Microbial Lipopeptide Supramolecular Self-Assemblies as a Methuosis-Like Cell Death Inducer with In Vivo Antitumor Activity.

Discovering new drugs and improving action mechanisms is a promising strategy to overcome chemotherapy ineffectiveness caused by cancer cell apoptosis resistance. Natural products (like cyclic lipopeptides, CLPs) are potential sources of nonapoptotic cell death inducers and can form diverse supramolecular structures, closely relating to their bioactivities. Herein, it is found for the first time that fatty chain is the key to maintain self-assembled form and antitumor activity of microbial-derived amphiphilic CLP bacillomycin Lb (B-Lb). Compared with B-Lb analogues assemblies without antitumor activity, B-Lb supramolecular self-assemblies (including nanomicelles, nanofibers, giant micrometer rods) can be generated in a multilevel and cross-scale manner and served as a methuosis-like cell death inducer triggered by cytoplasmic vacuolation through macropinocytosis in MDA-MB-231-Luc and MCF-7 cells and in vivo tumor-bearing mice. This study will promote constructing of customized CLP micro-/nanostructures with multipurposes and functions, and boost designing of new antitumor drugs as nonapoptotic cell death modulators based on structure-activity relationship.

New Insight into the Effects of Metformin on Diabetic Retinopathy, Aging and Cancer: Nonapoptotic Cell Death, Immunosuppression, and Effects beyond the AMPK Pathway.

Under metabolic stress conditions such as hypoxia and glucose deprivation, an increase in the AMP:ATP ratio activates the AMP-activated protein kinase (AMPK) pathway, resulting in the modulation of cellular metabolism. Metformin, which is widely prescribed for type 2 diabetes mellitus (T2DM) patients, regulates blood sugar by inhibiting hepatic gluconeogenesis and promoting insulin sensitivity to facilitate glucose uptake by cells. At the molecular level, the most well-known mechanism of metformin-mediated cytoprotection is AMPK pathway activation, which modulates metabolism and protects cells from degradation or pathogenic changes, such as those related to aging and diabetic retinopathy (DR). Recently, it has been revealed that metformin acts via AMPK- and non-AMPK-mediated pathways to exert effects beyond those related to diabetes treatment that might prevent aging and ameliorate DR. This review focuses on new insights into the anticancer effects of metformin and its potential modulation of several novel types of nonapoptotic cell death, including ferroptosis, pyroptosis, and necroptosis. In addition, the antimetastatic and immunosuppressive effects of metformin and its hypothesized mechanism are also discussed, highlighting promising cancer prevention strategies for the future.

Cucurbitacin I induces cancer cell death through the endoplasmic reticulum stress pathway.

Endoplasmic reticulum stress (ERS) is usually involved in tumor development and progression, and anticancer agents have recently been recognized to induce ERS. Cucurbitacin-I showed a potent anticancer action by inducing apoptosis through the inhibition of signal transducer and activator of transcription 3 pathway and triggering autophagic cell death. It is not known whether ERS mediates the cancer cell death induced by cucurbitacin-I. Here, we investigated the role of ERS in cucurbitacin-I-treated SKOV3 ovarian cancer cells and PANC-1 pancreatic cancer cells. We confirmed that cucurbitacin-I caused cell death and stirred excessive ERS levels by activating inositol requiring enzyme 1α (IRE1α) and protein kinase R-like endoplasmic reticulum kinase (PERK), as well as PERK downstream factors, including IRE1α and C/EBP homologous protein, but not activating transcription factor 6 (ATF6α) pathway, which was in parallel with the increased Bax and caspase-12-dependent ERS-associated apoptosis, autophagy and autophagy flux levels and caspase-independent nonapoptotic cell death. Furthermore, 4-phenylbutyrate, an ERS inhibitor, suppressed cucurbitacin-I-induced apoptosis, autophagy, autophagy flux, and autophagic cell death. Simultaneously, there are positive correlations among ERS and cucurbitacin-I-induced reactive oxygen species and Ca 2+ . Our results suggested that cucurbitacin-I-induced cancer cell death through the excessive ERS and CHOP-Bax and caspase-12-dependent ERS-associated apoptosis, as well as ERS-dependent autophagy, autophagy flux, and caspase-independent nonapoptotic cell death. These novel signaling insights may be useful for developing new, effective anticancer strategies in oncotherapy.

The mechanism of SO2 -induced stomatal closure differs from O3 and CO2 responses and is mediated by nonapoptotic cell death in guard cells.

