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[CANCER RESEARCH 47, 4613-4617, September 1, 1987]
Role of Free Radicals in an Adriamycin-resistant Human Small CellLung Cancer Cell Line1
C. Meijer, N. H. Mulder, H. Timmer-Bosscha, J. G. Zijlstra, and E. G. E. de Vries2
Division of Medical Oncology, Department of Internal Medicine, University Hospital, Groningen, The Netherlands
ABSTRACT
In two Adriamycin (Adr) resistant sublines ((•!X4-Adr, and GLQ-Vdri) of a human small cell lung carcinoma cell line, (¿IX'4,cross-
resistance for radiation was found. GIXVAdr, has an acquired Adrresistance factor of 44 after culturing without Adr for 20 days and GLC4-Adr2, the same subline cultured without Adr for 3 months, has a decreasedbut stable resistance factor of 8.
One of the assumed mechanisms of Adr is that the effect is mediatedthrough the formation of free radicals. Therefore free radical scavengingmight play a role in these Adr resistant cell lines.
Adr, H2O2,and X-ray induced cytotoxicity were evaluated. Glutathione(GSH) levels and activities of associated enzymes were determined aswell as Adr, I l;O,, and X-ray induced DNA breaks and repair. GSHlevel was decreased in GLG-Adn, but restored to the normal level inGI X4-Adr;. Superoxide dismutase, catalase, glutathione-peroxidase, andglutathione 5-transferase were not elevated in the resistant sublines. Adrinduced a decreased amount of DNA breaks in GIXV Adr, compared toGIX '4. For X-ray and IIX), a comparable amount of DNA damage was
found. GIX>Adr, was able to repair DNA breaks induced by Adr, X-ray, and II2O2 better than GLC4.
In conclusion, no increased enzyme capacity for detoxification of freeradicals could be detected in the cytosol of the resistant cells. Theresistance against free radicals in the GLCVAdr, line may at least in partbe a result of increased DNA repair.
INTRODUCTION
The development of acquired resistance to antineoplasticdrugs represents a significant problem in the treatment ofpatients with cancer. One of the most useful agents available inmedical oncology is Adr.3 Some of the assumed working mech
anisms of Adr resistance are an altered membrane transport(1-3), a more effective DNA repair (4), or a changed intracel-lular enzyme capacity to detoxify free radicals (5, 6). Severalstudies describe an increased GSH level associated with Adrresistance (7) but the role of free radical scavenging in resistanceto Adr has as yet not been elucidated.
In order to study the mechanisms causing Adr resistance wedeveloped resistance in vitro in a human small cell lung carcinoma cell line (8) by stepwise increase of the Adr incubationconcentration in the culture medium. The resistant cell lineshows cross-resistance for radiation (X-ray). A cytotoxic mechanism common for both Adr and radiation could be damage byfree radicals. Therefore this cell line could serve as a model forevaluation of the role of free radicals in drug resistance.
For this purpose Adr, hydrogen peroxide (H2O2), and X-ray
Received 12/19/86; revised 5/7/87; accepted 6/8/87.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.1Supported by Grant GUKC 86-1 from the Dutch Cancer Foundation K.W.I-'.
2To whom requests for reprints should be addressed, at Department of Internal
Medicine, University Hospital, Oostersingel 59, 9713 EZ Groningen, The Netherlands.
3The abbreviations used are: Adr, Adriamycin: GSH, glutathione; PBS, phosphate buffered saline (0.14 M NaCI, 2.7 HIMMl, 6.4 mM Na:HP()4 2IU). and1.5 mM KH2PO4, pH 7.4); GSSG, oxidized glutathione; GSH-Px, glutathioneperoxidase; GSH-R, glutathione reducÃase;SOD, Superoxide dismutase; GST,glutathione 5-transferase; BSO. D.L-buthionine-5,A-sulfoximine; ICM>,drug concentration inhibiting colony formation by 50%; CA, clonogenic assay; AS-SH.acid-soluble sulfhydryl compounds.
induced cytotoxicity were evaluated. GSH levels and activitiesof associated enzymes were determined, and Adr, H2O2, and X-ray induced DNA damage and repair were compared.
