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Clinical Cancer Research Vol. 6, 1590-1597, April 2000
© 2000 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

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

Maurizio Fanciulli, Tiziana Bruno, Aldo Giovannelli, Francesco P. Gentile, Monica Di Padova, Oriana Rubiu and Aristide Floridi2

Laboratory of Cell Metabolism and Pharmacokinetics, Center for Experimental Research, Regina Elena Cancer Institute, Rome, Italy [M. F., T. B., M. D. P., A. F.]; Department of Experimental Medicine, University of L’Aquila, 67100 L’Aquila, Italy [A. G., A. F.]; and Laboratories of Biophysics [A. G.] and Medical Physics [F. P. G., O. R.], Center for Experimental Research, Regina Elena Cancer Institute, 00158 Rome, Italy


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The relationship between modification of energy metabolism and extent of drug resistance was investigated in two sublines (LoVoDX and LoVoDX10) from human LoVo colon carcinoma cells that exhibit different degrees of resistance to doxorubicin. Results indicated that the extent of alteration in energy metabolism strictly correlated with degree of resistance. In LoVoDX cells, only 14CO2 production was enhanced, whereas in the more resistant LoVoDX10 cells, both 14CO2 and aerobic lactate production were stimulated. The basal and glucose-supported efflux rate and the amount of drug extruded by LoVoDX10 cells were significantly higher than in the resistant LoVoDX cells. Because the expression of surface P-170 glycoprotein was similar in both cell lines, this phenomenon was attributed to increased efflux pump activity resulting from greater ATP availability. Inhibition of 14CO2 production, aerobic glycolysis, and clonogenic activity by lonidamine (LND) increased with enhancement of the energy metabolism. Moreover, LND, by affecting energy-yielding processes, reduced intracellular ATP content, lowered the energy supply to the ATP-driven efflux pump, and inhibited, almost completely, doxorubicin extrusion by resistant LoVo cells. These findings strongly suggest that LND, currently used in tumor therapy, reduces drug resistance by restoring the capacity to accumulate and retain drug of cells with the MDR phenotype that overexpress P-170.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The major obstacle to effectiveness in cancer chemotherapy is the development of drug resistance, which remains one of the main reasons of treatment failure. Some tumors are inherently resistant to cytotoxic drugs; others initially respond, but become resistant during treatment as consequence of the selection of preexisting resistant cell population and/or drug induced mutations. The diminished sensitivity to the original drug is often associated with a cross-resistance to other drugs, diverse in their chemical structure and targets, and this phenomenon is referred to as MDR3 (1) .

One of the most important mechanisms of MDR involves a decrease of drug content within the cell resulting from an increased expression of a Mr 170,000 membrane glycoprotein, termed P-170 (2, 3, 4, 5) . This protein, a product of the MDR-1 gene, acts as an energy-dependent efflux pump that reduces the intracellular concentration of antitumor drugs by exporting them across the plasma membrane.

Neoplastic transformation modifies cellular energy metabolism (6 , 7) . In normal differentiated cells, oxidative phosphorylation is the major metabolic pathway for ATP synthesis. In cancer cells, even in the presence of oxygen, glucose catabolism is elevated with associated higher lactate production, which increases with malignancy (8, 9, 10, 11) . Elevate aerobic glycolysis raises the concentration of glucose 6-phosphate, which not only provides a cellular supply of ATP but also produces high levels of metabolites for lipid, protein, and nucleic acid biosynthesis, all of which are necessary for cell growth and replication (12) .

The development of MDR generally correlates with enhanced energy metabolism, consistent with a higher energy requirement for drug efflux at the expense of ATP hydrolysis by P-170. However, in a variety of doxorubicin-resistant cell lines, there does not appear to be a single or common metabolic alteration, with resistant cell lines developing metabolic alterations on the basis of histogenetic derivation and/or pattern of malignancy (13, 14, 15, 16, 17, 18) .

