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Silencing HOXD10 by promoter region hypermethylation activates ERK signaling in hepatocellular carcinoma
Yulin Guo1,2,
Yaojun Peng1,
Dan Gao1,3,
Meiying Zhang1,3,
Weili Yang1,3,
Enqiang Linghu1,
James G. Herman4,
François Fuks5,
Guanglong Dong2 &
Mingzhou Guo ORCID: orcid.org/0000-0002-9445-99841
Clinical Epigenetics volume 9, Article number: 116 (2017) Cite this article
Hepatocellular carcinoma is the fifth most common malignancy and the third leading cause of cancer-related death worldwide. Dysregulation of HomeoboxD10 (HOXD10) was found to suppress or promote cancer progression in different cancer types. The function and regulation of HOXD10 remain unclear in human hepatocellular carcinoma (HCC).
Primary HCC samples (117), normal liver tissue samples (15), and 13 HCC cell lines (SNU182, SNU449, HBXF344, SMMC7721, Huh7, HepG2, LM3, PLC/PRF/5, BEL7402, SNU387, SNU475, QGY7703, and Huh1) were included in this study. Methylation-specific PCR, flow cytometry, western blot, transwell, siRNA, and chromatin immunoprecipitation assays were employed.
HOXD10 was methylated in 76.9% (90/117) of human primary HCC samples. HOXD10 methylation was significantly associated with vessel cancerous embolus, tumor cell differentiation, and the 3-year overall survival rate (all P < 0.05). The expression of HOXD10 was regulated by promoter region methylation. HOXD10 suppressed colony formation, cell proliferation, cell invasion and migration, and induced G2/M phase arrest and apoptosis in HCC cells. HOXD10 suppressed HCC cell xenograft growth in mice. HOXD10 suppresses HCC growth by inhibiting ERK signaling.
HOXD10 is frequently methylated in human HCC, and the expression of HOXD10 is regulated by promoter region methylation. HOXD10 suppresses HCC cell growth both in vitro and in vivo. HOXD10 suppresses human HCC by inhibiting ERK signaling.
Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third leading cause of cancer-related death worldwide [1]. In China, HCC is the fourth most commonly diagnosed cancer in men, and it is the third leading cause of cancer death for both men and women [2]. The 5-year survival rate remains below 12% [3]. The mechanisms underlying the development and progression of HCC remain unclear. Chromosomal amplifications (1q, 6p, 8q, 17q, and 20q) and deletions (4q, 8p, 11q, 13q, 16q, and 17p) are frequent events in HCC [4]. In 5–10% of HCC patients, high-level amplifications have been described in 6p21 (vascular endothelial growth factor A, VEGFA) and 11q13 (cyclin D1, CCND1) [5]. Accumulating evidence has shown that epigenetic as well as genetic alterations play important roles in the development of many cancers [6,7,8]. Epigenetic inactivation of tumor suppressor genes has been frequently found in HCC [9]. Homeobox (HOX) genes encode homeoproteins, which share a common homeodomain and serve as important transcription factors targeting downstream proteins [10]. Homeoproteins play an important role in development and carcinogenesis by modulating cell growth, migration, cell cycle, and apoptosis [11,12,13,14]. Homeobox D10 (HOXD10) is a member of the homeobox gene family. HOXD10 expression levels and functions vary by cancer type [14,15,16,17]. In this study, we analyzed the regulation and the function of HOXD10 in human HCC.
Human tissue samples and cell lines
Primary human hepatocellular carcinoma samples (117 cases) and normal liver tissue samples (15 cases) were collected from the Chinese PLA General Hospital in Beijing between 1 July 2010 and 1 January 2014. The median age of the cancer patients was 55 years old (range 29–71), and the ratio of males/females was 6.3:1. All cancer samples were classified according to TNM staging (AJCC 2010). Forty cases of available matched cancer adjacent tissue paraffin samples were included in this study. All samples were collected following the guidelines approved by the Institutional Review Boards of the Chinese PLA General Hospital with written informed consent from patients. HCC cell lines, which included SNU182, SNU387, HBXF344, SNU475, HepG2, PLC/PRF/5, Huh7, BEL7402, LM3, SNU449, SMMC7721, QGY7703, and Huh1 were previously established from human primary HCC [18]. All cells were maintained in 90% RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum.
5-Aza-2′-deoxycytidine treatment
HCC cell lines were split to a low density (30% confluence) 12 h before treatment. Cells were treated with 5-aza-2′-deoxycytidine (5-aza) (Sigma, St. Louis, MO) at a concentration of 2 μM. Growth medium conditioned with 5-aza at a concentration of 2 μM was exchanged every 24 h for a total of 96 h of treatment.
RNA isolation and semi-quantitative RT-PCR
Total RNA was isolated by Trizol reagent (Life Technologies, Gaithersburg, MD). First-strand cDNA was synthesized according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). PCR primers for HOXD10 are listed in Additional file 1: Table S1. The primer sets for HOXD10 were designed to span intronic sequences between adjacent exons in order to control for genomic DNA contamination. RT-PCR was amplified for 33 cycles. GAPDH was amplified for 25 cycles as an internal control.
Bisulfite modification, methylation-specific PCR (MSP), and bisulfite sequencing
DNA was prepared by the proteinase K method. Bisulfite treatment was carried out as previously described [19]. MSP primers were designed according to genomic sequences around transcription start sites (TSS) and synthesized to detect unmethylated (U) and methylated (M) alleles. Bisulfite sequencing (BSSQ) was performed as previously described [20]. BSSQ products were amplified by primers flanking the targeted regions including MSP products. All primers are listed in Additional file 1: Table S1.
Immunohistochemistry (IHC) was performed in HCC tissue samples and paired adjacent tissue samples. The HOXD10 antibody was diluted to 1:100 (Abcam, Cambridge, UK). The staining intensity and extent of the staining area were scored using the German semi-quantitative scoring system as previously described [20, 21].
Construction of HOXD10 expression vector and transfection assay
Full-length HOXD10 cDNA (GenBank accession number NM_002148.3) was cloned into the pcDNA3.1 expression vector. Transient transfection was performed using Lipofectamine 3000 (Intrivogen, Carlsbad, CA) according to the manufacturer's instructions.
Cell viability detection
Cells were plated into 96-well plates at a density of 2 × 103 cells/well, and cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (KeyGEN Biotech, Nanjing, China) at 0, 24, 48, and 72 h. Absorbance was measured using a microplate reader (Thermo Multiskan MK3, MA, USA) at a wavelength of 490 nm.
Colony formation assay
Cells were seeded into 6-well culture plates at a density of 800 cells per well in triplicate and cultured for 2 weeks. For Huh7 and SMMC7721 cells, growth medium was conditioned with G418 (Invitrogen, Carlsbad, CA) at 300 and 50 μg/ml, respectively, and was exchanged every 24 h. Cells were then fixed with 75% ethanol for 30 min, stained with 0.2% crystal violet (Beyotime, Nanjing, China) for 20 min and counted.
For cell cycle analysis, cells were serum starved 12 h for synchronization and then re-stimulated with 10% FBS for 24 h. Cells were fixed with 70% ethanol and prepared for cell cycle detection using the Cell Cycle Detection Kit (KeyGen Biotech, Nanjing, China). Cells were then sorted by a FACSCalibur (BD Biosciences, San Jose, CA) and analyzed by the Modfit software (Verity Software House, ME, USA). For apoptosis analysis, the Annexin V-FITC/PI Apoptosis Detection Kit (KeyGen Biotechnology, China) was used according to manufacturer's instructions. Each sample was analyzed by flow cytometry with a FACScan Flow Cytometer (Becton-Dickinson Biosciences, Mansfield, MA).
Transwell assay
Cells were suspended in serum-free medium. Cells (2 × 104) were placed into the upper chamber of an 8-μm pore size transwell apparatus (Corning, NY, USA) and incubated for 24 h. Cells that migrated to the lower surface of the membrane were stained with crystal violet and counted in three independent high-power fields (×200). For invasion analysis, cells (3 × 104) were seeded into the upper chamber of a transwell apparatus coated with Matrigel (BD Biosciences, San Jose, CA) and incubated for 48 h. Cells that invaded into the lower membrane surface were stained with crystal violet and counted in three independent high-power fields (×200).
Chromatin immunoprecipitation (ChIP) was performed in HOXD10 highly expressed Huh1 cells using HOXD10 monoclonal antibody (Life Span Bio Sciences, Inc., WA, USA) or normal rabbit IgG (negative control) according to the EpiTect ChIP One Day Kit (Qiagen, Hilden, Germany). Two primers encompassing HOXD10 binding sites in different regions of the IGFBP3 promoter were designed as shown in Additional file 1: Table S1.
SiRNA knockdown technique
SiRNAs targeting HOXD10 and the RNAi negative control duplex were used in this study. The sequences of the siRNAs are listed in Additional file 1: Table S1 (Gene Pharma Co, Shanghai, China). The RNAi oligonucleotide and RNAi negative control duplex were transfected into HOXD10 highly expressing QGY7703 and Huh1 cells.
Proteins from HCC cells were collected 48 h after transfection. For extracellular signal-regulated kinase (ERK) signaling analysis, cells were starved with serum-free medium for 24 h after transfection. These cells were then stimulated with medium containing 10% serum for 15 to 60 min before collection. Western blot was performed as described previously [21]. Antibodies were diluted according to manufacturer's instructions. The primary antibodies were as follows: HOXD10 (Life Span Bio Sciences, Inc., WA, USA), IGFBP3 (Protein Tech Group, Chicago, IL, USA), ERK1/2 (Bioworld Tech, MN, USA), p-ERK1/2 (Bioworld Tech, MN, USA), MMP2 (Protein Tech Group, Chicago, IL, USA), MMP9 (Protein Tech Group, Chicago, IL, USA), cyclinB1 (Protein Tech Group, Chicago, IL, USA), cdc-2 (Protein Tech Group, Chicago, IL, USA), bcl-2 (Protein Tech Group, Chicago, IL, USA), cleaved caspase 3 (Protein Tech Group, Chicago, IL, USA), and β-actin (Bioworld Tech, MN, USA).
HOXD10 unexpressed and re-expressed SMMC7721 cell xenograft mouse model
