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Nanoparticle tracking analysis (NTA) of exosomes and microvesicles (EMVs) released from PC3 cells in the presence of a range of EMV inhibitors. Plots presenting NTA analysis show the concentration of vesicles (0-900 nm in diameter) released from PC3 cells in the absence of any EMV inhibitors (A); In (B) the EMVs are shown to comprise exosomes and microvesicles (MVs) by electron microscopy, by Western blotting (for CD63 expression), and by the degree of phosphatidylserine (PS) exposition. NTA analysis for released EMVs from PC3 cells are presented in the presence of Cl-amidine (C); bisindolylmaleimide-I (D); and imipramine (E). Vesicles outside the size range of 0-900 nm were excluded to avoid including larger vesicles such as MV aggregates or apoptotic bodies. The experiment was repeated three times in total (error bars ± SEM, indicated in red).
Source publication
Microvesicle (MV) release from tumour cells influences drug retention, contributing to cancer drug resistance. Strategically regulating MV release may increase drug retention within cancer cells and allow for lower doses of chemotherapeutic drugs. The contribution of exosomes to drug retention still remains unknown. Potential exosome and MV (EMV) b...
Contexts in source publication
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... were collected from cells after 24 h and analysed by nanoparticle tracking analysis NTA ( Figure 1A). The vesicles collected at 25,000× g were confirmed ( Figure 1B) to comprise EMVs by separate isolation of MVs (centrifugation at 10,000× g) and of exosomes (100,000× g) as determined by electron microscopy (MVs ≥ 150 nm; exosomes ≤150 nm), by the expression of CD63 (negligible in MVs, strong in exosomes), and by a higher PS exposition in MVs compared to exosomes. ...
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... were collected from cells after 24 h and analysed by nanoparticle tracking analysis NTA ( Figure 1A). The vesicles collected at 25,000× g were confirmed ( Figure 1B) to comprise EMVs by separate isolation of MVs (centrifugation at 10,000× g) and of exosomes (100,000× g) as determined by electron microscopy (MVs ≥ 150 nm; exosomes ≤150 nm), by the expression of CD63 (negligible in MVs, strong in exosomes), and by a higher PS exposition in MVs compared to exosomes. Cells were then treated with a range of pharmacological agents and EMV counts determined by NTA analysis (Figures 1C-E and 2A). ...
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... 18, 1007 3 of 11 I, Cl-amidine (and imipramine) reduced numbers of both MVs and exosomes; while this was not the case with Y27632, calpeptin, MβCD, cytochalasin D, and chlorpromazine, which showed a specific tendency to reduce only one vesicle subtype. Figure 1. Nanoparticle tracking analysis (NTA) of exosomes and microvesicles (EMVs) released from PC3 cells in the presence of a range of EMV inhibitors. ...
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... summary, Cl-amidine, and bisindolylmaleimide-I gave the highest inhibition of EMV release followed by imipramine and D-pantethine as detected by NanoSight analysis (Figures 1 and 2A). However, D-pantethine significantly affected cell viability, which was found to be only 25% after 24 h ( Figure 2B). ...
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... were collected from cells after 24 h and analysed by nanoparticle tracking analysis NTA ( Figure 1A). The vesicles collected at 25,000× g were confirmed ( Figure 1B) to comprise EMVs by separate isolation of MVs (centrifugation at 10,000× g) and of exosomes (100,000× g) as determined by electron microscopy (MVs ≥ 150 nm; exosomes ≤150 nm), by the expression of CD63 (negligible in MVs, strong in exosomes), and by a higher PS exposition in MVs compared to exosomes. ...
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... were collected from cells after 24 h and analysed by nanoparticle tracking analysis NTA ( Figure 1A). The vesicles collected at 25,000× g were confirmed ( Figure 1B) to comprise EMVs by separate isolation of MVs (centrifugation at 10,000× g) and of exosomes (100,000× g) as determined by electron microscopy (MVs ≥ 150 nm; exosomes ≤150 nm), by the expression of CD63 (negligible in MVs, strong in exosomes), and by a higher PS exposition in MVs compared to exosomes. Cells were then treated with a range of pharmacological agents and EMV counts determined by NTA analysis (Figures 1C-E and 2A). ...
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... 18, 1007 3 of 11 I, Cl-amidine (and imipramine) reduced numbers of both MVs and exosomes; while this was not the case with Y27632, calpeptin, MβCD, cytochalasin D, and chlorpromazine, which showed a specific tendency to reduce only one vesicle subtype. Figure 1. Nanoparticle tracking analysis (NTA) of exosomes and microvesicles (EMVs) released from PC3 cells in the presence of a range of EMV inhibitors. ...
