Michel Manfait

Selective analysis of antitumor drug interaction with living cancer cells as probed by surface-enhanced Raman spectroscopy.

A new technique for the selective measurement of small amounts of antitumor drugs in the nucleus and cytoplasm of a living cancer cell, based on surface-enhanced Raman spectroscopy (SERS), is proposed. The ability to detect SERS signals from very dilute (up to 10−10M) solutions of doxorubicin or adriamycin (DOX), and 4′O-tetrahydropyranyl-adriamycin (THP-ADM), as well as from their complexes with targets in vitro and in vivo, has been demonstrated. SERS spectra were obtained from a population as well as from single living erythroleukaemic K562 cells treated with DOX. The results of the measurements on the population of cells containing DOX in nuclei or in the cytoplasm are well correlated with the microscopic SERS measurements on the single cells treated with DOX, obtained by selectively recording signals from the living cell nucleus or from the cytoplasm. Possibilities for the application of this new technique in different aspects of cancer research are discussed.

Separation of picrocrocin, cis-trans-crocins and safranal of saffron using high-performance liquid chromatography with photodiode-array detection

High-performance liquid chromatography with photodiode-array detection was used to separate picrocrocin (bitter-tasting component, glucoside of safranal), cis/trans-crocins (carotenoids, glucosyl esters of crocetin) and safranal (flavour, monoterpene aldehyde) of saffron. All components of pure red Greek saffron were extracted from dried stigma with 50% methanol. These compounds were detected, separated collected and identified simultaneously using a Merck LiChroCART 125-4 Superspher 100 RP-18 (4 microns) column and as mobile phase a linear gradient from 20% to 100% acetonitrile in water in 20 min with a detection wavelength at 308 nm.

Embolization of hepatocellular carcinoma with drug-eluting beads: Doxorubicin tissue concentration and distribution in patient liver explants

BACKGROUND & AIMS To follow the local tissue delivery of doxorubicin in HCC explants from patients embolized with drug-eluting beads and to compare it with histologic modifications. METHODS Six patients with HCC underwent chemoembolization with doxorubicin-eluting beads (caliber 100-300 μm, dose 75-150 mg) followed by liver transplantation at different time points (8 h to 36 days). On sections of the explanted liver, the tissue concentration of doxorubicin was determined radially around bead-occluded vessels with microspectrofluorimetry. The intra/peritumoral location of the beads and the modifications of the surrounding tissue were determined on an adjacent hematein-eosin-saffron-stained section and compared to drug measurements. RESULTS Doxorubicin was detected in the tissue surrounding the beads at all times of explantation. The drug impregnates an area of at least 1.2 mm in diameter around the occluded vessel. The tissue concentration of drug ranges from 5 μM at 8 h to 0.65 μM at 1 month. In patient transplanted at 8 h, no major tissue modification was observed and we found 42% of the beads occluding intratumoral vessels. Drug concentration was not different around intratumoral and peritumoral occluded vessels. After 9-14 days, necrosis was present around 37% of vessels and at 32-36 days, around 40% of vessels. Necrotic tissue was associated with a deeper penetration and a higher concentration of the drug than non necrotized areas, though statistically significant only at 32-36 days. CONCLUSIONS Doxorubicin-eluting beads provide a sustained delivery of drug for a period of 1 month and local tissue concentrations above cytotoxic threshold in HCC-bearing livers.

Elevation of glucosylceramide in multidrug-resistant cancer cells and accumulation in cytoplasmic droplets.

