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The high incidence and mortality of cancer make it a global health issue. However, conventional cancer therapies have several disadvantages, especially serious side effects due to low selective toxicity to cancer cells. Gold nanoparticles (AuNPs) are an excellent drug carrier, enhance drug delivery efficiency, and hold promise for photothermal and radiation therapies. (-)-Epigallocatechin-3-gallate (EGCG) is the major polyphenolic antioxidant constituent of green tea, has a potent antitumor effect, and binds specifically to the 67 kDa laminin receptor, which is overexpressed on the surface of several cancer cell lines such as HeLa and MDA-MB-231 cells. We synthesized EGCG-modified AuNPs (EGCG-AuNPs) using ratios (

Introduction: Pancreatic cancer (PC) is believed to be a difficult disease because of its radio-resistance and metastases. Radiotherapy reported to modify tumor microenvironment (TME) and to affect PC progression. Exosomes containing such as microRNAs (miRNAs), messenger RNAs (mRNAs), and proteins of cells, play essential roles in cell-to-cell communications. However, characteristics and mechanisms for radiation responsive exosomes remain unclear. Here, we first report mechanisms of exosomes-mediated radiation response and regulation in PC metastasis with focusing on intra/intercellular communications via miRNA. Methods: MIAPaCa-2 cells, human pancreatic carcinoma, was used. To isolate exosomes, cell culture media was ultracentrifuged at 100,000 x g for 90 min at 4°C. Exosomes derived from non-irradiated culture media was named ‘0 Gy-Exo’ and those from irradiated with 5 Gy culture media is called ‘5 Gy-Exo’. Exosome sizes and features were evaluated by transmission electron microscopy (TEM). Intracellular reactive oxygen species (ROS) levels induced by exosomes and by X-ray irradiation were evaluated by the C-H2DCF dye staining. To detect DNA damage, cells were fixed and stained by γ-H2AX antibody. The expression of superoxide dismutase 1 (SOD1) was evaluated by using immunoblots. Total RNA was extracted from 0 Gy-Exo and 5 Gy-Exo, and then comprehensive miRNA expression analysis was performed to analyze 2,565 human miRNA sequences. The Gene Expression Omnibus database (GSE163133) and the TargetScan were used for specific miRNAs identification. Living MIAPaCa-2 cells in the presence or absence of those exosomes were injected in the spleen of Balb/c nude mice to establish liver metastatic models. Eight weeks after the injection, mice were sacrificed, and then the spleen and the liver were harvested as a whole organ. H-E staining and immunohisitochemical analyses using S100A4 and SMAD4 antibodies were performed to detect liver metastases. Results: Isolated exosomes were shaped in the form of closed, round vesicles with a diameter of 10~100 nm. Intracellular ROS levels and the numbers of γ-H2AX foci in the cells with 5 Gy-Exo were significantly increased compared to those the control (without adding exosomes, p Conclusion: Exosomes increased radiation sensitivity through increase of intracellular ROS levels via exosomal miRNAs and also could affect liver metastases in pancreatic cancer models. Citation Format: Ai Nakaoka, Makiko Nakahana, Sachiko Inubushi, Yasuyuki Shimizu, Kazuma Iwashita, Naritoshi Mukumoto, Kana Kobayashi, Takeaki Ishihara, Daisuke Miyawaki, Ryohei Sasaki. Exosomes enhance the radiation sensitivity via miR-6823-5p and modulate metastases in pancreatic cancer model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3796.

Background/aim Sarcomas are considered a heterogeneous disease with incomplete understanding of its molecular basis. In the present study, to further understand general molecular changes in sarcoma, patient-derived xenograft (PDX) mouse models of the three most common soft-tissue sarcomas: myxofibrosarcoma, undifferentiated pleomorphic sarcoma (UPS) and liposarcoma were established and the methylation status of histone H3 lysine marks was studied. Materials and methods Immunoblotting and immunohistochemical staining were used to quantify the extent of methylation of histone H3K4me3 and histone H3K9me3. Results In all 3 sarcoma types in PDX models, histone H3K4me3 and H3K9me3 were found highly over-methylated compared to normal muscle tissue. Conclusion Histone H3 lysine over-methylation may be a general basis of malignancy of the major sarcoma types.

