Atractylenolide I ameliorates cancer cachexia through inhibiting biogenesis of IL-6 and tumour-derived extracellular vesicles.

BACKGROUND: Atractylenolide I (AI) is a natural sesquiterpene lactone isolated from Atractylodes macrocephala Koidz, known as Baizhu in traditional Chinese medicine. AI has been found to ameliorate cancer cachexia in clinic cancer patients and in tumour-bearing mice. Here, we checked the influence of AI on biogenesis of IL-6 and extracellular vesicles (EVs) in cancer cachexia mice and then focused on studying mechanisms of AI in inhibiting the production of tumour-derived EVs, which contribute to the ameliorating effects of AI on cancer cachexia. METHODS: C26 tumour-bearing BALB/c mice were applied as animal model to examine the effects of AI (25 mg/kg) in attenuating cachexia symptoms, serum IL-6 and EVs levels. IL-6 and EVs secretion of C26 tumour cells treated with AI (0.31-5 µM) was further observed in vitro. The in vitro cultured C2C12 myotubes and 3T3-L1 mature adipocytes were used to check the potency of conditioned medium of C26 cells treated with AI (0.625-5 µM) in inducing muscle atrophy and lipolysis. The glycolysis potency of C26 cells under AI (0.31-5 µM) treatment was evaluated by measuring the extracellular acidification rate using Seahorse XFe96 Analyser. Levels of related signal proteins in both in vitro and in vivo experiments were examined using western blotting to study the possible mechanisms. STAT3 overexpression or knockout C26 cells were also used to confirm the effects of AI (5 µM). RESULTS: AI ameliorated cancer cachexia symptoms (P < 0.05), improved grip strength (P < 0.05) and decreased serum EVs (P < 0.05) and IL-6 (P < 0.05) levels of C26 tumour-bearing mice. AI directly inhibited EVs biogenesis (P < 0.001) and IL-6 secretion (P < 0.01) of cultured C26 cells. The potency of C26 medium in inducing C2C12 myotube atrophy (+59.54%, P < 0.001) and 3T3-L1 adipocyte lipolysis (+20.73%, P < 0.05) was significantly attenuated when C26 cells were treated with AI. AI treatment inhibited aerobic glycolysis and the pathway of STAT3/PKM2/SNAP23 in C26 cells. Furthermore, overexpression of STAT3 partly antagonized the effects of AI in suppressing STAT3/PKM2/SNAP23 pathway, EVs secretion, glycolysis and the potency of C26 medium in inducing muscle atrophy and lipolysis, whereas knockout of STAT3 enhanced the inhibitory effect of AI on these values. The inhibition of AI on STAT3/PKM2/SNAP23 pathway was also observed in C26 tumour tissues. CONCLUSIONS: AI ameliorates cancer cachexia by decreasing the production of IL-6 and EVs of tumour cells. The decreasing effects of AI on EVs biogenesis are based on its inhibition on STAT3/PKM2/SNAP23 pathway.

Extracellular Vesicles May Predict Response to Radioembolization and Sorafenib Treatment in Advanced Hepatocellular Carcinoma: An Exploratory Analysis from the SORAMIC Trial.

PURPOSE: SORAMIC is a randomized controlled trial in patients with advanced hepatocellular carcinoma (HCC) undergoing sorafenib ± selective internal radiation therapy (SIRT). We investigated the value of extracellular vesicle (EV)-based proteomics for treatment response prediction. EXPERIMENTAL DESIGN: The analysis population comprised 25 patients receiving SIRT+sorafenib and 20 patients receiving sorafenib alone. Patients were classified as responders or nonresponders based on changes in AFP and imaging or overall survival. Proteomic analysis was performed on plasma EVs by LC/MS, followed by bioinformatics analysis. Clinical relevance of candidate EV proteins was validated by survival and receiver-operating characteristic analysis with bootstrap internal sampling validation. Origin of circulating EV was explored by IHC staining of liver and tumor tissues and transcriptomics of blood cells. RESULTS: Proteomic analysis identified 56 and 27 EV proteins that were differentially expressed in plasma EVs between responders and nonresponders receiving SIRT+sorafenib and sorafenib alone, respectively. High EV-GPX3/ACTR3 and low EV-ARHGAP1 were identified as candidate biomarkers at baseline from the 13 responders to SIRT+sorafenib with statistically significant AUC = 1 for all and bootstrap P values 2.23 × 10-5, 2.22 × 10-5, and 2.23 × 10-5, respectively. These patients showed reduced abundance of EV-VPS13A and EV-KALRN 6 to 9 weeks after combined treatment with significant AUC and bootstrap P values. In reverse, low GPX3 and high ARHGAP1 demonstrated better response to sorafenib monotherapy with AUC = 0.9697 and 0.9192 as well as bootstrap P values 8.34 × 10-5 and 7.98 × 10-4, respectively. HCC tumor was the likely origin of circulating EVs. CONCLUSIONS: In this exploratory study, EV-based proteomics predicted response to SIRT+sorafenib and sorafenib-only treatment in patients with advanced HCC of metabolic origin.

