Bioenergetics of Rat Prostate CancerCell Migration

Stylianos Kouvroukoglou,1 Clair L. Lakkis,2 John D. Wallace,2Kyriacos Zygourakis,1 and Daniel E. Epner2*

1Department of Chemical Engineering, Institute of Biosciences and Bioengineering, RiceUniversity, Houston, Texas

2Medical Service, Houston Veterans Affairs Medical Center and Department of Medicine,Division of Hematology/Oncology, Baylor College of Medicine, Houston, Texas

BACKGROUND. Increased cell motility and increased glycolysis are two well-known hall-marks of cancer. We undertook these studies to determine whether increased glycolysis isrequired for prostate cancer cell locomotion.METHODS. We studied the highly metastatic MatLu cell line, which is a variant of theDunning R-3327 rat prostate adenocarcinoma model. Using videomicroscopy and computerimage analysis, we compared the speed of migration of cells grown in serum-free medium ineither the presence or absence of glucose.RESULTS. We found that cells grown in glucose-free, conditioned medium maintainedspeeds of migration and intracellular ATP levels for 24 hr which were equivalent to those ofcells grown in conditioned medium containing glucose. In contrast, migration was signifi-cantly inhibited by growth in glucose-free, unconditioned medium. We also tested the effectof antimycin A and rotenone, two inhibitors of mitochondrial electron transport, on cellmigration and ATP levels. Antimycin A had no significant effect on either feature, whilerotenone slightly inhibited cell migration without affecting ATP levels.CONCLUSIONS. 1) Glycolysis is not necessary for rat prostate cancer cell locomotion in thepresence of conditioned medium. 2) MatLu cells grown in the absence of both serum andconditioned medium require glucose to maintain cellular ATP levels and cell migration.3) MatLu cells in conditioned medium adapt to inhibition of glycolysis or mitochondrialrespiration by increasing the activity of the uninhibited pathway. Prostate 34:137–144, 1998.© 1998 Wiley-Liss, Inc.

KEY WORDS: videomicroscopy; glycolysis; cell motility

INTRODUCTION

Several decades ago, Otto Warburg [1] first de-scribed one of the classical hallmarks of cancer: in-creased aerobic glycolysis. Glycolysis is the metabolicpathway by which glucose is converted to pyruvatewith the generation of two mol of ATP per mol ofglucose. Tumors convert much of the pyruvate gener-ated from glycolysis to lactic acid even in the presenceof oxygen, unlike most normal tissues, which do soonly when starved of oxygen. The reason for this dra-matic metabolic alteration in cancer cells remains amystery to this day.

In normal tissues, pyruvate formed from glycolysis

is further oxidized to carbon dioxide and water inmitochondria, yielding 38 mol of ATP per mol of glu-cose. Mitochondrial respiration is therefore consid-ered to be more efficient than glycolysis [2]. Why thendo tumors paradoxically have such high rates of lacticacid production from glycolysis? Several theories havebeen proposed in an attempt to answer this question.One reason may be that the maximal rate of ATP gen-eration from glycolysis is greater than that of mito-

*Correspondence to: Daniel E. Epner, VA Medical Center, MedicalService (111H), 2002 Holcombe Blvd., Houston, TX 77030. E-mail:depner@bcm.tmc.eduReceived 4 October 1996; Accepted 13 January 1997

The Prostate 34:137–144 (1998)

© 1998 Wiley-Liss, Inc.

chondrial respiration [2]. Cancer cells may need tohave high rates of glycolysis even under aerobic con-ditions in order to meet increased energy require-ments of cancer-specific activities such as invasion andmetastasis. Other investigators, including Warburg,[1] have suggested that elevated aerobic glycolysis incancer cells represents a permanent adaptation to an-aerobic conditions that prevail within rapidly expand-ing tumors. Ephraim Racker [3] proposed that in-creased glycolysis in tumors results from increasedATPase activity, which leads to increased availabilityof ADP and inorganic phosphate. Inorganic phosphateis present in limiting amounts in most cells and is oneof the key determinants of glycolytic activity [2].