Background/aim Exosomes are produced by normal and cancer cells. Exosomes are found in the serum of cancer patients and have been used for diagnosis and prognosis. Recently tears from non-cancer patients have been found to contain exosomes. In the present report we describe tears from advanced breast-cancer patients. Materials and methods We found oncogenic miRNAs in the exosomes isolated from tear fluids obtained from five patients with metastatic breast cancer and compared them with tear exosomes form eight healthy volunteers. Results Tear exosomes had a significantly higher quantity of exosome markers than serum exosomes (CD9, CD63). Tear exosomes were subjected to quantitative reverse-transcription polymerase reaction (qRT-PCR), and western blot analysis to elucidate the status of miRNAs, previously reported in serum from patients with metastatic breast cancer. qRT-PCR and western-blot analysis revealed that breast-cancer-specific miR-21 and miR-200c were highly expressed in tear exosomes from metastatic breast cancer patients in contrast to tear exosomes from healthy volunteers. Conclusion Tear exosomes can be a potential source of diagnostic and prognostic biomarkers for metastatic breast cancer, and possibly other cancers or diseases.

BACKGROUND/AIM In the present study, the breast cancer patient-derived orthotopic xenograft (PDOX) model was used to identify an effective drug for a highly aggressive triple negative breast cancer (TNBC). MATERIALS AND METHODS The TNBC tumor from a patient was implanted in the right 4th inguinal mammary fat pad of nude mice to establish a PDOX model. Three weeks later, 19 mice were randomized into the untreated-control group (n=10) and the eribulin treatment group (n=9, eribulin, 0.3 mg/kg, i.p., day 1). RESULTS On day 8, eribulin significantly inhibited tumor volume compared to the control group (p

Background/aim Matrix-producing breast carcinoma (MPBC) is a rare and usually aggressive triple-negative breast cancer (TNBC). In the present report, we determined the drug sensitivity for a triple-negative MPBC using a patient-derived orthotopic xenograft (PDOX) model. Materials and methods The PDOX model was established in the left 2nd mammary by surgical orthotopic implantation (SOI). MPBC PDOX models were randomized into 4 groups (6 mice per group) when the tumor volume became 80 mm3: G1, control group; G2, cisplatinum group [intraperitoneal (i.p.) injection, weekly, for 2 weeks]; G3, paclitaxel group (i.p., weekly, for 2 weeks); G4, eribulin group [intravenous (i.v.) injection, weekly, for 2 weeks]. All mice were sacrificed on day 15. Tumor volume and body weight were measured one time per week. Results The MPBC PDOX model was resistant to cisplatinum (p=0.800). Paclitaxel suppressed tumor growth compared to the control group (p=0.009). However, only eribulin regressed the tumor (p=0.001). Conclusion Eribulin has clinical potential for triple-negative MPBC patients.

Small extracellular vesicles (sEVs) are reliable biomarkers for early cancer detection; however, conventional detection methods such as immune-based assays and microRNA analyses are not very sensitive and require sample pretreatments and long analysis time. Here, we developed a molecular imprinting-based dynamic molding approach to fabricate antibody-conjugated signaling nanocavities capable of size recognition. This enabled the establishment of an easy-to-use, rapid, sensitive, pretreatment-free, and noninvasive sEV detection platform for efficient sEV detection-based cancer diagnosis. An apparent dissociation constant was estimated to be 2.4 × 10-16 M, which was ∼1000 times higher than that of commercial immunoassays (analysis time, 5 min/sample). We successfully used tears for the first time to detect cancer-related intact sEVs, clearly differentiating between healthy donors and breast cancer patients, as well as between samples collected before and after total mastectomy. Our nanoprocessing strategy can be easily repurposed for the specific detection of other types of cancer by changing the conjugated antibodies, thereby facilitating the establishment of liquid biopsy for early cancer diagnosis.

Background/aim obesity is a world-wide recalcitrant problem leading to many diseases. Dietary methionine restriction (MR) has been shown to prevent obesity, but it is an onerous regimen. The present study aimed to determine the effects of oral recombinant methionase (o-rMETase), on preventing obesity in mice on a high-fat diet. Materials and methods Male C57BL/6J mice in the control group were fed a control diet (CD) (+6.5% fat) for 25 days, and others were fed a high-fat (HF) diet (+34.3% fat) for 25 days. Then, the mice were divided into three dietary groups: 1) HF + phosphate buffered saline (PBS) group; 2) HF + o-rMETase group; and 3) untreated non-HF group. Results The mice on the CD increased in body weight by 14% during experimental period of 25 days; in contrast the mice in the HF+PBS group increased by 33%; however, the mice on the HF+o-rMETase group increased only by 14% (p=0.02, HF+PBS vs HF+o-rMETase). Conclusion The HF+ o-rMETase group had the same weight increase as untreated mice on a normal fat diet, demonstrating the potential for o-rMETase to eliminate the need for dieting to maintain normal body weight.