Stably transfected SMMC7721 cell line with pLenti6 vector or pLenti6-HOXD10 vector (6 × 106 cells diluted in phosphate-buffered saline and matrigel mixed at the ratio of 1:1) were injected subcutaneously into the dorsal right side of 4-week-old female Balb/c nude mice. Each group includes six mice. Tumor volume was measured every 3 days. Tumor volume was calculated according to the formula: V = L × W2/2, where V represents volume (mm3), L represents biggest diameter (mm), and W represents smallest diameter (mm). Mice were sacrificed on the 39th day after inoculation and tumor was weighted. All procedures were approved by the Animal Ethics Committee of the Chinese PLA General Hospital.
SPSS 17.0 software (IBM, NY, USA) was used for data analysis. Categorical variables were analyzed using the chi-squared test or Fisher's exact test. The two-tailed independent sample t test was applied to determine the statistical significance of the differences between the two experimental groups. For matched HCC and adjacent tissue samples, paired Student's t test was employed. Survival rates were calculated by the Kaplan–Meier method, and differences in survival curves were evaluated using the log-rank test. Cox proportional hazards models were fit to determine independent associations of HOXD10 methylation with 3-year OS outcomes. Two-sided tests were used to determine the significance, and P < 0.05 was considered as statistically significant.
HOXD10 is silenced by promoter region hypermethylation in HCC cells
Semi-quantitative RT-PCR was employed to detect the expression of HOXD10 in HCC cells. Loss of HOXD10 expression was found in SNU182, SNU449, HBXF344, SMMC7721, Huh7, HepG2, LM3, PLC/PRF/5, and BEL7402 cells. Reduced expression of HOXD10 was found in SNU387 and SNU475 cells. HOXD10 was highly expressed in QGY7703 and Huh1 cells (Fig. 1a). MSP was employed to detect promoter region methylation. MSP primers were designed around the transcription start site in the CpG islands within the HOXD10 gene promoter region. Complete methylation was found in SNU182, SNU449, HBXF344, SMMC7721, Huh7, HepG2, LM3, PLC/PRF/5, and BEL7402 cells, partial methylation was found in SNU387 and SNU475 cells, and unmethylation was found in QGY7703 and Huh1 cells (Fig. 1b). These results demonstrate that loss or reduced expression of HOXD10 is correlated with promoter region methylation.
HOXD10 expression and methylation status in human HCC cells. a Semi-quantitative RT-PCR shows HOXD10 expression levels in HCC cell lines. HBXF344, SNU182, SNU387, SNU449, SNU475, SMMC7721, BEL7402, Huh1, Huh7, LM3, PLC/PRF/5, HepG2, and QGY7703 are HCC cell lines. 5-AZA: 5-aza-2′-deoxycytidine; GAPDH: internal control of RT-PCR; (−): absence of 5-AZA; (+): presence of 5-AZA. b MSP results of HOXD10 in HCC cell lines. U: unmethylated alleles; M: methylated alleles; IVD: in vitro methylated DNA, serves as methylation control; NL: normal peripheral lymphocytes DNA, serves as unmethylated control; H2O: double distilled water. c BSSQ results of HOXD10 in SMMC7721, Huh7 and Huh1 cells. Double-headed arrow: MSP PCR product size was 100 bp and bisulfite sequencing focused on a 254 bp region of the CpG island (from − 375 to − 122) around the HOXD10 transcription start site. Filled circles: methylated CpG sites, open circles: unmethylated CpG sites. TSS: transcription start site
To further validate that the expression of HOXD10 was regulated by promoter region methylation, HCC cells were treated with the DNA methylation transferase inhibitor, 5-aza-2′-deoxycytidine (5-aza). Re-expression of HOXD10 was found in SNU182, SNU449, HBXF344, SMMC7721, Huh7, HepG2, LM3, PLC/PRF/5, and BEL7402 cells, and increased expression of HOXD10 was found in SNU387 and SNU475 cells. No expression change was found in QGY7703 and Huh1 cells (Fig. 1a). These results suggest that HOXD10 expression is regulated by promoter region methylation in HCC cells. To further validate the MSP results and examine the methylation density in the promoter region, BSSQ was performed in SMMC7721, Huh7, and Huh1 cells. BSSQ results were consistent with the MSP results, showing dense promoter region methylation in SMMC7721 and Huh7 cells and no methylation in Huh1 cells (Fig. 1c).
HOXD10 is frequently methylated in human primary HCC, and reduced expression of HOXD10 is associated with promoter region hypermethylation
To determine the methylation status of HOXD10 in human primary HCC, 117 cases of primary HCC and 15 cases of normal liver tissue samples were examined by MSP. HOXD10 was methylated in 76.9% (90/117) of primary HCC samples, but no methylation was detected in normal liver tissue samples (Fig. 2a). As shown in Table 1, methylation of HOXD10 was more frequently in patients with vessel cancerous embolus (p < 0.05) and poorly differentiated tumors (P < 0.05), but no association was found between HOXD10 methylation and age, gender, HBV infection, cirrhosis, tumor size, number of lesions, TNM stage, and lymph node metastasis (all P > 0.05).The median follow-up period for patients was 23 months (range, 0–76 months).Four patients in the unmethylated group and 17 patients in the methylated group were lost to follow-up. Kaplan–Meier plots indicated that methylation of HOXD10 was associated with poor 3-year overall survival (OS) (P = 0.048, Fig. 2b). While, according to Cox proportional hazards model analysis, HOXD10 methylation was not an independent prognostic factor for 3-year OS after adjusting for tumor differentiation, vessel cancerous embolus, and TNM stage (P = 0.127, Table 2).
The expression and methylation status of HOXD10 in primary HCC. a Representative MSP results of HOXD10 in normal liver tissue samples and primary HCC samples. N: normal liver tissue samples; HCC: primary HCC samples. b The 3-year overall survival curves for patients in the methylated and unmethylated groups (P < 0.05). c Representative IHC results show HOXD10 expression in HCC tissue and adjacent tissue samples (top: ×200; bottom: ×400). d The expression of HOXD10 and DNA methylation status is shown as a bar diagram. Reduced expression of HOXD10 was significantly associated with promoter region hypermethylation. *P < 0.05, r = 0.33. e HOXD10 expression scores are shown as box plots; horizontal lines represent the median score; the bottom and top of the boxes represent the 25th and 75th percentiles, respectively; vertical bars represent the range of data. Expression of HOXD10 was significantly different between adjacent tissue and HCC tissue in 40-matched HCC samples. ***P < 0.001
Table 1 Clinical factors and HOXD10 methylation in 117 cases of HCC samples
Table 2 Univariate and multivariate analysis of HOXD10 methylation status with 3-year overall survival (OS) in HCC patients
The expression of HOXD10 was evaluated by immunohistochemistry (IHC) in 40 cases of available matched primary HCC and adjacent tissue samples. Staining of HOXD10 was mainly localized in the nucleus, and its expression was significantly reduced in primary HCC compared to adjacent tissue samples (P < 0.001, Fig. 2c, e). In 40 cases of available primary HCC, loss or reduced expression of HOXD10 was found in 28 cases. Of these 28 case samples, 26 cases were methylated and 2 cases were unmethylated. Loss or reduced expression of HOXD10 was significantly associated with promoter region hypermethylation (P < 0.05, r = 0.33, Fig. 2d). These results indicate that HOXD10 expression is regulated by promoter region methylation in primary HCC.
HOXD10 suppresses proliferation of HCC cells
To evaluate the effects of HOXD10 on HCC cell proliferation, cell viability was determined by the MTT assay. The OD value was 0.63 ± 0.05 vs. 0.50 ± 0.05 (P < 0.01) and 0.68 ± 0.01 vs. 0.53 ± 0.02 (P < 0.05) before and after restoration of HOXD10 expression in HOXD10 unexpressed SMMC7721 and Huh7 cells, respectively. The OD value was reduced significantly (Fig. 3a). In HOXD10 highly expressed QGY7703 cells, the OD value was 0.45 ± 0.03 vs. 0.50 ± 0.04 before and after knockdown of HOXD10 expression. The OD value was increased significantly (P < 0.05, Fig. 3a). These results suggest that HOXD10 suppresses HCC cell viability. Colony formation assays were performed to evaluate the effect of HOXD10 on clonogenicity. As shown in Fig. 3b, the colony number was 155.67 ± 14.64 vs. 61.0 ± 7.55 in SMMC7721 cells (P < 0.01) and 139.67 ± 15.18 vs. 97.33 ± 10.07 in Huh7 cells (P < 0.05) before and after re-expression of HOXD10, showing significant reduction in colony formation with HOXD10 re-expression. In HOXD10 highly expressed QGY7703 cells, the colony number was 51.67 ± 6.43 vs. 91.67 ± 20.50 before and knockdown of HOXD10. The colony number was significantly increased after knockdown of HOXD10 expression (P < 0.05, Fig. 3b). These results demonstrate that HOXD10 suppresses HCC cell growth.
The effect of HOXD10 on HCC cell proliferation and apoptosis. a Growth curves represent the cell viability analyzed by the MTT assay in HOXD10 re-expressed and unexpressed SMMC7721 and Huh7 cells, as well as in QGY7703 cells before and after knockdown of HOXD10. Each experiment was repeated in triplicate. *P < 0.05, **P < 0.01. b Colony formation results show that colony number was reduced by re-expression of HOXD10 in SMMC7721 and Huh7 cells, while increased by knockdown of HOXD10 in QGY7703 cells. Each experiment was repeated in triplicate. Average number of tumor clones is represented by bar diagram. *P < 0.05, **P < 0.01. c Flow cytometry results show induction of apoptosis by re-expression of HOXD10 in SMMC7721 and Huh7 cells, while reduction of apoptosis was found after knockdown of HOXD10 in QGY7703 cell. * P < 0.05. d Western blots show the effects of HOXD10 on the levels of cleaved caspase 3 and bcl-2 expression in SMMC7721, Huh7, and QGY7703 cells. VECTOR: control vector, Re-HOXD10: HOXD10 expressing vector, β-actin: internal control
HOXD10 induces cell apoptosis
