Dose Enhancement Ratio Research Articles

Theranostic Gold Nanoparticles Modified for Durable Systemic Circulation Effectively and Safely Enhance the Radiation Therapy of Human Sarcoma Cells and Tumors

Radiation therapy (RT) is an integral component of the treatment of many sarcomas and relies on accurate targeting of tumor tissue. Despite conventional treatment planning and RT, local failure rates of 10% to 28% at 5 years have been reported for locally advanced, unresectable sarcomas, due in part to limitations in the cumulative RT dose that may be safely delivered. We describe studies of the potential usefulness of gold nanoparticles modified for durable systemic circulation (through polyethylene glycosylation; hereinafter "P-GNPs") as adjuvants for RT of sarcomas. In studies of two human sarcoma-derived cell lines, P-GNP in conjunction with RT caused increased unrepaired DNA damage, reflected by approximately 1.61-fold increase in γ-H2AX (histone phosphorylated on Ser(139)) foci density compared with RT alone. The combined RT and P-GNP also led to significantly reduced clonogenic survival of tumor cells, compared to RT alone, with dose-enhancement ratios of 1.08 to 1.16. In mice engrafted with human sarcoma tumor cells, the P-GNP selectively accumulated in the tumor and enabled durable imaging, potentially aiding radiosensitization as well as treatment planning. Mice pretreated with P-GNP before targeted RT of their tumors exhibited significantly improved tumor regression and overall survival, with long-term survival in one third of mice in this treatment group compared to none with RT only. Interestingly, prior RT of sarcoma tumors increased subsequent extravasation and in-tumor deposition of P-GNP. These results together suggest P-GNP may be integrated into the RT of sarcomas, potentially improving target imaging and radiosensitization of tumor while minimizing dose to normal tissues.

Dependence of Monte Carlo microdosimetric computations on the simulation geometry of gold nanoparticles

Recently, interactions of x-rays with gold nanoparticles (GNPs) and the resulting dose enhancement have been studied using several Monte Carlo (MC) codes (Jones et al 2010 Med. Phys. 37 3809–16, Lechtman et al 2011 Phys. Med. Biol. 56 4631–47, McMahon et al 2011 Sci. Rep. 1 1–9, Leung et al 2011 Med. Phys. 38 624–31). These MC simulations were carried out in simplified geometries and provided encouraging preliminary data in support of GNP radiotherapy. As these studies showed, radiation transport computations of clinical beams to obtain dose enhancement from nanoparticles has several challenges, mostly arising from the requirement of high spatial resolution and from the approximations used at the interface between the macroscopic clinical beam transport and the nanoscopic electron transport originating in the nanoparticle or its vicinity. We investigate the impact of MC simulation geometry on the energy deposition due to the presence of GNPs, including the effects of particle clustering and morphology. Dose enhancement due to a single and multiple GNPs using various simulation geometries is computed using GEANT4 MC radiation transport code. Various approximations in the geometry and in the phase space transition from macro- to micro-beams incident on GNPs are analyzed. Simulations using GEANT4 are compared to a deterministic code CEPXS/ONEDANT for microscopic (nm–µm) geometry. Dependence on the following microscopic (µ) geometry parameters is investigated: µ-source-to-GNP distance (µSAD), µ-beam size (µS), and GNP size (µC). Because a micro-beam represents clinical beam properties at the microscopic scale, the effect of using different types of micro-beams is also investigated. In particular, a micro-beam with the phase space of a clinical beam versus a plane-parallel beam with an equivalent photon spectrum is characterized. Furthermore, the spatial anisotropy of energy deposition around a nanoparticle is analyzed. Finally, dependence of dose enhancement on the number of GNPs in a finite cluster of nanoparticles is determined. Simulations were performed for 100 nm GNPs irradiated in water phantom by various monoenergetic (11 keV–1 MeV) and spectral (50 kVp) sources. The dose enhancement ratio (DER) is very sensitive to the specific simulation geometry (µSAD, µS, µC parameters) and µ-source type. For a single GNP the spatial distribution of DER is found to be nearly isotropic with limited magnitude and relatively short range (∼100–200 nm for DER significantly greater than 1). For a cluster of GNPs both the magnitude and range are found much greater (∼1–2 µm). The relation between DER for a cluster of GNPs and a single GNP is strongly nonlinear. Relatively strong dependence of DER on the simulation micro-geometry cautions future studies and the interpretation of existing MC results obtained in different simulations geometries. The nonlinear relation between DER for a single and multiple GNPs suggests that parameters such as the number of adjacent nanoparticles per cell and the distances between the GNPs and the cellular target may be important in assessing the biological effectiveness associated with GNP.

