Gabriel Charest

Ectodomain shedding of furin: kinetics and role of the cysteine-rich region

Furin, a member of the subtilisin‐like pro‐protein convertase family, is a type I membrane protein that undergoes ectodomain shedding. Metabolic labeling of cells stably expressing furin demonstrated that the shed form of furin is detected after 30 min. Moreover, sequence analysis revealed that specific residues of the cysteine‐rich region of furin aligned with those of tumor necrosis factor receptor, which is also shed. Introduction within furins cysteine‐rich region of mutations that impair TNFR1 shedding also abolished furin shedding. Our results show that shedding of furin occurs rapidly and further suggest that specific cysteine residues may impart a conformation to the enzyme, thereby affecting its susceptibility to proteolysis.

Glioblastoma Treatment: Bypassing the Toxicity of Platinum Compounds by Using Liposomal Formulation and Increasing Treatment Efficiency With Concomitant Radiotherapy

PURPOSE Treatments of glioblastoma with cisplatin or oxaliplatin only marginally improve the overall survival of patients and cause important side effects. To prevent adverse effects, improve delivery, and optimize the tumor response to treatment in combination with radiotherapy, a potential approach consists of incorporating the platinum agent in a liposome. METHODS AND MATERIALS In this study, cisplatin, oxaliplatin, carboplatin, Lipoplatin (the liposomal formulation of cisplatin), and Lipoxal (the liposomal formulation of oxaliplatin) were tested on F98 glioma orthotopically implanted in Fischer rats. The platinum compounds were administered by intracarotid infusion and were assessed for the ability to reduce toxicity, improve cancer cell uptake, and increase survival of animals when combined or not combined with radiotherapy. RESULTS The tumor uptake was 2.4-fold more important for Lipoxal than the liposome-free oxaliplatin. Lipoxal also improved the specificity of oxaliplatin as shown by a higher ratio of tumor to right hemisphere uptake. Surprisingly, Lipoplatin led to lower tumor uptake compared with cisplatin. However, Lipoplatin had the advantage of largely reducing the toxicity of cisplatin and allowed us to capitalize on the anticancer activity of this agent. CONCLUSION Among the five platinum compounds tested, carboplatin showed the best increase in survival when combined with radiation for treatment of glioma implanted in Fischer rats.

Identification of furin pro-region determinants involved in folding and activation.

The pro-region of the subtilisin-like convertase furin acts early in the biosynthetic pathway as an intramolecular chaperone to enable proper folding of the zymogen, and later on as an inhibitor to constrain the activity of the enzyme until it reaches the trans -Golgi network. To identify residues that are important for pro-region function, we initially identified amino acids that are conserved among the pro-regions of various mammalian convertases. Site-directed mutagenesis of 17 selected amino acids within the 89-residue pro-region and biosynthetic labelling revealed that I60A-furin and H66A-furin were rapidly degraded in a proteasome-dependent manner, while W34A-furin and F67A-furin did not show any autocatalytic activation. Intriguingly, the latter mutants proteolytically cleaved pro-von Willebrand factor precursor to the mature polypeptide, suggesting that the mutations permitted proper folding, but did not allow the pro-region to exercise its role in inhibiting the enzyme. Homology modelling of furins pro-region revealed that residues Ile-60 and His-66 might be crucial in forming the binding interface with the catalytic domain, while residues Trp-34 and Phe-67 might be involved in maintaining a hydrophobic core within the pro-region itself. These results provide structural insights into the dual role of furins pro-region.

Cyclooxygenase-2 inhibitor prevents radiation-enhanced infiltration of F98 glioma cells in brain of Fischer rat

