Inhibition of Notch signaling pathway enhanced the radiosensitivity of breast cancer cells.

This study aimed to investigate the effect of inhibiting the Notch signaling pathway on the radiosensitivity of breast cancer cells. Human breast cancer cell lines (MCF-7 and T47D) were selected and treated with radiation of different doses. Cells were treated with Gamma secretase inhibitor (GSI) to analyze the effects of GSI on the Notch signaling, which were detected by Immunofluorescence assay, RT-qPCR, and Western blot analysis. Besides, Transwell assay, Scratch test, colony formation assay, MTT assay, and flow cytometry were conducted to show the effects of GSI on the invasion and migration, survival fraction, cell viability, and apoptosis of MCF-7 and T47D cells after radiation therapy. Moreover, cell transfection with a dominant negative mutant of RBPJ, the key transcription factor of Notch signaling pathway, were also applied to show the inhibition of Notch signaling pathway. Initially, we found that the 4 Gy radiation activated Notch signaling pathway, and enhanced the invasion and migration of MCF-7 and T47D cells. However, GSI inhibited the Notch signaling pathway, and reversed the enhancement of radiation on the migration and invasion, promoted the enhancement of apoptosis and inhibition of proliferation of MCF-7 and T47D cells induced by radiation. Except that, we also determined that GSI and dnRBPJ suppressed the upregulation of Notch signaling after radiation therapy. Our study demonstrated that inhibition of the Notch signaling pathway enhanced the radiosensitivity of breast cancer cells, which may provide evident for a beneficial adjuvant therapy in the breast cancer treatment.

Contrasting roles for C/EBPα and Notch in irradiation-induced multipotent hematopoietic progenitor cell defects.

Ionizing radiation (IR) is associated with reduced hematopoietic function and increased risk of hematopoietic malignancies, although the mechanisms behind these relationships remain poorly understood. Both effects of IR have been commonly attributed to the direct induction of DNA mutations, but evidence supporting these hypotheses is largely lacking. Here we demonstrate that IR causes long-term, somatically heritable, cell-intrinsic reductions in hematopoietic stem cell (HSC) and multipotent hematopoietic progenitor cell (mHPC) self-renewal that are mediated by C/EBPα and reversed by Notch. mHPC from previously irradiated (>9 weeks prior), homeostatically restored mice exhibit gene expression profiles consistent with their precocious differentiation phenotype, including decreased expression of HSC-specific genes and increased expression of myeloid program genes (including C/EBPα). These gene expression changes are reversed by ligand-mediated activation of Notch. Loss of C/EBPα expression is selected for within previously irradiated HSC and mHPC pools and is associated with reversal of IR-dependent precocious differentiation and restoration of self-renewal. Remarkably, restoration of mHPC self-renewal by ligand-mediated activation of Notch prevents selection for C/EBPα loss of function in previously irradiated mHPC pools. We propose that environmental insults prompt HSC to initiate a program limiting their self-renewal, leading to loss of the damaged HSC from the pool while allowing this HSC to temporarily contribute to differentiated cell pools. This "programmed mediocrity" is advantageous for the sporadic genotoxic insults animals have evolved to deal with but becomes tumor promoting when the entire HSC compartment is damaged, such as during total body irradiation, by increasing selective pressure for adaptive oncogenic mutations.

Radiosurgery inhibition of the Notch signaling pathway in a rat model of arteriovenous malformations.

