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Estimating river discharge using climate model output can aid in analyzing the potential impacts of climate change on water-related disasters. This study aimed to explore the uncertainty in simulated streamflow using the non-hydrostatic regional climate model (NHRCM) outputs in Thailand. The NHRCM was simulated at 5- and 2 km resolutions. To estimate runoff, two land surface models (LSMs) were employed: the Meteorological Research Institute–Simple Biosphere Model (MRI-SiB) in NHRCM and the Simple Biosphere including Urban Canopy (SiBUC). The NHRCM rainfalls captured the seasonal pattern of rainfall in the upper Ping River Basin, although they were underestimated. The 2 km NHRCM had less rainfall, but it captured the local topography better than the 5 km model. This difference in rainfall affected the simulated streamflow. Furthermore, the uncertainty of the simulated streamflow was influenced by the different runoff schemes used by the LSMs. For instance, MRI-SiB incorporates a direct infiltration structure from the surface to the second soil layer and estimates subsurface runoff using hydraulic diffusion and gravitational flow, while SiBUC uses a gravitational-only subsurface runoff approach. These variations led to significant disparities in surface and subsurface runoff. Future work should enhance the accuracy of rainfall from climate models and runoff from LSMs for assessing the potential impacts of climate change on water-related disasters.

To manage flood disaster with an exceeding designed level, flood risk control based on appropriate risk assessment is essential. To make an integrated economic risk assessment by flood disaster, a flood risk curve, which is a relation between flood inundation damage and its exceedance probability, plays an important role. This research purposes a method to develop a flood risk curve by utilizing a probability distribution function of annual maximum rainfall through rainfall-runoff and inundation simulations so that risk assessment can consider climate and socio-economic changes. Among a variety of uncertainties, the method proposed in this study considered spatio-temporal rainfall distributions that have high uncertainty for damage estimation. The method was applied to the Yura-gawa river basin (1882 km2) in Japan; and the annual economic benefit of an existing dam in the basin was successfully quantified by comparing flood risk curves with/without the dam.

There is much research assessing the impact of climate change on the hydrologic cycle. However, it has often focused on a specific hydrologic process, without considering the interaction among hydrologic processes. In this study, a distributed hydrologic model considering the interaction between flow routing and land surface processes was developed, and its effect on river discharge estimation was investigated. The model enables consideration of flow routing, irrigation withdrawal from rivers at paddy fields, crop growth depending on water and energy status, and evapotranspiration based on meteorological, soil water and vegetation status. To examine the effects of hydrologic process interaction on river discharge estimation, a developed model was applied to the Chao Phraya river basin using near surface meteorological data collected by the Japanese Meteorological Research Institute's Atmospheric General Circulation Model (MRI-AGCM3.2S) with TL959 spatial resolution as forcing data. Also, a flow routing model, which was part of the developed model, was applied independently, using surface and subsurface runoff data from the same GCM. In the results, the developed model tended to estimate a smaller river discharge than was estimated by the river routing model, because of the irrigation effect. In contrast, the annual maximum daily discharge calculated by the developed model was 24% greater than that by the flow routing model. It is assumed that surface runoff in the developed model was greater than that in the flow routing model because the soil water content was maintained at a high level through irrigation withdrawal. As for drought discharge, which is defined as the 355th largest daily discharge, the developed model gave a discharge 2.7-fold greater than the flow routing model. It seems that subsurface runoff in the developed model was greater than that in the flow routing model. The results of this study suggest that considering hydrologic interaction in a numerical model could affect both flood and drought estimation.

Immunotherapy improves survival outcomes in cancer patients, but there is still an unmet clinical need in the treatment of brain metastases. Here, we used a mouse model to investigate the antitumor effect of programmed death-ligand 1 (PD-L1) and vascular endothelial growth factor (VEGF) dual blockade on metastatic brain tumors and evaluated immune responses during treatment. After establishing hematogenous brain metastasis by transplanting murine bladder carcinoma MBT2 cells stably expressing secNLuc reporter via the internal carotid artery of C3H/HeNCrl mice, we observed the formation of metastases not only in the brain parenchyma but also in the ventricles. The observed pathological areas showed that metastases in the ventricle were histologically larger than that in the brain parenchyma. Regarding the total tumor burden in the whole brain as revealed by Nluc activities, the combination of anti-PD-L1 antibody and anti-VEGF antibody showed a stronger anti-tumor effect than each single agent. Anti-PD-L1 antibody alone enhanced CD8 + T cell priming in regional lymph nodes, increased the proportion of activated CD8 + T cells in whole brain, and increased the density of CD8 + cells in the brain parenchyma. Furthermore, anti-VEGF antibody alone decreased microvessel density (MVD) in ventricular metastases, and the combination treatment increased intratumoral CD8 + cell density in the brain parenchyma and ventricular metastases. These results suggest that PD-L1 blockade enhanced cancer immunity not only in brain metastases lesions but also in the regional lymph nodes of the metastases, and that the addition of VEGF blockade increased the antitumor effect by increasing the infiltration of activated CD8 + T cell and decreasing MVD., Competing Interests: Declarations Conflicts of interest Chinami Masuda, Shinichi Onishi, Keigo Yorozu, Mitsue Kurasawa, Mamiko Morinaga, Daiko Wakita, Masamichi Sugimoto are employees of Chugai Pharmaceutical Co., Ltd. Research involving human participants and/or animals All animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals at Chugai Pharmaceutical Co. Ltd., and all animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Chugai Pharmaceutical Co., Ltd. Informed consent This article does not contain any studies involving human participants performed by any of the authors., (© 2024. The Author(s).)

Myasthenia gravis (MG) is a chronic autoimmune disease characterized by muscle weakness and fatigue. It is caused by pathological autoantibodies against components expressed at neuromuscular junctions, such as acetylcholine receptor (AChR). Interleukin-6 (IL-6) has been suggested to play a role in the pathogenesis of MG, and IL-6 receptor (IL-6R) antibody treatment may provide a novel therapeutic option. In this study, we investigated the effects of IL-6R antibody treatment in an experimental autoimmune MG (EAMG) mouse model. We demonstrated that IL-6R antibody treatment improved muscle weakness, reduced IgG deposition at neuromuscular junctions, and the levels of AChR autoantibodies in serum. In addition, follicular helper T cells and Th17, plasma cells in lymph nodes were lower in IL-6R antibody treated mice. Our findings suggest that IL-6R blockade may be a novel and effective therapeutic strategy for the treatment of MG., Competing Interests: SM, KS, SO, YK, MB, MK, HT-S, KY, YM, and MN-S were employed by Chugai Pharmaceutical Co., Ltd., (Copyright © 2024 Miyake, Serizawa, Onishi, Katsura, Baba, Kurasawa, Tomizawa-Shinohara, Yorozu, Matsumoto and Noguchi-Sasaki.)

