Inhibition of extracellular vesicle release enhances cancer chemotherapy and inhibition of outer membrane vesicle release sensitises bacteria to antibiotic and bacteriophage therapy

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