Valrubicin-loaded immunoliposomes for specific vesicle-mediated cell death in the treatment of hematological cancers.

We created valrubicin-loaded immunoliposomes (Val-ILs) using the antitumor prodrug valrubicin, a hydrophobic analog of daunorubicin. Being lipophilic, valrubicin readily incorporated Val-lLs that were loaded with specific antibodies. Val-ILs injected intravenously rapidly reached the bone marrow and spleen, indicating their potential to effectively target cancer cells in these areas. Following the transplantation of human pediatric B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), or acute myeloid leukemia (AML) in immunodeficient NSG mice, we generated patient-derived xenograft (PDX) models, which were treated with Val-ILs loaded with antibodies to target CD19, CD7 or CD33. Only a small amount of valrubicin incorporated into Val-ILs was needed to induce leukemia cell death in vivo, suggesting that this approach could be used to efficiently treat acute leukemia cells. We also demonstrated that Val-ILs could reduce the risk of contamination of CD34+ hematopoietic stem cells by acute leukemia cells during autologous peripheral blood stem cell transplantation, which is a significant advantage for clinical applications. Using EL4 lymphoma cells on immunocompetent C57BL/6 mice, we also highlighted the potential of Val-ILs to target immunosuppressive cell populations in the spleen, which could be valuable in impairing cancer cell expansion, particularly in lymphoma cases. The most efficient Val-ILs were found to be those loaded with CD11b or CD223 antibodies, which, respectively, target the myeloid-derived suppressor cells (MDSC) or the lymphocyte-activation gene 3 (LAG-3 or CD223) on T4 lymphocytes. This study provides a promising preclinical demonstration of the effectiveness and ease of preparation of Val-ILs as a novel nanoparticle technology. In the context of hematological cancers, Val-ILs have the potential to be used as a precise and effective therapy based on targeted vesicle-mediated cell death.

Valproic Acid Treatment after Traumatic Brain Injury in Mice Alleviates Neuronal Death and Inflammation in Association with Increased Plasma Lysophosphatidylcholines.

The histone deacetylase inhibitor (HDACi) valproic acid (VPA) has neuroprotective and anti-inflammatory effects in experimental traumatic brain injury (TBI), which have been partially attributed to the epigenetic disinhibition of the transcription repressor RE1-Silencing Transcription Factor/Neuron-Restrictive Silencer Factor (REST/NRSF). Additionally, VPA changes post-traumatic brain injury (TBI) brain metabolism to create a neuroprotective environment. To address the interconnection of neuroprotection, metabolism, inflammation and REST/NRSF after TBI, we subjected C57BL/6N mice to experimental TBI and intraperitoneal VPA administration or vehicle solution at 15 min, 1, 2, and 3 days post-injury (dpi). At 7 dpi, TBI-induced an up-regulation of REST/NRSF gene expression and HDACi function of VPA on histone H3 acetylation were confirmed. Neurological deficits, brain lesion size, blood-brain barrier permeability, or astrogliosis were not affected, and REST/NRSF target genes were only marginally influenced by VPA. However, VPA attenuated structural damage in the hippocampus, microgliosis and expression of the pro-inflammatory marker genes. Analyses of plasma lipidomic and polar metabolomic patterns revealed that VPA treatment increased lysophosphatidylcholines (LPCs), which were inversely associated with interleukin 1 beta (Il1b) and tumor necrosis factor (Tnf) gene expression in the brain. The results show that VPA has mild neuroprotective and anti-inflammatory effects likely originating from favorable systemic metabolic changes resulting in increased plasma LPCs that are known to be actively taken up by the brain and function as carriers for neuroprotective polyunsaturated fatty acids.

Important Factors Affecting Induction of Cell Death, Oxidative Stress and DNA Damage by Nano- and Microplastic Particles In Vitro.

