[CANCER RESEARCH 52, 6885-6889, December 15, 1992]

Relationship of Cellular Glutathione to the Cytotoxicity and Resistance of Seven Platinum Compounds 1

Coby Mei jer , N a n n o H . M u l d e r , H e t t y T i m m e r - B o s s c h a , Wire J . Slui ter , Gert J a n M e e r s m a , and E l i s a b e t h G. E. de Vries 2

Divisions of Medical Oncology [C. M., N. 1t. M., H. T-B., G. J. M., E. G. E. de V.] and Endocrinology [W. J. S.], Department of Internal Medicine, University Hospital, Oostersinge159, 9713 EZ Groningen, the Netherlands

ABSTRACT

The role of glutathione (GSH) in the effectiveness of and resistance to 7 platinum compounds [5 Pt(II) and 2 Pt(IV) drugs] was evaluated in a 8.6-fold cisplatin (CDDP)-resistant human small cell lung cancer cell line (GLC4/CDDP), the parent GLC4 line, a 3.7-fold CDDP-resistant human embryonal carcinoma cell line (Tera-CP), and the parent Tera line (NTera2/D 1). Resistance factors for both CDDP-resistant cell lines were determined after continuous incubation (4 days) with CDDP.

Continuous incubation with the other studied platinum drugs revealed complete cross-resistance for carboplatin (CBDCA) and zeniplatin but less for enloplatin (ENLO) and iproplatin in both models. Tetraplatin and lobaplatin showed, respectively, partial and complete cross-resis- tance in GLC4/CDDP but no cross-resistance in Tera-CP.

GSH level, but not glutathione S-transferase activity, of the 4 cell lines correlated with platinum drug concentrations inhibiting cell sur- vival by 50% after continuous incubation (r = 0.86, P < 0.05). GSH depletion by aL-buthionine-S,R-sulfoximine (BSO) increased sensitiv- ity, as measured after a 4-h exposure to the drugs, of GLC4/CDDP for CDDP 2.0-fold, for CBDCA 1.7-fold, for zeniplatin 1.7-fold, and almost to the level of the sensitive GLC4 for ENLO, whereas no effect was observed for iobaplatin and the Pt(IV) compounds iproplatin and tetra- platin. BSO-modulating effect was higher in the sensitive GLC4 line for most compounds; therefore reduction of resistance could be achieved only for CDDP and ENLO. In contrast to GLC4, no modulation occurred in Tera. In Tera-CP BSO increased sensitivity for CDDP 1.5-fold, for CBDCA 1.9-fold, and for zeniplatin 1.2-fold; no effect was observed for ENLO, lobaplatin, and the Pt(IV) compounds.

Reduction of CDDP resistance by BSO was known to occur with identical cellular platinum levels and higher Pt-DNA binding in GLC4/ CDDP. However, pretreatment with BSO followed by 4 h ENLO incu- bation increased cellular platinum levels in both GLC4 and GLC4/ CDDP while Pt-DNA binding remained unchanged.

In conclusion, GSH reflected sensitivity to platinum-containing drugs. However, since the involvement of GSH differed between the models and the various platinum drugs, the effect of modulation with BSO was unpredictable.

I N T R O D U C T I O N

The development of acquired resistance to the useful anti- neoplastic agent C D D P 3 and the severity of toxicity associated

Received 6/22/92; accepted 10/2/92. 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 accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was partly presented at the 82nd Annual Meeting of the American Association for Cancer Research, Houston, TX, May 15-18, 1991, and partly supported by Grants GUKC 90-18 and 91-09 from the Dutch Cancer Foundation.

