AtHMGB15 regulates tapetal apoptosis in pollen development and actin dynamics during pollen germination in arabidopsis.

KEY MESSAGE: ARID-HMG DNA binding protein, AtHMGB15, regulates pollen development and pollen germination in Arabidopsis. Previous studies have shown that ARID-HMG DNA binding protein, AtHMGB15 regulate pollen development and pollen germination in Arabidopsis. Here, we performed transcriptome and cytological studies to understand the role of AtHMGB15 in regulating pollen wall morphology and the pollen tube germination rate. Our result showed abnormal vacuolization in the tapetal cells during anther maturation and prolonged PCD in AtHMGB15 loss-of-function mutant. The tapetum has the ability to perform both secretory and biosynthetic activities critical for pollen maturation and pollen viability. Interestingly, expression of PCD executer genes CEP1, MC9 and RNS3 were significant down-regulation of in athmgb15-4. The growth of pollen tubes is regulated by the actin cytoskeleton dynamics. To address the defect in pollen tube growth of athmgb15, we monitored the actin network in growing pollen tubes of wildtype and athmgb15-4 using Rhodamine-phalloidin fluorescence. Our results indicate a highly fragmented actin distribution in athmgb15-4 pollen tubes with a lesser number of long actin fibers and significantly low f-actin concentration at the apex. q-RTPCR further indicates significant downy-regulation of actin regulatory proteins VLN2 and PRF4. Collectively, our results suggest that AtHMGB15 being a nuclear architectural protein orchestrates high-order chromatin organization to promote the transcription of genes responsible for pollen development and pollen germination.

Inhibition of PIKfyve Leads to Lysosomal Disorders via Dysregulation of mTOR Signaling.

PIKfyve is an endosomal lipid kinase that synthesizes phosphatidylinositol 3,5-biphosphate from phosphatidylinositol 3-phsphate. Inhibition of PIKfyve activity leads to lysosomal enlargement and cytoplasmic vacuolation, attributed to impaired lysosomal fission processes and homeostasis. However, the precise molecular mechanisms underlying these effects remain a topic of debate. In this study, we present findings from PIKfyve-deficient zebrafish embryos, revealing enlarged macrophages with giant vacuoles reminiscent of lysosomal storage disorders. Treatment with mTOR inhibitors or effective knockout of mTOR partially reverses these abnormalities and extend the lifespan of mutant larvae. Further in vivo and in vitro mechanistic investigations provide evidence that PIKfyve activity is essential for mTOR shutdown during early zebrafish development and in cells cultured under serum-deprived conditions. These findings underscore the critical role of PIKfyve activity in regulating mTOR signaling and suggest potential therapeutic applications of PIKfyve inhibitors for the treatment of lysosomal storage disorders.

Myelin lesion in the aspartoacylase (Aspa) knockout rat, an animal model for Canavan disease.

Canavan disease (CD) is a fatal hereditary neurological disorder caused by a mutation in the aspartoacylase (ASPA) gene and characterized by neurological signs and vacuolation in the central nervous system (CNS). The mutation inhibits the hydrolysis of N-acetyl-aspartate (NAA) resulting in accumulation of NAA in the CNS. A new Aspa-knockout rat was generated by transcription activator-like effector nuclease (TALEN) technology. Herein we describe the pathological and morphometrical findings in the brain and spinal cords of Aspa-knockout rats. Although Aspa-knockout rats did not show any neurological signs, vacuolation with swollen axons, hypomyelination, and activated swollen astrocytes were observed mainly in the brainstem reticular formation, ascending and descending motor neuron pathway, and in the olfactory tract. Morphometrical analysis revealed no obvious change in the number of neurons. These changes in the CNS are similar to human CD, suggesting that this animal model would be useful for further study of treatment and understanding the pathophysiology of human CD.

Rutin Linoleate Triggers Oxidative Stress-Mediated Cytoplasmic Vacuolation in Non-Small Cell Lung Cancer Cells.

