Construction and Mechanism of IL-15-Based Coactivated Polymeric Micelles for NK Cell Immunotherapy.

Natural killer (NK) cells are an important contributor to cancer immunotherapy, but their antitumor efficacy remains suboptimal. While cytokine-based priming shows promise in enhancing NK-cell activity, its clinical translation faces many challenges, including coactivation of multiple cytokines, poor pharmacokinetics, and limited mechanistic understanding. Here, this work develops a polymeric micelle-based IL-15/IL-2 codelivery system (IL-15/2-PEG-PTMC) for NK-cell activation. In vivo studies demonstrate that half-life of IL-15 and IL-2 and the recruitment of NK cell within tumor tissue are significantly increased after PEG-PTMC loading. Coupled with the coactivation effect of IL-15 and IL-2 conferred by this system, it noticeably delays the growth of tumors compared to conventional NK-cell activation approach, that is free IL-15 and IL-2. It is also surprisingly found that cholesterol metabolism is highly involved in the NK cell activation by IL-15/2-PEG-PTMC. Following stimulation with IL-15/2-PEG-PTMC or IL-15, NK cells undergo a series of cholesterol metabolism reprogramming, which elevates the cholesterol levels on NK cell membrane. This in turn promotes the formation of lipid rafts and activates immune synapses, effectively contributing to the enhancement of NK cell's antitumor activity. It is believed that it will open a new avenue for improving the efficacy of NK cell immunotherapy by regulating cholesterol metabolism.

Aeromonas spp. from hospital sewage act as a reservoir of genes resistant to last-line antibiotics.

BACKGROUND: Aeromonas species are opportunistic pathogens distributed widely in the ecosystem. They are known to be capable of acquiring antibiotic resistance genes, including those encoding proteins against last-line antibiotics, such as the tmexCD-toprJ, mcr and carbapenemase genes. We investigated the genomic and phenotypic characteristics of tmexCD-toprJ-positive Aeromonas strains collected from human, animals, and water samples, particularly those from hospital wastewater in China. METHODS: Samples were collected from living animals, meat, water and human. Aeromonas strains in these samples were isolated in selective media. Antimicrobial resistance profiles of all Aeromonas strains were tested by the broth microdilution method. The presence of tmexCD-toprJ was verified by polymerase chain reaction (PCR). All tmexCD-toprJ-positive (n = 36) and selected tmexCD-toprJ-negative (n = 18) Aeromonas strains were subjected to whole genome sequencing. Carriage of antimicrobial resistance genes, the genetic environment of tmexCD-toprJ and genetic diversity of tmexCD-toprJ-positive Aeromonas strains were determined by bioinformatics analysis. Phylogenetic tree of the Aeromonas strains was built by using the Harvest Suite. FINDINGS: Among the 636 Aeromonas strains isolated from different sources, 36 were positive for tmexCD-toprJ, with the highest prevalence of tmexCD-toprJ being found in fishes (8.8%, 95 CI% 3.6-17.2%), followed by hospital wastewater (6.5%, 95 CI% 4.3-9.3%), river water (2.0%, 0.1-10.9) and duck (1.2%, 95 CI% 3.6-17.2%). All tmexCD-toprJ-positive Aeromonas strains carried multiple antimicrobial resistance genes and exhibited resistance to different classes of antibiotics. Co-existence of tmexCD-toprJ, mcr and blaKPC-2 were identified in 21 strains. The tmexCD-toprJ-positive Aeromonas strains were genetically diverse and found to belong to four different species that could be clustered into three major lineages. The tmexCD-toprJ gene clusters were predominantly located in the chromosome (35/36) of Aeromonas spp., with only one strain carrying the plasmid-borne tmexCD-toprJ cluster. The tmexCD-toprJ genes were associated with seven different types of genetic environments, each of which carried distinct types of mobile elements that may be responsible for mediating transmission of this gene cluster.

Potential role of selenium in alleviating obesity-related iron dyshomeostasis.

