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Molecular and Cellular Endocrinology
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Molcedar and Cellular Endocrinology
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Lack of long-lasting effects of mitotane adjuvant therapy in a mouse xenograft model of adrenocortical carcinoma
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Mabrouka Doghman, Enzo Lalli *
Institut de Pharmacologie Moléculaire et Cellulaire CNRS, Valbonne, France
Associated International Laboratory (LIA) NEOGENEX CNRS, Valbonne, France University of Nice-Sophia-Antipolis, Valbonne, France
ARTICLE INFO
Article history: Received 15 March 2013 Received in revised form 18 July 2013 Accepted 19 July 2013 Available online 30 July 2013
Keywords:
Adrenal cortex Cancer Mitotane
Xenografts
ABSTRACT
Mitotane is a widely used drug in the therapy of adrenocortical carcinoma (ACC). It is important to set up preclinical protocols to study the possible synergistic effects of its association with new drugs for ACC therapy. We assessed the efficacy of different routes of administration of mitotane (i.p. and oral) in inhib- iting growth of H295R ACC cell xenografts in an adjuvant setting. Both formulations of mitotane could inhibit H295R xenografts growth only at short times after carcinoma cells inoculation, even though plasma mitotane levels approached or fell within the therapeutic range in humans. Our results show that mitotane adjuvant therapy is inadequate to antagonize long-term growth of H295R cancer cells xeno- grafts and that care should then be taken in the design of preclinical protocols to evaluate the perfor- mance of new drugs in association with mitotane.
2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Adrenocortical cancer (ACC) is a rare, life-threatening endocrine malignancy (Fassnacht et al., 2011). Mitotane (o,p’-DDD), a com- pound related to the insecticide DDT, is a drug widely used, alone or in combination with chemotherapeutic agents, in the therapy of advanced ACC because of its selective adrenolytic properties (Maluf et al., 2011; De Francia et al., 2012). To exert its activity, this drug requires metabolic transformation into an acyl chloride, which takes place in the mitochondria of steroidogenic cells. Mitotane is also used as an adjuvant in ACC patients who have undergone surgical resection of their primary tumor. A large retro- spective study has shown that adjuvant mitotane may prolong recurrence-free survival in patients with radically resected ACC (Terzolo et al., 2007), although other studies have not confirmed a therapeutic benefit for it (Bertherat et al., 2007; Grubbs et al., 2010; reviewed in Maluf et al., 2011). Consequently, current ther- apeutic protocols envisage the use of adjuvant mitotane in patients with an incomplete (Rx/R1) resection, possibly in conjunction with tumor bed irradiation, and in high-risk (defined as having >10% Ki-67 labeling index) patients with complete (R0) surgical resec- tion of their primary ACC (Fassnacht et al., 2011). A prospective,
randomized clinical trial (ADIUVO; NCT00777244) is currently underway to assess the efficacy of adjuvant mitotane to prolong recurrence-free and overall survival in patients with low-interme- diate risk (<10% Ki-67 labeling index) (https://www.epiclin.it/adi- uvo). However, mitotane therapy is often associated to important secondary effects (especially neurological and gastrointestinal), which in some cases force the patients to suspend the treatment. Moreover, due to the peculiar pharmacokinetics of mitotane, only a subset of patients reach plasma mitotane levels in the therapeu- tic range (14-20 mg/L) (Haak et al., 1994).
Xenograft models of ACC have proven valuable to evaluate the efficacy of drugs targeting tumor cells (reviewed in Luconi and Mannelli, 2012). We have then set up to evaluate the effect of mitotane administered in an adjuvant setting in a xenograft model of the H295R ACC cell line implanted in immunodeficient mice. Two routes of administration of mitotane were compared: intra- peritoneal (i.p.) and oral and plasma mitotane levels were mea- sured in samples taken at the end of the experiment. The results of our study show that both formulations of mitotane lack long- lasting effect on H295R xenograft growth.
2. Materials and methods
2.1. Chemicals
Mitotane (o,p’-DDD) was purchased from Sigma-Aldrich (cata- log number 25925). Stock solutions were prepared in ethanol at a concentration of 10 mM.
Abbreviations: ACC, adrenocortical carcinoma; NOD/SCID, non-obese diabetic/ severe combined immunodeficiency.
* Corresponding author. Address: Institut de Pharmacologie Moléculaire et Cellulaire CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France. Tel .: +33 (0)4 93 95 77 55; fax: +33 (0)4 93 95 77 08.
