Patent Application
20060013801
There are few if any treatments for radiation exposure that have quantitative dose modifying benefits when given hours a day after exposure. Similarly, even those drugs that have benefits when given before radiation, typically are ethicacious only for a few hours and have transient side effects that prevent the subject from full function, for example, hypotension and peripheral neuropathy.
Radiation-induced soft tissue fibrosis is a consequence of acute and chronic inflammatory responses. While modern radiation techniques have improved therapeutic gain and reduced the incidence of severe radiation-induced fibrosis, radiation-related side effects still occur when aiming for optimal tumor control. It has been shown that radiation-induced soft tissue damage is expected in about 10% of patients when radiation is optimized to achieve 90% tumor control.
Soft tissue fibrosis occurs in the late stage of radiation-induced tissue damage. It is caused by multiple factors and is poorly understood. However, the early stage of radiation-induced soft tissue damage is characterized by infiltration of various inflammatory cells and overproduction of cytokines. The late stage is pathologically characterized by active fibroblast proliferation with atypical fibroblasts, and excessive extracellular matrix production. Radiation injury is similar in some ways to normal tissue injury. Surgical injury, for example, is a process that features a relatively short period of brisk cytokine production, angiogenesis, fibroblast, and epithelial cell proliferation. The atypical proliferation results in granulation, which abruptly stops, allowing mature scar to develop. IL-1 is an important signal controlling this process. Radiation-induced soft tissue fibrosis has many of the same features of normal tissue repair, but is less brisk and may remain active for years at subclinical levels. The continuous inflammation results in continuously active deposition of collagen.
Radiation pneumonitis is a distinct clinical entity that differs from other pulmonary symptoms such as allergic pneumonitis, chemical pneumonitis, or pneumonia by various infectious agents. Recent research has supported the mechanism of cellular interaction between lung parenchymal cells and circulating immune cells mediated through a variety of cytokines including pro-inflammatory cytokines, chemokines, adhesion molecules, and pro-fibrotic cytokines. Identifying reliable biomarkers for radiation pneumonitis will allow identifying individuals at risk for pneumonitis before or during the early stage of therapy.
Radiation pulmonary injury manifested as subacute pneumonitis and late fibrosis has long been recognized in patients receiving radiotherapy to the chest region. Lung injury by radiation is a major obstacle prohibiting the high dose radiation required for eradicating cancer of the thoracic region. Radiation pneumonitis is a distinct clinical entity and there has been increasing awareness and recognition of its impact on the treatment of thoracic malignancy. It manifests unique clinical and radiographic characteristics that separate it from other pulmonary symptoms such as allergic pneumonitis, chemical pneumonitis, or pneumonia by various infectious agents of viral, bacterial, fungal, or parasitical origins.
Radiation pneumonitis is a type of inflammatory response of the lung tissue in response to radiation insult. Indeed, at the cellular level, radiation pneumonitis is characterized by lymphocytic alveolitis, a result of inflammatory infiltrates of mononuclear cells from the vascular compartment into the alveolar spaces. As expected at sites of inflammation, an active interaction between cellular and humoral factors are involved including immune cells, parenchymal cells, macrophages, chemokines, adhesion molecules, lymphocytes, inflammatory cytokines and fibrotic cytokines. Research in radiation pulmonary injury has supported involvement of inflammatory cytokines, chemokines, and fibrotic cytokines. Although investigation of adhesion molecules in radiation lung injury is still underway, these molecules are expected to be involved to serve as prerequisites for leukocyte adhesion to endothelial cells of blood vessels and consequently for transmigration to tissues at sites of inflammation. At the time of clinical symptoms, radiographic infiltrates are often observed in lung volumes, which generally conform to the radiation treatment ports on chest radiographs. The alveolar spaces are filled with patchy infiltrates on chest CT scans and the patients often experience worsening dyspnea. These mononuclear infiltrates may be cleared from alveolar spaces rapidly in response to steroids, likely due to rapid apoptosis of lymphocytes by steroids, and patients often experience marked improvement of dyspnea. With longer follow-up, almost all patients develop radiographic evidences of lung fibrosis.