Obesity is a serious health problem in modern life and increases the risk of many comorbidities including iron dyshomeostasis. In contrast to malnourished anemia, obesity-related iron dyshomeostasis is mainly caused by excessive fat accumulation, inflammation, and disordered gut microbiota. In obesity, iron dyshomeostasis also induces disorders associated with gut microbiota, neurodegenerative injury, oxidative damage, and fat accumulation in the liver. Selenium deficiency is often accompanied by obesity or iron deficiency, and selenium supplementation has been shown to alleviate obesity and overcome iron deficiency. Selenium inhibits fat accumulation and exhibits anti-inflammatory activity. It regulates gut microbiota, prevents neurodegenerative injury, alleviates oxidative damage to the body, and ameliorates hepatic fat accumulation. These effects theoretically meet the requirements for the inhibition of factors underlying obesity-related iron dyshomeostasis. Selenium supplementation may have a potential role in the alleviation of obesity-related iron dyshomeostasis. This review verifies this hypothesis in theory. All the currently reported causes and results of obesity-related iron dyshomeostasis are reviewed comprehensively, together with the effects of selenium. The challenges and strategies of selenium supplementation are also discussed. The findings demonstrate the possibility of selenium-containing drugs or functional foods in alleviating obesity-related iron dyshomeostasis.

Fecal Carriage of Escherichia coli Harboring the tet(X4)-IncX1 Plasmid from a Tertiary Class-A Hospital in Beijing, China.

The emergence of the mobile tigecycline-resistance gene, tet(X4), poses a significant threat to public health. To investigate the prevalence and genetic characteristics of the tet(X4)-positive Escherichia coli in humans, 1101 human stool samples were collected from a tertiary class-A hospital in Beijing, China, in 2019. Eight E. coli isolates that were positive for tet(X4) were identified from clinical departments of oncology (n = 3), hepatology (n = 2), nephrology (n = 1), urology (n = 1), and general surgery (n = 1). They exhibited resistance to multiple antibiotics, including tigecycline, but remained susceptible to meropenem and polymyxin B. A phylogenetic analysis revealed that the clonal spread of four tet(X4)-positive E. coli from different periods of time or departments existed in this hospital, and three isolates were phylogenetically close to the tet(X4)-positive E. coli from animals and the environment. All tet(X4)-positive E. coli isolates contained the IncX1-plasmid replicon. Three isolates successfully transferred their tigecycline resistance to the recipient strain, C600, demonstrating that the plasmid-mediated horizontal gene transfer constitutes another critical mechanism for transmitting tet(X4). Notably, all tet(X4)-bearing plasmids identified in this study had a high similarity to several plasmids recovered from animal-derived strains. Our findings revealed the importance of both the clonal spread and horizontal gene transfer in the spread of tet(X4) within human clinics and between different sources.

The Role of Vasoactive Intestinal Peptide and Mast Cells in the Regulatory Effect of Lactobacillus casei ATCC 393 on Intestinal Mucosal Immune Barrier.

Vasoactive intestinal peptide (VIP) plays an important role in the neuro-endocrine-immune system. Mast cells (MCs) are important immune effector cells. This study was conducted to investigate the protective effect of L. casei ATCC 393 on Enterotoxigenic Escherichia coli (ETEC) K88-induced intestinal mucosal immune barrier injury and its association with VIP/MC signaling by in vitro experiments in cultures of porcine mucosal mast cells (PMMCs) and in vivo experiments using VIP receptor antagonist (aVIP) drug. The results showed that compared with the ETEC K88 and lipopolysaccharides (LPS)-induced model groups, VIP pretreatment significantly inhibited the activation of MCs and the release of ß-hexosaminidase (ß-hex), histamine and tryptase. Pretreatment with aVIP abolished the protective effect of L. casei ATCC 393 on ETEC K88-induced intestinal mucosal immune barrier dysfunction in C57BL/6 mice. Also, with the blocking of VIP signal transduction, the ETEC K88 infection increased serum inflammatory cytokines, and the numbers of degranulated MCs in ileum, which were decreased by administration of L. casei ATCC 393. In addition, VIP mediated the regulatory effect of L. casei ATCC 393 on intestinal microbiota in mice. These findings suggested that VIP may mediate the protective effect of L.casei ATCC 393 on intestinal mucosal immune barrier dysfunction via MCs.