Plants closing stomata in the presence of harmful gases is believed to be a stress avoidance mechanism. SO2 , one of the major airborne pollutants, has long been reported to induce stomatal closure, yet the mechanism remains unknown. Little is known about the stomatal response to airborne pollutants besides O3 . SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1) and OPEN STOMATA 1 (OST1) were identified as genes mediating O3 -induced closure. SLAC1 and OST1 are also known to mediate stomatal closure in response to CO2 , together with RESPIRATORY BURST OXIDASE HOMOLOGs (RBOHs). The overlaying roles of these genes in response to O3 and CO2 suggested that plants share their molecular regulators for airborne stimuli. Here, we investigated and compared stomatal closure event induced by a wide concentration range of SO2 in Arabidopsis through molecular genetic approaches. O3 - and CO2 -insensitive stomata mutants did not show significant differences from the wild type in stomatal sensitivity, guard cell viability, and chlorophyll content revealing that SO2 -induced closure is not regulated by the same molecular mechanisms as for O3 and CO2 . Nonapoptotic cell death is shown as the reason for SO2 -induced closure, which proposed the closure as a physicochemical process resulted from SO2 distress, instead of a biological protection mechanism.

Identification of a Bcl-xL homolog from orange-spotted grouper (Epinephelus coioides) involved in SGIV-induced nonapoptotic cell death.

Bcl-2 family proteins play essential roles in modulating immune response and controlling cells' fate. Bcl-xL is one of anti-apoptotic protein in this family. In this study, a new Bcl-xL homolog (EcBcl-xL) was identified and characterized from orange-spotted grouper, Epinephelus coioides. EcBcl-xL encoded a 221 amino acid peptides that shared 86% identity to Larimichthys crocea Bcl-xL protein, contained four conserved BH domains and one transmembrane region. The predicted three-dimensional structure of EcBcl-xL was similar with Homo sapiens Bcl-xL. EcBcl-xL widely expressed in all tested tissues with highest expression in head kidney. Its expression level was significantly up-regulated after SGIV infection in vivo. Furthermore, overexpression of EcBcl-xL could inhibit SGIV-induced nonapoptotic cell death and suppressed viral genes transcriptions in GS cells. Our findings suggested that EcBcl-xL might play a role during virus infection through modulating SGIV-induced nonapoptotic cell death.

V-ATPase V0 subunit activation mediates maduramicin-induced methuosis through blocking endolysosomal trafficking in vitro and in vivo.

Methuosis, a novel cell death phenotype, is characterized by accumulation of cytoplasmic vacuolization upon external stimulus. Methuosis plays a critical role in maduramicin-induced cardiotoxicity despite the underlying mechanism is largely unknown. Herein, we aimed to investigate the origin and intracellular trafficking of cytoplasmic vacuoles, as well as the molecular mechanism of methuosis caused by maduramicin (1 µg/mL) in myocardial cells. H9c2 cells and broiler chicken were used and were exposed to maduramicin at doses of 1 µg/mL in vitro and 5 ppm-30 ppm in vivo. Morphological observation and dextran-Alexa Fluor 488 tracer experiment showed that endosomal compartments swelling and excessive macropinocytosis contributed to madurdamcin-induced methuosis. Cell counting kit-8 assay and morphology indicated pharmacological inhibition of macropinocytosis largely prevent H9c2 cells from maduramicin-triggered methuosis. In addition, late endosomal marker Rab7 and lysosomal associated membrane protein 1 (LAMP1) increased in a time-dependent manner after maduramicin treatment, and the recycling endosome marker Rab11 and ADP-ribosylation factor 6 (Arf6) were decreased by maduramicin. Vacuolar-H+-ATPase (V-ATPase) was activated by maduramicin, and pharmacological inhibition and genetic knockdown V0 subunit of V-ATPase restore endosomal-lysosomal trafficking and prevent H9c2 cells methuosis. Animal experiment showed that severe cardiac injury included the increase of creatine kinase (CK) and creatine kinase-MB (CK-MB), and vacuolar degeneration resembled methuosis in vivo after maduramicin treatment. Taken together, these findings demonstrate that targeting the inhibition of V-ATPase V0 subunit will prevent myocardial cells methuosis by restoring endosomal-lysosomal trafficking.

Inherited Retinal Degeneration: PARP-Dependent Activation of Calpain Requires CNG Channel Activity.

