Protective Autophagy in 5-Fluorouracil-Resistant Colorectal Cancer Cells and ERK-RSK-ABCG2 Linkage / Autofagia Protectora en Células de Cáncer Colorrectal Resistentes al 5-Fluorouracilo y Enlace ERK-RSK-ABCG2

SUMMARY: To evaluate the anti-cancer effects of yeast extract on resistant cells, autophagy and necroptosis were investigated in 5-fluorouracil (5-FU)-resistant colorectal cancer cells. Further underlying characteristics on drug resistance were evaluated, focused on ERK-RSK-ABCG2 linkage. SNU-C5 and 5-FU resistant SNU-C5 (SNU-C5/5-FUR) colorectal cancer cells were adopted for cell viability assay and Western blotting to examine the anti-cancer effects of yeast extract. Yeast extract induced autophagy in SNU-C5 cells with increased Atg7, Atg12-5 complex, Atg16L1, and LC3 activation (LC3-II/LC3-I), but little effects in SNU-C5/5-FUR cells with increased Atg12-5 complex and Atg16L1. Both colorectal cancer cells did not show necroptosis after yeast extract treatment. Based on increased ABCG2 and RSK expression after yeast extract treatment, drug resistance mechanisms were further evaluated. As compared to wild type, SNU-C5/5-FUR cells showed more ABCG2 expression, less RSK expression, and less phosphorylation of ERK. ABCG2 inhibitor, Ko143, treatment induces following changes: 1) more sensitivity at 500 mM 5-FU, 2) augmented proliferation, and 3) less phosphorylation of ERK. These results suggest that protective autophagy in SNU-C5/5-FUR cells with increased ABCG2 expression might be candidate mechanisms for drug resistance. As the ERK responses were different from each stimulus, the feasible mechanisms among ERK-RSK-ABCG2 should be further investigated in 5-FU-resistant CRC cells.

Para evaluar los efectos anticancerígenos del extracto de levadura en células resistentes, se investigaron la autofagia y la necroptosis en células de cáncer colorrectal resistentes al 5-fluorouracilo (5-FU). Además se evaluaron otras características subyacentes de la resistencia a los medicamentos centrándose en el enlace ERK-RSK-ABCG2. Se usaron células de cáncer colorrectal SNU-C5 (SNU-C5/5-FUR) resistentes a SNU-C5 y 5- FU para el ensayo de viabilidad celular y la transferencia Western para examinar los efectos anticancerígenos del extracto de levadura. El extracto de levadura indujo autofagia en células SNU-C5 con mayor activación de Atg7, complejo Atg12-5, Atg16L1 y LC3 (LC3-II/LC3-I), pero pocos efectos en células SNU-C5/5-FUR con aumento de Atg12-5 complejo y Atg16L1. Ambas células de cáncer colorrectal no mostraron necroptosis después del tratamiento con extracto de levadura. Se evaluaron los mecanismos de resistencia a los medicamentos. en base al aumento de la expresión de ABCG2 y RSK después del tratamiento con extracto de levadura.En comparación con las de tipo salvaje, las células SNU-C5/5-FUR mostraron más expresión de ABCG2, menos expresión de RSK y menos fosforilación de ERK. El tratamiento con inhibidor de ABCG2, Ko143, induce los siguientes cambios: 1) más sensibilidad a 5-FU 500 mM, 2) proliferación aumentada y 3) menos fosforilación de ERK. Estos resultados sugieren que la autofagia protectora en células SNU-C5/5-FUR con mayor expresión de ABCG2 podría ser un mecanismo candidato para la resistencia a los medicamentos. Como las respuestas de ERK fueron diferentes de cada estímulo, los mecanismos factibles entre ERK-RSK- ABCG2 deberían investigarse más a fondo en células CCR resistentes a 5-FU.

