ALDH2 activation attenuates oxygen–glucose deprivation/reoxygenation-induced cell apoptosis, pyroptosis, ferroptosis and autophagy

Purpose It is previously reported that aldehyde dehydrogenase 2 family member (ALDH2) shows neuroprotective effects in cerebral ischemia/reperfusion injury. However, whether the protective effects are through mediating the programmed cell death is yet to be fully elucidated. Methods In vitro oxygen–glucose deprivation/reoxygenation (OGD/R) model was established in HT22 cells and mouse cortical neurons. Subsequently, ALDH2 expression were assessed by qRT-PCR and western blot. The methylation status was examined by methylation-specific PCR (MS-PCR). Then, ALDH2 expression was promoted and suppressed to explore the role of ALDH2 in OGD/R-treated cells. CCK-8 assay was applied to detect cell viability, and flow cytometry was applied to evaluate cell apoptosis. Western blot was applied to detect the apoptosis-related proteins (Caspase 3, Bcl-2 and Bax), necroptosis-related proteins (RIP3 and MLKL), pyroptosis-related proteins (NLRP3 and GSDMD), ferroptosis-related protein (ACSL4 and GPX4), and autophagy-related proteins (LC3B, and p62). IL-1β and IL-18 production was evaluated by ELISA assay. Reactive oxygen species production and Fe2+ content were evaluated by the corresponding detection kit. Results In OGD/R-treated cells, ALDH2 expression was decreased, which was due to the hypermethylation of ALDH2 in the promoter region. ALDH2 overexpression improved cell viability and ALDH2 knockdown suppressed cell viability in OGD/R-treated cells. We also found that ALDH2 overexpression attenuated OGD/R-induced cell apoptosis, pyroptosis, ferroptosis and autophagy, while ALDH2 knockdown facilitated the OGD/R-induced cell apoptosis, pyroptosis, ferroptosis and autophagy. Conclusions Collectively, our results implied that ALDH2 attenuated OGD/R-induced cell apoptosis, pyroptosis, ferroptosis and autophagy to promote cell viability in HT22 cells and mouse cortical neurons (AU)

BAG3 protects chondrocytes against lumbar facet joint osteoarthritis by regulating autophagy and apoptosis

Bcl2-associated athanogene3 (BAG3) protein, mainly induced by stressful stimuli, has been confirmed to participate in apoptosis and autophagy. In recent studies, BAG3 has gradually become a key molecule in tumors. However, the role of BAG3 in the progression of lumbar facet joint osteoarthritis (FJOA) and whether it can regulate chondrocyte apoptosis and autophagy are still unknown. In both human and FJOA rat models, we observed an upregulation of BAG3 and apoptosis and autophagy-related proteins compared with healthy tissues. Then, we established the chondrocytes injury model in vitro by using IL-1β to stimulate human SW1353 cells. Western blot analysis data showed significant expression of BAG3, apoptosis, and autophagy-related proteins in SW1353 cells. Finally, by knocking down and overexpressing BAG3, we discovered possible anti-apoptotic and autophagy-promoted effects of BAG3 in FJOA through various experimental methods. This study demonstrated that BAG3 actively participates in regulating chondrocyte apoptosis and autophagy in FJOA and may be a highly interesting target for pharmacological interventions. (AU)

STAT1/IFIT2 signaling pathway is involved in PD-L1-mediated epithelial-to-mesenchymal transition in human esophageal cancer

BackgroundWe have previously reported significant change of epithelial to mesenchymal transition (EMT) phenotype of Eca-109 cells upon PD-L1 operation, and the cytoplasmic domain of PD-L1 played an essential role in promoting EMT of esophageal cancer cells. However, the underlying mechanism of how PD-L1 regulated EMT in esophageal cancer remained unclear.MethodsThe overexpression and knockdown expression models of PD-L1 and IFIT2 were established by using lenti-virus transfection and RNAi method. Western blotting, qRT-PCR, CCK8 assay, transwell assay and wound healing assay were chosen to investigate their impact on the cells. The expression levels of IFIT2 and EMT markers in esophageal cancer tissues were examined by immunohistochemical staining. The rescue experiments were further applied to investigate the role of STAT1/IFIT2 signal pathway in the PD-L1-mediated EMT. Luciferase reporter assays were performed to examine the IFIT2 promoter activities upon knockdown expression of PD-L1 to identify the putative targeted region of IFIT2 promoter.ResultsThe STAT1/IFIT2 signal pathway was activated when PD-L1 was knockdown in human esophageal cancer cells. Decreased IFIT2 expression significantly increased the cellular abilities of viability, invasion and migration by using RNAi method in human esophageal cancer cells. Decreased IFIT2 expression in esophageal cancer tissues significantly correlated with EMT status, and could be used as an independent prognostic predictor for the patients. Rescue experiments in PD-L1 knockdown cells further confirmed that STAT1/IFIT2 pathway was involved in the PD-L1 mediated EMT of esophageal cancer cells. Moreover, the luciferase reporter assay also confirmed that in esophageal cancer cells, the promoter region of IFIT2 (-3K~-1K) remains more active in PD-L1 knockdown expression cells compared with controls.