To explore the role of HOXD10 in apoptosis, flow cytometry was performed. The percentages of apoptotic cells were 9.30 ± 0.15% vs. 11.00 ± 0.57% in SMMC7721 cells (P < 0.05) and 4.86 ± 0.26% vs. 6.57 ± 0.55% in Huh7 cells (P < 0.05) before and after re-expression of HOXD10. The percentage of apoptotic cells was increased significantly (Fig. 3c) after re-expression of HOXD10. In HOXD10 highly expressed QGY7703 cells, the percentage of apoptotic cells was 13.56 ± 2.03% before knockdown of HOXD10 and 9.83 ± 0.36% after knockdown of HOXD10. The percentage of apoptotic cells was reduced significantly (P < 0.05, Fig. 3c). To further validate HOXD10-induced apoptosis in HCC cells, cleaved capase-3 and bcl-2 levels were examined by western blot. The levels of cleaved capase-3 were increased and the levels of bcl-2 were reduced after re-expression of HOXD10 in SMMC7721 and Huh7 cell. In HOXD10 highly expressed QGY7703 cells, the levels of cleaved capase-3 were reduced and the levels of bcl-2 were increased after knockdown of HOXD10 (Fig. 3d). These results suggest that HOXD10 induces apoptosis in HCC cells.
HOXD10 induces G2/M phase arrest
The role of HOXD10 in the cell cycle was analyzed by flow cytometry. As shown in Fig. 4a, the distribution of cell phase in HOXD10 unexpressed and re-expressed SMMC7721 cells was 61.35 ± 1.75% vs. 56.86 ± 0.69% in G0/G1 phase (P < 0.05), 29.55 ± 0.59% vs. 27.04 ± 1.43% in S phase, and 9.10 ± 1.76% vs. 16.09 ± 0.80% in G2/M phase (P < 0.05). In Huh7 cells, the cell phase distribution was 63.95 ± 0.92% vs. 52.27 ± 0.23% in G0/G1 phase (P < 0.01), 24.85 ± 1.09% vs. 26.16 ± 0.38% in S phase, and 11.20 ± 0.36% vs. 21.58 ± 0.15% in G2/M phase (P < 0.01) before and after restoration of HOXD10 expression (Fig. 4a). G2/M phase was increased significantly after re-expression of HOXD10 in HCC cells. To further validate these results, siRNA knockdown technique was employed. The cell phase distribution was 53.00 ± 0.61% vs. 61.28 ± 0.38% in G0/G1 phase (P < 0.01), 27.30 ± 0.52% vs. 26.40 ± 0.40% in S phase, and 19.67 ± 0.12% vs. 12.20 ± 0.61% in G2/M phase before and after knockdown of HOXD10 in HOXD10 highly expressed QGY7703 cells (Fig. 4a). The percentage of G2/M phase was reduced significantly after knockdown of HOXD10 (P < 0.01). These results suggest that HOXD10 induces G2/M phase arrest in HCC cells.
The effect of HOXD10 on HCC cell cycle. a Cell phase distribution in HOXD10 unexpressed and re-expressed SMMC7721 and Huh7 cells, as well as in QGY7703 cells before and after knockdown of HOXD10. The ratio is presented by bar diagram. Each experiment was repeated three times. *P < 0.05, **P < 0.01. b The expression of HOXD10, cyclinB1, and cdc-2 was detected by western blot in HOXD10 unexpressed and re-expressed SMMC7721 and Huh7 cells, as well as in QGY7703 cells before and after knockdown of HOXD10. β-actin: internal control
The induction of G2/M checkpoint arrest by HOXD10 was further validated by detecting G2/M phase-related proteins. The expression levels of cyclinB1 and cdc-2 were dramatically reduced after re-expression of HOXD10 in SMMC7721 and Huh7 cells, and the levels of cyclinB1 and cdc-2 expression were increased obviously after knockdown of HOXD10 in HOXD10 highly expressed QGY7703 cells (Fig. 4b). Above results suggest that HOXD10 inhibits cell proliferation in HCC.
HOXD10 suppresses cell invasion and migration in HCC
The transwell assay was employed to evaluate the effects of HOXD10 on cell invasion. The number of cells for each high-power field under the microscope was 140.00 ± 10.00 vs. 70.33 ± 11.68 in SMMC7721 cells and 159.00 ± 14.73 vs. 93.33 ± 7.51 in Huh7 cells before and after restoration of HOXD10 expression. The number of invasive cells was reduced significantly after re-expression of HOXD10 in SMMC7721 and Huh7 cells (all P < 0.001, Fig. 5a). In HOXD10 highly expressed QGY7703 cells, the number of invasive cells for each high-power field under the microscope was 78.00 ± 6.08 vs 129.30 ± 10.07 before and after knockdown of HOXD10. The number of invasive cells was increased significantly (P < 0.01, Fig. 5a). To explore the mechanism of HOXD10 in HCC cell invasion, MMP2 and MMP9 were examined by western blot. The expression levels of MMP2 and MMP9 were reduced after re-expression of HOXD10 in SMMC7721 and Huh7 cells. While, the expression levels of MMP2 and MMP9 was increased obviously after knockdown of HOXD10 in HOXD10 highly expressed QGY7703 cells (Fig. 5c). The results suggest that HOXD10 inhibits cell invasion in HCC cells.
HOXD10 inhibits cell invasion and migration in HCC cells. a Cell invasion in HOXD10 unexpressed and expressed SMMC7721 and Huh7 cells, as well as in QGY7703 cells before and after knockdown of HOXD10. The number of cell invading to the lower chamber is presented by bar diagram. Each experiment was repeated three times. **P < 0.01, ***P < 0.001. b Cell migration in HOXD10 unexpressed and expressed SMMC7721 and Huh7 cells, as well as in QGY7703 cells before and after knockdown of HOXD10. The migratory cell number is presented by bar diagram. Each experiment was repeated three times. *P < 0.05, **P < 0.01, ***P < 0.001. c The expression levels of HOXD10, MMP2, and MMP9 were detected by western blot
Next, the transwell assay was performed in the absence of extracellular matrix (ECM) gel coating to explore the effects of HOXD10 on cell migration. The numbers of migrated cells for each high-power field under the microscope were 251.00 ± 11.36 vs. 197.67 ± 11.02 in SMMC7721 cells (P < 0.05) and 402.00 ± 20.52 vs. 203.00 ± 12.29 (P < 0.001) in Huh7 cells before and after restoration of HOXD10 expression. The number of migrated cells was 221.70 ± 19.76 vs. 317.00 ± 24.64 before and after knockdown of HOXD10 in HOXD10 highly expressed QGY7703 cells (P < 0.01, Fig. 5b). The results indicate that HOXD10 inhibits cell migration in HCC cells.
HOXD10 inhibits ERK signaling in HCC cells
HOXD10 has been demonstrated to act as a transcription factor targeting the promoter region of IGFBP3 in gastric cancer [15, 22]. IGFBP3 may activate different signaling pathways in different cancers [23]. In the NSCLC cell line H1299, IGFBP3 interacts with and inactivates ERK1/2 by inhibiting ERK1/2 phosphorylation [24]. In human HCC, the signaling pathway involving HOXD10 remains unclear. To further understand the mechanism of HOXD10 in HCC and determine whether it binds to IGFBP3, ChIP assays were performed. The IGFBP3 promoter region was pulled down by the HOXD10 antibody in HOXD10 highly expressing Huh1 cells (Fig. 6a). Results of the ChIP assay suggest that HOXD10 interacts with the promoter region of IGFBP3. To further analyze the role of HOXD10, the expression levels of IGFBP3 were examined by western blot in HOXD10 unexpressed and re-expressed SMMC7721 and Huh7 cells. The expression of IGFBP3 increased after re-expression of HOXD10, suggesting that HOXD10 upregulates IGFBP3 in HCC cells (Fig. 6b).
HOXD10 interacts with IGFBP3 and inhibits ERK1/2 phosphorylation in HCC. a ChIP results show that HOXD10 binds to the promoter region of IGFBP3. b Western blots show the levels of HOXD10, IGFBP3, ERK1/2 and p-ERK1/2 in SMMC7721 and Huh7 cells. β-actin: internal control. −: no serum stimulation. +: serum stimulation. c The expression levels of HOXD10 after knockdown by siRNA in Huh1 cells. HOXD10-Si: siRNA, NC: negative control. d The levels of HOXD10, IGFBP3, ERK1/2, and p-ERK1/2 were detected by western blot after knockdown of HOXD10 in Huh1 cells. β-actin: internal control. −: no serum stimulation. +: serum stimulation
Next, we detected phosphorylation status of ERK1/2 by western blot to further explore the mechanism of IGFBP3 in regulation of downstream signaling. After 24 h of serum starvation followed by 10% serum stimulation for 45 min, the levels of phosphorylated ERK1/2 were reduced in HOXD10 re-expressed SMMC7721 cells compared to HOXD10 unexpressed SMMC7721 cells. Similarly, the levels of phosphorylated ERK1/2 were reduced by HOXD10 in Huh7 cells after 10% serum stimulation for 30 min. These results indicate that HOXD10 inhibits ERK1/2 phosphorylation by upregulating IGFBP3 expression (Fig. 6b). To further validate the inhibitory effect of HOXD10 on ERK signaling, siRNA knockdown technique was used in HOXD10 highly expressing Huh1 cells. HOXD10-SiR1 effectively knocked down HOXD10 in Huh1 cells (Fig. 6c). The levels of phosphorylated ERK1/2 were increased by knockdown of HOXD10 in 10% FBS-stimulated Huh1 cells (Fig. 6d). These results suggest that HOXD10 inhibits ERK signaling by upregulating IGFBP3 in HCC.
HOXD10 suppresses human HCC cell tumor growth in xenograft mice
To further investigate the role of HOXD10 in human HCC, a xenograft mouse model was employed. HOXD10 unexpressed and re-expressed SMMC7721 cells were inoculated into nude mice subcutaneously. The tumor volume in HOXD10 unexpressed and re-expressed SMMC7721 cell transplanted xenograft mice was 527.22 ± 271.23 mm3 vs. 212.00 ± 75.93 mm3. The tumor volume was smaller in HOXD10 re-expressed SMMC7721 cell xenograft mice compared to HOXD10 unexpressed SMMC7721 cell xenograft mice (P < 0.001, Fig. 7a). The tumor weights were 0.17 ± 0.09 g vs. 0.06 ± 0.02 g (P < 0.05) in HOXD10 unexpressed and re-expressed SMMC7721 cell xenografts. The tumor weight was significantly reduced after re-expression of HOXD10 (P < 0.05, Fig. 7b). The results demonstrate that HOXD10 suppresses HCC cell growth in vivo.
HOXD10 suppresses human HCC cell tumor growth in xenograft mice. a Tumor growth curve for xenograft mice subcutaneously burdened with SMMC7721 cells stable transfected with HOXD10 or GFP control. ***P < 0.001. b Tumor weights of nude mice 39 days after inoculation with SMMC7721 cells in which HOXD10 is re-expressed or unexpressed. *P < 0.05
HCC is a complex disease. Hepatocarcinogenesis involves hepatocyte injury, inflammation, proliferation, and genomic instability, which lead to alteration in several oncogenic pathways, including EGFR, AKT, WNT, and ERK signaling [25,26,27,28]. Four clusters and 39 Hox genes have been identified in humans. Hox genes are conserved across species and encode transcription factors that are defined by the DNA-binding domain called the homeodomain. Hox proteins can function as monomers or homodimers to directly drive the transcription of downstream targets. The effects exerted by Hox genes are varied in different pathways with notable tissue specificity. Hox genes may serve as oncogenes or tumor suppressor genes in different cancer types [10]. The expression of HOXD10 was lost during the malignant progression of breast cancer, while the expression of HOXD10 was increased in human head and neck cancer [29, 30]. The expression and regulation of HOXD10 in human HCC remains unclear.
