Investigation of squalene-doxorubicin distribution and interactions within single cancer cell using Raman microspectroscopy.

Intracellular distribution of doxorubicin (DOX) and its squalenoylated (SQ-DOX) nanoparticles (NPs) form in murine lung carcinoma M109 and human breast carcinoma MDA-MB-231 cells was investigated by Raman microspectroscopy. Pharmacological data showed that DOX induced higher cytotoxic effect than SQ-DOX NPs. Raman data were obtained using single-point measurements and imaging on the whole cell areas. These data showed that after DOX treatment at 1 µM, the spectral features of DOX were not detected in the M109 cell cytoplasm and nucleus. However, the intracellular distribution of SQ-DOX NPs was higher than DOX in the same conditions. In addition, SQ-DOX NPs were localized into both cell cytoplasm and nucleus. After 5 µM treatment, Raman bands of DOX at 1211 and 1241 cm-1 were detected in the nucleus. Moreover, the intensity ratio of these bands decreased, indicating DOX intercalation into DNA. However, after treatment with SQ-DOX NPs, the intensity of these Raman bands increased. Interestingly, with SQ-DOX NPs, the intensity of 1210/1241 cm-1 ratio was higher suggesting a lower fraction of intercalated DOX in DNA and higher amount of non-hydrolyzed SQ-DOX. Raman imaging data confirm this subcellular localization of these drugs in both M109 and MDA-MB-231 cells. These finding brings new insights to the cellular characterization of anticancer drugs at the molecular level, particularly in the field of nanomedicine.

Structural comparison of AP endonucleases from the exonuclease III family reveals new amino acid residues in human AP endonuclease 1 that are involved in incision of damaged DNA.

Oxidatively damaged DNA bases are substrates for two overlapping repair pathways: DNA glycosylase-initiated base excision repair (BER) and apurinic/apyrimidinic (AP) endonuclease-initiated nucleotide incision repair (NIR). In the BER pathway, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases, whereas in the NIR pathway, the same AP endonuclease incises DNA 5' to an oxidized base. The majority of characterized AP endonucleases possess classic BER activities, and approximately a half of them can also have a NIR activity. At present, the molecular mechanism underlying DNA substrate specificity of AP endonucleases remains unclear mainly due to the absence of a published structure of the enzyme in complex with a damaged base. To identify critical residues involved in the NIR function, we performed biochemical and structural characterization of Bacillus subtilis AP endonuclease ExoA and compared its crystal structure with the structures of other AP endonucleases: Escherichia coli exonuclease III (Xth), human APE1, and archaeal Mth212. We found conserved amino acid residues in the NIR-specific enzymes APE1, Mth212, and ExoA. Four of these positions were studied by means of point mutations in APE1: we applied substitution with the corresponding residue found in NIR-deficient E. coli Xth (Y128H, N174Q, G231S, and T268D). The APE1-T268D mutant showed a drastically decreased NIR activity and an inverted Mg(2+) dependence of the AP site cleavage activity, which is in line with the presence of an aspartic residue at the equivalent position among other known NIR-deficient AP endonucleases. Taken together, these data show that NIR is an evolutionarily conserved function in the Xth family of AP endonucleases.

Efficient "green" encapsulation of a highly hydrophilic anticancer drug in metal-organic framework nanoparticles.

Metal-organic frameworks (MOFs) are coordination polymers of interest for biomedical applications. Of particular importance, nanoparticles made of iron(III) trimesate (MIL-100, MIL standing for Material Institut Lavoisier) (nanoMOFs) can be conveniently synthesised under mild and green conditions. They were shown to be biodegradable, biocompatible and efficient to encapsulate a variety of active molecules. We have addressed here the challenges to encapsulate a highly hydrophilic anticancer prodrug, phosphated gemcitabin (Gem-MP) known for its instability and inability to bypass cell membranes. MIL-100 nanoMOFs acted as efficient "nanosponges", soaking Gem-MP from its aqueous solution with almost perfect efficiency (>98%). Maximal loadings reached ∼30 wt% reflecting the strong interaction between the drug and the iron trimesate matrices. Neither degradation nor loss of crystalline structure was observed after the loading process. Storage of the loaded nanoMOFs in water did not result in drug release over three days. However, Gem-MP was released in media containing phosphates, as a consequence to particle degradation. Drug-loaded nanoMOFs were effective against pancreatic PANC-1 cells, in contrast to free drug and empty nanoMOFs. However, an efflux phenomenon could contribute to reduce the efficacy of the nanocarriers. Size optimization and surface modification of the nanoMOFs are expected to further improve these findings.

