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Background: This study investigated the characteristics of optically stimulated luminescence dosimeters (OSLDs) with fully filled deep electron/hole traps in the kV energy ranges.Materials and Methods: The experimental group consisted of InLight nanoDots, whose deep electron/hole traps were fully filled with 5 kGy pre-irradiation (OSLDexp), whereas the non-preirradiated OSLDs were arranged as a control group (OSLDcont). Absorbed doses for 75, 80, 85, 90, 95, 100, and 105 kVp with 200 mA and 40 ms were measured and defined as the unit doses for each energy value. A bleaching device equipped with a 520-nm long-pass filter was used, and the strong beam mode was used to read out signal counts. The characteristics were investigated in terms of fading, dose sensitivities according to the accumulated doses, and dose linearity.Results and Discussion: In OSLDexp, the average normalized counts (sensitivities) were 12.7%, 14.0%, 15.0%, 10.2%, 18.0%, 17.9%, and 17.3% higher compared with those in OSLDcont for 75, 80, 90, 95, 100, and 105 kVp, respectively. The dose accumulation and bleaching time did not significantly alter the sensitivity, regardless of the filling of deep traps for all radiation qualities. Both OSLDexp and OSLDcont exhibited good linearity, by showing coefficients determination (R2) > 0.99. The OSL sensitivities can be increased by filling of deep electron/hole traps in the energy ranges between 75 and 105 kVp, and they exhibited no significant variations according to the bleaching time.

Background: The hemi-body electron beam irradiation (HBIe–) technique has been proposed for the treatment of mycosis fungoides. It spares healthy skin using an electron shield. However, shielding electrons is complicated owing to electron scattering effects. In this study, we developed a thimble-like head bolus shield that surrounds the patient’s entire head to prevent irradiation of the head during HBIe–.Materials and Methods: The feasibility of a thimble-like head bolus shield was evaluated using a simplified Geant4 Monte Carlo (MC) simulation. Subsequently, the head bolus was manufactured using a three-dimensional (3D) printed mold and Ecoflex 00-30 silicone. The fabricated head bolus was experimentally validated by measuring the dose to the Rando phantom using a metal-oxide-semiconductor field-effect transistor (MOSFET) detector with clinical configuration of HBIe–.Results and Discussion: The thimble-like head bolus reduced the electron fluence by 2% compared with that without a shield in the MC simulations. In addition, an improvement in fluence degradation outside the head shield was observed. In the experimental validation using the inhouse- developed bolus shield, this head bolus reduced the electron dose to approximately 2.5% of the prescribed dose.Conclusion: A thimble-like head bolus shield for the HBIe– technique was developed and validated in this study. This bolus effectively spares healthy skin without underdosage in the region of the target skin in HBIe–.

6,7,4'-THIF does not bind to FKBP or cyclophilin A. The interaction between 6,7,4'-THIF and FKBP or cyclophilin A was confirmed using 6,7,4'-THIF-Sepharose 4B beads. 6,7,4'-THIF-Sepharose 4B beads were incubated with each cell lysate overnight. Total experimental procedures are described in Materials and Methods.

Pin1 is overexpressed in esophageal cancer cells. The protein expression level of Pin1 was compared between normal (HaCaT) cells and esophageal cancer cells (KYSE 30, 450 and 510). The same amount of protein was analyzed by Western blot as described in Materials and Methods.

Intake of soy isoflavones is inversely associated with the risk of esophageal cancer. Numerous experimental results have supported the anticancer activity of soy isoflavones. This study aimed to determine the anti-esophageal cancer activity of 6,7,4′-trihydroxyisoflavone (6,7,4′-THIF), a major metabolite of daidzein, which is readily metabolized in the human body. Notably, 6,7,4′-THIF inhibited proliferation and increased apoptosis of esophageal cancer cells. On the basis of a virtual screening analysis, Pin1 was identified as a target protein of 6,7,4′-THIF. Pull-down assay results using 6,7,4′-THIF Sepharose 4B beads showed a direct interaction between 6,7,4′-THIF and the Pin1 protein. Pin1 is a critical therapeutic and preventive target in esophageal cancer because of its positive regulation of β-catenin and cyclin D1. The 6,7,4′-THIF compound simultaneously reduced Pin1 isomerase activity and the downstream activation targets of Pin1. The specific inhibitory activity of 6,7,4′-THIF was analyzed using Neu/Pin1 wild-type (WT) and Neu/Pin1 knockout (KO) MEFs. 6,7,4′-THIF effected Neu/Pin1 WT MEFs, but not Neu/Pin1 KO MEFs. Furthermore, the results of a xenograft assay using Neu/Pin1 WT and KO MEFs were similar to those obtained from the in vitro assay. Overall, we found that 6,7,4′-THIF specifically reduced Pin1 activity in esophageal cancer models. Importantly, 6,7,4′-THIF directly bound to Pin1 but not FKBP or cyclophilin A, the same family of proteins. Because Pin1 acts like an oncogene by modulating various carcinogenesis-related proteins, this study might at least partially explain the underlying mechanism(s) of the anti-esophageal cancer effects of soy isoflavones. Cancer Prev Res; 10(5); 308–18. ©2017 AACR.