We have described the influence of selected factors that increase the toxicity of nanoplastics (NPs) and microplastics (MPs) with regard to cell viability, various types of cell death, reactive oxygen species (ROS) induction, and genotoxicity. These factors include plastic particle size (NPs/MPs), zeta potential, exposure time, concentration, functionalization, and the influence of environmental factors and cell type. Studies have unequivocally shown that smaller plastic particles are more cytotoxic, penetrate cells more easily, increase ROS formation, and induce oxidative damage to proteins, lipids, and DNA. The toxic effects also increase with concentration and incubation time. NPs with positive zeta potential are also more toxic than those with a negative zeta potential because the cells are negatively charged, inducing stronger interactions. The deleterious effects of NPs and MPs are increased by functionalization with anionic or carboxyl groups, due to greater interaction with cell membrane components. Cationic NPs/MPs are particularly toxic due to their greater cellular uptake and/or their effects on cells and lysosomal membranes. The effects of polystyrene (PS) vary from one cell type to another, and normal cells are more sensitive to NPs than cancerous ones. The toxicity of NPs/MPs can be enhanced by environmental factors, including UV radiation, as they cause the particles to shrink and change their shape, which is a particularly important consideration when working with environmentally-changed NPs/MPs. In summary, the cytotoxicity, oxidative properties, and genotoxicity of plastic particles depends on their concentration, duration of action, and cell type. Also, NPs/MPs with a smaller diameter and positive zeta potential, and those exposed to UV and functionalized with amino groups, demonstrate higher toxicity than larger, non-functionalized and environmentally-unchanged particles with a negative zeta potential.

Cytotoxic sigma-2 ligands trigger cancer cell death via cholesterol-induced-ER-stress.

Sigma-2-ligands (S2L) are characterized by high binding affinities to their cognate sigma-2 receptor, overexpressed in rapidly proliferating tumor cells. As such, S2L were developed as imaging probes (ISO1) or as cancer therapeutics, alone (SV119 [C6], SW43 [C10]) and as delivery vehicles for cytotoxic drug cargoes (C6-Erastin, C10-SMAC). However, the exact mechanism of S2L-induced cytotoxicity remains to be fully elucidated. A series of high-affinity S2L were evaluated regarding their cytotoxicity profiles across cancer cell lines. While C6 and C10 displayed distinct cytotoxicities, C0 and ISO1 were essentially non-toxic. Confocal microscopy and lipidomics analysis in cellular and mouse models revealed that C10 induced increases in intralysosomal free cholesterol and in cholesterol esters, suggestive of unaltered intracellular cholesterol trafficking. Cytotoxicity was caused by cholesterol excess, a phenomenon that contrasts the effects of NPC1 inhibition. RNA-sequencing revealed gene clusters involved in cholesterol homeostasis and ER stress response exclusively by cytotoxic S2L. ER stress markers were confirmed by qPCR and their targeted modulation inhibited or enhanced cytotoxicity of C10 in a predicted manner. Moreover, C10 increased sterol regulatory element-binding protein 2 (SREBP2) and low-density lipoprotein receptor (LDLR), both found to be pro-survival factors activated by ER stress. Furthermore, inhibition of downstream processes of the adaptive response to S2L with simvastatin resulted in synergistic treatment outcomes in combination with C10. Of note, the S2L conjugates retained the ER stress response of the parental ligands, indicative of cholesterol homeostasis being involved in the overall cytotoxicity of the drug conjugates. Based on these findings, we conclude that S2L-mediated cell death is due to free cholesterol accumulation that leads to ER stress. Consequently, the cytotoxic profiles of S2L drug conjugates are proposed to be enhanced via concurrent ER stress inducers or simvastatin, strategies that could be instrumental on the path toward tumor eradication.

LPS and ATP-induced Death of PMA-differentiated THP-1 Macrophages and its Validation.

Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced phorbol-12-myristate-13-acetate (PMA)-differentiated lipid deposition in human monocyte (THP-1) macrophage model to observe cell death. LPS combined with ATP is a classic inflammatory induction method, often used to study pyroptosis, but apoptosis and necroptosis also respond to stimulation by LPS/ATP. Under normal circumstances, phosphatidylserine is only localized in the inner leaflet of the plasma membrane. However, in the early stages of pyroptosis, apoptosis, and necroptosis, the cell membrane remains intact and exposed to phosphatidylserine, and in the later stages, the cell membrane loses its integrity. Here, flow cytometry was used to analyze Annexin V and 7-Aminoactinomycin D (AAD) double staining to detect the cell death from the whole cells. The results show that substantial cells died after stimulation with LPS/ATP. Using scanning electron microscopy, we observe the possible forms of cell death in individual cells. The results indicate that cells may undergo pyroptosis, apoptosis, or necroptosis after stimulation with LPS/ATP. This protocol focuses on observing the death of macrophages after stimulation with LPS/ATP. The results showed that cell death after LPS and ATP stimulation is not limited to pyroptosis and that apoptosis and necrotic apoptosis can also occur, helping researchers better understand cell death after LPS and ATP stimulation and choose a better experimental method.