Lipid-Conjugation of Endogenous Neuropeptides: Improved Biotherapy against Human Pancreatic Cancer.

Neuropeptides are small neuronal signaling molecules that act as neuromodulators for a variety of neural functions including analgesia, reproduction, social behavior, learning, and memory. One of the endogenous neuropeptides-Met-Enkephalin (Met-Enk), has been shown to display an inhibitory effect on cell proliferation and differentiation. Here, a novel lipid-modification approach is shown to create a small library of neuropeptides that will allow increased bioavailability and plasma stability after systemic administration. It is demonstrated, on an experimental model of human pancreatic adenocarcinoma, that lipid conjugation of Met-Enk enhances its tumor suppression efficacy compared to its nonlipidated counterparts, both in vitro and in vivo. More strikingly, the in vivo studies show that a combination therapy with a reduced concentration of Gemcitabine has suppressed the tumor growth considerably even three weeks after the last treatment.

Gemcitabine-based therapy for pancreatic cancer using the squalenoyl nucleoside monophosphate nanoassemblies.

Gemcitabine is currently the most effective agent against advanced pancreatic cancer. However, the major therapeutic hurdles using gemcitabine include rapid inactivation by blood deaminases and fast development of cell chemoresistance, induced by down-regulation of deoxycytidine kinase or nucleoside transporters. To overcome the above drawbacks we designed recently a novel nanomedicine strategy based on squalenoyl prodrug of 5'-monophosphate gemcitabine (SQdFdC-MP). This amphiphilic conjugate self-organized in water into unilamellar vesicles with a mean diameter of 100 nm. In this study the antitumor efficacy of SQdFdC-MP nanoassemblies (NAs) on chemoresistant and chemosensitive pancreatic adenocarcinoma models have been investigated. Cell viability assays showed that SQdFdC-MP NAs displayed higher antiproliferative and cytotoxic effects, particularly in chemoresistant pancreatic tumor cells. In in vivo studies, SQdFdC-MP NAs decreased significantly the growth (∼70%) of human MiaPaCa2 xenografts, also preventing tumor cell invasion, whereas native dFdC did not display any anticancer activity when tumor growth inhibition was only 35% with SQdFdC NAs. These results correlated with a reduction of Ki-67 antigen and the induction of apoptosis mediated by caspase-3 activation in tumor cells. These findings demonstrated the feasibility of utilizing SQdFdC-MP NAs to make tumor cells more sensitive to gemcitabine and thus providing an efficient new therapeutic alternative for pancreatic adenocarcinoma.

Therapeutic modalities of squalenoyl nanocomposites in colon cancer: an ongoing search for improved efficacy.

Drug delivery of combined cytotoxic and antivascular chemotherapies in multidrug nanoassemblies may represent an attractive way to improve the treatment of experimental cancers. Here we made the proof of concept of this approach on the experimental LS174-T human colon carcinoma xenograft nude mice model. Briefly, we have nanoprecipitated the anticancer compound gemcitabine conjugated with squalene (SQ-gem) together with isocombretastatin A-4 (isoCA-4), a new isomer of the antivascular combretastatin A-4 (CA-4). It was found that these molecules spontaneously self-assembled as stable nanoparticles (SQ-gem/isoCA-4 NAs) of ca. 142 nm in a surfactant-free aqueous solution. Cell culture viability tests and apoptosis assays showed that SQ-gem/isoCA-4 NAs displayed comparable antiproliferative and cytotoxic effects than those of the native gemcitabine or the mixtures of free gemcitabine with isoCA-4. Surprisingly, it was observed by confocal microscopy that the nanocomposites made of SQ-gem/isoCA-4 distributed intracellularly as intact nanoparticles whereas the SQ-gem nanoparticles remained localized onto the cell membrane. When used to deliver these combined chemotherapeutics to human colon cancer model, SQ-gem/isoCA-4 nanocomposites induced complete tumor regression (by 93%) and were found superior to all the other treatments, whereas the overall tolerance was better than the free drug treatments. This approach could be applied to other pairs of squalenoylated nanoassemblies with other non-water-soluble drugs, thus broadening the application of the "squalenoylation" concept in oncology.

A unique squalenoylated and nonpegylated doxorubicin nanomedicine with systemic long-circulating properties and anticancer activity.

