Sorafenib inhibits cell growth but fails to enhance radio- and chemosensitivity of glioblastoma cell lines

Abstract

Background: Glioblastomas (GBM) are the most common malignant type of primary brain tumor. GBM are intensively treated with surgery and combined radiochemotherapy using X-irradiation and temozolomide (TMZ) but they are still associated with an extremely poor prognosis, urging for the development of new treatment strategies. To improve the outcome of GBM patients, the small molecule multi-kinase inhibitor sorafenib has moved into focus of recent research. Sorafenib has already been shown to enhance the radio- and radiochemosensitivity of other tumor entities. Whether sorafenib is also able to sensitize GBM cells to radio- and chemotherapy is still an unsolved question which we have addressed in this study.

Methods: The effect of sorafenib on signaling, proliferation, radiosensitivity, chemosensitivity and radiochemosensitivity was analyzed in six glioblastoma cell lines using Western blot, proliferation- and colony formation assays.

Results: In half of the cell lines sorafenib clearly inhibited MAPK signaling. We also observed a strong blockage of proliferation, which was, however, not associated with MAPK pathway inhibition. Sorafenib had only minor effects on cell survival when administered alone. Most importantly, sorafenib treatment failed to enhance GBM cell killing by irradiation, TMZ or combined treatment, and instead rather caused resistance in some cell lines.

Conclusion: Our data suggest that sorafenib treatment may not improve the efficacy of radiochemotherapy in GBM.

Keywords: X-irradiation; glioblastoma; radiochemosensitivity; sorafenib; temozolomide.

Figure 1. Effect of sorafenib on proliferation & clonogenicity

A. To determine the effect of sorafenib on proliferation U87MG, DKMG and LN229 cells were incubated with different concentrations of sorafenib for 3 days as indicated. The number of cells measured in treated cultures was divided by the number of cells determined for untreated cultures, both counted after 3 days of incubation. The relative cell number is depicted. B. Proliferation of U87MG and DKMG cells in the presence of sorafenib (n=2). Twenty-four hours after seeding the cells were treated with 5 μg/ml sorafenib either for 24 h (media change, MC) or for up to 9 d. C. Relative cytotoxicity as determined by colony forming assay. Cells were treated with 5 μg/ml sorafenib for 24 h and cultivated for 10-25 days to allow for colony formation.

Figure 2. Effect of sorafenib on MAPK signaling

A. Phosphorylation of ERK1/2 (T202/Y204) and MEK1/2 (S217/S221) was determined by Western blot analysis using phosphospecific antibodies. Cells were treated with 5 μg/ml sorafenib for 2 h. The detection of total ERK, MEK and β-actin served as controls. MGMT was detected using MGMT-specific antibodies while lysates from Jurkat cells were used as a positive control. B. Quantification of ERK1/2 phosphorylation after sorafenib treatment. Corrected pERK1/2 levels (pERK/ERK) of sorafenib-treated samples were normalized to the corrected pERK1/2 levels of untreated samples. Depicted are the results of three independent Western blots.

Figure 3. Effect of sorafenib on cellular radiosensitivity

Cells were treated with 5 μg/ml sorefenib for 2 h before irradiation with 0, 2, 4 and 6 Gy. Cell survival was assessed by colony forming assay. A. Relative cell survival of U87MG cells. B. Relative surviving fraction of all six GBM cell lines after 6 Gy (SF6_IR).

Figure 4. Effect of sorafenib on chemosensitivity

Cells were treated with different concentrations of TMZ as indicated for 1-3 days according to their doubling time. The cells were also treated with 5 μg/ml sorafenib for the first 24 h. The medium was changed and cell survival was assessed by colony forming assay. A. Relative cell survival of U87MG cells. B. Relative survival fraction of all six GBM cell lines after 10 μM TMZ (SF10_TMZ).

Figure 5. Impact of sorafenib on combined treatment with irradiation and TMZ

Cells were treated with 10 μM TMZ with or without 5 μg/ml sorafenib 2 h before irradiation. Cellular survival was analyzed by colony formation. A, B. Relative cell survival of (A) LN229 and (B) U87MG cells. C. Relative surviving fraction at 6 Gy (SF6) with combined sorafenib and/or TMZ treatment as detected for all six cell lines.