Monte Carlo investigation of the increased radiation deposition due to gold nanoparticles using kilovoltage and megavoltage photons in a 3D randomized cell model

Investigation of increased radiation dose deposition due to gold nanoparticles (GNPs) using a 3D computational cell model during x-ray radiotherapy. Two GNP simulation scenarios were set up in Geant4; a single 400 nm diameter gold cluster randomly positioned in the cytoplasm and a 300 nm gold layer around the nucleus of the cell. Using an 80 kVp photon beam, the effect of GNP on the dose deposition in five modeled regions of the cell including cytoplasm, membrane, and nucleus was simulated. Two Geant4 physics lists were tested: the default Livermore and custom built Livermore/DNA hybrid physics list. 10(6) particles were simulated at 840 cells in the simulation. Each cell was randomly placed with random orientation and a diameter varying between 9 and 13 μm. A mathematical algorithm was used to ensure that none of the 840 cells overlapped. The energy dependence of the GNP physical dose enhancement effect was calculated by simulating the dose deposition in the cells with two energy spectra of 80 kVp and 6 MV. The contribution from Auger electrons was investigated by comparing the two GNP simulation scenarios while activating and deactivating atomic de-excitation processes in Geant4. The physical dose enhancement ratio (DER) of GNP was calculated using the Monte Carlo model. The model has demonstrated that the DER depends on the amount of gold and the position of the gold cluster within the cell. Individual cell regions experienced statistically significant (p < 0.05) change in absorbed dose (DER between 1 and 10) depending on the type of gold geometry used. The DER resulting from gold clusters attached to the cell nucleus had the more significant effect of the two cases (DER ≈ 55). The DER value calculated at 6 MV was shown to be at least an order of magnitude smaller than the DER values calculated for the 80 kVp spectrum. Based on simulations, when 80 kVp photons are used, Auger electrons have a statistically insignificant (p < 0.05) effect on the overall dose increase in the cell. The low energy of the Auger electrons produced prevents them from propagating more than 250-500 nm from the gold cluster and, therefore, has a negligible effect on the overall dose increase due to GNP. The results presented in the current work show that the primary dose enhancement is due to the production of additional photoelectrons.

SU‐E‐T‐255: Monte Carlo Quantification of Dose Distributions Around Thin Lead Foils

Purpose: Patients are increasingly undergoing radiotherapy procedures, in which small metals are implanted in the body for target localization in IGRT or dose enhancement. Previously we proposed the polymer gel dosimetry technique for measurement of 3D dose distributions around small metallic materials including thin foils. The purpose of this study is to determine the effect of separation between thin metallic objects and 3D dose distributions using a Monte Carlo method and to provide theoretical data which will be compared with the measurement data. Methods: DOSxyz was used to perform Monte Carlo simulations of a two lead foil setup. The Mohan 24MV spectrum was used as a radiation source with 5cm x 5cm collimation at a 100 SSD. The foils had dimensions 1cm × 1cm × 0.5mm and had separations of 2mm, 5mm, 7mm, 10mm and 15mm. The phantom size was 16cm × 16cm × 28cm. Simulations were performed on custom Intel I7 machines with various histories (100 million to 1 billion). Percent depth dose curves were determined, then the minimum dose between the foils was recorded and compared to the dose without the foil. The plots were normalized against the maximum dose point. Results: The minimum dose enhancement ratios between two foils were 1.47, 1.29, 1.18, 1.13 and 1.08 for the separation of 2, 5, 7, 10, and 15 mm, respectively. The dose enhancement ratio exponentially decreased with the separation. The Monte Carlo simulation results were in agreement with experimental data previously obtained with films. Conclusion: The degree of dose enhancement around thin lead foils was determined by the Monte Carlo method. It is expected that other high Z materials such as gold will behave in similar manner. The results would be used to identify optimal placement of materials for measurement by a polymer‐gel dosimeter.

ALK Inhibitor PF02341066 (Crizotinib) Increases Sensitivity to Radiation in Non–Small Cell Lung Cancer Expressing EML4-ALK

Crizotinib (PF02341066) is a tyrosine kinase inhibitor of anaplastic lymphoma kinase (ALK) that has been shown to selectively inhibit growth of cancer cells that harbor the EML4-ALK fusion found in a subset of patients with non-small cell lung cancer (NSCLC). While in clinical trials, PF02341066 has shown a significant therapeutic benefit as a single agent; the effectiveness of combining it with other therapeutic modalities including ionizing radiation remains unknown. To further elucidate the role of PF02341066 in tumor inhibition, we examined its effects alone and in combination with radiation on downstream signaling, apoptosis, and radiosensitivity in two NSCLC cell lines in vitro: H3122, which harbors the EML4-ALK fusion, and H460, which does not. We also examined the in vivo effects of PF02341066 in H3122 mouse xenografts. In the H3122 cell line, PF02341066 inhibited phosphorylation of ALK and its downstream effectors: AKT, ERK, and STAT3. H3122 cells treated with a combination of PF02341066 and radiation showed an increase in cellular apoptosis and were sensitized to radiation therapy (dose enhancement ratio, 1.43; P < 0.0001). Moreover, in an H3122 xenograft model, the combined treatment resulted in greater tumor growth inhibition than either treatment alone (P < 0.05). None of these effects was observed in the EML4-ALK-negative H460 cells. Our findings indicate that PF02341066 acts as a radiation sensitizer in cells harboring the EML4-ALK fusion, providing a rationale for a clinical trial combining ALK inhibitor with radiation in the NSCLCs expressing ALK.

