lnt. J . Cancer: 48, 265-269 (1991) 0 1991 Wiley-Liss, Inc.
Publication of the International Union Against Cancer Publication de I'Union lnternationale Contre le Cancer
COMPARATIVE CYTOTOXICITY OF CI-973, CISPLATIN, CARBOPLATIN AND TETRAPLATIN IN HUMAN OVARIAN CARCINOMA CELL LINES R.P. PEREZ, P.J. O'DWYER,L.M. HANDEL,R.F. OZOLSand T.C. HAMILTON' Department of Medical Oncology, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111, USA. The clinical efficacy of cisplatin-based chemotherapy for ovarian cancer is frequently compromised by drug resistance or dose-limiting renal and neurologic toxicities. Cl-973 (NKI2 I). a 2-methyl-I ,+butanediamine analogue of carboplatin, has shown little nephro- and neuro-toxicity in pre-clinical model systems and in phase-I trials. I t s in vitro spectrum of activity against ovarian cancer cell lines has not been previously characterized. The in vitro activities of Cl-973, cisplatin, carboplatin and tetraplatin were compared in several platinum-sensitive and -resistant human ovarian carcinoma cell lines. Cytotoxicity was assessed by inhibition of clonogenic survival in soft agar with continuous drug exposure. On a molar basis, cisplatin and tetraplatin were the most potent analogues, while carboplatin was consistently less potent. Cisplatin, carboplatin and Cl-973 elicited a very similar response pattern by Spearman rank correlation, distinct from that seen with tetraplatin. The magnitude of resistance t o Cl-973 was comparable t o cisplatin in 5 cell lines but was substantially lower in the highly cisplatin-resistant 2780-CP70 and OVCAR-I0 cell lines. These results suggest that Cl-973 and tetraplatin may have potential utility in some cases of cisplatin-resistant ovarian cancer. In addition, our data are consistent with the existence of at least 2 platinum-resistance phenotype-ne with moderate levels of resistance t o cisplatin, carboplatin and Cl-973 but highly resistant t o tetraplatin, the other highly resistant t o cisplatin and carboplatin but only partially cross-resistant with tetraplatin and Cl-973. The recognition of different resistance phenotypes may facilitate the study of cellular resistance mechanisms t o cisplatin and newer platinum analogues.
Cisplatin has become one of the most widely used chemotherapeutic agents for the treatment of solid tumors (Rosenberg, 1985). Unfortunately, the clinical utility of cisplatin in many diseases, including ovarian cancer, has been limited by the frequent development of resistance and/or dose-limiting toxicities (0201s and Young, 1984; Loehrer and Einhorn, 1984). Numerous platinum analogues have been developed and tested in an effort to overcome these problems. Carboplatin, the only other platinum analogue approved for clinical use, produces myelosuppression as its dose-limiting toxicity and is relatively free of neuro- and nephrotoxicity, compared with cisplatin (Canetta et al., 1990). Unfortunately, nearly complete cross-resistance between cisplatin and carboplatin is evident both in vitro (Behrens et al., 1987; Hills et al., 1990; Schurig et al., 1990) and in clinical ovarian cancer (Gore et al., 1989; Eisenhauer et al., 1990). Therefore, the evaluation of platinum analogues remains an area of considerable interest. The present study was undertaken to compare the cytotoxicities of CI-973 (NK- 121), cisplatin, carboplatin and tetraplatin (Fig. 1) against a panel of platinum-sensitive and -resistant human ovarian cancer cell lines. Preliminary reports suggest that CI-973 has significant anti-tumor activity and minimal nephrotoxicity in some pre-clinical model systems (Kraker et al., 1988). It is presently being evaluated in phase-I clinical trials. In this report, the activity of CI-973 was compared with cisplatin, carboplatin and tetraplatin in ovarian carcinoma cell lines selected for platinum resistance in vitro or obtained from patients refractory to cisplatin- or carboplatinbased chemotherapy.
MATERIAL AND METHODS
Ovarian carcinoma cell lines A2780, an ovarian cancer cell line from an untreated patient, was originally provided by Dr. S . Aaronson (NCI, Bethesda, MD) (Eva et al., 1982). 278OCp and 2780TP cell lines were produced by intermittent, incremental exposure of the sensitive parental A2780 cell line to various concentrations of cisplatin or tetraplatin, respectively. The OVCAR-3, -4 and -10 cell lines were developed from patients who were clinically refractory to platinum-based chemotherapy (Hamilton et al., 1983, 1989). The PEOl and PE04 cell lines were derived from the same patient before and after the onset of clinical resistance to platinum-containing combination chemotherapy (Langden et al., 1988). The treatment status of patients from whom these cell lines were derived and the cloning efficiencies of the individual cell lines are summarized in Table I. All cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS (GIBCO, Grand Island, NY), 0.28 units/ml insulin (Squibb-Novo, Princeton, NJ), 100 Fg/ml streptomycin, 100 unitdm1 penicillin, and 0.3 mg/ml glutamine. Cells were grown at 37"C, in a humidified atmosphere of 5% CO, in air. Drugs CI-973 was provided by Parke-Davis (Ann Arbor, MI). Cisplatin and carboplatin were furnished by Bristol-Myers (Evansville, IN). Tetraplatin was obtained from the Investigational Drug Branch, Division of Cancer Treatment, National Cancer Institute (Bethesda, MD). The clinical formulations of all drugs were used. Drugs were reconstituted in sterile water according to the manufacturers' directions and diluted to their final concentrations immediately before use. Cytotoxicity assay Cytotoxicity was assayed in a soft agarose bilayer system (Behrens et al., 1987; Ozols et al., 1980). Briefly, single-cell suspensions in 0.3% agarose (in media) were plated in the presence or absence of drugs over chilled 0.6% agarose feeder layers. Cells were plated at concentrations of 10-30,OOO cells/ ml, depending on the cloning efficiency of the individual cell lines, to yield approximately 2,000 colonies per 10 cm2 in untreated controls. Colonies were counted with an Artek Omnicon FAS IV Image Analysis System (Chantilly, VA) following incubation of the plated cells under routine culture conditions for 7 to 14 days. Within experiments, percent clonogenic survival was determined as the mean number of colonies from triplicate platings at each drug concentration relative to untreated controls. Data analysis The IC,,s of the platinum analogues in individual cell lines were determined from survival curves generated for each ex'To whom correspondence and reprint requests should be sent. Received November 5, 1990 and in revised form January 7, 1991
266
PEREZ ET A L . NH3
\
Pt
/“I
NHi C ‘l
Carboolatiq
dl Tetradatin €u!3 FIGURE1 - Structures of cisplatin, carboplatin, tetraplatin, and CI973.
