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Purpose Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are widely used for the treatment of advanced estrogen receptor (ER)-positive breast cancer. To develop a treatment strategy for cancers resistant to CDK4/6 inhibitors, here, we established palbociclib-resistant sublines and analyzed their resistance mechanisms. Methods Palbociclib-resistant sublines were established from T47D and MCF7 cells. Sensitivity to other drugs was assessed via the WST assay. Altered expression/phosphorylation of proteins related to signal transduction and cell cycle regulation was examined using western blotting. Copy number alterations and mutations in the retinoblastoma (RB1) gene were also analyzed. Results Although an increase in CDK6 and decrease in retinoblastoma protein (Rb) expression/phosphorylation were commonly observed in the resistant sublines, changes in other cell cycle-related proteins were heterogeneous. Upon extended exposure to palbociclib, the expression/phosphorylation of these proteins became altered, and the long-term removal of palbociclib did not restore the Rb expression/phosphorylation patterns. Consistently a copy number decrease, as well as RB1 mutations were detected. Moreover, although the resistant sublines exhibited cross-resistance to abemaciclib, their response to dinaciclib was the same as that of wild-type cells. Of note, the cell line exhibiting increased mTOR phosphorylation also showed a higher sensitivity to everolimus. However, the sensitivity to chemotherapeutic agents was unchanged in palbociclib-resistant sublines. Conclusion ER-positive breast cancer cells use multiple molecular mechanisms to survive in the presence of palbociclib, suggesting that targeting activated proteins may be an effective strategy to overcome resistance. Additionally, palbociclib monotherapy induces mutations and copy number alterations in the RB1 gene.

RNA in extracellular vesicles (EVs) are uptaken by cells, where they regulate fundamental cellular functions. EV-derived mRNA in recipient cells can be translated. However, it is still elusive whether “naked nonvesicular extracellular mRNA” (nex-mRNA) that are not packed in EVs can be uptaken by cells and, if so, whether they have any functions in recipient cells. Here, we show the entrance of nex-mRNA in the nucleus, where they exert a translation-independent function. Human nex-interleukin-1β (IL1β)-mRNA outside cells proved to be captured by RNA-binding zinc finger CCCH domain containing protein 12D (ZC3H12D)-expressing human natural killer (NK) cells. ZC3H12D recruited to the cell membrane binds to the 3′-untranslated region of nex-IL1β-mRNA and transports it to the nucleus. The nex-IL1β-mRNA in the NK cell nucleus upregulates antiapoptotic gene expression, migration activity, and interferon-γ production, leading to the killing of cancer cells and antimetastasis in mice. These results implicate the diverse actions of mRNA., Nonvesicular extracellular RNA (nex-RNA) that are not packed in extracellular vesicles is detected outside the cell, but it is poorly understood. Here the authors report that nex-RNA is captured by a zinc finger protein and transported to the nucleus to enhance antimetastatic characters of the cell.

Improved prognosis for triple-negative breast cancer (TNBC) has plateaued and the development of novel therapeutic strategies is required. This study aimed to explore the anti-tumor effect of combined eribulin and HDAC inhibitor (vorinostat: VOR, pan-HDAC inhibitor and ricolinostat: RICO, selective HDAC6 inhibitor) treatment for TNBC. The effect of eribulin in combination with an HDAC inhibitor was tested in three TNBC cell lines (MDA-MB-231, Hs578T, and MDA-MB-157) and their eribulin-resistant derivatives. The expression of acetylated α-tubulin was analyzed by Western blotting for TNBC cells and immunohistochemical analyses for clinical specimens obtained from breast cancer patients who were treated with eribulin. The simultaneous administration of low concentrations (0.2 μM) of VOR or RICO enhanced the anti-tumor effect of eribulin in MDA-MB-231 and Hs578T cells but not in MDA-MB-157 cells. Meanwhile, pretreatment with 5 μM of VOR or RICO enhanced eribulin sensitivity in all three cell lines. Low concentration of VOR or RICO increased acetylated α-tubulin expression in MDA-MB-231 and Hs578T cells. In contrast, whereas 5 μM of VOR or RICO increased the expression of acetylated α-tubulin in MDA-MB-157 cells, low concentrations did not. Eribulin increased the expression of acetylated α-tubulin in MDA-MB-231 and Hs578T cells but not in MDA-MB-157 cells. These phenomena were also observed in eribulin-resistant cells. Immunohistochemical analyses revealed that the expression of acetylated α-tubulin was increased after eribulin treatment in TNBC. HDAC6 inhibition enhances the anti-tumor effect of eribulin through the acetylation of α-tubulin. This combination therapy could represent a novel therapeutic strategy for TNBC.

Background/aim The prognostic nutritional index (PNI) and neutrophil-to-lymphocyte ratio (NLR) are prognostic markers for operable breast cancer. However, their importance in patients with metastatic breast cancer (MBC) remains unclear. This study aimed to evaluate these parameters as prognostic markers in MBC patients treated with eribulin. Patients and methods A total of 60 patients with MBC treated with eribulin were included. Results Although high PNI and low NLR were correlated with better progression-free survival (PFS) and overall survival (OS), PNI had stronger impact as prognostic marker than NLR (PNI: HR=0.35, p=0.0008 for PFS and HR=0.27, p=0.0068 for OS; NLR: HR=0.71, p=0.081 for PFS and HR=0.63, p=0.14 for OS). Multivariate analysis demonstrated that PNI was an independent predictor of PFS (HR=0.30, p=0.0009). Conclusion PNI could be a more reliable prognostic marker for MBC patients treated with eribulin than NLR.

The tyrosine kinase inhibitors (TKIs) sorafenib, lenvatinib, vandetanib, and cabozantinib are currently used for thyroid cancer treatment; however, the differences in their clinical efficacy and toxicity remain unclear. This meta-analysis assessed the efficacy and toxicity of these four TKIs based on 34 studies. The pooled incidence of partial response (PR), stable disease (SD), TKI-related adverse events (AEs), and pooled median progression-free survival (PFS) were calculated with 95% confidence intervals (CI). Complete response to TKIs was extremely rare (0.3%). The highest PR rate and longest PFS were observed for lenvatinib in differentiated thyroid cancer (69%, 95% CI: 57-81 and 19 months, 95% CI: 9-29, respectively) and vandetanib in medullary thyroid cancer (40%, 95% CI: 25-56 and 31 months, 95% CI: 19-43, respectively). Although the discontinuation rate due to AEs was similar for each TKI, there was a difference in the most frequently observed AE for each TKI (hand-foot syndrome for sorafenib, hypertension and proteinuria for lenvatinib, and QTc prolongation for vandetanib). The identified differences in the TKI efficacy and AE profiles may provide a better understanding of thyroid cancer treatment. Although TKIs are promising agents for thyroid cancer treatment, they are unlikely to lead to a cure. Thus, even in the TKI era, a multimodal treatment including surgery, radioiodine therapy, external beam radiotherapy, and TKIs is required to optimize patient chances of improved survival.

