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Botulinum Toxin Confers Radioprotection in Murine Salivary Glands
Poster Viewing Abstracts S639
Volume 87 Number 2S Supplement 2013 three regimens of whole thorax x-ray doses and divided into subgroups: 12 Gy (n Z 3), 16 Gy (n Z 3) and 20 Gy (n Z 3) and monitored for 16 weeks post radiation. Immunohistochemistry technique was employed to localize and quantify the distribution of CD24, CD44v6, CD73, Clara and pneumocytes type II cells, as stem-like cells, in the upper, middle and lower lobes of the lung in each group. Results: The upper lobe was found to harbor statistically significant (p < 0.05) more stem-like cells compared to the middle/lower lobes. The middle and lower lobes contained comparable percentage of different stem-like cells. All stem-like cells tested were distributed unsystematically in the lung tissue of this experimental model with no specific identifiable niches. Conclusions: Our findings show that the upper lobe harbors more population of stem-like cells compared to the lower lobe, which may explain the variation in regional radiosensitivity, with the lower lung lobe being more prone to radiation injury compared to the upper lobe as predicted from several experimental and clinical data. This variability in stem-like cells distribution seems to be related to local blood perfusion and gas exchange. No specific stem cell niche could be identified in our study. These results may support the development of new targeted radioprotection strategies to reduce incidences of radiation-induced lung injury during radiation therapy. Acknowledgment: The project was supported partially by CIHR-MOP11491, and Richard and Edith Strauss Postdoctoral Fellowship. Author Disclosure: O. Maria: None. A. Maria: None. N. Ybarra: None. K. Jeyaseelan: None. S. Lee: None. J. Perez: None. S.M. Lehnert: None. M. Serban: None. J. Seuntjens: None. I. El Naqa: None.
3140 The Mechanism of a Novel Radioprotector, 8-Quinolinol Derivative KH-3 I. Takahashi,1,2 A. Morita,2 S. Aoki,3 B. Wang,4 Y. Hosoi,2 Y. Kaneyasu,1 M. Kenjyo,1 T. Kimura,1 Y. Murakami,1 and Y. Nagata1; 1Department of Radiation Oncology, Hiroshima University, Hiroshima, Japan, 2 Department of Radiation Medicine, RIRBM, Hiroshima University, Hiroshima, Japan, 3Department of Medicinal and Life Science, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Tokyo, Japan, 4 Radiation Risk Reduction Research Program, National Institute of Radiological Sciences, Chiba, Japan Purpose/Objective(s): Radiation therapy for cancer often has severe side effects that limit its efficacy. Because these side effects are in part determined by p53-mediated apoptosis, temporary suppression of p53 has been suggested as a therapeutic strategy to relieve the damage of normal tissues during treatment of p53-deficient tumors. On the other hand, it is known that dissociation of a zinc ion, which is coordinated to metal ion binding site of p53, could induce p53 denaturation, hence we evaluated some zinc chelators as radioprotective p53 inhibitor. As a result, we found 5-chloro8-quinolinol (KH-3) as a radioprotector that can protect mice from a sublethal dose of 7.5 Gy total-body irradiation (TBI). In this study, we investigated the effects and mechanism of KH-3 comparatively to a known radioprotective p53 inhibitor, PFTm. Materials/Methods: Imprinting control region (ICR) female mice, aged 8week old, were total-body irradiated with an X-ray generator (Pantak320S, Shimadzu) operated at 200 kV at a dose rate of 0.66 Gy/min. KH-3 (35 mg/kg) or vehicle was ip injected 30 minutes before 7.5 Gy TBI. In cell analysis, highly radiosensitive cell line MOLT-4, derived from human T-cell leukemia, was used and 10 Gy-irradiated. The suppressive effect of KH-3 and the reference compound PFTm on radiation-induced MOLT-4 apoptosis was evaluated using Annexin V-FITC or MitoTracker Red staining. To examine the specificity of KH-3 for p53-mediated apoptosis, we used MOLT-4 cells and their derivatives, namely, p53-knockdown transformant and p53 revertant which re-expressed p53 using shRNAresistant FLAG-tagged p53-expressing plasmid. To elucidate the mechanism of action of KH-3 in p53-mediated apoptotic pathway, we mainly investigated the expression of p53 target-genes, p21 and PUMA, using quantitative real-time PCR and immunoblotting analyses. Results: For the 30-day survival study, half of the animals in the KH-3treated group survived, whereas all the mice receiving only TBI died
within 14 days (p < 0.01). Cell death analysis revealed that KH-3 had a higher inhibitory effect on cell death and lower toxicity than that of PFTm in the MOLT-4 cells. KH-3 was ineffective against the radiation-induced apoptosis of p53-knockdown transformant, but suppressed that of p53 revertant. Results indicated that suppression of radiation-induced apoptosis by KH-3 was specifically mediated through p53 signaling pathways. Furthermore, KH-3 modulated p53 target-gene expression without affecting p53 expression. In particular, p21, which has anti-apoptotic activity, was markedly up-regulated by KH-3. Conclusions: KH-3 is a novel kind of radioprotector that modulates p53 transcription. The radioprotective effect of KH-3 may be associated with its ability to up-regulate p21 expression. Author Disclosure: I. Takahashi: None. A. Morita: None. S. Aoki: None. B. Wang: None. Y. Hosoi: None. Y. Kaneyasu: None. M. Kenjyo: None. T. Kimura: None. Y. Murakami: None. Y. Nagata: None.
