Modulation of cytotoxic drug activity by mitotane and lonidamine in human adrenocortical carcinoma cells
RAFFAELLA VILLA1, LINDA ORLANDI1, ALFREDO BERRUTI2, LUIGI DOGLIOTTI2 and NADIA ZAFFARONI!
1Divisione di Oncologia Sperimentale C, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano;
2Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Clinica Medica e Oncologia Medica, Azienda Ospedaliera San Luigi, Orbassano, Italy
Received October 29, 1998; Accepted November 23, 1998
Abstract. The ability of mitotane, a DDT derivative with adrenotoxic activity, and lonidamine, an energolytic derivative of indazole-carboxylic acid, to modulate the cytotoxic activity of doxorubicin, epidoxorubicin, cisplatin and VP16 was investigated in a human adrenocortical carcinoma cell line (SW13). A marked variability in cellular response to a 1-h treatment with the individual anticancer agents was observed. The concentrations able to inhibit SW13 cell proliferation by 50% (IC50) were 0.45 µg/ml and 0.4 ug/ml for doxorubicin and epidoxorubicin, respectively, thus indicating a relative sensitivity to anthracyclines. Conversely, the SW13 cell line displayed a marked resistance to cisplatin (IC50, 13.9 ug/ml) and VP16 (IC3), 15 µg/ml). When cells were exposed to anticancer drugs and mitotane simultaneously or in sequence, a positive modulation of anthracycline cytotoxic effects was observed. Although to a lesser extent, mitotane also increased cisplatin activity. Conversely, no potentiation was observed when mitotane was combined with VP16. Lonidamine slightly increased the cytotoxicity of epirubicin and cisplatin as individual agents. Moreover, a supra-additive effect of the three-drug (epidoxorubicin-cisplatin-lonidamine) combination was observed.
Introduction
Adrenocortical carcinoma is a rare and extremely aggressive disease with an annual incidence of 0.5 to 2 per million people. According to recently reported clinical studies, only 20-25% of all patients survive more than 5 years from the diagnosis, and the mean survival is around 14 months (1).
Correspondence to: Dr Nadia Zaffaroni, Divisione di Oncologia Sperimentale C, Istituto Nazionale Tumori, Via Venezian 1, 20133 Milano, Italy
Key words: adrenocortical carcinoma, mitotane, lonidamine, drug combinations
Radical surgical excision is the only potentially curative treatment, but often patients present with metastatic disease, which is associated with a very poor prognosis (2).
An important factor contributing to the poor prognosis is the refractory nature of the malignancy to chemotherapeutic agents. Clinical reports have indicated that doxorubicin (DX), carmustine, 5-fluorouracil (5-FU) and methotrexate, singly or in association, produced very modest responses (3-5). Recent studies have reported remissions after cisplatin (CDDP)- and etoposide (VP16)-containing schemes (6,7; Burgess MA, et al, Proc ASCO 12: abs. 188, 1993). However, the rarity of the tumor makes it difficult to perform controlled studies to assess the actual impact of any therapy on survival.
Since 1960, mitotane (o,p’-DDD, MIT), an adrenalytic drug, has been considered the compound of choice for the treatment of adrenocortical carcinoma. MIT has demonstated its efficacy as an adjuvant therapy (8,9) and also for inoperable and metastatic disease (10), mainly when high MIT serum levels were achieved (11), by inducing long-term remissions (8,9) and improving clinical outcome (8,9). However, the adjuvant use of MIT in prolonging the disease- free interval or survival has been recently questioned by some authors (12-14).
The recent finding that MIT is capable of reversing in vitro multidrug resistance mediated by MDR1/p170 has raised interest in the use of the drug in association with anticancer drugs in an attempt to overcome chemoresistance of this tumor type (15,16). Moreover, association of MIT with a combination chemotherapy containing CDDP indicated a significant improvement in the response rate compared to that obtained with CDDP alone (17). Thus far, no study has been performed on adrenocortical carcinoma to investigate the role of modulators other than MIT. In this regard, the energolytic compound lonidamine (LND) (18) could be considered an attractive candidate. In fact, experimental studies have demonstrated its ability to increase the cell killing effect of alkylating agents (19,20) and anthracyclines (21) and to reverse DX resistance in MDR1-expressing cells (22). The potential of LND as an enhancer of drug activity has also been demonstrated in clinical studies on breast and ovarian cancer patients (23,24).