Multidrug‐resistant (MDR) cancer cells have been shown to have an accumulation of glucosylceramide (GlcCer). In this study, we aim at localizing, at subcellular level, where these lipids accumulate. Neutral lipids and phospholipid containing organelles have been identified using confocal fluorescence microscopy and microspectrofluorometry by monitoring the emission of the fluorescent probe Nile‐red. Data from confocal fluorescence microscopy analysis shows accumulation of neutral lipids in cytoplasmic droplets of MDR human carcinoma MCF7R cells. Microspectrofluorometric measurements show an increase of the gold‐yellow emission intensity in MCF7R cells, corresponding to neutral lipids. Similar observations were made in human MDR vincristine‐HL60 and doxorubicin‐KB selected cells. Total cellular glucosylceramide (GlcCer) measurements using [3H]‐palmitic acid and thin layer chromatography show a significant increase of GlcCer in MCF7R cells. Moreover, MCF7R cells treated with fluorescent GlcCer‐bodipy exhibit an accumulation of this lipid in cytoplasmic droplets. Treatment of MCF7R cells with 1‐phenyl‐2‐palmitoylamino‐3‐morpholino‐1‐propanolol (PPMP), a potent inhibitor of GlcCer synthase, attenuates the Nile‐red fluorescence emission emanating from these structures and reverses MDR. Moreover, Golgi compartments stained with fluorescent PPMP‐bodipy, show an increase in the Golgi compartments density. Treatment of MCF7R cells with cyclosporine A (CSA), tamoxifen (TMX) and 3′‐azido‐3′deoxythymidine (AZT) leads to the same effect observed in the presence of PPMP. Treatment of MCF7 and MCF7R with the β‐glucosidase inhibitor conduritol β‐epoxide (CBE) significantly increases resistance to daunorubicin only in MCF7R cells. These data demonstrate also that: (i) CSA, an inhibitor of MDR, has an additional target in addition to P‐glycoprotein; and (ii) TMX (used in breast cancer treatment and prevention) and AZT (used in the treatment of HIV) could have side effects by disturbing lipid metabolism and inhibiting many cellular functions required in normal cells.

Micro-Raman Spectroscopy for Optical Pathology of Oral Squamous Cell Carcinoma

Micro-Raman spectra of formalin-fixed oral squamous normal and carcinoma tissues, stored at room temperature for 2 months, have been recorded. Spectra were recorded both in the epithelial and subepithelial regions of the tissues. No noticeable spectral contamination due to formalin was observed. Very significant differences between spectra of normal epithelial and malignant epithelial samples were found. No such differences in spectra of subepithelial malignant and subepithelial normal samples could be observed. This study shows that spectra from the epithelial region changes drastically because of malignancy-induced biochemical changes in this region. Major differences between normal and malignant spectra seem to arise from the protein composition, conformational/structural changes, and possible increase in protein content in malignant epithelia. The differences between normal epithelial and subepithelial spectra, as expected, arise mainly from the collagen in subepithelial tissue. Principal component analysis of the combined sets of spectra—epithelial and subepithelial, normal and malignant— showed that very good discrimination can be achieved by Raman microspectroscopy. This study thus validates the suitability of formalin-fixed tissues for optical pathology in oral malignancy.

Quantitative study of doxorubicin in living cell nuclei by microspectrofluorometry

Doxorubicin-DNA association has been studied by quantitative microspectrofluorometry. Fluorescence emission spectra from a microvolume of single living cell nuclei treated with doxorubicin have been analyzed in terms of difference in spectral shape and fluorescence yield between free and DNA-bound drug. Contribution of each spectral component to the total signal was calculated by least-squares linear regression. With this method of analysis, total drug concentration has been determined with an error of less than 10%. Moreover, the uptake into the nucleus has been studied in a non destructive way, avoiding use of 14C-labelled drug. Kinetic studies of drug accumulation into the nuclei were conducted on sensitive and resistant cells.

Monitoring of bacterial growth and structural analysis as probed by FT-IR spectroscopy

Fourier-transform infrared spectroscopy was used to explore structural changes in bacteria under different incubation conditions. In particular, differences between Bradyrhizobium japonicum (BRJ) grown in liquid and on solid media were investigated, as well as the rearrangement of BRJ after transfer from one medium to the other. The FT-IR absorption bands located between 1200 and 900 cm-1 region, vary in spectral shape and intensity when BRJ were suspended in solution medium or plated on solid medium. In agreement with the electronic micrograph data, these spectroscopic changes are due to the changes involving the bacterial wall (peptidoglycan) when BRJ are plated in agar medium. By means of this FT-IR ultrastructural study of Bradyrhizobium japonicum bacteria, it has been possible to follow and to evaluate the rate of the molecular change in bacteria without any destructive interference. This indicates that FT-IR spectroscopy can prove to be a valuable technique in the monitoring of metabolic events in bacterial cells relevant to agriculture as well as environmental and health sciences.

