Pro-apoptotic Signaling Pathways Research Articles

The Long Non-Coding RNA NR3C2-8:1 Promotes p53-Mediated Apoptosis through the miR-129-5p/USP10 Axis in Amyotrophic Lateral Sclerosis

Motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is a form of apoptosis, but the mechanisms underlying this neuronal cell death remain unclear. Numerous studies demonstrate abnormally elevated and active p53 in the central nervous system of ALS patients. Activation of p53-regulated pro-apoptotic signaling pathways may trigger motor neuron death. We previously reported decreased expression of the long non-coding RNA NR3C2-8:1 (Lnc-NR3C) in leukocytes of ALS patients. Here, we show lnc-NR3C promotes p53-mediated cell death in ALS by upregulating USP10 and promoting lnc-NR3C-triggered p53 activation, resulting in cell death. Conversely, lnc-NR3C knockdown inhibited USP10-triggered p53 activation, thereby protecting cells against oxidative stress. As a competitive endogenous RNA, lnc-NR3C competitively binds miR-129-5p, regulating the usp10/p53 axis. Elucidating the link between Lnc-NR3C and the USP10/p53 axis in an ALS cell model reveals a role for long non-coding RNAs in activating apoptosis. This provides new therapeutic opportunities in ALS.

Astaxanthin-loaded alginate-chitosan gel beads activate Nrf2 and pro-apoptotic signalling pathways against oxidative stress

Oxidative stress (OS) plays a crucial role in disease development. Astaxanthin (ATX), a valuable natural compound, may reduce OS and serve as a treatment for diseases like neurodegenerative disorders and cancer. Nuclear factor-erythroid 2-related factor 2 (Nrf2) regulates antioxidant enzymes and OS management. We evaluated ATX’s antioxidant activity via Alg-CS/ATX gel beads in vitro. ATX-encapsulated alginate-chitosan (Alg-CS/ATX) gel beads were synthesized and structurally/morphologically characterized by SEM, FT-IR, and XRD. Their biological effects were examined in human umbilical vein endothelial cells (HUVECs) treated with H2O2 through MTT assay, Annexin V/PI, cell cycle studies, and western blotting. Alg-CS effectively carried ATX, with high capacity and reduced pore size. Alg-CS/ATX displayed an 84% encapsulation efficiency, maintaining stability for 30 days. In vitro studies showed a 1.4-fold faster release at pH 5.4 than at neutral pH, improving ATX’s therapeutic potential. HUVECs treated with Alg-CS/ATX showed enhanced viability via increased Nrf2 expression. Alg-CS gel beads exhibit significant potential as a biocompatible vehicle for delivering ATX to combat OS with considerable opportunity for clinical applications.

Elucidating the Molecular Mechanism by Which Gallic Acid Alleviates Oxidative Stress and Inflammatory Response of Acrylamide-Induced Renal Injury in a Rat Model

This study investigates the molecular effects of Acrylamide (ACR)-induced kidney damage and the potential protective role of Gallic acid (GA). Forty male rats were divided into five groups: Control, ACR, ACR+GA50, ACR+GA100, and GA100. The ACR groups received a daily oral dose of 50 mg/kg, while GA groups received 50 or 100 mg/kg oral doses for 14 consecutive days. On the 15th day, the animals were euthanized, and kidney samples were collected. The MDA, GSH, SOD, GPx, and CAT oxidative stress parameters were measured. The renal inflammatory response was evaluated by measuring the level of TNF-α, IL-1β, IL-6, NF-κB, COX-2, and IL-10. The downstream pro-apoptotic signaling pathway was resolved by measuring the levels of p38 MAPK and p53. The ACR induced renal oxidative stress with aggravated lipid peroxidation as revealed by the reduction in the levels GSH, SOD, GPx, and CAT of antioxidants while over-increase in the level of MDA, respectively. The levels of IL-1β, IL-6, NF-kB, COX-2 pro-inflammatory mediators as well as the p38 MAPK and p53 pro-apoptotic intermediates were further elevated. This increase in inflammatory response was met with marked decrease in anti-inflammatory IL-10 level. However, GA treatments- in dose dependent manner- had been demonstrated to effectively mitigate oxidative stress and reduce inflammatory responses, while also enhancing the cellular anti-inflammatory defense mechanisms. The GA can be considered as a novel protective antioxidant, anti-apoptotic drug against ACR-induced nephrotoxic insult. Further study should be performed to estimate the exact effective dose.

