- Email: [email protected]
7 Deficiency
Proceedings of the 51st Annual ASTRO Meeting for 7 days. For IR dose response, xenografts were injected with R2636, and 6 hrs later 0, 1, 2, 5, 10, or 20 Gy was given. Xenografts were imaged for HSV-1 luciferase expression. For viral particle quantification, xenografts were injected with HSV-1 and 20 Gy given at times prior or after viral injection. Xenografts were quantified for infectious viral particles. Results: In non-irradiated R2636 injected xenografts, HSV-1 luciferase expression (indicating viral replication) increased up to day 4 post injection after which it declined. In general, all irradiated glioma xenografts had higher levels of luciferase. Notably IR given 6 hours post R2636 injection resulted in the highest luciferase expression. We next determined if IR increased viral gene expression increased infectious viral particles within xenografts. Similar to the in-situ imaging results, IR delivered 6 hrs post viral injection resulted in the highest recovery of infectious viral particles. Interestingly, the greatest xenograft regression was also seen with IR given 6 hr post R2636 injection, which correlated directly with luciferase expression. In IR dose response studies, HSV-1 luciferase expression increased with doses of IR from 0 to 5 Gy after which the detection of luciferase leveled off up to 20 Gy. A patient on trial has shown dramatic MRI response to such treatment. Conclusions: We have shown that the interaction of IR with the HSV-1 lytic cycle can be manipulated for a therapeutic gain. In our preclinical model of glioma, the oncolytic potential of attenuated HSV-1 is optimized by delivering IR at a specific time within viral replicative cycle. A clinical trial is underway to show the safety and feasibility of combining IR and oncolytic HSV-1, and we will present an initial patient’s response to combining oncolytic HSV-1 and focally delivered 5 Gy. Author Disclosure: S.J. Advani, None; J.M. Markert, None; R.F. Sood, None; G.Y. Gillespie, None; B. Roizman, None; R. Weichselbaum, None; J.B. Fiveash, None.
2841
Targeting Heat Shock Protein 90 Inhibits Epithelial Mesenchymal Transition, Abrogates IL-6 Signaling, and Sensitizes Hormone-refractory Prostate Cancer to Irradiation
M. Chen, C. Wu, W. Chen Chang Gung Memorial Hospital, Putz City, Taiwan Purpose/Objective(s): Hormone therapy for prostate cancer eventually fails, leading to hormone-refractory disease (HRPC). Given the poor results and survival rates of patients with hormone refractory prostate cancer, new strategies are needed to improve the current therapeutic regimes and/or develop novel treatments. A better understanding of the molecular mechanisms involved in the process of becoming hormone-refractory is clearly needed if we are to develop more rational anti-tumor therapies. Materials/Methods: Three hormone- refractory (HR) cell lines, including 2 androgen receptor (AR)-positive cell lines (established by long term androgen deprivation), and AR-negative PC-3 cells, were used for experiments. Changes in the phenotype, tumor behavior, and signaling pathway in HR were investigated in vitro and in vivo. The effects of AR, IL-6 signaling and HSP90 function on the HR process, including epithelial mesenchymal transition (EMT), were determined by Western blotting, migration assay, measurement of apoptosis, and by the growth kinetics of xenografted tumors. Clonogenic assay and tumor growth retardation were used to investigate the radiosensitizing effect of HSP90 inhibitor. Results: Accelerated tumor growth and increased invasiveness of HR cells were linked with EMT changes, demonstrated by the loss of epithelial marker and an increase in EMT-related transcription factors, including HIF-1a, TGF-b1, and VEGF. Aggressive tumor behavior noted in HR was not induced when AR expression alone was altered in PC-3 and 22RV1-HR cells. Activated IL6R/STAT3 signaling, with only partially dependent on IL-6, plays an important role on HR invasive behavior and treatment resistance. When the increased chaperone function of HSP-90 in HR was inhibited with 17-DMAG, inhibited STAT3 signaling was associated with decreased EMT-related protein expression and slower xenograft tumor growth noted in HR with AR or not. Moreover, HSP90 inhibition attenuated DNA repair, and consequently cells were more likely to die from irradiation, as determined by clonogenic assay and growth delay for HR prostate cancer. The radiosensitization effect induced by HSP90 inhibitor is AR-independent, at least partly. Conclusions: Activated IL-6R/STAT3 signaling and subsequent EMT changes might be the major mechanism responsible to the aggressive behavior of HR. In addition to suppressing tumor growth, blocking HSP-90 may sensitize HR cells to irradiation by compromising DNA-repair. Therefore, HSP-90 inhibitor could be a promising strategy for the treatment of HR prostate cancer. Author Disclosure: M. Chen, None; C. Wu, None; W. Chen, None.
