Sensitizer Enhancement Ratio Research Articles

In vitro and in silico study of biological effects on cancer cells in the presence of metallic materials during radiotherapy.

X-ray therapy aims to eliminate tumours while minimizing side effects. Intense mucositis is sometimes induced when irradiating the oral cavity with a dental metal crown (DMC). However, the underlying mechanisms of such inducing radiosensitization by DMC remain uncertain. This study explored the radiosensitizing mechanisms around DMCs in an interdisciplinary approach with cell experiments and Monte Carlo simulation with the PHITS code. Clonogenic survival and nuclear 53BP1 foci of a cell line derived from cervical cancer cells (HeLa cells) were measured post-irradiation with therapeutic X-rays near high-Z materials such as Pb or Au plates, and the experimental sensitizer enhancement ratio (SER) was obtained. Meanwhile, the dose enhancement ratio (DER) and relative biological effectiveness for DNA damage yields were calculated using the PHITS code, by considering the corresponding experimental condition. The experiments show the experimental SER values for cell survival and 53BP1 foci near metals are 1.2-1.4, which agrees well with the calculated DER values. These suggest that the radiosensitizing effects near metal are predominantly attributed to the dose increase. In addition, as a preclinical evaluation, the spatial distributions of DER near DMC are calculated using Computed Tomography Digital Imaging and Communications in Medicine (CT-DICOM) data and a simple tooth model. As a result, the DER values evaluated using the CT-DICOM data were lower than those from a simple tooth model. These findings highlight the challenge of evaluating radiosensitizing effects near DMCs using Digital Imaging and Communications in Medicine (DICOM) images due to volume-averaging effects and emphasize the need for a high-resolution (<1mm) dose assessment method unaffected by these effects.

"Abraxane-Like" Radiosensitizer for In Situ Oral Cancer Therapy.

Radiotherapy plays a vital role in cancer therapy. However, the hypoxic microenvironment of tumors greatly limits the effectiveness, thus it is crucial to develop a simple, efficient, and safe radiosensitizer to reverse hypoxia and ameliorate the efficacy of radiotherapy. Inspired by the structure of canonical nanodrug Abraxane, herein, a native HSA-modified CaO2 nanoparticle system (CaO2-HSA) prepared by biomineralization-induced self-assembly is developed. CaO2-HSA will accumulate in tumor tissue and decompose to produce oxygen, altering the hypoxic condition inside the tumor. Simultaneously, ROS and calcium ions will lead to calcium overload and further trigger immunogenic cell death. Notably, its sensitizing enhancement ratio (SER = 3.47) is much higher than that of sodium glycididazole used in the clinic. Furthermore, in animal models of in situ oral cancer, CaO2-HSA can effectively inhibit tumor growth. With its high efficacy, facile preparation, and heavy-metal free biosafety, the CaO2-HSA-based radiosensitizer holds enormous potential for oral cancer therapy.

MiR-450b-5p enhances the radiosensitivity of HR+ and HER2− breast cancer by targeting CDK6

Abstract Background The sensitivity of breast cancer cells to radiation is a key cause of locoregional recurrence after postoperative radiotherapy. Several studies have reported that microRNAs (miRNAs) are involved in the radiosensitivity of human breast cancer cells. One miRNA microarray study showed that miR-450b-5p was overexpressed 13.3-fold in patients with estrogen receptor–positive (ER+) and human epidermal growth factor receptor 2–negative (HER2−) breast cancer and no local relapse compared with local relapse patients. However, its underlying mechanism of action remains unknown. Methods The predicted target mRNAs of miR-450b-5p were screened using the TargetScan, miRDB, and miRWalk databases. Western blotting, quantitative polymerase chain reaction, and dual-luciferase reporter assays explored the association between cyclin-dependent kinase 6 (CDK6) and miR-450b-5p. The cell counting kit-8 assay and flow cytometry detected the proliferation of transfected MCF7 cells. Colony formation and xenograft tumors detected the radiosensitivity of the transfected MCF7 cells. Results Bioinformatics analysis, Western blotting, quantitative polymerase chain reaction, and dual-luciferase reporter assays demonstrated that CDK6 was the target gene of miR-450b-5p. Furthermore, in vitro and in vivo experiments showed that miR-450b-5p inhibited MCF7 cell proliferation and cell cycle progression, increased the sensitizer enhancement ratio, and decreased the volume of xenograft tumors after irradiation by regulating CDK6. Conclusions This study demonstrates that miR-450b-5p enhances the radiosensitivity of hormone receptor–positive (HR+) and HER2− breast cancer cells and elucidates its mechanism. miR-450b-5p may be considered a therapeutic target in HR+ and HER2− breast cancer treated with radiotherapy.

