Ultrastructural detection of photosensitizing molecules by fluorescence photoconversion of diaminobenzidine.

Photodynamic therapy is a moderately invasive therapeutic procedure based on the action of photosensitizers (PSs). These compounds are able to absorb light, and dissipate energy through photochemical processes leading to the production of oxidizing chemical species (singlet oxygen, free radicals or reactive oxygen species) which can damage the cell molecular structures eventually inducing cell death. To increase the entering through the plasma membrane, a PS with suitable chemical structure can be modified by addition of chemical groups (e.g., acetate or phosphate): this affects both the fluorescence emission and of the photosensitizing properties of the native PS. The modified compounds behave as fluorogenic substrates (FSs), since inside the cell the bound groups can be enzymatically removed and the fluorescence and photosensitizing properties of the native molecules are restored. With the aim to detect the subcellular localization of photoactive molecules at transmission electron microscopy, we loaded cultured HeLa cells with two different FSs, Rose Bengal acetate (RB-Ac) or Hypocrellin B acetate (HypB-Ac), and took advantage of the photophysical properties of the intracellularly restored PS molecules to obtain the photoconversion of diaminobenzidine (DAB) into an electrondense product. We demonstrated that RB-Ac and HypB-Ac are mostly internalized by endocytosis, and are converted into the native PSs already at the cell surface. Endocytosed PS molecules apparently follow the endosomes-lysosome route, being found in endosomes, lysosomes and multivescicular bodies; PS molecules were also detected in the cytosol. This ultrastructural localization of the photoactive molecules is fully consistent with the multiorganelle photodamage observed after irradiation in culture of RB-Ac- or HypB-Ac-loaded cells. Due to the very short half-life of the oxidizing chemical species and their limited mobility, DAB deposits do localize in close proximity of the very place where photoactive molecules elicited the production of reactive oxygen species upon light irradiation. Therefore, DAB photoconversion promises to be a suitable tool for directly visualizing in single cells the PS molecules at high resolution, helping to elucidate their mode of penetration into the cell as well as their dynamic intracellular redistribution and organelle targeting.

Hypocrellin-B acetate as a fluorogenic substrate for enzyme-assisted cell photosensitization.

Photosensitizing molecules (PSs) undergo chemico-physical changes upon addition of suitable substituents, influencing both their photophysical properties and their ability to accumulate into cells. Once inside the cells, the modified PS acts as a fluorogenic substrate: the added substituent is removed by a specific enzyme, restoring the native PS in subcellular sensitive sites. We investigated the photophysical properties and interaction with HeLa cells of Hypocrellin-B (HypB), as native molecule and upon acetate-group addition (HypB-Ac). Chemical modification alters both absorption and fluorescence features of HypB; consequently, the dynamics of the enzyme hydrolysis of HypB-Ac can be monitored through restoring the native HypB spectral properties. At the cellular level, only the HypB emission signal was detected within 5 min of incubation with either HypB or HypB-Ac, allowing a direct comparison of the time courses of their intracellular accumulation. Plateau values were reached within 15 min of incubation with both compounds, the emission signals being significantly higher in HypB-Ac than in HypB treated cells. Consistently, imaging showed a rapid appearance of red fluorescence in the cytoplasm, with more abundant bright spots in HypB-Ac treated cells. Both compounds did not induce dark toxicity at concentrations up to 1 × 10(-6) M, while upon irradiation at 480 nm phototoxicity was significantly higher for cells exposed to HypB-Ac than for HypB-loaded cells. These findings suggest an improved efficacy of acetylated HypB to be internalized by cells through membrane trafficking, with a preferential interaction of the photoactive molecules on sensitive intracellular sites. After irradiation, in HypB-Ac treated cells, prominent disorganization of several cytoplasmic organelles such as the endoplasmic reticulum, Golgi apparatus, lysosomes, microfilaments and microtubules were observed.

New light in flavin autofluorescence.