MATERIALS AND METHODS
Chemicals. Adr was provided by Farmitalia Carlo Erba, Milano,Italy. RPMI was obtained from GIBCO, Paisley, Scotland. Fetal calfserum was from Flow Lab, Irvine, Scotland. GSH, GSSG, GSH-R,SOD, xanthine, xanthine-oxidase, cytochrome c, catalase, 5,5-dithiobis(2-nitrobenzoic acid), and l-ch!oro-2,4-dinitrobenzene were obtainedfrom Sigma, St. Louis, MO. H2O2 and NADPH were from Merck,Darmstadt, West Germany. 2-Vinylpyridine was from EGA-Chemie,West Germany. BSO was obtained from Chemalog, South Plainfield,NJ. Ethidium bromide was from Serva, Heidelberg, West Germany.
Cell Lines, Clonogenic Assay. GLC4, a human small cell lung carcinoma cell line, was derived from a pleura! effusion in our laboratoryand kept in continuous culture in RPMI 1640 with 10% heat-inactivated fetal calf serum. A subline of the parent line, GLC4-Adr,, wasmade resistant in vitro by stepwise increase in the concentration of Adrin the growth medium resulting in a resistance factor of 44x in CA.GLCVAdr, was maintained in continuous culture in the same way asGLC4, only in continuous presence of Adr (8). (Before measurementswere performed, GLC4-Adn was cultured without Adr for 20 days).This subline, after acquiring an Adr resistance of 44-fold, was also keptin continuous culture without Adr and named GLC4-Adr2. The resistance factor of this cell line started at 44x and decreased in time to astable resistance of 8x after 3 months of Adr free culture. Fig. 1 showsthe IC;o value for Adr in the Adr resistant cell line after various daysof drug free culture. Cells were cultured at 37*C in a humidified
atmosphere with 5% CO2.The CA with l h and continuous incubation was performed as
described earlier (9) to determine drug sensitivity of Adr and H2O2 andX-ray survival. For l h incubation the drug was washed out. Forcontinuous incubation cells with the appropriate agent were plated asan upper layer. Cells were irradiated at room temperature (Philips X-ray source at 200 Kv, 15 mA, 0.5-mm Cu/Al filter; dosimetry wascarried out).
Sulfhydryl Compounds and Enzyme Assays. For GLC4 and GLC4-Adr, cells in the logarithmic phase of growth were harvested, 4 daysafter passage. For GLC4-Adr2, the cells were also collected after 4 daysbut of a cell culture not exposed to Adr for at least 3 months. Cellswere washed with PBS and resuspended in a relevant concentration inPBS or another solution mentioned in the assay procedures. Cells werevortexed and sonicated. The cell suspension was centrifuged at 4"C
(100,000 x K. l h). The supernatant was assayed.All measurements were performed under rm;„conditions.Sulfhydryl Compounds. For GSH determination, cells were resus
pended in cold 5% trichloroacetic acid, vortexed, and centrifuged at4°C(10,000 x g, 15 min). The supernatant was assayed for total GSH
by the enzyme recycling method under conditions similar to thosedescribed by Tietze (10) (detection limit, 25 ng). Oxidized glutathione(GSSG) was quantitated following derivati/ut ion of reduced GSH with2-vinylpyridine according to Griffith (11). Reduced GSH was calculatedby subtracting oxidized GSH from total GSH. AS-SH were determinedby the method of Ellman (12). 5,5-Dithiobis (2-nitrobenzoic acid),Ellman's reagent, was used as substrate and absorbance was measured
at412 nm.Enzyme Assays. GSH-Px was measured by the method of Paglia and
Valentine (13) and glutathione reducÃase(GSH-R) activity by a modification of Carlberg and Mannervik (14). In both assays the consump-
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FREE RADICALS IN ADRIAMYCIN-RESISTANT LUNG CANCER
1600
1200
800
¿00
20 60 80 100 120 10)
days of drug tree culture
Fig. 1. KM, for Adr in the Adr resistant cell line (O) after various days of drugfree culture, estimated by clonogenic assay with continuous drug incubation. D,ICsoofGLC4.
