Franck Sureau

SNARF-1 as an intracellular pH indicator in laser microspectrofluorometry : a critical assessment

The use of SNARF-1-AM (seminaphtorhodafluor-1-acetoxymethylester) to measure the internal pH of a single living cell by laser microspectrofluorometry has been analyzed with a lymphocyte murine B cell line A20. After incubation of the cells at 37 degrees C in the presence of 10 microM SNARF-1-AM, the internal concentration of SNARF-1 was approximately 200 microM. The enhancement of fluorescent intensity of the probe is concomitant with its leakage out of the cells. During the measurement period, this induces a continuous increase of the contribution of the external probe to the total fluorescence intensity. This prevented classical spectrofluorometry measurements, but did not preclude microspectrofluorometry measurements of internal pH. The ratio R was calculated from fluorescence intensities at 635 and 590 nm and used as an indicator of the intracellular pH. Calibration curves of the intracellular pH were obtained in the presence of nigericin and valinomycin. It appeared that both the fluorescence intensity and the ratio R were lower inside the cell than those values obtained in aqueous solutions. Possible interactions with the main biological macromolecules (i.e., DNA, proteins, membranes) were investigated as well as a possible compartmentation of the probe in cellular organelles. The modifications of probe characteristics inside the cells were attributed to the binding of the probe to cellular proteins. The intracellular pH of A20 cells, measured by SNARF-1 on 84 cells, was found to be 7.18 +/- 0.10 (with an external pH of 7.40 +/- 0.05), which corresponded with values obtained by conventional fluorometric methods.

Hypocrellin A Photosensitization Involves an Intracellular pH Decrease in 3T3 Cells

Abstract— The fluorescent pH probe carboxy‐seminaphtorhoda‐fluor‐1 (C‐Snarf‐1) has been used for laser microspectrofluorometric assays of intracellular pH in 3T3 mouse fibroblasts treated with hypocrellin A. These results are compared to those previously obtained with the structurally related hydroxylated polycyclic quinone, hypericin (Sureau et al, J. Am. Chem. Soc. 118, 9484‐9487, 1996). A mean local intracellular pH drop of 0.6 units has been observed in the presence of 1 μM hypocrellin A after 90 s of exposure to 0.1 μW of laser irradiation at 514.5 nm. The time evolution of the cytoplasm acidification for hypocrellin A‐treated cells is faster than that for cells treated by hypericin. Thus, release of protons from an excited state of hypocrellin A appears to be more efficient than that from hypericin. In addition, the pH dependence of the quenching of C‐Snarf‐1 fluorescence in 3T3 cells under continuous irradiation has been observed. It is shown here that under continuous illumination, a pH decrease is able to induce a modification of the intracellular binding equilibrium of C‐Snarf‐1 that results in an increase of C‐Snarf‐1 fluorescence intensity. This latter observation suggests that the protons generated upon the photoexcitation of hypericin or its analogs may be involved in the production of other photoreactive species. Finally, we suggest that, just as for hypericin, this pH drop may be involved in the antiviral and antitumor activity of hypocrellin A.

Microspectrofluorometry of the protonation state of ellipticine, an antitumor alkaloid, in single cells

The protonation state and intracellular distribution of ellipticine were investigated in single human mammary T47D cells by confocal laser microspectrofluorimetry. In the cell nucleus, only the protonated form of ellipticine was detected as a direct consequence of its apparent pK increase upon DNA binding. Both protonated and neutral forms were present in the aqueous cytoplasm, where the pH is close to the drug pK. When cells were incubated in high concentrations of K+, a condition that depolarizes the plasma membrane potential, ellipticine cellular accumulation was reduced. In the cytoplasm, ellipticine was mainly bound to mitochondria, and its protonation equilibrium was shifted toward the neutral form. The fluorescence spectrum of ellipticine bound to mitochondria was insensitive to valinomycin, whereas it was markedly shifted toward the protonated form after carbonyl cyanide p-trifluoromethoxy-phenylhydrazone or nigericin addition. Similar studies with ellipticine bound to isolated mitochondria suggest that it behaves as a fluorescent probe of mitochondrial pH in both isolated mitochondria and single living cells.

The effect of hypericin and hypocrellin-A on lipid membranes and membrane potential of 3T3 fibroblasts.

