives: To investigate the inhibitory effects of STM2457, which is a novel METTL3 (m 6 A writer) inhibitor, both as a monotherapy and in combination with anlotinib, in the treatment of oral squamous cell carcinoma (OSCC) both in vitro and in vivo., Materials and Methods: The efficacy of STM2457 or STM2457 plus anlotinib was evaluated using two OSCC cell lines by CCK8, transwell, colony formation, would-healing, sphere formation, cell cycle, apoptosis assays, and nude mice tumor xenograft techniques. The molecular mechanism study was carried out by western blotting, qRT-PCR, MeRIP-qPCR, immunofluorescence, and immunohistochemistry., Results: STM2457 combined with anlotinib enhanced inhibition of cellular survival/proliferation and promotion of apoptosis in vitro. Moreover, this combinatorial approach exerted a notable reduction in stemness properties and EMT (epithelial-mesenchymal transition) features of OSCC cells. Remarkably, in vivo studies validated the efficacy of the combination treatment. Mechanistically, our investigations revealed that the combined action of STM2457 and anlotinib exerted downregulatory effects on EGFR (epidermal growth factor receptor) expression in OSCC cells., Conclusions: The combination of STM2457 and anlotinib targeting EGFR exerted a multiple anti-tumor effect. In near future, anlotinib combined with STM2457 may provide a novel insight for the treatment of OSCC., (© 2024 Wiley Periodicals LLC.)

Venetoclax is a BCL2-targeted drug employed in treating various cancers, particularly hematologic malignancies. Venetoclax combination therapies are increasingly recognized as promising treatment strategies for acute myeloid leukemia (AML). In this study, we conducted an unbiased drug screen and identified anlotinib, a promising multi-targeted receptor tyrosine kinase inhibitor with oral activity currently utilized in the treatment of solid tumor, as a potent enhancer of venetoclax's anticancer activity in AML. Our investigation encompassed AML cell lines, primary cells, and mouse models, demonstrating effective low-dose combination therapy of anlotinib and venetoclax with minimal cytopenia or organ damage. Proteomic analysis revealed abnormal mitotic signals induced by this combination in AML cells. Mechanistically, anlotinib synergized with venetoclax by suppressing ARPP19 protein, leading to sustained activation of PP2A-B55δ. This inhibited AML cells from entering the mitotic phase, culminating in mitotic catastrophe and apoptosis. Additionally, we identified a specific synthetic lethal vulnerability in AML involving an ARPP19 mutation at S62 phosphorylation. These findings underscore the therapeutic potential of anlotinib and venetoclax combination therapy in AML, warranting further clinical investigation., Competing Interests: Declaration of competing interest The authors declare no potential conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

To explore the effects of β-Sitosterol upon hepatocellular carcinoma cell proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition (EMT), and to investigate the underlying mechanism using network pharmacology. Human hepatocellular carcinoma cell lines (Huh-7 and HCCLM3) were expose to gradient concentrations of β-Sitosterol (5 μg/mL, 10 μg/mL, and 20 μg/mL). Cell viability and proliferation were assessed using MTT, CCK-8, colony formation, and EdU assays.Flow cytometry was employed to evaluate cell cycle and apoptosis. Scratch and Transwell assays were performed, respectively, to detect cell migration and invasion. The levels of apoptosis-associated proteins (BAX, BCL2, and cleaved caspase3) as well as EMT-associated proteins (E-cadherin, N-cadherin, Snail, and Vimentin) were detected in Huh-7 and HCCLM3 cell lines using Western blot analysis. The drug target gene for β-Sitosterol was screened via PubChem and subsequently evaluated for expression in the GSE112790 dataset. In addition, the expression level of glycogen synthase kinase 3 beta (GSK3B) within the Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database was analyzed, along with its correlation to the survival outcomes of patients with hepatocellular carcinoma. The diagnostic efficiency of GSK3B was assessed by analyzing the ROC curve. Subsequently, Huh-7 and HCCLM3 cell lines were transfected with the overexpression vector of GSK3B and then treated with β-Sitosterol to further validate the association between GSK3B and β-Sitosterol. GSK3B demonstrated a significantly elevated expression in patients with hepatocellular carcinoma, which could predict hepatocellular carcinoma patients' impaired prognosis based on GEO dataset and TCGA database. GSK3B inhibitor (CHIR-98014) notably inhibited cell proliferation and invasion, promoted cell apoptosis and cell cycle arrest at G0/G1 phase in hepatocellular carcinoma cells. β-Sitosterol treatment further promoted the efffects of GSK3B inhibitor on hepatocellular carcinoma cells. GSK3B overexpression has been found to enhance the proliferative and invasive capabilities of hepatocellular carcinoma cells. Furthermore it has been observed that GSK3B overexpression, it has been obsear can partially reverse the inhibitory effect of β-Sitosterol upon hepatocellular. β-Sitosterol suppressed hepatocellular carcinoma cell proliferation and invasion, and enhanced apoptosis via inhibiting GSK3B expression., (© 2024. The Author(s).)

Small peptides are nutrients and bioactive molecules that have dual regulatory effects on nutrition and physiology. They are of great significance for maintaining the intestinal health and production performance of broilers. We here cultured the primary small intestinal epithelial cells (IEC) of chicken in a medium containing L-Leu (Leu) and L-Leu-L-Leu (Leu-Leu) for 24 h. The untreated cells were considered as the control group. The growth, proliferation, and apoptosis of IEC were examined. By combining RNA-seq and label-free sequencing technology, candidate genes, proteins, and pathways related to the growth, proliferation, and apoptosis of IEC were screened. Immunofluorescence detection revealed that the purity of the isolated primary IEC was >90%. The Leu-Leu group significantly promoted IEC growth and proliferation and significantly inhibited IEC apoptosis, and the effect was better than those of the Leu and control groups. Using transcriptome sequencing, four candidate genes, CCL20, IL8L1, IL8, and IL6, were screened in the Leu group, and one candidate gene, IL8, was screened in the Leu-Leu group. Two candidate genes, IL6 and RGN, were screened in the Leu-Leu group compared with the Leu group. Nonquantitative proteomic marker sequencing results revealed that through the screening of candidate proteins and pathways, found one growth-related candidate protein PGM3 and three proliferation-related candidate proteins RPS17, RPS11, and RPL23, and two apoptosis-related candidate proteins GPX4 and PDPK1 were found in the Leu-Leu group compared with Leu group. In short, Leu-Leu could promote IEC growth and proliferation and inhibit IEC apoptosis. On combining transcriptome and proteome sequencing technologies, multiple immune- and energy-related regulatory signal pathways were found to be related to IEC growth, proliferation, and apoptosis. Three candidate genes of IL8, IL6, and RGN were identified, and six candidate proteins of PGM3, RPS17, RPS11, RPL23, GPX4, and PDPK1 were involved in IEC growth, proliferation, and apoptosis. The results provide valuable data for preliminarily elucidating small peptide-mediated IEC regulation pathways, improving the small peptide nutrition theoretical system, and establishing small peptide nutrition regulation technology., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell death can be classified into two primary categories: accidental cell death and regulated cell death (RCD). Within RCD, there are distinct apoptotic and non-apoptotic cell death pathways. Among the various forms of non-apoptotic RCD, paraptosis stands out as a unique mechanism characterized by distinct morphological changes within cells. These alterations encompass cytoplasmic vacuolization, organelle swelling, notably in the endoplasmic reticulum and mitochondria, and the absence of typical apoptotic features, such as cell shrinkage and DNA fragmentation. Biochemically, paraptosis distinguishes itself by its independence from caspases, which are conventionally associated with apoptotic death. This intriguing cell death pathway can be initiated by various cellular stressors, including oxidative stress, protein misfolding, and specific chemical compounds. Dysregulated paraptosis plays a pivotal role in several critical cancer-related processes, such as autophagic degradation, drug resistance, and angiogenesis. This review provides a comprehensive overview of recent advancements in our understanding of the mechanisms and regulation of paraptosis. Additionally, it delves into the potential of paraptosis-related compounds for targeted cancer treatment, with the aim of enhancing treatment efficacy while minimizing harm to healthy cells., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease., (© 2023. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Background: Ulcerative colitis (UC) is a chronic and debilitating inflammatory bowel disease that impairs quality of life. Cuproptosis, a recently discovered form of cell death, has been linked to many inflammatory diseases, including UC. This study aimed to examine the biological and clinical significance of cuproptosis-related genes in UC., Methods: Three gene expression profiles of UC were obtained from the Gene Expression Omnibus (GEO) database to form the combined dataset. Differential analysis was performed based on the combined dataset to identify differentially expressed genes, which were intersected with cuproptosis-related genes to obtain differentially expressed cuproptosis-related genes (DECRGs). Machine learning was conducted based on DECRGs to identify signature genes. The prediction model of UC was established using signature genes, and the molecular subtypes related to cuproptosis of UC were identified. Functional enrichment analysis and immune infiltration analysis were used to evaluate the biological characteristics and immune infiltration landscape of signature genes and molecular subtypes., Results: Seven signature genes (ABCB1, AQP1, BACE1, CA3, COX5A, DAPK2, and LDHD) were identified through the machine learning algorithms, and the nomogram built from these genes had excellent predictive performance. The 298 UC samples were divided into two subtypes through consensus cluster analysis. The results of the functional enrichment analysis and immune infiltration analysis revealed significant differences in gene expression patterns, biological functions, and enrichment pathways between the cuproptosis-related molecular subtypes of UC. The immune infiltration analysis also showed that the immune cell infiltration in cluster A was significantly higher than that of cluster B, and six of the characteristic genes (excluding BACE1) had higher expression levels in subtype B than in subtype A., Conclusions: This study identified several promising signature genes and developed a nomogram with strong predictive capabilities. The identification of distinct subtypes of UC enhances our current understanding of UC's underlying pathogenesis and provides a foundation for personalized diagnosis and treatment in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tang, Pu, Liu and Li.)