A new paradigm for diagnosis of neurodegenerative diseases: peripheral exosomes of brain origin.

Neurodegenerative diseases are a heterogeneous group of maladies, characterized by progressive loss of neurons. These diseases involve an intricate pattern of cross-talk between different types of cells to maintain specific signaling pathways. A component of such intercellular cross-talk is the exchange of various types of extracellular vesicles (EVs). Exosomes are a subset of EVs, which are increasingly being known for the role they play in the pathogenesis and progression of neurodegenerative diseases, e.g., synucleinopathies and tauopathies. The ability of the central nervous system exosomes to cross the blood-brain barrier into blood has generated enthusiasm in their study as potential biomarkers. However, the lack of standardized, efficient, and ultra-sensitive methods for the isolation and detection of brain-derived exosomes has hampered the development of effective biomarkers. Exosomes mirror heterogeneous biological changes that occur during the progression of these incurable illnesses, potentially offering a more comprehensive outlook of neurodegenerative disease diagnosis, progression and treatment. In this review, we aim to discuss the challenges and opportunities of peripheral biofluid-based brain-exosomes in the diagnosis and biomarker discovery of Alzheimer's and Parkinson's diseases. In the later part, we discuss the traditional and emerging methods used for the isolation of exosomes and compare their advantages and disadvantages in clinical settings.

Jianpi Jiedu Recipe inhibits colorectal cancer liver metastasis via regulating ITGBL1-rich extracellular vesicles mediated activation of cancer-associated fibroblasts.

BACKGROUND: Extracellular vesicles (EVs) contribute greatly to the formation of pre-metastatic niche and tumor metastasis. Our previous study has revealed that tumor-derived ITGBL1 (integrin beta- like 1)-rich EVs activate fibroblasts through the NF-κB signaling to promote colorectal cancer (CRC) metastasis. Targeting ITGBL1-loaded EVs may be a new and effective therapy for treating CRC metastasis. Simultaneously, our preliminary clinical trial has demonstrated that Jianpi Jiedu Recipe (JPJDR) was an ideal alternative traditional Chinese medicine for the prevention and treatment of CRC metastasis. However, the underlying mechanism of JPJDR in the prevention of CRC metastasis is not clear. In this study, we will investigate the regulatory effect of JPJDR on ITGBL1 levels in CRC-derived EVs, and to detect how JPJDR regulate ITGBL1-rich EVs mediated activation of fibroblasts to inhibit CRC metastasis. METHODS: EVs derived from CRC cells with/without JPJDR treatment were obtained by ultracentrifugation, following by characterization with electron microscopy, LM10 nanoparticle characterization system and western blot. The migration and growth of CRC cells were tested by transwell assay, wound healing assay and colony formation assay. The effect of JPJDR on the fibroblasts-activation associated inflammatory factors including IL-6, IL-8 and α-SMA was detected by real-time PCR. The levels of IL-6, IL-8 and α-SMA in the cell culture supernatant were detected by ELISA. The protein expressions of TNFAIP3, ITGBL1, p-NF-κB, IκBα and ß-actin were detected by western blot. Liver metastasis model in mice was established by injecting MC38 single cell suspension into the spleen of mice to observe the effect of JPJDR on CRC liver metastasis. Immunohistochemistry were applied to detect the expression of ITGBL1 and TNFAIP3 in the liver metastatic tissues. Tissue immunofluorescence detection was performed to observe the regulatory effect of JPJDR on the ITGBL1-NF-κB signaling pathway. Cancer-associated fibroblasts (CAFs) in the liver metastatic tissues were sorted and characterized by platelet-derived growth factor receptor ß (PDGFRß) with flow cytometry, following by the detection of inflammatory factors including IL-6, IL-8 and α-SMA using real-time PCR. RESULTS: JPJDR reduced the ITGBL1 levels in CRC cells-derived EVs. JPJDR inhibited the migration and growth of CRC cells via regulating ITGBL1-rich EVs mediated fibroblasts activity. Mechanically, JPJDR decreased fibroblasts activation by regulating ITGBL1-rich EVs mediated TNFAIP3-NF-κB signaling. Further in vivo experiments demonstrated that JPJDR reduced CRC liver metastasis by regulating ITGBL1-rich EVs secretion from CRC and blocked the fibroblasts activation by regulating ITGBL1-TNFAIP3- NF-κB signaling. CONCLUSION: Our research demonstrated that JPJDR preventd CRC liver metastasis via down-regulating CRC-derived ITGBL1-loaded EVs mediated activation of CAFs, providing the experimental evidence for the clinical application of JPJDR in the prevention and treatment of CRC metastasis. More importantly, our study confirmed the great benefits of therapeutic targeting the EVs-mediated metastasis and warranted future clinical validation.

