Cyclooxygenase-2 inhibitor, nimesulide, improves radiation treatment against non-small cell lung cancer both in vitro and in vivo

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I. J. Radiation Oncology

● Biology ● Physics

Volume 60, Number 1, Supplement, 2004

aberrations and complex exchanges, all of which are known to contribute to the development of human cancers including glioblastoma. Materials/Methods: Two human glioblastoma cell lines were used in the experiment, M059J cells lacking the catalytic subunit of DNA-PK, and their isogenic but DNA-PK proficient counterpart, M059K. Cell death was induced by staurosporine (STS) and determined by MTT assay, TUNEL detection and annexin V- and propidium iodide staining. Results: We found that M059K cells were much more sensitive to STS treatment than M059J cells, as demonstrated by MTT assay, TUNEL detection and annexin V- and propidium iodide staining. A possible mechanism responsible for the different sensitivity in these two cell lines was explored by the examination of Bcl-2, Bax, Bak and Fas. The cell death stimuli increased anti-apoptotic Bcl-2 and decreased pro-apoptotic Bcl-2 members (Bak and Bax) and Fas in glioblastoma cells deficient in DNA-PK. Activation of DNA-PK is known to promote cell death of human tumor cells via modulation of p53, which can down-regulate the anti-apoptotic Bcl-2 member proteins, induce pro-apoptotic Bcl-2 family members and promote a Bax-Bak interaction. Our experiment also demonstrated that the mode of glioblastoma cell death induced by STS consisted of both apoptosis and necrosis and the percentage of cell death in both modes was similar in glioblastoma cell lines either lacking DNA-PK or containing intact DNA-PK. Conclusions: Our findings suggest that DNA-PK has a positive role in the regulation of apoptosis in human glioblastomas. The aberrant expression of Bcl-2 family members and Fas was, at least in part, responsible for decreased sensitivity of DNA-PK deficient glioblastoma cells to cell death stimuli.

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Early Detection of Cytogenetic Damage from Radiation or Chemical Agents by Flow-Cytometric Analysis of Micronucleated CD71-Positive Reticulocytes in Human Peripheral Blood

Y. Chen,1 S. D. Dertinger,2 K. Pandya,3 D. K. Torous,2 K. J. Brewer,4 T. Smudzin,1 R. K. Miller4 Radiation Oncology, University of Rochester, Rochester, NY, 2Litron Laboratories, Rochester, NY, 3Hematology/Oncology, University of Rochester, Rochester, NY, 4Obstetrics and Gynecology, University of Rochester, Rochester, NY 1

Purpose/Objective: Micronuclei (MN) are formed upon cell division in cells with DNA double-stand break(s) or dysfunctional mitotic spindle apparatus. Based on the detailed understanding of MN origin, the rodent-based micronucleus test has become the most widely utilized in vivo system for evaluating chemicals’ clastogenic and aneugenic potential. The rodent-based tests are most typically performed as reticulocyte-(RET) based assays. Target cells for RET-based micronucleus assays were traditionally obtained from the bone marrow compartment. The application of this endpoint to peripheral blood has been limited due to the expected low sensitivity as a result of the effective splenic clearance of MN-containing reticulocytes (MN-RET) from circulation. Litron Laboratories has developed a flow cytometry-based method for quantifying the incidence of MN-RET in peripheral blood compartment of humans. This high-throughput scoring system utilizes a nucleic acid dye in combination with an erythrocyte maturity marker (fluorescent antibody against CD71). Together, these reagents allow for quantification of MN in the most immature fraction of RETs (MN-RETCD71⫹), thereby helping to reduce the effect of splenic filtration. This technology is capable of high rates of analysis and may offer rapid, efficient, and quantitative assessment of cytogenetic damages in humans by radiation or chemoradiation. We report a pilot study applying this methodology to the early detection of genotoxicity from radiation or chemotherapeutic agents using human peripheral blood samples. Materials/Methods: Eighteen patients receiving either radiation (n ⫽ 9), full-dose chemotherapy (n ⫽ 4), or combination chemoradiation (n ⫽ 5) for cancer treatments consented to an IRB-approved clinical protocol, which allows up to five daily blood sampling for the assay. Two ml of peripheral blood was drawn before treatment, and daily after treatment at approximately 24-hour intervals over the first course of the first week of therapy. Blood samples were fixed, and processed, stored, and stained according to the standard procedures developed by Litron Laboratories. The frequency of MN-RETCD71⫹ was measured using a single-laser flow cytometer providing 488nm excitation. Results: The majority of cancer patients receiving radiotherapy or chemotherapy demonstrated increased MN-RETCD71⫹ frequencies over the course of therapy. The increase was observed at as early as 24 hours after treatments, with the peak occurring between day 2 and day 4 after radiation or chemotherapy. The peak fold increases of MN-RETCD71⫹ ranged from 1.3 to 10.0 fold for radiation only, from 1.7 to 16.7 fold for chemoradiation, and from 10.7 to 26.7 fold for full-dose chemotherapy. There was an exponential increase of MN-RETCD71⫹ as a function of bone marrow suppression, which was measured by the percent reduction of peripheral RETs. There was also a positive correlation in MN-RETCD71⫹ increase with the volume of irradiated bone. Conclusions: This study demonstrates the feasibility of high-throughput flow cytometric analysis of MN-RETCD71⫹ in detecting cytogenetic damage by ionizing radiation and chemotherapeutic agents as early as 24 hours after exposure. The method is quantitative, sensitive, rapid, and is feasible with only 2 ml of human peripheral blood samples. This technology offers potential in the management of genotoxic exposure in the early detection, assessment of severity, monitoring status, and gauging therapeutic interventions. The work was supported by a NIEHS grant to Litron Laboratories (S.D.D.), number R44 ES010752-02.