T790M mutation sensitizes non-small cell lung cancer cells to radiation via suppressing SPOCK1

BackgroundApproximately 50% of patients harbor the T790M mutation after developing first-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance. Evidence has showed the major treatment failure is local relapses and limited metastases. Several studies have demonstrated the value of radiotherapy in metastatic non-small cell lung cancer (NSCLC) with the EGFR T790M mutation after the development of TKI resistance. The aim of this study was to explore the role of radiation in T790M-mutant NSCLC and the value of early radiotherapy for NSCLC with T790M-mediated EGFR-TKI resistance. MethodsGefitinib-resistant NSCLC cell lines were established via stepwise exposure to increasing concentrations of gefitinib (PC-9-GR). Droplet digital PCR was used to determine the relative T790M subclone abundance. In vitro and in vivo models were established using different mixtures of PC-9-GR and PC-9 cells. Differentially expressed genes were identified using RNA sequencing. Two research models were constructed (salvage and prophylactic radiotherapy) to determine the effects of early radiotherapy on gefitinib-resistant cells. ResultsPC-9-GR cells exhibited higher radiosensitivity than PC-9 cells (sensitivity enhancement ratio = 1.5). Salvage radiation reduced the number of T790M-mutant subclones, and the relative T790M abundance was significantly lower than that without radiation at 90 days (10.94% vs. 21.54%). Prophylactic radiation prevented the development of T790M subclones. These results were also confirmed in vivo. qRT-PCR revealed threefold elevation of miR-1243 in PC-9-GR cells, and the increased radiosensitivity of PC-9-GR cells was inhibited when miR-1243 was knocked down. RNA sequencing revealed that SPOCK1 was downregulated in PC-9-GR cells. Interestingly, bioinformatic analysis showed that SPOCK1 was a target gene of miR-1243. SPOCK1 knockdown markedly increased the radiosensitivity of PC-9 cells. ConclusionGefitinib-resistant NSCLC with the T790M mutation had higher radiosensitivity than that without the mutation, possibly mediated by SPOCK1. Early radiotherapy can eliminate T790M subclones, providing evidence for the benefit of early local treatment in patients with TKI-resistant NSCLC.

Effect of Raltitrexed on ECA109 Cellular Radiosensitivity and its Mechanism in Esophageal Cancer.

To investigate the effect of raltitrexed + X-ray irradiation on esophageal cancer ECA109 cells and analyze the potential action mechanism. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to analyze the inhibitory effect of raltitrexed on cell proliferation. The effect of raltitrexed on radiosensitivity was studied through a clone-forming experiment. The scratch assay and invasion test were performed to understand the cell migration and invasion abilities. The apoptosis rate change was measured using a flow cytometer, and Western Blotting was used to determine the expression of B cell lymphoma-2 (Bcl-2) and Bcl2-associated X protein (Bax) in each group. Raltitrexed significantly inhibited ECA109 proliferation in a time-dose-dependent manner; there were significant differences among different concentrations and times of action. The results of the clone-forming experiment showed a sensitization enhancement ratio of 1.65, and this demonstrated a radiosensitization effect. After the combination of raltitrexed with X-ray, the cell migration distance was shortened, and the number of cells penetrating the membrane was reduced. Raltitrexed can inhibit the growth of esophageal cancer ECA109 cells and has a radiosensitization effect.

Engineering biomimetic nanosystem targeting multiple tumor radioresistance hallmarks for enhanced radiotherapy.

Tumor cells establish a robust self-defense system characterized by hypoxia, antioxidant overexpression, DNA damage repair, and so forth to resist radiotherapy. Targeting one of these features is insufficient to overcome radioresistance due to the feedback mechanisms initiated by tumor cells under radiotherapy. Therefore, we herein developed an engineering biomimetic nanosystem (M@HHPt) masked with tumor cell membranes and loaded with a hybridized protein-based nanoparticle carrying oxygens (O2) and cisplatin prodrugs (Pt(IV)) to target multiple tumor radioresistance hallmarks for enhanced radiotherapy. After administration, M@HHPt actively targeted and smoothly accumulated in tumor cells by virtue of its innate homing abilities to realize efficient co-delivery of O2 and Pt(IV). O2 introduction induced hypoxia alleviation cooperated with Pt(IV) reduction caused glutathione consumption greatly amplified radiotherapy-ignited cellular oxidative stress. Moreover, the released cisplatin effectively hindered DNA damage repair by crosslinking with radiotherapy-produced DNA fragments. Consequently, M@HHPt-sensitized radiotherapy significantly suppressed the proliferation of lung cancer H1975 cells with an extremely high sensitizer enhancement ratio of 1.91 and the progression of H1975 tumor models with an excellent tumor inhibition rate of 94.7%. Overall, this work provided a feasible strategy for tumor radiosensitization by overcoming multiple radioresistance mechanisms.