The microtubule targeting agent ST-401 triggers cell death in interphase and prevents the formation of polyploid giant cancer cells.

Microtubule targeting agents (MTAs) are commonly prescribed to treat cancers and predominantly kill cancer cells in mitosis. Significantly, some MTA-treated cancer cells escape death in mitosis, exit mitosis and become malignant polyploid giant cancer cells (PGCC). Considering the low number of cancer cells undergoing mitosis in tumor tissues, killing them in interphase may represent a favored antitumor approach. We discovered that ST-401, a mild inhibitor of microtubule (MT) assembly, preferentially kills cancer cells in interphase as opposed to mitosis, a cell death mechanism that avoids the development of PGCC. Single cell RNA sequencing identified mRNA transcripts regulated by ST-401, including mRNAs involved in ribosome and mitochondrial functions. Accordingly, ST-401 induces a transient integrated stress response, reduces energy metabolism, and promotes mitochondria fission. This cell response may underly death in interphase and avoid the development of PGCC. Considering that ST-401 is a brain-penetrant MTA, we validated these results in glioblastoma cell lines and found that ST-401 also reduces energy metabolism and promotes mitochondria fission in GBM sensitive lines. Thus, brain-penetrant mild inhibitors of MT assembly, such as ST-401, that induce death in interphase through a previously unanticipated antitumor mechanism represent a potentially transformative new class of therapeutics for the treatment of GBM.

Sodium arsenite and arsenic trioxide differently affect the oxidative stress of lymphoblastoid cells: An intricate crosstalk between mitochondria, autophagy and cell death.

Although the toxicity of arsenic depends on its chemical forms, few studies have taken into account the ambiguous phenomenon that sodium arsenite (NaAsO2) acts as a potent carcinogen while arsenic trioxide (ATO, As2O3) serves as an effective therapeutic agent in lymphoma, suggesting that NaAsO2 and As2O3 may act via paradoxical ways to either promote or inhibit cancer pathogenesis. Here, we compared the cellular response of the two arsenical compounds, NaAsO2 and As2O3, on the Burkitt lymphoma cell model, the Epstein Barr Virus (EBV)-positive P3HR1 cells. Using flow cytometry and biochemistry analyses, we showed that a NaAsO2 treatment induces P3HR1 cell death, combined with drastic drops in ΔΨm, NAD(P)H and ATP levels. In contrast, As2O3-treated cells resist to cell death, with a moderate reduction of ΔΨm, NAD(P)H and ATP. While both compounds block cells in G2/M and affect their protein carbonylation and lipid peroxidation, As2O3 induces a milder increase in superoxide anions and H2O2 than NaAsO2, associated to a milder inhibition of antioxidant defenses. By electron microscopy, RT-qPCR and image cytometry analyses, we showed that As2O3-treated cells display an overall autophagic response, combined with mitophagy and an unfolded protein response, characteristics that were not observed following a NaAsO2 treatment. As previous works showed that As2O3 reactivates EBV in P3HR1 cells, we treated the EBV- Ramos-1 cells and showed that autophagy was not induced in these EBV- cells upon As2O3 treatment suggesting that the boost of autophagy observed in As2O3-treated P3HR1 cells could be due to the presence of EBV in these cells. Overall, our results suggest that As2O3 is an autophagic inducer which action is enhanced when EBV is present in the cells, in contrast to NaAsO2, which induces cell death. That's why As2O3 is combined with other chemicals, as all-trans retinoic acid, to better target cancer cells in therapeutic treatments.

The stress-responsive cytotoxic effect of diesel exhaust particles on lymphatic endothelial cells.