Lung cancer (LC) represents one of the most prevalent health issues globally and is a leading cause of tumor-related mortality. Despite being one the most attractive compounds of plant origin due to its numerous biological properties, the therapeutic applications of rutin (RUT) are limited by its disadvantageous pharmacokinetics. Thus, the present study aimed to evaluate in vitro the application of two RUT fatty acids bioconjugates, rutin oleate (RUT-O) and rutin linoleate (RUT-L), as potential improved RUT-based chemotherapeutics in non-small cell lung cancer (NSCLC) treatment. The results indicate that both compounds lacked cytotoxic potential in EpiAirway™ tissues at concentrations up to 125 µM. However, only RUT-L exerted anti-tumorigenic activity in NCI-H23 NSCLC cells after 24 h of treatment by reducing cell viability (up to 47%), proliferation, and neutral red uptake, causing cell membrane damage and lactate dehydrogenase (LDH) leakage, affecting cytoskeletal distribution, inducing cytoplasmic vacuolation, and increasing oxidative stress. The cytopathic effects triggered by RUT-L at 100 and 125 µM are indicators of a non-apoptotic cell death pathway that resembles the characteristics of paraptosis. The novel findings of this study stand as a basis for further investigations on the anti-cancer properties of RUT-L and their underlying mechanisms.

Cellular and Molecular Responses to Mitochondrial DNA Deletions in Kearns-Sayre Syndrome: Some Underlying Mechanisms.

Kearns-Sayre syndrome (KSS) is a rare multisystem mitochondrial disorder. It is caused by mitochondrial DNA (mtDNA) rearrangements, mostly large-scale deletions of 1.1-10 kb. These deletions primarily affect energy supply through impaired oxidative phosphorylation and reduced ATP production. This impairment gives rise to dysfunction of several tissues, in particular those with high energy demand like brain and muscles. Over the past decades, changes in respiratory chain complexes and energy metabolism have been emphasized, whereas little attention has been paid to other reports on ROS overproduction, protein synthesis inhibition, myelin vacuolation, demyelination, autophagy, apoptosis, and involvement of lipid raft and oligodendrocytes in KSS. Therefore, this paper draws attention towards these relatively underemphasized findings that might further clarify the pathologic cascades following deletions in the mtDNA.

Enhancing cryopreservation survival in Petunia × Calibrachoa 'Light Yellow' callus: Insights into material characteristics and genetic integrity.

Petunia × Calibrachoa 'Light Yellow' (× Petchoa 'Light Yellow') is a kind of perennial herbaceous flower obtained through intergeneric hybridization of Petunia and Calibrachoa with high ornamental value and wide application, facing challenges in seed acquisition. Expanding propagation through tissue culture is an economically efficient means. Hence, establishing an effective procedure for the storage of callus is essential for × Petchoa 'Light Yellow'. Cryopreservation is an effective method for the in vitro propagation and long-term preservation of × Petchoa 'Light Yellow' germplasms. For formulating the optimization of the vitrification procedure, first, an orthogonal experimental design was employed to pinpoint critical steps in the vitrification protocol (pre-culture, osmoprotection, dehydration, and dilution) for Petunia × Calibrachoa callus tissues and then five additional factors (pre-culture, osmoprotection I and II, dehydration, and dilution) were optimized to further reduce the sample water content and enhance cell viability levels. The vitrification procedure was described as follows: callus tissues were precultured in MS solid medium with 0.3 M sucrose for 5 d, incubated with osmoprotection solution I and II for 15 min at 25 °C, respectively, cryoprotected with PVS2 for 30 min at 0 °C, and rapidly immersed in liquid nitrogen. Cryopreserved callus tissues were then diluted in MS liquid medium with 1.2 M sucrose for 20 min at 25 °C and recovered on MS solid medium with 0.5 mg/L 6-BA and 0.1 mg/L NAA, and sucrose. The cell viability measured by TTC staining was approximately 16 %-18 % after 72 h-recovery. Following 45 days, the relative survival of callus reached up to 49.48 %. Furthermore, EST-SSR analysis showed no significant difference in the genetic stability of cryopreserved callus compared to the control. Based on the cryopreservation of × Petchoa 'Light Yellow' callus, we further evaluated the response of callus water contents to the osmotic stress in the optimized and original protocols (CK) for a higher cryopreservation survival. A comparative analysis of water content demonstrated that the procedure of gradual and gentle dehydration significantly improved water content and cell survival. Ultrastructural changes between cryopreserved and non-cryopreserved callus were examined and high vacuolation emerged as a key determinant, indicating its substantial impact on the low survival of cryopreserved cells, which should help us to understand the effectiveness of osmotic protectants in dehydration.

Cell death induced by nicotine in human neuroblastoma SH-SY5Y cells is mainly attributed to cytoplasmic vacuolation originating from the trans-Golgi network.