Progress in study on the final executor of necroptosis MLKL and its inhibitors / 中南大学学报(医学版)

Necroptosis is one of the regulated cell death, which involves receptor interacting protein kinase (RIPK) 1/RIPK3/mixed lineage kinase domain like protein (MLKL) signaling pathway. Among them, MLKL is the final execution of necroptosis. The formation of RIPK1/RIPK3/MLKL necrosome induces the phosphorylated MLKL, and the activated MLKL penetrates into the membrane bilayer to form membrane pores, which damages the integrity of the membrane and leads to cell death. In addition to participating in necroptosis, MLKL is also closely related to other cell death, such as NETosis, pyroptosis, and autophagy. Therefore, MLKL is involved in the pathological processes of various diseases related to abnormal cell death pathways (such as cardiovascular diseases, neurodegenerative diseases and cancer), and may be a therapeutic target of multiple diseases. Understanding the role of MLKL in different cell death can lay a foundation for seeking various MLKL-related disease targets, and also guide the development and application of MLKL inhibitors.

Resveratrol Prevents Vibrio vulnificus-Induced Sepsis by Attenuating Necroptosis / 生物医学与环境科学（英文）

OBJECTIVE@#This study investigated how the natural phytophenol and potent SIRT1 activator resveratrol (RSV) regulate necroptosis during Vibrio vulnificus (V. vulnificus)-induced sepsis and the potential mechanism.@*METHODS@#The effect of RSV on V. vulnificus cytolysin (VVC)-induced necroptosis was analyzed in vitro using CCK-8 and Western blot assays. Enzyme-linked immunosorbent assays and quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry and survival analyses were performed to elucidate the effect and mechanism of RSV on necroptosis in a V. vulnificus-induced sepsis mouse model.@*RESULTS@#RSV relieved necroptosis induced by VVC in RAW264.7 and MLE12 cells. RSV also inhibited the inflammatory response, had a protective effect on histopathological changes, and reduced the expression level of the necroptosis indicator pMLKL in peritoneal macrophages, lung, spleen, and liver tissues of V. vulnificus-induced septic mice in vivo. Pretreatment with RSV downregulated the mRNA of the necroptosis indicator and protein expression in peritoneal macrophages and tissues of V. vulnificus-induced septic mice. RSV also improved the survival of V. vulnificus-induced septic mice.@*CONCLUSION@#Our findings collectively demonstrate that RSV prevented V. vulnificus-induced sepsis by attenuating necroptosis, highlighting its potency in the clinical management of V. vulnificus-induced sepsis.

Effects and mechanisms of total flavones of Abelmoschus manihot in inhibiting podocyte necroptosis and renal fibrosis in diabetic kidney disease / 中国中药杂志