Lactobacillus rhamnosus from human breast milk ameliorates ulcerative colitis in mice via gut microbiota modulation.

Gut microbiota imbalance is one of the major causes of ulcerative colitis (UC). L. rhamnosus SHA113 (LRS), a strain isolated from healthy human milk, influences the regulation of gut flora. This study aims to determine whether this strain can ameliorate UC by modulating gut microbiota. Mouse models of UC were established using C57BL/6Cnc mice with intragastric administration of 3.0% (w/v) dextran sodium sulfate (DSS). LRS was used to treat the mouse models of UC with 109 cfu mL-1 cell suspension via intragastric administration. To verify the effect of gut microbiota on UC, fecal microbiota collected from the mice after the treatment with LRS were also used to treat the UC mouse models (FMT). The severity of UC was evaluated based on body weight, colon length, disease activity index (DAI), and hematoxylin-eosin staining. The microbial composition was analyzed by 16S rRNA sequencing. The mRNA expression levels of cytokines, mucins, tight junction proteins, and antimicrobial peptides in the gastrointestinal tract were detected by quantitative real-time polymerase chain reaction. The short-chain fatty acid (SCFAs) in the cecal contents of all mice were quantitatively detected by gas chromatography and mass spectrometry. Both LRS and FMT exerted excellent therapeutic effects on UC, as evidenced by the reduction in body weight loss, colon length, and colon structural integrity, as well as the increase in the DAI (disease activity index). LRS and FMT treatments showed similar effects: (1) an increase of total SCFA production in the cecal contents and the abundance of gut microbial diversity and flora composition; (2) decreases in two genera (Parabacteroides and Escherichia/Shigella) related to the DAI and the enhancement of SCFAs and IL-10 positively related genera in the gut microbiota (Bilophila, Roseburia, Akkermansia, and Bifidobacterium); (3) downregulation of the expression of tumor necrosis factor-α, interleukin IL-6, and IL-1ß, and upregulation of the expression of the anti-inflammatory cytokine IL-10; and (4) upregulation of the expression of mucins (Muc1-4) and tight junction protein ZO-1. Overall, L. rhamnosus SHA113 relieves UC via the regulation of gut microbiota: increases in SCFA-producing genera and decreases in UC-related genera. In addition, a single strain is sufficient to induce a significant change in the gut microbiota and exert therapeutic effects on UC.

Effects of Bacillus subtilis iturin A on HepG2 cells in vitro and vivo.

Iturin A with cyclic peptide and fatty acid chain isolated from Bacillus subtilis fermentation shows a variety of biological activities. Among them, the anticancer activity attracted much attention. However, the molecular mechanism of its inhibitory effect on hepatocellular carcinoma was still unclear. Thus its effect on hepatocellular carcinoma was tested in this research. It was found that iturin A could enter HepG2 cells immediately and cause reactive oxygen species burst, disrupt cell cycle and induce apoptosis, paraptosis and autophagy in vitro. The iturin A without fatty acid chain showed no antitumor activity. Amphiphilic is critical to the activity of iturin A. The anticancer activity of iturin A to hepatocellular carcinoma was also verified in mice models carrying xenograft tumors constructed by HepG2 cells. At a dosage of 3 mg/kg/day, iturin A significantly inhibited the further increase of the tumor weight by 58.55%, and reduced the expression of Ki67 in tumor. In the tumor treated with iturin A, lymphocyte infiltration was found, and the expressions of TGF-ß1and PD-L1 were decreased, which indicated that the tumor immune microenvironment was improved. Besides, iturin A showed no significant harm on the health of mice except slight disturbance of liver function. These results suggested that iturin A had significant antitumor effect in vitro and vivo, and provide a basis for the application of iturin A as anticancer agent.

Novel Biomedical Functions of Surfactin A from Bacillus subtilis in Wound Healing Promotion and Scar Inhibition.