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Cyclooxygenase-2 Inhibitor, Nimesulide, Improves Radiation Treatment Against Non-Small Cell Lung Cancer Both In Vitro and In Vivo

K. R. Grimes,1 G. W. Warren,2 D. K. St. Clair,3,1 W. H. St. Clair4,1 Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, KY, 2College of Medicine, University of Kentucky, Lexington, KY, 3Graduate Center of Toxicology, University of Kentucky, Lexington, KY, 4Radiation Medicine, University of Kentucky, Lexington, KY 1

Purpose/Objective: Lung cancer is the leading cause of cancer related deaths in the United States. Despite improvements in radiation, surgery, and chemotherapy the 5 year survival statistics of non-small cell lung cancer (NSCLC) have improved little over the past two decades. It has been proposed that NF-␬B is a participant in the cytoprotection against several redox-mediated

Proceedings of the 46th Annual ASTRO Meeting

therapeutic agents including ionizing radiation. Cyclooxygenase-2 (COX-2) inhibition has become an attractive target for enhancing the efficacy of radiation and chemotherapy. Numerous mechanistic pathways have been proposed as the means through which COX-2 inhibition enhances the efficacy of radiation. We hypothesize that the COX-2 inhibitor nimesulide will improve the efficacy of radiation therapy (RT) via the suppression of the NF-␬B mediated cytoprotective pathways. Materials/Methods: In this study we used the COX-2 inhibitor nimesulide to improve the efficacy of RT when measured by tumor regrowth assays in vivo and clonegenic survival in vitro. For the in vivo assay A549 tumor cells representing NSCLC were subcutaneously injected into the right flank of female athymic nude mice (n ⫽ 10/group). Mice were given nimesulide via drinking water at a concentration of 5 and 10␮g/g body weight (b.w.) where the water was replenished every day and tumors were measured biweekly. For our in vitro study, clonegenic survival assays were evaluated to determine the effect of nimesulide, radiation, and the combination. The mechanism by which nimesulide improved the efficacy of radiation treatment was measured by Western blot analysis of NF-␬B target genes. A detailed time course of 1– 48 h was performed to determine the effect of nimesulide alone as well as a combination of nimesulide concentrations from 100–300␮M with x-radiation doses of 3 and 6 Gy. Results: In vivo, mice that received combined treatments of 5␮g/g b.w. nimesulide and 30 Gy radiation (3 Gy/fraction, 10 days) had significant reduction in tumor size in comparison to the 30 Gy radiation control group (p ⬍ 0.05). Whereas nimesulide at 10␮g/g b.w. showed no significant change in comparison to radiation alone. In vitro nimesulide alone produced a significant decrease in clonegenic survival at doses from 300 –500␮M. When combined, nimsulide demonstrated an additive effect in combination with radiation. Nimesulide alone reduced MnSOD protein levels. Radiation at 3 and 6 Gy caused and elevation of MnSOD protein levels which was inhibited by prior treatment of nimesulide suggesting an inhibition of radiation induced NF-␬B target genes. Conclusions: These results support the hypothesis that COX-2 inhibitors like nimesulide can increase the efficacy of radiation therapy. Interestingly, the lower dose of nimesulide increased the in vivo efficacy of RT whereas the higher dose produced no effect when compared to radiation alone. In vitro, our results suggest that the radiosensitization of A549 tumor cells by nimesulide is mediated by the suppression of NF-␬B radiation-induced cytoprotective genes.

