Journal of Cancer Research and Clinical Oncology

, Volume 144, Issue 8, pp 1475–1485 | Cite as

Effects of sequentially applied single and combined temozolomide, hydroxychloroquine and AT101 treatment in a long-term stimulation glioblastoma in vitro model

  • Vivian Adamski
  • Christina Schmitt
  • Florian Ceynowa
  • Rainer Adelung
  • Ralph Lucius
  • Michael Synowitz
  • Kirsten Hattermann
  • Janka Held-FeindtEmail author
Original Article – Cancer Research



Glioblastoma multiforme (GBM) is a poorly curable disease due to its heterogeneity that enables single cells to survive treatment regimen and initiate tumor regrowth. Although some progress in therapy has been achieved in the last years, the efficient treatment of GBMs is still a clinical challenge. Besides the standard therapeutic drug temozolomide (TMZ), quinoline-based antimalarial drugs such as hydroxychloroquine (HCQ) and BH3 mimetics such as AT101 were considered as possible drugs for GBM therapy.


We investigated the effects of sequentially applied single and combined TMZ, HCQ and AT101 treatments in a long-term stimulation GBM in vitro model. We performed all investigations in parallel in human astrocytes and two differentially TMZ-responsive human GBM cell lines and adjusted used drug concentrations to known liquor/plasma concentrations in patients. We determined amounts of dead cells and still remaining growth rates and depicted our results in a heatmap-like summary to visualize which sequential long-term treatment schedule seemed to be most promising.


We showed that sequential stimulations yielded higher cytotoxicity and better tumor growth control in comparison to single TMZ treatment. This was especially the case for the sequences TMZ/HCQ and TMZ + AT101/AT101 which was as effective as the non-sequential combination TMZ + AT101. Importantly, those affected both less and more TMZ-responsive glioma cell lines, whilst being less harmful for astrocytes in comparison to single TMZ treatment.


Sequential treatment with mechanistically different acting drugs might be an option to reduce side effects in long-term treatment, for example in local administration approaches.


R-(−)-gossypol Alternative drugs Sequential treatment Quinoline-based drugs BH3 mimetics Autophagocytosis 



CCAAT-enhancer-binding protein homologous protein


Combination index




Dulbecco’s modified Eagle’s medium


Dimethyl sulfoxide


Endoplasmatic reticulum


Fetal bovine serum


Growth arrest and DNA-damage-inducible protein


Glioblastoma multiforme




Poly-ADP ribose polymerase







We thank Fereshteh Ebrahim, Brigitte Rehmke, Judith Becker and Sonja Dahle for expert technical assistance.

Author contributions

JHF, KH conceived and designed the study; VA, CS, JHF and KH performed the experiments and analyzed the data; FC, RA, RL and MS contributed materials and assisted in data analysis; JHF and KH wrote the paper, and all authors revised the manuscript.


This study was funded by the German Research Foundation (DFG) as part as of the Research Training Group “Materials4Brain” (GRK2154; P3, P7 and P8).

Compliance with ethical standards

Conflict of interest

Author Vivian Adamski declares that she has no conflict of interest. Author Christina Schmitt declares that she has no conflict of interest. Author Florian Ceynowa declares that he has no conflict of interest. Author Rainer Adelung declares that he has no conflict of interest. Author Ralph Lucius declares that he has no conflict of interest. Author Michael Synowitz declares that he has no conflict of interest. Author Kirsten Hattermann declares that she has no conflict of interest. Author Janka Held-Feindt declares that she has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

432_2018_2680_MOESM1_ESM.tif (677 kb)
Supplementary material 1 Figure 1 Supplement : Treatment schedules for the long-term GBM in vitro model. A. Cells were treated for 3 days with the gold standard therapeutic temozolomide (TMZ), switching on day 3 either to the combined treatment of TMZ plus an alternative drug [shown as TMZ+AT101 or TMZ+hydroxychloroquine(HCQ)], or to the single alternative drug (AT101 or HCQ), respective staying with the TMZ treatment. B. Cells were treated for 3 days with one of the single alternative drugs (AT101 or HCQ), switching on day 3 to either the combined therapy with TMZ, to the gold standard therapeutic TMZ or staying with the single alternative drug. C. Cells were treated for 3 days with the combined treatment of TMZ plus one alternative drug, switching on day 3 to either one of the single drugs TMZ, AT101 or HCQ respective staying with the combined therapy until day 6 (TIF 676 KB)
432_2018_2680_MOESM2_ESM.tif (1.3 mb)
Supplementary material 2 Figure 2 Supplement : Effect of selected (non)-sequential treatments on growth rates of astrocytes and two different GBM cell lines treated according to the treatment schedules (ref. Fig. 1 Supplement). The relative growth rates for day 3 and day 6 of SVGA (A), A172 (B) and LN229 (C) were determined compared to the number of cells at day 0 of the treatment. Growth rates of cell treated with the gold standard TMZ are highlighted with dashed lines. Means are significantly different for *p<0.05, **p<0.01 respective ***p<0.001 (TIF 1339 KB)