Patients with a history of cesarean section and a highly adherent bladder may have difficulty in bladder dissection and may suffer damage. It may also be difficult to orient the patient for repair in the event of damage. In such cases, dissection from the side between the bladder and cervix is a good way to avoid complication. This video reviews the steps of the HUBB technique procedure, provides tips and tricks for performing a successful adhesiolysis, and illustrates the procedure's adaptability. The first is the identification of the uterine artery and its position. Step2, a Laparoscopic retrovesical lower uterine segment bypass is created. Step3 a Hung Up the Bladder Bypass is created, the bladder is completely dissected. Step4, an adhesiolysis and leak test is performed. In this report, we would like to introduce a technique for creating a retrovesical lower uterine segment bypass using a laparoscope and traction on the bypass to dissect the bladder., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

We challenged to create a mouse model of neuromyelitis optica spectrum disorder (NMOSD) induced by AQP4 peptide immunization. Intradermal immunization with AQP4 p201-220 peptide induced paralysis in C57BL/6J mice, but not in AQP4 KO mice. AQP4 peptide-immunized mice showed pathological features similar to NMOSD. Administration of anti-IL-6 receptor antibody (MR16-1) inhibited the induction of clinical signs and prevented the loss of GFAP/AQP4 and deposition of complement factors in AQP4 peptide-immunized mice. This novel experimental model may contribute to further understanding the pathogenesis of NMOSD, elucidating the mechanism of action of therapeutic agents, and developing new therapeutic approaches., Competing Interests: Declaration of Competing Interest All authors are employees of Chugai Pharmaceutical Co., Ltd., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Gene amplification and protein overexpression of human epidermal growth factor receptor type 2 (HER2) are specific targets for HER2-targeting drugs in breast, gastric, salivary gland, and colorectal cancers. The histopathological determination of HER2 status is crucial for treatment, highlighting the importance of improving HER2 detection accuracy in clinical practice. We prepared tissue microarray (TMA) slides for use as control slides for the standardization of gastric HER2 testing. Four human gastric cancer cell lines with HER2 scores of 3+, 2+, 1+, and 0 were xenografted in NOG mice. The TMA slides were constructed using samples from three different areas in these tumors. Staining properties were determined using six clinical kits for HER2. In TMA, HER2-positive tumors with HER2 scores of 3+ and 2+ showed good staining with all diagnostic kits, and the tissue images were similar to those of clinical samples. Xenograft tumor slides could potentially be used as external controls to standardize staining conditions for a variety of kits and may improve the accuracy of HER2 detection in clinical practice., (© 2022 The Authors. Pathology International published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Background/aim: Although CDK4/6 inhibitors have been increasingly used in combination with hormonal agents to treat hormone-receptor positive and human epithelial growth factor receptor 2-negative breast cancer, the mechanism of CDK4/6 inhibitor resistance and its impact on established therapy for post-resistance, especially bevacizumab combined with chemotherapy, are unclear., Materials and Methods: Sensitivity of RB knockout MCF7 clones to CDK4/6 inhibitors was evaluated in vitro. One RB knockout clone was subcutaneously implanted in nude mice and the effects of bevacizumab on volume and microvessel density (MVD) of tumors were investigated., Results: Palbociclib did not exhibit antitumor efficacy against the RB knockout tumor, in contrast to the parental MCF7 xenograft model. Bevacizumab significantly exhibited antitumor efficacy and suppressed the MVD both in RB knockout and parental MCF7 xenograft models., Conclusion: Bevacizumab inhibited tumor growth by suppressing MVD in the CDK4/6 inhibitor-resistant tumor acquired due to RB loss, suggesting its efficacy also in patients after treatment with CDK4/6 inhibitors., Competing Interests: All the Authors are employees of Chugai Pharmaceutical Co., Ltd., Tokyo, Japan., (Copyright 2022, International Institute of Anticancer Research.)

We reported the development of an effective cancer treatment using a multidisciplinary treatment, including photodynamic therapy (PDT) with indocyanine green (ICG) liposomes and a combination of Lentinula edodes mycelia (LEM) and hydrogen gas inhalation therapy. ICG liposomes were prepared by adding 5 mg of ICG to 50 mL liposomes. Later, 25 mL of ICG liposomes were diluted with 250 mL of 5% glucose solution and administered intravenously to the patient. We selected the multi-laser delivery system (MLDS), a laser irradiator for performing PDT. Further, the patients received a combination of LEM and hydrogen gas inhalation therapy throughout the treatment. We reported two cases of PDT therapy, one with middle intrathoracic esophagus carcinoma and the other with hypopharyngeal cancer. In the first case, the MLDS laser was directly attached to the endoscope and directed to the cancer area with wavelengths of 810 nm. After the treatment, a biopsy demonstrated no tumor recurrence. In the second case, the patient was treated with endovascular PDT using ICG liposomes and MLDS fiber optics. Later, tumor shrinkage was demonstrated after the first round and disappeared after six months. In conclusion, the present findings suggest that the effect of PDT using ICG liposomes with LEM and hydrogen gas may eradicate cancer without burdening patients by enhancing tumor immunity.

Background: The glycoengineered, humanized anti-CD20 antibody obinutuzumab is indicated for previously untreated or relapsed/refractory CD20-positive follicular lymphoma (FL). However, the effectiveness of obinutuzumab retreatment in relapsed/refractory FL after prior obinutuzumab-containing therapy is unclear. To address this issue, we investigated the antitumor activity of obinutuzumab plus bendamustine in obinutuzumab-resistant tumors established from a human non-Hodgkin lymphoma xenograft model., Materials and Methods: Obinutuzumab-resistant tumors (SU-DHL-4-OR-18-8) were established from an SU-DHL-4 xenograft model by repeated administration of obinutuzumab. Antitumor activity was evaluated based on tumor volume after treatment with obinutuzumab on Day 1, 8, and 15 and/or bendamustine on Day 1 and 2. Intratumoral natural killer (NK) cells/macrophages were evaluated by immunohistochemistry and flow cytometry., Results: In SU-DHL-4-OR-18-8 xenografted tumors, intratumoral NK cells/macrophages after obinutuzumab treatment were significantly decreased compared with parent tumors on Day 4. The endoplasmic reticulum stress sensor phospho-IRE1 was also decreased. In SU-DHL-4-OR-18-8 tumors, bendamustine treatment increased phospho-IRE1 on Day 4 and intratumor NK cells/macrophages on Day 10. Obinutuzumab combined with bendamustine significantly increased antitumor activity compared with each single agent on Day 29, with an increase in chemoattractant CCL6 expression on Day 10., Conclusions: Coadministration of bendamustine in obinutuzumab retreatment may be effective against obinutuzumab-resistant tumors., (© 2022. Japanese Society of Hematology.)

Brain metastases are common complication in cancer patients. Immune checkpoint inhibitors show therapeutic benefits also in patients with central nervous system (CNS) metastases. However, their antitumor effects on metastatic tumors and their underlying mechanisms are still poorly understood. In this study we investigated the antitumor effect of anti-programmed death-ligand 1 (PD-L1) antibody on metastatic brain tumors and evaluated immune responses during treatment. We employed a hematogenous brain metastasis xenograft model using immunodeficient mice with murine lymphocyte infusions. A human non-small-cell lung cancer (NSCLC) cell line stably expressing NanoLuc ® reporter (Nluc-H1915) was inoculated from the internal carotid artery of SCID mice. After metastases were established (24 days after inoculation), splenocytes prepared from H1915-immunized BALB/c mice were injected intravenously and mouse IgG or anti-PD-L1 antibody treatment was started (day 1). Evaluated by Nluc activity, tumor volume in the brain on day 14 was significantly lower in anti-PD-L1-treated mice than in mouse IgG-treated mice. Furthermore CD8 + cells were primarily infiltrated intratumorally and peritumorally and anti-PD-L1 treatment induced a significantly higher proportion of Granzyme B (GzmB) + cells among CD8 + T cells. The antitumor effect of anti-PD-L1 antibody on brain metastasis is thought to be achieved by the enhanced activation of infiltrated CD8 + T cells into metastatic brain tumor. These results suggest that anti-PD-L1 antibody-containing regimens may be promising treatment options for cancer patients with brain metastases., (© 2021. The Author(s).)