Hypericin (HY) and Hypocrellin-A (HA) photosensitization induce rapid depolarization of plasma membrane in 3T3 cells as revealed by confocal microspectrofluorimetry using diO-C5(3) fluorescent probe. HY and HA are also able to rigidify the lipid membrane of DMPC liposomes as indicated by the decrease of pyrene excimer fluorescence used as a marker of the lipid membrane fluidity. We have also observed a nonspecific inhibition of Na+,K+-ATPase activity due to the HY and HA photosensitization. The described effects are concentration- and light dose-dependent and generally more pronounced for HA than for HY. All these observations suggest that the lipid membranes can play an important role in the photosensitization process induced by HY and HA at the cellular level. It can be hypothesized that for HA and HY the secondary mechanism following type I or type II photosensitization process can be the peroxidation of membrane lipids as well, and thus intracellular membranes seem to be one of the most important targets of these photosensitizers.

Mechanistic Issues of the Interaction of the Hairpin-Forming Domain of tBid with Mitochondrial Cardiolipin

Background The pro-apoptotic effector Bid induces mitochondrial apoptosis in synergy with Bax and Bak. In response to death receptors activation, Bid is cleaved by caspase-8 into its active form, tBid (truncated Bid), which then translocates to the mitochondria to trigger cytochrome c release and subsequent apoptosis. Accumulating evidence now indicate that the binding of tBid initiates an ordered sequences of events that prime mitochondria from the action of Bax and Bak: (1) tBid interacts with mitochondria via a specific binding to cardiolipin (CL) and immediately disturbs mitochondrial structure and function idependently of its BH3 domain; (2) Then, tBid activates through its BH3 domain Bax and/or Bak and induces their subsequent oligomerization in mitochondrial membranes. To date, the underlying mechanism responsible for targeting tBid to mitochondria and disrupting mitochondrial bioenergetics has yet be elucidated. Principal Findings The present study investigates the mechanism by which tBid interacts with mitochondria issued from mouse hepatocytes and perturbs mitochondrial function. We show here that the helix αH6 is responsible for targeting tBid to mitochondrial CL and disrupting mitochondrial bioenergetics. In particular, αH6 interacts with mitochondria through electrostatic interactions involving the lysines 157 and 158 and induces an inhibition of state-3 respiration and an uncoupling of state-4 respiration. These changes may represent a key event that primes mitochondria for the action of Bax and Bak. In addition, we also demonstrate that tBid required its helix αH6 to efficiently induce cytochrome c release and apoptosis. Conclusions Our findings provide new insights into the mechanism of action of tBid, and particularly emphasize the importance of the interaction of the helix αH6 with CL for both mitochondrial targeting and pro-apoptotic activity of tBid. These support the notion that tBid acts as a bifunctional molecule: first, it binds to mitochondrial CL via its helix αH6 and destabilizes mitochondrial structure and function, and then it promotes through its BH3 domain the activation and oligomerization of Bax and/or Bak, leading to cytochrome c release and execution of apoptosis. Our findings also imply an active role of the membrane in modulating the interactions between Bcl-2 proteins that has so far been underestimated.

Interaction dynamics of hypericin with low-density lipoproteins and U87-MG cells.

The natural photosensitizer hypericin exhibits potent properties for tumor diagnosis and photodynamic therapy. Fluorescent properties of hypericin along with various technical approaches have been used for dynamic studies of its interaction with low-density lipoprotein and U87 glioma cells. Evidences for hypericin release from low-density lipoprotein towards cells plasmatic membrane are addressed. Subsequent subcellular bulk flow redistribution leading to non-specific staining of intracellular membranes compartment were observed within cells. It was shown, that monomers of hypericin are the only redistributive forms. Increasing concentration of hypericin leads to the formation of non-fluorescent aggregates within low-density lipoprotein as well as within the U87 cells, and can preclude its photosensitizing activities. However, the aggregation process can only account for a part of the observed emission decrease. As shown by the excited state lifetime measurements, this fluorescence quenching actually results from a combination of aggregation process and energy transfer from monomers to aggregates. In all experiments, hydrophobic character of hypericin appears as the driving force of its redistribution process.