periment. Data in tables and figures represent the mean (*SD) of 3 to 6 individual experiments, unless otherwise indicated. Resistance in Tables I1 and IV and Figure 3 is arbitrarily expressed relative to A2870 to facilitate comparison between cell lines. The sensitivity patterns of pairs of platinum analogues were analyzed by Spearman Rank-Order Coefficient determination (Bruning and Kintz, 1987). Briefly, sensitivities of the cell lines to platinum analogues were numerically ranked, based on the IC,, data in Table 11, and correlation coefficients were determined for pairs of analogues. Significance was tested against the hypothesis that there was no correlation between pairs of analogues ( i . e . , rho = 0) according to tables of the exact distribution of Spearman’s rank correlation coefficient (Kendall, 1963). RESULTS
The cytotoxicity curves of cisplatin, carboplatin, tetraplatin
and CI-973 against the A2780 cell line are shown in Figure 2. All 4 analogues produced dose-dependent cytotoxicity , with relatively steep dose-response curves. The molar order of POtency in A2780 was tetraplatin > cisplatin = CI-973 > carboplatin. Cytotoxicity of the platinum analogues was also assessed in several additional cell lines (Table 11). Resistance is arbitrarily expressed relative to the untreated A2780 cell line, despite the fact that most of the other lines were not derived from A2780. There was substantial variability in sensitivity of the cell lines to individual platinum analogues, in both absolute and relative terms. The A2780 and PEOl cell lines were consistently most sensitive to the analogues. Complete cross-resistance was evident between cisplatin and carboplatin, while more complex patterns were seen with tetraplatin and CI-973. Tetraplatin was more potent than cisplatin in the A2780, 2780-CP70, and OVCAR-10 cell lines. The latter 2 cell lines were somewhat less resistant to tetraplatin (12- and 10-fold, respectively) than to cisplatin (46- and 70-fold). The OVCAR-3, OVCAR-4, PEO1, and PE04 cell lines were more resistant to tetraplatin than to cisplatin. There was essentially no difference in tetraplatin sensitivity in the PEOl and PE04 cell lines, although the latter cell line was 5-fold resistant to cisplatin (relative to PEO1). The sensitivities of the A2780 and OVCAR-10 cell lines to CI-973 were similar to tetraplatin. The OVCAR-3, OVCAR-4, PEOl , and PE04 cell lines were substantially less resistant to CI-973 than to tetraplatin. Conversely, the 2780CP70 cell line was somewhat more resistant to CI-973 (21fold) than to tetraplatin (12-fold). The sensitivity patterns of the cell lines to pairs of analogues were analyzed by Spearman rank-order correlation (Table 111). A high correlation coefficient is consistent with a similar pattern of sensitivity (1 = identical). Cisplatin and carboplatin exhibited identical sensitivity patterns in these cell lines. The pattern of sensitivity to CI-973 correlated highly with both cisplatin and carboplatin, but not with tetraplatin. Conversely, the pattern of sensitivity to tetraplatin was distinct from that obtained with other analogues. Although the overall pattern of sensitivity of CI-973 was similar to cisplatin and carboplatin, the magnitude of resistance to CI-973 in the highly cisplatin-resistant OVCAR-10 cell line was comparatively small, similar to tetraplatin. This cell line was unique in its very high level of resistance to cisplatin and
TABLE I - CELL LINES Cell line
Cloning efficiency’
A2780 2780-CP70 2780-TP7.O 2780-TP 10.5 2780-TP50 OVCAR-3 OVCAR-4 OVCAR-10
.20 .29 .15 .19 .23 .06 .02 .18
Treatment*
Untreated
------ ---
Comment
-- ------I n vitro resistance selected by intermittent,
incremental exposure to cisplatin or tetraplatin
CTX/ADR/CDDP CTWADWCDDP CDDP, CBDCA
-----------------
Progressive disease after CDDP-based
PEO 1
.03
5FU/CLB/CDDP
PE04
.07
SFU/CLB/CDDP
chemotherapy, high-dose CDDP (400 mg/m*) and high-dose CBDCA (800 mg/m2) Cell lines obtained from the same patient before and after the onset of clinical resistance
‘Cloning efficiency = number colonies per platehumber cells plated.-’Treatment status of patient at the time cell line was established. Abbreviations: CTX, cyclophosphamide; ADR, doxorubicin; CDDP, cisplatin; CBDCA, carboplatin; 5FU, 5fluorouracil; and CLB, chlorambucil.
PLATINUM ANALOGUES IN OVARIAN CANCER CELLS
267
TABLE I1 - CYTOTOXICITY OF PLATINUM ANALOGUES Cell line
A2780 GO(J*M)' Rel. resist.2 2780-CP70 IC50(J*M) Rel. resist. OVCAR-3 ICSO(CLM) Rel. resist. 0V CAR -4 ICSO(J*M) Rel. resist. OVCAR-10 Ic5O(bM) Rel. resist. PEO 1 ICSO(P?f) Re1 resist. PE04 IC50(PM) Rel. resist.