Improved prognosis for triple-negative breast cancer (TNBC) has currently plateaued and the development of novel therapeutic strategies is required. Therefore, we aimed to explore the anti-tumor effect of eribulin and paclitaxel combination therapy for TNBC. The effect of eribulin and paclitaxel in combination was tested, with both concurrent and sequential administration, using four TNBC cell lines (MDA-MB-231, Hs578T, MDA-MB-157, and Mx-1) in vitro and in an MDA-MB-231 BALB/c-nu/nu mouse xenograft model. The expression of epithelial-mesenchymal phenotypic markers was analyzed by western blotting and immunohistochemical analyses. Simultaneous administration of eribulin and paclitaxel resulted in a synergistic anti-tumor effect with MDA-MB-231 and Hs578T cells, but not MDA-MB-157 and Mx-1 cells, in vitro. Moreover, pre-treatment with one drug significantly enhanced sensitivity to the subsequently administrated second drug in MDA-MB-231 and Hs578T cells. Eribulin increased E-cadherin expression and decreased the expression of mesenchymal markers in MDA-MB-231 and Hs578T cells. In contrast, paclitaxel increased the expression of mesenchymal markers. When epithelial-mesenchymal transition was induced by TGF-β1, eribulin sensitivity was enhanced. In contrast, a TGF-β receptor kinase inhibitor decreased eribulin sensitivity. In MDA-MB-231 tumor-bearing mice, concurrent administration of low doses of eribulin and paclitaxel significantly inhibited tumor growth compared to that with either monotherapy. Moreover, single administration of eribulin before the initiation of paclitaxel treatment decreased vimentin expression and reduced the average tumor volume in a mouse xenograft model. Eribulin and paclitaxel show synergistic anti-tumor effect by altering the epithelial-mesenchymal phenotype. This combination therapy could represent a novel therapeutic strategy for TNBC.

Background: Therapeutic options for treating advanced or metastatic medullary thyroid carcinoma (MTC) and anaplastic thyroid carcinoma (ATC) are still limited in Japan, even though vandetanib for MTC and lenvatinib for MTC and ATC have been approved. Sorafenib is an oral multikinase inhibitor approved for the treatment of patients with radioactive iodine-refractory differentiated thyroid cancer (DTC). An uncontrolled, open-label, multicenter, single-arm, Phase 2 clinical study was conducted to evaluate the safety and efficacy of sorafenib in Japanese patients with MTC and ATC. Methods: Japanese patients with histologically confirmed ATC and locally advanced or metastatic MTC were enrolled from April to September 2014. The primary endpoint was to evaluate the safety of sorafenib. Treatment efficacy variables including progression-free survival (PFS), overall survival (OS), objective response rate (ORR), disease control rate (DCR), and maximum reduction in tumor size were evaluated as secondary endpoints. Patients received sorafenib 400 mg orally twice daily on a continuous basis and then continued treatment until the occurrence of disease progression, unacceptable toxicity, or withdrawal of consent. Results: A total of 20 patients were screened, and 18 (8 with MTC and 10 with ATC) were enrolled. The most common drug-related adverse events were palmar-plantar erythrodysesthesia (72%), alopecia (56%), hypertension (56%), and diarrhea (44%). In the ATC patients, median PFS was 2.8 months [confidence interval 0.7–5.6], and median OS was 5.0 months [confidence interval 0.7–5.7]; ORR and DCR were 0% and 40%, respectively. In the MTC population, neither median PFS nor OS had been reached at the time of this analysis; ORR was 25% and DCR was 75%. Conclusions: The toxicities reported in this study were consistent with the known safety profile of sorafenib. Sorafenib seems to be effective in the treatment of advanced MTC but not ATC, and could be a new treatment option for locally advanced or metastatic MTC and radioactive iodine-refractory DTC.

Anaplastic thyroid cancer (ATC) is a rare and extremely aggressive malignancy, with a median survival of less than 6 months due to rapid progression and resistance to multimodal therapies. Effective treatment strategies have not been identified. A prospective clinical study was performed to objectively evaluate outcomes of treatment with paclitaxel.An investigator-initiated, multicenter, nonrandomized, open-label, single-arm study to evaluate the feasibility and efficacy of weekly paclitaxel (80 mg/m(2)) administration for patients with pathologically confirmed ATC was conducted in a nationwide organization.Feasibility was analyzed in 56 patients. More than one course of treatment was performed in 52 (93%) patients retaining sufficient dose intensity (84%). No patient had to terminate the treatment because of an adverse event. The median overall survival was 6.7 months [confidence interval 4.4-9.0]. The 6-month survival was 54%. Among the 42 patients with an evaluable lesion, none demonstrated complete remission, 9 (21%) showed partial remission, 22 (52%) achieved stable disease, and 8 (19%) exhibited progressive disease; 3 did not complete the initial treatment course. The objective response rate was 21%, and the clinical benefit rate was 73%. The median time to progression was 1.6 months. Statistically, no additional effect of concomitant radiation was demonstrated in 6 patients receiving combined therapy. Eight patients, in whom a complete post-treatment surgical removal of the tumor was feasible, survived significantly longer (median 7.6 months [CI 8.1-23.0]) than the other 34 patients in whom the tumor could not be completely removed after chemotherapy (5.4 months [CI 3.0-7.8], p = 0.018).The study demonstrates objective and accurate information concerning the feasibility and efficacy of a standardized treatment with weekly paclitaxel administration for ATC patients.Weekly paclitaxel administration for ATC patients can be of clinical benefit in a neo-adjuvant setting.

Anaplastic thyroid carcinoma (ATC) accounts for only 1 to 2% of all thyroid carcinomas, but it is one of the most lethal neoplasms in humans. To obtain further insights into this "orphan disease," we have established the ATC Research Consortium of Japan (ATCCJ) in 2009. It represents a multicenter registry for ATC that have been treated in Japan. To date, 67 institutions have taken part in the collaborative research system and over 1,200 cases have been accumulated in its database. Using this big data, several retrospective studies were carried out to evaluate 1) prognostic factors to determine initial treatment policy, 2) significance of extended radical surgery for Stage IVB cases, 3) characteristics of ATC incidentally found on pathological examination and 4) pathological features of ATC with long-term survival. Moreover, the ATCCJ has conducted an investigator-initiated, nationwide, prospective clinical trial since 2012; namely, the feasibility, safety and efficacy study of weekly paclitaxel administration for patients with ATC (UMIN: 000008574). Revised Japanese guidelines for treatment of thyroid tumors are going to adopt the recommendations from the results of this research. Since 2016, the ATCCJ has started the phase II study assessing the efficacy and safety of lenvatinib, a newly developed tyrosine kinase inhibitor for ATC (UMIN: 000020773). Our nationwide clinical trial network will strengthen the activity to recruit orphan disease patients and may discover new strategies to conquer this dismal malignancy in the near future.

Pemetrexed (MTA) is a folate antimetabolite used for treating non-small cell lung cancer. To elucidate the mechanisms of pemetrexed resistance in lung cancer, we established pemetrexed-resistant sublines in PC9 (mutant EGFR) and H1993 (wild-type EGFR) lung adenocarcinoma cell lines (PC9-MTA, H1993-MTA). Gene expression profile comparison by microarray analyses revealed enhanced fibroblast growth factor 2 (FGF2) and FGF receptor 1 (FGFR1) expression, confirmed by Western blotting, enzyme-linked immunosorbent assay, and reverse transcription-polymerase chain reaction. ERK phosphorylation was increased in PC9-MTA but decreased in H1993-MTA along with decreased downstream signaling molecule phosphorylation. Cellular morphological change from epithelial to spindle-shape together with increased mesenchymal marker protein expression was observed in H1993-MTA. SiRNA-mediated FGF2 knockdown partially restored pemetrexed sensitivity in both lines, whereas anti-FGFR1 inhibitor PD173074 restored pemetrexed sensitivity in PC9-MTA. FGF2 or FGFR1 inhibition decreased pERK levels in PC9-MTA but increased pEGFR levels together with downstream signaling molecule activation and reversed epithelial-mesenchymal transition marker protein expression in H1993-MTA. Although thymidylate synthase strongly facilitates the development of pemetrexed resistance, our results reveal involvement of the FGF2-FGFR1 pathway in pemetrexed resistance in lung cancer cells and suggest that cellular function alterations induced by FGF2-FGFR1 pathway activation depend on the innate feature of cancer cells.