Surfactin produced by Bacillus subtilis is a powerful biosurfactant in food, cosmetics, and pesticide industries. However, its suitability in wound healing applications is uncertain. In this article, we determined the effects of surfactin A from B. subtilis on wound healing, angiogenesis, cell migration, inflammatory response, and scar formation. The results indicated that 80.65 ± 2.03% of surfactin A-treated wounds were closed, whereas 44.30 ± 4.26% of the vehicle-treated wound areas remained open on day 7 (P < 0.05). In mechanisms, it upregulated the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF), accelerated keratinocyte migration through mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways, and regulated the secretion of proinflammatory cytokines and macrophage phenotypic switch. More attractive, surfactin A showed a seductive capability to inhibit scar tissue formation by affecting the expression of α-smooth muscle actin (α-SMA) and transforming growth factor (TGF-ß). Overall, the study revealed a new function and potential of surfactin A as an affordable and efficient wound healing drug.

Blocking ACAT-1 Activity for Tumor Therapy with Fluorescent Hyperstar Polymer-Encapsulated Avasimible.

Targeting the distinct cholesterol metabolism of tumor cells is proposed as a novel way to treat tumors. Blocking acyl-CoA cholesterol acyltransferase-1 (ACAT-1) by the inhibitor avasimible (Ava), which elevates intracellular free cholesterol levels, is shown to effectively induce apoptosis. However, Ava faces disadvantages of poor water solubility, a short half-life, and no capability for fluorescence detection, which have greatly limited its application. Herein, a fluorescent hyperstar polymer (FHSP) is developed to encapsulate Ava to improve its ability to inhibit HeLa cells and K562 cells. The results of this study show that the obtained Ava-FHSP micelles possess a high drug loading capacity of 22.7% and bright green fluorescence. Ava and Ava-FHSP are cytotoxic to both HeLa and K562 cells and cause reductions in cell size, nuclear lysis, and chromatin condensation and hindered proliferation of both cell types by causing S phase cell cycle arrest. Further mechanistic analysis indicates that Ava-FHSP reduces the protein and messenger RNA expression of ACAT-1 and significantly increases intracellular free cholesterol levels, which can increase endoplasmic reticulum stress and finally cause cell apoptosis. All these results suggest that this fluorescent hyperstar polymer represents a potential therapeutic tumor strategy by changing the cholesterol metabolism of tumor cells.

miRNA-mediated macrophage behaviors responding to matrix stiffness and ox-LDL.

Atherosclerosis is one of the leading causes of morbidity and mortality, mainly due to the immune response triggered by the recruitment of monocytes/macrophages in the artery wall. Accumulating evidence have shown that matrix stiffness and oxidized low-density lipoproteins (ox-LDL) play important roles in atherosclerosis through modulating cellular behaviors. However, whether there is a synergistic effect for ox-LDL and matrix stiffness on macrophages behavior has not been explored yet. In this study, we developed a model system to investigate the synergistic role of ox-LDL and matrix stiffness on macrophage behaviors, such as migration, inflammatory and apoptosis. We found that there was a matrix stiffness-dependent behavior of monocyte-derived macrophages stimulated with ox-LDL. What's more, macrophages were more sensitive to ox-LDL on the stiff matrices compared to cells cultured on the soft matrices. Through next-generation sequencing, we identified miRNAs in response to matrix stiffness and ox-LDL and predicted pathways that showed the capability of miRNAs in directing macrophages fates. Our study provides a novel understanding of the important synergistic role of ox-LDL and matrix stiffness in modulating macrophages behaviors, especially through miRNAs signaling pathways, which could be potential key regulators in atherosclerosis and immune-targeted therapies.

Grape seed proanthocyanidins suppressed macrophage foam cell formation by miRNA-9 via targeting ACAT1 in THP-1 cells.

