PDT induced bystander effect on human xenografted colorectal tumors as evidenced by sodium MRI.

BACKGROUND: Previous in vivo studies on photodynamic therapy (PDT)-treated, high cellular density tumors showed evidences of a bystander effect accompanying the therapy, cellular death continuing beyond the limits of the photochemical reactions in time and space. This process is generated by the initially damaged cells on the light pathway. The aim of this study was to determine if the bystander effect may be induced as well in colorectal xenografted tumors (less compact structure) and if the cellular signaling depends primarily on cellular proximity or not. METHODS: The photosensitizer was a glycoconjugated, meso substituted porphyrin derivative synthesized at Institut Curie. The longitudinal follow-up of the tumors was carried out by (23)Na/(1)H MRI, ideal imaging modality for mapping the extracellular compartment. Two regimens were followed in order to target either blood vessels alone or blood vessels and cancer cells simultaneously. RESULTS: The antivascular PDT did not succeed to arrest the tumors growth at the end of the follow-up. For double targeting PDT, we managed to stop the tumoral evolution. Sodium MRI evidenced a bystander effect. CONCLUSION: The results obtained showed that the bystander effect is more difficult to induce for the type of colorectal tumors used in this work. It needs a double treatment, 4 days apart, in order to be promoted.

Glycodendrimeric phenylporphyrins as new candidates for retinoblastoma PDT: blood carriers and photodynamic activity in cells.

Photodynamic therapy (PDT) has recently been proposed as a possible indication in the conservative treatment of hereditary retinoblastoma. In order to create photosensitizers with enhanced targeting ability toward retinoblastoma cells, meso-tetraphenylporphyrins bearing one glycodendrimeric moiety have been synthesized. The binding properties to plasma proteins and photodynamic activity of two monodendrimeric porphyrins bearing three mannose units via monoethylene glycol (1) or diethylene glycol (2) linkers have been compared to that of the non-dendrimeric tri-substituted derivative [TPP(p-Deg-O-α-ManOH)(3)]. The dendrimeric structure was found to highly increase the binding affinity to plasma proteins and to modify to some extent plasma distribution. HDL and to a lesser extent LDL have been shown to be the main carriers of dendrimeric and non-dendrimeric compounds. The phototoxicity observed for the two glycodendrimers (1) and (2) (LD(50)=0.5 µM) in Y79 cells is of the same order of magnitude that for TPP(p-Deg-O-α-ManOH)(3) (LD(50)=0.7 µM), with a similar cellular uptake level for (1) and a lower for (2). A serum content increase from 2% to 20% (v/v) in the incubation medium was found to inhibit both cellular uptake and photoactivity of dendrimeric derivatives, whereas those of TPP(p-Deg-O-α-ManOH)(3) remained little affected. Specificities of glycodendrimeric porphyrins, combining a lower cellular uptake together with a higher affinity toward plasma proteins, make these derivatives possible candidates for a vascular targeting PDT.

Synthesis of chiral ß-aminoalcohol palladium complexes exhibiting cytotoxic properties.

Original palladium complexes involving (-)-ephedrine, (-)-norephedrine, L-prolinol, L-valinol and L-isoleucinol have been rapidly prepared in neutral or basic medium and simply purified. They have been fully characterized by classical analytical methods and four of them were characterized by X-Ray analysis. In parallel with the experimental work, HF-DFT(B3LYP/PCM) computations were performed to obtain additional structural information. Their antiproliferative properties have been evaluated and some complexes showed small activities especially towards HT29 human cancer cells.

23Na MRI longitudinal follow-up of PDT in a xenograft model of human retinoblastoma.