Inherited retinal degenerations (IRDs) are a group of blinding diseases, typically involving a progressive loss of photoreceptors. The IRD pathology is often based on an accumulation of cGMP in photoreceptors and associated with the excessive activation of calpain and poly (ADP-ribose) polymerase (PARP). Inhibitors of calpain or PARP have shown promise in preventing photoreceptor cell death, yet the relationship between these enzymes remains unclear. To explore this further, organotypic retinal explant cultures derived from wild-type and IRD-mutant mice were treated with inhibitors specific for calpain, PARP, and voltage-gated Ca2+ channels (VGCCs). The outcomes were assessed using in situ activity assays for calpain and PARP and immunostaining for activated calpain-2, poly (ADP-ribose), and cGMP, as well as the TUNEL assay for cell death detection. The IRD models included the Pde6b-mutant rd1 mouse and rd1*Cngb1-/- double-mutant mice, which lack the beta subunit of the rod cyclic nucleotide-gated (CNG) channel and are partially protected from rd1 degeneration. We confirmed that an inhibition of either calpain or PARP reduces photoreceptor cell death in rd1 retina. However, while the activity of calpain was decreased by the inhibition of PARP, calpain inhibition did not alter the PARP activity. A combination treatment with calpain and PARP inhibitors did not synergistically reduce cell death. In the slow degeneration of rd1*Cngb1-/- double mutant, VGCC inhibition delayed photoreceptor cell death, while PARP inhibition did not. Our results indicate that PARP acts upstream of calpain and that both are part of the same degenerative pathway in Pde6b-dependent photoreceptor degeneration. While PARP activation may be associated with CNG channel activity, calpain activation is linked to VGCC opening. Overall, our data highlights PARP as a target for therapeutic interventions in IRD-type diseases.

Stratification of lung squamous cell carcinoma based on ferroptosis regulators: Potential for new therapeutic strategies involving ferroptosis induction.

OBJECTIVES: Lung squamous cell carcinoma (LSCC) exhibits poor response to treatment compared with other lung cancer subtypes, resulting in worse prognosis. Therefore, new therapeutic strategies are required for advanced LSCC. Ferroptosis is a recently discovered nonapoptotic cell death caused by intracellular lipid peroxidation that can bring about effective cell death in cancer cells resistant to apoptosis. Hence, ferroptosis is a potential therapeutic strategy for refractory cancer. MATERIALS AND METHODS: In this study, we performed clinicopathological and molecular analyses on tumor specimens from 270 patients with squamous cell lung cancer, focusing on the expression of glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1), which are known to be key regulators of ferroptosis, and the accumulation of 4-hydroxynoneral (4-HNE), a lipid peroxidation marker. RESULTS: Immunohistochemistry revealed that patients with low 4-HNE accumulation and low levels of GPX4 or FSP1 had significantly worse prognoses than other patients (P = 0.001). This stratification was an independent prognostic predictor (P = 0.003). A dramatic cell death synergistic effect was observed on LSCC-derived LK-2 and EBC1 cells treated with GPX4 and FSP1 inhibitors. This effect was completely inhibited by treatment with the ferroptosis inhibitor. Notably, this was not the case in LK-2 cells treated with the apoptosis inhibitor, and in these cells, ferroptosis was induced. CONCLUSION: Ferroptosis regulators GPX4 and FSP1 are associated with lung squamous cell cancer cancer's prognosis. We present the clinicopathological and molecular basis of novel therapeutic strategies for refractory LSCC.

Glioblastoma cytotoxicity conferred through dual disruption of endolysosomal homeostasis by Vacquinol-1.

BACKGROUND: Increased membrane trafficking is observed in numerous cancer types, including glioblastoma. Targeting the oncogenic driven acquired alterations in membrane trafficking by synthetic cationic amphiphilic small molecules has recently been shown to induce death of glioblastoma cells, although the molecular targets are unknown. METHODS: The mechanism of action of the cationic amphiphilic drug Vacquinol-1 (Vacq1)-induced cytotoxicity was investigated using cell biology, biochemistry, functional experiments, chemical biology, unbiased antibody-based post-translation modification profiling, and mass spectrometry-based chemical proteomic analysis on patient-derived glioblastoma cells. RESULTS: Vacq1 induced two types of abnormal endolysosomal vesicles, enlarged vacuoles and acidic vesicle organelles (AVOs). Mechanistically, enlarged vacuoles were formed by the impairment of lysosome reformation through the direct interaction and inhibition of calmodulin (CaM) by Vacq1, while AVO formation was induced by Vacq1 accumulation and acidification in the endosomal compartments through its activation of the v-ATPase. As a consequence of v-ATPase activation, cellular ATP consumption markedly increased, causing cellular energy shortage and cytotoxicity. This effect of Vacq1 was exacerbated by its inhibitory effects on calmodulin, causing lysosomal depletion and a failure of acidic vesicle organelle clearance. CONCLUSION: Our study identifies the targets of Vacq1 and the mechanisms underlying its selective cytotoxicity in glioblastoma cells. The dual function of Vacq1 sets in motion a glioblastoma-specific vicious cycle of ATP consumption resulting in cellular energy crisis and cell death.

