Exposure to Ionizing Radiation Causes Long-Term Increase in Serum Estradiol and Activation of PI3K-Akt Signaling Pathway in Mouse Mammary Gland

Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC 20057-1468, USA.
International journal of radiation oncology, biology, physics (Impact Factor: 4.26). 02/2012; 84(2):500-7. DOI: 10.1016/j.ijrobp.2011.12.033
Source: PubMed


Exposure to ionizing radiation is an established risk factor for breast cancer. Radiation exposure during infancy, childhood, and adolescence confers the highest risk. Although radiation is a proven mammary carcinogen, it remains unclear where it acts in the complex multistage process of breast cancer development. In this study, we investigated the long-term pathophysiologic effects of ionizing radiation at a dose (2 Gy) relevant to fractionated radiotherapy.
Adolescent (6-8 weeks old; n = 10) female C57BL/6J mice were exposed to 2 Gy total body γ-radiation, the mammary glands were surgically removed, and serum and urine samples were collected 2 and 12 months after exposure. Molecular pathways involving estrogen receptor-α (ERα) and phosphatidylinositol-3-OH kinase (PI3K)-Akt signaling were investigated by immunohistochemistry and Western blot.
Serum estrogen and urinary levels of the oncogenic estrogen metabolite (16αOHE1) were significantly increased in irradiated animals. Immunostaining for the cellular proliferative marker Ki-67 and cyclin-D1 showed increased nuclear accumulation in sections of mammary glands from irradiated vs. control mice. Marked increase in p85α, a regulatory sub-unit of the PI3K was associated with increase in Akt, phospho-Akt, phospho-BAD, phospho-mTOR, and c-Myc in irradiated samples. Persistent increase in nuclear ERα in mammary tissues 2 and 12 months after radiation exposure was also observed.
Taken together, our data not only support epidemiologic observations associating radiation and breast cancer but also, specify molecular events that could be involved in radiation-induced breast cancer.

