Induction of apoptotic cell death in human bladder cancer cells by ethanol extract of Zanthoxylum schinifolium leaf,through ROS-dependent inactivation of the PI3K/Akt signaling pathway

BACKGROUND/OBJECTIVESZanthoxylum schinifolium is traditionally used as a spice for cooking in East Asian countries. This study was undertaken to evaluate the anti-proliferative potential of ethanol extracts of Z. schinifolium leaves (EEZS) against human bladder cancer T24 cells.MATERIALS/METHODSSubsequent to measuring the cytotoxicity of EEZS, the anti-cancer activity was measured by assessing apoptosis induction, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP). In addition, we determined the underlying mechanism of EEZS-induced apoptosis through various assays, including Western blot analysis.RESULTSEEZS treatment concentration-dependently inhibited T24 cell survival, which is associated with apoptosis induction. Exposure to EEZS induced the expression of Fas and Fas-ligand, activated caspases, and subsequently resulted to cleavage of poly (ADP-ribose) polymerase. EEZS also enhanced the expression of cytochrome c in the cytoplasm by suppressing MMP, following increase in the ratio of Bax:Bcl-2 expression and truncation of Bid. However, EEZS-mediated growth inhibition and apoptosis were significantly diminished by a pan-caspase inhibitor. Moreover, EEZS inhibited activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway, and the apoptosis-inducing potential of EEZS was promoted in the presence of PI3K/Akt inhibitor. In addition, EEZS enhanced the production of ROS, whereas N-acetyl cysteine (NAC), a ROS scavenger, markedly suppressed growth inhibition and inactivation of the PI3K/Akt signaling pathway induced by EEZS. Furthermore, NAC significantly attenuated the EEZS-induced apoptosis and reduction of cell viability.CONCLUSIONSTaken together, our results indicate that exposure to EEZS exhibits anti-cancer activity in T24 bladder cancer cells through ROS-dependent induction of apoptosis and inactivation of the PI3K/Akt signaling pathway.     

Piperlongumine increases the sensitivity of bladder cancer to cisplatin by mitochondrial ROS

BackgroundThe development of cisplatin resistance often results in cisplatin inefficacy in advanced or recurrent bladder cancer. However, effective treatment strategies for cisplatin resistance have not been well established.MethodsGene expression was measured by qRT‐PCR and Western blotting. CCK‐8 assay was performed to detect cell survival. The number of apoptotic cells was determined using the Annexin V‐PI double‐staining assay. The level of reactive oxygen species (ROS) was measured using 2’,7''‐dichlorodihydrofluorescein diacetate fluorescent dye, and the ATP level was detected using an ATP measurement kit.ResultsThe expression of receptor‐interacting protein kinase 1 (RIPK1), a key regulator of necroptosis, gradually decreased during cisplatin resistance. We first used piperlongumine (PL) in combination with cisplatin to act on cisplatin‐resistant BC cells and found that PL‐induced activation of RIPK1 increased the sensitivity of T24 resistant cells to cisplatin treatment. Furthermore, we revealed that PL killed T24 cisplatin‐resistant cells by triggering necroptosis, because cell death could be rescued by the mixed lineage kinase domain‐like (MLKL) protein inhibitor necrotic sulfonamide or MLKL siRNA, but could not be suppressed by the apoptosis inhibitor z‐VAD. We further explored the specific mechanism and found that PL activated RIPK1 to induce necroptosis in cisplatin‐resistant cells by stimulating mitochondrial fission to produce excessive ROS.ConclusionsOur results demonstrated the role of RIPK1 in cisplatin‐resistant cells and the sensitization effect of the natural drug PL on bladder cancer. These may provide a new treatment strategy for overcoming cisplatin resistance in bladder cancer.     

Glycine induces enhancement of bactericidal activity of neutrophils

Severe bacterial infections are frequently accompanied by depressed neutrophil functions. Thus, agents that increase the microbicidal activity of neutrophils could add to a direct antimicrobial therapy. Lysophosphatidylcholine augments neutrophil bactericidal activity via the glycine (Gly)/glycine receptor (GlyR) α2/TRPM2/p38 mitogen-activated protein kinase (MAPK) pathway. However, the direct effect of glycine on neutrophil bactericidal activity was not reported. In this study, the effect of glycine on neutrophil bactericidal activity was examined. Glycine augmented bactericidal activity of human neutrophils (EC₅₀ = 238 μM) in a strychnine (a GlyR antagonist)-sensitive manner. Glycine augmented bacterial clearance in mice, which was also blocked by strychnine (0.4 mg/kg, s.c.). Glycine enhanced NADPH oxidase-mediated reactive oxygen species (ROS) production and TRPM2-mediated [Ca²⁺]_i increase in neutrophils that had taken up E. coli. Glycine augmented Lucifer yellow uptake (fluid-phase pinocytosis) and azurophil granule-phagosome fusion in neutrophils that had taken up E. coli in an SB203580 (a p38 MAPK inhibitor)-sensitive manner. These findings indicate that glycine augments neutrophil microbicidal activity by enhancing azurophil granule-phagosome fusion via the GlyRα2/ROS/calcium/p38 MAPK pathway. We suggest that glycine could be a useful agent for increasing neutrophil bacterial clearance.     