Targeting nonapoptotic pathways with functionalized nanoparticles for cancer therapy: current and future perspectives.

Apoptotic death evasion is a hallmark of cancer progression. In this context, past decades have witnessed cytotoxic agents targeting apoptosis. However, owing to cellular defects in the apoptotic machinery, tumors develop resistance to apoptosis-based cancer therapies. Hence, targeting nonapoptotic cell-death pathways displays enhanced therapeutic success in apoptosis-defective tumor cells. Exploitation of multifunctional properties of engineered nanoparticles may allow cancer therapeutics to target yet unexplored pathways such as ferroptosis, autophagy and necroptosis. Necroptosis presents a programmed necrotic death initiated by same apoptotic death signals that are caspase independent, whereas autophagy is self-degradative causing vacuolation, and ferroptosis is an iron-dependent form driven by lipid peroxidation. Targeting these tightly regulated nonapoptotic pathways may emerge as a new direction in cancer drug development, diagnostics and novel cancer nanotherapeutics. This review highlights the current challenges along with the advancement in this field of research and finally summarizes the future perspective in terms of their clinical merits.

Maduramicin triggers methuosis-like cell death in primary chicken myocardial cells.

Maduramicin frequently induces severe cardiotoxicity in broiler chickens as well as in humans who consume maduramicin accidentally. Apoptosis and non-apoptotic cell death occur concurrently in the process of maduramicin-induced cardiotoxicity; however, the underlying mechanism of non-apoptotic cell death is largely unknown. Here, we report the relationship between maduramicin-caused cytoplasmic vacuolization and methuosis-like cell death as well as the underlying mechanism in primary chicken myocardial cells. Maduramicin induced a significant increase of cytoplasmic vacuoles with a degree of cell specificity in primary chicken embryo fibroblasts and chicken hepatoma cells (LMH), along with a decrease of ATP and an increase of LDH. The accumulated vacuoles were partly derived from cellular endocytosis rather than the swelling of endoplasm reticulum, lysosomes, and mitochondria. Moreover, the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) did not prevent maduramicin-induced cytoplasmic vacuolization. DNA ladder and cleavage of PARP were not observed in chicken myocardial cells during maduramicin exposure. Pretreatment with 3-methyladenine (3-MA) and cholorquine (CQ) of chicken myocardial cells did not attenuate cytoplasmic vacuolization and cytotoxicity, although LC3 and p62 were activated. Bafilomycin A1 almost completely prevented the generation of cytoplasmic vacuoles and significantly attenuated cytotoxicity induced by maduramicin, along with downregulation of K-Ras and upregulation of Rac1. Taken together, "methuosis" due to excessive cytoplasmic vacuolization mediates the cardiotoxicity of maduramicin. This provides new insights for understanding a nonclassical form of cell death in the field of drug-induced cytotoxicity.

On the role of sphingolipids in cell survival and death.

Sphingolipids, universal components of biological membranes of all eukaryotic organisms, from yeasts to mammals, in addition of playing a structural role, also play an important part of signal transduction pathways. They participate or, also, ignite several fundamental subcellular signaling processes but, more in general, they directly contribute to key biological activities such as cell motility, growth, senescence, differentiation as well as cell fate, i.e., survival or death. The sphingolipid metabolic pathway displays an intricate network of reactions that result in the formation of multiple sphingolipids, including ceramide, and sphingosine-1-phosphate. Different sphingolipids, that have key roles in determining cell fate, can induce opposite effects: as a general rule, sphingosine-1-phosphate promotes cell survival and differentiation, whereas ceramide is known to induce apoptosis. Furthermore, together with cholesterol, sphingolipids also represent the basic lipid component of lipid rafts, cholesterol- and sphingolipid-enriched membrane microdomains directly involved in cell death and survival processes. In this review, we briefly describe the characteristics of sphingolipids and lipid membrane microdomains. In particular, we will consider the involvement of various sphingolipids per se and of lipid rafts in apoptotic pathway, both intrinsic and extrinsic, in nonapoptotic cell death, in autophagy, and in cell differentiation. In addition, their roles in the most common physiological and pathological contexts either as pathogenetic elements or as biomarkers of diseases will be considered. We would also hint how the manipulation of sphingolipid metabolism could represent a potential therapeutic target to be investigated and functionally validated especially for those diseases for which therapeutic options are limited or ineffective.