Ros-responsive dextran-phenylboronic acid-silibinin nanoparticles for targeting delivery of BAPTA-AM to effectively eliminate calcium overload-mediated inflammatory cascades and mitochondrial apoptosis: Alleviating acute liver injury

Calcium overload in hepatocytes mediates the crosstalk and amplification loop with oxidative stress and severe inflammation that initiate pro-apoptotic and necrotic signaling pathways in acute liver injury (ALI). In this study, an N-acetylgalactosamine-modified amphiphilic dextran-silibinin conjugate (GalNAc-Dex-Sil) with ROS-sensitive phenylboronic acid (PBA) linkage was synthesized. The as-prepared amphiphilic polymer was self-assembled into GalNAc-Dex-Sil nanoparticles, which efficiently encapsulated the membrane-permeable calcium chelator BAPTA-AM (BA), achieving GalNAc-mediated hepatocyte-specific targeting and ROS-responsive release in injured hepatocytes through PBA cleavage. Accordingly, a single low-dose injection of formulation (BA: 150 μg·kg−1) significantly alleviated hepatocyte oxidative stress and inflammation, rescued dying hepatocytes, prevented further hepatocyte damage, and ultimately restored liver function in the ALI mouse model by 1) rapidly and effectively eliminating the overloaded calcium and scavenging excessive ROS via released BA to block the feed-forward vicious cycles of the cell damage, 2) inhibiting the inflammatory cascade by decreasing the expression of the key factor of the NF-κB/NLPR3 axis, 3) inhibiting the key branch of the mitochondrial apoptotic pathway (Cyt C/Bcl-2 family/caspase-3 axis) and death receptor pathway (TNFα/Caspase-8 axis), and 4) stabilizing hepatocyte membranes through the synergistic hepatoprotective effect of silibinin. Taken together, this formulation exhibited great potential as a potent and effective ALI treatment strategy.

Linagliptin in combination with insulin suppresses apoptotic unfolded protein response in ovaries exposed to type 1 diabetes.

Type 1 diabetes mellitus (T1DM) is one of the main causes of ovarian atresia, but its molecular effect on the ovaries is not fully understood. Accumulating evidence suggests that T1DM causes excessive endoplasmic reticulum(ER) stress and insufficient adaptive unfolded protein response that triggers proapoptotic signaling pathways in ovarian tissue. In addition, problems such as amenorrhea and infertility, which are frequently seen in women with T1DM, continue despite the intensification of insulin therapy and improvement of metabolic control. Therefore new, and adjunctive treatments for women with T1DM need to be explored. We aimed to examine how the use of linagliptin, which has blood sugar-lowering effects and high antioxidant activity, together with insulin affects the expression levels of proteins and genes that play a role in ERstress in type 1 diabetic mouse ovaries. Eighty-four Balb/C 6-week-old female mice were randomly divided into seven groups: control, vehicle, diabetes + insulin, diabetes + linagliptin, diabetes + linagliptin + insulin, diabetes + TUDCA, and diabetes + TUDCA + insulin. TUDCA (an inhibitor of ER stress) groups are positive control groups created to compare linagliptin groups in terms of ER stress. Linagliptin and TUDCA were given by oral gavage and 1U insulin was administered subcutaneously for 2 weeks. A significant decrease was observed in the MDA and NOX1 levels and the number of atretic follicles in the ovaries of the diabetes + linagliptin + insulin group compared to the diabetes + insulin group. The use of linagliptin and insulin increased the expression of pro-survival XBP1s transmembrane protein and decreased the expression of proapoptotic ATF4, pJNK1/2, cleaved caspase 12, and cleaved caspase 3 in mouse ovaries. Our study provides new therapeutic evidence that linagliptin administered in addition to insulin induces ER stress mechanism-dependent survival in ovaries with type 1 diabetes.