Other studies support the possibility that glycolysisis the primary energy source for the migration of bothnormal and cancer cells [4–6]. Migration is but one ofthe many requirements for cancer cell invasion andmetastasis [7–9]. In the present study, we used a newlydeveloped videomicroscopy assay to determinewhether glycolysis is always required for maintenanceof cancer cell migration. This computer-automated as-say provided detailed information about both cellmorphology and migratory behavior of individualcells and cell populations over extended periods oftime. Our cell-migration studies were combined withmeasurements of cellular ATP levels to address thepossibility that inhibition of glycolysis leads to deple-tion of cellular ATP levels, which would inhibit notonly cell migration, but also many other vital pro-cesses.

MATERIALS AND METHODS

Cell Culture

MatLu cells were used for all experiments. MatLu isa highly metastatic variant of the Dunning R-3327 ratprostate cancer model which has been characterizedpreviously [10]. Cells were routinely grown at 37°C ina mixture of 5% carbon dioxide/95% air in completemedium, which consisted of RPMI-1640 medium(Gibco BRL, Grand Island, NY) supplemented with10% fetal bovine serum (Sigma Chemical Co., St.Louis, MO), penicillin 100 units/ml, streptomycin sul-fate 100 mg/ml, and dexamethasone 250 nM (SigmaChemical Co.). During experiments, cell were grownin serum-free medium in either the presence or ab-sence of glucose. Glucose-free medium consisted ofRPMI-1640 without glucose (Gibco BRL, catalog num-ber 11879), 0.1% bovine serum albumin (BSA), 250 nMdexamethasone, 100 units/ml penicillin G, and 100mg/ml streptomycin sulfate. Glucose medium wasmade by adding 2 g/l cell culture-grade glucose

(Sigma Chemical Co.) to glucose-free medium, fol-lowed by sterile filtering.

Preparation of Conditioned Medium

MatLu cells were grown to 90% confluence in 150-cm2 cell-culture flasks in complete medium. One daylater, the complete medium was aspirated, cells werewashed twice with phosphate-buffered saline, and 50ml of glucose-free medium were added to the flask.Conditioned medium was removed from the cells 48hr later and stored at 4°C. We verified that no glucosewas present in the conditioned medium by directlymeasuring glucose concentration with a YSI 2700 se-lect biochemistry analyzer (Yellow Springs InstrumentCo., Inc., Yellow Springs, Ohio).

Measurement of Cell Migration Speed [11]

Acquisition of a sequence of digital images. Oneday prior to each experiment, 20,000 cells were seededin each of the three wells of a strip cut from a 12-welltissue culture plate (Corning Glass Works, Corning,NY). The bottom area of each well was 1.21 cm2. Cellswere grown in complete medium until the time of theexperiment. At the beginning of each experiment,complete medium was aspirated from each well, cellswere washed twice with glucose-free medium, and 3ml of various test media were added to each well. Thewell strip was then immediately placed in a microin-cubator that was sealed and positioned on the motor-ized stage (LEP, Hawthorne, NY) of an inverted mi-croscope. The incubator was maintained at 37°C, andcells were intermittently perfused with 5% CO2, 95%air throughout the course of the experiment.

