Estradiol induces JNK-dependent apoptosis in glioblastoma cells

Abstract

Estrogens exert multiple regulatory actions on cellular events in a variety of tissues including the brain. In the present study, the signaling mechanisms of the concentration-dependent effects of 17-β-estradiol (estradiol) on glioblastoma cells were investigated. Cell viability was evaluated by the trypan blue exclusion assay. Cell growth and kinase activities were evaluated by immunocytochemistry and Western blotting. The results showed that high concentrations of estradiol inhibit growth and induce apoptosis in C6 rat glioma and T98G human glioblastoma cells. The blockade of the c-jun NH(2)-terminal kinase (JNK) signaling pathway prevented these effects of estradiol, indicating the critical role of the JNK/c-jun signaling cascade in glioblastoma cell growth inhibition and cell death in response to high concentrations of estradiol. Collectively, these findings highlight the potential of new discoveries in sensitizing estrogen-sensitive tumors to chemotherapeutic drugs, and may lead to the development of new JNK-based effective therapies.

Figure 1

Estradiol induced cytotoxicity in C6 and T98G cells. Cells were pretreated with JNK inhibitor, SP600125 (20 μM) or vehicle (control) under low growth-stimulated conditions for 30 min prior to treatment with increasing concentrations of estradiol for 24 h. Viable cells were detected by the trypan blue exclusion assay. Data points are an average of the results obtained from three separate experiments.

Figure 2

Estradiol inhibited cell proliferation in C6 and T98G cells. Cells were pretreated with SP600125 (20 μM) or vehicle (control) for 30 min, and incubated for 18 h in the presence or absence of estradiol (20 μM) under low growth-stimulated conditions. BrdU incorporation in C6 cells (upper panel) and PCNA expression analysis in T98G cells (lower panel) were carried out. Images were captured using a brightfield microscope at a magnification of ×600.

Figure 3

Estradiol induced JNK activation. (A) Immunocytochemical analysis showing c-jun phosphorylation (p-c-Jun) in C6 cells (upper panel) and in T98G cells (lower panel) pretreated with 20 μM SP600125 or vehicle (control) for 30 min, and incubated with 20 μM estradiol for 18 h under low growth-stimulated conditions. Phosphorylation of c-jun was visualized by anti-phosphorylated-c-jun antibody. Cells were visualized by using biotinylated secondary antibody, streptavidin/HRP and AEC chromogen. Images were captured using a brightfield microscope at a magnification of ×600. (B) Western blot analysis for the level of phosphorylated c-Jun (p-c-Jun) in C6 cells (right panel) and in T98G cells (left panel) pretreated with 20 μM SP600125 or vehicle (control) for 30 min, and incubated with 20 μM estradiol, for 18 h under low growth-stimulated conditions. Cell lysates were separated by 10% SDS-PAGE, and Western blotted with polyclonal antibody against the phosphorylated form of c-jun (upper panels). The corresponding native proteins of JNKs from the same lysates were blotted in separate membranes and shown in the lower panels. The blots were then visualized by using alkaline phosphatase conjugated secondary antibodies and BCIP/NBT as substrates.