ARTICLE |
Correspondence to: Michel Manfait, Université de Reims ChampagneArdenne, UFR de Pharmacie, IFR53, Laboratoire de Spectroscopie Biomoléculaire, 51 rue Cognacq Jay, 51096 Reims Cedex France..
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Summary |
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We used confocal microspectrofluorometry to investigate intracellular distribution of pirarubicin or THP-DOX in parental K562, CEM, and LR73 tumor cells and their corresponding multidrug-resistant (MDR) strains. Each spectrum of a recorded image was considered as a combination of cell autofluorescence and fluorescence of the drug. In the cytoplasm of parental K562, CEM, and LR73 cells, THP-DOX fluorescence emission profile was similar to that of free drug in aqueous buffer. The (I550nm/I600nm) ratio was 0.50 ± 0.1. However, in the cytoplasm of resistant cells the 550-nm band profile was modified. The I550nm/I600nm ratio was 0.85 ± 0.2 in MDR K562 cells, which is significantly different from the ratio in sensitive cells (p<0.01). This appeared first to correspond to accumulation and self-oligomerization of THP-DOX in cytoplasmic organelles of MDR cells. Transfection of LR73 cells with the mdr1 gene conferred this characteristic on the resistant LR73R cells. Bodipy-ceramide, a trans-Golgi probe, was co-localized with the typical fluorescence emission peak at 550 nm observed in the cytoplasm of MDR cells. This organelle has been shown to be more acidic in MDR cells. Moreover, this specific pattern was similar to that observed when anthracycline is complexed with sphingomyelin. The typical fluorescence emission peak at 550 nm decreased in MDR cells incubated simultaneously in the presence of the drug and quinine, verapamil, or S9788. Growth inhibitory effect and nuclear accumulation of THP-DOX data obtained on LR73R and LR73D cell lines showed that only during reversion of resistance by verapamil and S9788 was an increase of nuclear THP-DOX accumulation observed. Our data suggest that characteristics of molecular environment, such as higher pH gradient or lipid structures, would be potential mechanisms of multidrug-resistance via the sequestration of anthracyclines. (J Histochem Cytochem 46:13691376, 1998)
Key Words: multidrug resistance, anthracyclines, microspectrofluorometry, reversal, cytoplasmic environment
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Introduction |
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Studies of chemoresistance in tumor cells have revealed the existence of several mechanisms for cell survival in the presence of cytostatic drugs (
Nucleocytoplasmic distribution and compartmentalization of anthracyclines have already been investigated, using confocal fluorescence microscopy, on both sensitive and resistant tumor cells (
In our previous works, using confocal laser microspectrofluorometry (
Here we report on the spectroscopic evidence of a specific interaction of pirarubicin (THP-DOX) in cytoplasmic organelles of MDR cells. This specific spectral pattern disappeared in resistant cells incubated simultaneously in the presence of the drug and MDR modulators. Data obtained on growth inhibitory effect and nuclear accumulation of THP-DOX in the presence of MDR modulators, particularly quinine, poses intriguing questions regarding the role of the cytoplasmic environment of anthracyclines in MDR cells.
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Materials and Methods |
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Chemicals
Anticancer Drugs.
Pirarubicin or tetrahydropyranyl-doxorubicin (THP-DOX) was provided by Laboratoires Bellon (Neuilly, France). Doxorubicin (DOX) was purchased from Farmitalia (Milan, Italy). Vinblastine (VLB) was purchased from Sigma (St Quentin Fallavier, France). THP-DOX and DOX are prepared in PBS at a concentration of 1 mM and stored at -20C.
Resistance Modulators. S9788, a new triazinoaminopiperidine derivative, was generously given by Institut de Recherche Internationale Servier (Paris, France). Verapamil (VPL) and quinine (QUI) were purchased respectively from Biosedra (Malakoff, France) and Sigma. S9788 and QUI were prepared respectively in water and methanol at a concentration of 10 mM. VPL was provided in aqueous solution form at 5 mM. All stock solutions of modulators were kept at -20C.
Other Chemicals. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) or MTT and trypsin were purchased from Sigma. The trans-Golgi probe DMB-ceramide (C5-DMB-Cer) was purchased from Molecular Probes (Eugene, OR).