ClonoScreen3D – A Novel 3-Dimensional Clonogenic Screening Platform for Identification of Radiosensitizers for Glioblastoma

PurposeGlioblastoma (GBM) is a lethal brain tumor. Standard of care treatment comprising surgery, radiation and chemotherapy results in median survival rates of 12-15 months. Molecular targeted agents identified using conventional two-dimensional (2D) in vitro models of GBM have failed to improve outcome in patients, rendering such models inadequate for therapeutic target identification. A previously developed 3D GBM in vitro model that recapitulates key GBM clinical features and responses to molecular therapies was investigated for utility for screening novel radiation-drug combinations using gold-standard clonogenic survival as readout. ResultsPatient-derived GBM cell lines were optimized for inclusion in a 96-well plate 3D clonogenic screening platform, ClonoScreen3D. Radiation responses of GBM cells in this system were highly reproducible and comparable to those observed in low-throughout 3D assays. The screen methodology provided quantification of candidate drug single agent activity (EC50) and the interaction between drug and radiation (radiation interaction ratio, RIR). The PARP inhibitors talazoparib, rucaparib and olaparib, each showed a significant interaction with radiation by ClonoScreen3D and were subsequently confirmed as true radiosensitizers by full clonogenic assay. Screening a panel of DNA damage response inhibitors revealed the expected propensity of these compounds to interact significantly with radiation (13/15 compounds). A second screen assessed a panel of compounds targeting pathways identified by transcriptomic analysis and demonstrated single agent activity and a previously unreported interaction with radiation of dinaciclib and cytarabine (RIR 1.28 and 1.90, respectively). These compounds were validated as radiosensitizers in full clonogenic assays (sensitizer enhancement ratio 1.47 and 1.35, respectively). ConclusionsThe ClonoScreen3D platform was demonstrated to be a robust method to screen for single agent and radiation-drug combination activity. Using gold-standard clonogenicity, this assay is a tool for identification of radiosensitizers. We anticipate this technology will accelerate identification of novel radiation-drug combinations with genuine translational value.

Utility of realistic microscopy-based cell models in simulation studies of nanoparticle-enhanced photon radiotherapy

To enhance the effect of radiation on the tumor without increasing the dose to the patient, the combination of high-Z nanoparticles with radiotherapy has been proposed. In this work, we investigate the effects of the physical parameters of nanoparticles (NPs) on the Dose Enhancement Factor (DEF), and on the Sensitive Enhancement Ratio (SER) by applying a version of the Linear Quadratic Model. A method for constructing voxelized realistic cell geometries in Monte Carlo simulations from confocal microscopy images was developed and applied to Gliobastoma Multiforme cell lines (U87 and U373). The comparison of simulations with realistic geometry and spherical geometry shows that there is significant impact on the survival curves obtained for the same irradiation conditions. Using this model, the DEF and the SER are determined as a function of the concentration, size and distribution of gold nanoparticles within the cell. For small NPs, d AuNP = 10 nm, no clear trend in the DEF and SER was observed when the number of NPs within the cell increases. Experimentally, the variable number of NPs measured inside the U373 cells (ranging between 1.48 × 105 and 1.19 × 106) also did not influence much the observed cell survival upon irradiation of the cells with a Co-60 source. The same lack of trend is obtained when the Au content in the cell is kept constant, 0.897 mg/g, but the size of the NPs is changed. However, if the number of NPs is kept constant (7.91 × 105) and the size changes, there is a critical diameter above which the dose effect increases significantly. Using the realistic geometries, it was verified that the key parameter for the DEF and the SER enhancement is the volume fraction of Au in the cell, with NP size being a more important parameter than the number of NPs.

LKB1 Mutations Enhance Radiosensitivity in Non-Small Cell Lung Cancer Cells by Inducing G2/M Cell Cycle Phase Arrest.

