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Noscapine Enhances Tumor Radioresponse in the GL261 Glioma Model: Implications for Glioma Therapy
Proceedings of the 49th Annual ASTRO Meeting a T7 phage-displayed cDNA library. After three rounds of biopanning, single clones of phages were screened with Enzyme-Linked Immunosorbent Assay (ELISA) to isolate clones that specifically bind to the peptide. cDNA from the positives were amplified and sequenced to identify the peptide-binding proteins. Interaction of the proteins with the peptide was studied with both of phage-displayed protein and purified GST-fused proteins. Expression pattern of peptide-binding proteins was studied with immunohistochemical staining and western blotting. Results: From the screening of a phage-displayed human cDNA library, Tax-interacting protein 1 (TIP-1) was identified as the major protein that can specifically bind to the HVGGSSV peptide. Specific binding of TIP-1 to the peptide was found to be dose-dependent as determined by ELISA using both the phage-displayed protein and purified protein from recombinant bacteria. Expression of the TIP-1 was found to correlate with peptide binding in cell culture. TIP-1 antibody staining showed that this protein is radiation-inducible in endothelial cells in culture. Irradiated tumors were sectioned and stained with TIP-1 antibody. This showed minimal staining following sham irradiation (0 Gy) and substantial increase in staining both tumor vasculature endothelium and tumor cells following treatment with 4 Gy. Western blot data confirmed that radiation can induce TIP-1 expression in LLC cells in culture. Conclusions: TIP-1 was identified as a putative radiation inducible receptor that binds the HVGGSSV recombinant peptide isolated from phage displayed peptide library. Targeted drug delivery to TIP-1 in irradiated tumor endothelium may improve efficacy of radio-chemotherapy regimens.
Author Disclosure: H. Wang, None; A. Fu, None; Z. Han, None; D. Hallahan, None.
2705
Inhibition of PI3K/Akt Signaling Impairs DNA Repair in Glioblastoma Cells Following Ionizing Radiation
A. Maity, Z. Jiang, A. Fernandes, A. K. Gupta, G. D. Kao Univ. of Pennsylvania School of Medicine, Philadelphia, PA Purpose/Objective(s): Radiation therapy is a mainstay in the treatment of glioblastomas; nonetheless, these tumors generally recur locally. Activation of the PI3K/Akt pathway, which occurs frequently in glioblastomas due to loss of the tumor suppressor PTEN, correlates with radioresistance. To directly test the link between PTEN expression and radioresistance, we utilized a derivative of PTEN-deficient U251 glioblastoma cells engineered to inducibly express PTEN protein upon exposure to doxycycline (U251PTEN cells). A counterpart cell line, U251-C124S, in which catalytically inactive (phosphatase-dead) PTEN (cysteine 124 mutated to serine) is expressed in response to doxycycline was used as a control. Materials/Methods: Clonogenic radiation cell survival assays, immunofluorescence for gamma-H2AX foci, and Western blotting were performed. Results: U251-PTEN cells showed high basal levels of activation of Akt (i.e. high levels of phospho-Akt), but induction of PTEN led to substantially decreased Akt activation and was associated with radiosensitization. To investigate whether the PTEN-induced radiosensitization was attributable to impaired sensing versus repair of DNA damage, we assessed numbers of gamma-H2AX foci after ionizing radiation in uninduced cells versus cells induced for PTEN. Initial post-radiation levels of gamma-H2AX were not decreased in PTEN-induced cells; however, the resolution of the gamma-H2AX foci was significantly delayed. In contrast to these results, induction of phosphatase-dead PTEN protein in U251-C124S cells showed no appreciable effect on either radiosensitivity or resolution of gamma-H2AX foci. Finally, exposure of cells to the PI3K inhibitor LY294002 also markedly delayed resolution of gamma-H2AX after irradiation, similar to that observed with PTEN induction. Conclusions: Together these results support a direct link between PTEN loss, Akt activation, repair of DNA damage, and radioresistance in glioblastoma. Targeting Akt activation may modulate DNA repair to improve the efficacy of radiation therapy. Author Disclosure: A. Maity, None; Z. Jiang, None; A. Fernandes, None; A.K. Gupta, None; G.D. Kao, None.