Previous studies have shown that high blood glucose-induced chronic microinflammation can cause inflammatory podocyte injury in patients with diabetic kidney disease(DKD). Therein, necroptosis is a new form of podocyte death that is closely associated with renal fibrosis(RF). To explore the effects and mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese herbal medicine Abelmoschus manihot for treating kidney diseases, on podocyte necroptosis and RF in DKD, and to further reveal its scientific connotation with multi-pathway and multi-target, the authors randomly divided all rats into four groups: a namely normal group, a model group, a TFA group and a rapamycin(RAP) group. After the modified DKD rat models were successfully established, four group rats were given double-distilled water, TFA suspension and RAP suspension, respectively by gavage every day. At the end of the 4th week of drug treatment, all rats were sacrificed, and the samples of their urine, blood and kidneys were collected. And then, the various indicators related to podocyte necroptosis and RF in the DKD model rats were observed, detected and analyzed, respectively. The results indicated that, general condition, body weight(BW), serum creatinine(Scr), urinary albumin(UAlb), and kidney hypertrophy index(KHI) in these modified DKD model rats were both improved by TFA and RAP. Indicators of RF, including glomerular histomorphological characteristics, fibronectin(FN) and collagen type Ⅰ(collagen Ⅰ) staining extent in glomeruli, as well as the protein expression levels of FN, collagen Ⅰ, transforming growth factor-β1(TGF-β1) and Smad2/3 in the kidneys were improved respectively by TFA and RAP. Podocyte damage, including foot process form and the protein expression levels of podocin and CD2AP in the kidneys was improved by TFA and RAP. In addition, tumor necrosis factor-α(TNF-α)-mediated podocyte necroptosis in the kidneys, including the morphological characteristics of podocyte necroptosis, the extent and levels of the protein expression of TNF-α and phosphorylated mixed lineage kinase domain like pseudokinase(p-MLKL) was improved respectively by TFA and RAP. Among them, RAP had the better effect on p-MLKL. More importantly, the activation of the receptor interacting serine/threonine protein kinase 1(RIPK1)/RIPK3/MLKL signaling axis in the kidneys, including the expression levels of its key signaling molecules, such as phosphorylated receptor interacting serine/threonine protein kinase 1(p-RIPK1), p-RIPK3, p-MLKL and cysteinyl aspartate specific proteinase-8(caspase-8) was improved respectively by TFA and RAP. Among them, the effect of TFA on p-RIPK1 was superior. On the whole, in this study, the authors demonstrated that TFA alleviates podocyte necroptosis and RF in DKD through inhibiting the activation of the TNF-α-mediated RIPK1/RIPK3/MLKL signaling axis in diabetic kidneys. The authors' findings provide new pharmacological evidence to reveal the scientific connotation of TFA in treating RF in DKD in more depth.

The role of regulated cell death in renal ischemia-reperfusion injury / 生理学报

Renal ischemia-reperfusion injury (IRI) is histologically characterized by tubular cell death. Diverse pathways of regulated cell death (RCD) have been reported to contribute to renal IRI in recent studies. In this review, we discuss the signaling pathways, regulators and crosstalk of RCD, including necroptosis, ferroptosis and pyroptosis, and their role in renal IRI in order to pave the way for new therapeutic opportunities.

Acute kidney injury and regulated cell death / 生理学报

Acute kidney injury (AKI) refers to a clinical syndrome in which renal function declines rapidly in a short period of time caused by various pathological factors. During the development of AKI, renal tubules with the functions of reabsorption and excretion are prone to cell death due to external pathological stimuli, which is an important cause of impaired renal function. In recent years, a variety of new cell death pathways have been gradually recognized. Researchers have now found that regulated cell death (RCD), such as necroptosis, pyroptosis and ferroptosis, are important regulatory mechanisms of AKI. This article will summarize the research advances of various types of RCD involved in the process of AKI, aiming to deepen the understanding of AKI and provide innovative thoughts for the clinical treatment of AKI.

Liver fibrosis inhibits lethal injury through D-galactosamine/lipopolysaccharide-induced necroptosis / 中华肝脏病杂志

Objective: To explore the new mechanism of liver fibrosis through D-galactosamine/lipopolysaccharide (D-GalN/LPS)-induced necroptosis as an entry point to inhibit lethal injury. Methods: The carbon tetrachloride (CCl4)-induced mouse model of liver fibrosis was established. At 6 weeks of fibrosis, the mice were challenged with a lethal dose of D-GalN/LPS, and the normal mice treated with the same treatment were used as the control. The experiment was divided into four groups: control group (Control), acute injury group (D-GalN/LPS), liver fibrosis group (Fib), and liver fibrosis + acute challenge group (Fib + D-GalN/LPS). Quantitative PCR and immunofluorescence were used to analyze the expression of necroptosis key signal molecules RIPK1, RIPK3, MLKL and/or P-MLKL in each group. Normal mice were treated with inhibitors targeting key signaling molecules of necroptosis, and then given an acute challenge. The inhibitory effect of D-GalN/LPS-induced-necroptosis on acute liver injury was evaluated according to the changes in transaminase levels and liver histology. Liver fibrosis spontaneous ablation model was established, and then acute challenge was given. Necroptosis key signal molecules expression was analyzed in liver tissue of mice in each group and compared by immunohistochemistry. The differences between groups were compared with t-test or analysis of variance. Results: Quantitative PCR and immunofluorescence assays result showed that D-GalN/LPS-induced significant upregulation of RIPK1, RIPK3, MLKL and/or P-MLKL. Necroptosis key signal molecules inhibition had significantly reduced D-GalN/LPS-induced liver injury, as manifested by markedly reduced serum ALT and AST levels with improvement in liver histology. Necroptosis signaling molecules expression was significantly inhibited in fibrotic livers even under acute challenge conditions. Additionally, liver fibrosis with gradual attenuation of fibrotic ablation had inhibited D-GalN/LPS-induced necroptosis. Conclusion: Liver fibrosis may protect mice from acute lethal challenge injury by inhibiting D-GalN/LPS-induced necroptosis.

