Activation Of Stress Signaling Pathways Research Articles

ABT199/venetoclax synergism with thiotepa enhances the cytotoxicity of fludarabine, cladribine and busulfan in AML cells.

ABT199/venetoclax, an inhibitor of the pro-survival BCL-2 protein, has improved AML treatment. Its efficacy in hematopoietic stem cell transplantation (HSCT), when combined with other chemotherapeutic drugs, has not been thoroughly investigated. The present study demonstrates the synergistic cytotoxicity of ABT199/venetoclax with the DNA alkylator thiotepa (Thio) in AML cells. Cleavage of Caspase 3, PARP1 and HSP90, as well as increased Annexin V positivity, suggest potent activation of apoptosis by this two-drug combination; increased levels of γ-H2AX, P-CHK1 (S317), P-CHK2 (S19) and P-SMC1 (S957) indicate an enhanced DNA damage response. Likewise, the increased level of P-SAPK/JNK (T183/Y185) and decreased P-PI3Kp85 (Y458) suggest enhanced activation of stress signaling pathways. These molecular readouts were synergistically enhanced when ABT199/venetoclax and Thio were combined with fludarabine, cladribine and busulfan. The five-drug combination decreased the levels of BCL-2, BCL-xL and MCL-1, suggesting its potential clinical relevance in overcoming ABT199/venetoclax resistance. Moreover, this combination is active against P53-negative and FLT3-ITD-positive cell lines. Enhanced activation of apoptosis was observed in leukemia patient-derived cell samples exposed to the five-drug combination, suggesting a clinical relevance. The results provide a rationale for clinical trials using these two- and five-drug combinations as part of a conditioning regimen for AML patients undergoing HSCT.

Synergistic Anti-Tumor Effects in RAS Mut Multiple Myeloma By Targeting MAP4K2 and RAS Pathways

Mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2) is an important key regulator of the stress-activated MAPK core signaling pathways. Our previous work demonstrated that MAP4K2 is highly expressed in multiple myeloma (MM) cells, and MM cells carrying a RAS mutation are more vulnerable to MAP4K2 inhibition, associated with the downregulation of critical transcriptional factors including IKZF1/3, BCL-6, and c-MYC proteins ( Li et al. Blood 2021). Targeting the aberrant RAS/MAPK signaling, MEK inhibitors have been shown to induce cytotoxicity in MM (De la Puente, P. et al. Blood Cancer J. 2016). Thus, we used a recently developed MEK inhibitor (MEKi) Trametinib to investigate the therapeutic potential of combined MEK and MAP4K2 signaling inhibition in RAS-driven MM. Tet-on sh-MAP4K2 lentivirus was introduced into RAS Mut MM cells to establish the inducible MAP4K2 knockdown cells upon doxycycline treatment. To examine the combined effects, Tet-on sh-MAP4K2 RAS Mut MM cells were treated with doxycycline to silence MAP4K2 expression, followed by treatment of MEKi Trametinib at different dosages. Cell proliferation, apoptosis, and cell cycle were analyzed five days later. Data showed that MAP4K2 silencing combined with MEK inhibition significantly decreased cell proliferation to 10% compared with MEKi alone 32% ( p<0.01) by MTS assays, and enhanced cell apoptosis (MEKi alone vs. with MAP4K2 silencing: 26% vs 91%, p<0.01). Cell cycle assay demonstrated that MEKi induced G1 arrest in RAS Mut MM cells (MEKi alone:76% vs vehicle control: 54%, p<0.01), which was also significantly higher in combination with MAP4K2 silencing (92%). Western blot assay confirmed that MAP4K2 silencing combined with MEKi significantly enhanced the downregulation of IKZF1 and c-MYC compared to the MEKi treatment alone. These data suggest that the MEKi and MAP4K2 inhibition combination has synergetic anti-MM effects. To further confirm the synergetic effects, we tested the combined effects of MEKi with the MAP4K2 kinase inhibitor TL4-12. H929 (N-Ras Mut) cells were treated by vehicle control, TL4-12 (2 uM) alone, MEKi (200 nM) alone, or TL4-12 + MEKi for 4 days, followed by an apoptosis assay. The combination of TL4-12 and MEKi strongly enhanced the cytotoxicity effects compared to the single-agent treatments. Consistently, the carboxyfluorescein diacetate succinimidyl ester (CFSE) cell proliferation assay confirmed that dual treatment significantly delayed H929 cell proliferation compared to the single-agent treatments (percentage of proliferating cells: TL4-12 alone: 86.2%; MEKi alone: 89.03%; dual treatment: 18.9%. p<0.01). Western blotting assay further confirmed that dual treatment of MEKi and MAP4K2i enhanced the decrease in MEK1/2 and ERK1/2 phosphorylation and downregulation of transcriptional factors IKZF1 and c-MYC. Taken together, our findings demonstrate that the combination of MAP4K2 inhibition with MEKi results in synergetic anti-MM effects in RAS mutation myeloma, therefore could be a potent novel therapeutic regimen for patients with relapsed/refractory multiple myeloma.