The current study was undertaken to examine the relationship 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 agents of cells with the MDR phenotype, due to an overexpression of P-170, would be expected to increase as a function of the energy metabolism. Therefore, the effect of LND, which inhibits both energy-yielding processes (19, 20, 21) , was also evaluated.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
D-[6-14C] glucose was purchased from Amersham Pharmacia Biotech; ATP and reagents for enzymatic assay were obtained from Roche Molecular Biochemicals. N-Tris(hydroxymethyl)-2-aminoethanesulfonic acid was purchased from Sigma. Ham’s F-12 culture medium, FCS, and glutamine were purchased from BioWhittaker. Doxorubicin was furnished by Farmitalia. Hyamine was purchased from Packard Instruments Italia, and Aquassure scintillation liquid was purchased from NEN Life Science Products. All other reagents were of analytical grade and purchased from BDH Italia.

Cell Lines.
Doxorubicin-sensitive and -resistant human LoVo colon carcinoma cells were kindly supplied by Dr. M. Colombo (Istituto Nazionale Tumori, Milano, Italy). The doxorubicin-sensitive cell line (LoVo) was propagated as a monolayer culture in Ham’s F-12 medium supplemented with 10% FCS, vitamins, antibiotics, and glutamine. The doxorubicin-resistant line (LoVoDX) were grown in the same culture medium supplemented with 1.2 µg/ml doxorubicin. The resistant subclone LoVoDX10 was induced/selected as described by Yang and Trujillo (22) by exposing LoVoDX cells to 12 pulse drug treatments with doxorubicin (10 µg/ml). For each pulse treatment, LoVoDX cells (2 x 105) were plated on to a 25 cm2 Corning flask. At exponential growth phase, cells were exposed to 10 µg/ml doxorubicin for 1 h and then immediately replated at a density of 500 cells/60-mm Petri dish in doxorubicin-free growth medium. After a 2-week incubation at 37°C, colonies were isolated by trypsinization and grown to confluence prior to further pulse treatment. The LoVoDX10 subclone was established 7 months following initial treatment. LoVoDX10 cells were routinely grown in Ham’s F-12 medium supplemented with 10 µg/ml doxorubicin. Prior to experimentation, cells were grown in drug-free medium for 2 weeks.

Drug sensitivity of LoVo, LoVoDX, and LoVoDX10 was evaluated by calculating the drug dose responsible for 50% cell killing (IC50 value). Briefly, cells plated in triplicate at a density of 2 x 105 cells/60-mm-diameter tissue culture dish were incubated for 24 h in Ham’s F-12 medium supplemented with 10% FCS, and the appropriate doxorubicin concentrations were added. After 8 days, dishes were washed with PBS, and cells were detached by trypsinization, stained with trypan blue, and counted in a Fuchs-Rosenthal chamber. All cell lines were examined simultaneously, and all experiments were performed at least four times.

Drug.
LND, obtained from the F. Angelini Research Institute, was dissolved in DMSO at a concentration of 10 mg/ml (31.1 mM) immediately prior to use and sterilized by filtration through a 0.22 µm Millex GV filter (Millipore).

Determination of P-170 Glycoprotein by Flow Cytometry.
The monoclonal antibody Mab57, kindly provided by Dr. M. Cianfriglia (Istituto Superiore di Sanità, Rome, Italy), which recognizes an external domain of P-170 (23) , was used to detect the P-170 expression as reported previously (24) . Briefly, 5 µl of propidium iodide (1 mg/ml) was added to each sample prior to fluorescence-activated cell sorting analysis to exclude nonviable cells. Samples were analyzed in a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Logarithmic green fluorescence of FITC-bound Mab57 was collected after filtration through a BP 530/15 filter, and the linear red propidium iodide fluorescence was collected after filtration through an LP 620 filter. Ten thousand duplicated events for each sample were accumulated and data were analyzed using Becton Dickinson LYSIS II-C32 software.

