Top

Published in:

Open Access 01-12-2016 | Research

Effect of low doses of actinomycin D on neuroblastoma cell lines

Authors: Constanza L. Cortes, Sonia R. Veiga, Eugènia Almacellas, Javier Hernández-Losa, Joan C. Ferreres, Sara C. Kozma, Santiago Ambrosio, George Thomas, Albert Tauler

Published in: Molecular Cancer | Issue 1/2016

Abstract

Background

Neuroblastoma is a malignant embryonal tumor occurring in young children, consisting of undifferentiated neuroectodermal cells derived from the neural crest. Current therapies for high-risk neuroblastoma are insufficient, resulting in high mortality rates and high incidence of relapse. With the intent to find new therapies for neuroblastomas, we investigated the efficacy of low-doses of actinomycin D, which at low concentrations preferentially inhibit RNA polymerase I-dependent rRNA trasncription and therefore, ribosome biogenesis.

Methods

Neuroblastoma cell lines with different p53 genetic background were employed to determine the response on cell viability and apoptosis of low-dose of actinomycin D. Subcutaneously-implanted SK-N-JD derived neuroblastoma tumors were used to assess the effect of low-doses of actinomycin D on tumor formation.

Results

Low-dose actinomycin D treatment causes a reduction of cell viability in neuroblastoma cell lines and that this effect is stronger in cells that are wild-type for p53. MYCN overexpression contributes to enhance this effect, confirming the importance of this oncogene in ribosome biogenesis. In the wild-type SK-N-JD cell line, apoptosis was the major mechanism responsible for the reduction in viability and we demonstrate that treatment with the MDM2 inhibitor Nutlin-3, had a similar effect to that of actinomycin D. Apoptosis was also detected in p53^−/−deficient LA1-55n cells treated with actinomycin D, however, only a small recovery of cell viability was found when apoptosis was inhibited by a pan-caspase inhibitor, suggesting that the treatment could activate an apoptosis-independent cell death pathway in these cells. We also determined whether actinomycin D could increase the efficacy of the histone deacetylase inhibitor, SAHA, which is in being used in neuroblastoma clinical trials. We show that actinomycin D synergizes with SAHA in neuroblastoma cell lines. Moreover, on subcutaneously-implanted neuroblastoma tumors derived from SK-N-JD cells, actinomycin D led to tumor regression, an effect enhanced in combination with SAHA.

Conclusions

The results presented in this work demonstrate that actinomycin D, at low concentrations, inhibits proliferation and induces cell death in vitro, as well as tumor regression in vivo. From this study, we propose that use of ribosome biogenesis inhibitors should be clinically considered as a potential therapy to treat neuroblastomas.

Available only for authorised users

Maris JM, Hogarty MD, Bagatell R, Cohn SL. Neuroblastoma. Lancet. 2007;369(9579):2106–20. doi:10.1016/S0140-6736(07)60983-0.CrossRefPubMed

Brodeur GM. Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer. 2003;3(3):203–16.CrossRefPubMed

Casciano I, Mazzocco K, Boni L, Pagnan G, Banelli B, Allemanni G, et al. Expression of DeltaNp73 is a molecular marker for adverse outcome in neuroblastoma patients. Cell Death Differ. 2002;9(3):246–51.CrossRefPubMed

Cohn SL, Pearson ADJ, London WB, Monclair T, Ambros PF, Brodeur GM, et al. The International Neuroblastoma Risk Group (INRG) classification system: an INRG Task Force report. J Clin Oncol. 2009;27(2):289–97.CrossRefPubMedPubMedCentral

Strieder V, Lutz W. E2F proteins regulate MYCN expression in neuroblastomas. J Biol Chem. 2003;278(5):2983–9.CrossRefPubMed

Mosse YP, Wood A, Maris JM. Inhibition of ALK signaling for cancer therapy. Clin Cancer Res. 2009;15(18):5609–14.CrossRefPubMed

Holzel M, Huang S, Koster J, Ora I, Lakeman A, Caron H, et al. NF1 is a tumor suppressor in neuroblastoma that determines retinoic acid response and disease outcome. Cell. 2010;142(2):218–29.CrossRefPubMedPubMedCentral

Goldman SC, Chen CY, Lansing TJ, Gilmer TM, Kastan MB. The p53 signal transduction pathway is intact in human neuroblastoma despite cytoplasmic localization. Am J Pathol. 1996;148(5):1381–5.PubMedPubMedCentral

Fesik SW. Promoting apoptosis as a strategy for cancer drug discovery. Nat Rev Cancer. 2005;5(11):876–85.CrossRefPubMed

10.