CDDP
CBDCA
Tetraplatin
CI-973
0.1 1('-c .05) 1
0.37(*.05) 1
0.03(+-.02) 1
0.25(5.23) 1
5.03(rt 1.69)
8.65(f 2.85) 23
0.37(2.29) 12
5.35( 24.32) 21
0.21(?.07) 2
-------
1.02(*.34) 34
0.63(5.26) 2.5
0.18(rt.05) 1.6
-------
0.47(+-.15) 16
0.97( f.33) 4
7.67(+5.31) 70
23(+ 11) 62
0.30(+-.14) 10
2.70( f.5) 11
0.07(*.02) 0.6
0.27(2.02) 0.7
0.32(& .07) 11
0.21(2.04) 1
0.32(rt: .07) 3
1.29( .39) 3
0.27(&.14) 9
0.73(&.25) 3
46
*
iIC,,(pM) expressed as mean (+SO) of 3 to 6 separate e x pximents. Within experiments, triplicate platings were done at each concentration.--Resistance relative to A2780.
-
100
80 -
-0-
60 -1
TABLE I11 - SPEARMAN RANK CORRELATION BETWEEN ANALOGUE PAIRS CDDP CBDCA
Tetrapiatin Cl-973
40 -
Combination
rho
D
CDDP-CBDCA CDDP-CI973 CDDP-tetraplatin CBDCA-C1973 CBDCA-tetraplatin Tetraplatin-CI973
1.000 .857 .036 ,900 .200 .208
,0083 ,012 ,482 .042 ,392 .331
carboplatin. We subsequently evaluated the sensitivities of cisplatin, tetraplatin and (21-973 in variant 2780 cell lines selected for resistance to tetraplatin in vitro (Table IV). Relatively high levels of resistance to both tetraplatin and CI-973 were seen in all 3 cell lines. There was substantial difference in cisplatin sensitivity between the 2780-TP7.0 and -TP10.5 cell lines.
20 0 .01
1
.1
10
Concentration (pM) FIGURE2 - Clonogenic survival of A2780 cells following continuous treatment with platinum analogues. Each point is the mean of at least 3 independent experiments.
8o
r
m-
A2780
2780-CF70 OVCARJ O V W 4 OVCAR10
PEOl
EM
Cell Line
FIGURE3 - Resistance of ovarian cancer cell lines to platinum analogues, arbitrarily expressed relative to A2780.
DISCUSSION
We observed substantial variability in the sensitivity of the cell lines tested to the individual platinum analogues. On a molar basis, cisplatin and tetraplatin were consistently the most potent analogues. Cisplatin was the most potent analogue in 3 cell lines (OVCAR-3, OVCAR-4, and PEOl), while tetraplatin was most potent in 3 other cell lines (A2780, 2780-CP70, and OVCAR-10). Their potency was roughly equivalent in the PEO4 cell line. In general, carboplatin was the least potent analogue. These results are consistent with an earlier report from our laboratory in which the activities of cisplatin, carboplatin, and tetraplatin were assessed in the A2780 and 2780CP8 cell lines (Behrens et al., 1987). Analysis of our data by the Spearman rank correlation test clearly showed that the pattern of sensitivity of our cell lines to cisplatin and carboplatin was identical, while the pattern of tetraplatin sensitivity was dissimilar. These results confirm and extend earlier findings from our laboratory in the A2780 and 2780-CP8 cell lines (Behrens et al., 1987). Similar results were obtained by Hills et al. (1990), who assessed the in vitro cytotoxicity of cisplatin, carboplatin, and tetraplatin against a panel of 10 human ovarian carcinoma cell lines. The patterns
268
PEREZ ET AL. TABLE IV - CYTOTOXICITY OF TETRAPLATIN AND Cl-973 IN TETRAPLATIN-RESISTANT 1780 VARIANT CELL LINES Cell line
2780-Tw.0 ICs0(pM)' Rel. resist.* 2780-TP 10.5 ICSO(P?) Rel. resist.
Templatin
a-973
3
1.29(&.58) 19
3.80(*2) 15
2.63(%.51) 24
2.07(4.31) 31
6.10(+ .45) 24
2.57( %. 54) 38
3.37( +- .94) 13
CDDP-
0.34(+ .04)
-
' I C 5 , J ~ expressed ~) as mean (LSD)of 3 to 6 sparate ex nmnts Within expnments, triplicate platings were done dt edLh concentration. 'Gesistance relative to A2180
of sensitivity to cisplatin and carboplatin were highly correlated in these cell lines (Spearman correlation coefficients > 0.9), while the pattern of tetraplatin sensitivity did not correlate with that obtained with the other analogues. The mechanism(s) responsible for these different sensitivity patterns is unknown. Tetraplatin may require reduction to an active form (Eastman, 1987) and is reportedly more active against plateau-phase cells than exponentially growing cells in culture (Bhuyan et al., 1988; Bhuyan and Groppi, 1989). Differential sensitivity to these platinum analogues might reflect differences in cellular transport or metabolism, or it may reflect the growth kinetics of various cell lines. Sensitivity to CI-973 correlated with sensitivity to cisplatin and carboplatin, but not tetraplatin. On a molar basis, CI-973 was not the most active analogue in any of the cell lines tested. However, the magnitude of resistance of individual cell lines to CI-973 was generally comparable with or lower than to cisplatin. These results are consistent with preliminary observations in cisplatin sensitive and resistant L1210 cells (Kraker et al., 1988). In particular, the magnitude of resistance to CI-973 was much lower than to cisplatin in the highly cisplatinresistant 2780-CWO and OVCAR-10 cell lines. The relatively low degree of cross-resistance in the OVCAR-10 cell line is especially interesting, since this cell line was derived from a patient who was clinically refractory to high-dose cisplatin and high-dose carboplatin. CI-973 is presently being evaluated in phase-I clinical trials (Hudes et al., 1990; Fukuoka et al., 1989; Theriault et al., 1990; Newman et al., 1990). Overall, clinical toxicity has been minimal. Myelosuppression, predominantly neutropenia, has been dose limiting. Thrombocytopenia, which often complicates clinical carboplatin therapy, was not a prominent toxicity. CI-973 appears to have minimal, if any, neuro- or nephrotoxicity. Preliminary pharmacokinetic data (O'Dwyer, personal communication) suggest that the concentrations of CI-973 which were cytotoxic in our in vitro assay are compa-