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    • "Exposure to ionizing radiation (IR) at non-lethal doses initiates a cascade of complex cellular response, which others and we have reported to be a continuous process [11],[58]–[61]. Additionally, the chronic cellular response after radiation exposure has been reported to be dependent on radiation quality [11],[58]–[60]. Heavy ion radiation in radiobiological terms is high-LET and hence is more damaging than low-LET γ-ray and x-ray radiation and is predicted to be a major risk factor to astronauts’ health during prolonged space missions [62]–[64]. "
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    ABSTRACT: Tissue consequences of radiation exposure are dependent on radiation quality and high linear energy transfer (high-LET) radiation, such as heavy ions in space is known to deposit higher energy in tissues and cause greater damage than low-LET γ radiation. While radiation exposure has been linked to intestinal pathologies, there are very few studies on long-term effects of radiation, fewer involved a therapeutically relevant γ radiation dose, and none explored persistent tissue metabolomic alterations after heavy ion space radiation exposure. Using a metabolomics approach, we report long-term metabolomic markers of radiation injury and perturbation of signaling pathways linked to metabolic alterations in mice after heavy ion or γ radiation exposure. Intestinal tissues (C57BL/6J, female, 6 to 8 wks) were analyzed using ultra performance liquid chromatography coupled with electrospray quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS) two months after 2 Gy γ radiation and results were compared to an equitoxic (56)Fe (1.6 Gy) radiation dose. The biological relevance of the metabolites was determined using Ingenuity Pathway Analysis, immunoblots, and immunohistochemistry. Metabolic profile analysis showed radiation-type-dependent spatial separation of the groups. Decreased adenine and guanosine and increased inosine and uridine suggested perturbed nucleotide metabolism. While both the radiation types affected amino acid metabolism, the (56)Fe radiation preferentially altered dipeptide metabolism. Furthermore, (56)Fe radiation caused upregulation of 'prostanoid biosynthesis' and 'eicosanoid signaling', which are interlinked events related to cellular inflammation and have implications for nutrient absorption and inflammatory bowel disease during space missions and after radiotherapy. In conclusion, our data showed for the first time that metabolomics can not only be used to distinguish between heavy ion and γ radiation exposures, but also as a radiation-risk assessment tool for intestinal pathologies through identification of biomarkers persisting long after exposure.
    PLoS ONE 01/2014; 9(1):e87079. DOI:10.1371/journal.pone.0087079 · 3.23 Impact Factor
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    • "Since IR was shown to activate Akt signaling [23], [24], we tested the physiological relevance of NBS1 to activate Akt activity through IR. IR increased the levels of NBS1 and phosphorylated Akt (pAkt Ser-473) in two different cell lines (Fig. 6B). "
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    ABSTRACT: Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome. The NBS gene product, NBS1 (p95 or nibrin), is a part of the Mre11-Rad50-NBS1 complex. SIN1 is a component of the mTOR/Rictor/SIN1 complex mediating the activation of Akt. Here we show that NBS1 interacted with mTOR, Rictor, and SIN1. The specific domains of mTOR, Rictor, or SIN1 interacted with the internal domain (a.a. 221-402) of NBS1. Sucrose density gradient showed that NBS1 was located in the same fractions as the mTOR/Rictor/SIN1 complex. Knockdown of NBS1 decreased the levels of phosphorylated Akt and its downstream targets. Ionizing radiation (IR) increased the NBS1 levels and activated Akt activity. These results demonstrate that NBS1 interacts with the mTOR/Rictor/SIN1 complex through the a.a. 221-402 domain and contributes to the activation of Akt activity.
    PLoS ONE 06/2013; 8(6):e65586. DOI:10.1371/journal.pone.0065586 · 3.23 Impact Factor
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    • "Reproductive hormones LH and FSH are the central molecules in an upregulated pathway network (Figure  5A) and are known to regulate estrogen [65], which is strongly associated with risk of developing breast cancer [66]. Indeed, in an earlier study, we have shown that exposure to a radiation dose (2 Gy) used in the current study significantly increased serum estrogen level, urinary oncogenic estrogen metabolite level, mammary gland estrogen receptor α (ERα) level, and cellular proliferation in mice [50]. Estrogen has also been reported to influence TGFβ activity negatively [67], and interestingly our downregulated data sets mapped to a pathway, which has TGFβ as the nodal molecule (Figure  10A). "
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    ABSTRACT: Background Breast tissue is among the most sensitive tissues to the carcinogenic actions of ionizing radiation and epidemiological studies have linked radiation exposure to breast cancer. Currently, molecular understanding of radiation carcinogenesis in mammary gland is hindered due to the scarcity of in vivo long-term follow up data. We undertook this study to delineate radiation-induced persistent alterations in gene expression in mouse mammary glands 2-month after radiation exposure. Methods Six to eight week old female C57BL/6J mice were exposed to 2 Gy of whole body γ radiation and mammary glands were surgically removed 2-month after radiation. RNA was isolated and microarray hybridization performed for gene expression analysis. Ingenuity Pathway Analysis (IPA) was used for biological interpretation of microarray data. Real time quantitative PCR was performed on selected genes to confirm the microarray data. Results Compared to untreated controls, the mRNA levels of a total of 737 genes were significantly (p<0.05) perturbed above 2-fold of control. More genes (493 genes; 67%) were upregulated than the number of downregulated genes (244 genes; 33%). Functional analysis of the upregulated genes mapped to cell proliferation and cancer related canonical pathways such as ‘ERK/MAPK signaling’, ‘CDK5 signaling’, and ‘14-3-3-mediated signaling’. We also observed upregulation of breast cancer related canonical pathways such as ‘breast cancer regulation by Stathmin1’, and ‘HER-2 signaling in breast cancer’ in IPA. Interestingly, the downregulated genes mapped to fewer canonical pathways involved in cell proliferation. We also observed that a number of genes with tumor suppressor function (GPRC5A, ELF1, NAB2, Sema4D, ACPP, MAP2, RUNX1) persistently remained downregulated in response to radiation exposure. Results from qRT-PCR on five selected differentially expressed genes confirmed microarray data. The PCR data on PPP4c, ELF1, MAPK12, PLCG1, and E2F6 showed similar trend in up and downregulation as has been observed with the microarray. Conclusions Exposure to a clinically relevant radiation dose led to long-term activation of mammary gland genes involved in proliferative and metabolic pathways, which are known to have roles in carcinogenesis. When considered along with downregulation of a number of tumor suppressor genes, our study has implications for breast cancer initiation and progression after therapeutic radiation exposure.
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