Heat stress, as a kind of oxidative stress, induces cell apoptosis. Apoptosis is a form of programmed cell death, and mitochondria play an important role in apoptosis. Manganese (Mn) has an antioxidant capacity by enhancing the activity of manganese superoxide dismutase (MnSOD). To investigate the potential effect of Mn on heat stress-induced apoptosis and mitochondrial function, we examined crucial related factors in the context of heat stress using primary chick embryonic myocardial cells pretreated with Mn for 24 h. The results showed that Mn restored the heat stress-induced decrease in cell viability and reduced the activities of caspase-3 (P < 0.05). The repression of the Δψm and intracellular ATP content caused by heat stress was reversed dramatically in the Mn pretreatment group (P < 0.05). Additionally, Mn inhibited heat stress-induced mitochondrial fission, as shown by decreased mitochondrial fission-related protein dynamin-related protein 1 (Drp1) expression and increased mitochondrial fusion-related protein optic atrophy 1 (Opa1) and mitofusin 1 (Mfn1) (P < 0.05) in primary chick embryonic myocardial cells. It was concluded that Mn attenuates the mitochondrial-mediated apoptosis pathway and sustains mitochondrial structure and function under heat stress in primary chick embryonic myocardial cells., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Programmed cell death (PCD) is an essential biological process involved in many human pathologies. According to the continuous discovery of new PCD forms, a large number of proteins have been found to regulate PCD. Notably, post-translational modifications play critical roles in PCD process and the rapid advances in proteomics have facilitated the discovery of new PCD proteins. However, an integrative resource has yet to be established for maintaining these regulatory proteins. Here, we briefly summarize the mainstream PCD forms, as well as the current progress in the development of public databases to collect, curate and annotate PCD proteins. Further, we developed a comprehensive database, with integrated annotations for programmed cell death (iPCD), which contained 1,091,014 regulatory proteins involved in 30 PCD forms across 562 eukaryotic species. From the scientific literature, we manually collected 6493 experimentally identified PCD proteins, and an orthologous search was then conducted to computationally identify more potential PCD proteins. Additionally, we provided an in-depth annotation of PCD proteins in eight model organisms, by integrating the knowledge from 102 additional resources that covered 16 aspects, including post-translational modification, protein expression/proteomics, genetic variation and mutation, functional annotation, structural annotation, physicochemical property, functional domain, disease-associated information, protein-protein interaction, drug-target relation, orthologous information, biological pathway, transcriptional regulator, mRNA expression, subcellular localization and DNA and RNA element. With a data volume of 125 GB, we anticipate that iPCD can serve as a highly useful resource for further analysis of PCD in eukaryotes.

In the field of therapeutic protein production, process intensification strategies entailing higher starting cell seeding densities, can potentially increase culture productivity, lower cost of goods and improve facility utilization. However, increased cell densities often trigger apoptotic cell death at the end of the cell culture process and thus reduce total viable cell count. Apoptosis-resistant Chinese hamster ovary cell lines may offer the possibility to diminish this undesired outcome of the intensified production process. In this study, we have generated and tested Bax/Bak double-knock-out (DKO) apoptosis resistant hosts to express standard and bispecific antibodies, as well as complex molecules in intensified production processes both as pools and single cell clones, and at different scales. In all cases, therapeutic proteins expressed from clones or pools generated from the Bax/Bak DKO hosts showed not only better viability but also enabled extended productivity in the later stages of the 14-day intensified production process. The product qualities of the produced molecules were comparable between Bax/Bak DKO and wild type cells. Overall, we showed that Bax/Bak DKO apoptosis-resistant host cell lines significantly improve viability and volumetric productivity of the intensified production cultures without altering product qualities., (© 2021 American Institute of Chemical Engineers.)

Background: H. pylori is closely related to the occurrence and development of various digestive gastritis, peptic ulcer and mucosa-associated lymphoid tissue (MALT) lymphoma. H. pylori is also a class I carcinogen of gastric cancer. VacA is the only exocrine toxin of H. pylori, which plays a very important role in the pathogenesis of H. pylori. The production of VacA in natural circumstances is complex with heavy workload and low yield. Therefore, it is very important to obtain recombinant VacA protein which is stable and biologically active. This study therefore aims to explore the expression, purification and stable storage of VacA toxin of H. pylori in E.coli, and to provide experimental basis for further exploration of the role of VacA in H. pylori -induced inflammation of cancer., Results: A 2502-bp fragment and VacA gene were identified. An 89.7-kDa VacA 34-854 recombinant protein was expressed and purified from the recombinant engineering bacteria and was preserved stably in 50 mM acetic acid buffer (pH 2.9). The amount of the recombinant protein was larger in the inclusion bodies than in the supernatant. In addition, after a 24-h culture with VacA recombinant protein, GES-1 cells demonstrated evidence of apoptosis including early nuclear immobilization and clustering under inverted microscope and TEM. It was found that VacA recombinant protein induced apoptosis by TUNEL assay., Conclusions: A VacA recombinant protein that is stably and highly expressed and possesses pro-apoptotic activity is successfully constructed. The protein is stably preserved in 50 mM acetic acid buffer (pH 2.9)., (© 2021. The Author(s).)