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The Wnt pathway is a promising therapeutic and preventive target in various human cancers. The transcriptional complex of β-catenin–T-cell factor (Tcf), a key mediator of canonical Wnt signaling, has been implicated in human colon cancer development. Current treatment of colon cancer depends on traditional cytotoxic agents with limited effects. Therefore, the identification of natural compounds that can disrupt the β-catenin–TcF complex to suppress cancer cell growth with fewer adverse side effects is needed. To identify compounds that inhibit the association between β-catenin and Tcf, we used computer docking to screen a natural compound library. Esculetin, also known as 6,7-dihydroxycoumarin, is a derivative of coumarin and was identified as a potential small-molecule inhibitor of the Wnt–β-catenin pathway. We then evaluated the effect of esculetin on the growth of various human colon cancer cell lines and its effect on Wnt–β-catenin signaling in cells and in an embryonic model. Esculetin disrupted the formation of the β-catenin–Tcf complex through direct binding with the Lys312, Gly307, Lys345, and Asn387 residues of β-catenin in colon cancer cells. In addition, esculetin effectively decreased viability and inhibited anchorage-independent growth of colon cancer cells. Esculetin potently antagonized the cellular effects of β-catenin–dependent activity, and in vivo treatment with esculetin suppressed tumor growth in a colon cancer xenograft mouse model. Our data indicate that the interaction between esculetin and β-catenin inhibits the formation of the β-catenin–Tcf complex, which could contribute to esculetin's positive therapeutic and preventive effects against colon carcinogenesis. Cancer Prev Res; 6(12); 1356–64. ©2013 AACR.

Supplemental Figure Legend

6,7,4'-THIF inhibits esophageal cancer cell growth more effectively than 7,8,4'-THIF. 6,7,4'-THIF shows a better inhibitory effect on (A) KYSE 30, (B) 450 or (C) 510 esophageal cancer cell growth compared to 7,8,4'-THIF. Cell growth was estimated by MTS assay after 72 h of treatment. Total experimental procedures are described in Materials and Methods.

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PDF file - 2098KB, In silico model of curcumin binding with CDKs at the ATP binding site. To better understand how curcumin interacts with CDK1/4, we performed a computational docking model using the Glide docking program from Schrodinger Suite 2013. The binding affinity between curcumin and CDK1 and 4 was predicted by a score of −8.94 and −7.63 kcal/mol, respectively. In the docked models, curcumin can bind at the ATP binding pocket of CDK1 or 4 and form some hydrogen bonds and other interactions. (Some images were generated with the UCSF Chimera program 3). A) Binding of curcumin at the ATP binding pocket of CDK1 or 4. B) Ligand Interaction Diagram (LID) for curcumin binding with CDKs. C) LID legend.

PDF file - 259KB, Curcumin inhibits CDK2 kinase activity ex vivo. After immunoprecipitation of the CDK2 proteins from HCT116 cell lysates using a specific CDK2 antibody, the effect of curcumin on CDK2 kinase activity was measured. Data are represented as means plus-minus S.D. as determined from 3 independent experiments and the asterisk (*) indicates a significant (p < 0.001) difference compared to untreated cell lysate group.

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PDF file - 75K, Aseculetin had no significant cytotoxicity on the viability of normal HCEC cells.

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PDF file - 29K, The protein levels of total and phosphorylated BRAF, MEK1/2, and RSK2 in SK-MEL-5, SK-MEL-28, and SK-MEL-2 melanoma cells and NHDF cells, a normal control cell line.

Supplementary Figures and Legends - PDF file 2854K, Supplementary Figure 1. Effect of USP8 knockdown on cell viability Supplementary Figure 2. Effect of USP8 knockdown on EGFR wildtype cells and apoptosis of NSCLC and normal cells. Supplementary Figure 3. Effect of USP8 knockdown on mRNA levels of RTKs. Supplementary Figure 4. Effect of USP8 inhibitor treatment on apoptosis of NSCLC and normal cells. Supplementary Figure 5. Fluorescence Intensity Profiling Supplementary Figure 6. Effect of USP8 knockdown or an USP8 inhibitor on insulin receptor (IR) expression. Supplementary Figure 7. An USP8 inhibitor reduces ERKs and STAT3 phosphorylation levels in tumors

