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) biogenesis inhibitors, tested on human prostate cancer (PC3) cells for their capacity to inhibit EMV release, were also tested on PC3 and MCF-7 (breast cancer) cells for improving chemotherapy. Agents inhibiting EMV release most significantly, whilst maintaining cell viability, were chloramidine (Cl-amidine; 50 µM) and bisindolylmaleimide-I (10 µM). Apoptosis mediated by the chemotherapy drug 5-fluorouracil (5-FU) was significantly enhanced in PC3 cells in the presence of both these EMV inhibitors, resulting in a 62% (Cl-amidine + 5-FU) and 59% (bisindolylmaleimide-I + 5-FU) decrease in numbers of viable PC3 cells compared to 5-FU alone after 24 h. For MCF-7 cells, there were similar increased reductions of viable cells compared to 5-FU treatment alone ranging from 67% (Cl-amidine + 5-FU) to 58% (bisindolylmaleimide-I + 5-FU). Using combinatory treatment, the two EMV inhibitors further reduced the number of viable cancer cells tested. Neither inhibitor affected cell viability. Combining selected EMV inhibitors may pose as a novel strategy to enhance the efficacy of chemotherapeutic drug-mediated apoptosis.
Exosomes and microvesicles (EMVs) are lipid bilayer-enclosed structures released from cells and participate in cell-to-cell communication via transport of biological molecules. EMVs play important ...roles in various pathologies, including cancer and neurodegeneration. The regulation of EMV biogenesis is thus of great importance and novel ways for manipulating their release from cells have recently been highlighted. One of the pathways involved in EMV shedding is driven by peptidylarginine deiminase (PAD) mediated post-translational protein deimination, which is calcium-dependent and affects cytoskeletal rearrangement amongst other things. Increased PAD expression is observed in various cancers and neurodegeneration and may contribute to increased EMV shedding and disease progression. Here, we review the roles of PADs and EMVs in cancer and neurodegeneration.
Exosomes and microvesicles (EMV) are lipid bilayer-enclosed structures, released by cells and involved in intercellular communication through transfer of proteins and genetic material. EMV release is ...also associated with various pathologies, including cancer, where increased EMV release is amongst other associated with chemo-resistance and active transfer of pro-oncogenic factors. Recent studies show that EMV-inhibiting agents can sensitize cancer cells to chemotherapeutic agents and reduce cancer growth
. Cannabidiol (CBD), a phytocannabinoid derived from
, has anti-inflammatory and anti-oxidant properties, and displays anti-proliferative activity. Here we report a novel role for CBD as a potent inhibitor of EMV release from three cancer cell lines: prostate cancer (PC3), hepatocellular carcinoma (HEPG2) and breast adenocarcinoma (MDA-MB-231). CBD significantly reduced exosome release in all three cancer cell lines, and also significantly, albeit more variably, inhibited microvesicle release. The EMV modulating effects of CBD were found to be dose dependent (1 and 5 μM) and cancer cell type specific. Moreover, we provide evidence that this may be associated with changes in mitochondrial function, including modulation of STAT3 and prohibitin expression, and that CBD can be used to sensitize cancer cells to chemotherapy. We suggest that the known anti-cancer effects of CBD may partly be due to the regulatory effects on EMV biogenesis, and thus CBD poses as a novel and safe modulator of EMV-mediated pathological events.
Outer membrane and membrane vesicles (OMV/MV) are released from bacteria and participate in cell communication, biofilm formation and host-pathogen interactions. Peptidylarginine deiminases (PADs) ...are phylogenetically conserved enzymes that catalyze post-translational deimination/citrullination of proteins, causing structural and functional changes in target proteins. PADs also play major roles in the regulation of eukaryotic extracellular vesicle release. Here we show phylogenetically conserved pathways of PAD-mediated OMV/MV release in bacteria and describe deiminated/citrullinated proteins in
and their derived OMV/MVs. Furthermore, we show that PAD inhibitors can be used to effectively reduce OMV/MV release, both in Gram-negative and Gram-positive bacteria. Importantly, this resulted in enhanced antibiotic sensitivity of both
and
to a range of antibiotics tested. Our findings reveal novel strategies for applying pharmacological OMV/MV-inhibition to reduce antibiotic resistance.
Membrane vesicles (MVs) released from bacteria participate in cell communication and host-pathogen interactions. Roles for MVs in antibiotic resistance are gaining increased attention and in this ...study we investigated if known anti-bacterial effects of cannabidiol (CBD), a phytocannabinoid from
, could be in part attributed to effects on bacterial MV profile and MV release. We found that CBD is a strong inhibitor of MV release from Gram-negative bacteria (
VCS257), while inhibitory effect on MV release from Gram-positive bacteria (
subsp
Rosenbach) was negligible. When used in combination with selected antibiotics, CBD significantly increased the bactericidal action of several antibiotics in the Gram-negative bacteria. In addition, CBD increased antibiotic effects of kanamycin in the Gram-positive bacteria, without affecting MV release. CBD furthermore changed protein profiles of MVs released from
after 1 h CBD treatment. Our findings indicate that CBD may pose as a putative adjuvant agent for tailored co-application with selected antibiotics, depending on bacterial species, to increase antibiotic activity, including via MV inhibition, and help reduce antibiotic resistance.