Selective Targeting of Brain Tumors with Gold Nanoparticle-Induced Radiosensitization

Successful treatment of brain tumors such as glioblastoma multiforme (GBM) is limited in large part by the cumulative dose of Radiation Therapy (RT) that can be safely given and the blood-brain barrier (BBB), which limits the delivery of systemic anticancer agents into tumor tissue. Consequently, the overall prognosis remains grim. Herein, we report our pilot studies in cell culture experiments and in an animal model of GBM in which RT is complemented by PEGylated-gold nanoparticles (GNPs). GNPs significantly increased cellular DNA damage inflicted by ionizing radiation in human GBM-derived cell lines and resulted in reduced clonogenic survival (with dose-enhancement ratio of ∼1.3). Intriguingly, combined GNP and RT also resulted in markedly increased DNA damage to brain blood vessels. Follow-up in vitro experiments confirmed that the combination of GNP and RT resulted in considerably increased DNA damage in brain-derived endothelial cells. Finally, the combination of GNP and RT increased survival of mice with orthotopic GBM tumors. Prior treatment of mice with brain tumors resulted in increased extravasation and in-tumor deposition of GNP, suggesting that RT-induced BBB disruption can be leveraged to improve the tumor-tissue targeting of GNP and thus further optimize the radiosensitization of brain tumors by GNP. These exciting results together suggest that GNP may be usefully integrated into the RT treatment of brain tumors, with potential benefits resulting from increased tumor cell radiosensitization to preferential targeting of tumor-associated vasculature.

Abstract 1597: C646, a selective small molecule inhibitor of p300, radiosensitizes cancer cells through enhancement of mitotic catastrophe.

Abstract Background: The histone acetyltransferases (HATs) including p300, CBP and TIP60 are transcriptional co-activators implicated in many gene regulatory pathways and protein acetylation events. We have previously shown that siRNA-mediated ablation of p300 and CBP sensitized cancer cells to ionizing radiation (IR) (Ogiwara, Oncogene 2011), and that garcinol, curcumin and anacardic acid, natural compounds with multi-HATs inhibitory activity, had radiosensitizing effect in cancer cells (Oike, Int J Radiat Oncol Biol Phys 2012). However, radiosensitization by selective small molecule inhibitors targeting activity of a specific HAT, that can have much potential for clinical use, have not been well investigated. C646 is a selective small molecule inhibitor of p300 identified by structure-based and active site-directed in silico screening (Bowers, Chem Biol 2010). In this study, we investigated the in vitro effect of C646 on radiosensitization and cell death in human cancer cells. Materials and Methods: The radiosensitizing effect in A549 (lung adenocarcinoma), H460 (lung squamous cell carcinoma) and H157 (lung large cell carcinoma) cells were assessed by colony formation assay, while that in MRC-5 (fibroblast) cells lacking in colony-forming ability was examined by ATP assay. Cell cycle distribution in A549 cells was analyzed using flow cytometry. The effect on mitotic catastrophe, apoptosis and senescence in A549 cells was evaluated by DAPI, annexin V and senescence-associated β-galactosidase staining, respectively. C646 was used at concentrations less than IC50. Results: C646 radiosensitized A549, H460 and H157 cancer cells with dose enhancement ratio at 10% surviving fraction of 1.4, 1.2 and 1.2, respectively. Meanwhile, C646 unaffected the radiosensitivity of MRC-5 cells. Cell cycle analysis showed that the hyperploid cell population (&amp;gt; 4N DNA cells) increased in C646-treated A549 cells after IR. Analyses of mode of cell death revealed that C646 enhanced mitotic catastrophe, but not apoptosis nor senescence, induced by IR in A549 cells. Conclusion: These data indicate that C646 is a radiosensitizer which enhances mitotic catastrophe induced by IR. Citation Format: Takahiro Oike, Mayumi Komachi, Hideaki Ogiwara, Jun Yokota, Takashi Nakano, Takashi Kohno. C646, a selective small molecule inhibitor of p300, radiosensitizes cancer cells through enhancement of mitotic catastrophe. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1597. doi:10.1158/1538-7445.AM2013-1597

Impact of beam quality on megavoltage radiotherapy treatment techniques utilizing gold nanoparticles for dose enhancement

This study determines the optimal clinical scenarios for gold nanoparticle dose enhancement as a function of irradiation conditions and potential biological targets using megavoltage x-ray beams. Four hundred and eighty clinical beams were studied for different potential cellular or sub-cellular targets. Beam quality was determined based on a 6 MV linac with and without a flattening filter for various delivery conditions. Dose enhancement ratios DER = DGNP/Dwater were calculated for all cases using the GEANT4 Monte Carlo code and the CEPXS/ONEDANT radiation transport deterministic code. Dose enhancement using GEANT4 agreed with CEPXS/ONEDANT. DER for unflattened beams is ∼2 times larger than for flattened beams. The maximum DER values were calculated for split-IMRT fields (∼6) and for out-of-field areas of an unflattened linac (∼17). In-field DER values, at the surface of gold nanoparticles, ranged from 2.2 to 4.2 (flattened beam) and from 3 to 4.7 (unflattened beams). For a GNP cluster with thicknesses of 10 and 100 nm, the DER ranges from 14% to 287%. DER is the greatest for split-IMRT, larger depths, out-of-field areas and/or unflattened linac. Mapping of a GNP location in tumor and normal tissue is essential for efficient and safe delivery of nanoparticle-enhanced radiotherapy.