In the present study, we evaluated, on the SW13 adreno- cortical carcinoma cell line, the possibility to improve the antitumor activity of conventional anticancer drugs by the use of modulators, such as MIT and LND, in order to provide a biological basis for the rational design of new combination therapies for this tumor type.
Materials and methods
Cell line. The human adrenocortical carcinoma cell line SW13 (obtained from American Type Culture Collection) was used in the study. Its biological characteristics have been previously described (25). The cell line was maintained in logarithmic phase at 37°℃ in a 5% CO2 humidified atmosphere in air, using Leibovitz’s medium, supplemented with 10% fetal calf serum, 2 uM L-glutamine and 0.1% gentamycin. During the exponential growth phase, the doubling time was 36 h. All experiments were performed within the tenth passage after thawing.
Drugs. DX (Pharmacia Upjohn, Milan, Italy), EPI (Pharmacia- Upjohn), CDDP (Bristol Myers, Evansville, IL), VP16 (Bristol Myers) and MIT (Sigma Chemical Co., St. Louis, MO) were reconstituted in sterile water and then diluted with 0.9% sodium chloride to the desired concentration immediately before each experiment. LND, obtained from F. Angelini Research Institute (Rome, Italy), was dissolved in a 2.3% N- methyl-D-glucamine solution and then diluted with fresh medium immediately before each experiment. Control samples were always run with glucamine alone. At the indicated doses and exposure times, the solvent was neither cytotoxic against SW13 cells nor did it influence the activity of drugs in combination.
Cell survival assay. The sulforhodamine B (SRB) assay was performed as described by Perez et al (26) with minor modifications. Briefly, 7x103 cells in 0.1 ml culture medium were plated in each well (0.16 cm2) of a 96-well plate and allowed to attach for 24 h. Cells were exposed to DX (0.05- 10 µg/ml), EPI (0.05-10 µg/ml), CDDP (1-15 µg/ml) or VP16 (1-15 µg/ml) for 1 h, to LND (10-75 µg/ml) for 24 h, and to MIT (5-50 µg/ml) for 1, 24 or 72 h. In combination experiments with MIT, cells were simultaneosly treated with individual drugs and MIT for 1 h or sequentially exposed to drug for 1 h and then to MIT for 24 h. In combination experiments with LND, cells were exposed to EPI, or CDDP, and LND for 1 h and then post-incubated with LND for 24 h. In the three-drug combination experiments, cells were exposed to EPI and LND for 1 h, then to CDDP and LND for an additional hour, and post-incubated with LND for 24 h. At the end of the different treatments, cells were washed and incubated at 37℃ for 3 days. Cells were then fixed by gentle addition of 50 ul of cold (4℃) 50% trichloroacetic acid to each well, followed by incubation at 4℃ for 1 h. Plates were washed five times with deionized water and allowed to air dry. Cells were stained with 50 ul of an SRB solution (0.4% SRB w/v in 1% acetic acid v/v) to each well for 10 min, then quickly washed five times with 1% acetic acid to remove unbound dye and allowed to air dry. Bound dye was solubilized with 10 mmol/l Tris base (pH 10.5) prior to
3
Absorbance (O.D.)
2
1
0
0
2
4
6
8
10
Cells/well (x 1000)
reading plates on a microplate reader. Optical density (OD) was read at 550 nm. Each experimental point was run in triplicate. The results were expressed as the absorbance values of treated samples compared with those of controls. In vitro activities of drugs were expressed in terms of concentrations able to inhibit cell proliferation by 50% (IC50).