2842
Ionizing Radiation Mediates ER Stress-induced Autophagy via PERK-eIF2a in Caspase 3/7 Deficiency
K. Kim, L. Moretti, D. Jung, B. Lu Vanderbilt University Ingram Cancer Center, Nashville, TN Purpose/Objective(s): Endoplasmic reticulum (ER) stress is caused by the accumulation of unfolded or misfolded proteins which induces activation of ER mediated cell death mechanisms. We have previously shown that caspase-3/7 inhibition resulted in an increase in autophagy and in cancer radiosensitivity both in vitro and in vivo when blocked apoptosis. In the current study, we further investigated the specific mechanism by which radiation triggers autophagy and results in enhanced radiosensitivity in caspase-3/7 deficient cells via the involvement of Endoplasmic Reticulum (ER) stress. Materials/Methods: Clonogenic assay was used to determine radiosensitivity. Protein levels of PERK, eIF2a and Caspase-3 were determined by western blotting. Autophagy was determined with the punctate GFP-LC3 fluorescence, which observed under a confocal fluorescence microscope. siRNA elF2a (mouse), siRNA PERK (mouse), siRNA ATF6a (mouse), siRNA IRE1a (mouse)and siRNA control were transfected with 25nM of siRNAs using Lipofectamine 2000. Results: PERK and eIF2a, a downstream of PERK, regulates radiation-induced autophagy and radiosensitivity in caspase-3/7 double-knockout cells. This enhanced radiosensitization was associated with an increase in characteristic punctate localization of GFPLC3. Irradiation of these cells was found to induce elevated expression of phosphorylated-elf2a, a protein normally cleaved by activated caspase-3. Similar results were seen following administration of tunicamycin (TM), a well known ER stressor. Importantly, we found that the administration of TM with radiation in MCF-7 breast cancer cells, which are lacking functional caspase-3 and relatively resistant to many anti-cancer agents, enhances radiation sensitivity.
S559
I. J. Radiation Oncology d Biology d Physics
S560
Volume 75, Number 3, Supplement, 2009
Conclusions: Our findings reveal ER stress as a novel mechanism of radiation-induced autophagy in caspase-3/7 deficient cells and as a potential strategy to maximize efficiency of radiation therapy in breast cancer. Author Disclosure: K. Kim, None; L. Moretti, None; D. Jung, None; B. Lu, None.