Cell Lines
LR73 Chinese hamster ovary-sensitive carcinoma cells were transfected with the mammalian expression plasmid pDREX4 containing the biologically active cDNA insert of phage-DR11 (LR73R) (
K562 is a human erythroleukemic cell line established from a patient with chronic myelogenous leukemia in blast transformation (
CEM is a human leukemic lymphoblastic cell line (
Cytotoxic Effect of THP-DOX
LR73 cells were maintained at 37C in the absence of THP-DOX in a 96-multiwell dish (103 cells/well). After 1 day, THP-DOX (0.1 nM100 nM) was added to each sample in the presence or absence of different concentrations of each modulator. After 48 hr, LR73 cells were washed and incubated for 2 days in fresh medium before MTT assay measurements. Cell viability was then determined by addition of 20 µl of 2.5 mg/ml MTT for 3 hr at 37C. Then the medium was discarded and 200 µl DMSO was added to each well. Optical densities were measured at 540 nm using a Series 750 microplate reader (Cambridge Technology; Watertown, MA). IC50 (concentration that induces 50% inhibition of cell growth) values were calculated from the ratio of optical densities of treated cells to that of control cells (
Confocal Laser Scanning Microspectrofluorometry
This technique allowed the acquisition and analysis of fluorescence signals from a microvolume of living cancer cells treated with the anthracycline. The original microspectrofluorometer M51 (Dilor; Lille, France) is equipped with an ionized argon laser (2065; Spectra Physics, Les Ulis, France). For our measurements the 457.9-nm excitation line was used. The optical microscope BX40 (Olympus; Tokyo, Japan) equipped with a x100 phase contrast water-immersion objective UVFL-100PL permitted a micrometer spatial resolution (
Confocal Spectral Image Acquisition
Confocal spectral imaging analysis is an effective probe for antitumor drug distribution in single living cells. This approach is based on a line illumination system that employs a two-dimensional detector and a system of two synchronized scanning mirrors, which provides simultaneous spectral accumulation from hundreds of points on the sample in real confocal mode for each point. While the laser beam is being moved by the first scanning mirror along the X axis in the plane of the cell, the emitted light from the line is time-encoded on the first scanning mirror and transmitted to the pinhole diaphragm for confocal filtration. After passing through the diaphragm, the signal is decoded by the second scanning mirror vibrating in phase with the first one. Then the light is dispersed into spectra by the grating spectrograph and focused onto a two-dimensional CCD detector. The sample holder is moved with an automatic scanning stage along the Y axis with a minimal step size of 0.1 µm. A set of 50 x 50 spectra are recorded at different locations of the cell and a whole spectral image is generated by the line-by-line scanning system. The scanning of the sample stage and mirrors of the optical scanners and all operations connected with recording of spectra are computer-controlled. All treatment and analysis of spectral images were acquired with an RISC 6000 model 520 workstation (IBM).
Determination of Nuclear Concentration of THP-DOX
The fluorescence emission spectrum originating from nuclei of LR73-treated cells F(), can be expressed as a sum of the spectral contributions of free THP-DOX, DNA-bound THP-DOX, and signal of nuclear autofluorescence (
F() = Cf.Ff(
) + Cb.Fb(
) + Cn .Fn(
)
where Ff and Fb are the fluorescence spectra of free and bound drug referred to a unitary concentration. Taking this concentration into account, Cf and Cb represent intranuclear concentrations of free and bound drug, respectively. Cn is the contribution of autofluoresence responsible for the intrinsic nuclear spectrum Fn. In aqueous solution, each of these contributions has a characteristic spectral shape. The fluorescence yield in the free form was 40 times higher than that of the bound-DNA form. These spectral contributions lead to the concentrations of free and DNA-bound THP-DOX. The sum of the values obtained gives the total nuclear concentration of THP-DOX (
Co-localization of THP-DOX and DMBCeramide in LR73D Cells
Cells were incubated in the presence of C5-DMBCer complexed with defatted BSA (DF-BSA) for 30 min at 2C, washed, and incubated with 5 µM THP-DOX in HMEMB for 2 hr at 37C. The complex contained 5 µM of both the fluorescent lipid and DF-BSA and was prepared in 10 mM 4-(2- hydroxyethyl)-1-piperazineethane sulfonic acid-buffered Eagle's MEM, pH 7.4, containing 0.5 mM choline chloride, ethanolamine, serine, and (myo)inositol (HMEMB) (