Our attention was captured by the interesting debate on the identification of lipofuscins, lipofuscin-like lipopigments, or flavins as the responsible for intracellular autofluorescence (AF) detected in epithelial cancer stem cells when exciting at 480-490 nm. Evidence was provided leading to ascribe AF emission to flavins accumulating in cytoplasmic structures, bounded to membranes and bearing ATP-dependent ABCG2 transporters. Flavins were then proposed as an intrinsic AF biomarker of cancer stem cells, with advantageous implications on cell invasiveness and chemoresistance investigations. It is however worth recalling the huge amount of literature on flavins and NAD(P)H as AF biomarkers of cell energetic metabolism and redox state, an aspect that should not be overlooked in the renewed course to extend the potential of flavins as AF biomarkers, entailing also a recent proposal of Flavin-based fluorescent proteins as substitutes of Green fluorescent proteins.

Fluorescence excimer formation imaging: a new technique to investigate association to cells and membrane behavior of glycolipids.

A new fluorescence ratio imaging technique aiming to monitor the lateral distribution of pyrene-labeled lipids in the membranes through visualization of the excimer/monomer (E/M) intensity ratio, has been set up, studying the association of a fluorescent derivative of GM1 ganglioside (pyreneGM1) to rat cerebellar granule cells in culture. The imaging results show that the mean E/M ratio value, under experimental conditions leading to the association of pyreneGM1 with the plasma membrane, is significantly higher in neuritic processes than in cell bodies and, moreover, locally distributed in patches. Fluorescence antisotropy imaging of the fluorescent probe TMA-DPH (trimethylaminodiphenylhexatriene) shows the presence of domains having different fluidity and that the average fluidity is higher in cell bodies than in neuritic processes. Additional experiments using TMA-DPH as a marker of fluid-phase pinocytosis, suggest that the rate of endocytosis is comparable in the two regions of the cell. Taken together, on the one hand these data indicate that the fluorescent ganglioside is present in a more clustered state at the level of neuritic processes than in cell bodies and locally distributed in patches of different size and enrichment. On the other hand, they point out the potentiality of the excimer formation imaging technique to study membrane behavior and dynamics of pyrene-labeled lipids, with a particular insight into their aggregative properties.

Changes in cell cycle and chromatin distribution in C6 glioma cells treated by dibutyryl cyclic AMP.

C6 glioma cells in culture were treated with 1 mM dibutyryl cyclic AMP (Db-cAMP) for 5, 8, 24 and 72 h. The cells were labelled with [3H]-thymidine before either the end, or the beginning, of the Db-cAMP treatment. The cell cycle passage was monitored by the simultaneous determination of DNA content and DNA synthesis in propidium iodide stained autoradiograms. The data revealed an early (t less than or equal to 3-8 h) and moderate inhibitory effect of Db-cAMP on all phases of the cell cycle except mitosis; some cells (2%) were completely blocked in the S phase. Later (8 less than t less than 24-72 h), the cycling of a substantial part of the population became inhibited in G1 phase. Microdensitometric texture analysis of Feulgen-stained nuclei, performed 24 h after administration of Db-cAMP, showed a higher inhomogeneity of the DNA distribution in cell nuclei, caused by the condensation of a part of the chromatin. This may reflect either changes in genome expression taking part in the process of cAMP induced differentiation or transit of some cells into quiescent G0 or S0 phases.

Characterisation of biotinylated liposomes for in vivo targeting applications.

Liposomes containing monosialoganglioside (GM1) or polyethylene glycol (PEG) lipid derivatives have prolonged circulation in the blood. This favours liposome extravasation to tumour sites. In this report it is shown that inclusion of GM1, PEG550-DPPE or PEG2000-DPPE in liposomes containing biotin-DPPE significantly diminished the ability of vesicles to bind to streptavidin in vitro. Steric inhibition due to the bulky head group of these lipids was least for biotin-DPPE liposomes containing GM1. Biodistribution studies in C26 tumour-bearing mice showed that GM1-liposomes containing small amounts of biotin-DPPE have long circulation life-times in the blood. Using fluorescent microscopic techniques, liposomes containing both GM1 and biotin-DPPE were detected within extra-vascular spaces in tumours. In addition it was shown that biotin-DPPE in GM1-liposomes bound streptavidin in situ. These results suggest that GM1-liposomes containing biotin-DPPE have potential use as diagnostic or therapeutic reagents in pre-targeting applications dependent on the high-affinity interaction of biotin with streptavidin.