Venetoclax is a highly selective BCL2 inhibitor widely used in the treatment of leukemia, especially chronic lymphocytic leukemia and acute myeloid leukemia (AML). However, long-term use of venetoclax may lead to secondary drug resistance, which constitutes an important obstacle to prolonging the duration of the therapeutic response. Here, we show that the acquired resistance to venetoclax in human AML cell lines depends on NF-κB activation rather than on the upregulation of anti-apoptotic BCL2L1 expression. Moreover, alkaliptosis induced by the small molecular compound JTC801, but not necroptosis and ferroptosis, inhibits the growth of venetoclax-resistant AML cells in vitro and in xenograft mouse models. Mechanistically, NF-κB-mediated CA9 downregulation is required for intracellular pH upregulation, thereby inducing alkaliptosis in venetoclax-resistant cells. These findings provide a new strategy to selectively remove venetoclax-resistant AML cells., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest or financial interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Methylglyoxal (MGO), a cytotoxic byproduct of glycolysis in biological systems, can induce endothelial cells dysfunction, implicated in diabetic vascular complications. Pterostilbene (PTS), a naturally occurring resveratrol derivative, is involved in various pharmacological activities. This study aimed to explore the effects of PTS on MGO induced cytotoxicity in human umbilical vein endothelial cells (HUVECs) and the underlying mechanisms for the first time. In the current study, it has been demonstrated that PTS could enhance the level of glyoxalase 1 (GLO-1) and elevate glutathione (GSH) content to active the glyoxalase system, resulting in elimination of the toxic MGO as well as advanced glycation end products (AGEs) in HUVECs. Meanwhile, PTS could also suppress oxidative stress and thus exert cytoprotective effects by elevating Nrf2 nuclear translocation and the corresponding down-stream antioxidant enzymes in MGO induced HUVECs. In addition, PTS could alleviate MGO induced apoptosis in HUVECs via inhibition of oxidative stress and associated downstream mitochondria-dependent signaling apoptotic cascades, as characterized by preventing caspases family activation. Taken together, these findings suggest that PTS could protect against MGO induced endothelial cell cytotoxicity by regulating glyoxalase, oxidative stress and apoptosis, suggesting that PTS could be beneficial in the treatment of diabetic vascular complications., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Stem cell therapy is widely employed in treating osteoarthritis (OA), and bone marrow-derived mesenchymal stem cells (BMSCs) has gradually become the most attractive new method for treating OA due to the benefit for cartilage tissue repair. However, the apoptosis in the neural stem cell transplantation severely decreases repairing efficacy. Icariin has been reported to exert multiple effects on BMSCs, including its proliferation, osteogenic, and chondrogenic differentiation. However, its effects on the injury induced by oxygen, glucose and serum deprivation (OGD) remains unknown. We prospectively investigated the role of ICA on rabbit BMSCs under conditions of OGD. Firstly, BMSCs were cultured under conditions of OGD, ICA relieved OGD-induced cell damage by promoting cell proliferation and suppressing apoptosis. Secondly, Markers of endoplasmic reticulum stress (ERs), ER stress IRE-1 pathway, and autophagy were both inhibited by ICA via inhibition of phosphor-extracellular regulated protein kinases (p-ERKs), p-P38, p-c-Jun N-terminal kinase (p-JNK) or si-MAPK. Finally, decrease of ERs marker levels enhanced protective effect of ICA against OGD-induced injury by limiting apoptosis and autophagy. Moreover, an autophagy inhibitor (3-methyladenine: 3-MA) contributed to a synergistic effect in conjunction with ICA, in promoting cell proliferation, suggesting that ICA exerts anti-ERs and anti-autophagy effects in OGD-treated BMSCs. Therefore, ICA protected rabbit BMSCs from OGD-induced apoptosis through inhibitory regulation of ERs-mediated autophagy related to the MAPK signaling pathway, which provided insights for a potential therapeutic strategy in OA., Competing Interests: Declaration of competing interest All authors declare having no conflict of interest related to this research., (Copyright © 2020 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) have emerged as critical regulators of neuronal survival during cerebral ischaemia/reperfusion injury. Accumulating evidence has shown that miR-199a-5p plays a crucial role in regulating apoptosis and survival in various cell types. However, whether miR-199a is involved in regulating neuronal survival during cerebral ischaemia/reperfusion injury remains unknown. In this study, we aimed to explore the biological role of miR-199a-5p in regulating neuronal injury induced by oxygen-glucose deprivation/reoxygenation (OGD/R), an in vitro cellular model of cerebral ischaemia and reperfusion injury. We found that miR-199a-5p expression was significantly altered in neurons in response to OGD/R treatment. Overexpression of miR-199a-5p facilitated OGD/R-induced apoptosis and reactive oxygen species (ROS) production, whereas miR-199a-5p inhibition alleviated OGD/R-induced apoptosis and ROS production. Notably, our results identified Brahma-related gene 1 (Brg1) as a target gene of miR-199a-5p. Moreover, inhibition of miR-199a-5p promoted the activation of nuclear factor-erythroid-2-related factor-2 (Nrf2)/heme oxygenase-1 (HO-1) signalling via targeting Brg1. However, silencing of Brg1 markedly reversed the miR-199a-5p inhibition-mediated neuroprotective effect. Taken together, our results suggest that downregulation of miR-199a-5p protects neurons from OGD/R-induced neuronal injury through upregulating Brg1 to activate Nrf2/HO-1 signalling. The miR-199a-5p/Brg1/Nrf2/HO-1 regulation axis may play an important role in regulating neuronal survival during cerebral ischaemic/reperfusion injury in vivo., (© 2020 John Wiley & Sons Australia, Ltd.)

Glycogen synthase kinase beta (GSK3β) is considered as a promising target for lung cancer treatment and its inhibitor lithium chloride (LiCl) is widely regarded as having potent anti-proliferative and apoptosis-modulating activities. Proliferating cell nuclear antigen (PCNA), as an auxiliary protein for DNA polymerase delta, which regulates DNA replication and repair, has been reported to play an important role in regulating apoptosis. Here, we showed that GSK3β interacted with PCNA in H1299 lung adenocarcinoma cells using GST pull-down and co-immunoprecipitation experiments. We discovered that their interaction can be enhanced within the first 3 h after UVC irradiation and decreased gradually with time. Overexpression of PCNA protein decreased GSK3β Ser9 phosphorylation, whereas knockdown of PCNA using small interfering RNA (siRNA) increased Ser9 phosphorylated GSK3β, which was attenuated by phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 after UVC irradiation, indicating the involvement of the PI3K-AKT pathway. Functional analyses suggested that downregulation of PCNA sensitized H1299 cells to LiCl-induced apoptosis. Thus, our results unraveled a novel regulatory of GSK3β by PCNA and provided a promising direction for treatment of lung cancer.

Objectives: The influences of COL1A1 and MZB1 on the proliferation, migration, invasion and apoptosis abilities of rectum adenocarcinoma was aimed to explore in this study., Methods: Gene expression levels in rectum adenocarcinoma and adjacent tissues were analyzed by differential analysis. Weighted gene correlation network analysis (WGCNA) was employed to investigate rectal adenocarcinoma (READ) hub genes. MCODE was performed to screen the modules of protein-protein interaction network in Cytoscape software. Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool is considered to be the most effective tool in gene ontology (GO) enrichment and Kyoto Gene and Genomics Encyclopedia (KEGG) pathway analysis. Survival analysis was performed using READ patient information from TCGA-READ project database. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) and western blot were employedto examinemRNA and protein expressions of COL1A1 and MZB1 in tumor tissues and cell lines. After transfecting various interference sequences by liposome-mediated transfection, the influence of COL1A1 and MZB1 on the proliferation, apoptosis, migration and invasion of rectal cancer cells were observed by plate clone formation assay, flow cytometry, wound healing assay and transwell assay respectively. Moreover, xenograft tumor growth assay in vivo validated the results., Results: Higher expression levels of COL1A1 and lower expression levels of MZB1 were discovered in tumor tissues of patients with colorectal adenocarcinoma. Overexpression of MZB1 and silencing COL1A1 significantly inhibited proliferation, migration and invasion, while cell apoptosis was promoted. Overexpression of MZB1 and silencing COL1A1 inhibited the orthotopic growth of tumor in vivo., Conclusion: COL1A1 promoted proliferation, migration and invasion but inhibited apoptosis of rectal adenocarcinoma cells while MZB1 was totally on the contrary., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Mitochondria play important roles in the development of diabetic kidney disease (DKD). The SS peptide is a tetrapeptide that is located and accumulated in the inner mitochondrial membrane; it reduces reactive oxygen species (ROS) and prevents mitochondrial dysfunction. Podocytes are key cellular components in DKD progression. However, whether the SS peptide can exert renal protection through podocytes and the mechanism involved are unknown. In the present study, we explored the mechanisms of the SS peptide on podocyte injury in vivo and in vitro. Compared with the control group, the glomerular podocyte number and expression of WT1 were significantly reduced and TUNEL-positive podocytes were significantly increased in renal tissues in the diabetic rat. These effects were further exacerbated by hypochlorite-modified albumin (HOCl-alb) challenge but prevented by SS-31. In vitro, SS-31 blocked apoptosis in podocyte cell line induced by HOCl-alb. SS-31 prevented oxidative stress and mitochondria-dependent apoptosis signalling by HOCl-alb in vivo and in vitro, as evidenced by the release of cytochrome c (cyt c), binding of apoptosis activated factor-1 (Apaf-1) and caspase-9, and activation of caspases. These data suggest that SS-31 may prevent podocyte apoptosis, exerting renal protection in diabetes mellitus, probably through an apoptosis-related signalling pathway involving oxidative stress and culminating in mitochondria.

The ubiquitin-proteasome system (UPS) is a tight homeostatic control mechanism of intracellular protein degradation and turnover involved in many human diseases. Proteasome inhibitors were initially developed as anticancer agents with potential benefits in the suppression of tumor growth. However, clinical trials of patients with solid tumors fail to demonstrate the same efficacy of these proteasome inhibitors. Here, we show that Parkin, an E3 ubiquitin ligase, is implicated in tumorigenesis and therapy resistance of hepatocellular carcinoma (HCC), the most common type of primary liver cancer in adults. Lower Parkin expression correlates with poor survival in patients with HCC. Ectopic Parkin expression enhances proteasome inhibitor-induced apoptosis and tumor suppression in HCC cells in vitro and in vivo. In contrast, knockdown of Parkin expression promotes apoptosis resistance and tumor growth. Mechanistically, Parkin promotes TNF receptor-associated factor (TRAF) 2 and TRAF6 degradation and thus facilitates nuclear factor-kappa-B (NF-κB) inhibition, which finally results in apoptosis. These findings reveal a direct molecular link between Parkin and protein degradation in the control of the NF-κB pathway and may provide a novel UPS-dependent strategy for the treatment of HCC by induction of apoptosis.