Immunocytochemical determinations. Cells were trypsinized, washed with phosphate-buffered saline, centrifuged onto coverslips (500 g, 3 min) and air dried. The avidin-biotin peroxidase method was used to detect protein expression (Vectastain ABC Kit, Vector Laboratories Inc., Burlingame, CA). For p170, samples were incubated at 4℃ for 2 h with 10 µg/ml of monoclonal antibody C219, 1:10 dilution (CIS Diagnostici, Vercelli, Italy). For glutathione-S-transferase Tt (GSTI), samples were incubated at 4℃ for 2 h with rabbit polyclonal antibody GSTT, 1:50 dilution (Novocastra, Newcastle Upon Tyne, UK). For p53, samples were incubated at room temperature for 1 h with 2 ug/ml of monoclonal antibody Pab1801, 1:50 dilution (Oncogene Science, Manhasset, NY). For bcl-2, samples were incubated at room temperature for 1 h with mouse monoclonal antibody anti-bcl-2, clone 124, 1:40 dilution (Dako, Carpenteria, CA). For all determinations, negative controls were obtained by omission of the primary antibody. Positive cells were quantified by two independent observers by evaluating at least 5x103 cells and expressed as the percentage ratio over the total number of scored cells.
Results
Before starting the cytotoxicity studies, and with the purpose to calibrate the SRB assay on the SW13 cell line, cells were plated at a large range of seeding densities and cultured for 7 days (Fig. 1). A linear relation between plated cell number and OD at 550 nm was observed at up to 7x103 cells/well. This seeding density was consequently used in chemo- sensitivity experiments.
A marked difference in response to a 1-h treatment with the individual drugs was observed, thus suggesting a
DOXORUBICIN
100
CISPLATIN
100
percentage of control (O.D.)
percentage of control (O.D.)
50
50
E
0
0.0
0.2
0.4
0.6
0.8
1.0
0
0
5
10
15
EPIDOXORUBICIN
VP 16
100
100
percentage of control (O.D.)
percentage of control (O.D.)
50
50
0
0.0
0.2
0
0.4
0.6
0.8
1.0
0
5
10
15
ug/ml
ug/ml
different inherent sensitivity profile of SW13 cells to each compound. Specifically, cells were relatively sensitive to DX and EPI (Fig. 2), as indicated by the ICsg values of 0.45 and 0.40 µg/ml, respectively. Conversely, adrenocortical cells displayed a very modest susceptibility to CDDP and VP16 (Fig. 3). In fact, no antiproliferative effect of the drugs was recorded at concentrations lower than 10 µg/ml, and IC50 values were 13.9 and 15.0 µg/ml for CDDP and VP16, respectively.
In order to explain differences observed in individual drug sensitivity, an immunocytochemical determination of biological markers potentially involved in cellular response to treatment was performed (Table I). SW13 cells did not express the anti-apototic protein bcl-2, whereas they were largely positive to p53 staining. The immunoreactivity for p170 was evident in a limited fraction of SW13 cells, whereas the totality of cells expressed GSTn.
As regards modulators, a dose- and time-dependent activity of MIT (Fig. 4) was observed. Specifically, the IC50) value of 45.0 µg/ml obtained for a 1-h treatment decreased to 26.0 and 2.5 µg/ml when exposure time was prolonged to 24
| MoAb | Biological marker | Positive cells (%) |
|---|---|---|
| 1801 | p53 | 60 |
| Clone 124 | bc12 | 0 |
| C219 | p170 | 40 |
| GSTR | GSTR | 100 |
and 72 h, respectively. Conversely, a 24-h exposure to LND (Fig. 4) had a negligible antiproliferative effect at all concentrations tested.
In a further step of the study, we analyzed the ability of MIT and LND to modulate the cytotoxic activity of individual antitumor agents. As regards MIT, 15 µg/ml for 1 h and 5 µg/ml for 24 h were used as the maximal non-toxic
MITOTANE
100
percentage of control (O.D.)
50
I
I
0
0
15
30
45
LONIDAMINE
100
percentage of control (O.D.)
50
0
0
25
50
75
ug/ml
100
percentage of control (O.D.)