Background/aim We have recently shown that oral recombinant methionase (o-rMETase) prevents obesity and diabetes onset in mice on a high-fat (HF) diet. The present study aimed to determine if o-rMETase can inhibit the onset of nonalcoholic fatty liver disease (NAFLD) onset in mice on a high-fat diet. Materials and methods Male C57BL/6J mice in the control group were fed a normal-fat diet (NFD) (+6.5% fat), and other mice were fed a high-fat (HF) diet (+34.3% fat). Then, the mice on the HF diet were divided into two dietary groups: i) HF+phosphate buffered saline (PBS) group, and ii) HF+o-rMETase group. Result The fatty change score in the livers of mice treated with HF+PBS increased to an average of 2.6 during the experimental period of 8 weeks. In contrast, the fatty change in the livers of mice on the HF+o-rMETase group had an average score of 0.92 (p=0.04, HF+PBS vs HF+o-rMETase). Conclusion o-rMETase inhibited the onset of NAFLD as well as prevented obesity and the onset of diabetes on a high-fat diet, offering a possibility of a new paradigm to prevent liver cirrhosis or liver cancer via NAFLD.

Background/Aim: Surgical orthotopic implantation (SOI) is used to establish patient-derived orthotopic xenograft (PDOX) and other orthotopic mouse models. Orthotopic liver models can be challenging, as the liver parenchyma is prone to bleeding. The present report describes a sutureless method to implant tumors in the liver that reduces bleeding and procedural time. Materials and Methods: Human HCC cell-line (Huh-7-GFP) and CM2, a patient-derived colon-cancer liver metastasis, were used for sutureless SOI of tumor fragments in the liver of nude mice. A small cavity was formed on the liver surface. A solitary tumor fragment was implanted in the cavity without suturing to create hemostasis. Results: Six weeks after sutureless SOI, the tumor volume of Huh-7-GFP (n=5) was 584.41±147.64 mm(3) and the tumor volume of CM2 (n=5) was 1336.54±1038.20 mm(3). The engraftment rate was 100%. Conclusion: This novel method for establishing orthotopic liver-implantation mouse models is suitable for studies of liver cancer and liver metastases due to its simple procedure and potential high engraftment rate.

Background/aim The discovery of the nude mouse model enabled the experimental growth of human-patient tumors. However, the low establishment rate of tumors in nude and other immunodeficient strains of mice has limited wide-spread clinical use. Materials and methods In order to increase the establishment rate of surgical specimens of patient tumors, we transplanted tumors to nude mice subcutaneously along with large amounts of surrounding tissue of the tumor. Results The new transplantation method increased the establishment rate in nude mice to 66% compared to the old method of implanting the surgical tumor specimen with surrounding tissue removed (14%). High stage and presence of metastasis in the patient donor are positively correlated to tumor engraftment in nude mice. Conclusion The new method can potentially allow most cancer patients who undergo surgery or biopsy to have their own mouse model for drug-sensitivity testing.

Methionine addiction, found in all types of cancer investigated, is because of the overuse of methionine by cancer cells for excess transmethylation reactions. In the present study, we compared the histone H3 lysine-methylation status and degree of malignancy between methionine-addicted cancer cells and their isogenic methionine-independent revertants, selected by their growth in low concentration of methionine. The methionine-independent revertans can grow on low levels of methionine or independently of exogenous methionine using methionine precursors, as do normal cells. In the methionine-independent revertants, the excess levels of trimethylated histone H3 lysine marks found in the methionine-addicted parental cancer cells were reduced or lost, and their tumorigenicity and experimental metastatic potential in nude mice were also highly reduced. Methionine addiction of cancer is linked with malignancy and hypermethylation of histone H3 lysines. The results of the present study thus provide a unique framework to further understand a fundamental basis of malignancy.

Background/Aim: Methionine addiction is a fundamental and general hallmark of cancer, termed the Hoffman effect. Methionine addiction is due to excessive use of and dependence on methionine by cancer cells. In the present report, we correlated the extent of methionine addiction and degree of malignancy with the amount and stability of methylated histone H3 lysine marks. Materials and Methods: We established low- and high-malignancy variants from a parental human pancreatic-cancer cell line and compared their sensitivity to methionine restriction and histone H3 lysine methylation status. Results: A low-malignancy, low-methionine-addiction revertant of the parental pancreatic-cancer cell line had less methylated H3K9me3 and was less sensitive to methionine restriction effected by recombinant methioninase (rMETase) than the parental cell line. A high-malignancy variant of the pancreatic cancer cell line had increased methylated H3K9me3 and was more sensitive to methionine restriction by rMETase with regard to inhibition of proliferation and to instability of histone H3 lysine methylation than the parental cell line. Orthotopic malignancy in nude mice was reduced in the low-methionine-addiction revertant and greater in the high-malignancy variant than in the parental cell line. Conclusion: The present study indicates that the degree of malignancy is linked to the extent of methionine addiction and the level and instability of trimethylation of histone H3, suggesting these phenomena are linked as a fundamental basis of oncogenic transformation.