Background: Carcinoma showing thymus-like differentiation (CASTLE) is an extremely rare malignant neoplasm of the thyroid that originates from ectopic thymic tissue. No sufficient evidence exists regarding the efficacy of chemotherapy for cases with distant metastases or advanced disease because of the rarity of the disease itself. Patient: We report a case of CASTLE with lung metastasis that showed good responses to first-line (cisplatin, doxorubicin, vincristine, and cyclophosphamide) and second-line (carboplatin and paclitaxel) chemotherapies. Summary: This is the first case of CASTLE reported to show a good response to two serial chemotherapies. Conclusion: This case suggests that CASTLE is a chemosensitive tumor and that chemotherapy should be attempted in patients with advanced or metastatic CASTLE.

This study aimed to elucidate the mechanisms underlying the resistance of breast cancer to eribulin. Seven eribulin-resistant breast cancer cell lines (MCF7/E, BT474/E, ZR75-1/E, SKBR3/E, MDA-MB-231/E, Hs578T/E, and MDA-MB-157/E) were established. mRNA and protein expression of ATP-binding cassette subfamily B member 1 (ABCB1) and subfamily C member 11 (ABCC11) increased in all eribulin-resistant cell lines compared to the parental cell lines. When ABCB1 or ABCC11 expression was inhibited by small interfering RNA in MCF7/E, BT474/E, and MDA-MB-231/E cells, eribulin sensitivity was partially restored. Moreover, eribulin resistance was attenuated additively by inhibiting ABCB1 and ABCC11 in MCF7/E cells. Additionally, overexpression of exogenous ABCB1 or ABCC11 in HEK293T cells conferred resistance to eribulin. MCF7/E and MDA-MB-231/E cells showed cross-resistance to paclitaxel, doxorubicin, and fluorouracil. Inhibition of ABCB1 partially restored paclitaxel and doxorubicin sensitivity. Partial restoration of fluorouracil sensitivity was induced by inhibiting ABCC11 in MCF7/E and MDA-MB-231/E cells. Both ABCB1 and ABCC11 are involved in the development of eribulin resistance in breast cancer cells in vitro regardless of the breast cancer subtype. Thus, ABCB1 and ABCC11 expression may be used as a biomarker for predicting the response to eribulin in patients with breast cancer. Concomitant inhibition of ABCB1 and ABCC11 might help enhance the antitumor effects of eribulin.

Eribulin (ERI), an inhibitor of microtubule dynamics, is a key drug for metastatic breast cancer. While HDAC inhibitors have been shown to presents antitumor effect for various malignancies. Among HDAC family, HDAC6 is known as a deacetylase of tubulin. To develop a novel therapeutic strategy for triple negative breast cancer (TNBC), we tested the combinational effect of ERI and HDAC inhibitor for TNBC cells in vitro. Two TNBC cell lines (MDA-MB-231, Hs578T) and their ERI-resistant cells (MDA-MB-231/E, Hs578T/E) established in our laboratory were used in the study. Vorinostat (VOR), a pan-HDAC inhibitor, and ricolinostat (RICO), a selective HDAC6 inhibitor were used. The sensitivity to VOR or RICO was not different between the parental and the ERI-resistant cells. In the protein expression analysis, HDAC6 expression was not altered between parental and ERI-resistant cells. Prior treatment with VOR or RICO increased the expression of acetylatedα-tubulin in both parental and ERI-resistant TNBC cell lines. When low concentration of VOR (0.5 μM) or RICO (0.5 μM) was added simultaneously with ERI, the sensitivity to ERI was enhanced significantly in both parental MDA-MB-231 and Hs578T cell lines. Furthermore, when both parental cell lines were treated with VOR (5 μM) or RICO (5 μM) for 48 hrs prior to addition of ERI, the sensitivity to ERI was significantly enhanced. The enhancement of sensitivity to ERI both by the simultaneous addition with low concentration of VOR or RICO and by the prior treatment with VOR or RICO was observed in the ERI-resistant cells as well. Moreover, inhibition of HDAC6 by small interfering RNA partially enhanced the sensitivity to ERI in the both parental cell lines. Our study demonstrated the possibility that the combination of ERI with HDAC inhibitor might enhance the antitumor effect of ERI and overcome ERI resistance through acetylation of α-tubulin by HDAC6 inhibition in TNBC cells. Citation Format: Takaaki Oba, Hiroto Izumi, Ken-ichi Ito. Preclinical analysis of synergistic antitumor effect of eribulin and HDAC inhibitor for triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2924.

Hippocampal inhibitory interneurons have a central role in the control of network activity, and excitatory synapses that they receive express Hebbian and anti-Hebbian long-term potentiation (LTP). Because many interneurons in the hippocampus express nicotinic acetylcholine receptors (nAChRs), we explored whether exposure to nicotine promotes LTP induction in these interneurons. We focussed on a subset of interneurons in the stratum oriens/alveus that were continuously activated in the presence of nicotine due to the expression of non-desensitizing non-alpha7 nAChRs. We found that, in addition to alpha2 subunit mRNAs, these interneurons were consistently positive for somatostatin and neuropeptide Y mRNAs, and showed morphological characteristics of oriens-lacunosum moleculare cells. Activation of non-alpha7 nAChRs increased intracellular Ca(2+) levels at least in part via Ca(2+) entry through their channels. Presynaptic tetanic stimulation induced N-methyl-D-aspartate receptor-independent LTP in voltage-clamped interneurons at -70 mV when in the presence, but not absence, of nicotine. Intracellular application of a Ca(2+) chelator blocked LTP induction, suggesting the requirement of Ca(2+) signal for LTP induction. The induction of LTP was still observed in the presence of ryanodine, which inhibits Ca(2+) -induced Ca(2+) release from ryanodine-sensitive intracellular stores, and the L-type Ca(2+) channel blocker nifedipine. These results suggest that Ca(2+) entry through non-alpha7 nAChR channels is critical for LTP induction. Thus, nicotine affects hippocampal network activity by promoting LTP induction in oriens-lacunosum moleculare cells via continuous activation of non-alpha7 nAChRs.

Purpose: The Y-box binding protein 1 (YB-1) regulates expression of P-glycoprotein encoded by the MDR1 gene. There have been no previous studies regarding the involvement of YB-1 in the development of resistance to paclitaxel. The present study was done to examine how paclitaxel affects the localization and expression of YB-1 in breast cancer. Experimental Design: We evaluated the expression and localization of YB-1 and P-glycoprotein in breast cancer tissues obtained from 27 patients before and after treatment with paclitaxel. The effect of paclitaxel on localization of cellular YB-1 was examined by using GFP-YB-1. Interaction of YB-1 with the Y-box motif of the MDR1 promoters was studied by electrophoretic mobility shift assay. The effects of paclitaxel on MDR1 promoter activity were examined by luciferase assay. Results: Of 27 breast cancer tissues treated with paclitaxel, nine (33%) showed translocation of YB-1 from the cytoplasm to the nucleus together with increased expression of P-glycoprotein during the course of treatment. Twelve breast cancer tissues (44%) showed neither translocation of YB-1 nor increased expression of P-glycoprotein. Nuclear translocation of YB-1 was correlated significantly with increased expression of P-glycoprotein (P = 0.0037). Confocal analysis indicated that paclitaxel induced nuclear translocation of green fluorescent fused YB-1 in MCF7 cells. Furthermore, binding of YB-1 to the Y-box of MDR1 promoter was increased in response to treatment with paclitaxel. In addition, MDR1 promoter activity was significantly up-regulated by paclitaxel in MCF7 cells (P < 0.001). Conclusions: The results of the present study suggested that YB-1 may be involved in the development of resistance to paclitaxel in breast cancer.

