In vitro phototoxicity of glycoconjugated porphyrins and chlorins in colorectal adenocarcinoma (HT29) and retinoblastoma (Y79) cell lines.

BACKGROUND: Retinoblastoma is the most common malignant intraocular tumor in children. The current treatment gives a good vital prognostic but there are several drawbacks to the arsenal of "classical antitumoral" therapies. Photodynamic therapy (PDT) could be an exciting non-toxic and non-mutagenic alternative protocol. METHOD: In this paper, we report about the screening of the in vitro photocytotoxicity of hydrophenylporphyrins and chlorins and their glycoconjugated derivatives in a human retinoblastoma cell line (Y79) and for comparison in a colorectal adenocarcinoma cell line (HT29). RESULTS: Despite lower photodynamic activity than that observed for hydroxylated photosensitizers, in particular Foscan(®) glycoconjugated derivatives display phototoxicity (IC50 2.4-0.05µM ±10%) against Y79 cells with examples of significant intrinsic cytotoxicity. Amongst them the triglucosyl porphyrin 10 is highly photocytotoxic (IC50 0.9µM ±10%) but is fully devoid of cytotoxicity (IC50>15µM). The photoactivity is highly modulated by the presence of a diethyleneglycol spacer between the chromophore and the glycoside (compounds 14-17, IC50 0.5, 0.6, 0.05 and 0.35µM ±10%) and by the anomeric configuration of the sugar (compound 15 and 17, IC50 0.6 and 0.05µM ±10% respectively). One of the main problems for the use of Foscan(®) is its poor solubility which might be improved by glycoconjugation. Moreover Foscan has been shown to induce necrosis after PDT leading to a possible ulceration of surrounding tissues unsuitable for a conservative treatment. A preferential mitochondrial subcellular localization which has been previously reported for some glycoconjugated photosensitizers could enhance the contribution of apoptosis process. CONCLUSION: Tri-α-O-galactosyl porphyrin 16 is a better candidate than Foscan(®) for a clinical application of PDT for a conservative therapy of retinoblastoma.

Synthesis, cellular internalization and photodynamic activity of glucoconjugated derivatives of tri and tetra(meta-hydroxyphenyl)chlorins.

Glucoconjugated tri and tetra(meta-hydroxyphenyl)chlorins have been synthesized in order to explore how glucoconjugation of the macrocycle affects the photoactivity of the molecule. Internalization processes, photosensitizing efficacy of TPC(m-O-GluOH)(3) and TPC(m-O-GluOH)(4), in HT29 human adenocarcinoma cells have been compared to those of tetra(meta-hydroxyphenyl) chlorin (m-THPC, Foscan). The tetra glucoconjugated chlorin, TPC(m-O-GluOH)(4), was found to be poorly internalized and weakly photoactive. In contrast, the asymmetric and more amphiphilic compound TPC(m-O-GluOH)(3), exhibited superior phototoxicity compared to m-THPC. Drug concentration, temperature and sodium azide effects indicated that TPC(m-O-GluOH)(3) internalization partly proceeds via an active receptor-mediated endocytosis mechanism. Cellular uptake appeared as a saturable process and remained 30% lower than for mTHPC. However, a maximum phototoxicity in HT29 cells (survival fraction of 2+/-0.6%) were observed for concentration as low as 2 microM. A 4-fold higher concentration of m-THPC was necessary to observe the same level of photoactivity. This higher phototoxicity has been correlated to a greater mitochondrial affinity. On the basis of these results, work is in progress to further evaluate the potential of glycosylated chlorins in photodynamic therapy (PDT).

