[Show abstract][Hide abstract] ABSTRACT: Sphere formation assay is widely used in selection and enrichment of normal stem cells or cancer stem cells (CSCs), also known as tumor initiating cells (TICs), based on their ability to grow in serum-free suspension culture for clonal proliferation. However, there is no standardized parameter to accurately score the spheres, which should be reflected by both the number and size of the spheres. Here we define a novel parameter, designated as Standardized Sphere Score (SSS), which is expressed by the total volume of selected spheres divided by the number of cells initially plated. SSS was validated in quantification of both tumor spheres from cancer cell lines and embryonic bodies (EB) from mouse embryonic stem cells with high sensitivity and reproducibility.
[Show abstract][Hide abstract] ABSTRACT: Radiotherapy is a treatment choice for local control of breast cancer. However, intrinsic radioresistance of cancer cells limits therapeutic efficacy. We have recently validated that SCF (SKP1, Cullins, and F-box protein) E3 ubiquitin ligase is an attractive radiosensitizing target. Here we tested our hypothesis that MLN4924, a newly discovered investigational small molecule inhibitor of NAE (NEDD8 Activating Enzyme) that inactivates SCF E3 ligase, could act as a novel radiosensitizing agent in breast cancer cells. Indeed, we found that MLN4924 effectively inhibited cullin neddylation, and sensitized breast cancer cells to radiation with a sensitivity enhancement ratio (SER) of 1.75 for SK-BR-3 cells and 1.32 for MCF7 cells, respectively. Mechanistically, MLN4924 significantly enhanced radiation-induced G2/M arrest in SK-BR-3 cells, but not in MCF7 cells at early time point, and enhanced radiation-induced apoptosis in both lines at later time point. However, blockage of apoptosis by Z-VAD failed to abrogate MLN4924 radiosensitization, suggesting that apoptosis was not causally related. We further showed that MLN4924 failed to enhance radiation-induced DNA damage response, but did cause minor delay in DNA damage repair. Among a number of tested SCF E3 substrates known to regulate growth arrest, apoptosis and DNA damage response, p21 was the only one showing an enhanced accumulation in MLN4924-radiation combination group, as compared to the single treatment groups. Importantly, p21 knockdown via siRNA partialy inhibited MLN4924-induced G2/M arrest and radiosensitization, indicating a causal role played by p21. Our study suggested that MLN4924 could be further developed as a novel class of radiosensitizer for the treatment of breast cancer.
[Show abstract][Hide abstract] ABSTRACT: Radiotherapy is a treatment choice for local control of breast cancer, particularly after the removal of tumor tissues by surgery. However, intrinsic radioresistance of cancer cells limits therapeutic efficacy. Here, we determined in breast cancer cells the potential radiosensitizing activity of SM-164, a small molecule compound, that mimics the activity of SMAC, a mitochondrial protein released during apoptosis to activate caspases by inhibiting cellular inhibitor of apoptosis proteins, cIAP-1, and XIAP. We found that SM-164 at nanomolar concentrations promoted degradation of cIAP-1, disrupted the inhibitory binding of XIAP to active caspase-9, and sensitized breast cancer cells to radiation with a sensitization enhancement ratio (SER) of 1.7-1.8. In one line of breast cancer cells resistant to SM-164 as a single agent, SM-164 radiosensitization was mediated by intrinsic apoptosis pathway through activation of caspases-9 and -3. In a line of breast cancer cells sensitive to SM-164 as a single agent, SM-164 radiosensitization was mediated by both extrinsic and intrinsic apoptosis pathways through activation of caspases-9, -8, and -3. Consistently, blockage of caspase activation, through siRNA knockdown or treatment with a pan-caspase inhibitor z-VAD-fmk, inhibited apoptosis and abrogated SM-164 radiosensitization. Our study demonstrates that IAPs are valid radiosensitizing targets in breast cancer cells and SM-164 could be further developed as a novel class of radiosensitizers for the treatment of radioresistant breast cancer.
No preview · Article · Sep 2011 · Breast Cancer Research and Treatment