Novel Bacteriophage Specific against Staphylococcus epidermidis and with Antibiofilm Activity

Staphylococcus epidermidis has emerged as the most important pathogen in infections related to indwelling medical devices, and although these infections are not life-threatening, their frequency and the fact that they are extremely difficult to treat represent a serious burden on the public health system. Treatment is complicated by specific antibiotic resistance genes and the formation of biofilms. Hence, novel therapeutic strategies are needed to fight these infections. A novel bacteriophage CUB-EPI_14 specific to the bacterial species S. epidermidis was isolated from sewage and characterized genomically and phenotypically. Its genome contains a total of 46,098 bp and 63 predicted genes, among which some have been associated with packaging and lysis-associated proteins, structural proteins, or DNA- and metabolism-associated proteins. No lysogeny-associated proteins or known virulence proteins were identified in the phage genome. CUB-EPI_14 showed stability over a wide range of temperatures (from −20 °C to 50 °C) and pH values (pH 3–pH 12) and a narrow host range against S. epidermidis. Potent antimicrobial and antibiofilm activities were observed when the phage was tested against a highly susceptible bacterial isolate. These encouraging results open the door to new therapeutic opportunities in the fight against resilient biofilm-associated infections caused by S. epidermidis.     

Oxaliplatin-loaded nanoemulsion containing Teucrium polium L. essential oil induces apoptosis in Colon cancer cell lines through ROS-mediated pathway

Oxaliplatin (Oxa)-associated adverse side effects have considerably limited the clinical use of the drug in colon cancer therapy. Mutant p53 has diverse mutational profiles in colon cancer, and it influences the potencies of various chemotherapeutic drugs, including Oxa. Thus, it would be highly beneficial to identify an alternative therapeutic strategy that not only reduces the toxicity of Oxa, but also exerts a synergistic effect against colon cancers, regardless of their p53 profiles. The present study was aimed at preparing and optimizing Teucrium polium L. essential oil nanoemulsion (TPO-NANO) and investigating its effect on the sensitivity of colon cancer cells with differences in p53 status (HCT116 wild-type and HT-29 mutant-type) to Oxa. The viability of treated cells was determined and the combination index (CI) was calculated. Morphological changes were determined under inverted microscopy, while percentage apoptosis was assayed using flow cytometry. Intracellular ROS and the protein levels of p53 and Bax were measured. The colony-forming potential of treated cells was determined using colony assay. The size of TPO-NANO was markedly increased from 12.90 ± 0.04 nm to 14.47 ± 0.53 nm after loading Oxa (p ≤ 0.05). The combination (Oxa + TPO-NANO) produced a synergetic effect in HCT116 and HT-29, with CI of 0.94 and 0.88, respectively. Microscopic examination and flow cytometric analysis revealed that cells treated with Oxa + TPO-NANO had a higher percentage of apoptosis than cells exposed to monotherapy. Cumulatively, Oxa exerted an apoptotic effect on wild or mutant p53 colon cancer cells when combined with TPO-NANO, through a mechanism involving ROS-mediated mitochondrial apoptosis.     

Acoustic triggered nanobomb for US imaging guided sonodynamic therapy and activating antitumor immunity

We fabricated an ultrasound activated ‘nanobomb’ as a noninvasive and targeted physical therapeutic strategy for sonodynamic therapy and priming cancer immunotherapy. This ‘nanobomb’ was rationally designed via the encapsulation of indocyanine green (ICG) and perfluoropentane (PFP) into cRGD peptide-functionalized nano-liposome. The resulting Lip-ICG-PFP-cRGD nanoparticle linked with cRGD peptide could actively targeted ID8 and TC-1 cells and elicits ROS-mediated apoptosis after triggered by low-intensity focused ultrasound (LIFU). Moreover, the phase change of PFP (from droplets to microbubbles) under LIFU irradiation can produce a large number of microbubbles, which act as intra-tumoral bomber and can detonate explode tumor cells by acoustic cavitation effect. Instant necrosis of tumor cells further induces the release of biologically active damage-associated molecular patterns (DAMPs) to facilitate antitumor immunity. More important, the ‘nanobomb’ in combination with anti-PD-1checkpoint blockade therapy can significantly improve the antitumor efficacy in a subcutaneous model. In addition, the liposomes may also be used as an imaging probe for ultrasound (US) imaging after being irradiated with LIFU. In summary, the US imaging-guided, LIFU activated ROS production and explosion ‘nanobomb’ might significantly improve the antitumor efficacy and overcome drug resistance through combination of SDT and immunotherapy, we believe that this is a promising approach for targeted therapy of solid tumor including ovarian cancer.     