3141 Botulinum Toxin Confers Radioprotection in Murine Salivary Glands Y. Zeidan, N. Xiao, H. Cao, C. Kong, Q. Le, and D. Sirjani; Stanford University Affiliated Hospitals, Stanford, CA Purpose/Objective(s): Xerostomia is a common radiation toxicity which negatively impacts the quality of life of HNC patients. Current treatment strategies offer partial relief at best. Besides their wide use in cosmetic procedures, botulinum toxins (BTX) have been successfully used in treating radiation sequelae as radiation-induced cystitis, proctitis, fibrosis and facial pain. The purpose of this study is to evaluate the effect of botulinum toxin on radiation-induced salivary gland damage. Materials/Methods: We used a previously established model for murine salivary gland irradiation. Submandibular glands of C5BL6 mice (n Z 6/ group) were injected with saline or botulinum toxin 72 hrs prior to receiving 1500 cGy of focal gamma radiation. Saliva flow was measured at 3 and 7 days post treatment. Submandibular glands were collected at various time points for immunohistochemistry, confocal microscopy and Western blotting analysis. TUNEL staining was used to evaluate cell death. A cytokine array, consisting of 40 different mouse cytokines was used to evaluate cytokine profiles after radiation treatment. Results: Irradiated mice showed a 50% reduction in saliva flow whereas mice pre-injected with BTX had 25% reduction in saliva flow (p < 0.05) after 3 days. Cell death detected by TUNEL staining was similar in SMG sections from both groups. However, neutrophil infiltrate, detected via myeloperoxidase staining, was 2 to 3 fold lower for the BTX treated mice. A cytokine array showed 2 fold upregulation of LPS induced-chemokine (LIX/CXCL5), 3 days after irradiation. Interestingly, BTX pretreatment reduced LIX levels by 40%. At 4 weeks post irradiation, saline (control) group showed 40% reduction in basal SMG weight as compared to 20% in the BTX group. Histologically, BTX pretreated glands showed relative preservation of acinar volume post radiation. Conclusions: Taken together, these data suggest that BTX pretreatment ameliorates radiation-induced salivary dysfunction. Moreover, we demonstrate a novel role for CXCL5 in the acute phase of radiationinduced salivary gland damage. These results carry important clinical implications for treatment of xerostomia in HNC patients. Author Disclosure: Y. Zeidan: None. N. Xiao: None. H. Cao: None. C. Kong: None. Q. Le: None. D. Sirjani: None.