While current fast-developing new techniques have significantly improved radiotherapeutic gains, radiation-related normal tissue damage still remains unavoidable especially when aiming for optimal tumor control. Normal tissue tolerance, in particular, soft tissue fibrosis, is one of the major dose-limiting factors influencing radiation therapy. It has been reported that radiation-induced soft tissue damage is expected in ten percent of patients when radiation dose is optimized to maximum tumor control. Therefore, a better understanding of the molecular basis of radiation-induced normal damage could provide an effective means for the prevention, or even reversal of radiation-related complications in the clinical radiotherapy. Furthermore, due to the unsatisfactory outcomes of present combination of radiotherapy and chemotherapy, especially with multiple-areas and prolong schedule procedure, much emphasis also are needed to placed on developing better and less side-effects treatment procedure for normal tissue protection.
We have discovered that IL-1 is a major contributor to acute and late radiation complications to the bone marrow, bowel, and lungs and soft tissues. We have shown that humans that have high circulating levels of IL-1 before any radiation is delivered develop radiation pneumonitis. In addition, we have found that the absence of IL-1 alpha results in a low propensity for the development of fibrosis following radiation. However, we have also discovered that the elevation of IL-1 persists or rises at later times after radiation.
We have further found that blocking IL function with circulating proteins or drugs is a useful method for the prevention of toxicity to normal tissue and is ethicacious after radiation for the prevention of the progression of toxicity over time.
As a result, the present invention provides for the prevention of and therapy for radiation pneumonitis, dermatitis, soft tissue fibrosis and central nervous system toxicity in patients undergoing therapeutic radiation. In addition, it provides for pre-treatment of those responding to nuclear bio terrorism or other nuclear or radiological accidents. Thus, with the present invention, subjects may be treated in order to prevent toxicity from nuclear bio terrorism or other nuclear or radiological accidents. More particularly, we have discovered a method for profallactically treating radiation toxicity in normal tissue of a subject comprising administering an anti-radiation toxicity effective amount of a cytokine blocking agent through the subject.
More specifically, we have discovered a method for profallactically treating radiation pneumonitis, dermatitis, soft tissue fibrosis or central nervous system toxicity in a subject comprising administering an anti-radiation pneumonitis, dermatitis, soft tissue fibrosis or central nervous system toxicity effective amount of a cytokine blocking agent to the subject.
We have further discovered that the administration of an anti-radiation induced soft tissue effective amount of a COX-2 enzyme inhibitor significantly reduces the amount of tissue damage due to radiation.
We have investigated the role of specific COX-2 inhibitors (Celebrex) in radiation induced soft tissue damage, and explored the relationship between chemokine and its receptor mRNA expression and radiation-induced skin damage in mammary tumor-bearing mice. Here we report that 50 mg/kg Celebrex, given daily with gavage for 15 doses in three weeks, significantly reduced single dose of radiation (60 Gy) induced normal skin damage in MCa-35 mammary tumor-bearing mice. Decreased skin damages are associated with the reduction of the radiation-induced chemokines, Rantes, MCP1, and their related receptor mRNA expression in skin, but not in tumor tissues.
Materials and Methods: Prospective blood sampling, scoring of respiratory symptoms, and chest imaging were conducted for patients receiving thoracic radiation for malignancy. Serial plasma specimens were analyzed for circulating cytokine changes before, during radiation, and up to 12 weeks post-radiation. Radiation pneumonitis was diagnosed using NCI common Toxicity Criteria. Cytokine analysis was assayed for interleukin a (IL-1α), interleukin 6 (IL-6), Monocyte Chemotactic Protein 1 (MCP-1), E-Selectin, L-Selectin, Transforming Growth Factor β1 (TGF-β1), and Basic Fibroblast Growth Factor (bFGF) using Enzyme Linked Immmunosorbant Assay (ELISA).