Abnormal lipid metabolism in macrophages leads to atherosclerosis (AS). Excessive LDL cholesterol uptake by macrophages in the aortic endothelium leads to formation of foam cells. Previous studies suggested that proanthocyanidins effectively suppress this process, while the in-depth mechanism has not been elucidated. In mononuclear THP-1 cells, we found that the oligomeric fraction of proanthocyanidins was more effective in suppressing foam cell formation and 25 µg ml-1 for 48 h were the optimum conditions. Under these model conditions, we investigated gene expression and for the first time reported expression of regulatory microRNA (miRNA). It was found that the proanthocyanidins restrained macrophage foaming mainly by lowering the expression levels of cholesterol influx-related receptors CD36 and SR-A, and promoting the expression of cholesterol efflux-related receptor ABCA1. Further, it was latest revealed that proanthocyanidins could notably inhibit the expression of ACAT1, a key gene for intracellular cholesterol esterification. Further investigation was performed on the expression of regulatory miRNAs (miR-134 for CD36, miR-134, miR-155 for SR-A, miR-155, let-7g for LOX-1, miR-9 for ACAT1, miR-27a, miR-19b, miR-10b and miR-33a for ABCA1). The relative expression of miR-9, a miRNA targeting ACAT1, was decreased after the treatment of proanthocyanidins. It was most likely that proanthocyanidins suppressed the expression of ACAT1 via up-regulating the expression of miR-9, thus lessening the intracellular lipid accumulation and eventually inhibiting macrophage foam cell formation. This assumption was further verified by use of miR-9 mimic and its inhibitor.

Artemisia sphaerocephala Krasch polysaccharide mediates lipid metabolism and metabolic endotoxaemia in associated with the modulation of gut microbiota in diet-induced obese mice.

Artemisia sphaerocephala Krasch polysaccharide (ASKP) has been proved to have many bioactivities. To determine the underlying mechanisms on anti-obesogenic effect of ASKP in mice, parameters related to obesity, gut microbiota composition, and the correlation between the parameters and specific bacterial taxa were investigated. The results showed that ASKP significantly alleviated high-fat-diet-induced obesity in mice with the amelioration of dyslipidemia, and metabolic endotoxaemia. Relative expression analyses of genes indicated that ASKP administration modulated hepatic lipid metabolism with the downregulation of related genes, including ACC-1, FAS, SREBP-1c, and PPARγ. 16S rRNA analysis showed that ASKP mediated the gut dysbiosis induced by high-fat diet, such as the reduction of Proteobacteria, AF12, and Helicobacter. Spearman's correlation showed that some specific genera, such as Odoribacter, AF12, and Rikenella, were strongly associated with obesity-related parameters. Our results demonstrated that ASKP could serve as a potential prebiotic agent in the prevention of diet-induced obesity.

Prediction of new targets and mechanisms for quercetin in the treatment of pancreatic cancer, colon cancer, and rectal cancer.

Quercetin has been widely found to exhibit anticancer activity with low toxicity and prevalence in foods. Quercetin has been reported to inhibit digestive system cancers including pancreatic cancer (PAAD) and colon cancer (COAD), but rectal cancer (READ) has not been reported. The reported mechanisms and targets are divergent. In this study, new targets and mechanisms were predicted for the influence of quercetin on PAAD, COAD, and READ using bioinformatics methods. The results showed that quercetin may target CD36 and reduce the death rate caused by PAAD by enhancing the cell adhesion, mediating the uptake of fatty acids (FAs), regulating thrombospondin-1, and stimulating the immune response. Quercetin may lower the death rate from READ by targeting SLCO1B1 and producing enhanced effects from use of this compound, inhibiting cell growth, and inducing apoptosis in tumor cells. ACADS, ALDH3B2, UGT2A3, AMH, CDKN2A, FOSL1, CD36, CFL2, CYP3A4, and MAF were identified as targets for quercetin to reduce the death rate caused by COAD. Glutathione metabolism was mainly involved in the effect of quercetin on COAD, including the enhancement of the oxidation of fatty acids, the metabolism of anticancer medications, and the stiffness of cells, and the reduction of chemical carcinogenesis, the level of anti-Müllerian hormone, the proliferation of cancer cells and transcriptional misregulation, and mediation of the activity of glutathione transferases. The combined analyses of three databases can be referred to and used to seek medications and targets that can be applied to other diseases.

Reverse cholesterol transport-related miRNAs and their regulation by natural functional compounds.