AZD5438, an Inhibitor of Cdk1, 2, and 9, Enhances the Radiosensitivity of Non-Small Cell Lung Carcinoma Cells

Radiation therapy (RT) is one of the primary modalities for treatment of non-small cell lung cancer (NSCLC). However, due to the intrinsic radiation resistance of these tumors, many patients experience RT failure, which leads to considerable tumor progression including regional lymph node and distant metastasis. This preclinical study evaluated the efficacy of a new-generation cyclin-dependent kinase (Cdk) inhibitor, AZD5438, as a radiosensitizer in several NSCLC models that are specifically resistant to conventional fractionated RT. The combined effect of ionizing radiation and AZD5438, a highly specific inhibitor of Cdk1, 2, and 9, was determined invitro by surviving fraction, cell cycle distribution, apoptosis, DNA double-strand break (DSB) repair, and homologous recombination (HR) assays in 3 NSCLC cell lines (A549, H1299, and H460). For invivo studies, human xenograft animal models in athymic nude mice were used. Treatment of NSCLC cells with AZD5438 significantly augmented cellular radiosensitivity (dose enhancement ratio rangeing from 1.4 to 1.75). The degree of radiosensitization by AZD5438 was greater in radioresistant cell lines (A549 and H1299). Radiosensitivity was enhanced specifically through inhibition of Cdk1, prolonged G(2)-M arrest, inhibition of HR, delayed DNA DSB repair, and increased apoptosis. Combined treatment with AZD5438 and irradiation also enhanced tumor growth delay, with an enhancement factor ranging from 1.2-1.7. This study supports the evaluation of newer generation Cdk inhibitors, such as AZD5438, as potent radiosensitizers in NSCLC models, especially in tumors that demonstrate variable intrinsic radiation responses.

TU-C-BRB-11: In Vitro Dose Enhancement from Gold Nanoparticles under Different Clinical MV Photon Beam Configurations

Purpose: To quantify the relative in vitro dose enhancement due to the presence of goldnanoparticles (GNPs) in a clinical 6 MV beam. It is expected that depths and delivery modes that produce larger proportions of low energy photons will have a larger effect on cell samples containing GNP. Methods: HeLa cells were combined with 50 nm GNPs at a concentration of 0 or 0.05 mg/ml. The cells were irradiated in clinical solid water at depths of 1.5, 5, 10, 15 and 20 cm (SAD setup). Conventional beams with square aperture sizes 5, 10 and 15 cm at isocenter, IMRT and flattening filter free (FFF) beams were used. DNA double strand breaks (DSBs) were evaluated by H2AX staining. Results: Statistically significant dose enhancement was observed for all depths and delivery modes. Dose enhancement ratios varied between 1.1 and 1.7. Relative to the shallowest depth, dose enhancement was observed to increase as a function of increasing depth for all deliveries. For the conventional (open field) delivery, dose enhancement was seen to increase as a function of field size. The IMRT delivery chosen for this study did not significantly increase the dose enhancement at each depth, but did in the aggregate. For FFF delivery, a substantial increase in gH2AX foci was found, relative to the conventional field delivery. Without GNP, no enhancement was observed as a function of depth, indicating that the enhancing effect with GNP is due to the change in energy spectrum under each delivery condition. Conclusions: The measured relative dose enhancement validates the theoretically predicted trends in dose enhancement as a function of depth and delivery mode for a clinical MV beam. The results of this study open new possibilities for the clinical development of goldnanoparticle aided radiation therapy.

SU-E-T-35: Optimal Clinical Megavoltage X-Ray Beam Quality for Contrast Enhanced RT (CERT)