MPA-capped CdTequantum dots induces endoplasmic reticulum stress-mediated autophagy and apoptosis through generation of reactive oxygen species in human liver normal cell and liver tumor cell

The rapid developments in nanotechnology have brought increased attention to the safety of Quantum Dots (QDs). Exploring their mechanisms of toxicity and characterizing their toxic effects in different cell lines will help us better understand and apply QDs appropriately. This study aims to elucidate the importance of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress-induced autophagy for CdTe QDs toxicity, that is, the importance of the nanoparticles in mediating cellular uptake and consequent intracellular stress effects inside the cell. The results of the study showed that cancer cells and normal cells have different cell outcomes as a result of intracellular stress effects. In normal human liver cells (L02), CdTe QDs leads to ROS generation and prolong ER stress. The subsequent autophagosome accumulation eventually triggers apoptosis by activating proapoptotic signaling pathways and the expression of proapoptotic Bax. In contrast, in human liver cancer cells (HepG2 cells), expression of UPR restrains proapoptotic signaling and downregulates Bax, and activated protective cellular autophagy, as a result of protecting these liver cancer cells from CdTe QDs-induced apoptosis. In summary, we assess the safety of CdTe QDs and recounted the molecular mechanism underlying its nanotoxicity in normal and cancerous cells. Notwithstanding, additional detailed studies on the deleterious effects of these nanoparticles in the organisms of interest are required to ensure low-risk application.

Non-specific porins from the outer membrane of <i>Yersinia pseudotuberculosis</i> effect on mice brain cortex tissues

It was found that with a single immunization of OmpF and OmpC mice with Yersinia pseudotuberculosis porins, against the background of an increasing titer of specific antibodies, pathological changes develop in the deep layers of the cerebral cortex, characterized by dystrophic changes in cells. At the same time, an increased level of caspase 3 expression is observed in neurons, which indicates the induction of proapoptotic signaling pathways. The results obtained indicate the potential ability of nonspecific pore-forming proteins of the outer membrane of Gram-negative bacteria to initiate the development of degenerative changes in brain cells.

Curcumin as an anti-proliferative agent in breast cancer through RassF1a, Bax, and caspase-3 protein

Background. Curcumin is a polyphenol that has pharmacological activity that can inhibit tumor cell growth and induce apoptosis through various mechanisms. However, the specifc mechanism of curcumin cytotoxicity remains controversial because of many anti- and pro-apoptotic signaling pathways in various cell types.This study aims to examine the relationship among curcumin on RASSF1A, Bax protein levels, and caspase-3 activity in supporting the apoptotic mechanism in CSA03 breast cancer cells.Material and Methods. Curcumin administration to cancer cells is based on differences in dosage with 24-hour incubation. Cytotoxicity after curcumin administration was determined using MTS. RASSF1A and Bax protein levels were tested through ELISA. Caspase-3 activity was used to determine apoptosis and was tested using fow cytometry.Results. The results indicated that curcumin had a cytotoxicity effect of 40.85 µg/mL. At concentrations of 40 µg/mL and 50 µg/mL, curcumin increases levels of protein RASSF1A (∆ = 26.53% and 47.35%, respectively), Bax (∆ = 48.79% and 386.15%), and caspase-3 (∆ = 1,678.51% and 1,871.889%) signifcantly.Conclusions. Curcumin exhibits anti-proliferative activity and apoptotic (Caspase-3) effects through activation of RASSF1A and Bax.