rable to the peak plasma levels achieved at the initial clinical dose levels in the Fox Chase Cancer Center phase-I trial (2-3 Fg/ml, approx. 4-7 FM). Maximally achievable peak plasma levels have yet to be determined. In addition, the in vitro pharmacokinetics of CI-973 are not known. Thus, it is not possible at present to determine how closely the concentrations required for cytotoxicity in our assays approximate to clinical conditions. Our results are consistent with the existence of at least 2 platinum resistance phenotypes in human ovarian cancer cells. The first phenotype, seen in the OVCAR-10 cell line, is characterized by very high levels of resistance to cisplatin and carboplatin, with more moderate resistance to tetraplatin and CI-973. It is interesting that comparable levels of crossresistance between CI-973 and tetraplatin were also seen in cell lines selected for tetraplatin resistance in vitro. An alternative phenotype is apparent in the OVCAR-3, OVCAR-4 and PE04 cell lines. These cell lines have low levels of resistance to cisplatin and carboplatin, while the levels of resistance to tetraplatin are comparatively high. In these cell lines, the pattern of sensitivity and the magnitudes of resistance to CI-973 were similar to cisplatin and carboplatin, and markedly different from tetraplatin. The resistance profile of the 2780-CP70 cell line more closely approximated the former phenotype. Our in vitro observations are consistent with clinical evidence of nearly complete cross-resistance between cisplatin and carboplatin (Gore et al., 1989; Eisenhauer et al., 1990), although some patients who fail conventional-dose chemotherapy may respond to high-dose therapy (Ozols et al., 1985, 1987; Rothenberg et al., 1988). These patients must have had relatively low levels of resistance (s2-fold) in order to respond to a mere doubling of the dose of cisplatin or carboplatin. Whether higher percentages of cisplatin-refractory patients will respond to further carboplatin dose escalations remains to be seen. Tetraplatin and CI-973 are both currently in phase-I trials, and no data are yet available regarding their activity in patients who are refractory to conventional or high-dose cisplatin. The existence of different platinum resistance phenotypes suggests that different resistance mechanisms may be operative. The recognition and characterization of platinumresistance phenotypes may ultimately yield insights into the cellular mechanisms of platinum resistance and provide a basis for more rational chemotherapy in ovarian cancer. ACKNOWLEDGEMENTS
Work from our laboratory was supported by institutional grants NIH CA 00927, NIH RR0895, and appropriations from the Commonwealth of Pennsylvania. Additional support was obtained from the Pew Charitable Trusts 88-01522-000. Dr. Perez is supported by fellowships from the American Cancer Society (Clinical Oncology Fellowship 89-142) and US Bioscience.
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PLATINUM ANALOGUES IN OVARIAN CANCER CELLS
WESTIN,E., WONG-STAAL, F., GALLO,R. and AARONSON, S., Cellular genes analogous to retroviral onc genes are transcribed in human tumor cells. Nature (Lond.),295, 116-119 (1982). FUKUOKA, M., NIITANI,H., HASEGAWA, K., MAJIMA,H., HINO, M., S., FUJITA,H., OHTA,K., FURUSE,K., KIMURA,I. and TSUKAGOSHI, KATOH,T., Phase-I study of new platinum compound, NK121. Proc. Amer. Soc. d i n . Oncol., 8, 62, 240a (1989). GORE,M., FRYATT,I., WILTSHAW,E., DAWSON,T., ROBINSON,B. and CALVERT,A., Cisplatidcarboplatin cross-resistance in ovarian cancer. Brit. J . Cancer, 60, 767-769 (1989). HAMILTON,T., LAI, G., ROTHENBERG, M., Foro, A., YOUNG,R. and OZOLS,R., Mechanisms of resistance to cisplatin and alkylating agents. In: R. Ozols (ed.), Drug resistance in cancer therapy, pp. 151-169, Kluwer, Boston (1989). HAMILTON, T., YOUNG,R., MCKOY,W., GROTZINGER, K., GREEN,J., CHU, E., WHANG-PENG, J., ROGAN,A,, GREEN,W. and OZOLS,R., Characterizationof a human ovarian carcinoma cell line (N1H:OVCAR-3) with androgen and estrogen receptors. Cancer Res., 43, 5379-5389 (1983). HILLS,C., KELLAND, L., ABEL,G., SIRACKY, J., WILSON,A. and HARRAP, K., Biological properties of ten human ovarian carcinoma cell lines: calibration in v i m against four platinum complexes. Brit. J . Cancer, 59, 527-534 (1990). HUDES,G., O’DWYER,P., COHEN,I., KOWAL,C. and WHITFIELD,L., Phase-Vpharmacokinetic study of CI-973 on a 5-day bolus schedule. Proc. Amer. Assoc. Cancer Res., 31, 179, 1065a (1990). KENDALL,M., Rank correlarion methods, 3rd ed., Griffin, London (1 963). KRAKER, A,, MOORE,C., LEOPOLD,W. and TAKAHASHI, K., Pre-clinical characterization of the in vitro and in vivo activity of [l,l-cyclobutane(NKdicarboxylato(2-)-0,0’] (2-methyl-1 ,4-butanediamine-N,Nf)Pt 121/CI-973).Proc. Amer. Assoc. Cancer Res., 29, 344, 1370a (1988). LANGWN,S., LAWRIE, S., HAY, F., HAWKES,M., MCDONALD,A., I., SCHOL,D., HILGERS,J., LEONARD,R. and SMYTH,J., HAYWARD,