Radiosensitivity remains an important factor affecting the clinical outcome of radiotherapy for non-small cell lung cancer (NSCLC). Liver kinase B1 (LKB1) as a tumor suppressor, is one of the most commonly mutated genes in NSCLC. However, the role of LKB1 on radiosensitivity and the possible mechanism have not been elucidated in the NSCLC. In this study, we investigated the regulatory function of LKB1 in the radiosensitivity of NSCLC cells and its possible signaling pathways. After regulating the expression of LKB1, cell proliferation was determined by Cell Counting Kit-8 (CCK-8) assay. The flow cytometry assay was used to analyse cell cycle distribution. Survival fraction and sensitization enhancement ratio (SER) were generated by clonogenic survival assay. Western blot analysis was used to assess expression levels of LKB1, p53, p21, γ-H2AX and p-Chk2. Our study found that when the NSCLC cells were exposed to ionizing radiation, LKB1 could inhibit NSCLC cell proliferation by promoting DNA double strand break and inducing DNA repair. In addition, LKB1 could induce NSCLC cells G1 and G2/M phase arrest through up-regulating expression of p53 and p21 proteins. This current study demonstrates that LKB1 enhances the radiosensitivity of NSCLC cells via inhibiting NSCLC cell proliferation and inducing G2/M phase arrest, and the mechanism of cell cycle arrest associated with signaling pathways of p53 and p21 probably.

Clonogenic assay and computational modeling using real cell images to study physical enhancement and cellular sensitization induced by metal nanoparticles under MV and kV X-ray irradiation.

This study was initiated due to the physically unexplainable tumor controls resulting from metal nanoparticle (MNP) experiments even under MV X-ray irradiation. A more accurate explanation of the mechanism of radiosensitization induced by MNP is warranted, considering both its physical dose enhancement and biological sensitization, as related research is lacking. Thus, we aimed to examine the intricate dynamics involved in MNP-induced radiosensitization. We conducted specifically designed clonogenic assays for the A549 lung cancer cell line with MNP irradiated by 6 MV and 300 kVp X-rays. Two types of MNP were employed: one based on iron oxide, promoting ferroptosis, and the other on gold nanoparticles known for inducing a significant dose enhancement, particularly at low-energy X-rays. We introduced the lethality enhancement factor (LEF) as the fraction in the cell killing attributed to biological sensitization. Subsequently, Monte Carlo simulations were conducted to evaluate the radial dose profiles for each MNP, corresponding to the physical enhancement. Finally, the local effect model was applied to the clonogenic assay results on real cell images. The LEF and the dose enhancement in the cytoplasm were incorporated to increase the accuracy in the average lethal events and, consequently, in the survival fraction. The results reveal an increased cell killing for both of the MNP under MV and kV X-ray irradiation. In both types of MNP, the LEF reveals a biological sensitization evident. The sensitizer enhancement ratio, derived from the calculations, exhibited only 3% and 1% relative differences compared to the conventional linear-quadratic model for gold and ferroptosis inducer nanoparticles, respectively. These findings indicate that MNPs sensitize cells via radiation through mechanisms akin to ferroptosis inducers, not exclusively relying on a physical dose enhancement. Their own contributions to survival fractions were successfully integrated into computational modeling.

