Radiation-induced Akt activation modulates radioresistance in human glioblastoma cells

Abstract

Background: Ionizing radiation (IR) therapy is a primary treatment for glioblastoma multiforme (GBM), a common and devastating brain tumor in humans. IR has been shown to induce PI3K-Akt activation in many cell types, and activation of the PI3K-Akt signaling pathway has been correlated with radioresistance.

Methods: Initially, the effects of IR on Akt activation were assessed in multiple human GBM cell lines. Next, to evaluate a potential causative role of IR-induced Akt activation on radiosensitivity, Akt activation was inhibited during IR with several complementary genetic and pharmacological approaches, and radiosensitivity measured using clonogenic survival assays.

Results: Three of the eight cell lines tested demonstrated IR-induced Akt activation. Further studies revealed that IR-induced Akt activation was dependent upon the presence of a serum factor, and could be inhibited by the EGFR inhibitor AG1478. Inhibition of PI3K activation with LY294002, or with inducible wild-type PTEN, inhibition of EGFR, as well as direct inhibition of Akt with two Akt inhibitors during irradiation increased the radiosensitivity of U87MG cells.

Conclusion: These results suggest that Akt may be a central player in a feedback loop whereby activation of Akt induced by IR increases radioresistance of GBM cells. Targeting the Akt signaling pathway may have important therapeutic implications when used in combination with IR in the treatment of a subset of brain tumor patients.

Figure 1

The effect of IR on Akt phosphorylation differs in human GBM cell lines. A. U87MG, MO59J and LN18 cells were irradiated with 6 Gy and harvested after the indicated times. Cell lysates were prepared and subjected to Western blot analysis with the indicated antibody. B. H4, A174, DBTRG-05MG, LN229, and HS683 cells were irradiated with 6 Gy and harvested after 1 hr. Cell lysates were prepared and subjected to Western blot analysis with the indicated antibody.

Figure 2

IR induces Akt activation in U87MG cells through EGFR in a serum factor-dependent manner. A. U87MG cells were cultured in the absence or presence of 10% FBS for 18-20 hr, then irradiated at 6 Gy. Cell lysates were harvested 1 hr later and subjected to Western blot analysis with the indicated antibody. The ratio of P-Akt-Ser473 to total Akt pooled from three different experiments were shown in the lower panel. Results represent mean ± SEM, ***:p < 0.001 compared to 0 Gy; #: p < 0.05 compared to 10% FBS (one-way ANOVA) B. Cells were treated with 5 μM AG1478 for 1 hr, then were irradiated for 1 hr at the indicated dosage. Cell lysates were prepared and subjected to Western blot analysis with the indicated antibody. The fold induction of normalized P-Akt-Ser473 induced by 6 Gy pooled from two different experiments were shown in the lower panel. Results represent mean ± SEM, **:p < 0.01(Student's t-test).

Figure 3

Inhibition of PI3K-Akt signaling with PI3K inhibitor LY294002 or EGFR inhibitor AG1478 increases the radiosensitivity of U87MG cells. A. U87MG cells were treated with 20 μM LY294002 for 1 hr prior to IR, and then irradiated with 6 Gy. Total cell lysate was harvested 1 hr after IR and subjected to Western blot analysis with the indicated antibody. Cells without IR treatment were used as a control. B. Cells were treated with vehicle (control) or 20 μM LY294002 for 1 hr, then irradiated with indicated dosage. 4 hr after IR, cells were fed with drug-free medium, and incubated for another 20 hr at 37°C, after which they were trypsinized and seeded for clonogenic survival assays. Colony-forming efficiency was determined 14 d later. Results were pooled from three different experiments. C. Cells were treated with vehicle (control) or 5 μM AG1478 for 16 hr, then irradiated with indicated dosage. 4 hr after IR, cells were fed with drug-free medium, and incubated for another 20 hr at 37°C, after which they were trypsinized and seeded for clonogenic survival assay. Colony-forming efficiency was determined 14 d later.

Figure 4

Expression of PTEN increases the radiosensitivity of U87MG cells. A. Genetically modified U87MG cells were treated with vehicle or 1 μg/mL doxycycline for 24 hr before harvest. Cell lysates were subjected to Western blot analysis with indicated antibody. B. Genetically modified U87MG cells were treated with 1 μg/mL doxycycline for 24 hr, and then irradiated with the indicated does. Afterwards, cells were incubated for 4 hr at 37°C, and trypsinized and seeded for clonogenic assay. Colony-forming efficiency was determined 14 d later.

Figure 5

Akt inhibitors increase the radiosensitivity of U87MG cells. A. U87MG cells were treated with vehicle or 10 μM SH-5 for 16 hrs, and then irradiated with 6 Gy. Total cell lysate was harvested 1 hr after IR and subjected to Western blot analysis with the indicated antibody. Cells without IR treatment were used as a control. The relative ratio of P-Akt-Ser473 to total Akt pooled from two different experiments are shown in the right panel. Results represent mean ± SEM, ***:p < 0.001 compared to vehicle (one-way ANOVA) B. Cells were treated with vehicle (control) or 10 μM SH-5 for 16 hrs, then irradiated with indicated dosage. 4 hr after IR, cells were fed with drug-free medium, and incubated for another 20 hr at 37°C, after which they were trypsinized and seeded for clonogenic survival assay. Colony-forming efficiency was determined 14 d later. C. U87MG cells were treated with vehicle or 1 μM MK-2206 for 1 hr, and then irradiated with 6 Gy. Total cell lysate was harvested 1 hr after IR and subjected to Western blot analysis with the indicated antibody. Cells without IR treatment were used as a control. D. Cells were treated with vehicle (control) or 1 μM MK-2206 for 1 hr, then irradiated with indicated dosage. 4 hr after IR, cells were fed with drug-free medium, and incubated for another 20 hr at 37°C, after which they were trypsinized and seeded for clonogenic survival assay. Colony-forming efficiency was determined 14 d later.