Intramolecular Cyclization of N-phenyl N'(2-chloroethyl)ureas leads to Active N-phenyl-4,5-dihydrooxazol-2-amines Alkylating β-Tubulin Glu198 and Prohibitin Asp40

International audienceThe cyclization of anticancer drugs into active intermediates has been reported mainly for DNA alkylating molecules including nitrosoureas. We previously defined the original cytotoxic mechanism of anticancerous phenyl '(2-chloroethyl)ureas (CEUs) that involves their reactivity towards cellular proteins and not against DNA; two CEUs subsets have been shown to alkylate β-tubulin and prohibitin leading to inhibition of cell proliferation by G/M or G/S cell cycle arrest. In this study, we demonstrated that cyclic derivatives of CEUs, -phenyl-4,5-dihydrooxazol-2-amines (Oxas) are two to threefold more active than CEUs and share the same cytotoxic properties in B16F0 melanoma cells. Moreover, the CEU original covalent binding by an ester linkage on β-tubulin Glu198 and prohibitin Asp40 was maintained with Oxas. Surprisingly, we observed that Oxas were spontaneously formed from CEUs in the cell culture medium and were also detected within the cells. Our results suggest that the intramolecular cyclization of CEUs leads to active Oxas that should then be considered as the key intermediates for protein alkylation. These results could be useful for the design of new prodrugs for cancer chemotherapy

Antibody Pretargeting Based on Bioorthogonal Click Chemistry for Cancer Imaging and Targeted Radionuclide Therapy

International audienceBioorthogonal click chemistry-employing antibody-conjugated trans-cyclooctenes (TCO) and tetrazine (Tz)-based radioligands able to covalently bind in vivo-appeared recently as a potential alternative to circumvent the hematotoxicity induced by radioimmunotherapy of solid tumors. This Review focuses on the recent advances concerning TCO/Tz pretargeting in both cancer imaging and targeted-radionuclide therapy for prospective clinical transfer. We exhaustively identified 25 PubMed publications reporting preclinical imaging and 5 therapy studies with full mAbs as targeting vectors, since its first application in 2010. The fast, safe, modulable, and specific TCO/Tz pretargeting showed high potential as a theranostic tool to get more personalized and precise cancer care. The recent optimizations reported here highlighted a possible first clinical evaluation of IEDDA pretargeting in the coming years

Identification et caractérisation de protéines impliquées dans la dissémination du mélanome (rôle de l'Annexine A1)

Nous avons voulu identifier et caractériser des protéines jouant un rôle dans la formation des métastases du mélanome cutané. Pour ce faire, nous avons réalisé des comparaisons protéomiques de deux lignées génétiquement semblables : B16F10 et B16B16, qui induisent toutes deux la formation d'une tumeur primitive, alors que seules les tumeurs B16B16 génèrent des métastases. Parmi les 37 protéines différentielles identifiées, l'Annexine A1 (ANXA1) est plus exprimée dans les cellules et les tumeurs B16B16. Cette protéine est impliquée dans de nombreuses régulations cellulaires, notamment dans la migration et l'extravasation des neutrophiles en se fixant sur les récepteurs aux peptides formylés (FPR). Nous montrons que la diminution du taux d'ANXA1 par ARN interférents entraîne la réduction de la capacité invasive des cellules B16B16 in vitro, et du nombre de métastases in vivo. Des peptides, soit agonistes des FPR (fMLP), soit correspondants à la région N-terminale d'ANXA1, stimulent l'invasion et la sécrétion de MMP9 des cellules B16B16. L'axe ANXA1-FPR, habituellement rencontré dans les lignages myélocytaires, pourrait participer au processus de dissémination du mélanome. Cette hypothèse est en partie corroborée par une étude rétrospective montrant que la présence d'ANXA1 dans les tumeurs primitives humaines est associée à un risque de métastases 6 fois plus élevé. Nousavons également montré que la Synténine, habituellement intracellulaire et favorisant la dissémination du mélanome, est significativement plus sécrétée dans les surnageants des cellules les moins agressives, B16F10. Elle interagirait avec des protéines d'adhésion, renforçant ainsi les liens matrice-cellules ou cellules-cellules.Proteins involved in melanoma dissemination have been studied and protein profiles of two genetically similar melanoma cell lines have been compared. Both B16F10 and B16B16 cells induced primary tumours after subcutaneous graft, but pulmonary metastases were observed only in mice bearing B16B16 melanoma. Among the 37 identified proteins, we focused on Annexin A1 (ANXA1) which expression increased in both B16B16cell culture and tumours. This protein is involved in leukocyte migration and extravasation, particularly by its binding to the formyl peptide receptor (FPR). Here, using RNA interference strategies, we have shoxn that ANXA1 reduction decreased both in vitro cell invasion and metastasis formation in mice. Moreover, treatment of B16B16 cells by fMLP (FPR agonists) or by ANXA1 N-terminal peptide enhanced cell invasion and MMP9 secretion. ANXA1-FPR binding, usually involved in leukocyte regulation, should also contribute to melanoma dissemination processes. This is partly confirmed by a retrospective study in human primary tumours, showing that ANXA1 expression increased 6 fold the metastasis risk. We have also shown that Syntenin, an adaptater protein, was more present in B16F10 secretomes. Intracellular Syntenin was previously described as a marker for melanoma aggressiveness. Its extracellular localisation should protect melanoma cells from invasion, probably by reinforcing cell-cell adhesion or cell-matrix adhesion.CLERMONT FD-BCIU-Santé (631132104) / SudocSudocFranceF

