Correlation to Drug Resistance and Influence of Lonidamine1

1590 Vol. 6, 1590–1597, April 2000 Clinical Cancer Research

Energy Metabolism of Human LoVo Colon Carcinoma Cells: Correlation to Drug Resistance and Influence of Lonidamine1

Maurizio Fanciulli, Tiziana Bruno, INTRODUCTION Aldo Giovannelli, Francesco P. Gentile, The major obstacle to effectiveness in cancer chemother- Monica Di Padova, Oriana Rubiu, and apy is the development of drug resistance, which remains one of Aristide Floridi2 the main reasons of treatment failure. Some tumors are inher- ently resistant to cytotoxic drugs; others initially respond, but Laboratory of Cell Metabolism and Pharmacokinetics, Center for Experimental Research, Regina Elena Cancer Institute, Rome, Italy become resistant during treatment as consequence of the selec- [M. F., T. B., M. D. P., A. F.]; Department of Experimental Medicine, tion of preexisting resistant cell population and/or drug induced University of L’Aquila, 67100 L’Aquila, Italy [A. G., A. F.]; and mutations. The diminished sensitivity to the original drug is Laboratories of Biophysics [A. G.] and Medical Physics [F. P. G., O. R.], Center for Experimental Research, Regina Elena Cancer often associated with a cross-resistance to other drugs, diverse in Institute, 00158 Rome, Italy their chemical structure and targets, and this phenomenon is referred to as MDR3 (1). One of the most important mechanisms of MDR involves a ABSTRACT decrease of drug content within the cell resulting from an The relationship between modification of energy me- increased expression of a Mr 170,000 membrane glycoprotein, tabolism and extent of drug resistance was investigated in termed P-170 (2–5). This protein, a product of the MDR-1 gene, two sublines (LoVoDX and LoVoDX10) from human LoVo acts as an energy-dependent efflux pump that reduces the intra- colon carcinoma cells that exhibit different degrees of resist- cellular concentration of antitumor drugs by exporting them ance to doxorubicin. Results indicated that the extent of across the plasma membrane. alteration in energy metabolism strictly correlated with de- Neoplastic transformation modifies cellular energy metab- gree of resistance. In LoVoDX cells, only 14CO production 2 olism (6, 7). In normal differentiated cells, oxidative phospho- was enhanced, whereas in the more resistant LoVoDX10 14 rylation is the major metabolic pathway for ATP synthesis. In cells, both CO2 and aerobic lactate production were stim- ulated. The basal and glucose-supported efflux rate and the cancer cells, even in the presence of oxygen, glucose catabolism is elevated with associated higher lactate production, which amount of drug extruded by LoVoDX10 cells were significantly higher than in the resistant LoVoDX cells. Because increases with malignancy (8–11). Elevate aerobic glycolysis the expression of surface P-170 glycoprotein was similar in raises the concentration of glucose 6-phosphate, which not only both cell lines, this phenomenon was attributed to increased provides a cellular supply of ATP but also produces high levels efflux pump activity resulting from greater ATP availability. of metabolites for lipid, protein, and nucleic acid biosynthesis, 14 Inhibition of CO2 production, aerobic glycolysis, and clo- all of which are necessary for cell growth and replication (12). nogenic activity by lonidamine (LND) increased with en- The development of MDR generally correlates with enhancement of the energy metabolism. Moreover, LND, by hanced energy metabolism, consistent with a higher energy affecting energy-yielding processes, reduced intracellular requirement for drug efflux at the expense of ATP hydrolysis by ATP content, lowered the energy supply to the ATP-driven P-170. However, in a variety of doxorubicin-resistant cell lines, efflux pump, and inhibited, almost completely, doxorubicin there does not appear to be a single or common metabolic extrusion by resistant LoVo cells. These findings strongly alteration, with resistant cell lines developing metabolic alter- suggest that LND, currently used in tumor therapy, reduces ations on the basis of histogenetic derivation and/or pattern of drug resistance by restoring the capacity to accumulate and malignancy (13–18). retain drug of cells with the MDR phenotype that overex- The current study was undertaken to examine the relation- press P-170. ship between the modification of energy metabolism and extent of drug resistance in two sublines from human LoVo colon carcinoma cells that exhibit different degrees of resistance to doxorubicin. Moreover, the sensitivity to energy-depleting Received 7/16/99; revised 12/17/99; accepted 12/20/99. agents of cells with the MDR phenotype, due to an overexpres- The costs of publication of this article were defrayed in part by the sion of P-170, would be expected to increase as a function of the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to energy metabolism. Therefore, the effect of LND, which inhibits indicate this fact. both energy-yielding processes (19–21), was also evaluated. 1 This study was funded in part by the Ministero dell’Universita`e Ricerca Scientifica e Tecnologica (60%) and also by the Ministero della Sanita`. M. D. P. is a Fellow of Federazione Italiana Ricerca sul Cancro. 2 To whom requests for reprints should be addressed, at Department of Experimental Medicine, University of L’Aquila, Via Vetoio, Coppito 2, 67100 L’Aquila, Italy. Phone: 39-0862-433521; Fax: 39-0862-433523; 3 The abbreviations used are: MDR, multidrug resistance; LND, E-mail: [email protected]. lonidamine.