BACKGROUND: Photodynamic therapy is an established cancer treatment in which a photosensitizing agent is activated by exposure to light thus generating cytotoxic reactive oxygen species that cause cellular damage. METHODS: A new photosensitizer synthesized at Curie Institute was used to treat retinoblastoma xenografts in mice, a glycoconjugated meso substituted porphyrin derivative, that showed some retinoblastoma cell affinity. The longitudinal follow-up of the tumors was carried out by (23)Na MRI (without adding exogenous contrast agents) to map the extracellular compartment and to characterize cell packing. Two regimens were followed to target either blood vessels alone or blood vessels and cancer cells simultaneously. RESULTS AND CONCLUSIONS: Only the protocol targeting both cancer cells and blood vessels effectively induces cellular death, confirmed by histology at the end of the experiment. Sodium MRI evidences a huge change in the cellular density of tumors only 24h after a double targeting (vascular and cellular) PDT treatment. We suggest that this change was possibly due to a bystander effect that can be promoted by the intercellular signaling favored by the high cellular density of retinoblastoma. These results indicate that non-invasive (23)Na imaging (which detects the tumor response to treatment from very early stages) in association with non-mutagenic therapies represents an effective option for tailored and individualized clinical treatments.

Unusual cellular uptake of cytotoxic 4-hydroxymethyl-3-aminoacridine.

Aminoacridine derivatives display interesting chemical and biological properties in the field of antitumor agents. The synthesis of 4-hydroxymethyl-3-aminoacridine and its iodo labelled analogue allows the study of cell distribution using two innovative, complementary and powerful techniques, real time fluorescence microscopy and dynamic secondary ion mass spectrometry (SIMS). All the data point to lysosomal localization of the active molecule.

Melanoma affinity in mice and immunosuppressed sheep of [(125)I]N-(4-dipropylaminobutyl)-4-iodobenzamide, a new targeting agent.

The increasing incidence of melanoma and the lack of effective therapy have prompted the development of new vectors, more specific to the pigmented tumor, for early detection and treatment. Targeted agents have to exhibit a rapid, high tumor uptake, long tumor retention and rapid clearance from nontarget organs. This joint work presents results obtained with a new melanoma targeting agent, [(125)I]-N-(4-dipropylaminobutyl)-4-iodobenzamide or [(125)I]BZ18. After labeling with a high specific activity, the biodistribution of the compound was investigated in two animal models, the mouse and the sheep. Melanotic tumor retention was observed lasting several days. We visualized the internalization of the agent inside the melanosomes by secondary ion mass spectroscopy imaging, we measured the affinity constants of [(125)I]BZ18 on a synthetic melanin model and we demonstrated a radiotoxic effect of this labeled agent on B16F0 melanoma cell culture due to its cellular internalization. From this work, [(125)I]BZ18 appeared a promising melanoma targeting agent in the nuclear medicine field.

Intracellular precipitation of hydroxyapatite mineral and implications for pathologic calcification.

In contrast to physiologic biomineralization occurring in bones, teeth and otoconia in vertebrates, calcification of soft tissues occurs in many pathologic conditions. Although similarities have been noted between the two processes, and despite the important clinical consequences of ectopic calcification, the molecular mechanisms regulating ectopic calcification are poorly understood. Although calcification is mainly extracellular, intracellular calcification has been reported and might indeed contribute to pathologic calcification of soft tissues. To better understand the process of intracellular calcification as a potential origin for pathologic calcification, and to examine the role of proteoglycans in this process, we investigated a pattern of intracellular nucleation and growth of hydroxyapatite in Madin-Darby Canine Kidney (MDCK) epithelial cells using electron microscopy, secondary ion mass spectroscopy (NanoSIMS), cytochemical staining, immunolabeling and biochemical analysis. We report here that under mineralizing cell culture conditions where beta-glycerophosphate (betaGP) was added as an exogenous organic source of phosphate, betaGP-cleaving alkaline phosphatase activity increased and hydroxyapatite crystals subsequently nucleated within intracellular, membrane-bounded compartments. The small, leucine-rich proteoglycan decorin was also upregulated and associated with mineral in these cultures. Such information provides insight into the mechanisms leading to pathologic calcification and describes a process whereby hydroxyapatite deposition in cells might lead to ectopic calcification.

A new O6-alkylguanine-DNA alkyltransferase inhibitor associated with a nitrosourea (cystemustine) validates a strategy of melanoma-targeted therapy in murine B16 and human-resistant M4Beu melanoma xenograft models.