50
₹
0
0.0
0.1
0.2
0.3
0.4
0.5
Epidoxorubicin (ug/ml)
| IC50 (ug/ml) | DMF | |
|---|---|---|
| DX alone | 0.45±0.04 | - |
| + MIT- (1 h) | 0.26±0.02€ | 1.70 |
| ·MITP (24 h) | 0.24±0.03€ | 1.87 |
| EPI alone | 0.40±0.03 | - |
| + MITª (1 h) | 0.26±0.05€ | 1.53 |
| ·MIT® (24 h) | 0.25±0.03€ | 1.60 |
| CDDP alone | 13.90±1.30 | - |
| + MITª (1 h) | 10.69±1.25 | 1.30 |
| ->MIT™ (24 h) | 10.10±1.43 | 1.38 |
| VP16 alone | 15.00±1.54 | - |
| + MIT- (1 h) | 14.05±1.62 | 1.07 |
| ->MITb (24 h) | 13.75±1.31 | 1.09 |
15 µg/ml; b5 µg/ml; “p<0.05, Student’s t-test; Data represent the mean value ± SD of three independent experiments. DMF, dose-modifying factor: IC50 in the absence of MIT/IC50 in the presence of MIT.
| IC50 (µg/ml) | DMF | ||
|---|---|---|---|
| - LND | + LNDª | ||
| EPI | 0.40±0.03 | 0.29±0.03 | 1.37 |
| CDDP | 13.90±1.30 | 9.86±1.26 | 1.41 |
“50 µg/ml for 1 h in combination with EPI or CDDP followed by a 24-h postincubation with LND alone; Data represent the mean value ± SD of three independent experiments. DMF, dose-modifying factor: IC50 in the absence of LND/IC50 in the presence of LND.
concentrations of modulator in drug-combination experiments (Table II). Simultaneous exposure to DX and MIT for 1 h significantly increased the cytotoxic activity of the anthra- cycline. Similar results were obtained when SW13 cells were first exposed to DX for 1 h and then post-incubated with MIT for 24 h. The extent of potentiation was comparable following the two treatments. Although to a lesser extent, simultaneous or sequential exposure to MIT also increased EPI cytotoxicity. Conversely, a very modest enhancement of CDDP activity and lack of interference with VP16 cyto- toxicity by MIT were recorded.
As regards LND (Table III), combined exposure to EPI and a non-cytotoxic dose (50 µg/ml) of the modulator for 1 h, followed by a 24-h post-incubation with LND alone, increased the activity of the anthracycline. Similar results were obtained after sequential exposure to CDDP and LND. However,
when we assessed the activity of the three drugs in combination (i.e., EPI and LND for 1 h followed by CDDP and LND for 1 h, and then LND alone for an additional 24 h), a cytotoxic effect markedly greater than that expected by simple additivity of the effects of the three individual agents was observed (Fig. 5).
Discussion
The present study was designed to evaluate the potential of chemical modulators, such as MIT and LND, to enhance the cytotoxic activity of conventional anticancer agents in the human adrenocortical carcinoma cell line SW13, with the final aim to provide a preclinical rationale for the development of new treatment strategies.
Chemosensitivity experiments performed with drugs frequently used in chemotherapy regimens for adrenocortical carcinoma (DX, CDDP, VP16) showed that the SW13 cell line was characterized by different sensitivity profiles to individual agents. In an attempt to explain such differences, we examined the expression of proteins involved in cellular response to treatment. Specifically, the overexpression of p170, which is a common feature in adrenocortical carcinoma surgical specimens (27), could justify the marked resistance of SW13 cells to VP16 but not the relative sensitivity to DX and EPI. As a consequence, it could be hypothesized that in these cells p170 is not completely functional and that other mechanisms, such as alterations in the target enzyme topoisomerase II (28), could be responsible for VP16 resistance.