The efficacy of programmed cell death‑ligand 1 (PD‑L1)/programmed cell death protein 1 (PD‑1) blockade therapy has been demonstrated but is limited in patients with PD‑L1 low or immune desert tumors. This limitation can be overcome by combination therapies that include anti‑vascular endothelial growth factor (VEGF) therapy. Such combinations have been investigated in clinical trials for a number of cancer types; however, evidence on the mechanisms underlying their effects in these types of patients is still not sufficient. Therefore, the present study investigated the efficacy and effects on CD8 + T cell and C‑X‑C motif chemokine receptor 3 (CXCR3) ligand expression in tumors by combining anti‑PD‑L1 and anti‑VEGF antibodies using an OV2944‑HM‑1 mouse model with PD‑L1 low and immune desert‑like phenotypes. Although the model exhibited anti‑PD‑L1 insensitivity, anti‑PD‑L1 antibody treatment combined with anti‑VEGF antibody inhibited tumor growth compared with anti‑VEGF monotherapy, which itself inhibited tumor growth compared with the control treatment on Day 25. In combination‑treated mice, a higher percentage of CD8 + T cells and higher levels of CXCR3 ligands were observed in tumor tissues compared with those in the anti‑VEGF antibody treatment group, which was not significantly different from control treatment on Day 8. The increase in the intratumoral percentage of CD8 + T cells following the combination treatment was reversed by CXCR3 blocking to the same level as the control. In an anti‑PD‑L1 insensitive model with PD‑L1 low and immune desert‑like phenotypes, although anti‑PD‑L1 antibody alone was not effective, anti‑PD‑L1 antibody in combination with anti‑VEGF antibody exhibited antitumor combination efficacy with an increase of CD8 + T cell infiltration, which was suggested to be dependent on the increase of intratumoral CXCR3 ligands. This mechanism could explain the efficacy of anti‑PD‑L1 antibody and anti‑VEGF antibody combination therapy in the clinical setting.

Immune-related pneumonitis is an important toxicity associated with checkpoint inhibitor therapy with anti-PD-1 or anti-PD-L1 antibodies, often necessitating discontinuation of treatment. Development of methods to mitigate checkpoint inhibitor-related pneumonitis is required.The contributions of PD-L1, PD-L2, and VEGF to the pathogenesis of pneumonitis were examined in an IL2- plus IL18-induced mouse pneumonitis model (IL pneumonitis model). Furthermore, the incidences of pneumonitis were retrospectively examined in patients with non-small cell lung cancer treated with the anti-PD-L1 mAb atezolizumab plus chemotherapy, with or without the anti-VEGF mAb bevacizumab, in the phase III IMpower150 trial. PD-1 signal blockade by anti-PD-L1 and anti-PD-L2 antibodies aggravated pneumonitis in the IL pneumonitis model. An anti-VEGF antibody prevented PD-1 signal blockade from aggravating pneumonitis in this model. PD-1 signal blockade induced interstitial T-cell infiltration in the lungs, but VEGF blockade did not affect this T-cell infiltration. The anti-VEGF antibody protected against vascular-to-alveolar leakage of protein and fluid due to PD-1 signal blockade in a murine model. In the IMpower150 trial, incidence rates of pneumonitis of any grade were 4.3% in the group without bevacizumab and 2.8% in the group with bevacizumab. In patients with pneumonitis, outcomes of "Not recovered/Not resolved" were reported for 29.4% in the group without bevacizumab compared with 9.1% in the group with bevacizumab. Our findings suggest that anti-VEGF antibodies in combination with checkpoint inhibitors may be a treatment method that can control checkpoint inhibitor-related pneumonitis., (©2021 The Authors; Published by the American Association for Cancer Research.)

There are various important factors in reducing the risk of cancer development and progression; these factors may correct an unbalanced intake of nutrients to maintain the living body's homeostasis, detoxify toxic materials, acting as an external factor, and maintain and strengthen the body's immune function. In a normal cell environment, nutrients, such as carbohydrates, lipids, proteins, vitamins, and minerals, are properly digested and absorbed into the body, and, as a result, an environment in which cancer can develop and progress is prevented. It is necessary to prevent toxic materials from entering the body and to detoxify poisons in the body. If these processes occur correctly, cells work normally, and genes cannot be damaged. The most important factor in the fight against cancer and prevention of the development and progression of cancer is the immune system. This requires a nutritional state in which the immune system works well, allowing the intestinal microbiome to carry out all of its roles. In order to grow intestinal microbiota, the consumption of prebiotics, such as organic vegetables, fruits, and dietary fiber, and probiotics of effective intestinal microbiota, such as fermented foods and supplements, is required. Symbiosis, in which these organisms work together, is an effective means of reducing the risk of cancer. In addition, fecal microbiota transplantation (FMT) using ultrafine bubble water, produced specially by the Association for Clinical Research of Fecal Microbiota Transplantation Japan, is also useful for improving the nutritional condition and reducing the risk of cancer.

Anti-PD-L1 antibodies benefit many cancer patients, even those with "non-inflamed tumor". Determining which patients will benefit remains an important clinical goal. In a non-inflamed tumor mouse model, we found that PD-L1 was highly expressed on antigen-presenting cells (APCs) especially on CD103 + CD11c + dendritic cells in tumor-draining lymph nodes (dLNs), suppressing T-cell priming by APCs. In this model, anti-PD-L1 antibodies enhanced T-cell priming and increased CXCR3 + activated T-cells in dLNs, which was followed by the trafficking of T-cells to tumors in response to CXCR3 ligands. As predictive biomarker, each APCs-related gene expression (AP score) alone or T-cells trafficking-related chemokine gene expression (T score) alone were still less than perfect among the 17 mouse models examined. However a combining score of AP score and T score (AP/T score) precisely identified anti-PD-L1-sensitive tumors. In the phase 3 trial of atezolizumab vs docetaxel in advanced NSCLC patients (OAK), the AP/T score could identify atezolizumab-treated NSCLC patients who achieved significant improvement in overall survival. This biomarker concept would be a clinically valuable for prediction of anti-PD-L1 antibody efficacy.

Brain metastases are common in patients with non-small-cell lung cancer (NSCLC). The efficacy of bevacizumab, an anti-vascular endothelial growth factor (VEGF) humanized antibody, has been demonstrated in patients with nonsquamous NSCLC. We established a transplantable NSCLC cell line (Nluc-H1915) that stably expresses NanoLuc® reporter and confirmed the correlation between total Nluc activity in tumor and tumor volume in vivo. SCID mice inoculated with these cells through the internal carotid artery formed reproducible brain metastases, in which human VEGF was detected. Next, after metastases were established in the model mice (15-17 days), they were intraperitoneally administered weekly doses of human immunoglobulin G (HuIgG) or bevacizumab. Nluc activity in the brain was significantly lower in bevacizumab-treated mice than in HuIgG-treated mice. Additionally, bevacizumab concentration in the brain was higher in mice with brain metastasis than in normal mice, and bevacizumab was primarily observed in brain metastasis lesions. The microvessel density in brain metastasis was lower in bevacizumab-treated mice than in HuIgG-treated mice. We believe bevacizumab's anti-proliferative effect on brain metastasis is due to anti-angiogenic activity achieved by its penetration into brain metastases; this suggests that a bevacizumab-containing regimen may be a promising treatment option for patients with NSCLC brain metastasis.

