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Temozolomide increases heat shock proteins in extracellular vesicles released from glioblastoma cells

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Abstract

Background

Glioblastoma (GBM) is the most malignant and the fastest-progressing type of primary brain tumours. Temozolomide (TMZ) is a chemotherapeutic drug for the treatment of GBM. Extracellular vesicles (EVs) have been recently confirmed to have a substantial role in the GBM, and their contents released from GBM cells have been considered a target for treatment. The purpose of this study is to evaluate the impact of TMZ on heat shock proteins (HSPs) derived from EVs originated from GBM cell lines (U87-MG and LN229) and the significance of EVs in response to chemotherapy in GBM.

Methods and results

NTA, ELISA, and immunoblotting were used to characterization studies of EVs and results showed that U87-MG cells released many EVs compared to LN229 cells. The effect of TMZ treatments on HSPs expression levels were assessed with immunoblotting and was found to be led to increases in HSF-1, Hsp90, Hsp70, Hsp60 and Hsp27 expression in GBM cells and their EV contents, which these increases are related to therapeutic resistance. What is more, in Real-time PCR studies showing which signalling pathways might be associated with these increases, it was observed that TMZ triggered the expression of RAD51 and MDM2 genes in cells and EV contents. More strikingly, we discover a correlation between EV and parental cells in regard of mRNA and protein level in both cell lines as a result of TMZ treatment.

Conclusions

Our data suggest of EVs in the treatment of GBM may have potential biomarkers that can be used to investigate the treatment response.

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Data availability

The related detailed data from this study is available from the corresponding author.

Code availability

Not applicable.

Abbreviations

ALIX:

Apoptosis-linked gene 2-interacting protein X

EV:

Extracellular vesicle

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

GBM:

Glioblastoma

HSF:

Heat shock factor

Hsp:

Heat shock protein

IC50 :

The half-maximal inhibitory concentration

MDM2:

Murine double-minute 2

NTA,:

Nanoparticle tracking analysis

RAD51,:

RAD51 recombinase

TMZ,:

Temozolomide

TSG101,:

Tumor susceptibility gene 101

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Acknowledgements

We cordially thank to PhD candidate Aslıhan Şengelen and PhD candidate Yunus Aksüt, MSc Ayşen Güngör for their assistance to during experimental works.

Funding

This study was funded by Istanbul University Scientific Research Projects (BAP) Coordination Unit, Turkey (Grant Number: 33460) and The Scientific and Technological Research Council of Turkey (TUBITAK, Grant Number: 216S973).

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Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Institute of Graduate Studies in Sciences, Istanbul University, Istanbul, Turkey

    Ezgi Kıyga

  2. Basic Medical Sciences Department of Molecular Biology and Genetics, School of Medicine, Koç University, Istanbul, Turkey

    Zelal Adıgüzel

  3. Department of Molecular Biology and Genetics, Faculty of Science, Istanbul University, Vezneciler, 34134, Istanbul, Turkey

    Evren Önay Uçar

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  1. Ezgi Kıyga
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Contributions

EK designed the study. EK, and ZA performed the study and EK analysed the results. EK wrote the manuscript. ZA and EOU drafted the manuscript. All authors read and approved the final manuscript.

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Correspondence to Ezgi Kıyga or Zelal Adıgüzel.

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Supplementary Information

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11033_2022_7714_MOESM1_ESM.tif

Supplementary file1 (TIF 800 kb). Supplementary material 1 Effects of TMZ on the viability of human glioma cancer cells U87-MG and LN229. TMZ treatments were associated with an increase in cell death in a dose- and time-dependent manner in (a) U87-MG and (b) LN229 cell lines

11033_2022_7714_MOESM2_ESM.xlsx

Supplementary file2 (XLSX 16 kb). Supplementary material 2 This is a list all genes analysed in this study and their fold change after the administration of 200 μM TMZ

11033_2022_7714_MOESM3_ESM.tif

Supplementary file3 (TIF 1858 kb). Supplementary material 3 Quality of EV-RNAs isolated were evaluated with Agilent Bioanalyzer

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Kıyga, E., Adıgüzel, Z. & Önay Uçar, E. Temozolomide increases heat shock proteins in extracellular vesicles released from glioblastoma cells. Mol Biol Rep 49, 8701–8713 (2022). https://doi.org/10.1007/s11033-022-07714-5

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