Tumor-derived extracellular vesicles as messengers of natural products in cancer treatment.

Extracellular vesicles (EVs) are kinds of two-layer vesicles secreted by cells. They play significant roles in mediating component exchange between cells, signal transduction, and pathological development. Among them, the tumor-derived EVs (TDEVs) are found related to the tumor microenvironment and cancer development. TDEVs can be designed as a natural drug carrier with high tumor targeting and permeability. In recent years, drug delivery systems (DDS) based on TDEVs for cancer treatments have received considerable attention. In this review, the biological characteristics of TDEVs are introduced, especially the effect on the tumor. Furthermore, the various approaches to constructing DDS based on TDEVs are summarized. Then we listed examples of TDEVs successfully constructing treatment systems. The use of chemical drugs, biological drugs, and engineered drugs as encapsulated drugs are respectively introduced, particularly the application progress of active ingredients in traditional Chinese medicine. Finally, this article introduces the latest clinical research progress, especially the marketed preparations and challenges of clinical application of TDEVs.

Abnormal Lysosomal Positioning and Small Extracellular Vesicle Secretion in Arterial Stiffening and Calcification of Mice Lacking Mucolipin 1 Gene.

Recent studies have shown that arterial medial calcification is mediated by abnormal release of exosomes/small extracellular vesicles from vascular smooth muscle cells (VSMCs) and that small extracellular vesicle (sEV) secretion from cells is associated with lysosome activity. The present study was designed to investigate whether lysosomal expression of mucolipin-1, a product of the mouse Mcoln1 gene, contributes to lysosomal positioning and sEV secretion, thereby leading to arterial medial calcification (AMC) and stiffening. In Mcoln1-/- mice, we found that a high dose of vitamin D (Vit D; 500,000 IU/kg/day) resulted in increased AMC compared to their wild-type littermates, which was accompanied by significant downregulation of SM22-α and upregulation of RUNX2 and osteopontin in the arterial media, indicating a phenotypic switch to osteogenic. It was also shown that significantly decreased co-localization of lysosome marker (Lamp-1) with lysosome coupling marker (Rab 7 and ALG-2) in the aortic wall of Mcoln1-/- mice as compared to their wild-type littermates. Besides, Mcoln1-/- mice showed significant increase in the expression of exosome/ sEV markers, CD63, and annexin-II (AnX2) in the arterial medial wall, accompanied by significantly reduced co-localization of lysosome marker (Lamp-1) with multivesicular body (MVB) marker (VPS16), suggesting a reduction of the lysosome-MVB interactions. In the plasma of Mcoln1-/- mice, the number of sEVs significantly increased as compared to the wild-type littermates. Functionally, pulse wave velocity (PWV), an arterial stiffening indicator, was found significantly increased in Mcoln1-/- mice, and Vit D treatment further enhanced such stiffening. All these data indicate that the Mcoln1 gene deletion in mice leads to abnormal lysosome positioning and increased sEV secretion, which may contribute to the arterial stiffness during the development of AMC.

Arterial Medial Calcification through Enhanced small Extracellular Vesicle Release in Smooth Muscle-Specific Asah1 Gene Knockout Mice.