MATERIALS AND METHODS tion through a BP 530/15 filter, and the linear red propidium 14 Chemicals. D-[6- C] glucose was purchased from Am- iodide fluorescence was collected after filtration through an LP ersham Pharmacia Biotech; ATP and reagents for enzymatic 620 filter. Ten thousand duplicated events for each sample were assay were obtained from Roche Molecular Biochemicals. accumulated and data were analyzed using Becton Dickinson N-Tris(hydroxymethyl)-2-aminoethanesulfonic acid was pur- LYSIS II-C32 software. 14 chased from Sigma. Ham’s F-12 culture medium, FCS, and Assay of CO2 Production, Aerobic Glycolysis, ATP glutamine were purchased from BioWhittaker. Doxorubicin was Content, and Enzyme Activities. Doxorubicin-sensitive and furnished by Farmitalia. Hyamine was purchased from Packard -resistant subclones of human LoVo colon carcinoma in expo- Instruments Italia, and Aquassure scintillation liquid was pur- nential growth (5th day of culture) were detached by EDTA chased from NEN Life Science Products. All other reagents (0.02% in Ham’s F-12 medium for 5 min), recovered by cen- trifugation (600 ϫ g for 5 min), washed twice in NKT buffer were of analytical grade and purchased from BDH Italia. (105 mM NaCl, 5 mM KCl, 50 mM N-tris(hydroxymethyl)- Cell Lines. Doxorubicin-sensitive and -resistant human 2-aminoethanesulfonic acid, pH 7.40), counted in a Coulter LoVo colon carcinoma cells were kindly supplied by Dr. M. counter (model ZM, Coulter Electronics), and resuspended in Colombo (Istituto Nazionale Tumori, Milano, Italy). The doxo- NKT medium at a concentration of 5 ϫ 107 cells/ml. Determi- rubicin-sensitive cell line (LoVo) was propagated as a mono- nation of 14CO production from [6-14C]glucose, aerobic gly- layer culture in Ham’s F-12 medium supplemented with 10% 2 colysis, ATP content, and enzyme activity were performed as FCS, vitamins, antibiotics, and glutamine. The doxorubicin- described previously (16, 17). resistant line (LoVoDX) were grown in the same culture me- Dose-dependent Proliferation Analysis for LND Cyto- ␮ dium supplemented with 1.2 g/ml doxorubicin. The resistant toxicity. For these experiments, 1 ϫ 105 cells were plated subclone LoVoDX10 was induced/selected as described by onto 25-cm2 culture flasks (Corning). On the 5th day of Yang and Trujillo (22) by exposing LoVoDX cells to 12 pulse culture, during exponential growth, freshly prepared drug ␮ drug treatments with doxorubicin (10 g/ml). For each pulse was added to the flasks, which were then incubated for 24 h. ϫ 5 2 treatment, LoVoDX cells (2 10 ) were plated on to a 25 cm Appropriate DMSO controls evaluated under the identical Corning flask. At exponential growth phase, cells were exposed experimental conditions exhibited no toxicity. Following in- ␮ to 10 g/ml doxorubicin for 1 h and then immediately replated cubation, medium was discarded, and cells were detached at a density of 500 cells/60-mm Petri dish in doxorubicin-free with 0.02% EDTA for 1 min and counted in a Coulter growth medium. After a 2-week incubation at 37°C, colonies Counter. Aliquots of cell suspension of known concentration were isolated by trypsinization and grown to confluence prior to were then dispersed into 80-mm plastic Petri dishes (five further pulse treatment. The LoVoDX10 subclone was estab- dishes for each point), and colonies were allowed to grow for lished 7 months following initial treatment. LoVoDX10 cells 15–18 days. In each experiment plating efficiency of at least were routinely grown in Ham’s F-12 medium supplemented five controls was assayed simultaneously. The proliferating with 10 ␮g/ml doxorubicin. Prior to experimentation, cells were fractions for different drug concentrations were normalized grown in drug-free medium for 2 weeks. with respect to the individual control for each experiment. All

