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C-Cbl reverses HER2-mediated tamoxifen resistance in human breast cancer cells
Wei Li1,2,
Ling Xu1,2,
Xiaofang Che1,2,
Haizhou Li3,
Ye Zhang1,2,
Na Song1,2,
Ti Wen1,2,
Kezuo Hou1,2,
Yi Yang4,
Lu Zhou1,2,
Xing Xin1,2,
Lu Xu1,2,
Xue Zeng1,2,
Sha Shi1,2,
Yunpeng Liu1,2,
Xiujuan Qu1,2 &
Yuee Teng ORCID: orcid.org/0000-0002-9198-97951,2
Tamoxifen is a frontline therapy for estrogen receptor (ER)-positive breast cancer in premenopausal women. However, many patients develop resistance to tamoxifen, and the mechanism underlying tamoxifen resistance is not well understood. Here we examined whether ER-c-Src-HER2 complex formation is involved in tamoxifen resistance.
MTT and colony formation assays were used to measure cell viability and proliferation. Western blot was used to detect protein expression and protein complex formations were detected by immunoprecipitation and immunofluorescence. SiRNA was used to examine the function of HER2 in of BT474 cells. An in vivo xenograft animal model was established to examine the role of c-Cbl in tumor growth.
MTT and colony formation assay showed that BT474 cells are resistant to tamoxifen and T47D cells are sensitive to tamoxifen. Immunoprecipitation experiments revealed ER-c-Src-HER2 complex formation in BT474 cells but not in T47D cells. However, ER-c-Src-HER2 complex formation was detected after overexpressing HER2 in T47D cells and these cells were more resistant to tamoxifen. HER2 knockdown by siRNA in BT474 cells reduced ER-c-Src-HER2 complex formation and reversed tamoxifen resistance. ER-c-Src-HER2 complex formation was also disrupted and tamoxifen resistance was reversed in BT474 cells by the c-Src inhibitor PP2 and HER2 antibody trastuzumab. Nystatin, a lipid raft inhibitor, reduced ER-c-Src-HER2 complex formation and partially reversed tamoxifen resistance. ER-c-Src-HER2 complex formation was disrupted by overexpression of c-Cbl but not by the c-Cbl ubiquitin ligase mutant. In addition, c-Cbl could reverse tamoxifen resistance in BT474 cells, but the ubiquitin ligase mutant had no effect. The effect of c-Cbl was validated in BT474 tumor-bearing nude mice in vivo. Immunofluorescence also revealed ER-c-Src-HER2 complex formation was reduced in tumor tissues of nude mice with c-Cbl overexpression.
Our results suggested that c-Cbl can reverse tamoxifen resistance in HER2-overexpressing breast cancer cells by inhibiting the formation of the ER-c-Src-HER2 complex.
Breast cancer is one of the most common malignant tumors in women and the second major cause of cancer-related death in women [1]. The estrogen receptor (ER) is a member of the nuclear hormone receptor family, which plays an important role in cell proliferation, differentiation, and tumor formation. Approximately 60%–70% of breast tumors are ER-positive at diagnosis, and anti-estrogen therapies, such as tamoxifen, are very important in premenopausal women breast cancer management [2]. However, nearly 50% of breast cancer patients develop resistance to endocrine therapy, leading to tumor progression and reduced patient survival [2]. Therefore, research on the mechanism of tamoxifen resistance is important to improve the prognosis of breast cancer patients.
Human epidermal growth factor receptor 2 (HER2)/neu overexpression or amplification is found in approximately 15%–30% of breast cancers, and increased expression of this receptor correlates with poor clinical outcome and resistance to endocrine therapy [2, 3]. The crosstalk between ER and HER2 pathways plays an important role in both intrinsic and acquired resistance to endocrine therapy [4], and thus current research in drug resistance has mainly focused on these pathways.
Evidence indicates that the non-genomic actions of estrogen are mediated by membrane-associated ERα, which resides in or near the cell membrane and interacts with several growth factor signaling pathways [5]. A previous study showed that extranuclear ER colocalizes with the HER2 receptor in membrane signaling domains that modulate downstream nuclear events leading to the growth of breast cancer cells [3].
Lipid rafts provide a functional platform for the interaction of protein molecules through dynamic aggregation [6]. Lipid rafts are essential for the plasma membrane localization of ER and play a critical role in its membrane-initiated effects [7, 8]. In HER2-overexpressing breast cancer cells, HER2 receptors are present on the cell surface as monomers, homodimers, and heterodimers [9]. Signal activation and transduction require the localization of HER2 to lipid rafts, and lipid raft aggregation is necessary for HER2 activation and function. Combination of lipid rafts inhibition, tamoxifen was more effective in inhibiting the proliferation of melanoma cells [10]. Cholesterol-rich lipid rafts were highly amplified in TAM resistant cell lines, disrupted lipid rafts acted cooperatively with TAM to reduce prosurvival mediators [11]. However, whether HER2 interacts with ER in lipid rafts and whether this interaction is involved in tamoxifen resistance remains unclear.
Research has shown that the molecular mechanisms of estrogen involve the ability of the 17beta-estradiol (E2)-ER complex to induce gene transcription through specific coregulators (i.e. coactivators or corepressors) [12, 13] and to evoke membrane-initiated activation of specific rapid phosphorylation cascades, such as Src/ERK/MAPK [13, 14]. A previous study showed that E2-induced Src activation requires the formation of a protein complex that contains at least ERα and c-Src [15]. In previous reports, membrane-associated ERα was shown to interact with HER2 in the presence of E2 [16], and HER2 interacts with the non-receptor tyrosine kinase c-Src [17]. No studies have examined the complex formation between HER2, ERα and c-Src.
Previous studies show that the ubiquitin ligases c-Cbl and Cbl-b are important regulators of lipid rafts [18]. Cbl-b inhibits the aggregation of T cell receptors by preventing lipid raft aggregation, and we previously showed that Cbl-b inhibits aggregation of lipid rafts [18]. Recent work from our group also showed that c-Cbl and Cbl-b overexpression inhibits lipid raft aggregation in gastric cancer cells [19].
In this study, we hypothesized that the ER-c-Src-HER2 complex formation is involved in HER2-mediated tamoxifen resistance and that lipid rafts are important sites for the formation of these complexes. We also investigated the potential role of c-Cbl in modulating the ER-c-Src-HER2 complex formation and function through its established effects on lipid raft formation.
Reagents and antibodies
Anti-HER2 was purchased from NeoMarker (Waltham, MA, USA), anti-ER, anti-PR, anti-c-Src, anti-p-HER2 (Tyr1248), anti-p-c-Src (Tyr416), and anti-p-ER (Ser118) were purchased from CST (Danvers, MA, USA). Anti-actin, anti-c-Cbl, anti-Caveolin1 and anti-HER2 labeled with phycoerythrin were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Tamoxifen, 17-β estradiol, Nystatin, PP2were purchased from Sigma (St. Louis, MO, USA). The c-Cbl-3 × flag-CMV9 plasmid was constructed by Taihe Biotechnology (Beijing, China). The HER2-pEGFP-N1 plasmid was constructed by GENEWIZ (Suzhou, China). The PSVL-cbl-70Z plasmid was kindly gift from Professor Kiyonao Sada (Kobe University Graduate School of Medicine). The lentiviral system was purchased from Genechem (Shanghai, China).
Cells and cell culture
The breast cancer BT474 and T47D cell lines were obtained from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). BT474 (catalogue number TCHu143) is an ER-positive and HER2-overexpressing cell line. BT474 cells were cultured in RPMI 1640 medium (Hyclone, GE, USA) containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (0.1 mg/mL). T47D (catalogue number TCHu87) is an ER-positive and low HER2-expressing cell line. T47D cells were maintained in DMEM (Hyclon, GE, USA) containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (0.1 mg/mL). Cells were cultured at 37 °C in 5% CO2. The cells were routinely subcultured every 3–5 days, and cells used for experiments were from the logarithmic growth phase. All cell lines were mycoplasma negative by PCR reaction.
Cell viability assay
Cell proliferation was measured with the 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Cells (5 × 103 cells/well) were seeded in 96-well plates for 24 h and then treated with E2 (10 nmol/L) and/or tamoxifen (1 μmol/L). Absorbance was measured at 570 nM at 48 and 72 h. The data represents the mean ± SD of at least nine wells from three independent experiments.
Colony formation analysis
Cells were plated in 6-well plates (800 cells/well), and after 24 h the cells were treated with E2 (10 nmol/L) and/or tamoxifen (1 μmol/L). Treated cells were then cultured for another 14 days. After removing the medium from the wells, colonies were stained with Giemsa and images were captured.
Western blot analysis was performed as described in our previous studies [20]. Cells were solubilized in 1% Triton lysis buffer and quantified with the Kaumas blue method. Cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane. The PVDF membranes were blocked by 5% skim milk powder in TBST buffer for 2 h at room temperature. The membranes were then incubated with primary antibodies overnight (HER2 1:1000, p-HER 1:500, ER 1:1000, p-ER 1:500, c-Src 1:1000, p-c-Src 1:250, c-Cbl 1:1000, Caveolin1 1:250, Actin 1:1000) at 4 °C, followed by incubation with secondary antibodies(CST, 1:2000) for 30 min at room temperature. The proteins were detected with enhanced chemiluminescence reagent (SuperSignal Western Pico Chemiluminescent Substrate; Pierce, USA) and visualized with the Electrophoresis Gel Imaging Analysis System (Hercules, California, USA).
Immunoprecipitates was performed as described in our previous studies [21]. Briefly, the collected cell lysates were incubated with anti-c-Src antibody or immunoglobulin-G (CST), and precleared protein G-agarose beads for 6 h at room temperature. Immunoprecipitates were washed four times in mild lysis buffer and then subjected to western blot analysis using anti-HER2(1:1000), anti-ER(1:1000), and anti-c-Src antibodies(1:1000).
Inhibition of lipid rafts
Lipid rafts are dynamic assemblies of proteins and lipids that harbour many receptors and regulatory molecules and so act as a platform for signal transduction, nystatin inhibits lipid rafts by affecting lipid metabolism [22]. To inhibit the function of lipid rafts, cells were treated with nystatin (5 μg/mL) for 2 h before the experiments.
HER2 siRNA
Three siRNAs against HER2 and a scrambled control were purchased from VIEWSOLID BIOTECH (Beijing, China) The si-HER2 target sequences were as follows:5′-GUUGGAUGAUUGACUCUGATT-3′,5′-GGAGACCCGCUGAACAAUATT-3′, and 5′-GCUCAUC GCUCACAACCAATT-3′. SiRNA transfection was performed using Lipofectamin 2000 (Invitrogen, Carlsbad, CA, USA), according to the protocol previously [21].
Cell transfection and lentiviral infection