In this study, we demonstrated that the expression of HOXD10 is reduced/lost frequently in HCC, and the expression of HOXD10 is regulated by promoter region methylation. HOXD10 methylation was associated with vessel cancerous embolus, tumor cell differentiation, and the 3-year survival rate. The results suggest that HOXD10 methylation may serve as a poor prognostic marker of HCC. Following up of this cohort, we only obtained 3-year OS data. Analyzing by Cox proportional hazards model, HOXD10 methylation was not an independent prognostic marker for 3-year OS after adjusting for tumor differentiation, vessel cancerous embolus, and TNM stage. Increasing the cohort number is necessary in our future study. To further clarify the function of HOXD10 in HCC, we analyzed the effects of HOXD10 on cell proliferation, apoptosis, cell cycle, cell invasion, and migration in HCC cells. In our study, HOXD10 suppressed HCC cell proliferation, induced apoptosis, and G2/M phase arrest and inhibited cell invasion and migration. Li et al. found that miR-224 directly targeted HOXD10, which triggered the down-stream p-PAK4/MMP-9 signaling pathway, subsequently contributing to the regulation of cell migration and invasion [31]. Our study found that HOXD10 methylation is associated with vessel cancerous embolus and HOXD10 suppresses HCC cell invasion and migration. These results suggest that HOXD10 is a tumor suppressor in human HCC. The role of HOXD10 in HCC was validated by xenograft mice model in vivo.
HOXD10 targeted the IGFBP3 gene promoter region and upregulated its expression in gastric cancer [22]. Therefore, we analyzed the expression of IGFBP3 in HCC cells by western blot. The expression of IGFBP3 was upregulated by HOXD10. IGFBP3 was previously reported to interact with and inactivate ERK1/2 by inhibiting ERK1/2 phosphorylation in human non-small cell lung cancer [24]. We analyzed the effects of HOXD10 on the ERK pathway in HCC. The levels of phosphorylated ERK1/2 were reduced by HOXD10. These results were validated using the siRNA knockdown technique.
HOXD10 is frequently methylated in human HCC, and the expression of HOXD10 is regulated by promoter region methylation. Methylation of HOXD10 was associated with vessel cancerous embolus, tumor cell differentiation, and the 3-year survival rate in human HCC. HOXD10 suppresses HCC cell growth both in vitro and in vivo. HOXD10 suppresses human HCC by inhibiting ERK signaling.
5-aza:
5-Aza-2′-deoxycytidine
bcl-2:
B cell lymphoma-2 B
BSSQ:
Bisulfite sequencing
CCND1 :
Cyclin D1
cdc-2:
Cyclin-dependent kinase 1
ChIP:
ERK:
Extracellular signal-regulated kinase
GADPH:
Glyceraldehyde-3-phosphate dehydrogenase
HBV:
Hepatitis B virus
HCC:
HOX :
Homeobox
IGFBP3:
Insulin-like growth factor-binding protein 3
IHC:
MMP2:
Matrix metalloprotein 2
Matrix metalloprotein9
MTT:
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
VEGFA :
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El-Serag HB. Hepatocellular carcinoma. N Engl J Med. 2011;365:1118–27.
Farazi PA, DePinho RA. Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer. 2006;6:674–87.
Villanueva A, Newell P, Chiang DY, Friedman SL, Llovet JM. Genomics and signaling pathways in hepatocellular carcinoma. Semin Liver Dis. 2007;27:55–76.
Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA Jr, Kinzle KW, et al. Cancer genome landscapes. Science. 2013;339:1546–58.
Guo M, Ren J, Brock MV, Herman JG, Carraway HE. Promoter methylation of HIN-1 in the progression to esophageal squamous cancer. Epigenetics. 2008;3:336–41.
Guo M, Ren J, House MG, Qi Y, Brock MV, Herman JG. Accumulation of promoter methylation suggests epigenetic progression in squamous cell carcinoma of the esophagus. Clin Cancer Res. 2006;12:4515–22.
Revill K, Wang T, Lachenmayer A, Kojima K, Harrington A, Li J, et al. Genome-wide methylation analysis and epigenetic unmasking identify tumor suppressor genes in hepatocellular carcinoma. Gastroenterology. 2013;145:1424–35. e1421-1425
Shah N, Sukumar S. The Hox genes and their roles in oncogenesis. Nat Rev Cancer. 2010;10:361–71.
Bhatlekar S, Fields JZ, Boman BM. HOX genes and their role in the development of human cancers. J Mol Med (Berl). 2014;92:811–23.
Cui Y, Gao D, Linghu E, Zhan Q, Chen R, Brock MV, et al. Epigenetic changes and functional study of HOXA11 in human gastric cancer. Epigenomics. 2015;7:201–13.
Hong CS, Jeong O, Piao Z, Guo C, Jung MR, Choi C, et al. HOXB5 induces invasion and migration through direct transcriptional up-regulation of beta-catenin in human gastric carcinoma. Biochem J. 2015;472:393–403.
Yang H, Zhou J, Mi J, Ma K, Fan Y, Ning J, et al. HOXD10 acts as a tumor-suppressive factor via inhibition of the RHOC/AKT/MAPK pathway in human cholangiocellular carcinoma. Oncol Rep. 2015;34:1681–91.
Wang L, Chen S, Xue M, Zhong J, Wang X, Gan L, et al. Homeobox D10 gene, a candidate tumor suppressor, is downregulated through promoter hypermethylation and associated with gastric carcinogenesis. Mol Med. 2012;18:389–400.
Hakami F, Darda L, Stafford P, Woll P, Lambert DW, Hunter KD. The roles of HOXD10 in the development and progression of head and neck squamous cell carcinoma (HNSCC). Br J Cancer. 2014;111:807–16.
Mo RJ, Lu JM, Wan YP, Hua W, Liang YX, Zhuo YJ, et al. Decreased HoxD10 expression promotes a proliferative and aggressive phenotype in prostate cancer. Curr Mol Med. 2017;17:70-8.
Li X, Yu J, Brock MV, Tao Q, Herman JG, Liang P. Epigenetic silencing of BCL6B inactivates p53 signaling and causes human hepatocellular carcinoma cell resist to 5-FU. Oncotarget. 2015;6:11547–60.
Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A. 1996;93:9821–6.
Jia Y, Yang Y, Liu S, Herman JG, Lu F, Guo M. SOX17 antagonizes WNT/beta-catenin signaling pathway in hepatocellular carcinoma. Epigenetics. 2010;5:743–9.
Yan W, Wu K, Herman JG, Brock MV, Fuks F, Yang L, et al. Epigenetic regulation of DACH1, a novel Wnt signaling component in colorectal cancer. Epigenetics. 2013;8:1373–83.
Xue M, Fang Y, Sun G, Zhuo W, Zhong J, Qian C, et al. IGFBP3, a transcriptional target of homeobox D10, is correlated with the prognosis of gastric cancer. PLoS One. 2013;8:e81423.
Shahjee HM, Bhattacharyya N. Activation of various downstream signaling molecules by IGFBP-3. J Cancer Ther. 2014;5:830–5.
Kim JH, Choi DS, Lee OH, Oh SH, Lippman SM, Lee HY. Antiangiogenic antitumor activities of IGFBP-3 are mediated by IGF-independent suppression of Erk1/2 activation and Egr-1-mediated transcriptional events. Blood. 2011;118:2622–31.
Finn RS. Development of molecularly targeted therapies in hepatocellular carcinoma: where do we go now? Clin Cancer Res. 2010;16:390–7.
Zhang X, Yang Y, Liu X, Herman JG, Brock MV, Licchesi JD, et al. Epigenetic regulation of the Wnt signaling inhibitor DACT2 in human hepatocellular carcinoma. Epigenetics. 2013;8:373–82.
Chen Y, Liu Q, Wu M, Li M, Ding H, Shan X, et al. GAB2 promotes cell proliferation by activating the ERK signaling pathway in hepatocellular carcinoma. Tumour Biol. 2016;37:11763-73.
Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet. 2012;379:1245–55.
Sharpe DJ, Orr KS, Moran M, White SJ, McQuaid S, Lappin TR, et al. POU2F1 activity regulates HOXD10 and HOXD11 promoting a proliferative and invasive phenotype in head and neck cancer. Oncotarget. 2014;5:8803–15.
Carrio M, Arderiu G, Myers C, Boudreau NJ. Homeobox D10 induces phenotypic reversion of breast tumor cells in a three-dimensional culture model. Cancer Res. 2005;65:7177–85.
Li Q, Ding C, Chen C, Zhang Z, Xiao H, Xie F, et al. MiR-224 promotion of cell migration and invasion by targeting Homeobox D 10 gene in human hepatocellular carcinoma. J Gastroenterol Hepatol. 2014;29:835–42.
We sincerely thank Xiaomo Su for managing the experimental materials.
This work was supported by the following grants: National Basic Research Program of China (973 Program No.2012CB934002), National Key research and development Programme 2016YFC1303600, National Key Scientific Instrument Special Programme of China (Grant No. 2011YQ03013405), National Science Foundation of China (NSFC Nos. 81672318, 81402345, U1604281, 8167100001), Beijing Science Foundation of China (BJSFC No. 17G10035), Beijing Natural Science Foundation (7171008), and The Science and Technology Commission of Beijing Municipality (Z151100004015209).
The datasets analyzed during the current study available from the corresponding author on reasonable request.
Department of Gastroenterology and Hepatology, Chinese PLA General Hospital, #28 Fuxing Road, Beijing, 100853, China
Yulin Guo, Yaojun Peng, Dan Gao, Meiying Zhang, Weili Yang, Enqiang Linghu & Mingzhou Guo
Department of General Surgery, Chinese PLA General Hospital, #28 Fuxing Road, Beijing, 100853, China
Yulin Guo & Guanglong Dong
Medical College of NanKai University, #94 Weijin Road, Tianjin, 300071, China
Dan Gao, Meiying Zhang & Weili Yang
The Hillman Cancer Center, University of Pittsburgh Cancer Institute, 5117 Centre Avenue, Suite 2.18/Research, Pittsburgh, PA, 15213, USA
James G. Herman
Laboratory of Cancer Epigenetics, Free University of Brussels (U.L.B.), 808 Route de Lennik, 1070, Brussels, Belgium
François Fuks
Yulin Guo
Yaojun Peng
Dan Gao
Meiying Zhang
Weili Yang
Enqiang Linghu
Guanglong Dong
Mingzhou Guo
YG and YP performed the research and analyzed the data. YG and MG wrote the manuscript. MG made substantial contributions to conception and design of the study. DG, MZ, and WY helped in collecting samples. EL and FF (François Fuks) provided manuscript and experimental advices. GD supervised the study. All authors read and approved the final manuscript.
Correspondence to Guanglong Dong or Mingzhou Guo.
This study was approved by the institutional review board of the Chinese PLA General Hospital.
Primer sequences. (DOCX 13 kb)
Guo, Y., Peng, Y., Gao, D. et al. Silencing HOXD10 by promoter region hypermethylation activates ERK signaling in hepatocellular carcinoma. Clin Epigenet 9, 116 (2017). https://doi.org/10.1186/s13148-017-0412-9
HOXD10
IGFBP3
ERK1/2
Cancer epigenetics and diagnostics