Protective role of downregulated MLK3 in myocardial adaptation to chronic hypoxia

A series of protective responses could be evoked to achieve compensatory adaptation once cardiomyocytes are subjected to chronic hypoxia. MLK3/JNK/c-jun signaling pathway was previously demonstrated to be involved in this process. In the present study, we aim to further examine the performance of MLK3 in hypoxic H9C2 cells and potential mechanism. Myocardial samples of patients with congenital heart disease (CHD) were collected. H9C2 cells were cultured in hypoxic conditions for various durations. MLK3 was silenced by transfection of shRNA to evaluate its role in cell viability. We found expression of MLK3 protein was lower in patients with cyanotic CHD. In hypoxic H9C2 cells, its expression was gradually decreased in a time-dependent manner. However, there was no significant difference about expression of MLK3 mRNA. According to the results of MTT, LDH, and TUNEL, faster cell growth curve, lower death rate, and less apoptotic cells could be observed in MLK-shRNA group compared with scramble-shRNA group. Silencing of MLK3 significantly reduced expression of cleaved caspase-3, cleaved PARP, Bad, and Bax, together with increased expression of Bcl-2 and ration of Bcl-2/Bax. Both ratio of phospho-JNK/total JNK and ratio of phospho-c-jun/total c-jun were significantly decreased once MLK3 was silenced. At various reoxygenation time, MLK3 shRNA could significantly promote cell survival and decrease cell death according to MTT and LDH. Our results suggested that chronic hypoxia could reduce MLK3 expression in a posttranscriptional regulatory manner. Downregulation of MLK3 protects H9C2 cells from hypoxia-induced apoptosis and H/R injury via blocking the activation of JNK and c-jun

CC genotype of anti-apoptotic gene BCL-2 (-938 C/A) is an independent prognostic marker of unfavorable clinical outcome in patients with non-small-cell lung cancer

Background: B cell lymphoma 2 (BCL-2) gene is a well-known regulator of apoptosis and a key element in cancer development and progression. A regulatory (−938C>A, rs2279115) single-nucleotide polymorphism in the inhibitory P2 BCL-2 gene promoter generates significantly different BCL-2 promoter activities and has been associated with different clinical outcomes in various malignancies. The aim of the present study was to analyze the possible influence of the (−938C>A) SNP on the risk and survival of Indian patients suffering from NSCLC. Materials and methods: A hospital-based case–control study of 155 age- and sex-matched patients diagnosed with NSCLC and 155 cancer-free controls was conducted and genotyped by performing PIRA–PCR to elucidate the putative association between clinical outcome and genotypes of BCL-2 (−938C>A, rs2279115). The association of the polymorphism with the survival of NSCLC patients was analyzed by Kaplan–Meier curves. Results: In Indian NSCLC, patients increased risk of developing NSCLC was found to be associated with BCL-2 (−938) CC genotype, [OR 3.68 (1.92−6.79), RR 1.87 (1.35−2.57) and RD 31.03 (16.79−45.27) p 0.00006 for CC and OR 2.08 (1.18−3.66), RR 1.36 (1.08−1.71) and RD 17.74 (4.68−30.81) p 0.01 for AC genotype]. Patients homozygous for C allele exhibited a significant poor overall survival compared with patients displaying AC + CC or AC or AA genotype [median survival (months) 8 vs. 11 vs. 14 vs. 35.5 (p < 0.0001)]. In addition, significant associations were observed between TNM stage, histological type, distant metastases status, family history of any cancer, gender and age group of NSCLC patients with BCL-2 (−938C>A) polymorphism. Conclusion: Genetic polymorphism in the inhibitory P2 promoter region of anti-apoptotic BCL-2 genes contributes to the risk of developing non-small-cell lung cancer in Indian population. BCL-2 (−938CC) genotype was an independent adverse prognostic factor for patients with NSCLC (AU)

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Secretory leukocyte protease inhibitor is a proliferation and survival factor for pancreatic cancer cells