Drug sensitivity of LoVo, LoVoDX, and LoVoDX10 was experiments were performed at least three times, with five evaluated by calculating the drug dose responsible for 50% cell samples for each drug concentration. The fitting and analysis

killing (IC50 value). Briefly, cells plated in triplicate at a density of curves was as reported previously (25, 26). of 2 ϫ 105 cells/60-mm-diameter tissue culture dish were incu- Analysis of Doxorubicin Distribution and Efflux. The bated for 24 h in Ham’s F-12 medium supplemented with 10% intrinsic fluorescence emission of doxorubicin, when exited at 488 FCS, and the appropriate doxorubicin concentrations were nm, was used to monitor intracellular drug distribution and to added. After 8 days, dishes were washed with PBS, and cells measure drug efflux kinetics in drug-sensitive and -resistant human were detached by trypsinization, stained with trypan blue, and LoVo colon cancer cells. For this purpose, cells were plated onto counted in a Fuchs-Rosenthal chamber. All cell lines were 35-mm dishes and used on the 5th day of culture. Cells were rinsed examined simultaneously, and all experiments were performed with fresh medium and incubated with 20 ␮M doxorubicin, in the

at least four times. presence or absence of 0.2 mM LND, for1hat37°C in 5% CO2. Drug. LND, obtained from the F. Angelini Research Cells were then washed twice with NKT buffer and mounted on the Institute, was dissolved in DMSO at a concentration of 10 stage of a Zeiss Axioscope upright microscope interfaced with a mg/ml (31.1 mM) immediately prior to use and sterilized by real time laser confocal fluorescence microscope (Odyssey, Noran filtration through a 0.22 ␮m Millex GV filter (Millipore). Instruments, Redwood City, CA) equipped with an Argon laser. Determination of P-170 Glycoprotein by Flow Cytom- Cells were visualized by a ϫ 40 water immersion objective (Zeiss, etry. The monoclonal antibody Mab57, kindly provided by numerical aperture ϭ 0.75) and continuously perfused with NKT Dr. M. Cianfriglia (Istituto Superiore di Sanita`, Rome, Italy), buffer, with or without 6 mM glucose, with a glass pipette posi- which recognizes an external domain of P-170 (23), was used to tioned close to the cell field and connected to a gravity-driven detect the P-170 expression as reported previously (24). Briefly, perfusion system. The effect of glucose on drug extrusion kinetics 5 ␮l of propidium iodide (1 mg/ml) was added to each sample was studied by switching from control to the glucose-containing prior to fluorescence-activated cell sorting analysis to exclude reservoir. Image acquisition and analysis were performed using nonviable cells. Samples were analyzed in a FACScan flow IMAGE-1 software (Universal Imaging Corp.). Cells were exited cytometer (Becton Dickinson, San Jose, CA). Logarithmic green at 488 nm, and fluorescence emission was monitored at ␭Ͼ515 fluorescence of FITC-bound Mab57 was collected after filtra- nm with a confocal slit of 100 mm. The laser beam was set at

Table 1 Doxorubicin sensitivity of LoVo, LoVoDX, and LoVoDX10 carcinoma cells Experiments were performed as described in “Materials and Meth-

ods.” IC50 values correspond to the drug concentration responsible for 50% inhibition of cell survival. The data shown (representing the mean Ϯ SD) were from four independent experiments performed in triplicate. ␮ Cells IC50 ( M) Resistance over LoVo LoVo 0.05 Ϯ 0.01 LoVoDX 2.10 Ϯ 0.04 42 Ϯ LoVoDX10 10.20 0.10 204