RESULTS
Radiation survival curves of GLC4, GLC4-Adri, and GLC4-Adr2 are shown in Fig. 2. The radiation doses inhibiting thecolony formation by 50%, ICso, were respectively, 95, 162, andI IS rads.
Colony survival of the three cell lines after incubation withvarious concentrations of H2O2 is shown in Fig. 3. The IC50forGLC4 was 9.0 x 10~5M, for GLC4-Adr, 37.6 x 10~5M, and forGLC4-Adr2 21.4 x IO'5 M. For GLC4-Adr, a 4.2-fold and for
GLC4-Adr2 a 2.4-fold increase in IC50 was found.Table 1 shows the levels of sulfhydryl compounds in the three
cell lines. GSH level of the GLC4-Adr! cell line was significantlydecreased compared to the parent cell line, whereas in GLC4-Adr2 the GSH level was restored. For the AS-SH compoundsthe same decrease was found for GLC4-Adri but GLC4-Adr2showed a significant increase in the amount of AS-SH compounds. GSSG was decreased for GLC4-Adr2 compared toGLC4. A shift in the ratio GSH:GSSG was seen for GLC4-Adr,
tion of NADPH was followed by analyzing the absorbance change at340 inn.
SOD activity was measured by the method of McCord and Fridovich(IS). The inhibition of formation of reduced cytochrome by SOD wasmeasured at 550 nm.
For the GST activity by the method of Habig (16), l-chloro-2,4-dinitrobenzene was used as substrate and the enzyme reaction wasmeasured by the absorbance change at 340 nm.
The catalase activity was measured following the decomposition ofi M >: mediated by catalase in a cell suspension of 10s cells/ml at 240
nm according to Beers and Sizer (17).For protein determination the Lowry assay was used (18).All enzyme measurements were performed on a Zeiss PMQ spectro-
photometer.BSO Treatment. Cells were cultured as described earlier for 22 h in
the presence of 25 >/MBSO. After 3, 6, and 22 h cell viability wasdetermined using the trypan blue dye exclusion assay and GSH levelswere assayed.
Intracellular Adr Level. Intracellular Adr levels were determined afterl h incubation with Adr. Cells were washed three times with ice-coldPBS, extracted with 0.3 N HCl-50% ethanol and centrifuged. Adrfluorescence was measured in the supernatant at excitation and emission wavelengths of 474 and 549 nm, respectively, in a Kontronspectrofluorometer (19).
DNA Damage and Repair. Strand breaks in DNA were measured byalkaline unwinding and determination of ethidium bromide fluorescence on a spectrofluorometer with excitation wavelength at 525 nmand emission wavelength at 580 nm (20). For Adr and H.-().. inducedDNA damage, cells were incubated with Adr and I !.•<)..for 1 h. To stopthe incubation period for Adr the cells were washed three times withice-cold PBS. For HjO? experiments, HiO2 was scavenged by resus-pending the cells in RPMI supplemented with catalase (100 U/ml) for5 min. For determination of X-ray induced DNA damage, cells wereirradiated at 4"C before the fluorometric assay was performed.
For repair measurements the DNA damage was induced the sameway. After stopping the incubation, cells were resuspended in freshmedium and allowed to repair at 37"C, 5% CO2 during a period of 30
to 90 min.Results were expressed as D (% double-stranded DNA) = (F—Fm¡n)/
(/W - ^nin) x 100, where F is the fluorescence of the sample, Fminthe
background fluorescence determined in samples that were sonicated atthe beginning of the unwinding period in order to induce maximalunwinding, and /•',„.„is the fluorescence of samples kept at pH 11.0,
which is below the pH level needed to induce unwinding of DNA.Statistics. Statistical significance was determined by use of the un
paired Student's t test.
For multiple comparisons to a single mean statistical significancewas determined by use of Dunnett's test (21).
- X-ray Iradl
Fig. 2. Clonogenic assay after X-ray with GLC4 (O O), GLC4-Adr,(• •),and GLC«-Adr2(• •)(N > 6). For GLC4-Adr„values weresignificantly different at 100 rads, P < 0.05; 200 rads, P < 0.05; and 300 rads, P<0.01; versus GLC4.