Plasmid transfection was performed using Lipofectamine 2000, according to the protocol previously [21]. For in vivo experiments, c-Cbl was overexpressed with a lentiviral system. Lentiviral production and infection were performed following the standard procedure recommended by the company (Shanghai Genechem Co., Ltd.). At 72 h, the virus-infected cells were used for experimental analysis.
In vivo xenograft animal model
Pathogen-free female BALB/c nude mice (4 weeks of age) were purchased from WeiTongLiHua (Beijing, China). The animals were housed in accordance with institutional ethical guidelines of animal care. Mice were housed in specific pathogen-free conditions, three per cage, and maintained at constant temperature (22 °C) and humidity. At 5 weeks of age, the mice were randomized into two groups of six mice each. To establish xenograft tumors, a breast cancer cell suspension (1 × 107 cells in 0.2 mL of PBS was injected subcutaneously in the right flank of each nude mouse. One group was subcutaneously inoculated with BT474 cells infected with lentiviral vector control and the second group was inoculated with BT474 cells infected with c-Cbl lentivirus. Tumor growth was measured with fine calipers twice each week and the tumor volume was calculated by the formula shown below:
$$ V=\left(L\times {W}^2\right)/ 2 $$
We observed 100% tumor incidence for both groups. When the volume of xenografts reached 50–100 mm3, each group was divided into two treatment subgroups: vehicle control or tamoxifen treatment (three mice per group). The control groups received 200 μL saline daily from days 1–7. The tamoxifen gavage group received tamoxifen (20 mg/kg) daily from days 1–7. Tumor volume and body weight were measured twice per week. Treatments continued for 7 days, and then mice were observed for 1 week. At the end of the experiment, all mice were humanely euthanized and necropsied. Tumor tissues were harvested, rinsed in saline, weighed, and immediately formalin-fixed.
Tumor samples were formalin-fixed, paraffin-embedded and then cut into 4 μm sections. All sections were de-paraffinized in xylene and hydrated through a graduated alcohol series and then immunofluorescence was performed based on standard procedures [21]. The sections were permeabilized with 0.2% Triton X-100 for 5 min, blocked with 5% bovine serum albumin for 1 h and then incubated with phycoerythrin-labeled anti-HER2 (Sc-33684PE), anti-c-Src (SC8056), and anti-ER (SC543) antibodies (all purchased from Santa Cruz) overnight at 4 °C. The next day, Alexa Fluor 405-conjugated goat anti-mouse IgG or Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes) antibodies were added in blocking solution for 1 h at room temperature in the dark. DAPI (4′6′-diamidino-2 phenylindole) was used to stain nuclei for 1 min. After mounting with the Slow Fade Antifade Kit (Molecular Probes, Eugene, OR, USA), the sections were visualized by fluorescence microscopy (BX61, Olympus, Japan).
All experiments were performed at least in triplicate and repeated in three independent studies. The data are expressed as the mean ± SD. Differences between groups were compared using the Student's t-test. SPSS Statistics 17.0.1 (SPSS, Chicago, IL, USA) was used for statistical analysis. P < 0.05 was considered significant.
HER2-overexpressing breast cancer cells are resistant to tamoxifen
We first performed molecular characterization of the BT474 and T47D breast cancer cell lines, as shown in Fig. 1a. BT474 cells showed positive ER expression and high HER2 expression, whereas T47D cells showed positive ER expression and low HER2 expression. We next examined tamoxifen sensitivity of the two cell lines. Cell viability assays and colony formation assays demonstrated that BT474 cells were more resistant to tamoxifen than T47D cells (Fig. 1b–d), indicating that breast cancer cells with HER2 overexpression were resistant to tamoxifen.
BT474 cells are resistant to tamoxifen. a Characterization of BT474 and T47D cells. Western blot analysis of ER, HER2, PR, and c-Src and their phosphorylation status in both cell lines, and Actin is as a loading control. b BT474 and T47D cells were seeded in 96-well plates at a density of 5000 cells/well and then treated with 4-OH tamoxifen (0–10 μmol/L). After 3 days, MTT assays were performed. c BT474 and T47D cells were seeded in 96-well plates and 24 h after plating, cells were treated with vehicle (CON), 17β-E2 (E2) (10 nmol/L), tamoxifen (TAM) (1 μmol/L), or combination (E2/TAM). After 48 h, MTT assays were performed. The bar graph shows the sensitivity of cells to the indicated treatments. Data represent the mean ± SD of at least three independent experiments. d Effect of tamoxifen on colony formation in BT474 cells. After 14 days, colonies in 6-well plates were imaged and quantified by counting the number of colonies. At least nine wells per treatment group were quantified per value reported. The results show the mean from three independent experiments. *P < 0.05
Detection of the ER-c-Src-HER2 complex in tamoxifen-resistant BT474 cells
Western blot analysis showed that the level of phosphorylated HER2 (p-HER2) is higher in BT474 cells compared with T47D cells (Fig. 2a). Treatment of E2 cells also resulted in activation of HER2 in T47D. However, the activation of HER2 in T47D cells did not result in resistance to tamoxifen, and this may be due to the low expression of HER2 in these cells.
ER-c-Src-HER2 complex formation in the tamoxifen-resistant breast cancer cell line BT474. a BT474 or T47D cells (approximately 60% confluent) were treated with 17β-E2 (10 nmol/L) and/or TAM (1 μmol/L) or left untreated (vehicle control) for 4 h. Lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. b BT474 and (c) T47D cells were treated with 17β-E2 (10 nmol/L) and/or tamoxifen (TAM) (1 μmol/L) for 4 h, and then lysates were immunoprecipitated using a c-Src antibody or IgG and subjected to immunoblot analysis using the indicated antibodies
We next evaluated whether ER, c-Src and HER2 formed a complex in breast cancer cells. Immunoprecipitation analysis confirmed the ER-c-Src-HER2 complex in tamoxifen-resistant BT474 cells, whereas the complex was not detected in tamoxifen-sensitive T47D cells (Fig. 2b and c). To determine the impact of estrogen or tamoxifen on complex formation, cells were treated with estrogen or tamoxifen alone or in combination for 4 h and then evaluated for the formation of the ER-c-Src-HER2 complex. Estrogen, tamoxifen and the combination treatment all promoted the formation of the ER-c-Src-HER2 complex in BT474 cells. In T47D cells, estrogen and/or tamoxifen treatment did not promote the formation of the ER-c-Src-HER2 complex. However, tamoxifen did induce binding between ER and c-Src. These results indicated that the level of p-HER2 is elevated in BT474 cells and that HER2, c-Src, and ER can form a complex in BT474 cells.
Overexpression of HER2 leads to ER-c-Src-HER2 formation in T47D cells
We next examined the role of HER2 in impacting ER-c-Src-HER2 complex formation and function. We transfected T47D cells with a HER2 expression vector and confirmed elevated HER2 levels (Fig. 3a). Overexpression of HER2 in T47D cells led to ER-c-Src-HER2 complex formation (Fig. 3b). Furthermore, overexpression of HER2 resulted in the T47D cells becoming more resistant to tamoxifen (Fig. 3c). These results indicated that the level of HER2 in T47D cells is related to tamoxifen resistance.
Overexpression of HER2 leads to tamoxifen resistance in T47D cells. a T47D cells were transfected with HER2-pEGFP-N1 plasmid (OE-HER2) or pEGFP-N1 vector as a control (Vector). Immunoblot analysis was performed using the indicated antibodies. b T47D cells were transfected with the HER2 overexpression vector for 48 h, and immunoprecipitation and immunoblot analysis were performed with the indicated antibodies. c MTT assays in HER2-overexpressing T47D cells treated with 17β-E2 and/or tamoxifen. T47D cells were transfected with pEGFP-N1 vector or HER2-pEGFP-N1 (OE-HER2) for 24 h, and then treated with vehicle (CON), 17β-E2 (E2) (10 nmol/L), tamoxifen (TAM) (1 μmol/L) or combination treatment (E2/TAM) for another 48 h. MTT assays were then performed. *P < 0.05
Inhibition of HER2, c-Src reduced ER-c-Src-HER2 complex formation and reversed the resistance of BT474 cells to tamoxifen
To examine the role of HER2 in the formation of the ER-c-Src-HER2 complex, we used siRNA to knockdown the expression of HER2 in BT474 cells. Knockdown of HER2 in BT474 cells (Fig. 4a) inhibited ER-c-Src-HER2 complex formation, the amount of HER2 and ER bound to c-Src was decreased (Fig. 4b). The phosphorylations of HER2, c-Src, and ER were also reduced in HER2 siRNA-transfected BT474 cells (Fig. 4c). We also observed that BT474 cells with HER2 knockdown showed increased sensitivity to tamoxifen compared with controls using cell viability and colony formation assays (Fig. 4d and e). Together these results indicated that suppression of HER2 expression partially reversed the resistance of BT474 cells to tamoxifen. In HER2 si-RNA transfected cells, c-Src-ER complex formation also be inhibited.
Knockdown of HER2 expression in BT474 cells suppresses the formation of the ER-c-Src-HER2 complex and partially restores tamoxifen sensitivity. a Western blot analysis of BT474 cells transfected with siRNA targeting HER2. b BT474 cells transfected with HER2 siRNA were subjected to immunoprecipitation and immunoblotting as indicated. c Western blot analysis of phosphorylated HER2, c-Src, and ER in BT474 cells knocked down for HER2 expression for 48 h and then treated with 17β-E2 (10 nmol/L) and/or tamoxifen (TAM) (1 μmol/L) for 4 h. d MTT assays in BT474 cells transfected with siRNA targeting HER2 for 24 h and then treated with 17β-E2 (10 nmol/L) and/or tamoxifen (TAM) (1 μmol/L) for another 48 h. e Colony formation assays of BT474 cells transfected with siRNA targeting HER2 for 24 h and treated with 17β-E2 (10 nmol/L) and/or tamoxifen (TAM) (1 μmol/L) for 14d
We next examined the ER-c-Src-HER2 complex formation in BT474 cells treated with the src inhibitor PP2 or the HER2 antibody trastuzumab and found that complex formation was decreased in response to both treatments (Fig. 5a, c). Furthermore, these agents could partially reverse tamoxifen resistance in BT474 cells (Fig. 5b, d). We also examined the levels of c-Cbl protein in cells treated with PP2 or trastuzumab along with TAM, but we did not observe any obvious difference between control and treatment groups (Fig. 5e).
PP2 and trastuzumab inhibit the formation of ER-c-Src-HER2 and partially restore tamoxifen sensitivity. a BT474 cells were treated with PP2 (10 μM) for 4 h and subjected to immunoprecipitation and immunoblotting as indicated. b MTT assays in BT474 cells treated with PP2 (10 μM) for 4 h and then treated with 17β-E2 (10 nmol/L) and/or tamoxifen (1 μmol/L) for 48 h. *P < 0.05. c BT474 cells were treated with trastuzumab (10 μg/ml) for 4 h and subjected to immunoprecipitation and immunoblotting as indicated. d MTT assays in BT474 cells treated with trastuzumab (10 μg/ml) for 4 h and then treated with vehicle (CON), 17β-E2 (E2) (10 nmol/L), tamoxifen (TAM) (1 μmol/L) or combination treatment (E2/TAM) for 48 h. *P < 0.05. e Western blot analysis of c-Cbl protein in BT474 cells treated with tamoxifen (TAM) (1 μmol/L), TAM (1 μmol/L) combined with PP2 (10 μM), or TAM (1 μmol/L) combined with trastuzumab (10 μg/ml)