Diesel exhaust particles (DEPs) are very small (typically < 0.2 µm) fragments that have become major air pollutants. DEPs are comprised of a carbonaceous core surrounded by organic compounds such as polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs. Inhaled DEPs reach the deepest sites in the respiratory system where they could induce respiratory/cardiovascular dysfunction. Additionally, a previous study has revealed that a portion of inhaled DEPs often activate immune cells and subsequently induce somatic inflammation. Moreover, DEPs are known to localize in lymph nodes. Therefore, in this study we explored the effect of DEPs on the lymphatic endothelial cells (LECs) that are a constituent of the walls of lymph nodes. DEP exposure induced cell death in a reactive oxygen species (ROS)-dependent manner. Following exposure to DEPs, next-generation sequence (NGS) analysis identified an upregulation of the integrated stress response (ISR) pathway and cell death cascades. Both the soluble and insoluble components of DEPs generated intracellular ROS. Three-dimensional Raman imaging revealed that DEPs are taken up by LECs, which suggests internalized DEP cores produce ROS, as well as soluble DEP components. However, significant cell death pathways such as apoptosis, necroptosis, ferroptosis, pyroptosis, and parthanatos seem unlikely to be involved in DEP-induced cell death in LECs. This study clarifies how DEPs invading the body might affect the lymphatic system through the induction of cell death in LECs.

Identification of ROS and KEAP1-related genes and verified targets of α-hederin induce cell death for CRC.

In this study, we analyzed and verified differentially expressed genes (DEGs) in ROS and KEAP1 crosstalk in oncogenic signatures using GEO data sets (GSE4107 and GSE41328). Multiple pathway enrichment analyses were finished based on DEGs. The genetic signature for colorectal adenocarcinoma (COAD) was identified by using the Cox regression analysis. Kaplan-Meier survival and receiver operating characteristic curve analysis were used to explore the prognosis value of specific genes in COAD. The potential immune signatures and drug sensitivity prediction were also analyzed. Promising small-molecule agents were identified and predicted targets of α-hederin in SuperPred were validated by molecular docking. Also, expression levels of genes and Western blot analysis were conducted. In total, 48 genes were identified as DEGs, and the hub genes such as COL1A1, CXCL12, COL1A2, FN1, CAV1, TIMP3, and IGFBP7 were identified. The ROS and KEAP1-associated gene signatures comprised of hub key genes were developed for predicting the prognosis and evaluating the immune cell responses and immune infiltration in COAD. α-hederin, a potential anti-colorectal cancer (CRC) agent, was found to enhance the sensitivity of HCT116 cells, regulate CAV1 and COL1A1, and decrease KEAP1, Nrf2, and HO-1 expression significantly. KEAP1-related genes could be an essential mediator of ROS in CRC, and KEAP1-associated genes were effective in predicting prognosis and evaluating individualized CRC treatment. Therefore, α-hederin may be an effective chemosensitizer for CRC treatments in clinical settings.

Neuroprotective Effect of Sterculia setigera Leaves Hydroethanolic Extract.

Plants are a valuable source of information for pharmacological research and new drug discovery. The present study aimed to evaluate the neuroprotective potential of the leaves of the medicinal plant Sterculia setigera. In vitro, the effect of Sterculia setigera leaves dry hydroethanolic extract (SSE) was tested on cultured cerebellar granule neurons (CGN) survival when exposed to hydrogen peroxide (H2O2) or 6-hydroxydopamine (6-OHDA), using the viability probe fluorescein diacetate (FDA), a lactate dehydrogenase (LDH) activity assay, an immunocytochemical staining against Gap 43, and the quantification of the expression of genes involved in apoptosis, necrosis, or oxidative stress. In vivo, the effect of intraperitoneal (ip) injection of SSE was assessed on the developing brain of 8-day-old Wistar rats exposed to ethanol neurotoxicity by measuring caspase-3 activity on cerebellum homogenates, the expression of some genes in tissue extracts, the thickness of cerebellar cortical layers and motor coordination. In vitro, SSE protected CGN against H2O2 and 6-OHDA-induced cell death at a dose of 10 µg/mL, inhibited the expression of genes Casp3 and Bad, and upregulated the expression of Cat and Gpx7. In vivo, SSE significantly blocked the deleterious effect of ethanol by reducing the activity of caspase-3, inhibiting the expression of Bax and Tp53, preventing the reduction of the thickness of the internal granule cell layer of the cerebellar cortex, and restoring motor functions. Sterculia setigera exerts neuroactive functions as claimed by traditional medicine and should be a good candidate for the development of a neuroprotective treatment against neurodegenerative diseases.

