Skip to main content

Advertisement

SpringerLink
Log in

Mitotane enhances doxorubicin cytotoxic activity by inhibiting P-gp in human adrenocortical carcinoma cells

  • Original Article
  • Published:
Endocrine Aims and scope Submit manuscript

Abstract

Mitotane is currently employed as adjuvant therapy as well as in the medical treatment of adrenocortical carcinoma (ACC), alone or in combination with chemotherapeutic agents. It was previously demonstrated that mitotane potentiates chemotherapeutic drugs cytotoxicity in cancer cells displaying chemoresistance due to P-glycoprotein (P-gp), an efflux pump involved in cancer multidrug resistance. The majority of ACC expresses high levels of P-gp and is highly chemoresistent. The aim of our study was to explore in vitro whether mitotane, at concentrations lower than those currently reached in vivo, may sensitize ACC cells to the cytotoxic effects of doxorubicin and whether this effect is due to a direct action on P-gp. NCI-H295 and SW13 cell lines as well as 4 adrenocortical neoplasia primary cultures were treated with mitotane and doxorubicin, and cell viability was measured by MTT assay. P-gp activity was measured by calcein and P-gp-Glo assays. P-gp expression was evaluated by Western blot. We found that very low mitotane concentrations sensitize ACC cells to the cytotoxic effects of doxorubicin, depending on P-gp expression. In addition, mitotane directly inhibits P-gp detoxifying function, allowing doxorubicin cytotoxic activity. These data provide the basis for the greater efficacy of combination therapy (mitotane plus chemotherapeutic drugs) on ACC patients. Shedding light on mitotane mechanisms of action could result in an improved design of drug therapy for patients with ACC.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. M. Terzolo, F. Daffara, A. Ardito, B. Zaggia, V. Basile, L. Ferrari, A. Berruti, Management of adrenal cancer: a 2013 update. J. Endocrinol. Invest. 37, 207–217 (2014)

    Article  CAS  PubMed  Google Scholar 

  2. M. Fassnacht, M. Kroiss, B. Allolio, Update in adrenocortical carcinoma. J. Clin. Endocrinol. Metab. 98, 4551–4564 (2013)

    Article  CAS  PubMed  Google Scholar 

  3. M. Terzolo, A. Angeli, M. Fassnacht, F. Daffara, L. Tauchmanova, P.A. Conton, R. Rossetto, L. Buci, P. Sperone, E. Grossrubatscher, G. Reimondo, E. Bollito, M. Papotti, W. Saeger, S. Hahner, A.C. Koschker, E. Arvat, B. Ambrosi, P. Loli, G. Lombardi, M. Mannelli, P. Bruzzi, F. Mantero, B. Allolio, L. Dogliotti, A. Berruti, Adjuvant mitotane treatment for adrenocortical carcinoma. N. Engl. J. Med. 356, 2372–2380 (2007)

    Article  CAS  PubMed  Google Scholar 

  4. S.D. Flynn, J.R. Murren, W.M. Kirby, J. Honig, L. Kann, B.K. Kinder, P-glycoprotein expression and multidrug resistance in adrenocortical carcinoma. Surgery. 112, 981–986 (1992)

    CAS  PubMed  Google Scholar 

  5. F.J. Sharom, ABC multidrug transporters: structure, function and role in chemoresistance. Pharmacogenomics. 9, 105–127 (2008)

    Article  CAS  PubMed  Google Scholar 

  6. L. Bao, A. Haque, K. Jackson, S. Hazari, K. Moroz, R. Jetly, S. Dash, Increased expression of P-glycoprotein is associated with doxorubicin chemoresistance in the metastatic 4T1 breast cancer model. Am. J. Pathol. 178, 838–852 (2011)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. T. Stankovic, E. Marston, Molecular mechanisms involved in chemoresistance in paediatric acute lymphoblastic leukaemia. Srp. Arh. Celok. Lek. 136, 187–192 (2008)