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... summary, Cl-amidine, and bisindolylmaleimide-I gave the highest inhibition of EMV release followed by imipramine and D-pantethine as detected by NanoSight analysis (Figures 1 and 2A). However, D-pantethine significantly affected cell viability, which was found to be only 25% after 24 h ( Figure 2B). ...
Citations
... One might speculate that an approach to reduce the detrimental effects of cancer-released EVs could be to inhibit their biogenesis, secretion, or uptake by the recipient cells. Several studies have already shown that modulating certain proteins or lipids (e.g., Rab27a, nSMase2, farnesyl transferase, Ras, ceramide) involved in the biogenesis and secretion mechanisms of EVs effectively reduced the level of tumor EVs in the extracellular environment, thereby increasing sensitivity to chemotherapy and reducing tumor progression [6][7][8][9][10][11][12][13] . Unfortunately, none of these strategies have progressed beyond in vivo modeling. ...
Dysregulated intercellular communication is a key feature driving cancer progression. Recently, extracellular vesicles (EVs) have added a new channel to this dense communication network. Despite solid evidence that EVs are central mediators of dysregulated signaling in onco-pathological settings, this has yet to be translated into clinically actionable strategies. The heterogeneity of EV cargo molecules, plasticity of biogenesis routes, and large overlap with their role in physiological communication, complicate a potential targeting strategy. However, recent work has linked EV biology to perhaps the "most druggable" proteins - G protein-coupled receptors (GPCRs). GPCR targeting accounts for ~60% of drugs in development and more than a third of all currently approved drugs, spanning almost all areas of medicine. Although several GPCRs have been linked to cancer initiation and progression, relatively few agents have made it into oncological regimes, suggesting that their potential is underexploited. Herein, we examine the molecular mechanisms linking GPCRs to EV communication in cancer settings. We propose that GPCRs hold potential in the search for EV-targeting in oncology.
... The results from the in vitro and in vivo studies suggest engineered sEVs carrying drugs as a future therapy for neuroendocrine PCa [104]. A research team demonstrated that inhibition of sEV release by treating PC-3 cells with chloramidine and bisindolylmaleimide-I increased 5-flurouracil sensitization and induced apoptosis in these cells [105]. High-throughput screening shows that the treatment of CD63labelled C4-2B cells with Manumycin A and tipifarnib significantly reduced sEV by 50 to 70% compared to control cells [106]. ...
... Additionally, inhibitors such as glyburide (glibenclamide) target ATP-sensitive K+ channels, while others, such as indomethacin, target ATP-binding cassette transporters. These inhibitors regulate cellular cholesterol and phospholipid concentrations, ultimately inhibiting the release of MVs and exosomes 93 . Simvastatin, an HMG-CoA reductase inhibitor, prevents the synthesis of cholesterol and decreases the intracellular concentrations of exosome-associated proteins such as ALIX, CD63, and CD81 94 . ...
Extracellular vesicles (EVs), including exosomes, are increasingly recognized as potent mediators of intercellular communication due to their capacity to transport a diverse array of bioactive molecules. They assume vital roles in a wide range of physiological and pathological processes and hold significant promise as emerging disease biomarkers, therapeutic agents, and carriers for drug delivery. Exosomes encompass specific groups of membrane proteins, lipids, nucleic acids, cytosolic proteins, and other signaling molecules within their interior. These cargo molecules dictate targeting specificity and functional roles upon reaching recipient cells. Despite our growing understanding of the significance of exosomes in diverse biological processes, the molecular mechanisms governing the selective sorting and packaging of cargo within exosomes have not been fully elucidated. In this review, we summarize current insights into the molecular mechanisms that regulate the sorting of various molecules into exosomes, the resulting biological functions, and potential clinical applications, with a particular emphasis on their relevance in cancer and other diseases. A comprehensive understanding of the loading processes and mechanisms involved in exosome cargo sorting is essential for uncovering the physiological and pathological roles of exosomes, identifying therapeutic targets, and advancing the clinical development of exosome-based therapeutics.
... 29 Research suggests that the diversion of EVs from cancer cells contributes to their resistance to chemotherapeutic agents through increased active drug efflux. 22,[28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46] Recent pharmacological studies have shown that inhibiting EV release could be a new strategy to make cancer cells more susceptible to anticancer drug treatment. 32,37-39 While significant progress has been made in exosome/sEV research in recent years, no medications that specifically target the inhibition of sEV secretion have been approved for human use. ...