Nanoparticles (NPs) are useful for radiotherapy. Currently, efforts are underway globally for the development of novel titanium dioxide NPs (TiO2-NPs) that exhibit both contrast effects and anti-tumor effects. In this study, the image contrast properties of TiO2-NPs were evaluated using a clinical magnetic resonance imaging (MRI) system and a clinical computed tomography (CT) scanner, as the use of TiO2-NPs as an anti-cancer agent has been reported in several reports. An obvious difference in visualization was observed between the control and TiO2-NP samples on T2-weighted images. These results suggest that TiO2 can potentially be used as a novel theranostic drug with radiosensitizing ability and radiological diagnostic ability, through modification of chemical groups on its surface, and as a component of drug delivery systems.

Purpose: Effective methods to ameliorate radiation enteropathy have not been developed. To address this issue, we investigated the reduced form of coenzyme Q10 (rCoQ10) as a potential radioprotector in a mouse model. Methods and Materials: rCoQ10 was added to a standard laboratory mouse diet at a final concentration of 1.0% 9 days before irradiation and 30 days thereafter or dissolved in corn oil and administered transorally. Accumulated amounts of coenzyme Q10 (CoQ10) or coenzyme Q9 in the intestine were measured by high-performance liquid chromatography. Reactive oxygen species (ROS), apoptosis, and morphologic changes in the intestine were assessed by immunohistochemistry after administration of 13 Gy of x-ray to the mouse abdomen. Body weight and survival were monitored for 30 days after irradiation. Cytotoxicity using 3 human cancer cell lines and the tumor growth-inhibiting effect in a xenograft were investigated to determine whether rCoQ10 interferes with radiation-specific cytotoxic effects on tumor growth. Results: CoQ10 was greatly accumulated in all sections of the intestine after both massive transoral dosing and dietary administration, whereas coenzyme Q9 was not. Administration of rCoQ10 suppressed ROS production and inhibited apoptosis in the crypts, resulting in preservation of villi structures after irradiation. Notably, 92% of mice fed the rCoQ10-supplemented diet were healthy and alive 30 days after irradiation, whereas 50% of control mice died (P < .05). Moreover, rCoQ10 did not interfere with radiation-specific cytotoxic effects on tumors either in vitro or in vivo. Conclusions: Administration of rCoQ10 led to its accumulation in the intestine and induced radioprotective effects by inhibiting ROS-mediated apoptosis, thereby preserving intestinal structures. Our results indicated that rCoQ10 supplementation effectively ameliorated radiation enteropathy.

Exosomes are small (30-100 nm) membrane vesicles that serve as regulatory agents for intercellular communication in cancers. Currently, exosomes are detected by immuno-based assays with appropriate pretreatments like ultracentrifugation and are time consuming (>12 h). We present a novel pretreatment-free fluorescence-based sensing platform for intact exosomes, wherein exchangeable antibodies and fluorescent reporter molecules were aligned inside exosome-binding cavities. Such antibody-containing fluorescent reporter-grafted nanocavities were prepared on a substrate by well-designed molecular imprinting and post-imprinting modifications to introduce antibodies and fluorescent reporter molecules only inside the binding nanocavities, enabling sufficiently high sensitivity to detect intact exosomes without pretreatment. The effectiveness of the system was demonstrated by using it to discriminate between normal exosomes and those originating from prostate cancer and analyze exosomes in tear drops.

Methionine addiction is a fundamental and general hallmark of cancer discovered by us almost a half-century ago [Proc Natl Acad Sci U S A 73 (1976) 1523-1527]. Methionine addiction is defined as the requirement, specific for cancer cells of all types, for exogenous methionine despite the normal ability to synthesize methionine from homocysteine. The methionine addiction of cancer is termed the Hoffman-effect, analogous to the Warburg-effect of the high glucose requirement of cancer cells. Methionine addiction is due to excess transmethylation reactions resulting in high methionine flux in cancer cells, which causes them to selectively arrest under methionine restriction due to depletion of free methionine and S-adenosyl methionine. Recently we have shown methionine-addicted cancer cells over-methylate histone H3 lysine marks which are not over-methylated in normal cells or in low-malignancy methionine-independent revertants derived from methionine-addicted cancer cells. In the present report, we show that in patient-derived xenograft (PDX) mouse models of the most common soft tissue sarcomas: myxofibrosarcoma, undifferentiated pleomorphic sarcoma (UPS) and liposarcoma, histone H3K4me3 and H3K9me3 are super over-methylated compared to normal muscle tissue. This new result is discussed along with our previous reports, regarding the potential of histone H3 over-methylation as a basis of malignancy.

