GYNECOLOGIC ONCOLOGY

39, 119-122 (1990)

Comparison of Cisplatin and Carboplatin Cytotoxicity in Human Ovarian Cancer Cell Lines Using the MTT Assay JAMES FANNING,

D.O.,*?’ WILLIAM C. BIDDLE, PH.D.,t MARTIN GOLDROSEN,PH.D.,t KENT CIWKARD, M.D., WLLA CRICKARD,$ M. STEVEN PIVER, M.D.,* AND KENNETH A. FOON, M.D.t

*Department of Gynecologic Oncology and TDivision of Clinical Immunology, Roswell Park Cancer Institute, Buffalo, New York 14263, and 8Department of Obstetrics and Gynecology, State University of New York, Buffalo, New York 14222

ReceivedFebruary 6, 1990

advanced epithelial ovarian cancer. Since cisplatin is associated with nephrotoxicity and neurotoxicity, platinum analogs have been developed to reduce these side effects. Recently, the new platinum analog carboplatin was introduced into clinical trials; it is associated with less nephrotoxicity and neurotoxicity, but with more myelosuppression [2]. However, there has been very limited in vitro investigation into the cytotoxicity of cisplatin and carboplatin in human ovarian cancer cell lines. There are several assays available for quantitating the number of viable cancer cells present following therapeutic interventions. Traditional assays consist of counting total viable cells using a hemocytometer or colony counting. However, these procedures are cumbersome and require the processing of a large number of samples. Also, colony counting may be misleading because each colony may contain a significantly different number of cells. Radionuclide incorporation assays, such as [3] thymidine incorporation, are widely used but are time consuming and expensive, Recently, a rapid calorimetric assay has been described [3]. This assay involves the ability of viable cells to convert a soluble tetrazolium salt, 3,4,5-dimethylthiazol-2,5diphenyl tetrazolium bromide (MTT), into an insoluble formazan precipitate. The purple-colored formazan crystals may be dissolved in INTRODUCTION organic solvents and the optical density of the resulting solution measured on a multiwell spectrophometer Approximately 20,000 new cases of ovarian cancer (ELISA plate reader). The M’IT assay can be used to were diagnosed and 12,000 women died of ovarian cancer in the United States in 1989. Ovarian cancer is the most evaluate chemosensitivity [43 and is suited to clinical common cancer to be diagnosed in advanced stages laboratories for routine testing of chemosensitivity of (73%) [ 11. Cisplatin-containing combination chemother- autologous tumor cells to various drugs. apy has become the standard postoperative therapy for The purpose of this study was to compare the cytotoxicity of cisplatin and carboplatin in a panel of human ’ To whom requests for reprints should be addressed. ovarian cancer cell lines using the MTT assay.

In this study, we compared the cytotoxicity of cisplatm and carboplatin against a panel of human ovarian cancer cell Lines using the MTI assay, a rapid colorhuetric test that can be.used to evaluate the number of residual viable tumor cells following chemotherapy. The established human ovarian cancer cell line OVCAR3 and the recently isolated and characterized A721, A90, A286, Al, and A121A cell lines were evaluated for chemosensitivity. Each cell line was treated separately with cisplatin and carboplatin at concentrations ranging from 500 to 0.16 &ml. Various chemotherapeutic exposure periods (1, 4, 24, and 48 hr) were tested to determine maximal efficacy. All cell lines were more susceptible to cisplatin than carboplatin at all drug concentrations and all exposure periods tested (P = 0.005). The overall median 50% inhibitory concentration (ID,) for cisplathr was 107 pg/ml compared with 490 pg/ml for carboplatln (P = 0.005). For both cisplathr and carboplatin a 24-hr exposure was significantly more cytotoxic than a 1-hr exposure (P = 0.003 and P = 0.006, respectively). These in vitro results suggest that cisplatin is significantly more cytotoxlc than carboplatin against human ovarian cancer cell lines and that cisplatin should not be replaced by carboplatin in the treatment of advanced epithelial ova&n cancer until randomized trials using maximum dosing of the cisplatin-containing regimen are performed. Q i~~~~eadaoich~, Inc.

119 oo9o-8258/90$1.50 Copyright 8 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ET AL.