Biodistribution of meta-tetra(hydroxyphenyl)chlorin incorporated into surface-modified nanocapsules in tumor-bearing mice.

meta-Tetra(hydroxyphenyl)chlorin (mTHPC), a second generation photosensitizer used in photodynamic therapy (PDT), was incorporated into long circulating carriers with the aim of improving the tumor selectivity by limiting the reticuloendothelial system (RES) uptake. Biodistribution of mTHPC (0.06 mg kg(-1) was studied directly in nude mice bearing HT29 human tumor by optical fiber fluorimetry and tissue drug contents were determined by HPLC after extraction. The drug was incorporated in the oily core of nanocapsules surrounded by poly(D,L lactic acid) (PLA NCs), PLA grafted with polyethylene glycol (PLA-PEG) or PLA coated with poloxamer 188 (polox PLA). Compared to PLA NCs, incorporation of mTHPC in surface-modified nanocapsules resulted in strong modifications of the drug biodistribution and tumoral retention with a three-fold increase of drug level as early as 24 h post-administration. A reduced liver uptake was observed at early times post-administration indicating that surface-modified NCs are effective in limiting the RES uptake and could be potential carriers to enhance the therapeutic ratio of lipophilic photosensitizers. Furthermore, in situ fluorescence measurements and concentration data were found in broad agreement showing that optical fiber fluorimetry is a very sensitive method that can be used to follow the biodistribution of fluorescent drugs in real-time.

Diaminopurine-acridine heterodimers for specific recognition of abasic site containing DNA. Influence on the biological activity of the position of the linker on the purine ring.

Three acridine-diaminopurine heterodimers tethered by a linker containing an N,N'-substituted guanidine were prepared. The molecules differ by the site of introduction of the linker on the 2,6-diaminopurine. The interactions of the new heterodimers with abasic site containing oligonucleotide were compared, and their cytotoxicity was measured in the presence or absence of the antitumor alkylating agent BCNU.

Potentiation of BCNU cytotoxicity by molecules targeting abasic lesions in DNA.

We describe the synthesis, DNA binding measurements and pharmacological properties of a series of new heterodimeric molecules, in which a 2,6-diaminopurine is linked to a 9-aminoacridine chromophore. The linking chain contains a central N,N'-disubstituted guanidine, connected to the two chromophores by polymethylenic units of variable length.

Diphenyl quinolines and isoquinolines: synthesis and primary biological evaluation.

The synthesis of a series of 35 substituted 3,4-diphenyl quinolines and isoquinolines is described. The majority of these molecules differ from all other triphenylethylene based antiestrogens by a different spatial location of the aminoalkyl side chain. The binding affinity of the most representative molecules (8, 9, 19, 20, 21, 23 and 25), including analogues 8 and 21 without the side chain, for the estrogen receptor alpha (ER) was determined. The ability of these molecules to induce the progesterone receptor was also studied. Antiproliferative activity was evaluated on MCF-7 human breast cancer cells, while intrinsic cytotoxic/cytostatic properties resulting from interaction with other targets than ER were assayed on L1210 murine leukemia cells. Introduction of an aminoalkylamino side chain at carbon 2 confers strong cytotoxic properties to diphenylquinolines 9 and 10 as well as pure antiestrogenic activities. However, cytotoxicity is so high with respect to antiestrogenicity that the latter was clearly observable only in one case (9b). The structure of compound 9b was determined by X-ray crystallography. Molecular modeling of its docking within the hormone-binding domain of the receptor was subsequently undertaken. According to our results, the design of molecules with the side chain bound to the ethylene part of the triphenyl ethylene skeleton might generate compounds of potential pharmacological interest.

Direct in vivo observation of 5-fluorouracil release from a prodrug in human tumors heterotransplanted in nude mice: a magnetic resonance study.

A glucuro-conjugated carbamate derivative of 5-fluorouracil (5-FU), originally designed as a prodrug for antibody-directed enzyme prodrug therapy (ADEPT) application, has been used for direct in vivo observation of in situ 5-FU generation in two human colon tumors heterotransplanted in nude mice. Because of the very fast elimination of glucuro-conjugated drugs, this observation required intratumoral injection. These tumors, when becoming necrotic, are rich enough in beta-glucuronidase to allow (19)F magnetic resonance spectroscopy monitoring, at the tumor level, of both prodrug elimination and 5-FU liberation without preliminary treatment by a specifically targeted enzyme conjugate. Convenient tumors have been selected by magnetic resonance imaging (MRI) on the basis of a correlative study between MRI and conventional histology. This contribution is the first report evidencing such a direct intra-tumoral conversion of a glucuro-conjugated prodrug into the expected active drug. This method, which should allow overall estimation of the beta-glucuronidase content of tumors, might also be helpful for selecting tumors as specific targets for non-toxic glucuro-conjugated prodrugs without prior treatment with a fusion protein.