Methods
Patient Characteristics
Patients who were to receive thoracic radiation for malignancy were eligible. Blood, thoracic imaging, and respiratory symptom scoring were collected prospectively. Twenty-four patients had follow-up longer than 12 months and their characteristics are shown in Table 1.
Abbreviations: NCI CTC, National Cancer Institute common toxicity criteria; nos, not otherwise specified.
Clinical and Radiographic Evaluation
History and physical examinations emphasizing the respiratory symptoms were performed periodically. Clinical evaluation for pulmonary symptoms was evaluated and graded using the LENT/SOMA scoring system for the lung. This system includes the RTOG treatment side effect scoring of subjective clinical symptoms, and an objective assessment of serial chest X-rays and CT scan changes. Pneumonitis grading was also defined according to NCI Common Toxicity Criteria.
Circulating Cytokinie Analysis
Plasma samples were collected before therapy and weekly, during therapy. Specimens were collected in sodium heparin as well as EDTA up to 12 weeks post-therapy. Platelet-free plasma was produced by centrifugation at 1200 rpm at 0° C. for 10 minutes. The plasma was stored in aliquots at −20° C. Heparinized plasma was used for the analysis of most cytokines and EDTA plasma was used for the analysis of bFGF. Cytokines were analyzed using Enzyme-Linked Immunosorbent Assay (ELISA). The methodology of ELISA analysis was according to manufacturers' instructions as previously described.
Twenty-four patients had clinical follow-up longer than 12 months after radiation. Thirteen developed symptomatic pneumonitis (NCI grade 2). The peak incidence of symptoms was between 6- and 13 weeks post radiotherapy. Six patients had only radiographic infiltrates. (NCI grade 1). Five patients did not have clinical or radiographic pneumonitis. Both IL-1α and IL-6 levels were significantly higher before, during, and after radiation for those who developed pneumonitis. The pattern of changes of MCP-1, E-Selectin, L-Selectin, TGF-β1, and bFGF varied but none of these cytokines correlated with radiation pneumonitis.
Analysis of a panel of circulatory cytokines with different putative function in radiation pulmonary injury showed that pre-treatment IΛ-1α and IL-6, as well as mid and post-treatment levels were significantly higher for patients who subsequently developed radiation pneumonitis.
Radiation Pneumonitis
Symptomatic radiation pneumonitis is characterized by an annoying cough that is either non-productive or with clear sputum. This period is generally accompanied by markedly worsening dyspnea in an otherwise healthy appearing individual. Generally there are also radiographic infiltrates on chest x-ray and CT scan that usually conforms to radiation ports. The individual in general is afebrile or has a low-grade temperature, and is without an increase of blood neutrophil counts. Clinical symptoms are rapidly relieved with low dose steroid treatment. Of the 24 patients with follow-up longer than 12 months, 13 developed clinical symptoms consistent with radiation pneumonitis (NCI grade 2 pneumonitis). Six had radiographic infiltrates only, without clinical symptoms (NCI grade 1). Five did not have either radiographic infiltrates or clinical symptoms. The timescale of occurrence of pneumonitis is shown in
Pro-inflammatory Cytokinies Markers: IL-1α and IL-6
We analyzed pro-inflammatory cytokine IL-1α, and IL-6 levels before radiation treatment, weekly during treatment, and up to 12 weeks following radiation.
Pro-fibrotic Cytokine Markers: bFGF and TGF-β1
Chemokine and Adhesion Molecule Markers: MCP-1, L-Selectin, and E-Selectin
Plasma levels of MCP-1 (Monocyte Chemotactic Protein 1), L-Selectin, and S-Selectin (
Radiation pneumonitis and fibrosis can be regarded as the consequences of a wound-healing inflammatory reaction to radiation damage of lung tissues. Research in immunological regulation of inflammation has revealed the complex interaction between local tissues and immune cells mediated through chemokines, adhesion molecules, inflammatory cytokine, and fibrotic cytokines.