E-mail address: ninino@ipmc.cnrs.fr (E. Lalli).
2.2. Cell culture
H295R cells were cultured in a humidified atmosphere contain- ing 5% CO2 at 37 ℃ in DMEM/F12 additioned with 2% NuSerum (BD), 1% ITS+ (BD) and penicillin/streptomycin (Invitrogen).
2.3. In vitro proliferation tests
H295R cells were seeded in complete medium in 96-well plates (5 × 103 cells/well) and treated either with vehicle (ethanol) or with doses of mitotane ranging from 10-9 to 10-4 M. After 6 days, cell proliferation was measured with the MTS-based CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega). Data are representative of four experiments, each one performed in trip- licate. IC50 for mitotane was calculated using the GraphPad Prism software.
2.4. Xenografts and treatments
For each animal, 6 x 106 H295R cells were inoculated subcuta- neously into the right flank of four-week old female NOD/SCID/ynull mice, as previously described (Doghman et al., 2010; Doghman and Lalli, 2012). Out of a total of 30 mice injected, 10 animals were ran- domly assigned to each of three groups receiving, respectively:
- Mitotane (440 mg/kg) i.p. (drug dissolved in 10% Tween-80 in PBS) (Barlaskar et al., 2009).
- Mitotane (440 mg/kg) by oral gavage (drug dissolved in olive oil).
- Placebo by oral gavage (olive oil) only.
Treatments were started the day after injection of tumor cells and continued for 5 days/week. No significant weight loss was no- ticed in any group of mice during the treatment period. Tumor growth was monitored (starting from day 13 after H295R cells inoculation) once per week by measuring with a Vernier caliper and calculating tumor volume by the formula: length x width x height x T/6 (Doghman et al., 2010; Doghman and Lalli, 2012). During the protocol period, two mice were lost in the control group and one in each of the mitotane-treated groups. All protocols were performed according to the institutional animal care and use committee guidelines. ANOVA with Bonfer- roni’s correction for multiple testing was used to assess the signif- icance of differences in xenografts growth among groups of animals.
2.5. Measurement of plasma mitotane levels
It was performed by HRA Pharma using HPLC chromatography on blood samples taken after animal euthanasia at the end of the protocol.
2.6. Steroid measurements in tissues
At the end of the treatment protocols, xenografts were excised, snap frozen in liquid nitrogen and stored at -20 ℃. Steroids were ether extracted from the tissues, as described (Chao et al., 2011) and aldosterone, cortisol and DHEA-S measured in the extracts by enzyme-linked immunoassays, as described (Doghman et al., 2007).
3. Results
3.1. Mitotane inhibits proliferation of H295R cell in vitro
High concentrations of mitotane significantly reduced H295R cell proliferation in vitro (Fig. 1). The calculated IC50 for mitotane in these experiments was 22.8 uM, which closely matches the IC50 value (24 µM) previously calculated by Schteingart et al. (1993).
3.2. Effect of adjuvant mitotane administered to mice bearing H295R xenografts
We tested two routes of administration of mitotane (i.p. and oral) for their relative efficacy to reduce H295R xenografts growth in immunodeficient mice in an adjuvant setting. Mitotane admin- istration was started the day after tumor cells inoculation in mice and continued 5 times/week at a dose of 440 mg/kg for a period of about 7 weeks. Another group of mice was treated with placebo only. At an early time point (day 13) after H295R cells inoculation both groups of mice treated with mitotane bore significantly smal- ler xenografts compared with the placebo-treated group (Fig. 2). However, the effect of oral mitotane treatment became non-signif- icant by day 20 after H295R cells inoculation, while the effect of i.p. mitotane lasted until day 34. Starting from day 41 and up to con- clusion of the protocol (day 48), xenograft volumes were similar in placebo-treated and in both groups of mitotane-treated animals.
3.3. Mitotane plasma levels
Mitotane plasma levels at the end of the treatment period reached a mean concentration of 12.3 + 2.4 mg/L in the i.p. mito- tane-treated group and of 20.1 + 1.7 mg/L in the oral mitotane- treated group (Fig. 3).
3.4. Steroid measurements in H295R xenografts
H295R cells produce a variety of steroid hormones (Wang and Rainey, 2012). To assay for the effect of mitotane in inhibiting ste- roid synthesis in the H295R xenografts, we assayed their content in aldosterone, cortisol and DHEA-S by enzyme-linked immunoas- says. No difference in steroid content was evident in xenografts from both mitotane-treated groups of animals compared to pla- cebo (Fig. 4).