Assay of 14CO2 Production, Aerobic Glycolysis, ATP Content, and Enzyme Activities.
Doxorubicin-sensitive and -resistant subclones of human LoVo colon carcinoma in exponential growth (5th day of culture) were detached by EDTA (0.02% in Ham’s F-12 medium for 5 min), recovered by centrifugation (600 x g for 5 min), washed twice in NKT buffer (105 mM NaCl, 5 mM KCl, 50 mM N-tris(hydroxymethyl)-2-aminoethanesulfonic acid, pH 7.40), counted in a Coulter counter (model ZM, Coulter Electronics), and resuspended in NKT medium at a concentration of 5 x 107 cells/ml. Determination of 14CO2 production from [6-14C]glucose, aerobic glycolysis, ATP content, and enzyme activity were performed as described previously (16 , 17) .

Dose-dependent Proliferation Analysis for LND Cytotoxicity.
For these experiments, 1 x 105 cells were plated onto 25-cm2 culture flasks (Corning). On the 5th day of culture, during exponential growth, freshly prepared drug was added to the flasks, which were then incubated for 24 h. Appropriate DMSO controls evaluated under the identical experimental conditions exhibited no toxicity. Following incubation, medium was discarded, and cells were detached with 0.02% EDTA for 1 min and counted in a Coulter Counter. Aliquots of cell suspension of known concentration were then dispersed into 80-mm plastic Petri dishes (five dishes for each point), and colonies were allowed to grow for 15–18 days. In each experiment plating efficiency of at least five controls was assayed simultaneously. The proliferating fractions for different drug concentrations were normalized with respect to the individual control for each experiment. All experiments were performed at least three times, with five samples for each drug concentration. The fitting and analysis of curves was as reported previously (25 , 26) .

Analysis of Doxorubicin Distribution and Efflux.
The intrinsic fluorescence emission of doxorubicin, when exited at 488 nm, was used to monitor intracellular drug distribution and to measure drug efflux kinetics in drug-sensitive and -resistant human LoVo colon cancer cells. For this purpose, cells were plated onto 35-mm dishes and used on the 5th day of culture. Cells were rinsed with fresh medium and incubated with 20 µM doxorubicin, in the presence or absence of 0.2 mM LND, for 1 h at 37°C in 5% CO2. Cells were then washed twice with NKT buffer and mounted on the stage of a Zeiss Axioscope upright microscope interfaced with a real time laser confocal fluorescence microscope (Odyssey, Noran Instruments, Redwood City, CA) equipped with an Argon laser. Cells were visualized by a x 40 water immersion objective (Zeiss, numerical aperture = 0.75) and continuously perfused with NKT buffer, with or without 6 mM glucose, with a glass pipette positioned close to the cell field and connected to a gravity-driven perfusion system. The effect of glucose on drug extrusion kinetics was studied by switching from control to the glucose-containing reservoir. Image acquisition and analysis were performed using IMAGE-1 software (Universal Imaging Corp.). Cells were exited at 488 nm, and fluorescence emission was monitored at {lambda} > 515 nm with a confocal slit of 100 mm. The laser beam was set at 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 x 256 pixels) from 100 images, taken by exposing the cells to light for 4 s, was stored on the computer hard disc. In some experiments real time 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 in the function of time from manually positioned areas on nuclear and cytoplasmatic regions of each cell, using the brightness versus time function of IMAGE-1. Data were displayed as space averaged fluorescence intensity. At least 20 cells were measured for each set of experimental data, and mean values were plotted and normalized with respect to the first determination.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Resistance Profile of LoVo Subclones.
The IC50 values for sensitive cells (LoVo) and two doxorubicin-resistant subclones (LoVoDX and LoVoDX10) were 0.05, 2.10, and 10.20 µM, respectively, following 8 days of doxorubicin treatment (Table 1)Citation . These values indicated that LoVoDX and LoVoDX10 were 42 and 204 times more resistant to doxorubicin, respectively, than the parental LoVo cell line.


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  		Table 1 Doxorubicin sensitivity of LoVo, LoVoDX, and LoVoDX10 carcinoma cells

Experiments were performed as described in "Materials and Methods." IC50 values correspond to the drug concentration responsible for 50% inhibition of cell survival. The data shown (representing the mean ± SD) were from four independent experiments performed in triplicate.