Inhibition of lipid rafts reduced ER-c-Src-HER2 complex formation and reversed the resistance of BT474 cells to tamoxifen
Nystatin inhibits lipid rafts by affecting lipid metabolism. Thus, we next examined the impact of inhibiting lipid rafts by nystatin on ER-c-Src-HER2 complex formation. Treatment of BT474 cells with nystatin reduced the levels of HER2 and ER bound to c-Src (Fig. 6a) and decreased the phosphorylation of HER2 compared with the control group (Fig. 6b). Furthermore, treatment of BT474 cells with nystatin increased the sensitivity of these cells to tamoxifen compared with the untreated cells (Fig. 6c). Colony formation assays showed that BT474 cells with nystatin treatment exhibited fewer colonies than the control group, and the number was further decreased in cells treated with tamoxifen and nystatin combined (Fig. 6d). Together this indicates that nystatin leads to a reversal of tamoxifen resistance in BT474 cells.
Inhibition of lipid rafts suppresses the formation of the ER-c-Src-HER2 complex and partially restores tamoxifen sensitivity. a BT474 cells were treated with vehicle (Con) or nystatin (N) (5 μg/ml) for 2 h and subjected to immunoprecipitation and immunoblotting analysis as indicated. b Cells were seeded in 6-well plates (2.5 × 104 cells/well) for 24 h and then treated with vehicle or nystatin(5 μg/ml) for 2 h, followed by treatment with vehicle, E2(10 nmol/L), TAM(1 μmol/L), or the combination for 4 h. c Cells were seeded in 96-well plates (5000 cells/well). At 24 h after plating, cells were treated with vehicle or nystatin (5 μg/ml) for 2 h, and then treated with vehicle, E2(10 nmol/L), TAM(1 μmol/L), or the combination. After 48 h, MTT assays were performed. The bar graph shows the sensitivity of BT474 cells to tamoxifen before and after treatment with nystatin. Data represent the mean ± SD of at least three independent experiments. P values were determined by the Student's t test. (*p < 0.05) d BT474 cells were treated with nystatin (5 μg/ml) for 2 h, and then treated with E2 (10 nmol/L) and/or TAM (1 μmol/L). Cells were plated in triplicate. On day 14, images were captured and the number of colonies was determined. Data represent the mean ± SD of at least three independent experiments. P values were determined by the Student's t test. (*p < 0.05)
Overexpression of c-Cbl reversed tamoxifen resistance in HER2 overexpressing breast cancer cells
We found that c-Cbl expression was higher in T47D cells than in BT474 cells (Fig. 7a), indicating a difference in c-Cbl expression levels between tamoxifen resistant and sensitive cell lines. We next examined the potential role of c-Cbl in impacting ER-c-Src-HER2 complex formation by transfecting BT474 cells with a c-Cbl expression vector (Fig. 7a). Overexpression of c-Cbl in BT474 cells reduced ER-c-Src-HER2 complex formation (Fig. 7b) and the phosphorylation of HER2 and ER (Fig. 7c). Furthermore, overexpression of c-Cbl partly reversed the resistance of BT474 cells to tamoxifen (Fig. 7d and e). To determine whether the ubiquitin ligase activity was required for c-Cbl-mediated effects, we used a plasmid expressing the c-Cbl 70Z mutant (Fig. 8a) that lacks ubiquitin ligase activity [23]. Transfection of the c-Cbl 70Z mutant in the BT474 cells did not impact the formation of the ER-c-Src-HER2 complex (Fig. 8b) or tamoxifen resistance (Fig. 8c).
Overexpression of c-Cbl reverses HER2-mediated tamoxifen resistance. a c-Cbl protein level was detected in BT474 and T47D cells. And then, BT474 cells were transfected with 3 × flag-CMV-9-c-Cbl (OE-c-Cbl) or 3 × flag-CMV-9 vector(Vector), and then examined the c-Cbl level by immunoblot analysis. b Immunoprecipitation after overexpression c-Cbl 48 h in BT474. c BT474 cells were transfected with control vector or c-Cbl overexpression plasmids, 24 h later, followed by vehicle, estrogen(10 nmol/L), and tamoxifen (1 μmol/L) or combination treatment for 4 h. Cell lysates were examined by immunoblot analysis using the indicated antibodies. d BT474 cells were transfected with plasmids expressing c-Cbl for 24 h, and then exposed to vehicle, estrogen(10 nmol/L), or tamoxifen(1 μmol/L) treatment for another 48 h. Total viable cell number was measured by MTT assays. Data represent the average of three independent replicates ± SD. (*p < 0.05) (e) Colony formation assays in BT474 cells transfected with vector or plasmids expressing c-Cbl. BT474 cells were transfected with vector or plasmids expressing c-Cbl for 24 h and then treated with E2 (10 nmol/L) and/or TAM (1 μmol/L) for 14 d. On day 14, colonies were fixed and stained with Giemsa. *P < 0.05
Overexpression of c-Cbl 70Z in BT474 cells. a BT474 cells were transfected with PSVL-70Z-c-Cbl (OE-70Zc-Cbl) or PSVL vector (Vector) and c-Cbl level was examined by immunoblot analysis. b BT474 cells were transfected with PSVL vector (Vec) or PSVL-70Zc-Cbl (70Z) for 48 h and then examined by immunoprecipitation and immunoblot analysis. c BT474 cells were transfected with PSVL-70Zc-Cbl for 24 h and then treated with vehicle, estrogen (10 nmol/L), or tamoxifen (1 μmol/L) for another 48 h. Total viable cell number was measured by MTT assays. Data represent the average of three independent replicates ± SD. *P < 0.05
To clarify the role of c-Cbl in tamoxifen resistance, we established a nude mouse xenograft model. We used a lentivirus system to generate BT474 cells that stably overexpressed the c-Cbl protein (OE-c-Cbl cells), and these cells were inoculated subcutaneously into nude mice. BT474 cells were injected into the control group mice. After subcutaneous tumor formation, each group was divided into two subgroups and treated with vehicle or tamoxifen for 7 days (Fig. 9a). Tumor volume was obviously smaller in the c-Cbl overexpression group after tamoxifen treatment compared with control group with tamoxifen (Fig. 9b). The growth curve of transplanted tumors showed that after overexpression c-Cbl, the xenografts were more sensitive to tamoxifen, and the difference was statistically significant (p < 0.05, Fig. 9c). These results suggested that overexpression of c-Cbl reversed the resistance of BT474 cells to tamoxifen in vitro and in vivo.
In vivo xenograft nude mouse model. a Female athymic mice were injected with BT474 cells or BT474 cells infected with lentivirus expressing c-Cbl (BT474-OE-c-Cbl cells) and then randomized to vehicle or 20 mg/kg TAM groups. Treatment was administered for 7 days. Three mice were included in each treatment group. b Xenograft tumors stripped from the nude mice (c) Tumor growth curve. Tumors were measured twice per week with calipers. Each data point represents the mean tumor volume in mm3 ± SEM. (*P > 0.05,** P < 0.05)
We evaluated the expressions of HER2, c-SRC and ER in the mouse tumor samples using immunofluorescence staining. We found that ER, c-Src and HER2 proteins showed co-localization in subcutaneous xenograft tumors formed by wild-type BT474 cells, but the amount of co-localization was reduced in OE-cbl-BT474 subcutaneous xenograft tumors (Fig. 10).
Immunofluorescence of tumor sections from xenograft mice. Formalin-fixed, paraffin-embedded tumors were cut into 4 μm sections and then stained with anti-HER2 (red), anti-c-Src (blue), and anti-ER (green) as indicated. Magnification 200X
Innate and acquired tamoxifen resistance is an important problem in ER+ breast cancer. HER2 is overexpressed in approximately 15%–30% of human breast cancers and plays a role in tamoxifen resistance [24]. In the previous study, the mechanisms of tamoxifen resistance have been explord, the reports included that pharmacologic mechanisms, loss or modification in estrogen receptor expression, alterations in co-regulatory proteins, autophagy, microRNA, and tumor microenvironment [25,26,27]. However, the underlying mechanism remains unclear.
BT474 is an ER- positive invasive human breast ductal carcinoma cell line with very high HER2 expression, which is very sensitive to Herceptin but resistant to tamoxifen [28]. We confirmed HER2-overexpressing breast cancer BT474 cells are relatively resistant to tamoxifen. The ER-c-Src-HER2 complex was detected in BT474 cells; this complex participated in tamoxifen resistance, inhibiting the formation of this complex reversed tamoxifen resistance. In Fig. 4, E2 along does not have much effect on cell proliferation after knockdown HER2, but this does not mean that the effect of tamoxifen is independent of ER in BT474 cells. Many previous studies have confirmed the reactivity of BT474 cells to estrogen [29,30,31]. In our study, the MTT assays were conducted in BT474 cells transfected with siRNA targeting HER2 24 h, and treated with 17β-E2 (10 nmol/L) and/or tamoxifen (TAM) (1 μmol/L) for another 48 h. BT474 is a cell that doubles for a long time. It takes about 96 h to multiply. So in this experiment, the effect of estrogen on the proliferation of BT474 cells is not yet fully reflected.
Lipid rafts are required for HER2 activation and signal transduction [32]. However, whether lipid rafts play a role in the formation of an ER-c-Src-HER2 complex has not been reported. In this study, we showed that lipid rafts are essential for the formation of the ER-c-Src-HER2 complex, as inhibition of lipid raft formation by nystatin reduced ER-c-Src-HER2 complex formation and reversed tamoxifen resistance. Our findings indicated that formation of the ER-c-Src-HER2 complex may play a key role in the crosstalk between ER and HER2 signaling pathways, and our results provide new insight into the mechanism of tamoxifen resistance.
Studies have shown that c-Cbl selectively regulates receptor translocation into lipid rafts [33], and c-Cbl is involved in the regulation of lipid rafts in several cell types [34, 35]. Overexpression of c-Cbl depleted Lck from lipid rafts in Jurkat cells [36]. Our previous study showed that the Cbl ubiquitin ligase family inhibits the function of lipid rafts [9, 16]. In vitro and in vivo experiments confirmed that overexpression of c-Cbl reduced ER-c-Src-HER2 complex formation and reversed tamoxifen resistance in BT474 cells. We also found that the ubiquitin ligase activity of c-Cbl may play a key role in the reversal of tamoxifen resistance. One hand, c-Cbl may reduce the formation of ER-c-Src-HER2 complex through inhibition of lipid rafts aggregation; the other hand, c-Cbl may promote ER-c-Src-HER2 complex ubiquitination through its ubiquitin ligase activity. Therefore, c-Cbl may be used as a therapeutic target in breast cancer patients resistant to tamoxifen, and the regulation of c-Cbl expression should be study furthermore.