Characterization of Acidic Vesicles in Multidrug-resistant and Sensitive Cancer Cells by Acridine Orange Staining and Confocal Microspectrofluorometry:

To study the pH gradient status through membranes of acidic vesicles, either in sensitive or in multidrug-resistant living cancer cells, we monitored the fluorescence-emission spectra of acridine orange. Successive stainings with a pH-sensitive dye and AO showed that low-pH organelles were stained red by AO. In these compartments, high AO concentrations are driven by the pH gradient through membrane vesicles. The resulting rise in the dyes oligomeric/monomeric ratio induced an increase in the red/green (655-nm/530-nm) emission intensity ratio. Therefore, the accumulation of AO in acidic organelles was appraised by determination of the contribution of the red emission intensity (R%) in each emission spectrum, using laser scanning confocal microspectrofluorometry. In vesicles of multidrug-resistant K562-R cells, R% is significantly higher (72 ± 10%) than the value (48 ± 8%) from K562-sensitive cells (p<0.001). This result is interpreted as a more important accumulation of AO in acidic cytoplasmic structures of resistant cells, which induces a shift from AO monomers (green emission) to self-associated structures (red emission). Equilibration of the pH gradient through acidic organelles was performed by addition of weak bases and carboxylic ionophores. Ammonium chloride (0.1 mM), methylamine (0.1 mM), monensine (10 μM), or nigericine (0.3 μM) all suppressed the initial difference of local AO accumulation between both cell lines. These agents decreased the red emission intensity for the resistant cell line but not for the sensitive one. The same effects were induced by 50 μM verapamil, a pleiotropic drug-resistance modulator. Our data allow the hypothesis of a higher pH gradient through membranes of acidic organelles, which would be a potential mechanism of multidrug resistance via the sequestration of weak bases inside these organelles.

Drug-eluting Beads for Liver Embolization: Concentration of Doxorubicin in Tissue and in Beads in a Pig Model

PURPOSE To evaluate the local tissue concentrations of the antineoplastic agent doxorubicin and the amount of drug still present inside drug delivery embolization beads at different time points after embolization and to compare doxorubicin levels with histologic modifications around the beads in a pig liver model. It was hypothesized that doxorubicin-eluting beads maintain cytotoxic concentrations of drug locally over a period of several weeks, as suggested by in vitro elution tests. MATERIALS AND METHODS Left lobe hepatic artery embolization was performed in 10 pigs with 100-300-microm or 700-900-microm beads loaded with 37.5 mg doxorubicin/mL. Control unloaded 100-300-microm beads were injected in five pigs. Livers were sampled 28 days or 90 days after embolization. The amount of drug retained inside the beads was assessed with infrared microspectroscopy. Doxorubicin concentration and distribution in the tissue around the beads were determined with microspectrofluorimetry and compared with tissue modifications on hematein eosin saffron-stained sections. RESULTS Doxorubicin-eluting beads eluted 43% of their initial drug load after 28 days and 89% after 90 days. Doxorubicin was present in tissues around the beads at both time points, with a significant decrease over time (P = .0004). The drug was detected at distances as far as 600 microm from the bead edge. Doxorubicin tissue concentrations ranged from 0.55 microM to 6.80 microM, [corrected] which are cytotoxic levels in hepatocyte cell cultures. High concentrations of drug were associated with coagulative necrosis of liver parenchyma. Doxorubicin-eluting beads 100-300 microm in size induced more necrosis than 700-900-microm beads (P = .0036). CONCLUSIONS Doxorubicin-eluting beads deliver high concentrations of the drug over a period of at least 3 months at several hundred micrometers from the bead, leading to significant cytotoxic effects.

Studies of chemical fixation effects in human cell lines using Raman microspectroscopy

AbstractThe in vitro study of cellular species using Raman spectroscopy has proven a powerful non-invasive modality for the analysis of cell constituents and processes. This work uses micro-Raman spectroscopy to study the chemical fixation mechanism in three human cell lines (normal skin, normal bronchial epithelium, and lung adenocarcinoma) employing fixatives that preferentially preserve proteins (formalin), and nucleic acids (Carnoy’s fixative and methanol–acetic acid). Spectral differences between the mean live cell spectra and fixed cell spectra together with principal components analysis (PCA), and clustering techniques were used to analyse and interpret the spectral changes. The results indicate that fixation in formalin produces spectral content that is closest to that in the live cell and by extension, best preserves the cellular integrity. Nucleic acid degradation, protein denaturation, and lipid leaching were observed with all fixatives and for all cell lines, but to varying degrees. The results presented here suggest that the mechanism of fixation for short fixation times is complex and dependent on both the cell line and fixative employed. Moreover, important spectral changes occur with all fixatives that have consequences for the interpretation of biochemical processes within fixed cells. The study further demonstrates the potential of vibrational spectroscopy in the characterization of complex biochemical processes in cells at a molecular level. FigureChemical preservation of cells for Raman microspectroscopy is shown to be strongly dependent on the cell type and the fixative used, in a variety of cell lines, with formalin fixation show to result in spectral content most comparable to that in the live cell