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Characterization and properties of nine human ovarian adenocarcinoma cell lines. Cancer Res., 48, 61666172 (1988). LOEHRER, P. and EINHORN,L., Cisplatin. Ann. int. Med., 100, 704-713 (1984). NEWMAN, R., BENEVENUTO, J., RABER,M., THERIAULT,R. and WHITFIELD, L., A phase-I clinical pharmacologic study of CI-973. Proc. Amer. Assoc. Cancer Res., 31, 181, 1073a (1990). OZOLS,R., OSTCHEGA, Y., CURT,G. and YOUNG,R., High-dose carboplatin in refractory ovarian cancer patients. Journ. clin. Oncol., 5, 197201 (1987). OZOLS,R., OSTCHEGA,Y., MYERS,C. and YOUNG,R.,High-dose cisplatin in hypertonic saline in refractory ovarian cancer. Journ. clin. Oncol., 3, 1246-1250 (1985). OZOLS,R., WILLSON,J., GROTZINGER, K. and YOUNG,R., Cloning of human ovarian cancer cells in soft agar from malignant effusions and peritoneal washings. Cancer Res., 40, 2743-2747 (1980). OZOLS, R. and YOUNG,R., Chemotherapy of ovarian cancer. Semin. Oncol., 11, 251-263 (1984). ROSENBERG, B., Fundamental studies with cisplatin. Cancer, 55, 23042316 (1985). ROTHENBERG, M., OSTCHEGA, Y.,STEINBERG, S., YOUNG,R., HUMMEL, S. and OZOLS,R., High-dose carboplatin with diethyldithiocarbamate chemoprotection in treatment of women with relapsed ovarian cancer. J . nat. Cancer Inst., 80, 1488-1492 (1988). SCHURIG,J., ROSE,W., CATINO,J., GAVER,R., LONG,B., MADISSOO, H. and CANETTA,R., Pharmacologic characteristics of carboplatin: preclinical experience. In: P. Bunn, R. Canetta, R. Ozols and M. Rozencweig (eds.), Carboplatin: current perspectives and future directions, pp. >17, Saunders, Philadelphia (1990). THERIAULT, R., ESPERZA,L., RABER,M., NEWMAN,R., KRAKOFF,I., COHEN,I. and KOWAL,C., Phase-I evaluation of [SP-4-3-(R)]-[l,lcyclobutanedicamboxylato(2-)] (2-methyl- 1,4-butanediamine-N,N’) platinum (CI-973). Proc. Amer. Assoc. Cancer Res., 31, 197, 1170a (1990).
Publication of the International Union Against Cancer Publication de I'Union lnternationale Contre le Cancer
COMPARATIVE CYTOTOXICITY OF CI-973, CISPLATIN, CARBOPLATIN AND TETRAPLATIN IN HUMAN OVARIAN CARCINOMA CELL LINES R.P. PEREZ, P.J. O'DWYER,L.M. HANDEL,R.F. OZOLSand T.C. HAMILTON' Department of Medical Oncology, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111, USA. The clinical efficacy of cisplatin-based chemotherapy for ovarian cancer is frequently compromised by drug resistance or dose-limiting renal and neurologic toxicities. Cl-973 (NKI2 I). a 2-methyl-I ,+butanediamine analogue of carboplatin, has shown little nephro- and neuro-toxicity in pre-clinical model systems and in phase-I trials. I t s in vitro spectrum of activity against ovarian cancer cell lines has not been previously characterized. The in vitro activities of Cl-973, cisplatin, carboplatin and tetraplatin were compared in several platinum-sensitive and -resistant human ovarian carcinoma cell lines. Cytotoxicity was assessed by inhibition of clonogenic survival in soft agar with continuous drug exposure. On a molar basis, cisplatin and tetraplatin were the most potent analogues, while carboplatin was consistently less potent. Cisplatin, carboplatin and Cl-973 elicited a very similar response pattern by Spearman rank correlation, distinct from that seen with tetraplatin. The magnitude of resistance t o Cl-973 was comparable t o cisplatin in 5 cell lines but was substantially lower in the highly cisplatin-resistant 2780-CP70 and OVCAR-I0 cell lines. These results suggest that Cl-973 and tetraplatin may have potential utility in some cases of cisplatin-resistant ovarian cancer. In addition, our data are consistent with the existence of at least 2 platinum-resistance phenotype-ne with moderate levels of resistance t o cisplatin, carboplatin and Cl-973 but highly resistant t o tetraplatin, the other highly resistant t o cisplatin and carboplatin but only partially cross-resistant with tetraplatin and Cl-973. The recognition of different resistance phenotypes may facilitate the study of cellular resistance mechanisms t o cisplatin and newer platinum analogues.
Cisplatin has become one of the most widely used chemotherapeutic agents for the treatment of solid tumors (Rosenberg, 1985). Unfortunately, the clinical utility of cisplatin in many diseases, including ovarian cancer, has been limited by the frequent development of resistance and/or dose-limiting toxicities (0201s and Young, 1984; Loehrer and Einhorn, 1984). Numerous platinum analogues have been developed and tested in an effort to overcome these problems. Carboplatin, the only other platinum analogue approved for clinical use, produces myelosuppression as its dose-limiting toxicity and is relatively free of neuro- and nephrotoxicity, compared with cisplatin (Canetta et al., 1990). Unfortunately, nearly complete cross-resistance between cisplatin and carboplatin is evident both in vitro (Behrens et al., 1987; Hills et al., 1990; Schurig et al., 1990) and in clinical ovarian cancer (Gore et al., 1989; Eisenhauer et al., 1990). Therefore, the evaluation of platinum analogues remains an area of considerable interest. The present study was undertaken to compare the cytotoxicities of CI-973 (NK- 121), cisplatin, carboplatin and tetraplatin (Fig. 1) against a panel of platinum-sensitive and -resistant human ovarian cancer cell lines. Preliminary reports suggest that CI-973 has significant anti-tumor activity and minimal nephrotoxicity in some pre-clinical model systems (Kraker et al., 1988). It is presently being evaluated in phase-I clinical trials. In this report, the activity of CI-973 was compared with cisplatin, carboplatin and tetraplatin in ovarian carcinoma cell lines selected for platinum resistance in vitro or obtained from patients refractory to cisplatin- or carboplatinbased chemotherapy.