Mutation of the class I beta-tubulin gene has been reported to be one of the mechanisms that cause resistance to paclitaxel. To assess the relationship between paclitaxel-resistance and class I beta-tubulin gene mutation in breast cancer, Japanese patients with breast cancer were screened for the class I beta-tubulin gene mutation. Total RNA was isolated from 82 breast cancer specimens and the corresponding normal tissues. Twenty-four of the 82 patients were treated with paclitaxel preoperatively and 12 of them did not respond to the treatment. Of the 82 breast cancer patients, 15 (18.3%) had silent polymorphism in exon 4, Leu217Leu (CTG/CTA). However, no mutations showing amino acid substitution of the beta-tubulin gene were detected in any of the patients, including 12 patients who did not respond to paclitaxel. Class I beta-tubulin gene mutation with amino acid substitution was not detected in 82 breast cancer specimens. Our results suggest that mutation of the class I beta-tubulin gene is unlikely to play an important role in the mechanism of resistance to paclitaxel in breast cancer.

The present study has investigated the role of ATP in the induction of synaptic plasticity, using local application of ATP by picopump administration into the stratum radiatum of guinea pig hippocampal region CA1. Excitatory postsynaptic currents (EPSCs) evoked by stimulation of Schaffer collateral/commissural afferents synapsing on CA1 pyramidal cells of hippocampal slices were monitored in voltage-clamp mode, using whole-cell recording. Brief local application of ATP (1 mM) induced an inward current, usually consisting of early- and late-phase components. Because the late-phase component of an ATP-induced current was largely inhibited by Ca2+-free solution, this component is supposed to depend on extracellular Ca2+. After local application of ATP, long-term synaptic modification of EPSCs was induced: LTP was detected in neurons exhibiting a small late Ca2+ current, while LTD was obtained from recordings showing a large late Ca2+ current in response to ATP application. There was a statistically significant correlation between the magnitude of long-term plastic changes and the size of Ca2+ currents in response to ATP application. Furthermore, there was significant difference between the average size of the Ca2+ current in the LTP group and the size in the LTD group. These results suggest that a small Ca2+ influx in response to ATP application induces LTP, whereas a large one induces LTD in guinea pig hippocampal CA1 neurons. Hippocampus 2003;13:81–92. © 2003 Wiley-Liss, Inc.

1. Temperature-dependent properties of synaptic transmission were studied by recording orthodromic responses of the population spike and excitatory postsynaptic potential in CA1 pyramidal neurons of guinea pig hippocampal slices.

Purpose: The ten-eleven translocation (TET) proteins are critical regulators in epigenetic modification. It is well known that TET proteins catalyze the oxidation of 5-methylcytosine (5-mC) to generate 5-hydroxymethylcytosine (5-hmC), and 5-hmC levels reflect diverse functions of TET proteins. It has been reported that the expression of 5-hmC in cancer tissues is decreased compared with that in normal tissues in several organs. However, 5-hmC expression in lung cancer has not been fully analyzed. To examine whether or not 5-hmC was associated with phenotypes or progression of lung cancer, we evaluated 5-hmC expression in a variety of histological types of lung cancers. Materials and methods: The expression of 5-hmC was analyzed by immunohistochemistry in 109 paired specimens of lung cancer and adjacent normal tissues obtained from the patients underwent operation at Shinshu University Hospital. Eighty-nine adenocarcinomas, 17 squamous cell carcinomas, two large cell carcinomas, and one small cell carcinoma were analyzed in the study. Results: 5-hmC expression in female was significantly lower than that in male, and was lower in smokers than non-smokers. 5-hmC expression was positive in 34.1±22.0% of lung cancer tissues and 69.4±18.8% of normal tissues. The expression of 5-hmC was significantly decreased in the lung cancer tissues compared with that in the adjacent normal tissues (p Conclusions: Our results indicate a possibility that 5-hmC may be related to carcinogenesis and progression or differentiation of lung cancer. Citation Format: Yoshiaki Tominaga, Ken-ichi Ito, Jun Nakayama. Decreased expression of 5-hydroxymethylcytosine is associated with grade of malignancy in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2393. doi:10.1158/1538-7445.AM2017-2393

Purpose: There is an urgent need to develop novel therapeutic strategies for triple negative breast cancer (TNBC). To explore the antitumor effect of combination of eribulin (ERI) and paclitaxel (PTX) for TNBC, we conducted preclinical experiments. Materials & methods: Combinational effects of ERI and PTX were analyzed both in vitro and in vivo using two TNBC cell lines (MDA-MB-231, Hs578T). Results: When ERI was added simultaneously with PTX to TNBC cells, the sensitivity to PTX was increased synergistically. On the other hand, synergistic increase of sensitivity to ERI was observed in the presence of PTX. When the TNBC cells were treated with each drug subsequently, pretreatment with ERI for 96 hours enhanced the sensitivity to PTX administrated subsequently. In contrast, pretreatment of TNBC cells with PTX enhanced the sensitivity to the subsequently administrated ERI. When the TNBC cells were treated with ERI, the expression of epithelial marker, E-cadherin was increased, while the expression of mesenchymal markers, pSmad2, ZEB1, Slug, and vimentin were decreased. In contrast, when the cells were treated with PTX, the expressions of mesenchymal markers were increased. When epithelial-mesenchymal transition (EMT) was induced by TGF-β in the TNBC cells, the sensitivity to ERI was enhanced, while TGF-β receptor inhibitor (LY2157299) reduced the sensitivity to ERI. Furthermore, knockdown of ZEB1 by small interfering RNA conferred resistance to ERI in both TNBC cell lines. In the mouse xenograft model bearing MDA-MB-231, ERI plus PTX combination therapy significantly inhibited tumor growth compared with either PTX or ERI monotherapy. Furthermore, prior treatment with ERI significantly enhanced the antitumor activity of PTX in the mouse xenograft model. Conclusions: Our findings demonstrate an EMT-mediated synergistic antitumor effect by combination of two tubulin inhibitors. This combination therapy may serve as a novel therapeutic strategy for TNBC. Citation Format: Takaaki Oba, Ken-ichi Ito. Preclinical analyses of synergistic effect of eribulin and paclitaxel for triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1051. doi:10.1158/1538-7445.AM2017-1051

Background The Y-box binding protein 1 (YB-1) possesses pleiotropic functions through its interactions with various cellular proteins, and its high expression levels make it a potential useful prognostic biomarker for cancer cells. Eukaryotic DNA topoisomerases, such as DNA topoisomerase 1 (TOPO1) and DNA topoisomerase 2 (TOPO2), are the essential DNA metabolism regulators that usually overexpressed in cancer cells, and multiple proteins have been reported to regulate the enzyme activity and the clinical efficacy of their inhibitors. The present study unraveled the interaction of YB-1 with TOPO1, and further investigated the related function and potential mechanisms during the interaction. Methods The direct association of TOPO1 with specific domain of YB-1 was explored by co-immunoprecipitation and GST pull-down assays. The interaction function was further clarified by DNA relaxation assays, co-immunoprecipitation and WST-8 assays with in vitro gain- and loss- of function models. Results We found that YB-1 interacts directly with TOPO1 (but not with TOPO2) and promotes TOPO1 catalytic activity. Interactions between YB-1 and TOPO1 increased when cancer cells were treated with the TOPO1 inhibitor, camptothecin (CPT), but not with the TOPO2 inhibitor, adriamycin (ADM). Furthermore, we found that the interaction is prevented by pretreatment with the antioxidant agent, N-acetyl cysteine, and that YB-1 downregulation renders cells resistant to CPT. Conclusions Our findings suggest that nuclear YB-1 serves as an intracellular promoter of TOPO1 catalytic activity that enhances CPT sensitivity through its direct interaction with TOPO1.