2 To whom requests for reprints should be addressed. 3 The abbreviations used are: CDDP, cisplatin, cis-diamminedichloroplatinum-

(II); GSH, glutathione; GST, glutathione S-transferase; CBDCA, carboplatin, cis- diamminecyclobutane-l,l-dicarboxylatoplatinum(II); ZENI, zeniplatin, CL286,558; ENLO, enloplatin, CL287,110; CHIP, iproplatin, cis-dichlorobisisopropylamine- trans-hydroxyplatinum(IV); TETRA, tetraplatin, tetrachloro(DL-trans)-l,2-diammi- necyclohexaneplatinum(IV); LOBA, iobaplatin, D19466, 1,2-diamminomethyl- cyclobutaneplatinum(II)qactate; DACH, diamminecyclohexane; ICso, concentration inhibiting cell survival by 50%; BSO, DL-buthionine-S,R-suifoximine; FCS, fetal calf serum; PBS, phosphate-buffered saline (0.14 M NaCI, 2.7 mM KCI, 6.4 mM Na2HPO4.2H20, and 1.5 mM KI-I2PO4, pH 7.4); DME, Dulbecco's modified Eagle's medium; RF, resistance factor; DMF, dose modifying factor.

with this drug encouraged the development of new platinum- containing drugs.

In vitro studies have demonstrated that cellular resistance to C D D P is multifactorial. Mechanisms involved include: altered membrane transport; inactivation of the drug by cellular thiols such as GSH and metallothioneins; decreased Pt -DNA binding; and/or an increased Pt -DNA repair (1). The role of the GSH system in C D D P resistance has received much attention, be- cause CDDP as well as other platinum-containing compounds are electrophilic agents which are most reactive towards sulfur- containing nucleophiles. A correlation between the GSH level and sensitivity to, e.g., CDDP has been reported in human tumor cell lines expressing a range of in vitro sensitivities to CDDP (2, 3). Elevated GSH levels have been found in C D D P resistant cell lines (4-13), and BSO, a specific inhibitor of the GSH synthesis, can act as an useful modulator of resistance to nucleophilic agents such as CDDP in vitro (6, 9).

The exact mechanism, however, by which GSH influences platinum-induced cytotoxicity and participates in resistance to various platinum-containing drugs is, as yet, not fully eluci- dated and may differ between various test models. The present study evaluated the role of GSH in the effectiveness of and the resistance to seven platinum-containing drugs in two models of CDDP resistance: a human embryonal carcinoma cell line, Tera, representing a sensitive tumor in the clinic; and a human small cell lung cancer cell line GLC4, representing a sensitive tumor type prone to resistance in the clinic, as well as their sublines with in vitro-acquired C D D P resistance, Tera-CP and GLC4/CDDP. Compared to the parent line, GSH levels were 1.4- and 2.5-fold higher in, respectively, Tera-CP and GLC4/ CDDP, and GST activity was 1.5-fold higher in Tera-CP but the same in GLC4/CDDP. Platinum drug cytotoxicity was tested in the absence and presence of BSO pretreatment. Five Pt(II) drugs, namely, CDDP, the less toxic CBDCA, three plat- inum compounds recently introduced in clinical studies (ZENI, ENLO, and LOBA), as well as two Pt(IV) drugs, CHIP and one of the DACH compounds (TETRA) were studied (Fig. 1). For ENLO, the platinum drug most effectively modulated by BSO in this study, the role of GSH was further investigated by mea- suring the effect of BSO-induced GSH depletion on cellular platinum levels and Pt -DNA binding.

MATERIALS AND M E T H O D S

Chemicals. CDDP, CBDCA, and CHIP were kindly provided by Bristol Myers S.A.E. (Weesp, the Netherlands), ZENI and ENLO by Lederle Laboratories (Pearl River, NY), LOBA by ASTA (Frankfurt, Germany), and TETRA by Upjohn (Kalamazoo, MI). RPMI 1610 was obtained from Life Technologies (Paisley, United Kingdom) and FCS from Sanbio (Uden, the Netherlands). DME and Ham's F-12 medium were purchased from Flow Laboratories (Irvine, United Kingdom). GSH, BSO, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were obtained from Sigma (St. Louis, MO), and dimethyl sulfoxide from Merck (Darmstadt, Germany).

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RELATIONSHIP OF GSH TO CYTOTOXICITY OF PLATINUM COMPOUNDS

.3. \ /c,

.,,/P\c. CDDP

.].\ /o-~ ^ CBOCA

Ho .z , o

ZENI

ENLO

./ \ o ~ 1t2

LOBA

NH2 CI CI

TETRA

~... f12 OH "\1/cl ~ - - . / ~ \ c l CHIP

N2 OH

Fig. ]. Structural formulas of the platinum drugs.