RIP1-dependent linear and nonlinear recruitments of caspase-8 and RIP3 respectively to necrosome specify distinct cell death outcomes

There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways. By employing the SWATH-MS technique, we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes. Combining SWATH-MS-based network modeling and experimental validation, we found that when RIP1 level is below ~1000 molecules/cell (mpc), the cell solely undergoes TRADD-dependent apoptosis. When RIP1 is above ~1000 mpc, pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount, which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical observations that RIP1 is required for necroptosis but its increase down-regulates necroptosis. Higher amount of RIP1 (>~46,000 mpc) suppresses apoptosis, leading to necroptosis alone. The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic. Our study provides a resource for encoding the complexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes, enabling RIP1 to play diverse roles in governing cell fate decisions.

The neuroprotection of cerebrolysin after spontaneous intracerebral hemorrhage through regulates necroptosis via Akt/ GSK3B signaling pathway

ABSTRACT Purpose: Spontaneous intracerebral hemorrhage (ICH) is a major cause of death and disability with a huge economic burden worldwide. Cerebrolysin (CBL) has been previously used as a nootropic drug. Necroptosis is a programmed cell death mechanism that plays a vital role in neuronal cell death after ICH. However, the precise role of necroptosis in CBL neuroprotection following ICH has not been confirmed. Methods: In the present study, we aimed to investigate the neuroprotective effects and potential molecular mechanisms of CBL in ICH-induced early brain injury (EBI) by regulating neural necroptosis in the C57BL/6 mice model. Mortality, neurological score, brain water content, and neuronal death were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, Evans blue extravasation, Western blotting, and quantitative real-time polymerase chain reaction (PCR). Results: The results show that CBL treatment markedly increased the survival rate, neurological score, and neuron survival, and downregulated the protein expression of RIP1 and RIP3, which indicated that CBL-mediated inhibition of necroptosis, and ameliorated neuronal death after ICH. The neuroprotective capacity of CBL is partly dependent on the Akt/GSK3β signaling pathway. Conclusions: CBL improves neurological outcomes in mice and reduces neuronal death by protecting against neural necroptosis.

Research progress of necroptosis on bone related diseases / 中国骨伤

As a new type of cell death, necroptosis is initiated by tumor necrosis factor receptor 1(TNFR1), and then activated receptor-interacting protein kinase 1(RIP1) and receptor-interacting protein kinase 3 (RIP3), following by the activation of mixed lineage kinase domain-like protein(MLKL) to deliver cell death signal. When necroptosis happens, damage associated molecular patterns (DAMPs) enter into extracellular area through the ruptured cytomembrane, followed by the disordered tissue hemeostasis. In recent years, many researches showed that necroptosis playimportant roles in a few bone related diseases, such as osteoporosis, osteonecrosis, osteosarcoma, etc. Thus, we try to briefly review the researches in this field.