Purpose: To determine the optimal beam quality of MV-x-rays for Contrast Enhanced RT (CERT) based on analysis of a broad database of clinical beams. Methods: EGSnrc was used to calculate spectra and doses for various irradiation conditions (6MV EX linac): flattened (STD) vs unflattened (FFF) beams, open/IMRT fields, in-/out-off-field areas as a function of depth in water and field size. Spectral differences were quantified and related to dose enhancement (DE) effects by introducing two metrics: an energy-dependent dose enhancement ratio DER(E) and an effective DER for the entire spectrum. Using these two metrics the dosimetric impact of spectral changes were studied for different materials (gadolinium-oxysulfide (Gd2O2S) and gold). Results: DER proved to be a good predictor for high-DE effects for the whole spectrum of each beam. However, the DER(E) metric revealed energy-specific effects hidden in the overall analysis. Spectral analysis with DER(E) showed that all DE effects are directly related to the spectral region <∼200KeV. The highest DE effects appear for FFF spectra and out-of-field cases. Differences in DER values can reach over one order of magnitude, depending on the case and the heterogeneity of the medium. For shallower depths behavior of FFF/STD beams is practically the same but as depth increases DE effects of FFF beams undergo more changes than STD beams. Conclusions: DER increases with depth, field size, distance from the CAX and OB vs IMRT due to the increased scatter and concomitant softening of the beam. Differences in DER can be more than one order of magnitude, depending on the case and the medium. The greatest dose enhancement is achieved with FFF (lower effective energy) and with other beams of large scatter content. Complex IMRT is better for CERT compared to conformal plans because of much greater photon scatter from various sources increasing the fluence below 200KeV.

Abstract 2804: Radiosensitization by the Dual PI3K/mTOR inhibitor NVP-BEZ235

Abstract The PI3K/Akt pathway has been implicated in cancer progression and in the survival of cancer cells following DNA damage induced by radiation. Hence, we chose to study the radiosensitizing ability of the Novartis compound, NVP-BEZ235, a dual PI3K/mTOR inhibitor. Clonogenic cell survival assays following ionizing radiation, cell cycle analysis, immunofluorescence for γ-H2AX foci, and Western blotting were performed using standard protocols.Treatment of SQ20B head and neck squamous cell carcinoma cells and U251 glioblastoma cells with NVP-BEZ235 led to a decrease in phosphorylation of Akt at serine 473 and of S6 and 4E-BP1, both of which are downstream markers of mTOR activation. Pre-treatment with the drug (25-50 nM) led to radiosensitization of these cell lines with a dose enhancement ratio (DER) ranging from 1.32 - 1.72. Knockdown of Akt1, p110alpha, or mTOR in these cell lines, also resulted in radiosensitization, but not to the same extent as with NVP-BEZ235. Of note, treatment with rapamycin led to minimal radiosensitization of these cells. Reduced survival with NVP-BEZ235 does not result from cell cycle redistribution during the treatment intervals tested, although combining NVP-BEZ235 with radiation enhances the G2/M delay seen after radiation. We examined how cells died after radiation and found no suggestion of increased apoptosis or necrosis. We did find evidence of increased conversion of microtubule-associated protein light chain 3 (LC3)-I to LC3-II, suggestive of autophagy. In split course experiments, in which cells were either irradiated with a single dose of radiation or with two half-doses separated by time, as the interval between doses increased, so did clonogenic survival. This increase in survival was abolished by pre-treatment with NVP-BEZ235, suggesting that the drug interfered with DNA damage repair. This was confirmed by assessing levels of gamma-H2AX after ionizing radiation. Initial post-radiation levels of gamma-H2AX foci were similar in NVP-BEZ235-pretreated and control cells; however, the resolution of these foci was significantly delayed in the BEZ235-treated cells. We then examined DNA-PKcs, a protein important for DNA damage repair, and found that the drug abrogated the increased phosphorylation normally seen after radiation. These results indicate that NVP-BEZ235 is a potent radiosensitizer of HNC cells in vitro. As siRNA against either p110alpha or mTOR leads to radiosensization, both of these targets of NVP-BEZ235 may be critical mediators of its to radiosensitizing ability; however, a second mechanism involved, may be via delayed repair of DNA damage following radiation, via interference of DNA-PKcs phosphorylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2804. doi:1538-7445.AM2012-2804

Abstract 5721: Garcinol, a histone acetyltransferase inhibitor, radiosensitizes cancer cells by inhibiting non-homologous end joining

Abstract Purpose: Non-homologous end joining (NHEJ), a major pathway to repair DNA double-strand breaks (DSBs) generated by ionizing radiation (IR), requires chromatin remodeling at DSB sites through acetylation of histones by histone acetyltransferases (HATs). However, effects of compounds with HAT inhibitory activities on DNA damage response (DDR), including NHEJ and cell cycle checkpoint, as well as on the radiosensitivity of cancer cells remain largely unclear. Here, we investigated whether garcinol, an HAT inhibitor found in the rinds of Garcinia indica fruit (called mangosteens), has effects on DDR and whether it can be used for radiosensitization. Methods and Materials: The ability of garcinol to inhibit DSB repair, including NHEJ, was examined by two assays; the conventional neutral comet assay and a cell-based assay recently developed by us, in which the activity of NHEJ against DSBs on chromosomal DNA was evaluated. The effects on cell cycle checkpoint after IR were assessed by phosphorylation levels of checkpoint kinases CHK1 and CHK2, and histone H3. The radiosensitizing effect was assessed by clonogenic survival assay. The effects on apoptosis and senescence were examined by annexin V and Senescence-associated β-galactosidase (SA-β-Gal) staining, respectively. Results: Garcinol was shown to inhibit DSB repair, including NHEJ, without affecting cell cycle checkpoint. Garcinol radiosensitized A549 lung and HeLa cervical carcinoma cells with a dose enhancement ratio at 10% surviving fraction of 1.6 and 1.5, respectively. Cellular senescence induced by IR was enhanced by garcinol. Conclusion: These results suggest that garcinol is a radiosensitizer that inhibits NHEJ and facilitates senescence, without impairing activation of cell cycle checkpoint. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5721. doi:1538-7445.AM2012-5721