The MatLu cell cultures were visualized using atransmitted light inverted microscope (Seiler Instru-ment Co., St. Louis, MO) with a 20× objective and anRGB color video camera (model TK 107-OU, JVC). Thecomposite video signal from the video camera was fedto a Macintosh IIci computer (Apple Computer, Inc.,Cupertino, CA) for digitization. Digitized images with256 gray-levels were acquired with a frame-grabberboard (QuickCapture, Data Translation, Inc., Marl-boro, MA) with a resolution of 640 × 480 pixels (cor-responding to a 0.4 × 0.3 mm field of view). A secondcomputer (Macintosh SE/30, Apple Computer, Inc.)controlled the microscope motorized stage, regulatedthe gas flow through the incubator, and monitored itstemperature. A program we developed using Lab-View 2.1 (National Instruments, Austin, TX) orches-trated the entire process, providing complete automa-tion. At 20-min intervals, this control computer movedthe motorized stage to a preprogrammed location ineach well and triggered the acquisition of a 2 × 3 mo-saic of adjacent images, using the motorized stage con-

138 Kouvroukoglou et al.

troller to change the field of view between acquisi-tions. After acquiring six adjacent images for eachwell, the computer spliced them together to formlarger composite images (1,280 × 1,440 pixels coveringan area of 0.8 × 0.9 mm) that were stored on erasableoptical cartridges for postprocessing. This procedurewas used to monitor 30–40 MatLu cells per experimentunder various conditions for 24 hr. All experimentswere repeated three or more times.

Image analysis. The sequences of digital images ac-quired during these experiments were processed andanalyzed using the NIH-Image software package(NIH-Image was developed by Wayne Rashband, Na-tional Institutes of Health, and is available free ofcharge over the Internet by anonymous ftp from zip-py.nimh.nih.gov). The images were first convolvedwith a 17 × 17 ‘‘Mexican hat’’ filter, and the filteredimages were segmented, using thresholding, to obtainbinary images and identify the individual cells. Thecoordinates of the centroid of the nucleus of all cells ineach image were then computed and stored. Finally,the trajectories followed by every individual cell dur-ing an experiment were reconstructed using a modi-fied nearest-neighbor algorithm [11]. The algorithmwas coded in FORTRAN language and was executedon an IBM RS/6000 computer.

Once the trajectories of all cells were reconstructed,we calculated two measures of the motility of MatLucell populations. The first measure was the speed ofmigration, computed from the average distance thatMatLu cells traveled in any 20-min interval. Thisspeed varied with time over the course of each experi-ment. The second measure was the time-averagedspeed of migration, calculated using the average dis-tance covered by all MatLu cells in a population dur-ing the duration of an experiment.

Measurement of Intracellular ATP Levels

Twenty thousand cells were seeded per well in 12-well plates 1 day before experiments, using the samemethod as used for the videomicroscopy experimentsdescribed above. At the beginning of experiments,cells were washed twice with phosphate-buffered sa-line, and 2 ml of the appropriate test medium wereadded to each well.

At the time of the assay, medium was aspiratedfrom each well, and intracellular ATP levels weremeasured with the Bioluminescent Somatic Cell AssayKit (Sigma Chemical Co.). This kit employs the mostwidely used and sensitive method for ATP determi-nation, the luciferin-luciferase bioluminescent assay[12], which is sensitive enough to measure ATP re-leased by fewer than 10 cells. Light intensity is linearly

related to ATP concentration for up to 105 cells [13].Cells were not trypsinized at the time of the assay butwere instead lysed directly in wells after beingwashed twice with phosphate-buffered saline. An in-ternal standard was used for each sample to allowconversion of light units to ATP quantity. Readingswere obtained with a model TD-20e luminometer(Turner Designs, Sunnyvale, CA) with a 10-sec delaytime, 30-sec integrate time, and no predelay time. Allexperiments were repeated at least three times onseparate wells. Percent ATP content was calculated bydividing average ATP content of test samples by av-erage ATP content of control samples (grown in fullmedium).