Proteasome activation is critical for cell death induced by inhibitors of polo-like kinase 1 (PLK1) in multiple cancers.

Inhibitors of polo-like kinase (PLK) are currently being evaluated as anticancer drugs. However, the molecular mechanism of PLK inhibitor-induced cell death is not fully understood. In this study, we found that GW843682X and BI2536, two inhibitors of PLK1, significantly induced cell death in multiple type cells. The induction of cell death was related to the preferring expression of PLK1. However, in human umbilical vascular endothelial cells (HUVEC) and human colorectal carcinoma cells, which expressed higher levels of both PLK1 and PLK2, PLK1 inhibitors induced very low levels of cell death. Clinical analysis reveals PLK1 presence in 26 of 30 NPC tumor tissues. In in vivo NPC lung metastasis nude mouse models, PLK1 inhibitors decreased NPC progress. Mechanistically, the PLK1 inhibitor did not activate p53, and the cell death was not reversed by p53 inhibition. Moreover, PLK1 inhibitor-induced cell death was PARP- and caspase-independent. Although PLK1 inhibitors induced down-regulation of calpain inhibitor calpastatin and calpain was activated by PLK1 inhibition, calpain blocking did not reverse cell death induced by PLK1 inhibitors, suggesting the non-involvement of calpain. Surprisingly, we found that PLK1 inhibitors induced the activation of proteasome, and the treatment of cells with PLK1 inhibitors reduced the levels of ubiquitinated proteins. And proteasome inhibitors reversed cell death induced by PLK1 inhibitors in various cell types in which PLK1 was preferentially expressed. Moreover, PLK1 inhibition reversed the degradation of proteins including p53, caspase 8, PARP and calpastatin. These results suggest that the activation of proteasome is critical for cell death induced by PLK1 inhibition.

Short-chain fatty acids induced lung tumor cell death and increased peripheral blood CD4+ T cells in NSCLC and control patients ex vivo.

Background: Despite therapy advances, one of the leading causes of cancer deaths still remains lung cancer. To improve current treatments or prevent non-small cell lung cancer (NSCLC), the role of the nutrition in cancer onset and progression needs to be understood in more detail. While in colorectal cancer, the influence of local microbiota derived SCFAs have been well investigated, the influence of SCFA on lung cancer cells via peripheral blood immune system should be investigated more deeply. In this respect, nutrients absorbed via the gut might affect the tumor microenvironment (TME) and thus play an important role in tumor cell growth. Objective: This study focuses on the impact of the short-chain fatty acid (SCFA) Sodium Butyrate (SB), on lung cancer cell survival. We previously described a pro-tumoral role of glucose on A549 lung adenocarcinoma cell line. In this study, we wanted to know if SB would counteract the effect of glucose and thus cultured A549 and H520 in vitro with and without SB in the presence or absence of glucose and investigated how the treatment with SB affects the survival of lung cancer cells and its influence on immune cells fighting against lung cancer. Methods: In this study, we performed cell culture experiments with A549, H520 and NSCLC-patient-derived epithelial cells under different SB levels. To investigate the influence on the immune system, we performed in vitro culture of peripheral mononuclear blood cells (PBMC) from control, smoker and lung cancer patients with increasing SB concentrations. Results: To investigate the effect of SB on lung tumor cells, we first analyzed the effect of 6 different concentrations of SB on A549 cells at 48 and 72 hours cell culture. Here we found that, SB treatment reduced lung cancer cell survival in a concentration dependent manner. We next focused our deeper analysis on the two concentrations, which caused the maximal reduction in cell survival. Here, we observed that SB led to cell cycle arrest and induced early apoptosis in A549 lung cancer cells. The expression of cell cycle regulatory proteins and A549 lung cancer stem cell markers (CD90) was induced. Additionally, this study explored the role of interferon-gamma (IFN-γ) and its receptor (IFN-γ-R1) in combination with SB treatment, revealing that, although IFN-γ-R1 expression was increased, IFN-γ did not affect the efficacy of SB in reducing tumor cell viability. Furthermore, we examined the effects of SB on immune cells, specifically CD8+ T cells and natural killer (NK) cells from healthy individuals, smokers, and NSCLC patients. SB treatment resulted in a decreased production of IFN-γ and granzyme B in CD8+ T cells and NK cells. Moreover, SB induced IFN-γ-R1 in NK cells and CD4+ T cells in the absence of glucose both in PBMCs from controls and NSCLC subjects. Conclusion: Overall, this study highlights the potential of SB in inhibiting lung cancer cell growth, triggering apoptosis, inducing cell cycle arrest, and modulating immune responses by activating peripheral blood CD4+ T cells while selectively inducing IFN-γ-R1 in NK cells in peripheral blood and inhibiting peripheral blood CD8+ T cells and NK cells. Thus, understanding the mechanisms of action of SB in the TME and its influence on the immune system provide valuable insights of potentially considering SB as a candidate for adjunctive therapies in NSCLC.

