CANCER BIOTHERAPYVolume 9, Number 2, 1994Mary Ann Liebert, Inc., Publishers

In vitro Effects of Pentoxifylline and Doxorubicin onCell Survival and DNA Damage in Sensitive and MDR-P388 Leukemia Cells

Arundhati Viladkar and Manik ChitnisChemotherapy Division, Cancer Research Institute, Tata Memorial Centre, Parel, Bombay-400012, INDIA

The utility of chemosensitizers to improve efficacy of chemotherapy is now gaining importance. Thisreport investigated whether an active hemorheological agent, pentoxifylline (PTX), can circumvent drugresistance in parental (P388/S) and multidrug resistant (P388/DOX) P388 leukemia cells. For detectionofdoxorubicin (DOX) resistance and reversal of this resistance by PTX, the incorporation ofnucleic acidprecursor was measured after addition ofDOX and PTX, respectively. The effect ofPTX on the inductionof DNA strand breaks by DOX was also examined. Increased fragmentation of DNA was illustrated inP388/DOX leukemia cells exposed to the combination ofDOX and PTX. The most prominent feature ofthe multidrug-resistant cell is the reduced accumulation of the drug intracellularly. P388/DOX cellsshowed less accumulation of DOX in the cell as compared to that of the parental cell line. Furtherstudies demonstrated that PTX significantly enhanced the intracellular accumulation ofDOX in both thecell lines. These studies warrant the use of PTX as an adjuvant in cancer chemotherapy.

INTRODUCTION

Resistance of malignant tumors tochemotherapeutic agents remains the major causeof failure in cancer therapy. Recently,significant progress has been made inunderstanding several mechanisms of drugresistance. One of them is the cross-resistance toa wide range of compounds which have no

obvious structural or functional similarities (e.g.alkaloids, anthracyclines, antibiotics). This kind

Address reprint requests to Dr. AS. Juvekar, ScientificOfficer, Chemotherapy & Stem Cell Biology Division,Cancer Research Institute, Tata Memorial Centre, Parel,Bombay 400 012, INDIA

of resistance has been termed multidrugresistance (MDR)13. The most frequentlyreported alteration of MDR cells is theoverexpression of P-glycoprotein (P-gp)originally described by Juliano, Ling4 andRiordan, Ling5. Besides overexpression of P-gp,increased levels of GSH6"8, altered activity oftopoisomerase II9 and an increased tolerance to

Superoxide and hydroxyl radical generation dueto higher levels of GSH peroxidase10,11 have beenreported to be possible mediators of MDR.

A number of agents have been identifiedwhich are capable of overcoming MDR in vitroand in animal models. These chemosensitizersare believed to function by blocking the P-gpmediated efflux of the cytotoxic drugs which

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results in increased intracellular drugaccumulation and thus cytotoxicity. Amongagents shown to possess this property arecalcium channel blockers (e.g. Verapamil)12"14,analgesics (e.g. dipyrone)15, tricyclicantidepressants16 and antihypertensive agents(e.g. reserpine)17.

In the present paper, we have usedpentoxifylline (PTX) for improving thecytotoxicity of doxorubicin (DOX) in P388murine leukemia sensitive (P388/S) and resistantto DOX (P388/DOX). The effect of PTX on theintracellular drug accumulation was also studiedin an attempt to understand the mechanism ofPTX-mediated enhancement of DOXcytotoxicity. Earlier, we have reported the effectof PTX on the potentiation of vincristinecytotoxicity in P388/DOX cells18 and its effecton circumvention of DOX resistance in humanchronic myeloid leukemia cells19.

MATERIALS AND METHODS

Chemicals

DOX was a gift from Dr. Mathew Suffness,Chief Natural Products Branch, Division ofCancer Treatment (NIH, Bethesda, Maryland,USA). PTX was provided by Hoechst IndiaLimited, Bombay. Minimum Essential Medium(MEM) and Fetal Calf Serum (FCS) were

purchased from HiMedia Laboratories Pvt. Ltd.,Bombay, India and Difco Laboratories, USArespectively. Methyl 3H-thymidine (3H-tdr; sp.activity 18,500 mCi/mmole), 3H-uridine (3H-urd;sp. activity 14 (Ci/mmole) and 14C-leucine (14C-leu; sp. activity 1.6 Ci/mmole) were purchasedfrom Bhabha Atomic Research Centre, Bombay.