When choosing between two options, correlates of their value are represented in neural activity throughout the brain. Whether these representations reflect activity that is fundamental to the computational process of value comparison, as opposed to other computations covarying with value, is unknown. We investigated activity in a biophysically plausible network model that transforms inputs relating to value into categorical choices. A set of characteristic time-varying signals emerged that reflect value comparison. We tested these model predictions using magnetoencephalography data recorded from human subjects performing value-guided decisions. Parietal and prefrontal signals matched closely with model predictions. These results provide a mechanistic explanation of neural signals recorded during value-guided choice and a means of distinguishing computational roles of different cortical regions whose activity covaries with value. © 2012 Nature America, Inc. All rights reserved.


Why Should Several Big Schools Pay A Company $10K A Year To Monitor Their Athletes' Twitter Accounts?
At Varsity Monitor, we monitor the social media interaction of athletes for questionable conduct that could negatively affect their "Personal Brand". We monitor for actions that could endanger their future career and sponsorship opportunities as well as damage the brand of their team, league & institution.
Varsity Monitor doesn't contract with pro clubs, though. Or sports agencies. It deals with colleges—it lists North Carolina, Texas, Oklahoma, and Nebraska among its featured clients. According to the Times, schools pay Varsity Monitor $7,000 to $10,000 a year to watch their athletes. God forbid something endanger their Personal Brand (by which the school really means its own brand), you know?
With Varsity Monitor, it's not one size fits all…. Some of our clients use our service to monitor only publicly available data, others desire a more in depth approach.
Only Varsity Monitor offers the choice between monitoring all athlete social media accounts or only the accounts set to public. The choice is yours - and we never require passwords and fully adhere to every social networks TOS.
You see, schools? Varsity Monitor lets you choose whether they spy on your athletes. They won't spy on your athletes unless you let them, in which case they're totally down with it.
Apparently colleges don't think the player/school balance is tilted enough in their favor. So now some former motor-oil salesman (according to Varsity Monitor honcho Sam Carnahan's LinkedIn page, he used to work in marketing at Castrol BP) gets paid state funds to dispatch email alerts every time some 19-year-old mentions the word "crunk." The internet is a terrible place.