Exosomes are recognized as important mediators of cell-to-cell communication, facilitating carcinogenesis. Although there have been significant advancements in exosome research in recent decades, no drugs that target the inhibition of sEV secretion have been approved for human use. For this study, we employed GW4869 and Nexinhib20 as inhibitors of exosome synthesis and trafficking combined. First, we found that Nexinhib20 and GW4869 effectively inhibited RAB27A and neutral sphingomyelinase 2 (nSMase2) nsMase2. Interestingly, the inhibition of nsMase2 and RAB27A decreased expression of CD9, CD63 and Tsg101, both at RNA and protein levels. We used a combination treatment strategy of cisplatin/etoposide plus GW4869 or Nexinhib20 on small cell lung cancer (SCLC) cell lines. The combination treatment of GW4869 or Nexinhib20 effectively enhanced the inhibitory effects of first-line chemotherapy on the SCLC cells. Furthermore, we demonstrated that reducing exo-some release through GW4869 and Nexinhib20 treatment effectively reduced cellular proliferation and significantly induced apoptosis in SCLC cells. Also, we showed that combining exosome inhibition with chemotherapy has a significant synergistic effect on cellular proliferation. We also found increased p53 and p21 expressions with western blot and significantly changing Bax, BCL2, caspase-3 and caspase-9 expressions. Inhibiting the exosome pathway offers opportunities for developing novel, effective treatment strategies for SCLC.
... Indeed, increased expression of PADs has been correlated with an increased release of EVs, which have a role in tumors by contributing to disease progression and tumor chemoresistance. Inhibitors of PAD enzymes have been shown to have synergistic effects in both reducing EV release from cancer cells and inhibiting exosomes, as well as in microvesicle-mediated tumor chemoresistance [50,51]. ...
... Proteomic analysis after immunoprecipitation showed the citrullination of mitochondrial and cytoskeletal proteins and proteins associated with stress and invadopodia, including prohibitin, a mitochondrial protein involved in chemoresistance, and histone H3. Several processes were found to be affected in GBM cells following the inhibition of pan-PAD enzymes by Cl-amidine, namely, a consistent reduction in EV release and modulation of the microRNA composition, favoring anti-oncogenic components over pro-oncogenic ones, as well as a validated reduction in prohibitin and histone H3 citrullination, both in single and combined treatments with temoxolodime [50]. However, cell viability was not affected by the administration of Cl-amidine in GBM cells. ...
The present review aims to describe the state of the art of research studies investigating the citrullination post-translational modification in adult and pediatric brain tumors. After an introduction to the deimination reaction and its occurrence in proteins and polypeptide chains, the role of the citrullination post-translational modification in physiological as well as pathological states, including cancer, is summarized, and the recent literature and review papers on the topic are examined. A separate section deals with the specific focus of investigation of the citrullination post-translational modification in relation to brain tumors, examining the state of the art of the literature that mainly concerns adult and pediatric glioblastoma and posterior fossa pediatric tumors. We examined the literature on this emerging field of research, and we apologize in advance for any possible omission. Although only a few studies inspecting citrullination in brain tumors are currently available, the results interestingly highlighted different profiles of the citrullinome associated with different histotypes. The data outlined the importance of this post-translational modification in modulating cancer invasion and chemoresistance, influencing key factors involved in apoptosis, cancer cell communication through extracellular vesicle release, autophagy, and gene expression processes, which suggests the prospect of taking citrullination as a target of cancer treatment or as a source of potential diagnostic and prognostic biomarkers for potential clinical applications in the future.
... Thus, targeting tumor EV biogenesis can be also adopted as a successful strategy to reduce chemoresistance. In this respect, different substances, including indomethacin, bisindolylmaleimide-I, chloramidine, calpeptin, and cannabidiol have been shown to enhance tumor response to chemotherapy through their proposed effect on blocking EV production, thereby increasing drug retention within tumor cells [136][137][138][139][140]. ...
Simple Summary
Cancer metastasis accounts for almost 90% of cancer deaths worldwide. The efficiency of current surgical, radiotherapeutic, and chemotherapeutic approaches is limited, and new, more powerful therapeutic strategies are urgently needed. Tumor metastasis as well as primary tumor invasiveness strongly rely on the ability of cancer cells to migrate at distant sites or locally in the surrounding tissue. Here, we give an overview of the underlying mechanisms, with special emphasis on the role of extracellular vesicles (EVs). EVs are nanoparticles released by cells implied in cell–cell communication. They can travel in the bloodstream and in other body fluids, thus transporting molecules to specific cells and inducing specific responses. Based on the recent literature, we discuss the role of vesicles in cancer cell migration and metastasis as well as their anti-cancer therapeutic significance, focusing on tumor-derived EVs. Moreover, we propose EV encapsulation as an alternative route with specific effects on target cells.