Objective: To detect the expression of long non-coding ribonucleic acid (lncRNA) plasmacytoma variant translocation gene 1 (PVT1) in uveal melanoma (UM) tissues, and to investigate its influence on the proliferation and apoptosis of UM cells as well as its mechanism., Patients and Methods: 40 cases of UM tissues and 40 cases of adjacent tissues surgically resected in our hospital from October 2015 to April 2018 were collected. The expression level of lncRNA PVT1 in these tissues was determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Stable knockdown of lncRNA PVT1 was constructed in human UM cell line OCM-1 using small interfering RNA (siRNA). The impact of lncRNA PVT1 on UM cell proliferation was detected by Cell Counting kit-8 (CCK-8) and colony formation assay. Flow cytometry was applied to measure the apoptotic level of UM cells in the blank control group and lncRNA PVT1 knockdown group. Meanwhile, the expression level of enhancer of zeste homologue 2 (EZH2) was determined by Western blotting., Results: The expression level of lncRNA PVT1 in UM tissues was remarkably higher than that in the adjacent tissues (p<0.05). UM cell proliferation was notably repressed after lncRNA PVT1 knockdown by siRNA. Flow cytometry results indicated that the number of apoptotic UM cells in lncRNA PVT1 knockdown group significantly increased compared with that in the blank control group (p<0.05). The protein expression of EZH2 was suppressed after lncRNA PVT1 knockdown (p<0.05)., Conclusions: LncRNA PVT1 knockdown in UM cells can repress the proliferation of UM cells and promote their apoptosis by regulating EZH2 expression.

Changes in the expression and subcellular localization of high mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) molecule, have been implicated in tumorigenesis and tumor cell death in response to cancer therapy. Specifically, HMGB1 release has been shown to occur with a specific form of induced cell death known as necroptosis. In the present study, we examined the role of HMGB1 in the necroptosis of acute myeloid leukemia (AML) cells. In two AML cell lines and primary AML cells from two patients, etoposide induced necroptosis via cIAP1/2 degradation when caspase activity was inhibited by Z-VAD-fmk, but treatment with extracellular HMGB1 prevented this necroptosis. Interestingly, HMGB1 did not prevent the degradation of cIAP1/2, but rather activated the nuclear factor kappa B pathway. The results of the present study provide evidence that extracellular HMGB1 is not only an important DAMP molecule released by cells upon necrosis, but also a regulatory factor that prevents necroptosis in AML cells., (Copyright © 2019 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Background: Cancer cells avidly consume glucose and convert it to lactate, resulting in a low pyruvate level. This phenomenon is known as the Warburg effect, and is important for cell proliferation. Although cMyc has often been described as an oncoprotein that preferentially contributes to the Warburg effect and tumor proliferation, mechanisms of action remain unclear. Histone deacetylase 3 (HDAC3) regulates gene expression by removing acetyl groups from lysine residues, as well as has an oncogenic role in apoptosis and contributes to the proliferation of many cancer cells including cholangiocarcinoma (CCA). HDAC inhibitors display antitumor activity in many cancer cell lines. Cancer cells maintain low levels of pyruvate to prevent inhibition of HDAC but the mechanisms remain elusive. The purpose of our study was to explore the role of cMyc in regulating pyruvate metabolism, as well as to investigate whether the inhibitory effect of pyruvate on HDAC3 could hold promise in the treatment of cancer cells., Methods: We studied pyruvate levels in CCA cell lines using metabolite analysis, and analyzed the relationship of pyruvate levels and cell proliferation with cell viability analysis. We cultivated CCA cell lines with high or low levels of pyruvate, and then analyzed the protein levels of HDAC3 and apoptotic markers via Western Blotting. We then explored the reasons of low levels of pyruvate by using seahorse analysis and 13 C 6 metabolites tracing analysis, and then confirmed the results using patient tissue protein samples through Western Blotting. Bioinformatics analysis and transfection assay were used to confirm the upstream target of the low levels of pyruvate status in CCA. The regulation of cMyc by HDAC3 was studied through immunoprecipitation and Western Blotting., Results: We confirmed downregulated pyruvate levels in CCA, and defined that high pyruvate levels correlated with reduced cell proliferation levels. Downregulated pyruvate levels decreased the inhibition to HDAC3 and consequently protected CCA cells from apoptosis. Synergistically upregulated LDHA, PKM2 levels resulted in low levels of pyruvate, as well as poor patient survival. We also found that low levels of pyruvate contributed to proliferation of CCA cells and confirmed that the upstream target is cMyc. Conversely, high activity of HDAC3 stabilized cMyc protein by preferential deacetylating cMyc at K323 site, which further contributed to the low pyruvate levels. Finally, this creates a positive feedback loop that maintained the low levels of pyruvate and promoted CCA proliferation., Conclusions: Collectively, our findings identify a role for promoting the low pyruvate levels regulated by c-Myc, and its dynamic acetylation in cancer cell proliferation. These targets, as markers for predicting tumor proliferation in patients undergoing clinical treatments, could pave the way towards personalized therapies.

Accumulating evidence suggests that brahma-related gene 1 (Brg1) is a critical regulator of cell apoptosis and oxidative stress in response to various insults; however, whether Brg1 regulates neuronal apoptosis and oxidative stress during cerebral ischemia/reperfusion injury remains unclear. This study aimed to investigate the expression, biological function, and regulatory mechanism of Brg1 in regulating neuronal apoptosis and oxidative stress induced by oxygen-glucose deprivation/reoxygenation (OGD/R), an in vitro cellular model of cerebral ischemia and reperfusion injury. The results showed that Brg1 expression was altered in response to OGD/R treatment. Overexpression of Brg-1 increased cell viability, attenuated apoptosis, and reduced reactive oxygen species (ROS) production in neurons, thus showing a protective effect against OGD/R-induced injury. In contrast, knockdown of Brg1 significantly inhibited cell viability, increased apoptosis, and promoted ROS production in neurons following OGD/R treatment. Moreover, these results showed that Brg1 promoted the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and increased the expression of heme oxygenase-1 (HO-1). However, knockdown of Nrf2 or HO-1 significantly abrogated Brg1-mediated neuroprotective effects. Taken together, these results demonstrate that Brg1 ameliorates OGD/R-induced neuronal injury in vitro by promoting the activation of Nrf2/HO-1 signaling. The study highlights a potential role of Brg1 in regulating cerebral ischemia/reperfusion injury in vivo., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Background: As indoleamine-2,3-dioxygenase 1 (IDO1) is critical in tumor immune escape, we determined to study the regulatory mechanism of miR-218 on IDO1 in cervical cancer., Methods: Real-time PCR (RT-qPCR) was carried out to measure the expression of miR-218. RT-qPCR and Western blot were performed to detect the expression of IDO1 in cervical cancer. Dual-luciferase reporter assay was used to determine the binding of miR-218 on the IDO1 3'UTR. Cell viability, apoptosis, and related factors were determined using cell counting kit-8 (CCK-8), Annexin-V/PI (propidium) assay, enzyme-linked immunosorbnent assay (ELISA), RT-qPCR, and Western blot assays after miR-218 mimics has been transfected to HeLa cervical cancer cells., Results: MiR-218 was downregulated in cervical cancer. The expression of miR-218 was negatively correlated with IDO1 in cervical cancer tissues and cells. IDO1 is a direct target of miR-218. MiR-218 overexpression was found to inhibit cell viability and promoted apoptosis via activating the expression of Cleaved-Caspase-3 and to inhibit the expression of Survivin, immune factors (TGF-β, VEGF, IL-6, PGE2, COX-2), and JAK2/STAT3 pathway., Conclusion: MiR-218 inhibits immune escape of cervical cancer cells by direct downregulating IDO1.