2843
TLC388, a Novel Topotecan Derivative, as a Chemoradiosensitizer
1
S. Hsu , Y. Tseng2, J. Liu2, S. Shih2, C. Ou2, L. Chang2, Y. Chen2, K. Hong1, G. Melillo3, L. Yang4 TLC Biopharmaceuticals, Inc, South San Francisco, CA, 2Taiwan Liposome Company, Ltd., Taipei, Taiwan, 3Science Applications International Corporation–Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD, 4Radiobiology Laboratory, California Pacific Medical Center Research Institute, San Francisco, CA 1
Purpose/Objective(s): TLC388 Hydrochloride (TLC388 HCl) is a novel derivative of Topotecan hydrochloride (TPT HCl). It has the basic molecular structure of TPT with a unique modification in the lactone ring. The intact lactone ring of the camptothecin scaffold is the pharmacologically active moiety. As a consequence, the in vitro studies showed that, when compared with TPT, TLC388 exhibits 2.5-fold increase in Topo I inhibitory potency by DNA relaxation assays and enhanced cytotoxicities in 14 out of 18 tested tumor cell lines. The highest cytotoxity enhancement factor is 116.7 obtained from colon cancer cell line HT29. In addition, a preliminary mechanistic study also provided strong evidences that TLC388 is a potent inhibitor to HIF1 alpha pathway. Materials/Methods: TLC388 also exhibited potent radiosensitizing effects on human cancer cells. To further investigate TLC388 radiosensitization mechanism, we examined if TLC388 could enhance the effect of DNA double strand breaks (DSBs) mediated by radiation. There was a three-fold increase in DSBs by x-ray when cells were treated with TLC388 for 24 hours before radiation. We noted that the repair of DSBs was significantly slower in the group treated with TLC388 plus x-ray radiation than that in the group treated with radiation alone. The data suggest that TLC388 might act as an inhibitor of DSB repair. The inhibitory effects of TLC388 on DSB repair could constitute one of the molecular bases for its strong chemoradiosensitization of H23 human non-small cell lung cancer cells. Results: In the animal studies, the anti-tumor activity of TLC388 was demonstrated in mouse syngeneic model and xenograft model. The values of the Efficacy Enhance Ratio (EER) to TPT obtained from 6 xenograft models range from 1.2 to 3. In addition, the combination of TLC388 with radiation treatments showed a dramatic increase in radiosensitivity of the cancer cells during both in vitro and in vivo studies. This result suggests a potential utility of TLC388 as a chemo-radiosensitizer for cancer treatment. Conclusions: In summary, the non-clinical studies performed to date have shown that TLC388 is a potent chemoradiosensitizing agent for improved cancer therapy. TLC388 has been cleared for Investigative New Drug (IND) filing by US FDA in June 2008 and it is currently in phase 1 trial at several US and Taiwan clinical sites. Author Disclosure: S. Hsu, Shu Chi Hsu, A. Employment; Y. Tseng, Yulong Tseng, A. Employment; J. Liu, Jun-Jen Liu, A. Employment; S. Shih, Sheue-Fang Shih, A. Employment; C. Ou, Ching-Ju Ou, A. Employment; L. Chang, Lo Chang, A. Employment; Y. Chen, Yu-Ru Chen, A. Employment; K. Hong, Keelong Hong, A. Employment; G. Melillo, Giovanni Melillo, C. Other Research Support; L. Yang, Li-Xi Yang, F. Consultant/Advisory Board.
2844
Role of Thioredoxin Reductase 1 in Curcumin-mediated Radiosensitization of Oral Squamous Cell Carcinoma
L. Hertan, P. Javvadi, S. Tuttle, C. Koumenis Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA Purpose/Objectives(s): Thioredoxin Reductase 1 (TxnRd1) is an essential selenoprotein with well-defined roles in redox regulation of cell growth, death (apoptosis), transcription, DNA repair, and angiogenesis. TxnRd1 is overexpressed in many forms of human cancers and elevated levels of TxnRd1 have been associated with poor prognosis due to enhanced cellular resistance to chemo- and radiation therapy. Chemical inhibitors of TxnRd1 are yielding promising results in preclinical studies and clinical trials as cancer therapeutic agents. Curcumin is a naturally occurring plant polyphenolic antioxidant with potent chemopreventive and antitumorigenic properties in preclinical animal models and a demonstrated low toxicity profile in Phase I and II clinical trials. Recently, curcumin was found to bind covalently to the conserved c-terminal cysteine and selenocysteine amino acids in TxnRd family proteins, converting their function from anti-oxidant to pro-oxidant. We are currently examining the role of TxnRd1 in curcumin-induced toxicity and radiosensitization in the oral squamous cell carcinoma lines, FaDu and SCC1, and in a premalignant leukoplakia derived cell line, MSK-Leuk 1. Material/Methods: We have compared TxnRd1 levels in squamous cell lines of different transformation status and we used shRNA to human TxnRd1 to stably knock down TxnRd1 in the FaDu cells. In addition, we have also developed a tetracyclineinducible system expressing TxnRd1 shRNA under the control of doxycycline. Furthermore, we are overexpressing TxnRd1 in the MSK-Leuk 1 cells. Results: Immunoblot analysis demonstrated that TxnRd1 protein levels in FaDu and SCC1 cells were significantly higher compared to levels observed in MSK-Leuk 1 cells or normal keratinocytes. Moreover, curcumin’s ability to radiosensitize exhibited a close correlation with baseline expression of TxnRd1 in these cells. FaDu TxnRd1 knockdown cells were considerably less sensitive to the radiosensitizing effects of curcumin compared to FaDu cells transfected with a non-targeting shRNA. The preliminary results from these experiments suggest that TxnRd1 regulates the radiosensitizing properties of curcumin. Moreover, we are examining whether overexpression of TxnRD1 in MSK-Leuk 1 affects curcumin-mediated radiosensitization. Conclusions: Because curcumin is a natural bioactive food component with no overt systemic toxicities, we expect promising results from our preclinical experiments to be readily translated into a combined chemo-radiation therapy protocol for treating head and neck cancer. Supported by NIH grant CA104922-03. Author Disclosure: L. Hertan, None; P. Javvadi, None; S. Tuttle, None; C. Koumenis, None.