10 50 100
»•tone Hj02 HO"5 M)
Fig. 3. Clonogenic assay after l h incubation with 11.(), with GLC4(G 0), GLCrAdr, (• •),and GLC4-Adr2 (• •)(N a 6). For GLC4-Adr,, values were significantly different at 10 x 10~5M, P < 0.01; 50 x 10"' M,P < 0.01; and 100 x 10'' M, P < 0.05; versi«GLC4. For GLC4-Adr2 a significantdifference was f«undvertëGLC4 at 10 x 10"' M, P < 0.01.
Table 1 Sulfhydryl compounds in the three different cell lines
SulfhydrylcompoundTotal
GSH, /ig/mg protein
GSSG, ng/mgproteinRatio
of reducedGSH:GSSG
AS-SH compounds. Mg/mgproteinNo.
ofexperimentsGLC4yv=i5
6.2±i.rN= 3 532 ±70
12:1
N=3 7.4 ±0.5GLC4-Adr,3.7
±1.1P < 0.0l"
596 ±107NS
6:14.9
±1.0GLC„-Adr27.4
±2.5NS'
320 ±50P < 0.0523:112.3
±0.9P < 0.01
" Mean ±SD.* GLC4-Adr, and GLC4, P values.' GLC4-Adr2 and GLC4, /' values; NS, not significant.
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FREE RADICALS IN ADRIAMYCIN-RESISTANT LUNG CANCER
in favor of GSSG, but in GLC4-Adr2 the ratio has readjusted
in favor of GSH.Table 2 shows the results of the enzyme assays in the three
cell lines. A decreased level of GSH-Px activity was found inGLC4-Adr,. The GSH-R activity was raised in both GLC4-Adr,and GLC4-Adr2. No differences in the levels of GST activityand SOD activity were measured. Only one-third of the catalaseactivity of GLC4 was found in both GLC4-Adr! and GLC4-Adr2.There was no difference in cellular protein content between thethree cell lines.
Table 3 shows cell viability and GSH levels of GLC4 andGLC4-Adr, after incubation with 25 UMBSO. After 6 h cultur-ing with 25 /IM BSO a not detectable level of GSH is reachedfor GLC4-Adr,, whereas GLC4 contains still 46% of the controlvalue. Viability for GLC4-Adr, at this point of measure is stilluneffected as measured by trypan blue dye exclusion, but aftera longer period of GSH depletion (i = 22 h) viability decreases.
Intracellular Adr concentration for GLC4 cells after l hincubation with 5 jig/ml Adr is 44.4 ng/105 cells. For GLC4-Adr, and GLC4-Adr2 the intracellular Adr level at this drugconcentration is, respectively, 29.3 and 30.3 ng/105 cells (N =
6, P < 0.01).Fig. 4 shows the Adr induced DNA damage for both GLC4
and GLC4-Adri. After l h incubation with Adr for GLC4-Adriless DNA breaks at all Adr concentrations were observed. Adrdoes not attribute to the fluorescence in these experiments asthere is no difference between the background fluorescence ofthe control and the Adr treated sample.
For X-ray and H2O2 no difference in DNA breaks was observed between both cell lines (Figs. 5 and 6). However, in theresistant GLC4-Adr] cell line repair was more pronounced andfaster. For Adr repair measurements, GLC4 and GLC4-Adriwere incubated with, respectively, 4 and 10 /j\i Adr to start ata comparable level of DNA breaks. At this concentration GLC4-Adrr is capable to repair the induced DNA damage whereasGLC4 showed no repair capacity. Most probably due to thetime lost during the washing procedure (necessary to stop the
Table 2 Enzyme measurements in the three different cell lines
EnzymeGSH-PxnmolNADPH/min/mgproteinGSH-R
nmolNADPH/min/mgproteinGST
nmolCDNB/min/mgproteinCatalase
pmolHiOj/min/mgproteinSOD
U/mgproteinProtein
mg/10' cellsNo.