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Results |
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Cytoplasmic Localization of THP-DOX in MDR Cell Lines
For spectral measurements, cells at 100,000 cells/ml density were incubated in the presence of 1 µM THP-DOX for 2 hr at 37C. Figure 1A shows the spectra recorded from the cytoplasmic region of K562 and K562R cells. After subtraction of autofluorescence, analysis of the spectral contributions of THP-DOX showed that, in the cytoplasm of K562 cells, THP-DOX emission was similar to that in PBS. In this case, the intensity ratio of the 550- and 600-nm bands (I550nm/I600nm) was 0.5 ± 0.1 in K562 cells and 0.5 in PBS. However in the cytoplasm of K562-resistant cells, the I550nm/I600nm was 0.85 ± 0.2, which was significantly different from the ratio in sensitive cells (p<0.01). We observed the same spectral profile in CEMR, LR73R, and LR73D cells. It is important to note that the LR73R cell line was transfected with the mdr1 gene. Data on the emission intensity ratios (I550nm/I600nm) corresponding to sensitive and resistant K562, CEM, and LR73 cells are summarized in Figure 2A.
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Using the scanning system, a set of 50 x 50 spectra were recorded at different locations in the tumor cells and the integrated intensities of spectra allow a fluorescence spectral image equivalent to that expected with a conventional microscope to be obtained. In each of the 50 x 50 spectra, the contribution of the model spectra, i.e., (a) fluorescence spectrum of untreated cells, (b) fluorescence spectrum of THP-DOX in the cytoplasm of a sensitive cell, and (c) fluorescence spectrum of THP-DOX in the cytoplasm of resistant cells, were determined (Figure 1).
Taking into account the scale and by employing a pseudocolor intensity representation, the integrated intensities of these three contributions allowed us to obtain three images: (a) autofluorescence of the cell (data not shown), (b) THP-DOX fluorescence in a sensitive-like environment, and (c) THP-DOX fluorescence in a resistant-like environment. We investigated the distribution of THP-DOX in the resistant cell lines K562R, CEMR, LR73D, and LR73R. As expected, Figure 3 shows that in K562R cells the contribution of THP-DOX in a resistant-like environment was more important than the contribution of the free drug. In contrast, the images from K562 cells showed a significant contribution of free THP-DOX in the cytoplasm and no spectral contribution of THP-DOX as recorded in a resistant-like environment. A similar pattern of THP-DOX distribution was observed in the sensitive and resistant CEM and LR73 cells.
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To identify the organelle in which THP-DOX is localized in MDR cells and especially the 550-nm emission band, LR73D cells were incubated in the presence of C5-DMBCer and THP-DOX (see Materials and Methods). The results in Figure 4 show that THP-DOX fluorescence emission in both the sensitive- and resistant-like environments (Figure 1A, Spectrum 1) appears to be co-localized. This co-localization (cf. Figure 4E and Figure 4F) is probably due to the existence of an equilibrium between the free and bound forms of the drug. Distribution of the monomeric form of C5-DMBCer (green fluorescence) appears to be more diffuse in the cytoplasm, whereas the oligomeric form (red fluorescence) is more localized in the trans-Golgi apparatus. In fact, DMBceramide in the cis-Golgi (monomer) is converted to DMBsphingomyelin, which accumulates in the trans-Golgi. This red fluorescence was partially co-localized with THP-DOX fluorescence in a resistant-like environment (cf. images 4D and 4E). Figure 1B shows that this specific pattern of fluorescence emission (Figure 1A, Spectrum 1) was similar to that obtained when THP-DOX is complexed to sphingomyelin (Figure 1B, Spectrum 1). THP-DOX complexed to sphingomyelin was prepared as described by
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Effect of the Reversing Agents on THP-DOX Distribution in LR73 Cells
To study the effect of MDR modulators on the subcellular distribution of THP-DOX in MDR cells, LR73R and LR73D at 100,000 cells/ml were incubated simultaneously with 1 µM of THP-DOX and 5 µM QUI, VPL, or S9788, maintained for 2 hr at 37C, and then washed with PBS at 4C. The effect of VPL, S9788, and QUI on emission intensity ratio (I550nm/I600nm) of THP-DOX are summarized in Figure 2B. The results show that modulators were able to decrease the fluorescence intensity ratio (I550nm/I600nm).
