Cell-type-specific sensitivity of bortezomib in the methotrexate-resistant primary central nervous system lymphoma cells

  • Azusa Hayano
  • Yasuo Takashima
  • Ryuya YamanakaEmail author
Original Article



Methotrexate (MTX) is used in first-line treatment of primary central nervous system lymphoma (PCNSL), but most cases result in relapse-acquired resistance to MTX. However, only few studies have reported on internal changes and chemotherapies in PCNSL.


In this study, we generated two MTX-resistant PCNSL cell lines, designated MTX-HKBML and MTX-TK, in addition to a MTX-resistant Burkitt lymphoma cell line, designated MTX-RAJI. We examined gene expression changes and drug sensitivity to a proteasome inhibitor, bortezomib, in these cells.


Cytotoxic tests revealed that the 50% inhibitory concentration for MTX in MTX-HKBML is markedly higher than that in the other two cell lines. Expression of the genes in MTX and folate metabolisms, including gamma-glutamyl hydrolase and dihydrofolate reductase, are upregulated in both MTX-HKBML and MTX-TK, whereas the gene expression of folylpolyglutamate synthetase, thymidylate synthase, and methylenetetrahydrofolate dehydrogenase 1 were upregulated and downregulated in MTX-HKBML and MTX-TK, respectively, on the other hand, bortezomib sensitivity was observed in MTX-TK, as compared with control TK, but not in MTX-HKBML.


These results indicate the cell-type-specific changes downstream of metabolic pathways for MTX and folate, bortezomib sensitivity, and purine and pyrimidine syntheses, in each PCNSL cell line. The MTX-resistant lymphoma cell lines established may be useful for in vitro relapse models for MTX and development of salvage chemotherapy and drug discovery.


Primary central nervous system lymphoma Methotrexate Bortezomib Folate metabolism Purine and pyrimidine syntheses 



The study was supported by the MEXT KAKENHI Grant numbers 16H05441, 16K10766, and 18K09001.

Author contributions

AH and RY designed the experiments. AH and YT performed the experiments. AH, YT, and RY analyzed data. AH, YT, and RY wrote the manuscript.

Compliance with ethical standards

Conflict of interest

The authors have declared that no conflict of financial and non-financial interest exists.

Supplementary material

10147_2019_1451_MOESM1_ESM.pdf (311 kb)
Supplementary file1 (PDF 312 kb)