Tetrapyrrolic Glycosylated Macrocycles for an Application in PDT.

The synthesis and characterization of amphiphilic glycoconjugated porphyrins, benzochlorin, and azaporphyrins were reported. Among these molecules, several were found to be efficient photosensitizers in an in vitro assay using the human tumoral cell line HT29. Moreover, glycosylated benzochlorin and azaporphyrins, whose absorption bands in the red region of the visible spectrum are substantially increased as compared to porphyrins, display a good photocytotoxicity on tumor cells after irradiation with wavelength above 590 nm. © 1999 Society of Photo-Optical Instrumentation Engineers.

Complementary advantages of fluorescence and SIMS microscopies in the study of cellular localization of two new antitumor drugs.

Low light level fluorescence microscopy studies have been carried out on MCF7-P human mammary tumor cells to localize the intracellular distribution of two new anticancer drugs, Pazelliptine and Intoplicine, which are currently under clinical evaluation. These two molecules are thought to act at the nuclear level, through DNA topoisomerase interactions. Because fluorescence of these compounds appears strongly quenched by intercalation in double strand DNA, secondary ion mass spectrometry (SIMS) imaging was used to check the presence of the drugs in the nuclear compartment. In spite of chemical structure similitudes, pazelliptine and intoplicine appear to be distributed in quite different ways within the cells. Incubation for 1 and 24 hours also allowed us to bring to light strong differences in the distribution kinetics. Pazelliptine quickly enters into the nucleoli but is no longer present in the nucleus after 24 hours incubation. Intoplicine was not detected by fluorescence in the nucleus, however SIMS microscopy allowed us to show its accumulation within this cellular compartment as a function of time of exposure. This study shows the complementarity of fluorescence and SIMS microscopies.

Effect of methionine on glycolysis in tumor cells: in vivo and in vitro NMR studies.

Inhibition of glycolysis by methionine is a phenomenon previously shown in transformed cells growing in culture. In a recent paper, [Collet V. et al., Q. Magn. Res. Biol. Med. 11, 127-134 (1995)] we investigated this effect in vivo by 13C nuclear magnetic resonance spectroscopy, but the results did not clearly support this hypothesis. In this work, in vivo 13C NMR spectroscopy has been performed on tumors developing in nude mice following the injection of two types of cells established in culture: (1) rat kidney cells transformed by Kirsten murine sarcoma virus, (NRK-K), i.e. the same tumor cell line as that used in the original paper; and (2) a well dedifferentiated human prostate adenocarcinoma cell line (PC3). Furthermore, in vitro experiments were performed with the same tumor cell lines. The effect of methionine on glycolysis was assayed by biochemical monitoring of lactate production in the supernatant of these cells grown in vitro. Lastly, 1H in vitro NMR spectroscopy of the PC3 line performed on perchloric extracts of both supernatants and cells growing in the presence of (1-13C) glucose, allowed simultaneous detection of glucose and lactate as well as estimation of the lactate-specific enrichment. The in vitro experiments confirmed the inhibiting effect of methionine on glycolysis and demonstrated the absence of a significant modification of the pentose phosphate pathway activity by this aminoacid. In contrast, none of the in vivo experimental results were compatible with this phenomenon, which is probably affected by more general physiological events.

Synthesis and biological properties of new benz[h]isoquinoline derivatives.