Ferroptosis is a form of regulated cell death resulting from iron accumulation and lipid peroxidation. While impaired ferroptosis is tightly linked to human diseases and conditions, the mechanism and regulation of ferroptosis remain largely unknown. Here, we demonstrate that STAT3 is a positive regulator of ferroptosis in human pancreatic ductal adenocarcinoma (PDAC) cell lines. Activation of the MAPK/ERK pathway, but not inhibition of system Xc - , was required for STAT3 activation during erastin-induced ferroptosis. Importantly, pharmacological inhibition and genetic silencing of STAT3 through small molecules (e.g., cryptotanshinone and S3I-201) or siRNA blocked erastin-induced ferroptosis in PDAC cells. Mechanically, STAT3-mediated cathepsin B expression was required for ferroptosis. Consequently, inhibition of lysosome-dependent cell death by pharmacological blockade of cathepsin activity (using CA-074Me) or vacuolar type H+-ATPase (using bafilomycin A1) limited erastin-induced ferroptosis. These studies indicate that ferroptosis is a lysosomal cell death process., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Ferroptosis is a form of regulated cell death triggered by lipid peroxidation after inhibition of the cystine/glutamate antiporter system X c - . However, key regulators of system X c - inhibitors (e.g., erastin, sulfasalazine, and sorafenib), but not other ferroptosis inducers including RSL3, FIN56, and buthionine sulfoximine. Mechanistically, AMP-activated protein kinase (AMPK)-mediated phosphorylation of BECN1 at Ser90/93/96 is required for BECN1-SLC7A11 complex formation and lipid peroxidation. Inhibition of PRKAA/AMPKα by siRNA or compound C diminishes erastin-induced BECN1 phosphorylation at S93/96, BECN1-SLC7A11 complex formation, and subsequent ferroptosis. Accordingly, a BECN1 phosphorylation-defective mutant (S90,93,96A) reverses BECN1-induced lipid peroxidation and ferroptosis. Importantly, genetic and pharmacological activation of the BECN1 pathway by overexpression of the protein in tumor cells or by administration of the BECN1 activator peptide Tat-beclin 1, respectively, increases ferroptotic cancer cell death (but not apoptosis and necroptosis) in vitro and in vivo in subcutaneous and orthotopic tumor mouse models. Collectively, our work reveals that BECN1 plays a novel role in lipid peroxidation that could be exploited to improve anticancer therapy by the induction of ferroptosis.- activity via binding to its core component, SLC7A11 (solute carrier family 7 member 11). Knockdown of BECN1 by shRNA inhibits ferroptosis induced by system X c - inhibitors (e.g., erastin, sulfasalazine, and sorafenib), but not other ferroptosis inducers including RSL3, FIN56, and buthionine sulfoximine. Mechanistically, AMP-activated protein kinase (AMPK)-mediated phosphorylation of BECN1 at Ser90/93/96 is required for BECN1-SLC7A11 complex formation and lipid peroxidation. Inhibition of PRKAA/AMPKα by siRNA or compound C diminishes erastin-induced BECN1 phosphorylation at S93/96, BECN1-SLC7A11 complex formation, and subsequent ferroptosis. Accordingly, a BECN1 phosphorylation-defective mutant (S90,93,96A) reverses BECN1-induced lipid peroxidation and ferroptosis. Importantly, genetic and pharmacological activation of the BECN1 pathway by overexpression of the protein in tumor cells or by administration of the BECN1 activator peptide Tat-beclin 1, respectively, increases ferroptotic cancer cell death (but not apoptosis and necroptosis) in vitro and in vivo in subcutaneous and orthotopic tumor mouse models. Collectively, our work reveals that BECN1 plays a novel role in lipid peroxidation that could be exploited to improve anticancer therapy by the induction of ferroptosis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Proton pump inhibitors (PPIs) play a role in antitumor activity, with studies showing specialized impacts of PPIs on cancer cell apoptosis, metastasis, and autophagy. In this study, we demonstrated that pantoprazole (PPI) increased autophagosomes formation and affected autophagic flux depending on the pH conditions. PPI specifically elevated SQSTM1 protein levels by increasing SQSTM1 transcription via NFE2L2 activation independent of the specific effect of PPI on autophagic flux. Via decreasing proteasome subunits expression, PPI significantly impaired the function of the proteasome, accompanied by the accumulation of undegraded poly-ubiquitinated proteins. Notably, PPI-induced autophagy functioned as a downstream response of proteasome inhibition by PPI, while suppressing protein synthesis abrogated autophagy. Blocking autophagic flux in neutral pH condition or further impairing proteasome function with proteasome inhibitors, significantly aggravated PPI cytotoxicity by worsening protein degradation ability. Interestingly, under conditions of mitochondrial stress, PPI showed significant synergism when combined with Bcl-2 inhibitors. Taken together, these findings provide a new understanding of the impact of PPIs on cancer cells' biological processes and highlight the potential to develop more efficient and effective combination therapies.

As the major and preferred treatment for ovarian cancer ascites, chemotherapy can reduce or inhibit recurrent ascites (hereafter re-ascites); however, some patients still experience re-ascites. Therefore, this study investigated cases in which epithelial ovarian cancer (EOC) patients experienced re-ascites. In re-ascites cases, CA125, MDR1, LC-3, and Beclin-1 were highly expressed. In addition, CASP-9 and c-CASP-3 expression levels were decreased, and serum CA125 levels (highest 4348 U/ml) were increased compared to chemosensitive cases. The results suggest that high expression levels of Beclin-1 and LC-3, thus increasing the level of autophagy and inhibiting apoptosis in the no-chemotherapy group. In the chemosensitive group, survivin expression was decreased and CASP-9 expression was increased, which led to c-CASP-3 activation and increased tumor cell apoptosis. The results of the cell lines confirm that inhibition of autophagy can increase the sensitivity of ovarian cancer cells to CDDP and promote CDDP-induced cell death. Re-ascites, which appears after chemotherapy, may be associated with drug resistance. In addition, increased autophagy may protect tumor cells from chemotherapeutic drugs, thus inhibiting tumor cell death.

It has been well known that androgen receptor (AR) is critical to prostate cancer development and progression. It has also been documented that AR is expressed in more than 60% of breast tumors, which promotes the growth of estrogen receptor-negative (ER - )/AR-positive (AR + ) breast cancer cells. Thus, AR might be a potential therapeutic target for AR-positive/ER-negative breast cancer patients. Previously we reported that in prostate cancer cells proteasome-associated deubiquitinase ubiquitin-specific protease 14 (USP14) stabilized AR protein level by removing its ubiquitin chain. In the current study, we studied the USP14-AR protein interaction and cell proliferation status after USP14 reduction or inhibition in breast cancer cells, and our results support the conclusion that targeting USP14 is a novel strategy for treating AR-responsive breast cancer. We found that inhibition of USP14 accelerated the K48-ubiquitination and proteasome-mediated degradation of AR protein. Additionally, both genetic and pharmacological inhibition of USP14 significantly suppressed cell proliferation in AR-responsive breast cancer cells by blocking G 0 /G 1 to S phase transition and inducing apoptosis. Moreover, AR overexpression inhibited USP14 inhibition-induced events, suggesting that AR deubiquitination by USP14 is critical for breast cancer growth and USP14 inhibition is a possible strategy to treat AR-positive breast cancer.

Objective: To explore the effect of intervention of E-cadherin (E-cad) and B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) mediated by transcription activator-like effector nuclease (TALEN) on the biological behaviors of nasopharyngeal carcinoma cells.
 Methods: Multi-locus gene targeting vectors pUC-DS1-CMV-E-cad-2A-Neo-DS2 and pUC-DS1-Bmi-1 shRNA-Zeo-DS2 were constructed, and the E-cad and Bmi-1 targeting vectors were transferred with TALEN plasmids to CNE-2 cells individually or simultaneously. The integration of target genes were detected by PCR, the expressions of E-cad and Bmi-1 were detected by Western blot. The changes of cell proliferation were detected by cell counting kit-8 (CCK-8) assay. The cell cycle and apoptosis were detected by flow cytometry. The cell migration and invasion were detected by Transwell assay.
 Results: The E-cad and Bmi-1 shRNA expression elements were successfully integrated into the genome of CNE-2 cells, the protein expression level of E-cad was up-regulated, and the protein expression level of Bmi-1 was down-regulated. The intervention of E-cad and Bmi-1 didn't affect the proliferation, cell cycle and apoptosis of CNE-2 cells, but it significantly inhibited the migration and invasion ability of CNE-2 cells. Furthermore, the intervention of E-cad and Bmi-1 together significantly inhibited the migration ability of nasopharyngeal carcinoma cells compared with the intervention of E-cad or Bmi-1 alone (all P<0.01).
 Conclusion: The joint intervention of E-cad and Bmi-1 mediated by TALEN can effectively inhibit the migration and invasion of nasopharyngeal carcinoma cells in vitro, which may lay the preliminary experimental basis for gene therapy of human cancer.