Cardiovascular disease (CVD) is the biggest killer globally and atherosclerosis (AS) is the major trigger to this pathology. Abnormal cholesterol homeostasis is the starting point of AS, especially the aggregation of macrophage foam cells in the intra-arterial subcutaneous region. Reverse cholesterol transport (RCT) can remove excess cholesterol from macrophages and transport it to the liver for excretion, making this process vital to alleviate AS. MicroRNAs (miRNAs) are small, noncoding RNAs that play critical roles in various diseases including AS, by regulating post-transcriptional gene expression. Many natural compounds can exert anti-atherosclerotic effects by regulating different miRNAs that are implicated in RCT. Hence, targeting these miRNAs using natural functional compounds may be a safe, novel, and promising strategy to prevent and treat AS. This review describes the miRNAs involved in RCT and the potential uses of natural compounds to target RCT-related miRNAs to modulate AS.

Anti-tumor potential of cell free culture supernatant of Lactobacillus rhamnosus strains isolated from human breast milk.

Lactobacilli rhamnosus has been characterized as a probiotic and plays an important role in human health by stimulating the supplement of nutrients, preventing the colonization of pathogens, and influencing the immune system. This study investigated the anticancer activity of the three Lactobacillus rhamnosus strains SHA111, SHA112, and SHA113 isolated from human breast milk. The cell-free supernatant of a liquid culture of the three strains showed excellent antioxidant activities against DPPH free radicals, superoxide anion radicals, and hydroxyl radicals; furthermore, significant anticancer activity was found on cervix cancer cells (HeLa) via cytotoxicity and induction of apoptosis. RT-qPCR and western blot analysis showed the induction of apoptosis was achieved via the up-regulation of BAD, BAX, Caspase3, Caspase8, Caspase9, and down-regulation of BCL-2 genes in HeLa cells. The results suggest that these strains have potential anticancer capability.

pH-responsive dithiomaleimide-amphiphilic block copolymer for drug delivery and cellular imaging.

A drug delivery system that is integrated with fluorescent imaging is an emerging platform for tumor diagnostic and therapy. A pH-responsive fluorescent polymer that can respond to the surrounding medium is a desired component with which to construct an advanced drug delivery system with bioimaging characteristics and controllable drug releasing. In this work, we synthesized novel amphiphilic block copolymers of poly(ethylene glycol)-b-poly(2-(diisopropylamino) ethyl methacrylate-co-dithiomaleimide) (PEG-b-poly(DPA-co-DTM)) and poly(ethylene glycol)-b-poly(2-(dibutylamino) ethyl methacrylate-co-dithiomaleimide) (PEG-b-poly(DBA-co-DTM)) with pH-responsiveness and fluorescence. The block copolymers exhibited relatively stable fluorescence properties in different solvent and excitation-independent fluorescence behaviours. By copolymerizing the responsive segments in the molecule chain, the doxorubicin (DOX)-loaded micelles could be triggered to disassemble, thus releasing DOX at the corresponding pH values and yielding a pH-responsive drug release. Targeted deliveries of the drug within the cell were demonstrated by using the carrier responding to different pH values. The best antitumor effect was obtained by PEG-b-poly(DPA-co-DTM), which immediately released DOX as soon as it entered the tumor cells, as a result of responding to the regional pH level (pH = 6.3). The pH-responsive copolymers showed excellent biocompatibilities, as nearly 85% of cells with these fluorescent micelles survive when the testing concentration goes up to 200 µg mL-1. In all, these pH-responsive and dithiomaleimide-based fluorescent block copolymers hold great potential in future cancer diagnostic and therapeutic techniques.

Synthesis of silver nanoparticles and its contribution to the capability of Bacillus subtilis to deal with polluted waters.

Bacillus subtilis widely exists in environment and shows a capability to deal with heavy metals and dyes in polluted waters by adsorption or biological oxidation and reduction. Little is known about the roles of lipopeptides in this capability of B. subtilis. In this study, we found that the lipopeptides produced by B. subtilis could reduce silver ions to silver nanoparticles (AgNPs) and iturin was identified as the major effective fraction. Furthermore, the synthesized AgNPs was successfully used to catalyze the reduction of organic dyes and reduce Pb2+ contamination in water. The formation of AgNPs was confirmed by the features analyzed by UV-vis spectroscopy, dynamic light scattering, high-resolution transmission electron microscopy (HR-TEM), and selected area electron diffraction (SAED). The formed AgNPs showed crystalline, with small size (~ 20 nm) and spherical shape. The biosynthesis of AgNPs was significantly accelerated by UV irradiation. A pH of 10 resulted in the highest formation rate, while pH 9.2 provided the most stability of AgNPs. In mechanisms, tyrosine and the polypeptide were identified as the major groups in iturin-A to form AgNPs via Ar-OH groups. The study revealed that iturin played important roles for the capability of B. subtilis to treat polluted water via a possible way by synthesizing AgNPs and then catalyzing the reduction of organic dyes and reducing the contamination of Pb2+.