  1. Adamski V, Hempelmann A, Flüh C, Lucius R, Synowitz M, Hattermann K, Held-Feindt J (2017) Dormant human glioblastoma cells acquire stem cell characteristics and are differentially affected by temozolomide and AT101 treatment strategies. Oncotarget 8:108064–108078CrossRefPubMedPubMedCentralGoogle Scholar
  2. Antonietti P, Linder B, Hehlgans S, Mildenberger IC, Burger MC, Fulda S, Steinbach JP, Gessler F, Rödel F, Mittelbronn M, Kögel D (2017) Interference with the HSF1/HSP70/BAG3 pathway primes glioma cells to matrix detachment and BH3 mimetic-induced apoptosis. Mol Cancer Ther 16:156–168CrossRefPubMedGoogle Scholar
  3. Arevalo Á, Erices J, Uribe D, Howden J, Niechi I, Muñoz S, Martín R, Monrás C (2017) Current therapeutic alternatives and new perspectives in glioblastoma multiforme. Curr Med Chem 24:2781–2795CrossRefPubMedGoogle Scholar
  4. Bae SH, Park M-J, Lee MM, Kim M, Lee S-H, Cho SY, Kim Y-H, Kim YJ, Park C-K, Kim C-Y (2014) Toxicity profile of temozolomide in the treatment of 300 malignant glioma patients in Korea. J Korean Med Sci 29:980–984CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bonovia R, Inda M, Cavenee W, Furnari F (2011) Heterogeneity maintenance in glioblastoma: a social network. Cancer Res 71:4055–4060CrossRefGoogle Scholar
  6. Bricenõ E, Calderon A, Sotelo J (2007) Institutional experience with chloroquine as an adjuvant to the therapy for glioblastoma multiforme. Surg Neurol 67:388–391CrossRefPubMedGoogle Scholar
  7. Bushunow P, Reidenberg MM, Wasenko J, Winfiled J, Lorenzo B, Lemke S, Himpler B, Corona R, Coyle T (1999) Gossypol treatment of recurrent adult malignant gliomas. J Neurooncol 43:79–86CrossRefPubMedGoogle Scholar
  8. Chou TC (2010) Drug combination studies and their synergy quantification using the Chou–Talalay method. Cancer Res 70:440–446CrossRefPubMedPubMedCentralGoogle Scholar
  9. Chou TC, Talalay P (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22:27–55CrossRefPubMedGoogle Scholar
  10. Golden EB, Cho H-Y, Hofman FM, Louie S, Schönthal AH, Chen TC (2015) Quinoline-based antimalarial drugs: a novel class of autophagy inhibitors. Neurosurg Focus 38:E12CrossRefPubMedGoogle Scholar
  11. Gunja N, Roberts D, McCoubrie D, Lamberth P, Jan A, Simes DC, Hackett P, Buckley NA (2009) Survival after massive hydroxychloroquine overdose. Anaesth Intensive Care 37:130–133PubMedGoogle Scholar
  12. Heist RS, Fain J, Chinnasami B, Khan W, Molina JR, Sequist LV, Temel JS, Fidias P, Brainerd V, Leopold L, Lynch TJ (2010) Phase I/II study of AT-101 with topotecan in relapsed and refractory small cell lung cancer. J Thorac Oncol 5:1637–1643CrossRefPubMedGoogle Scholar
  13. Henriksen S, Tylden GD, Dumoulin A, Sharma BN, Hirsch HH, Hanssen Rinaldo C (2014) The human fetal glial cell line SVG p12 contains infectious BK polyomavirus. J Virol 88:7556–7768CrossRefPubMedPubMedCentralGoogle Scholar
  14. Hsu SPC, Kuo JS, Chiang HC, Wang HE, Wang YS, Huang CC, Huang YC, Chi MS, Mehta MP, Chi KH (2018) Temozolomide, sirolimus and chloroquine is a new therapeutic combination that synergizes to disrupt lysosomal function and cholesterol homeostasis in GBM cells. Oncotarget 9:6883–6896PubMedPubMedCentralGoogle Scholar
  15. Jarzabek MA, Amberger-Murphy V, Callanan JJ, Gao C, Zagozdzon AM, Shiels L, Wang J, Ligon KL, Rich BE, Dicker P, Gallagher WM, Prehn JH, Byrne AT (2014) Interrogation of gossypol therapy in glioblastoma implementing cell line and patient-derived tumor models. Br J Cancer 11:2275–2286CrossRefGoogle Scholar