Cholestane-3ß,5α,6ß-triol Induces Multiple Cell Death in A549 Cells via ER Stress and Autophagy Activation.

Cholestane-3ß,5α,6ß-triol (CT) and its analogues are abundant in natural sources and are reported to demonstrate cytotoxicity toward different kinds of tumor cells without a deep probe into their mechanism of action. CT is also one of the major metabolic oxysterols of cholesterol in mammals and is found to accumulate in various diseases. An extensive exploration of the biological roles of CT over the past few decades has established its identity as an apoptosis inducer. In this study, the effects of CT on A549 cell death were investigated through cell viability assays. RNA-sequencing analysis and western blot of CT-treated A549 cells revealed the role of CT in inducing endoplasmic reticulum (ER) stress response and enhancing autophagy flux, suggesting a putative mechanism of CT-induced cell-death activation involving reactive oxygen species (ROS)-mediated ER stress and autophagy. It is reported for the first time that the upregulation of autophagy induced by CT can serve as a cellular cytotoxicity response in accelerating CT-induced cell death in A549 cells. This research provides evidence for the effect of CT as an oxysterol in cell response to oxidative damage and allows for a deep understanding of cholesterol in its response in an oxidative stress environment that commonly occurs in the progression of various diseases.

Excessive intragastric alcohol administration exacerbates hepatic encephalopathy and provokes neuronal cell death in male rats with chronic liver disease.

Hepatic encephalopathy (HE) is defined as decline in neurological function during chronic liver disease (CLD). Alcohol is a major etiological factor in the pathogenesis of fibrosis/cirrhosis and has also been documented to directly impact the brain. However, the role of alcohol in the development of HE in CLD remains unclear. Here, we investigated the impact of excessive alcohol administration on neurological deterioration in rats with CLD. Starting day 7 post-BDL surgery, rats were administered alcohol twice daily (51% v/v ethanol, 3 g/kg, via gavage) for 4 weeks. Motor coordination was assessed weekly using rotarod and anxiety-like behavior was evaluated with open field and elevated plus maze at 5 weeks. Upon sacrifice, brains were collected for western blot and immunohistochemical analyses to investigate neuronal integrity and oxidative stress status. Alcohol worsened motor coordination performance and increased anxiety-like behavior in BDL rats. Impairments were associated with decreased neuronal markers of NeuN and SMI311, increased apoptotic markers of cleaved/pro-caspase-3 and Bax/Bcl2, increased necroptosis markers of pRIP3 and pMLKL, decreased total antioxidant capacity (TAC), and increased 4-hydroxynonenal (4-HNE)modified proteins in the cerebellum of BDL-alcohol rats when compared to respective controls. Immunofluorescence confirmed the colocalization of cleaved caspase-3 and pMLKL in the granular neurons of the cerebellum of BDL-alcohol rats. Excessive alcohol consumption exacerbates HE which leads to associated apoptotic and necroptotic neuronal loss in the cerebellum of BDL-alcohol rats. Additionally, higher levels of 4-HNE and decreased TAC in the cerebellum of BDL-alcohol rats suggest oxidative stress is the triggering factor of apoptotic and necroptotic neuronal loss/injury.