  1. 1.
    Panages KS, Elkin EB, DeAngelis LM et al (2005) Trends in survival from primary central nervous system lymphoma, 1975–1999: a population-based analysis. Cancer 104:2466–2472CrossRefGoogle Scholar
  2. 2.
    Ricard D, Idbaih A, Ducray F et al (2012) Primary brain tumours in adults. Lancet 379:1984–1996CrossRefGoogle Scholar
  3. 3.
    Louis DN, Perry A, Reifenberger G et al (2016) The 2016 World Health Organization classification of tumors of the central nervous system: a summary. Acta Neuropathol 131:803–820CrossRefGoogle Scholar
  4. 4.
    Ferreri AJ, Reni M, Foppoli M et al (2009) High-dose cytarabine plus high-dose methotrexate versus high-dose methotrexate alone in patients with primary CNS lymphoma: a randomised phase 2 trial. Lancet 374:1512–1520CrossRefGoogle Scholar
  5. 5.
    Thiel E, Korfel A, Martus P et al (2010) High-dose methotrexate with or without whole brain radiotherapy for primary CNS lymphoma (G-PCNSL-SG-1): a phase 3, randomised, non-inferiority trial. Lancet Oncol 11:1136–1147CrossRefGoogle Scholar
  6. 6.
    Morris PG, Correa DD, Yahalom J et al (2013) Rituximab, methotrexate, procarbazine, and vincristine followed by consolidation reduced-dose whole-brain radiotherapy and cytarabine in newly diagnosed primary CNS lymphoma: final results and long-term outcome. J Clin Oncol 31:3971–3979CrossRefGoogle Scholar
  7. 7.
    Reni M, Ferreri AJ, Villa E (1999) Second-line treatment for primary central nervous system lymphoma. Br J Cancer 9:530–534CrossRefGoogle Scholar
  8. 8.
    Rushworth D, Mathews A, Alpert A et al (2015) Dihydrofolate reductase and thymidylate synthase transgenes resistant to methotrexate interact to permit novel transgene regulation. J Biol Chem 290:22970–22976CrossRefGoogle Scholar
  9. 9.
    Mattaini KR, Sullivan MR, Vander Heiden MG (2016) The importance of serine metabolism in cancer. J Cell Biol 214:249–257CrossRefGoogle Scholar
  10. 10.
    Gorlick R, Goker E, Trippett T et al (1996) Intrinsic and acquired resistance to methotrexate in acute leukemia. N Engl J Med 335:1041–1048CrossRefGoogle Scholar
  11. 11.
    Zhao R, Goldman ID (2003) Resistance to antifolates. Oncogene 22(47):7431–7457CrossRefGoogle Scholar
  12. 12.
    Walling J (2006) From methotrexate to pemetrexed and beyond. A review of the pharmacodynamic and clinical properties of antifolates. Investig New Drugs 24:37–77CrossRefGoogle Scholar
  13. 13.
    Jin G, Huang J, Hu Z et al (2010) Genetic variants in one-carbon metabolism-related genes contribute to NSCLC prognosis in a Chinese population. Cancer 116:5700–5709CrossRefGoogle Scholar
  14. 14.
    Davis RE, Brown KD, Siebenlist U et al (2001) Constitutive nuclear factor kappaB activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells. J Exp Med 194:1861–1874CrossRefGoogle Scholar
  15. 15.
    Strauss SJ, Higginbottom K, Jüliger S et al (2007) The proteasome inhibitor bortezomib acts independently of p53 and induces cell death via apoptosis and mitotic catastrophe in B-cell lymphoma cell lines. Cancer Res 67:2783–2790CrossRefGoogle Scholar
  16. 16.
    Odqvist L, Montes-Moreno S, Sánchez-Pacheco RE et al (2014) NFkappaB expression is a feature of both activated B-cell-like and germinal center B-cell-like subtypes of diffuse large B-cell lymphoma. Mod Pathol 27:1331–1337CrossRefGoogle Scholar
  17. 17.
    Richardson PG, Barlogie B, Berenson J et al (2003) A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med 348:2609–2617CrossRefGoogle Scholar
  18. 18.
    Fisher RI, Bernstein SH, Kahl BS et al (2006) Multicenter phase II study of bortezomib in patients with relapsed or refractory mantle cell lymphoma. J Clin Oncol 24:4867–4874CrossRefGoogle Scholar
  19. 19.
    Dunleavy K, Pittaluga S, Czuczman MS et al (2009) Differential efficacy of bortezomib plus chemotherapy within molecular subtypes of diffuse large B-cell lymphoma. Blood 113:6069–6076CrossRefGoogle Scholar
  20. 20.
    Ribrag V, Gisselbrecht C, Haioun C et al (2009) Efficacy and toxicity of 2 schedules of frontline rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone plus bortezomib in patients with B-cell lymphoma: a randomized phase 2 trial from the French Adult Lymphoma Study Group (GELA). Cancer 115:4540–4546CrossRefGoogle Scholar
  21. 21.
    Ruan J, Martin P, Furman RR et al (2011) Bortezomib plus CHOP-rituximab for previously untreated diffuse large B-cell lymphoma and mantle cell lymphoma. J Clin Oncol 29:690–697CrossRefGoogle Scholar
  22. 22.
    Offner F, Samoilova O, Osmanov E et al (2015) Frontline rituximab, cyclophosphamide, doxorubicin, and prednisone with bortezomib (VR-CAP) or vincristine (R-CHOP) for non-GCB DLBCL. Blood 126:1893–1901CrossRefGoogle Scholar