Benzamide derivative radiotracers targeting melanin for melanoma imaging and therapy: Preclinical/clinical development and combination with other treatments

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Evaluation of biological effectiveness of 65 MeV therapeutic proton beams using the GATE platform

International audienceIntroduction: In order to optimize hadrontherapy treatments, it is needed to consider the biological effect on irradiated tumor cells. The biological dose can be evaluated through biophysical models, such as the Microdosimetric Kinetic Model (MKM)[1-5]. This model is implemented to take into account a corrected lineal energy spectrum obtained from Monte Carlo simulations and the model parameters of irradiated cells obtained from experiments. The purpose of this study is to validate the implementation of the MKM model into GATE through the simulation of the 65 MeV therapeutic proton beam (MediCyc) of Antoine Lacassagne centre for the estimation of the relative biological effectiveness (RBE)

Targeted hypoxia-activated phosphoramide mustards toward tumor proteoglycans in chondrosarcoma

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Proteoglycan-targeting applied to hypoxia-activated prodrug therapy in chondrosarcoma: first proof-of-concept

International audienceDue to its abundant chondrogenic matrix and hypoxic tissue, chondrosarcoma is chemo-and radio-resistant. Our group has developed a proteoglycan targeting strategy by using a quaternary ammonium (QA) function as a carrier of DNA alkylating agents to chondrosarcoma environment. Here, we assessed the relevance of this strategy applied to hypoxia-activated prodrugs, by conjugating a QA to 2-nitroimidazole phosphoramidate. This derivative, named as 8-QA, was evaluated respectively to its non-QA equivalent and to a QA-conjugated but non-hypoxia activated. Firstly binding to aggrecan was confirmed from dissociation constant determined by Surface Plasmon Resonance. In vitro, in HEMC-SS chondrosarcoma cells cultured in monolayer and in spheroids, 8-QA showed higher cytotoxic activity in hypoxia versus normoxia, and led to a strong accumulation of cells in S phase and apoptosis. In vivo, a HEMC-SS xenograft model was implanted on SCID mice and characterized for hypoxia by photoacoustic imaging as well as proteoglycan content. When HEMC-SS bearing mice were given 8-QA at 47 μmol/kg according to a q4d x 6 schedule, a significant 62.1% inhibition of tumor growth was observed, without associated hematological side effects. Mechanistic studies of treated tumors highlighted decrease in mitotic index associated to increase in both p21 and p53S15 markers. Interestingly, 8-QA treatment induced an increase of DNA damages as measured by γH2AX predominantly found in pimonidazole-positive hypoxic areas. These preclinical results are the first to demonstrate the interest of addressing hypoxia-activated prodrugs selectively to proteoglycan of chondrogenic tumor tissue, as a promising therapeutic strategy

Determination of eumelanin and pheomelanin in melanomas using solid-phase extraction and high performance liquid chromatography -diode array detection (HPLC-DAD) analysis