In order to determine the importance of the pyrrolic nitrogen atom in a series of suitably substituted gamma-carbolines derivatives for their cytotoxic and antitumor properties, a series of structurally related benz[h]isoquinolines were synthesized. When compared to the "parent" gamma-carbolines, these new compounds showed greatly decreased effects on topoisomerases I and II. Whereas the 8-hydroxylated derivatives retained a significant cytotoxicity, all the new compounds were devoid of antitumor effect in the P388 murine ip-ip system.

Efficient KEX2-like processing of a glucoamylase-interleukin-6 fusion protein by Aspergillus nidulans and secretion of mature interleukin-6.

We have designed an expression vector for the secretion of human interleukin-6 (hIL-6) in which the mature protein is fused through a spacer peptide, containing a KEX-2 like protein processing signal, to the entire Aspergillus niger glucoamylase (glaA) gene. Transformation of Aspergillus nidulans with this vector results in fungal strains secreting equimolar amounts of the glucoamylase and IL-6 proteins. The KEX2-type processing signal, Lys-Arg, is recognized and cleaved efficiently by an enzyme present in A. nidulans resulting in the secretion of an authentic mature hIL-6 protein at levels of up to 5 mg/l.

Heterologous gene expression by filamentous fungi: secretion of human interleukin-6 by Aspergillus nidulans.

Expression vectors for human interleukin-6 (hIL6) contain an expression cassette consisting of the Aspergillus niger glaA promoter and the Aspergillus nidulans argB terminator. The secretion signals were either those of glaA or that of the authentic hIL6 peptide. The constructs under study were introduced into A. nidulans and A. niger by means of cotransformation. No IL6 activity could be detected in the medium of a cotransformed A. niger strain, although transcripts corresponding with the IL6 cDNA were present. Evidence is presented that this apparent lack of IL6 expression is due to extracellular proteolytic activity. In the media of a cotransformed A. nidulans strain grown on starch, IL6 activity was detected by means of a bioassay. Up to 25 ng/ml of biologically active hIL6 could be secreted by A. nidulans transformed with the plasmid containing the mature hIL6-encoding gene fused to the glaA signal peptide nucleotide sequences. hIL6 of the expected 23-kDa size was also observed by Western-blot analysis of the medium. There was no evidence for glycosylation of the protein.

A practical test for in vitro evaluation of photosensitization: assessment with hematoporphyrin derivative (HPD).

A simple procedure is described for the determination of the photosensitizing potency of drugs, using three leukemic cell lines, two of lymphocytic origin, L1210 and P388 and one of erythroid type, Friend-745. The procedure allows one to investigate several aspects of the photosensitization properties of tested compounds such as cellular localization and direct (trypan blue exclusion) or delayed (clonogenicity) photomediated toxicities. The method was assessed using crude hematoporphyrin derivative (HPD) as well as dihematoporphyrin ether (DHE) or commercially available Photofrin II. Results were compared to those obtained with normal cells, e.g spleen lymphocytes and erythropoietic stem cells (CFU-e), and discussed in the light of the relative response of normal versus transformed cells.

Transformation of Aspergillus niger with the homologous nitrate reductase gene.

A homologous transformation for Aspergillus niger was developed based on the nitrate reductase structural gene niaD. This system offered certain advantages over existing A. niger systems, such as the ease of recipient mutant isolation, absence of abortive transformants, convenient enzyme assay, ease of transformant stability testing, and complete absence of background growth. Transformation frequencies of up to 100 transformants per microgram DNA were obtained with the vector pSTA10 which carries the niaD gene of A. niger. Southern blotting analysis indicated that vector DNA had integrated into the genome of A. niger. Mitotic stability studies demonstrated that while some transformants were as stable as the wild-type (wt), others were markedly less so. No correlation was seen between plasmid integration, mitotic stability and nitrate reductase activity, which was markedly different from wt in only three of the transformants examined.

31P NMR spectra of rodent and avian fibroblasts transformed by Rous or Kirsten sarcoma viruses.