Dissection of MKK6 and p38 Signaling Using Light-Activated Protein Kinases.

Stress-activated signaling pathways orchestrate cellular behaviors and fates. Studying the precise role(s) of stress-activated protein kinases is challenging, because stress conditions induce adaptation and impose selection pressure. To meet this challenge, we have applied an optogenetic system with a single plasmid to express light-activated p38α or its upstream activator, MKK6, in conjunction with live-cell fluorescence microscopy. In starved cells, decaging of constitutively active p38α or MKK6 by brief exposure to UV light elicits rapid p38-mediated signaling, release of cytochrome c from mitochondria, and apoptosis with different kinetics. In parallel, light activation of p38α also suppresses autophagosome formation, similarly to stimulation with growth factors that activate PI3K/Akt/mTORC1 signaling. Active MKK6 negatively regulates serum-induced ERK activity, which is p38-independent as previously reported. Here, we reproduce that result with the one plasmid system and show that although decaging active p38α does not reduce basal ERK activity in our cells, it can block growth factor-stimulated ERK signaling in serum-starved cells. These results clarify the roles of MKK6 and p38α in dynamic signaling programs, which act in concert to actuate apoptotic death while suppressing cell survival mechanisms.

Effect of Lutein on Ocular Goblet Cell, IFN-γ, and IL-17 Concentration in Dry Eye-Induced Mice Model.

Introduction Dry eye disease affects a substantial number of individuals globally and significantly impacts their quality of life and productivity. Understanding the underlying mechanisms and managing dry eye disease poses substantial challenges. Recent research has highlighted the involvement of various inflammatory mediators in the pathogenesis of dry eye disease, including the cytokines interferon (IFN)-γ and interleukin (IL)-17.Activation of stress signaling pathways and residential immune cells on the ocular epithelial surface ignites epithelial changes, destabilizes tear film, amplifies inflammation and creates an endless loop. Lutein is a β-carotenoid antioxidant which has been proven to be beneficial in many ocular diseasesdue to its protective and anti-inflammatory effect induced by various stimulators. Lutein also acts as a direct and indirectantioxidant agent,suppressing oxidative stress and mitigating oxidative damage. The purpose of this research is to investigate the potential therapeutic effects of lutein in a mouse model of dry eye, aiming to elucidate its impact on ocular manifestation, goblet cells count, IFN-γ and IL-17 level. Methods Desiccating stress was induced in C57BL/6 mice. In a separate group, lutein was administered subcutaneously on a daily basis throughout the experimental period. Clinical manifestations of dry eye, including ocular surface changes, were documented photographically. Goblet cell concentration was assessed through Periodic Acid-Schiff (PAS) staining, and the levels of IFN-γ and IL-17 were measured using enzyme-linked immunosorbent assay (ELISA). Data obtained from these assessments were compared between the experimental groups to determine the potential effects of lutein on dry eye pathology and cytokine levels. Results Significant differences were observed in clinical observations and goblet cell concentrations among the groups; however, no statistically significant differences were found in the levels of IFN-γ and IL-17 between the groups. The untreated group exhibited significantly higher opacities and irregularities compared to the lutein-treated group, whereas the mean goblet cell count was highest in the lutein-treated group. Conclusion Lutein administration improves clinical observations and goblet cell concentrations in a mouse model of dry eye. The treated group exhibited improved ocular surface integrity, but no significant differences in the tested cytokine levels were observed. These findings suggest that lutein supplementation could be a promising therapeutic option for managing dry eye disease. Further research is needed to understand the underlying mechanisms and long-term effects of lutein in dry eye management.

Mutual repression between JNK/AP-1 and JAK/STAT stratifies senescent and proliferative cell behaviors during tissue regeneration.