Inflammatory Cytokines and Radiation Pneumonitis
We have shown that lung radiation is associated with a temporal expression of IL-1α, TGF-β1, collagen I, collagen III, and collagen IV gene expression in fibrosis-prone mice (C57BL/6). Among the panel of cytokines potentially involved in the inflammatory response to radiation lung injury, IL-1α and IL-6 were the only two cytokines that correlated significantly with radiation pneumonitis (
The rise of IL-6 after completion of radiation was observed. It coincided with the period of clinical symptomatic pneumonitis and this deserves further investigation (
Pro-fibrotic Cytokines and Radiation Pneumonitis
Pro-fibrotic cytokines participate in radiation lung injury, especially during the development of lung fibrosis phase, which generally starts at 4 to 6 months after treatment and continues without a clear end point. Lung fibrosis is equivalent to the scar after the initial inflammatory phase of lung reaction to radiation injury. Although radiographic fibrosis in general is not observed until 4 to 6 months after completion of radiation, it has been reported that circulatory TGF-β1 changes may serve as an early predictor for radiation pneumonitis and its expression increases with radiation in animal research models. Two pro-fibrotic cytokines, bFGF and TGF-β1, and their changes in the association to radiation pneumonitis (
We have discussed that circulatory measure of IL-1α and IL-6 turned are significantly associated with radiation pneumonitis. Thus, patients with higher baseline levels of inflammatory cytokines are more vulnerable to radiation lung injury.
Figure Legends:
Materials and Methods
Mice Strains and Radiation Treatment
Six to 7 week-old female C3H/HeN, BALB/c and C57BL/6 mice were used (Jackson Laboratories, Bar Harbor, Me.). The right hind leg (10 mice per group) was given 10, 20, 30, 40, 60, or 80 Gy in a single radiation dose with a Shephered Irradiator, a 6000 Ci Cs source, together with collimating equipment. The left, non-irradiated hind leg was used as the non-irradiated control. Mice were sacrificed at different time points after radiation (0.5, 1, 2, 4, 8, 12hrs, day 1, day 7, and day 14). At least 10 mice were used at each time point. Tissues from 3 mice were used for histology, and the remaining animals were used for mRNA analysis. Skin and muscle tissues from control and irradiated legs were dissected, and total RNA was isolated. Guidelines for the humane treatment of animals were followed as approved by the University of Rochester Committee on Animal Resources.
Tumor Tissue RNA Isolation and RNase Protection Assays
Skin and muscle tissues from each treatment group (7-10 mice) were pooled and total RNA was isolated by pulverizing the frozen tissue and dissolving it in TRI Reagent (Molecular Research Center, Ohio) according to the manufacturer's specifications. To determine the integrity of isolated RNA, 2 μg of RNA from each sample was fractionated on a formaldehyde gel and visualized by staining in ethidium bromide. RNase protection was performed using established multi-probes template sets (PharMingen, SanDiago, Calif.) as described previously. The interleukin (IL) sets include: IL-1α:, IL-1β, IL-1Rα, IL-6, IL-10 and IL-12. Two internal controls, L32 and GAPDH, were used as loading controls. The cocktail constructs were used to prepare P-UTP labeled antisense cRNA probes using the PharMingen in vitro transcription kits (PharMingen, SanDiago, Calif.). Probes were hybridized with 30 μg of total RNA at 50° C. for 16 hr. RNase A (1 mg/ml) and RNase T1 (2000 U/ml) were then added to digest single-stranded RNA. After digestion, the RNA was precipitated and resuspended in gel loading buffer, heated at 95° C. for 5 min, and run in 7% denaturing polyacrylamide gel (National Diagnostics, Ga.). The gel was run for 2-3 hr at 60v, dried on Whatman filter paper, and placed on a phosphorimager screen for quantitative analysis using a Cyclone Phosphorimager device (HP Company, Conn.). Area integration of each mRNA-protected fragment was normalized against the protected internal control band (GAPDH) in the corresponding lane to calculate the ratio of targeted/GAPDH mRNA. In order to compare the basal levels with radiation-induced levels for each interleukine mRNA tested, relative mRNA levels (folds) were plotted. Some gels are shown with over-exposure of the control lanes to highlight differences in IL-1α/β expression.