Effects of DTIC, DM-COOK and ICRF-159 on the number of circulating Lewis lung carcinoma cells detected by flow cytometry.

Circulating tumor cells can be detected by means of flow cytometry in the blood of mice bearing i.m. Lewis lung carcinoma. This technique can be applied in the case of aneuploid tumors and does not require either concentration of nucleated cells or other processing of the blood samples. It offers the advantages of simplicity and speed and allows quantitative measurement of the number of circulating tumor cells. It can be applied to the study of the effects of drug treatment on the number of circulating tumor cells, for those drugs which do not cause alterations in the nuclear DNA content of normal diploid blood cells. The number of circulating tumor cells determined by flow cytometry is markedly reduced by treatment with ICRF-159, by dimethyltriazene DM-COOK, and also by its clinically used analog, DTIC.

Comparative studies on the effects of doxorubicin and differentiation inducing agents on B16 melanoma cells.

The differentiation-inducing activity of doxorubicin on B16 melanoma cells grown in vitro was compared with that of other known differentiation inducers, such as theophylline, retinoic acid, and melanocyte-stimulating hormone (MSH). At drug concentrations resulting in cytostatic effects, doxorubicin and theophylline induced morphological changes (dendritic-like structures with a terminal melanin granule) with an enhancement of total melanin content and tyrosinase activity. Retinoic acid did not alter melanin content and cell morphology, although it affected cell growth. MSH enhanced total melanin content and tyrosinase activity, with no significant morphological changes. Flow cytometric analysis showed that MSH led to an accumulation of cells in G1 phase whereas doxorubicin induced an accumulation of cells in G2 + M. Studies on DNA content in doxorubicin-treated cells, selected on the basis of a morphologically differentiated pattern, showed a clustering of these cells in G2 + M, probably due to a cytokinesis block. Thus doxorubicin can induce cell differentiation comparable with other differentiation inducers.

Sinusoidal 50 Hz magnetic fields do not affect structural morphology and proliferation of human cells in vitro.

The effects of electromagnetic fields on several processes related to cell physiology and proliferation are currently being investigated. Although the results are still not conclusive and even conflicting, there seems to be a fairly good agreement on the early effects of electromagnetic fields on the generation of free radicals and on Ca++-intracellular concentration and transport. To evaluate the long-lasting consequences of these precocious events, we examined the effects of short- and long-term magnetic field exposure on structural organization (cytokeratin or actin detection), proliferation (bromodeoxyuridine incorporation and propidium iodide staining), colony forming ability and viability (trypan blue exclusion test) of highly proliferating MCF-7 cells (from human breast carcinoma) and on slowly proliferating normal human fibroblasts (from healthy donors). Cells were exposed to either 20 or 500 microT sinusoidally oscillating (50Hz) magnetic fields for different lengths of time (1 to 4 days). Short (1 day)- and long (4 days)-time exposure to the two intensities did not affect MCF-7 growth and viability, colony number and size, or cellular distribution along the cell cycle; neither were the cell morphology and the intracellular distribution and amount of cytokeratin modified. Similarly, no modifications in the actin distribution and proliferative potential were observed in normal human fibroblasts. These findings suggest that under our experimental conditions, continuous exposure to magnetic fields does not result in any appreciable effect in both normal and tumor cells in vitro.

Equilibrium among hematoporphyrin derivative components--II. Effect of esterase activity.