Eleveld TF, Oldridge DA, Bernard V, Koster J, Daage LC, Diskin SJ, et al. Relapsed neuroblastomas show frequent RAS-MAPK pathway mutations. Nat Genet. 2015;47(8):864–71. doi:10.1038/ng.3333.CrossRefPubMed

11.

Schramm A, Koster J, Assenov Y, Althoff K, Peifer M, Mahlow E, et al. Mutational dynamics between primary and relapse neuroblastomas. Nat Genet. 2015;47(8):872–7.CrossRefPubMed

12.

Poortinga G, Quinn LM, Hannan RD. Targeting RNA polymerase I to treat MYC-driven cancer. Oncogene. 2015;34(4):403–12. doi:10.1038/onc.2014.13.CrossRefPubMed

13.

Gentilella A, Kozma SC, Thomas G. A liaison between mTOR signaling, ribosome biogenesis and cancer. Biochimica et biophysica acta. 2015. doi:10.1016/j.bbagrm.2015.02.005.

14.

Ruggero D, Pandolfi PP. Does the ribosome translate cancer? Nat Rev Cancer. 2003;3(3):179–92.CrossRefPubMed

15.

Ruggero D. Revisiting the nucleolus: from marker to dynamic integrator of cancer signaling. Sci Signal. 2012;5(241):e38.CrossRef

16.

Thomas G. An encore for ribosome biogenesis in the control of cell proliferation. Nat Cell Biol. 2000;2(5):E71–2.CrossRefPubMed

17.

van Riggelen J, Yetil A, Felsher DW. MYC as a regulator of ribosome biogenesis and protein synthesis. Nat Rev Cancer. 2010;10(4):301–9.CrossRefPubMed

18.

Boon K, Caron HN, van Asperen R, Valentijn L, Hermus MC, van Sluis P, et al. N-myc enhances the expression of a large set of genes functioning in ribosome biogenesis and protein synthesis. EMBO J. 2001;20(6):1383–93.CrossRefPubMedPubMedCentral

19.

Bywater MJ, Poortinga G, Sanij E, Hein N, Peck A, Cullinane C, et al. Inhibition of RNA polymerase I as a therapeutic strategy to promote cancer-specific activation of p53. Cancer Cell. 2012;22(1):51–65. doi:10.1016/j.ccr.2012.05.019.CrossRefPubMedPubMedCentral

20.

Hollstein U. Actinomycin. Chemistry and mechanism of action. Chem Rev. 1974;74(6):625–52.CrossRef

21.

Malogolowkin M, Cotton CA, Green DM, Breslow NE, Perlman E, Miser J, et al. Treatment of Wilms tumor relapsing after initial treatment with vincristine, actinomycin D, and doxorubicin. A report from the National Wilms Tumor Study Group. Pediatr Blood Cancer. 2008;50(2):236–41. doi:10.1002/pbc.21267.CrossRefPubMed

22.

Jaffe N, Paed D, Traggis D, Salian S, Cassady JR. Improved outlook for Ewing’s sarcoma with combination chemotherapy (vincristine, actinomycin D and cyclophosphamide) and radiation therapy. Cancer. 1976;38(5):1925–30.CrossRefPubMed

23.

Perry RP, Kelley DE. Inhibition of RNA synthesis by actinomycin D: characteristic dose–response of different RNA species. J Cell Physiol. 1970;76(2):127–39.CrossRefPubMed

24.

Donati G, Peddigari S, Mercer CA, Thomas G. 5S ribosomal RNA is an essential component of a nascent ribosomal precursor complex that regulates the Hdm2-p53 checkpoint. Cell Rep. 2013;4(1):87–98.CrossRefPubMedPubMedCentral

25.

Merkel O, Wacht N, Sifft E, Melchardt T, Hamacher F, Kocher T, et al. Actinomycin D induces p53-independent cell death and prolongs survival in high-risk chronic lymphocytic leukemia. Leukemia. 2012;26(12):2508–16. doi:10.1038/leu.2012.147.CrossRefPubMed

26.

Fouladi M, Park JR, Stewart CF, Gilbertson RJ, Schaiquevich P, Sun J, et al. Pediatric phase I trial and pharmacokinetic study of vorinostat: a Children’s Oncology Group phase I consortium report. J Clin Oncol. 2010;28(22):3623–9.CrossRefPubMedPubMedCentral

27.

Francisco R, Perez-Perarnau A, Cortes C, Gil J, Tauler A, Ambrosio S. Histone deacetylase inhibition induces apoptosis and autophagy in human neuroblastoma cells. Cancer Lett. 2012;318(1):42–52.CrossRefPubMed

28.