3142 Reduction of Late Pulmonary Fibrosis in Thoracic-Irradiated C57bl/ 6hnsd Mice by a Water Soluble Radiation Mitigator MMS-350 M. Epperly, J. Goff, R. Kalash, T. Dixon, D. Franicola, M. Sprachman, P. Wipf, H. Berhane, and J. Greenberger; University of Pittsburgh, Pittsburgh, PA Purpose/Objective(s): Radiation induced pulmonary fibrosis is a major complication of radiation therapy and identification of therapeutic drugs to ameliorate this complication remains a challenge. MMS-350, a novel water-soluble sulfoxide that has been developed as a second generation,
Volume 87 Number 2S Supplement 2013 three regimens of whole thorax x-ray doses and divided into subgroups: 12 Gy (n Z 3), 16 Gy (n Z 3) and 20 Gy (n Z 3) and monitored for 16 weeks post radiation. Immunohistochemistry technique was employed to localize and quantify the distribution of CD24, CD44v6, CD73, Clara and pneumocytes type II cells, as stem-like cells, in the upper, middle and lower lobes of the lung in each group. Results: The upper lobe was found to harbor statistically significant (p < 0.05) more stem-like cells compared to the middle/lower lobes. The middle and lower lobes contained comparable percentage of different stem-like cells. All stem-like cells tested were distributed unsystematically in the lung tissue of this experimental model with no specific identifiable niches. Conclusions: Our findings show that the upper lobe harbors more population of stem-like cells compared to the lower lobe, which may explain the variation in regional radiosensitivity, with the lower lung lobe being more prone to radiation injury compared to the upper lobe as predicted from several experimental and clinical data. This variability in stem-like cells distribution seems to be related to local blood perfusion and gas exchange. No specific stem cell niche could be identified in our study. These results may support the development of new targeted radioprotection strategies to reduce incidences of radiation-induced lung injury during radiation therapy. Acknowledgment: The project was supported partially by CIHR-MOP11491, and Richard and Edith Strauss Postdoctoral Fellowship. Author Disclosure: O. Maria: None. A. Maria: None. N. Ybarra: None. K. Jeyaseelan: None. S. Lee: None. J. Perez: None. S.M. Lehnert: None. M. Serban: None. J. Seuntjens: None. I. El Naqa: None.
3140 The Mechanism of a Novel Radioprotector, 8-Quinolinol Derivative KH-3 I. Takahashi,1,2 A. Morita,2 S. Aoki,3 B. Wang,4 Y. Hosoi,2 Y. Kaneyasu,1 M. Kenjyo,1 T. Kimura,1 Y. Murakami,1 and Y. Nagata1; 1Department of Radiation Oncology, Hiroshima University, Hiroshima, Japan, 2 Department of Radiation Medicine, RIRBM, Hiroshima University, Hiroshima, Japan, 3Department of Medicinal and Life Science, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Tokyo, Japan, 4 Radiation Risk Reduction Research Program, National Institute of Radiological Sciences, Chiba, Japan Purpose/Objective(s): Radiation therapy for cancer often has severe side effects that limit its efficacy. Because these side effects are in part determined by p53-mediated apoptosis, temporary suppression of p53 has been suggested as a therapeutic strategy to relieve the damage of normal tissues during treatment of p53-deficient tumors. On the other hand, it is known that dissociation of a zinc ion, which is coordinated to metal ion binding site of p53, could induce p53 denaturation, hence we evaluated some zinc chelators as radioprotective p53 inhibitor. As a result, we found 5-chloro8-quinolinol (KH-3) as a radioprotector that can protect mice from a sublethal dose of 7.5 Gy total-body irradiation (TBI). In this study, we investigated the effects and mechanism of KH-3 comparatively to a known radioprotective p53 inhibitor, PFTm. Materials/Methods: Imprinting control region (ICR) female mice, aged 8week old, were total-body irradiated with an X-ray generator (Pantak320S, Shimadzu) operated at 200 kV at a dose rate of 0.66 Gy/min. KH-3 (35 mg/kg) or vehicle was ip injected 30 minutes before 7.5 Gy TBI. In cell analysis, highly radiosensitive cell line MOLT-4, derived from human T-cell leukemia, was used and 10 Gy-irradiated. The suppressive effect of KH-3 and the reference compound PFTm on radiation-induced MOLT-4 apoptosis was evaluated using Annexin V-FITC or MitoTracker Red staining. To examine the specificity of KH-3 for p53-mediated apoptosis, we used MOLT-4 cells and their derivatives, namely, p53-knockdown transformant and p53 revertant which re-expressed p53 using shRNAresistant FLAG-tagged p53-expressing plasmid. To elucidate the mechanism of action of KH-3 in p53-mediated apoptotic pathway, we mainly investigated the expression of p53 target-genes, p21 and PUMA, using quantitative real-time PCR and immunoblotting analyses. Results: For the 30-day survival study, half of the animals in the KH-3treated group survived, whereas all the mice receiving only TBI died
within 14 days (p < 0.01). Cell death analysis revealed that KH-3 had a higher inhibitory effect on cell death and lower toxicity than that of PFTm in the MOLT-4 cells. KH-3 was ineffective against the radiation-induced apoptosis of p53-knockdown transformant, but suppressed that of p53 revertant. Results indicated that suppression of radiation-induced apoptosis by KH-3 was specifically mediated through p53 signaling pathways. Furthermore, KH-3 modulated p53 target-gene expression without affecting p53 expression. In particular, p21, which has anti-apoptotic activity, was markedly up-regulated by KH-3. Conclusions: KH-3 is a novel kind of radioprotector that modulates p53 transcription. The radioprotective effect of KH-3 may be associated with its ability to up-regulate p21 expression. Author Disclosure: I. Takahashi: None. A. Morita: None. S. Aoki: None. B. Wang: None. Y. Hosoi: None. Y. Kaneyasu: None. M. Kenjyo: None. T. Kimura: None. Y. Murakami: None. Y. Nagata: None.