2706
Noscapine Enhances Tumor Radioresponse in the GL261 Glioma Model: Implications for Glioma Therapy
Y. Lukyanov1, E. W. Newcomb1, M. Alonso-Basanta1, T. Schnee1, Y. Shao1, W. H. McBride2, S. C. Formenti1 1
New York University School of Medicine, New York, NY, 2University of California, Los Angeles, Los Angeles, CA
Purpose/Objective(s): There is a pressing need for adjunctive therapies to enhance the effectiveness of radiotherapy in high grade glioma. Recently we showed that a novel microtubule binding agent, noscapine, demonstrated antiangiogenic activity in vitro (Newcomb et al., Int J Oncol 2006;28:1121–30). When compared to taxane noscapine has a low toxicity profile and similar antiangiogenic properties, and has demonstrated antitumor activity in several animal models of cancer, We tested whether noscapine enhances the effects of ionizing radiation in the GL261 syngeneic murine glioma tumor model.
S591
I. J. Radiation Oncology d Biology d Physics
S592
Volume 69, Number 3, Supplement, 2007
Materials/Methods: Mice with established hind limb GL261 tumors were randomized into four treatment groups (5 mice/group) when tumors reached 7–8 mm-diameter in size: 1) sham treatment; 2) noscapine (150 mg/kg by gavage once daily); 3) a single fraction of radiation (25 Gy): 4 the combination of radiation and noscapine. Radiation was delivered to the hindlimb of anesthetized mice positioned on a dedicated plexiglass tray using a 60Co source, with the rest of the body blocked by lead. Noscapine treatment was initiated 3 days prior to radiotherapy to allow for an effective plasma concentration before the delivery of radiation. Tumor growth/response was measured twice weekly until tumor volume reached approximately 4000 mm3, at which point mice were sacrificed. Results: Nocapine treatment enhanced the antitumor effect of radiotherapy on the growth of the GL261 tumors. There was no significant difference between control and noscapine only treatment groups, with tumor’ mean volume of 1965 ± 503 mm3 on day 4 in controls and 1965 ± 486 mm3 on day 8 in noscapine treated mice. Radiation treatment alone produced a growth delay of about one week, with mice having tumors of 2167 ± 784 mm3 mean volume on day 14. The combination of radiation plus noscapine produced the most significant growth delay. The mean volume of the tumors treated with radiation plus noscapine was 2214 ± 469 mm3 on day 23. The mean survival time was 10 ± 1.5 days for the control group, 14 ± 2.1 days for the noscapine group, 21 ± 6.2 days for the radiation group, and 33 ± 6.1 days for the radiation plus noscapine group. The difference in overall survival in the combination group compared to control groups was significant (p \ 0.0001, Log rank test). To further analyze the antitumor effects of the different treatments, the time in days for tumors needed to reach a four-fold volume increase (500–2000 mm3) was determined. This time was 7.7 ± 0.8 days for the control group 8.4 ± 1.2 days for the noscapine group, 10.9 ± 1.4 days for the irradiation group, and 18.3 ± 4.9 days for the noscapine plus irradiation group. The difference in growth delay between the combination versus single treatments was significant (p \ 0.01, Student’s t test). Conclusions: Noscapine effectively enhanced tumor radioresponse in the GL261 glioma model. Author Disclosure: Y. Lukyanov, None; E.W. Newcomb, None; M. Alonso-Basanta, None; T. Schnee, None; Y. Shao, None; W.H. McBride, None; S.C. Formenti, None.