Lutz W, Stohr M, Schurmann J, Wenzel A, Lohr A, Schwab M. Conditional expression of N-myc in human neuroblastoma cells increases expression of alpha-prothymosin and ornithine decarboxylase and accelerates progression into S-phase early after mitogenic stimulation of quiescent cells. Oncogene. 1996;13(4):803–12.PubMed

29.

Moll UM, Petrenko O. The MDM2-p53 interaction. Mol Cancer Res. 2003;1(14):1001–8.PubMed

30.

Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS, et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy. 2008;4(2):151–75.CrossRefPubMedPubMedCentral

31.

Marks PA, Xu WS. Histone deacetylase inhibitors: potential in cancer therapy. J Cell Biochem. 2009;107(4):600–8.CrossRefPubMedPubMedCentral

32.

Yoon MK, Ha JH, Lee MS, Chi SW. Structure and apoptotic function of p73. BMB Rep. 2015;48(2):81–90.CrossRefPubMedPubMedCentral

33.

Lau LM, Nugent JK, Zhao X, Irwin MS. HDM2 antagonist Nutlin-3 disrupts p73-HDM2 binding and enhances p73 function. Oncogene. 2008;27(7):997–1003. doi:10.1038/sj.onc.1210707.CrossRefPubMed

34.

Peirce SK, Findley HW. High level MycN expression in non-MYCN amplified neuroblastoma is induced by the combination treatment nutlin-3 and doxorubicin and enhances chemosensitivity. Oncol Rep. 2009;22(6):1443–9.CrossRefPubMed

35.

Mischo HE, Hemmerich P, Grosse F, Zhang S. Actinomycin D induces histone gamma-H2AX foci and complex formation of gamma-H2AX with Ku70 and nuclear DNA helicase II. J Biol Chem. 2005;280(10):9586–94. doi:10.1074/jbc.M411444200.CrossRefPubMed

36.

Matthay KK, Villablanca JG, Seeger RC, Stram DO, Harris RE, Ramsay NK, et al. Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. Children’s Cancer Group. N Engl J Med. 1999;341(16):1165–73. doi:10.1056/NEJM199910143411601.CrossRefPubMed

37.

Van Maerken T, Rihani A, Dreidax D, De Clercq S, Yigit N, Marine JC, et al. Functional analysis of the p53 pathway in neuroblastoma cells using the small-molecule MDM2 antagonist nutlin-3. Mol Cancer Ther. 2011;10(6):983–93. doi:10.1158/1535-7163.MCT-10-1090.CrossRefPubMed

38.

Ozaki T, Hosoda M, Miyazaki K, Hayashi S, Watanabe K, Nakagawa T, et al. Functional implication of p73 protein stability in neuronal cell survival and death. Cancer Lett. 2005;228(1–2):29–35. doi:10.1016/j.canlet.2004.12.050.CrossRefPubMed

39.

Fumagalli S, Ivanenkov VV, Teng T, Thomas G. Suprainduction of p53 by disruption of 40S and 60S ribosome biogenesis leads to the activation of a novel G2/M checkpoint. Genes Dev. 2012;26(10):1028–40. doi:10.1101/gad.189951.112.CrossRefPubMedPubMedCentral

40.

Gomez-Santos C, Ferrer I, Santidrian AF, Barrachina M, Gil J, Ambrosio S. Dopamine induces autophagic cell death and alpha-synuclein increase in human neuroblastoma SH-SY5Y cells. J Neurosci Res. 2003;73(3):341–50. doi:10.1002/jnr.10663.CrossRefPubMed

41.

Gamble LD, Kees UR, Tweddle DA, Lunec J. MYCN sensitizes neuroblastoma to the MDM2-p53 antagonists Nutlin-3 and MI-63. Oncogene. 2012;31(6):752–63.CrossRefPubMedPubMedCentral

42.

Hill CR, Cole M, Errington J, Malik G, Boddy AV, Veal GJ. Characterisation of the Clinical Pharmacokinetics of Actinomycin D and the Influence of ABCB1 Pharmacogenetic Variation on Actinomycin D Disposition in Children with Cancer. Clin Pharmacokinet. 2014;53(8):741–51.CrossRefPubMedPubMedCentral

43.

Witt O, Milde T, Deubzer HE, Oehme I, Witt R, Kulozik A, et al. Phase I/II intra-patient dose escalation study of vorinostat in children with relapsed solid tumor, lymphoma or leukemia. Klin Padiatr. 2012;224(6):398–403.CrossRefPubMed

44.

Ouwehand K, de Ruijter AJ, van Bree C, Caron HN, van Kuilenburg AB. Histone deacetylase inhibitor BL1521 induces a G1-phase arrest in neuroblastoma cells through altered expression of cell cycle proteins. FEBS Lett. 2005;579(6):1523–8. doi:10.1016/j.febslet.2005.01.058.CrossRefPubMed

45.