Our results showed that in HER2 overexpressing breast cancer cells, ER-c-Src-HER2 complex formation resulted in HER2 signaling pathway activation and tamoxifen resistance. c-Cbl reduced ER-c-Src-HER2 complex formation by inhibiting lipid rafts, resulting in the reversal of tamoxifen resistance (Fig. 11). Together these findings indicate that ER-c-Src-HER2 complex formation plays a key role in promoting the activation of HER2 and tamoxifen resistance.
Proposed model for the role of c-Cbl in tamoxifen resistance of HER2-overexpressing breast cancer cells. In HER2-overexpressing breast cancer cells, ER-c-Src-HER2 complex formation results in HER2 signaling pathway activation and tamoxifen resistance. c-Cbl reduces ER-c-Src-HER2 complex formation by inhibiting lipid rafts and restores the sensitivity to tamoxifen
Cbl:
Casitas B-lineage lymphoma
17beta-estradiol
ERα:
Estrogen receptor alpha
HER2:
Human epidermal growth factor receptor 2
Berry DA, Cronin KA, Plevritis SK, Fryback DG, Clarke L, Zelen M, et al. Effect of screening and adjuvant therapy on mortality from breast cancer. New Engl J Med. 2005;353:1784–92.
Pietras RJ. Interactions between estrogen and growth factor receptors in human breast cancers and the tumor-associated vasculature. Breast J. 2003;9:361–73.
Marquez DC, Chen HW, Curran EM, Welshons WV, Pietras RJ. Estrogen receptors in membrane lipid rafts and signal transduction in breast cancer. Mol Cell Endocrinol. 2006;246:91–100.
Nahta R, O'Regan RM. Therapeutic implications of estrogen receptor signaling in HER2-positive breast cancers. Breast Cancer Res Treat. 2012;135:39–48.
Mauvais-Jarvis F, Clegg DJ, Hevener AL. The role of estrogens in control of energy balance and glucose homeostasis. Endocr Rev. 2013;34:309–38.
Hicks DA, Nalivaeva NN, Turner AJ. Lipid rafts and Alzheimer's disease: protein-lipid interactions and perturbation of signaling. Front Physiol. 2012;3:189.
Marin R, Ramirez C, Morales A, Gonzalez M, Alonso R, Diaz M. Modulation of Abeta-induced neurotoxicity by estrogen receptor alpha and other associated proteins in lipid rafts. Steroids. 2008;73:992–6.
Acconcia F, Marino M. The effects of 17β-estradiol in Cancer are mediated by estrogen receptor signaling at the plasma membrane. Front Physiol. 2011;2:30.
Ravacci GR, Brentani MM, Tortelli T Jr, Torrinhas RS, Saldanha T, Torres EA, et al. Lipid raft disruption by docosahexaenoic acid induces apoptosis in transformed human mammary luminal epithelial cells harboring HER-2 overexpression. J Nutr Biochem. 2013;24:505–15.
Mohammad N, Malvi P, Meena AS, Singh SV, Chaube B, Vannuruswamy G, et al. Cholesterol depletion by methyl-β-cyclodextrin augments tamoxifen induced cell death by enhancing its uptake in melanoma. Mol Cancer. 2014;13:204.
Khadka NK, Cheng X, Ho CS, Katsaras J, Pan J. Interactions of the anticancer drug tamoxifen with lipid membranes. Biophys J. 2015;108:2492–501.
Smith CL, O'Malley BW. Coregulator function: a key to understanding tissue specificity of selective receptor modulators. Endocr Rev. 2004;25:45–71.
Acconcia F, Kumar R. Signaling regulation of genomic and nongenomic functions of estrogen receptors. Cancer Lett. 2006;238:1–14.
Yang Z, Barnes CJ, Kumar R. Human epidermal growth factor receptor 2 status modulates subcellular localization of and interaction with estrogen receptor in breast Cancer cells. Clin Cancer Res. 2004;10:3621–8.
Sun J, Zhou W, Kaliappan K, Nawaz Z, Slingerland JM. ERα phosphorylation at Y537 by Src triggers E6-AP-ERα binding, ERα Ubiquitylation, promoter occupancy, and target gene expression. Mol Endocrinol. 2012;26:1567–77.
Yoshimaru T, Komatsu M, Miyoshi Y, Honda J, Sasa M, Katagiri T. Therapeutic advances in BIG3-PHB2 inhibition targeting the crosstalk between estrogen and growth factors in breast cancer. Cancer Sci. 2015;106:550–8.
Alajati A, Guccini I, Pinton S, Garcia-Escudero R, Bernasocchi T, Sarti M, et al. Interaction of CDCP1 with HER2 enhances HER2-driven tumorigenesis and promotes trastuzumab resistance in breast cancer. Cell Rep. 2015;11:564–76.
Xu L, Qu X, Zhang Y, Hu X, Yang X, Hou K, et al. Oxaliplatin enhances TRAIL-induced apoptosis in gastric cancer cells by CBL-regulated death receptor redistribution in lipid rafts. FEBS Lett. 2009;583:943–8.
Xu L, Qu X, Hu X, Zhu Z, Li C, Li E, et al. Lipid raft-regulated IGF-1R activation antagonizes TRAIL-induced apoptosis in gastric cancer cells. FEBS Lett. 2013;587(23):3815.
Li H, Xu L, Zhao L, Ma Y, Zhu Z, Liu Y, et al. Insulin-like growth factor-I induces epithelial to mesenchymal transition via GSK-3β and ZEB2 in the BGC-823 gastric cancer cell line. Oncol Lett. 2015;9:143–8.
Li H, Xu L, Li C, Zhao L, Ma Y, Zheng H, et al. Ubiquitin ligase Cbl-b represses IGF-I-induced epithelial mesenchymal transition via ZEB2 and microRNA-200c regulation in gastric cancer cells. Mol Cancer. 2014;13:136.
Varshney P, Yadav V, Saini N. Lipid rafts in immune signalling: current progress and future perspective. Immunology. 2016;149:13–24.
Qu X, Sada K, Kyo S, Maeno K, Miah SM, Yamamura H. Negative regulation of FcepsilonRI-mediated mast cell activation by a ubiquitin-protein ligase Cbl-b. Blood. 2004;103:1779–86.
Meattini I, Livi L, Saieva C, Franceschini D, Scotti V, Mangoni M, et al. Prognostic role of human epidermal growth factor receptor 2 status in premenopausal early breast cancer treated with adjuvant tamoxifen. Clinical breast cancer. 2013;13:247–53.
Viedma-Rodriguez R, Baiza-Gutman L, Salamanca-Gomez F, Diaz-Zaragoza M, Martinez-Hernandez G, Ruiz Esparza-Garrido R, et al. Mechanisms associated with resistance to tamoxifen in estrogen receptor-positive breast cancer (review). Oncol Rep. 2014;32:3–15.
Li Y, Jiang B, Zhu H, Qu X, Zhao L, Tan Y, et al. Inhibition of long non-coding RNA ROR reverses resistance to tamoxifen by inducing autophagy in breast cancer. Tumour Biol. 2017;39:1010428317705790.
Liu ZR, Song Y, Wan LH, Zhang YY, Zhou LM. Over-expression of miR-451a can enhance the sensitivity of breast cancer cells to tamoxifen by regulating 14-3-3zeta, estrogen receptor alpha, and autophagy. Life Sci. 2016;149:104–13.
Chen B, Wang Y, Kane SE, Chen S. Improvement of sensitivity to tamoxifen in estrogen receptor-positive and Herceptin-resistant breast cancer cells. J Mol Endocrinol. 2008;41:367–77.
Milosevic J, Klinge J, Borg AL, Foukakis T, Bergh J, Tobin NP. Clinical instability of breast cancer markers is reflected in long-term in vitro estrogen deprivation studies. BMC Cancer. 2013;13:473.
Rangel N, Villegas VE, Rondón-Lagos M. Profiling of gene expression regulated by 17β-estradiol and tamoxifen in estrogen receptor-positive and estrogen receptor-negative human breast cancer cell lines. Breast Cancer (Dove Med Press). 2017;9:537–50.
Liu T, Nath K, Liu W, Zhou R, Chen IW. A study of the relationship of metabolic MR parameters to estrogen dependence in breast Cancer xenografts. NMR Biomed. 2015;28(9):1087–96.
Alawin OA, Ahmed RA, Ibrahim BA, Briski KP, Sylvester PW. Antiproliferative effects of gamma-tocotrienol are associated with lipid raft disruption in HER2-positive human breast cancer cells. J Nutr Biochem. 2016;27:266–77.
Chakraborty S, ValiyaVeettil M, Sadagopan S, Paudel N, Chandran B. C-Cbl-mediated selective virus-receptor translocations into lipid rafts regulate productive Kaposi's sarcoma-associated herpesvirus infection in endothelial cells. J Virol. 2011;85:12410–30.
Haglund K, Ivankovic-Dikic I, Shimokawa N, Kruh GD, Dikic I. Recruitment of Pyk2 and Cbl to lipid rafts mediates signals important for actin reorganization in growing neurites. J Cell Sci. 2004;117:2557–68.
Qu X, Miah SM, Hatani T, Okazaki M, Hori-Tamura N, Yamamura H, et al. Selective inhibition of Fcepsilon RI-mediated mast cell activation by a truncated variant of Cbl-b related to the rat model of type 1 diabetes mellitus. J Biochem. 2005;137:711–20.
Hawash IY, Kesavan KP, Magee AI, Geahlen RL, Harrison ML. The Lck SH3 domain negatively regulates localization to lipid rafts through an interaction with c-Cbl. J Biol Chem. 2002;277:5683–91.
The authors thank Jia Liu (Animal Center of China Medical University) for kindly providing technical support. We thank Gabrielle White Wolf, PhD, from Edanz Group (https://www.edanzediting.com) for editing a draft of this manuscript.
This work is supported by the National Natural Science Foundation of China (No. 81172535, 81672605, 81673025, and 31770663), the Natural Science Foundation of Liaoning Province (2015020458), the Education Department of Liaoning Province (L2015558), and Jinzhou Science and Technology Project (17B1E37).
All data generated or analyzed during this study are included in this published article (and its Additional file 1: Figure S1).
Department of Medical Oncology, the First Hospital of China Medical University, Shenyang, 110001, Liaoning, China
Wei Li, Ling Xu, Xiaofang Che, Ye Zhang, Na Song, Ti Wen, Kezuo Hou, Lu Zhou, Xing Xin, Lu Xu, Xue Zeng, Sha Shi, Yunpeng Liu, Xiujuan Qu & Yuee Teng
Key Laboratory of Anticancer Drugs and Biotherapy of Liaoning Province, the First Hospital of China Medical University, NO. 155, North Nanjing Street, Heping District, Shenyang, 110001, Liaoning, China
Jinzhou Center Hospital, Jinzhou, 121000, Liaoning, China
Haizhou Li
Laboratory Animal Center, China Medical University, Shenyang, 110001, Liaoning, China
Wei Li
Ling Xu
Xiaofang Che
Ye Zhang
Na Song
Ti Wen
Kezuo Hou
Lu Zhou
Xing Xin
Lu Xu
Xue Zeng
Sha Shi
Yunpeng Liu
Xiujuan Qu
Yuee Teng
YT and XQ designed the research and participated in manuscript editing and review. WL and LX (Ling Xu) performed the data acquisition and prepared the manuscript. XC and HL performed statistical analysis. YZ and NS performed data analysis and interpretation. TW and KH carried out the statistical analysis. YY assisted with the animal experiments. LZ and XX performed western blot. LX(Lu Xu), XZ, and SS assisted in cell culture experiments. YL participated in manuscript editing and review. All authors read and approved the final manuscript.
Correspondence to Xiujuan Qu or Yuee Teng.
We required ethics approval for the use of cell lines used in this study. Ethical approval was given by the Medical Science Research Ethics Committee of the First Affiliated Hospital of China Medical University (reference number 201121). The animal experiments were approved by the Animal Experimental Ethical Inspection Committee of Laboratory Animal Center, China Medical University (reference number 15018 M).
Figure S1. Quantification of p-HER2 and p-ER blots in Fig. 7c. BT474 cells were transfected with control vector or c-Cbl overexpression plasmids for 24 h, followed by vehicle (CON), estrogen (10 nmol/L) (E2), tamoxifen (1 μmol/L) (TAM) or combination treatment (E2/TAM) for 4 h. (A) The relative density of phospho-HER2. *P < 0.05. (B) The relative density of phospho-ER. *P < 0.05. (JPG 799 kb)
Li, W., Xu, L., Che, X. et al. C-Cbl reverses HER2-mediated tamoxifen resistance in human breast cancer cells. BMC Cancer 18, 507 (2018). https://doi.org/10.1186/s12885-018-4387-5
C-Cbl