    Article  PubMed  Google Scholar 

  8. M.C. Zatelli, A. Luchin, D. Piccin, F. Tagliati, A. Bottoni, C. Vignali, M. Bondanelli, E.C. Degli Uberti, Cyclooxygenase-2 inhibitors reverse chemoresistance phenotype in medullary thyroid carcinoma by a permeability glycoprotein-mediated mechanism. J. Clin. Endocrinol. Metab. 90, 5754–5760 (2005)

    Article  CAS  PubMed  Google Scholar 

  9. M.C. Zatelli, A. Luchin, F. Tagliati, S. Leoni, D. Piccin, M. Bondanelli, R. Rossi, E.C. Degli Uberti, Cyclooxygenase-2 inhibitors prevent the development of chemoresistance phenotype in a breast cancer cell line by inhibiting glycoprotein p-170 expression. Endocr. Relat. Cancer 14, 1029–1038 (2007)

    Article  CAS  PubMed  Google Scholar 

  10. M.C. Zatelli, D. Molè, F. Tagliati, M. Minoia, Ambrosio, E. Degli Uberti, Cyclo-oxygenase 2 modulates chemoresistance in breast cancer cells involving NF-kappaB. Cell. Oncol. 31, 457–465 (2009)

    CAS  PubMed  Google Scholar 

  11. S.E. Bates, C.Y. Shieh, L.A. Mickley, H.L. Dichek, A. Gazdar, D.L. Loriaux, A.T. Fojo, Mitotane enhances cytotoxicity of chemotherapy in cell lines expressing a multidrug resistance gene (mdr-1/P-glycoprotein) which is also expressed by adrenocortical carcinomas. J. Clin. Endocrinol. Metab. 73, 18–29 (1991)

    Article  PubMed  Google Scholar 

  12. M. Fassnacht, M. Terzolo, B. Allolio, E. Baudin, H. Haak, A. Berruti, S. Welin, C. Schade-Brittinger, A. Lacroix, B. Jarzab, H. Sorbye, D.J. Torpy, V. Stepan, D.E. Schteingart, W. Arlt, M. Kroiss, S. Leboulleux, P. Sperone, A. Sundin, I. Hermsen, S. Hahner, H.S. Willenberg, A. Tabarin, M. Quinkler, C. de la Fouchardière, M. Schlumberger, F. Mantero, D. Weismann, F. Beuschlein, H. Gelderblom, H. Wilmink, M. Sender, M. Edgerly, W. Kenn, T. Fojo, H.H. Müller, B. Skogseid, FIRM-ACT Study Group: combination chemotherapy in advanced adrenocortical carcinoma. N. Engl. J. Med. 366, 2189–2197 (2012)

    Article  CAS  PubMed  Google Scholar 

  13. I. Veytsman, L. Nieman, T. Fojo, Management of endocrine manifestations and the use of mitotane as a chemotherapeutic agent for adrenocortical carcinoma. J. Clin. Oncol. 27, 4619–4629 (2009)

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. M.C. Zatelli, E. Gentilin, F. Daffara, F. Tagliati, G. Reimondo, G. Carandina, Ambrosio, M. Terzolo, E.C. Degli Uberti, Therapeutic concentrations of mitotane (o,p’-DDD) inhibit thyrotroph cell viability and TSH expression and secretion in a mouse cell line model. Endocrinology 151, 2453–2461 (2010)

    Article  CAS  PubMed  Google Scholar 

  15. E. Gentilin, F. Tagliati, M. Terzolo, M. Zoli, M. Lapparelli, M. Minoia, M.R. Ambrosio, E.C. degli Uberti, M.C. Zatelli, Mitotane reduces human and mouse ACTH-secreting pituitary cells viability and function. J. Endocrinol. 218, 275–285 (2013)