The ATP-binding cassette (ABC) proteins comprise a large superfamily of transmembrane transporters that utilize the energy of ATP hydrolysis to translocate their substrates across biological membranes. Multidrug resistance protein (MRP) 2 (ABCC2) belongs to subfamily C of the ABC superfamily and, when overexpressed in tumor cells, confers resistance to a wide variety of anticancer chemotherapeutic agents. MRP2 is also an active transporter of organic anions such as methotrexate (MTX), estradiol glucuronide (E217betaG), and leukotriene C4 and is located on the apical membrane of polarized cells including hepatocytes where it acts as a biliary transporter. We recently identified a highly conserved tryptophan residue in the related MRP1 that is critical for the substrate specificity of this protein. In the present study, we have examined the effect of replacing the analogous tryptophan residue at position 1254 of MRP2. We found that only nonconservative substitutions (Ala and Cys) of Trp1254 eliminated [3H]E217betaG transport by MRP2, whereas more conservative substitutions (Phe and Tyr) had no effect. In addition, only the most conservatively substituted mutant (W1254Y) transported [3H]leukotriene C4, whereas all other substitutions eliminated transport of this substrate. On the other hand, all substitutions of Trp1254 eliminated transport of [3H]MTX. Finally, we found that sulfinpyrazone stimulated [3H]E217betaG transport by wild-type MRP2 4-fold, whereas transport by the Trp1254 substituted mutants was enhanced 6-10-fold. In contrast, sulfinpyrazone failed to stimulate [3H]MTX transport by either wild-type MRP2 or the MRP2-Trp1254 mutants. Taken together, our results demonstrate that Trp1254 plays an important role in the ability of MRP2 to transport conjugated organic anions and identify this amino acid in the putative last transmembrane segment (TM17) of this ABC protein as being critical for transport of MTX.

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) play a crucial role in the induction of lung cancer, and NNAL-O-glucuronide formation and elimination are important steps in detoxification of these compounds. In the present study, we investigated the ATP-binding cassette (ABC) protein, MRP1 (ABCC1), as a candidate transporter responsible for NNAL-O-glucuronide export. MRP1 mediates the active transport of numerous GSH-, sulfate-, and glucuronide-conjugated organic anions and can transport certain xenobiotics by a mechanism that may involve co-transport with GSH. Using membrane vesicles prepared from transfected cells, we found that MRP1 transports [3H]NNAL-O-glucuronide but is dependent on the presence of GSH (K m 39 μm, V max 48 pmol mg−1 min−1). We also found that the sulfur atom in GSH was dispensable because transport was supported by the GSH analog, γ-glutamyl-α-aminobutyryl-glycine. Despite stimulation of NNAL-O-glucuronide transport by GSH, there was no detectable reciprocal stimulation of [3H]GSH transport. Moreover, whereas the MRP1 substrates leukotriene C4(LTC4) and 17β-estradiol 17β-(d-glucuronide) (E217βG) inhibited GSH-dependent uptake of [3H]NNAL-O-glucuronide, only [3H]LTC4 transport was inhibited by NNAL-O-glucuronide (+GSH) and the kinetics of inhibition were complex. A mutant form of MRP1, which transports LTC4but not E217βG, also did not transport NNAL-O-glucuronide suggesting a commonality in the binding elements for these two glucuronidated substrates, despite their lack of reciprocal transport inhibition. Finally, the related MRP2 transported NNAL-O-glucuronide with higher efficiency than MRP1 and unexpectedly, GSH inhibited rather than stimulated uptake. These studies provide further insight into the complex interactions of the MRP-related proteins with GSH and their conjugated organic anion substrates, and extend the range of xenotoxins transported by MRP1 and MRP2 to include metabolites of known carcinogens involved in the etiology of lung and other cancers.

In the present study, we used inositol-1,4,5-trisphosphate (IP3) type 1 receptor (IP3R1) knockout mice to examine the role of this receptor in the induction of LTP, LTD, and DP at mossy fiber-CA3 synapses. No difference in synaptically induced field-EPSPs was seen between the wild-type (IP3R1(+/+)) and IP3R1 knockout mice (IP3R1(-/-)), showing that basic synaptic transmission does not involve IP3R1 activation. Tetanus induced LTP in both wild-type and IP(3)R1(-/-) mice, but the magnitude of LTP was significantly greater in IP3R1(-/-) mice (149.8+/-3.5%, mean+/-S.E.M., n=15) than in wild-type mice (132.4+/-1.5%, n=17), suggesting that the IP3R1 has a suppressive effect on LTP induction. To determine whether this effect involved N-methyl-D-aspartate receptor (NMDAR)-dependent LTP, the effect of tetanus was tested in the present of the NMDAR antagonist, D,L-AP5 (50 microM); under these conditions, the LTP in both IP3R1(-/-) and IP3R1(+/+) mice was not significantly reduced. In addition, group I mGluR activation was shown to be necessary for LTP induction, as the LTP was almost blocked by the group I mGluR antagonist, RS-4CPG (500 microM) in both IP3R1(-/-) (117.6+/-1.7%, n=8) and IP3R1(+/+) (116.9+/-1.8%, n=5) mice. The IP3R1 also plays an essential role in LTD induction, as low-frequency stimulation (LFS) failed to induce LTD in the mutant mice (104.5+/-2.1%, n=10). DP was induced in both IP3R1(-/-) and wild-type mice.

A train of low-frequency afferent stimuli (LFS, 1 Hz, 1000 pulses), given 60 min prior to a tetanus (100 Hz, 100 pulses), suppresses the induction of long-term potentiation (LTP) in which a short-term potentiation decreases gradually back to the pre-tetanic level within 40–50 min (LTP suppression). We investigated the effects of adenosine A1 or A2 receptor antagonists (8-cyclopentyltheophylline (8-CPT) and CP-66713, respectively) on LTP suppression in CA1 neurons of guinea pig hippocampal slices. When the LFS was delivered in the presence of 8-CPT (1 μM), LTP suppression was not significantly affected. However, when LFS was delivered in the presence of CP-66713 (10 μM), LTP suppression was inhibited, leading to successful LTP induction. These results indicate that endogenous adenosine, acting via A2 receptors, is involved in the mechanism of LTP suppression.

1. Using simultaneous recordings of the field EPSP and the population spike in the CA1 neurons of guinea pig hippocampal slices, we confirmed that delivery of a high-frequency stimulation (tetanus: 100 pulses at 100 Hz) produced robust long-term potentiation of synaptic efficacy (LTP) in two independent components, a synaptic component that increases field excitatory postsynaptic potentials (EPSPs) and a component that results in a larger population spike amplitude for a given EPSP size (E-S potentiation).

1. Long-term potentiation (LTP) of synaptic efficacy comprises two components: a synaptic component consisting of increased field excitatory postsynaptic potentials (EPSPs), and a component consisting of a larger population spike amplitude for a given EPSP size (E-S potentiation). In hippocampal CA1 neurons, delivery of three weak bursts (5 pulses at 100 Hz, 20 min intervals) induced LTP in both the EPSP and E-S components. In the same cells, reversal of LTP (depotentiation, DP) in the field EPSP and the E-S component was achieved by delivering three trains of low-frequency stimuli (LFS; 200 pulses at 1 Hz, 20 min intervals). 2. The effects of adenosine A1 and A2 receptor antagonists on the synaptic and E-S components of LTP and DP in CA1 neurons were studied by perfusing guinea-pig hippocampal slices with either 8-cyclopentyltheophylline (8-CPT) or CP-66713. 3. When bursts or LFS were applied to CA1 inputs in the presence of the A1 receptor antagonist 8-CPT, the field EPSP was enhanced in LTP and attenuated in DP, while the E-S relationship was not significantly affected in either LTP or DP. 4. When similar experiments were performed using the A2 receptor antagonist CP-66713, the field EPSP was blocked in LTP, but facilitated in DP, while E-S potentiation was enhanced during both LTP and DP. 5. The results show that A1 and A2 adenosine receptors modulate both the synaptic and E-S components of the induction and reversal of LTP. Based on these results, we discuss the key issue of the contribution of these receptors to the dynamics of neuronal plasticity modification in hippocampal CA1 neurons.