text:
/**@file
 *
 * Implementation of non-inline methods from the Measure family of classes.
 */

#include "SimTKcommon/basics.h"
#include "SimTKcommon/Simmatrix.h"
#include "SimTKcommon/internal/Measure.h"
#include "SimTKcommon/internal/MeasureImplementation.h"

#include <cassert>
#include <algorithm>

#if defined(__clang__)
    #if __has_warning("-Winstantiation-after-specialization")
        // Avoid the harmless warning "explicit instantiation of 'Zero' that
        // occurs after an explicit specialization has no effect"
        #pragma clang diagnostic ignored "-Winstantiation-after-specialization"
    #endif
#endif

namespace SimTK {

// These are here just to make sure they compile.
template class Measure_<Real>::Constant;
template class Measure_<Real>::Zero;
template class Measure_<Real>::One;
template class Measure_<Real>::Differentiate;
template class Measure_<Real>::Integrate;
template class Measure_<Real>::Result;
template class Measure_<Real>::Variable;
template class Measure_<Real>::Extreme;
template class Measure_<Real>::Delay;

template class Measure_<Vec3>::Constant;
template class Measure_<Vec3>::Zero;
template class Measure_<Vec3>::One;
template class Measure_<Vec3>::Differentiate;
template class Measure_<Vec3>::Integrate;
template class Measure_<Vec3>::Result;
template class Measure_<Vec3>::Variable;
template class Measure_<Vec3>::Extreme;
template class Measure_<Vec3>::Delay;

template class Measure_<Vector>::Constant;
template class Measure_<Vector>::Zero;
template class Measure_<Vector>::One;
template class Measure_<Vector>::Differentiate;
template class Measure_<Vector>::Integrate;
template class Measure_<Vector>::Result;
template class Measure_<Vector>::Variable;
template class Measure_<Vector>::Extreme;
template class Measure_<Vector>::Delay;

} // namespace SimTK

redpajama_set_name: RedPajamaGithub


This is a placeholder page for Paul Gum, which means this person is not currently on this site. We do suggest using the tools below to find Paul Gum.
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Student hangs self in protest, after losing iPhone - Glitz Plus Ent.
Archie Day, a 20-year-old university student, hanged himself in his room just hours after 'sobbing uncontrollably' because he had lost his iPhone, during a night of drinking.
Day, who is reported to be a v-blogger and journalism student was so upset at the loss of the Apple iPhone after a night out in Manchester, according to Daily Mail UK.
The day after the drunken night, which happened in October 2018, Archie Day was found dead by a duty manager at Peel Park halls of residence at Salford University in Greater Manchester.
Mohammed Zafar, a security guard at Peel Park said he could hear Archie Day crying from the other side of the door when he was called up.
Tests showed Day had 225 milligrams per 100 milliliters of blood in his system. The alcohol limit for driving is 80mg.