Tumor SystemThe parental P388 murine leukemia cell line(P388/S) was originally obtained from theNational Cancer Institute, NIH, Bethesda, USAand was maintained in vivo in DBA/2 mice byweekly intraperitoneal inoculation of 106cells/animal in accordance with the NIHprotocol20. The subline of P388 leukemiaresistant to DOX (P388/DOX) was developed as

described by Johnson RK et al.21. DOXresistance was checked periodically and thissubline was completely refractory to DOX invivo at a dose of 1 mg/kg over a schedule of 1-9days.

Preparation of Cell SuspensionsAscites tumor cells from mice bearing P388/Sand P388/DOX leukemia were collected on 7 or8 days post-transplantation. The contaminatingred blood cells in the ascitic fluid were lysedwith 0.83% ammonium chloride buffered with0.17 mol/1 Tris-HCl, pH 7.2. The cells were

pelleted after centrifugation at 300 g, washedtwice with MEM and resuspended in fresh MEMsupplemented with 10% FCS, 40 pg/mlgentamycin and 10 U/ml penicillin to give a finalconcentration of 2xl06 cells/ml. The entireprocedure was performed under sterile conditionsat 4°C.

Evaluation of Drug Cytotoxicity byRadiolabelled Macromolecular PrecursorIncorporation AssayDoxorubicin and pentoxifylline solutions were

prepared freshly before use. Suspensions ofP388/S and P388/DOX tumor cells (2xl06cells/ml) were prepared as described earlier.Known aliquots of cell suspensions in MEMcontaining 10% FCS and antibiotics at a finaldensity of 2xl06 cells/ml were placed in 25 mlglass stoppered Erlenmeyer flasks. Freshlyprepared drug solutions were added either singlyor in combination. An equal volume of thevehicle was dispensed in the control flasks.After gently mixing the cell suspensions toassure homogenous distribution of drug/s or

vehicle, appropriate radiolabelled precursors wereadded. 3H-thymidine, 3H-uridine and 14C-leucinewere used as precursors of DNA, RNA andprotein biosyntheses, respectively. The finalconcentration of radioactivity in cell suspensionswas 0.5 pCi/ml. The cell suspensions wereincubated at 37°C in 5% C02 for the desiredtime intervals. After the incubation period,aliquots of cell suspensions were withdrawn intriplicate and processed in a Millipore sampling

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manifold over Whatman GF/C fibreglass filterpaper discs22. The radioactivity retained on thefilter was counted in a liquid scintillation counter(LKB Rack Beta 1215 Model II) equipped withan automatic quench compensation.Determination of Intracellular DoxorubicinAccumulation in P388/S and P388/DOX Cellsin vitro

The P388/S and P388/DOX leukemia cells wereobtained and processed as described earlier.

Cell suspensions were distributed in differentErlenmeyer flasks and were incubated at 37°Cfor 3 h after adding DOX alone or incombination with PTX in a shaking water bath.Appropriate controls were also run

simultaneously. Following incubations, the cellsuspensions were centrifuged, washed twice withchilled saline. The intracellular DOX wasextracted in ethanolic HC1 (0.3 HC1 in 50%ethanol). The DOX content in the extractobtained after centrifugation, was estimatedspectrophotometrically at 494 nm. Theconcentration of DOX was computed from thestandard curve prepared with DOX andexpressed as pg/106 cells23'24.

Detection of DNA Strand Breaks

Induction of DNA strand breaks was assayedover an alkaline sucrose density gradient.P388/S and P388/DOX cells (105 cells/ml) werecultured in supplemented MEM for 24 h periodalong with 3H-thymidine (0.5 pCi/ml). The cellswere then pelleted by centrifugation and washedwith chilled MEM. They were resuspended infresh supplemented MEM and treated for 1 hwith different drug concentrations. Thereafter,they were centrifuged and washed again withcold MEM. An aliquot of cell suspensioncontaining 105 cells was then loaded onto 0.2 mlof lysing medium (0.5 NaOH, 0.1 M EDTA)which had been prelayered over 4.0 ml of a 5-20percent continuous sucrose density gradient madein 0.1 M NaOH, 0.9 M NaCl and 0.01 MEDTA. The gradients were allowed to stand atroom temperature in dark for 30 minutes toallow complete lysis of tumor cells and then

centrifuged in a TST 60 rotor at 28,000 rpm for2 h at 0°C, with the brakes off.

Fractions were collected from the bottom ofthe tubes on GF/C glass fibre filter paper discswhich were then washed with 5% TCA for 20minutes. The filter discs were dried by washingtwice with 95% ethanol. Radioactivity in theacid insoluble fractions was assayed as describedearlier22.