Glioblastoma multiforme (GBM) is the most aggressive form of adult primary malignant brain tumour with poor prognosis. Extracellular vesicles (EVs) are a key-mediator through which GBM cells promote ...a pro-oncogenic microenvironment. Peptidylarginine deiminases (PADs), which catalyze the post-translational protein deimination of target proteins, are implicated in cancer, including via EV modulation. Pan-PAD inhibitor Cl-amidine affected EV release from GBM cells, and EV related microRNA cargo, with reduced pro-oncogenic microRNA21 and increased anti-oncogenic microRNA126, also in combinatory treatment with the chemotherapeutic agent temozolomide (TMZ). The GBM cell lines under study, LN18 and LN229, differed in PAD2, PAD3 and PAD4 isozyme expression. Various cytoskeletal, nuclear and mitochondrial proteins were identified to be deiminated in GBM, including prohibitin (PHB), a key protein in mitochondrial integrity and also involved in chemo-resistance. Post-translational deimination of PHB, and PHB protein levels, were reduced after 1 h treatment with pan-PAD inhibitor Cl-amidine in GBM cells. Histone H3 deimination was also reduced following Cl-amidine treatment. Multifaceted roles for PADs on EV-mediated pathways, as well as deimination of mitochondrial, nuclear and invadopodia related proteins, highlight PADs as novel targets for modulating GBM tumour communication.
Glioblastoma multiforme (GBM) is the most common and aggressive form of primary malignant brain tumor in adults, with poor prognosis. Extracellular vesicles (EVs) are key-mediators for cellular ...communication through transfer of proteins and genetic material. Cancers, such as GBM, use EV release for drug-efflux, pro-oncogenic signaling, invasion and immunosuppression; thus the modulation of EV release and cargo is of considerable clinical relevance. As EV-inhibitors have been shown to increase sensitivity of cancer cells to chemotherapy, and we recently showed that cannabidiol (CBD) is such an EV-modulator, we investigated whether CBD affects EV profile in GBM cells in the presence and absence of temozolomide (TMZ). Compared to controls, CBD-treated cells released EVs containing lower levels of pro-oncogenic miR21 and increased levels of anti-oncogenic miR126; these effects were greater than with TMZ alone. In addition, prohibitin (PHB), a multifunctional protein with mitochondrial protective properties and chemoresistant functions, was reduced in GBM cells following 1 h CBD treatment. This data suggests that CBD may, via modulation of EVs and PHB, act as an adjunct to enhance treatment efficacy in GBM, supporting evidence for efficacy of cannabinoids in GBM.
Extracellular vesicle (EV) release from tumour cells plays an important role in cancer drug resistance. It is essential that chemotherapeutic drugs are retained within target cells for increased ...efficacy in inducing apoptosis and as microvesiculation influences drug retention, this study has focused on modulating microvesiculation. Based on EV biogenesis pathways, a range of potential inhibitors were tested on PC3, prostate cancer and MCF7, breast cancer cells. Flow cytometry (FCM), nanoparticle tracking analysis (NTA) and fluorescent microscopy were used for the investigation. All inhibitors tested were shown to inhibit EV release with minimal effect on the cell viability. Peptidyl Arginine Deiminase (PAD) inhibitor, Chloramidine (Cl-amidine, Cl-am) and Protein Kinase C inhibitor, Bisindolylmaleimide-I (Bis-I) exhibited extensive EV inhibition in PC3 and MCF-7 cells. This effect was further tested using an Annexin-V-based apoptosis assay and VB-48 vitality assay through image FCM. There was a significant increase in 5-fluorouracil (5-FU) activity in the presence of the inhibitors which was more apparent when a combination of inhibitors was applied. The hypothesis was further tested using a second, chemotherapeutic drug, Doxorubicin (Dox) on PC3 cells. Pre-treatment of PC3 cells with Cl-am prior to chemotherapy, exhibited increased activity of Dox and increased drug retention was shown by fluorescent microscopy, HPLC, FCM and fluorescence spectrophotometry. Overall the findings demonstrated the implication of EV inhibitors in improving chemotherapy while at the same time reducing its side effects. Cannabidiol (CBD) has been well known for its psychotherapeutic function and has also been used in some cancers, including in Glioblastoma multiforme (GBM), although the exact pathways that could explain its clear mode of action have not been revealed. This study presents CBD as a potent inhibitor of EVs for the first time. Its potential role has here been examined in many types of cancer including, prostate, liver, breast and GBM across the study. Successful EV inhibition was shown to elicit a favourable response in Temozolomide (TMZ)-mediated therapy against GBM cells, which sensitized some GBM cells to TMZ-mediated chemotherapy. Outer membrane vesicles (OMVs) are released by bacterial cells upon activation. Isolation and characterisation of these vesicles was carried out using differential centrifugation, ultrafiltration and nanoparticle tracking analysis. The presence of an arginine deiminase enzyme, corresponding to mammalian Peptidyl Arginine Deiminase (PAD), was confirmed in E. coli