text:
Il Terremoto Tour è stata una tournée della rock band italiana Litfiba, svoltasi in Italia e nel resto d'Europa nel corso del 1993. Così come il disco di riferimento, il tour è ricordato comunemente come quello più pesante a livello musicale nella carriera deila band.

Dopo uno showcase di presentazione dell'album tenuto al Rolling Stone di Milano il 13 gennaio, la band ai primi di febbraio si spostò in Costa Azzurra per mettere a punto lo show. Per una settimana provarono all'interno de La Palestre, una struttura presso Le Cannet, e l'11 febbraio tennero la prova generale gratuita aperta al pubblico. Dopo quattro giorni la prima parte del tour partì ufficialmente dal palasport di Trieste, toccando diciotto città italiane, con date raddoppiate a Milano, Torino e Firenze.
La data di Firenze del 25 febbraio fu registrata dalla Rai e trasmessa in diretta radio e successivamente in TV su Raiuno nello speciale "Notte Rock RAI".

Il tour invernale riscosse un grosso successo, facendo registrare incassi per ben 8 miliardi di lire con una media di settemila spettatori per concerto. Un risultato inedito per una rock band italiana.

Nel mese di aprile il tour proseguì in Europa, con date in Francia, Belgio, Lussemburgo e Svizzera, per chiudersi a Roma, al concerto del Primo Maggio, durante il quale Pelù si scagliò in diretta televisiva contro Papa Giovanni Paolo II, accusandolo di interessarsi troppo ad argomenti relativi al sesso. Facendo riferimento alle condanne della chiesa su l'uso del profilattico come mezzo contraccettivo ma, che era soprattutto un metodo valido per combattere le malattie sessualmente trasmissibili, come l'AIDS. Nella stessa serata si fece notare per un gesto provocatorio durante l'intervista con Vincenzo Mollica. Nel tentativo di lanciare un messaggio di prevenzione per la lotta all'AIDS infilò un preservativo sul microfono del giornalista, che in diretta non reagì ma in realtà si arrabbiò moltissimo con il cantante, interpretando il gesto come una provocazione nei suoi confronti. L'uscita del cantante fiorentino provocò una grossa reazione pubblica sui giornali e aumentò l'attenzione e le critiche nei confronti della band e sull'imminente trance estiva del tour che sarebbe partita a luglio.

Già a fine giugno però la band partì nuovamente per l'estero per esibirsi in alcuni festival, tra cui quello di Roskilde, in Danimarca. Considerato tra i più rinomati e longevi tra i festival rock in Europa, evento unico e storico per un gruppo rock italiano.
In un cartellone che comprendeva tra gli altri Jamiroquai, Anthrax e New Order; i Litfiba si esibirono nel tardo pomeriggio del 3 luglio sul Green Stage, appena prima degli svedesi Electric Boys e dei Porno for Pyros.

I concerti italiani ripartirono il 22 luglio da Monza per proseguire tutta l'estate fino al concerto conclusivo del 12 settembre alla Festa dell'Unità di Reggio Emilia.

La trance estiva può essere segnalata come l'unica vera occasione in cui la band è riuscita a fare il grande salto per esibirsi in stadi di media dimensione, come le date tenute allo Stadio Brianteo di Monza, all'Olimpico di Roma e al Comunale di Torino. L'apertura della scaletta venne cambiata rispetto a quella della prima parte del tour, con l'esibizione di Sotto il vulcano, che vedeva la band impegnata a suonare il brano celata dietro un tendone con proiezioni di giochi di luce e ombre, poi come già fatto in precedenza, con l'inizio del secondo brano, Resta, il tendone cadeva svelando i musicisti sul palco. Un'altra novità del live fu l'inedito Africa, che venne proposto per la prima volta dal vivo al pubblico come netto messaggio antirazzista.

Il concerto del 5 settembre tenuto alla festa dell'Unità di Bologna, venne registrato e pubblicato l'anno successivo con il titolo Colpo di coda, in doppio CD/MC e triplo vinile. Un'edizione in cofanetto a tiratura limitata del disco comprendeva un booklet con 99 foto frutto di un reportage sulla tournée estiva ad opera di Alex Majoli. Colpo di Coda sarebbe dovuto uscire anche in versione video VHS, ma la registrazione rimase "ostaggio" di un contenzioso legale tra la CGD e i Litfiba, che nel frattempo avevano cambiato casa discografica passando alla EMI. Il live rimase inedito per più di vent'anni, fino al 2015, quando venne finalmente pubblicato in DVD nel cofanetto Tetralogia degli elementi.

Lo spettacolo
Con l'esplosione a livello internazionale del fenomeno grunge, e soprattutto con una palese ispirazione al sound dei Metallica, i Litfiba imboccarono intenzionalmente una strada più vicina all'hard rock. Rispetto alla tournée di El Diablo, caratterizzata da un sound latin rock, per il Terremoto Tour misero a punto uno spettacolo che abbinava sonorità di grande potenza. Non a caso si decise di fare a meno di Candelo Cabezas alle percussioni e della chitarra ritmica di Poggipollini (che lasciò subito dopo l'uscita del disco), puntando a uno stile più essenziale ma di grande impatto. Per la prima volta la band poteva vantare uno sponsor ufficiale della tournée: Elephant Beer, una birra legata alla casa di produzione danese Carlsberg. Certamente questo aiuto economico permise di poter approntare una proposta scenografica e di produzione audio/luci di livello superiore al passato.
Tutti i brani dell'album vennero riproposti nel concerto, con l'aggiunta di alcuni brani tratti dal loro repertorio, che meglio si adattavano al sound e agli arrangiamenti in stile hard rock. Ad esempio vennero recuperati da 17 Re, brani come Resta, Gira nel mio cerchio e Cane, che già originariamente avevano un sound molto rock. Naturalmente vennero anche proposti molti brani dal precedente disco El Diablo e altri successi come Tex e Cangaceiro.

La tournée fu caratterizzata da una generale atmosfera di grande tensione, dovuta sia alle sonorità molto dure dello spettacolo, che attirò una nuova frangia di fans metallari, sia alle tematiche trattate nelle canzoni. Si era nel pieno dello scandalo di Tangentopoli, e l'indignazione pubblica per la corruzione della politica italiana e per le stragi di mafia, era molto viva e sentita anche tra il pubblico giovanile. Ogni sera Piero Pelù si lanciava in invettive di denuncia e accuse contro i protagonisti della scena politica italiana. Ad esempio, nella registrazione radio del concerto di Firenze, nell'intro di "Dimmi il nome" si sente Pelù urlare ironicamente: "Hanno arrestato a Bettino!" (Craxi).

Scaletta
 Resta
 Amigo
 Soldi
 Maudit
 Firenze sogna
 Bambino
 Il mistero di Giulia
 Proibito
 Gira nel mio cerchio
 Fata Morgana
 Prima guardia
 Resisti
 Dinosauro
 Dimmi il nome
 Cane
 El diablo
 Gioconda
 Il vento
 Il volo
 Sotto il vulcano
 Ci sei solo tu
 Tex'n Duet
 Cangaceiro

Note
Durante il tour estivo la scaletta fu leggermente modificata, con Sotto il vulcano come brano di apertura, l'inserimenti di Siamo umani e Woda-Woda tratti da El Diablo e il debutto dal vivo dell'inedito Africa.