The low susceptibility to CDDP observed in SW13 cells could be related to the high level of expression of GSTI, an enzyme which plays a critical role in cellular detoxification processes (29). The cytotoxic effect of CDDP has also been related to the induction of apoptosis in many experimental models. In some instances, such programmed cell death was mediated by an induction of wild-type p53 expression consequent to DNA damage (30). We detected in the SW13 cell line an appreciable fraction of p53-expressing cells. Since the wild-type p53 has a short half-life and mutant p53 has a half- life of several hours, which results in intracellular accumulation sufficient for immunohistochemical detection, it is likely that the immunoreactivity observed in our cells was due to presence of the mutant protein. It can thus be hypothesized that the cells are not susceptible to CDDP-induced apoptosis and consequently are resistant to the drug. A significant association between the presence of mutations in the p53 gene and/or overexpression of the p53 protein and resistance to clinical CDDP treatment has been recently reported (31).
As regards modulators, a dose- and time-dependent antiproliferative effect of MIT was observed, thus reflecting the clinical activity of the compound when used as a single agent (8,9), even though the efficacy of MIT in providing objective tumor regression has been recently questioned (13,14). In combination experiments, a MIT concentration achievable in human plasma (11) enhanced DX and EPI cytotoxicity independently of the schedule of drug administration. In agreement with previous results of other authors (27) showing the in vitro reversal of multidrug resistance by MIT through the inhibition of p170-mediated drug transport, our findings suggest the modulation of p170
as the mechanism responsible for the increase in anthra- cycline activity induced by MIT. Unfortunately, such an hypothesis is not consistent with the lack of enhancement in VP16 activity after combined/sequential exposure with MIT. Moreover, the possibility that MIT modulates cellular targets other than p170 is supported by the evidence of a slight increase in CDDP cytotoxic activity in combination experiments. Results from a clinical phase II study (17) suggested that the association of MIT with a combination chemotherapy containing CDDP may improve the response rate in comparison to CDDP alone. More recently, important clinical responses were obtained by Berruti et al (32) in patients treated with a combination of MIT, CDDP, DX and VP16 in a large multicenter phase II trial.
As regards LND, a negligible effect of the energolytic compound on SW13 cell proliferation was consistently observed at all the concentrations tested. However, as expected on the basis of previous results from experimental (19-21) and clinical (23,24) studies in other tumor types, we found that LND was able to increase the cytotoxic activity of EPI and CDDP when singly tested. Such a potentiating effect, is supposed to be due to the inhibition of cellular energy-dependent repair processes by LND that resulted in the fixation of drug-induced DNA damage and enhanced cell kill. Moreover, it was of particular relevance since, contrarily to that reported in other experimental studies (19-21), it was obtained with a clinically relevant LND concentration, i.e. within plasma peak levels (33).
Since preclinical (34) and clinical (35) evidence has shown great efficacy of the EPI, CDDP and LND combination in breast cancer tumors, we evaluated the activity of the three- drug regimen also in adrenocortical cells. A supra-additive interaction was observed when SW13 cells were exposed to EPI followed by CDDP in the presence of LND and then post-incubated with LND. Such results indicate the potential clinical usefulness of an EPI-CDDP-LND combination even for adrenocortical cancer and as a possible alternative to MIT-based combination therapies. In fact, given its peculiar action mechanism and unique spectrum of normal tissue toxicity (36), LND appears particularly promising for combination regimens.
In conclusions, we believe that in the case of rare tumors, such as adrenocortical cancer, for which it is very difficult to collect surgical specimens to obtain primary cultures, an established cell line can be a useful model to optimize therapeutic protocols on the basis of preclinical evidence on the mechanisms of cellular response to anticancer agents.
Acknowledgements
The study was supported in part by a grant from the Associazione Italiana per la Ricerca sul Cancro. We thank Mrs B. Canova and Miss B. Johnston for editorial assistance.
References
1. Samaan NA and Hickey RC: Adrenal cortical carcinoma. Semin Oncol 14: 292-296, 1987.
2. Lee JE, Berger DH, El-Naggar AK, Hickey RC, Vassilopoulou- Sellin R, Gagel RF, Burgess MA and Evans DB: Surgical management, DNA content, and patient survival in adreno- cortical carcinoma. Surgery 118: 1090-1098, 1995.