Anionic emulsion sizes consisting of rosin triglyceride esters and partly fortified rosin acids (i.e., rosin-ester sizes), have recently been used as internal sizes for alkaline papermaking. In this study, handsheets were prepared from pulp suspensions with alum and a rosin-ester size under various conditions, and sizing degree and size and aluminum contents of the handsheets were determined. Aluminum compounds originating from alum added to the pulp suspensions behave as retention aids of the rosin-ester size even in alkaline papermaking under limited conditions. Carboxyl groups in pulp are the primary retention sites of aluminum compounds in pulp suspensions. They form cationic sites on pulp fibers, and thus the anionic size emulsion particles are adsorbed on pulp fibers by electrostatic interactions. However, the cationic charges of aluminum compounds on pulp fibers decrease and finally disappear completely with the lapse of time after the alum addition by forming ionic bonds between the cationic sites and OH− ions. Thus, pH values of the pulp suspensions and timing of the size addition strongly influence the retention values of the rosin-ester size and the resultant sizing features.

Aromatase inhibitors (AIs) exert antiproliferative effects by reducing local estrogen production from androgens in postmenopausal women with hormone-responsive breast cancer. Previous reports have shown that androgen metabolites generated by the aromatase-independent enzymes, 5α-androstane-3β, 17β-diol (3β-diol), androst-5-ene-3β, and 17β-diol (A-diol), also activate estrogen receptor (ER) α. Estradiol (E2) can also reportedly be generated from estrone sulfate (E1S) pooled in the plasma. Estrogenic steroid-producing aromatase-independent pathways have thus been proposed as a mechanism of AI resistance. However, it is unclear whether these pathways are functional in clinical breast cancer. To investigate this issue, we assessed the transcriptional activities of ER in 45 ER-positive human breast cancers using the adenovirus estrogen-response element-green fluorescent protein assay and mRNA expression levels of the ER target gene, progesterone receptor, as indicators of ex vivo and in vivo ER activity, respectively. We also determined mRNA expression levels of 5α-reductase type 1 (SRD5A1) and 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD type 1; HSD3B1), which produce 3β-diol from androgens, and of steroid sulfatase (STS) and 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD type 1; HSD17B1), which produce E2 or A-diol from E1S or dehydroepiandrosterone sulfate. SRD5A1 and HSD3B1 expression levels were positively correlated with ex vivo and in vivo ER activities. STS and HSD17B1 expression levels were positively correlated with in vivo ER activity alone. Elevated expression levels of these steroid-metabolizing enzymes in association with high in vivo ER activity were particularly notable in postmenopausal patients. Analysis of the expression levels of steroid-metabolizing enzymes revealed positive correlations between SRD5A1 and HSD3B1, and STS and HSD17B1. These findings suggest that the SRD5A1-HSD3B1 as well as the STS-HSD17B pathways, could contributes to ER activation, especially postmenopause. These pathways might function as an alternative estrogenic steroid-producing, aromatase-independent pathways.

An attempt was made to simulate carbon gasification with the aid of molecular orbital theory. A simple Huckel method was applied to calculate molecular orbitals of a large polynuclear aromatic molecule, which was employed as a model crystallite for carbon. A free valence at each carbon atom in model crystallites of varying size and shape was determined, and this value was taken as a measure of reaction probability for the Monte Carlo simulation. Simulation of carbon gasification was performed by removing the carbon atoms from the model crystallites according to their corresponding reaction probabilities. This simulation made it possible to visualize how the shape of carbon crystallite is changed during gasification. It was found that the specific gasification rate depends on molecular size, but is essentially independent of the geometry of the starting model crystallite.

Copolymers of O-acryloyl acetophenone oxime (AOI) with styrene, ethyl acrylate, glycidyl methacrylate and methyl methacrylate become insoluble when irradiated by UV light in the presence of p-benzoquinone. Post-heating of the films after irradiation also induces further the insolubilization. In the photocrosslinking, it is deduced that p-benzoquinone serves not only as sensitizer for the decomposition of AOI groups, but also as crosslinker during the photo-irradiation and heating after irradiation

The formation of amines by the photolysis of O-acyloximes derived from acetophenone oxime, benzophenone oxime, fluorenone oxime, and 2-acetonaphthone oxime and the curing of epoxides by the resulting amines were investigated. The quantum yields of their photolysis at 366 nm in polystyrene films depend on their structures, and those for bifunctional ones bearing phenyl or naphthyl moieties were found to be very high, at 0.88 and 0.76, respectively. The yields of the resulting amines in polymer matrices were higher than those in solution. The yields for O-acyloximes bearing a naphthyl moiety were ca. 90% and higher than for those bearing a phenyl moiety. Although the resulting amines were efficient photo-cross-linkers for poly(glycidyl methacrylate), they were not effective for curing of epoxy resins (oligomers). On the other hand, polymer amines with a glass transition temperature lower than room temperature, which were obtained by the photolysis of copolymers bearing acyloxyimino groups, were found to be effective photo-cross-linkers for the epoxy resins. Keywords: photo-base generator, acyloxyimino group, photo-initiated and thermal curing, poly(glycidyl methacrylate), epoxy resin.

Background: Eribulin is a key drug for the patients with metastatic breast cancer; however, the mechanisms of how breast cancer cells become resistant to eribulin have not been fully elucidated. The purpose of this study is to analyze the mechanisms of eribulin resistance in breast cancer cells. Cell lines: Eribulin resistant cell lines were established for three estrogen receptor (ER) positive cell lines (MCF7/E, BT474/E,ZR75-1/E), three triple negative cell lines (MDA-MB-231/E, Hs578T/E, MDA-MB-157/E), and one HER2 positive cell line (SKBR3/E) by exposing to stepwise increased doses of eribulin. Results: The mRNA and protein expressions of both ABCC11 and ABCB1 were increased in all eribulin resistant cell lines regardless of their subtype. Inhibition of either ABCC11 or ABCB1 by small interfering RNA (siRNA) partially restored the sensitivity to eribulin in MCF7/E, BT474/E, and ZR75-1/E cells. Moreover, dual inhibition of ABCC11 and ABCB1 additively restored the sensitivity to eribulin in MCF7/E cells. On the other hand, overexpression of exogenous ABCC11 caused eribulin resistance in MCF7 and MDA-MB-231 cells (resistance ratio; MCF7; 13.8±0.85, MDA-MB-231; 11.4±0.74). Interestingly, inhibition of ER expression by siRNA or down regulation of ER by fluvestrant decreased ABCC11 expression and increased the sensitivity to eribulin in MCF7/E cells. Discussion: Our data demonstrates that ABCC11/MRP8 and ABCB1/MDR1 confer eribulin resistance to breast cancer cells regardless of their subtype; moreover, our data suggests the possibility of involvement of ER pathway in the regulation of ABCC11 expression in the ER positive breast cancer cells. Although further studies should be required, the expression of ABCC11/ABCB1 may be used as biomarkers for prediction of response to eribulin in breast cancer. In addition, the inhibition of ABCC11/ ABCB1 might be of service in a novel strategy to enhance the antitumor effect of eribulin. Citation Format: Takaaki Oba, Ken-ichi Ito, Shun’ichiro Taniguchi. ABCC11/MRP8 and ABCB1/MDR1 confer eribulin resistance to breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2112.

The effects of both activation and blockade of dopamine (DA) D1 receptors on long-term depression (LTD) of synaptic transmission were examined in CA1 neurons of rat hippocampal slices. Low frequency stimulation (LFS) consisting of 450 pulses at 1 Hz induced LTD (−14.3%, mean, n = 10) in the slope of the field excitatory postsynaptic potential. SKF-38393 (3–10 μM), an agonist of DA D1 receptors, significantly enhanced LFS-induced LTD (−31.1%, n = 11). SCH-23390 (2 μM), an antagonist of DA D1 receptors, blocked the induction of LTD by LFS (2.5%, n = 6). These results indicate that DA D1 receptors play an important role in the modulation of LFS-induced LTD in rat hippocampal CA1 neurons.