Humans are usually exposed to nicotine through the use of tobacco products. Although it is generally believed that nicotine is relatively harmless in tobacco consumption, it is, in fact, a toxic substance that warrants careful consideration of its potential toxicity. However, the current understanding of the neurotoxicity of nicotine is still very limited. In this study, we aim to reveal the toxic risk of nicotine to key target neuronal cells and its potential toxic mechanisms. The results showed that nicotine induced cell death, ROS increase, mitochondrial membrane potential decrease, and DNA damage in SH-SY5Y human neuroblastoma cells at millimolar concentrations, but did not cause toxic effects at the physiological concentration. These toxic effects were accompanied by cytoplasmic vacuolation. The inhibition of cytoplasmic vacuolation by bafilomycin A1 greatly reduced nicotine-induced cell death, indicating that cytoplasmic vacuolation is the key driving factor of cell death. These cytoplasmic vacuoles originated from the trans-Golgi network (TGN) and expressed microtubule-associated protein 1 light chain 3-II (LC3-II) and lysosomal associated membrane protein 1(LAMP1). The presence of LC3-II and LAMP1 within these vacuoles serves as evidence of compromised TGN structure and function. These findings provide valuable new insights into the potential neurotoxic risk and mechanisms of nicotine.

Species differences in small intestinal exposure-related epithelial vacuolation in rats and dogs treated with a heteroaryldihydropyrimidine molecule.

Small intestinal epithelial vacuolation induced by a heteroaryldihydropyrimidine compound (HAP-1) was observed in rats but not in dogs at termination in screening toxicity studies, despite the plasma exposure being higher in dogs. To understand the species differences, investigational studies with multiple time points following single dose (SD) and 7-day repeated dose (RD) were conducted in both species at doses resulting in comparable plasma exposures. In rats, epithelial vacuolation in the duodenum and jejunum were observed at all time points. In dogs, transient vacuolation was noted at 8 h post-SD (SD_8h) and 4 h post-RD (RD_4 h), but not at termination (RD_24 h). Special stains demonstrated lipid accumulation within enterocytes in both species and intracytoplasmic inclusion bodies in rats. Transmission electron microscopy identified these inclusion bodies as endoplasmic reticulum (ER) membranous structures. Transcriptomic analysis on jejunal mucosa at SD_8 h and RD_24 h revealed perturbations of lipid metabolism-related genes at SD_8 h in both species, but not at RD_24 h in dogs. ER stress-related gene changes at both time points were observed in rats only. Despite comparable HAP-1 plasma exposures, the duodenum and jejunum tissue concentrations of HAP-1 and acyl glucuronide metabolite were >5- and >30-fold higher in rats than in dogs, respectively. In vitro, similar cytotoxicity was observed in rat and dog duodenal organoids treated with HAP-1. In conclusion, HAP-1-induced intestinal epithelial vacuolation was related to lipid metabolism dysregulation in both species and ER-related injuries in rats only. The species differences were likely related to the difference in intestinal exposure to HAP-1 and its reactive metabolite.

Prenatal developmental sequences of the esophageal epithelium in the New Zealand white rabbits: Light and electron microscopic analysis.

Several morphogenetic sequences occur during esophageal development and birth defects occur due to defects in foregut morphogenesis. This work aimed to record the cellular events in the morphogenesis of rabbits' esophageal epithelium. On the 16th day of gestation, the esophageal epithelium varied from stratified ciliated columnar to stratified squamous type. The surface epithelium presented mucous cells with mucigen granules of various sizes occupying their supranuclear cytoplasm. Cytoplasmic vacuolation was evident in all layers of the esophageal epithelium at this age. On the 18th gestational day, some light cells could be detected in the middle portion of the epithelium, while others occupied the whole epithelial length. On the 21st day, mucous cells are more frequently observed at the apical esophageal part as well as at the surface epithelium. Numerous elongated dark cells could be distinguished embedded between the basal cells. On the 24th gestational day the number of the mucous cells reached its peak. Reaching the 30th gestational day, several lamellar bodies, a keratinized layer and mitotic divisions could be demonstrated, and the number of both mucous and dark cells was greatly decreased. Collectively, detection of surface mucous and dark cells together with the non-cornified surface in some regions of the rabbit esophageal epithelium at the end of gestation ensure a postnatal development to reach the adult epithelium essential to sustain the passage of the harsh raw food. Future immunohistochemical studies are recommended to investigate the components of secretions in mucous cells and functional studies to highlight the dark cells significance. RESEARCH HIGHLIGHTS: Esophageal epithelium of fetal rabbit was analyzed by light and transmission microscopy. Surface epithelium presented mucous cells with mucigen granules of various sizes. They reached their maximum number on 24th day then decreased. On the 16th day, cytoplasmic vacuolation was evident in all epithelial layers. On the 21st day, numerous elongated dark cells could be distinguished embedded between the basal cells. Before birth, several lamellar bodies, a keratinized layer and mitotic divisions could be demonstrated, and the number of both mucous and dark cells was greatly decreased.

