Increased betulinic acid induced cytotoxicity and radiosensitivity in glioma cells under hypoxic conditions

Abstract

Background: Betulinic acid (BA) is a novel antineoplastic agent under evaluation for tumor therapy. Because of the selective cytotoxic effects of BA in tumor cells (including gliomas), the combination of this agent with conservative therapies (such as radiotherapy and chemotherapy) may be useful. Previously, the combination of BA with irradiation under hypoxic conditions had never been studied.

Methods: In this study, the effects of 3 to 30 μM BA on cytotoxicity, migration, the protein expression of PARP, survivin and HIF-1α, as well as radiosensitivity under normoxic and hypoxic conditions were analyzed in the human malignant glioma cell lines U251MG and U343MG. Cytotoxicity and radiosensitivity were analyzed with clonogenic survival assays, migration was analyzed with Boyden chamber assays (or scratch assays) and protein expression was examined with Western blot analyses.

Results: Under normoxic conditions, a half maximal inhibitory concentration (IC50) of 23 μM was observed in U251MG cells and 24 μM was observed in U343MG cells. Under hypoxic conditions, 10 μM or 15 μM of BA showed a significantly increased cytotoxicity in U251MG cells (p = 0.004 and p = 0.01, respectively) and U343MG cells (p < 0.05 and p = 0.01, respectively). The combination of BA with radiotherapy resulted in an additive effect in the U343MG cell line under normoxic and hypoxic conditions. Weak radiation enhancement was observed in U251MG cell line after treatment with BA under normoxic conditions. Furthermore, under hypoxic conditions, the incubation with BA resulted in increased radiation enhancement. The enhancement factor, at an irradiation dose of 15 Gy after treatment with 10 or 15 μM BA, was 2.20 (p = 0.02) and 4.50 (p = 0.03), respectively. Incubation with BA led to decreased cell migration, cleavage of PARP and decreased expression levels of survivin in both cell lines. Additionally, BA treatment resulted in a reduction of HIF-1α protein under hypoxic conditions.

Conclusion: Our results suggest that BA is capable of improving the effects of tumor therapy in human malignant glioma cells, particularly under hypoxic conditions. Further investigations are necessary to characterize its potential as a radiosensitizer.

Figure 1

Cytotoxicity in U251MG and U343MG cell lines after treatment with BA. Both glioma cell lines were treated with increasing doses of BA from 3 - 30 μM. The half maximal inhibitory concentration (IC₅₀) with an incubation time of 24 h was 24 μM in U343MG cells and 23 μM in U251MG cells. Data represent mean values (± SD) of three independent experiments.

Figure 2

Effects of BA on clonogenic survival of glioma cells under normoxic or hypoxic conditions. Clonogenic survival in U251MG (A) and U343MG (B) cells after treatment with 10 or 15 μM BA under normoxic or hypoxic conditions. Under hypoxia, when compared to normoxic conditions, BA showed increased cytotoxicity in both glioma cell lines. Data represent mean values (± SD) of three independent experiments (* p < 0.05).

Figure 3

Effects of BA on cell migration of glioma cells. Migration rates of U251MG and U343MG cells treated with BA analyzed by Boyden chamber assays (A) and scratch assays (B) under normoxic conditions. Compared to DMSO-treated control cells, incubation with 5, 10 and 20 μM BA led to a decrease in cell migration rates in both glioma cell lines. Similarly, cells had a reduced migration rate after BA treatment as measured by the scratch assay. Data represent the average values (± SD) of three independent experiments.

Figure 4

Effects of BA and irradiation on protein expression levels of glioma cells. BA treatment affects the cleavage of PARP, the expression of survivin and hypoxia-induced HIF-1α protein levels in U251MG (left) and U343MG (right) cells. Cell lines were untreated (UT), treated with DMSO or with increasing doses of BA from 10, 20 or 25 μM under normoxic conditions (A, B) and untreated (UT), treated with DMSO or with doses of 10 or 15 μM BA plus irradiation at 15 Gy under hypoxic conditions (C, D). Actin served as an internal loading control. The Western blot shows one representative result out of three independent experiments.

Figure 5

Effects of BA on radiosensitivity of glioma cells. U251MG (left) and U343MG (right) cells were either treated with 10, 20 or 25 μM BA and irradiated with a dose of 2 and 6 Gy under normoxic conditions (A, B), or treated with 5, 10 or 15 μM BA and irradiated with a dose of 6 and 15 Gy under hypoxic conditions (C, D) and compared to DMSO-treated control cells. Data represent the mean values (± SD) of three independent experiments.