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

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 mu M BA on cytotoxicity, migration, the protein expression of PARP, survivin and HIF-1 alpha, 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 (IC(50)) of 23 mu M was observed in U251MG cells and 24 mu M was observed in U343MG cells. Under hypoxic conditions, 10 mu M or 15 mu M 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 mu M 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-1 alpha 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.