Cell Line Characteristics. The human small cell lung carcinoma cell line GLC4 and the human embryoual carcinoma cell line Tera (NTera2/ D1), as well as their CDDP-resistant sublines GLC4/CDDP and Tera-CP were cultured in RPMI 1640 with 10% heat-inactivated FCS at 37~ and 5% CO2 (4-7). In GLC4, GLC4/CDDP, Tera, and Tera-CP cellular doubling times were, respectively, 16, 19, 14, and 14 h; GSH levels were 4.6 + 0.5 (SD), 11.5 + 2.4, 1.9 + 0.2, and 2.6 _ 0.4 ug GSH/mg protein; GST activity was 49 + 9, 50 + 6, 95 • 8, and 146 _+ 8 nmol CDNB/min/mg protein; and cellular protein contents were 1.80 • 0.37, 1.33 • 0.19, 2.33 • 0.62, and 2.41 • 0.65 mg/107 cells. All were measured as described previously (5).

BSO Treatment. Cells were cultured as described above for 48 h in the presence of 50 pM BSO. At 48 h GSH was <5% in all cell lines, without growth delay or loss of viability (trypan blue dye exclusion assay).

Drug Sensitivity with the Microculture Tetrazolium Assay. For GLC4 and GLC4/CDDP, respectively, 5,000 and 15,000 cells and for both Tera and Tera-CP 10,000 cells, in exponential phase of growth, were incubated continuously with the platinum drugs in a total volume of 0.1 ml medium (DME/Ham's F-12 with 20% FCS) for GLC4 and GLC4/CDDP and 0.2 ml medium (DME/Ham's F-12 with 20% FCS) for Tera and Tera-CP in 96-well culture plates (Nunc, Life Technolo- gies). For experiments after 48 h BSO pretreatment, the cells were exposed to the drugs for 4 h in 0.1 ml culture medium (RPMI 1640 with 10% FCS) without BSO. After incubation, cells were washed three times (DME/Ham's F-12 with 20% FCS) by removing the medium after centrifugation of the microtiter plate (10 min, 180 • g) followed by addition of fresh medium (final culture volume was 0.1 ml for GLC4 and GLC4/CDDP and 0.2 ml for Tera and Tera-CP). After a culture

period of 4 days the microculture tetrazolium assay was performed as described before (6). At least three independent experiments were performed.

Platinum Measurements. For cellular platinum measurements 2.5 • 10 6 cells were incubated for 4 h with ENLO concentrations ranging from 0.45 to 2.7 mM. The cells were washed three times with PBS at 4~ The pellets were dissolved in 0.5 m165% HNO3 in an oven at 70~ for 2 h, and the amount of platinum in the samples was measured (atomic absorption spectrophotometry) as described before (6). Pt- DNA was measured after treating 5 • 107 GLC4 and GLC4/CDDP cells with ENLO concentrations ranging from 0.45 to 2.7 mM for 4 h. The cells were washed three times with PBS at 4~ DNA was isolated, and the DNA content (absorption at 260 nm) and the amount of plat- inum in the samples were measured (atomic absorption spectrophotom- etry) as described before (6). At least three independent experiments were performed for both cellular platinum levels and Pt-DNA binding at each concentration.

Statistics. Statistical significance was determined with the unpaired Student's t test and for the BSO experiments with the paired Student's t test. Correlation coefficients were determined by Spearman rank anal- ysis and averaged through z transformation.

P < 0.05 was considered significant.

R E S U L T S

Survival curves of GLC4, GLC4/CDDP, Tera, and Tera-CP after cont inuous incubation with C D D P are shown in Fig. 2. Resistance factors were calculated from the drug concentrat ions inhibiting cell growth by 50%. ICso values were 1.2, 10.3, 0.69, and 2.6 #M for, respectively, GLC4, GLC4/CDDP, Tera, and Tera-CP, corresponding with a resistance factor of 8.6 for GLC4/CDDP and 3.7 for Tera-CP.

Table 1 shows the cross-resistance for the plat inum-contain- ing drugs tested in the two C D D P resistance models. After continuous incubation complete cross-resistance for CBDCA and ZENI, but less for ENLO and the Pt(IV) compound CHIP, was observed in both resistant cell lines. TETRA showed partial cross-resistance in GLC4/CDDP and no cross-resistance in Tera-CP. LOBA showed full cross-resistance in GLC4/CDDP, whereas no cross-resistance could be observed in Tera-CP.