Channeling Nicotinamide Phosphoribosyltransferase (NAMPT) to Address Life and Death.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in NAD+ biosynthesis via salvage of NAM formed from catabolism of NAD+ by proteins with NADase activity (e.g., PARPs, SIRTs, CD38). Depletion of NAD+ in aging, neurodegeneration, and metabolic disorders is addressed by NAD+ supplementation. Conversely, NAMPT inhibitors have been developed for cancer therapy: many discovered by phenotypic screening for cancer cell death have low nanomolar potency in cellular models. No NAMPT inhibitor is yet FDA-approved. The ability of inhibitors to act as NAMPT substrates may be associated with efficacy and toxicity. Some 3-pyridyl inhibitors become 4-pyridyl activators or "NAD+ boosters". NAMPT positive allosteric modulators (N-PAMs) and boosters may increase enzyme activity by relieving substrate/product inhibition. Binding to a "rear channel" extending from the NAMPT active site is key for inhibitors, boosters, and N-PAMs. A deeper understanding may fulfill the potential of NAMPT ligands to regulate cellular life and death.

Mechanism of metal ion-induced cell death in gastrointestinal cancer.

Gastrointestinal (GI) cancer is one of the most severe types of cancer, with a significant impact on human health worldwide. Due to the urgent demand for more effective therapeutic strategies against GI cancers, novel research on metal ions for treating GI cancers has attracted increasing attention. Currently, with accumulating research on the relationship between metal ions and cancer therapy, several metal ions have been discovered to induce cell death. In particular, the three novel modes of cell death, including ferroptosis, cuproptosis, and calcicoptosis, have become focal points of research in the field of cancer. Meanwhile, other metal ions have also been found to trigger cell death through various mechanisms. Accordingly, this review focuses on the mechanisms of metal ion-induced cell death in GI cancers, hoping to provide theoretical support for further GI cancer therapies.

Beta hydroxybutyrate induces lung cancer cell death, mitochondrial impairment and oxidative stress in a long term glucose-restricted condition.

BACKGROUND: Metabolic plasticity gives cancer cells the ability to shift between signaling pathways to facilitate their growth and survival. This study investigates the role of glucose deprivation in the presence and absence of beta-hydroxybutyrate (BHB) in growth, death, oxidative stress and the stemness features of lung cancer cells. METHODS AND RESULTS: A549 cells were exposed to various glucose conditions, both with and without beta-hydroxybutyrate (BHB), to evaluate their effects on apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS) levels using flow cytometry, and the expression of CD133, CD44, SOX-9, and ß-Catenin through Quantitative PCR. The activity of superoxide dismutase, glutathione peroxidase, and malondialdehyde was assessed using colorimetric assays. Treatment with therapeutic doses of BHB triggered apoptosis in A549 cells, particularly in cells adapted to glucose deprivation. The elevated ROS levels, combined with reduced levels of SOD and GPx, indicate that oxidative stress contributes to the cell arrest induced by BHB. Notably, BHB treatment under glucose-restricted conditions notably decreased CD133 expression, suggesting a potential inhibition of cell survival through the downregulation of CD133 levels. Additionally, the simultaneous decrease in mitochondrial membrane potential and increase in ROS levels indicate the potential for creating oxidative stress conditions to impede tumor cell growth in such environmental settings. CONCLUSION: The induced cell death, oxidative stress and mitochondria impairment beside attenuated levels of cancer stem cell markers following BHB administration emphasize on the distinctive role of metabolic plasticity of cancer cells and propose possible therapeutic approaches to control cancer cell growth through metabolic fuels.

Alpha-synuclein interaction with mitochondria is the final mechanism of ferroptotic death induced by erastin in SH-SY5Y cells.