60–90% intensity. Fluorescence was monitored for 50 min at one digitized image/min to minimize doxorubicin bleaching and irradiation toxicity by a customized protocol within Image-1. An averaged image (256 ϫ 256 pixels) from 100 images, taken by exposing the cells to light for 4 s, was stored Fig. 1 Flow cytometric analysis of P-170 expression in the sensitive LoVo cell line and the doxorubicin-resistant LoVoDX and LoVoDX10 on the computer hard disc. In some experiments real time cell lines. acquisition (video rate, 25 Hz) was used to better resolve the drug extrusion kinetics. In this case, images were stored on a video recorder (Sony VO 9600P, Tokyo, Japan). Both sets of images were analyzed off-line, extracting fluorescence values ϫ 7 14 (307 cpm/2 10 cells/h). CO2 production by resistant Lo- in the function of time from manually positioned areas on VoDX cells was significantly higher (2852 cpm/2 ϫ 107 cells/h) nuclear and cytoplasmatic regions of each cell, using the and was dramatically reduced by LND (1692 cpm/2 ϫ 107 brightness versus time function of IMAGE-1. Data were cells/h). Oxidative degradation of glucose and the effect of LND displayed as space averaged fluorescence intensity. At least increased as cells became more resistant (5832 versus 2,349 20 cells were measured for each set of experimental data, and cpm/2 ϫ 107 cells/h). mean values were plotted and normalized with respect to the The rate of aerobic lactate production was similar for both first determination. sensitive and LoVoDX cells and was inhibited by approximately

40% in the presence of LND. Aerobic glycolysis by LoVoDX10 RESULTS cells was, in contrast, significantly higher than that of drug- ⌬ϭ Resistance Profile of LoVo Subclones. The IC50 values sensitive and drug-resistant LoVoDX cells ( 94 and 65%, for sensitive cells (LoVo) and two doxorubicin-resistant sub- respectively) and was inhibited by LND to an extent similar to ⌬ϭϪ clones (LoVoDX and LoVoDX10) were 0.05, 2.10, and 10.20 that of glucose oxidation ( 56%). ␮M, respectively, following 8 days of doxorubicin treatment The enhancement of energy-yielding pathways in resistant

(Table 1). These values indicated that LoVoDX and LoVoDX10 cells should theoretically correlate with increased levels of in- were 42 and 204 times more resistant to doxorubicin, respec- tracellular ATP. In LoVoDX and LoVoDX10 cells, ATP content tively, than the parental LoVo cell line. was 26 and 46% higher than in drug-sensitive cells, respec-

To establish whether the greater resistance of LoVoDX10 tively. LND did not significantly affect ATP concentration in could be attributed to increased expression of P-170, flow cy- LoVo-sensitive cells, but inhibition increased with resistance. tometric analysis of cell surface P-170 expression was per- ATP content was reduced by 22 and 66% in LoVoDX and in

formed in sensitive and doxorubicin-resistant LoVo cells (Fig. LoVoDX10 cells, respectively. 1). High P-170 expression was common to both resistant sub- Table 3 shows the activity of hexokinase, isocitric de- clones, in contrast to the doxorubicin-sensitive line, which did hydrogenase, and citrate synthase in sensitive and resistant not bind Mab57. human LoVo carcinoma cells. No differences in hexokinase Glucose Metabolism, ATP Content, Enzyme Activities, activity were observed in LoVo and LoVoDX cells. In Lo-

and Clonogenic Activity. It has been demonstrated that the VoDX10 cells, which exhibited higher lactate production, appearance of the MDR phenotype is associated with major hexokinase activity was markedly increased (⌬ϭ110%). modifications in energy metabolism (13–18). To investigate Higher glucose utilization in resistant cells via tricarboxylic whether enhancement of energy-yielding processes correlated acid cycle was confirmed by enhanced enzymatic activities of with the extent of drug resistance, oxidative and glycolytic two regulatory enzymes. The activity of isocitric dehydro- glucose metabolism and the effect of LND were evaluated. genase was elevated from 5 to 12 and 38 nmol/min/mg of 14 Table 2 shows CO2, aerobic lactate production and ATP protein, and the activity of citrate synthase increased from 78 content of sensitive and doxorubicin-resistant LoVo cells, in the to 115 and 474 nmol/min/mg of protein in LoVoDX and

absence and presence of LND. LoVoDX10 cells, respectively. 14 14 CO2 production by LoVo cells from [6- C]glucose was Fig. 2 shows the clonogenic activity curves of sensitive and very low (369 cpm/2 ϫ 107 cells/h) and unaffected by LND resistant LoVo cells, treated for 24 h with different LND con-