text:
Vocalists LP Joe & Eddie
Joe & Eddie
LP (Item 618305) Sunset, Mid 60s — Condition: Very Good+
$6.99 ...
Sunset (label)
Vocalists (CD, LP)
Folk/Country (CD, LP)
Billie Holiday & AL Hibbler
Sunset, Late 40s. Very Good+
LP...$6.99
... LP, Vinyl record album
Make Love to Me
Liberty, 1957. Very Good
Hard to imagine that Julie London would ever have to ask anyone to make love to her – at least not when she looks as sexy as this on the cover! The album's a key early classic for Liberty – still very fragile, personal, and up-close enough to make you think you might just be able to ... LP, Vinyl record album
Muse, 1978. Very Good+
One of his best for Muse, with a beautiful set of jazzy vocal grooves that includes his reading of "Stolen Moments" (later made famous by United Future Organization), plus "D.C. Farewell" and an excellent version of "Waters of March". Warm and breezy, with that ... LP, Vinyl record album
Lonely Girl
Liberty, 1956. Very Good-
Looking like she does on the cover, it's hard to imagine Julie London being a lonely girl – as she's very seductively posed, with that slinky bare shoulders look that no doubt helped many of her early Liberty sides sell so well! But apart from the cheesecake, the record's one of Julie's ... LP, Vinyl record album
Sunday In New York & Other Songs About New York
Atlantic, 1963. Very Good
One of our favorite-ever albums from Mel Torme – a set that sparkles with the same smooth swing as his best late 50s dates for Bethlehem records! Like those sets, this one features Torme's voice really taking off over some tight jazzy charts – arranged by Johnny Williams, Shorty Rogers ... LP, Vinyl record album
RCA, 1969. Very Good+
One of Nina's greatest records – recorded with her cool combo that included Weldon Irvine! Weldon's on organ on the set, and he co-wrote the two-part track "Revolution" with Simone, one of the album's strongest numbers! That track is set amidst covers of other hip 60s material, ... LP, Vinyl record album
Live In Paris
Impulse, 1986. Very Good
One of Dee Dee Bridgewater's greatest jazz albums, and a very important record in her career – as it marked a shift back to straight jazz vocals, after years of working in a more R&B inflected mode. The album's recorded in Paris, where Dee Dee's spent an awful lot of time over the years, ... LP, Vinyl record album
Capitol, 1958. Very Good
One of Frank Sinatra's last great ballad albums for Capitol – a sad and dreamy batch of tunes that kicks off with the incredible "Only The Lonely" – one of those Sinatra torch songs that's so great, so sad, nobody else has ever been able to record a better version, nor have ... LP, Vinyl record album
Nat King Cole Sings/George Shearing Plays
A nice little session that's one of Nat's last great vocal albums – thanks to some solid jazz backing by George Shearing. Shearing's group here is a super-hip west coast Latin one, with Emil Richards on vibes, Al Hendrickson on guitar, Al McKibbon on bass, and Shelly Manne on drums. Most ... LP, Vinyl record album
Act One Sing Too
Philips, Mid 60s. Very Good+
Great work from Polly Bergen – breathy and beautiful, but still plenty lively throughout – thanks to some backings from Jerry Fielding! The record's got a sound that's a fair bit more modern than some of Bergen's other albums from earlier years – and Fielding's use of bright ... LP, Vinyl record album
By Myself
Liberty, Mid 60s. Very Good
Genius Hits The Road
ABC, 1960. Very Good-
A stunning standout from Ray Charles' years on ABC Records – and a set that brings in a bit of hard-swinging jazz to the country-styled groove Ray was hitting at the time! Backings are by Ralph Burns, who keeps things pretty hip – not as modern as some of his 50s charts for other ... LP, Vinyl record album

redpajama_set_name: RedPajamaCommonCrawl


In a BDE based, Client-server application, I use a Server with BDE tables and remote providers.
The client connect to it with MIDAS tech. using Remote datamodule, TClientDatasets.
The problem is, that even if only one user is on. the dataset cannot always apply updates.
Actually there are'nt any exception, only a REconcileError event is called by the dataset, and the records wont be applied to the database..
If a (read only) query is open, using the same table, it occures more often.
I'd appriciate any ideas, what can be the reason for that.


From Barolo legend Renzo Seghesio, we offer this stunning Barbera d'Alba Superior. Made from handpicked, organic grapes from his Panjana della Ginestra vineyard in Monforte d'Alba – one of Barolo's greatest vineyards – this wine also comes from the excellent 2016 vintage, a year that produced aromatic, well-balanced wines. This is a seriously delicious wine with lively, mouth-watering acidity and an energetic minerality that invigorates the palate. The interplay between fruit and earth and the cool-toned, sweet but savoury flavours enhances food rather than dominates it. This is an immensely satisfying, affordable, knife-and-fork wine that can be enjoyed now but is so well structured that it can handle some serious cellaring.
Barbera is the archetypical Piedmontese wine. The people of Piedmont and next door, in Lombardy, grow up in Barbera's company; drinking it characterizes a Milanese as much as the Duomo. Grown mainly in Lombardy, Emilia-Romagna and Sardinia, no one disputes that Piedmont, especially Alba, is its spiritual home and from where the best wines are grown. Usually found as a joyous, everyday trattoria-style wine, in the right hands it can be made into something rich, complex and age-worthy; some believe it's in the same class as Barolo or Brunello because of its incredible ability to display site-specificity (aka terroir). An authentic, great Barbera is distinguished by this sense of place as well as high acidity, intense purple hues, recognizable aromas of red fruits, underbrush, delicate spices and a long, dry finish. Such is today's wine.
For more than a century the Seghesio family has been crafting their traditionally made wines in famed Monforte d'Alba; but the estate's tiny production has kept it a relative secret. Renzo bottled his first wines in 1968; his Barolo bears the name 'Pajana,' which might be called a 'cru-within-a-cru' in that it's considered a sub-section of Ginestra, one of the most famous and greatest vineyards of Barolo. Renzo is also a stubborn purist who farms organically, works his small vineyard holdings manually and ferments using wild yeasts. He served as the mayor of Monforte d'Alba for 25 years and also mapped the areas vineyards as an amateur cartographer. This man truly lives and breathes his home terroir.
The wines of Renzo Seghesio have been considered classic, iconic expressions of traditional Barbera and Nebbiolo (Barolo) for decades, but sadly (or happily, depending on your viewpoint) there is little information about them that is readily available and finding their wines can be difficult. However, when you thumb through Sheldon Wasserman's landmark tome, The Noble Red Wines of Italy (published way back in 1991), you can read his notes on these wines stretching back decades to his first tasting trips to the Langhe, and he's not the only notable to be impressed, Burton Anderson's Guide to the Wines of Italy is another, and there are more. It's an indication of the esteem these wines have enjoyed among Barbera and Nebbiolo connoisseurs and insiders for decades. But despite its embrace by the Italian wine cognoscenti the estate is one of those rare Langhe wineries that has managed to stay under the radar, which is lucky for us because it helps to keep prices reasonable.
So back to today's wine; Renzo Seghesio's Barbera d'Alba Superior 2016. The vines for this wine occupy less than two hectares of the Ginestra vineyard. They face southeast at an elevation of 500 meters and grow in sandy, calcareous soils. The grapes were hand-picked at the end of September and fermented with wild, indigenous yeasts in steel barrels for 18 days. This was followed by a second malolactic fermentation and aging in old 30 hectoliter Slavonian oak barrels for another 10 months. The wine was then bottled without fining or filtering and aged an additional six months before release.
In the glass it is an intense ruby red and displays aromas of violets, liquorice and spice. On the palate it is dry, mouth-wateringly delicious, balanced, full bodied, structured, complex and refreshing. Fresh blackberry, cherry and hints of plum are followed by a long, persistent finish with spicy after-tones. Serve this wine at 16-17° in Bordeaux stems with seasoned cheese or roasted venison. Cin Cin!
PS: To read a review of the excellent 2015 vintage of today's wine go to Peter Vetch's blog Pop and Pour.


Phew! Your kids have already completed their exams and the school term has come to an end! As parents, do you feel that sometimes you are more anxious and worried about the final exams than your children? Many of us feel forced to give our kids extra help in the form of tutors to keep ahead of their other classmates or simply because all our colleagues and friends are doing it too!
Amidst all the rush to secure the most qualified tutors, theAsianparent talks to a working mother who does not believe that tuition is necessary for her children! Not only are Mrs Choo's 5 children studying in top local universities, junior colleges and secondary schools, they have always done well academically. She shares with us some of her tips and secrets to her kids' academic success.
1. Determine if your kids really need tuition, but know when to stop!
There are 2 main reasons why kids may require tuition. The first is known as 'Foundation Tutoring', where children are unable to keep up in class. Furthermore, falling behind in class may result in them losing interest in the subject altogether! In this case, tuition is necessary to help your child get back on track. So, find a qualified tutor who will be able to inspire your child to learn to love the subject. Most importantly, the end goal of tuition is to make sure that your child does not need tuition in the future! The second reason is that your child may not be fulfilling their potential and may need extra coaching a few months before a major exam. The goal of the tutor here will be to help your child consolidate their knowledge and answer any questions they may come across when doing practice papers.
How can learning be fun, you ask? Well, the key here is to improvise and use whatever you can find to make new learning tools for your kids. For example, Mrs Choo made simple flashcards to teach Mandarin words to her children, printed out handy timetable charts for memory on the go, used jigsaw puzzles as a tool to learn spelling and colourful ice-cream sticks to help her younger kids visualize Math problems. The list goes on, so be creative!
This is Mrs Choo's sneaky way of getting her youngest child interested in Science without her even knowing it! Mrs Choo subscribed to channels such as National Geographic and magazines like Horrible Science, which is a science magazine full of fun facts and experiments kids can try at home. (Definitely not as "Horrible" as its name suggests!) So by the time her daughter was introduced to Science at Primary Three, she had already cultivated a liking for the subject. She also subscribes to Reader's Digest and Time for her older children.
We know you've heard this one before. But the truth is that it works! Besides exposing your kids to reading at a young age, the library can also be a place where you discover your children's other interests. Perhaps your daughter tends to borrow books on Arts and Crafts or your son somehow seems to gravitate towards the Cooking section? Your child may have interests they're not telling anyone about, so observing the types of books that attract them may give you a clue!
Discuss this with your children and agree upon a fixed study time, say from 7 to 8.30pm every school day. During this time, all distractions like their gaming consoles and mobile phones should be put away. Mrs Choo saw that the fixed timing idea works well on her younger kids as it provides them with a daily structure, making them more likely to focus as they know they can take a break after study time is over.
Probably a month or so before an exam, Mrs Choo sits down with her kids to list out the topics tested in the exam, fit the exam dates onto their calendar and draw up a revision schedule. Another important thing is to set goals for the coming exam. Now, remember to be realistic here! Look at your child's current grades and encourage him to aim to improve by one grade.
"If you do well in the exam, Mummy will buy you that new toy you wanted." Sound familiar? Many of us know that threats only serve to stress kids out so we resort to promises of rewards in order to "motivate" our children before an exam. But did you know that this is doesn't work well too? We've heard stories of kids not being able to focus in the exam because their parents promised them a holiday to Switzerland and they were too worried that they would not do well enough to go!
With five children to raise, Mrs Choo knew that she could not afford tuition for all her kids. So she got her older children who were able to handle their work independently, to teach their younger siblings. This reinforced their knowledge and her younger kids found learning more enjoyable. But only do this if your older children are done with their own work, you wouldn't want to cause them unnecessary stress!
9. Stay away from 'kiasu' parents!
Okay, we've all been guilty of comparing our kids to our relatives' and colleagues' children. Whose child goes to better schools, has better grades and has the most expensive tuition fees… there's just no end to it! Instead of getting stressed up by these 'kiasu' parents, talk to your child's teacher or someone whom you feel has been coaching his or her kids well.
Learning opportunities are everywhere so don't just restrict your kids to their study room at home! Check out our list of educational places in Singapore you can bring your kids to have some fun!