Aim: Iron overload is a life-threatening disorder that can increase the risks of cancer, cardiovascular disease, and liver cirrhosis. There is also a risk of iron overload in patients with chronic kidney disease. In patients with renal failure, iron storage is increased due to inadequate iron utilization associated with decreased erythropoiesis and also to the inflammatory status. To evade the risk of iron overload, an accurate and versatile indicator of body iron storage in patients with iron overload is needed. In this study, we aimed to find useful iron-related parameters that could accurately reflect body iron storage in mice in order to construct a murine model of iron overload., Methods: To select an appropriate indicator of body iron status, a variety of parameters involved in iron metabolism were evaluated. Noninvasively measured parameters were R1, R2, and R2 ∗ derived from magnetic resonance imaging (MRI). Invasively measured parameters included serum hepcidin levels, serum ferritin levels, and liver iron contents. Histopathological analysis was also conducted., Results/conclusion: Among the several parameters evaluated, the MRI T2 ∗ relaxation time was able to detect iron storage in the liver as sensitively as serum ferritin levels. Moreover, it is expected that using an MRI parameter will allow accurate evaluation of body iron storage in mice over time., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2019 Yukari Matsuo-Tezuka et al.)

Although bevacizumab maintenance following bevacizumab in combination with chemotherapy has demonstrated significant prolongation of progression-free survival in clinical studies in patients with ovarian cancer, the majority of the cancer cases in the study were of the serous histotype; therefore, data regarding clear cell carcinoma is limited. Furthermore, the efficacy of bevacizumab beyond progression has not yet been demonstrated in ovarian cancer. A xenograft model using the human ovarian clear cell carcinoma cell line RMG-I was used to investigate the antitumor effects and the mechanisms of bevacizumab in maintenance treatment and bevacizumab when administered beyond disease progression. In the RMG-I model, bevacizumab maintenance following bevacizumab in combination with paclitaxel exhibited increased tumor suppression, compared with its absence, and inhibited the increase of microvessel density (MVD) in tumors. Following disease progression during bevacizumab maintenance, continued bevacizumab treatment in combination with PEGylated liposomal doxorubicin as a secondary chemotherapeutic agent had increased efficacy, compared with PEGylated liposomal doxorubicin alone, and resulted in lower MVD accompanied with lower levels of insulin-like growth factor binding protein-3, which is reported to have angiogenic activity. Continuous suppression of angiogenesis by bevacizumab may contribute to the superior efficacy of bevacizumab maintenance and bevacizumab beyond progression in ovarian cancer.

We report on the application of radon inhalation therapy to patients with 4 types of cancer: colon, uterine, lung, and liver cell. The radon treatments were given to improve the efficacy of chemotherapy and were potent in all 4 cases. Marker values decreased and disease symptoms were alleviated. We include a lengthy discussion on the mechanism that may be responsible for the observed results. While employing the radon generator to treat the patient with hepatocellular carcinoma, we discovered that a concentration of 6 MBq/m 3 was very effective, while 1 MBq/m 3 was marginal. This implies different, and rather high, radon concentration thresholds for the treatment of different types of cancer. The evidence from these 4 cases suggests that radon inhalation may be beneficial against various cancer types as an important adjuvant therapy to conventional chemotherapy and for local high-dose radiotherapy, which would address the problem of distant metastasis. A previous case report on 2 patients with advanced breast cancer, who refused chemotherapy or radiotherapy, indicates that radon may be effective as a primary therapy for cancer. Clinical trials should be carried out to determine the best radon concentrations for treatment of other types of cancer, at different stages of progression., Competing Interests: Declaration of Conflicting Interests: The author(s) declare that there are no potential conflicts of interests with respect to the report, the authorship, and/or the publication of this article.

Purpose: Trastuzumab emtansine (T-DM1) provides clinical benefit in breast cancers overexpressing human epidermal growth factor receptor 2 (HER2). However, its efficacy against biliary tract cancers (BTC) has not been evaluated. In this study, the effectiveness of T-DM1 in various BTC cell lines and xenograft models with different levels of HER2 expression was investigated., Methods: HER2 expression status in xenografts and patient tissue microarrays was assessed by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH). Cell-surface HER2 expression levels and cell growth inhibition in response to T-DM1 were examined in 17 BTC cell lines. The antitumor activity of T-DM1 was evaluated in four xenograft mouse models with different levels of HER2 expression. The effects of T-DM1 on HER2 signaling, antibody-dependent cell-mediated cytotoxicity (ADCC), cell cycle, and apoptosis were assessed in vitro., Results: Cell-surface expression of HER2 was observed in both gallbladder carcinoma and cholangiocarcinoma tissues. The anti-proliferative activity of T-DM1 was higher in BTC cell lines and breast cancer cell lines with higher levels of HER2 expression. The HER2 status (IHC score|HER2-to-CEP17 ratio by FISH testing) of each BTC xenograft was 3 +|8.3 for KMCH-1, 2 +|4.7 for Mz-ChA-1, 1 +/0|1.4 for OCUG-1, and 0|1.1 for KKU-100, and T-DM1 showed antitumor activity in proportion to the HER2 status. T-DM1 inhibited HER2 signaling and induced ADCC, mitotic arrest, and apoptosis in KMCH-1 cells., Conclusions: T-DM1 exhibited preclinical activity in HER2-overexpressing BTC. Further evaluation in clinical studies is warranted.

Anti-PD-L1 antibodies inhibit interactions between PD-L1 and PD-1 and interactions between PD-L1 and B7-1, thereby reinvigorating anticancer immunity. Although there are numerous ongoing clinical studies evaluating combinations of standard chemotherapies and anti-PD-L1 antibodies, irinotecan has not yet been investigated in this context so there is little information about its compatibility with anti-PD-L1 antibodies. Here we investigated the efficacy of anti-PD-L1 antibody in combination with irinotecan and the role of irinotecan in the tumor-immunity cycle in an FM3A murine tumor model. Despite a transient decrease in lymphocytes in the peripheral blood after irinotecan treatment, the antitumor activity of anti-PD-L1 antibody plus irinotecan was significantly greater than each agent alone. Irinotecan in combination with anti-PD-L1 antibody enhanced proliferation of CD8 + cells in both tumors and lymph nodes, and the number of tumor-infiltrating CD8 + cells was higher than either irinotecan or anti-PD-L1 antibody monotherapy. Irinotecan was found to decrease the number of Tregs in lymph nodes and tumors, and specific depletion of Tregs by anti-folate receptor 4 antibodies was found to enhance the proliferation of CD8 + cells in this model. In addition, irinotecan augmented MHC class I expression on tumor cells and concurrently increased PD-L1 expression on tumor cells and tumor-infiltrating immune cells. These results indicate that irinotecan may enhance the effect of T cell activation caused by anti-PD-L1 treatment by reducing Tregs and augmenting MHC class I-mediated tumor antigen presentation, and concurrent upregulation of PD-L1 expression can be blocked by the anti-PD-L1 antibody. These interactions may contribute to the superior combination effect., Competing Interests: CONFLICTS OF INTEREST All authors are employees of Chugai Pharmaceutical Co., Ltd.