Garcinol, a Histone Acetyltransferase Inhibitor, Radiosensitizes Cancer Cells by Inhibiting Non-Homologous End Joining

Non-homologous end joining (NHEJ), a major pathway used to repair DNA double-strand breaks (DSBs) generated by ionizing radiation (IR), requires chromatin remodeling at DSB sites through the acetylation of histones by histone acetyltransferases (HATs). However, the effect of compounds with HAT inhibitory activities on the DNA damage response (DDR), including the NHEJ and cell cycle checkpoint, as well as on the radiosensitivity of cancer cells, remains largely unclear. Here, we investigated whether garcinol, a HAT inhibitor found in the rinds of Garcinia indica fruit (called mangosteens), has effects on DDR, and whether it can be used for radiosensitization. The following assays were used to examine the effect of garcinol on the inhibition of DSB repair, including the following: a conventional neutral comet assay; a cell-based assay recently developed by us, in which NHEJ repair of DSBs on chromosomal DNA was evaluated; the micrococcal nuclease sensitivity assay; and immunoblotting for autophosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We assessed the effect of garcinol on the cell cycle checkpoint after IR treatment by analyzing the phosphorylation levels of checkpoint kinases CHK1 and CHK2 and histone H3, and by cell cycle profile analysis using flow cytometry. The radiosensitizing effect of garcinol was assessed by a clonogenic survival assay, whereas its effects on apoptosis and senescence were examined by annexin V and senescence-associated β-galactosidase (SA-β-Gal) staining, respectively. We found that garcinol inhibits DSB repair, including NHEJ, without affecting cell cycle checkpoint. Garcinol radiosensitized A549 lung and HeLa cervical carcinoma cells with dose enhancement ratios (at 10% surviving fraction) of 1.6 and 1.5, respectively. Cellular senescence induced by IR was enhanced by garcinol. These results suggest that garcinol is a radiosensitizer that inhibits NHEJ and facilitates senescence without impairing activation of the cell cycle checkpoint.

TGFβ Inhibition Radiosensitizes Murine Glioblastoma Cells and Decreases Neurosphere-forming Capacity

Transforming growth factor-β (TGFβ) is a pleotropic cytokine in the tumor microenvironment that can promote malignant behaviors, including invasion and motility. Glioblastomas produce abundant TGFβ, are routinely treated with radiation, and have a very poor prognosis. A role for TGFβ in the DNA damage response has recently been discovered in which TGFβ inhibition in vitro and in vivo compromises ATM-kinase activity induced by ionizing radiation (Cancer Research 66:10861 - 68; 62:5627 - 31). These data suggest that TGFβ could protect cancer cells from the cytotoxic effects of radiation by promoting ATM dependent responses; if so, TGFβ inhibitors, which are in clinical trials, might increase therapeutic response to radiation. We used the murine glioblastoma cell line, GL261, to test the effects of TGFβ inhibition by LY364947 (a small molecule inhibitor of the TGFβ type I receptor kinase) on proliferation, radiosensitivity, and neurosphere-forming capacity. Experiments were performed in triplicate and differences tested by ANOVA. GL261 cells produce 0.9 ng/mL per 106 cells of total TGFβ in media conditioned for 24 hr, the majority of which is latent TGFβ2. They also respond to exogenous TGFβ1 with an increase in Smad2 phosphorylation. Despite intact TGFβ receptor kinase activity, GL261 cells displayed no growth modulation response to exogenous TGFβ1 (0.5 - 2ng/mL) treatment or to inhibition by LY364947 (400nM). Nonetheless, inhibition of TGFβ with LY364947 for 24 hours prior to radiation treatment significantly increased GL261 radiosensitivity in the clonogenic assay (p<0.001), with a 1.25 dose enhancement ratio at 10% surviving fraction. This correlated with a significant 55% decrease in γH2AX foci following radiation treatment with 2Gy (p<0.0001). Irradiation of GL261 cells with 2Gy also decreased primary neurosphere-forming capacity by 28% (p<0.001, ANOVA), but had no effect on secondary neurosphere formation. Treatment with LY364947 alone had no effect on neurosphere formation. In contrast, treatment for 24 hours prior to irradiation decreased the primary neurosphere-forming capacity of irradiated GL261 cells by an additional 47% (p<0.001) and decreased secondary neurosphere formation by 68% (p<0.001). Given the radiosensitization and specifically the response of the neurosphere assay, which is thought to measure the glioma initiating cell population, our results suggest that inhibition of TGFβ in combination with radiation represents a promising therapeutic strategy. By targeting the putative stem cells long term benefit of TGFβ inhibition in glioblastoma may be achieved compared to poor response rates seen with the standard regimen of chemotherapy and radiotherapy.