Chemoresistance to O(6)-alkylating agents is a major barrier to successful treatment of melanoma. It is mainly due to a DNA repair suicide protein, O(6)-alkylguanine-DNA alkyltransferase (AGT). Although AGT inactivation is a powerful clinical strategy for restoring tumor chemosensitivity, it was limited by increased toxicity to nontumoral cells resulting from a lack of tumor selectivity. Achieving enhanced chemosensitization via AGT inhibition preferably in the tumor should protect normal tissue. To this end, we have developed a strategy to target AGT inhibitors. In this study, we tested a new potential melanoma-directed AGT inhibitor [2-amino-6-(4-iodobenzyloxy)-9-[4-(diethylamino) ethylcarbamoylbenzyl] purine; IBgBZ] designed as a conjugate of O(6)-(4-iododbenzyl)guanine (IBg) as the AGT inactivator and a N,N-diethylaminoethylenebenzamido (BZ) moiety as the carrier to the malignant melanocytes. IBgBZ demonstrated AGT inactivation ability and potentiation of O(6)-alkylating agents (cystemustine, a chloroethylnitrosourea) in M4Beu highly chemoresistant human melanoma cells both in vitro and in tumor models. The biodisposition study on mice bearing B16 melanoma, the standard model for the evaluation of melanoma-directed agents, and the secondary ion mass spectrometry imaging confirmed the concentration of IBgBZ in the tumor and in particular in the intracytoplasmic melanosomes. These results validate the potential of IBgBZ as a new, more tumor-selective, AGT inhibitor in a strategy of melanoma-targeted therapy.

Retrograde delivery of photosensitizer (TPPp-O-beta-GluOH)3 selectively potentiates its photodynamic activity.

Photodynamic therapy involves administration of a photosensitizing drug and its subsequent activation by visible light of the appropriate wavelength. Several approaches to increasing the specificity of photosensitizers for cancerous tissues and, in particular, through their conjugation to ligands that are directed against tumor-associated antigens have been investigated. Here, we have studied the delivery of the photocytotoxic porphyrin compound TPP(p-O-beta-D-GluOH)3 into tumor cells that overexpress the glycosphingolipid Gb3, using the Gb3-binding nontoxic B-subunit of Shiga toxin (STxB) as a vector. To allow for site-directed chemical coupling, an STxB variant carrying a free sulfhydryl moiety at its C-terminal end has been used. Binding affinity, cellular uptake, singlet oxygen quantum yield, and phototoxicity of the conjugate have been examined. Despite some effect of coupling on both the photophysical properties of TPP(p-O-beta-D-GluOH)3 and the affinity of STxB for its receptor, the conjugate exhibited a higher photocytotoxic activity than the photosensitizer alone and was exquisitely selective for Gb3-expressing tumor cells. Furthermore, our data strongly suggest that STxB-mediated retrograde delivery of the photosensitizer to the biosynthetic/secretory pathway is critical for optimal cytotoxic activity. In conclusion, a strong rationale for using retrograde delivery tools such as STxB in combination with photosensitizing agents for the photodynamic therapy of tumors is presented.

[Kinetics of intracolloidal iodine within the thyroid of newborn rats. Direct imagery using secondary ion mass spectrometry]. / Cinétique intracolloïdale de l'iode dans la thyroïde de rat nouveau-né. Imagerie directe par microscopie ionique analytique.

The spatiotemporal distribution of cellular uptake site of radiotoxics is essential data for microdosimetric studies. As early as 1950, the heterogeneity of iodine incorporation within the thyroid has been shown using autoradiography. The objective of this study is to describe the kinetic cellular distribution of newly organified iodine in the thyroid of newborn rats using secondary ion mass microscopy (NanoSIMS50). Ionic images obtained at high mass resolution and with a lateral resolution of about 50 nm show that the early distribution of iodine is heterogeneous from one follicle to another, from one thyrocyte to another inside the same follicle, and that this distribution varies as a function of time. The obtained kinetic profile will allow us to refine the studies concerning the aetiopathology of thyroid cancers of the Chernobyl children.

Morphological and chemical studies of pathological human and mice brain at the subcellular level: correlation between light, electron, and nanosims microscopies.