Effect of different types of immunosuppressive therapy on the parameters of TNF receptor expression in patients with rheumatoid arthritis

Background. The balance of TNF receptor expression on immune cells is a key factor determining cytokine-induced activation of proapoptotic or proliferative signaling pathways. As a result, the changes in cytokine level and in expression of its receptors may be one of the mechanisms that regulate the level of systemic and local inflammation in rheumatoid arthritis (RA) and determine the degree of therapy effectiveness. The aim. To study the effect of rheumatoid arthritis therapy on the change in the patterns of TNF receptors expression in terms of co-expression and the number of receptors on the main subpopulations of immunocompetent cells.Materials and methods. A comparative analysis of the profiles of TNF receptors type 1 and 2 (TNFR1/2) co-expression was carried out in patients with RA (n = 16) before and after having inpatient effective therapy and in comparison with a group of healthy individuals (n = 21). We compared the number of receptors and the proportion of cells expressing the corresponding receptor using flow cytometry and studied the subpopulations of regulatory T cells, T cells, B cells, and monocytes. Results. In patients with RA, there is a significant redistribution of TNFR1 and TNFR2 expression on immunocompetent cells, while the intensity of changes is associated not only with disease severity indicators, but also with the therapy received. The key adaptive mechanism of the TNF system in long-term treatment refractory course of RA is a change in the proportion of double-positive TNFR1+TNFR2+ cells, while the effectiveness of therapy and clinical indicators of the disease severity are associated with individual variability in the parameters of type 2 receptors expression. Conclusions. The data obtained confirm the existence of a relationship between an imbalance in the expression of type 1 and type 2 TNF receptors on immunocompetent cells and the effectiveness of response to therapy. The identified patterns of typical changes in TNFR1/2 co-expression in RA can be used as potential therapeutic targets and predictive factors for the effectiveness of therapy.

Filamin A regulates caspase-3 cleavage in platelets in a protein kinase C (PKC)-dependent manner.

Apoptosis is a critical process for the maintenance of cell populations, and involves mitochondrial depolarization, the sequential cleavage of caspase-9 and -3, followed by the externalization of phosphatidylserine (PS) on the plasma membrane. The actin cytoskeleton and its accessory proteins are known regulators of apoptotic signaling in nucleated cells but their roles in platelet apoptosis are undefined. Filamin A (FLNA) is a ubiquitously expressed actin-crosslinking protein that also serves as an intracellular signaling scaffold. Here we used platelets from mice with a platelet-specific FLNA deficiency (Flnafl/Y, Pf4-cre/+, termed platelet-specific knockout) to test the role of FLNA in platelet apoptosis. Treatment with the BH3-mimetic drug ABT-737 induced caspase-3 cleavage and PS exposure in platelets from floxed mice (Flnafl/Y, termed control) but these effects were essentially abrogated in FLNA-null platelets (platelet-specific knockout). Protein kinase C (PKC), a known FLNA ligand, was also activated by ABT-737, and PKC's phosphorylation of its downstream substrates was attenuated in FLNA-null platelets. The PKC inhibitor bisindolylmaleimide (BIM) also reduced caspase-3 cleavage, thus essentially phenocopying the FLNA-null platelets. Notably, the caspase-3 cleavage defect in FLNA-null platelets was rescued by the PKC-activating phorbol ester PMA, suggesting that FLNA and PKC share a common pathway in regulating platelet apoptosis. Mitochondrial depolarization and caspase-9 cleavage were unaffected by BIM treatment, suggesting that PKC specifically controls the downstream caspase-3 point of the pro-apoptotic signaling pathway. These data point to a novel role for FLNA in the regulation of platelet apoptosis, thus providing an improved understanding of how circulating platelet counts are maintained.