We identified that the chemical linkage of the anticancer drug doxorubicin onto squalene, a natural lipid precursor of the cholesterol's biosynthesis, led to the formation of squalenoyl doxorubicin (SQ-Dox) nanoassemblies of 130-nm mean diameter, with an original "loop-train" structure. This unique nanomedicine demonstrates: (i) high drug payload, (ii) decreased toxicity of the coupled anticancer compound, (iii) improved therapeutic response, (iv) use of biocompatible transporter material, and (v) ease of preparation, all criteria that are not combined in the currently available nanodrugs. Cell culture viability tests and apoptosis assays showed that SQ-Dox nanoassemblies displayed comparable antiproliferative and cytotoxic effects than the native doxorubicin because of the high activity of apoptotic mediators, such as caspase-3 and poly(ADP-ribose) polymerase. In vivo experiments have shown that the SQ-Dox nanomedicine dramatically improved the anticancer efficacy, compared with free doxorubicin. Particularly, the M109 lung tumors that did not respond to doxorubicin treatment were found inhibited by 90% when treated with SQ-Dox nanoassemblies. SQ-Dox nanoassembly-treated MiaPaCa-2 pancreatic tumor xenografts in mice decreased by 95% compared with the tumors in the saline-treated mice, which was significantly higher than the 29% reduction achieved by native doxorubicin. Concerning toxicity, SQ-Dox nanoassemblies showed a fivefold higher maximum-tolerated dose than the free drug, and moreover, the cardiotoxicity study has evidenced that SQ-Dox nanoassemblies did not cause any myocardial lesions, such as those induced by the free doxorubicin treatment. Taken together, these findings demonstrate that SQ-Dox nanoassemblies make tumor cells more sensitive to doxorubicin and reduce the cardiac toxicity, thus providing a remarkable improvement in the drug's therapeutic index.

Multifunctional squalene-based prodrug nanoparticles for targeted cancer therapy.

Fluorescent and biotinylated squalene-gemcitabine prodrug nanoparticles exhibiting high drug payloads have been prepared and successfully used to target different cancer cell lines, resulting in increased cell uptake and improved anticancer efficiency, which represents the first targeted system derived from the squalenoylation approach.

Effects of silencing the RET/PTC1 oncogene in papillary thyroid carcinoma by siRNA-squalene nanoparticles with and without fusogenic companion GALA-cholesterol.

BACKGROUND: RET/PTC1 is the most prevalent type of gene rearrangement found in papillary thyroid carcinoma (PTC). Previously, we introduced a new noncationic nanosystem for targeted RET/PTC1 silencing by efficient delivery of small interfering RNA (siRNA) using the "squalenoylation" approach. With the aim of improving these results further, we designed new squalenoyl nanostructures consisting of the fusogenic peptide GALA-cholesterol (GALA-Chol) and squalene (SQ) nanoparticles (NPs) of siRNA RET/PTC1. METHODS: The siRNA RET/PTC1-SQ bioconjugate was synthesized. The corresponding NPs were prepared with or without GALA-Chol by nanoprecipitation and then characterized for their size and zeta potential. The effects of NPs on BHP 10-3 SCmice and TPC-1 cell viability (MTT assay), gene and protein silencing (reverse transcription-quantitative polymerase chain reaction [rt-qPCR], Western blot), and cellular uptake (fluorescent microscopy) were studied. In vivo gene silencing efficiency of siRNA RET/PTC1-SQ NPs was assessed by administration in nude mice via either intratumoral (i.t.) or intravenous (i.v.) routes. Tumor growth was followed for 19 days. Tumors were then collected, and RET/PTC1 gene and protein inhibitions were assessed by RT-qPCR and Western blot. RESULTS: The combination of siRNA RET/PTC1-SQ bioconjugate and GALA-Chol leads to stable NPs of ∼200 nm diameter. In vitro, the results revealed that combining GALA-Chol with siRNA RET/PTC1-SQ NPs decreased cell viability, enhanced cellular internalization, and induced gene silencing efficiency in both human PTC (BHP 10-3 SCmice and TPC-1) cell lines. On the contrary, in vivo, the siRNA RET/PTC1-SQ GALA-Chol NPs were not found to be efficient either in gene silencing or in tumor growth inhibition, compared to siRNA RET/PTC1-SQ NPs both via i.t. and i.v. routes (p<0.001). CONCLUSIONS: Conversely to siRNA RET/PTC1-SQ NPs, the siRNA RET/PTC1-SQ GALA-Chol NPs are efficient in vitro but not in vivo. Finally, NPs of siRNA RET/PTC1-SQ were found to be efficient silencers of the RET/PTC1 fusion oncogene in in vivo applications even at a concentration lower than used in a previously published study.