Band
Ghigo Renzulli: chitarra
Piero Pelù: voce
Antonio Aiazzi: tastiere
Roberto Terzani: basso
Franco Caforio: batteria

Date

Note

Bibliografia
 Cotto, M.; Pelù, P. (2000) Perfetto difettoso, Milano, Mondadori. ISBN 88-04-47871-3.
 Guglielmi F. (2000) A denti stretti. La vera storia dei Litfiba, Firenze, Giunti Editore. ISBN 88-09-01729-3.

Filmografia
 Terremoto Tour (2015), regia di Adam Dubin e Piero Pelù

Voci correlate
 Colpo di coda
 Tetralogia degli elementi

Tour dei Litfiba
Tour musicali del 1993

redpajama_set_name: RedPajamaWikipedia


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Back in 1993, Ice Cube rapped in "It Was A Good Day": "Even saw the lights of the Goodyear Blimp / And it read "Ice Cube's a Pimp." While the rapper has said that the events he wrote about in the iconic song were fiction, in 2014, it could become a reality.
As TIME previously reported,the Good Day Blimp project was trying to convince the Goodyear corporation to fly their blimp over South Central Los Angeles on January 20, 2014, the day stand-up-comedian Donovan Strain determined was the actual day in Ice Cube's song. The project is both an homage to the song as well as a fundraising drive for a nonprofit organization called A Place Called Home, which works to provide a safe, positive, family environment for at-risk youth. The Good Day Blimp project sought to raise money for the charity — their goal is $25,000 — on the condition that Goodyear flies the blimp.
Now it looks like the Goodyear folks are on board, too. On the project's website, they posted a letter sent by Goodyear to Ice Cube agreeing to "recognize the 'good day'" on their blimp and requested the rapper to "ride along" with the kids from the charity when the blimp goes up today.
"@GoodyearBlimp: .@LateNightJimmy we wanted to be the first to share this news with you. pic.twitter.com/KBhd8fqOk7" yahoo!
YOU KNOW I ACCEPT YOUR @RideAlong ON THE @GoodyearBlimp THANKS @jimmyfallon LETS RAISE SOME MONEY FOR @apch2830 GOODYEAR STEPPED UP!!!
While it's not clear whether Goodyear will actually use the words "Ice Cube's A Pimp" on their "iconic airship," as Ice Cube's song said, it's definitely going to be a good day for the children of South Central Los Angeles and A Place Called Home.