Neurodegenerative diseases induce morphological and chemical alterations in well-characterized regions of the brain. Understanding their pathological processes requires the use of methods that assess both morphological and chemical alterations in the tissues. In the past, microprobe approaches such as scanning electron microscopy combined with an X-ray spectrometer, Proton induced X-ray emission, secondary ion mass spectrometry (SIMS), and laser microprobe mass analysis have been used for the study of pathological human brain with limited success. At the present, new SIMS instruments have been developed, such as the NanoSIMS-50 ion microprobe, that allow the simultaneous identification of five elements with high sensitivity, at subcellular spatial resolution (about 50-100 nm with the Cs(+) source and about 150-200 nm with O(-) source). Working in scanning mode, 2D distribution of five elements (elemental maps) can be obtained, thus providing their exact colocalization. The analysis can be performed on semithin or ultrathin embedded sections. The possibility of using transmission electron microscopy and SIMS on the same ultrathin sections allows the correlation between structural and analytical observations at subcellular and ultrastructural level to be established. Our observations on pathological brain areas allow us to establish that the NanoSIMS-50 ion microprobe is a highly useful instrument for the imaging of the morphological and chemical alterations that take place in these brain areas. In the human brain our results put forward the subcellular distribution of iron-ferritin-hemosiderin in the hippocampus of Alzheimer disease patients. In the thalamus of transgenic mice, our results have shown the presence of Ca-Fe mineralized amyloid deposits.

Amino- and glycoconjugates of pyrido[4,3,2-kl]acridine. Synthesis, antitumor activity, and DNA binding.

A series of amino- and glycoconjugates of pyrido[4,3,2-kl]acridine and pyrido[4,3,2-kl]acridin-4-one have been prepared. The most active molecules, the amino conjugates 7 and 11, display a cytostatic activity against HT-29 cancer cells at micromolar concentration. This activity correlates well with a strong DNA binding. The molecules, amino or glycoconjugates, bind DNA by intercalation, the amino or glyco substituent being located in one groove. None of the molecules inhibits topoisomerase activity.

3,4-Dihydro-1H-[1,3]oxazino[4,5-c]acridines as a new family of cytotoxic drugs.

A series of [1,3]oxazino fused acridines has been prepared as precursors of cytotoxic 3-amino-4-hydroxymethylacridine 2. Their cytotoxic activity has been evaluated against HT29 colon carcinoma cell line and was shown to be dependent on the nature of the substituent located on position 2 of the oxazine ring. Additionally, the nitrophenyl derivative 3f is activated by nitroreductase, indicating its potency as prodrug for either gene-directed or antibody-directed enzyme prodrug therapies.

Isoxazole-type derivatives related to combretastatin A-4, synthesis and biological evaluation.

Novel combretastatin analogues bearing various five-membered heterocycles with consecutive oxygen and nitrogen atoms, in place of the olefinic bridge of CA4, have been synthesized (isoxazole, isoxazoline, oxadiazole, etc). These compounds have been evaluated for cytotoxicity and their ability to inhibit the tubulin assembly. On the basis of the relative position of the aromatic A- and B-rings on the heterocyclic moiety, they could be split in two classes, the alpha,gamma- or alpha,beta-diaryl heterocyclic derivatives. In the first series, the 3,5-diaryloxadiazole 9a displayed comparable antitubulin activity to that of CA4, but was devoid of cytotoxic effects. Among the alpha,beta-diaryl heterocyclic derivatives, the 4,5-diarylisoxazole 35 exhibited greater antitubulin activity than that of CA4 (0.75 vs 1.2 microM), but modest antiproliferative activity. These data showed that minor alteration in the chemical structure of the heterocyclic ring and its relative orientation with regard to the two phenyl rings of CA4 could dramatically influence the tubulin binding properties.

Nitrobenzylcarbamate prodrugs of cytotoxic acridines for potential use with nitroreductase gene-directed enzyme prodrug therapy.

