Designer exosomes for targeted and efficient ferroptosis induction in cancer via chemo-photodynamic therapy.

Background: Efficient and specific induction of cell death in liver cancer is urgently needed. In this study, we aimed to design an exosome-based platform to deliver ferroptosis inducer (Erastin, Er) and photosensitizer (Rose Bengal, RB) into tumor tissues with high specificity. Methods: Exosome donor cells (HEK293T) were transfected with control or CD47-overexpressing plasmid. Exosomes were isolated and loaded with Er and RB via sonication method. Hepa1-6 cell xenograft C57BL/6 model was injected with control and engineered exosomes via tail vein. In vivo distribution of the injected exosomes was analyzed via tracking the fluorescence labeled exosomes. Photodynamic therapy was conducted by 532 nm laser irradiation. The therapeutic effects on hepatocellular carcinoma and toxic side-effects were systemically analyzed. Results: CD47 was efficiently loaded on the exosomes from the donor cells when CD47 was forced expressed by transfection. CD47 surface functionalization (ExosCD47) made the exosomes effectively escape the phagocytosis of mononuclear phagocyte system (MPS), and thus increased the distribution in tumor tissues. Erastin and RB could be effectively encapsulated into exosomes after sonication, and the drug-loaded exosomes (Er/RB@ExosCD47) strongly induced ferroptosis both in vitro and in vivo in tumor cells after irradiation of 532 nm laser. Moreover, compared with the control exosomes (Er/RB@ExosCtrl), Er/RB@ExosCD47 displayed much lower toxicity in liver. Conclusion: The engineered exosomes composed of CD47, Erastin, and Rose Bengal, induce obvious ferroptosis in hepatocellular carcinoma (HCC) with minimized toxicity in liver and kidney. The proposed exosomes would provide a promising strategy to treat types of malignant tumors.

Zinc enhances the cellular energy supply to improve cell motility and restore impaired energetic metabolism in a toxic environment induced by OTA.

Exogenous nutrient elements modulate the energetic metabolism responses that are prerequisites for cellular homeostasis and metabolic physiology. Although zinc is important in oxidative stress and cytoprotection processes, its role in the regulation of energetic metabolism remains largely unknown. In this study, we found that zinc stimulated aspect in cell motility and was essential in restoring the Ochratoxin A (OTA)-induced energetic metabolism damage in HEK293 cells. Moreover, using zinc supplementation and zinc deficiency models, we observed that zinc is conducive to mitochondrial pyruvate transport, oxidative phosphorylation, carbohydrate metabolism, lipid metabolism and ultimate energy metabolism in both normal and toxic-induced oxidative stress conditions in vitro, and it plays an important role in restoring impaired energetic metabolism. This zinc-mediated energetic metabolism regulation could also be helpful for DNA maintenance, cytoprotection and hereditary cancer traceability. Therefore, zinc can widely adjust energetic metabolism and is essential in restoring the impaired energetic metabolism of cellular physiology.

Bufalin, a bufanolide steroid from the parotoid glands of the Chinese toad, suppresses hERG K+ currents expressed in HEK293 cells.

In this study, we investigated the effect of bufalin on the human ether-à-go-go-related gene (hERG) K+ channels using the perforated patch recording technique. We measured a half-maximal inhibitory concentration (IC50 ) of 24.83 µM and maximal inhibitory effect of 39.45 ± 1.14% with bufalin. These findings suggest that bufalin is a potent hERG K+ channel blocker and may provide a new way for understanding Chan Su-induced arrhythmia.

Effects of ultra-high dilutions of sodium butyrate on viability and gene expression in HEK 293 cells.

BACKGROUND: Several recent studies reported the capability of high diluted homeopathic medicines to modulate gene expression in cell cultures. In line with these studies, we examined whether ultra-high dilutions (30C and 200C) of sodium butyrate (SB) can affect the expression levels of genes involved in acquisition of a senescence-associated secretory phenotype (SASP) in human embryonic kidney (HEK) 293 cells. METHODS: Cell viability was evaluated using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The expression levels of TNF-α, interleukin (IL)-2, IL-4, IL-6 and IL-10 genes were determined by real-time PCR assay. RESULTS: Exposure to both 30C and 200C during 48 h led to a significant decrease of the level of expression of TNF-α gene, while expression of IL-2 gene was increased when exposed to 30C, and expression of IL-10 gene was decreased when exposed to 200C. No changes in expression levels of all genes studied were observed in cells treated with both 30C and 200C remedies of SB during the 24 h. CONCLUSION: Observed changes in gene expression levels after exposure to 30C and 200C remedies of SB during 48 h suggest that extremely low concentrations of this agent can modulate the transcriptome of HEK 293 cells. These results are in line with findings from other studies confirming the ability of homeopathic remedies to modulate gene expression in cell cultures.

Unbiased screen identifies aripiprazole as a modulator of abundance of the polyglutamine disease protein, ataxin-3.