 
To establish whether the greater resistance of LoVoDX10 could be attributed to increased expression of P-170, flow cytometric analysis of cell surface P-170 expression was performed in sensitive and doxorubicin-resistant LoVo cells (Fig. 1)Citation . High P-170 expression was common to both resistant subclones, in contrast to the doxorubicin-sensitive line, which did not bind Mab57.



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  		Fig. 1. Flow cytometric analysis of P-170 expression in the sensitive LoVo cell line and the doxorubicin-resistant LoVoDX and LoVoDX10 cell lines.

 
Glucose Metabolism, ATP Content, Enzyme Activities, and Clonogenic Activity.
It has been demonstrated that the appearance of the MDR phenotype is associated with major modifications in energy metabolism (13, 14, 15, 16, 17, 18) . To investigate whether enhancement of energy-yielding processes correlated with the extent of drug resistance, oxidative and glycolytic glucose metabolism and the effect of LND were evaluated. Table 2Citation shows 14CO2, aerobic lactate production and ATP content of sensitive and doxorubicin-resistant LoVo cells, in the absence and presence of LND.


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  		Table 2 Effect of LND on 14CO2 and aerobic lactate production and ATP content by sensitive and doxorubicin-resistant (DX and DX10) LoVo carcinoma cells

The cells (2 x 107) were incubated at 37°C for 1 h with or without 0.2 mM LND. The final concentration of glucose was 6 mM. 14CO2, aerobic lactate production, and ATP content are expressed as cpm/2 x 107 cells/h, µmol/2 x 107 cells/h, and nmol/2 x 107 cells, respectively. Mean values ± SD from seven different experiments performed in duplicate are presented.

 
14CO2 production by LoVo cells from [6-14C]glucose was very low (369 cpm/2 x 107 cells/h) and unaffected by LND (307 cpm/2 x 107 cells/h). 14CO2 production by resistant LoVoDX cells was significantly higher (2852 cpm/2 x 107 cells/h) and was dramatically reduced by LND (1692 cpm/2 x 107 cells/h). Oxidative degradation of glucose and the effect of LND increased as cells became more resistant (5832 versus 2,349 cpm/2 x 107 cells/h).

The rate of aerobic lactate production was similar for both sensitive and LoVoDX cells and was inhibited by approximately 40% in the presence of LND. Aerobic glycolysis by LoVoDX10 cells was, in contrast, significantly higher than that of drug-sensitive and drug-resistant LoVoDX cells ({Delta} = 94 and 65%, respectively) and was inhibited by LND to an extent similar to that of glucose oxidation ({Delta} = -56%).

The enhancement of energy-yielding pathways in resistant cells should theoretically correlate with increased levels of intracellular ATP. In LoVoDX and LoVoDX10 cells, ATP content was 26 and 46% higher than in drug-sensitive cells, respectively. LND did not significantly affect ATP concentration in LoVo-sensitive cells, but inhibition increased with resistance. ATP content was reduced by 22 and 66% in LoVoDX and in LoVoDX10 cells, respectively.

Table 3Citation shows the activity of hexokinase, isocitric dehydrogenase, and citrate synthase in sensitive and resistant human LoVo carcinoma cells. No differences in hexokinase activity were observed in LoVo and LoVoDX cells. In LoVoDX10 cells, which exhibited higher lactate production, hexokinase activity was markedly increased ({Delta} = 110%). Higher glucose utilization in resistant cells via tricarboxylic acid cycle was confirmed by enhanced enzymatic activities of two regulatory enzymes. The activity of isocitric dehydrogenase was elevated from 5 to 12 and 38 nmol/min/mg of protein, and the activity of citrate synthase increased from 78 to 115 and 474 nmol/min/mg of protein in LoVoDX and LoVoDX10 cells, respectively.


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  		Table 3 Activities of hexokinase, isocitric dehydrogenase and citrate synthase of sensitive and doxorubicin-resistant (DX and DX10) LoVo carcinoma cells

Values ± SD are expressed as nmol/min/mg of protein. Mean value, averaged from seven different cell preparations, is presented.