Abstract
The infiltration of primary tumors and metastasis formation at distant sites strongly impact the prognosis and the quality of life of cancer patients. Current therapies including surgery, radiotherapy, and chemotherapy are limited in targeting the complex cell migration mechanisms responsible for cancer cell invasiveness and metastasis. A better understanding of these mechanisms and the development of new therapies are urgently needed. Extracellular vesicles (EVs) are lipid-enveloped particles involved in inter-tissue and inter-cell communication. This review article focuses on the impact of EVs released by tumor cells, specifically on cancer cell migration and metastasis. We first introduce cell migration processes and EV subtypes, and we give an overview of how tumor-derived EVs (TDEVs) may impact cancer cell migration. Then, we discuss ongoing EV-based cancer therapeutic approaches, including the inhibition of general EV-related mechanisms as well as the use of EVs for anti-cancer drug delivery, focusing on the harnessing of TDEVs. We propose a protein-EV shuttle as a route alternative to secretion or cell membrane binding, influencing downstream signaling and the final effect on target cells, with strong implications in tumorigenesis. Finally, we highlight the pitfalls and limitations of therapeutic EV exploitation that must be overcome to realize the promise of EVs for cancer therapy.
... The HeLa cells used were routinely screened for Mycoplasma using the MycoProbe Mycoplasma Detection Kit (R and D Systems) and found to be free of contamination. Exponentially growing cells were counted and viability determined using the Guava ViaCount Reagent by flow cytometry (ViaCount assay; EMD Millipore) as described before [19,60]. After 3 days in culture, cells were routinely split 1 : 4 at about 80 % confluence, and only cultures with at least 95 % viability were used in experiments. ...
... The m/sEV samples were diluted 1/1500 using EV-free RPMI, and the size distribution and concentration then determined by nanoparticle tracking analysis (NTA) using a Nanosight NS300 (Nanosight, U.K.) as described before [19,43,60,62]. The particles were quantified for size and number and represented on a frequency size distribution graph. ...
... This study provides a platform, through modulation of mEV release, for limiting the spread of non-enveloped viruses. This will require further elucidation of pathways of mEV biogenesis which we have been actively pursuing [19,60] leading to discovery of specific pharmacological inhibitors of mEV release [19,60,62,123]. In this study we described calpeptin inhibiting infection levels of CVB1 in HeLa cells treated with CVB1, CVB1 i -mEVs or in combination. ...
Like most non-enveloped viruses, CVB1 mainly uses cell lysis to spread. Details of a nonlytic virus transmission remain unclear. Extracellular Vesicles (EVs) transfer biomolecules between cells. We show that CVB1 entry into HeLa cells results in apoptosis and release of CVB1-induced 'medium-sized' EVs (CVB1i-mEVs). These mEVs (100-300 nm) harbour CVB1 as shown by immunoblotting with anti-CVB1-antibody; viral capsids were detected by transmission electron microscopy and RT-PCR revealed CVB1 RNA. The percentage of mEVs released from CVB1-infected HeLa cells harbouring virus was estimated from TEM at 34 %. Inhibition of CVB1i-mEV production, with calpeptin or siRNA knockdown of CAPNS1 in HeLa cells limited spread of CVB1 suggesting these vesicles disseminate CVB1 virions to new host cells by a nonlytic EV-to-cell mechanism. This was confirmed by detecting CVB1 virions inside HeLa cells after co-culture with CVB1i-mEVs; EV release may also prevent apoptosis of infected cells whilst spreading apoptosis to secondary sites of infection.
... Roles for citrullination/deimination have been studied in acute central nervous system (CNS) injury, including neonatal hypoxic-ischaemic brain injury [13,14], spinal cord injury [15] and traumatic brain injury [16,17], as well as in brain cancer [18,19] and neurodegenerative diseases, namely Parkinson's disease (PD) [20][21][22][23], Alzheimer's disease (AD) [24][25][26][27], prion disease [28], amyotrophic lateral sclerosis (ALS) [29] and multiple sclerosis (MS) [30][31][32]. Furthermore, a link between citrullination and extracellular vesicle (EV) signatures has been established in several disease models [33,34], including in the CNS [13,19,21,35]. ...