Measurement of Oxygen Consumption

Cells were grown to 90% confluence in 75-cm2 tis-sue-culture flasks in complete medium, washed twicewith phosphate-buffered saline, trypsinized, sus-pended in 5 ml complete medium, counted, andplaced in a 37°C water bath. Aliquots containing 3million cells were placed in a separate tube, centri-fuged, and washed twice with phosphate-buffered sa-line prior to being suspended in 3 ml of serum-free testmedium. Test conditions included: 1) glucose-free, 2)glucose, 3) glucose plus 1 mmol antimycin A, and 4)glucose plus 1 mmol rotenone. Oxygen consumptionwas immediately measured using a Clark-type elec-trode (model 5300 biological oxygen monitor, YellowSprings Instrument Co., Inc., Yellow Springs, OH) in-serted into a water-jacketed sealed glass chamber in abath assembly connected to a thermostatic circulatorset at 37°C, as previously described [14]. Readingswere taken at 30-sec intervals for 10 min to determinerate of change in percent saturation/min. These rateswere converted to mg-atoms of oxygen/min as previ-ously described [15]. All measurements were repeatedat least three times on separate wells.

Measurement of Lactic Acid Production

One day prior to each experiment, 20,000 cells wereseeded in each well of 12-well tissue culture plates(Corning Glass Works). Cells were grown in completemedium until the time of the experiment. At the be-ginning of each experiment, complete medium wasaspirated from each well, cells were washed twicewith glucose-free medium, and 3 ml of various testmedia were added to each well. Test media were thesame as those listed above for measurement of oxygenconsumption. Eight hours later, the medium was re-moved, allowed to reach room temperature for 15min, and immediately analyzed for lactic acid contentwith a YSI 2700 select biochemistry analyzer (Yellow

Prostate Cancer Cell Migration 139

Springs Instrument Co., Inc.). All measurements wererepeated on at least three separate wells.

RESULTS

Migration and ATP Content of Cells inGlucose-Free Medium

Figure 1 shows the temporal evolution of the speedof migration for three typical experiments whereMatLu cells were cultured in either full medium, se-rum-free medium containing glucose, or serum-free,glucose-free medium. Cells were analyzed under glu-cose-free conditions to determine whether eliminationof glycolysis, which requires glucose, would affect cellmigration. The speeds of migration for each of theexperiments in Figure 1 were computed from the av-erage distance covered by 30–40 MatLu cells in 20-minintervals. The ultimate migration speed of MatLu cellsgrown in glucose-free medium was significantly lowerthan that of cells grown in glucose-containing me-dium. In comparison, cells maintained in completemedium had significantly higher migration speedsthan cells grown under either serum-free condition. Inno case did the growth medium change color over thecourse of the experiment, indicating that the metabo-lism of glucose did not significantly acidify the me-dium.

To determine whether inhibition of migration inglucose-free medium was due to depletion of cellular

ATP, we measured intracellular ATP levels of cellsgrown in the presence or absence of glucose for vari-ous times. Within 5 hr, ATP levels of cells grown in theabsence of glucose dropped to about 20% of ATP lev-els of cells grown in serum-containing medium (Fig.2A). On the other hand, ATP levels of cells grown inthe presence of glucose exhibited a smaller and moregradual decrease over 24 hr (Fig. 2A). There was aclose correlation between cellular ATP content 8 hrafter the start of an experiment and average migrationspeed, as shown in Figure 3A. These results indicatedthat MatLu cells grown in the absence of serum re-quired glucose to prevent rapid ATP depletion andassociated inhibition of migration.

Migration and ATP Content of Cells inConditioned, Glucose-Free Medium

We next determined whether glucose was requiredfor maintenance of ATP content and migration even inthe presence of growth factors, which were absentfrom the serum-free media used in the above experi-ments. To do so, we measured ATP content and mi-gration of MatLu cells grown in conditioned mediumwith or without added glucose. While conditionedmedium from MatLu cells has never been character-ized, we hypothesized that MatLu cells produce sev-eral autocrine growth factors, as do other prostate can-cer cell lines [16–27].