Development of a new LDL-based transport system for hydrophobic/amphiphilic drug delivery to cancer cells

Low-density lipoproteins (LDL), a natural in vivo carrier of cholesterol in the vascular system, play a key role in the delivery of hydrophobic/amphiphilic photosensitizers to tumor cells in photodynamic therapy of cancer. To make this delivery system even more efficient, we have constructed a nano-delivery system by coating of LDL surface by dextran. Fluorescence spectroscopy, confocal fluorescence imaging, stopped-flow experiments and flow-cytometry were used to characterize redistribution of hypericin (Hyp), a natural occurring potent photosensitizer, loaded in LDL/dextran complex to free LDL molecules as well as to monitor cellular uptake of Hyp by U87-MG cells. It is shown that the redistribution process of Hyp between LDL molecules is significantly suppressed by dextran coating of LDL surface. The modification of LDL molecules by dextran does not inhibit their recognition by cellular LDL receptors and U-87 MG cellular uptake of Hyp loaded in LDL/dextran complex appears to be similar to that one observed for Hyp transported by unmodified LDL particles. Thus, it is proposed that dextran modified LDL molecules could be used as a basis for construction of a drug transport system for targeted delivery of hydrophobic/amphiphilic drugs to cancer cells expressing high level of LDL receptors.

Kinetics of Hypericin Association With Low-Density Lipoproteins

Steady‐state and time‐resolved fluorescence spectroscopy have been used for the study of the incorporation kinetics of hypericin (Hyp) into low‐density lipoproteins (LDL). Biphasic kinetics of Hyp association with LDL was observed when solutions of Hyp and LDL were mixed at various concentration ratios. The rapid phase of Hyp incorporation is completed within seconds, while the slow phase lasts several minutes. The relative contributions of the individual phases show that a higher amount of Hyp molecules (65%) are incorporated into LDL in the second phase. The kinetics of the incorporation of Hyp into LDL particles preloaded with Hyp (Hyp/LDL = 25:1) was also investigated. The decreased intensity of Hyp fluorescence is a sign of the formation of Hyp aggregates after penetration of additional Hyp molecules into Hyp/LDL = 25:1 complex. The time dependence of Hyp fluorescence was measured after mixing the complex Hyp/LDL = 200:1 with appropriate amounts of free LDL molecules. For each final Hyp/LDL ratio, an increase in the intensity and lifetime of Hyp fluorescence was observed, suggesting a monomerization of Hyp aggregates. The half‐time of Hyp transfer from Hyp/LDL complex to LDL particles is similar to the half‐time of the slow phase of Hyp incorporation into free LDL particles.

Advanced Microfluorescence Methods in Monitoring Intracellular Uptake of “Antisense” Oligonucleotides

Antisense strategy represents a promising molecular tool for efficient and selective chemotherapeutic action. It belongs among oligonucleotide strategies that employ specific single-stranded sequences of deoxyriboand ribonucleotides or their synthetic analogs to block or suppress expression of a pathogen in its early stage. This approach is also promising for studies of the biological function of the gene. However, the routine use of modified oligonucleotides in practice is complicated by non-ideal properties of currently available oligonucleotide analogs. A successful medical treatment requires not only proper binding of the modified oligonucleotide to its cellular target but also its efficient cellular uptake, stability and appropriate distribution in the intracellular environment. The latter processes can be effectively studied by various microfluorescence techniques. The paper reviews the current situation in the application of advanced microfluorescence methods in this field and gives a brief description of the oligonucleotide strategy and possibilities to support the cellular uptake, theoretical and technical basics of current fluorescence microimaging and fluorescence microspectroscopy including time-resolved measurements. Second part of the paper describes experiment preparation, surveys the most interesting studies published so far and outlines the perspectives.

Berberine as a photosensitizing agent for antitumoral photodynamic therapy: Insights into its association to low density lipoproteins

Recent years have seen a growing interest in Berberine, a phytochemical with multispectrum therapeutic activities, as anti-tumoral agent for photodynamic therapy (PDT). In this context, low density lipoproteins (LDL) play a key role in the delivery of the photosensitizer in tumor cells. We correlate the physicochemical parameters of the berberine association to LDL with the influence of LDL-delivery on its accumulation in a glioma cell line and on its photo-induced activity in view of antitumor PDT. Our results evidence an important binding of 400 berberine molecules per LDL. Changes in berberine and apoprotein fluorescence suggest different fixation types, involving various LDL compartments including the vicinity of the apoprotein. The berberine association to LDL does not affect their recognition by the specific B/E receptors, of which over-expression increases the cellular uptake of LDL-preloaded berberine. Fluorescence microscopy evidences the mitochondrial labeling of the glioma model cells, with no significant modification upon LDL-delivery. Moreover, the cellular delivery of berberine by LDL increases its photocytotoxic effects on such cells. So, this research illustrates the potential of berberine as a photosensitizing agent for PDT, in particular due to their behavior towards LDL as plasma vehicles, and gives insights into its mechanisms of cell uptake.