The differences in the sedimenting profiles ofDNA from the treated and untreated tumor cellsgives a direct estimate of single and doublestranded breaks in DNA25, induced by DOXalone and in combination with PTX.

Statistics

The amount of labelled precursor incorporatedwas expressed as the percentage of counts in thesample at the end of the time period. Thepercentage inhibition ws calculated as

% Inhibition = 100 (1-

cpmLicpmc

where cpmt is counts per minute in the treatedsample and cpmc is counts per minute in thecontrol sample. Student's paired 't' test was

utilized to find values of differences ininhibition of radioactive labelled precursorincorporated in tumor cells treated with DOXalone vs DOX + PTX.

RESULTS

In Vitro Cytotoxicity AssayTable 1 demonstrates the effect of differentconcentrations of PTX alone and in combinationwith DOX (17.2 µ ) in P388/S cells at the endof 4 hours incubation at 37°C. DOX elicited32.2 percent inhibition of DNA biosynthesis, theeffect of which was further potentiated to 45,49.1 and 49 percent when used in combinationwith 1, 10 and 100 µ PTX respectively.

Similarly potentiating effect of PTX on DOXcytotoxicity in P388/DOX cells is shown inTable 2. DOX at 17.2 µ gave 11.3 percentinhibition of DNA biosynthesis which was

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Table 1. Effect of different concentrations of pentoxifyllinealone and in combination with doxorubicin on the DNAbiosynthesis in murine P388 lymphocytic leukemia cells,treated at 37°C for 4 hours.

3H-TdR pulse-

90 mins

Drug/s Concentrations % Inhibition of -TdRIncorporation

FTXPTXPTXDOXPTX + DOXPTX + DOXPTX + DOX

1 µ 10 µ 100 µ 17.2 µ 1 µ + 17.2 µ 10 µ + 17.2 µ 100 µ + 17.2 µ

11.2 ±3.113.2 ±2.413.0 ± 1.132.2 ± 2.2

*45.0 ± 4.3*49.1 ± 2.2•49.0 ± 4.2

Mean ± SD of 4 observations* ( < 0.02)** ( < 0.05) by student's paired test

further enhanced to 40.8, 43.2 and 53.5 percentwhen used in combination with 1, 10 and 100µ PTX respectively.

The inhibition of 3H-uridine incorporation as

a function of inhibition of RNA biosynthesis inP388/S and P388/DOX cells treated with DOXalone or in combination with PTX was alsoutilized as a measure of drug cytotoxicity. Table3 illustrates the effects of DOX and PTX aloneand in combination on the RNA biosynthesis of

Table 2. Effect of pentoxifylline alone and in combination withdoxorubicin on the DNA biosynthesis in P388/DOX murineleukemia cells treated at 37°C for 4 hours.

^-TdR pulse-

90 mins

Drug/s Concentration % Inhibition of 3H-TdRIncorporation

PTXPTXPTXDOXPTX + DOXPTX + DOXPTX + DOX

1 µ 10 µ 100 µ 17.2 µ 1 µ + 17.2 µ 10 µ + 17.2 µ 100 µ + 17.2 µ

13.1 ±1.713.4 ±4.114.0 ± 5.311.3 ± 6.2

*40.8 ± 16.3*43.2± 17.7

***52.5 ± 10.8

* < 0.05** < 0.02*** < 0.001

P388/S and P388/DOX leukemia cells. DOX atthe concentrations of 8.6 and 17.2 µ indicated30.3 and 44.3 percent inhibition of RNAbiosynthesis respectively in P388/S cells whichwas further enhanced to 57.5 and 56.1 percentrespectively when combined with 100 µ PTX,while a 13.9 and 27.5 percent inhibition of RNAbiosynthesis by the same concentrations of DOXin P388/DOX cells was increased to 56.3 and58.9 percent when DOX and PTX were used incombination.

14C-leucine incorporation in cellular proteinswas utilized as an index of protein biosynthesis.The effect of DOX alone and in combinationwith PTX on protein biosynthesis in P388/S andP388/DOX leukemia cells is illustrated in Table4. DOX at 8.6 and 17.2 µ elicited 46 and57.7 percent inhibition of protein biosynthesisrespectively in P388/S cells which was increasedto 65 and 73.6 percent respectively whencombined with PTX, while the same

concentrations of DOX indicated 12.3 and 26.8percent inhibition in P388/DOX cells which wereenhanced to 38.8 and 38.5 percent respectivelywhen combined with PTX.