Objectives Methionine addiction is a fundamental and general hallmark of cancer caused by enhanced methyl flux. In the present study, we effected a novel methionine-methylation blockade to target a patient-derived orthotopic xenograft model of pancreatic cancer. Methods The pancreatic cancer patient-derived orthotopic xenograft mouse models were randomized into 6 groups of 8 mice each and treated for 2 weeks: untreated control; azacitidine; oral recombinant methioninase (o-rMETase); o-rMETase plus cycloleucine; o-rMETase plus cycloleucine plus azacitidine (triple-methyl blockade therapy); and gemcitabine (positive control). Results Triple-methyl blockade therapy arrested tumor growth (mean relative tumor volume, 1.03 [standard deviation, 0.36]) and was significantly more effective compared with azacitidine (P = 0.0001); o-rMETase (P = 0.007); or o-rMETase plus cycloleucine (P = 0.04). Gemcitabine alone also inhibited but did not arrest tumor growth (mean relative tumor volume, 1.50 [standard deviation, 0.30]). The percentage of cancer cells that were negative for 5-methylcytosine staining in immunohistochemistry, indicating reduction of DNA methylation, increased with triple-methyl blockade therapy (37.5%), compared with gemcitabine (1.8%); o-rMETase (2.8%); azacitidine (9.0%); or o-rMETase plus cycloleucine (10.6%). Conclusions This new concept of triple-methyl blockade therapy has clinical potential for pancreatic cancer, which is currently a recalcitrant disease.

Background/aim Methionine addiction, a fundamental and general hallmark of cancer, is due to the excess use of methionine for transmethylation, and is described as the Hoffman-effect. Methionine-addicted cancer cells can revert at low frequency to methionine independence when selected under methionine-restriction. We report here that highly-malignant methionine-addicted H460 human lung-cancer cells, when selected for methionine independence, have greatly-reduced tumorigenic potential. Materials and methods Methionine-addicted H460 parental cancer cells and methionine-independent revertant H460-R1 cells were injected in nude mice subcutaneously. Results When the parental H460 methionine-addicted cells were injected in nude mice at 2.5×105, 1×105 and 5×104, the cells could form tumors. In contrast, the H460-R1 methionine-independent revertant cells could not form tumors when the above-listed cell numbers were injected in nude mice. Conclusion There is a tight linkage between methionine addiction and malignancy.

Methionine addiction is a fundamental and general hallmark of cancer and is an area of current intense interest. Methionine addiction results from the overuse of methionine by cancer cells for excess transmethylation reactions. In order to identify excess transmethylation reactions in cancer and further understand the basis of methionine addiction, we compared the histone H3 lysine methylation status between methionine-addicted cancer cells, normal cells and rare revertants of methionine-addicted cancer cells which regained methionine independence and lost malignancy. The levels of H3K4me3, H3K9me3 and H3K36me3 were strongly elevated in methionine-addicted cancer cells in vitro compared to methionine-independent revertants isolated from the cancer cells. Tumorigenicity and experimental metastatic potential in nude mice were highly reduced in the methionine-independent revertants compared to the parental cells. Our previous studies showed that methionine restriction (MR) selectively arrests methionine-addicted cancer cells due to loss of histone H3 lysine methylation which was stable in normal cells under MR. Our previous and present results suggest that overmethylation of histone H3 lysine is linked with methionine addiction of cancer, required for the growth of cancer cells in vitro and in vivo, and necessary for malignancy as the histone lysine overmethylation disappears when the methionine-addicted cells revert to methionine independence and reduced malignancy. Methionine addiction has revealed fundamental molecular changes necessary for malignancy and presents great potential as a pan-cancer therapeutic target.

Background/aim The direct placement of patient tumors in 2-D culture on plastic or glass surfaces has inhibited the establishment of patient-derived cancer cells (PDCCs). The aim of the present study was to develop universal and efficient methods to prepare PDCCs. Materials and methods Fragments of patient-derived xenograft (PDX) tumors established form colon cancer liver metastasis (1 mm3) were placed on Gelfoam and cultured in DMEM. Results PDX tumor fragments were cultured on Gelfoam. Cancer cells migrated from the explant and formed distinct 3-D structures in the Gelfoam. Each of the three PDCCs showed a distinct morphology. The cultures were essentially all cancer cells without fibroblasts, the opposite of what usually occurs in 2-D culture on plastic or glass. Gelfoam cultures could be readily passaged from one Gelfoam cube to anothers suggesting indefinite culture potential. Conclusion A potentially universal method to establish PDCC using PDX tumors and 3-D Gelfoam histoculture was developed.