There was a significant correlat ion between the amount of GSH/mg cellular protein of the four cell lines and the ICso values after continuous incubation with the various plat inum drugs in the cell lines (average correlation coefficient: r = 0.86; P < 0.05) (Fig. 3). All correlations between the amount of GSH/mg cellular protein of the four cell lines and the ICso values for the individual drugs were significant (Spearman rank correlat ion coefficient range, 0.67-0.97; P < 0.05). There was

100-

80-

60-

! il 40-

20-

! 1 ! ! !

2 4 6 8 10 CDDP (1~)

Fig. 2. Survival of GLC4 (O), GLC4/CDDP (Q), Tera (A) and Tera-CP (A) after continuous incubation with CDDP. Bars, SEM, n > 3).

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R E L A T I O N S H I P OF GSH TO C Y T O T O X I C I T Y OF P L A T I N U M C O M P O U N D S

Table 1 Cytotoxicity after 4 days of continuous incubation (n >_ 3)

IC5o (~M)

GLC4 GLC4/CDDP RF Tera Tera-CP

CDDP 1.2 10.3 8.6 0.69 2.6 CBDCA 22.7 189 8.3 5.6 33.9 ZENI 1.1 12.1 11.0 0.59 1.8 LOBA 0.58 4.2 7.2 0.34 0.47 ENLO 29.7 78.4 2.6 7.2 14.1 CHIP 12.1 30.0 2.5 2.5 5.3 TETRA 0.81 2.9 3.6 0.87 1.1

IC50 Pt dn,lOs ~ld) 1000

alter the CDDP- induced cellular pla t inum content in GLC4 and G L C J C D D P [data published previously (6)]. However, pre-

RF t reatment with BSO increased ENLO-induced cellular plati- 3.7 num content in both cell lines (Fig. 4). The amount of P t -DNA 6.1 binding induced by C D D P [data published previously (5)] as 3.1 well as E N L O was for both drugs significantly lower in GLC4/ 1.4 2.0 C D D P than in GLC4. Following BSO pretreatment, an in- 2.1 creased CDDP- induced P t -DNA binding in G L C 4 / C D D P was 1.3 observed whereas the P t -DNA binding in GLC4 remained the

same. For ENLO, no effect of BSO pretreatment on P t -DNA binding could be observed in GLC4 and G L C 4 / C D D P (Fig. 5).

100

0.1

X �9 6 �9

i +' | r-i r-i

r,- O J6, Ik 0.~

Fig. 3. Correlation between the GSH content of the 4 cell lines and the IC5o values for the various platinum drugs after continuous incubation. Cell lines (left to right): Tera (1.9 gg GSH/mg protein); Tera-CP (2.6 #g GSH/mg protein); GLC4 4.6 #g GSH/mg protein); GLC4/CDDP (11.5 #g GSH/mg protein), e, CDDP; �9 CBDCA; *, ZENI; [~, LOBA; x, ENLO; A, CHIP; A, TETRA.

D I S C U S S I O N

In the present study we evaluated the role of G S H in the effectiveness of and resistance to C D D P as well as six other pla t inum analogues in two models of C D D P resistance in order to obtain more insight in the interaction of G S H with the different drugs and the involvement of G S H in resistance to these drugs.

In accordance with the literature (8-10, 14-18) complete cross-resistance to C B D C A was found in both CDDP-res i s tan t cell lines. Cross-resistance to C B D C A has been described for normal (10, 14-16) and elevated cell G S H status (8-10).