MATERIAL AND METHODS
Ovarian carcinoma cell lines A2780, an ovarian cancer cell line from an untreated patient, was originally provided by Dr. S . Aaronson (NCI, Bethesda, MD) (Eva et al., 1982). 278OCp and 2780TP cell lines were produced by intermittent, incremental exposure of the sensitive parental A2780 cell line to various concentrations of cisplatin or tetraplatin, respectively. The OVCAR-3, -4 and -10 cell lines were developed from patients who were clinically refractory to platinum-based chemotherapy (Hamilton et al., 1983, 1989). The PEOl and PE04 cell lines were derived from the same patient before and after the onset of clinical resistance to platinum-containing combination chemotherapy (Langden et al., 1988). The treatment status of patients from whom these cell lines were derived and the cloning efficiencies of the individual cell lines are summarized in Table I. All cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS (GIBCO, Grand Island, NY), 0.28 units/ml insulin (Squibb-Novo, Princeton, NJ), 100 Fg/ml streptomycin, 100 unitdm1 penicillin, and 0.3 mg/ml glutamine. Cells were grown at 37"C, in a humidified atmosphere of 5% CO, in air. Drugs CI-973 was provided by Parke-Davis (Ann Arbor, MI). Cisplatin and carboplatin were furnished by Bristol-Myers (Evansville, IN). Tetraplatin was obtained from the Investigational Drug Branch, Division of Cancer Treatment, National Cancer Institute (Bethesda, MD). The clinical formulations of all drugs were used. Drugs were reconstituted in sterile water according to the manufacturers' directions and diluted to their final concentrations immediately before use. Cytotoxicity assay Cytotoxicity was assayed in a soft agarose bilayer system (Behrens et al., 1987; Ozols et al., 1980). Briefly, single-cell suspensions in 0.3% agarose (in media) were plated in the presence or absence of drugs over chilled 0.6% agarose feeder layers. Cells were plated at concentrations of 10-30,OOO cells/ ml, depending on the cloning efficiency of the individual cell lines, to yield approximately 2,000 colonies per 10 cm2 in untreated controls. Colonies were counted with an Artek Omnicon FAS IV Image Analysis System (Chantilly, VA) following incubation of the plated cells under routine culture conditions for 7 to 14 days. Within experiments, percent clonogenic survival was determined as the mean number of colonies from triplicate platings at each drug concentration relative to untreated controls. Data analysis The IC,,s of the platinum analogues in individual cell lines were determined from survival curves generated for each ex'To whom correspondence and reprint requests should be sent. Received November 5, 1990 and in revised form January 7, 1991
266
PEREZ ET A L . NH3
\
Pt
/“I
NHi C ‘l
Carboolatiq
dl Tetradatin €u!3 FIGURE1 - Structures of cisplatin, carboplatin, tetraplatin, and CI973.
periment. Data in tables and figures represent the mean (*SD) of 3 to 6 individual experiments, unless otherwise indicated. Resistance in Tables I1 and IV and Figure 3 is arbitrarily expressed relative to A2870 to facilitate comparison between cell lines. The sensitivity patterns of pairs of platinum analogues were analyzed by Spearman Rank-Order Coefficient determination (Bruning and Kintz, 1987). Briefly, sensitivities of the cell lines to platinum analogues were numerically ranked, based on the IC,, data in Table 11, and correlation coefficients were determined for pairs of analogues. Significance was tested against the hypothesis that there was no correlation between pairs of analogues ( i . e . , rho = 0) according to tables of the exact distribution of Spearman’s rank correlation coefficient (Kendall, 1963). RESULTS
The cytotoxicity curves of cisplatin, carboplatin, tetraplatin
and CI-973 against the A2780 cell line are shown in Figure 2. All 4 analogues produced dose-dependent cytotoxicity , with relatively steep dose-response curves. The molar order of POtency in A2780 was tetraplatin > cisplatin = CI-973 > carboplatin. Cytotoxicity of the platinum analogues was also assessed in several additional cell lines (Table 11). Resistance is arbitrarily expressed relative to the untreated A2780 cell line, despite the fact that most of the other lines were not derived from A2780. There was substantial variability in sensitivity of the cell lines to individual platinum analogues, in both absolute and relative terms. The A2780 and PEOl cell lines were consistently most sensitive to the analogues. Complete cross-resistance was evident between cisplatin and carboplatin, while more complex patterns were seen with tetraplatin and CI-973. Tetraplatin was more potent than cisplatin in the A2780, 2780-CP70, and OVCAR-10 cell lines. The latter 2 cell lines were somewhat less resistant to tetraplatin (12- and 10-fold, respectively) than to cisplatin (46- and 70-fold). The OVCAR-3, OVCAR-4, PEO1, and PE04 cell lines were more resistant to tetraplatin than to cisplatin. There was essentially no difference in tetraplatin sensitivity in the PEOl and PE04 cell lines, although the latter cell line was 5-fold resistant to cisplatin (relative to PEO1). The sensitivities of the A2780 and OVCAR-10 cell lines to CI-973 were similar to tetraplatin. The OVCAR-3, OVCAR-4, PEOl , and PE04 cell lines were substantially less resistant