3. Haq MM, Legha SS, Samaan NA, Bodey GP and Burgess MA: Cytotoxic chemotherapy in adrenal cortical carcinoma. Cancer Treat Rep 64: 909-913, 1980.
4. Van Slooten H and van Oosterom AT: CAP (cyclo- phosphamide, doxorubicin and cisplatin) regimen in adrenal cortical carcinoma. Cancer Treat Rep 67: 377-379, 1983.
5. Schlumberger M. Brugieres L, Gicquel, Travagli JP, Droz JP and Parmentier C: 5-Fluorouracil, doxorubicin, and cisplatin as treatment for adrenal cortical carcinoma. Cancer 67: 2997-3000, 1991.
6. Burgess MA: Adrenal cortical carcinoma: the role of adjuvant post- operative chemotherapy. Minerva Endocrinol 20: 101-104, 1995.
7. Zidan J, Shpendler M and Robinson E: Treatment of metastatic adrenal cortical carcinoma with etoposide (VP-16) and cisplatin after failure with o.p’ DDD. Clinical case reports. Am J Clin Oncol 19: 229-231, 1996.
8. Luton JP, Cerdas S, Billaud L, Thomas GC and Bertagna X: Clinical features of adrenocortical carcinoma, prognostic factors and the effect of mitotane therapy. N Engl J Med 322: 1195-1201, 1990.
9. Kasperlik-Zaluska AA, Migdalska BM and Makowska AM: Impact of adjuvant mitotane on the clinical course of patients with adrenocortical cancer. Two years later. Cancer 78: 1520-1521, 1996.
10. Kasperlik-Zaluska AA, Migdalska BM, Zgliczynski S and Makowska AM: Adrenocortical carcinoma. A clinical study and treatment results of 52 patients. Cancer 75: 2587-2591, 1995.
11. Haak HR, Hermans J, van de Velde CJH, Lentjes EGWM, Goslings BM, Fleuren GJ and Krans HMJ: Optimal treatment of adrenocortical carcinoma with mitotane: results in a consecutive series of 96 patients. Br J Cancer 69: 947-951, 1994.
12. Vassilopoulou-Sellin R, Guinee VF, Klein MJ, Taylor SH, Hess KR, Schultz PN and Samaan NA: Impact of adjuvant mitotane on the clinical course of patients with adrenocortical cancer. Cancer 71: 3119-3123, 1993.
13. Vassilopoulou-Sellin R and Guinee VF: Mitotane in adreno- cortical carcinoma (Letter). Br J Cancer 70: 779, 1994.
14. Barzon L, Fallo F, Sonino N, Daniele O and Boscaro M: Adrenocortical carcinoma: experience in 45 patients. Oncology 54: 490-496, 1997.
15. Flynn SD, Murren JR, Kirby WM, Honig J, Kan L and Kinder BK: P-glycoprotein expression and multidrug resistance in adreno- cortical carcinoma. Surgery 112: 981-986, 1992.
16. Bates SE, Shieh CY, Mickley LA, Dichek HL, Gazdar A, Loriaux L and Fojo AT: Mitotane enhances cytotoxicity of chemotherapy in cell lines expressing a multidrug resistance gene (mdr-1/P glycoprotein) which is also expressed in adrenocortical carcinomas. J Clin Endocrinol Metab 73: 18-29, 1991.
17. Bukowski RM, Wolfe M, Levine HS, Crawford DE, Stephens RL, Gaynor E and Harker G: Phase II trial of mitotane and cisplatin in patients with adrenal carcinoma: a Southwest Oncology Group Study. J Clin Oncol 11: 161-165, 1993.
18. Silvestrini B. Palazzo G and De Gregorio M: Lonidamine and related compounds. Prog Med Chem 21: 111-135, 1984.
19. Silvestrini R, Zaffaroni N, Villa R, Orlandi L and Costa A: Enhancement of cisplatin activity by lonidamine in human ovarian cancer cells. Int J Cancer 52: 813-817, 1992.
20. Ning S and Hahn GM: Cytotoxicity of lonidamine alone and in combination with other drugs against murine RIF-1 and human HT1080 cells in vitro. Cancer Res 50: 7867-7870, 1990.