  16. Kanzawa T, Germano IM, Komata T, Ito H, Kondo Y, Kondo S (2004) Role of autophagy in temozolomide induced cytotoxicity for malignant glioma cells. Cell Death Differ 11:448–457CrossRefPubMedGoogle Scholar
  17. Katayama M, Kawaguchi T, Berger MS, Pieper RO (2007) DNA damaging agent-induced autophagy produces a cytoprotective adenosine triphosphate surge in malignant glioma cells. Cell Death Differ 14:548–558CrossRefPubMedGoogle Scholar
  18. Keshmiri-Neghab H, Goliaei B, Nikoofar A (2014) Gossypol enhances radiation-induced autophagy in glioblastoma multiforme. Gen Physiol Biophys 33:433–442CrossRefPubMedGoogle Scholar
  19. Krakstad C, Chekenya M (2010) Survival signaling and apoptosis resistance in glioblastomas: opportunities for targeted therapeutics. Mol Cancer 9:135CrossRefPubMedPubMedCentralGoogle Scholar
  20. Lee SY (2016) Temozolomide resistance in glioblastoma multiforme. Genes Dis 3:198e210Google Scholar
  21. Lee SW, Kim HK, Lee NH, Yi HY, Kim HS, Hong SH, Hong YK, Joe YA (2015) The synergistic effect of combination temozolomide and chloroquine treatment is dependent on autophagy formation and p53 status in glioma cells. Cancer Lett 360:195–204CrossRefPubMedGoogle Scholar
  22. Li S, Jiang T, Li G, Wang Z (2008) Impact of p53 status to response of temozolomide in low MGMT expression glioblastomas: preliminary results. Neurol Res 30:567–570CrossRefPubMedGoogle Scholar
  23. Lu Y, Li J, Dong C-E, Hunag J, Zhou H-B, Wang W (2017) Recent advances in gossypol derivatives and analogs: a chemistry and biology view. Future Med Chem 9:1243–1275CrossRefPubMedGoogle Scholar
  24. Ohgaki H, Kleihues P (2005) Epidemiology and etiology of gliomas. Acta Neuropathol 109:93–108CrossRefPubMedGoogle Scholar
  25. Opydo-Chanek M, Gonzola O, Marzo I (2017) Multifaceted anticancer activity of BH3 mimetics: current evidence and future prospects. Biochem Pharmacol 136:12–23CrossRefPubMedGoogle Scholar
  26. Ostermann S, Csajka C, Buclin T, Leyvraz S, Lejeune F, Decosterd LA, Stupp R (2004) Plasma and cerebrospinal fluid population pharmacokinetics of temozolomide in malignant glioma patients. Clin Cancer Res 10:3728–3736CrossRefPubMedGoogle Scholar
  27. Parazzoli G, Prados J, Ortiz R, Caba O, Cabeza L, Berdasco M, Gónzalez B, Melguizo C (2015) Temozolomide resistance in glioblastoma cell lines: implication of MGMT, MMR, P-glycoprotein and CD133 expression. PLoS One 10:e0140131CrossRefGoogle Scholar
  28. Rangwala R, Leone R, Chang YC, Fecher LA, Schuchter LM, Kramer A, Tan KS, Heitjan DF, Rodgers G, Gallagher M, Piao S, Troxel AB, Evans TL, DeMichele AM, Nathanson KL, O’Dwyer PJ, Kaiser J, Pontiggia L, Davis LE, Amaravadi RK (2014) Phase I trial of hydroxychloroquine with dose-intense temozolomide in patients with advanced solid tumors and melanoma. Autophagy 10:1369–1379CrossRefPubMedPubMedCentralGoogle Scholar
  29. Rosenfeld MR, Ye X, Supko JG, Desideri S, Grossman SA, Brem S, Mikkelson T, Wang D, Chang YC, Hu J, McAfee Q, Fisher J, Troxel AB, Piao S, Heitjan DF, Tan KS, Pontiggia L, O’Dwyer PJ, Davis LE, Amaravadi RK (2014) A phase I/II trial of hydroxychloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme. Autophagy 10:1359–1368CrossRefPubMedPubMedCentralGoogle Scholar