    Article  CAS  PubMed  Google Scholar 

  16. E. Gentilin, D. Molè, T. Gagliano, M. Minoia, M.R. Ambrosio, E.C. Degli Uberti, M.C. Zatelli, Inhibitory effects of mitotane on viability and secretory activity in mouse gonadotroph cell lines. Reprod. Toxicol. 45, 71–76 (2014)

    Article  CAS  PubMed  Google Scholar 

  17. M.C. Zatelli, R. Rossi, E.C. Degli Uberti, Androgen influences Transforming Growth Factor Beta1 (TGF b1) gene expression in human adrenocortical cells. J. Clin. Endocrinol. Metab. 85, 847–852 (2000)

    CAS  PubMed  Google Scholar 

  18. R. Rossi, M.C. Zatelli, A. Valentini, P. Cavazzini, F. Fallo, L. del Senno, E.C. degli Uberti, Evidence for androgen receptor gene expression and growth inhibitory effect of dihydrotestosterone on human adrenocortical cells. J. Endocrinol. 159, 373–380 (1998)

    Article  CAS  PubMed  Google Scholar 

  19. M.C. Zatelli, D. Piccin, F. Tagliati, A. Bottoni, A. Luchin, C. Vignali, A. Margutti, M. Bondanelli, G.C. Pansini, Pelizzo, M.D. Culler, E.C. Degli Uberti, Selective activation of somatostatin receptor subtypes differentially modulates secretion and viability in human medullary thyroid carcinoma primary cultures: potential clinical perspectives. J. Clin. Endocrinol. Metab. 91, 2218–2224 (2006)

    Article  CAS  PubMed  Google Scholar 

  20. M.C. Zatelli, F. Tagliati, D. Piccin, J.E. Taylor, M.D. Culler, M. Bondanelli, E.C. Degli Uberti, Somatostatin receptor subtype 1 selective activation reduces cell growth and calcitonin secretion in a human medullary thyroid carcinoma cell line. Biochem. Biophys. Res. Commun. 29, 828–834 (2002)

    Article  Google Scholar 

  21. T. Gagliano, M. Bellio, E. Gentilin, D. Molè, F. Tagliati, M. Schiavon, N.G. Cavallesco, L.G. Andriolo, M.R. Ambrosio, F. Rea, E. degli Uberti, M.C. Zatelli, mTOR, p70S6 K, AKT, and ERK1/2 levels predict sensitivity to mTOR and PI3 K/mTOR inhibitors in human bronchial carcinoids. Endocr. Relat. Cancer 20, 463–475 (2013)

    Article  CAS  PubMed  Google Scholar 

  22. F. Tagliati, M.C. Zatelli, A. Bottoni, D. Piccin, A. Luchin, M.D. Culler, E.C. degli Uberti, Role of complex Cyclin D1/Cdk4 in somatostatin subtype 2 receptor-mediated inhibition of cell proliferation of a medullary thyroid carcinoma cell line in vitro. Endocrinology 147, 3530–3538 (2006)

    Article  CAS  PubMed  Google Scholar 

  23. K. Fujimoto, G. Fujii, M. Mutoh, M. Yasunaga, H. Tanaka, M. Wada, Suppression of intestinal polyp development in Apc(Min/+) mice through inhibition of P-glycoprotein using verapamil. Eur. J. Cancer Prev. 22, 8–10 (2013)

    Article  CAS  PubMed  Google Scholar 

  24. R.A. Decker, P. Elson, T.F. Hogan, D.L. Citrin, D.W. Westring, T.K. Banerjee, K.W. Gilchrist, J. Horton, Eastern Cooperative Oncology Group study 1879: mitotane and adriamycin in patients with advanced adrenocortical carcinoma. Surgery. 110, 1006–1013 (1991)

    CAS  PubMed  Google Scholar 

  25. C. Hantel, S. Jung, T. Mussack, M. Reincke, F. Beuschlein, Liposomal polychemotherapy improves adrenocortical carcinoma treatment in a preclinical rodent model. Endocr. Relat. Cancer 21, 383–394 (2014)