Determination of Intracellular DOXAccumulation

The intracellular accumulation of DOX inP388/S and P388/DOX cells, when exposed toDOX alone and in combination with PTX isshown in Table 5. As clear from Table 5, thereduced level of intracellular DOX was observedin P388/DOX cells as compared to DOX levelsin P388/S cells, at both (8.6 and 17.2 µ )extracellular concentrations of DOX utilized,wherein 0.63 and 0.83 µg/106 cells DOXrespectively was observed in P388/S cells and0.52 and 0.56 pg/106 cells intracellular DOXrespectively was present in P388/DOX tumorcells. When the tumor cells were exposed toDOX in the presence of PTX (100 µ ), a.significant increase in the intracellular DOXlevel was observed at both extracellular DOXconcentrations in P388/S and P388/DOX cells.PTX elicited 32 and 8 percent higher level ofDOX in P388/S cells at 8.6 and 17.2 µ ofextracellular DOX concentrations respectively

146

Drugs Concentration

PTXDOXDOXPTX + DOXPTX + DOX

% inhibition ± SEP388/S P388/DOX

100 µ 8.6 µ 17.2 µ 100 µ + 8.6 µ 100 µ + 17.2 µ

11.6 ± 1.630.3 ± 0.6544.3 ± 2.8

*57.5 ± 4.1**56.1 ±6.0

= 4* <0.02

** <0.05

13.2 ±2.813.9 ±327.5 ± 5.2

*56.3 ± 4.3**58.9 ± 3.2

= 3* <0.05

** <0.01

Table 3. Effect ofpentoxifylline alone and incombination with doxorubicinon RNA biosynthesis ofP388/S and P388/DOX murineleukemia cells treated at 37°Cfor 4 hours.

while the same concentrations of DOX incombination with PTX elicited 50 and 68 percenthigher levels of DOX in P388/DOX cells.

Induction of DNA Strand Breaks by PTX andIt's Effect on the Strand Breaking Ability ofDOX

sucrose fractions (from fraction 12 onwards,Figure 1), indicating the presence of shorterstrands of lower molecular weight DNA. Thecombination of PTX and DOX elicited anincrease in the induction of DNA strand breaks,wherein a shift to the right of the labelled DNApeak was observed.

From the experimental data of P388/S cells,DNA strand breaks were dose-dependent andmore predominant than P388/DOX cells(unpublished observation). The profile ofinduced strand breaks in P388/DOX cells treatedwith DOX (1.7 µ ) alone and in combinationwith PTX (100 µ ) is illustrated in Figure 1. Inthe presence of PTX, a greater amount oflabelled DNA was retained in the lower density

DISCUSSION

Multidrug resistance (MDR) due tooverexpression of P-glycoprotein (P-gp) is nowa well described experimental phenomenon26.Because of the potential clinical importance ofPgp-MDR, substantial efforts are being broughtto bear on development of pharmacologiemethods to circumvent it. Chemosensitizers are

"O-Leu pulse labelling-

for 1 hour

Drugs

DOXPTX + DOXDOXPTX + DOX

Concentrations % inhibition ± SDP388/S P388/DOX

8.6 µ 100 µ + 8.6 µ 17.2 µ 100 µ + 17.2 µ

46.0 ± 14**65.0 ± 7.7

57.7 ± 17.5*73.6 ± 9.3

* <0.01** <0.05

12.3 ±1.2*38.8±7.126.8 ± 6.3

**38.5 ± 5.6

* <0.05* <0.02

Table 4. Effect ofpentoxifylline alone and incombination with doxorubicinon protein biosynthesis ofP388 murine leukemia cellssensitive and resistant todoxorubicin at the end of 4hours incubation.

147

membrane active agents that can modulate or

reverse this form of MDR. Although variousmechanisms have been discussed to account forthis diverse group of compounds to reverse

MDR27, the mode of action of thesechemosensitizers is not completely understood.However, it is accepted that MDR is due to

overexpression of P-gp, which functions as a

drug transporter and that chemosensitizersinterfere with this protein and thereby restore

chemosensitivity28.In our investigation we utilized PTX as a

chemosensitizer based on its various mechanismsof action. Pentoxifylline is a nontoxicmethylxanthine which blocks DNA repair. It is

also reported that PTX can decrease tumor cellinduced pulmonary embolization andthrombocytopenia in experimental animals andprevent the adherence of tumor cell complexes to

capillary walls in the microcirculation29. Tomitaet al. have shown the synergistic effects ofcisplatin and PTX using human osteosarcomacells, by the inhibition of DNA repairmechanisms30. An alternative mechanism is thatmethylxanthines act in the G2 phase of the cellcycle to induce cells to undergo mitosis beforethe completion of DNA repair31,32. The G2 phaseof the cell cycle has been generally observed to

lengthen in many normal or malignant cellsexposed to radiation, alkylating agents or other