Enhancement of Radiosensitivity and the Potential Mechanism on Human Esophageal Carcinoma Cells by Tetrandrine

The purposes of this study were to test the effect of tetrandrine, alone or combined with radiation, on human esophageal cancer cell line TE1 (TE1 cells) and investigate the potential antitumor mechanism. Human esophageal cancer cell line TE1 was tested by methyl thiazolyl tetrazolium assay for cell proliferation, colony-forming assay for cell radiosensitivity, flow cytometry assay for cell cycle distribution, and western blot assay for cell cycle protein expression. When treated alone, tetrandrine had a time- and concentration-dependent cytotoxic effect on TE1 cells. The dose-enhancement ratio for combined tetrandrine and radiation was markedly increased when compared with tetrandrine alone. Further, expression of cyclin B1 protein increased after addition of tetrandrine when compared with radiation only. Radiation-induced G2 arrest was abrogated with treatment of tetrandrine. In conclusion, tetrandrine can enhance the radiosensitivity of TE1 cells and this may involve relief of radiation-induced G2/M arrest in TE1 cells.

SU‐E‐T‐323: Investigation of the Effect of the Gold‐Tissue Interface on the Radiation Dose Enhancement Due to Gold Nanoparticles

Purpose: Injection of gold nanoparticles into a tumor site has been found promising in achieving selective radiation dose enhancement within the tumor due to goldˈs high atomic number Z=79, and favorable biological compatibility. So far the amplitude of the dose enhancement has been modeled in an approximation of average distribution of separate gold atoms rather than nanoparticles, thus neglecting the effect of the gold‐tissue interface. Evaluation of the dose deposition at the interface allowed us to estimate the dose enhancement more adequately. Method and Materials: Monte Carlo N‐Particle radiation transport code version 5 (MCNP5) was used to study the dose enhancement in a 1‐dimensional approximation, with a layer of gold placed within tumor. Dimension of gold along the beam direction was set to 100 nm, while in the transverse directions to 10×10 cm2. Mono‐energetic photon sources, from 20 keV to 4 MeV, were directed onto the tumor, and dose enhancements relative to the uniform all‐tumor case were obtained in the vicinity of the gold layer. Results: The dose deposited in the tumor in the vicinity of the gold layer varies with the energy of the incoming photon beam. The highest enhancement, up to several hundred percents, was obtained for the lowest energy; with much more modest enhancement of 10 to 20% at MeV energies. The region of the dose enhancement fall‐off around the particle extends to ∼3microns, therefore for realistic nanoparticle concentrations ∼1% dose enhancement regions will overlap. Conclusions: Our approach of evaluating the gold‐tissue interface around one particle allowed for a simple way of obtaining dose distributions and more adequate values of dose enhancement factors. Use of gold particles can be justified even for MeV photon therapies where only modest, 10 to 20 % dose enhancement ratios are achieved, if a tumor site is close to a critical structure.

MLN8054, A Small Molecule Inhibitor of Aurora Kinase A, Sensitizes Androgen-Resistant Prostate Cancer to Radiation

To determine whether MLN8054, an Aurora kinase A (Aurora-A) inhibitor causes radiosensitization in androgen-insensitive prostate cancer cells in vitro and in vivo. In vitro studies consisted of culturing PC3 and DU145 prostate cancer cells and then immunoblotting Aurora A and phospho-Aurora A after radiation and/or nocodazole with MLN8054. Phases of the cell cycle were measured with flow cytometry. PC3 and DU145 cell lines were measured for survival after treatment with MLN8054 and radiation. Immunofluorescence measured γ-H2AX in the PC3 and DU145 cells after treatment. In vivo studies looked at growth delay of PC3 tumor cells in athymic nude mice. PC3 cells grew for 6 to 8 days in mice treated with radiation, MLN8054, or combined for 7 more days. Tumors were resected and fixed on paraffin and stained for von Willebrand factor, Ki67, and caspase-3. In vitro inhibition of Aurora-A by MLN8054 sensitized prostate cancer cells, as determined by dose enhancement ratios in clonogenic assays. These effects were associated with sustained DNA double-strand breaks, as evidenced by increased immunofluorescence for γ-H2AX and significant G2/M accumulation and polyploidy. In vivo, the addition of MLN8054 (30 mg/kg/day) to radiation in mouse prostate cancer xenografts (PC3 cells) significantly increased tumor growth delay and apoptosis (caspase-3 staining), with reduction in cell proliferation (Ki67 staining) and vascular density (von Willebrand factor staining). MLN8054, a novel small molecule Aurora-A inhibitor showed radiation sensitization in androgen-insensitive prostate cancer in vitro and in vivo. This warrants the clinical development of MLN8054 with radiation for prostate cancer patients.