3141 Botulinum Toxin Confers Radioprotection in Murine Salivary Glands Y. Zeidan, N. Xiao, H. Cao, C. Kong, Q. Le, and D. Sirjani; Stanford University Affiliated Hospitals, Stanford, CA Purpose/Objective(s): Xerostomia is a common radiation toxicity which negatively impacts the quality of life of HNC patients. Current treatment strategies offer partial relief at best. Besides their wide use in cosmetic procedures, botulinum toxins (BTX) have been successfully used in treating radiation sequelae as radiation-induced cystitis, proctitis, fibrosis and facial pain. The purpose of this study is to evaluate the effect of botulinum toxin on radiation-induced salivary gland damage. Materials/Methods: We used a previously established model for murine salivary gland irradiation. Submandibular glands of C5BL6 mice (n Z 6/ group) were injected with saline or botulinum toxin 72 hrs prior to receiving 1500 cGy of focal gamma radiation. Saliva flow was measured at 3 and 7 days post treatment. Submandibular glands were collected at various time points for immunohistochemistry, confocal microscopy and Western blotting analysis. TUNEL staining was used to evaluate cell death. A cytokine array, consisting of 40 different mouse cytokines was used to evaluate cytokine profiles after radiation treatment. Results: Irradiated mice showed a 50% reduction in saliva flow whereas mice pre-injected with BTX had 25% reduction in saliva flow (p < 0.05) after 3 days. Cell death detected by TUNEL staining was similar in SMG sections from both groups. However, neutrophil infiltrate, detected via myeloperoxidase staining, was 2 to 3 fold lower for the BTX treated mice. A cytokine array showed 2 fold upregulation of LPS induced-chemokine (LIX/CXCL5), 3 days after irradiation. Interestingly, BTX pretreatment reduced LIX levels by 40%. At 4 weeks post irradiation, saline (control) group showed 40% reduction in basal SMG weight as compared to 20% in the BTX group. Histologically, BTX pretreated glands showed relative preservation of acinar volume post radiation. Conclusions: Taken together, these data suggest that BTX pretreatment ameliorates radiation-induced salivary dysfunction. Moreover, we demonstrate a novel role for CXCL5 in the acute phase of radiationinduced salivary gland damage. These results carry important clinical implications for treatment of xerostomia in HNC patients. Author Disclosure: Y. Zeidan: None. N. Xiao: None. H. Cao: None. C. Kong: None. Q. Le: None. D. Sirjani: None.
3142 Reduction of Late Pulmonary Fibrosis in Thoracic-Irradiated C57bl/ 6hnsd Mice by a Water Soluble Radiation Mitigator MMS-350 M. Epperly, J. Goff, R. Kalash, T. Dixon, D. Franicola, M. Sprachman, P. Wipf, H. Berhane, and J. Greenberger; University of Pittsburgh, Pittsburgh, PA Purpose/Objective(s): Radiation induced pulmonary fibrosis is a major complication of radiation therapy and identification of therapeutic drugs to ameliorate this complication remains a challenge. MMS-350, a novel water-soluble sulfoxide that has been developed as a second generation,