2841
Targeting Heat Shock Protein 90 Inhibits Epithelial Mesenchymal Transition, Abrogates IL-6 Signaling, and Sensitizes Hormone-refractory Prostate Cancer to Irradiation
M. Chen, C. Wu, W. Chen Chang Gung Memorial Hospital, Putz City, Taiwan Purpose/Objective(s): Hormone therapy for prostate cancer eventually fails, leading to hormone-refractory disease (HRPC). Given the poor results and survival rates of patients with hormone refractory prostate cancer, new strategies are needed to improve the current therapeutic regimes and/or develop novel treatments. A better understanding of the molecular mechanisms involved in the process of becoming hormone-refractory is clearly needed if we are to develop more rational anti-tumor therapies. Materials/Methods: Three hormone- refractory (HR) cell lines, including 2 androgen receptor (AR)-positive cell lines (established by long term androgen deprivation), and AR-negative PC-3 cells, were used for experiments. Changes in the phenotype, tumor behavior, and signaling pathway in HR were investigated in vitro and in vivo. The effects of AR, IL-6 signaling and HSP90 function on the HR process, including epithelial mesenchymal transition (EMT), were determined by Western blotting, migration assay, measurement of apoptosis, and by the growth kinetics of xenografted tumors. Clonogenic assay and tumor growth retardation were used to investigate the radiosensitizing effect of HSP90 inhibitor. Results: Accelerated tumor growth and increased invasiveness of HR cells were linked with EMT changes, demonstrated by the loss of epithelial marker and an increase in EMT-related transcription factors, including HIF-1a, TGF-b1, and VEGF. Aggressive tumor behavior noted in HR was not induced when AR expression alone was altered in PC-3 and 22RV1-HR cells. Activated IL6R/STAT3 signaling, with only partially dependent on IL-6, plays an important role on HR invasive behavior and treatment resistance. When the increased chaperone function of HSP-90 in HR was inhibited with 17-DMAG, inhibited STAT3 signaling was associated with decreased EMT-related protein expression and slower xenograft tumor growth noted in HR with AR or not. Moreover, HSP90 inhibition attenuated DNA repair, and consequently cells were more likely to die from irradiation, as determined by clonogenic assay and growth delay for HR prostate cancer. The radiosensitization effect induced by HSP90 inhibitor is AR-independent, at least partly. Conclusions: Activated IL-6R/STAT3 signaling and subsequent EMT changes might be the major mechanism responsible to the aggressive behavior of HR. In addition to suppressing tumor growth, blocking HSP-90 may sensitize HR cells to irradiation by compromising DNA-repair. Therefore, HSP-90 inhibitor could be a promising strategy for the treatment of HR prostate cancer. Author Disclosure: M. Chen, None; C. Wu, None; W. Chen, None.