Ferroptosis has been characterized as a form of iron-dependent regulated cell death accompanied by an accumulation of reactive oxygen species and lipid oxidation products along with typical morphological alterations in mitochondria. Ferroptosis is activated by diverse triggers and inhibited by ferrostatin-1 and liproxstatin-1, apart from iron chelators and several antioxidants, and the process is implicated in multiple pathological conditions. There are, however, certain ambiguities about ferroptosis, especially regarding the final executioner of cell death subsequent to the accumulation of ROS. This study uses a typical inducer of ferroptosis such as erastin on SH-SY5Y cells, and shows clearly that ferroptotic death of cells is accompanied by the loss of mitochondrial membrane potential and intracellular ATP content along with an accumulation of oxidative stress markers. All these are prevented by ferrostatin-1 and liproxstatin-1. Additionally, cyclosporine A prevents mitochondrial alterations and cell death induced by erastin implying the crucial role of mitochondrial permeability transition pore (mPTP) activation in ferroptotic death. Furthermore, an accumulation of α-synuclein occurs during erastin induced ferroptosis which can be inhibited by ferrostatin-1 and liproxstatin-1. When the knock-down of α-synuclein expression is performed by specific siRNA treatment of SH-SY5Y cells, the mitochondrial impairment and ferroptotic death of the cells induced by erastin are markedly prevented. Thus, α-synuclein through the involvement of mPTP appears to be the key executioner protein of ferroptosis induced by erastin, but it needs to be verified if it is a generalized mechanism of ferroptosis by using other inducers and cell lines.

Both hemoglobin and hemin cause damage to retinal pigment epithelium through the iron ion accumulation.

Subretinal hemorrhages result in poor vision and visual field defects. During hemorrhage, several potentially toxic substances are released from iron-based hemoglobin and hemin, inducing cellular damage, the detailed mechanisms of which remain unknown. We examined the effects of excess intracellular iron on retinal pigment epithelial (RPE) cells. A Fe2+ probe, SiRhoNox-1 was used to investigate Fe2+ accumulation after treatment with hemoglobin or hemin in the human RPE cell line ARPE-19. We also evaluated the production of reactive oxygen species (ROS) and lipid peroxidation. Furthermore, the protective effect of-an iron chelator, 2,2'-bipyridyl (BP), and ferrostatin-1 (Fer-1) on the cell damage, was evaluated. Fe2+ accumulation increased in the hemoglobin- or hemin-treated groups, as well as intracellular ROS production and lipid peroxidation. In contrast, BP treatment suppressed RPE cell death, ROS production, and lipid peroxidation. Pretreatment with Fer-1 ameliorated cell death in a concentration-dependent manner and suppressed ROS production and lipid peroxidation. Taken together, these findings indicate that hemoglobin and hemin, as well as subretinal hemorrhage, may induce RPE cell damage and visual dysfunction via intracellular iron accumulation.

Selected Flavonols Targeting Cell Death Pathways in Cancer Therapy: The Latest Achievements in Research on Apoptosis, Autophagy, Necroptosis, Pyroptosis, Ferroptosis, and Cuproptosis.

The complex and multi-stage processes of carcinogenesis are accompanied by a number of phenomena related to the potential involvement of various chemopreventive factors, which include, among others, compounds of natural origin such as flavonols. The use of flavonols is not only promising but also a recognized strategy for cancer treatment. The chemopreventive impact of flavonols on cancer arises from their ability to act as antioxidants, impede proliferation, promote cell death, inhibit angiogenesis, and regulate the immune system through involvement in diverse forms of cellular death. So far, the molecular mechanisms underlying the regulation of apoptosis, autophagy, necroptosis, pyroptosis, ferroptosis, and cuproptosis occurring with the participation of flavonols have remained incompletely elucidated, and the results of the studies carried out so far are ambiguous. For this reason, one of the therapeutic goals is to initiate the death of altered cells through the use of quercetin, kaempferol, myricetin, isorhamnetin, galangin, fisetin, and morin. This article offers an extensive overview of recent research on these compounds, focusing particularly on their role in combating cancer and elucidating the molecular mechanisms governing apoptosis, autophagy, necroptosis, pyroptosis, ferroptosis, and cuproptosis. Assessment of the mechanisms underlying the anticancer effects of compounds in therapy targeting various types of cell death pathways may prove useful in developing new therapeutic regimens and counteracting resistance to previously used treatments.