Photofrin II is the hematoporphyrin-derivative fraction enriched in covalently-linked oligomers, characterized by a high degree of folding. Interaction with hydrophobic structures, such as biomolecules and cell structures, results in a modification of the equilibria among the different species, as a consequence of an unfolding effect exerted towards the electrostatic aggregates. The effect of esterase activity was evaluated, taking into account the nature suggested for the covalent linkage of the oligomers (ether and/or ester). The study was performed in Photofrin II aqueous solution by means of absorption and fluorescence spectral analysis. The results showed that the esterase is active only towards the unfold oligomers: that is, in Photofrin II solution supplemented with albumin. In these conditions, spectral analysis revealed the presence of a monomerization process, which is clearly evident during the first four hours of incubation. The monomerization effect induced by the enzyme was also proven by both equilibrium-dialysis measurements and zinc ion complexation. Zinc ion complexes with high affinity for monomeric species, giving rise to a very distinct emission band at 580 nm. The amount of ester linkage shown in the oligomers through enzyme hydrolysis appeared to be less than might have been expected, owing to the inhibiting effect of the monomer produced on the enzyme. The results are a step toward clarifying the intracellular and intratissue turnover of the drug observed after administration.

Modulation of porphyrin derivatives accumulation in C6 glioma cells by drugs acting on beta-adrenergic receptors. A spectrofluorometric study.

The pharmacological modulation of the uptake of porphyrin derivatives in cultured C6 glioma cells was investigated by means of spectrofluorometric analysis both in single cells and in cell homogenates. The influence of drugs acting as beta-receptor agonists or antagonists was studied in cells grown to semiconfluency. Isoproterenol (ISO), a beta-receptor agonist, enhanced the intracellular fluorescence intensity of both Photofrin and protoporphyrin IX (PpIX). A treatment with a beta-receptor antagonist I-propranolol (PRO), simultaneous with ISO, resulted in an intracellular Photofrin fluorescence signal comparable to that of the control cells, indicating the specificity of the pharmacological action. The pharmacological treatment seemed particularly effective with the aggregated species. This is suggested by the relative increase of the band at 670 nm, being greater than that in the 630 nm band in the emission spectra of Photofrin and PpIX, and by the comparison of the fluorescence intensity on cell homogenates measured both in the absence and in the presence of cetyltrimethyl-ammonium bromide as a detergent.

Distribution of di-sulfonated aluminum phthalocyanine and Photofrin II in living cells: a comparative fluorometric study.

Microspectrofluorometric and fluorescence imaging techniques have been employed to study the internalization and intracellular distribution of both Photofrin II, an experimental drug used in photodynamic therapy, and di-sulfonated aluminum phthalocyanine, a very promising photosensitizer. The results obtained by microscopic techniques in living cells have been compared with those obtained in solution on cell extracts. Experimental results indicated that the complexity of the drug-cell interaction can be explained according to the chemico-physical nature of the drugs. In particular, the presence of both monomeric and aggregated fractions, which are supposed to be internalized through different mechanisms, accounts for the intracellular distributions observed for both drugs, depending on the treatment conditions. Equilibria among the drug fractions take place within the cells, resulting in the persistence of the intracellular fluorescence. On the whole, the behavior of the two drugs appears very similar, except for some aspects related to the intracellular distribution, which can be explained in terms of different degree of lipophilicity of the drugs.

Dependence of fibroblast autofluorescence properties on normal and transformed conditions. Role of the metabolic activity.