Abstract A160: The FGFR axis is a potential radiosensitization target

Abstract Background: Establishing methods that sensitize cancers to photons is important for improving the treatment outcome of radiotherapy. Although sequencing has been widely used to identify druggable mutations in various cancers, this strategy have not been used extensively to identify new targets for radiotherapy sensitization. Thus, we performed clinical sequencing of radiotherapy-treated patient samples and investigated whether inhibition of the identified candidate targets resulted in radiosensitization in vitro and in vivo. Material and Methods: Ninety-eight patients with newly diagnosed and pathologically confirmed cervical cancer who had been treated with definitive radiotherapy were analyzed. DNA was extracted from formalin fixed and paraffin embedded pre-treatment tumor biopsy specimens. The exons of 409 cancer-related genes were sequenced using a next generation sequencer. Identified mutations were analyzed for correlations with clinical outcome. The radiosensitization induced by inhibition of the candidate target was investigated in in vitro clonogenic assays and nude mouse xenograft models using radioresistant cancer cell lines and small molecule inhibitors. The in vitro sensitizer enhancement ratio (SER) was calculated as the ratio of radiation dose required to reduce clonogenic cell survival by 50% without drug to that with drug. Results: Recurrent mutations were identified in PIK3CA (35.7%), ARID1A (25.5%), NOTCH1 (19.4%), FGFR3 (16.3%), FBXW7 (19.4%), TP53 (13.3%), EP300 (12.2%), and FGFR4 (10.2%). The prevalence of mutations in FGFR family genes combined (24.5%) was almost as high as that for well-studied PIK3CA and ARID1A. Fifty-five percent (21/38) of the identified FGFR mutations were located in the tyrosine kinase domain. Five-year progression-free survival (PFS) rates for FGFR mutation-positive patients (n = 24) were significantly worse than those for FGFR mutation-negative patients (n = 74) (43.9% vs. 68.5%). Multivariate analysis identified FGFR mutations as significant predictors of worse 5 year PFS, independent of clinicopathological variables. To investigate whether the FGFR axis can be targeted by drugs to increase radiosensitization, the radiosensitizing effect of a human-administrable selective pan-FGFR inhibitor LY2874455 was investigated. LY2874455 (at 100 nM) showed a potent in vitro radiosensitizing effect (SER: 1.62, 1.53, and 1.31 for A549, H1299, and H1703 cells, respectively). In A549 xenograft models, the combination of X-rays (10 Gy on day 1) and LY2874455 (3 mg/kg body weight, once daily, on Day 1–7) led to significantly greater suppression of tumor growth than either treatment with X-rays or LY2874455 alone, with no evidence of toxicities. LY2874455 treatment inhibited ERK signaling, the main target of the FGFR axis, both in vitro and in vivo. Conclusion: These data indicate that targeting the FGFR axis with LY2874455 could potentially be used to sensitize cancers to radiotherapy, which warrants clinical validation. Citation Format: Takahiro Oike, Eisuke Horigome, Akiko Adachi, Narisa Dewi Maulany Darwis, Atsushi Shibata, Tatsuya Ohno. The FGFR axis is a potential radiosensitization target [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A160.

Enhancing nasopharyngeal carcinoma cell radiosensitivity by suppressing AKT/mTOR via CENP-N knockdown

ObjectiveInvestigating the impact of centromere protein N (CENP-N) on radiosensitivity of nasopharyngeal carcinoma (NPC) cells.MethodsUsing immunohistochemistry and immunofluorescence to detect CENP-N expression in tissues from 35 patients with radiosensitive or radioresistant NPC. Assessing the effect of combined CENP-N knockdown and radiotherapy on various cellular processes by CCK-8, colony formation, flow cytometry, and Western blotting. Establishing a NPC xenograft model. When the tumor volume reached 100 mm3, a irradiation dose of 6 Gy was given, and the effects of the combined treatment were evaluated in vivo using immunofluorescence and Western blotting techniques.ResultsThe level of CENP-N was significantly reduced in radiosensitive tissues of NPC (p < 0.05). Knockdown of CENP-N enhanced NPC radiosensitivity, resulting in sensitizing enhancement ratios (SER) of 1.44 (5-8 F) and 1.16 (CNE-2Z). The combined treatment showed significantly higher levels of proliferation suppression, apoptosis, and G2/M phase arrest (p < 0.01) compared to either CENP-N knockdown alone or radiotherapy alone. The combined treatment group showed the highest increase in Bax and γH2AX protein levels, whereas the protein Cyclin D1 exhibited the greatest decrease (p < 0.01). However, the above changes were reversed after treatment with AKT activator SC79. In vivo, the mean volume and weight of tumors in the radiotherapy group were 182 ± 54 mm3 and 0.16 ± 0.03 g. The mean tumor volume and weight in the combined treatment group were 84 ± 42 mm3 and 0.04 ± 0.01 g.ConclusionKnockdown of CENP-N can enhance NPC radiosensitivity by inhibiting AKT/mTOR.

