Potentiation of radiation sensitivity in breast tumor cells by the vitamin D3 analogue, EB 1089, through promotion of autophagy and interference with proliferative recovery

Abstract

Abstract1,25-Dihydroxyvitamin D 3 and vitamin D 3 analogues, such as EB 1089, potentiate the response to ionizing radiation in breast tumor cells. The current studies address the basis for this interaction by evaluating DNA damage and repair, the effect of interference with reactive oxygen generation, the involvement of p53 and caspase-3, signaling through c-myc, as well as the induction of senescence and multiple modes of cell death. EB 1089 failed to increase the extent of radiation-induced DNA damage or to attenuate the rate of DNA repair. The reactive oxygen scavengers N-acetyl-L-cysteine and reduced glutathione failed to protect the cells from the promotion of cell death by EB 1089 and radiation. Whereas MCF-7 cells expressing caspase-3 showed significant apoptosis with radiation alone as well as with EB 1089 followed by radiation, EB 1089 maintained its ability to confer susceptibility to radiation-induced cell killing, in large part by interference with proliferative recovery. In contrast, in breast tumor cells lacking p53, where radiation promoted extensive apoptosis and the cells failed to recover after radiation treatment, EB 1089 failed to influence the effect of radiation. EB 1089 treatment interfered with radiationinduced suppression of c-myc; however, induction of c-myc did not prevent senescence by radiation alone or radiation-induced cell death promoted by EB 1089. EB 1089 did not increase the extent of micronucleation, indicative of mitotic catastrophe, induced by radiation alone. However, EB 1089 did promote extensive autophagic cell death in the irradiated cells. Taken together, these studies suggest that the effect of EB 1089 treatment on the radiation response is related in part to enhanced promotion of autophagic cell death and in part to interference with the proliferative recovery that occurs with radiation alone in p53 wild-type breast tumor cells.

Dual functions of autophagy in the response of breast tumor cells to radiation

“…2,17 In contrast, treatment with the vitamin D analogs, EB1089 or ILX-23-7553, prior to radiation was found to change the response to irradiation from growth arrest to cell death. [2][3][4][5] Our studies further excluded apoptosis and mitotic catastrophe as the modes of cell death when EB1089 was combined with radiation and were consistent with cell death occurring through autophagy. 2,3 Autophagy reflects a cellular response to stress in which the fusion of autophagosomes and lysosomes allows the degradation of subcellular organelles to generate energy and metabolic precursors; autophagy may be cytoprotective or cytotoxic, depending on the cells and the nature of the stressful challenge.…”

“…1 Although strategies have been developed in efforts to enhance the response to chemotherapy and radiation by interfering with cytoprotective signaling, there have been few direct attempts to address the problem of disease recurrence. One promising approach based on our previous work [2][3][4][5] may be to utilize vitamin D 3 or its analogs in combination with conventional therapies. 1,25D 3 , a steroid hormone with a central role in regulating calcium homeostasis, has been shown to produce pronounced effects on cell growth, differentiation and survival.…”

“…One promising approach based on our previous work [2][3][4][5] may be to utilize vitamin D 3 or its analogs in combination with conventional therapies. 1,25D 3 , a steroid hormone with a central role in regulating calcium homeostasis, has been shown to produce pronounced effects on cell growth, differentiation and survival. [6][7][8] Because of the possibility of hypercalcemia with the utilization of 1,25D 3 at high doses, 9 analogs of 1,25D 3 such as EB1089 have been developed that demonstrate similar growth regulatory effects, while having a reduced impact on calcium metabolism.…”

“…[6][7][8] Because of the possibility of hypercalcemia with the utilization of 1,25D 3 at high doses, 9 analogs of 1,25D 3 such as EB1089 have been developed that demonstrate similar growth regulatory effects, while having a reduced impact on calcium metabolism. 6,10 Additionally, vitamin D 3 (cholecalciferol), and the active form 1,25 dihydroxyvitamin D 3 (1,25D 3 ) have been used experimentally in combination with a number of established treatments in preclinical cancer studies. radiation (IR) was administered either as a single dose of 10 Gy or more clinically relevant fractionated doses of 5¾2 Gy, instead undergoing a form of senescence arrest.…”

Radiation-induced autophagy in normal and cancer cells: Towards novel cytoprotection and radio-sensitization policies?

“…DeMasters et al showed an autophagic cell death response in irradiated MCF-7 and MCF-7/ caspase-3 breast tumor cells, an effect that was intensified by the vitamin D3 analogue EB 1089. 80 In the same study it was observed that the percentage of cells undergoing mitotic catastrophe and apoptosis was a small fraction of the overall cell population, while over 50% of the cells had increased presence of acidic vesicular organells. Similarly, Paglin et al showed that 48 hours after radiation with 10 Gy, autophagic vacuoles were present in 76% of the MCF-7 cells.…”