Ischemia reperfusion injury (IRI) during liver-metastasis resection for treatment of colon cancer may increase the risk of further metastasis. Peroxisome proliferator-activated receptor-γ (PPARγ) activation has been observed to exert a protective effect against IRI and IRI-induced metastasis of hepatocellular carcinoma. The present study aimed to investigate the effect of the PPARγ agonist pioglitazone on tumor metastasis and liver injury following IRI in a mouse model of colon cancer. Pioglitazone (30 mg/kg weight) was administered orally 1.5 h before and 2 h after the initiation of ischemia and was orally administrated daily to mice from day 0–21. SL4-cancer cells expressing red fluorescent protein (SL4-RFP) (1 × 106) were injected into the spleen. Fifteen minutes after injection, the hepatoduodenal ligament was clamped with a vessel clip, and released 5 min later. Liver, blood and tumor samples were taken from mice in order to determine if inflammation was induced by IRI. The effect of pioglitazone on liver metastasis was assessed. Furthermore, the effect of pioglitazone to control the inflammatory response during IRI progression was examined. Liver metastasis along with MMP-9 activation and the production of inflammatory cytokines were resistant to pioglitazone. Our results indicate that liver metastasis and associated inflammation in mice were resistant to pioglitazone.

Methionine addiction is a fundamental and general hallmark of cancer. Methionine addiction prevents cancer cells, but not normal cells from proliferation under methionine restriction (MR). Previous studies reported that MR altered the histone methylation levels in methionine-addicted cancer cells. However, no study has yet compared the status of histone methylation status, under MR, between cancer cells and normal cells. In the present study, we compared the histone methylation status between cancer cells and normal fibroblasts of H3K4me3 and H3K9me3, using recombinant methioninase (rMETase) to effect MR. Human lung and colon cancer cell lines and human normal foreskin fibroblasts were cultured in control medium or medium with rMETase. The viability of foreskin fibroblasts was approximately 10 times more resistant to rMETase than the cancer cells in vitro. Proliferation only of the cancer cells ceased under MR. The histone methylation status of H3K4me3 and H3K9me3 under MR was evaluated by immunoblotting. The levels of the H3K4me3 and H3K9me3 were strongly decreased by MR in the cancer cells. In contrast, the levels of H3K4me3 and H3K9me3 were not altered by MR in normal fibroblasts. The present results suggest that histone methylation status of H3K4me3 and H3K9me3 under MR was unstable in cancer cells but stable in normal cells and the instability of histone methylation status under MR may determine the high methionine dependency of cancer cells to survive and proliferate.

Background/aim A more realistic mouse model of bladder cancer is necessary to develop effective drugs for the disease. Tumor models enhanced by bright fluorescent-reporter genes to follow the disease in real-time would enhance the ability to accurately predict the efficacy of various therapeutics on this particularly-malignant human cancer. Materials and methods A highly-fluorescent green fluorescent protein (GFP)-expressing bladder cancer model was orthotopically established in nude mice using the UM-UC-3 human bladder-cancer cell line (UM-UC-3-GFP). Fragments from a subcutaneous tumor of UM-UC-3-GFP were surgically implanted into the nude mouse bladder. Non-invasive and intra-vital fluorescence imaging was obtained with a simple imaging box. Results The GFP-expressing orthotopic bladder tumor was imaged in real-time non-invasively as well as intra-vitally, with the two methods correlating at r=0.99. Conclusion This is the first non-invasive-fluorescence-imaging orthotopic model of bladder cancer and can be used for rapidly screening novel effective agents for this recalcitrant disease.

Methionine addiction is a fundamental and general hallmark of cancer. Gene expression analysis showed that methionine restriction (MR) of methionine-addicted cancer cells increases TNF-related apoptosis-induced ligand receptor-2 (TRAIL-R2) expression. Here, we determined the effects of MR on TRAIL-R2 targeted therapy in pancreatic cancer by the TRAIL-R2 agonist tigatuzumab. Human pancreatic cancer cell lines were cultured in control or methionine-free medium. The effects of MR on TRAIL-R2 expression and sensitivity to tigatuzumab were evaluated in vitro. An orthotopic pancreatic cancer mouse model was established to evaluate the efficacy of MR using oral recombinant methioninase (o-rMETase), and the efficacy of tigatuzumab and their combination. MR enabled tigatuzumab-induced apoptosis, by increasing TRAIL-R2 expression in pancreatic cancer cells in vitro. The protein expression level of the melanoma-associated antigen MAGED2, which reduces TRAIL-R2 expression, was decreased by MR. In the orthotopic pancreatic cancer mouse model, o-rMETase increased TRAIL-R2 expression level in the tumors and enabled the antitumor efficacy of tigatuzumab. MR, effected by o-rMETase, enabled the efficacy of the TRAIL-R2 agonist tigatuzumab by increasing TRAIL-R2 expression in pancreatic cancer. Our results suggest that o-rMETase has clinical potential for treating pancreatic cancer.