Epoetin beta pegol (continuous erythropoiesis receptor activator; C.E.R.A.), or methoxy-polyethylene glycol-modified epoetin beta, is a long-acting erythropoiesis stimulating agent (ESA) that effectively maintains hemoglobin levels. It promotes proliferation of erythroid progenitor cells in hematopoietic organs and leads to increased reticulocyte and hemoglobin levels. However, the detailed erythropoietic effects of various ESAs on their target organs have yet to be clarified, and new approaches are needed to analyze tissue iron localization with structural information. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) techniques are widely used in basic pharmaceutical research. High-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) imaging enables the spatial mapping and identification of biomolecules. In this study, mice administered with C.E.R.A. were fed a diet containing the stable iron isotope 57 Fe. The 57 Fe-heme + isotopic fine structure peak (m/z 617.1772) was separated from the non-labeled heme + isotopic peak (Δ0.0029) by FTICR-MS with a resolving power of more than 500,000. We optimized the platform to analyze the distribution of 57 Fe-heme in the spleen using MALDI FTICR-MS imaging. The combination of the ultrahigh resolution power of FTICR-MS and a stable isotope labeling technique has the potential to be very effective in basic pharmaceutical research. Graphical Abstract ᅟ.

In a Phase III trial for HER2-positive breast cancer (the CLEOPATRA study), the triple-drug combination arm of pertuzumab plus trastuzumab plus docetaxel showed significantly longer progression-free survival and overall survival than did the trastuzumab plus docetaxel arm. In this study, we investigated the mechanism of action of the triple-drug combination therapy in vivo . For this purpose, we established a mouse xenograft model using KPL-4, a HER2-positive human breast cancer cell line, in which the triple-drug combination treatment dramatically induced tumor regression compared with double-drug combinations (trastuzumab plus docetaxel, pertuzumab plus docetaxel, or pertuzumab plus trastuzumab). Four days after the triple-drug treatment was started, strong reduction in the phosphorylation of HER2, epidermal growth factor receptor (EGFR), HER3, extracellular signal-regulated kinase (ERK), and AKT in tumor tissues was seen, despite only weak suppression of phosphorylation seen with the single- or double-drug treatments. Histopathological analysis and flow cytometric analysis showed that the triple-drug treatment enhanced apoptosis after mitotic arrest induced by docetaxel. Furthermore, infiltration of mononuclear cells around the tumor cells was strongly induced by the triple-drug combination treatment. These results suggested that the mechanism underlying the synergistic efficacy of the triple-drug combination was attributable, at least in part, to the docetaxel-mediated apoptosis being promoted by enhanced inhibition of HER2-HER3-AKT signaling as well to the intratumor infiltration of mononuclear cells induced by anti-HER2 antibodies being enhanced by docetaxel.

Erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), shows superior efficacy in patients with non-small cell lung cancer (NSCLC) harboring activating EGFR mutations (EGFR Mut+). However, almost all tumors eventually develop resistance to erlotinib. Recently, the Phase II JO25567 study reported significant prolongation of progression-free survival (PFS) by erlotinib plus bevacizumab combination compared with erlotinib in EGFR Mut+ NSCLC. Herein, we established a preclinical model which became refractory to erlotinib after long-term administration and elucidated the mode of action of this combination. In this model, tumor regrowth occurred after remarkable shrinkage by erlotinib; regrowth was successfully inhibited by erlotinib plus bevacizumab. Tumor vascular endothelial growth factor (VEGF) was greatly reduced by erlotinib in the erlotinib-sensitive phase but significantly increased in the erlotinib-refractory phase despite continued treatment with erlotinib. Although EGFR phosphorylation remained suppressed in the erlotinib-refractory phase, phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated AKT, and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) were markedly higher than in the erlotinib-sensitive phase; among these, pERK was suppressed by erlotinib plus bevacizumab. MVD was decreased significantly more with erlotinib plus bevacizumab than with each drug alone. In conclusion, the erlotinib plus bevacizumab combination demonstrated promising efficacy in the B901L xenograft model of EGFR Mut+ NSCLC. Re-induction of VEGF and subsequent direct or indirect VEGF-dependent tumor growth was suggested as a major mechanism of erlotinib resistance, and erlotinib plus bevacizumab achieved remarkably prolonged antitumor activity in this model.

Erythropoiesis-stimulating agents are among the therapeutic options for renal anemia. Under erythropoietic stimulation, the synthesis of hemoglobin requires a large amount of iron, which is supplied both from absorption of dietary iron and the mobilization of stored iron. However, under iron-loading conditions, dietary iron absorption is suppressed via down-regulation of duodenal iron transporters. Because the contribution of dietary iron is essential for erythropoiesis, we aimed to investigate the conditions in which dietary iron is efficiently used for erythropoiesis. To quantify the contribution of dietary iron for erythropoiesis, we labelled dietary iron using the stable iron isotope 57 Fe, and assessed 57 Fe-derived hemoglobin synthesis under erythropoietic stimulation by C.E.R.A. (Continuous Erythropoietin Receptor Activator) and iron loading in mice. Our results show that the contribution of dietary iron to erythropoiesis is not changed by different increments of C.E.R.A. dose. However, iron loading suppressed the contribution of dietary iron to erythropoiesis. To confirm these results under conditions mimicking clinical settings, we compared single and intermittent administration of C.E.R.A. in mice under both physiological and iron-loaded conditions, and found that the contribution of dietary iron to erythropoiesis is more strongly affected by body iron status than by erythropoietic stimulation.

Background: Bevacizumab in combination with chemotherapeutics has shown significant survival benefit in clinical studies in patients with non-small cell lung cancer (NSCLC). Since bevacizumab was administered as standard treatment until disease progression, the importance of bevacizumab during the maintenance phase was not prospectively investigated in these studies., Materials and Methods: Three human NSCLC cell line xenograft models were used to investigate antitumor effect of bevacizumab and tumor microvessel density (MVD) was analyzed., Results: In A549 and NCI-H292 models, bevacizumab maintenance following combination with paclitaxel exhibited stronger tumor suppression than its absence. In an NCI-H292 model, bevacizumab maintenance continuously inhibited increase of MVD. In an NCI-H2228 model following induction treatment with pemetrexed and bevacizumab, maintenance with pemetrexed plus bevacizumab, had stronger efficacy than pemetrexed alone and led to lower MVD and level of thymidylate synthase., Conclusion: Continuous suppression of angiogenesis by bevacizumab may contribute to the superior efficacy of maintenance treatment containing bevacizumab., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Vascular endothelial growth factor (VEGF)-neutralizing therapy with bevacizumab has become increasingly important for treating colorectal cancer. It was demonstrated that second-line chemotherapy together with bevacizumab after disease progression (PD) on first-line therapy including bevacizumab showed clinical benefits in metastatic colorectal and breast cancers (ML18147 trial, TANIA trial). One of the rationales for these trials was that the refractoriness to first-line therapy is caused by resistance to not so much bevacizumab as to the chemotherapeutic agents. Nevertheless, resistance to bevacizumab cannot be ruled out because VEGF-independent angiogenesis has been reported to be a mechanism of resistance to anti-VEGF therapy. In this study, we used a xenograft model with the human colon cancer HT-29 cells to investigate the mechanisms underlying the effect of continued administration of bevacizumab plus capecitabine even after resistance to bevacizumab was acquired. The combination of capecitabine plus bevacizumab exhibited significantly stronger antitumor and anti-angiogenic activities than did monotherapy with either agent. Capecitabine treatment significantly increased the intratumoral VEGF level compared with the control group; however, the combination with bevacizumab neutralized the VEGF. Among angiogenic factors other than VEGF, intratumoral galectin-3, which reportedly promotes angiogenesis both dependent on, and independently of VEGF, was significantly decreased in the capecitabine group and the combination group compared with the control group. In an in vitro experiment, 5-fluorouracil (5-FU), an active metabolite of capecitabine, inhibited galectin-3 production by HT-29 cells. These results suggested that capecitabine has a dual mode of action: namely, inhibition of tumor cell growth and inhibition of galectin-3 production by tumor cells. Thus, capecitabine and bevacizumab may work in a mutually complementary manner in tumor angiogenesis inhibition to overcome the resistance caused by angiogenic factors other than VEGF. These results suggest the clinical relevance and the mechanism of action of treatment with bevacizumab in combination therapy beyond PD.