Efficacy of 15 nm Gold Nanoparticles for Image-Guided Gliosarcoma Radiotherapy

Targeted brain cancer treatments are sorely needed to improve long-term prognosis, particularly for gliosarcoma and glioblastoma patients. Gold nanoparticles (GNPs) have unique properties including high atomic number, biocompatibility, and small size for cancer cell internalization. GNPs are consequently an ideal candidate for improved cancer targeting using image-guided radiotherapy. This work investigated 15 nm AuroVistTM GNPs for image-guided gliosarcoma radiotherapy and identified optimum GNP concentrations. The GNPs were found to be 15–20 nm using optical surface plasmon resonance absorption, with a (41.3 ± 0.3) nm hydrodynamic diameter. Confocal imaging showed that 50–500 µg/mL of the GNPs was well-internalized into the 9L cells within 24–48 h. γ-H2AX assays showed that 50–500 µg/mL of the GNPs radiosensitized the 9L cells irradiated with 125 and 150 kVp X-rays. However, only 500 µg/mL of the GNPs produced significant long-term dose enhancement with 150 kVp X-rays (with a sensitization enhancement ratio at 10% survival of 1.43, and 1.13 with 50 µg/mL) using clonogenic assay. CT imaging of the GNPs in the 9L tumors in Fischer rats further showed that GNP concentrations above 500 µg/mL were required to distinguish the tumor from the brain, and the GNPs were detected 48 h after injection. These promising results indicate that the GNPs can be used for selective gliosarcoma treatment with image-guided X-ray radiotherapy at concentrations above 500 µg/mL.

Most DNA repair defects do not modify the relationship between relative biological effectiveness and linear energy transfer in CRISPR-edited cells.

Cancer is a highly heterogeneous disease, driven by frequent genetic alterations which have significant effects on radiosensitivity. However, radiotherapy for a given cancer type is typically given with a standard dose determined from population-level trials. As a result, a proportion of patients are under- or over-dosed, reducing the clinical benefit of radiotherapy. Biological optimization would not only allow individual dose prescription but also a more efficient allocation of limited resources, such as proton and carbon ion therapy. Proton and ion radiotherapy offer an advantage over photons due to their elevated Relative Biological Effectiveness (RBE) resulting from their elevated Linear Energy Transfer (LET). Despite significant interest in optimizing LET by tailoring radiotherapy plans, RBE's genetic dependence remains unclear. The aim of this study is to better define the RBE/LET relationship in a panel of cell lines with different defects in DSB repair pathways, but otherwise identical biological features and genetic background to isolate these effects. Normal human cells (RPE1), genetically modified to introduce defects in DNA double-strand break (DSB) repair genes, ATM, BRCA1, DCLRE1C, LIG4, PRKDC and TP53, were used to map the RBE-LET relationship. Cell survival was measured with clonogenic assays after exposure to photons, protons (LET 1 and 12keV/µm) and alpha particles (129keV/µm). Gene knockout sensitizer enhancement ratio (SER) values were calculated as the ratio of the mean inactivation dose (MID) of wild-type cells to repair-deficient cells, and RBE values were calculated as the ratio of the MID of X-ray and particle irradiated cells. 53BP1 foci were used to quantify radiation-induced DSBs and their repair following irradiation. Deletion of NHEJ genes had the greatest impact on photon sensitivity (ATM-/- SER=2.0 and Lig4-/- SER=1.8), with genes associated with HR having smaller effects (BRCA1-/- SER=1.2). Wild-type cells showed RBEs of 1.1, 1.3, 5.0 for low- and high-LET protons and alpha particles respectively. SERs for different genes were independent of LET, apart from NHEJ knockouts which proved to be markedly hypersensitive across all tested LETs. Due to this hypersensitivity, the impact of high LET was reduced in cell models lacking the NHEJ repair pathway. HR-defective cells had moderately increased sensitivity across all tested LETs, but, notably, the contribution of HR pathway to survival appeared independent of LET. Analysis of 53BP1 foci shows that NHEJ-defective cells had the least DSB repair capacity after low LET exposure, and no visible repair after high LET exposure. HR-defective cells also had slower repair kinetics, but the impact of HR defects is not as severe as NHEJ defects. DSB repair defects, particularly in NHEJ, conferred significant radiosensitivity across all LETs. This sensitization appeared independent of LET, suggesting that the contribution of different DNA repair pathways to survival does not depend on radiation quality.

An in situ self‐assembled peptide derivative for inhibition of glutathione synthesis and selective enhancement of tumor radiotherapy