Abstract Purpose: Radiation induces a neuro-inflammation that is characterized by the expression of genes known to increase the invasion of cancer cells. In Fischer rats, brain irradiation increases the infiltration of cancer cells and reduced the median survival of the animals. In this study, we have determined whether these adverse effects of radiation can be prevented with the cyclooxygenase-2 (COX-2) inhibitor meloxicam. Materials and methods: Brain of Fischer rats treated or not with meloxicam were irradiated (15 Gy) and then implanted with the F98 glioma cells. The median survival of the animals, the infiltration of F98 cells, and the expression of inflammatory cytokines and pro-migration molecules were measured. Results: Meloxicam reduced by 75% the production of prostaglandin E2 (bioproduct of COX-2) in irradiated brains validating its anti-inflammatory effect. Median survival was increased to control levels by the treatment of meloxicam following brain irradiation. This protective effect was associated with a reduction of the infiltration of F98 cells in the brain, a complete inhibition of radiation-enhancement of matrix metalloproteinase-2, and a significant reduction of tumor necrosis factor α (TNF-α) and tumor growth factor β1 (TGF-β1) expression. Using invasion chambers, interleukin-1β (IL-1β) stimulated by 5-fold the invasiveness of F98 cells, but this stimulation was completely inhibited by meloxicam. This suggests that a cooperation between IL-1β and COX-2 are involved in radiation-enhancement of F98 cell invasion. Conclusions: Our results indicate the importance of reducing the inflammatory response of normal brain tissue following irradiation in an effort to extend median survival in F98 tumor-bearing rats.

Polymer gel in rat skull to assess the accuracy of a new rat stereotactic device for use with the Gamma Knife.

PurposeAccurate targeting is crucial for the irradiation of a small-volume animal brain. We propose an original method, based on a polymer gel, to determine the accuracy and reproducibility of irradiation using a new stereotactic frame.MethodsAn in-house designed rat stereotactic frame, compatible with the Gamma Knife, was constructed. The rat brain was then removed through a small burr hole and the intracranial cavity was washed and filled with a polymer gel. This “gel brain” was irradiated by Gamma Knife and the irradiated volumes and coordinates were measured after the irradiation.ResultsThe position of the polymerized areas revealed that the stereotactic frame was able to accurately reproduce the same position of irradiation in each animal. The small standard deviation demonstrated the high reproducibility.ConclusionThe polymer gel confirmed the ability of the rat stereotactic frame to accurately and reproducibly position a small animal for precise radiosurgery procedures.

Applications of Gamma Knife Radiosurgery for Experimental Investigations in Small Animal Models

The Gamma Knife (GK) was not originally designed for experimentations in small animals. In fact, there are no compatible custom accessories or stereotactic frames on the market for the spatial positioning of small animals in the GK. In addition, the GK is intensively used for patient treatments, and consequently the access for research with small animals is limited. On the other hand, devices specially designed for the irradiation of small animals are available on the market. For examples, small animal irradiators can be purchasable from Best Theratronics (Theratronics, 2011), Rad Source Technologies (Rad Source Technologies, 2011), Precision X-Ray Inc. (Precision X-Ray, 2011) and Xstrahl-Gulmay Medical Inc. (Xstrahl, 2011). Compared to the GK, these small animal irradiators have the following advantages: lower cost, smaller size, some are shielded and thus don’t require a special shielded room, some can be combined with an imaging device that allows to image the animal and immediately irradiate the region of interest and, if needed, repeat imaging. Then, since irradiators designed for small animal already exist, why use a GK for animal experimentations? The answer should include technical as well as conceptual aspects. The most important benefit of using the GK for small animals is related to the difference between conventional external radiotherapy and GK radiosurgery (GKRS). Radiation deposition with a GK is produced by multiple concentric beams that allow high dose deposition in a very small volume. These converging beams of ionizing radiation in a precise volume allow a rapid fall-off of dose near the edges which limit adverse effects on the surrounding adjacent tissue.

Infiltration of F98 glioma cells in Fischer rat brain is temporary stimulated by radiation

Abstract Purpose: Irradiation of brain stimulates the expression of inflammatory mediators, some of which can modify the ability of cancer cells to infiltrate the brain. In the present study, the time window during which this stimulation occurs was determined. Materials and methods: Brain of Fischer rat was irradiated (15 Gy) and expression of pro-inflammatory mediators IL-1β, IL-6 and TNF-α was measured from 4 h to 20 days post-irradiation. Level of the matrix metalloproteinase 2 (MMP-2) and prostaglandin E2 (PGE2) which can favor cancer cell infiltration were also measured. The F98 glioma cells were implanted either during (4 h post-irradiation) or after (10 days post-irradiation) the pro-inflammatory phase. Infiltration distance of F98 cells in brain parenchyma and the median survival time of the animals were determined. Results: Expression of IL-1β, IL-6 and TNF-α was significantly increased in the irradiated brains with a peak at 4 h post-irradiation. Implantation of F98 glioma cells 4 h post-irradiation reduced the median survival time of Fischer rats to 18 days, compared to 25 days when the F98 were implanted in non-irradiated brain. Irradiation of the brain increased the distance of infiltration of F98 cells and was associated with increased levels of MMP-2 and PGE2. Conversely, F98 cells implanted 10 days post-irradiation have infiltrated the brain over a shorter distance and the median survival time of rats was increased to 35 days. Conclusions: Cancer recurrence is frequently observed in GBM patients. A better understanding of the inflammatory response observed in irradiated brain could contribute to develop new therapeutic modalities to further increase the efficiency of radiotherapy.