Dual tumor- and subcellular-targeted photodynamic therapy using glucose-functionalized MoS2 nanoflakes for multidrug-resistant tumor ablation

Photodynamic therapy (PDT) is emerging as an efficient strategy to combat multidrug-resistant (MDR) cancer. However, the short half-life and limited diffusion of reactive oxygen species (ROS) undermine the therapeutic outcomes of this therapy. To address this issue, a tumor-targeting nanoplatform was developed to precisely deliver mitochondria- and endoplasmic reticulum (ER)-targeting PDT agents to desired sites for dual organelle-targeted PDT. The nanoplatform is constructed by functionalizing molybdenum disulfide (MoS2) nanoflakes with glucose-modified hyperbranched polyglycerol (hPG), and then loading the organelle-targeting PDT agents. The resultant nanoplatform Cy7.5-TG@GPM is demonstrated to mediate both greatly enhanced internalization within MDR cells and precise subcellular localization of PDT agents, facilitating in situ near-infrared (NIR)-triggered ROS generation for augmented PDT and reversal of MDR, causing impressive tumor shrinkage in a HeLa multidrug-resistant tumor mouse model. As revealed by mechanistic studies of the synergistic mitochondria- and ER-targeted PDT, ROS-induced ER stress not only activates the cytosine-cytosine-adenosine-adenosine thymidine/enhancer-binding protein homologous protein (CHOP) pro-apoptotic signaling pathway, but also cooperates with ROS-induced mitochondrial dysfunction to trigger cytochrome C release from the mitochondria and induce subsequent cell death. Furthermore, the mitochondrial dysfunction reduces ATP production and thereby contributes to the reversal of MDR. This nanoplatform, with its NIR-responsive properties and ability to target tumors and subcellular organelles, offers a promising strategy for effective MDR cancer therapy.

Hypothermia Prevents Cardiac Dysfunction during Acute Ischemia Reperfusion by Maintaining Mitochondrial Bioenergetics and by Promoting Hexokinase II Binding to Mitochondria.

Background Hypothermia (H), cardioplegia (CP), and both combined (HCP) are known to be protective against myocardial ischemia reperfusion (IR) injury. Mitochondria have molecular signaling mechanisms that are associated with both cell survival and cell death. In this study, we investigated the dynamic changes in proapoptotic and prosurvival signaling pathways mediating H, CP, or HCP-induced protection of mitochondrial function after acute myocardial IR injury. Methods Rats were divided into five groups. Each group consists of 3 subgroups based on a specific reperfusion time (5, 20, or 60 min) after a 25-min global ischemia. The time control (TC) groups were not subjected to IR but were perfused with 37 °C Krebs-Ringer's (KR) buffer, containing 4.5 mM K+, in a specific perfusion protocol that corresponded with the duration of each IR protocol. The IR group (control) was perfused for 20 min with KR, followed by 25-min global ischemia, and then KR reperfusion for 5, 20, or 60 min. The treatment groups were exposed to 17 °C H, 37 °C CP (16 mM K+), or HCP (17 °C + CP) for 5 min before ischemia and for 2 min on reperfusion before switching to 37 °C KR perfusion for the remainder of each of the reperfusion times. Cardiac function and mitochondrial redox state (NADH/FAD) were monitored online in the ex vivo hearts before, during, and after ischemia. Mitochondria were isolated at the end of each specified reperfusion time, and changes in O2 consumption, membrane potential (ΔΨm), and Ca2+ retention capacity (CRC) were assessed using complex I and complex II substrates. In another set of hearts, mitochondrial and cytosolic fractions were isolated after a specified reperfusion time to conduct western blot assays to determine hexokinase II (HKII) and Bax binding/translocation to mitochondria, cytosolic pAkt levels, and cytochrome c (Cyto-c) release into the cytosol. Results H and HCP were more protective of mitochondrial integrity and, concomitantly, cardiac function than CP alone; H and HCP improved post-ischemic cardiac function by (1) maintaining mitochondrial bioenergetics, (2) maintaining HKII binding to mitochondria with an increase in pAkt levels, (3) increasing CRC, and (4) decreasing Cyto-c release during reperfusion. Bax translocation/binding to mitochondria was unaffected by any treatment, regardless of cardiac functional recovery. Conclusions Hypothermia preserved mitochondrial function and cardiac function, in part, by maintaining mitochondrial bioenergetics, by retaining HKII binding to mitochondria via upstream pAkt, and by reducing Cyto-c release independently of Bax binding to mitochondria.