 
Fig. 2Citation shows the clonogenic activity curves of sensitive and resistant LoVo cells, treated for 24 h with different LND con- 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 µM obtained, respectively. The effect of LND on clonogenic activity of LoVoDX10 cells was far more evident. The curve had a shoulder (n = 2.2), prior to an exponential decrease (D0 = 110 µM). The presence of a shoulder indicated that 24 h treatment with LND at concentrations up to 75 µM did not affect proliferation.



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  		Fig. 2. Dose-response curves for exponentially growing sensitive and doxorubicin-resistant LoVoDX and LoVoDX10 cell lines exposed to increasing LND concentrations. The time of exposure was always 24 h. Vertical bars, single experimental value ± SD.

 
Doxorubicin Distribution and Efflux.
The data we report indicate that extent of resistance correlates with enhanced energy metabolism. Doxorubicin efflux is energy-dependent (4) . Because LoVoDX10 should extrude drug to a greater extent and/or rate than LoVoDX cells, the kinetics of drug efflux and intracellular distribution were evaluated.

Sensitive and resistant cells were loaded with 20 µM doxorubicin, washed to remove the uninternalized drug, mounted on a microscope stage, and perfused at a constant rate with glucose-free NKT medium. Images were acquired for 10 min, and then perfusion was switched to glucose-supplemented NKT medium. Calibration of cellular doxorubicin concentration was not possible under our experimental conditions. Therefore, the variation of intracellular fluorescence over time with respect to fluorescence at the beginning of each experiment (F/F0) was evaluated. F/F0 was measured in nucleus and cytoplasm of each cell and mean results plotted against time. Each data set represents the mean of at least 20 cells ± SD.

In LoVo-sensitive cells, perfusion with glucose-free and glucose-supplemented medium did not modify cytoplasmatic or nuclear fluorescence. This indicated that all drug was retained by the cells over the time course (Fig. 3, A–C)Citation . The intracellular distribution of doxorubicin was similar in both nucleus and cytoplasm (Fig. 3, B and C)Citation , as confirmed by the ratio of nuclear to cytoplasmatic fluorescence (Fn/Fc), which, when plotted against time, remained constant (Fig. 3A,Citation inset). The values of Fn/Fc calculated from 120 cells, at the beginning of each experiment, were plotted in a histogram of the number of the cells over Fn/Fc and peaked at 1.061 ± 0.028 (not shown).



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  		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 µM doxorubicin as reported in "Materials and Methods." Drug extrusion was measured from the nuclear ({circ}) and cytoplasmatic (•) 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.

 
Resistant LoVo cells exhibited a markedly different behavior. Fig. 3, D–F,Citation shows doxorubicin efflux and distribution in LoVoDX cells. Cells perfused with glucose-free medium eliminated up to 20% of the doxorubicin into the medium. After 2–3 min of perfusion with glucose, the rate of outward transport increased to a plateau, i.e., the same efflux rate as without glucose after 25–30 min. At the end of the experiment, approximately 40% of the drug had been retained by cells. The concentration of doxorubicin in the cytoplasm was higher than in the nucleus (Fig. 3E).Citation A histogram of Fn/Fc, calculated from 110 cells in multiple experiments, was symmetrical around a mean value of 0.616 ± 0.018 (not shown). The decrease of nuclear doxorubicin was less than that of cytoplasmatic decrease as demonstrated by the ratio Fn/Fc, which increased with time (Fig. 3D,Citation inset).

In LoVoDX10 cells, perfused without glucose, outward drug transport was 2-fold greater than that observed in LoVoDX cells. The efflux rate increased dramatically when perfusion was switched to glucose-supplemented medium, and after 45 min, the cytoplasmatic drug fraction was almost completely (91%) released into the medium. The release of doxorubicin accumulated within the nucleus proceeded at the same rate of cytoplasmatic release for the first 20 min, slowing thereafter, resulting in an increased ratio of nuclear to cytoplasmatic fluorescence (Fig. 3G,Citation inset). At the end of the experiment, 80% of nuclear doxorubicin had been released into the medium. As for LoVoDX cells, the concentration of doxorubicin was higher in the cytoplasm, and the Fn/Fc ratio calculated from 110 cells in several experiments was symmetrical around a mean value (± SD) of 0.620 ± 0.022 (not shown).

