Abstract
Glioblastoma (GBM) is an aggressive brain tumor with temozolomide (TMZ)-based chemotherapy as the main therapeutic strategy. Doxorubicin (DOX) is not used in gliomas due to its low bioavailability in the brain; however, new delivery strategies and low doses may be effective in the long term, especially as part of a drug cocktail. Our aim was to evaluate the chronic effects of low doses of DOX and TMZ in GBM. Human U87-ATCC cells and a primary GBM culture were chronically treated with TMZ (5 μM) and DOX (1 and 10 nM) alone or combined. DOX resulted in a reduction in the number of cells over a period of 35 days and delayed the cell regrowth. In addition, DOX induced cell senescence and reduced tumor sphere formation and the proportion of NANOG- and OCT4-positive cells after 7 days. Low doses of TMZ potentiated the effects of DOX on senescence and sphere formation. This combined response using low doses of DOX may pave the way for its use in glioma therapy, with new technologies to overcome its low blood–brain barrier permeability.
Similar content being viewed by others
References
Maher EA, Furnari FB, Bachoo RM et al (2001) Malignant glioma: genetics and biology of a grave matter. Genes Dev 15:1311–1333
Stupp R, Mason WP, van den Bent MJ et al (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:987–996
Ewald JA, Desotelle JA, Wilding G, Jarrard DF (2010) Therapy-induced senescence in cancer. J Natl Cancer Inst 102:1536–1546
Dimri GP, Lee X, Basile G et al (1995) A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 92:9363–9367
Dumont P, Burton M, Chen QM et al (2000) Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast. Free Radic Biol Med 28:361–373
Lundberg AS, Hahn WC, Gupta P, Weinberg RA (2000) Genes involved in senescence and immortalization. Curr Opin Cell Biol 12:705–709
Volonte D, Zhang K, Lisanti MP, Galbiati F (2002) Expression of caveolin-1 induces premature cellular senescence in primary cultures of murine fibroblasts. Stress-induced premature senescence upregulates the expression of endogenous caveolin-1. Mol Biol Cell 13:2502–2517
Reddy JP, Li Y (2011) Oncogene-induced senescence and its role in tumor suppression. J Mammary Gland Biol Neoplasia 16:247–256
Banumathy G, Adams PD (2010) Cellular senescence and tumor suppression. Chapter 5:109–125
Ohtani N, Hara E (2013) Roles and mechanisms of cellular senescence in regulation of tissue homeostasis. Cancer Sci 104:525–530
Ohanna M, Cheli Y, Bonet C et al (2013) Secretome from senescent melanoma engages the STAT3 pathway to favor reprogramming of naive melanoma towards a tumor-initiating cell phenotype. Oncotarget 4:2012–2224
Ohanna M, Giuliano S, Bonet C et al (2011) Senescent cells develop a parp-1 and nuclear factor-κB-associated secretome (PNAS). Genes Dev 25:1245–1261
Vjetrovic J, Shankaranarayanan P, Mendoza-Parra MA, Gronemeyer H (2014) Senescence-secreted factors activate Myc and sensitize pretransformed cells to TRAIL-induced apoptosis. Aging Cell 13:487–496
Acosta JC, Gil J (2012) Senescence: a new weapon for cancer therapy. Trends Cell Biol 22:211–219
Beier D, Röhrl S, Pillai DR et al (2008) Temozolomide preferentially depletes cancer stem cells in glioblastoma. Cancer Res 68:5706–5715
Bleau A-M, Hambardzumyan D, Ozawa T et al (2009) PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells. Cell Stem Cell 4:226–235
Menna P, Recalcati S, Cairo G, Minotti G (2007) An introduction to the metabolic determinants of anthracycline cardiotoxicity. Cardiovasc Toxicol 7:80–85
Kurata K, Yanagisawa R, Ohira M et al (2008) Stress via p53 pathway causes apoptosis by mitochondrial Noxa upregulation in doxorubicin-treated neuroblastoma cells. Oncogene 27:741–754
Lesniak MS, Upadhyay U, Goodwin R et al (2005) Local delivery of doxorubicin for the treatment of malignant brain tumors in rats. Anticancer Res 25:3825–3831
Minotti G, Menna P, Salvatorelli E et al (2004) Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev 56:185–229
Cheng Y, Morshed R, Cheng SH et al (2013) Nanoparticle-programmed self-destructive neural stem cells for glioblastoma targeting and therapy. Small 9:4123–4129
Gonçalves C, Martins-Neves SR, Paiva-Oliveira D et al (2015) Sensitizing osteosarcoma stem cells to doxorubicin-induced apoptosis through retention of doxorubicin and modulation of apoptotic-related proteins. Life Sci 130:47–56