The synthesis, solvolytic behaviour and cytotoxicity of novel 4-nitrobenzyl carbamates and carbonates derived from 3-amino-4-hydroxymethylacridine 1 are described. Compounds 2 and 6 are both substrates for Escherichia coli nitroreductase and the highly active lead structure 1 is liberated upon incubation of the two compounds in the presence of NTR and its cofactor NADH. Additionally, the cytostatic activity of 2 and 6 against human HT29 colon carcinoma cell lines is decreased 80-fold and 360-fold, respectively, indicating their suitability and potency as prodrugs for either gene-directed enzyme prodrug therapy or antibody-directed enzyme prodrug therapy.

An alternative way of the synthesis of 1-substituted 9-methoxy-5-methyl-6H-pyrido[4,3-b]carbazole derivatives.

A keystone of this work was a modification of synthesis of the title compounds, which were used as substrates for the preparation of amides 5, 9-methoxyolivacine (4a) and ethyl 9-methoxy-5-methyl-6H-pyrido[4,3-b]carbazole-1-carboxylate (4b) were obtained in good overall yields (4a--72%, and 4b--31%) on alternative ways of the synthesis. The pilot results of the cytostatic activity of iminium salts 12a (IC50 = 8 microM) and 12b (IC50 = 2 microM) were determined on L1210 mouse leukaemia cells.

Progress in analytical imaging of the cell by dynamic secondary ion mass spectrometry (SIMS microscopy).

This paper reviews the most recent methodological advances in the field of biological imaging using dynamic secondary ion mass spectrometry (SIMS). After a short reminder of the basic principle of SIMS imaging, the latest high-resolution dynamic SIMS equipment is briefly described. This new ion nanoprobe (CAMECA NanoSIMS 50) has a lateral resolution of less than 50 nm with primary Cs+ ion, the ability to detect simultaneously 5 different ions from the same micro-volume and a very good transmission even at high mass resolution (60% at M/DeltaM=5000). Basic considerations related to sample preparation, mass resolution and primary ion implantation are given. The decisive capability of this new instrument, and more generally of high-resolution dynamic SIMS imaging in biology, are illustrated with the most recent examples of utilization.

Cytostatic activity of 1,10-phenanthroline derivatives generated by the clip-phen strategy.

The cytostatic activities of a series of twelve 1,10-phenanthroline (Phen) derivatives and of their copper complexes were studied on L1210 murine leukemia cells. Large increases in the biological activity were observed for compounds of the 3-Clip-Phen series, in which two Phen moieties were bridged at their C3 positions by an alkoxy linker, the 3-pentyl-Clip-Phen derivative showing an IC(50) value of 130 nM while Phen shows an IC(50) value of 2500 nM under the same conditions. IC(50) values seemed to be modulated not only by the position, the nature, and the length of the linker of Clip-Phen but also by hydrophobicity. Since copper complexes of Phen are chemical nucleases and nucleic acids are thus a potential target for these compounds, the corresponding copper complexes were also studied. Copper complexation of the 3-Clip-Phen ligands did not increase their biological activities. Attempts to vectorize 3-Clip-Phen derivatives with a DNA binder such as spermine or with a cell-penetration peptide failed to increase their biological activity relative to the original 3-Clip-Phen series.

Synthesis, structure, and cytostatic properties of new olivacine derivatives.

Starting from 2-(6-methoxy-1-methyl-9H-carbazol-2-yl)ethylamine and 6-methylpicolinic acid, 9-methoxy-5-methyl-1-(6-methylpyridin-2-yl)-6H-pyrido[4,3-b]carbazole 10 and its 6-alkylderivatives 12-17 were obtained. The newly obtained compounds showed significant cytostatic activity against cultured L1210 cells and high cytotoxicity towards various human tumor cell lines.

Synthesis and cytostatic properties of some 6H-Indolo[2, 3-b][1, 8]naphthyridine derivatives.

The new and efficient synthesis of the title heterocyclic ring system is described starting from suitable 2-chloro-1, 8-naphthyridines. The synthesized 6H-indolo[2, 3-b][1, 8]naphthyridine derivatives were tested in vitro on 55 tumor cell lines for their anticancer properties. The presence of the acetylamino moiety at position 3 in the main ring system proved to be crucial for the cytostatic activity of this class of compounds.