Characterization of LysoTracker Red uptake by in vitro model cells of the outer blood-retinal barrier: Implication of lysosomal trapping with cytoplasmic vacuolation and cytotoxicity.

Lysosomal trapping, a physicochemical process in which lipophilic cationic compounds are sequestered in lysosomes, can affect drug disposition and cytotoxicity. To better understand lysosomal trapping at the outer blood-retinal barrier (BRB), we investigated the distribution of LysoTracker Red (LTR), a probe compound for lysosomal trapping, in conditionally immortalized rat retinal pigment epithelial (RPE-J) cells. LTR uptake by RPE-J cells was dependent on temperature and attenuated by ammonium chloride and protonophore, which decreased the pH gradient between the lysosome and cytoplasm, suggesting lysosomal trapping of LTR in RPE-J cells. The involvement of lysosomal trapping in response to cationic drugs, including neuroprotectants such as desipramine and memantine, was also suggested by an inhibition study of LTR uptake. Chloroquine, which is known to show ocular toxicity, induced cytoplasmic vacuolization in RPE-J cells with a half-maximal effective concentration of 1.35 µM. This value was 59 times lower than the median lethal concentration (= 79.1 µM) of chloroquine, suggesting that vacuolization was not a direct trigger of cell death. These results are helpful for understanding the lysosomal trapping of cationic drugs, which is associated with drug disposition and cytotoxicity in the outer BRB.

Subnuclear renal tubular vacuoles in alcohol use disorder.

Subnuclear vacuoles in the proximal renal tubules have been reported as a histologic sign of ketoacidosis. Originally described in diabetic ketoacidosis, renal vacuoles can be found in other ketogenic states such as alcoholic ketoacidosis (AKA), starvation, and hypothermia, underpinned by deranged fatty acid metabolism. A retrospective analysis of 133 deaths associated with alcohol use disorder (AUD) examined at autopsy between 2017 and 2020 was undertaken. This study aimed to determine the prevalence of subnuclear vacuoles in deaths of those with AUD and their specificity for deaths from AKA, and to elucidate what demographic, biochemical, and pathologic findings are associated with subnuclear vacuoles. In each case, vitreous humor biochemistry including electrolytes, glucose, and beta-hydroxybutyrate (BHB) was analyzed alongside postmortem hemoglobin A1c and renal and liver histology. Renal histology was graded for the presence of vacuoles as absent (0), scanty (1), or easily identifiable (2). Liver histology was graded for steatosis and for fibrosis if Masson trichrome staining was available. Vacuoles were commonly seen in the deaths of those with AUD. They were seen in deaths due to AKA but were not specific to that cause of death. With vacuoles present, lower vitreous sodium (139 vs. 142 mmol/L; p = 0.005), higher vitreous BHB (1.50 vs. 1.39 mmol/L; p = 0.04), severe hepatic steatosis, and severe hepatic fibrosis were seen, compared with those without renal vacuoles.

Lysosomal trapping of 4-dimethylamino-1-{3-(1-methyl-1H-imidazole-2-yl)propanoyl}piperidine, a hydrophilic and weakly basic amine, in human aortic vascular smooth muscle cells.