Erythropoiesis requires large amounts of iron for hemoglobin synthesis. There are two sources of iron for erythropoiesis, dietary and stored iron; however, their relative contributions to erythropoiesis remain unknown. In this study, we used the stable iron isotope (57)Fe to quantify synthesis of hemoglobin derived from dietary iron. Using this method, we investigated the activities of dietary iron absorption and the utilization of dietary iron for erythropoiesis in responses to stimulated erythropoiesis and to interventions to alter body iron status. Under iron-loaded conditions, the activity of dietary iron absorption was clearly lowered in response to up-regulation of hepcidin, although the estimated activity of iron release from stored iron was not compared with that under control conditions. This result was supported by the observation that two duodenal iron transporters, divalent metal transporter 1 (DMT1) and ferroportin, were downregulated by iron loading, although the levels of expression of ferroportin in iron storage tissues were not changed by iron loading under erythropoietic stimulation by epoetin-β pegol (C.E.R.A., a long-acting erythropoiesis-stimulating agent). These results indicate that the dietary iron absorption system is more sensitive to body iron status than are reticuloendothelial iron- release mechanisms. Our data indicated that there could be a regulatory mechanism favoring use of stored iron over dietary iron under iron-loaded conditions., (Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Iron, an essential element for various biological processes, can induce oxidative stress. We hypothesized that iron utilization for erythropoiesis, stimulated by epoetin beta pegol (C.E.R.A.), a long-acting erythropoiesis-stimulating agent, contributes to the reduction of iron-induced oxidative stress. We first investigated the sensitivity of several biomarkers to detect oxidative stress in mice by altering the amount of total body iron; we then investigated whether C.E.R.A. ameliorated oxidative stress through enhanced iron utilization. We treated db/db mice with intravenous iron-dextran and evaluated several biomarkers of iron-induced oxidative stress. In mice loaded with 5 mg/head iron, hepatic iron content was elevated and the oxidative stress marker d-ROMs (serum derivatives of reactive oxygen metabolites) was increased, whereas urinary 8-hydroxy-2'-deoxyguanosine and serum malondialdehyde were not, indicating that d-ROMs is a sensitive marker of iron-induced oxidative stress. To investigate whether C.E.R.A. ameliorated oxidative stress, db/db mice were intravenously administered iron-dextran or dextran only, followed by C.E.R.A. Hemoglobin level increased, while hepatic iron content decreased after C.E.R.A., Treatment: Serum d-ROMs decreased after C.E.R.A. treatment in the iron-dextran-treated group. Our results suggest that C.E.R.A. promotes iron utilization for erythropoiesis through mobilization of hepatic iron storage, leading to a decrease in serum oxidative stress markers in iron-loaded db/db mice.

Purpose: Alectinib is a highly selective next-generation anaplastic lymphoma kinase (ALK) inhibitor. Although alectinib shows inhibitory activity against various crizotinib-resistant ALK mutations in studies using cell-free kinase assays and Ba/F3 cell-based assays, it has not been tested for efficacy against non-small cell lung cancer (NSCLC) with the ALK mutations., Methods: We conducted in vitro and in vivo investigations into the antitumor activity of alectinib against an ALK-positive NSCLC cell line, SNU-2535, which harbors an ALK G1269A mutation. The clinical efficacy of alectinib against a NSCLC patient harboring ALK G1269A mutation was evaluated in the phase I part of the North American study., Results: Alectinib exhibited antiproliferative activity against SNU-2535 cells in vitro with IC50 of 33.1 nM. Alectinib strongly inhibited phosphorylation of ALK and its downstream signaling molecules ERK1/2, AKT, and STAT3. In a mouse xenograft model, once-daily oral administration of alectinib for 21 days resulted in strong tumor regression. In addition, administration of alectinib for 100 days achieved continuous tumor regression without tumor regrowth in all mice. Notably, eradication of tumor cells was observed in half of the mice. In the clinical study, a patient with ALK G1269A mutation showed partial response to alectinib with a duration of response of 84 days., Conclusion: These results indicated that alectinib has potent antitumor activity against NSCLC cells harboring the crizotinib-resistant mutation ALK G1269A. It is expected that alectinib would provide a valuable therapeutic option for patients with NSCLC having not only native ALK but also crizotinib-resistant ALK mutations.

Erlotinib (ERL), an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, shows notable efficacy against non-small cell lung cancer (NSCLC) harboring EGFR mutations. Bevacizumab (BEV), a humanized monoclonal antibody to vascular endothelial cell growth factor (VEGF), in combination with ERL (BEV+ERL) significantly extended progression-free survival in patients with EGFR-mutated NSCLC compared with ERL alone. However, the efficacy of BEV+ERL against EGFR-mutated NSCLC harboring T790M mutation or MET amplification, is unclear. Here, we examined the antitumor activity of BEV+ERL in four xenograft models of EGFR-mutated NSCLC (three harboring ERL resistance mutations). In the HCC827 models (exon 19 deletion: DEL), ERL significantly inhibited tumor growth by blocking EGFR signal transduction. Although there was no difference between ERL and BEV+ERL in maximum tumor growth inhibition, BEV+ERL significantly suppressed tumor regrowth during a drug-cessation period. In the HCC827-EPR model (DEL+T790M) and HCC827-vTR model (DEL+MET amplification), ERL reduced EGFR signal transduction and showed less pronounced but still significant tumor growth inhibition than in the HCC827 model. In these models, tumor growth inhibition was significantly stronger with BEV+ERL than with each single agent. In the NCI-H1975 model (L858R+T790M), ERL did not inhibit growth or EGFR signal transduction, and BEV+ERL did not inhibit growth more than BEV. BEV alone significantly decreased microvessel density in each tumor. In conclusion, addition of BEV to ERL did not enhance antitumor activity in primarily ERL-resistant tumors with T790M mutation; however, BEV+ERL enhanced antitumor activity in T790M mutation- or MET amplification-positive tumors as long as their growth remained significantly suppressed by ERL., (© 2015 UICC.)