Objectives: A variety of inflammatory cytokines have been demonstrated to participate in tumorigenesis and progression. Secretory leukocyte protease inhibitor (SLPI) has been demonstrated to show a broad-spectrum of anti-inflammatory effects. This study investigates the expression of SLPI in human pancreatic cancer tissues and cells as well as its biological effects in human pancreatic cancer cells. Methods: Reverse transcription-polymerase chain reaction, immunohistochemistry, and Western blot were used to detect SLPI mRNA and protein levels in human pancreatic cancer tissues, adjacent tissues, and pancreatic cancer Bxpc-3 and Panc-1 cells. Knockout of SLPI expression was established by recombinant viral vector expressing short hairpin RNA (shRNA) targeting SLPI. Cell viability was analyzed by MTT assay. Cell apoptosis was detected by Hochest33258 staining and flow cytometry assay. Results: Higher SLPI expression was observed in pancreatic tissues, Bxpc-3 cells, and Panc-1 cells compared to the peritumoral tissues (p < 0.01). SLPI expression in Bxpc-3 and Panc-1 cells was effectively silenced by shRNA (p < 0.001). Silencing of SLPI expression significantly reduced cell viability, inhibited cell proliferation, and induced cell apoptosis (p < 0.001). Conclusions: Abnormal over-expression of SLPI in pancreatic cancer cells may be associated with the development of disease through its roles in promoting cancer cell survival and proliferation as well as anti-apoptosis. SLPI can be used as a target for developing targeted therapy of pancreatic cancer (AU)

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BIK/NBK gene as potential marker of prognostic and therapeutic target in breast cancer patients

PURPOSE: The purpose of this study is to determine the association between the BIK/NBK gene expression and estrogen receptor alpha expression. MATERIALS AND METHODS: We determined the association of BIK/NBK gene expression by real time quantitative reverse transcription polymerase chain reaction and estrogen receptor alpha expression by immunohistochemistry in samples of breast cancer tissue. RESULTS: We found a statistically significant correlation of BIK/NBK gene expression with the estrogen receptor alpha expression (ρ = 0.751, p = 0.004). For verify differences of BIK/NBK gene expression among ERα+ and ERα- breast cancer tissues, Mann-Whitney U test was performed, obtaining significant differences. CONCLUSIONS: BIK/NBK gene expression may have important clinical implications and provide predictive, prognostic or therapeutic marker in breast cancer patients according to the estrogen receptor alpha expression (AU)

hRpn13, a newly identified component of the 19S particle, regulates proliferation, differentiation, and function in the human osteoblast-like cell line MG63

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The 26S proteasome is a key component of the ubiquitin-proteasome system, a process responsible for the majority of cellular protein degradation. The function of the proteasomal ubiquitin receptor hRpn13, a component of the 26S proteasome, is not completely understood. To investigate the role of hRpn13 in the ubiquitin-proteasome system in osteoblasts, the effects of suppressing and overexpressing the hRpn13 gene on proliferation, differentiation, and function of human osteoblast-like MG63 cells were examined. After knockdown of hRpn13 by small interfering RNA, changes in osteoblast proliferation were evaluated by methyl-thiazolyl-tetrazolium assay. There was an increase in markers for osteoblast proliferation, specifically alkaline phosphatase activity, and elevated protein levels of osteocalcin, proliferating cell nuclear antigen (PCNA), and ubiquitin. Furthermore, hRpn13 knockdown also resulted in a decrease in the ratio between the gene expressions of RANKL and OPG, key players in the pathogenesis of bone diseases that influence the normal balance between bone formation and resorption. In contrast, overexpression of hRpn13 inhibited the proliferation of MG63 cells, and decreased alkaline phosphatase activity as well as protein levels of osteocalcin, PCNA, and ubiquitin while the ratio of RANKL to OPG expression increased. To confirm the function of hRpn13 in the ubiquitin-proteasome pathway, osteoblast proliferation enhancement and ubiquitin accumulation after hRpn2 knockdown was assessed. The results suggest that overexpression of hRpn13 negatively influences proliferation and osteogenic differentiation in MG63 cells. The evidence implies that hRpn13 modulates the influence of osteoblasts on osteoclasts by controlling the stability of regulatory proteins in osteoblasts. In summary, overexpression of hRpn13 promoted the activity of the ubiquitin-proteasome system (AU)

Estabilización clínica en enfermedades neurodegenerativas: estudio clínico en fase II / Clinical stabilisation in neurodegenerative diseases: clinical study in phase II

