Abstract
Background: Medulloblastoma is one of the most common pediatric brain cancers. Radiotherapy is part of the standard of treatment for medulloblastoma however response rate still lags compared to other pediatric malignancies due to radiation resistance, or radioresistance, and can have long-term side effects. Radiation can activate the transcription factor NF-κB, triggering a cascade of signaling pathways resulting in the production of cytokines and other pro-inflammatory molecules. This inflammatory response can damage healthy tissues and contribute to side effects associated with radiation therapy. Therefore, inhibition of this inflammatory protein may mitigate these side-effects. In this study, we investigated the effects of supplementing radiotherapy with TAT-NBD, a small peptide which inhibits NF-κB activation, on tumor response.
Objectives: To determine if the addition of TAT-NBD peptide to radiation treatment affects treatment outcomes for wildtype and radioresistant pediatric cerebellar medulloblastoma cells (Daoy).
Methods: A clonogenic assay was performed to evaluate cell survival after treatment. Daoy cells (wildtype and radioresistant) were plated and grouped into six experimental groups: (1) wildtype cells with radiation but no TAT-NBD, (2) wildtype cells with radiation and 10 uM TAT-NBD, (3) wildtype cells with radiation and 100 uM TAT-NBD, (4) radioresistant cells with radiation but no TAT-NBD, (5) radioresistant cells with radiation and 10 um TAT-NBD, and (6) radioresistant cells with radiation and 100 uM TAT-NBD. After a day of growth, cells were placed in fresh DMEM containing the appropriate amount of TAT-NBD concentration and irradiated (160kvP, 25mA) at 0, 2, 4, 6, or 8 Gy. Following radiation, cells remained in the media for 24 hours before it was removed and replaced with fresh DMEM media. The cells were then allowed to grow for a week before being analyzed using ImageJ. Colony formation assay was used to examine the clonogenic survival of cells. The number of colonies were counted and analyzed statistically using t-tests.
Results: Our results showed that TAT-NBD significantly enhanced the radiation sensitivity of both wildtype and radioresistant Daoy cells, as evidenced by decreased cell survival and reduced colony-forming ability. In wildtype Daoy cells, the D10 value, radiation dose required to reduce cell population to 10%, was reduced from 5.03 Gy±0.12 with no TAT-NBD to 4.85 Gy±0.04 with 10 uM TAT-NBD and further reduced to 4.28 Gy±0.08 with 100 uM TAT-NBD. In radioresistant Daoy cells, the D10 value was reduced from 6.15 Gy±0.27 with no TAT-NBD to 4.77 Gy±0.08 with 10 uM TAT-NBD and further reduced to 4.59 Gy±0.56 with 100 uM TAT-NBD. P-value comparison of no drug and 10 uM TAT-NBD addition to radioresistant cells and comparison of no drug to 100uM TAT-NBD addition to radioresistant strains was less than 0.0001 which indicates that TAT-NBD significantly lowered D10.
Conclusion: Overall, our findings suggest that supplementing radiation treatment with TAT-NBD could be a promising strategy to improve the therapeutic outcome of medulloblastoma patients by increasing Daoy cell sensitivity to radiation therapy and overcoming radioresistance. Further studies are warranted to validate the efficacy and safety of this combination therapy in preclinical and clinical settings.
