Evaluation of Synergy Effects
Cells (3,000 per well) were seeded in 100 ml cell culture media in 96-well plates and adhered for 24 hours before different concentrations of cisplatin alone, compound 12 or a mixture of a fixed concentration ratio of the two compounds were added. After incubation for another 72 hours before living cells were quantified with the MTT assay. Values for inhibitory concentrations 50 (IC50) and combination index 95 (CI95) were calculated with CompuSyn software 1.0 (ComboSyn, Inc., USA), and results expressed as mean of three independent experiments.
Ultraviolet (UV) Cell Survival Assay
A549 and HCT116 cell lines we used were obtained from the ATCC Cell Biology Collection. Cells (A549 or HCT116 cell lines) were seeded in 12-well or 24-well plates with 50,000 or 20,000 cells per well in a final volume of 750 or 400 ml of DMEM media (Invitrogen, Cergy Pontoise, France) containing L-glutamine, penicillin (200 IU/ml), streptomycin (200 mg/ml) and 10% fetal bovine serum (Invitrogen, Cergy Pontoise, France), and incubated overnight at 37uC in presence of 5% CO2. Media was removed and cells were washed twice with PBS 1X and exposed to different doses of UVC irradiation in a Spectrolinker XL 1000 (Spectronics Corporation). Media with or without potential inhibitors was added and cells were incubated for another 72 hours before living cells were quantified with methylthiazoletetrazolium (MTT) assay as previously described [57].
Abstract
Background: Treatment of head and neck cancer with radiation often results in damage to surrounding normal tissues such as salivary glands. Permanent loss of function in the salivary glands often leads patients to discontinue treatment due to incapacitating side effects. It has previously been shown that IGF-1 suppresses radiation-induced apoptosis and enhances G2/M arrest leading to preservation of salivary gland function. In an effort to recapitulate the effects of IGF-1, as well as increase the likelihood of translating these findings to the clinic, the small molecule therapeutic Roscovitine, is being tested. Roscovitine is a cyclin-dependent kinase inhibitor that acts to transiently inhibit cell cycle progression and allow for DNA repair in damaged tissues. Methodology/Principal Findings: Treatment with Roscovitine prior to irradiation induced a significant increase in the percentage of cells in the G2/M phase, as demonstrated by flow cytometry. In contrast, mice treated with radiation exhibit no differences in the percentage of cells in G2/M when compared to unirradiated controls. Similar to previous studies utilizing IGF-1, pretreatment with Roscovitine leads to a significant up-regulation of p21 expression and a significant decrease in the number of PCNA positive cells. Radiation treatment leads to a significant increase in activated caspase-3 positive salivary acinar cells, which is suppressed by pretreatment with Roscovitine. Administration of Roscovitine prior to targeted head and neck irradiation preserves normal tissue function in mouse parotid salivary glands, both acutely and chronically, as measured by salivary output. Conclusions/Significance: These studies suggest that induction of transient G2/M cell cycle arrest by Roscovitine allows for suppression of apoptosis, thus preserving normal salivary function following targeted head and neck irradiation. This could have an important clinical impact by preventing the negative side effects of radiation therapy in surrounding normal tissues.
Citation: Martin KL, Hill GA, Klein RR, Arnett DG, Burd R, et al. (2012) Prevention of Radiation-Induced Salivary Gland Dysfunction Utilizing a CDK Inhibitor in a Mouse Model. Editor: Eric Y. Chuang, National Taiwan University, Taiwan Received August 8, 2012; Accepted November 5, 2012; Published December 7, 2012 Copyright: ?2012 Martin 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. Funding: The Flow Cytometry Core Facility is supported in part from a Cancer Center Support Grant CCSG- CA 023074. This work was supported by the National Institutes of Health R01 (DE18888). No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Introduction
It is estimated that over 40,000 cases of head and neck cancer will be diagnosed in the United States in 2012 [1]. Head and neck cancer is the 6th most common in the world, having a higher incidence in developing countries [2]. These cancers are often associated with increased tobacco and alcohol use [2]. The standard of care for these cancers includes surgical resection of the tumor and a combination of chemotherapy and ionizing radiation. However, irradiation of the head and neck region often exposes surrounding non-diseased tissues to incidental radiation, resulting in secondary side effects. Intensity-modulated radiation therapy (IMRT) is a type of radiotherapy used to spare normal tissues, like the salivary glands, in order to reduce the secondary side effects [3]. IMRT has made improvements in salivary gland sparing; however, depending on tumor location and grade, radiationinduced damage to the salivary glands still occurs resulting in salivary gland dysfunction.
Dysfunction of the salivary glands following radiation occurs in two stages, acute and chronic. Clinically, acute salivary gland dysfunction occurs within days and is characterized by loss of salivary flow, loss of acinar cells, glandular shrinkage, and changes in saliva composition. Chronic salivary gland dysfunction occurs months to years following radiotherapy and is characterized by reduced salivary flow and changes in saliva composition [4]. Affected patients suffer from xerostomia (dry mouth), oral mucositis, difficulty speaking, increased oral pathologies, difficulty chewing and swallowing food, as well as malnutrition due to loss of salivary flow [4]. Due to the dysfunction of the salivary glands, patients must resort to temporary treatments for xerostomia to maintain adequate nutrition and hydration. Overall there is a significant reduction in quality of life for those undergoing treatment. The mechanisms responsible for the elevated radiosensitivity of salivary glands are not well understood [4].