Arterial medial calcification (AMC) involves an increased small extracellular vesicle (sEV) secretion and apatite calcium precipitation in the arterial wall. The mechanisms mediating AMC remain poorly understood. In the present study, smooth muscle-specific acid ceramidase (Ac) gene knockout mice (Asah1fl/fl/SMCre) were used to demonstrate the role of lysosomal ceramide signaling pathway in AMC. Asah1fl/fl/SMCre mice were found to have more severe AMC in both aorta and coronary arteries compared to their littermates (Asah1fl/fl/SMwt and WT/WT mice) after receiving a high dose vitamin D. These mice also had pronounced upregulation of osteopontin and RUNX2 (osteogenic markers), CD63, AnX2 (sEV markers) and ALP expression (mineralization marker) in the arterial media. In cultured coronary arterial smooth muscle cells (CASMCs) from Asah1fl/fl/SMCre mice, high dose of Pi led to a significantly increased calcium deposition, phenotypic change and sEV secretion compared to WT CASMCs, which was associated with reduced lysosome-multivesicular body (MVB) interaction. Also, GW4869, sEV release inhibitor decreased sEV secretion and calcification in these cells. Lysosomal transient receptor potential mucolipin 1 (TRPML1) channels regulating lysosome interaction with MVBs were found remarkably inhibited in Asah1fl/fl/SMCre CASMCs as shown by GCaMP3 Ca2+ imaging and Port-a-Patch patch clamping of lysosomes. Lysosomal Ac in SMCs controls sEV release by regulating lysosomal TRPML1 channel activity and lysosome-MVB interaction, which importantly contributes to phenotypic transition and AMC.

Adipocyte-derived extracellular vesicles modulate appetite and weight through mTOR signalling in the hypothalamus.

AIM: Type 2 diabetes and obesity are diseases related to surplus energy in the body. Abnormal interaction between the hypothalamus and adipose tissues is a key trigger of energy metabolism dysfunction. Extracellular vesicles (EVs) regulate intercellular communication by transporting intracellular cargo to recipient cells thereby altering the function of recipient cells. This study aimed to evaluate whether adipocyte-derived EVs can act on hypothalamic neurons to modulate energy intake and to identify the EV-associated non-coding RNAs. METHODS: Confocal imaging was used to trace the uptake of labelled adipocyte-derived exosomes by hypothalamic anorexigenic POMC neurons. The effects of adipocyte-derived EVs on the mammalian target of rapamycin (mTOR) signalling pathway in POMC neurons were evaluated based on mRNA and protein expression in vitro using quantitative real-time PCR and western blotting. In addition, adipocyte-derived EVs were injected into recipient mice, and changes in mice body weight and daily food intake were monitored. The biological effects of the EV-associated MALAT1 on POMC neurons were explored. RESULTS: Adipocyte-derived EVs were successfully transferred into POMC neurons in vitro. Results showed that adipocytes of obese mice secreted MALAT1-containing EVs, which increased appetite and weight when administered to lean mice. Conversely, adipocyte-derived EVs from lean mice decreased food intake and weight when administered to obese mice. CONCLUSION: Adipocyte-derived EVs play important roles in mediating the interaction between adipocytes and hypothalamic neurons. Adipocyte-derived EVs can regulate POMC expression through the hypothalamic mTOR signalling in vivo and in vitro, thereby affecting body energy intake.

Impact of polyphenols on extracellular vesicle levels and effects and their properties as tools for drug delivery for nutrition and health.

Polyphenols are found in plant-derived foods and beverages and display numerous protective effects against cancers, cardiovascular, metabolic and neurodegenerative diseases. Extracellular vesicles (EVs), microparticles, exosomes, and apoptotic bodies, originated by different cell types are emerging as a novel mean of cell-to-cell communication in physiology and pathology and represent a new way to convey fundamental information between cells. Polyphenols can act on signaling pathways that interfere with the biogenesis of EVs. Thus, they are able to control EV release from cells and their content and therefore their functional properties. Both EVs and polyphenols are therapeutic tools that can be used against several diseases. In this context, the combination of both tools can increase their therapeutic potential. Three types of strategies can be used: (i) plants are able to produce EVs that encapsulate natural components from vegetables, polyphenols for instance, (ii) mammalian cells can be treated with polyphenols and the subsequent EVs produced are enriched in these components, and (iii) EVs from mammalian cells can be uploaded with polyphenols. We review the novel aspects of the interplay between polyphenols and EVs that could trigger and improve the health benefits in cancer, cardiovascular, metabolic and neurodegenerative diseases.