Novel isoprenoyl nanoassembled prodrug for paclitaxel delivery.

A new paclitaxel (Ptx) prodrug was designed by coupling a single terpene unit (MIP) to the hydroxyl group in position 2' of the drug molecule. Using a squalene derivative of polyethylene glycol (SQ-PEG) as surface active agent, the resulting bioconjugate (PtxMIP) self-assembled in water leading to the formation of stable nanoparticles (PtxMIP_SQ-PEG NPs) with an impressively high drug loading (82%). In vivo, the anticancer activity of this novel Ptx nanoassembled prodrug was compared to the conventional Cremophor-containing formulation (Taxol) on a murine model of breast cancer lung metastasis induced by intravenous injection of 4T1 tumor cells, genetically modified to stably express firefly luciferase. Cell growth was assessed noninvasively by bioluminescence imaging (BLI) which enabled monitoring tumor metastatic burden in the same animals. PtxMIP_SQ-PEG nanoparticles slowed metastatic spread and were better tolerated than the Cremophor-containing formulation (i.e., free drug), thus demonstrating the potential of terpene-based nanoassembled prodrugs in the improvement of the therapeutic index of Ptx in balb/c mice.

Polymer prodrug nanoparticles based on naturally occurring isoprenoid for anticancer therapy.

The synthesis of a novel class of polymer prodrug nanoparticles with anticancer activity is reported by using squalene, a naturally occurring isoprenoid, as a building block by the reversible addition-fragmentation (RAFT) technique. The RAFT agent was functionalized by gemcitabine (Gem) as anticancer drug, and the polymerization of squalenyl-methacrylate (SqMA) led to well-defined macromolecular prodrugs comprising one Gem at the extremity of each polymer chain. The amphiphilic nature of the resulting Gem-PSqMA conjugates allowed them to self-assemble into long-term stable and narrowly dispersed nanoparticles with significant anticancer activity in vitro on various cancer cell lines. To confer stealth properties on these nanoparticles, their PEGylation was successfully performed, as confirmed by X-ray photoelectron spectroscopy (XPS) and complement activation assay. It was also shown that the PEGylated nanoparticles could be internalized in cancer cells to a greater extent than their non-PEGylated counterparts.

Polyisoprenoyl gemcitabine conjugates self assemble as nanoparticles, useful for cancer therapy.

A series of new polyisoprenoyl prodrugs of gemcitabine, which can be formulated as nanoassemblies are described. These prodrugs were designed to improve gemcitabine efficacy and to overcome the limitations due to the systemic toxicity of this anticancer compound. In vitro biological assessment showed that these polyisoprenoyl gemcitabine nanoassemblies displayed notable cytotoxicity on several cancer cell lines, including murine melanoma cell line B16F10, human pancreatic carcinoma cell line MiaPaCa-2, human lung carcinoma cell line A549 and human breast adenocarcinoma cell line MCF7. Interestingly, it was observed that the anticancer efficacy of these nanoassemblies was dependant on the size of polyisoprenoyl moiety. The polyisoprenoyl prodrug of gemcitabine containing three isoprene units (2d) was the more active on all the cancer cell lines tested. The antitumor efficacy of the nanoassemblies (NAs) constructed with the most active prodrug 2d was further evaluated on a human pancreatic (MiaPaCa-2) carcinoma xenograft model in mice. The prodrug 2d NAs showed an increased antitumor efficacy as compared to free gemcitabine or to squalene-gemcitabine (SQ-gem, 2a) nanoassemblies. Interestingly, MiaPaCa-2 tumors that did not respond to gemcitabine were inhibited by 76% after treatment with prodrug 2d NAs, whereas SQ-gem-treated MiaPaCa-2 tumor xenografts decreased only by 41% compared to saline or to gemcitabine-treated mice. Together, these findings demonstrated that the modulation of the length of nanoassemblies polyisoprenoyl moiety made tumor cells more sensitive to gemcitabine treatment without flagrant toxicity, thus providing a significant improvement in the drug therapeutic index.

Improving the antitumor activity of squalenoyl-paclitaxel conjugate nanoassemblies by manipulating the linker between paclitaxel and squalene.