The dependence of autofluorescence properties on the metabolic and functional engagement and on the transformation condition was studied on single cells. Normal Galliera rat fibroblasts at low subculture passage (cell strain), at high subculture passage (stabilized cell line), and transformed cell line derived from a rat sarcoma were used as a cell model. The study was performed by microspectrofluorometric and fluorescence imaging technique. The autofluorescence properties of cells were studied by excitation at two wavelengths, namely 366 nm and 436 nm, that are known to favor the emission of different fluorophores. Spectral shape analysis indicated that under excitation at 366 nm autofluorescence is ascribable mainly to coenzyme molecules, particularly to reduced pyridine nucleotides, while under excitation at 436 nm, flavin and lipopigment emission is favored. The energetic metabolic engagement of the different cell lines was analyzed in terms both of parameters related to anaerobic-aerobic pathways (biochemical assay) and of mitochondrial features (supravital cytometry). The results showed that the cell strain and the stabilized and transformed cell lines can be distinguished from one another on the basis of both overall fluorescence intensity and the relative contributions of spectral components. These findings indicated a relationship between autofluorescence properties and energetic metabolism engagement of the cells that, in turn, is dependent on the proliferative activity and the transformed condition of the cells. In that it is a direct expression of the energetic metabolic engagement, autofluorescence can be assumed as an intrinsic parameter of the cell biological condition, suitable for diagnostic purposes.

Enzyme-assisted cell photosensitization: a proposal for an efficient approach to tumor therapy and diagnosis. The rose bengal fluorogenic substrate.

Rose bengal, a xanthene derivative among the most efficient producer of singlet oxygen, was submitted to a chemical modification consisting in the introduction of an acetate group into the aromatic ring fluorophore structure. The acetate group acts as a quencher, thus inactivating both fluorescence and photosensitization properties of the molecule. In the modified structure, rose bengal acts as a fluorogenic substrate giving rise to the cellular reaction termed fluorochromasia. The acetate group is recognized by a carboxylic esterase activity that splits it. Removal of the quencher group results in restoring the native structure of photosensitizer inside the cells. The intracellular turnover of rose bengal acetate was studied in rat glioma-derived cultures cells, in terms of the balance of the processes of influx and enzyme hydrolysis of the fluorogenic substrate, and of the efflux of the fluorescent product. A large intracellular accumulation of photosensitizer is obtained when treatments are performed with the fluorogenic substrate, even at the drug concentration at which rose bengal does not enter the cells. The intracellular localization allows rose bengal to exert a more effective photosensitization effect. Provided that the quencher group is selected according to the metabolic properties of the tumor cells, the use of fluorogenic substrates as photosensitizer precursors could improve fluorescence diagnosis and the photodynamic therapy of tumors, exploiting the biological properties that distinguish pathological from normal conditions.

Free radical-dependent DNA lesions are involved in the delayed cardiotoxicity induced by adriamycin in the rat.

The role of free radicals in the genesis of adriamycin (ADR)-induced delayed cardiotoxicity and the cardioprotective effects of the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) were investigated in an in vivo rat model. As ADR and free radicals are no longer present in the myocardium by the time the delayed effects of the drug become apparent, ADR has been proposed to act by causing early radical-dependent DNA lesions, resulting in impaired synthesis of critical target proteins. DNA lesions were detected 10 days after ADR treatment (3X3 mg/kg i.v.) and were still present at the time of onset of the delayed cardiomyopathy. PBN, administered by a slow-release osmotic pump to maintain constant plasma levels throughout the time of persistence of ADR in the myocardium (approximately 2 weeks), prevented the development of DNA lesions, as well as the late contractile and electrical impairment induced by the anthracycline, thus supporting the hypothesis that free radicals play a causal role in both phenomena.

Metabolic energy availability and proliferative activity in adriamycin-treated cells.

Metabolic energy availability and proliferative activity were studied in HeLa cells treated with Adriamycin (Adr). Conditions (ID50) that induced growth delay, without significant cell lethality, did not appreciably alter ATP content, over 72 hours after treatment. Energy-dependent membrane permeability properties, assessed with the vital dye fluorescein, were similarly unaffected. The cytostatic effect was confirmed by the incorporation rate of 3H-thymidine and 14C-leucine, and by flow cytometric analysis of cell cycle progression. Early depletion in cellular ATP occurred when Adr induced an evident cytocidal effect.

Topoisomerase II alpha and beta in human tumor cells grown in vitro and in vivo.