Purpose: Common treatment modalities for non–small cell lung cancer (NSCLC) involve the EGF receptor-tyrosine kinase inhibitors (EGFR-TKIs) like gefitinib and erlotinib. However, the vast majority of treated patients acquire resistance to EGFR-TKIs, due, in large part, to secondary mutations in EGFR or amplification of the MET gene. Our purpose was to test ubiquitin-specific peptidase 8 (USP8) as a potential therapeutic target for gefitinib-resistant and -sensitive non–small cell lung cancer (NSCLC).Experimental Design: Testing the effect of knockdown of USP8 and use of a synthetic USP8 inhibitor to selectively kill gefitinib-resistant (or -sensitive) NSCLCs with little effect on normal cells in cell culture and a xenograft mouse model.Results: Knockdown of ubiquitin-specific peptidase 8 (USP8) selectively kills gefitinib-resistant NSCLCs while having little toxicity toward normal cells. Genetic silencing of USP8 led to the downregulation of several receptor tyrosine kinases (RTK) including EGFR, ERBB2, ERBB3, and MET. We also determined that a synthetic USP8 inhibitor markedly decreased the viability of gefitinib-resistant and -sensitive NSCLC cells by decreasing RTK expression while having no effect on normal cells. Moreover, treatment with a USP8 inhibitor led to significant reductions in tumor size in a mouse xenograft model using gefitinib-resistant and -sensitive NSCLC cells.Conclusions: Our results show for the first time that the inhibition of USP8 activity or reduction in USP8 expression can selectively kill NSCLC cells. We propose USP8 as a potential therapeutic target for gefitinib-resistant and -sensitive NSCLC cells. Clin Cancer Res; 19(14); 3894–904. ©2013 AACR.

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the first step of polyamine biosynthesis that is associated with cell growth and tumor formation. Existing catalytic inhibitors of ODC have lacked efficacy in clinical testing or displayed unacceptable toxicity. In this study, we report the identification of an effective and nontoxic allosteric inhibitor of ODC. Using computer docking simulation and an in vitro ODC enzyme assay, we identified herbacetin, a natural compound found in flax and other plants, as a novel ODC inhibitor. Mechanistic investigations defined aspartate 44 in ODC as critical for binding. Herbacetin exhibited potent anticancer activity in colon cancer cell lines expressing high levels of ODC. Intraperitoneal or oral administration of herbacetin effectively suppressed HCT116 xenograft tumor growth and also reduced the number and size of polyps in a mouse model of APC-driven colon cancer (ApcMin/+). Unlike the well-established ODC inhibitor DFMO, herbacetin treatment was not associated with hearing loss. Taken together, our findings defined the natural product herbacetin as an allosteric inhibitor of ODC with chemopreventive and antitumor activity in preclinical models of colon cancer, prompting its further investigation in clinical trials. Cancer Res; 76(5); 1146–57. ©2015 AACR.

The effect of herbacetin or DFMO on hearing in C57BL/6 mice as measured by PPI. Vehicle, herbacetin (100 mg/kg B.W. p.o. or 2 mg/kg B.W. i.p.) or DFMO (1 g/kg B.W. p.o.) was administered by gavage or intraperitoneal injection 5 times per week for 5 weeks. (A) DFMO significantly decreased hearing (measured as prepulse inhibition or PPI) on day 35. Hearing as measured by PPI was not affected by oral administration (B) or intraperitoneal injection (C) of herbacetin. Data are shown as mean values {plus minus} S.E. The asterisk (*) indicates a significant decrease (p < 0.05) in hearing in DFMO-treated mice compared to the vehicle-treated group.

Supplemental Materials and Methods

The effect of herbacetin on AP-1 upstream proteins. Colon cancer cells were treated with herbacetin for 48 h and expression of total and phosphorylated ERKs and RSK and total ODC was determined by Western blotting. Beta-Actin was used as a loading control. Similar results were obtained from 3 independent experiments and representative blots are shown.

PDF file - 148K, Screening of HI-TOPK compounds for inhibition of TOPK kinase activity.

Supplemental Figure Legend

Effect of herbacetin on activity of various kinases. Herbacetin has no effect on (A) CHK1, (B) EGFR, (C) GSK3beta, (D) JNK1, (E) JNK2, (F) ERK1, (G) ERK2, (H) MEK1, (I) AKT1, (J) AKT2, (K) RSK2, (L) p38 or (M) PI3-K kinase activity or on (N) AdoMetDC activity. The effect of herbacetin on kinase or enzyme activity was determined by using an in vitro kinase or enzyme assay. All data are represented as mean values {plus minus} S.D. of values from duplicates experiments.

PDF file - 105K, Chemical structure of 36 HI-TOPK compounds.

PDF file - 63K, Effect of HI-TOPK-032 on AP1, NF-kB or COX2 reporter activity in colon cancer cells.