ofexperimentsN=3JV=
3JV=6N
=6N=3N=
15GLC416.6
±4.0°10.1
±1.677
±80.8
±0.36.1
±2.31.89
±0.4NSGLC4-Adr,9.8
±1.9P<0.05*15.8
±3.7/><0.0579
±5NS0.3
±0.1P<0.016.3
±0.8NS1.96
±0.7NSGLC4-Adr214.1
±2.7NSC17.3
±2.6P<0.0173
±4NS0.3
+0.1P<0.015.2
+3.3NS1.96
±0.4
•Mean ±SD.* GLd-Adr, and GLC4, P values.' GLC4-Adr2 and GLC4, P values; NS, not significant
Table 3 Cell viability and GSH levels after incubation with 25 >iMBSO (N --
Detection limit, 0.1 pg GSH/mg protein2)
Incubationtime(h)0
3622Cell
viability(%)91
9695
96GLC,GSH
0*g/mgprotein)7.1
5.53.3NDGLC4-Adr,Cell
viability(%)89
858369GSH
Oig/mgprotein)4.1
1.0ND"
ND
%D
70
60-
50-
to-
30-
20-
10-
%D
70-
60-
50-
¿.0-
30
20-
10-
,1-
2 t 10 20 U) 30 60 90
UMAdr Kmm)Fig. 4. DNA strand breaks as determined by alkaline unwinding technique,
GLC, (• •)and GLC4-Adr, (O O) were incubated with different doses ofAdr for l h (N > 3). Repair measurements were done with 4 <iMfor GLC4 and10 Õ<Mfor GLC4-Adr, (N a 3). D, % double-stranded DNA. (For GLC4-Adr,values were significantly different versus GLC4 for DNA breaks, at 2 ¿IM,P <0.025; at 4 ^M, P < 0.0025; at 10 »M,P < 0.0005; at 20 pM, P < 0.0005; forDNA repair at t = 30 min, P < 0.0005; t = 60 min, P < 0.0005, i = 90 min, P <0.0005. Values for %D of the GLC4-Adr,, at t = 30, 60, and 90 min weresignificantly increased versus t = O min, P < 0.0125. This was not the case forGLC4).
%D
70
60
50
40-
30-
20-
10-
%D
70
60
50-
40
30-
20
10
225 450 675 900
X-Ray(rad)
30 60 90
t(min)
•ND, not detectable.
Fig. 5. DNA strand breaks as determined by alkaline unwinding technique,GLC4 (• •)and GLC4-Adr, (O O) were irradiated with different doses(Ara 3). Repair measurements were performed with 675 rads for both cell lines(.\ s 5). D, % double-stranded DNA. (Values were significantly different forDNA repair: at / = 60 min, P < 0.0025).
Adr incubation) the DNA damage increased (due to intracellular Adr) before the DNA repair could be measured.
DISCUSSION
Both Adr and X-ray resistance are present in GLC4-Adr] andboth treatment modalities have been thought to damage DNAby the action of free radicals as part of their mode of action.Therefore an increased capacity to detoxify free radicals in theresistant line could explain both the increased Adr and X-rayresistance. To investigate the effect of free radicals we incubatedthese cell lines with H2O2. H2O2 forms free radicals in thepresence of Fe2+ described as the Fenton reaction and intracel-lulary H2O2 is metabolized by GSH-Px using GSH as substrateand catalase.
In the clonogenic assay GLC4-Adr! was significantly moreresistant to H2O2 than the parent cell line. The degree ofresistance to H2O2 is in the same order of magnitude as for X-
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FREE RADICALS IN ADRIAMYCIN-RESISTANT LUNG CANCER
%D
70-
60
50
40
30
20-
10-
O 25 5 10concH;0;(10'5M|
Fig. 6. DNA strand breaks as determined by alkaline unwinding technique,GLC4 (• •)and GLCrAdri (O O) were incubated with different doses ofH2O2 for l h (n > 3). Repair measurements were done with 2.5 x 10"* M H2O2
for both cell lines (N > 3). D, % double-stranded DNA. (Values were significantlydifferent for DNA repair at r MImin, P < 0.005; r = 60 min, P < 0.025; t =90 min, P < 0.025).
rays. These data also suggest that the resistant sublines arebetter capable to survive a free radical attack. This could however not be explained by changes in the levels of sulfhydrylcompounds or scavenging enzymes. On the contrary GSH levelswere significantly decreased in GLC4-Adr,, augmenting thedeficient GSH-Px activity.