Table 2 Effect of BSO-induced GSH depletion on the cytotoxicity of the drugs for GLC+ and GLC4/CDDP after a 4-h incubation (n >_ 3)

neither a positive correlation between GST activity and ICso values for the individual pla t inum drugs after cont inuous incu- bation in the four cell lines nor an averaged correlation (r = -0 .13 , not significant). CDDP

CBDCA Table 2 shows the effect of BSO-induced G S H depletion on ZENI

the cytotoxicity of the tested drugs for GLC4 and G L C 4 / C D D P LOBA

expressed as ICso after 4 h incubation with the various pla t inum ENLO CHIP

drugs. BSO-induced G S H depletion increased sensitivity of TETRA G L C 4 / C D D P for C D D P , CBDCA, and ZENI and did this almost to the level of the parent GLC4 for ENLO. No effect was observed for the two Pt(IV) drugs. Except for C D D P and E N L O , dose modifying factors were higher in the sensitive GLC4 cell line. Also an increased sensitivity for the two Pt(IV) compounds, C H I P and TETRA, could be observed in this cell line. No effect of G S H depletion on the sensitivity for LOBA was found in either cell line. Reduction of resistance could be achieved for C D D P and ENLO. Table 3 shows the effect of

CDDP BSO-induced G S H depletion on the cytotoxicity of the drugs CBDCA for Tera and Tera-CP expressed as ICso after 4 h incubation ZENI with the various pla t inum drugs. BSO-induced G S H depletion LOBA

ENLO increased sensitivity of Tera-CP for C D D P , CBDCA, and CHIP Z E N I whereas no effect was observed for E N L O , LOBA, and TETRA the two Pt(IV) drugs. In the sensitive Tera cell line, G S H de- pletion had no effect on the sensitivity of any pla t inum drug. Reduct ion of resistance could be achieved for C D D P , CBDCA, and ZENI. Table 4 gives a survey of the resistance factors found in the two tested models of C D D P resistance after the various experiments.

For E N L O , the most promising BSO-modulated pla t inum drug in this study, the role of G S H was further evaluated by measuring the effect of BSO-induced G S H depletion on cellular p la t inum levels and P t -DNA binding in GLC4 and GLC4/ C D D P . The cellular p la t inum content, corrected for cellular protein or cellular volume, did not differ between GLC4 and G L C 4 / C D D P after incubation with C D D P [data published pre- viously (5)] or ENLO. Pre t rea tment of cells with BSO did not

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I C s o (].tM) ICso (#M)

GLC4 G L C 4 GLC4/CDDP GLC4/CDDP control + BSO DMF control + BSO DMF

3.0 1.9 1.6 a 27.3 14.0 2.0 a 62.1 31.8 2.0 a 695 404 1.7 a

7.1 3.7 1.9 a 41.1 23.8 1.7 a 3.7 2.8 1.3 b 20.0 26.2 0.8 b

115 41.5 2.8 a 472 148 3.2 a 41.6 14.3 2.9 a 161 122 1.3/'

2.6 1.7 1.5 a 15.1 11.7 1.3 b a p < 0.05 with BSO pretreatment versus control. b Not significant with BSO pretreatment versus control.

Table 3 Effect of BSO-induced GSH depletion on the cytotoxicity of the drugs for Tera and Tera-CP after a 4-h incubation (n >_ 3)

ICso (UM) ICso (uM)

Tera Tera Tera-CP Tera-CP control + BSO DMF control + BSO DMF

2.3 2.2 1.0 a 6.7 4.4 1.5 b 44.9 34.5 1.3 a 90.0 47.7 1.9/' 4.2 4.8 0.9 a 5.6 4.7 1.2/' 3.4 2.9 1.2 a 7.2 5.9 1.2 a

82.7 82.0 1.0 a 95.6 88.6 1.1 a 17.0 15.5 1.1 a 21.1 16.6 1.3 a 3.2 2.4 1.3 a 3.6 3.1 1.2 a

a Not significant with BSO pretreatment versus control. b p < 0.05 with BSO pretreatment versus control.

Table 4 Survey of the resistance factors for the two models of CDDP resistance after the continuous and 4-h incubation experiments

GLC4-GLC4/CDDP Tera-Tera-CP

Continous 4-h Continuous 4-h incubation incubation incubation incubation

CDDP 8.6 9.1 3.7 2.9 CBDCA 8.3 11.2 6.1 2.0 ZEN1 11.0 5.8 3.1 1.3 LOBA 7.2 5.4 1.4 2.1 ENLO 2.6 4.1 2.0 1.2 CHIP 2.5 3.9 2.1 1.2 TETRA 3.6 5.8 1.3 1.1

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RELATIONSHIP OF GSH TO CYTOTOXICITY OF PLATINUM COMPOUNDS

1.28

GO 1.00 Q 0

qD 0.78 0 0 V m

It o.8o ~ m

o E r o.28

0.00 0.48 0.9

,:.Z

:!:! !:!: ~-~.