In contrast to the above findings, we found thatglucose was not required for maintenance of ATP lev-els or migration of cells grown in conditioned me-dium. As shown in Figure 2B, ATP levels droppedwithin 2 hr of removal of serum regardless of whetherglucose was present. Following that drop, ATP levelsof cells grown in conditioned medium remained stablefor 24 hr, even when glucose was absent. Glucose alsohad no effect on the time-averaged speed of migrationof cells grown in conditioned medium (Fig. 3B). Cellmigration was maintained at high levels even in thecomplete absence of glucose, which is required forglycolysis. We confirmed that glucose was not presentin glucose-free, conditioned medium by direct mea-surement (see Materials and Methods). In addition,we found that no measurable lactic acid was producedby cells grown in glucose-free medium (Table I), fur-ther supporting the conclusion that glycolytic activitywas eliminated.

Glucose-free medium contained no sugars and nolipids. The only energy source for cells grown in glu-cose-free medium was amino acids, which were me-tabolized in mitochondria by the tricarboxylic acidcycle [2]. Mitochondrial metabolism of amino acids, asreflected by oxygen consumption, increased signifi-

Fig. 1. Temporal evolution of the speed of migration of MatLucells cultured in full medium with serum and glucose (Serum),serum-free medium with glucose (Glucose-containing), or mediumwith neither glucose nor serum (Glucose-free).

140 Kouvroukoglou et al.

cantly when cells were grown in the absence of glu-cose (Table I).

Migration and ATP Content of Cells TreatedWith Inhibitors of Electron Transport

We next measured the effect of two inhibitors ofmitochondrial respiration, antimycin A and rotenone,on cancer cell migration. Antimycin A acts by inhib-iting mitochondrial electron transport from FADH2,and rotenone by inhibiting electron transport fromNADH [2]. Neither compound is known to inhibit gly-colysis. As shown in Figure 3C, antimycin A had noeffect on the time-averaged speed of migration, whilerotenone caused a small decrease in migration speedthat was not statistically significant at the 95% level.Neither compound caused depletion of cellular ATPlevels. Again, the migration speed results correlated

well with the measured cellular ATP content 8 hr afterthe start of experiments.

We used two methods to confirm that antimycin Aand rotenone significantly inhibited mitochondrialrespiration at the concentrations used. First, we foundthat ATP levels of cells grown in glucose-free, condi-tioned medium in the presence of either inhibitor werecompletely depleted within 1 hr (data not shown). Sec-ond, we found that oxygen consumption, which is adirect reflection of mitochondrial respiration, was sig-nificantly reduced by both compounds, while lacticacid production from glycolysis increased (Table I).

DISCUSSION

We have shown that glycolysis is not required forlocomotion of rat prostate cancer cells grown in thepresence of conditioned medium (Fig. 3B). Our results

Fig. 2. ATP content of MatLu cells at various times after growth in (A) serum-free medium with or without glucose, or (B) conditionedmedium with or without glucose. Percent ATP content was calculated as described in Materials and Methods. Bars represent standarderror. Values were corrected for cell numbers, which were determined from growth curves established in independent experiments.

TABLE I. Metabolism of MatLu Cells in Various Media*

Contents of mediumLactate production,

mmol/106 cells/hr (SD)Oxygen consumption,

mg-atoms/106 cells/hr (SD)Glucose Antimycin A Rotenone

− − − 0 0.94 (0.04)+ − − 1.67 (0.19) 0.49 (0.02)+ + − 2.56 (0.38) 0.09 (0.02)+ − + 2.86 (0.29) 0.16 (0.01)

*Conditioned medium was used in all cases.

