Inhibition of the PI3K but not the MEK/ERK pathway sensitizes human glioma cells to alkylating drugs

Abstract

BackgroundIntrinsic chemoresistance of glioblastoma (GBM) is frequently owed to activation of the PI3K and MEK/ERK pathways. These signaling cascades are tightly interconnected however the quantitative contribution of both to intrinsic resistance is still not clear. Here, we aimed at determining the activation status of these pathways in human GBM biopsies and cells and investigating the quantitative impact of both pathways to chemoresistance.MethodsReceptor tyrosine kinase (RTK) pathways in temozolomide (TMZ) treatment naive or TMZ resistant human GBM biopsies and GBM cells were investigated by proteome profiling and immunoblotting of a subset of proteins. Resistance to drugs and RTK pathway inhibitors was assessed by MTT assays. Apoptotic rates were determined by Annexin V staining and DNA damage with comet assays and immunoblotting.ResultsWe analyzed activation of RTK pathways by proteome profiling of tumor samples of patients which were diagnosed a secondary GBM and underwent surgery and patients which underwent a second surgery after TMZ treatment due to recurrence of the tumor. We observed substantial activation of the PI3K and MEK/ERK pathways in both groups. However, AKT and CREB phosphorylation was reduced in biopsies of resistant tumors while ERK phosphorylation remained unchanged. Subsequent proteome profiling revealed that multiple RTKs and downstream targets are also activated in three GBM cell lines. We then systematically describe a mechanism of resistance of GBM cell lines and human primary GBM cells to the alkylating drugs TMZ and cisplatin. No specific inhibitor of the upstream RTKs sensitized cells to drug treatment. In contrast, we were able to restore sensitivity to TMZ and cisplatin by inhibiting PI3K in all cell lines and in human primary GBM cells. Interestingly, an opposite effect was observed when we inhibited the MEK/ERK signaling cascade with two different inhibitors.ConclusionsTemozolomide treatment naive and TMZ resistant GBM biopsies show a distinct activation pattern of the MEK/ERK and PI3K signaling cascades indicating a role of these pathways in resistance development. Both pathways are also activated in GBM cell lines, however, only the PI3K pathway seems to play a crucial role in resistance to alkylating agents and might serve as drug target for chemosensitization.Electronic supplementary materialThe online version of this article (10.1186/s12935-018-0565-4) contains supplementary material, which is available to authorized users.

Chemotherapy Sensitizing Effects of Methadone in Glioblastoma Cells Are Drug- and Cell Line-dependent

“…When we tested MET in combination with doxorubicin in a panel of GBM cell lines and primary cells we found a sensitizing effect only for the A172 cell line and to some extend in primary GBM cells. We have previously shown that P-gp is not expressed in U87, U251 and U373 cell lines [18] which might explain why these cells could not be sensitized to doxorubicin by MET, while inhibition of doxorubicin e ux has previously been reported for the A172 cell line [10]. Our ndings are in line with a study which also reported no sensitizing effect of MET on various doxorubicintreated canine tumor cells [12].…”

“…All other cells and cell lines were cultivated in Dulbecco's Modi ed Eagle Medium (DMEM) low glucose (Biowest, Nuaillé, France) containing 10% FCS (Biochrom, Berlin, Germany), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Biowest, Nuaillé, France). Primary GBM cells derived from a primary GBM tumor biopsy were obtained from the University Hospital Cologne, genetically characterized and cultured as previously described [18].…”

Thioredoxin Confers Intrinsic Resistance to Cytostatic Drugs in Human Glioma Cells

“…GBM cell lines and high-grade glioma tissue have been reported to express significant amounts of Trx [ 16 , 28 , 29 ]. In order to confirm this, we determined Trx and TXNIP expression in three established GBM cell lines—U87, U251, and U373—and primary GBM cells, which have previously been shown by us to be intrinsically resistant to TMZ and cisplatin [ 30 ]. Trx-1 and TXNIP protein was expressed in all cells as determined by Western blotting ( Figure 1 a).…”

“…Yet, TMZ is the only substance for which a statistically significant and clinically relevant benefit in both overall survival and progression-free survival has been demonstrated [ 4 ]. However, it is very difficult to handle in the laboratory setting as it must be given to GBM cells in concentrations as high as or even beyond its solubility [ 30 ]. Thus, we also used cisplatin as a model substance with a similar molecular mechanism of action: both substances interfere with DNA, resulting in strand breaks and apoptosis.…”

“…Overexpression of TXNIP and consequently reduced Trx-1 expression significantly sensitized cells towards cisplatin when compared to empty vector transfected cells ( Figure 3 b–e). It has previously been shown by us and others that pharmacological inhibition of phosphatidylinositide 3-kinase (PI3K) sensitizes GBM cells to cisplatin and TMZ [ 30 ]. Reports from the literature indicate that PI3K/AKT signaling and the Trx system can be interconnected on various levels [ 31 ].…”