14 Table 2 Effect of LND on CO2 and aerobic lactate production and ATP content by sensitive and doxorubicin-resistant (DX and DX10) LoVo carcinoma cells ϫ 7 14 The cells (2 10 ) were incubated at 37°C for 1 h with or without 0.2 mM LND. The final concentration of glucose was 6 mM. CO2, aerobic lactate production, and ATP content are expressed as cpm/2 ϫ 107 cells/h, ␮mol/2 ϫ 107 cells/h, and nmol/2 ϫ 107 cells, respectively. Mean values Ϯ SD from seven different experiments performed in duplicate are presented. 14 ⌬ ⌬ ⌬ Cells LND CO2 (%) Lactate (%) ATP (%) LoVo Ϫ 369 Ϯ 56 3.4 Ϯ 0.6 42.4 Ϯ 4.2 ϩ 307 Ϯ 24 (NS)c Ϫ17 2.1 Ϯ 0.5a Ϫ38 36.9 Ϯ 4.8 (NS)c Ϫ13 LoVoDX Ϫ 2852 Ϯ 193 4.0 Ϯ 0.3 53.4 Ϯ 3.5 ϩ 1692 Ϯ 100* Ϫ41 2.4 Ϯ 0.2a Ϫ40 41.4 Ϯ 2.7a Ϫ22 Ϫ Ϯ Ϯ Ϯ LoVoDX10 5832 535 6.6 0.6 61.8 2.5 ϩ 2349 Ϯ 249* Ϫ60 2.9 Ϯ 0.4b Ϫ56 21.3 Ϯ 1.8b Ϫ66 a P Ͻ 0.05 vs. control. b P Ͻ 0.01 vs. control. c NS, not significant.

Table 3 Activities of hexokinase, isocitric dehydrogenase and citrate

synthase of sensitive and doxorubicin-resistant (DX and DX10) LoVo carcinoma cells Values Ϯ SD are expressed as nmol/min/mg of protein. Mean value, averaged from seven different cell preparations, is presented.

Enzyme LoVo LoVoDX LoVoDX10 Hexokinase 42 Ϯ 344Ϯ 4 (NS)a 84 Ϯ 6b Isocitric dehydrogenase 5 Ϯ 112Ϯ 3b 38 Ϯ 5b Citrate synthase 78 Ϯ 4 115 Ϯ 7c 474 Ϯ 9b a NS, not significant. b P Ͻ 0.0001 vs. sensitive cells. c P Ͻ 0.005 vs. sensitive cells.

centration in the exponential phase of growth. The colony- forming ability of LoVo and LoVoDX cells was poorly affected by LND, with both curves decreasing exponentially and D0 values of 522 and 360 ␮M obtained, respectively. The effect of

LND on clonogenic activity of LoVoDX10 cells was far more Fig. 2 Dose-response curves for exponentially growing sensitive and ϭ evident. The curve had a shoulder (n 2.2), prior to an doxorubicin-resistant LoVoDX and LoVoDX10 cell lines exposed to exponential decrease (D ϭ 110 ␮M). The presence of a shoul- increasing LND concentrations. The time of exposure was always 24 h. 0 Ϯ der indicated that 24 h treatment with LND at concentrations up Vertical bars, single experimental value SD. to 75 ␮M did not affect proliferation. Doxorubicin Distribution and Efflux. The data we report indicate that extent of resistance correlates with enhanced nuclear fluorescence. This indicated that all drug was retained energy metabolism. Doxorubicin efflux is energy-dependent (4). by the cells over the time course (Fig. 3, A–C). The intracellular