AbstractBackgroundInhibition of glutathione (GSH) synthesis in cancer cells considerably improves the efficacy of reactive oxygen species (ROS)‐related tumor therapy. Self‐assembled peptide derivatives can facilitate the efficient delivery and accumulation of small molecular drugs in cancer cells.MethodsSelf‐assembling modules were covalently linked to the GSH‐biosynthesis inhibitor l‐buthionine sulfoximine (BSO) by solid‐phase synthesis to form the self‐assembling peptide derivative Nap‐DFDFpY‐GG‐BSO (Nano‐BSO@ in situ). Subsequently, its enzyme‐instructed self‐assembly in vitro and on cell surfaces were confirmed, and its intracellular GSH depletion and radiotherapy‐sensitizing effects were determined.ResultsNano‐BSO@ in situ successfully self‐assembles into a hydrogel with a nanofibrous microstructure upon incubation with alkaline phosphatase (ALP) at a critical concentration of 9.84 μM. Furthermore, it selectively self‐assembles in situ on HeLa cells with high ALP expression. At a concentration of 50 μM, Nano‐BSO@ in situ decreases intracellular GSH levels by 80%, ∼2.3 times more than free BSO. Meanwhile, pretreatment of HeLa cells with 50 μM Nano‐BSO@ in situ for 24 h results in a radiotherapy sensitization enhancement ratio to γ‐rays of 2.09.ConclusionsA novel in situ self‐assembling peptide derivative for GSH depletion and selective enhancement of tumor radiotherapy was constructed. The excellent GSH‐depletion ability and remarkable radiotherapy‐enhancement performance indicate that Nano‐BSO@ in situ is a promising selective sensitizer for ROS‐related treatment of tumor cells with high ALP expression.

Cetuximab enhances radiosensitivity of esophageal squamous cell carcinoma cells by G2/M cycle arrest and DNA repair delay through inhibiting p-EGFR and p-ERK.

Although radiotherapy has improved local control in esophageal squamous cell carcinoma (ESCC), a considerable number of patients still experience relapse due to resistance. In this study, we aimed to evaluate the effects of cetuximab on radiosensitivity in two ESCC cell lines (ECA109 and TE-13) and to investigate their underlying mechanisms. Cells were pretreated with or without cetuximab before irradiation. The MTT assay and clonogenic survival assay were performed to evaluate cell viability and radiosensitivity. Flow cytometry was performed to analyze cell cycle distribution and apoptosis. The γH2AX foci were counted to determine cellular DNA-repairing capacity via immunofluorescence assay. The phosphorylation of key molecules involved in the epidermal growth factor receptor (EGFR) signaling pathway and DNA double-strand break (DSB) repair were measured by western blot. Cetuximab alone was not sufficient to suppress cell viability, but significantly enhanced radiation-induced inhibition of clonogenic survival in ECA109 and TE-13. The radiation sensitivity enhancement ratio (SER) was 1.341 and 1.237 for ECA109 and TE-13, respectively. ESCC cells treated with cetuximab were arrested at the G2/M phase in response to radiation. No significant increase in apoptotic rate was observed in irradiated cells that were treated with cetuximab. The average number of γH2AX foci increased in the combination group (cetuximab and radiation). Cetuximab suppressed phosphorylation of EGFR and downstream ERK, but had no significant effect on AKT. These results indicate the potential for use of cetuximab as an effective radiosensitizer in ESCC. Cetuximab promotes G2/M cycle arrest and reduces DSB repair, as well as inhibiting EGFR and downstream ERK pathways in ESCC.

Curcumol β-cyclodextrin inclusion complex enhances radiosensitivity of esophageal cancer under hypoxic and normoxic condition.

Radiotherapy is an indispensable treatment for esophageal cancer (EC), but radioresistance is not uncommon. Curcumol, as an active extract from traditional Chinese medicines, has been reported to have antitumor activity in various types of human tumor cells. However, its reversal of radioresistance has been rarely reported. In the present study, curcumol was prepared as an inclusion complex with β-cyclodextrin. EC cell lines were treated with radiation and curcumol β-cyclodextrin inclusion complex (CβC), and the effect of radiosensitization of CβC was investigated in vitro and in vivo. The in vitro experiments included cell proliferation assay, clonogenic survival assay, apoptosis assay, cell cycle assay, and western blot assay. The in vitro data revealed that CβC and irradiation synergistically inhibited the proliferation, reduced the colony formation, promoted the apoptosis, increased the G2/M phase, inhibited DNA damage repair, and reversed the hypoxia-mediated radioresistance of EC cells to a greater extent than did CβC alone or irradiation alone. The sensitization enhancement ratios (SERs) were 1.39 for TE-1 and 1.48 for ECA109 under hypoxia. The SERs were 1.25 for TE-1 and 1.32 for ECA109 under normoxia. The in vivo data demonstrated that the combination of CβC and irradiation could inhibit tumor growth to the greatest extent compared with either monotherapy alone. The enhancement factor was 2.45. This study demonstrated that CβC could enhance radiosensitivity of EC cells under hypoxic and normoxic condition. Thus, CβC can be used as an effective radiosensitizer for EC.