Blood Cytokines Assays (ELISA)
Blood samples were collected from 3 mice strains at various time points after radiation. After centrifugation for 30 minutes at 4° C., plasmas were aliquated and stored at −70° C. until analysis. Immunoenzymetric assays for murine IL-1β (Endogen Inc, Cambridge, Mass.) were performed according to the manufacturer's instructions. A standard curve with cytokine-positive control was run in each assay and the lower limit of detection was determined to be 3.5 pg/ml. Most of non-irradiated mice had circulating IL-1β protein levels near the limit of detection.
In Situ Hybridization
Localization of the IL-1β gene in soft tissue was determined by in situ localization and was performed as previously published. Briefly, leg tissues were fixed in 10% formalin and 2% paraformadhyde by cutting the whole leg into 3-5 pieces. Tissue sections were then placed on specially prepared slides (acid washed and T3-aminopropyl trietlioxysilane coated) and were deparaffinized and rebydrated. Proteinase K-digested sections were hybridized with appropriate amounts of IL-1β riboprobe. Sections to be examined were hybridized with anti-sense RNA under conditions of probe excess, and, after washing, they were prepared for autoradiography using NBTII emulsion (Kodak, Rochester, N.Y.). After autoradiography and staining, the slides were analyzed by bright and dark field microscopy. Backgrounds for these studies were determined using the sense stand RNA probe. As positive controls for hybridization, some sections were hybridized with constitutively expressed mRNA (GAPDH) and were analyzed for cell specific expression of the molecule of interest. Cell types and locations of IL-1β over-expression were identified histologically.
Statistical Analysis
Cytokine mRNA expression levels from skin and muscle in non-irradiated versus irradiated tissues were compared using the unpaired Student's t-test, or Mann-Whitney Rank Sum test as appropriate. Differences were considered significant for p<0.05.
Results
Pathological Observation
At early time points after irradiation of the skin, the gross appearance was only mildly different from one strain of mice to another.
The histological changes mirrored the clinical examinations, with some qualitative differences. As an example, 30 Gy irradiated tissues at various times after treatment are shown in
Expression of IL-1β mRNA
In order to determine the molecular correlation of radiation-induced soft tissue damage, we examined mRNA expression of interleukins, IL-1α, IL-1β, and IL-1Ra by RNase protection assay in skin and muscle tissues from the 3 mice strains before and after different doses of radiation. As shown in
Expression of IL-1α mRNA
As shown in
Expression of IL-1Ra mRNA
Like IL-1β mRNA, IL-1Ra mRNA was highly expressed in skin tissue, and no substantial difference in the basal levels of IL-1Ra mRNA was seen among the three strains (
Discusssion
Murine models were used to simulate the situation that occurs in human skin after irradiation. This enabled us to examine the molecular characteristics of soft tissue fibrosis. Doses that caused little or no fibrosis (<30Gy), as well as highly fibrogenic doses (60-80Gy) were used in the 3 mice strains. We expected that, if radiation-induced cytokine mRNA expression is a causal event, then high doses would induce higher levels of cytokine mRNA, explaining strain variation in fibrosis sensitivity. Two key questions were asked in this study: 1) Is there a difference in basal mRNA expression of certain cytokines in skin or muscle tissues among 3 mouse strains with different fibrosis sensitivities? 2) Does this difference in mRNA expression contribute to the various radiation-related fibrosis responses in the three mouse strains? We demonstrated that: 1) skin tissues express higher levels of several interleukins than muscle tissues, independent of mouse strain. This is consistent with prominent initial fibrosis occurring in the subepidermal regions, with less and later development of fibrosis in muscle tissue. 2) C3H/HeN mice have the lowest predisposition for developing fibrosis and did not express IL-1α mRNA in their skin. The most fibrosis sensitive strain, C57BL/6, had high basal and radiation-induced levels of this cytokine. Muscle, which is more fibrosis resistant than skin, also had lower or undetectable IL-1α expression compared to skin. 3) Radiation induced elevation of IL-1β mRNA was biphasic with an early peak (1 to 4 hr) and another at a later time (3 to 14 day). The first phase was absent in the fibrosis sensitive strain, and it was intermediate in the strain with intermediate fibrosis sensitivity. 4) Cytokine responses in muscle were more blunted, compared to those in skin, and required higher radiation doses. 5) Cytokine responses after local radiation could be large enough to be detected in the circulation. 6) The cells synthesizing the greatest quantities of IL-1β appear to be the keratinocytes and stromal cells of the epidermis and dermis. Taken together we propose that these patterns suggest that brisk IL-1α responses to radiation and high basal IL-1α mRNA levels are associated with a higher risk for late radiation fibrosis. An early pulse of IL-1β expression after irradiation appears to correlate with a lower risk for developing radiation soft tissue fibrosis. The data also provided evidence that circulating levels of cytokines might be a useful marker of local cytokine production following radiation.