Introducción. Los trastornos neurodegenerativos, como enfermedad de Parkinson (EP), enfermedad de Alzheimer (EA) y esclerosis múltiple (EM), son patologías progresivas cuyos tratamientos actuales no han demostrado eficacia para detener su progresión (estabilización clínica). Aunque tienen características clínicas muy distintas, comparten mecanismos fisiopatológicos de progresión. Desarrollamos un compuesto diseñado para obtener estabilización clínica, al controlar el daño celular por apoptosis aberrante, oxidación, depósito de metales y proteínas anormales y de vías enzimáticas fisiopatológicas, como la de las caspasas y el sistema MAPK. Pacientes y métodos. Incluimos 42 pacientes con EA, EP y EM. Les administramos el compuesto cada 12 horas y los citamos trimestralmente para evaluación clínica y revisión de exámenes de laboratorio generales. Resultados. Se realizó seguimiento de 3 a 24 meses (media: 8,85 ± 5,99 meses). No se registraron efectos clínicos adversos y sólo aisladas y leves alteraciones en los resultados de laboratorio, sin importancia clínica. Obtuvimos estabilización clínica en todos los pacientes (100%) con EM y mejoría en las puntuaciones de la Expanded Disability Status Scale en 4 pacientes (40%); estabilización clínica en 17 pacientes (100%) con EP y mejoría en puntuación de la Unified Parkinson’s Disease Rating Scale en 15 (88,2%); y estabilización clínica en los 12 pacientes (100%) con EA y aumento de la calificación del test minimental en 9 (75%). Conclusión. El compuesto es seguro y una opción terapéutica prometedora, al observarse una evidente tendencia hacia la estabilización clínica por medio de su utilización. Debe realizarse un estudio experimental para establecer el alcance terapéutico del compuesto, su posible efecto preventivo y valorar otras indicaciones (AU)

Introduction. Neurodegenerative disorders, such as Parkinson’s disease (PD), Alzheimer’s disease (AD) and multiple sclerosis (MS), are progressive pathological conditions in which current treatments have not proved to be effective at curbing their progress (clinical stabilisation). Although they have very different clinical characteristics, they share the same pathophysiological mechanisms of progression. We developed a compound designed to obtain clinical stabilisation which acts by controlling cell damage due to aberrant apoptosis, oxidation, abnormal deposits of metals and proteins, and pathophysiological enzymatic pathways, such as that of caspases and the MAPK system. Patients and methods. Forty-two patients with AD, PD and MS were included in the study. The compound was administered to them every 12 hours and they were given appointments every three months for a clinical evaluation and a review of general lab analyses. Results. Subjects were submitted to a follow-up of between 3 and 24 months (mean: 8.85 ± 5.99 months). No clinical side effects were recorded and there were only some slight alterations in the lab test results, although they were not clinically relevant. Clinical stabilisation was achieved in all the patients (100%) with MS and the scores on the Expanded Disability Status Scale improved in four patients (40%); clinical stabilisation in 17 patients (100%) with PD and improvements in the score on the Unified Parkinson’s Disease Rating Scale in 15 of them (88.2%); and clinical stabilisation in 12 patients (100%) with AD, and an increase in the score obtained on the minimental test in nine cases (75%). Conclusions. The compound is safe and a promising therapeutic option, since there is a clear tendency towards clinical stabilisation when it is being used. An experimental study needs to be conducted in order to determine the therapeutic scope of the compound and its possible preventive effects, as well as to evaluate other indications (AU)

Apoptosis regulators as targets for cancer therapy

Apoptosis serves to remove excess or damaged cells and its dysregulation may lead to a number of pathological disorders including cancer. Studies during the last 20 years have unravelled much of the molecular mechanisms that control apoptosis. Whether a cell dies in response to diverse apoptotic stimuli, including DNA-damaging agents, is determined largely by interactions between proteins of the Bcl-2 family. A death signal is transmitted through the BH3-only proteins to Bax and Bak which in turn permeabilise the outer mitochondrial membrane allowing the release of apoptogenic factors, which triggers activation of cell-deathpromoting caspases. These proteolytic enzymes are tightly controlled by members of the inhibitor of apoptosis (IAP) family. Activation of the caspase cascade via cell death receptors also represents a key apoptotic pathway in both normal and tumour cells. Basic knowledge of these apoptosis regulators provides the basis for novel therapeutic strategies aimed at promoting tumour cell death or enhancing susceptibility to apoptotic inducers. This review focuses on these strategies (AU)

C4BP and CD46 (MCP) in the control of inflammation and autoimmunity

En este Panorama se discuten varias publicaciones recientes acerca de las funciones de las proteinas reguladoras del Complemento C4BP y MCP (CD46), y sus posibles implicaciones en la regulación de los procesos inflamatorios, la respuesta immune adaptativa, y la autoimmunidad (AU)

Several recent publications showing novel functions of the Complement Regulatory Proteins C4BP and MCP (CD46) arereviewed. Their potential consequences in the regulation of inflammation,adaptive immune responses, and autoimmunity are discussed (AU)