The cellular content and the cell-cycle distribution of the 170 kD- and 180 kD-isoforms of DNA topoisomerase II were investigated in human tumor cells with specific monoclonal antibodies and by immunofluorescent detection with flow cytometry. Levels of topo II alpha were almost three-fold higher than the beta-isozyme in exponentially growing cells in vitro. In contrast, topo II alpha but not beta, was markedly reduced in plateau-phase cells. Tumor cells from surgical biopsies, mainly in G0/G1 phase, exhibited a 95% beta- versus 5% alpha-isoform expression. These results support the hypothesis that topo II alpha is mainly related to DNA synthesis, and topo II beta to DNA transcription.

The spin trap alpha-phenyl-tert-butyl nitrone protects against myelotoxicity and cardiotoxicity of adriamycin while preserving the cytotoxic activity.

The possibility of preventing the myelo- and cardiotoxicity of adriamycin (ADR), was explored in in vivo experiments in the rat. Assuming that free radicals play a key role in the ADR organotoxicity, a new anti-radical approach was set up by administering a spin trapping compound (PBN) which is taken up by the cells and specifically interacts with radicals. ADR was given i.v., 3 mg/kg every 3rd day for 3 times. PBN was continuously administered throughout the persistence time of ADR in myocardium (15 days). Serial ECG and leukocyte counting were performed during 10 weeks, then hearts were isolated and Langendorff-perfused; functional parameters (heart rate, contractility, coronary flow) were evaluated. PBN improved ECG and prevented the myelotoxicity, while functional parameters were not significantly different from those of control. Cytotoxicity was evaluated in vitro in 3 different human tumour cell lines; PBN did not modify the cytotoxicity of ADR, thus excluding a free radical involvement in this activity. The present results suggest that a proper administration schedule of spin traps might be a promising approach for improving the therapeutic index of ADR.

Effects of hematoporphyrin-derivative on mouse erythroleukemia cells in the absence of light irradiation.

This paper concerns a general study on the effects of hematoporphyrin-derivative (HpD) on mouse erythroleukemia (MEL) cells, in the absence of light irradiation. In particular, HpD intrinsic cytotoxicity was evaluated at different doses and the results correlated with those referring to membrane functional and morphological changes. HpD uptake and release processes were also studied and compared with the above-mentioned results. In order to have an overall picture of HpD-cell interactions, time-resolved fluorescence measurements were performed on both undifferentiated and differentiated MEL cells. The results obtained indicate that, even at HpD doses exhibiting neither any cytotoxicity nor any morphological damage (1-10 micrograms/ml), membrane permeability alterations are observed. Thirty minutes of treatment are sufficient for HpD to develop its toxic effect: indeed, no differences in HpD influence on cell viability can be observed after 30 min, 60 min or 5 days of treatment. HpD cytotoxicity is reduced by high protein content in the incubation culture medium. The presence of both monomeric species and 580 nm emitting species was observed at cellular level. The latter is likelier in undifferentiated MEL cells, which also exhibit higher overall HpD uptake, as compared with differentiated MEL cells.

The time-dependent behaviour of hematoporphyrin-derivative in saline: a study of spectral modifications.

Hematoporphyrin-Derivative (HpD), a widely-used tumor-specific photosensitizer, is a complex mixture of porphyrins whose composition has yet to be clarified. This paper reports on the behaviour of HpD in saline. From a spectroscopic point of view, the fresh solution is characterized by two main absorption peaks, attributable to monomeric and dimeric forms. With aging, a new porphyrin species (NPS) appears. To define the NPS, absorption, excitation and emission spectra were measured in different conditions and time-resolved fluorescence measurements were also performed. This species exhibits an absorption/excitation peak at 405 nm, an emission peak at 575 nm and a fluorescence decay time of approximately 3.5 ns. Its formation is strongly influenced by many environmental factors: in particular, gases diluted in the solution, temperature, pH and concentration. The presence of Oxygen and a pH value outside the 6-8 range may be considered inhibiting factors. The NPS seems to be quite important in the understanding of HpD tumor-specificity, since the presence of an emission band similar to the NPS one seems to be favoured in tumor cells as compared with normal cells.