Propentofylline inhibits glioblastoma cell invasion and survival by targeting the TROY signaling pathway

Abstract

Glioblastoma (GBM) is the most common primary tumor of the CNS and carries a dismal prognosis. The aggressive invasion of GBM cells into the surrounding normal brain makes complete resection impossible, significantly increases resistance to the standard therapy regimen, and virtually assures tumor recurrence. Median survival for newly diagnosed GBM is 14.6 months and declines to 8 months for patients with recurrent GBM. New therapeutic strategies that target the molecular drivers of invasion are required for improved clinical outcome. We have demonstrated that TROY (TNFRSF19), a member of the TNFR super-family, plays an important role in GBM invasion and resistance. Knockdown of TROY expression inhibits GBM cell invasion, increases sensitivity to temozolomide, and prolongs survival in an intracranial xenograft model. Propentofylline (PPF), an atypical synthetic methylxanthine compound, has been extensively studied in Phase II and Phase III clinical trials for Alzheimer's disease and vascular dementia where it has demonstrated blood-brain permeability and minimal adverse side effects. Here we showed that PPF decreased GBM cell expression of TROY, inhibited glioma cell invasion, and sensitized GBM cells to TMZ. Mechanistically, PPF decreased glioma cell invasion by modulating TROY expression and downstream signaling, including AKT, NF-κB, and Rac1 activation. Thus, PPF may provide a pharmacologic approach to target TROY, inhibit cell invasion, and reduce therapeutic resistance in GBM.

Keywords: Glioblastoma; Invasion; Resistance; Survival; TROY.

Figure 1

PPF treatment decreases TROY expression in GBM cells. (A) Western blot analysis of TROY expression in T98G cells. Cells were treated with PPF at indicated concentrations for 6 hrs, lysed, and cell lysates immunoblotted with anti-TROY antibody. Immunoblotting of α–Tubulin in cell lysates is included as a loading control. (B) Western blot analysis of TROY expression in T98G, GBM43, and GBM10 cells. Cells were treated with PPF at indicated concentrations for 24 hrs, lysed, and cell lysates immunoblotted with anti-TROY and α–Tubulin antibodies. (C) Western Blot analysis of TROY expression in GBM10 and GBM43 primary glioma cells. Cells were treated with 5 μM PPF, lysed at the indicated time-points, and the lysates immunoblotted with an anti-TROY antibody. Immunoblotting of β-Actin in cell lysates is included as a loading control. (D) Western Blot analysis of T98G, GBM10 and GBM43 glioma cells treated with 5 μM PPF for 24 hours. Lysates were immunoblotted with anti-TROY, anti-TNFR1, anti-EGFR, and β-Actin antibodies.

Figure 2

PPF does not affect the proliferation in GBM cells. (A) T98G, GBM43, and GBM10 cells were incubated with increasing concentrations of PPF (0, 5, 50, and 500 μM). After 0, 48, 96 and 144 hours of treatment, cell were trypsinized and counted using an automated cell counter. (B) GBM43, GBM10, and T98G glioma cells were treated with increasing doses of PPF (0.5, 1, 5, 10, and 20 μM) in triplicate. The Cell Titer Glo (Promega) reagent was used to measure survival. Raw values were normalized on a plate-by-plate basis such that 100% cell viability was equivalent to the mean of vehicle wells and 0% cell viability was equivalent to the mean of the MG132 positive control. The normalized data was used to assess viability of glioma cells after PPF treatment.

Figure 3

PPF sensitizes GBM cells to TMZ and radiotherapy (A) A clonogenic assay was used to assess T98G and GBM43 cells survival after TMZ and radiation treatment. Cells were pre-treated with 5μM PPF for 24 hours, and then either treated with 250 μM TMZ for 24 hours or exposed to 2 Gy radiation. Graph depicts the surviving fraction in the treated cells compared to vehicle (VC) treated or non-treated (NT) cells, **p<.01. (B) T98G glioma cells were treated with vehicle, PPF (5 μM), TMZ (250 μM), and PPF in combination with TMZ. TMZ-induced apoptosis was assayed by immunoblot analysis of cell lysates with an antibody to cleaved PARP. Immunoblotting for α-Tubulin was included as a loading control. (C) T98G, GBM10, and GBM43 cells were treated with PPF (5 μM), lysed, and then immunoblotted to assess the activation of AKT and NF-κB. Immunoblotting for β-Actin is included as a loading control.

Figure 4

PPF suppresses GBM cell invasion and Rac1 activation. (A) T98G, GBM10, and GBM43 glioma cells were treated with 5 μM PPF and invasion was assayed over 24 hours utilizing a Matrigel invasion assay, *p<.05. (B) T98G glioma cells were serum starved, pre-incubated with 5 μM PPF or vehicle for 1 hour, and then stimulated with 10% FBS for 2–10 mins. Cell lysates were harvested and equal concentrations of protein were assessed for Rac1 activation.

Figure 5

PPF suppresses GBM cell membrane ruffling. (A) GBM43 cells were preincubated with 5 μM PPF or vehicle for 1 hour prior to 10% FBS stimulation for 5 min. After FBS stimulation, cells were fixed, permeabilized, and stained for F-actin. For each experimental condition, at least 12 images were taken randomly. Arrowhead indicates membrane ruffles. (B) Graph depicts the average lamellipodia in T98G, GBM10, and GBM43 cells in the presence or absence of 10% FBS with or without 5 μM PPF as indicated. Lamellipodia were traced using Image J software. For each cell, the fraction of the cell perimeter that displayed lamellipodia was calculated, *p<.05.