Effect of cell culture models on the evaluation of anticancer activity and mechanism analysis of the potential bioactive compound, iturin A, produced by Bacillus subtilis.

Two-dimensional (2D) cell culture is widely used to evaluate the potential of food compounds in anticancer activity in vitro. However, 3D culture is rarely used. In this study, we compared the obtained anticancer activity and mechanisms of iturin A, a multiple functional compound produced by Bacillus subtilis, in 2D and 3D cultures of HepG2 cells. 3D culture resulted in a much higher 50% inhibitory concentration (55.26 µM) compared to 2D culture (11.91 µM). Reactive oxygen species accumulation, autophagy, apoptosis characterized by cytochrome c release, high apoptotic protein expression and caspase activation were detected in both 2D and 3D cultures. Induction of paraptosis was also detected in 2D culture and the cytoplasmic vacuoles occurred in large numbers. Compared with 2D culture, 3D culture can simulate the microenvironment in vivo and provide more accurate data. Therefore, 3D culture was recommended for the evaluation of anticancer activity of food compounds towards solid tumors.

Iturin A-like lipopeptides from Bacillus subtilis trigger apoptosis, paraptosis, and autophagy in Caco-2 cells.

This study revealed that iturin A-like lipopeptides produced by Bacillus subtillis induced both paraptosis and apoptosis in heterogeneous human epithelial colorectal adenocarcinoma (Caco-2) cells. Autophagy was simultaneously induced in Caco-2 cells treated with iturin A-like lipopeptides at the early stage and inhibited at the later stage. A western blot analysis showed that the lipopeptides induced apoptosis in Caco-2 cells via a mitochondrial-dependent pathway, as indicated by upregulated expression of the apoptotic genes bax and bad and downregulated expression of the antiapoptotic gene bcl-2. The induction of paraptosis in Caco-2 cells was indicated by the occurrence of many cytoplasmic vacuoles accompanied by endoplasmic reticulum (ER) dilatation and mitochondrial swelling and dysfunction. ER stress also occurred with significant increases in reactive oxygen species and Ca2+ levels in cells. Autophagy was detected by a transmission electron microscopy analysis and by upregulated expression of LC3-II and downregulated expression of LC3-I. The inhibition of autophagy at the later stage was shown by upregulated expression of p62. This study revealed the capability of iturin A-like B. subtilis lipopeptides to simultaneously execute antitumor potential via multiple pathways.

Potential of iturins as functional agents: safe, probiotic, and cytotoxic to cancer cells.

The iturin produced by Bacillus subtilis was previously found to exhibit significant antifungal activities, showing potential for application to food to protect it from spoilage and decay. However, its safe level must be evaluated before it can be used in foods consumed by mammals. Therefore, we carried out acute toxicity (7 days) and subacute toxicity (28 days) evaluations by intragastric administration of the iturin at 5000 mg per kg (bw) and 2000 mg per kg (bw) to Kunming mice. At the end of the subacute toxicity evaluation, the blood parameters of the mice were evaluated, and the visceral organs and intestinal flora were examined. No acute toxicity was found for 5000 mg per kg (bw) iturin and no significant negative effects were found for 2000 mg per kg (bw) iturin on blood parameters or organ functions. A more important observation was that the application of the iturin at the concentration of 2000 mg per kg (bw) showed great enhancement of the abundance of probiotics in the intestinal microflora. The iturin also showed inhibitory effects on cancer cells HepG2, Caco-2, MCF-7, A549, and BIU-87, at the level of 30 µM. The results indicate that the B. subtilis iturin has the potential to be developed as a functional food additive with antitumor potential and the capability to enhance the abundance of probiotics in the intestinal microflora. This study indicates a new possible source of functional food additives.