Growth Hormone Protects the Intestine Preserving Radiotherapy Efficacy on Tumors: A Short-Term Study

RESEARCH ARTICLE Growth Hormone Protects the Intestine Preserving Radiotherapy Efficacy on Tumors: A Short-Term Study Victor Caz1, Marcos Elvira1, Maria Tabernero1, Antonio G. Grande2, Bricia Lopez-Plaza3, Enrique de Miguel1, Carlota Largo1*, Monica Santamaria1* 1 Experimental Surgery Department, La Paz Hospital Research Institute, Madrid, Spain, 2 Department of Radiation Oncology, La Paz University Hospital, Madrid, Spain, 3 Department of Nutrition, La Paz Hospital Research Institute, Madrid, Spain * (MS); (CL) Abstract OPEN ACCESS Citation: Caz V, Elvira M, Tabernero M, Grande AG, Lopez-Plaza B, de Miguel E, et al. (2015) Growth Hormone Protects the Intestine Preserving Radiotherapy Efficacy on Tumors: A Short-Term Study. PLoS ONE 10(12): e0144537. doi:10.1371/ journal.pone.0144537 Editor: David D. Roberts, Center for Cancer Research, National Cancer Institute, UNITED STATES Received: August 3, 2015 Accepted: November 19, 2015 Published: December 15, 2015 Copyright: © 2015 Caz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The efficacy of radiotherapy on tumors is hampered by its devastating adverse effects on healthy tissue, particularly that of the gastrointestinal tract. These effects cause acute symptoms that are so disruptive to patients that they can lead to interruption of the radiotherapy program. These adverse effects could limit the intensity of radiation received by the patient, resulting in a sublethal dose to the tumor, thus increasing the risk of tumor resistance. The lack of an effective treatment to protect the bowel during radiation therapy to allow higher radiation doses that are lethal to the tumor has become a barrier to implementing effective therapy. In this study, we present a comparative analysis of both intestinal and tumor tissue in regard to the efficacy and the preventive impact of a short-term growth hormone (GH) treatment in tumor-bearing rats as a protective agent during radiotherapy. Our data show that the exogenous administration of GH improved intestinal recovery after radiation treatment while preserving the therapeutic effect against the tumor. GH significantly increased proliferation in the irradiated intestine but not in the irradiated tumors, as assessed by Positron Emission Tomography and the proliferative markers Ki67, cyclin D3, and Proliferating Cell Nuclear Antigen. This proliferative effect was consistent with a significant increase in irradiated intestinal villi and crypt length. Furthermore, GH significantly decreased caspase3 activity in the intestine, whereas GH did not produce this effect in the irradiated tumors. In conclusion, short-term GH treatment protects the bowel, inducing proliferation while reducing apoptosis in healthy intestinal tissue and preserving radiotherapy efficacy on tumors. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by Instituto de Salud Carlos III-Fondos Feder grant FIS PI13/01990 to MS. This work was also supported by a contract (CES10/011) from the Instituto de Salud Carlos III, I3SNS to MS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction Radiotherapy is currently used for over 70% of cancer patients during the course of their treatment [1]. Its usefulness is limited, however, by the high incidence of radiation-induced adverse effects, which primarily affect tissues with high cellular turnover, such as tissues of the gastrointestinal tract, bone marrow, and skin [2]. Specifically, abdominal irradiation can produce PLOS ONE | DOI:10.1371/journal.pone.0144537 December 15, 2015 1 / 19 Radiation Protection with Growth Hormone and Antitumoral Therapy Competing Interests: The authors have declared that no competing interests exist. radiation enteritis, and recent reports indicate that as many as 75% of those receiving radiotherapy suffer significant adverse effects from this therapy [3,4]. As a result, the radiation dose is reduced—or in extreme cases interrupted—to preserve the patient’s status, thus decreasing radiotherapy efficacy and inducing tumor resistance. To prevent this loss of efficacy, much effort has been made in the past 5–10 years to develop medical countermeasures against the adverse effects of radiation, and many of these efforts have been directed toward reducing gastrointestinal radiation damage [1,2,5]. Several approaches have been investigated to reduce the negative impact of anticancer therapy on the intestine, including dietary modification [6] and the use of trophic factors [3,7]. We have previously shown the beneficial effects of GH treatment for acute radiation-induced injury in the small intestine of rats exposed to a sublethal dose of radiation. These beneficial effects were attributed to the proliferative and antiapoptotic effects of GH on the ileal crypts [8]. In the irradiated ileum of rats, we have shown that GH administration upregulates mRNA and protein expression of intestinal trefoil factor [9], a protective peptide against radiationinduced intestinal mucositis [10]. Other authors have shown additional positive GH effects: Specifically, GH counteracts the loss of progenitor cells in irradiated bone marrow [11] and GH administration selectively augments the early-outgrowth endothelial progenitor cell population in healthy individuals, indicating possible implications for the use of GH in future regenerative cell-based therapies [12]. Furthermore, the use of GH delays and decreases the severity of radiation-induced dermatitis [13]. The benefits of GH as an anticachectic agent have also been reported [14], stimulating liver protein synthesis in an animal model without changing tumor growth [15]. Despite the benefits related to these GH therapies, their effects on irradiated tumors are still poorly understood. The studies on these therapies’ effects on nonirradiated tumors are controversial, primarily because of their potential effect on the proliferation and survival of cancer cells [16,17]. The objective of this study was to investigate whether GH treatment would protect the intestine against acute injury induced by radiation without impairing the therapeutic effects of radiotherapy in terms of cell viability, cell proliferation, and apoptosis. Methods and Materials Cell culture and in vitro analysis The rat cell lines, DHD/K12/Trb (colonic adenocarcinoma, 90062901; DHD) and MCA-RH 7777 (hepatocarcinoma, 90021504; RH), were obtained from the European Collection of Cell Cultures (ECACC) and were cultured according to their instructions. To assess cell proliferation, cells (50–150 x103) were seeded into 6-well culture plates in standard culture medium and treated or not with GH (0.2 μg/ml). At the indicated times (...truncated)