International audienceDetermination of eumelanin and pheomelanin in melanomas that exhibit different pigmentation was carried using a solid-phase extraction (SPE) preparation method based on weak anion exchange chemistry. This extraction significantly enhanced the chromatographic profile obtained by reverse phase high performance liquid chromatography-diode array detection (RP-HPLC-DAD). The SPE method was developed using aqueous standards of melanin markers: thiazole-2,4,5-tricarboxylic acid (TTCA), thiazole-4,5-dicarboxylic acid (TDCA), pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA) and non-pigmented cell lines spiked with those markers. An excellent average recovery, above 90%, was obtained for the four markers with a relative standard deviation below 7%. We have also optimized the stationary phase and the mobile phase (phosphate concentration and pH) to improve sensitivity and to reduce the analysis time. Elution of the four markers is achieved in 5 minutes and total analysis of biological samples is completed in 15 minutes. The quantification limits for TDCA, TTCA, PDCA and PTCA are 60, 50, 47 and 48 ng/mL respectively. Furthermore, DAD detection improves the marker identification in complex matrices through the analysis of UV spectra. We have successfully applied this method to melanoma tumors and cells. Murine B16BL6 tumor are highly pigmented with mostly eumelanin (98.1 % of eumelanin) while human SK-MEL-3 tumor contain about 30 % pheomelanin. B16BL6 and B16F10 are eumelanic cells lines and NHEM melanocytes contain about 24 % of pheomelanin

Alkylation of beta-tubulin on Glu 198 by a microtubule disrupter

International audienceWe have shown that beta-tubulin was alkylated by a microtubule disrupter, N-4-iodophenyl-N'-(2-chloroethyl)urea (ICEU), on a glutamic acid residue at position 198 and not on the previously proposed reactive cysteine 239. ICEU belongs to the 4-substituted-phenyl- N'-(2-chloroethyl) urea class that alkylates mainly cellular proteins. Previous studies have shown that the tertbutyl (tBCEU) and iodo (ICEU) derivatives induce microtubule disruption because of beta-tubulin alkylation. tBCEU was supposed to bind covalently to cysteine 239 of beta-tubulin, but this binding site was not clearly confirmed (Cancer Res 60: 985-992, 2000). We have isolated and analyzed beta-tubulin after two-dimensional gel electrophoresis of proteins from B16 cells incubated with ICEU. Alkylated beta-tubulin had a lower apparent molecular weight and a more basic isoelectric point than the unmodified protein. Labeled N-4-[I-125]CEU was effectively bound to the modified beta-tubulin but using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, we demonstrated that none of the cysteine residues of beta-tubulin was linked to the alkylating agent. In contrast, peptide masses at m/z 4883 and 1792 in trypsin or Asp-N digestions of beta-tubulin confirmed binding of iodophenylethylureido moiety to peptides [175-213] or [197-208] respectively. Fragmentation analyses by electrospray mass spectrometry using triply charged ions of peptide [175-213] identified a glutamic acid at position 198 as target for alkylation via an ester bond with ICEU. This amino acid located in the intermediate domain of the beta-tubulin should play an essential role in the conformational structure necessary for the interaction between dimers in the protofilament

Tspan8 Drives Melanoma Dermal Invasion by Promoting ProMMP-9 Activation and Basement Membrane Proteolysis in a Keratinocyte-Dependent Manner

International audienceMelanoma is the most aggressive skin cancer with an extremely challenging therapy. The dermal-epidermal junction (DEJ) degradation and subsequent dermal invasion are the earliest steps of melanoma dissemination, but the mechanisms remain elusive. We previously identified Tspan8 as a key actor in melanoma invasiveness. Here, we investigated Tspan8 mechanisms of action during dermal invasion, using a validated skin-reconstruct-model that recapitulates melanoma dermal penetration through an authentic DEJ. We demonstrate that Tspan8 is sufficient to induce melanoma cells' translocation to the dermis. Mechanistically, Tspan8+ melanoma cells cooperate with surrounding keratinocytes within the epidermis to promote keratinocyte-originated proMMP-9 activation process, collagen IV degradation and dermal colonization. This concurs with elevated active MMP-3 and low TIMP-1 levels, known to promote MMP-9 activity. Finally, a specific Tspan8-antibody reduces proMMP-9 activation and dermal invasion. Overall, our results provide new insights into the role of keratinocytes in melanoma dermal colonization through a cooperative mechanism never reported before, and establish for the first time the pro-invasive role of a tetraspanin family member in a cell non-autonomous manner. This work also displays solid arguments for the use of Tspan8-blocking antibodies to impede early melanoma spreading and therefore metastasis