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Dual treatment with shikonin and temozolomide reduces glioblastoma tumor growth, migration and glial-to-mesenchymal transition

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Abstract

Purpose

Glioblastomas (GBM) comprise 17% of all primary brain tumors. These tumors are extremely aggressive due to their infiltrative capacity and chemoresistance, with glial-to-mesenchymal transition (GMT) proteins playing a prominent role in tumor invasion. One compound that has recently been used to reduce the expression of these proteins is shikonin (SHK), a naphthoquinone with anti-tumor properties. Temozolomide (TMZ), the most commonly used chemotherapeutic agent in GBM treatment, has so far not been studied in combination with SHK. Here, we investigated the combined effects of these two drugs on the proliferation and motility of GBM-derived cells.

Methods

The cytotoxic and proliferative effects of SHK and TMZ on human GBM-derived cells were tested using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Ki67 staining and BrdU incorporation assays. The migration capacities of these cells were evaluated using a scratch wound assay. The expression levels of β3 integrin, metalloproteinases (MMPs) and GMT-associated proteins were determined by Western blotting and immunocytochemistry.

Results

We found that GBM-derived cells treated with a combination of SHK and TMZ showed decreases in their proliferation and migration capacities. These decreases were followed by the suppression of GMT through a reduction of β3 integrin, MMP-2, MMP-9, Slug and vimentin expression via inactivation of PI3K/AKT signaling.

Conclusion

From our results we conclude that dual treatment with SHK and TMZ may constitute a powerful new tool for GBM treatment by reducing therapy resistance and tumor recurrence.

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Acknowledgements

This study was supported by the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Rio de Janeiro (FAPERJ), Pró-Saúde Associação Beneficente de Assistência Social e Hospitalar and Ary Frauzino Foundation for Cancer Research. It was also supported by the Portuguese agencies FEDER funds through the Operational Programme Factors Competitiveness COMPETE, and National Funds through the FCT Fundação para a Ciência e Tecnologia.

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Authors and Affiliations

  1. Brain’s Biomedicine Laboratory, Paulo Niemeyer State Brain Institute, Secretaria de Estado de Saúde do Rio de Janeiro, Rua do Resende 156, Rio de Janeiro, 20231-092, Rio de Janeiro, Brazil

    Diana Matias, Joana Balça-Silva, Luiz Gustavo Dubois, Valéria Pereira Ferrer, Luciane Rosário & Vivaldo Moura-Neto

  2. Institute of Biomedical Sciences at Federal University of Rio de Janeiro (ICB/UFRJ), Rio de Janeiro, 21941-902, Brazil

    Diana Matias & Bruno Pontes

  3. Center for Neuroscience and Cell Biology and Institute for Biomedical Imaging and Life Sciences (CNC.IBILI), Rua Larga Faculdade de Medicina, Pólo I, 1° andar, 3004-504, Coimbra, Portugal

    Joana Balça-Silva, Anália do Carmo, Ana Bela Sarmento-Ribeiro & Maria Celeste Lopes

  4. Faculty of Medicine at University of Coimbra (FMUC), Pólo III - Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-354, Coimbra, Portugal

    Joana Balça-Silva & Ana Bela Sarmento-Ribeiro

  5. Hospital Center and University of Coimbra (CHUC), Praceta Prof. Mota Pinto, 3000-075, Coimbra, Portugal

    Anália do Carmo & Ana Bela Sarmento-Ribeiro

  6. Paulo de Góes Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, 21941-902, Brazil

    Juliana Echevarria-Lima

  7. Faculty of Pharmacy at University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548, Coimbra, Portugal

    Maria Celeste Lopes

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  1. Diana Matias
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Correspondence to Vivaldo Moura-Neto.

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Supplementary Fig. 1

mRNA expression levels for MGMT. U118-MG and GBM02 cells were cultured under standard conditions (at 37 °C, 5% CO2, 10% FBS). Total RNA was extracted using a PureLink RNA Mini Kit following the manufacturer’s instructions. Five micrograms of total RNA and a High-Capacity cDNA Reverse Transcription Kit were used to perform cDNA synthesis. Astrocytes were used as positive control. The qPCR reaction was performed in quadruplicate using the MGMT Taqman Probe (Hs04419844_s1) and GAPDH (array plate) as endogenous control. To calculate the relative fold variation in mRNA expression, the 2-ΔΔCT method was used. The results show that GBM02 cells exhibit ~zero MGMT expression and U118-MG cells ~15% MGMT expression relative to astrocytes. The MGMT expression differed statistically between the two GBM cell lines. Each value represents the ±SD, *p < 0.05. (GIF 14 kb)

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Matias, D., Balça-Silva, J., Dubois, L.G. et al. Dual treatment with shikonin and temozolomide reduces glioblastoma tumor growth, migration and glial-to-mesenchymal transition. Cell Oncol. 40, 247–261 (2017). https://doi.org/10.1007/s13402-017-0320-1

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