Esculetin has therapeutic potential via the proapoptotic signaling pathway in A253 human submandibular salivary gland tumor cells.

Esculetin is a natural lactone that is commonly derived from coumarins. According to previous experiments using human cancer cells, esculetin has potent antitumor activity; it also inhibits proliferation and induces the apoptosis of cancer cells. In the present study, the anti-proliferative effect of esculetin on the submandibular salivary gland tumor cell line, A253, was evaluated via in vitro and in vivo analyses. Furthermore, the anti-cancer effects of esculetin in A253 cells and a xenograft model of salivary gland tumors were determined using 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide and TUNEL assay, apoptosis protein array, quantitative polymerase chain reaction and western blot analysis. Esculetin (50-150 µM) was demonstrated to have an anti-proliferative effect in the A253 cell line in vitro; this observed effect was dependent on the dose and duration of treatment. Esculetin also increased the levels of Bax, cleaved caspase-3, cleaved-9 and cleaved poly (ADP-ribose) polymerase apoptosis-related proteins, and decreased the expression levels of the Bcl-2 anti-apoptotic protein. With respect to apoptosis regulation, esculetin significantly decreased the proliferation of tumor cells in a xenograft model (100 mg/kg/day) for 18 days. Overall, esculetin could be a potential oral anticancer drug against salivary gland cancer.

Targeting Differential Roles of Tumor Necrosis Factor Receptors as a Therapeutic Strategy for Glaucoma.

Glaucoma is an irreversible sight-threatening disorder primarily due to elevated intraocular pressure (IOP), leading to retinal ganglion cell (RGC) death by apoptosis with subsequent loss of optic nerve fibers. A considerable amount of empirical evidence has shown the significant association between tumor necrosis factor cytokine (TNF; TNFα) and glaucoma; however, the exact role of TNF in glaucoma progression remains unclear. Total inhibition of TNF against its receptors can cause side effects, although this is not the case when using selective inhibitors. In addition, TNF exerts its antithetic roles via stimulation of two receptors, TNF receptor I (TNFR1) and TNF receptor II (TNFR2). The pro-inflammatory responses and proapoptotic signaling pathways predominantly mediated through TNFR1, while neuroprotective and anti-apoptotic signals induced by TNFR2. In this review, we attempt to discuss the involvement of TNF receptors (TNFRs) and their signaling pathway in ocular tissues with focus on RGC and glial cells in glaucoma. This review also outlines the potential application TNFRs agonist and/or antagonists as neuroprotective strategy from a therapeutic standpoint. Taken together, a better understanding of the function of TNFRs may lead to the development of a treatment for glaucoma.

STUDY THE ROLE OF CYTOCHROME C COMPLEX WITH CARDIOLIPIN IN THE CATALYSIS OF LIPID PEROXIDATION AND THE INITIATION OF APOPTOSIS: CALCULATION OF KINETIC CONSTANTS AND QUANTUM YIELDS BASED ON THE KINETICS OF ACTIVATED CHEMILUMINESCENCE

Cytochrome c in a cell, in an aqueous medium or in a non-polar environment can be both in a free state and in complex with cardiolipin. In the latter case, it is partially denatured, characterized by a specific conformation, has peroxidase activity and is an important component of proapoptotic signaling pathways [1,2,9]. Differences between native cytochrome c and the cytochrome c—cardiolipin complex are recorded by the absorption and fluorescence spectra, as well as by the pronounced peroxidase activity of the latter and chemiluminescence accompanying the free radical processes it triggers. Based on this, software was developed for automatic calculation of kinetic parameters and quantum yields, lipid peroxidation of activated chemiluminescence and visualization of model curves.