Zheng X, Cui D, Xu S et al (2010) Doxorubicin fails to eradicate cancer stem cells derived from anaplastic thyroid carcinoma cells: characterization of resistant cells. Int J Oncol 37:307–315
Atashpour S, Fouladdel S, Movahhed TK et al (2015) Quercetin induces cell cycle arrest and apoptosis in CD133(+) cancer stem cells of human colorectal HT29 cancer cell line and enhances anticancer effects of doxorubicin. Iran J Basic Med Sci 18:635–643
Almubarak M, Newton M, Altaha R (2008) Reinduction of bevacizumab in combination with pegylated liposomal doxorubicin in a patient with recurrent glioblastoma multiforme who progressed on bevacizumab/irinotecan. J Oncol 2008:1–4
Kikuchi T, Saito R, Sugiyama S et al (2008) Convection-enhanced delivery of polyethylene glycol-coated liposomal doxorubicin: characterization and efficacy in rat intracranial glioma models. J Neurosurg 109:867–873
da Ros M, Iorio AL, Consolante D et al (2016) Morphine modulates doxorubicin uptake and improves efficacy of chemotherapy in an intracranial xenograft model of human glioblastoma. Am J Cancer Res 6:639–648
Chen H, Qin Y, Zhang Q et al (2011) Lactoferrin modified doxorubicin-loaded procationic liposomes for the treatment of gliomas. Eur J Pharm Sci 44:164–173
Qin Y, Chen H, Zhang Q et al (2011) Liposome formulated with TAT-modified cholesterol for improving brain delivery and therapeutic efficacy on brain glioma in animals. Int J Pharm 420:304–312
Baltes S, Freund I, Lewis AL et al (2010) Doxorubicin and irinotecan drug-eluting beads for treatment of glioma: a pilot study in a rat model. J Mater Sci Mater Med 21:1393–1402
Sun TM, Wang YC, Wang F et al (2014) Cancer stem cell therapy using doxorubicin conjugated to gold nanoparticles via hydrazone bonds. Biomaterials 35:836–845
Beier D, Hau P, Proescholdt M et al (2007) CD133+ and CD133− glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles. Cancer Res 67:4010–4015
Fan X, Salford LG, Widegren B (2007) Glioma stem cells: evidence and limitation. Semin Cancer Biol 17:214–218
Yuan X, Curtin J, Xiong Y et al (2004) Isolation of cancer stem cells from adult glioblastoma multiforme. Oncogene 23:9392–9400
Clarke MF, Fuller M (2006) Stem cells and cancer: two faces of eve. Cell 124:1111–1115
Denysenko T, Gennero L, Roos MA et al (2010) Glioblastoma cancer stem cells: heterogeneity, microenvironment and related therapeutic strategies. Cell Biochem Funct 28:343–351
Singh SK, Clarke ID, Hide T, Dirks PB (2004) Cancer stem cells in nervous system tumors. Oncogene 23:7267–7273
Du Z, Jia D, Liu S et al (2009) Oct4 in expressed in human gliomas and promotes colony formation in glioma cells. Glia 57:724–733
Nern C, Sommerlad D, Acker T, Plate KH (2009) Brain Tumor Stem Cells. pp 241–259
Bao S, Wu Q, McLendon RE et al (2006) Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444:756–760
Eramo A, Ricci-Vitiani L, Zeuner A et al (2006) Chemotherapy resistance of glioblastoma stem cells. Cell Death Differ 13:1238–1241
Kang M-K, Kang S-K (2007) Tumorigenesis of chemotherapeutic drug-resistant cancer stem-like cells in brain glioma. Stem Cells Dev 16:837–847
Ledur PF, Villodre ES, Paulus R et al (2012) Extracellular ATP reduces tumor sphere growth and cancer stem cell population in glioblastoma cells. Purinergic Signal 8:39–48
Zhuang W, Li B, Long L et al (2011) Induction of autophagy promotes differentiation of glioma-initiating cells and their radiosensitivity. Int J Cancer 129:2720–2731
Beier D, Schulz JB, Beier CP (2011) Chemoresistance of glioblastoma cancer stem cells—much more complex than expected. Mol Cancer 10:1–11
Sesen J, Dahan P, Scotland SJ et al (2015) Metformin inhibits growth of human glioblastoma cells and enhances therapeutic response. PLoS One 10:1–24
Yu Z, Zhao G, Li P et al (2016) Temozolomide in combination with metformin act synergistically to inhibit proliferation and expansion of glioma stem-like cells. Oncol Lett 11:2792–2800
Yuan S, Wang F, Chen G et al (2013) Effective elimination of cancer stem cells by a novel drug combination strategy. Stem Cells 31:23–34
Silva AO, Felipe KB, Villodre ES et al (2016) A guide for the analysis of long-term population growth in cancer. Tumor Biol 37:1–7
Filippi-Chiela EC, Oliveira MM, Jurkovski B, et al (2012) Nuclear morphometric analysis (NMA): screening of senescence, apoptosis and nuclear irregularities. PLoS One