These data indicate a relationship between drug extrusion and energy metabolism. Therefore, the effect of LND, which inhibits both oxidative and glycolytic metabolism (19, 20, 21 , 27) , on drug efflux and distribution was evaluated. Fig. 4ACitation shows that in sensitive cells, LND did not affect intracellular doxorubicin concentration but modified its distribution, resulting in an elevated cytoplasmatic concentration (Fig. 4, A–C)Citation .



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  		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 µM doxorubicin and 0.2 mM LND, as reported in "Materials and Methods." Other conditions were as reported in Fig. 3Citation . The experiments were repeated with four different cell preparations.

 
LND had a dramatic effect on the efflux of doxorubicin from resistant cells. Both basal and glucose-supported drug extrusion were almost completely inhibited. Indeed, after 50 min, 100 and 85% of the drug was still retained by LoVoDX (Fig. 4D)Citation and LoVoDX10 cells (Fig. 4G),Citation respectively. The nuclear drug concentration in LoVoDX (Fig. 4D,Citation inset, E, and F)Citation and LoVoDX10 cells (Fig. 4G,Citation inset, H, and I)Citation was higher than that of cytoplasm. The Fn/Fc ratio, evaluated from 130 cells pooled from several experiments, indicated a largely symmetrical distribution peaking around a mean ± SD of 1.27 ± 0.06 (not shown), i.e., a value quite similar to that found for the sensitive cells.

Table 4Citation shows the effect of LND exposure on doxorubicin sensitivity of LoVo-resistant cells. In LoVoDX cells, 8 days of treatment with 50 µM LND, i.e., a concentration that by itself was unable to affect cell survival, lowered the IC50 value from 2.1 to 0.5 µM doxorubicin. LoVoDX10 cells were more sensitive to LND; therefore, to avoid any effect on cell survival, a concentration 8 times lower (6.25 µM) was used. Nevertheless, despite this low concentration, LND was still able to reduce IC50 concentration from the 10.2 to 2.5 µM.


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  		Table 4 Effect of LND on doxorubicin sensitivity of LoVoDX and LoVoDX10 carcinoma cells

Cell number was evaluated after 8 days of treatment as described in "Materials and Methods." The data shown represent the mean ± SD of seven independent experiments performed in triplicate.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data reported in this communication provide, to our knowledge, the first experimental evidence that the extent of alterations in energy metabolism strictly correlate with degree of drug resistance.

In doxorubicin-sensitive cells, glycolysis was the main energy-yielding process, as indicated by the very low amount of glucose metabolized via the Krebs cycle. In resistant LoVoDX cells, the most relevant metabolic modification was a marked increase in 14CO2 production from [6-14C]glucose (Table 2)Citation , which releases 14CO2 only at the level of the Krebs cycle, whereas aerobic glycolysis remained essentially unmodified. In this regard, LoVoDX cells differ from adriamycin-resistant human MCF-7 breast cancer cells, which exhibit increased glycolysis (13, 14, 15, 16) and adriamycin-resistant Ehrlich ascites tumor cells, in which both respiration and glycolysis were enhanced (18) .

LoVoDX10 cells, which exhibit an even more resistant phenotype, had increased glycolytic and Krebs cycle regulatory enzyme activity and produced more 14CO2, and aerobic lactate increased (Table 2)Citation , leading to greater ATP availability, which, in turn, enhanced the activity of the ATP-driven efflux pump. The higher efflux rate and the almost complete extrusion of doxorubicin by LoVoDX10 cells could not be attributed to increased levels of P-170, which was similar in both of the resistant LoVo cell lines (Fig. 1)Citation .