N-Tertaining a New Signaling Paradigm for the Cardiomyocyte β 1 -Adrenergic Receptor.

β 1 -adrenergic receptors (β 1 ARs) are the principle mediators of catecholamine actions in cardiomyocytes. β 1 ARs rapidly adjust cardiac output and provide short-term hemodynamic support for the failing heart by activating a Gs-adenylyl cyclase pathway that increases 3'-5'-cyclic adenosine monophosphate and leads to the activation of protein kinase A and the phosphorylation of substrates involved in excitation-contraction coupling. However, chronic persistent β 1 AR activation in the setting of heart failure leads to a spectrum of maladaptive changes that contribute to the evolution of heart failure. The molecular basis for β 1 AR-driven maladaptive responses remains uncertain because chronic persistent β 1 AR activation has been linked to the activation of both proapoptotic and antiapoptotic signaling pathways. Of note, studies to date have been predicated on the assumption that β 1 ARs signal exclusively as full-length receptor proteins. Our recent studies show that β 1 ARs are detected as both full-length and N-terminally truncated species in cardiomyocytes, that N-terminal cleavage is regulated by O-glycan modifications at specific sites on the β 1 AR N-terminus, and that N-terminally truncated β 1 ARs remain signaling competent, but their signaling properties differ from those of the full-length β 1 AR. The N-terminally truncated form of the β 1 AR constitutively activates the protein kinase B signaling pathway and confers protection against doxorubicin-dependent apoptosis in cardiomyocytes. These studies identify a novel signaling paradigm for the β 1 AR, implicating the N-terminus as a heretofore-unrecognized structural determinant of β 1 AR responsiveness that could be pharmacologically targeted for therapeutic advantage.

Curcumin as an Anti-Proliferative Agent in Breast Cancer through RASSF1A, Bax, and Caspase-3 Protein

Introduction: Curcumin is a polyphenol that has pharmacological activity that can inhibit tumor cell growth and induce apoptosis through various mechanisms. However, the specific mechanism of curcumin cytotoxicity remains controversial because of many anti- and pro-apoptotic signaling pathways in various cell types. This study aims to examine the relationship among curcumin on RASSF1A, Bax protein levels, and caspase-3 activity in supporting the apoptotic mechanism in CSA03 breast cancer cells. Method: Curcumin administration to cancer cells is based on differences in dosage with 24-hour incubation. Cytotoxicity after curcumin administration was determined using MTS. RASSF1A and Bax protein levels were tested through ELISA. Caspase-3 activity was used to determine apoptosis and was tested using flow cytometry. Results: The results indicated that curcumin had a cytotoxicity effect of 40.85 µg/mL. At concentrations of 40 µg/mL and 50 µg/mL, curcumin increases levels of protein RASSF1A (∆ = 26.53% and 47.35%, respectively), Bax (∆ = 48.79% and 386.15%), and caspase-3 (∆ = 1,678.51% and 1,871.889%) significantly. Conclusions: Curcumin exhibits anti-proliferative activity and apoptotic (Caspase-3) effects through activation of RASSF1A and Bax.

Autophagic degradation of IRF3 induced by the small-molecule auranofin inhibits its transcriptional and proapoptotic activities