Epithelial repair relies on the activation of stress signaling pathways to coordinate tissue repair. Their deregulation is implicated in chronic wound and cancer pathologies. Using TNF-α/Eiger-mediated inflammatory damage to Drosophila imaginal discs, we investigate how spatial patterns of signaling pathways and repair behaviors arise. We find that Eiger expression, which drives JNK/AP-1 signaling, transiently arrests proliferation of cells in the wound center and is associated with activation of a senescence program. This includes production of the mitogenic ligands of the Upd family, which allows JNK/AP-1-signaling cells to act as paracrine organizers of regeneration. Surprisingly, JNK/AP-1 cell-autonomously suppress activation of Upd signaling via Ptp61F and Socs36E, both negative regulators of JAK/STAT signaling. As mitogenic JAK/STAT signaling is suppressed in JNK/AP-1-signaling cells at the center of tissue damage, compensatory proliferation occurs by paracrine activation of JAK/STAT in the wound periphery. Mathematical modelling suggests that cell-autonomous mutual repression between JNK/AP-1 and JAK/STAT is at the core of a regulatory network essential to spatially separate JNK/AP-1 and JAK/STAT signaling into bistable spatial domains associated with distinct cellular tasks. Such spatial stratification is essential for proper tissue repair, as coactivation of JNK/AP-1 and JAK/STAT in the same cells creates conflicting signals for cell cycle progression, leading to excess apoptosis of senescently stalled JNK/AP-1-signaling cells that organize the spatial field. Finally, we demonstrate that bistable separation of JNK/AP-1 and JAK/STAT drives bistable separation of senescent signaling and proliferative behaviors not only upon tissue damage, but also in RasV12, scrib tumors. Revealing this previously uncharacterized regulatory network between JNK/AP-1, JAK/STAT, and associated cell behaviors has important implications for our conceptual understanding of tissue repair, chronic wound pathologies, and tumor microenvironments.

Effects of Spatial Expression of Activating Transcription Factor 4 on the Pathogenicity of Two Phenotypes of Bovine Viral Diarrhea Virus by Regulating the Endoplasmic Reticulum-Mediated Autophagy Process.

The endoplasmic reticulum (ER) stress response is a highly conserved stress-defense mechanism and activates the adaptive unfolded protein response (UPR) to mitigate imbalance. The ER stress-activated signaling pathways can also trigger autophagy to facilitate cellular repair. Bovine viral diarrhea virus (BVDV) utilizes the host cellular ER as the primary site of the life cycle. However, the interplay between cellular ER stress and BVDV replication remains unclear. This report reveals that cytopathic (cp) and noncytopathic (ncp) BVDV have distinct strategies to regulate UPR mechanisms and ER stress-mediated autophagy for their own benefit. Immunoblot analysis revealed that cp and ncp BVDV differentially regulated the abundance of ER chaperone GRP78 for viral replication, while the protein kinase RNA-like ER kinase (PERK)-eukaryotic translation initiation factor 2 subunit α (eIF2α)-activating transcription factor 4 (ATF4) pathway of the UPR was switched on at different stages of infection. Pretreatment with ER stress inducer promoted virion replication, but RNA interference (RNAi) knockdown of ATF4 in BVDV-infected cells significantly attenuated BVDV infectivity titers. More importantly, the effector ATF4 activated by cp BVDV infection translocated into the nucleus to mediate autophagy, but ATF4 was retained in the cytoplasm during ncp BVDV infection. In addition, we found that cp BVDV core protein was localized in the ER to induce ER stress-mediated autophagy. Overall, the potential therapeutic target ATF4 may contribute to the global eradication campaign of BVDV. IMPORTANCE The ER-tropic viruses hijack the host cellular ER as the replication platform of the life cycle, which can lead to strong ER stress. The UPR and related transcriptional cascades triggered by ER stress play a crucial role in viral replication and pathogenesis, but little is known about these underlying mechanisms. Here, we report that cytopathic and noncytopathic BVDV use different strategies to reprogram the cellular UPR and ER stress-mediated autophagy for their own advantage. The cytopathic BVDV unconventionally downregulated the expression level of GRP78, creating perfect conditions for self-replication via the UPR, and the noncytopathic BVDV retained ATF4 in the cytoplasm to provide an advantage for its persistent infection. Our findings provide new insights into exploring how BVDV and other ER-tropic viruses reprogram the UPR signaling pathway in the host cells for replication and reveal the attractive host target ATF4 for new antiviral agents.

The Nuclear Transporter Importin 13 Can Regulate Stress-Induced Cell Death through the Clusterin/KU70 Axis.