2842
Ionizing Radiation Mediates ER Stress-induced Autophagy via PERK-eIF2a in Caspase 3/7 Deficiency
K. Kim, L. Moretti, D. Jung, B. Lu Vanderbilt University Ingram Cancer Center, Nashville, TN Purpose/Objective(s): Endoplasmic reticulum (ER) stress is caused by the accumulation of unfolded or misfolded proteins which induces activation of ER mediated cell death mechanisms. We have previously shown that caspase-3/7 inhibition resulted in an increase in autophagy and in cancer radiosensitivity both in vitro and in vivo when blocked apoptosis. In the current study, we further investigated the specific mechanism by which radiation triggers autophagy and results in enhanced radiosensitivity in caspase-3/7 deficient cells via the involvement of Endoplasmic Reticulum (ER) stress. Materials/Methods: Clonogenic assay was used to determine radiosensitivity. Protein levels of PERK, eIF2a and Caspase-3 were determined by western blotting. Autophagy was determined with the punctate GFP-LC3 fluorescence, which observed under a confocal fluorescence microscope. siRNA elF2a (mouse), siRNA PERK (mouse), siRNA ATF6a (mouse), siRNA IRE1a (mouse)and siRNA control were transfected with 25nM of siRNAs using Lipofectamine 2000. Results: PERK and eIF2a, a downstream of PERK, regulates radiation-induced autophagy and radiosensitivity in caspase-3/7 double-knockout cells. This enhanced radiosensitization was associated with an increase in characteristic punctate localization of GFPLC3. Irradiation of these cells was found to induce elevated expression of phosphorylated-elf2a, a protein normally cleaved by activated caspase-3. Similar results were seen following administration of tunicamycin (TM), a well known ER stressor. Importantly, we found that the administration of TM with radiation in MCF-7 breast cancer cells, which are lacking functional caspase-3 and relatively resistant to many anti-cancer agents, enhances radiation sensitivity.
S559
I. J. Radiation Oncology d Biology d Physics
S560
Volume 75, Number 3, Supplement, 2009
Conclusions: Our findings reveal ER stress as a novel mechanism of radiation-induced autophagy in caspase-3/7 deficient cells and as a potential strategy to maximize efficiency of radiation therapy in breast cancer. Author Disclosure: K. Kim, None; L. Moretti, None; D. Jung, None; B. Lu, None.
2843
TLC388, a Novel Topotecan Derivative, as a Chemoradiosensitizer
1
S. Hsu , Y. Tseng2, J. Liu2, S. Shih2, C. Ou2, L. Chang2, Y. Chen2, K. Hong1, G. Melillo3, L. Yang4 TLC Biopharmaceuticals, Inc, South San Francisco, CA, 2Taiwan Liposome Company, Ltd., Taipei, Taiwan, 3Science Applications International Corporation–Frederick, Inc., National Cancer Institute at Frederick, Frederick, MD, 4Radiobiology Laboratory, California Pacific Medical Center Research Institute, San Francisco, CA 1
Purpose/Objective(s): TLC388 Hydrochloride (TLC388 HCl) is a novel derivative of Topotecan hydrochloride (TPT HCl). It has the basic molecular structure of TPT with a unique modification in the lactone ring. The intact lactone ring of the camptothecin scaffold is the pharmacologically active moiety. As a consequence, the in vitro studies showed that, when compared with TPT, TLC388 exhibits 2.5-fold increase in Topo I inhibitory potency by DNA relaxation assays and enhanced cytotoxicities in 14 out of 18 tested tumor cell lines. The highest cytotoxity enhancement factor is 116.7 obtained from colon cancer cell line HT29. In addition, a preliminary mechanistic study also provided strong evidences that TLC388 is a potent inhibitor to HIF1 alpha pathway. Materials/Methods: TLC388 also exhibited potent radiosensitizing effects on human cancer cells. To further investigate TLC388 radiosensitization mechanism, we examined if TLC388 could enhance the effect of DNA double strand breaks (DSBs) mediated by radiation. There was a three-fold increase in DSBs by x-ray when cells were treated with TLC388 for 24 hours before radiation. We noted that the repair of DSBs was significantly slower in the group treated with TLC388 plus x-ray radiation than that in the group treated with radiation alone. The data suggest that TLC388 might act as an inhibitor of DSB repair. The inhibitory effects of TLC388 on DSB repair could constitute one