Because LoVoDX10 should extrude drug to a greater extent distribution of doxorubicin was similar in both nucleus and and/or rate than LoVoDX cells, the kinetics of drug efflux and cytoplasm (Fig. 3, B and C), as confirmed by the ratio of nuclear intracellular distribution were evaluated. to cytoplasmatic fluorescence (Fn/Fc), which, when plotted Sensitive and resistant cells were loaded with 20 ␮M doxo- against time, remained constant (Fig. 3A, inset). The values of rubicin, washed to remove the uninternalized drug, mounted on Fn/Fc calculated from 120 cells, at the beginning of each exper- a microscope stage, and perfused at a constant rate with glucose- iment, were plotted in a histogram of the number of the cells Ϯ free NKT medium. Images were acquired for 10 min, and then over Fn/Fc and peaked at 1.061 0.028 (not shown). perfusion was switched to glucose-supplemented NKT medium. Resistant LoVo cells exhibited a markedly different behav- Calibration of cellular doxorubicin concentration was not pos- ior. Fig. 3, D–F, shows doxorubicin efflux and distribution in sible under our experimental conditions. Therefore, the variation LoVoDX cells. Cells perfused with glucose-free medium elim- of intracellular fluorescence over time with respect to fluores- inated up to 20% of the doxorubicin into the medium. After 2–3 cence at the beginning of each experiment (F/F0) was evaluated. min of perfusion with glucose, the rate of outward transport F/F0 was measured in nucleus and cytoplasm of each cell and increased to a plateau, i.e., the same efflux rate as without mean results plotted against time. Each data set represents the glucose after 25–30 min. At the end of the experiment, approx- mean of at least 20 cells Ϯ SD. imately 40% of the drug had been retained by cells. The con- In LoVo-sensitive cells, perfusion with glucose-free and centration of doxorubicin in the cytoplasm was higher than in glucose-supplemented medium did not modify cytoplasmatic or the nucleus (Fig. 3E). A histogram of Fn/Fc, calculated from 110

Fig. 3 A representative experiment of extrusion kinetics and intracellular distribution of doxorubicin in sensitive LoVo (A–C), resistant LoVoDX ␮ (D–F), and resistant LoVoDX10 (G–I) cells. The cells were incubated with 20 M doxorubicin as reported in “Materials and Methods.” Drug extrusion was measured from the nuclear (E) and cytoplasmatic (F) regions. Perfusion with medium supplemented with 6 mM glucose was initiated as indicated by the arrow. Inset, ratio of nuclear to cyoplasmatic fluorescence. Bars, SE. The color scale indicates signal intensity with pseudocolors, minimum (blue) to maximum (orange) intensity. The experiments were repeated with four different cell preparations.

cells in multiple experiments, was symmetrical around a mean leased into the medium. As for LoVoDX cells, the concen- value of 0.616 Ϯ 0.018 (not shown). The decrease of nuclear tration of doxorubicin was higher in the cytoplasm, and the

doxorubicin was less than that of cytoplasmatic decrease as Fn/Fc ratio calculated from 110 cells in several experiments Ϯ Ϯ demonstrated by the ratio Fn/Fc, which increased with time (Fig. was symmetrical around a mean value ( SD) of 0.620 3D, inset). 0.022 (not shown).

In LoVoDX10 cells, perfused without glucose, outward These data indicate a relationship between drug extru- drug transport was 2-fold greater than that observed in Lo- sion and energy metabolism. Therefore, the effect of LND, VoDX cells. The efflux rate increased dramatically when which inhibits both oxidative and glycolytic metabolism (19– perfusion was switched to glucose-supplemented medium, 21, 27), on drug efflux and distribution was evaluated. Fig. and after 45 min, the cytoplasmatic drug fraction was almost 4A shows that in sensitive cells, LND did not affect intracel- completely (91%) released into the medium. The release of lular doxorubicin concentration but modified its distribution, doxorubicin accumulated within the nucleus proceeded at the resulting in an elevated cytoplasmatic concentration (Fig. 4, same rate of cytoplasmatic release for the first 20 min, A–C). slowing thereafter, resulting in an increased ratio of nuclear LND had a dramatic effect on the efflux of doxorubicin to cytoplasmatic fluorescence (Fig. 3G, inset). At the end of from resistant cells. Both basal and glucose-supported drug the experiment, 80% of nuclear doxorubicin had been re- extrusion were almost completely inhibited. Indeed, after 50

Fig. 4 A representative experiment of the effect of LND on the kinetics of extrusion and intracellular distribution of doxorubicin in sensitive (A–C), ␮ resistant LoVoDX (D–F), and resistant LoVoDX10 (G–I) cells. The cells were incubated with 20 M doxorubicin and 0.2 mM LND, as reported in “Materials and Methods.” Other conditions were as reported in Fig. 3. The experiments were repeated with four different cell preparations.