In the treatment of human epidermal growth factor receptor 2 (HER2)-positive advanced gastric or gastroesophageal junction cancer, it has been reported that the combination of trastuzumab with capecitabine plus cisplatin, or with 5-fluorouracil (5-FU) plus cisplatin, significantly increased overall survival compared with chemotherapy alone (ToGA trial). In addition, adjuvant therapy with capecitabine plus oxaliplatin (XELOX) improved the survival of patients who received curative D2 gastrectomy (CLASSIC trial). However, the efficacy of the combination of trastuzumab with XELOX for patients with HER2-positive gastric cancer remains unknown. The aim of this study, was to investigate the efficacy of the combination of trastuzumab with XELOX in a HER2-positive human gastric cancer xenograft model. Combination treatment with these three agents (trastuzumab 20 mg/kg, capecitabine 359 mg/kg and oxaliplatin 10 mg/kg), was found to exhibit a significantly stronger antitumor activity in NCI-N87 xenografts compared with either trastuzumab or XELOX alone. In this model, treatment with trastuzumab alone or trastuzumab plus oxaliplatin enhanced the expression of thymidine phosphorylase (TP), a key enzyme in the generation of 5-FU from capecitabine in tumor tissues. In in vitro experiments, trastuzumab induced TP mRNA expression in NCI-N87 cells. In addition, NCI-N87 cells co-cultured with the natural killer (NK) cell line CD16(158V)/NK-92 exhibited increased expression of TP mRNA. When NCI-N87 cells were cultured with CD16(158V)/NK-92 cells in the presence of trastuzumab, the mRNA expression of cytokines reported to have the ability to induce TP was upregulated in tumor cells. Furthermore, a medium conditioned by CD16(158V)/NK-92 cells also upregulated the expression of TP mRNA in NCI-N87 cells. These results suggest that trastuzumab promotes TP expression, either by acting directly on NCI-N87 cells, or indirectly via a mechanism that includes trastuzumab-mediated interactions between NK and NCI-N87 cells. Therefore, the combination of trastuzumab with XELOX may be a potent therapy for HER2-positive gastric cancer.

Background: Accurate and reliable assessment of human epidermal growth factor receptor type 2 (HER2) status is important for selecting patients with gastric cancer who may benefit from trastuzumab treatment. Here we examined the impact of formalin fixing conditions on HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in xenografted tumor tissues., Methods: Xenografted tumor tissues of the human gastric cancer cell lines NCI-N87, SCH, and SNU-16 were collected and kept at room temperature for 0, 6, or 24 h before being fixed with 10 % neutral buffered formalin (NBF) for 24 h or 5, 7, or 10 days and embedded in paraffin. Use of 10 % NBF, 20 % NBF, or nonbuffered formalin as fixative was investigated., Results: The HER2 IHC scores for NCI-N87, SCH, and SNU-16 tumors were 3+, 2+, and 1+, respectively, when specimens were fixed with 10 % NBF for 24 h immediately after resection of the tumors. Specimens left for longer than 6 h before fixation had shrinkage of the tumor periphery and decreased immunostaining intensity in this region in all specimens. In SCH and SNU-16 specimens, starting fixation 24 h after tumor tissue collection induced autolysis and reduction of the number of stained cells, and 10-day-fixation lowered the HER2 score. Prolongation of fixation time did not affect FISH results, but if samples were left for more than 6 h before fixation, the FISH score was strongly reduced in SCH specimens (2.3 to 1.3). Reduced IHC staining intensity was observed with 20 % NBF and nonbuffered formalin compared to 10 % NBF., Conclusions: The time to and length of fixation of tumor specimens can affect HER2 IHC and FISH scores. The fixative used can affect IHC results.

Epoetin beta pegol (C.E.R.A.) is a novel, long-acting, erythropoiesis-stimulating agent. We investigated iron utilization associated with erythropoiesis, and how it is affected by the long-term suppression of hepcidin following C.E.R.A. treatment in mice. C57BL/6N mice were administered a single intravenous injection of C.E.R.A. Hemoglobin (Hb) levels were significantly elevated following C.E.R.A. administration, while serum hepcidin levels were continuously suppressed. Expression levels of duodenal iron transporters, ferroportin (FPN) which is internalized by hepcidin binding and divalent metal transporter 1 (DMT1), increased after C.E.R.A. administration. To evaluate the contribution of dietary iron absorption to erythropoiesis following C.E.R.A. treatment, C.E.R.A.-treated mice were fed either an iron-deficient diet to restrict dietary iron absorption or a control diet. The restriction of dietary iron absorption resulted in impaired Hb elevation after C.E.R.A. administration. To investigate the utilization of stored iron for erythropoiesis following C.E.R.A. treatment, iron indices were analyzed in C.E.R.A.-treated mice given an intraperitoneal pre-treatment injection of iron-dextran. Serum hepcidin levels and splenic hemosiderin deposition decreased after C.E.R.A. administration. These results indicate that C.E.R.A. promotes utilization of iron for erythropoiesis both through enhancement of dietary iron absorption and mobilization of iron storage from reticuloendothelial cells.

Erythropoiesis-stimulating agents (ESA) are now central to the treatment of renal anemia and are associated with improved clinical outcomes. It is well known that erythropoietin (EPO) is a key regulator of erythropoiesis through its promotion of red blood cell production. In order to investigate the role of ESA on iron metabolism, we analyzed the regulation of the iron regulatory hormone hepcidin by ESA treatment in a bone marrow transplant model in mouse. After treating C57BL/6 mice with continuous erythropoietin receptor activator (C.E.R.A.), recombinant human epoetin-β (rhEPO), or recombinant human carbamylated epoetin-β (rhCEPO), we investigated serum hepcidin concentrations and parameters of erythropoiesis. Serum hepcidin concentrations after rhEPO treatment were analyzed in mice subjected to total body irradiation followed by bone marrow transplantation. C.E.R.A. administration caused long-term downregulation of serum hepcidin levels. Serum hepcidin levels in rhEPO-treated mice decreased significantly, whereas there was no change in rhCEPO-treated mice. The reduction in circulating hepcidin levels after rhEPO administration was not observed in irradiated mice. Finally, bone marrow transplantation recovered the response to rhEPO administration that downregulates hepcidin concentration in irradiated mice. These results indicate that ESA treatment downregulates serum hepcidin concentrations, mainly by indirect mechanisms affecting hematopoietic activity in bone marrow cells.