    Article  CAS  PubMed  Google Scholar 

  26. R. Villa, L. Orlandi, A. Berruti, L. Dogliotti, N. Zaffaroni, Modulation of cytotoxic drug activity by mitotane and lonidamine in human adrenocortical carcinoma cells. Int. J. Oncol. 14, 133–138 (1999)

    CAS  PubMed  Google Scholar 

  27. G. Poli, D. Guasti, E. Rapizzi, R. Fucci, L. Canu, A. Bandini, N. Cini, D. Bani, M. Mannelli, M. Luconi, Morphofunctional effects of mitotane on mitochondria in human adrenocortical cancer cells. Endocr. Relat. Cancer 20, 537–550 (2013)

    Article  CAS  PubMed  Google Scholar 

  28. P.M. van Koetsveld, G. Vitale, R.A. Feelders, M. Waaijers, D.M. Sprij-Mooij, R.R. de Krijger, E.J. Speel, J. Hofland, S.W. Lamberts, W.W. de Herder, L.J. Hofland, Interferon-β is a potent inhibitor of cell growth and cortisol production in vitro and sensitizes human adrenocortical carcinoma cells to mitotane. Endocr. Relat. Cancer 20, 443–454 (2013)

    Article  PubMed  Google Scholar 

  29. M.C. Zatelli, R. Rossi, L. del Senno, E.C. Degli Uberti, Role of transforming growth factor beta 1 (TGF beta 1) in mediating androgen-induced growth inhibition in human adrenal cortex in vitro. Steroids 63, 243–245 (1998)

    Article  CAS  PubMed  Google Scholar 

  30. M.C. Zatelli, M. Minoia, C. Martini, F. Tagliati, Ambrosio, M. Schiavon, M. Buratto, F. Calabrese, F. Calabrese, E. Gentilin, G. Cavallesco, L. Berdondini, F. Rea, E.C. Degli Uberti, Everolimus as a new potential antiproliferative agent in aggressive human bronchial carcinoids. Endocr. Relat. Cancer 28, 719–729 (2010)

    Article  Google Scholar 

  31. M.C. Zatelli, M. Minoia, C. Filieri, F. Tagliati, M. Buratto, Ambrosio, M. Lapparelli, M. Scanarini, E.C. Degli Uberti, Effect of everolimus on cell viability in nonfunctioning pituitary adenomas. J. Clin. Endocrinol. Metab. 95, 968–976 (2010)

    Article  CAS  PubMed  Google Scholar 

  32. D.R. Barpe, D.D. Rosa, P.E. Froehlich, Pharmacokinetic evaluation of doxorubicin plasma levels in normal and overweight patients with breast cancer and simulation of dose adjustment by different indexes of body mass. Eur. J. Pharm. Sci. 41, 458–463 (2010)

    Article  CAS  PubMed  Google Scholar 

  33. E. Bello-Reuss, S. Ernest, O.B. Holland, M.R. Hellmich, Role of multidrug resistance P-glycoprotein in the secretion of aldosterone by human adrenal NCI-H295 cells. Am. J. Physiol. Cell Physiol. 278, 1256–1265 (2000)

    Google Scholar 

  34. K. Ueda, D.P. Clark, C.J. Chen, I.B. Roninson, M.M. Gottesman, I. Pastan, The human multidrug resistance (mdr1) gene. cDNA cloning and transcription initiation. J. Biol. Chem. 262, 505–508 (1987)

    CAS  PubMed  Google Scholar 

  35. M. Dean, The Human ATP-Binding Cassette (ABC) Transporter Superfamily (National Library of Medicine, US, 2002)

    Google Scholar 

  36. H.R. Haak, A.P. van Seters, A.J. Moolenaar, G.J. Fleuren, Expression of P-glycoprotein in relation to clinical manifestation, treatment and prognosis of adrenocortical cancer. Eur. J. Cancer 29A, 1036–1038 (1993)