The cellular response to environmental stresses, such as heat and oxidative stress, is dependent on extensive trafficking of stress-signalling molecules between the cytoplasm and nucleus, which potentiates stress-activated signalling pathways, eventually resulting in cell repair or death. Although Ran-dependent nucleocytoplasmic transport mediated by members of the importin (IPO) super family of nuclear transporters is believed to be responsible for nearly all macromolecular transit between nucleus and cytoplasm, it is paradoxically known to be significantly impaired under conditions of stress. Importin 13 (IPO13) is a unique bidirectional transporter that binds to and releases cargo in a Ran-dependent manner, but in some cases, cargo release from IPO13 is affected by loading of another cargo. To investigate IPO13's role in stress-activated pathways, we performed cell-based screens to identify a multitude of binding partners of IPO13 from human brain, lung, and testes. Analysis of the IPO13 interactome intriguingly indicated more than half of the candidate binding partners to be annotated for roles in stress responses; these included the pro-apoptotic protein nuclear clusterin (nCLU), as well as the nCLU-interacting DNA repair protein KU70. Here, we show, for the first time, that unlike other IPOs which are mislocalised and non-functional, IPO13 continues to translocate between the nucleus and cytoplasm under stress, retaining the capacity to import certain cargoes, such as nCLU, but not export others, such as KU70, as shown by analysis using fluorescence recovery after photobleaching. Importantly, depletion of IPO13 reduces stress-induced import of nCLU and protects against stress-induced cell death, with concomitant protection from DNA damage during stress. Overexpression/FACS experiments demonstrate that nCLU is dependent on IPO13 to trigger stress-induced cell death via apoptosis. Taken together, these results implicate IPO13 as a novel functional nuclear transporter in cellular stress, with a key role thereby in cell fate decision.

ABT199/venetoclax potentiates the cytotoxicity of alkylating agents and fludarabine in acute myeloid leukemia cells.

The antineoplastic activity of pre-transplant regimens in hematopoietic stem cell transplantation (HSCT) is a critical factor for acute myeloid leukemia (AML) patients. There is an urgent need to identify novel approaches without jeopardizing patient safety. We hypothesized that combination of drugs with different mechanisms of action would provide better cytotoxicity. We, therefore, determined the synergistic cytotoxicity of various combinations of the alkylating agents busulfan (Bu) and 4-hydroperoxycyclophosphamide (4HC), the nucleoside analog fludarabine (Flu) and the BCL2 inhibitor ABT199/venetoclax in AML cells. [Bu+4HC] and [Bu+Flu] inhibited cell proliferation and activated apoptosis; addition of ABT199 to either combinations significantly increased these effects with combination indexes < 1. Apoptosis is suggested by cleavages of PARP1 and CASPASE 3, DNA fragmentation, increased reactive oxygen species, decreased mitochondrial membrane potential, and increased pro-apoptotic proteins in the cytoplasm. A similar enhancement of apoptosis was observed in patient-derived cell samples. ABT199/venetocalx upregulated anti-apoptotic MCL1 as a compensatory mechanism but addition of [Bu+4HC] or [Bu+Flu] negated this effect by CASPASE 3-mediated cleavage of MEK1/2 and its substrate MCL1. CASPASE 3 caused cleavage of pro-survival β-CATENIN, which likely contributed to the activation of stress signaling pathways involving SAPK/JNK and AMPK. The observed synergistic cytotoxicity was associated with an inhibition of pro-survival pathways involving STAT1, STAT5 and PI3K. These findings will be useful in designing clinical trials using these drug combinations as pre-transplant conditioning regimens for AML patients.

Oxidation of catalytic cysteine of human deubiquitinase BAP1 triggers misfolding and aggregation in addition to functional loss

Deubiquitinating enzymes (DUBs) form a large protease family involved in a myriad of biological and pathological processes, including ROS sensors. ROS-mediated inhibition of their DUB activities is critical for fine-tuning the stress-activated signaling pathways. Here, we demonstrate that the ubiquitin C-terminal hydrolase (UCH) domain of BAP1 (BAP1-UCH) is highly sensitive to moderate oxidative stress. Oxidation of the catalytic C91 significantly destabilizes BAP1-UCH and increases the population of partially unfolded form, which is prone to aggregation. Unlike other DUBs, the oxidation-induced structural and functional loss of BAP1-UCH cannot be fully reversed by reducing agents. The oligomerization of oxidized BAP1-UCH is attributed to inter-molecular disulfide bond formation. Hydrogen-deuterium mass exchange spectrometry (HDX-MS) reveals increased fluctuations of the central β-sheet upon oxidation. Our findings suggest that oxidation-mediated functional loss and increased aggregation propensity may contribute to oncogenesis associated with BAP1.