to CI-973 than to tetraplatin. Conversely, the 2780CP70 cell line was somewhat more resistant to CI-973 (21fold) than to tetraplatin (12-fold). The sensitivity patterns of the cell lines to pairs of analogues were analyzed by Spearman rank-order correlation (Table 111). A high correlation coefficient is consistent with a similar pattern of sensitivity (1 = identical). Cisplatin and carboplatin exhibited identical sensitivity patterns in these cell lines. The pattern of sensitivity to CI-973 correlated highly with both cisplatin and carboplatin, but not with tetraplatin. Conversely, the pattern of sensitivity to tetraplatin was distinct from that obtained with other analogues. Although the overall pattern of sensitivity of CI-973 was similar to cisplatin and carboplatin, the magnitude of resistance to CI-973 in the highly cisplatin-resistant OVCAR-10 cell line was comparatively small, similar to tetraplatin. This cell line was unique in its very high level of resistance to cisplatin and
TABLE I - CELL LINES Cell line
Cloning efficiency’
A2780 2780-CP70 2780-TP7.O 2780-TP 10.5 2780-TP50 OVCAR-3 OVCAR-4 OVCAR-10
.20 .29 .15 .19 .23 .06 .02 .18
Treatment*
Untreated
------ ---
Comment
-- ------I n vitro resistance selected by intermittent,
incremental exposure to cisplatin or tetraplatin
CTX/ADR/CDDP CTWADWCDDP CDDP, CBDCA
-----------------
Progressive disease after CDDP-based
PEO 1
.03
5FU/CLB/CDDP
PE04
.07
SFU/CLB/CDDP
chemotherapy, high-dose CDDP (400 mg/m*) and high-dose CBDCA (800 mg/m2) Cell lines obtained from the same patient before and after the onset of clinical resistance
‘Cloning efficiency = number colonies per platehumber cells plated.-’Treatment status of patient at the time cell line was established. Abbreviations: CTX, cyclophosphamide; ADR, doxorubicin; CDDP, cisplatin; CBDCA, carboplatin; 5FU, 5fluorouracil; and CLB, chlorambucil.
PLATINUM ANALOGUES IN OVARIAN CANCER CELLS
267
TABLE I1 - CYTOTOXICITY OF PLATINUM ANALOGUES Cell line
A2780 GO(J*M)' Rel. resist.2 2780-CP70 IC50(J*M) Rel. resist. OVCAR-3 ICSO(CLM) Rel. resist. 0V CAR -4 ICSO(J*M) Rel. resist. OVCAR-10 Ic5O(bM) Rel. resist. PEO 1 ICSO(P?f) Re1 resist. PE04 IC50(PM) Rel. resist.
CDDP
CBDCA
Tetraplatin
CI-973
0.1 1('-c .05) 1
0.37(*.05) 1
0.03(+-.02) 1
0.25(5.23) 1
5.03(rt 1.69)
8.65(f 2.85) 23
0.37(2.29) 12
5.35( 24.32) 21
0.21(?.07) 2
-------
1.02(*.34) 34
0.63(5.26) 2.5
0.18(rt.05) 1.6
-------
0.47(+-.15) 16
0.97( f.33) 4
7.67(+5.31) 70
23(+ 11) 62
0.30(+-.14) 10
2.70( f.5) 11
0.07(*.02) 0.6
0.27(2.02) 0.7
0.32(& .07) 11
0.21(2.04) 1
0.32(rt: .07) 3
1.29( .39) 3
0.27(&.14) 9
0.73(&.25) 3
46
*
iIC,,(pM) expressed as mean (+SO) of 3 to 6 separate e x pximents. Within experiments, triplicate platings were done at each concentration.--Resistance relative to A2780.
-
100
80 -
-0-
60 -1
TABLE I11 - SPEARMAN RANK CORRELATION BETWEEN ANALOGUE PAIRS CDDP CBDCA
Tetrapiatin Cl-973
40 -
Combination
rho
D
CDDP-CBDCA CDDP-CI973 CDDP-tetraplatin CBDCA-C1973 CBDCA-tetraplatin Tetraplatin-CI973
1.000 .857 .036 ,900 .200 .208
,0083 ,012 ,482 .042 ,392 .331
carboplatin. We subsequently evaluated the sensitivities of cisplatin, tetraplatin and (21-973 in variant 2780 cell lines selected for resistance to tetraplatin in vitro (Table IV). Relatively high levels of resistance to both tetraplatin and CI-973 were seen in all 3 cell lines. There was substantial difference in cisplatin sensitivity between the 2780-TP7.0 and -TP10.5 cell lines.
20 0 .01
1
.1
10
Concentration (pM) FIGURE2 - Clonogenic survival of A2780 cells following continuous treatment with platinum analogues. Each point is the mean of at least 3 independent experiments.
8o
r
m-
A2780
2780-CF70 OVCARJ O V W 4 OVCAR10
PEOl
EM
Cell Line
FIGURE3 - Resistance of ovarian cancer cell lines to platinum analogues, arbitrarily expressed relative to A2780.
DISCUSSION
We observed substantial variability in the sensitivity of the cell lines tested to the individual platinum analogues. On a molar basis, cisplatin and tetraplatin were consistently the most potent analogues. Cisplatin was the most potent analogue in 3 cell lines (OVCAR-3, OVCAR-4, and PEOl), while tetraplatin was most potent in 3 other cell lines (A2780, 2780-CP70, and OVCAR-10). Their potency was roughly equivalent in the PEO4 cell line. In general, carboplatin was the least potent analogue. These results are consistent with an earlier report from our laboratory in which the activities of cisplatin, carboplatin, and tetraplatin were assessed in the A2780 and 2780CP8 cell lines (Behrens et al., 1987). Analysis of our data by the Spearman rank correlation test clearly showed that the pattern of sensitivity of our cell lines to cisplatin and carboplatin was identical, while the pattern of tetraplatin sensitivity was dissimilar. These results confirm and extend earlier findings from our laboratory in the A2780 and 2780-CP8 cell lines (Behrens et al., 1987). Similar results were obtained by Hills et al. (1990), who assessed the in vitro cytotoxicity of cisplatin, carboplatin, and tetraplatin against a panel of 10 human ovarian carcinoma cell lines. The patterns
268
PEREZ ET AL. TABLE IV - CYTOTOXICITY OF TETRAPLATIN AND Cl-973 IN TETRAPLATIN-RESISTANT 1780 VARIANT CELL LINES Cell line
2780-Tw.0 ICs0(pM)' Rel. resist.* 2780-TP 10.5 ICSO(P?) Rel. resist.