A series of new lipid prodrugs of paclitaxel, which can be formulated as nanoassemblies, are described. These prodrugs which are designed to overcome the limitations due to the systemic toxicity and low water solubility of paclitaxel consist of a squalene chain bound to the 2'-OH of paclitaxel through a 1,4-cis,cis-dienic linker. This design allows the squalene-conjugates to self-assemble as nanoparticular systems while preserving an efficient release of the free drug, thanks to the dienic spacer. The size, steric hindrance, and functional groups of the spacer have been modulated. All these prodrugs self-assemble into nanosized aggregates in aqueous solution as characterized by dynamic light scattering and transmission electron microscopy and appear stable in water for several days as determined by particle size measurement. In vitro biological assessment shows that these squalenoyl-paclitaxel nanoparticles display notable cytotoxicity on several tumor cell lines including A549 lung cell line, colon cell line HT-29, or KB 3.1 nasopharyngeal epidermoid cell line. The cis,cis-squalenyl-deca-5,8-dienoate prodrug show improved activity over simple 2'-squalenoyl-paclitaxel prodrug highlighting the favourable effect of the dienic linker. The antitumor efficacy of the nanoassemblies constructed with the more active prodrugs has been investigated on human lung (A549) carcinoma xenograft model in mice. The prodrug bearing the cis,cis-deca-5,8-dienoyl linker shows comparable antitumor efficacy to the parent drug, but reveals a much lower subacute toxicity as seen in body weight loss. Thus, nanoparticles with the incorporated squalenoyl paclitaxel prodrug may prove useful for replacement of the toxic Cremophor EL.

Versatile and efficient targeting using a single nanoparticulate platform: application to cancer and Alzheimer's disease.

A versatile and efficient functionalization strategy for polymeric nanoparticles (NPs) has been reported and successfully applied to PEGylated, biodegradable poly(alkyl cyanoacrylate) (PACA) nanocarriers. The relevance of this platform was demonstrated in both the fields of cancer and Alzheimer's disease (AD). Prepared by copper-catalyzed azide-alkyne cycloaddition (CuAAC) and subsequent self-assembly in aqueous solution of amphiphilic copolymers, the resulting functionalized polymeric NPs exhibited requisite characteristics for drug delivery purposes: (i) a biodegradable core made of poly(alkyl cyanoacrylate), (ii) a hydrophilic poly(ethylene glycol) (PEG) outer shell leading to colloidal stabilization, (iii) fluorescent properties provided by the covalent linkage of a rhodamine B-based dye to the polymer backbone, and (iv) surface functionalization with biologically active ligands that enabled specific targeting. The construction method is very versatile and was illustrated by the coupling of a small library of ligands (e.g., biotin, curcumin derivatives, and antibody), resulting in high affinity toward (i) murine lung carcinoma (M109) and human breast cancer (MCF7) cell lines, even in a coculture environment with healthy cells and (ii) the ß-amyloid peptide 1-42 (Aß(1-42)), believed to be the most representative and toxic species in AD, both under its monomeric and fibrillar forms. In the case of AD, the ligand-functionalized NPs exhibited higher affinity toward Aß(1-42) species comparatively to other kinds of colloidal systems and led to significant aggregation inhibition and toxicity rescue of Aß(1-42) at low molar ratios.

Antitumor vectorized oligonucleotides in a model of ewing sarcoma: unexpected role of nanoparticles.

Oligonucleotides (ONs) such as antisense oligonucleotides (AS-ON) and siRNAs are used as experimental tools to study gene function and are currently being tested in clinical trials for use as therapeutic anticancer agents. However, their therapeutic use has been limited by their low physiological stability and their slow cellular uptake. The systemic delivery of sequence-specific AS-ON targeting the EWS/FLI1 gene product by a targeted, nonviral delivery system dramatically inhibits tumor growth in a murine model of Ewing's sarcoma. The nonviral delivery system uses a poly-iso-hexyl-cyanoacrylate (PIHCA)-containing polycation (chitosan) to bind and protect the AS-ON. No antitumor effect is observed using a control oligonucleotide sequence. We found here that injection of the free AS-ON stimulates tumor growth independently of its sequence and that this stimulation is abolished in the presence of nanosphere-chitosan, which exerts with the oligonucleotides a specific inhibitory effect on tumor growth. The stimulation of tumor growth is likely to be due to a polyanionic effect; indeed, a similar stimulatory response is observed upon treatment with dextran sulfate and heparin in vivo. These results suggest that ON loaded onto nanosphere-chitosan provides efficient and tumor-specific delivery, and provides protection against a polyanionic secondary effect.

Assessment of cellular actin dynamics by measurement of fluorescence anisotropy.