In vitro evaluation of photon and carbon ion radiotherapy in combination with cisplatin in head and neck squamous cell carcinoma cell lines.

Heavy ion radiotherapy, such as carbon ion radiotherapy (CIRT), has multiple advantages over conventional photon therapy. Cisplatin, as a classic anti-tumor drugs, has been tested and discovered as a photon radiosensitizer in several cell lines, including head and neck squamous cell carcinoma (HNSCC). Hence, the aim of our study is to evaluate whether cisplatin can sensitize CIRT towards HNSCC cell lines in vitro. Human nasopharyngeal carcinoma cell line CNE-2, human tongue squamous carcinoma cell line TCA 8113 and human hypopharynx squamous carcinoma cell line FADU were all irradiated with photon beam of 2, 4, 6, 8 Gy (physical dose) and carbon ion beam of 1, 2, 3, 4 Gy (physical dose) and treated with cisplatin. Cell survival was assessed by clonogenic survival assay. CIRT showed significantly stronger cytotoxic effect than standard photon radiotherapy. The relative biological effectiveness (RBE) of carbon ion beam at 10% survival ( ) was calculated 3.07 for CNE-2, 2.33 for TCA 8113 and 2.36 for FADU. Chemoradiotherapy (both photon radiotherapy and CIRT) was more effective than radiotherapy alone. In vitro sensitizer enhancement ratios (SERs) of cisplatin in CNE-2, TCA 8113 and FA DU cell lines after photon irradiation were 1.33, 1.14 and 1.21, while after carbon ion irradiation were 1.02, 1.00 and 0.96, showed that cisplatin sensitized photon irradiation but showed no sensitization effect in carbon ion irradiation in all tested cell lines. In conclusion, high linear energy transfer (LET) CIRT was more effective than photon irradiation to prevent the proliferation of HNSCC cell lines. Additional treatment with cisplatin could sensitize photon irradiation but showed no effect on carbon ion irradiation.

Albumin-Modified Gold Nanoparticles as Novel Radiosensitizers for Enhancing Lung Cancer Radiotherapy.

Considering the strong attenuation of photons and the potential to increase the deposition of radiation, high-atomic number nanomaterials are often used as radiosensitizers in cancer radiotherapy, of which gold nanoparticles (GNPs) are widely used. We prepared albumin-modified GNPs (Alb-GNPs) and observed their radiosensitizing effects and biotoxicity in human non-small-cell lung carcinoma tumor-bearing mice models. The prepared nanoparticles (Alb-GNPs) demonstrated excellent colloidal stability and biocompatibility at the mean size of 205.06 ± 1.03 nm. Furthermore, clone formation experiments revealed that Alb-GNPs exerted excellent radiosensitization, with a sensitization enhancement ratio (SER) of 1.432, which is higher than X-ray alone. Our in vitro and in vivo data suggested that Alb-GNPs enabled favorable accumulation in tumors, and the combination of Alb-GNPs and radiotherapy exhibited a relatively greater radiosensitizing effect and anti-tumor activity. In addition, no toxicity or abnormal irritating response resulted from the application of Alb-GNPs. Alb-GNPs can be used as an effective radiosensitizer to improve the efficacy of radiotherapy with minimal damage to healthy tissues.

Application of Gold Nanoparticles as Radiosensitizer for Metastatic Prostate Cancer Cell Lines

More than 50% of all prostate cancer (PCa) patients are treated by radiotherapy (RT). Radioresistance and cancer recurrence are two consequences of the therapy and are related to dose heterogeneity and non-selectivity between normal and tumoral cells. Gold nanoparticles (AuNPs) could be used as potential radiosensitizers to overcome these therapeutic limitations of RT. This study assessed the biological interaction of different morphologies of AuNPs with ionizing radiation (IR) in PCa cells. To achieve that aim, three different amine-pegylated AuNPs were synthesized with distinct sizes and shapes (spherical, AuNPsp-PEG, star, AuNPst-PEG, and rods, AuNPr-PEG) and viability, injury and colony assays were used to analyze their biological effect on PCa cells (PC3, DU145, and LNCaP) when submitted to the accumulative fraction of RT. The combinatory effect of AuNPs with IR decreased cell viability and increased apoptosis compared to cells treated only with IR or untreated cells. Additionally, our results showed an increase in the sensitization enhancement ratio by cells treated with AuNPs and IR, and this effect is cell line dependent. Our findings support that the design of AuNPs modulated their cellular behavior and suggested that AuNPs could improve the RT efficacy in PCa cells.