Proliferation (% of control)
150
100
50
0
Mitotane (M) 10 10 10 10 10 104
day 13
day 20
day 27
70
150
200
Volume (mm3)
60
50
150
100
40
30
*
100
20
50
*
**
**
50
10
0
0
0
day 34
day 41
day 48
500
600
700
Volume (mm3)
400
500
600
500
300
400
300
400
200
*
300
200
200
100
100
100
0
0
0
Plasma mitotane (mg/L)
25
20
15
10
5
0
i.p. mitotane
oral mitotane
4. Discussion
Current therapeutic protocols in ACC envisage the use of adju- vant mitotane to reduce the risk of relapse in patients even after a R0 surgical resection, especially in the presence of a high prolif- erative index in tumoral cells (Terzolo et al., 2007; Fassnacht et al., 2011). However, mitotane treatment is limited by potential serious side effects and by the difficulty in achieving therapeutic drug
levels in some patients. In addition, some studies reported no sig- nificant effect of adjuvant mitotane treatment in increasing recur- rence-free survival in ACC patients (reviewed in Maluf et al., 2011). Because of the widespread use of mitotane in ACC therapy, it is of interest to set up preclinical protocols to study the possible syner- gistic effects of its association with new drugs. To this end, we studied the effect of two routes of administration of mitotane (i.p. and oral) in an adjuvant setting on the growth of H295R cells xenografts in immunodeficient mice. Both treatments were active in slowing down tumor growth at short times after starting the protocol. However, they lost efficacy at later times, with the effect of i.p. mitotane administration lasting longer than oral administra- tion, even though mice treated with oral mitotane reached drug plasma levels within the therapeutic range for humans. These lev- els are of the same order of magnitude as the IC50 of mitotane ef- fect on H295R cells in vitro. Similarly, both mitotane treatment regimens did not modify the levels of aldosterone, cortisol and DHEA-S present in the xenografts, compared with placebo-treated animals. There are a number of explanations for the reduced effi- cacy of mitotane on H295R xenografts growth compared to the in vitro model, including the differential partitioning of the drug between the extracellular space and the tumor cells and its prefer- ential association to adipose tissue in vivo. Our results are diver- gent from a previous study where a single dose of 50 mg/kg adjuvant mitotane was shown to significantly reduce H295R
Aldosterone
Cortisol
DHEA-S
1.5
0.4
0.25
pg/mg tissue
ng/mg tissue
0.20
1.0
0.3
ng/mg tissue
0.15
0.2
0.5
0.10
0.1
0.05
0
0
0
placebo
i.p. mitotane oral mitotane
placebo
i.p. mitotane oral mitotane
placebo
i.p. mitotane oral mitotane
xenografts growth in nude mice when administered i.p. the same day as tumor cells injection (Lindhe and Skogseid, 2010). Details in the experimental protocols between the two studies [lower number of H295R cells inoculated in (Lindhe and Skogseid, 2010), differences in the dosages and times of mitotane adminis- tration and in the solvent used] may explain in part the differences in the outcome of the two studies. Mitotane alone (administered i.p.) also performed poorly in antagonizing growth of H295R xeno- grafts in another study (Barlaskar et al., 2009). It is unclear if in that study mitotane treatment was performed on established xeno- grafts or in an adjuvant setting, similarly to our work. In any case, our data clearly indicate that oral administration of mitotane in mice, mirroring the route of administration in ACC patients, is not effective in reducing H295R xenografts growth, even if drug plasma level reach the human therapeutic range.
5. Conclusions
Our results show that mitotane adjuvant therapy is inadequate to antagonize long-term growth of H295R cancer cell xenografts in immunodeficient mice and that care should then be taken in the design of preclinical protocols to evaluate the performance of new drugs (Kirschner, 2012) in association with mitotane in those animal models.
Acknowledgements
We thank the TrGET preclinical assay platform (Canceropole PACA) for the mouse xenograft assays and Dr. Rita Chadarevian (HRA Pharma) for mitotane measurements in mouse plasma. This work was supported by a grant from the European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement no. 259735 (ENS@T-CANCER)
References
Barlaskar, F., Spalding, A.C., Heaton, J.H., Kuick, R., Kim, A.C., Thomas, D.G., Giordano, T.J., Ben-Josef, E., Hammer, G.D., 2009. Preclinical targeting of the type I Insulin- like Growth Factor Receptor in adrenocortical carcinoma. J. Clin. Endocrinol. Metab. 94, 204-212.