The enhanced energy metabolism of LoVoDX10 cells made them more sensitive to LND, because the effect of LND on tumor energy metabolism does not depend either on the nature or the origin of the neoplastic cells, but is related mainly to metabolic capacity.

The marked inhibition of LoVoDX10 cell proliferation by LND can be attributed mainly to the decrease in ATP content, leading to an altered functional cellular state (28, 29, 30) . Such alterations can be reversed although ATP has been low for several hours but becomes irreversible only after 18–24 h (31) .

Low ATP content was also responsible for the increased doxorubicin content in LND-treated resistant LoVo cells. Decreased ATP availability may affect the P-170 activity by two mechanisms. In the first, reduced ATP production would not supply sufficient energy for extrusion by molecular pump. In the second, P-170 function may be affected by reduced phosphorylation, a critical factor for MDR. Indeed, human SW 620 colon carcinoma cells treated with sodium butyrate show decreased P-170 phosphorylation tightly correlated to reduced drug efflux. Decrease in P-170 phosphorylation is observed after 12 h treatment and further decreases at 24, 36, and 48 h (32) .

Because drug efflux by LoVo-resistant cells was already inhibited after 1 h of exposure to LND, an effect on P-170 phosphorylation may be excluded. Thus, the inability of LND-treated LoVo-resistant cells to extrude doxorubicin was likely to depend upon reduced energy supply to the ATP-driven efflux pump resulting from inhibition of glycolysis and respiration (Table 2)Citation .

An alternative pathway by which LND can increase the intracellular doxorubicin concentration in LoVo-resistant cells may be an acidic shift of cytosolic pH (33) , resulting from lactate accumulation. Indeed, Ben-Horin et al. (34) and Vivi et al. (35 , 36) report that the lower extracellular content of lactate in LND-treated adriamycin-sensitive and -resistant MCF-7 cells is dependent upon impaired outward lactate transport. However, such a mechanism may be excluded because low extracellular lactate in LND-treated cells is primarily attributed to inhibition of glycolysis by impairment of mitochondria-bound hexokinase (16 , 20 , 37) , confirmed in human gliomas transplanted in nude mice by Oudard et al. (38) , with extent of inhibition of tumor growth rate by LND strongly correlated with mitochondria-bound hexokinase activity. Moreover, LND decreases the acidity of vacuolar system organelles due to a decreased ATP content and increased ion membrane permeability (39) .

In conclusion, although our results neither prove nor disprove the existence of alternative pathways for MDR (e.g., a passive-trapping mechanism), they clearly demonstrated that inhibition of doxorubicin extrusion by LND in LoVo-resistant cells can be attributed to an effect on oxidative and glycolytic metabolism that results in reduced intracellular ATP content, thus lowering the energy supply to the ATP-driven efflux pump.

There are a number of potential therapeutic implications from this work. If MDR is associated with to P-170 overexpression, then LND, currently used in tumor therapy, may reduce or reverse drug resistance by restoring the capacity to accumulate and retain drug (40) . It is noteworthy that in LoVo cells, reduction of resistance (Table 4)Citation was obtained with an LND concentration similar to (LoVoDX) or remarkably lower than (LoVoDX10) the peak plasma concentration achievable in humans (18–40 µg/ml).

Moreover, LND, because of its effect on the energy-yielding processes, potentiates the therapeutic efficacy of other antineoplastic drugs or agents, including radiation and/or hyperthermia, while reducing toxic side effects (41, 42, 43, 44, 45) .


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Andrew R. Mackay (Department of Experimental Medicine, University of L’Aquila) for critical reading and English revision, A. Federico (Regina Elena Institute) for HPLC analysis, and R. Bruno for graphics work.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was funded in part by the Ministero dell’Università e Ricerca Scientifica e Tecnologica (60%) and also by the Ministero della Sanità. M. D. P. is a Fellow of Federazione Italiana Ricerca sul Cancro. Back

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; E-mail: caruso{at}univaq.it Back

3 The abbreviations used are: MDR, multidrug resistance; LND, lonidamine. Back

Received 7/16/99; revised 12/17/99; accepted 12/20/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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