Allium jesdianum Extract Induces Oxidative Stress and Necroptosis in Human Colorectal Cancer (HT-29) Cell Line

Abstract This study aimed to evaluate the toxic impact of hydro-alcoholic Allium jesdianum extract (AJE) on the growth of HT-29 human colorectal cancer cell line. Phytochemical analysis using gas chromatography and mass spectroscopy (GCMS) was done to determine the bioactive components of AJE. HT-29 cells exposed to 0 (control), 25, 50, and 100 ��g/mL of AJE for 48 hours. Cell survival, colony numbers, flow cytometry, oxidative stress, and gene expression were examined to evaluate the toxic impacts of the AJE. Twelve different phyto-constituents with peak areas were determined by the GCMS analysis. The major compounds were Allicin and α-Pinene. AJE considerably reduced the viability and colony numbers of the HT-29 cells. The AJE concentration-dependently increased necrosis, but not apoptosis in the HT-29 cells. AJE upregulated the expression of necroptosis-associated genes including RIPK1, RIPK3, and MLKL in a concentration-dependent manner. AJE also dose-dependently enhanced MDA contents and reactive oxygen species (ROS) level and diminished antioxidant enzyme level in the HT-29 cells. These data collectively indicated that AJE prevented the growth of the HT-29 cells by inducing oxidative stress, and activation necroptosis signaling pathways.

Controlled decompression alleviates early brain injury in rabbit intracranial hypertension model by regulating apoptosis/necroptosis

ABSTRACT Purpose To evaluate the effects of controlled decompression and rapid decompression, explore the potential mechanism, provide the theoretical basis for the clinical application, and explore the new cell death method in intracranial hypertension. Methods Acute intracranial hypertension was triggered in rabbits by epidural balloon compression. New Zealand white rabbits were randomly put into the sham group, the controlled decompression group, and the rapid decompression group. Brain water content, etc., was used to evaluate early brain injury. Western blotting and double immunofluorescence staining were used to detect necroptosis and apoptosis. Results Brain edema, neurological dysfunction, and brain injury appeared after traumatic brain injury (TBI). Compared with rapid decompression, brain water content was significantly decreased, neurological scores were improved by controlled decompression treatment. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and Nissl staining showed neuron death decreased in the controlled decompression group. Compared with rapid decompression, it was also found that apoptosis-related protein caspase-3/ tumor necrosis factor (TNF)-a was reduced markedly in the brain cortex and serum, and the expression levels of necroptosis-related protein, receptor-interacting protein 1 (RIP1)/receptor-interacting protein 1 (RIP3) reduced significantly in the controlled decompression group. Conclusions Controlled decompression can effectively reduce neuronal damage and cerebral edema after craniocerebral injury and, thus, protect the brain tissue by alleviating necroptosis and apoptosis.

Study on visfatin-induced inflammation and necroptosis via LOX-1 in human umbilical vein endothelial cells / 生物医学工程学杂志

The aim of the study is to identify the effects and underlying mechanisms of visfatin on inflammation and necroptosis in vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with visfatin or pretreated with Polyinosinic acid (LOX-1 inhibitor). By using the Western blot, RT-PCR, immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), MTT and flow cytometry technique, the occurrence of inflammation and necroptosis in HUVECs were evaluated. Our results showed that 100 ng/mL visfatin significantly increased the mRNA and protein expression of monocyte chemotactic protein 1 (MCP-1) and LOX-1 after 24 hours' treatment in HUVECs. However, pretreatment with Polyinosinic acid could significantly reduce the expression of MCP-1 compared with visfatin group. Additionally, 100 ng/mL visfatin could induce the production of necrotic features and increase the mRNA expression of BMF (one of the markers of necroptosis), while pretreating with Polyinosinic acid markedly downregulated the mRNA expression of BMF gene and promoted the cell proliferation. These results indicate that visfatin might induce inflammation and necroptosis via LOX-1 in HUVECs, suggesting that visfatin plays a central role in the development of atherosclerosis.