Templatin
a-973
3
1.29(&.58) 19
3.80(*2) 15
2.63(%.51) 24
2.07(4.31) 31
6.10(+ .45) 24
2.57( %. 54) 38
3.37( +- .94) 13
CDDP-
0.34(+ .04)
-
' I C 5 , J ~ expressed ~) as mean (LSD)of 3 to 6 sparate ex nmnts Within expnments, triplicate platings were done dt edLh concentration. 'Gesistance relative to A2180
of sensitivity to cisplatin and carboplatin were highly correlated in these cell lines (Spearman correlation coefficients > 0.9), while the pattern of tetraplatin sensitivity did not correlate with that obtained with the other analogues. The mechanism(s) responsible for these different sensitivity patterns is unknown. Tetraplatin may require reduction to an active form (Eastman, 1987) and is reportedly more active against plateau-phase cells than exponentially growing cells in culture (Bhuyan et al., 1988; Bhuyan and Groppi, 1989). Differential sensitivity to these platinum analogues might reflect differences in cellular transport or metabolism, or it may reflect the growth kinetics of various cell lines. Sensitivity to CI-973 correlated with sensitivity to cisplatin and carboplatin, but not tetraplatin. On a molar basis, CI-973 was not the most active analogue in any of the cell lines tested. However, the magnitude of resistance of individual cell lines to CI-973 was generally comparable with or lower than to cisplatin. These results are consistent with preliminary observations in cisplatin sensitive and resistant L1210 cells (Kraker et al., 1988). In particular, the magnitude of resistance to CI-973 was much lower than to cisplatin in the highly cisplatinresistant 2780-CWO and OVCAR-10 cell lines. The relatively low degree of cross-resistance in the OVCAR-10 cell line is especially interesting, since this cell line was derived from a patient who was clinically refractory to high-dose cisplatin and high-dose carboplatin. CI-973 is presently being evaluated in phase-I clinical trials (Hudes et al., 1990; Fukuoka et al., 1989; Theriault et al., 1990; Newman et al., 1990). Overall, clinical toxicity has been minimal. Myelosuppression, predominantly neutropenia, has been dose limiting. Thrombocytopenia, which often complicates clinical carboplatin therapy, was not a prominent toxicity. CI-973 appears to have minimal, if any, neuro- or nephrotoxicity. Preliminary pharmacokinetic data (O'Dwyer, personal communication) suggest that the concentrations of CI-973 which were cytotoxic in our in vitro assay are compa-
rable to the peak plasma levels achieved at the initial clinical dose levels in the Fox Chase Cancer Center phase-I trial (2-3 Fg/ml, approx. 4-7 FM). Maximally achievable peak plasma levels have yet to be determined. In addition, the in vitro pharmacokinetics of CI-973 are not known. Thus, it is not possible at present to determine how closely the concentrations required for cytotoxicity in our assays approximate to clinical conditions. Our results are consistent with the existence of at least 2 platinum resistance phenotypes in human ovarian cancer cells. The first phenotype, seen in the OVCAR-10 cell line, is characterized by very high levels of resistance to cisplatin and carboplatin, with more moderate resistance to tetraplatin and CI-973. It is interesting that comparable levels of crossresistance between CI-973 and tetraplatin were also seen in cell lines selected for tetraplatin resistance in vitro. An alternative phenotype is apparent in the OVCAR-3, OVCAR-4 and PE04 cell lines. These cell lines have low levels of resistance to cisplatin and carboplatin, while the levels of resistance to tetraplatin are comparatively high. In these cell lines, the pattern of sensitivity and the magnitudes of resistance to CI-973 were similar to cisplatin and carboplatin, and markedly different from tetraplatin. The resistance profile of the 2780-CP70 cell line more closely approximated the former phenotype. Our in vitro observations are consistent with clinical evidence of nearly complete cross-resistance between cisplatin and carboplatin (Gore et al., 1989; Eisenhauer et al., 1990), although some patients who fail conventional-dose chemotherapy may respond to high-dose therapy (Ozols et al., 1985, 1987; Rothenberg et al., 1988). These patients must have had relatively low levels of resistance (s2-fold) in order to respond to a mere doubling of the dose of cisplatin or carboplatin. Whether higher percentages of cisplatin-refractory patients will respond to further carboplatin dose escalations remains to be seen. Tetraplatin and CI-973 are both currently in phase-I trials, and no data are yet available regarding their activity in patients who are refractory to conventional or high-dose cisplatin. The existence of different platinum resistance phenotypes suggests that different resistance mechanisms may be operative. The recognition and characterization of platinumresistance phenotypes may ultimately yield insights into the cellular mechanisms of platinum resistance and provide a basis for more rational chemotherapy in ovarian cancer. ACKNOWLEDGEMENTS
Work from our laboratory was supported by institutional grants NIH CA 00927, NIH RR0895, and appropriations from the Commonwealth of Pennsylvania. Additional support was obtained from the Pew Charitable Trusts 88-01522-000. Dr. Perez is supported by fellowships from the American Cancer Society (Clinical Oncology Fellowship 89-142) and US Bioscience.
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