It was found that amines were formed efficiently by the photolysis of O-acyloximes followed by hydrolysis in polystyrene films and the relationship between structures of O-acyloximes and yields of amines were investigated. O-phenylacetyl acetophenone oxime (PaApO), O-pivaloyl acetophenone oxime (PApO), and O-benzoyl acetophenone oxime (BApO) as monofunctional O-acyloximes and O,O′-succinyl diacetophenone oxime (SDApO) and O,O′-glutaryl diacetophenone oxime (GDApO) as bifunctional O-acyloximes were examined. The yields of amines for PaApO and SDApO under N2 were ca. 70%, which was the highest among O-acyloximes examined in this experiment. On the other hand, the yields for PApO, BApO, and GDApO were less than 15% and it was verified that the hydrogen abstraction by imino radicals via 6-membered cyclic intermediates resulted in the lowering of yields. Although the effect of oxygen under photolysis on the yields of amine for PaApO was negligible under 50% conversion of PaApO, the yield decreased with further increase in the conversion and was 50% at 90% conversion. © 1994 John Wiley & Sons, Inc.

Insolubilization of poly(glycidyl methacrylate) (PGMA) and epoxy resins by amines formed by the photolysis of several O-acyloximes under air were investigated. The order of insolubilization rates of PGMA by monofunctional O-acyloximes was the same as that of the yields of amine. Bifunctional O-acyloximes were more effective than the monofunctional ones

Background: CSLEX is a type II carbohydrate antigen that interacts with the CSLEX-1 monoclonal antibody. CSLEX in combination with carbohydrate antigen 15-3 may be more useful than Carcinoembryonic Antigen with carbohydrate antigen 15-3 as tumor markers for monitoring of breast cancer. Methods: The serum levels of tumor markers, including CSLEX, were measured in 480 consecutive breast cancer patients with or without metastasis who visited the outpatient clinic of the Division of Breast and Endocrine Surgery, Shinshu University Hospital, between April 2007 and September 2007. Results: Serum levels of each of the tumor markers correlated significantly with the status of metastasis (P , 0.01). Combinations of Carcinoembryonic Antigen and carbohydrate antigen 15-3, Carcinoembryonic Antigen and Nation Cancer Center-Stomach-439, Carcinoembryonic Antigen and CSLEX, carbohydrate antigen 15-3 and Nation Cancer Center-Stomach-439, and carbohydrate antigen 15-3 and CSLEX levels also correlated significantly with the status of metastasis (P , 0.01). The sensitivity, specificity, positive predictive value, negative predictive value and accuracy were almost the same for CSLEX and Nation Cancer CenterStomach-439, which are both type II carbohydrate antigens. The cutoff indexes of serum CSLEX and Nation Cancer Center-Stomach-439 for detection of breast cancer metastasis were 38.8+52.7-fold and 22.1+27.8-fold, respectively (P ¼ 0.16). Conclusions: These data suggest that the diagnostic values of CSLEX and Nation Cancer Center-Stomach-439 are similar in single or combined use. However, the cutoff index of serum CSLEX tended to be higher than that of Nation Cancer Center-Stomach-439, which may make CSLEX more useful for detection of breast cancer metastasis.

Photolysis of an O-acyloxime in a polymer solid was applied to thermal cross-linking of poly(glycidyl methacrylate) films.

A bee venom, mast cell degranulating peptide (MCD), which induces long-term potentiation (LTP) of synaptic transmission in hippocampal slices, was found to possess multiple functions. They include (1) binding and thereby inhibiting a voltage-dependent K + -channel in brain membranes, (2) incorporation in a lipid bilayer to form voltage-dependent and cation-selective channels by itself, and (3) activation of a pertussis toxin (Ptx)-sensitive GTP-binding proteins. In this study, we prepared several derivatives and analogues of MCD and investigated which function is more closely related to the induction of LTP. Another bee venom, apamin, formed ion channels in a lipid bilayer which were indistinguishable from those formed by MCD. D-MCD, an optical isomer of MCD, activated a Ptx-sensitive GTP-binding protein. However, these peptides did not induce LTP in the hippocampal slices. A snake venom, dendrotoxin-I (DTX-I), bound to the same K + -channels as MCD and did induce LTP. These results suggest that the most potent aspect of MCD involved in LTP inducibility is its interaction with the voltage-dependent K + -channel.

The reduction of the long-term potentiated response induced by tetanus (depotentiation (DP) of LTP) was investigated by the delivery of a train of low-frequency afferent stimuli (depotentiating stimulation: DPS) after the tetanus (100 Hz, 100 pulses) in CA1 neurons of the guinea pig's hippocampal slice. The parameters of DPS (frequencies of 1, 2, 5 and 10 Hz; number of pulses of 200 and 1000; and the time-lag after tetanus of 20 and 100 min) were altered systematically and their effects on LTP were evaluated through the analysis of the slope of field EPSP (S-EPSP) and amplitude and peak latency of population spike (A- and L-PS). DPS of 1 Hz, 1000 pulses, given 20 min after tetanus, reduced the potentiated component of S-EPSP, A-PS and L-PS by 68.5%, 80.1% and 56.1%, respectively (mean, n = 6), whereas it reduced the control response by 4.3%, 7.1%, and 1.9%, respectively (n = 6). Significantly less effectiveness was observed for DPS at higher frequencies (2-10 Hz), with smaller numbers of pulses, featuring a longer time-lag after tetanus and under APV administration. When DPS was applied before tetanus, significantly less robust LTP was observed. However, these effects were blocked by the administration of APV during DPS.

A bee venom, mast-cell-degranulating (MCD) peptide, was synthesized by stepwise formation of the two disulfide bridges. This synthetic MCD peptide induced long-term potentiation (LTP) in the CA1 region of a hippocampus slice at concentrations ranging from 10(-7) to 10(-5) M. The potentiating effect of MCD was not lost by biotinylation of its N-terminus, and thus it became possible to investigate localization of its binding site at a cellular level in the hippocampus slice at a LTP-inducible concentration (1 microM). We found that even after brief application of N-biotinyl-MCD (1 microM) to perfusate, its presence was observed in pyramidal cell bodies in the CA1 and CA3 regions, but not in granule cells in the dentate gyrus of the hippocampus slice. This observation does not coincide with the localization of its high-affinity binding sites revealed by 125I-MCD binding, suggestive that another type(s) of MCD binding site, such as low-affinity binding sites, are present in the hippocampus.

A bee venom, mast cell degranulating peptide (MCD), was synthesized by stepwise formation of the two distinct disulfide bridges. This synthetic peptides with mast cell degrahulating activity MCD induced long-term potentiation (LTP) in the CAl region of hippocampus, while other peptides with mast cell degranulating activity apamin and mastoparan did not. This and other results using derivatives of the synthetic MCD suggest that a rigid structure is required for the LTP induction by MCD. Two antagonists of the N-methyl-D-aspartate (NMDA) receptor, AP5 and MK801, did not block the MCD induced-LTP. Moreover, the efficacy of the synaptic transmission in the slice which had been maximally activated by tetanic stimulation was further potentiated by MCD treatment. These results indicate that two quite distinct pathways for LTP formation exist in the CAl region of the hippocampus.