Do you suffer from aches and pains? Stress? Insomnia? Lack of concentration? Or do you just want to improve your overall performance and excel everyday with an energised and motivated mind? If so, then Yoga is for you!
Whether it's Yoga (flow), Aerial Yoga, Yogalates, Energyse Yoga or Hatha Yoga, you name it here at The Circle we live, breathe and sleep Yoga! Yoga has an incredible ability to connect your body to your mind. Despite the variety that Yoga offers, the idea is always the same - that it brings relief to any mental or physical problem - and we truly believe it to be an effective way to handle the pressures of everyday life.
This is a very active form of Yoga that incorporates movement. The flowing movements are great for burning calories! This one is also great for ensuring a relaxed and peaceful mind as well as reducing stress and anxiety.
Aerial Yoga is a practice of Yoga which uses low-handing aerial silks (hung like a hammock), which can help aid you in achieving postures and stretches in a beautifully relaxed way. This is perfect for beginners as you can let the hammock take the weight of your body therefore encouraging you to concentrate merely on your stretches.
This technique of Yogalates combines strength training of Pilates and the flexibility of Yoga. This is a very popular exercise as it allows you to work within your own limits, whilst really testing your flexibility and endurance.
EnergYse Yoga is the unique exercise that focuses on treating the mind, body and spirit to increase flexibility whilst ensuring heightened relaxation. This series is perfect to condition your body, improve strength, muscle tone, core stability and calm the mind. If you want to focus on all aspects then this is the class for you!
Hatha Yoga is a very calming and gentle approach to Yoga, designed to align and calm your body, mind, and spirit in preparation for meditation.
Like no other exercise, yoga finds a space between activity and meditation and draws on the benefits of both so come on down and explore all that The Circle has to offer!


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Bae Doona And Cho Seung Woo's "Forest Of Secrets" In Talks.
This page is for the walkthrough of Season 1. If you are looking for a different Season click the following links: Season 2 or go back to our main walkthrough directory. What is a walkthrough? A walkthrough is a chapter-by-chapter guide that will reveal the effects each choice has on your character and the story. Please note that these pages.
Stranger aka Forest of Secrets (Season) - South Korean Police Purcedural November 10, 2017 2:42 PM - Subscribe. A prosecutor without empathy and a bold female police are taking a murder case involving unrestrained political corruption. It is definitely a slow burn murder mystery. Netflix only at the moment. Stars: Seung-woo Cho, Doona Bae, Jae-woong Choi IMDB. posted by KaizenSoze (13 comments.
Netflix has released the trailer for season two of Stranger Things, which returns to the streaming service on October 27. They've also revealed new stills for the show. Stranger Things stars.
Name Air Dates; 1: Nazi Doomsday Forest: Jan 9, 2020: 2: Bermuda Triangle of Space: Jan 16, 2020: 3: Chernobyl's Nuclear Monsters: Jan 23, 2020: 4: Curse of.
EastEnders: Secrets from the Square Season 1 Episode 1 of 14 Mick and Linda Stacey Dooley is joined by some of Walford's biggest stars to relive their characters' most iconic moments, share behind.
Metacritic TV Episode Reviews, Secrets in the Forest, Psychic Kelli Faulkner helps police search for a man who disappeared in the woods after an argument with his wife.
The Cold Feet revival continues in the shape of series eight, but while the five friends are older, they're not necessarily wiser. Adam, Pete, Jenny, Karen and David are back with more tales of love, loss and the emotional twists and turns of middle-aged life.
Collect all 8 VHS Tapes in Stranger Things: The Game to unlock an exclusive trailer for Season 2 of Stranger Things. With all 8 VHS Tapes collected in Stranger Things: The Game, you'll be able to unlock an exclusive trailer for Season 2 of Stranger Things. To unlock and view this trailer, head to the Hawk Theater located in Downtown Hawkins.
Stranger. Season 1. Release year: 2017. With the help of a gutsy female detective, a prosecutor who has lost the ability to feel empathy tackles a murder case amid political corruption. 1. Episode 1 77m. As a teen, prosecutor Hwang Si-mok received surgical treatment for his abnormally developed brain, but the procedure left behind serious side effects. 2. Episode 2 69m. The convict's strong.
Stranger Things has so many eighties influences we could be here all day talking about them, but the creators also revealed they were inspired by certain horror-themed video game franchises too.
It seems like Stranger is winning a lot on this thread but the thread is, in fact, titled: Is Stranger (Forest of Secrets) the best crime drama of all time? Personally, I really liked both but Stranger is the more polished and cohesive story whereas Signal, despite its rough ending, carried more emotional value for me.
Stranger Things season 2 is coming after the first season made it's debut last July and people became instantly obsessed with the show. Set in a small town in Indiana in the 80's, it follows a group of kids (and a few adults) as they deal with an inter-dimensional portal that opens up and claims one of their friends. The show was a huge success and won a SAG Award for best ensemble in a.
Warning: Spoilers about Netflix's The Stranger finale ahead. The streaming series' 2020 finale, "Episode 8," however, reveals Adam's search was for nought all long. Corrine is dead, and.
As the town preps for Halloween, a high-scoring rival shakes things up at the arcade, and a skeptical Hopper inspects a field of rotting pumpkins.
Chapter description Fireheart feels so astonished that he can barely speak; and he asks Graypool if Mistyfoot and Stonefur really did come from ThunderClan.Graypool gives her chest a few licks, and mews that's exactly what she's telling the warrior.Fireheart is stunned, and questions if Oakheart had stolen them for RiverClan.The gray elder's fur bristles as she snarls that Oakheart was a noble.
Drama Korea Forest of Secrets Subtitle Indonesia - Drama terbaru dari bintang film yang tengah naik daun Bae Doo Na, Siap buat menghibur anda. Sinopsis Drama Korea Forest of Secrets Though he is rational, a childhood brain surgery leaves prosecutor Hwang Shi Mok (Cho Seung Woo) to lack emotion.
There is also something from the forest in her blood stream that is keeping her alive. Yes, I know it doesnt make any sense. Just roll with it. Franck visits a higher up. Sirani is still after the Steiners (Marion's father Bertrand and grandfather Gerard) but there's no evidence to link them to anything. Remember: They own everything (and everyone) in town. Cut over to the rock quarry. We see.
Season 2 is not needed in my opinion, it comes to a natural and satisfying conclusion at the end of the finale, and all the characters' plotlines are bought to an end. The only way a continuation could feasibly take place is through an ensemble soap drama, but as much as I would love to step back into the chaotic carnage of this world, it could only harm the show. A thrilling six hours (or.
The Society season 2 - Netflix release date and UK air date, cast, filming, theories, episode 1 details, plot and everything you need to know.
A source from cable network tvN has confirmed on September 3 that its critically acclaimed thriller Secret Forest, also known as Stranger, will be returning for a second season next year. The source said that writer Lee Soo-yeon will also be back to write the script for the second season, but another director will be at the helm of the production.
Stranger Things. was a gigantic hit last year, and I'm very happy to welcome back Emmy-award winners Brad North and Craig Henighan as they take you behind the scenes on the sound for the brand-new 2nd season. Here's the story behind their creative work, sonic approaches and ideas for Stranger Things 2 - and how they cleverly designed the sounds for everything from pollywogs to Demodogs, and.
The prosecutor's office and the police find themselves on opposing sides. The prosecutors, including elite prosecutor Woo Tae Ha, want discretionary power.