No disease-modifying treatment exists for the fatal neurodegenerative polyglutamine disease known both as Machado-Joseph disease and spinocerebellar ataxia type 3. As a potential route to therapy, we identified small molecules that reduce levels of the mutant disease protein, ATXN3. Screens of a small molecule collection, including 1250 Food and Drug Administration-approved drugs, in a novel cell-based assay, followed by secondary screens in brain slice cultures from transgenic mice expressing the human disease gene, identified the atypical antipsychotic aripiprazole as one of the hits. Aripiprazole increased longevity in a Drosophila model of Machado-Joseph disease and effectively reduced aggregated ATXN3 species in flies and in brains of transgenic mice treated for 10 days. The aripiprazole-mediated decrease in ATXN3 abundance may reflect a complex response culminating in the modulation of specific components of cellular protein homeostasis. Aripiprazole represents a potentially promising therapeutic drug for Machado-Joseph disease and possibly other neurological proteinopathies.

Newly identified allatostatin Bs and their receptor in the two-spotted cricket, Gryllus bimaculatus.

A cDNA encoding allatostatin Bs (ASTBs) containing the W(X)6W motif was identified using a database generated by a next generation sequencer (NGS) in the two-spotted cricket, Gryllus bimaculatus. The contig sequence revealed the presence of five novel putative ASTBs (GbASTBs) in addition to GbASTBs previously identified in G. bimaculatus. MALDI-TOF MS analyses revealed the presence of these novel and previously identified GbASTBs with three missing GbASTBs. We also identified a cDNA encoding G. bimaculatus GbASTB receptor (GbASTBR) in the NGS data. Phylogenetic analysis demonstrated that this receptor was highly conserved with other insect ASTBRs, including the sex peptide receptor of Drosophila melanogaster. Calcium imaging analyses indicated that the GbASTBR heterologously expressed in HEK293 cells exhibited responses to all identified GbASTBs at a concentration range of 10(-10)-10(-5)M.

GABA(A) receptor modulation by terpenoids from Sideritis extracts.

SCOPE: GABAA receptors are modulated by Sideritis extracts. The aim of this study was to identify single substances from Sideritis extracts responsible for GABAA receptor modulation. METHODS AND RESULTS: Single volatile substances identified by GC have been tested in two expression systems, Xenopus oocytes and human embryonic kidney cells. Some of these substances, especially carvacrol, were highly potent on GABAA receptors composed of α1ß2 and α1ß2γ2 subunits. All effects measured were independent from the presence of the γ2 subunit. As Sideritis extracts contain a high amount of terpenes, 13 terpenes with similar structure elements were tested in the same way. Following a prescreening on α1ß2 GABAA receptors, a high-throughput method was used for identification of the most effective terpenoid substances on GABA-affinity of α1ß2γ2 receptors expressed in transfected cell lines. Isopulegol, pinocarveol, verbenol, and myrtenol were the most potent modifiers of GABAA receptor function. CONCLUSION: Comparing the chemical structures, the action of terpenes on GABAA receptors is most probably due to the presence of hydroxyl groups and a bicyclic character of the substances tested. We propose an allosteric modulation independent from the γ2 subunit and similar to the action of alcohols and anesthetics.

Transient receptor potential vanilloid-1 participates in the inhibitory effect of ginsenoside Rg1 on capsaicin-induced interleukin-8 and prostaglandin E2 production in HaCaT cells.

OBJECTIVES: Ginsenoside Rg1 (GRg1), one of the major active constituents of Panax notoginseng, has shown anti-inflammatory and antinocioceptic activity, but its role in keratinocytes needs further study. We have examined the inhibitory effect of GRg1 on transient receptor potential vanilloid-1 (TRPV1) activation in keratinocyte HaCaT cells and explored its involved mechanism. METHODS: HEK 293T cells over-expressing exogenous TRPV1 were constructed and named HEK 293T-TRPV1 cells. The effects of GRg1 on production of interleukin-8 (IL-8) and prostaglandin E(2) (PGE(2) ), calcium influx, the expression of cyclooxygenase-2 (COX-2) and nuclear factor-κB (NF-κB) transcriptional activity in HEK 293T-TRPV1 and HaCaT cells were examined by ELISA, Fluo 3-AM fluorescence probe, Western blot and Dual-Luciferase Reporter Assay, respectively. KEY FINDINGS: The results showed that GRg1 blocked intracellular calcium by both capsaicin and proton activation in a TRPV1-dependent manner. Furthermore, GRg1 inhibited the expression of COX-2 and NF-κB transcriptional activity induced by capsaicin in keratinocytes. The inhibitory effect of GRg1 was similar to capsazepine, an antagonist of TRPV1. More importantly, GRg1 dose-dependently inhibited capsaicin-induced PGE(2) and IL-8 secretion in HaCaT cells and HEK 293T-TRPV1 cells. CONCLUSIONS: These data showed that GRg1 could inhibit TRPV1 mediated responses in HaCaT cells, indicating that GRg1 acted as a TRPV1 antagonist.