Thomas AA, Brennan CW, DeAngelis LM, Omuro AM (2014) Emerging therapies for glioblastoma. JAMA Neurol 71:1437–1444
Kondo S, Kondo Y, Hara H et al (1996) mdm2 gene mediates the expression of mdr1 gene and P-glycoprotein in a human glioblastoma cell line. Br J Cancer 74:1263–1268
Chen XJ, Zhang K, Xin Y, Jiang G (2014) Oncolytic adenovirus-expressed RNA interference of O-methylguanine DNA methyltransferase activity may enhance the antitumor effects of temozolomide. Oncol Lett 8:2201–2202
Lopez PLC, Filippi-Chiela EC, Silva AO et al (2012) Sensitization of glioma cells by x-linked inhibitor of apoptosis protein knockdown. Oncology 83:75–82
von Holst H, Knochenhauer E, Blomgren H et al (1990) Uptake of adriamycin in tumour and surrounding brain tissue in patients with malignant gliomas. Acta Neurochir 104:13–16
Zhang R, Saito R, Shibahara I et al (2016) Temozolomide reverses doxorubicin resistance by inhibiting P-glycoprotein in malignant glioma cells. J Neuro-Oncol 126:235–242
Sin S, Kim SY, Kim SSU (2012) Chronic treatment with ginsenoside Rg3 induces Akt-dependent senescence in human glioma cells. 1669–1674
Wu Y, Dong L, Bao S et al (2016) FK228 augmented temozolomide sensitivity in human glioma cells by blocking PI3K/AKT/mTOR signal pathways. Biomed Pharmacother 84:462–469
Staedler D, Idrizi E, Kenzaoui BH, Juillerat-Jeanneret L (2011) Drug combinations with quercetin: doxorubicin plus quercetin in human breast cancer cells. Cancer Chemother Pharmacol 68:1161–1172
Liffers S-T, Tilkorn DJ, Stricker I et al (2013) Salinomycin increases chemosensitivity to the effects of doxorubicin in soft tissue sarcomas. BMC Cancer 13:1–9
Strik HM, Marosi C, Kaina B, Neyns B (2012) Temozolomide dosing regimens for glioma patients. Curr Neurol Neurosci Rep 12:286–293
Hammond LA, Eckardt JR, Baker SD et al (1999) Phase I and pharmacokinetic study of temozolomide on a daily-for-5-days schedule in patients with advanced solid malignancies. J Clin Oncol 17:2604–2613
Gilbert CA, Daou MC, Moser RP, Ross AH (2010) γ-Secretase inhibitors enhance temozolomide treatment of human gliomas by inhibiting neurosphere repopulation and xenograft recurrence. Cancer Res 70:6870–6879
Liu G, Yuan X, Zeng Z et al (2006) Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma. Mol Cancer 5:1–12
Al-Lazikani B, Banerji U, Workman P (2012) Combinatorial drug therapy for cancer in the post-genomic era. Nat Biotechnol 30:679–692
Kummar S, Chen HX, Wright J et al (2010) Utilizing targeted cancer therapeutic agents in combination: novel approaches and urgent requirements. Nat Rev Drug Discov 9:843–856
Pan C, Kumar C, Bohl S et al (2009) Comparative proteomic phenotyping of cell lines and primary cells to assess preservation of cell type-specific functions. Mol Cell Proteomics 8:443–450
Allen M, Bjerke M, Edlund H et al (2016) Origin of the U87MG glioma cell line: good news and bad news. Sci Transl Med 8:1–5
Beier D, Schriefer B, Brawanski K et al (2012) Efficacy of clinically relevant temozolomide dosing schemes in glioblastoma cancer stem cell lines. J Neuro-Oncol 109:45–52
Acknowledgements
This work was supported by CAPES Probitec 004/2012 and FAPERGS Pronem 11/2072-2. ESV and PLCL received CAPES fellowships and GL receives CNPq fellowship.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
The ethical committee at the UFRGS (n. 420.856) and PUCRS (n. 429.849) approved the use of LS12 cells.
Electronic Supplementary Material
Fig. S1
Flow cytometry analysis. (JPEG 72 kb)
Fig. S2
Cumulative population doubling analysis of the primary GBM cell, LS12. (JPEG 252 kb)
Fig. S3
Apoptosis and necrosis analysis after treatment with DOX and TMZ after 24 h. (JPEG 46 kb)
Fig. S4
Nuclear morphometric analysis (NMA) after DOX 1 and 10 nM and TMZ 5 μM treatment. (JPEG 194 kb)
Fig. S5
Number of spheres and protein quantification after 7 days of treatment. (JPEG 205 kb)
Fig. S6
Radar graph was used in order to integrate all the results analysis using different combinations. (JPEG 262 kb)
Rights and permissions
About this article
Cite this article
Villodre, E.S., Kipper, F.C., Silva, A.O. et al. Low Dose of Doxorubicin Potentiates the Effect of Temozolomide in Glioblastoma Cells. Mol Neurobiol 55, 4185–4194 (2018). https://doi.org/10.1007/s12035-017-0611-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12035-017-0611-6