2707
Modulation of Therapeutic Sensitivity by the ATR Kinase
F. Bunz, D. Wilsker, P. J. Hurley, N. Sangster-Guity Johns Hopkins University School of Medicine, Baltimore, MD There has been considerable interest in the components of the DNA damage signaling network as potential targets for new forms of anti-cancer therapy. We have examined the contributions of the ataxia telangiectasia mutated- and rad3-related (ATR) protein kinase, to the resistance of cancer cells to ionizing radiation and to DNA damaging agents that are commonly employed as anticancer therapeutics. The biallelic disruption of ATR in human cancer cells strikingly enhanced the effects of several of the most commonly used therapeutic agents, concurrently impeding the progression of the cell cycle and reducing long-term cell survival. Most cancer cells intrinsically have defective responses to DNA damage. It is believed that these alterations, acquired during tumorigenesis, underlie the efficacy of current treatment modalities. There are many signaling molecules that could be targeted to potentially enhance the cell killing effect of DNA damaging agents; few of these potential targets have been validated in a rigorous genetic fashion. At the core of the DNA damage signaling network is a two-tiered protein kinase cascade, in which upstream signals from the phosphoinositide-3-kinase-like kinases are transduced to and amplified by the checkpoint kinases, Chk1 and Chk2. Chk1 has been a major focus of interest in the attempt to abrogate DNA damage signaling in cancer cells. Encouraging progress with Chk1 inhibitors has stimulated studies of additional targets. One promising drug target is ATR. ATR is upstream of Chk1 and signals directly to Chk1 in response to diverse forms of DNA damage. In our recent studies, we used ATR-mutant knockin cells that express very low levels of ATR to comparatively evaluate the roles of ATR in cell survival and cell cycle progression following treatment with a broad panel of widely used therapeutic agents. By employing these engineered cancer cells, we have shown that ATR promotes cell cycle progression in the presence of diverse forms of DNA damage, and that a loss of this activity leads to a form of cell cycle arrest known as S phase stasis. The induction of S phase stasis was associated with diminished clonogenic survival. Using a defined genetic system, rather than siRNA or nonspecific chemical inhibitors, is advantageous because ATR expression is not variable between experiments, making quantification and comparison of drug effects possible. We determined that ATR-mutant cancer cells displayed a marked sensitivity to DNA damaging agents and DNA synthesis inhibitors, but not to agents that impede growth by other mechanisms. Additionally, the degree of sensitization depended on the type of drug used and the type of DNA lesion imparted. ATR-mutant cells were most sensitive to agents that cause interstrand DNA crosslinks and were more moderately sensitized to agents that inhibit nucleotide synthesis or generate double strand DNA breaks. Author Disclosure: F. Bunz, None; D. Wilsker, None; P.J. Hurley, None; N. Sangster-Guity, None.
2708
Single-Dose Radiation Stimulates Induction of Stromal Derived Factor-1 Expression and Endothelial Cell Migration Independently of Hypoxia Activation
O. Lerman, M. Grieves, J. Levine, R. Schneider, S. Formenti New York University Medical Center, New York, NY Purpose/Objective(s): A component of the inflammatory response to ionizing radiation (IR) includes a pro-angiogenic effect. A pro-angiogenic effect of radiation therapy could be counterproductive in cancer but it can be exploited for treating impaired wound healing due to vascular insufficiency or ischemia. Here we demonstrate for the first time that ionizing radiation stimulates stromalcell derived factor-1 (SDF-1) production in endothelial cells, in a hypoxia inducible factor-1 (HIF-1) independent manner and mediates endothelial migration, an essential function for angiogenesis. Materials/Methods: Human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC) were grown in EGM-2 media (Clonetics). Cells were irradiated using a Varian 2300 Linear Accelerator using 6Mv photons, at a dose rate 200 cGy/min. SDF levels were quantified using the human SDF Quantikine ELISA (R&D). siRNAs were developed against HIF-1 and transfected into HUVEC with standard methods. Migration assays were conducted using a ChemoTx system. Data are expressed as the mean SEM. Data were analyzed with an unpaired two-tailed Student’s t-test or analysis of variance (ANOVA)
Author Disclosure: H. Wang, None; A. Fu, None; Z. Han, None; D. Hallahan, None.
2705
Inhibition of PI3K/Akt Signaling Impairs DNA Repair in Glioblastoma Cells Following Ionizing Radiation
A. Maity, Z. Jiang, A. Fernandes, A. K. Gupta, G. D. Kao Univ. of Pennsylvania School of Medicine, Philadelphia, PA Purpose/Objective(s): Radiation therapy is a mainstay in the treatment of glioblastomas; nonetheless, these tumors generally recur locally. Activation of the PI3K/Akt pathway, which occurs frequently in glioblastomas due to loss of the tumor suppressor PTEN, correlates with radioresistance. To directly test the link between PTEN expression and radioresistance, we utilized a derivative of PTEN-deficient U251 glioblastoma cells engineered to inducibly express PTEN protein upon exposure to doxycycline (U251PTEN cells). A counterpart cell line, U251-C124S, in which catalytically inactive (phosphatase-dead) PTEN (cysteine 124 mutated to serine) is expressed in response to doxycycline was used as a control. Materials/Methods: Clonogenic radiation cell survival assays, immunofluorescence for gamma-H2AX foci, and Western blotting were performed. Results: U251-PTEN cells showed high basal levels of activation of Akt (i.e. high levels of phospho-Akt), but induction of PTEN led to substantially decreased Akt activation and was associated with radiosensitization. To investigate whether the PTEN-induced radiosensitization was attributable to impaired sensing versus repair of DNA damage, we assessed numbers of gamma-H2AX foci after ionizing radiation in uninduced cells versus cells induced for PTEN. Initial post-radiation levels of gamma-H2AX were not decreased in PTEN-induced cells; however, the resolution of the gamma-H2AX foci was significantly delayed. In contrast to these results, induction of phosphatase-dead PTEN protein in U251-C124S cells showed no appreciable effect on either radiosensitivity or resolution of gamma-H2AX foci. Finally, exposure of cells to the PI3K inhibitor LY294002 also markedly delayed resolution of gamma-H2AX after irradiation, similar to that observed with PTEN induction. Conclusions: Together these results support a direct link between PTEN loss, Akt activation, repair of DNA damage, and radioresistance in glioblastoma. Targeting Akt activation may modulate DNA repair to improve the efficacy of radiation therapy. Author Disclosure: A. Maity, None; Z. Jiang, None; A. Fernandes, None; A.K. Gupta, None; G.D. Kao, None.