Huang L, Sowa Y, Sakai T, Pardee AB. Activation of the p21WAF1/CIP1 promoter independent of p53 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through the Sp1 sites. Oncogene. 2000;19(50):5712–9. doi:10.1038/sj.onc.1203963.CrossRefPubMed

46.

Peart MJ, Smyth GK, van Laar RK, Bowtell DD, Richon VM, Marks PA, et al. Identification and functional significance of genes regulated by structurally different histone deacetylase inhibitors. Proc Natl Acad Sci U S A. 2005;102(10):3697–702. doi:10.1073/pnas.0500369102.CrossRefPubMedPubMedCentral

47.

Hahn CK, Ross KN, Warrington IM, Mazitschek R, Kanegai CM, Wright RD, et al. Expression-based screening identifies the combination of histone deacetylase inhibitors and retinoids for neuroblastoma differentiation. Proc Natl Acad Sci U S A. 2008;105(28):9751–6. doi:10.1073/pnas.0710413105.CrossRefPubMedPubMedCentral

48.

De los Santos M, Zambrano A, Aranda A. Combined effects of retinoic acid and histone deacetylase inhibitors on human neuroblastoma SH-SY5Y cells. Mol Cancer Ther. 2007;6(4):1425–32. doi:10.1158/1535-7163.MCT-06-0623.CrossRefPubMed

49.

Carr-Wilkinson J, O’Toole K, Wood KM, Challen CC, Baker AG, Board JR, et al. High Frequency of p53/MDM2/p14ARF Pathway Abnormalities in Relapsed Neuroblastoma. Clin Cancer Res. 2010;16(4):1108–18.CrossRefPubMedPubMedCentral

50.

Morgado-Palacin L, Llanos S, Urbano-Cuadrado M, Blanco-Aparicio C, Megias D, Pastor J, et al. Non-genotoxic activation of p53 through the RPL11-dependent ribosomal stress pathway. Carcinogenesis. 2014;35(12):2822–30. doi:10.1093/carcin/bgu220.CrossRefPubMed

51.

Caporale DM, Bobbio A, Accordino R, Ampollini L, Internullo E, Cattelani L, et al. Ectopic mediastinal parathyroid adenoma. Acta Biomed. 2003;74(3):157–9.PubMed

52.

Real S, Meo-Evoli N, Espada L, Tauler A. E2F1 regulates cellular growth by mTORC1 signaling. PLoS One. 2011;6(1):e16163. doi:10.1371/journal.pone.0016163.CrossRefPubMedPubMedCentral

53.

Chou T-C. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev. 2006;58(3):621–81.CrossRefPubMed

Title: Effect of low doses of actinomycin D on neuroblastoma cell lines
Authors: Constanza L. Cortes
Sonia R. Veiga
Eugènia Almacellas
Javier Hernández-Losa
Joan C. Ferreres
Sara C. Kozma
Santiago Ambrosio
George Thomas
Albert Tauler
Publication date: 01-12-2016
Publisher: BioMed Central
Published in: Molecular Cancer / Issue 1/2016
Electronic ISSN: 1476-4598
DOI: https://doi.org/10.1186/s12943-015-0489-8

Webinar | 19-02-2024 | 17:30 (CET)

Keynote webinar | Spotlight on antibody–drug conjugates in cancer

Watch now

Antibody–drug conjugates (ADCs) are novel agents that have shown promise across multiple tumor types. Explore the current landscape of ADCs in breast and lung cancer with our experts, and gain insights into the mechanism of action, key clinical trials data, existing challenges, and future directions.

Dr. Véronique Diéras

Prof. Fabrice Barlesi

Developed by: Springer Medicine

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

Abstract

Background

Methods

Results

Conclusions

Please log in to get access to this content

Other articles of this Issue 1/2016

Identification of the dopamine transporter SLC6A3 as a biomarker for patients with renal cell carcinoma

NOTCH receptors in gastric and other gastrointestinal cancers: oncogenes or tumor suppressors?

Emerging roles of T helper 17 and regulatory T cells in lung cancer progression and metastasis

Estrogen receptor α in cancer associated fibroblasts suppresses prostate cancer invasion via reducing CCL5, IL6 and macrophage infiltration in the tumor microenvironment

Efficient elimination of pancreatic cancer stem cells by hedgehog/GLI inhibitor GANT61 in combination with mTOR inhibition

Less is more: low expression of MT1-MMP is optimal to promote migration and tumourigenesis of breast cancer cells

Keynote webinar | Spotlight on antibody–drug conjugates in cancer