Background/aim Matrix-producing breast carcinoma (MPBC) is a rare and usually aggressive triple-negative breast cancer (TNBC). In this study, we determined drug sensitivity for a triple-negative MPBC, without BRCA mutations, in a patient-derived orthotopic xenograft (PDOX) model. Materials and methods The MPBC PDOX model was established in the left 2nd mammary gland of nude mouse by implantation of the patient tumor using surgical orthotopic implantation (SOI). We randomized MPBC PDOX mice into 5 groups (n=5 mice/per treatment group) when the tumor volume reached 80 mm3: G1, control-no treatment; G2, bevacizumab [intra-peritoneal (i.p.), weekly, for 2 weeks]; G3, vinorelbine (i.p., weekly, for 2 weeks); G4, olaparib (oral., daily, for 2 weeks); G5, eribulin [intravenous (i.v.), weekly, for 2 weeks]. The mice in each treatment group were sacrificed on day 15. Tumor volume and body weight were measured once/week. Results The MPBC PDOX model was resistant to olaparib (p=0.22). The MPBC PDOX model treated with bevacizumab and vinorelbine showed significantly suppressed tumor growth compared to the untreated group (p=0.005 and 0.002, respectively). However, only eribulin regressed the tumor (p=0.0001). Eribulin was more effective than olaparib (p=0.0001), bevacizumab (p=0.0025) and vinorelbine (p=0.0061). Conclusion Eribulin has clinical potential as treatment for triple-negative MPBC patients that are resistant to a PARP inhibitor such as olaparib.

Purpose/Objectives: The mechanism of intercellular communication after radiation exposure in cancer cells remains fully undetermined. Exosomes are lipid bilayer-constituted, membrane-enclosed small vesicles that are recognized as mediators transporting a variety of intracellular components including miRNA. Here we identified the novel role of exosomes released from irradiated cells to neighboring cancer cells. Materials/Methods: Human pancreatic cancer cell line MIAPaCa-2 was used in this study. Purified exosome product (PEP) was obtained from cultured media by ultra-centrifugation. PEP was morphologically confirmed by transmission electron microscopy (TEM), and analyzed by NanoSight. Exosome-specific surface markers CD9 and CD63 were evaluated by western blotting. Endocytosis of irradiated exosomes was confirmed by fluorescent microscopy by using the PKH26 dye. Cell survival after irradiation was evaluated by a colony-forming assay. Intracellular reactive oxygen species (ROS) levels were determined using the oxidation-sensitive fluorescent probe dye C-H 2 DCF, and DNA damage was evaluated by detecting phosphorylated histone 2AX (γ-H2AX) foci by immunocytochemistry. MiRNAs were isolated from the exosomes after 5 Gy or 8 Gy of irradiation and comprehensive miRNA expression analysis was performed by miRNA microarray analyses. Expressions of Cu/Zn superoxide dismutase enzyme (SOD1) or Mn superoxide dismutase enzyme (SOD2), catalase, and glutathione peroxidase were studied to determine whether the exosomes received by the neighboring cells may have influence to them and lead to production of ROS or not. Results: Exosome characteristics were confirmed by multiple methods. The uptake of irradiated exosomes was significantly higher than that of nonirradiated exosomes. Notably, nonirradiated neighboring cells with irradiated exosomes induced higher intracellular levels of ROS, and a higher frequency of DNA damage. These neighboring cells also showed greater sensitivity to radiation. Seven upregulated and 5 downregulated miRNAs were identified that correlated with the miRNA microarray analyses results obtained after 5 Gy and 8 Gy radiation. Among them, miR-6823-5p was identified as a possible candidate for SOD1 inhibition, leading to intracellular ROS production and DNA damage. Conclusions : This is the first study to determine that irradiated exosomes can enhance the radiation effect via ROS production in cancer cells. This novel finding may lead to the understanding of the bystander effect of neighboring cancer cells.

Purpose To develop a novel biocompatible solid fiducial marker that prevents radiopaque imaging artifacts and also maintains high imaging contrast for kilovoltage x-ray image-guided radiation therapy. Methods The fiducial marker was made of pure zinc. An in-house water-equivalent phantom was designed to evaluate artifacts and visibility under various simulated treatment scenarios. Image artifacts were quantitatively assessed in terms of the metal artifact index (MAI) on kilovoltage computed tomography (CT) and cone-beam CT (CBCT) scans. Marker visibility was evaluated on two types of kilovoltage planar x-ray images in terms of the contrast-to-background ratio (CBR). Comparisons with a conventional gold fiducial marker were conducted. Results The use of zinc rather than a gold marker mitigates imaging artifacts. The MAI near the zinc marker decreased by 76, 79, and 77 % in CT, and by 77 (81), 74 (80), and 79 (85) % in CBCT full-fan (half-fan) scans, when using one-, two-, and three-marker phantom settings, respectively. The high-contrast part of the zinc marker exhibited CBRs above 2.00 for 28/32 exposures under four (lung, tissue, low-density bone, and high-density bone) different simulation scenarios, making its visibility comparable to that of the gold marker (30/32 exposures with CBRs > 2.00). Conclusions We developed a biocompatible, artifact-robust, and highly visible solid zinc fiducial marker. Although further evaluation is needed in clinical settings, our findings suggest its feasibility and benefits for kilovoltage x-ray image-guided radiation therapy.