min, 100 and 85% of the drug was still retained by LoVoDX despite this low concentration, LND was still able to reduce IC50 ␮ (Fig. 4D) and LoVoDX10 cells (Fig. 4G), respectively. The concentration from the 10.2 to 2.5 M. nuclear drug concentration in LoVoDX (Fig. 4D, inset, E, and

F) and LoVoDX10 cells (Fig. 4G, inset, H, and I) was higher DISCUSSION than that of cytoplasm. The Fn/Fc ratio, evaluated from 130 cells pooled from several experiments, indicated a largely symmet- The data reported in this communication provide, to our rical distribution peaking around a mean Ϯ SD of 1.27 Ϯ 0.06 knowledge, the first experimental evidence that the extent of (not shown), i.e., a value quite similar to that found for the alterations in energy metabolism strictly correlate with degree of sensitive cells. drug resistance. Table 4 shows the effect of LND exposure on doxorubicin In doxorubicin-sensitive cells, glycolysis was the main sensitivity of LoVo-resistant cells. In LoVoDX cells, 8 days of energy-yielding process, as indicated by the very low amount of treatment with 50 ␮M LND, i.e., a concentration that by itself glucose metabolized via the Krebs cycle. In resistant LoVoDX

was unable to affect cell survival, lowered the IC50 value from cells, the most relevant metabolic modification was a marked ␮ 14 14 2.1 to 0.5 M doxorubicin. LoVoDX10 cells were more sensitive increase in CO2 production from [6- C]glucose (Table 2), 14 to LND; therefore, to avoid any effect on cell survival, a which releases CO2 only at the level of the Krebs cycle, concentration 8 times lower (6.25 ␮M) was used. Nevertheless, whereas aerobic glycolysis remained essentially unmodified. In

Table 4 Effect of LND on doxorubicin sensitivity of LoVoDX and may be an acidic shift of cytosolic pH (33), resulting from LoVoDX10 carcinoma cells lactate accumulation. Indeed, Ben-Horin et al. (34) and Vivi et Cell number was evaluated after 8 days of treatment as described in al. (35, 36) report that the lower extracellular content of lactate Ϯ “Materials and Methods.” The data shown represent the mean SD of in LND-treated adriamycin-sensitive and -resistant MCF-7 cells seven independent experiments performed in triplicate. is dependent upon impaired outward lactate transport. However, ␮ ␮ ␮ ␮ M 50 M LoVoDX M 6.25 M LoVoDX10 such a mechanism may be excluded because low extracellular ϫ Ϫ6 ϫ Ϫ6 doxorubicin LND cells 10 doxorubicin LND cells 10 lactate in LND-treated cells is primarily attributed to inhibition Ϫ 2.79 Ϯ 0.31 Ϫ 3.29 Ϯ 0.07 of glycolysis by impairment of mitochondria-bound hexokinase ϩ Ϯ ϩ Ϯ 2.55 0.12 3.00 0.00 (16, 20, 37), confirmed in human gliomas transplanted in nude 2.1 Ϫ 1.42 Ϯ 0.13 10.2 Ϫ 1.72 Ϯ 0.13 0.2 ϩ 2.09 Ϯ 0.06 0.5 ϩ 2.28 Ϯ 0.30 mice by Oudard et al. (38), with extent of inhibition of tumor 0.5 ϩ 1.48 Ϯ 0.03 1.0 ϩ 2.02 Ϯ 0.06 growth rate by LND strongly correlated with mitochondria- 1.0 ϩ 0.84 Ϯ 0.08 2.5 ϩ 1.68 Ϯ 0.06 bound hexokinase activity. Moreover, LND decreases the acid- ϩ Ϯ ϩ Ϯ 2.0 0.56 0.08 5.0 1.48 0.05 ity of vacuolar system organelles due to a decreased ATP content and increased ion membrane permeability (39). In conclusion, although our results neither prove nor dis- prove the existence of alternative pathways for MDR (e.g., a this regard, LoVoDX cells differ from adriamycin-resistant hu- passive-trapping mechanism), they clearly demonstrated that man MCF-7 breast cancer cells, which exhibit increased glycol- inhibition of doxorubicin extrusion by LND in LoVo-resistant ysis (13–16) and adriamycin-resistant Ehrlich ascites tumor cells can be attributed to an effect on oxidative and glycolytic cells, in which both respiration and glycolysis were enhanced metabolism that results in reduced intracellular ATP content, (18). thus lowering the energy supply to the ATP-driven efflux pump.

