Local and temporal control of drug release has for long been a main focus in the development of novel drug carriers. Polymersomes, which can load both hydrophilic and hydrophobic species and, at the same time, be tailored to respond to a desired stimulus, have drawn much attention over the last decade. Here we describe polymersomes able to encapsulate up to 6% (w/w) of doxorubicin (DOX) together with 30% (w/w) of superparamagnetic iron oxide nanoparticles (USPIO; γ-Fe2O3). Upon internalization in HeLa cells and when a high frequency AC magnetic field (14mT at 750kHz) was applied, the developed delivery system elicited an 18% increase in cell toxicity, associated with augmented DOX release kinetics. In order to ensure that the observed cytotoxicity arose from the increased doxorubicin release and not from a pure magnetic hyperthermia effect, polymersomes loaded with magnetic nanoparticles alone were also tested. In this case, no increased toxicity was observed. We hypothesize that the magnetic field is inducing a very local hyperthermia effect at the level of the polymersome membrane, increasing drug release. This approach opens new perspectives in the development of smart delivery systems able to release drug upon demand and therefore, improving treatment control.

The plasma membrane of cells from rat submandibular glands was isolated and extensively sonicated. The homogenate was centrifuged at high speed in a discontinuous sucrose gradient. Light fractions contained vesicles analogous to rafts: they were rich in cholesterol, they contained GM1 and caveolin-1, and P2X 7 receptors were detected in these fractions. The location of the P2X 7 receptors in rafts was abolished when cellular cholesterol was removed by methyl-b-cyclodextrin (MCD). ATP activated neutral sphingomyelinase (N-SMase), which provoked a decrease of the cellular content of sphingomyelin and an increase of ceramide levels in these cells and in the rafts. Treatment with MCD and filipin (but not with a-cyclodextrin) abolished the increase of the intracellular concentration of calcium ([Ca 21 ] i ) in response to epinephrine but not to ATP. MCD and filipin also inhibited the activation by ATP of phospholipase A 2 (PLA 2 ). Inhibition of N-SMase with glutathione or GW4869 prevented the activation of PLA 2 by P2X 7 agonists without affecting [Ca 21 ] i levels. We conclude that P2X 7 receptors are present in both raft and nonraft compartments of plasma membranes; the receptors forming a nonselective cation channel are located in the nonraft fraction. P2X 7 receptors in the rafts are coupled to the activation of N-SMase, which increases the content of ceramides in rafts. This may contribute to the activation of PLA 2 in response to P2X 7 receptor occupancy.-Garcia-Marcos, M., E. Pérez-Andrés, S. Tandel, U. Fontanils, A. Kumps, E. Kabré, A. Gómez-Muñoz, A. Marino, J-P. Dehaye, and S. Pochet. Coupling of two pools of P2X 7 receptors to distinct intracellular signaling pathways in rat submandibular gland. J. Lipid Res. 2006. 47: 705-714.

Exogenous ATP stimulated phospholipase D (PLD), but not sphingomyelinase in rat submandibular gland (SMG) acini. PLD activation was dependent upon extracellular Ca2+ and did not involve intracellular Ca2+ mobilization or phosphoinositide-specific phospholipase C activation. ATP-stimulated PLD was attenuated by inhibition or downregulation of protein kinase C (PKC). PLD activation was fully blocked by the cytosolic phospholipase A2 (PLA2) inhibitor ONO-RS-082 and partially attenuated by the selective Ca2+-dependent cytosolic PLA2 inhibitor, arachidonyl trifluoromethylketone (AACOCF3), or by bromoenol lactone, an inhibitor of Ca2+-independent cytosolic PLA2. Magnesium, which decreases the concentration of ATP4−, and nickel, which blocks nonspecific cation channels coupled to purinergic receptors, inhibited PLD activation by ATP. Using reverse transcription-polymerase chain reaction and Northern blotting techniques, we demonstrated that the PLD isoform stimulated by ATP was PLD-2. Among various ATP analogs, only the P2Z/P2X7 purinergic receptor agonist benzoyl-benzoyl ATP stimulated PLD-2. The response to ATP was inhibited by the nonselective P2X purinergic antagonist suramin and by oxidized ATP, a potent P2Z/P2X7 receptor antagonist.It is concluded that in rat SMG acinar cells, PLD-2 is upregulated by exogenous ATP through a mechanism involving Ca2+ influx, cytosolic PLA2, and PKC. Also, the data suggest an involvement of P2X7 receptors in PLD-2 stimulation by ATP.

Encapsulating chemotherapy drugs in targeted nanodelivery systems is one of the most promising approaches to tackle cancer disease, avoiding side effects of common treatment. In the last decade, several nanocarriers with different nature have been tested, but polypeptide-based copolymers have attracted considerable attention for their biocompatibility, controlled and slow biodegradability as well as their low toxicity. In this work, we synthesized, characterized and evaluated poly(trimethylene carbonate)-bock-poly(L-glutamic acid) derived polymersomes, targeted to epidermal growth factor receptor (EGFR), loaded with plitidepsin and ultimately tested in HT29 and LS174T colorectal cancer cell lines for specificity and efficacy. Furthermore, morphology, physico-chemical properties and plitidepsin loading were carefully investigated. A thorough in vitro cytotoxicity analysis of the unloaded polymersomes was carried out for biocompatibility check, studying viability, cell membrane asymmetry and reactive oxygen species levels. Those cytotoxicity assays showed good biocompatibility for plitidepsin-unloaded polymersomes. Cellular uptake and cytotoxic effect of EGFR targeted and plitidepsin loaded polymersome indicated that colorectal cancer cell lines were.more sensitive to anti-EGFR-drug-loaded than untargeted drug-loaded polymersomes. Also, in both cell lines, the use of untargeted polymersomes greatly reduced plitidepsin cytotoxicity as well as the cellular uptake, indicating that the use of this targeted nanocarrier is a promising approach to tackle colorectal cancer disease and avoid the undesired effects of the usual treatment. Furthermore, in vivo assays support the in vitro conclusions that EGFR targeted polymersomes could be a good drug delivery system. This work provides a proof of concept for the use of encapsulated targeted drugs as future therapeutic treatments for cancer.

29Hypoxia Inducible Factor (HIF) is the master transcriptional regulator that orchestrates cellular 30 adaptation to low oxygen. HIF is tightly regulated via the stability of its α-subunit, which is 31 subjected to oxygen-dependent proline hydroxylation by Prolyl-Hydroxylase Domain 32 containing proteins (PHDs/EGLNs), and ultimately targeted for proteasomal degradation 33 through poly-ubiquitination by von-Hippel-Lindau protein (pVHL). However, sustained HIF-α 34 signalling is found in many tumours independently of oxygen availability pointing towards the 35 relevance of non-canonical HIF-α regulators. In this study, we establish the Ubiquitin Specific 36Protease 29 (USP29) as direct post-translational activator of HIF-α in a variety of cancer cell 37 lines. USP29 binds to HIF-α, decreases poly-ubiquitination and thus protects HIF-α from 38 proteasomal degradation. Deubiquitinating activity of USP29 is essential to stabilise not only 39 HIF-1α but also HIF-2α, via their C-termini in an oxygen/PHD/pVHL-independent manner. 40 Furthermore, in prostate cancer samples the expression of USP29 correlates with the HIF-41 target gene CA9 (carbonic anhydrase 9) as well as disease progression and severity. 42 43 44 45 46 47 48 49 50 51 52 53 54