Human ovarian cancer cells were washed with Hanks’ buffered salt solution (HBSS), trypsinized, washed in RPM1 medium, and counted and viability was determined by trypan blue exclusion. Ovarian cancer cells (2 x 104/well) were then added to 96-well plates and allowed to attach for 24 hr. Cisplatin and carboplatin (supplied by Bristol Meyers, Evansville, IN) were reconstituted in sterile water immediately prior to use and added to wells at final concentrations of 500, 100, 20, 4, 0.8, and 0.16 pg/ml. Each cell line was treated separately with cisplatin and carboplatin at equivalent concentrations for chemotherapeutic exposure periods of 1, 4, 24, and 48 hr. Following the chemotherapeutic exposure (1, 4, 24, or 48 hr) 10 ~1 of 5 mg/ml MTT was added to each well and incubated for 4 hr. After 4 hr at 37°C 120 ~1 of 5% formic isopropanol was added to each well and incubation was continued for 5 min. Optical density measurements were obtained on a multiwell spectrophotometer using a 570-nm filter. All assays were run in triplicate. Statistics. A comparison of the cytotoxicities of cisplatin and carboplatin was analyzed using a two-tailed t test. Results showing a p value less than 0.05 were considered to be statistically significant. RESULTS

MATERIALS

AND METHODS

Cisplatin was significantly more cytotoxic than carboplatin at each concentration at all four chemotherapeutic exposure periods in all six human ovarian cancer ilton, Philadelphia) and five new human ovarian cancer cell lines tested (P = 0.005). In all six cell lines after 1, cell lines (A721, A90, A286, Al, A121A) were used in 4, 24, and 48 hr exposure, cisplatin was more cytotoxic this study. The five new cell lines were established on than carboplatin. Also, in all six cell lines, cisplatin was an extracellular matrix produced by bovine cornea1 en- more cytotoxic than carboplatin at each concentration dothelial cells and have been previously described [5]. (500, 100, 20, 4, 0.8, and 0.16 pg/ml). The concentration Cell lines A121A, Al, and A721 had no previous che- of cisplatin required to produce 50% inhibition of cell motherapeutic exposure in vivo, while cell lines A286, growth (ID,,) was significantly less than that for carA90, and OVCAR-3 were treated with cisplatin in vivo boplatin (Fig. 1). For all cell lines at all four exposure prior to establishment in cell culture. Cell lines were periods the overall median 50% inhibitory concentration maintained in RPMI-1640 supplemented with 2 mM glu- for cisplatin was 107 pg/ml compared with 490 pg/ml tamine, antibiotics (100 units/ml penicillin, 100 pg/ml for carboplatin (P = 0.005). The median 50% inhibitory streptomycin, and 250 rig/ml of amphotericin B), 15 mM concentrations at 1 and 4 hr exposure for cisplatin were Hepes buffer, and fetal calf serum (5%) at 37°C in a 95% 301 and 288 pg/ml compared with ~500 pg/ml for carsir/5% CO* environment. All cell culture media and ad- boplatin (1 hr, P = 0.013; 4 hr, P = 0.006). The median ditives were obtained from Grand Island Biologic Cor- 50% inhibitory concentrations at 24 and 48 hr exposure for cisplatin were 34 and 42 pg/ml compared with 319 poration (Grand Island, NY). MTT chemosensitivity assay. The tetrazolium salt, and 390 pg/ml for carboplatin (24 hr, P = 0.0004; 48 3,4,5-dimethylthiazol-2,5-diphenyl tetrazolium bromide hr, P = 0.0001). (MTT), was obtained from Sigma Corporation (St. Louis, Figure 2 shows the percentage inhibition of cell growth MO). The formazan product dissolving reagent consisted at various concentrations and exposure periods for a of 5% formic acid, 95% isopropanol. Spectroscopy mea- representative cell line, A90. Cisplatin was more cytosurements were performed on a multiwell spectropho- toxic than carboplatin at each concentration and each tometer (ELISA plate reader, Titer Tech II) using a 570- exposure period. Similar results were obtained for the nm filter. other five cell lines.

Tumor cell lines. One established human ovarian cancer cell line (OVCAR-3, kindly provided by Dr. T. Ham-