It has been demonstrated both experimentally and clinically that high basal levels of fibrogenic cytokines and/or growth factors are related to a higher incidence of radiation- or chemotherapy-induced late tissue damage. Our recent animal studies also suggest that high blood TGF-β levels are associated with a high risk for radiation-induced tissue fibrosis. We measured local and circulating levels of interleukin mRNA in our 3 mice strains with different fibrosis sensitivities because higher basal mRNA levels of these cytokines may also be related to a higher risk of radiation-mediated normal tissue fibrosis. It is apparent from our data that C3H/HeN skin does not have detectable IL-1α mRNA. Low or undetectable skin IL-1α mRNA in C3H/HeN mice, a fibrosis resistance mouse strain, may be responsible for its resistant phenotype. In our radiation-induced lung fibrosis models, similar results were also observed. The correlation of low mRNA levels of skin and lung IL-1α with increased resistance of radiation-induced fibrosis warrants further investigation.
Radiation-induced expression of interleukin mRNA is organ-dependent. All interleukin responses were more pronounced in the skin than in muscle. Inducible levels of each cytokine, however, varied between skin and muscle tissues. For example, radiation induced an elevation of skin IL-1α mRNA, not muscle IL-1α mRNA, in C57BL/6 mice. Our previous data in cultured cell lines (keratinocytes, skin fibroblast, and squamous cell carcinoma cells) also demonstrated that different cell types not only express different levels of each cytokine, but also respond to radiation differently. Our data here may also provide some guidance for clinical radiation therapy. For example, avoidance of cutaneous radiation might prevent cytokine cascades that could result in late tissue fibrosis. This is because soft tissue fibrosis begins in the subepidermis, later extends through the dermis, and eventually involves the superficial and the deeper muscle layers. Clinically, the efficacy of megavoltage radiation is in large part due to the lower epidermal dosimetry. It is an intriguing notion that patients with elevated basal IL-1α mRNA might be treated prophylactically with anti-cytokine therapy to prevent fibrosis.
While radiation-induced alteration of interleukin mRNA in lung and other organs have been reported in several strains of mice, altered mRNA levels of cytokines in soft tissues from different strains of mice have not yet been reported. In this study, we collected and processed RNA samples of three strains in the same RNase protection gel, and we also compared the IL-1 mRNA expression difference between skin and muscle. We found that the patterns of cytokine mRNA expression were consistent with the degree of fibrotic response. In contrast, macroscopic and microscopic acute alterations were weak predictors of fibrosis sensitivity. The lack of correlation between acute reactions and late effects has been studied for decades, and the role that cytokines and growth factors play appears to finally help explain the phenomenon.