Conventional rigid 2D substrates cause complex contractile signals in monolayers of human induced pluripotent stem cell‐derived cardiomyocytes

Human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CM) in monolayers interact mechanically via cell–cell and cell–substrate adhesion. Spatiotemporal features of contraction were analysed in hiPSC‐CM monolayers (1) attached to glass or plastic (Young's modulus (E) >1 GPa), (2) detached (substrate‐free) and (3) attached to a flexible collagen hydrogel (E = 22 kPa). The effects of isoprenaline on contraction were compared between rigid and flexible substrates. To clarify the underlying mechanisms, further gene expression and computational studies were performed. HiPSC‐CM monolayers exhibited multiphasic contractile profiles on rigid surfaces in contrast to hydrogels, substrate‐free cultures or single cells where only simple twitch‐like time‐courses were observed. Isoprenaline did not change the contraction profile on either surface, but its lusitropic and chronotropic effects were greater in hydrogel compared with glass. There was no significant difference between stiff and flexible substrates in regard to expression of the stress‐activated genes NPPA and NPPB. A computational model of cell clusters demonstrated similar complex contractile interactions on stiff substrates as a consequence of cell‐to‐cell functional heterogeneity. Rigid biomaterial surfaces give rise to unphysiological, multiphasic contractions in hiPSC‐CM monolayers. Flexible substrates are necessary for normal twitch‐like contractility kinetics and interpretation of inotropic interventions. Key points Spatiotemporal contractility analysis of human induced pluripotent stem cell‐derived cardiomyocyte (hiPSC‐CM) monolayers seeded on conventional, rigid surfaces (glass or plastic) revealed the presence of multiphasic contraction patterns across the monolayer with a high variability, despite action potentials recorded in the same areas being identical.These multiphasic patterns are not present in single cells, in detached monolayers or in monolayers seeded on soft substrates such as a hydrogel, where only ‘twitch’‐like transients are observed.HiPSC‐CM monolayers that display a high percentage of regions with multiphasic contraction have significantly increased contractile duration and a decreased lusotropic drug response.There is no indication that the multiphasic contraction patterns are associated with significant activation of the stress‐activated NPPA or NPPB signalling pathways.A computational model of cell clusters supports the biological findings that the rigid surface and the differential cell–substrate adhesion underly multiphasic contractile behaviour of hiPSC‐CMs.

Stress sensing within the breast tumor microenvironment: how glucocorticoid receptors live in the moment.

The classification and treatment of breast cancer is largely defined by the expression of steroid hormone receptors (HRs), namely estrogen receptor (ER) and progesterone receptor (PR), and gene amplification/overexpression of human epidermal growth factor receptor 2 (HER2). More recently, studies of androgen receptor (AR), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) have revealed that targeting these related HRs may be a promising strategy for a more personalized approach to the treatment of specific subtypes of HR+ breast cancer. For example, GR expression is associated with a good prognosis in ER+ breast cancer, but predicts poor prognosis in triple-negative breast cancer (TNBC). GR, like ER, PRs, and AR, is a ligand-activated transcription factor, but also has significant ligand-independent signaling activities. GR transcriptional activity is classically regulated by circulating glucocorticoids (GCs; ligand-dependent). Recent studies demonstrate that GR transcriptional activity is also regulated by a variety of cellular stress stimuli that input to GR Ser134 phosphorylation via rapid activation of the p38 mitogen activated protein kinase (MAPK) signaling pathway (ligand-independent). Furthermore, ligand-independent GR activation promotes feedforward signaling loops that mediate sustained activation of stress signaling pathways to drive advanced cancer biology (i.e. migration, invasion, chemoresistance, survival, and cellular growth). In this review, we will focus on the role of GR as a key sensor and mediator of physiologic and tumor microenvironment (TME)-derived cellular stress signaling in TNBC and discuss how targeting GR and/or associated signaling pathways may provide a strategy to inhibit deadly TNBC progression.

