Stimulation of macrophages with lipopolysaccharide (LPS) leads to the production of cytokines that elicit massive liver apoptosis. We investigated the in vivo role of stress-responsive transcription factors (SRTFs) in this process focusing on the precipitating events that are sensitive to a cell-permeant peptide inhibitor of SRTF nuclear import (cSN50). In the absence of cSN50, mice challenged with LPS displayed very early bursts of inflammatory cytokines/chemokines, tumor necrosis factor ␣ (1 h), interleukin 6 (2 h), interleukin 1  (2 h), and monocyte chemoattractant protein 1 (2 h). Activation of both initiator caspases 8 and 9 and effector caspase 3 was noted 4 h later when full-blown DNA fragmentation and chromatin condensation were first observed (6 h). At this time an increase of pro-apoptotic Bax gene expression was observed. It was preceded by a decrease of anti-apoptotic Bcl2 and BclX L gene transcripts. Massive apoptosis was accompanied by microvascular injury manifested by hemorrhagic necrosis and a precipitous drop in blood platelets observed at 6 h. An increase in fibrinogen/fibrin degradation products and a rise in plasminogen activator inhibitor 1 occurred between 4 and 6 h. Inhibition of SRTFs nuclear import with the cSN50 peptide abrogated all these changes and increased survival from 7 to 71%. Thus, the nuclear import of SRTFs induced by LPS is a prerequisite for activation of the genetic program that governs cytokines/ chemokines production, liver apoptosis, microvascular injury, and death. These results should facilitate the rational design of drugs that protect the liver from inflammation-driven apoptosis.Programmed cell death (apoptosis) is the major mechanism of embryonic development and remodeling of tissues and organs, homeostatic control of immune cells that recognize self and non-self antigens, and removal of virally infected cells (1). Apoptosis of hepatocytes may occur in fulminant hepatitis, an inflammatory process that is caused by viral and non-viral agents (2). For example, recent gene therapy approaches to correct an inborn error of metabolism led to fulminant liver failure (3). This inflammation-related complication of gene therapy impedes broader application of viral vectors (4, 5). The sequence of intracellular signaling events that underlie inflammation-driven development of ultimately fatal liver apoptosis remains incompletely understood.Fulminant liver apoptosis has been studied in several animal models. These studies indicate that activation of T cells with concanavalin A (6) or with agonists that interact with T cell receptor such as staphylococcal enterotoxin B can lead to massive apoptosis (7,8). Staphylococcal enterotoxin B-induced apoptosis occurs under conditions of metabolic stress imposed by 2-amino-2-deoxy-D-galactosamine (D-Gal).1 Similarly, activation of macrophages with their Toll-like receptors (TLR) agonists, such as lipopolysaccharide (LPS, endotoxin), induces massive liver apoptosis when animals are treated with ethanol or D-Gal (9, 10). By reversibly dep...
Targeting of peptides, proteins, and other functional cargo into living cells is contingent upon efficient transport across the plasma membrane barrier. We have harnessed the signal sequence hydrophobic region (SSHR) to deliver functional cargoes to cultured cells and to experimental animals. We now report evidence that two chirally distinct forms of SSHR composed of all L or all D amino acids showed similar membrane-translocating activity as assessed by confocal microscopy, flow cytometry, and direct fluorescence measurement. An attached nuclear localization sequence ferried by the SSHR enantiomers displayed similar intracellular function by inhibiting inducible nuclear import of transcription factor nuclear factor B and suppressing nuclear factor B-dependent gene expression of cytokines. A nuclear localization sequence comprised of a positively charged cluster of amino acids was rapidly translocated by SSHR enantiomers to the interior of unilamellar phospholipid vesicles. These findings indicate that the SSHR translocates functional peptides directly through the plasma membrane phospholipid bilayer without involving chirally specific receptor/transporter mechanisms. This mechanism of SSHR translocation is suitable for facile delivery of biologically active peptides for cell-based and animal-based functional proteomic studies.The plasma membrane imposes tight control on the access of extracellular peptides and proteins to the cell interior. Through its mosaic structure of proteins and glycolipids embedded into the phospholipid bilayer, the plasma membrane provides a boundary for the 10,000 -15,000 proteins expressed in a typical mammalian cell (1, 2). Despite this barrier, transfer of information across the membrane is essential for cell development, function, and survival. Membrane receptors and transporters sense the extracellular environment of growth-promoting or -inhibiting ligands, short peptides, ions, and nutrients. This recognition allows their cellular uptake through specific receptor-mediated endocytosis or transporter-based translocation. To bypass these inherent mainstays of the plasma membrane functional integrity, we harnessed a signal sequence-derived hydrophobic region to deliver functional cargoes composed of peptides and proteins to probe and modulate intracellular signaling (3, 4). However, the mechanism of SSHR 1 -directed translocation of functional cargo across the plasma membrane remains unexplained.The overall structure of signal peptides is conserved in evolution between prokaryotes and eukaryotes, although the sequences of signal peptides are highly diverse (5). Their tripartite structure comprises an NH 2 -terminal region (n region), and a hydrophobic h region of variable length; this is followed by a cleavage site (c region) for signal peptidase. The hydrophobic region, which usually forms a helix, is endowed with a membrane-translocating activity (6). Its primary function is to guide a nascent polypeptide chain from the ribosomal tunnel through a translocon pore that is open latera...