Radiation increased IL-1 mRNA expression in two waves, the first at approximately 4 hours after therapy and another 3 to 14 days post-radiation. Examination of corresponding skin tissue morphology at each time point suggested that acute tissue response in preexisting cellular components may be responsible for the first peak of cytokine production. In situ hybridization studies suggest that keratinocytes, endothelial cells, and skin fibroblasts are the source of the early IL-1β mRNA expression. Infiltrating inflammatory cells and activated fibroblasts are probably responsible for the second peak in cytokine mRNA production. Several studies have demonstrated that pulses of IL-1, given within 24 hours of radiation, are radioprotective. Endogenous pulsing of IL-1β in C3H/HeN mice after radiation may therefore partly explain this strain's higher resistance to fibrosis compared to C57BL/6 mice.
In conclusion, we have shown that skin tissues produce more interleukin mRNA compared with muscle tissues. Skin IL-1α and IL-1Ra mRNA are upregulated in C57BL/6 mice, while IL-1β mRNA is increased in C3H/HeN and BALB/c mice within a few hours of local leg radiation. These results show that radiation-induced differential mRNA expression for interleukin and varied basal levels of interleukin mRNA participate in radiation-induced normal tissue damage.
Legends
elevation compared to baseline significant p<0.05.
Elevation of IL-1α during the first day after radiation was most pronounced in the fibrosis sensitive strain.
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Material and Methods
Tumor Models and Radiation Treatment
Isotransplantable murine MCa-35 mammary tumor cells was inoculated i.m. into right hind thighs of 6-7 week-old female C3H/HeN mice (NCI, Fredrick, Md.). Right hind thigh tumors were given 60 Gy (single dose using a Cs irradiator) when tumors reached 8-9 mm in diameter. Mice were sacrificed 20 days after radiation.
Tumors and the overlaying skin tissues were removed for histology and RNA preparation. Irradiated tissues (tumor and skin) were also collected for making paraffin blocks for immunohistochemical staining. Guidelines for the humane treatment of animals were followed as approved by the University of Rochester Committee on Animal Resources.
Celebrex Treatment
Celebrex (Pfizer Inc.) powder was dissolved in PBS. Due to partial dissolution, the agent was mixed very well every time before gavaging. 50 mg/kg (0.2 ml) Celebrex was given daily, and five days per week for constitutive three weeks. Four experiment groups were used. All mice were treated with single 60 Gy radiation in tumor-bearing leg. Group 1 was radiation alone; mice in group 2 were given 50 mg/kg Celebrex 2 hours before radiation (2 hr pre-radiation); mice in group 3 and 4 were received the same amount of Celebrex at day 2 or day 7 post-radiation. Mice in the group 4 were received total 10 doses, and rest treated mice were given total 15 doses. All mice were sacrificed 20 day after radiation.
Determination of Radiation Induced Skin Damage by 5-Scales Scoring System
Radiation induce skin damage was assessed using 5-scales Skin Scoring System. 20 days after single 60 Gy radiation, mice from each treatment group were determined blindly for the degree of skin damage by three investigators. Grade 1: normal skin; grade 2: slight hair loss in irradiated area; grade 3: radish and swollen tissue; grad 4: small area erosion; grade 5: small ulceration. Grades 2-3 is referred as mild, and grades 4-5 is considered as severe skin damage.
Tumor Tissue RNA Isolation and RNase Protection Assays
Total RNA was isolated from tumors and skin overlying tumors, respectively, with 9-10 mice in each treatment group by pulverizing the frozen tissue, and dissolved in TRI Reagent (Molecular Research Center, Ohio) according to the manufacturer's specifications. To determine the integrity of isolated RNA, 2 μg of RNA from each sample was fractionated on a formaldehyde gel and visualized by staining in ethidium bromide. RNase protection was performed using established multi-probe template sets (PharMingen, San Diego, Calif.) as described previously [Okunieff, 1998 #4388]. The chemokine multiple templet includes: MCP-1, MIP-1α, MIP-1β, MIP-2, Rantes, Eotaxin and IP-10. The C-C chemokine receptor multiple templete includes: CCR1, CCR2, CCR3, CCR4 and CCR5. The C-X-C chemokine receptor multiple templets includes: CXCR2 and CXCR4. Two internal standards, L32 and GAPDH, were used as loading controls. The cocktail constructs were used to prepare 32P-UTP labeled antisense cDNA probes using PharMingen in vitro transcription kits (PharMingen, San Diego, Calif.). Probes were hybridized with 30 μg of total RNA at 50° C. for 16 hrs RNase A (1 mg/ml), and RNase T1 (2000 U/ml) was then added to digest single-stranded RNA. After digestion, the RNA was precipitated and resuspended in gel loading buffer, heated at 95° C. for 5 min, and run on a 6M urea, 7% denaturing polyacrylamide gel (National Diagnostics, Ga.). The gel was dried on filter paper and placed on a phosphorimager screen for quantitative analysis of mRNA expression levels for each cytokine/chemokine. Area integration of each mRNA-protected fragment probe was normalized against the protected band for GAPDH or L32 mRNA in each corresponding lane to calculate the ratio of targeted mRNA/GAPDH mRNA expression. In order to compare the basal levels of each gene tested, relative levels (ratios) were plotted.