  30. Schelman WR, Mohammed TA, Traynor AM, Kolesar JM, Marnocha RM, Eickhoff J, Keppen M, Alberti DB, Wilding G, Takebe N, Liu G (2014) A phase I study of AT-101 with cisplatin and etoposide in patients with advanced solid tumors with an expanded cohort in extensive-stage small cell lung cancer. Investig New Drugs 32:295–302CrossRefGoogle Scholar
  31. Schweighardt B, Shieh JT, Atwood WJ (2001) CD4/CXCR4-independent infection of human astrocytes by a T-tropic strain of HIV-1. J Neurovirol 7:155–162CrossRefPubMedGoogle Scholar
  32. Shen S, Wu Y, Li K, Wang Y, Wu J, Zeng Y, Wu D (2018) Versatile hyaluronic acid modified AQ4N-Cu(II)-gossypol infinite coordination polymer nanoparticles: multiple tumor targeting, highly efficient synergistic chemotherapy, and real-time self-monitoring. Biomaterials 154:197–212CrossRefPubMedGoogle Scholar
  33. Sonpavde G, Matveev V, Burke JM, Caton JR, Fleming MT, Hutson TE, Galsky MD, Berry WR, Karlov P, Holmlund JT, Wood BA, Brookes M, Leopold L (2012) Randomized phase II trial of docetaxel plus prednisone in combination with placebo or AT-101, an oral small molecule Bcl-2 family antagonist, as first-line therapy for metastatic castration-resistant prostate cancer. Ann Oncol 23:1803–1808CrossRefPubMedGoogle Scholar
  34. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO, European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups, National Cancer Institute of Canada Clinical Trials Group (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:987–996CrossRefPubMedGoogle Scholar
  35. Voss V, Senft C, lang V, Ronellenfitsch MW, Steinbach JP, Seifert V, Kögel D (2010) The pan-Bcl-2 inhibitor (−)gossypol triggers autophagic cell death in malignant gliomas. Mol Cancer Res 8:1002–1016CrossRefPubMedGoogle Scholar
  36. Wang Y, Shi K, Zhang L, Hu G, Wan J, Tang J, Yin S, Duan J, Qin M, Wang N, Xie D, Gao X, Gao H, Zhang Z, He Q (2016) Significantly enhanced tumor cellular and lysosomal hydroxychloroquine delivery by smart liposomes for optimal autophagy inhibition and improved antitumor efficiency with liposomal doxorubicin. Autophagy 12:949–962CrossRefPubMedPubMedCentralGoogle Scholar
  37. Warnsmann V, Meyer N, Hamann A, Kögel D, Osiewacz (2018) A novel role of the mitochondrial permeability transition pore in (−)gossypol-induced mitochondrial dysfunction. Mech Ageing Dev 170:45–58CrossRefPubMedGoogle Scholar
  38. Yan Y, Xu Z, Dai S, Qian L, Sun L, Gong Z (2016) Targeting autophagy to sensitive glioma to temozolomide treatment. J Exp Clin Cancer Res 35:23CrossRefPubMedPubMedCentralGoogle Scholar
  39. Zerp SF, Stoter TR, Hoebers FJP, van den Brekel MWM, Dubbelman R, Kuipers GK, Lafleur MVM, Slotman BJ, Verheij M (2015) Targeting anti-apoptotic Bcl-2 by AT-101 to increase radiation efficacy: data from in vitro and clinical pharmacokinetic studies in head and neck cancer. Radiat Oncol 10:158CrossRefPubMedPubMedCentralGoogle Scholar
  40. Zhi K, Wang L, Zhang Y, Zhang X, Zhang L, Liu L, Yao J, Xiang W (2018) Preparation and evaluation of molecularly imprinted polymer for selective recognition and adsorption of gossypol. J Mol Recognit. PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of NeurosurgeryUniversity Medical Center Schleswig-Holstein UKSHKielGermany
  2. 2.Department of AnatomyUniversity of KielKielGermany
  3. 3.Institute for Materials ScienceUniversity of KielKielGermany

Personalised recommendations