Background & Aims: Induction of nonapoptotic cell death could be an approach to eliminate apoptosis-resistant tumors. We investigated necroptosis-based therapies in mouse models of pancreatic ductal adenocarcinoma cancer (PDAC)., Methods: We screened 273 commercially available kinase inhibitors for cytotoxicity against a human PDAC cell line (PANC1). We evaluated the ability of the aurora kinase inhibitor CCT137690 to stimulate necroptosis in PDAC cell lines (PANC1, PANC2.03, CFPAC1, MiaPaCa2, BxPc3, and PANC02) and the HEK293 cell line, measuring loss of plasma membrane integrity, gain in cell volume, swollen organelles, and cytoplasmic vacuoles. We tested the effects of CCT137690 in colon formation assays, and the effects of the necroptosis (necrostatin-1 and necrosulfonamide), apoptosis, autophagy, and ferroptosis inhibitors. We derived cells from tumors that developed in Pdx1-Cre;K-Ras G12D/+ ;p53 R172H/+ (KPC) mice. Genes encoding proteins in cell death pathways were knocked out, knocked down, or expressed from transgenes in PDAC cell lines. Athymic nude or B6 mice were given subcutaneous injections of PDAC cells or tail-vein injections of KPC tumor cells. Mice were given CCT137690 (80 mg/kg) or vehicle and tumor growth was monitored; tumor tissues were collected and analyzed by immunohistochemistry. We compared gene expression levels between human pancreatic cancer tissues (n = 130) with patient survival times using the online R2 genomics analysis and visualization platform., Results: CCT137690 induced necrosis-like death in PDAC cell lines and reduced colony formation; these effects required RIPK1, RIPK3, and MLKL, as well as inhibition of aurora kinase A (AURKA). AURKA interacted directly with RIPK1 and RIPK3 to reduce necrosome activation. AURKA-mediated phosphorylation of glycogen synthase kinase 3 beta (GSK3β) at serine 9 inhibited activation of the RIPK3 and MLKL necrosome. Mutations in AURKA (D274A) or GSK3β (S9A), or pharmacologic inhibitors of RIPK1 signaling via RIPK3 and MLKL, reduced the cytotoxic activity of CCT137690 in PDAC cells. Oral administration of CCT137690 induced necroptosis and immunogenic cell death in subcutaneous and orthotopic tumors in mice, and reduced tumor growth and tumor cell phosphorylation of AURKA and GSK3β. CCT137690 increased survival times of mice with orthotopic KPC PDACs and reduced tumor growth, stroma, and metastasis. Increased expression of AURKA and GSK3β mRNAs associated with shorter survival times of patients with pancreatic cancer., Conclusions: We identified the aurora kinase inhibitor CCT137690 as an agent that induces necrosis-like death in PDAC cells, via RIPK1, RIPK3, and MLKL. CCT137690 slowed growth of orthotopic tumors from PDAC cells in mice, and expression of AURKA and GSK3β associate with patient survival times. AURKA might be targeted for treatment of pancreatic cancer., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

This study aimed to investigate the effects of SOST and the Wnt/β-catenin signaling pathway on the proliferation, migration, invasion, and apoptosis of human retinoblastoma cells. Fifty-five retinoblastoma and 21 normal retinal tissue samples were collected as the case group and control group, respectively. HXO-RB44 and SO-RB50 cells were selected and assigned into blank, negative control (NC), siRNA 1, siRNA 2, siRNA 3, IWR-1-endo 1, IWR-1-endo 2 and IWR-1-endo 3 groups. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the expression of SOST, Wnt-1, and β-catenin in the collected tissue samples. MTT assay, flow cytometry, transwell assay and the starch test were employed to determine the cell proliferation, cell cycle, apoptosis, invasion and migration after transfection. The qRT-PCR and western blotting were also used to detect the mRNA and protein expressions of SOST, Wnt-1, β-catenin, C-myc, Cyclin D1, MMP-2 and MMP-9. The tumor formation in nude mice was conducted to evaluate the effects of SOST on the growth of a transplanted tumor. Compared with normal retinal tissues, the retinoblastoma tissues exhibited a downregulation of SOST but an upregulation of Wnt-1 and β-catenin. The proliferation, invasion and migration of HXO-RB44 and SO-RB50 cells in the SOST-siRNA group were significantly higher than the cells in the blank and NC groups. The expressions of Wnt-1, β-catenin, C-myc, Cyclin D1, MMP-2 and MMP-9 in the three SOST-siRNA groups were elevated, but the SOST decreased when compared with the blank and NC groups. SOST silencing promoted the growth of transplanted tumors in nude mice. These findings indicate that SOST silencing promotes the proliferation, invasion and migration, and decreases the apoptosis of human retinoblastoma cells by activating the Wnt/β-catenin signaling pathway.

Bone marrow injury remains a serious concern in traditional cancer treatment. Ferroptosis is an iron- and oxidative-dependent form of regulated cell death that has become part of an emerging strategy for chemotherapy. However, the key regulator of ferroptosis in bone marrow injury remains unknown. Here, we show that Fanconi anemia complementation group D2 (FANCD2), a nuclear protein involved in DNA damage repair, protects against ferroptosis-mediated injury in bone marrow stromal cells (BMSCs). The classical ferroptosis inducer erastin remarkably increased the levels of monoubiquitinated FANCD2, which in turn limited DNA damage in BMSCs. FANCD2-deficient BMSCs were more sensitive to erastin-induced ferroptosis (but not autophagy) than FANCD2 wild-type cells. Knockout of FANCD2 increased ferroptosis-associated biochemical events (e.g., ferrous iron accumulation, glutathione depletion, and malondialdehyde production). Mechanically, FANCD2 regulated genes and/or expression of proteins involved in iron metabolism (e.g., FTH1, TF, TFRC, HAMP, HSPB1, SLC40A1, and STEAP3) and lipid peroxidation (e.g., GPX4). Collectively, these findings indicate that FANCD2 plays a novel role in the negative regulation of ferroptosis. FANCD2 could represent an amenable target for the development of novel anticancer therapies aiming to reduce the side effects of ferroptosis inducers., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ferroptosis, a recently identified form of non-apoptotic cell death, is involved in several physiological and pathological processes. Although lipid peroxidation plays a central role in triggering ferroptosis, the essential regulator of lipid metabolism in ferroptosis remains poorly defined. Here, we show that acyl-CoA synthetase long-chain family member 4 (ACSL4) is required for ferroptotic cancer cell death. Compared with ferroptosis-sensitive cells (e.g., HepG2 and HL60), the expression of ACSL4 was remarkably downregulated in ferroptosis-resistant cells (e.g., LNCaP and K562). In contrast, the expression of other ACSLs, including ACSL1, ACSL3, ACSL5, and ACSL6, did not correlate with ferroptosis sensitivity. Moreover, knockdown of ACSL4 by specific shRNA inhibited erastin-induced ferroptosis in HepG2 and HL60 cells, whereas overexpression of ACSL4 by gene transfection restored sensitivity of LNCaP and K562 cells to erastin. Mechanically, ACSL4-mediated production of 5-hydroxyeicosatetraenoic acid (5-HETE) contributed to ferroptosis. Pharmacological inhibition of 5-HETE production by zileuton limited ACSL4 overexpression-induced ferroptosis. Collectively, these results indicate that ACSL4 is not only a sensitive monitor of ferroptosis, but also an important contributor of ferroptosis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Ferroptosis is a form of non-apoptotic cell death originally identified in cancer cells. However, the key regulator of ferroptosis in mitochondria remains unknown. Here, we show that CDGSH iron sulfur domain 1 (CISD1, also termed mitoNEET), an iron-containing outer mitochondrial membrane protein, negatively regulates ferroptotic cancer cell death. The classical ferroptosis inducer erastin promotes CISD1 expression in an iron-dependent manner in human hepatocellular carcinoma cells (e.g., HepG2 and Hep3B). Genetic inhibition of CISD1 increased iron-mediated intramitochondrial lipid peroxidation, which contributes to erastin-induced ferroptosis. In contrast, stabilization of the iron sulfur cluster of CISD1 by pioglitazone inhibits mitochondrial iron uptake, lipid peroxidation, and subsequent ferroptosis. These findings indicate a novel role of CISD1 in protecting against mitochondrial injury in ferroptosis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Hematoporphyrin monomethyl ether (HMME) combined with He-Ne laser irradiation is a novel and promising photodynamic therapy (PDT)-induced apoptosis that can be applied in vitro on canine breast cancer cells. However, the exact pathway responsible for HMME-PDT in canine breast cancer cells remains unknown. CHMm cells morphology and apoptosis were analyzed using optical microscope, terminal deoxynucleotidyl transferase dUTP nick end labeling fluorescein staining and DNA ladder assays. Apoptotic pathway was further confirmed by Real-time-polymerase chain reaction and Western blotting assays. Our results showed that HMME-PDT induced significant changes in cell morphology, such as formation of cytoplasmic vacuoles and the gradual rounding of cells coupled with decreased size and detachment. DNA fragmentation and cell death was shown to occur in a time-dependent manner. Furthermore, HMME-PDT increased the activities of caspase-9 and caspase-3, and released cytochrome c from mitochondria into the cytoplasm. HMME-PDT also significantly increased both mRNA and protein levels of Bax and decreased P53 gene expression in a time-dependent manner, while the mRNA and protein expression of Bcl-2 were repressed. These alterations suggest that HMME-PDT induced CHMm cell apoptosis via the mitochondrial apoptosis pathway and had anti-canine breast cancer effects in vitro.