[Background] ERα and HER2 are two major biomarkers of therapeutic efficacy in breast cancer patients. Y-box binding protein YB-1 is an oncoprotein involved in breast cancer and a transcription factor for HER2/ErbB2. However, regulatory mechanisms of ERα and HER2 expression by YB-1 are largely unknown. In our present study, we examined how nuclear YB-1 regulates expression of HER2 and ERα in breast cancer cells. [Materials and methods] We established YB-1/Tet-On system in which nuclear YB-1 expression was induced by doxycycline, and examined how YB-1 could modulate expression of HER2 and ERα in breast cancer cells. Furthermore, we established the ERα/Tet-On system, in which ERα expression was markedly induced after treatment with doxycycline. To assess whether the YB-1-HER2-ERα correlation was affected by menopause, we analyzed biopsy samples of 116 premenopausal and 114 postmenopausal patients, who had not received any therapeutic drugs, by IHC. [Results] We first examined whether YB-1 regulates expression of ERα and HER2 by using various human breast cancer cell lines, and observed following findings. (1) Nuclear YB-1 overexpression increased HER2 expression and decreases ERα expression, while HER2 expression was suppressed by estradiol and enhanced by anti-estrogen drugs. (2) ERα binding to YB-1 suppressed YB-1 binding to the HER2 promoter region. Furthermore, binding of YB-1 to ERα was enhanced by estradiol and suppressed by anti-estrogen drugs. (3) YB-1 induced proteasomal degradation of ERα when both interact directly. We have next examined whether expression of HER2, nuclear YB-1, and ERα are significantly associated by IHC analysis of biopsy samples of breast cancers. In breast cancers of postmenopausal, but not premenopausal patients, nuclear YB-1 expression was positively correlated with HER2 expression and negatively correlated with ERα expression. [Conclusions] In our study, we presented a new finding that YB-1 promoted proteasomal degradation of ERα by direct interaction and that YB-1-induced HER2 expression was suppressed by ERα. Furthermore, in breast tumors of postmenopausal patients, nuclear YB-1 expression was positively correlated with HER2 expression and negatively correlated with ERα expression. Therefore, this study could contribute to further development of optimized endocrine- and HER2- targeted therapeutics against breast cancer. Citation Format: Tomohiro Shibata, Kosuke Watari, Hiroto Izumi, Akihiko Kawahara, Satoshi Hattori, Chihiro Fukumitsu, Yuichi Murakami, Ryuji Takahashi, Uhi Toh, Ken-ichi Ito, Maki Tanaka, Masayoshi Kage, Michihiko Kuwano, Mayumi Ono. Direct binding of ERα to YB-1 suppresses HER2 expression in human breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C13.

Injection of stem cells into ischaemic areas of the heart is expected to be an effective method for myocardial regeneration. The embryogenic carcinoma (EC) cell line P19CL6 is known to differentiate into cardiomyocytes when cultured with dimethyl sulfoxide (DMSO) and is expected to be a promising source for regenerative therapy in cardiac disease. To establish a high-yield method of cardiomyocyte differentiation, P19CL6 cells were double-stimulated with 5-azacytidine. Double stimulation-induced cardiomyocytes were also transplanted into ectopic sites in mice and their function evaluated.To induce differentiation under adherent conditions, P19CL6 cells were incubated in growth medium with 10 microM 5-azacytidine for 24 h. After 5-azacytidine treatment, P19CL6 cells were incubated with 1% DMSO for nine days until they began to pulsate. Prior to transplantation, cells were treated again with 5-azacytidine. Differentiated cells were injected into the greater omentum, para-aorta region of the retroperitoneum and peri-femoral artery of adult BALB/c nude mice. Nine days after transplantation, irregularly pulsating tissues at a rate slower than the host heart were observed in the transplanted sites. Light microscopy showed formation of cardiac muscle tissues originating from P19CL6 cells. Differentiated cardiomyocytes were positive for cardiac troponin I, cadherin and alpha-smooth muscle actin, and the expressions of Csx/Nkx2.5 and GATA4 mRNAs were up-regulated. Electron microscopy demonstrated components specific to cardiomyocytes, such as Z-bands, desmosomes, fasciae adherens, myofibrils and mitochondria, which confirmed successful heterotopic cardiac muscle differentiation from P19CL6 cells.This study demonstrated high-yield cardiac muscle differentiation of P19CL6 by 5-azacytidine and DMSO double stimulation and successful formation of cardiac muscle-like tissue by ectopic transplantation of cardiomyocytes derived from P19CL6 into the retroperitoneal area as well as into the peripheral vessel area.

The Multidrug Resistance Protein, MRP1 (ABCC1) confers drug resistance and transports organic anions such as leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) (E(2)17betaG). Previous studies showed that portions of the first membrane spanning domain (MSD1) and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function. We have replaced 12 prolines in MSD1 and CL3 with alanine and determined the effects of these substitutions on MRP1 expression and transport activity. All singly substituted MRP1-Pro mutants could be expressed in HeLa cells, except MRP1-P104A. The expressed mutants also transported LTC(4) and E(2)17betaG, and their K(m) (LTC(4)) values were similar to wild-type MRP1. Expression of the double mutant MRP1-P42/51A was reduced by80% although it localized to the plasma membrane and transported organic anions. MRP1 expression was also reduced when the first transmembrane helix (amino acids 37-54) was deleted. In contrast, the phenotypes of the multiply substituted CL3 mutants MRP1-P196/205/207/209A and MRP1-P235/255A were comparable to wild-type MRP1. However, Pro(255)-substituted MRP1 mutants showed reduced immunoreactivity with a monoclonal antibody (MAb) whose epitope is located in CL3. We conclude that certain prolines in MSD1 and CL3 play a role in the expression and structure of MRP1.

A 66-year-old female, suffering from small-intestinal cancer underwent resection of the small intestine. Genome DNAs were extracted from the patient’s blood and small-intestinal cancer and were subjected to a polymerase chain reaction-single strand conformation polymorphism and nucleotide sequence analysis for exons of the p53 and PTEN/MMAC1 genes to search for any mutations. The sequence analysis revealed a point mutation of the p53 codon193 in the cancer DNA; however, no mutation of the PTEN/MMAC1 gene was observed in either the blood or cancer DNA. The p53 mutation, therefore, seems to be related to tumour progression of small-intestinal cancer; however, no relationship was found between the PTEN/MMAC1 gene and the small-intestinal cancer.

Recently, mutations of KCNQ2 or KCNQ3, members of the KCNQ-related K(+)-channel (KCNQ-channel) family, were identified as cause of benign familial neonatal convulsions (BFNC). However, the exact pathogenic mechanisms of age-dependent development and spontaneous remission of BFNC remain to be elucidated. To clarify the age-dependent etiology of BFNC, we determined age-dependent functional switching of KCNQ-channels, GABAergic- and glutamatergic-transmission in rat hippocampus. The effects of inhibitors of KCNQ-channel, GABA- and glutamate-receptors on propagation of neuronal-excitability and neurotransmitter release were determined by 64-channel multielectrode-dish (MED64), whole-cell recording, in vitro release technique and in vivo microdialysis biosensor, using rat hippocampus from day of birth (P0) to postnatal-day 56 (P56). Inhibition of KCNQ-channels enhanced depolarization-induced glutamate and GABA releases during P0-P7, but not during P14-P28. Inhibition of KCNQ-channels magnified neuronal-excitability propagation from P0 to P14: maximal at P3, but this effect disappeared by P28. GABA(A)-receptor inhibition surprisingly reduced neuronal-excitability propagation during P0-P3, but not at P7. AMPA/glutamate-receptors inhibition reduced propagation of neuronal-excitability throughout the study period. KCNQ-channels inhibition shortened spike-frequency adaptation, but this stimulation was more predominant during P7 than P14. During the first week of life, KCNQ-channels performed as a predominant inhibitory system, whereas after this period GABAergic-transmission switched from excitatory to inhibitory function. Contrary, glutamatergic-transmission has acquired as excitatory function from P0. These findings suggest that the pathogenic mechanisms of age-dependent development and spontaneous remission of BFNC are, at least partially, associated with the interaction between age-dependent reduction of inhibitory KCNQ-channel activity and age-dependent functional switching of the GABAergic-system from excitatory to inhibitory action in neonatal CNS.