MAP4K2 Silencing Overcomes IMiDs-Resistance in Multiple Myeloma

Recently, mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2) has emerged as an important key regulator of the stress-activated MAPK core signaling pathways. MAP4K2, also called Germinal Center Kinase (GCK), is predominantly and highly expressed in the germinal center of B cells. Recently we have shown that MAP4K2 knockdown in K- or N-RAS mutated MM cells induces MM cell growth inhibition, associated with the downregulation of critical transcriptional factors including IKZF1/3, BCL-6, and c-MYC proteins (Li et al. Blood 2021). Importantly, MAP4K2 silencing induces IKZF1 protein degradation without affecting IKZF1 mRNA level. Furthermore, IMiDs-resistant K-RAS Mut MM cells are sensitive to MAP4K2 inhibition induced IKZF1 degradation and cell growth suppression, suggesting that MAP4K2 inhibition overcomes IMiDs resistances in MM.To further validate MAP4K2 inhibition as a novel strategy to overcome IMiDs-resistance, we generated lenalidomide-resistant human myeloma cell lines. In this model, MM1S-LEN RES cells showed significantly decreased expression of CRBN protein compared to the parent cells. Accordingly, upon lenalidomide treatment, CRBN-mediated down-regulation of IKZF1, c-MYC, IKZF3 and IRF4 were abrogated in the MM1S-LEN RES cells. As expected, MM1S-LEN RES cells were resistant to lenalidomide induced growth inhibition in cell proliferation assay. In contrast, MAP4K2 inhibition using TL4-12 potently induced IKZF1, c-MYC, and IRF4 downregulation as well as cell proliferation inhibition, demonstrating that MAP4K2 regulates IKZF1 and cell growth independently of CRBN. These results indicate that MAP4K2 is a novel therapeutic target to overcome IMiDs-resistance MM.Iberdomide (CC-220) is an orally available IMiD® compound under development for the treatment of relapsed/refractory multiple myeloma. Previous biochemical and structural studies demonstrated that Iberdomide binds to cereblon with a higher affinity than lenalidomide or pomalidomide. Here, we evaluated the combination effects of Iberdomide with MAP4K2 silencing in MM. Tet-on sh-MAP4K2 lentivirus were introduced into RAS Mut MM cells to establish the inducible MAP4K2 knockdown cells upon doxycycline treatment. To address the combination effects, Tet-on sh-MAP4K2 RAS Mut MM cells were treated with doxycycline and different dosages of iberdomide. We found that MAP4K2 silencing strongly increased iberdomide-induced apoptosis (iberdomide alone vs. with MAP4K2 KD: 32% vs 92). Similar, in western blot assays, MAP4K2 silencing combined with Iberdomide significantly enhanced downregulation of IKZF1, c-MYC, and IRF4 compared to the iberdomide treatment alone. These data suggest that combination of iberdomide and MAP4K2 inhibition have synergetic anti-MM effects.Taken together, our findings demonstrate that MAP4K2 is a novel therapeutic target to bypass IMiDs resistance in RAS mutated MM. Combination of MAP4K2 inhibition with Iberdomide results in synergetic anti-cancer effects in MM, therefore could be a potent novel therapeutic regimen for patients with relapsed/refractory multiple myeloma. DisclosuresMarcireau: Sanofi: Current Employment.

New Succinimides with Potent Anticancer Activity: Synthesis, Activation of Stress Signaling Pathways and Characterization of Apoptosis in Leukemia and Cervical Cancer Cells.

Based on previously identified dicarboximides with significant anticancer and immunomodulatory activities, a series of 26 new derivatives were designed and synthesized by the Diels–Alder reaction between appropriate diene and maleimide or hydroxymaleimide moieties. The resulting imides were functionalized with alkanolamine or alkylamine side chains and subsequently converted to their hydrochlorides. The structures of the obtained compounds were confirmed by 1H and 13C NMR and by ESI MS spectral analysis. Their cytotoxicity was evaluated in human leukemia (K562, MOLT4), cervical cancer (HeLa), and normal endothelial cells (HUVEC). The majority of derivatives exhibited high to moderate cytotoxicity and induced apoptosis in K562 cells. Microarray gene profiling demonstrated upregulation of proapoptotic genes involved in receptor-mediated and mitochondrial cell death pathways as well as antiapoptotic genes involved in NF-kB signaling. Selected dicarboximides activated JNK and p38 kinases in leukemia cells, suggesting that MAPKs may be involved in the regulation of apoptosis. The tested dicarboximides bind to DNA as assessed by a plasmid DNA cleavage protection assay. The selected dicarboximides offer new scaffolds for further development as anticancer drugs.

Role of MicroRNA-145 in DNA Damage Signalling and Senescence in Vascular Smooth Muscle Cells of Type 2 Diabetic Patients.

Increased cardiovascular morbidity and mortality in individuals with type 2 diabetes (T2DM) is a significant clinical problem. Despite advancements in achieving good glycaemic control, this patient population remains susceptible to macrovascular complications. We previously discovered that vascular smooth muscle cells (SMC) cultured from T2DM patients exhibit persistent phenotypic aberrancies distinct from those of individuals without a diagnosis of T2DM. Notably, persistently elevated expression levels of microRNA-145 co-exist with characteristics consistent with aging, DNA damage and senescence. We hypothesised that increased expression of microRNA-145 plays a functional role in DNA damage signalling and subsequent cellular senescence specifically in SMC cultured from the vasculature of T2DM patients. In this study, markers of DNA damage and senescence were unambiguously and permanently elevated in native T2DM versus non-diabetic (ND)-SMC. Exposure of ND cells to the DNA-damaging agent etoposide inflicted a senescent phenotype, increased expression of apical kinases of the DNA damage pathway and elevated expression levels of microRNA-145. Overexpression of microRNA-145 in ND-SMC revealed evidence of functional links between them; notably increased secretion of senescence-associated cytokines and chronic activation of stress-activated intracellular signalling pathways, particularly the mitogen-activated protein kinase, p38α. Exposure to conditioned media from microRNA-145 overexpressing cells resulted in chronic p38α signalling in naïve cells, evidencing a paracrine induction and reinforcement of cell senescence. We conclude that targeting of microRNA-145 may provide a route to novel interventions to eliminate DNA-damaged and senescent cells in the vasculature and to this end further detailed studies are warranted.