Chemotherapy-related fatigue (CRF), one of the most severe adverse effects observed in cancer patients, has been theoretically related to oxidative stress, and antioxidant treatment might be one of the most valuable therapeutic approaches. However, there are still few effective pharmacological therapies. Yifei Sanjie pills (YFSJ), a classical formula used to treat lung cancer as complementary and alternative medicine, have been proved to alleviate CRF of lung cancer patients in clinical practices. However, the underlying mechanisms have not been clarified. In this study, our data showed that YFSJ alleviated CRF presented as reversing the decline of swimming time and locomotor activity induced by cisplatin (DDP). Moreover, YFSJ significantly reduces the accidence of mitophagy and mitochondrial damage and reduces apoptosis in skeletal muscle tissues caused by DDP. It probably works by decreasing the oxidative stress, inhibiting the activation of the AMPK/mTOR pathway, decreasing protein expression levels of Beclin1 and other autophagy-related proteins, and attenuating the activation of Cytochrome c (cyto. C), Cleaved Caspase-9 (c-Casp 9), and other apoptosis-related proteins. Furthermore, YFSJ enhanced DDP sensitivity by specifically promoting oxidative stress and activating apoptosis and autophagy in the tumor tissues of mice. It was also found that YFSJ reduced the loss of body weight caused by DDP, reversed the ascent of serum concentrations of alanine aminotransferase (ALT), aminotransferase (AST), and creatinine (CREA), increased the spleen index, and prolonged the survival time of mice. Taken together, these results revealed that YFSJ could alleviate CRF by reducing mitophagy and apoptosis induced by oxidative stress in skeletal muscle; these results also displayed the effects of YFSJ on enhancing chemotherapy sensitivity, improving quality of life, and prolonging survival time in lung cancer mice received DDP chemotherapy.
Colorectal cancer (CRC) is a severe threat to human health. Ginsenosides such as ginsenoside Rh4 have been widely studied in the antitumor field. Here, we investigated the antiproliferative activity and mechanism of Rh4 against CRC in vivo and in vitro. The CRC xenograft model showed that Rh4 inhibited xenograft tumor growth with few side effects ( p < 0.05 ). As determined by MTT colorimetric assays, Western blotting, and immunohistochemical analysis, Rh4 effectively inhibited CRC cell proliferation through autophagy and ferroptosis ( p < 0.05 ). Rh4 significantly upregulated autophagy and ferroptosis marker expression in CRC cells and xenograft tumor tissues in the present study ( p < 0.05 ). Interestingly, the ferroptosis inhibitor ferrostatin-1 (Fer-1) reversed Rh4-induced ferroptosis ( p < 0.05 ). Moreover, the autophagy inhibitor 3-methyladenine (3-MA) also reversed Rh4-induced ferroptosis ( p < 0.05 ). These results indicate that Rh4-induced ferroptosis is regulated via the autophagy pathway. In addition, Rh4 increased reactive oxygen species (ROS) accumulation, leading to the activation of the ROS/p53 signaling pathway ( p < 0.05 ). Transcriptome sequencing also confirmed this ( p < 0.05 ). Moreover, the ROS scavenger N-acetyl-cysteine (NAC) reversed the inhibitory effect of Rh4 on CRC cells ( p < 0.05 ). Therefore, this study proves that Rh4 inhibits cancer cell proliferation by activating the ROS/p53 signaling pathway and activating autophagy to induce ferroptosis, which provides necessary scientific evidence of the great anticancer potential of Rh4.
Luteolin, which is a natural flavonoid, has anti-inflammatory, antioxidant, and anticancer properties. Numerous studies have proven that luteolin inhibits the growth of many types of cancer cells by promoting apoptosis, autophagy, and cell cycle arrest in tumour cells. However, in vivo research on this topic has been limited. In addition, other studies have shown that luteolin exerts a good inhibitory effect on apoptosis-resistant cancer cells. While existing studies have not completely elucidated the mechanism underlying this phenomenon, we assume that luteolin, which is a natural compound that exerts its effects through various mechanisms, may have the potential to inhibit tumour growth. In our study, we proved that luteolin exerted a good inhibitory effect on the proliferation of colon cancer cells according to CCK8 and EdU fluorescence assays, and the same conclusion was drawn in animal experiments. In addition, we found that luteolin, which is an antioxidant, unexpectedly promoted oxidative stress as shown by measuring the levels of oxidative balance-related indicators, such as reactive oxygen species (ROS), SOD, H2O2 and GSH. However, the decreased oxidation of luteolin-treated HT-29 cells after treatment with the active oxygen scavenger NAC did not reverse the inhibition of cell growth. However, the Caspase1 inhibitor VX765 did reverse the inhibition of cell growth. Western blotting analysis showed that luteolin treatment increased the expression of Caspase1, Gasdermin D and IL-1β, which are members of the pyroptosis signalling pathway, in colon cancer cells. We further intuitively observed NLRP3/Gasdermin D colocalization in luteolin-treated HT-29 cells and mouse tumour tissues by immunofluorescence. These results suggest that luteolin inhibits the proliferation of colon cancer cells through a novel pathway called pyroptosis. This study provides a new direction for the development of natural products that inhibit tumour growth by inducing pyroptosis.
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