Some weakly basic compounds lead to cell death accompanied by cellular vacuolation. The novel analgesic agent, 4-dimethylamino-1-{3-(1-methyl-1H-imidazole-2-yl)propanoyl}piperidine (DMIP), is a hydrophilic and weakly basic compound that induces vacuolation in the vascular smooth muscle cells in dogs. Here, we investigated the vacuolation mechanism and the potential cytotoxicity of DMIP using human aortic vascular smooth muscle cells. When cells were treated with DMIP (0.1, 0.3, and 1 mM) for 6, 24, and 48 h, clear cytoplasmic vacuolation was observed at 1 mM after 24 and 48 h, along with an increase in the intracellular DMIP concentration. The vacuolation and intracellular DMIP were markedly reduced by bafilomycin A1, a vacuolar H+-ATPase inhibitor. The late endosome marker Rab7 and lysosome marker LAMP-2 were highly expressed but the early endosome marker Rab5 and autophagosome marker LC3 were not expressed specifically on the vacuolar membranes. These results suggested that the most vacuoles were enlarged late endosomes/lysosomes, resulting from the accumulation of DMIP by ion trapping. Moreover, DMIP did not affect lysosomal membrane integrity and was less cytotoxic than chloroquine, an inducer of phospholipidosis. The current study provides further insight into the mechanisms of vacuolation and lysosomal trapping induced by the hydrophilic and weakly basic amine DMIP.

Diplacone Isolated from Paulownia tomentosa Mature Fruit Induces Ferroptosis-Mediated Cell Death through Mitochondrial Ca2+ Influx and Mitochondrial Permeability Transition.

The recently defined type of cell death ferroptosis has garnered significant attention as a potential new approach to cancer treatment owing to its more immunogenic nature when compared with apoptosis. Ferroptosis is characterized by the depletion of glutathione (GSH)/glutathione peroxidase-4 (GPx4) and iron-dependent lipid peroxidation. Diplacone (DP), a geranylated flavonoid compound found in Paulownia tomentosa fruit, has been identified to have anti-inflammatory and anti-radical activity. In this study, the potential anticancer activity of DP was explored against A549 human lung cancer cells. It was found that DP induced a form of cytotoxicity distinct from apoptosis, which was accompanied by extensive mitochondrial-derived cytoplasmic vacuoles. DP was also shown to increase mitochondrial Ca2+ influx, reactive oxygen species (ROS) production, and mitochondrial permeability transition (MPT) pore-opening. These changes led to decreases in mitochondrial membrane potential and DP-induced cell death. DP also induced lipid peroxidation and ATF3 expression, which are hallmarks of ferroptosis. The ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 were effective in counteracting the DP-mediated ferroptosis-related features. Our results could contribute to the use of DP as a ferroptosis-inducing agent, enabling studies focusing on the relationship between ferroptosis and the immunogenic cell death of cancer cells.

Spontaneous early-onset neurodegeneration in the brainstem and spinal cord of NSG, NOG, and NXG mice.

The spectrum of background, incidental, and experimentally induced lesions affecting NSG and NOG mice has been the subject of intense investigation. However, comprehensive studies focusing on the spontaneous neuropathological changes of these immunocompromised strains are lacking. This work describes the development of spontaneous early-onset neurodegeneration affecting both juvenile and adult NSG, NOG, and NXG mice. The study cohort consisted of 367 NSG mice of both sexes (including 33 NSG-SGM3), 61 NOG females (including 31 NOG-EXL), and 4 NXG females. These animals were primarily used for preclinical CAR T-cell testing, generation of humanized immune system chimeras, and/or tumor xenograft transplantation. Histopathology of brain and spinal cord and immunohistochemistry (IHC) for AIF-1, GFAP, CD34, and CD45 were performed. Neurodegenerative changes were observed in 57.6% of the examined mice (affected mice age range was 6-36 weeks). The lesions were characterized by foci of vacuolation with neuronal degeneration/death and gliosis distributed throughout the brainstem and spinal cord. IHC confirmed the development of gliosis, overexpression of CD34, and a neuroinflammatory component comprised of CD45-positive monocyte-derived macrophages. Lesions were significantly more frequent and severe in NOG mice. NSG males were considerably more affected than NSG females. Increased lesion frequency and severity in older animals were also identified. These findings suggest that NSG, NOG, and NXG mice are predisposed to the early development of identical neurodegenerative changes. While the cause of these lesions is currently unclear, potential associations with the genetic mutations shared by NSG, NOG, and NXG mice as well as unidentified viral infections are considered.

Antiparasitic effect of copper alloy mesh on tomont stage of Cryptocaryon irritans in aquaculture.