Efficacy of Cigu Xiaozhi pill on non-alcoholic steatohepatitis-associated lipoapoptosis through stress-activated c-Jun N-terminal kinase signalling pathway.

To investigate the efficacy of Cigu Xiaozhi pill (, CGXZ) on non-alcoholic steatohepatitis (NASH)-associated lipoapoptosis through the stress-activated c-Jun N-terminal kinase (JNK)/ stress-activated protein kinase signalling pathway. Sixty male Sprague-Dawley rats were randomly divided into the following groups (10rats each): blank control, model, low-dose CGXZ, medium-dose CGXZ, high-dose CGXZ, and positive control (treated with SP600125, a JNK inhibitor). The NASH model was established and the histomorphological characteristics of haematoxylin and eosin-stained liver tissues were examined under a light microscope. Cell apoptosis in liver tissues was assessed via terminal deoxynucleotidyl transferase dUTP nick-end labelling assay. In addition, the mRNA and protein expression levels of p-JNK, p-c-Jun, caspase-8, Fas, and Fas-L were determined via fluorescence-based quantitative real-time PCR, immunohistochemical and Western blot assays. Histopathological examination of the liver showed that the model rats had moderate-to-severe steatosis with infiltration of inflammatory cells as well as significantly higher expression levels of the p-JNK, p-c-Jun, caspase-8, Fas, and Fas-L proteins, compared with those in the blank control group (P < 0.01). Hepatic lobules of the rats in the treatment groups showed significantly reduced vacuolar degeneration and steatosis as well as alleviated inflammatory cell infiltration. The high and medium-dose CGXZ groups exhibited significantly lower mRNA and protein expression levels of p-JNK, p-c-Jun, caspase-8, Fas, and Fas-L, compared with those in the model group (P < 0.05 or P < 0.01). CGXZ pill inhibited the onset of hepatocyte apoptosis by regulating the expression of p-JNK, p-c-Jun, caspase-8, Fas, and Fas-L, thereby exerting therapeutic effects against NASH.

Oxidative stress during insulin resistance in prediabetes : a review on role of antioxidants

Oxidative stress is one of the major causal factors behind insulin resistance during prediabetes. It can impair insulin signalling by triggering stress activated signaling pathways and can also directly oxidize and damage the proteins of these pathways. Although cells have impressive stock of antioxidant enzymes and minor antioxidant moieties, these agents may not be adequate to maintain the redox balance during oxidative stress. The important functional antioxidant vitamins for tissue protection from excess free radicals comprises vitamin C, E and A. Vitamin C can improve insulin resistance by altering endothelial function and reducing oxidative stress. Vitamin E is an excellent trap for peroxyl radicals which suppresses ROS production in the pancreas, preserves pancreatic β cell function and maintains structural integrity of pancreatic islet cells. Vitamin A prevents tissue oxidative damage as well as exhibits a regenerative role in the pancreas. Like vitamins, minerals play a crucial role during oxidative stress and insulin resistance. Zinc is crucial for insulin actions and carbohydrate metabolism. Chromium activates insulin receptors through the oligopeptidechromudulin, which occurs in the insulin sensitive cells that binds to the insulin receptor, thereby increasing insulin signal transduction and sensitivity. Selenium up regulates glutathione peroxidase activity can inhibit NF-kappa B activation. The risk of diabetes and its complications will be more with elevating oxidative stress. Interventions with these antioxidants have proven beneficial role in reducing oxidative stress which can be utilized for reduction of insulin resistance in prediabetes.

First person – Dimitrije Stanković

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms, helping early-career researchers promote themselves alongside their papers. Dimitrije Stanković is first author on ‘A Drosophila model to study retinitis pigmentosa pathology associated with mutations in the core splicing factor Prp8’, published in DMM. Dimitrije is a PhD student in the lab of Mirka Uhlirova at CECAD Research Center in Cologne, Germany, investigating the functional consequences of aberrant pre-mRNA splicing in the context of regulation of gene expression, activation of stress signaling pathways and DNA damage.

A Novel Salicylanilide Derivative Induces Autophagy Cell Death in Castration-Resistant Prostate Cancer via ER Stress-Activated PERK Signaling Pathway.