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Waterbath Versus Ultrasound Hyperthermia In Vivo: Implications for Clinical Hyperthermia

N. Thompson,1 A. Zeug,1 J. Allan,1 J. Locke1 Radiation Oncology, Washington University School of Medicine, Saint Louis, MO

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Purpose/Objective: Ultrasound is utilized clinically for both superficial and deep hyperthermia. In contrast, in vivo experimentation has primarily utilized waterbath limb immersion. We determined the response of xenotransplants to waterbath immersion versus focal ultrasound hyperthermia using immunhistochemistry, histopathology, and tumor growth delay assays. We also assessed thermal dosimetry of ultrasound hyperthemia and compared the local, regional, and systemic temperature changes. Materials/Methods: Nu/Nu mice were used with institutional approval. A suspension of 2 x 10∧6 HeLa cells were injected in the bilateral proximal thigh of mice. Each mouse was inoculated with two tumors grown to an average of 350mm∧3. Mice were randomly divided into treatment groups (41 C or 43 C for 60 min, and ultrasound or waterbath treatment delivery) or control (untreated) for tumor growth delay. Waterbath (WB) hyperthermia was given via limb immersion. Ultrasound (US) hyperthermia was given via a 1cm portal using the SAHUS system. Temperature was controlled via a feedback loop between the thermometry units and a personal computer running customized software. Hsp70 western and TUNEL analysis were performed 18 hours following hyperthermia treatment. Tumor growth was assessed a minimum of twice weekly with digital calipers. Results: Consistency of hyperthermia delivery was demonstrated with both WB and US. Intratumoral probe measured temperatures of 43.0 C ⫾ 0.4 C and 41.0 C ⫾ 0.5 C for animals prescribed to receive 43 C or 41 C US respectively. Contralateral unheated tumor temperature in SAHUS treated animals varied a maximum of 0.9 C from baseline. Intratumor temperature with 43 C waterbath was 40.8 ⫾ 0.4 C with contralateral tumor varying 2 to 5 C higher. Rectal temperatures (32 C) in ultrasound treated mice did not vary more than 1 C from baseline while animals treated with 43 C WB averaged 34.2 ⫾ 0.9 C. TUNEL analysis demonstrated significant differences (p ⫽ 0.027) between ultrasound and waterbath treatment of tumors at 43 C. Conversely, contralateral (non-treated) tumors in animals receiving 43 C demonstrated no significant differences between modalities. Western blot analysis revealed increased hsp70 induction at 43 C in waterbath versus focal ultrasound hyperthermia. T-tests revealed significant differences in tumor growth delay between tumors heated with waterbath versus ultrasound at 43 but not at 41 C. No animal was removed from analysis due to toxicity of treatment. Thermal dosimetry during US showed average of three independent probes placed 5mm from central axis was 42.6 C ⫾ 0.5 C. Conclusions: Consistent US hyperthermia can be achieved throughout a typical xenotransplanted tumor. Local versus systemic effects of hyperthermia may be involved in tumor growth delay. Waterbath hyperthermia demonstrated greater local response despite lower intratumoral temperature measurements. The mildly elevated systemic temperature’s occurring during WB, and the concurrent improved response, may be an important consideration for clinical trials of hyperthermia.

2052

Radiation Damage Protection by the Benzyl Styrl Sulfone Analog, Ex-Rad

A. A. Alfieri,1 L. Liu,1 A. Sharma,1 G. Gorla,1 S. Bell,2,3 R. M. Ramana,2 S. Cosenza,2 P. E. Reddy,2 C. Guha1 Radiation Oncology, Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, NY, 2Fels Institute for Cancer Research, Philadelphia, PA, 3Onconova Therapeutics, Inc., Princeton, NJ

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Purpose/Objective: To date, there is no effective orally available prophylactic drug to ameliorate radiation injury after accidental exposure to ionizing irradiation. Currently available agents have inherent toxicities. Benzyl styryl sulfones are a family of drugs that exhibit inhibitory properties to a variety of protein kinases, such as cyclin-dependent kinases, thereby arresting cycling cells at cell cycle checkpoints. In these studies, we investigated the protective effects of a water-soluble, orally available, benzyl sulfone analog (Novonex/Ex-Rad) after total body irradiation of mice. Materials/Methods: The 4-carboxystyrl-4-chlorobenzylsulfone (ON 01210, Novonex/Ex-Rad) is stable at pH 7.6 to 2.5mg/ml final concentration in PBS. A dose escalating toxicity, pharmacokinetic and tissue distribution assay was performed in C57Bl/6

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