Bertherat, J., Coste, J., Bertagna, X., 2007. Adjuvant mitotane in adrenocortical carcinoma. N. Engl. J. Med. 357, 1256-1257.
Chao, A., Schlinger, B.A., Remage-Healey, L., 2011. Combined liquid and solid-phase extraction improves quantification of brain estrogen content. Front. Neuroanat. 5, 57.
De Francia, S., Ardito, A., Daffara, F., Zaggia, B., Germano, A., Berruti, A., Di Carlo, F., 2012. Mitotane treatment for adrenocortical carcinoma: an overview. Minerva Endocrinol. 37, 9-23.
Doghman, M., Karpova, T., Rodrigues, G.A., Arhatte, M., De Moura, J., Cavalli, L.R., Virolle, V., Barbry, P., Zambetti, G.P., Figueiredo, B.C., Heckert, L.L., Lalli, E., 2007. Increased Steroidogenic Factor-1 dosage triggers adrenocortical cell proliferation and cancer. Mol. Endocrinol. 21, 2968-2987.
Doghman, M., El Wakil, A., Cardinaud, B., Thomas, E., Wang, J., Zhao, W., Peralta Del Valle, M.H.C., Figueiredo, B.C., Zambetti, G.P., Lalli, E., 2010. Regulation of insulin-like growth factor - mammalian target of Rapamycin signalling by microRNA in childhood adrenocortical tumors. Cancer Res. 70, 4666-4675.
Doghman, M., Lalli, E., 2012. Efficacy of the novel dual PI3-kinase/mTOR inhibitor NVP-BEZ235 in a preclinical model of adrenocortical carcinoma. Mol. Cell. Endocrinol. 364, 101-104.
Fassnacht, M., Libé, R., Kroiss, M., Allolio, B., 2011. Adrenocortical carcinoma: a clinician’s update. Nat. Rev. Endocrinol. 7, 323-335.
Grubbs, E.G., Callender, G.G., Xing, Y., Perrier, N.D., Evans, D.B., Phan, A.T., Lee, J.E., 2010. Recurrence of adrenal cortical carcinoma following resection: surgery alone can achieve results equal to surgery plus mitotane. Ann. Surg. Oncol. 17, 263-270.
Haak, H.R., Hermans, J., van de Velde, C.J., Lentjes, E.G., Goslings, B.M., Fleuren, G.J., Krans, H.M., 1994. Optimal treatment of adrenocortical carcinoma with mitotane: results in a consecutive series of 96 patients. Br. J. Cancer 69, 947- 951.
Kirschner, L.S., 2012. The next generation of therapies for adrenocortical cancers. Trends Endocrinol. Metab. 23, 343-350.
Lindhe, Ö., Skogseid, B., 2010. Mitotane effects in a H295R xenograft model of adjuvant treatment of adrenocortical cancer. Horm. Metab. Res. 42, 725-730. Luconi, M., Mannelli, M., 2012. Xenograft models for preclinical drug testing: implications for adrenocortical cancer. Mol. Cell. Endocrinol. 351, 71-77.
Maluf, D., de Oliveira, B.H., Lalli, E., 2011. Therapy of adrenocortical cancer: present and future. Am. J. Cancer Res. 1, 222-232.
Schteingart, D.E., Sinsheimer, J.E., Counsell, R.E., Abrams, G.D., Mcclellan, N., Djanegara, T., Hines, J., Ruangwises, N., Benitez, R., Wotring, L.L., 1993. Comparison of the adrenalytic activity of mitotane and a methylated homolog on normal adrenal cortex and adrenal cortical carcinoma. Cancer Chemother. Pharmacol. 31, 459-466.
Terzolo, M., Angeli, A., Fassnacht, M., Daffara, F., Tauchmanova, L., Conton, P.A., Rossetto, R., Buci, L., Sperone, P., Grossrubatscher, E., Reimondo, G., Bollito, E., Papotti, M., Saeger, W., Hahner, S., Koschker, A.C., Arvat, E., Ambrosi, B., Loli, P., Lombardi, G., Mannelli, M., Bruzzi, P., Mantero, F., Allolio, B., Dogliotti, L., Berruti, A., 2007. Adjuvant mitotane treatment for adrenocortical carcinoma. N. Engl. J. Med. 356, 2372-2380.
Wang, T., Rainey, W.E., 2012. Human adrenocortical carcinoma cell lines. Mol. Cell. Endocrinol. 351, 58-65.