Peters, BJ, Day JMD, Greenwood RC, Hilton DR, Gibson J, Franchi IA. 2017. Helium-oxygen-osmium isotopic and elemental constraints on the mantle sources of the Deccan Traps. Earth and Planetary Science Letters. 478:245-257.
The Deccan Traps, a 65 million-year-old continental flood basalt province located in western India, is the result of one of the largest short-lived magmatic events to have occurred on Earth. The nature and composition of its mantle source(s), however, have been difficult to resolve due to extensive assimilation of continental crust into the ascending Traps magmas. To circumvent this issue, using high-precision electron microprobe analysis, we have analyzed olivine grains from MgO-rich (up to 15.7wt.%) lavas that likely erupted before substantial crustal assimilation occurred. We compare olivine, pyroxene and plagioclase mineral chemistry and He–O–Os isotope compositions with bulk rock major-and trace-element abundances and 187Os/188Os for both bulk-rocks and mineral separates. Helium isotope compositions for the olivine grains generally show strong influence from crustal assimilation (<3 RA), but one ankaramite from the Pavagadh volcanic complex has a 3He/4He ratio of 10.7 RA, which is slightly lower than the range of 3He/4He measured for present-day Réunion Island volcanism (∼12–14 RA). Olivine-dominated mineral separates span a more restricted range in 187Os/188Os (0.1267 to 0.1443) compared with their host lavas (0.1186 to 0.5010), with the separates reflecting a parental magma composition less affected by lithospheric or crustal interaction than for the bulk-rocks. Despite significant He–Os isotopic variations, D17O is relatively invariant (−0.008 ±0.014 per mil)and indistinguishable from the bulk mantle, consistent with high-3He/4He hotspots measured to-date.
Compositions of olivine grains indicate the presence of up to 25% of a pyroxenite source for Deccan parental magmas, in good agreement with ∼20% predicted from isotopic data for the same samples. Modeled pyroxenite signatures appear like geochemical signatures expected to arise due to other types of mantle differentiation or due to assimilation of continental crust; however, we show that crustal assimilation cannot account for all of the compositional features of the olivine. Weak correlations exist between a global compilation of Xpx(Deccan: 0.2–0.7) and 3He/4He, δ18O (Deccan olivine: 4.9–5.2 per mil) and 187Os/188Os. Robust relationships between these parameters may be precluded due to a lack of two-reservoir source mixing, instead involving multiple mantle domains with distinct compositions, or because Xpxmay reflect both source features and crustal assimilation. Notwithstanding, geochemical similarities exist between Deccan Traps olivine (3He/4He =10.7 RA; 187Os/188Osi=0.1313 ±45, 2σ) and Réunion igneous rocks (3He/4He =12–14 RA; 187Os/188Osi=0.1324 ±14). These relationships imply that a characteristic geochemical 'fingerprint' may have persisted in the mantle plume that fed the Deccan Traps, since its inception at 65 Ma, to ongoing eruptions occurring on Réunion up to the present-day.


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La cripta del Museo San Francisco es el recinto ubicado en el subsuelo, de la Basílica de San Francisco, en la ciudad de La Paz, Bolivia.
Es parte de lo que fuera en Conjunto Conventual San Francisco que ocupara inicialmente dos manzanos del barrio de San Francisco. En este sitio están depositados los restos de personajes destacados por su importancia en el desarrollo de la historia de Bolivia.

El recinto
Mide aproximadamente 3 por 2 metros. El único acceso re realiza mediante unas escalinatas que se encuentran frente a retablo principal.

Al final del descenso los primeros restos que se aprecian son los de Diego de Baena y Antipara, ubicado en un promontorio al lado izquierdo de las escaleras y frente a la entrada a la Cripta. Baena, rico minero y principal impulsor de la refundación de la iglesia (1743), donó 600 monedas de oro para la reconstrucción del retablo mayor de la catedral. La administración de entonces aceptó la donación, por lo que sus restos aún yacen en el lugar.

Frente de la entrada principal, presidiendo las urnas mortuorias, se encuentra la virgen del Carmen.

Las urnas

A los lados se encuentran los restos de trece personajes que participaron en las revoluciones previas a la independencia boliviana, como las batallas posteriores.
Las urnas dentro de la cripta están dispuestas en el siguiente orden:
 Otto Phillip Braun, quien durante la guerra de independencia boliviana enseñó el manejo de las armas al grupo de nacionales.
 José Ballivián Segurola, capitán de los ejércitos bolivianos durante al Batalla de Ingavi, y expresidente de Bolivia.
 Eduardo Abaroa, que participó de la Guerra del Pacífico y de quién en la urna no yacen sus restos, sino arena de mar que fue entregada por el ejército chileno en su homenaje, según se lee en al leyenda inscrita.  Su urna está resguardada a cada lado por la Virgen del Carmen y la Virgen Dolorosa.
 Los miembros de la Junta Tuitiva de 1809 y de la revolución paceña de ese mismo año,  a quienes ejecutaron el 29 de enero de 1810, están ordenados de la siguiente forma: Mariano Graneros, Buenaventura Bueno, Basilio Catacora, Apolinar Jaén, Juan Bautista Sagárnaga, Pedro Domingo Murillo, Gregorio Ballivián, Manuel Victorio Ballivián Lanza, Juan Antonio Figueroa y Melchor Jiménez.
La cabeza de Pedro Domingo Murillo, que después de ser ahorcado fue decapitado,  fue trasladada a lo que hoy es la zona Faro Murillo en El Alto, de donde su hija, Tomasita, la recogió para enterrarla junto al resto de su cuerpo. En la actualidad, la cabeza y el cuerpo yacen cremados juntos dentro de la urna al interior de la cripta.

Véase también
 Centro Cultural Museo San Francisco
 Independencia de Bolivia

Enlaces externos
 Sitio Web del museo que comprende la iglesia y la cripta

San Francisco
Criptas

redpajama_set_name: RedPajamaWikipedia


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usually rare items are much rare drop than gems so it best to farm when u have full mil accessory it add 1 more item each time.
i'm a retard..i discovered only now that the fountain of yout let you trasmigrate character-.- there is a limit to how many times you can do it?
so basicaly you can relevel them till they have all stat to 100 and 999hp?
HP cap is 500 I think.
The game become trivial once you hit lv90 even on hard mode, esp if you have been using marwen's items during all the level ups.
Meister requires recruiting all the NG+ characters and having the regular roster as well since you need them for Asmodeus. Once you complete Sin Gate, Ragena Ruins, Asmodeus conversation / item creation quest in chapter 9, you fight her in ugh forgetting the name, seal place and then a conversation recruits her. Now you should have an event at the lord's manor to become a meister. The sad part is though is if you waited to long for this then it might be superseded by some storyline event that also takes place there, it's kind of low on the priority pole.
The thing that confuses me is that you said item creation quest in chapter 9. If it's a chapter 9 requirement (which there certainly was that trigger), how could I have done it earlier?
Alright, seems like NG++ isn't advisable, it doesn't seem like I'm more than 5 steps away from Asmodeus. If I somehow missed it (which couldn't be true, it should say "failed" probably), then I'll either backtrack or go into NG++. Thanks again, I think that answers everything.
Into Chapter 8 of Emilita's route: Story-wise it has been good so far. I felt that one part of the Leguna plot point would turn out like this as soon as the tournament was over, but the second development was kinda unexpected.
Eushully should make a fandisc and give me a Roka route . Having her as just a clone unit is dissastisfying >_<.
I mentioned it before, but Melodiana's character quests are really greatly done. Same as the last few events for Rag (e. g. the back-to-back talk with Yuela). I haven't seen the last bits of dragon and Paras because I cannot motivate to level them.
I don't really care about all the optional dungeons, however I might bring myself to - at least - finish the Yuidora mine sections because receiving an optional character serves as a well-enough motivation for me.
Ran into a new problem, how does one bypass the unbypassable flight barriers found in both Blockaded Pit and Verdant Cave?
gotta love yuela straight forward personality: "if we kill him now, nobody will know"
Teleport stones or displace from fox lady.
I think Roka is from the battle goddess games so if you want her you would have to play her game .
I think Eushully is releasing a new game next month. I am tempted to play it even if I do not understand anything just for the game play.
I really love Melodianna's development and thinks her "route" is even better than the heroine's. Watching her act as Wil's judge and guardian, taking caring of Wil. Watching them go on walks (walking around town with an angel will make every men jealous!). She slowly develops her feelings for Wil and muster the courage to follow her heart... Seeing her pout during the hot spring scene was cute, and seeing her disappointment when she learned of Wil being such a womanizer made me feel sorry for her.
Most of the heroines' route sort of "just happened". Yuela's route could have been a lot more epic if the invasion happened much earlier and lead to an all out war that spans several chapters.
I mentioned earlier that there are some loose ends/explained things in the game. Would still like to see them resolved. Yuela not being able to meet her sister was a let down. Would also like to see some more story in the NG++ dungeons (ie: the scenario when the heroines were captured), esp Asmodeus story line. I feel there should be a lot of "side effects" to Wil killing off the gathering of angels at the end of the game.
Last edited by Flying Dagger; 2012-03-29 at 14:09.
Or how about Rosanna in Emilita's route: "If you make Emilita cry I will use every connection, technology and knowledge that I have to make you dissappear from this world." One of scariest mothers in the VNs I have played so far.
they need better writing staff tho, so many loop holes.
I'm not sure about the first Battle Goddess game, but I know that one is able to romance Roka in Battle Goddess 2. When she first appears, she is seen wearing the armour that Wil crafted for her; I guess this means that Roka had met Wil after the first game but before the game? It's just too bad that none of the games in this series have been translated.
I really liked Melodiana until Eliza and the other angels showed up. For me, what made her special was the fact that she was angel, but after having so many other angels join your party starting with Eliza, I felt that she had lost a fair amount of her appeal.
I thought you could only displace to blue ... OH. Thank you! I actually haven't actively used teleport/egress/portal/door stones yet. Alright, time to continue venturing!
Eliza treats Wil like a toy. Melo actually loves him .
NG+ Eushully angels do not really count as they do not have any plot to them. Combat wise Melodianna suffers from rather poor growth even with the lv50 stat cap unlocking. She also lack high end combat skills .
How do I get the Bronze, Silver etc. keys required to access some doors? I've covered few dungeons like 95% so I'm 100% sure I haven't just missed an area where the key would be hidden. Beat all games etc. but to no avail ..
You need to craft them in your shop, but you get the receipt only in later chapters. Bronze key is sometimes around chapter 3 or 4 I think. Silver Key late game and gold key in New Game+. So just progress through the chapters and check the last tab in the Alchemy section after each chapter.
Yep, that's precisely why I prefer Melo to Eliza. It helps that I love the valkyrie-like armour she sports.
Leveling Aht is such a chore. It's like the parent needs to chew on the food himself into paste before feeding it to the kid. Not much of a tank as I believe it to be, is there any benefit from using Aht in the future at all?