To study cellular actin dynamics, a cell-free assay based on fluorescence anisotropy was developed. Using G-actin-Alexa as a probe, we found that anisotropy enhancement reflects F-actin elongation. Anisotropy enhancement varies with the concentration of magnesium and calcium cations and with ethylenediaminetetraacetate or well-known effectors of the polymerization. This assay gives the overall status of actin dynamics in cell extracts which are the closest conditions to in vivo, implying most of the regulating proteins that are missing in purified actin measurements. It can be used in a large-scale screening for chemical compounds which modulate actin polymerization.

Efficacy of siRNA nanocapsules targeted against the EWS-Fli1 oncogene in Ewing sarcoma.

The EWS-Fli1 fusion gene encodes for a chimeric oncogenic transcription factor considered to be the cause of the Ewing sarcoma. The efficiency of small interfering RNAs (siRNAs) targeted toward the EWS-Fli1 transcript (at the junction point type 1) was studied, free or encapsulated into recently developed polyisobutylcyanoacrylate aqueous core nanocapsules. Because this mRNA sequence is only present in cancer cells, it therefore constituted a relevant target. Studies of the intracellular penetration by confocal microscopy in NIH/3T3 EWS-Fli1 cells showed that nanocapsules improved the intracellular penetration of siRNA with mainly a cytoplasmic localization. These biodegradable siRNA-loaded nanocapsules were then tested in vivo on a mice xenografted EWS-Fli1-expressing tumor; they were found to trigger a dose-dependant inhibition of tumor growth after intratumoral injection. A specific inhibition of EWS-Fli1 was observed, too. These findings now open new prospects for the treatment of experimental cancers with junction oncogenes.

Uncoupling of the base excision and nucleotide incision repair pathways reveals their respective biological roles.

The multifunctional DNA repair enzymes apurinic/apyrimidinic (AP) endonucleases cleave DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases in the base excision repair pathway. Alternatively, in the nucleotide incision repair (NIR) pathway, the same AP endonucleases incise DNA 5' of a number of oxidatively damaged bases. At present, the physiological relevance of latter function remains unclear. Here, we report genetic dissection of AP endonuclease functions in base excision repair and NIR pathways. Three mutants of Escherichia coli endonuclease IV (Nfo), carrying amino acid substitutions H69A, H109A, and G149D have been isolated. All mutants were proficient in the AP endonuclease and 3'-repair diesterase activities but deficient in the NIR. Analysis of metal content reveals that all three mutant proteins have lost one of their intrinsic zinc atoms. Expression of the nfo mutants in a repair-deficient strain of E. coli complemented its hypersensitivity to alkylation but not to oxidative DNA damage. The differential drug sensitivity of the mutants suggests that the NIR pathway removes lethal DNA lesions generated by oxidizing agents. To address the physiological relevance of the NIR pathway in human cells, we used the fluorescence quenching mechanism of molecular beacons. We show that in living cells a major human AP endonuclease, Ape1, incises DNA containing alpha-anomeric 2'-deoxyadenosine, indicating that the intracellular environment supports NIR activity. Our data establish that NIR is a distinct and separable function of AP endonucleases essential for handling lethal oxidative DNA lesions.

Oncogene-targeted antisense oligonucleotides for the treatment of Ewing sarcoma.

The genetic hallmark of the Ewing sarcoma family of tumours (ESFT) is the presence of the t(11;22)(q24;q12) translocation, present in up to 85% of cases of ESFT, which creates the EWS/FLI1 fusion gene and results in the expression of a chimeric protein regulating many other genes. The inhibition of this protein by antisense strategies has shown its predominant role in the transformed phenotype of Ewing cells. In addition, the junction point at the mRNA level offers a target for short therapeutic nucleic acids that is present only in the cancer cells and not in the normal tissues of a patient. Several teams have, therefore, investigated the activity of antisense oligonucleotides and siRNAs targeted against the junction point in mRNA; thus, inhibiting EWS/FLI1 synthesis. Generally speaking, the molecules induce a cell growth inhibition in culture. Apoptosis has also been reported. One laboratory has reported the in vivo tumour inhibitory effect of phosphorothioate antisense oligonucleotide directed against the EWS part of EWS/FlI1 when injected intratumourally. Independently, a tumour inhibitory effect of oligonucleotides targeting the junction point has been demonstrated provided they are delivered by polymeric nanoparticles through the intratumoural route. Alongside this target, other genes participating to the maintenance of the transformed phenotype of Ewing cells have been downregulated by antisense strategies.