An Updated Review on SARS-CoV-2 Infection in Animals

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has lasted for two years and caused millions of infections and deaths in humans. Although the origin of SARS-CoV-2 infection in humans remains unknown, infection in animals has been frequently reported in varieties of animals all over the world. Both experimental and natural infections of SARS-CoV-2 in different animal species provide useful information on viral host range and pathogenicity. As the pandemic continues to evolve, SARS-CoV-2 infection in animals will be expanding. In this review, we summarized SARS-CoV-2 testing and infection in animals as well as SARS-CoV-2 strains and transmission in animals. Current data showed that at least 18 different animal species tested positive for SARS-CoV-2. These 18 animal species belong to pet, captive, farmed, and wild animals. Fifteen of the eighteen animal species were known to be positive for the Delta variant and ten animal species were infected with two different types of variants. Human-to-animal, animal-to-animal, and animal-to-human transmission events were suggested in different outbreaks involved in animal infection with SARS-CoV-2. Continued testing, immunization, and surveillance are warranted.     

HMGB1-Like Dorsal Switch Protein 1 Triggers a Damage Signal in Mosquito Gut to Activate Dual Oxidase via Eicosanoids

Several mosquitoes transmit human pathogens by blood feeding, with the gut being the main entrance for the pathogens. Thus, the gut epithelium defends the pathogens by eliciting potent immune responses. However, it was unclear how the mosquito gut discriminates pathogens among various microflora in the lumen. This study proposed a hypothesis that a damage signal might be specifically induced by pathogens in the gut. The Asian tiger mosquito, Aedes albopictus, encodes dorsal switch protein 1 (Aa-DSP1) as a putative damage-associated molecular pattern (DAMP). Aa-DSP1 was localized in the nucleus of the midgut epithelium in naïve larvae. Upon infection by a pathogenic bacterium, Serratia marcescens, Aa-DSP1 was released to hemocoel and activated phospholipase A₂ (PLA₂). The activated PLA₂ increased the level of prostaglandin E₂ (PGE₂) in the gut and subsequently increased Ca²⁺ signal to produce reactive oxygen species (ROS) via dual oxidase (Duox). Inhibition of Aa-DSP1 via RNA interference or specific inhibitor treatment failed to increase PGE₂/Ca²⁺ signal upon the bacterial infection. Thus, the inhibitors specifically targeting eicosanoid biosynthesis significantly prevented the upregulation of ROS production in the gut and enhanced mosquito mortality after the bacterial infection. However, such inhibitory effects were rescued by adding PGE₂. These suggest that Aa-DSP1 plays an important role in immune response of the mosquito gut as a DAMP during pathogen infection by triggering a signaling pathway, DSP1/PLA₂/Ca²⁺/Duox.     

Hydroxyl radicals release in rat striatum involves metabotropic glutamate receptors

Disruption of glutamate homeostasis frequently leads to oxidative stress and to the release of hydroxyl radicals (radical OH). Here, we investigated, via a microdialysis approach, the possible involvement of metabotropic glutamate receptors in the glutamate-induced release of hydroxyl radicals in adult rat striatum. Glutamate was applied at low amount, resulting in a moderate release that was not inhibited by dizocilpine (MK-801), a specific NMDA receptor antagonist. (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG), a broad spectrum metabotropic antagonist, that does not exert any effect on the basal release of radical OH suppressed their response to glutamate. (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), a non-selective metabotropic glutamate receptors agonist, promoted an radical OH release almost similar to that observed after glutamate, which was similarly impaired by co-infusion with MCPG. By contrast, infusion of (RS)-3,5-dihydroxyphenylglycine (DHPG), a more specific group I metabotropic glutamate receptors agonist, did not result in any appreciable radical OH response. Thus, beside NMDA receptors, some metabotropic glutamate receptors may also be involved in the glutamate-induced release of hydroxyl radicals.