31P NMR spectra of normal rodent and avian fibroblasts were compared to those of the same cells transformed either by the Rous sarcoma virus (RSV) or by the Kirsten sarcoma virus (Ki-MSV). Under physiological conditions, the spectra of living or perchloric acid extracted chicken embryo fibroblasts, rat cell line FR3T3 and mouse cell line C127 did not differ from those of their counterparts transformed by RSV or Ki-MSV. However, in the case of FR3T3 cells, on shifting from 37 degrees C to 20 degrees C, and particularly if PBS replaced serum growth medium, a different, though transitory, response of the transformed cells was detected. They then showed, within few minutes, a more rapid ATP depletion with accumulation of fructose 1,6-diphosphate (FDP), as compared to normal control cells.

The antiproliferative effect of murine interferon alpha/beta on early bone marrow-derived erythroid precursors (BFU/e).

We have examined the antiproliferative effect of murine interferon (MuIFN) alpha/beta on a primitive class of erythroid precursors, the burst-forming units (BFUe) which give rise to macroscopic colonies of erythroblasts in plasma clots. Cultures were examined respectively on day 9 and on day 15; in both instances there was a significant reduction in the number of erythropoietic bursts in cultures exposed to IFN. The effect of IFN was more pronounced when the cells were cultured in the presence of suboptimal concentrations of erythropoietin and it could to a significant extent be reversed by ouabain at 10(-15) M. The latter finding is in line with observations made by others that growth factors can act as IFN antagonists. A comparison of BFUe derived from mice of two different genotypes, BALB/c and C57BL/6, revealed no difference in relative sensitivity to the antiproliferative effect of IFN. This is in contrast with the previously described fivefold greater sensitivity of the BALB/c genotype when a more differentiated class of erythroid precursors, the colony-forming units or CFUe, are exposed to IFN. Since the BFUe, studied in the present work, are a more primitive class of erythroid precursors than the CFUe, it can be concluded that the genes modulating the difference in sensitivity to IFN action on erythroid precursors are only expressed after a certain stage of differentiation has been reached.

Strain dependence of the antiproliferative action of interferon on murine erythroid precursors.

Electrophoretically pure mouse interferon inhibits erythropoietin-dependent proliferation of committed erythroid precursors (CFU-E) obtained either from adult mouse bone marrow or from 14-day fetal mouse livers. The degree of inhibition is significantly influenced by the genotype of the cell donor; about ten times as much interferon is required to inhibit proliferation of CFU-E from C57BL/6 than is needed for comparable inhibition of CFU-E from BALB/c or Swiss mice. These strain-dependent results point to the existence of genes that influence the degree of the inhibitory effect of interferon on cell multiplication.

Effect of cortisol in vitro on haemoglobin synthesis by mouse foetal liver.

In cultures of foetal mouse liver, collected at various gestational days, the effect of cortisol was investigated on 59Fe uptake into haemoglobin heme and also on the differentiation of the erythroid stem cells or ERC (erythropoietin-responsive cells) into erythroblasts under the action of erythropoietin. Addition of cortisol to the culture medium did not modify in any way the responsiveness of the cell system to erythropoietin but exerted a sustaining action on the rate of haemoglobin synthesis during the second half of the incubation period. This action was restricted to cultures of 15- to 16-day livers and was particularly obvious in erythropoietin-supplemented samples. This selective effect of cortisol likely concerns only the later types of erythroblasts since after 24 h of incubation the polychromatophilic or orthochromatic stages predominate respectively in samples with, or without, erythropoietin. The timing of these results is discussed in the light of previous findings observed in vivo by other authors in the foetal rat.

Sensitivity of 15-day mouse fetal liver cells to erythropoietin (ESF). Its practical application to in vitro methods of ESF bioassay.

Demonstration was made of equal reactivity of 13-, 14- and 15-day mouse fetal livers to graded concentrations of Erythropoietin "in vitro". In consequence, replacement by 15-day fetal liver of 13-day liver, currently used as material in Erythropoietin bioassays "in vitro", is proposed as a technical improvement on the method.