Copper alloy sheets have been shown to prevent cryptocaryoniasis. Therefore, we studied the potential efficiency of copper alloy mesh (CAM) in aquaculture tanks to prevent cryptocaryoniasis outbreaks. The effectivenesses of CAM against the tomont stage of Cryptocaryon irritans and in protecting fish from cryptocaryoniasis were tested both in vitro and in vivo. The mortality rate of C. irritans tomonts increased as the contact time with CAM rose and peaked at 70 min (100% of mortality). Morphological changes were observed such as the shrinking of the protoplasm of the treated tomonts, resulting in a larger gap between the cytoplasm and the cyst wall. Mitochondrial dysfunction due to shrinkage in the inner portion, outer and inner mitochondrial membrane damage and cytoplasmic vacuolation was revealed by ultrastructural analysis. The use of CAM effectively preventing reinfection was also provided. In comparison with group B (infected fish without CAM), both groups A (uninfected fish as a control group) and C (infected fish treated with CAM) had a 100% survival rate until the end of the trial. CAM has the same anticryptocaryoniasis effect as copper alloy sheets but is more advantageous due to its lightweight, reduced labor cost and lower purchase cost. It is noticeable that CAM exposure also prevents the excessive accumulation of copper ions in aquaculture sea water.

There Are No Insurmountable Barriers: Passage of the Helicobacter pylori VacA Toxin from Bacterial Cytoplasm to Eukaryotic Cell Organelle.

The Gram-negative bacterium Helicobacter pylori is a very successful pathogen, one of the most commonly identified causes of bacterial infections in humans worldwide. H. pylori produces several virulence factors that contribute to its persistence in the hostile host habitat and to its pathogenicity. The most extensively studied are cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA). VacA is present in almost all H. pylori strains. As a secreted multifunctional toxin, it assists bacterial colonization, survival, and proliferation during long-lasting infections. To exert its effect on gastric epithelium and other cell types, VacA undergoes several modifications and crosses multiple membrane barriers. Once inside the gastric epithelial cell, VacA disrupts many cellular-signaling pathways and processes, leading mainly to changes in the efflux of various ions, the depolarization of membrane potential, and perturbations in endocytic trafficking and mitochondrial function. The most notable effect of VacA is the formation of vacuole-like structures, which may lead to apoptosis. This review focuses on the processes involved in VacA secretion, processing, and entry into host cells, with a particular emphasis on the interaction of the mature toxin with host membranes and the formation of transmembrane pores.

Complete subunit structure of serotype C and D botulinum progenitor toxin complex induces vacuolation in the specific epithelial cell line.

Clostridium botulinum produces seven botulinum neurotoxin (BoNT) serotypes. In nature, BoNT exists as a part of the progenitor toxin complex (PTC) through associations with neurotoxin associated proteins (NAPs), including nontoxic nonhemagglutinin and hemagglutinin (HA) complex, consists of HA-70, HA-17 and HA-33. Because PTC displays higher oral toxicity than pure BoNTs, NAPs play a critical role in food poisoning. In a previous study, we demonstrated that the NAP complex in mature large-sized PTC (L-PTC) from serotypes C and D concomitantly induced cell death and cytoplasmic vacuolation in the rat intestinal epithelial cell line IEC-6. Here, we found that the serotype D NAP complex induces only cytoplasmic vacuolation in the normal rat kidney cell line NRK-52E without reducing cell viability. NAP complexes from serotype A and B L-PTCs did not affect cell viability or cytoplasmic vacuolation in IEC-6 and NRK-52E cells. Furthermore, we assessed the effect of immature L-PTCs with fewer HA-33/HA-17 trimers (two HA-33 and one HA-17) than mature L-PTCs on cell viability and cytoplasmic vacuolation in IEC-6 and NRK-52E cells. As a result, mature L-PTCs with the maximum number of HA-33/HA-17 trimers displayed the greatest potency. Consequently, the reduction in cell viability and vacuolation induction are related to the number of HA-33/HA-17 trimers in PTC. The discovery of an epithelial cell model where botulinum PTC specifically induces vacuolization may help clarify the unknown cytotoxicity of PTC, which plays an important role in the trans-epithelial transport of the toxin.

Oxidative Products of Curcumin Rather Than Curcumin Bind to Helicobacter Pylori Virulence Factor VacA and Are Required to Inhibit Its Vacuolation Activity.