The ubiquitously expressed transcription factor interferon (IFN) regulatory factor 3 (IRF3) is critical for the induction of antiviral genes, e.g., type-I IFN. In addition to its transcriptional function, IRF3 also activates a nontranscriptional, proapoptotic signaling pathway. While the proapoptotic function of IRF3 protects against viral infections, it is also involved in harmful immune responses that trigger hepatocyte cell death and promote liver disease. Thus, we hypothesized that a small-molecule inhibitor of the proapoptotic activity of IRF3 could alleviate fatty-acid-induced hepatocyte cell death. We conducted a high-throughput screen, which identified auranofin as a small-molecule inhibitor of the proapoptotic activity of IRF3. In addition to the nontranscriptional apoptotic pathway, auranofin also inhibited the transcriptional activity of IRF3. Using biochemical and genetic tools in human and mouse cells, we uncovered a novel mechanism of action for auranofin, in which it induces cellular autophagy to degrade IRF3 protein, thereby suppressing IRF3 functions. Autophagy-deficient cells were unable to degrade IRF3 upon auranofin treatment, suggesting that the autophagic degradation of IRF3 is a novel approach to regulate IRF3 activities. Using a physiologically relevant in vitro model, we demonstrated that auranofin inhibited fatty-acid-induced apoptotic cell death of hepatocytes. In summary, auranofin is a novel inhibitor of IRF3 functions and may represent a potential therapeutic option in diseases where IRF3 is deleterious.

Divalent Metal Transporter 1 Knock-Down Modulates IL-1β Mediated Pancreatic Beta-Cell Pro-Apoptotic Signaling Pathways through the Autophagic Machinery.

Pro-inflammatory cytokines promote cellular iron-import through enhanced divalent metal transporter-1 (DMT1) expression in pancreatic β-cells, consequently cell death. Inhibition of β-cell iron-import by DMT1 silencing protects against apoptosis in animal models of diabetes. However, how alterations of signaling networks contribute to the protective action of DMT1 knock-down is unknown. Here, we performed phosphoproteomics using our sequential enrichment strategy of mRNA, protein, and phosphopeptides, which enabled us to explore the concurrent molecular events in the same set of wildtype and DMT1-silenced β-cells during IL-1β exposure. Our findings reveal new phosphosites in the IL-1β-induced proteins that are clearly reverted by DMT1 silencing towards their steady-state levels. We validated the levels of five novel phosphosites of the potential protective proteins using parallel reaction monitoring. We also confirmed the inactivation of autophagic flux that may be relevant for cell survival induced by DMT1 silencing during IL-1β exposure. Additionally, the potential protective proteins induced by DMT1 silencing were related to insulin secretion that may lead to improving β-cell functions upon exposure to IL-1β. This global profiling has shed light on the signal transduction pathways driving the protection against inflammation-induced cell death in β-cells after DMT1 silencing.

Mechanisms of the Cytotoxic Effect of Selenium Nanoparticles in Different Human Cancer Cell Lines.

In recent decades, studies on the functional features of Se nanoparticles (SeNP) have gained great popularity due to their high biocompatibility, stability, and pronounced selectivity. A large number of works prove the anticarcinogenic effect of SeNP. In this work, the molecular mechanisms regulating the cytotoxic effects of SeNP, obtained by laser ablation, were studied by the example of four human cancer cell lines: A-172 (glioblastoma), Caco-2, (colorectal adenocarcinoma), DU-145 (prostate carcinoma), MCF-7 (breast adenocarcinoma). It was found that SeNP had different concentration-dependent effects on cancer cells of the four studied human lines. SeNP at concentrations of less than 1 μg/mL had no cytotoxic effect on the studied cancer cells, with the exception of the A-172 cell line, for which 0.5 μg/mL SeNP was the minimum concentration affecting its metabolic activity. It was shown that SeNP concentration-dependently caused cancer cell apoptosis, but not necrosis. In addition, it was found that SeNP enhanced the expression of pro-apoptotic genes in almost all cancer cell lines, with the exception of Caco-2 and activated various pathways of adaptive and pro-apoptotic signaling pathways of UPR. Different effects of SeNP on the expression of ER-resident selenoproteins and selenium-containing glutathione peroxidases and thioredoxin reductases, depending on the cell line, were established. In addition, SeNP triggered Ca2+ signals in all investigated cancer cell lines. Different sensitivity of cancer cell lines to SeNP can determine the induction of the process of apoptosis in them through regulation of the Ca2+ signaling system, mechanisms of ER stress, and activation of various expression patterns of genes encoding pro-apoptotic proteins.