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Fig. 3. Time-averaged speed and ATP content of MatLu cells cultured in(A) serum-free medium without glucose, serum-free medium with glucose,or full medium with both glucose and serum; (B) conditioned medium withor without glucose; or (C) conditioned medium with glucose ± 1 µM anti-mycin A or 1 µM rotenone. Time-averaged speeds were for the entireexperiment, while ATP content refers to the 8-hr time point. Percent ATPcontent was calculated as described in Materials and Methods. n, number oftimes the experiment was repeated. Each experiment included 30–40 cells.Bars represent standard error. *Statistically significant difference betweenthis value and the other two (P < 0.05, ANOVA Fisher and Scheffe tests).

contrast earlier studies showing that glycolysis wasrequired for motility of normal and malignant cells[4–6]. There are several possible reasons for this ap-parent discrepancy. One is that we directly measuredcell trajectories and speeds for up to 36 hr using vid-eomicroscopy and computer image analysis, whileother authors measured cell migration with othermethods. Beckner et al. [4] measured migration with amodified Boyden chamber. Movement of cellsthrough a modified Boyden chamber relies not onlyon cell migration, but also on cell spreading, cellshape, and other unknown factors. Beckner et al. [4]also measured chemotaxis, while we measured che-mokinesis. Our conclusions may also differ from thoseof other authors due to variability across species andtissues, or differences that exist between benign andmalignant cells. Finally, we correlated cell migrationwith cellular ATP content in all of our studies, whichwas not done previously. When we grew cells underconditions that led to depletion of cellular ATP, mi-gration was inhibited. This inhibition was not specifi-cally due to inhibition of glycolysis, but to lack of ATP.The association between decreased glycolysis and de-creased cell migration reported by other authors [4–6]may be a reflection of ATP depletion.

MatLu cells obtained ATP from both glycolysis andmitochondrial respiration. However, when eitherpathway was inhibited, they adapted readily by in-creasing the activity of the other pathway, therebymaintaining ATP levels and locomotion. These resultssuggest that cancer cells can use ATP generated fromeither glycolysis or mitochondrial respiration to sus-tain migration. However, our results do not indicatewhether one pathway is used preferentially for cellmigration when both glycolysis and respiration areactive.

Conditioned medium had dramatic effects on cel-lular ATP content and migration. Cells grown in con-ditioned medium maintained high levels of migrationand intracellular ATP regardless of whether glucosewas present. In contrast, ATP levels and the migrationof cells grown in unconditioned, serum-free mediumdropped rapidly if glucose was absent. We hypoth-esize that MatLu cells produce an autocrine factor orfactors that regulate energy metabolism. Conditionedmedium from MatLu cells may also have broadlystimulatory effects on ATP content and migration ofother cell types.

Previous studies have shown that human and ro-dent prostate cancer tissues and cell lines produce avariety of autocrine factors [24,28], including epider-mal growth factor [22,23,25,26,29], transforminggrowth factor alpha [22,23,27,30], fibroblast growthfactor [31], interleukin 6 [17], androgen-inducedgrowth factor [18], neurotensin [19], parathyroid hor-

mone-related protein [20], insulin-like growth factors Iand II [21], and autocrine motility factor [16,32]. Infuture studies, we intend to identify the various fac-tors produced by prostate cancer cells that regulateenergy metabolism. Prior studies have also shown thatseveral growth factors have major effects on cell en-ergy metabolism [33–44]. Those studies and the cur-rent ones strongly suggest that growth-factor signal-transduction pathways and bioenergetic pathways areclosely integrated. In future studies, we intend to de-termine the molecular mechanisms by which thesetwo critical pathways interact in hopes of ultimatelyidentifying novel therapeutic targets for men with ad-vanced prostate cancer.

CONCLUSIONS

We draw three conclusions. 1) Glycolysis is not nec-essary for rat prostate cancer cell locomotion in thepresence of conditioned medium. 2) MatLu cellsgrown in the absence of both serum and conditionedmedium require glucose to maintain cellular ATP lev-els and cell migration. 3) MatLu cells in conditionedmedium adapt to inhibition of glycolysis or mitochon-drial respiratory by increasing the activity of the un-inhibited pathway.

ACKNOWLEDGMENTS

This work was supported by a Merit Award fromthe U.S. Department of Veterans Affairs, by AmericanCancer Society grant PRTA-14, by National ScienceFoundation grant BES-9511750, and by the Chao Fund,Baylor College of Medicine.

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