Quantitative Measurement of Total Structural and Perfused Vessels
Immunohistochemistry methods have previously been described in detail. Immediately following cryostat sectioning, tissue slices (normal muscle and tumor) were stained with CD31 antibody (PharMingen Calif.) for determination of total vasculature. The stained sections were imaged using an epi-fluorescence equipped microscope, digitized (3-CCD camera), background-corrected, and image-analyzed using Image Pro software (Media Cybernetics, Mass.) and a 450 MHz Pentium computer. Color images from individual microscope fields were automatically acquired and digitally combined to form four montages of the tumor cross-section (total area=15.5 mm2) using a motorized stage and controller. The image montages were processed to enhance the contrast between background and CD31 staining. From the enhanced images, locations of CD31-stained vessels were recorded. The quantitative vascular information was analyzed using custom Fortran programs to perform a “closest individual” analysis as previously described. Briefly, the distances from computer-superimposed sampling points to the nearest blood vessel were determined. The cumulative frequency distribution of these distances provided the probability of encountering vessels within any specified distance from the tumor cells. Median distances (μm) to the nearest vessel were used for statistical comparisons.
Statistical Analysis
mRNA levels (ratios) of tumors and skin from irradiated or non-irradiated mice were evaluated using the unpaired Students t-test or Mann-Whitney Rank Sum test as appropriate. Differences were considered significant for p<0.05.
Results
20 days after single 60 Gy irradiated MCa-35 tumor skin had varied lesions including edema, erosion and superficial necrosis in most of saline-treated control mice 20 days after radiation (
Because radiation inducing soft tissue damages has been reported to associate with the persistent overproduction of cytokine or chemokine in irradiated normal or tumor cells, we next examined the effects of Celebrex on the radiation-induced mRNA expression of chemokines including five C-C family members (Rantes, eotactin, MIP-1α, MIP-β and MCP-1), one C-X-C family (MIP-2) and one C family member (lymphotactin), as well as C-C receptors (CCR1, CCR2 and CCR5) and C-X-C receptors (CXCR2 and CXCR4) in tumor skin and tumor tissues by RNase protection assay. As shown in
Due to each individual mouse variation, there was 15-30% of Celebrex-treated mice still developed the moderate or severe skin damage after radiation. Radiation-induced skin damage was quantitatively determined by the skin scores from each individual mouse. In order to find out the relationship between overexpression of chemokines or their receptors mRNA and radiation-induced skin damages, the correlation of skin scores and skin tissue chemokine and chemokine receptor mRNA expression levels from each individual mouse were plotted and shown in
As shown in
Discussion
Thus we have discussed that: 1) Radiation induced Rantes/CCR5 and MCP-1/CCR2 mRNA expression was decreased by Celebrex; and 2) Celebrex-mediated reduction of chemokine and their receptor mRNA expression was correlated with ameliorated skin damage. 2
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US03/05624 | 2/25/2003 | WO | 7/15/2005 |
| Number | Date | Country | |
|---|---|---|---|
| 60360093 | Feb 2002 | US |