Metastatic castration-resistant prostate cancer (CRPC) is currently incurable. Cancer growth and progression is intimately affected by its interaction with host microenvironment. Cotargeting of the stroma and prostate cancer is therefore an emerging therapeutic strategy for metastatic CRPC. Cancer-induced osteoclastogenesis is known to contribute to CRPC bone metastasis. This study is to extend pharmacologic value of our synthesized LCC03, a derivative of 5-(2',4'-difluorophenyl)-salicylanilide that has previously testified for its osteoclastogenesis activity, by exploring its additional cytotoxic properties and underlying mechanism in CRPC cells. LCC03 was chemically synthesized and examined for cell growth inhibition in a serial of CRPC cell lines. We demonstrated that LCC03 dose-dependently suppressed proliferation and retarded cell-cycle progression in CRPC cells. The classical autophagy features, including autophagosome formation and LC3-II conversion, were dramatically shown in LCC03-treated CRPC cells, and it was associated with the suppressed AKT/mTOR signaling pathways, a major negative regulator of autophagy. Moreover, an expanded morphology of the endoplasmic reticulum (ER), increased expression of the ER stress markers GRP78 and PERK, and eIF2α phosphorylation were observed. Blockage of autophagy and PERK pathways using small molecule inhibitors or shRNA knockdown reversed LCC03-induced autophagy and cell death, thus indicating that the PERK-eIF2α pathway contributed to the LCC03-induced autophagy. Furthermore, treatment of tumor-bearing mice with intraperitoneal administered LCC03 suppressed the growth of CRPC xenografts in mouse bone without systemic toxicity. The dual action of 5-(2',4'-difluorophenyl)-salicylanilide on targeting both the osteoclasts and the tumor cells strongly indicates that LCC03 is a promising anticancer candidate for preventing and treating metastatic CRPC.

Normal Saline solutions cause endothelial dysfunction through loss of membrane integrity, ATP release, and inflammatory responses mediated by P2X7R/p38 MAPK/MK2 signaling pathways.

Resuscitation with 0.9% Normal Saline (NS), a non-buffered acidic solution, leads to increased morbidity and mortality in the critically ill. The goal of this study was to determine the molecular mechanisms of endothelial injury after exposure to NS. The hypothesis of this investigation is that exposure of endothelium to NS would lead to loss of cell membrane integrity, resulting in release of ATP, activation of the purinergic receptor (P2X7R), and subsequent activation of stress activated signaling pathways and inflammation. Human saphenous vein endothelial cells (HSVEC) incubated in NS, but not buffered electrolyte solution (Plasma-Lyte, PL), exhibited abnormal morphology and increased release of lactate dehydrogenase (LDH), adenosine triphosphate (ATP), and decreased transendothelial resistance (TEER), suggesting loss of membrane integrity. Incubation of intact rat aorta (RA) or human saphenous vein in NS but not PL led to impaired endothelial-dependent relaxation which was ameliorated by apyrase (hydrolyzes ATP) or SB203580 (p38 MAPK inhibitor). Exposure of HSVEC to NS but not PL led to activation of p38 MAPK and its downstream substrate, MAPKAP kinase 2 (MK2). Treatment of HSVEC with exogenous ATP led to interleukin 1β (IL-1β) release and increased vascular cell adhesion molecule (VCAM) expression. Treatment of RA with IL-1β led to impaired endothelial relaxation. IL-1β treatment of HSVEC led to increases in p38 MAPK and MK2 phosphorylation, and increased levels of arginase II. Incubation of porcine saphenous vein (PSV) in PL with pH adjusted to 6.0 or less also led to impaired endothelial function, suggesting that the acidic nature of NS is what contributes to endothelial dysfunction. Volume overload resuscitation in a porcine model after hemorrhage with NS, but not PL, led to acidosis and impaired endothelial function. These data suggest that endothelial dysfunction caused by exposure to acidic, non-buffered NS is associated with loss of membrane integrity, release of ATP, and is modulated by P2X7R-mediated inflammatory responses.

Quantitative Phosphoproteome Analysis of Clostridioides difficile Toxin B Treated Human Epithelial Cells

The large clostridial glucosylating toxin B (TcdB) is a major virulence factor of the nosocomial pathogen Clostridioides difficile. TcdB inhibits small GTPases by glucosylation leading to impaired downstream signaling. TcdB also possesses a glucosyltransferase independent effect described as pyknosis. To elucidate the impact of TcdB and its glucosylation-inactive mutant TcdBNXN on the kinome of human cells, SILAC labeled HEp-2 cells were treated with 2 nM TcdB for 8 h. Phosphopeptides were enriched using SCX chromatography, IMAC and TiO2 followed shotgun mass spectrometry analysis. Overall 4,197 phosphopeptides were identified; more than 1,200 phosphosites responded to treatment with TcdB or TcdBNXN. The data suggested that predominantly stress-activated MAPK-dependent signaling pathways were triggered by toxin B treatment.