:i:i i:i: :!:! :!:!

m ~ . R

1.8 2.7

E N L O (raM)

Fig. 4. Effect of pretreatment of GLC4 and GLC4/CDDP with 50 .aM BSO for 48 h on cellular platinum content after 4 h incubation with ENLO. Bars, SEM; n > 3. Significantly different values were observed for GLC4 versus GLC4 plus BSO and GLC4/CDDP versus GLC4/CDDP plus BSO at all but one tested concentra- tions: *, P < 0.05. I , GLC4; [ , GLC4 plus BSO; Fq, GLC4/CDDP; ~, GLC4/ CDDP plus BSO.

500

400" < Z a

300

G. 200 m

0 E

t t -

100 '

0 . . . . . .

0.45 0.9 1.8 2.7

E N L O (mM)

Fig. 5. Effect of pretreatment of GLC4 and GLC4/CDDP with 50 pM BSO for 48 h on Pt-DNA binding after 4 h incubation with ENLO. Bars, SEM; n _> 3. No significant changes were observed in either cell line following BSO treatment. I , GLC4; [], GLC4 plus BSO; [2, GLC4/CDDP; [~, GLC4/CDDP plus BSO.

CHIP and TETRA showed partial to no cross-resistance in GLC4/CDDP and Tera-CP. Platinum drugs containing the DACH carrier ligand, such as TETRA, received much attention because of their potential to overcome CDDP resistance (8, 1 l, 15, 19-21). For the DACH carrier ligand as mechanism of resistance a carrier ligand-specific decrease in accumulation (11, 15, 20, 22), increase in ability to tolerate specific Pt-DNA lesions (20, 22), preferential DNA repair (20, 22), and an effect on the inhibition of DNA synthesis (23) has been reported. Selective activity of TETRA might also be attributed to the circumvention of detoxification systems such as GSH metabo- lism (12). For Pt(IV) compounds, it has been assumed that reduction to Pt(II) is required for activity. GSH can activate Pt(IV) compounds resulting in their reaction with DNA (19). If this mechanism plays a role, this cannot be in disadvantage of sensitivity to Pt(IV) drugs in resistant cells with elevated GSH levels. Most studies with CDDP-resistant cell lines report par- tial or complete cross-resistance, with unchanged (14) or ele- vated GSH levels (8, 9), to CHIP (8, 9, 14, 17).

ZENI showed complete cross-resistance in both models whereas for ENLO partial cross-resistance was found. As ZENI and ENLO differ only in their carrier ligand, with structure and charge of ZENI most similar to CBDCA, carrier ligand speci- ficity of resistance may play a role for these compounds as it does for the DACH ligand. Interestingly, for LOBA, full cross- resistance was observed in GLC4/CDDP but none in Tera-CP. The existence of at least two platinum resistance phenotypes in human ovarian carcinoma cell lines has been reported (24). A difference in platinum resistance phenotype between GLC4/ CDDP and Tera-CP may explain their differential response to LOBA, although cross-resistance patterns for the other plati- num compounds hardly differ. As yet no data concerning GSH status and cross-resistance for ZENI, ENLO, and LOBA are reported.

CDDP can react with thiols such as GSH and therefore may play a role in determining cellular sensitivity to platinum-con- taining compounds by preventing platination of critical loci. We found a close correlation between the amount of GSH, but not between the GST activity, and sensitivity for the various platinum drugs in our cell lines.