Ferroptosis, a novel form of regulated cell death, is characterized by oxidative injury from iron accumulation and lipid peroxidation. In a natural product library screening for ferroptosis inhibitor, we found that baicalein is a potent inhibitor of erastin-induced ferroptosis in pancreatic cancer cells. Baicalein (also termed 5,6,7-trihydroxyflavone) is a flavonoid originally obtained from the roots of Scutellaria baicalensis and Scutellaria lateriflora. We showed that baicalein exhibits remarkable anti-ferroptosis activity compared with well-known ferroptosis inhibitors such as ferrostatin-1, liproxstatin-1, deferoxamine mesylate, and β-mercaptoethanol. At the biochemistry level, baicalein limits erastin-induced ferrous iron production, glutathione depletion, and lipid peroxidation. At the protein level, baicalein suppresses erastin-mediated degradation of glutathione peroxidase 4, a phospholipid hydroperoxidase that protects cells against membrane lipid peroxidation. Thus, baicalein enhances cellular anti-ferroptosis capacity and could be a potential therapeutic agent for ferroptosis-associated tissue injury., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aim: Apoptosis is one of the most important mechanisms underlying renal tubulointerstitial fibrosis. We identified a role of protein Peroxiredoxin 1 (Prx1) in protecting apoptosis occurred in tubular epithelial cells of the rat and human kidney., Methods: Immunohistochemistry (IHC) staining was used to detect Prx1 expression in kidney derived from unilateral-ureteral obstruction (UUO) rats or patients with obstructive nephropathy. Modulation of Prx1 expression by transfecting siRNA and overexpression plasmid approach were carried out in NRK-52E (rat kidney tubular epithelial cell line) cells. UUO-induced apoptosis was determined using TUNEL assay., Results: Immunohistochemistry staining showed that Prx1 expressed in the cytoplasm of renal tubular epithelial cells, in the kidneys of UUO rats. The reduction was confirmed by both IHC and real-time polymerase chain reaction following a course of renal tubulointerstitial fibrosis in UUO rats and a decrease of Prx1 occurred concomitantly with an elevation of TUNEL-positive cells. Fluorofenidone (AKF-PD), a new anti-tubulointerstitial fibrotic agent, attenuated Prx1 reduction in UUO rats. Furthermore, hydrogen peroxide (H2 O2 )-derived oxidative stress activated p38 MAPK, and induced apoptosis in NRK-52E cells; knockdown of Prx1 sensitized both events in NRK-52E cells, and overexpression of Prx1 diminished the apoptosis and the phosphorylation of p38 CONCLUSION: Downregulation of Prx1 occurred in renal tubular epithelial cells of UUO rats and patients with obstructive nephropathy. Prx1 may alleviate the pathogenesis by inhibiting H2 O2 -induced apoptosis via inhibiting the p38 MAPK pathway. Prx1 may represent a useful target for a protective therapy towards renal tubulointerstitial fibrosis., (© 2015 Asian Pacific Society of Nephrology.)

Galectin-1, a β-galactoside-binding protein implicated in cancer cell immune privilege, was highly expressed in activated pancreatic stellate cells (PSCs). This study was designed to investigate the relationship between PSC-derived galectin-1 and tumor immunity in pancreatic cancer. Isolated PSCs were identified as normal pancreas cells (hNPSCs) or pancreatic cancer cells (hCaPSCs) by immunohistochemical staining for α-SMA and vimentin, and galectin-1 expression was evaluated by Western blotting and quantitative RT-PCR. Apoptosis, caspase activity, and cytokine production (IL-6, IL-10, TNF-β, and IFN-γ) of T cells co-cultured with PSCs were evaluated, and immunohistochemical staining of galectin-1 was correlated with CD3 and clinicopathological variables in 66 pancreatic cancer and 10 normal pancreatic tissue samples. hCaPSCs exhibited higher galectin-1 expression than did hNPSCs, and hCaPSCs induced higher levels of apoptosis in T cells following co-culture. hCaPSCs activated caspase-9 and caspase-3 in the mitochondrial apoptotic pathway and stimulated secretion of Th2 cytokines (IL-6 and IL-10) but decreased secretion of Th1 cytokines (TNF-β and IFN-γ), compared with hNPSCs. Immunohistochemical staining indicated that galectin-1 and CD3 were more highly expressed in pancreatic cancer tissue. Galectin-1 expression was highest in poorly differentiated pancreatic cancer cells and lowest in well-differentiated pancreatic cancer cells and was associated with tumor size, lymph node metastasis, differentiation, and UICC stage. However, CD3 expression showed the opposite trend and was highest in well-differentiated pancreatic cancer cells and was associated with tumor differentiation and UICC stage. High expression of galectin-1 was associated with short survival, as was low expression of CD3. hCaPSC-derived galectin-1 enhanced apoptosis and anergy of T cells in pancreatic cancer, which contributes to the immune escape of pancreatic cancer cells.

Unresectable colorectal liver metastases remain a major unresolved issue and more effective novel regimens are urgently needed. While screening synergistic drug combinations for colon cancer therapy, we identified a novel multidrug treatment for colon cancer: chemotherapeutic agent melphalan in combination with proteasome inhibitor bortezomib and mTOR (mammalian target of rapamycin) inhibitor rapamycin. We investigated the mechanisms of synergistic antitumor efficacy during the multidrug treatment. All experiments were performed with highly metastatic human colon cancer CX-1 and HCT116 cells, and selected critical experiments were repeated with human colon cancer stem Tu-22 cells and mouse embryo fibroblast (MEF) cells. We used immunochemical techniques to investigate a cross-talk between apoptosis and autophagy during the multidrug treatment. We observed that melphalan triggered apoptosis, bortezomib induced apoptosis and autophagy, rapamycin caused autophagy and the combinatorial treatment-induced synergistic apoptosis, which was mediated through an increase in caspase activation. We also observed that mitochondrial dysfunction induced by the combination was linked with altered cellular metabolism, which induced adenosine monophosphate-activated protein kinase (AMPK) activation, resulting in Beclin-1 phosphorylated at Ser 93/96. Interestingly, Beclin-1 phosphorylated at Ser 93/96 is sufficient to induce Beclin-1 cleavage by caspase-8, which switches off autophagy to achieve the synergistic induction of apoptosis. Similar results were observed with the essential autophagy gene, autophagy-related protein 7, -deficient MEF cells. The multidrug treatment-induced Beclin-1 cleavage was abolished in Beclin-1 double-mutant (D133A/D146A) knock-in HCT116 cells, restoring the autophagy-promoting function of Beclin-1 and suppressing the apoptosis induced by the combination therapy. These observations identify a novel mechanism for AMPK-induced apoptosis through interplay between autophagy and apoptosis.

Despite the development of chemoresistance as a major concern in prostate cancer therapy, the underlying mechanisms remain elusive. In this report, we demonstrate that DU145-derived prostate cancer stem cells (PCSCs) progress slowly with more cells accumulating in the G1 phase in comparison to DU145 non-PCSCs. Consistent with the important role of the AKT pathway in promoting G1 progression, DU145 PCSCs were less sensitive to growth factor-induced activation of AKT in comparison to non-PCSCs. In response to etoposide (one of the most commonly used chemotherapeutic drugs), DU145 PCSCs survived significantly better than non-PCSCs. In addition to etoposide, PCSCs demonstrated increased resistance to docetaxel, a taxane drug that is commonly used to treat castration-resistant prostate cancer. Etoposide produced elevated levels of γH2AX and triggered a robust G2/M arrest along with a coordinated reduction of the G1 population in PCSCs compared to non-PCSCs, suggesting that elevated γH2AX plays a role in the resistance of PCSCs to etoposide-induced cytotoxicity. We have generated xenograft tumors from DU145 PCSCs and non-PCSCs. Consistent with the knowledge that PCSCs produce xenograft tumors with more advanced features, we were able to demonstrate that PCSC-derived xenograft tumors displayed higher levels of γH2AX and p-CHK1 compared to non-PCSC-produced xenograft tumors. Collectively, our research suggests that the elevation of DNA damage response contributes to PCSC-associated resistance to genotoxic reagents., (Copyright © 2014 Elsevier Inc. All rights reserved.)