A ZDHHC5-GOLGA7 Protein Acyltransferase Complex Promotes Nonapoptotic Cell Death.

Lethal small molecules are useful probes to discover and characterize novel cell death pathways and biochemical mechanisms. Here we report that the synthetic oxime-containing small molecule caspase-independent lethal 56 (CIL56) induces an unconventional form of nonapoptotic cell death distinct from necroptosis, ferroptosis, and other pathways. CIL56-induced cell death requires a catalytically active protein S-acyltransferase complex comprising the enzyme ZDHHC5 and an accessory subunit GOLGA7. The ZDHHC5-GOLGA7 complex is mutually stabilizing and localizes to the plasma membrane. CIL56 inhibits anterograde protein transport from the Golgi apparatus, which may be lethal in the context of ongoing ZDHHC5-GOLGA7 complex-dependent retrograde protein trafficking from the plasma membrane to internal sites. Other oxime-containing small molecules, structurally distinct from CIL56, may trigger cell death through the same pathway. These results define an unconventional form of nonapoptotic cell death regulated by protein S-acylation.

Natural products to prevent drug resistance in cancer chemotherapy: a review.

Chemotherapy is the standard internal medical treatment for cancer. However, the resistance of cancer cells to nearly all kinds of chemotherapeutic drugs and targeted drugs has become prevalent, and approximately 80-90% of deaths in cancer patients are directly or indirectly attributed to drug resistance. The progress of new drug research and development has also been impeded by the occurrence of drug resistance, which has emerged as a considerable challenge in cancer therapy. Fortunately, natural products with diverse chemical structures and pharmacological effects serve as effective substances against drug resistance. Since the discovery of a series of drug-resistant proteins, drug-efflux inhibition has been applied as the primary strategy to overcome drug resistance by maintaining the intracellular concentrations of chemotherapeutic drugs. Nonapoptotic cell death is considered an alternative strategy because most cases of drug resistance result in evasion and insensitivity to apoptosis. In this concise review, we summarize two strategies using natural products against drug resistance.

Cell Death in C. elegans Development.

Cell death is a common and important feature of animal development, and cell death defects underlie many human disease states. The nematode Caenorhabditis elegans has proven fertile ground for uncovering molecular and cellular processes controlling programmed cell death. A core pathway consisting of the conserved proteins EGL-1/BH3-only, CED-9/BCL2, CED-4/APAF1, and CED-3/caspase promotes most cell death in the nematode, and a conserved set of proteins ensures the engulfment and degradation of dying cells. Multiple regulatory pathways control cell death onset in C. elegans, and many reveal similarities with tumor formation pathways in mammals, supporting the idea that cell death plays key roles in malignant progression. Nonetheless, a number of observations suggest that our understanding of developmental cell death in C. elegans is incomplete. The interaction between dying and engulfing cells seems to be more complex than originally appreciated, and it appears that key aspects of cell death initiation are not fully understood. It has also become apparent that the conserved apoptotic pathway is dispensable for the demise of the C. elegans linker cell, leading to the discovery of a previously unexplored gene program promoting cell death. Here, we review studies that formed the foundation of cell death research in C. elegans and describe new observations that expand, and in some cases remodel, this edifice. We raise the possibility that, in some cells, more than one death program may be needed to ensure cell death fidelity.

Multidimensional profiling in the investigation of small-molecule-induced cell death.

Numerous morphological variations of cell death have been described. These processes depend on a complex and overlapping cellular signaling network, making molecular definition of the pathways challenging. This review describes one solution to this problem for small-molecule-induced death, the creation of high-dimensionality profiles for compounds that can be used to define and compare pathways. Such profiles have been assembled from gene expression measurements, protein quantification, chemical-genetic interactions, chemical combination interactions, cancer cell line sensitivity profiling, quantitative imaging, and modulatory profiling. We discuss the advantages and limitations of these techniques in the study of cell death.