IntroductionProtein deimination, defined as the post-translational conversion of protein-bound arginine to citrulline, is carried out by a family of 5 calcium-dependent enzymes, the peptidylarginine deiminases (PADs) and has been linked to various cancers. Cellular microvesicle (MV) release, which is involved in cancer progression, and deimination have not been associated before. We hypothesize that elevated PAD expression, observed in cancers, causes increased MV release in cancer cells and contributes to cancer progression.BackgroundWe have previously reported that inhibition of MV release sensitizes cancer cells to chemotherapeutic drugs. PAD2 and PAD4, the isozymes expressed in patients with malignant tumours, can be inhibited with the pan-PAD-inhibitor chloramidine (Cl-am). We sought to investigate whether Cl-am can inhibit MV release and whether this pathway could be utilized to further increase the sensitivity of cancer cells to drug-directed treatment.MethodsProstate cancer cells (PC3) were induced to release high levels of MVs upon BzATP stimulation of P2X7 receptors. Western blotting with the pan-protein deimination antibody F95 was used to detect a range of deiminated proteins in cells stimulated to microvesiculate. Changes in deiminated proteins during microvesiculation were revealed by immunoprecipitation and immunoblotting, and mass spectrometry identified deiminated target proteins with putative roles in microvesiculation.ConclusionWe report for the first time a novel function of PADs in the biogenesis of MVs in cancer cells. Our results reveal that during the stimulation of prostate cancer cells (PC3) to microvesiculate, PAD2 and PAD4 expression levels and the deimination of cytoskeletal actin are increased. Pharmacological inhibition of PAD enzyme activity using Cl-am significantly reduced MV release and abrogated the deimination of cytoskeletal actin. We demonstrated that combined Cl-am and methotrexate (MTX) treatment of prostate cancer cells increased the cytotoxic effect of MTX synergistically. Refined PAD inhibitors may form part of a novel combination therapy in cancer treatment.

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Microvesicles shed from cells carry constituents of the cell cytoplasm, including, of importance in multidrug resistance to cancer chemotherapy, drugs that the tumor cell attempts to efflux. To see whether such drugs could be used at lower concentrations with the same efficacy, it was first shown that microvesiculation of prostate cancer (PCa) cells, PC3, could be inhibited pharmacologically with calpeptin (calpain inhibitor) and by siRNA (CAPNS1). In cells treated with docetaxel (DTX), this inhibition resulted in a third-fold increase in intracellular concentrations of DTX. As a result, 20-fold lower concentrations of DTX (5 nM) could be used, in the presence of calpeptin (20 μM) inducing the same degree of apoptosis after 48 h in PC3 cells, as 100 nM of DTX alone. Inhibition of microvesiculation similarly improved combination chemotherapy (DTX and methotrexate). In a mouse xenograft model of PCa, DTX (0.1 mg/kg) together with calpeptin (10 mg/kg), administered i.p., significantly reduced tumor volumes compared to DTX alone (0.1 mg/kg) and brought about the same reductions in tumor growth as 10 mg/kg of DTX alone. As well as further reducing vascularization, it also increased apoptosis and reduced proliferation of PC3 cells in tumor xenografts.

Microvesicles (or MVs) are plasma membrane-derived vesicles released from most eukaryotic cells constitutively during early apoptosis or at higher levels after chemical or physical stress conditions. This review looks at some of the functions of MVs in terms of intercellular communication and ensuant signal transduction, including the transport of proteins (unconventional protein export) as well as of mRNA and microRNA. MVs also have roles in membrane repair, the removal of misfolded proteins, and in the control of apoptosis. We also discuss the role MVs have been shown to have in invasive growth and metastasis as well as in hypoxia in tumours and cerebral ischaemia. The association of MVs in infectious and autoimmune disease is also summarised together with their possible use as therapeutic agents.

Microvesicles are shed constitutively, or upon activation, from both normal and malignant cells. The process is dependent on an increase in cytosolic Ca2+, which activates different enzymes, resulting in depolymerization of the actin cytoskeleton and release of the vesicles. Drug resistance can be defined as the ability of cancer cells to survive exposure to a wide range of anti-cancer drugs, and anti-tumour chemotherapeutic treatments are often impaired by innate or acquired MDR (multidrug resistance). Microvesicles released upon chemotherapeutic agents prevent the drugs from reaching their targets and also mediate intercellular transport of MDR proteins.