The effect of herbacetin on cell cycle. The effect of herbacetin or DFMO on cell cycle was determined using HCT116, DLD1 or HT29 colon cancer cells. Cells were treated with herbacetin or DFMO for 48 h in medium supplemented with 10% FBS and then cells were serum starved for 24 h in medium supplemented with 0.5% FBS. Cells were stained with propidium iodide (PI) and cell cycle was analyzed by Fluorescence Activated Cell Sorting (FACS). All data are represented as mean values {plus minus} S.D. of triplicate values from 3 independent experiments. The asterisk (*) indicates a significant effect (p < 0.05) of herbacetin or DFMO compared to untreated control.

PDF file - 51K, Representative photographs of tumor-bearing athymic nude mouse treated or not treated with HI-TOPK-032.

PDF file - 77K, Effect of HI-TOPK-032 on transformation of JB6 cells.

PDF file - 27K, Expression of total and phosphorylated TOPK in colon cancer cell lines.

The effect of polyamine depletion with herbacetin on cancer cell doubling time. Colon cancer cells were stably transfected with shControl, shODC, Mock or SAT1. (A-B) The effect of herbacetin on HCT116 or HT29 colon cancer cell doubling time was assessed in shControl and shODC cells. (C-D) The effect of herbacetin on HCT116 or HT29 colon cancer cell doubling time was assessed in Mock and SAT1 cells. All data are represented as mean values {plus minus} S.D. of triplicate values from 3 independent experiments. The asterisk (*) indicates a significant effect (p < 0.05) of herbacetin compared to untreated controls.

List of potential ODC inhibitors generated by using computer screening.

The effect of herbacetin on AP-1 upstream protein expression. (A-B) Colon tumors were isolated from vehicle- and herbacetin-treated groups (A; treatment by I.P., B: treatment by oral administration) of mice for immunoblot analysis of AP-1 upstream proteins and (C) ODC expression by immunohistochemistry (IHC).

Effect of knocking down ODC expression in HCT116 colon cancer cells. Colon cancer cells were stably transfected with shMock or shODC. Confirmation of (A, upper left panel) knockdown of ODC protein expression by Western blot; (A, upper right panel) knockdown of ODC transcription level by RT-PCR; and (A, lower panel) knockdown of ODC activity measured as release of L-[1-C14] ornithine. (B) The effect of knocking down ODC expression on anchorage-independent HCT116 colon cancer cell growth. Colonies were counted using a microscope and the Image-Pro PLUS (v6) computer software program. (C) The effect of knocking down ODC expression on HCT116 colon cancer cell growth was analyzed over 1, 2 or 3 days using the MTS assay. (D) The effect of knocking down ODC expression on AP-1 reporter activity was measured using substrates in the reporter assay system. For A-D, data are represented as mean values {plus minus} S.D. of triplicate experiments and the asterisk (*) indicates a significant difference (p < 0.05) versus shMock cells. (E) Effect of knocking down ODC expression on the phosphorylation of ERK1/2 and RSK was determined by Western blotting. Similar results were observed from two independent experiments and representative blots are shown.

The effect of polyamines on intracellular herbacetin concentration. The effect of polyamines on the intracellular herbacetin level was determined using HCT116 colon cancer cells. Cells were treated with herbacetin and putrescine, spermidine or spermine for 5 h in medium supplemented with 10% FBS and intracellular herbacetin was extracted and analyzed by using HPLC. All data are represented as means {plus minus} S.D. of triplicate values from 3 independent experiments.

The objective of this study was to fabricate an anthropomorphic multimodality pelvic phantom to evaluate a deep-learning-based synthetic computed tomography (CT) algorithm for magnetic resonance (MR)-only radiotherapy.Polyurethane-based and silicone-based materials with various silicone oil concentrations were scanned using 0.35 T MR and CT scanner to determine the tissue surrogate. Five tissue surrogates were determined by comparing the organ intensity with patient CT and MR images. Patient-specific organ modeling for three-dimensional printing was performed by manually delineating the structures of interest. The phantom was finally fabricated by casting materials for each structure. For the quantitative evaluation, the mean and standard deviations were measured within the regions of interest on the MR, simulation CT (CTFor the prostate site, the mean MR intensities for the patient and phantom were 78.1 ± 13.8 and 86.5 ± 19.3, respectively. The mean intensity of the synthetic image was 30.9 Hounsfield unit (HU), which was comparable to that of the real CT phantom image. The original and synthetic CT intensities of the fat tissue in the phantom were -105.8 ± 4.9 HU and -107.8 ± 7.8 HU, respectively. For the target volume, the difference in DThis work demonstrated that the anthropomorphic phantom was physiologically and geometrically similar to the patient organs and was employed to quantitatively evaluate the deep-learning-based synthetic CT algorithm.