Curcumin is a hydrophobic polyphenol derived from turmeric with potent anti-oxidant, anti-microbial, anti-inflammatory and anti-carcinogenic effects. Curcumin is degraded into various derivatives under in vitro and in vivo conditions, and it appears that its degradation may be responsible for the pharmacological effects of curcumin. The primary risk factor for the cause of gastric cancer is Helicobacter pylori (H. pylori). A virulence factor vacuolating cytotoxic A (VacA) is secreted by H. pylori as a 88 kDa monomer (p88), which can be fragmented into a 33 kDa N-terminal domain (p33) and a 55 kDa C-terminal domain (p55). Recently it has been reported that curcumin oxidation is required to inhibit the activity of another major H.pylori toxin CagA. We performed molecular docking of curcumin and its oxidative derivatives with p33 and p55 domains of VacA. Further, we have examined the effect of the oxidation of curcumin on the vacuolation activity of VacA protein. We observed the binding of curcumin to the p55 domain of VacA at five different sites with moderate binding affinities. Curcumin did not bind to p33 domain of VacA. Remarkably, cyclobutyl cyclopentadione and dihydroxy cyclopentadione, which are oxidized products of curcumin, showed a higher binding affinity with VacA protein at all sites except one as compared to parent curcumin itself. However, cyclobutyl cyclopentadione showed a significant binding affinity for the active site 5 of the p55 protein. Active site five (312-422) of p55 domain of VacA plays a crucial role in VacA-mediated vacuole formation. Invitro experiments showed that curcumin inhibited the vacuolation activity of H. pylori in human gastric cell line AGS cells whereas acetyl and diacetyl curcumin, which cannot be oxidized, failed to inhibit the vacuolation in AGS cells after H. pylori infection. Here our data showed that oxidation is essential for the activity of curcumin in inhibiting the vacuolation activity of H. pylori. Synthesis of these oxidized curcumin derivatives could potentially provide new therapeutic drug molecules for inhibiting H. pylori-mediated pathogenesis.

Glabridin induces paraptosis-like cell death via ER stress in breast cancer cells.

Glabridin, a polyphenolic flavonoid isolated from the root of the glycyrrhiza glabra, has been demonstrated to have anti-tumor properties in human malignancies. This study found that glabridin decreased the viability of human breast cancer MDA-MB-231 and MCF7 cells in a dose-dependent manner that was not involved in the caspase-3 cascade. Glabridin promoted the formation of extensive cytoplasmic vacuolation by increasing the expression of endoplasmic reticulum (ER) stress markers BiP, XBP1s, and CHOP. The transmission electron microscopy and fluorescence with the ER chaperon KDEL suggested that the vacuoles were derived from ER. Glabridin-induced vacuolation was blocked when protein synthesis was inhibited by cycloheximide, demonstrating that protein synthesis is crucial for this process. Furthermore, we determined that glabridin causes loss of mitochondrial membrane potential as well as the production of reactive oxygen species, both of which lead to mitochondrial dysfunction. These features are consistent with a kind of programmed cell death described as paraptosis. This work reports for the first time that glabridin could induce paraptosis-like cell death, which may give new therapeutic approaches for apoptosis-resistant breast cancers.

Ursolic acid-piperazine-dithiocarbamate ruthenium(II) polypyridyl complexes induced necroptosis in MGC-803 cells.

Three ursolic acid-piperazine-dithiocarbamate ruthenium(II) polypyridyl complexes Ru1-Ru3 were designed and synthesized for evaluating antitumor activity. All the complexes exhibited high in vitro cytotoxicity against MGC-803, T24, HepG2, CNE2, MDA-MB-231, MCF-7, A549, and A549/DDP cell lines. Ru1, Ru2, and Ru3 were 11, 8 and 10 times, respectively, more active than cisplatin against A549/DDP. An in vivo study on MGC-803 xenograft mouse models demonstrated that representative Ru2 exhibited an effective inhibitory effect on tumor growth, showing stronger antitumor activity than cisplatin. Biological investigations suggested that Ru2 entered MGC-803 cells by a clathrin-mediated endocytic pathway, initially localizing in the lysosomes and subsequently escaping and localizing in the mitochondria. Mitochondrial swelling resulted in vacuolization, which induced vacuolation-associated cell death and necroptosis with the formation of necrosomes (RIP1-RIP3) and the uptake of propidium iodide. These results demonstrate that the potential of Ru2 as a chemotherapeutic agent to kill cancer cells via a dual mechanism represents an alternative way to eradicate apoptosis-resistant forms of cancer.