of the molecular bases for its strong chemoradiosensitization of H23 human non-small cell lung cancer cells. Results: In the animal studies, the anti-tumor activity of TLC388 was demonstrated in mouse syngeneic model and xenograft model. The values of the Efficacy Enhance Ratio (EER) to TPT obtained from 6 xenograft models range from 1.2 to 3. In addition, the combination of TLC388 with radiation treatments showed a dramatic increase in radiosensitivity of the cancer cells during both in vitro and in vivo studies. This result suggests a potential utility of TLC388 as a chemo-radiosensitizer for cancer treatment. Conclusions: In summary, the non-clinical studies performed to date have shown that TLC388 is a potent chemoradiosensitizing agent for improved cancer therapy. TLC388 has been cleared for Investigative New Drug (IND) filing by US FDA in June 2008 and it is currently in phase 1 trial at several US and Taiwan clinical sites. Author Disclosure: S. Hsu, Shu Chi Hsu, A. Employment; Y. Tseng, Yulong Tseng, A. Employment; J. Liu, Jun-Jen Liu, A. Employment; S. Shih, Sheue-Fang Shih, A. Employment; C. Ou, Ching-Ju Ou, A. Employment; L. Chang, Lo Chang, A. Employment; Y. Chen, Yu-Ru Chen, A. Employment; K. Hong, Keelong Hong, A. Employment; G. Melillo, Giovanni Melillo, C. Other Research Support; L. Yang, Li-Xi Yang, F. Consultant/Advisory Board.
2844
Role of Thioredoxin Reductase 1 in Curcumin-mediated Radiosensitization of Oral Squamous Cell Carcinoma
L. Hertan, P. Javvadi, S. Tuttle, C. Koumenis Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA Purpose/Objectives(s): Thioredoxin Reductase 1 (TxnRd1) is an essential selenoprotein with well-defined roles in redox regulation of cell growth, death (apoptosis), transcription, DNA repair, and angiogenesis. TxnRd1 is overexpressed in many forms of human cancers and elevated levels of TxnRd1 have been associated with poor prognosis due to enhanced cellular resistance to chemo- and radiation therapy. Chemical inhibitors of TxnRd1 are yielding promising results in preclinical studies and clinical trials as cancer therapeutic agents. Curcumin is a naturally occurring plant polyphenolic antioxidant with potent chemopreventive and antitumorigenic properties in preclinical animal models and a demonstrated low toxicity profile in Phase I and II clinical trials. Recently, curcumin was found to bind covalently to the conserved c-terminal cysteine and selenocysteine amino acids in TxnRd family proteins, converting their function from anti-oxidant to pro-oxidant. We are currently examining the role of TxnRd1 in curcumin-induced toxicity and radiosensitization in the oral squamous cell carcinoma lines, FaDu and SCC1, and in a premalignant leukoplakia derived cell line, MSK-Leuk 1. Material/Methods: We have compared TxnRd1 levels in squamous cell lines of different transformation status and we used shRNA to human TxnRd1 to stably knock down TxnRd1 in the FaDu cells. In addition, we have also developed a tetracyclineinducible system expressing TxnRd1 shRNA under the control of doxycycline. Furthermore, we are overexpressing TxnRd1 in the MSK-Leuk 1 cells. Results: Immunoblot analysis demonstrated that TxnRd1 protein levels in FaDu and SCC1 cells were significantly higher compared to levels observed in MSK-Leuk 1 cells or normal keratinocytes. Moreover, curcumin’s ability to radiosensitize exhibited a close correlation with baseline expression of TxnRd1 in these cells. FaDu TxnRd1 knockdown cells were considerably less sensitive to the radiosensitizing effects of curcumin compared to FaDu cells transfected with a non-targeting shRNA. The preliminary results from these experiments suggest that TxnRd1 regulates the radiosensitizing properties of curcumin. Moreover, we are examining whether overexpression of TxnRD1 in MSK-Leuk 1 affects curcumin-mediated radiosensitization. Conclusions: Because curcumin is a natural bioactive food component with no overt systemic toxicities, we expect promising results from our preclinical experiments to be readily translated into a combined chemo-radiation therapy protocol for treating head and neck cancer. Supported by NIH grant CA104922-03. Author Disclosure: L. Hertan, None; P. Javvadi, None; S. Tuttle, None; C. Koumenis, None.