Liposomal formulations of carboplatin injected by convection-enhanced delivery increases the median survival time of F98 glioma bearing rats

BackgroundEffectiveness of chemotherapy for treating glioblastoma (GBM) brain tumors is hampered by the blood–brain barrier which limits the entry into the brain of most drugs from the blood. To bypass this barrier, convection-enhanced delivery (CED) was proposed to directly inject drugs in tumor. However, the benefit of CED may be hampered when drugs diffuse outside the tumor to then induce neurotoxicity. Encapsulation of drugs into liposome aims at increasing tumor cells specificity and reduces neurotoxicity. However, the most appropriate liposomal formulation to inject drugs into brain tumor by CED still remains to be determined. In this study, four liposomal carboplatin formulations were prepared and tested in vitro on F98 glioma cells and in Fischer rats carrying F98 tumor implanted in the brain. Impact of pegylation on liposomal surface and relevance of positive or negative charge were assessed.ResultsThe cationic non-pegylated (L1) and pegylated (L2) liposomes greatly improved the toxicity of carboplatin in vitro compared to free carboplatin, whereas only a modest improvement and even a reduction of efficiency were measured with the anionic non-pegylated (L3) and the pegylated (L4) liposomes. Conversely, only the L4 liposome significantly increased the median survival time of Fisher rats implanted with the F98 tumor, compared to free carboplatin. Neurotoxicity assays performed with the empty L4′ liposome showed that the lipid components of L4 were not toxic. These results suggest that the positive charge on liposomes L1 and L2, which is known to promote binding to cell membrane, facilitates carboplatin accumulation in cancer cells explaining their higher efficacy in vitro. Conversely, negatively charged and pegylated liposome (L4) seems to diffuse over a larger distance in the tumor, and consequently significantly increased the median survival time of the animals.ConclusionsSelection of the best liposomal formulation based on in vitro studies or animal model can result in contradictory conclusions. The negatively charged and pegylated liposome (L4) which was the less efficient formulation in vitro showed the best therapeutic effect in animal model of GBM. These results support that relevant animal model of GBM must be considered to determine the optimal physicochemical properties of liposomal formulations.

Tumor Cell Invasion Induced by Radiation in Balb/C Mouse is Prevented by the Cox-2 Inhibitor NS-398

Radiation stimulates the expression of inflammatory mediators known to increase cancer cell invasion. Therefore, it is important to determine whether anti-inflammatory drugs can prevent this adverse effect of radiation. Since cyclooxygenase-2 (COX-2) is a central player in the inflammatory response, we performed studies to determine whether the COX-2 inhibitor NS-398 can reduce the radiation enhancement of cancer cell invasion. Thighs of Balb/c mice treated with NS-398 were irradiated with either daily fractions of 7.5 Gy for five consecutive days or a single 30 Gy dose prior to subcutaneous injection of nonirradiated MC7-L1 mammary cancer cells. Five weeks later, tumor invasion, blood vessel permeability and interstitial volumes were assessed using magnetic resonance imaging (MRI). Matrix metalloproteinase-2 (MMP-2) was measured in tissues by zymography at 21 days postirradiation. Cancer cell invasion in the mouse thighs was increased by 12-fold after fractionated irradiations (5 × 7.5 Gy) and by 17-fold after a single 30 Gy dose of radiation. This stimulation of cancer cell invasion was accompanied by a significant increase in the interstitial volume and a higher level of the protease MMP-2. NS-398 treatment largely prevented the stimulation of cancer cell invasion, which was associated with a reduction in interstitial volume in the irradiated thighs and a complete suppression of MMP-2 stimulation. In conclusion, this animal model using MC7-L1 cells demonstrates that radiation-induced cancer cell invasion can be largely prevented with the COX-2 inhibitor NS-398.