21. Del Bufalo D and Zupi G: In vitro potentiation of epirubicin activity by lonidamine in a human breast cancer cell line. Int J Oncol 4: 737-740, 1994.
22. Citro G, Cucco C, Verdina A and Zupi G: Reversal of adriamycin resistance by lonidamine in a human breast cancer cell line. Br J Cancer 64: 534-536, 1991.
23. Dogliotti L, Berruti A, Buniva T, Zola P, Baù MG, Farris A, Sarobba MG, Bottini A, Alquati P, Deltetto F, Gosso P. Monzeglio C, Moro G, Sussio M and Perroni D: Lonidamine significantly increases the activity of epirubicin in patients with advanced breast cancer: results from a multicenter prospective randomized trial. J Clin Oncol 14: 1165-1172, 1996.
24. De Lena M, Lorusso V, Bottalico C, Brandi M, De Mitrio A. Catino A, Guida M, Latorre A, Leone B and Gargano G: Revertant and potentiating activity of lonidamine in patients with ovarian cancer previously treated with platinum. J Clin Oncol 15: 3208-3213, 1997.
25. Leibovitz A, McCombs WM, Johnston D, McCoy CE and Stinson JC: New human cancer cell culture lines. SW13, small- cell carcinoma of the adrenal cortex. J Natl Cancer Inst 51: 691-697, 1973.
26. Perez RP, Godwin AK, Handel LM and Hamilton TC: A comparison of clonogenic, microtetrazolium and sulforho- damine B assays for determination of cisplatin cytotoxicity in human ovarian carcinoma cell lines. Eur J Cancer 29A: 395- 399, 1993.
27. Feller N, Hoekman K, Kuiper CM, Linn SC, Verheul HMW. Wolthers BG, Popp-Snijders C and Pinedo HM: A patient with adrenocortical carcinoma: characterization of its biological activity and drug resistance profile. Clin Cancer Res 3: 389-394, 1997.
28. Taki T, Ohnishi T, Arita N, Hiraga S and Hayakawa T: In vivo etoposide-resistant C6 glioma cell line: significance of altered DNA topoisomerase Il activity in multi-drug resistance. J Neuro-Oncol 36: 41-53, 1998.
29. Hamaguchi K, Godwin AK, Yakushiji M, O’Dwyer PJ, Ozols RF and Hamilton TC: Cross-resistance to diverse drugs is associated with primary cisplatin resistance in ovarian cancer cell lines. Cancer Res 53: 5225-5232, 1993.
30. Nelson WG and Kastan MB: DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways. Mol Cell Biol 14: 1815-1823, 1994.
31. Gallagher WM, Cairney M, Schott B, Roninson IB and Brown R: Identification of p53 genetic suppressor elements which confer resistance to cisplatin. Oncogene 14: 185-193, 1997.
32. Berruti A, Terzolo M, Pia A, Angeli A and Dogliotti L: Mitotane associated with etoposide, doxorubicin and cisplatin in the treatment of advanced adrenocortical carcinoma. Cancer 83: 2194-2200, 1998.
33. Besner JG, Leclaive R, Band PR, Deschamps M, de Sanctis AJ and Catanese B: Pharmacokinetics of lonidamine after oral administration in cancer patients. Oncology 41: 48-52, 1984.
34. Silvestrini R, Gornati D, Zaffaroni N, Bearzatto A and de Marco C: Modulation by lonidamine on the combined activity of cisplatin and epidoxorubicin in human breast cancer cells. Breast Cancer Res Treat 42: 103-112, 1997.
35. Dogliotti L, Danese S, Berruti A, Zola P, Buniva T, Bottini A, Richiardi G. Moro G, Farris A, Bau MG and Porcile G: Cisplatin, epirubicin, and lonidamine combination regimen as first-line chemotherapy for metastatic breast cancer: a pilot study. Cancer Chemother Pharmacol 41: 333-338, 1998.
36. Battelli T, Manocchi P and Guistini L: A long-term clinical experience with lonidamine. Oncology 41: 39-47, 1984.