MIR34A (microRNA 34a) is a tumor suppressor gene, but how it regulates chemotherapy response and resistance is not completely understood. Here, we show that the microRNA MIR34A-dependent high mobility group box 1 (HMGB1) downregulation inhibits autophagy and enhances chemotherapy-induced apoptosis in the retinoblastoma cell. HMGB1 is a multifaceted protein with a key role in autophagy, a self-degradative, homeostatic process with a context-specific role in cancer. MIR34A inhibits HMGB1 expression through a direct MIR34A-binding site within the HMGB1 3' untranslated region. MIR34A inhibition of HMGB1 leads to a decrease in autophagy under starvation conditions or chemotherapy treatment. Inhibition of autophagy promotes oxidative injury and DNA damage and increases subsequent CASP3 activity, CASP3 cleavage, and PARP1 [poly (ADP-ribose) polymerase 1] cleavage, which are important to the apoptotic process. Finally, upregulation of MIR34A, knockdown of HMGB1, or inhibition of autophagy (e.g., knockdown of ATG5 and BECN1) restores chemosensitivity and enhances tumor cell death in the retinoblastoma cell. These data provide new insights into the mechanisms governing the regulation of HMGB1 expression by microRNA and their possible contribution to autophagy and drug resistance.

Endothelial cell (EC) apoptosis plays a pivotal role in the progression of diabetic complications. Abundant studies have demonstrated the pivotal role of advanced glycation end products (AGEs) on the development of diabetes. The aim of the present study was to investigate the effect of calycosin, a phytoestrogen, on AGEs-induced human umbilical vein endothelial cell (HUVEC) apoptosis. Fluorescence polarization and fluorescence absorption assays indicated that calycosin interacted with AGEs in a time-dependent manner. Further studies found that calycosin entered the cells as detected by HPLC. The MTT method demonstrated that calycosin ameliorated AGEs-induced HUVEC apoptosis in a dose-dependent manner, and statistical significance was observed at 1 × 10(-8) M of calycosin; this behavior was further demonstrated by acridine orange/ethidium bromide staining in that the presence of calycosin dramatically reduced AGEs-induced red staining in HUVECs. Further studies found that pre-incubation with calycosin at 1 × 10(-8) M dramatically increased anti-apoptotic Bcl-2 while decreased pro-apoptotic Bax and Bad expressions as detected by immunocytochemistry, and the effect of calycosin on rebalancing the ratio of Bcl-2/Bax was more significant than that of its glycoside, calycosin-7-O-β-D-glucopyranoside (CG). Furthermore, calycosin slightly reversed AGEs-induced cell oxidative stress at 1 × 10(-8) M, but its antioxidative stress effect was less significant than that of CG. The present study strongly indicates that calycosin can enter the cell and modulate endothelial cell dysfunction by ameliorating AGEs-induced cell apoptosis.

Background: Apoptosis caused by endoplasmic reticulum (ER) stress contributes to atherothrombosis, the underlying cause of cardiovascular disease (CVD). T-cell death-associated gene 51 (TDAG51), a member of the pleckstrin homology-like domain gene family, is induced by ER stress, causes apoptosis when overexpressed, and is present in lesion-resident macrophages and endothelial cells., Methods and Results: To study the role of TDAG51 in atherosclerosis, male mice deficient in TDAG51 and apolipoprotein E (TDAG51(-/-)/ApoE(-/-)) were generated and showed reduced atherosclerotic lesion growth (56 ± 5% reduction at 40 weeks, relative to ApoE(-/-) controls, P<0.005) and necrosis (41 ± 4% versus 63 ± 8% lesion area in TDAG51(-/-)/ApoE(-/-) and ApoE(-/-), respectively; P<0.05) without changes in plasma levels of lipids, glucose, and inflammatory cytokines. TDAG51 deficiency caused several phenotypic changes in macrophages and endothelial cells that increase cytoprotection against oxidative and ER stress, enhance PPARγ-dependent reverse cholesterol transport, and upregulate peroxiredoxin-1 (Prdx-1), an antioxidant enzyme with antiatherogenic properties (1.8 ± 0.1-fold increase in Prdx-1 protein expression, relative to control macrophages; P<0.005). Two independent case-control studies found that a genetic variant in the human TDAG51 gene region (rs2367446) is associated with CVD (OR, 1.15; 95% CI, 1.07 to 1.24; P=0.0003)., Conclusions: These findings provide evidence that TDAG51 affects specific cellular pathways known to reduce atherogenesis, suggesting that modulation of TDAG51 expression or its activity may have therapeutic benefit for the treatment of CVD.

Cyclooxygenase-2 (COX-2) plays an important role in the carcinogenesis and progression of gastric cancer. It has been demonstrated that COX-2 overexpression depends on different cellular pathways, involving both transcriptional and post-transcriptional regulation. MicroRNAs (miRNAs) are small, noncoding RNAs that function as post-transcriptional regulators. Here, we characterize miR-101 expression and its role in the regulation of COX-2 expression, which in turn, will provide us with additional insights into the potential therapeutic benefits of exogenous miR-101 for treatment of gastric cancer. Our results showed that miR-101 levels in gastric cancer tissues were significantly lower than those in the matched normal tissue (P < 0.01). Furthermore, lower levels of miR-101 were associated with increased tumor invasion and lymph node metastasis (P < 0.05). We also found an inverse correlation between miR-101 and COX-2 expression in both gastric cancer specimens and cell lines. Significant decreases in COX-2 mRNA and COX-2 levels were observed in the pre-miR-101-infected gastric cancer cells. One possible mechanism of interaction is that miR-101 inhibited COX-2 expression by directly binding to the 3'-UTR of COX-2 mRNA. Overexpression of miR-101 in gastric cancer cell lines also inhibited cell proliferation and induced apoptosis in vitro, as well as inhibiting tumor growth in vivo. These results collectively indicate that miR-101 may function as a tumor suppressor in gastric cancer, with COX-2 as a direct target., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Pathogen-associated molecular pattern molecules (PAMPs) are derived from microorganisms and recognized by pattern recognition receptor (PRR)-bearing cells of the innate immune system as well as many epithelial cells. In contrast, damage-associated molecular pattern molecules (DAMPs) are cell-derived and initiate and perpetuate immunity in response to trauma, ischemia, and tissue damage, either in the absence or presence of pathogenic infection. Most PAMPs and DAMPs serve as so-called 'Signal 0s' that bind specific receptors [Toll-like receptors, NOD-like receptors, RIG-I-like receptors, AIM2-like receptors, and the receptor for advanced glycation end products (RAGE)] to promote autophagy. Autophagy, a conserved lysosomal degradation pathway, is a cell survival mechanism invoked in response to environmental and cellular stress. Autophagy is inferred to have been present in the last common eukaryotic ancestor and only to have been lost by some obligatory intracellular parasites. As such, autophagy represents a unifying biology, subserving survival and the earliest host defense strategies, predating apoptosis, within eukaryotes. Here, we review recent advances in our understanding of autophagic molecular mechanisms and functions in emergent immunity., (© 2012 John Wiley & Sons A/S.)

The balance between apoptosis ("programmed cell death") and autophagy ("programmed cell survival") is important in tumor development and response to therapy. Here, we show that high mobility group box 1 (HMGB1) and p53 form a complex that regulates the balance between tumor cell death and survival. We show that knockout of p53 in HCT116 cells increases expression of cytosolic HMGB1 and induces autophagy. Conversely, knockout of HMGB1 in mouse embryonic fibroblasts increases p53 cytosolic localization and decreases autophagy. p53 is thus a negative regulator of the HMGB1/Beclin 1 complex, and HMGB1 promotes autophagy in the setting of diminished p53. HMGB1-mediated autophagy promotes tumor cell survival in the setting of p53-dependent processes. The HMGB1/p53 complex affects the cytoplasmic localization of the reciprocal binding partner, thereby regulating subsequent levels of autophagy and apoptosis. These insights provide a novel link between HMGB1 and p53 in the cross-regulation of apoptosis and autophagy in the setting of cell stress, providing insights into their reciprocal roles in carcinogenesis.

Autophagy and apoptosis are tightly regulated biological processes that are crucial for cell growth, development and tissue homeostasis. UVRAG (UV radiation resistance-associated gene), a mammalian homolog of yeast Vps38, activates the Beclin 1/PtdIns3KC3 (class III phosphatidylinositol-3-kinase) complex, which promotes autophagosome formation. Moreover, UVRAG promotes autophagosome maturation by recruiting class C Vps complexes (HOPS complexes) and Rab7 of the late endosome. We found that UVRAG has anti-apoptotic activity during tumor therapy through interactions with Bax. UVRAG inhibits Bax translocation from the cytosol to mitochondria during chemotherapy- or UV irradiation-induced apoptosis of human tumor cells. Moreover, deletion of the UVRAG C2 domain abolishes Bax binding and anti-apoptotic activity. These results suggest that, in addition to its previously recognized pro-autophagy activity in response to starvation, UVRAG has cytoprotective functions in the cytosol that control the localization of Bax in tumor cells exposed to apoptotic stimuli.