We have evaluated the cytotoxic effect and nuclear accumulation of THP-DOX. For this, LR73D and LR73R cells were incubated with THP-DOX and reversing agents (S9788, QUI, and VPL) as described in Materials and Methods. The results obtained are summarized in Table 1. At modulator concentrations that induce less than 10% cell death, S9788 and verapamil appear more potent than QUI. Verapamil and S9788 decreased the IC50 to the same level as that in sensitive cells. The reversing activity of QUI was different in the two cell lines. Treatment with 20 µM QUI decreased the IC50 of THP-DOX in LR73D cells to the same level as that obtained in LR73 cells (the reversing factor was 43), whereas in LR73R cells the reversing factor was 14.4.
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To determine if co-incubation of MDR cells in the presence of THP-DOX and S9788, QUI, or VPL induces an alteration in the nucleocytoplasmic distribution of THP-DOX, we measured the nuclear concentration of THP-DOX by confocal laser microspectrofluorometry. In this study, LR73 cell lines were incubated simultaneously with 1 µM THP-DOX and an appropriate modulator for 2 hr at 37C. It should be noted that, for all experiments performed on all cell lines, the intercell variations were small, and for each experiment the nuclear drug concentration was measured in 2030 nuclei. Table 2 shows that VPL and S9788 were able to restore drug accumulation in nuclei of resistant cells, whereas QUI was unable to significantly increase nuclear THP-DOX accumulation even at a much higher concentration (20 µM). In addition, LR73D and LR73R cells were not affected in the same manner in the presence of VPL. Nuclear accumulation of THP-DOX in LR73D cells appeared to be less affected by VPL than in the transfected cells, whereas S9788 increased drug accumulation in the same manner in both resistant cell lines.
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Discussion |
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Cytoplasmic Interaction of THP-DOX in MDR Cells
We report here that the specific fluorescence spectral shape of THP-DOX recorded in the cytoplasm is different in MDR cells (I550nm/I600nm = 0.85 ± 0.2) compared to sensitive ones (I550nm/I600nm = 0.5 ± 0.1). Our spectral imaging data show that the observed phenomenon in MDR cells does not depend on the origin of the MDR cell line (K562, CEM, and LR73 cells) and on the drug used for resistance selection (doxorubicin or vinblastine). Moreover, transfection of LR73 cells with the mdr1 gene leads to the observation of this fluorescence spectral pattern. Bodipyceramide (green fluorescence), which is converted to bodipysphingomyelin in the cis-Golgi and accumulated in the trans-Golgi (red fluorescence), was co-localized with the typical fluorescence emission peak at 550 nm relative to the anthracycline in the cytoplasm of MDR cells. Neutral red, a vital probe of lysosomes, did not have the same cytoplasmic distribution as THP-DOX in resistant cells (data not shown). The same pattern of fluorescence emission at 550 nm was obtained in solution when THP-DOX was complexed with sphingomyelin (Figure 1B). In this case, the complex THP-DOXsphingomyelin was prepared as described by
Models of THP-DOX Interaction in the Cytoplasm of MDR Cells
Phospholipid Hypothesis.
Results obtained on THP-DOX when complexed to sphingomyelin suggest that a lipidic environment of MDR cells could play an important role in THP-DOX distribution.
pH Hypothesis.
pH could also play an important role in intracellular drug distribution. The pH-dependent character of anthracycline entrapping in unilamellar vesicles has been shown (
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Acknowledgments |
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We are grateful to the Institut de Recherche International Servier and Laboratoires Bellon (Paris, France) for their kind gifts of S9788 and pirarubicin, respectively. We thank Dr G.D. Sockalingum for his assistance in the preparation of the manuscript.
Received for publication February 24, 1998; accepted August 4, 1998.
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