Effects of temozolomide (TMZ) on the expression and interaction of heat shock proteins (HPS) and DNA repair proteins in human malignant glioma cells

GISELA N. CASTRO; NIUBYS CAYADO-GUTIÉRREZ; MARTIN ZOPPINO; MARIEL A FANELLI; F.D. CUELLO-CARRIÓN; MAYRA SOTTILE; SILVINA NADIN; DANIEL CIOCCA

Congreso; First Conference of the South American Chapter of Cell Stress Society International.; 2014

Temozolomide (TMZ) is the main drug administrated to patients suffering of high grade gliomas. Gliomas are highly invasive, lethal brain tumors. Although many therapeutic approaches have been explored, there has been no major improvement in survival over the last years. In a previous study in human gliomas we reported that heat shock proteins (HSPs) HSPB1 and HSPA together with β-catenin, P53, and MGMT were associated with high-grade astrocitomas, suggesting that these markers are associated with the disease outcome and the response to treatments.6 In addition, in oligodendroglial tumors HSPB1 appeared as a surrogate molecular marker of loss of heterozygosity (LOH) of 1p.The MMR system and MGMT enzyme are responsible for repairing the DNA damages induced by TMZ, it has been determined that cells that have a deficient MMR system and high expression levels of MGMT may be resistant to treatment. Nadin et al. reported in peripheral blood lymphocytes from healthy subjects exposed in vitro to hyperthermia and cisplatin, that the MMR proteins MLH1 and MSH2 colocalize with HSPB1 and HSPA.Tumor recurrence and resistance to TMZ remain major challenges in malignant gliomas, so it is necessary to delve into the molecular mechanisms of resistance / susceptibility associated to treatment.. OBJETIVEStudied the biological effects of TMZ administration three glioma cell lines. Analyze expression levels related to treatment resistance and DNA repair proteins: HSPA, HSPB1, MGMT and MSH2.MATERIALS AND METHODSThree human malignant glioma cell lines were used: Gli36, U87 y DBTRG 0.5 MG (DBTRG), were subjected to TMZ administration at concentrations ranged between 0-500 μM for 24 hours. Protein expression was assessed by immunocytochemistry (ICQ) and western blot (WB). Cytotoxicity assays (MTT), survival, senescence (β-galactosidase activity), apoptosis (TUNEL) and inmnoprecipitación comet assay was performed. The cells were examined under confocal microscope (Olympus, FV 1000, Japan) by indirect immunofluorescence. Hsp25/27 (1:50), MSH2 (1:50) and secondary antibodies conjugated to Alexa Fluor 555 (Invitrogen, 1:500) and Alexa Fluor 488 (Invitrogen, 1: primary antibodies were used : 500).CONCLUSIONThe three cell lines showed differences in viability, apoptosis and senescence after treatment with TMZ. Moreover we show biological effects of TMZ and the interaction between HSPB1 and MSH2 in glioma cells as a first step to better understand the mechanisms of TMZ sensitivity and to design novel therapies in neurooncology.