MTT ASSAY FOR CISPLATIN

CYTOTOXICITY

121

was as effective as cisplatin in stage IIB and III epithelial ovarian cancer [2]. In another study, cisplatin (100 mg/m2) and cyclophosphamide (600 mg/m2) were compared with carboplatin (300 mg/m’) and cyclophosphamide (600 mg/m2) in 39 patients with stage II-IV epithelial ovarian cancer. Although the response rate to the cisplatin combination was greater (79% versus 53%) and the 5-year survival was doubled, there was no statistically significant difference in median survival. A major limitation of this study was that the maximum tolerated dose of carboplatin and cyclophosphamide was comPL s 60 pared to 60% of the maximum tolerated dose of cisplatin and cyclophosphamide (1000 mg/m2 of cyclophospha60 / mide can be administered with 100 mg/m2 of cisplatin). Nonetheless, these studies have prompted some clinicians to replace cisplatin with carboplatin because of its decreased neurotoxicity and nephrotoxicity and its con36 .6 4 20 (00 500 .t6 .6 4 20 100 500 venience of administration. Because the in vitro results Concentration (pg/mL) of our study and several other investigations have clearly shown that cisplatin is significantly more cytotoxic than FIG. 2. Percentage inhibition of cell growth. PL, cisplatin; CA, carboplatin, we believe that carboplatin should not recarboplatin. place cisplatin in the treatment of advanced epithelial ovarian cancer until randomized trials using maximum There was no significant difference in cytotoxicity bedosing of the cisplatin-containing regimen are performed. tween a I-hr and a 4-hr chemotherapeutic exposure peIn all six human ovarian cancer cell lines, cisplatin riod for cisplatin (median at 1 hr, 301 pg/ml; at 4 hr, and carboplatin were more cytotoxic after a 24-hr ex288 pg/ml, P = 0.87) or carboplatin (median at 1 and 4 posure period versus a 1-hr exposure period. Similarly, hr, >500 hg/ml, P > 1). Similarly, there was no signifCurt et al. [91 found cisplatin and carboplatin to be more icant difference in cytotoxicity between a 24-hr and a cytotoxic after a 24-hr exposure period in the human 48hr chemotherapeutic exposure period for cisplatin ovarian cancer cell line A2780. Optimal effects with pro(median at 24 hr, 34 pg/ml; 48 hr, 42 pg/ml, P = 0.78) longed exposure may indicate that cellular platinum upor carboplatin (median at 24 hr, 319 pg/ml; 48 hr, 390 take is slow. Although there have been phase I trials, pg/ml, P = 0.33). However, there was a significant difthere have been no phase III trials in advanced epithelial ference in cytotoxicity between a 1-hr and a 24-hr che- ovarian cancer comparing bolus to 24-hr infusional admotherapeutic exposure period for cisplatin (median at ministration of cisplatin or carboplatin. 1 hr, 301 pg/ml; 24 hr, 34 pg/ml, P = 0.003) and carboplatin (median at 1 hr, ~500 pg/ml; 24 hr, 319 pg/ml, REFERENCES P = 0.006). DISCUSSION By use of the MTT assay, cisplatin was shown to be significantly more cytotoxic than carboplatin at each concentration and each chemotherapeutic exposure period tested in all six human ovarian cancer cell lines. 0~01s et al. [6] reported that cisplatin was more cytotoxic than carboplatin against the human ovarian cancer cell lines A2780 and 2780 [81 using a colony counting assay. Recently, Harstrick et al. [7] compared the cytotoxicity of cisplatin and carboplatin in four human testicular cell lines using [3] thymidine incorporation and an in vivo nude mouse model. In both the in vitro and in vivo models, cisplatin was significantly more cytotoxic than carboplatin. In one clinical study, first-line single-agent carboplatin

1. Ca 39, No. 1 (1989). 2. Adams, M., Kerby, I. J., Rocker, I., Evans, E. K., and Franks,

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C. R. A comparison of the toxicity and efficacy of cisplatin and carboplatin in advanced ovarian cancer, Acta Oncol. 28, 57 (1989). Mossman, T. Rapid calorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays, J. Zmmunol. Methods 65, 55 (1983). Twentyman, P. R., and Luscombe, M. A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity, &it. J. Cancer 56, 279 (1987). Crickard, K., Niedbala, M. J., Crickard, U., Yoonessi, M., Sandberg, A. A., Okuyama, K., Bemacki, R. J., and Satchidanand, S. K. Characterization of human ovarian and endometrial carcinoma cell lines established on extracellular matrix, Gynecof. Oncol. 32, 163 (1989). Ozols, R. F., Behrens, B. C., Ostchega, Y., and Young, R. C. High dose cisplatin and high dose carboplatin in refractory ovarian cancer, Cancer Treat. Rev. 12, 59 (1985).

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7. Harstrick, A., Casper, J., Guba, R., Wilke, H., Poliwoda, H., and Schmoll, H. J. Comparison of the antitumor activity of cisplatin, carboplatin and iproplatin against established human testicular cancer cell lines in vivo and in vitro, Cancer 63, 1079 (1989). 8. Anderson, H., Wags&& J., Crowther, D., Swindell, R., Lind, M. J., McGregor, J., Timms, M. S., Brown, D., and Palmer, P. Comparative toxicity of cisplatin, carboplatin (CBDCA) and ipro-

platin (CHIP) in combination with cyclophosphamide in patients with advanced epithelial ovarian cancer, Eur. J. Cancer Clin. Oncol. 24, 1471 (1988). 9. Curt, G. A., Grygiel, J. J., Corden, B. J., Ozols, R. F., Weiss, R. B., Tell, D. T., Myers, C. E., and Collins, J. M. A phase I and pharmacokinetic study of diamminecyclobutane-dicarboxylatoplatinum (NSC 241240), Cancer Res. 43, 4470 (1983).

Comparison of cisplatin and carboplatin cytotoxicity in human ovarian cancer cell lines using the MTT assay.

In this study, we compared the cytotoxicity of cisplatin and carboplatin against a panel of human ovarian cancer cell lines using the MTT assay, a rap...
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