  23. 23.
    Leonard JP, Kolibaba KS, Reeves JA et al (2017) Randomized phase II study of R-CHOP with or without bortezomib in previously untreated patients with non-germinal center B-cell-like diffuse large B-cell lymphoma. J Clin Oncol 35:3538–3546CrossRefGoogle Scholar
  24. 24.
    Huang L, Jiang Y, Chen Y (2017) Predicting drug combination index and simulating the network-regulation dynamics by mathematical modeling of drug-targeted EGFR-ERK signaling pathway. Sci Rep 7:40752CrossRefGoogle Scholar
  25. 25.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408CrossRefGoogle Scholar
  26. 26.
    Connelly S, DeMartino JK, Boger DL et al (2013) Biological and structural evaluation of 10R- and 10S-methylthio-DDACTHF reveals a new role for sulfur in inhibition of glycinamide ribonucleotide transformylase. Biochemistry 52:5133–5144CrossRefGoogle Scholar
  27. 27.
    Bleyer WA (1977) Methotrexate: clinical pharmacology, current status and therapeutic guidelines. Cancer Treat Rev 4:87–101CrossRefGoogle Scholar
  28. 28.
    Jolivet J, Cowan H, Curt A et al (1983) The pharmacology and clinical use of methotrexate. N Engl J Med 309:1094–1104CrossRefGoogle Scholar
  29. 29.
    Allegra CJ, Chabner BA, Drake JC et al (1985) Enhanced inhibition of thymidylate synthase by methotrexate polyglutamates. J Biol Chem 260:9720–9726Google Scholar
  30. 30.
    Chabner BA, Allegra CJ, Curt GA et al (1985) Polyglutamation of methotrexate. Is methotrexate a prodrug? J Clin Investig 76:907–912CrossRefGoogle Scholar
  31. 31.
    Göker E, Kheradpour A, Waltham M et al (1995) Acute monocytic leukemia: a myeloid leukemia subset that may be sensitive to methotrexate. Leukemia 9:274–276Google Scholar
  32. 32.
    Matherly LH, Taub JW, Ravindranath Y et al (1995) Elevated dihydrofolate reductase and impaired methotrexate transport as elements in methotrexate resistance in childhood acute lymphoblastic leukemia. Blood 85:500–509Google Scholar
  33. 33.
    Mpakou V, Papadavid E, Kontsioti F et al (2017) Apoptosis induction and gene expression profile alterations of cutaneous T-cell lymphoma cells following their exposure to bortezomib and methotrexate. PLoS One 12:e0170186CrossRefGoogle Scholar
  34. 34.
    Romaguera JE, Fayad LE, McLaughlin P et al (2010) Phase I trial of bortezomib in combination with rituximab-HyperCVAD alternating with rituximab, methotrexate and cytarabine for untreated aggressive mantle cell lymphoma. Br J Haematol 151:47–53CrossRefGoogle Scholar
  35. 35.
    Romaguera JE, Wang M, Feng L et al (2018) Phase 2 trial of bortezomib in combination with rituximab plus hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone alternating with bortezomib, rituximab, methotrexate, and cytarabine for untreated mantle cell lymphoma. Cancer 124:2561–2569CrossRefGoogle Scholar
  36. 36.
    Hodge DL, Yang J, Buschman MD et al (2009) Interleukin-15 enhances proteasomal degradation of bid in normal lymphocytes: implications for large granular lymphocyte leukemias. Cancer Res 69:3986–3994CrossRefGoogle Scholar
  37. 37.
    Bagacean C, Tempescul A, Patiu M et al (2016) Atypical aleukemic presentation of large granular lymphocytic leukemia: a case report. OncoTargets Ther 10:31–34CrossRefGoogle Scholar
  38. 38.
    Johnson-Farley N, Veliz J, Bhagavathi S et al (2015) ABT-199, a BH3 mimetic that specifically targets Bcl-2, enhances the antitumor activity of chemotherapy, bortezomib and JQ1 in "double hit" lymphoma cells. Leuk Lymphoma 56:2146–2152CrossRefGoogle Scholar
  39. 39.
    Gozzetti A, Cerase A (2014) Novel agents in CNS myeloma treatment. Cent Nerv Syst Agents Med Chem 14:23–27CrossRefGoogle Scholar
  40. 40.
    Kaspers GJL, Niewerth D, Wilhelm BAJ et al (2018) An effective modestly intensive re-induction regimen with bortezomib in relapsed or refractory paediatric acute lymphoblastic leukaemia. Br J Haematol 181:523–527CrossRefGoogle Scholar
  41. 41.
    Wang W, Swenson S, Cho HY et al (2019) Efficient brain targeting and therapeutic intracranial activity of bortezomib through intranasal co-delivery with NEO100 in rodent glioblastoma models. J Neurosurg 15:1–9Google Scholar
  42. 42.
    Foran E, Kwon DY, Nofziger JH et al (2016) CNS uptake of bortezomib is enhanced by P-glycoprotein inhibition: implications for spinal muscular atrophy. Neurobiol Dis 88:118–124CrossRefGoogle Scholar

Copyright information

© Japan Society of Clinical Oncology 2019

Authors and Affiliations

  1. 1.Laboratory of Molecular Target Therapy for Cancer, Graduate School of Medical ScienceKyoto Prefectural University of MedicineKyotoJapan

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