LoVoDX10 cells, which exhibit an even more resistant There are a number of potential therapeutic implications phenotype, had increased glycolytic and Krebs cycle regulatory from this work. If MDR is associated with to P-170 overexpres- 14 enzyme activity and produced more CO2, and aerobic lactate sion, then LND, currently used in tumor therapy, may reduce or increased (Table 2), leading to greater ATP availability, which, reverse drug resistance by restoring the capacity to accumulate in turn, enhanced the activity of the ATP-driven efflux pump. and retain drug (40). It is noteworthy that in LoVo cells, reduc- The higher efflux rate and the almost complete extrusion of tion of resistance (Table 4) was obtained with an LND concen-

doxorubicin by LoVoDX10 cells could not be attributed to tration similar to (LoVoDX) or remarkably lower than (Lo- increased levels of P-170, which was similar in both of the VoDX10) the peak plasma concentration achievable in humans resistant LoVo cell lines (Fig. 1). (18–40 ␮g/ml).

The enhanced energy metabolism of LoVoDX10 cells made Moreover, LND, because of its effect on the energy-yield- them more sensitive to LND, because the effect of LND on ing processes, potentiates the therapeutic efficacy of other an- tumor energy metabolism does not depend either on the nature tineoplastic drugs or agents, including radiation and/or hyper- or the origin of the neoplastic cells, but is related mainly to thermia, while reducing toxic side effects (41–45). metabolic capacity.

The marked inhibition of LoVoDX10 cell proliferation by LND can be attributed mainly to the decrease in ATP content, ACKNOWLEDGMENTS leading to an altered functional cellular state (28–30). Such We are grateful to Dr. Andrew R. Mackay (Department of Exper- alterations can be reversed although ATP has been low for imental Medicine, University of L’Aquila) for critical reading and English revision, A. Federico (Regina Elena Institute) for HPLC anal- several hours but becomes irreversible only after 18–24 h (31). ysis, and R. Bruno for graphics work. Low ATP content was also responsible for the increased doxorubicin content in LND-treated resistant LoVo cells. De- creased ATP availability may affect the P-170 activity by two REFERENCES mechanisms. In the first, reduced ATP production would not 1. Simon, S. M., and Schindler, M. Cell biologic mechanisms of mul- supply sufficient energy for extrusion by molecular pump. In the tidrug resistance in tumors. Proc. Natl. Acad. Sci. USA, 91: 3497–3504, second, P-170 function may be affected by reduced phospho- 1994. rylation, a critical factor for MDR. Indeed, human SW 620 2. Kartner, N. Riordan, J. R., and Ling, V. Cell surface P-glycoprotein associated with multidrug resistance n mammalian cell lines. Science colon carcinoma cells treated with sodium butyrate show de- (Washington DC), 221: 1285–1287,1983. creased P-170 phosphorylation tightly correlated to reduced 3. Center, M. S. Mechanisms regulating cell resistance to adriamycin. drug efflux. Decrease in P-170 phosphorylation is observed after Biochem. Pharmacol., 13: 145–147 1985. 12 h treatment and further decreases at 24, 36, and 48 h (32). 4. Scheper, R. J., Broxterman, H. J., Scheffer, G. M., Meijer, Because drug efflux by LoVo-resistant cells was already C. J. L. M., and Pinedo, H. M. Drug transporter protein in clinical inhibited after1hofexposure to LND, an effect on P-170 phos- multidrug resistance. Clin. Chim. Acta, 206: 25–27, 1992. phorylation may be excluded. Thus, the inability of LND-treated 5. Gottesman, M. M., and Pastan, I. Biochemistry of multidrug resistance mediated by the multidrug transportes. Annu. Rev. Biochem., 62: LoVo-resistant cells to extrude doxorubicin was likely to depend 385–427, 1993. upon reduced energy supply to the ATP-driven efflux pump result- 6. Warburg, O. On the origin of the cancer cell. Science (Washington ing from inhibition of glycolysis and respiration (Table 2). DC), 123: 309–314, 1956. An alternative pathway by which LND can increase the 7. Aisemberg, A. C. The Glycolysis and Respiration of Tumor. New intracellular doxorubicin concentration in LoVo-resistant cells York: Academic Press, 1961.

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Maurizio Fanciulli, Tiziana Bruno, Aldo Giovannelli, et al.

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