OBJECT: Notch signaling has been suggested to promote the development and maintenance of arteriovenous malformations (AVMs), but whether radiosurgery inhibits Notch signaling pathways in AVMs is unknown. The aim of this study was to examine molecular changes of Notch signaling pathways following radiosurgery and to explore mechanisms of radiosurgical obliteration of "nidus" vessels in a rat model of AVMs. METHODS: One hundred eleven rats received common carotid artery-to-external jugular vein anastomosis to form an arteriovenous fistula (AVF) model. Six weeks postoperatively, dilated small vessels and capillaries formed a nidus. The rats with AVFs received 25-Gy radiosurgery. The expression of Notch1 and Notch4 receptors and their ligands, Delta-like1 and Delta-like4, Jagged1, Notch downstream gene target HES1, and an apoptotic marker caspase-3 in nidus vessels in the AVF rats was examined immunohistochemically and was quantified using LAS-AF software at 7 time points over a period of 42 days postradiosurgery. The interaction events between Notch1 receptor and Jagged1, as well as Notch4 receptor and Jagged1, were quantified in nidus vessels in the AVF rats using proximity ligation assay at different time points over 42 days postradiosurgery. RESULTS: The expression of Notch1 and Notch4 receptors, Delta-like1, Delta-like4, Jagged1, and HES1 was observed in nidus vessels in the AVF rats pre- and postradiosurgery. Radiosurgery enhanced apoptotic activity (p < 0.05) and inhibited the expression of Notch1 and Notch4 receptors and Jagged1 in the endothelial cells of nidus vessels in the AVF rats at 1, 2, 3, 7, 21, 28, and 42 days postradiosurgery (p < 0.05). Radiosurgery suppressed the interaction events between Notch1 receptor and Jagged1 (p < 0.001) as well as Notch4 receptor and Jagged1 (p < 0.001) in the endothelial cells of nidus vessels in the AVF rats over a period of 42 days postradiosurgery. Radiosurgery induced thrombotic occlusion of nidus vessels in the AVF rats. There was a positive correlation between the percentage of fully obliterated nidus vessels and time after radiosurgery (r = 0.9324, p < 0.001). CONCLUSIONS: Radiosurgery inhibits endothelial Notch1 and Notch4 signaling pathways in nidus vessels while inducing thrombotic occlusion of nidus vessels in a rat model of AVMs. The underlying mechanisms of radiosurgery-induced AVM shrinkage could be a combination of suppressing Notch receptor signaling in blood vessel endothelial cells, leading to a reduction in nidus vessel size and thrombotic occlusion of nidus vessels.

Effect of radiation on the Notch signaling pathway in osteoblasts.

Notch signaling has been shown to be important in osteoblast differentiation. Therapeutic radiation has been shown to alter the skeletal system, yet little information is available on the changes in Notch signaling in irradiated osteoblasts. The purpose of this study was to analyze the effect of radiation therapy with 2 and 4 Gy on Notch signaling in osteoblasts. In order to assess the radiation damage on osteoblast differentiation, total RNA and protein were collected three days after exposure to radiation. The effects of radiation on Notch signaling at the early and terminal stages of osteoblastic MC3T3-E1 cell differentiation was analyzed by qRT-PCR and western blot analysis. Our study applied a previously established method to induce MC3T3-E1 cell differentiation into osteoblasts and osteoblast precursors. Our results showed that the expression of Notch receptors (Notch1-4), ligands (Jagged1, Jagged2 and Delta1), target of Notch signaling (Hes1) and markers (ALP, M-CSF, RANKL and OPG) were altered following 2 and 4 Gy of irradiation. The present research did not indicate a strong relationship between Notch1 regulation and suppression of osteoblast differentiation. We found Hes1 may play a role in the radiation effect on osteoblast differentiation. Our results indicate that radiated osteoblast precursors and osteoblasts promoted osteoclast differentiation and proliferation.

Radiation-induced Notch signaling in breast cancer stem cells.

PURPOSE: To explore patterns of Notch receptor and ligand expression in response to radiation that could be crucial in defining optimal dosing schemes for γ-secretase inhibitors if combined with radiation. METHODS AND MATERIALS: Using MCF-7 and T47D breast cancer cell lines, we used real-time reverse transcription-polymerase chain reaction to study the Notch pathway in response to radiation. RESULTS: We show that Notch receptor and ligand expression during the first 48 hours after irradiation followed a complex radiation dose-dependent pattern and was most pronounced in mammospheres, enriched for breast cancer stem cells. Additionally, radiation activated the Notch pathway. Treatment with a γ-secretase inhibitor prevented radiation-induced Notch family gene expression and led to a significant reduction in the size of the breast cancer stem cell pool. CONCLUSIONS: Our results indicate that, if combined with radiation, γ-secretase inhibitors may prevent up-regulation of Notch receptor and ligand family members and thus reduce the number of surviving breast cancer stem cells.