Abstract LB-361: Radiosensitization and decreased neurosphere-forming capacity with TGFβ inhibition in glioma cells

Abstract Transforming growth factor-β (TGFβ) is a pleotropic cytokine in the tumor microenvironment that can promote malignant behaviors, including invasion and motility, at late stages of tumorigenesis. Glioblastomas produce abundant TGFβ, are routinely treated with radiation, and have a very poor prognosis (median survival of only 14.6 months). An unexpected role for TGFβ in the DNA damage response has recently been discovered in which TGFβ inhibition in vitro and in vivo compromises ATM-kinase activity induced by ionizing radiation (Cancer Research 66:10861–68; 62:5627–31). These data suggest that TGFβ could actually protect cancer cells from the cytotoxic effects of radiation by promoting ATM dependent responses; if so, TGFβ inhibitors, which are in clinical trials, might increase therapeutic response to radiation. To test this idea, we asked whether TGFβ inhibition could radiosensitize glioblastoma cells. We used the murine glioblastoma cell line, GL261, to test the effects of TGFβ inhibition by LY364947 (a small molecule inhibitor of the TGFβ type I receptor kinase) on proliferation, radiosensitivity, and neurosphere-forming capacity. GL261 cells were found to produce 0.9 ng/mL per 106 cells of total TGFβ in media conditioned for 24 hr, the majority of which is latent TGFβ2. They also respond to exogenous TGFβ1 with an increase in Smad2 phosphorylation by Western blot. Despite intact TGFβ receptor kinase activity, GL261 cells displayed no growth modulation response to exogenous TGFβ1 (0.5–2ng/mL) treatment or to inhibition by LY364947 (400nM). Nonetheless, inhibition of TGFβ with LY364947 for 24 hours prior to radiation treatment significantly increased GL261 radiosensitivity in the clonogenic assay, with a 1.25 (p&amp;lt;0.001, ANOVA) dose enhancement ratio at 10% surviving fraction; in other words, an increase of 25% in clonogenic cell death. The increase in radiosensitivity correlated with a significant 55% decrease in γH2AX foci, which is a substrate of ATM, following radiation treatment with 2Gy (p&amp;lt;0.0001, ANOVA). We then assessed the response of glioma initiating cells (GIC) in a neurosphere assay. Irradiation of GL261 cells with 2Gy decreased neurosphere-forming capacity by 28% (p&amp;lt;0.001, ANOVA). Treatment with LY364947 alone had no effect on neurosphere formation, but treatment for 24 hours prior to irradiation decreased the neurosphere-forming capacity of irradiated GL261 cells by an additional 47% (p&amp;lt;0.001, ANOVA). Further studies using an orthotopic GL261 intracranial glioblastoma model are underway to test how these radiation effects mediated by TGFβ affect tumor growth in vivo. Given the radiosensitization and specifically the response of the GIC population, our results suggest that inhibition of TGFβ in combination with radiation represents promising therapeutic strategy in glioblastoma to improve poor response rates seen with the standard regimen of chemotherapy and radiotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-361. doi:10.1158/1538-7445.AM2011-LB-361

Inhibition of JAK2 Signaling by TG101209 Enhances Radiotherapy in Lung Cancer Models

Persistent STAT3 activation contributes to lung carcinogenesis. Survivin, one of STAT3-regulated genes, is antiapoptotic and confers cancer radioresistance. We tested whether TG101209, a small-molecule inhibitor of JAK2 (a STAT3-activating tyrosine kinase), affected survivin expression and sensitized lung cancer to radiation. We investigated whether inhibition of JAK2 signaling with TG101209 can be used to reduce survivin expression and enhance radiosensitivity of lung cancer cells in vitro and tumor growth delay in vivo. JAK2 downstream signaling, including PI3-K/Akt and Ras/MAPK/ERK pathways, was also explored. TG101209 inhibited STAT3 activation and survivin expression and sensitized HCC2429 (dose enhancement ratio = 1.34, p = 0.002) and H460 (dose enhancement ratio = 1.09, p = 0.006) cells to radiation in clonogenic assays. Radiation promoted phospho-Akt and phospho-ERK in H460 cells, while their levels were unchanged in HCC2429. After treatment with TG101209, phospho-ERK protein levels were reduced in both HCC2429 and H460 cells. HCC2429 cells transfected with KRAS-12V mutant were more resistant to radiation- and TG101209-induced apoptosis than wild-type control cells. In vivo, addition of TG101209 to radiation in lung xenografts produced a significant tumor growth delay (>10 days) compared with radiation alone and was well tolerated. Immunohistochemistry staining of tumor sections showed that TG101209 increased apoptosis and decreased cell proliferation and vascular density, suggesting that TG101209 also has antiangiogenic effects. TG101209 enhanced the effects of radiation in lung cancer in vitro and in vivo. This study suggests the potential utility of selecting lung cancer patients according to KRAS mutation status for future clinical trials testing combination of TG101209 and radiotherapy.