Background/Aim: We have recently shown that oral recombinant methionase (o-rMETase) prevents obesity in mice on a high-fat (HF) diet. The present study aimed to determine if o-rMETase can inhibit the onset of diabetes in mice on a HF diet. Materials and Methods: The mice on a HF diet were divided into two groups: 1) HF+phosphate buffered saline (PBS) group; 2) HF+o-rMETase group. Results: The blood glucose level in the HF+PBS group increased to average of 201 mg/dl during the experimental period of 8 weeks. In contrast, the blood glucose level in the HF+o-rMETase group maintained an average of 126 mg/dl (p

The precise mechanism of intercellular communication between cancer cells following radiation exposure is unclear. Exosomes are membrane‑enclosed small vesicles comprising lipid bilayers and are mediators of intercellular communication that transport a variety of intracellular components, including microRNAs (miRNAs or miRs). The present study aimed to identify novel roles of exosomes released from irradiated cells to neighboring cancer cells. In order to confirm the presence of exosomes in the human pancreatic cancer cell line MIAPaCa‑2, ultracentrifugation was performed followed by transmission electron microscopy and nanoparticle tracking analysis (NanoSight) using the exosome‑specific surface markers CD9 and CD63. Subsequent endocytosis of exosomes was confirmed by fluorescent microscopy. Cell survival following irradiation and the addition of exosomes was evaluated by colony forming assay. Expression levels of miRNAs in exosomes were then quantified by microarray analysis, while protein expression levels of Cu/Zn‑ and Mn‑superoxide dismutase (SOD1 and 2, respectively) enzymes in MIAPaCa‑2 cells were evaluated by western blotting. Results showed that the uptake of irradiated exosomes was significantly higher than that of non‑irradiated exosomes. Notably, irradiated exosomes induced higher intracellular levels of reactive oxygen species (ROS) and a higher frequency of DNA damage in MIAPaCa‑2 cells, as determined by fluorescent microscopy and immunocytochemistry, respectively. Moreover, six up‑ and five downregulated miRNAs were identified in 5 and 8 Gy‑irradiated cells using miRNA microarray analyses. Further analysis using miRNA mimics and reverse transcription‑quantitative PCR identified miR‑6823‑5p as a potential candidate to inhibit SOD1, leading to increased intracellular ROS levels and DNA damage. To the best of our knowledge, the present study is the first to demonstrate that irradiated exosomes enhance the radiation effect via increasing intracellular ROS levels in cancer cells. This contributes to improved understanding of the bystander effect of neighboring cancer cells.

In cancer research, small animal models, for example, mice, rats, or rabbits, facilitate the in-depth study of biological processes and the effects of radiation treatment that can lead to breakthrough discoveries. However, the physical quality of small animal irradiation systems has not been previously evaluated. In this study, we evaluate the quality of a small animal irradiation system using GAFCHROMICTM film and a Tough Water Phantom. The profiles and percentage depth dose curves for several irradiation conditions were measured to evaluate the quality of the irradiation system. The symmetry ratios when the table was rotated were 1.1 (no filter), 1.0 (0.5 mm Al filter), 1.0 (1.0 mm Al filter), 1.1 (2 mm Al filter), and 1.0 (filter consisting of 0.5 mm Al combined with 0.1 mm Cu). The results of measuring the percentage depth dose curve showed that the relative doses were 17.5% (10 mm depth), 12.4% (20 mm depth), 9.5% (30 mm depth), and 7.4% (40 mm filter) with no filters inserted, 78.0% (10 mm depth), 61.1% (20 mm depth), 46.9% (30 mm depth), and 35.3% (40 mm depth) when a 1.0 mm Al filter was inserted, and 94.4% (10 mm depth), 81.7% (20 mm depth), 68.1% (30 mm depth), and 54.7% (40 mm depth) when a filter consisting of 1.0 mm Al combined with 0.2 mm Cu was inserted. These physical assessments seem to be necessary especially in vivo experiments because those increase reliability of data obtained from small animal irradiation systems.