Drugs Concentrations DOX accumulationP388/S

(µ / * cells)P388/DOX

DOXDOXPTX +DOXPTX + DOX

8.6 µ 100 µ + 8.6 µ 17.2 µ100 µ + 17.2 µ

0.63 ± 0.02"0.82 ± 0.030.83 ± 0.02

b0.90 ± 0.008

0.52 ± 0.05 .78 ± 0.020.56 ± 0.03

"0.94 ± 0.03

* <0.05" <0.02CP<0.01By student's paired 'f test

Table 5. Accumulation of doxorubicin inthe presence ofdoxorubicin aloneand in combinationwith pentoxifyllineincubated for 3 h at37°C.

Figure 1. Induction of DNAsingle strand breaks inP388/DOX leukemia cellsexposed to doxorubicin aloneor in combination withpentoxifylline.

2 4 6 8 10 12 14 16 1« 20 22 24 0 2 4 6 8 10 12 14 16 18 20 22 24

FRACTION NUMBER

148

antineoplastic drugs33,34. According to thismodel, methylxanthines do not necessarily inhibitbiochemical DNA repair processes per se,instead, they act by reducing time available forrepair, possibly through a protein which isaltered directly or indirectly by DNA damageand which controls the transit from G2 tomitosis32,34. All these reports led us to use PTXas a chemosensitizer in our studies.

In our earlier reports, we have shownamelioration of vincristine and DOX resistanceby PTX in P388/S, P388/DOX and CML cellsrespectively18,19. Tables 1-4 demonstrate thepotentiating effect of PTX on DOX cytotoxicityin P388/S and P388/DOX cells. Doxorubicin, atthe concentrations employed in this study did notshow significant nucleic acid biosynthesisinhibition confirming the multidrug resistantnature of P388/DOX cell line. Fingert et al.35have shown the enhancement in thiotepa toxicityin human cancer cells when employed along withPTX. Our results are in accordance with theseobservations. The combination of PTX andDOX revealed a significant DNA (Tables 1 and2), RNA (Table 3) and protein (Table 4)biosynthesis inhibition. PTX inhibitsspontaneous and induced platelet aggregation ofmembrane bound phosphodiesterase36 implicatinga possible interaction of PTX with the peripheralmembrane glycoprotein.

In many studies, the MDR phenotype isaccompanied by decreased intracellular drugaccumulation which is associated with an

increase in expression of P-gp37· Doxorubicinaccumulation studies performed in the sensitiveand resistant P388 leukemic cells indicated areduced drug retention in P388/DOX cells as

compared to that observed in P388/S cells (Table5). The cytotoxicity results were corroboratedwith the intracellular DOX retention, whereinPTX elevated the accumulation of DOX in boththe cell lines. The increased cytotoxicity ofDOX-PTX combination may, therefore, be due tothe interference of PTX with the P-gp drugefflux pump, resulting in decreased drug effluxor increased drug accumulation.

Anthracycline antibiotics such as DOX are

among the most useful antitumor drugs.Interaction of anthracyclines between the double

stranded DNA produces protein-associated DNAstrand breaks38. In the present study, in vitroexposure of P388/DOX cells to PTX alone andin combination with DOX was performed toanalyse the extent of DNA strand breaks inducedby the drug employing alkaline sucrose densitygradient centrifugation. The extent of DNAdamage was found to be much greater in P388/Scells than P388/DOX cells. DOX alone did notinduce much change in sedimentation patterns inP388/DOX cells as compared to control. Thecombination of DOX and PTX resulted in theformation of single strand breaks, wherein a

higher incidence of unequal size DNA indicatedby closely sedimenting profile was evident. Thedifferences in sensitivity can perhaps be relatedto the intracellular levels of DOX. The netaccumulation of DOX being less in P388/DOXcontrol untreated cells which was furtherincreased after treatment with DOX plus PTXcombination.

To conclude, the results demonstrate theutility of PTX as an adjuvant in cancer

chemotherapy. Though further investigations are

required to understand the precise interactions ofPTX while potentiating the cytotoxic effects ofDOX, this study clearly suggests that a hightherapeutic index of DOX can be achieved whileminimizing its toxic side effects.

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