Background: Bevacizumab is a humanized monoclonal antibody to human vascular endothelial cell growth factor (VEGF) and has been used for many types of cancers such as colorectal cancer, non-small cell lung cancer, breast cancer, and glioblastoma. Bevacizumab might be effective against gastric cancer, because VEGF has been reported to be involved in the development of gastric cancer as well as other cancers. On the other hand, there are no established biomarkers to predict the bevacizumab efficacy in spite of clinical needs. Therefore, we tried to identify the predictive markers for efficacy of bevacizumab in gastric cancer patients by using bevacizumab-sensitive and insensitive tumor models., Methods: Nine human gastric and two colorectal cancer mouse xenografts were examined for their sensitivity to bevacizumab. We examined expression levels of angiogenic factors by ELISA, bioactivity of VEGF by phosphorylation of VEGFR2 in HUVEC after addition of tumor homogenate, tumor microvessel density by CD31-immunostaining, and polymorphisms of the VEGF gene by HybriProbe™ assay., Results: Of the 9 human gastric cancer xenograft models used, GXF97, MKN-45, MKN-28, 4-1ST, SC-08-JCK, and SC-09-JCK were bevacizumab-sensitive, whereas SCH, SC-10-JCK, and NCI-N87 were insensitive. The sensitivity of the gastric cancer model to bevacizumab was not related to histological type or HER2 status. All tumors with high levels of VEGF were bevacizumab-sensitive except for one, SC-10-JCK, which had high levels of VEGF. The reason for the refractoriness was non-bioactivity on the phosphorylation of VEGFR2 and micro-vessel formation of VEGF, but was not explained by the VEGF allele or VEGF165b. We also examined the expression levels of other angiogenic factors in the 11 gastrointestinal tumor tissues. In the refractory models including SC-10-JCK, tumor levels of another angiogenic factor, bFGF, were relatively high. The VEGF/bFGF ratio correlated more closely with sensitivity to bevacizumab than with the VEGF level., Conclusions: VEGF levels and VEGF/bFGF ratios in tumors were related to bevacizumab sensitivity of the xenografts tested. Further clinical investigation into useful predictive markers for bevacizumab sensitivity is warranted.

Background/aims: Recent studies have demonstrated that erythropoiesis-stimulating agents (ESAs) induce a tissue-protective effect in the kidney. In this study, we examined whether continuous erythropoietin receptor activator (CERA), a long-acting ESA, could prevent kidney injury, especially podocyte damage, in a rat model of nephrotic syndrome induced by puromycin aminonucleoside (PAN)., Methods: Rats were injected with CERA (30 µg/kg) or vehicle 4 h before the injection of PAN (50 mg/kg). Renal function, kidney injury, and podocyte damage were assessed at 7 days., Results: The levels of proteinuria, BUN, and plasma creatinine significantly increased in rats with PAN-induced nephrosis. Treatment with CERA significantly prevented these deteriorations induced by PAN. Glomerular lesions, especially vacuolation of podocytes, and the increase of desmin expression in PAN-treated rats were significantly ameliorated by treatment with CERA. Treatment with CERA also significantly prevented the decrease in the protein productions of nephrin and podocin in the kidneys of PAN-treated rats. We found persistent activation of the Akt signaling pathway in the kidneys of CERA-treated rats., Conclusion: CERA could ameliorate renal dysfunction in PAN-induced nephrosis, which might be due to the amelioration of podocyte injury. CERA inhibited the depletion of nephrin and podocin, key components of the glomerular filtration barrier, and alleviated proteinuria. Activation of the Akt signaling pathway might be involved in the renoprotective effect of CERA., (Copyright © 2012 S. Karger AG, Basel.)

Previous preclinical and clinical findings have suggested a potential role of epidermal growth factor receptor (EGFR) in osteoclast differentiation and the pathogenesis of bone metastasis in cancer. In this study, we investigated the effect of erlotinib, an orally active EGFR tyrosine kinase inhibitor (TKI), on the bone invasion of human non-small-cell lung cancer (NSCLC) cell line NCI-H292. First, we established a novel osteolytic bone invasion model of NCI-H292 cells which was made by inoculating cancer cells into the tibia of scid mice. In this model, NCI-H292 cells markedly activated osteoclasts in tibia, which resulted in osteolytic bone destruction. Erlotinib treatment suppressed osteoclast activation to the basal level through suppressing receptor activator of NF-κB ligand (RANKL) expression in osteoblast/stromal cell at the bone metastatic sites, which leads to inhibition of osteolytic bone destruction caused by NCI-H292 cells. Erlotinib inhibited the proliferation of NCI-H292 cells in in vitro. Erlotinib suppressed the production of osteolytic factors, such as parathyroid hormone-related protein (PTHrP), IL-8, IL-11 and vascular endothelial growth factor (VEGF) in NCI-H292 cells. Furthermore, erlotinib also inhibited osteoblast/stromal cell proliferation in vitro and the development of osteoclasts induced by RANKL in vitro. In conclusion, erlotinib inhibits tumor-induced osteolytic invasion in bone metastasis by suppressing osteoclast activation through inhibiting tumor growth at the bone metastatic sites, osteolytic factor production in tumor cells, osteoblast/stromal cell proliferation and osteoclast differentiation from mouse bone marrow cells.

Purpose: We investigated the antitumor activity of the combination of two different humanized monoclonal human epidermal growth factor receptor (HER) 2 antibodies, pertuzumab and trastuzumab, for gastric cancer., Experimental Design: Tumor mouse xenograft models were used to examine antitumor activity. Cell proliferation was examined using crystal violet staining. HER family proteins' expression was analyzed by ELISA and immunohistochemistry. Phosphorylated proteins and heterodimers were detected by Western blotting and in situ proximity ligation assay (PLA), respectively. Apoptosis activity was examined by caspase 3/7 activity. Antibody-dependent cellular cytotoxicity (ADCC) activity was detected by xCELLigence. Microvessel density was examined by CD31 staining., Results: Pertuzumab in combination with trastuzumab showed significant antitumor activity compared with each monotherapy in NCI-N87, an HER2-positive human gastric cancer xenograft model. The efficacy was stronger than that of the maximum effective dose with each monotherapy. Similar antitumor activity was shown in 4-1ST, another HER2-positive gastric cancer model, but not in MKN-28, an HER2-negative model. Combining pertuzumab with trastuzumab enhanced cell growth inhibition and apoptosis activity by inhibiting EGFR-HER2 heterodimerization and the phosphorylation of these receptors and their downstream factors. This effect was also seen in HER2-HER3 signaling. Furthermore, pertuzumab in combination with trastuzumab potentiated the ADCC activity of those antibodies and reduced tumor microvessel density., Conclusions: We showed the significantly enhanced efficacy of pertuzumab combining with trastuzumab for HER2 overexpressing gastric cancer through the potentiation of cell growth inhibition, apoptosis activity, cell killing activity by ADCC, and antiangiogenic activity. This study suggests the clinical benefit of combination therapy with pertuzumab and trastuzumab for patients with HER2-positive gastric cancers., (©2011 AACR.)

The pathological influences of inflammation on left ventricular hypertrophy (LVH) were studied in subtotal nephrectomized (SNx) rats after 0.3% NaCl loading for 5 weeks. We found that mild hypertension, increased plasma levels of creatinine, inorganic phosphate, asymmetric dimethylarginine (ADMA), and parathyroid hormone (PTH) were observed in the present SNx rats without LVH. In the present study, the NaCl-loaded SNx (SNx + NaCl) rats were characterized by significant LVH and hypertension with aggravated values of all the parameters. We further confirmed that glomerular sclerosis, tubulointerstitial fibrosis, and inflammatory cell infiltration into the tubulointerstitial area, observed in the SNx rats, were more severely caused in the SNx + NaCl rats. In addition, plasma interleukin-6 (IL-6) levels in the SNx + NaCl rats were significantly increased compared to those in the SNx rats. These findings indicated that NaCl-loaded SNx rats developed LVH and hypertension, which were accompanied with increased plasma levels of PTH, creatinine, inorganic phosphorus, ADMA, and IL-6. Thus, these results suggest that inflammation as well as endothelial dysfunction would be correlated with LVH as non-traditional risk factors at the early stage in the present renal failure model.