2706
Noscapine Enhances Tumor Radioresponse in the GL261 Glioma Model: Implications for Glioma Therapy
Y. Lukyanov1, E. W. Newcomb1, M. Alonso-Basanta1, T. Schnee1, Y. Shao1, W. H. McBride2, S. C. Formenti1 1
New York University School of Medicine, New York, NY, 2University of California, Los Angeles, Los Angeles, CA
Purpose/Objective(s): There is a pressing need for adjunctive therapies to enhance the effectiveness of radiotherapy in high grade glioma. Recently we showed that a novel microtubule binding agent, noscapine, demonstrated antiangiogenic activity in vitro (Newcomb et al., Int J Oncol 2006;28:1121–30). When compared to taxane noscapine has a low toxicity profile and similar antiangiogenic properties, and has demonstrated antitumor activity in several animal models of cancer, We tested whether noscapine enhances the effects of ionizing radiation in the GL261 syngeneic murine glioma tumor model.
S591
I. J. Radiation Oncology d Biology d Physics
S592
Volume 69, Number 3, Supplement, 2007
Materials/Methods: Mice with established hind limb GL261 tumors were randomized into four treatment groups (5 mice/group) when tumors reached 7–8 mm-diameter in size: 1) sham treatment; 2) noscapine (150 mg/kg by gavage once daily); 3) a single fraction of radiation (25 Gy): 4 the combination of radiation and noscapine. Radiation was delivered to the hindlimb of anesthetized mice positioned on a dedicated plexiglass tray using a 60Co source, with the rest of the body blocked by lead. Noscapine treatment was initiated 3 days prior to radiotherapy to allow for an effective plasma concentration before the delivery of radiation. Tumor growth/response was measured twice weekly until tumor volume reached approximately 4000 mm3, at which point mice were sacrificed. Results: Nocapine treatment enhanced the antitumor effect of radiotherapy on the growth of the GL261 tumors. There was no significant difference between control and noscapine only treatment groups, with tumor’ mean volume of 1965 ± 503 mm3 on day 4 in controls and 1965 ± 486 mm3 on day 8 in noscapine treated mice. Radiation treatment alone produced a growth delay of about one week, with mice having tumors of 2167 ± 784 mm3 mean volume on day 14. The combination of radiation plus noscapine produced the most significant growth delay. The mean volume of the tumors treated with radiation plus noscapine was 2214 ± 469 mm3 on day 23. The mean survival time was 10 ± 1.5 days for the control group, 14 ± 2.1 days for the noscapine group, 21 ± 6.2 days for the radiation group, and 33 ± 6.1 days for the radiation plus noscapine group. The difference in overall survival in the combination group compared to control groups was significant (p \ 0.0001, Log rank test). To further analyze the antitumor effects of the different treatments, the time in days for tumors needed to reach a four-fold volume increase (500–2000 mm3) was determined. This time was 7.7 ± 0.8 days for the control group 8.4 ± 1.2 days for the noscapine group, 10.9 ± 1.4 days for the irradiation group, and 18.3 ± 4.9 days for the noscapine plus irradiation group. The difference in growth delay between the combination versus single treatments was significant (p \ 0.01, Student’s t test). Conclusions: Noscapine effectively enhanced tumor radioresponse in the GL261 glioma model. Author Disclosure: Y. Lukyanov, None; E.W. Newcomb, None; M. Alonso-Basanta, None; T. Schnee, None; Y. Shao, None; W.H. McBride, None; S.C. Formenti, None.