Catalase activity was 3-fold reduced in GLC4-Adr] and GLC4-Adr2. GSH and GSH-Px levels in GLC4-Adr2 however wererestored. GR activity, measured to evaluate the oxidation-reduction cycle of GSH was found to be raised in both cell linescompared to the parent cell line, but SOD and GST were equalin all three cell lines. As all measurements providing data onsulfhydryl compounds and enzyme activities are done underoptimal conditions, it should be noticed that such measurements do not provide information on the actual rates of radicaldetoxification. Our data differ from other studies where increased GSH levels for Adr resistant cells are shown (22, 23).However, the restored GSH and GSH-Px levels of GLC4-Adr2draws attention to the importance of the moment at whichenzyme measurements are performed in relation to Adr withdrawal. The decreased GSH level for GLC4-Adr, can perhapsbe explained by the observed shift ¡nthe ratio GSH:GSSGdescribed by Akman et al. (24). Alterations in the GSH poolcan be brought about in two ways: (a) utilization of smallamounts of GSH which stimulates new GSH synthesis, wherebyboth oxidized and reduced GSH are raised and (/>)rapid depletion of GSH, not compensated by GSH synthesis. Rapid oxidation to GSSG causes a shift in the GSG:GSSG ratio favoringGSSG. As utilization of GSH in the Adr resistant cell lines ismost probably brought about by both conjugation of GSHequivalents and the described redox mechanism it should bemore appropriate to look at a shift of the total amount of GSH.In our cell lines total GSH levels suggest that Adr has adecreasing effect on the GSH pool under continuous pressureof Adr. In GLC4-Adr2, no longer influenced by Adr, this levelhas been fully restored.
The changes in levels of scavenging enzymes in these Adrresistant cell lines seem therefore to be an effect of Adr, andare no part of the resistance mechanism. Initially the evaluationof the role of free radicals has been performed on murine celllines. Ramu et al. (22) described Adr resistance in P388 mouseleukemia cells. They did not find an increased free radical
scavenging for the Adr resistant cell line despite a slight increasein the GSH level. Catalase activity was also decreased to one-third which they associated with the observed radiosensitivityof the P388 Adr resistant cell line.
No clear shift in the ratio GSH:GSSG was shown. In aresistant subline from a human ovarian cancer cell line anincreased GSH level and radiosensitivity but relatively smallchanges in enzyme activity were found (23, 25).
An increased level of an anionic isoenzyme of GST is thoughtto be responsible for a higher level of detoxification of Adr bysome investigators (26-28). GST in this study was identical inall cell lines.
In order to evaluate the cause of the reduced cytotoxicity byfree radicals in the resistant cell line despite a decreased detoxifying system we also studied the amount of DNA breaks andDNA break repair. The rapid and sensitive fluorescent assayfor DNA strand breaks described by Birnboim et al. (20) wasused. DNA damage was quantitatively identical in the resistantand the sensitive cell line for radiation as well as for H2O2. Thisalso suggests that there is no detoxification system inhibitingthe induction of cellular DNA damage. But the resistant cellline can repair free radical induced DNA damage better thanthe sensitive cell line. As Adr can induce DNA damage probablyalso partially through free radicals (29-31), this increased repaircould in part also explain the resistance to Adr.
In conclusion, although a decreased cytotoxicity by free radical inducers is found, no increased enzyme capacity for detoxification of free radicals could be detected in the cytosol of theresistant cells. The resistance against free radicals in the GLC4-Adri line may at least in part be a result of increased DNArepair.
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1987;47:4613-4617. Cancer Res C. Meijer, N. H. Mulder, H. Timmer-Bosscha, et al. Cell Lung Cancer Cell LineRole of Free Radicals in an Adriamycin-resistant Human Small
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