samples and its deiminating activity on various target proteins was shown in this study for the first time. Identification of PAD and PAD-mediated citrullinated/deiminatedproteins was identified using immunoprecipitation and Western blotting techniques. The same range of EV inhibitors as used for cancer cells, along with the PAD inhibitors, was used to investigate the effect on OMV biogenesis from Gram-negative, E.coli and Gram-positive S. aureus. EV inhibitors Bisindolylmaleimide-I, EGTA, Imipramine, Pantethine and Y27632 inhibited OMV release significantly. The role of EV inhibitors on antibiotic therapy was then further explored. The disc diffusion method and Minimum Inhibitory Concentration (MIC) was used to determine whether OMV inhibition affected antibiotic activity. E. coli samples were treated with OMV inhibitors and a range of antibiotics including colistin, erythromycin, rifampicin, kanamycin and vancomycin to identify an optimal OMV inhibitor. The OMV inhibitors had a selective response to antibiotics which facilitated the choice for the most sensitive antibiotic to use in further studies to examine the effect of OMV inhibition in lowering minimum inhibitory concentration. These findings, together with previous studies on the association of OMVs with bacteriophage particles, prompted an investigation of the role of OMV inhibition on phage therapy. E. coli samples were treated with lambda phage with/without pre-treatment of Cl-am. Bacterial titres were calculated after each experiment. Electron microscopy was used to visualise the formation of phage-E.coli-OMVs which confirmed the outcome of the experiments. The increase ofcancer cells withresistance to chemotherapy has increased over time which has added immense pressure on scientists to discover novel therapies within short periods. EVs play a major role in transferring a drug-resistance phenotype to drug-sensitive cells via cellular cargo. This also interferes with its negative role of drug efflux which together drive cancer metastasis. Increasing the dosage of chemotherapy or introducing novel therapeutic agents has hitherto not minimised this effect successfully. Therefore, EV inhibition, as introduced in this study, may have a positive influence in developing novel effective anti-cancer therapies. The use of OMV inhibition to sensitize bacterial cells to antibiotics is a novel idea which may become an effective solution to combat antibiotic resistance and permit the treatment of such diseases through effective phage therapy. Overall, the research ideas presented in the current study will benefit the implementation of new therapeutic strategies for cancer metastasis and bacterial colonisation, minimising multi drug resistance.
Glioblastoma multiforme (GBM) is the most common and aggressive form of primary malignant brain tumor in adults, with poor prognosis. Extracellular vesicles (EVs) are key-mediators for cellular ...communication through transfer of proteins and genetic material. Cancers, such as GBM, use EV release for drug-efflux, pro-oncogenic signaling, invasion and immunosuppression; thus the modulation of EV release and cargo is of considerable clinical relevance. As EV-inhibitors have been shown to increase sensitivity of cancer cells to chemotherapy, and we recently showed that cannabidiol (CBD) is such an EV-modulator, we investigated whether CBD affects EV profile in GBM cells in the presence and absence of temozolomide (TMZ). Compared to controls, CBD-treated cells released EVs containing lower levels of pro-oncogenic miR21 and increased levels of anti-oncogenic miR126; these effects were greater than with TMZ alone. In addition, prohibitin (PHB), a multifunctional protein with mitochondrial protective properties and chemoresistant functions, was reduced in GBM cells following 1 h CBD treatment. This data suggests that CBD may, via modulation of EVs and PHB, act as an adjunct to enhance treatment efficacy in GBM, supporting evidence for efficacy of cannabinoids in GBM.
Glioblastoma multiforme (GBM) is the most common and aggressive form of primary malignant brain tumor in adults, with poor prognosis. Extracellular vesicles (EVs) are key-mediators for cellular ...communication through transfer of proteins and genetic material. Cancers, such as GBM, use EV release for drug-efflux, pro-oncogenic signaling, invasion and immunosuppression; thus the modulation of EV release and cargo is of considerable clinical relevance. As EV-inhibitors have been shown to increase sensitivity of cancer cells to chemotherapy, and we recently showed that cannabidiol (CBD) is such an EV-modulator, we investigated whether CBD affects EV profile in GBM cells in the presence and absence of temozolomide (TMZ). Compared to controls, CBD-treated cells released EVs containing lower levels of pro-oncogenic miR21 and increased levels of anti-oncogenic miR126; these effects were greater than with TMZ alone. In addition, prohibitin (PHB), a multifunctional protein with mitochondrial protective properties and chemoresistant functions, was reduced in GBM cells following 1 h CBD treatment. This data suggests that CBD may, via modulation of EVs and PHB, act as an adjunct to enhance treatment efficacy in GBM, supporting evidence for efficacy of cannabinoids in GBM.
PDF