2707
Modulation of Therapeutic Sensitivity by the ATR Kinase
F. Bunz, D. Wilsker, P. J. Hurley, N. Sangster-Guity Johns Hopkins University School of Medicine, Baltimore, MD There has been considerable interest in the components of the DNA damage signaling network as potential targets for new forms of anti-cancer therapy. We have examined the contributions of the ataxia telangiectasia mutated- and rad3-related (ATR) protein kinase, to the resistance of cancer cells to ionizing radiation and to DNA damaging agents that are commonly employed as anticancer therapeutics. The biallelic disruption of ATR in human cancer cells strikingly enhanced the effects of several of the most commonly used therapeutic agents, concurrently impeding the progression of the cell cycle and reducing long-term cell survival. Most cancer cells intrinsically have defective responses to DNA damage. It is believed that these alterations, acquired during tumorigenesis, underlie the efficacy of current treatment modalities. There are many signaling molecules that could be targeted to potentially enhance the cell killing effect of DNA damaging agents; few of these potential targets have been validated in a rigorous genetic fashion. At the core of the DNA damage signaling network is a two-tiered protein kinase cascade, in which upstream signals from the phosphoinositide-3-kinase-like kinases are transduced to and amplified by the checkpoint kinases, Chk1 and Chk2. Chk1 has been a major focus of interest in the attempt to abrogate DNA damage signaling in cancer cells. Encouraging progress with Chk1 inhibitors has stimulated studies of additional targets. One promising drug target is ATR. ATR is upstream of Chk1 and signals directly to Chk1 in response to diverse forms of DNA damage. In our recent studies, we used ATR-mutant knockin cells that express very low levels of ATR to comparatively evaluate the roles of ATR in cell survival and cell cycle progression following treatment with a broad panel of widely used therapeutic agents. By employing these engineered cancer cells, we have shown that ATR promotes cell cycle progression in the presence of diverse forms of DNA damage, and that a loss of this activity leads to a form of cell cycle arrest known as S phase stasis. The induction of S phase stasis was associated with diminished clonogenic survival. Using a defined genetic system, rather than siRNA or nonspecific chemical inhibitors, is advantageous because ATR expression is not variable between experiments, making quantification and comparison of drug effects possible. We determined that ATR-mutant cancer cells displayed a marked sensitivity to DNA damaging agents and DNA synthesis inhibitors, but not to agents that impede growth by other mechanisms. Additionally, the degree of sensitization depended on the type of drug used and the type of DNA lesion imparted. ATR-mutant cells were most sensitive to agents that cause interstrand DNA crosslinks and were more moderately sensitized to agents that inhibit nucleotide synthesis or generate double strand DNA breaks. Author Disclosure: F. Bunz, None; D. Wilsker, None; P.J. Hurley, None; N. Sangster-Guity, None.
2708
Single-Dose Radiation Stimulates Induction of Stromal Derived Factor-1 Expression and Endothelial Cell Migration Independently of Hypoxia Activation
O. Lerman, M. Grieves, J. Levine, R. Schneider, S. Formenti New York University Medical Center, New York, NY Purpose/Objective(s): A component of the inflammatory response to ionizing radiation (IR) includes a pro-angiogenic effect. A pro-angiogenic effect of radiation therapy could be counterproductive in cancer but it can be exploited for treating impaired wound healing due to vascular insufficiency or ischemia. Here we demonstrate for the first time that ionizing radiation stimulates stromalcell derived factor-1 (SDF-1) production in endothelial cells, in a hypoxia inducible factor-1 (HIF-1) independent manner and mediates endothelial migration, an essential function for angiogenesis. Materials/Methods: Human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC) were grown in EGM-2 media (Clonetics). Cells were irradiated using a Varian 2300 Linear Accelerator using 6Mv photons, at a dose rate 200 cGy/min. SDF levels were quantified using the human SDF Quantikine ELISA (R&D). siRNAs were developed against HIF-1 and transfected into HUVEC with standard methods. Migration assays were conducted using a ChemoTx system. Data are expressed as the mean SEM. Data were analyzed with an unpaired two-tailed Student’s t-test or analysis of variance (ANOVA)