BSO was used to investigate a possible causal role of GSH in the resistance to platinum-containing drugs. The involvement of GSH differed between the models and the various platinum drugs. BSO did not affect the CDDP sensitive Tera, whereas in Tera-CP it reduced the resistance for the Pt(II) drugs CDDP, CBDCA, and ZENI. Because almost no cross-resistance could be observed after a 4 h incubation for ENLO, CHIP, and TETRA, no modulating effect of BSO could be expected. Re- duction of one-half of the CDDP resistance in Tera-CP may be ascribed to BSO-induced GSH depletion. In this model of CDDP resistance the increased GSH content, probably in com- bination with the increased GST activity although this cannot be proven statistically with the data from this study, seems to be a relevant mechanism, responsible for maximal 50% of the resistance, that is operational for CDDP and other platinum- containing drugs. In the GLC4-GLCJCDDP model the role of the increased GSH as a cause of resistance is less obvious. BSO-induced GSH depletion increased the sensitivity of the sensitive GLC4 line, for most platinum drugs tested, more ef- fectively than in the GLC4/CDDP line. Reduction of resistance could be achieved only for the Pt(II) compounds CDDP and ENLO. The fact that a 7.4-fold CDDP resistance remains after BSO pretreatment underscores the limited importance of GSH as CDDP resistance mechanism in this model. An effect of differences in the potential for rebound synthesis of GSH in various cell lines, in particular between resistant and parental cell lines (6, 25), cannot be excluded in our experiments as drug treatment after BSO exposure was performed in the absence of GSH inhibition. In the future, examination of the regulation of the GSH synthesis merits further investigation.

A substantial increase in CHIP but not in CDDP or CBDCA cytotoxicity was reported in two murine cell lines after GSH depletion (26). Mistry et al. (2) found a better effect of BSO for Pt(IV) than for Pt(II) drugs. They suggested a more important role for GSH as mechanism of resistance to Pt(IV) than to Pt(II) drugs. In our study, however, dose modifying factors did not differ between Pt(II) drugs and Pt(IV) drugs.

An earlier study with GLC4 and GLC4/CDDP suggested two roles for GSH in CDDP resistance, namely, a cytosolic elimi- nation resulting in less DNA platination and a nuclear effect on the formation and repair of Pt-DNA adducts (6). The amount of CDDP-induced Pt-DNA was lower in GLC4/CDDP than in

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RELATIONSHIP OF GSH TO CYTOTOXICITY OF PLATINUM COMPOUNDS

GLC4. BSO p r e t r e a t m e n t did not affect cel lular p l a t i n u m levels

but inc reased C D D P - i n d u c e d P t - D N A binding in G L C 4 /

C D D P but no t in GLC4. Fo r E N L O , the drug mos t eff ic ient ly

m o d u l a t e d by B S O in this s tudy, G L C 4 / C D D P showed also a

lower D N A p la t ina t ion c o m p a r e d to GLC4. Surpr is ingly , BSO

p r e t r e a t m e n t inc reased E N L O - i n d u c e d cel lular p l a t i n u m con-

ten t a l m o s t 2-fold, in G L C 4 and G L C 4 / C D D P , however , wi th-

ou t an effect on P t - D N A binding. Th i s m a y occur at longer drug

exposu re t ime, exp la in ing the increased cy to tox ic i ty in bo th cell

l ines af ter B S O p r e t r e a t m e n t . I nc reased cel lu lar d rug a c c u m u -

la t ion by BSO was also r epo r t ed for o the r n o n - p l a t i n u m drugs

such as d a u n o r u b i c i n and the c a m p t o t h e c i n ana logue CPT-11

(27, 28). T h e m e c h a n i s m beh ind this p h e n o m e n o n still mus t be

e lucidated .

In conc lus ion , in the p resen t s tudy G S H seems to be a good

re f lec tor o f sensi t ivi ty to p l a t i n u m - c o n t a i n i n g drugs and there-

fore a good ta rge t for m o d u l a t i o n in the clinic. Phase I s tudies

us ing BSO with n o n - p l a t i n u m drugs are u n d e r way (29, 30).

Howeve r , the fact tha t the i nvo lvemen t o f G S H differs be tween

var ious drugs and m o d e l s m a k e s a genera l causat ive role o f

G S H in sensi t ivi ty a n d / o r res i s tance to p l a t i n u m - c o n t a i n i n g

drugs ques t ionable and the effect o f m o d u l a t i o n with BSO un-

predic table . Desp i t e the fact tha t BSO can be an exce l len t m o d -

u la to r o f p l a t i n u m sensi t ivi ty in vitro, care shou ld be t aken no t

to genera l ize the op t ion o f G S H as ta rge t for m o d u l a t i o n in the

clinic.

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