    Article  CAS  PubMed  Google Scholar 

  37. S.D. Flynn, J.R. Murren, W.M. Kirby, J. Honig, L. Kann, B.K. Kinder, P-glycoprotein expression and multidrug resistance in adrenocortical carcinoma. Surgery. 112, 981–986 (1992)

    CAS  PubMed  Google Scholar 

  38. J. Abraham, S. Bakke, A. Rutt, B. Meadows, M. Merino, R. Alexander, D. Schrump, D. Bartlett, P. Choyke, R. Robey, E. Hung, S.M. Steinberg, S. Bates, T. Fojo, A phase II trial of combination chemotherapy and surgical resection for the treatment of metastatic adrenocortical carcinoma: continuous infusion doxorubicin, vincristine, and etoposide with daily mitotane as a P-glycoprotein antagonist. Cancer 94, 2333–2343 (2002)

    Article  CAS  PubMed  Google Scholar 

  39. F. Daffara, S. De Francia, G. Reimondo, B. Zaggia, M. Arosio, F. Porpiglia, M. Volante, A. Termine, F. Di Carlo, L. Dogliotti, A. Angeli, A. Berruti, M. Terzolo, Prospective evaluation of mitotane toxicity in adrenalcortical cancer patients treated adjuvantly. Endocr. Relat. Cancer 15, 1043–1053 (2008)

    Article  CAS  PubMed  Google Scholar 

  40. M. Lotrionte, G. Biondi-Zoccai, A. Abbate, G. Lanzetta, F. D’Ascenzo, V. Malavasi, M. Peruzzi, G. Frati, G. Palazzoni, Review and meta-analysis of incidence and clinical predictors of anthracycline cardiotoxicity. Am. J. Cardiol. 112, 1980–1984 (2013)

    Article  CAS  PubMed  Google Scholar 

  41. D. Ferone, C. Pivonello, G. Vitale, M.C. Zatelli, A. Colao, R. Pivonello, Molecular basis of pharmacological therapy in Cushing’s disease. Endocrine 46, 181–198 (2014)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the Italian Ministry of Education, Research and University (FIRB RBAP11884 M, RBAP1153LS, 2010TYCL9B_002), Fondazione Cassa di Risparmio di Ferrara, and Associazione Italiana per la Ricerca sul Cancro (AIRC) in collaboration with Laboratorio in rete del Tecnopolo “Tecnologie delle terapie avanzate” (LTTA) of the University of Ferrara.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Section of Endocrinology, Department of Medical Sciences, University of Ferrara, Via A. Moro, 8, 44124, Ferrara, Italy

    Teresa Gagliano, Erica Gentilin, Katiuscia Benfini, Carmelina Di Pasquale, Martina Tassinari, Simona Falletta, Federico Tagliati, Ettore degli Uberti & Maria Chiara Zatelli

  2. Laboratorio in Rete del Tecnopolo Tecnologie delle Terapie Avanzate (LTTA), University of Ferrara, Ferrara, Italy

    Ettore degli Uberti & Maria Chiara Zatelli

  3. Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Via A. Moro, 8, 44124, Ferrara, Italy

    Carlo Feo

Authors
  1. Teresa Gagliano
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Erica Gentilin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Katiuscia Benfini
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Carmelina Di Pasquale
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Martina Tassinari
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Simona Falletta
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Carlo Feo
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Federico Tagliati
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ettore degli Uberti
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Maria Chiara Zatelli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Maria Chiara Zatelli.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gagliano, T., Gentilin, E., Benfini, K. et al. Mitotane enhances doxorubicin cytotoxic activity by inhibiting P-gp in human adrenocortical carcinoma cells. Endocrine 47, 943–951 (2014). https://doi.org/10.1007/s12020-014-0374-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12020-014-0374-z

Keywords

Search

Navigation