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Sulfasalazine intensifies temozolomide cytotoxicity in human glioblastoma cells

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Abstract

Temozolomide (TMZ) is an alkylating agent used to treat glioblastoma. This tumor type synthesizes the antioxidant glutathione through system X −c , which is inhibited by sulfasalazine (SAS). We exposed A172 and T98G human glioblastoma cells to a presumably clinically relevant concentration of TMZ (25 µM) and/or 0.5 mM SAS for 1, 3, or 5 days and assessed cell viability. For both cell lines, TMZ alone did not alter viability at any time point, while the coadministration of TMZ and SAS significantly reduced cell viability after 5 days. The drug combination exerted a synergistic effect on A172 cells after 3 and 5 days. Therefore, this particular lineage was subjected to complementary analyses on the genetic (transcriptome) and functional (glutathione and proliferating cell nuclear antigen (PCNA) protein) levels. Cellular pathways containing differentially expressed genes related to the cell cycle were modified by TMZ alone. On the other hand, SAS regulated pathways associated with glutathione metabolism and synthesis, irrespective of TMZ. Moreover, SAS, but not TMZ, depleted the total glutathione level. Compared with the vehicle-treated cells, the level of PCNA protein was lower in cells treated with TMZ alone or in combination with SAS. In conclusion, our data showed that the association of TMZ and SAS is cytotoxic to T98G and A172 cells, thus providing useful insights for improving TMZ clinical efficacy through testing this novel drug combination. Moreover, the present study not only reports original information on differential gene expression in glioblastoma cells exposed to TMZ and/or SAS but also describes an antiproliferative effect of TMZ, which has not yet been observed in A172 cells.

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Funding

This work was supported by Grants from FAPESP (2011/50400-0; 2013/02618-1; 2013/07559-3) and FAEPEX/UNICAMP (379/13; 554/14; 621/14). RSI was a recipient of a scholarship from CAPES.

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    Authors and Affiliations

    1. Department of Anatomical Pathology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Av. Tessália Vieira De Camargo, 126, Campinas, SP, 13083-887, Brazil

      Raffaela Silvestre Ignarro, Gustavo Facchini & Fabio Rogerio

    2. Department of Medical Genetics, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Av. Tessália Vieira De Camargo, 126, Campinas, SP, 13083-887, Brazil

      André Schwambach Vieira & Iscia Lopes-Cendes

    3. Department of Clinical Pathology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Av. Tessália Vieira De Camargo, 126, Campinas, SP, 13083-887, Brazil

      Daniela Rodrigues De Melo & Roger Frigério Castilho

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    1. Raffaela Silvestre Ignarro
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    Correspondence to Fabio Rogerio.

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    Raffaela Silvestre Ignarro and Gustavo Facchini contributed equally to this work.

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    11010_2016_2742_MOESM1_ESM.xls

    Online Resource 1 Gene expression data in A172 human glioblastoma cells after 3 days of treatment with temozolomide (TMZ) and/or sulfasalazine (SAS). Columns in Tables 1–3 show the gene symbol, log2 fold change relative to 0.1 % DMSO-treated cells, p value and adjusted p value. Tables 4–7 contain the enriched pathways for the up- and downregulated genes for each group. Fold change for each gene in the enriched pathways is presented in parenthesis after the name of the gene (XLS 6870 kb)

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    Ignarro, R.S., Facchini, G., Vieira, A.S. et al. Sulfasalazine intensifies temozolomide cytotoxicity in human glioblastoma cells. Mol Cell Biochem 418, 167–178 (2016). https://doi.org/10.1007/s11010-016-2742-x

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