Abstract: Neonatal seizures are commonly associated with acute perinatal brain injury, while understanding regarding the downstream molecular pathways related to seizures remains unclear. Furthermore, effective treatment and reliable biomarkers are still lacking. Post-translational modifications can contribute to changes in protein function, and post-translational citrullination, which is caused by modification of arginine to citrulline via the calcium-mediated activation of the pep-tidylarginine deiminase (PAD) enzyme family, is being increasingly linked to neurological injury. Extracellular vesicles (EVs) are lipid-bilayer structures released from cells; they can be isolated from most body fluids and act as potential liquid biomarkers for disease conditions and response to treatment. As EVs carry a range of genetic and protein cargo that can be characteristic of pathological processes, the current study assessed modified citrullinated protein cargo in EVs isolated from plasma and CSF in a piglet neonatal seizure model, also following phenobarbitone treatment. Our findings provide novel insights into roles for PAD-mediated changes on EV signatures in neonatal seizures and highlight the potential of plasma-and CSF-EVs to monitor responses to treatment.
... The association of OME that inhibits SEVs uptake with GW4869 that limits SEV biogenesis reduces paclitaxel (PTX) amount in SEVs, thus increasing the therapeutic effect of PTX on BC cells [524]. As GW4869 seems promising, imipramine, a tricyclic antidepressant, is also a source of interest because of its inhibitory activity on SMPD1 [525,526]. ...
Breast cancer (BC) is the first worldwide most frequent cancer in both sexes and the most commonly diagnosed in females. Although BC mortality has been thoroughly declining over the past decades, there are still considerable differences between women diagnosed with early BC and when metastatic BC is diagnosed. BC treatment choice is widely dependent on precise histological and molecular characterization. However, recurrence or distant metastasis still occurs even with the most recent efficient therapies. Thus, a better understanding of the different factors underlying tumor escape is mainly mandatory. Among the leading candidates is the continuous interplay between tumor cells and their microenvironment, where extracellular vesicles play a significant role. Among extracellular vesicles, smaller ones, also called exosomes, can carry biomolecules, such as lipids, proteins, and nucleic acids, and generate signal transmission through an intercellular transfer of their content. This mechanism allows tumor cells to recruit and modify the adjacent and systemic microenvironment to support further invasion and dissemination. By reciprocity, stromal cells can also use exosomes to profoundly modify tumor cell behavior. This review intends to cover the most recent literature on the role of extracellular vesicle production in normal and cancerous breast tissues. Specific attention is paid to the use of extracellular vesicles for early BC diagnosis, follow-up, and prognosis because exosomes are actually under the spotlight of researchers as a high-potential source of liquid biopsies. Extracellular vesicles in BC treatment as new targets for therapy or efficient nanovectors to drive drug delivery are also summarized.
... Moreover, results of several studies have shown that agents like indomethacin, ketotifen, chloramidine, and bisindolylmaleimide-I combat drug resistance by enhancing cellular drug accumulation as a result of exosome inhibition. Therefore, exosome inhibitors are promising candidates to be used in combination regimens for treating cancer (Koch et al. 2016;Kosgodage et al. 2017;Khan et al. 2018). ...
... Many studies in this field attributed these antitumor effects of aprepitant to its effect on NK-1R and subsequently substance p (Javid et al. 2021). Since various studies have demonstrated that the combined use of exosome inhibitors with chemotherapeutic agents improves the response cancer cells to therapy (Kosgodage et al. 2017;Khan et al. 2018;Hekmatirad et al. 2021), our hypothesis is that this effect could be partly related, at least partly, due to the inhibitory effect of aprepitant on nSMase2. ...
Purpose:
Exosomes are membrane-derived nano-vesicles upregulated in pathological conditions like cancer. Therefore, inhibiting their release is a potential strategy for the development of more efficient combination therapies. Neutral sphingomyelinase 2 (nSMase2) is a key component in exosome release; however, a clinically safe yet efficient nSMase2 inhibitor remains to be used discovered. Accordingly, we made an effort to identify potential nSMase2 inhibitor(s) among the approved drugs.
Methods:
Virtual screening was performed and aprepitant was selected for further investigation. To evaluate the reliability of the complex, molecular dynamics were performed. Finally, using the CCK-8 assay in HCT116 cells, the highest non-toxic concentrations of aprepitant were identified and the nSMase2 activity assay was performed to measure the inhibitory activity of aprepitant, in vitro.
Results:
To validate the screening results, molecular docking was performed, and the retrieved scores were in line with the screening results. The root-mean-square deviation (RMSD) plot of aprepitant-nSMase2 showed proper convergence. Following treatment with different concentrations of aprepitant in both cell-free and cell-dependent assays, nSMase2 activity was remarkably decreased.
Conclusion:
Aprepitant, at a concentration as low as 15 µM, was able to inhibit nSmase2 activity in HCT116 cells without any significant effects on their viability. Aprepitant is therefore suggested to be a potentially safe exosome release inhibitor.