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Joseph W. "Joe" Shulter
San Antonio, TX Lawyer with 41 years of experience
(210) 344-4040 900 NE Loop 410
Free ConsultationBankruptcy
A San Antonio native, Joseph W. Shulter has been practicing law for over 34 years; he has been licensed since 1981. He has practiced consumer bankruptcy law for 27 years. Joseph W. Shulter has worked for several firms in San Antonio and has now decided to open his own practice. He is Board Certified, Consumer Bankruptcy -- Texas Board of Legal Specialization.
Joseph Joe Mugan
Las Vegas, NV Lawyer with 18 years of experience
(702) 405-8500 1835 Village Center Cir
UNLV William S. Boyd School of Law
Jo-Yee Joseph Lam
SF, CA Attorney with 20 years of experience
555 Battery St
SF, CA 94111
Claimed Lawyer ProfileOffers Video ConferencingQ&ALII PlatinumSocial Media
Kelley Anne Joseph
(888) 392-5784 8211 W. Broward Blvd.
Suite Ph 4
Free ConsultationOffers Video ConferencingVideo ConfPlantation, FL Attorney with 28 years of experience
Ms Joseph has over 25 years' experience litigating marital & family law cases throughout South Florida, around the state, and occasionally, around the country. Ms. Joseph is passionate about helping her clients navigate the legal process, yet mindful of the financial and emotional toll drawn out, and unnecessary litigation takes on the family. Ms. Joseph handles divorce, child custody, paternity, alimony & support, equitable distribution, injunctions, enforcement, modification; temporary relief, uncontested cases and more, every day. Ms Joseph works with respected experts, in complex & discreet settlement negotiations, prepares agreements, etc. Ms. Joseph has vast experience mediating and...
Casey W. Stevens
(770) 408-6364 2500 Northwinds Pkwy
Free ConsultationOffers Video ConferencingVideo ConfInsurance Claims, Personal Injury, Products Liability and Workers' Comp
Georgia State University College of Law
Casey W. Stevens worked in the insurance claims management business full time while attending Georgia State College of Law. Upon graduation in 1998, Attorney Stevens went to work for a large Atlanta insurance defense firm. In 2001, he started his own plaintiff's firm, representing the injured. Mr. Stevens has handled hundreds of injury cases, resulting in millions of dollars of settlements for his clients.
Casey Stevens handles ONLY personal injury cases that result from someone else's negligence. Our firm specializes in accident law, including automobiles, trucks, motorcycles, pedestrians, bicycles, ATV's, boats, small watercraft, and golf carts. ...
Gregory W. Stevens
Salt Lake City, UT Lawyer with 36 years of experience
(801) 990-3388 2825 E Cottonwood Pkwy
Free ConsultationBankruptcy, Criminal, Divorce and Family
"Success is not final. Failure is not fatal. It is the courage to continue that counts." Gregory W. Stevens, Esquire, is an attorney with a wealth of experience in private practice to help you get through life's challenges. He is dedicated to protecting your rights in the areas of bankruptcy law, criminal defense, and family law. He will provide you not only with his expertise, hard work, and tenacity, but also with integrity, empathy, and compassion. Through his law office in Salt Lake City, Mr. Stevens represents clients throughout the Wasatch Front and Back....
Michael H. Joseph
(914) 354-3809 203 E Post Rd
Free ConsultationOffers Video ConferencingVideo ConfWhite Plains, NY Lawyer with 23 years of experience
Business, Construction, Criminal and Personal Injury
Tulane University School of Law
Michael H. Joseph is an injury attorney serving New York City, NY, Manhattan, and White Plains. He is also a Partner at the Law Office of Michael H. Joseph, PLLC. He has devoted his career to representing people who were hurt in accidents at construction sites and other locations.
With more than a decade of experience representing clients, Mr. Joseph is an effective advocate in the courtroom, on appeal, at arbitrations, during mediations, and in administrative hearings. Since he understands how stressful and complicated life after an unexpected accident can be, Mr. Joseph vigorously advocates on behalf of injured individuals to...
Charles Joseph
(212) 651-4238 32 Broadway
Free ConsultationEmployment
Charles Joseph is the founding partner at Joseph & Kirschenbaum LLP. Charles founded Joseph & Kirschenbaum to help workers who had been wronged by unfair employment and wage practices. His firm is dedicated to helping employees win claims for discrimination, sexual harassment, wage theft, retaliation, whistleblowing, and other illegal employer activities. Charles has won numerous major legal victories for workers in all occupations and industries. His firm has recovered over $140 million for workers across the country.
Claimed Lawyer ProfileLII GoldBlawg SearchSocial Media
Brittany A. Stevens
New York, NY Attorney with 8 years of experience
Free ConsultationArbitration & Mediation, Civil Rights and Employment
University of San Francisco School of Law
Brittany A. Stevens has considerable experience representing employees in a wide range of employment discrimination matters. She manages a variety of cases involving sexual harassment, retaliation, and discrimination based on race, national origin, gender, pregnancy, age, disability, religion, caregiver status, and sexual orientation. She has handled numerous cases from inception through settlement.
Ms. Stevens zealously advocates for women's rights and is committed to helping women in the workplace. Ms. Stevens is a passionate litigator who believes strongly in civil rights and fairness throughout the work environment.
Ms. Stevens has obtained significant awards and settlements for her clients. Remarkably, Ms. Stevens received a...
Brandon Joseph
Dallas, TX Lawyer with 7 years of experience
(214) 692-8200 8115 Preston Rd
Brandon Joseph is an Attorney at McClure Law Group. As a persuasive advocate, Brandon's intense work ethic and passion for the law makes him a real asset to the McClure legal team and their clients.
Brandon has proven himself a force to be reckoned with in the courtroom. During his first month at McClure Law Group, Brandon successfully persuaded a court to reconsider and overturn its ruling which granted an emergency protective order. Since that time, Brandon has successfully prosecuted and defended against multiple family violence protective orders. Additionally, Brandon has received numerous victories in suits to modify prior court orders,...
Jason B. Stevens
(847) 902-5665 102 N. Evergreen Avenue
Free ConsultationArlington Heights, IL Lawyer with 19 years of experience
Criminal, DUI & DWI, Domestic Violence and Traffic Tickets
I have specialized in criminal, DUI & traffic defense for over 14 years. During that time period I have assisted clients extensively with felonies, misdemeanors, DUIs, simple traffic matters, CDL protection, driver's license reinstatement, and expungements.
I take pride in my Courtroom skills and long list of satisfied clients. My goal in each case I accept is to achieve the objective of my client. I return phone calls promptly and always give an honest straightforward assessment of each case no matter how big or small.
I offer a free no obligation consultation.
Marla A. Joseph
Jenkintown, PA Attorney
(215) 884-6664 261 Old York Rd
Free ConsultationCivil Rights, Employment, Personal Injury and Social Security Disability
Mr. Michael O. Stevens
Hillsboro, OR Attorney with 13 years of experience
(971) 533-6178 1915 NE Stucki Avenue
Free ConsultationConstruction, Employment, Insurance Claims and Personal Injury
Michael Stevens has a background in insurance defense representing individuals to large corporations in a wide variety of cases from personal injury to wrongful death and construction defects. He handled cases with values of as much as $4 million, and drafted many successful motions for summary judgment. An Oregon native, he graduated from Oregon State University with his bachelor's degree and earned his juris doctorate at Willamette University College of Law in Salem in 2009. At Willamette he was an editor and writer for Willamette Law Online and the Web & Publication Editor for the Willamette Sports Law Journal. He...
James Paul Joseph
Garden City, NY Lawyer with 29 years of experience
(516) 542-2000 666 Old Country Road
In 1998, I started my own family law practice. I have very high professional standards and only through my own firm could I ensure that my staff and my associates shared my values. By setting these standards and focusing exclusively in divorce and family law, Joseph Law Group, P.C. has gained a reputation as one of the preeminent family law firms on Long Island. We believe the divorce process must not just be about dissolving your marriage, but protecting and building your future. We carefully listen to your priorities and educate you on the best processes and...
Salem, NH Lawyer with 24 years of experience
(603) 893-0074 5 Manor Parkway
Appeals, Criminal and DUI & DWI
Attorney Mark Stevens is admitted to practice in all New Hampshire and Massachusetts courts. He is devoted entirely to criminal trials and appeals, with a concentration in defending clients accused of DWI, Aggravated DWI-DUI, OUI or any other type of criminal charges. Mark Stevens has 16 years experience in DWI trials and related administrative license suspension hearings, appeals, and post-conviction proceedings. His office is located in Salem, NH but can be reached 24/7 by phone @ 603.893.0074 The initial case evaluation and consultation are always free and can be done in person or by phone. Call now to schedule your...
Rachel R Stevens
Allentown, PA Lawyer with 14 years of experience
(484) 221-9267 PMB 218 915 Hamilton Street
Free ConsultationDUI & DWI, Personal Injury, Social Security Disability and Workers' Comp
Rachel Stevens graduated from Temple University's Beasley School of Law in Philadelphia where she was honored as a public interest fellow and also represented disabled workers in social security disability hearings for the school's legal aid office. Drawn to law by her passion for social justice, before coming to Temple Rachel was a community organizer (Student Area Coordinator) for two years for Amnesty International while an undergraduate at the University of Maryland and continues to be involved with Amnesty today. After graduating from Temple in 2008, she spent a year in California where she passed...
David Arthur Stevens
(404) 467-9017 513 Edgewood Avenue
Arbitration & Mediation, Civil Rights, Criminal and Social Security Disability
David A. Stevens is an attorney at the Atlanta veterans disability legal office, McElreath & Stevens, LLC. He has been practicing law since 2002, and is a member of the Atlanta Bar Association, the Dekalb Bar Association, the Georgia Trial Lawyers Association, and the National Organization of Social Security Claimants' Representatives. At the firm, Mr. Stevens is committed to helping veterans obtain the benefits they deserve. To find out about retaining the representation of Attorney Stevens, contact the firm today!
Dover, NH Lawyer
Christopher Joseph
Kansas City, MO Lawyer with 22 years of experience
(816) 673-3900 926 Cherry St.
Free ConsultationAppeals, Criminal and White Collar Crime
Mr. Joseph represents individuals and businesses during the investigation and prosecution of criminal charges in federal and state courts. He also represents businesses and individuals in complex civil litigation, primarily in federal court. He leads the firm's criminal and civil asset forfeiture practice groups.
Mr. Joseph received his juris doctorate in 2000 from the University of Kansas School of Law. He was invited to membership in the Order of the Coif, a national honor society for law school graduates with exceptional academic achievement. Mr. Joseph graduated summa cum laude from Wichita State University with a Bachelor of Arts degree in...
HILTON HEAD ISLAND, SC Attorney with 27 years of experience
(843) 842-3500 HILTON HEAD ISLAND, SC 29938
With over 30 years' experience practicing in the family law courts, Bob Stevens' practice focuses exclusively on matrimonial and family law. He represents individuals and families in matters of uncontested separation and divorce as well as asset division and distribution, often involving complex financial issues in cases of family-owned businesses or professional practices, such as dental, medical, and legal practices. Bob also handles child custody and visitation disputes as well as the determination and modification of alimony and child support. In addition to representing individuals in litigation, Bob assists clients pursuing alternative dispute resolution methods such as mediation and arbitration....
Andrew H Stevens
Columbus, OH Lawyer with 10 years of experience
(614) 826-3100 1689 E Main Street, Suite 201
Free ConsultationBusiness, Communications, IP and Securities
Andrew H. Stevens is a technology and corporate attorney licensed in Ohio and Washington. Drew has worked with companies of all sizes, including startups, family-owned businesses, middle market companies, Fortune 500s, and multinational corporations. Although Drew works with a number of software, SaaS, and technology companies, he also works with construction, telecommunications, manufacturing, finance, sports and entertainment, and real estate development businesses.
Drew offers business counsel in a variety of practice areas, including intellectual property, mergers and acquisitions, securities, venture capital, and cybersecurity and data privacy. Drew works closely with growing companies, to help protect intellectual property assets, structure business ventures...
Marc A Joseph
1207 N Franklin St #214,
Free ConsultationCriminal
Marc A. Joseph, P.A. has defended thousands of clients and prosecuted none. Unlike former prosecutors, I have never worked one day to put someone in jail or prison. I will provide the level of uncompromising legal representation that makes a difference in my ability to help my clients avoid criminal convictions and maximum penalties for the offenses they have been accused of committing. Marc A. Joseph is passionate about protecting the rights of the accused in Florida criminal cases! Many defendants are wrongly accused, others know they have made mistakes, but look for an attorney who will...
Chris Stevens
Winston-Salem, NC Attorney with 6 years of experience
(336) 373-1130 514 S Stratford Road
With his history of activism and public service, it's not surprising that Chris Stevens was drawn to a law career where he could help people. He is passionate about helping those who are in need, especially military veterans who are fighting the Department of Veterans Affairs to get the benefits to which they are entitled.
Chris earned his juris doctor from the UNC School of Law. He graduated from Appalachian State University with a bachelor of science in business administration, double majoring in economics and marketing. While in law school, Chris won the Eugene Gressman & Daniel H. Pollitt Appellate...
Sherley Joseph-Roper
Snellville, GA Attorney with 13 years of experience
(770) 874-0174 2330 Scenic Highway S
Offers Video ConferencingVideo ConfElder, Estate Planning and Real Estate
Sherley Joseph-Roper obtained her law degree from St. John's University School of law in 2003. She earned a B.A. in Sociology from Barnard College at Columbia University with a degree in Sociology, and a Master of Social Work from Clark Atlanta University. Very early in her career, she developed a passion for improving the lives of families and children, strengthening communities, and promoting public safety. Her unique background is informed by a rich professional career involving family counseling, social services and government benefits, criminal prosecution, regulatory compliance in financial and real estate matters, commercial liability and personal injury litigation. She...
Jeriamiah L. Joseph
Kalamazoo, MI Attorney with 17 years of experience
(888) 635-3824 491 West South Street
Free ConsultationMedical Malpractice
Jeriamiah L. Joseph is an Associate at Willis Law. He is admitted to practice law in Indiana and Michigan and is a member of both the Indiana and Michigan State Bar Associations. Jeriamiah earned his Juris Doctorate from the University of Toledo College of Law, graduating magna cum laude. He also graduated from Tiffin University with a B.A. in Criminal Justice, graduating magna cum laude. Prior to joining Willis Law, Jeriamiah practiced insurance defense work and family law in Indiana for over six years. Jeriamiah and his wife, Betsy, have three children. When out of the office, Jeriamiah enjoys...

redpajama_set_name: RedPajamaCommonCrawl


Yamaha has prepared an array of top-quality woodwind mouthpieces to meet the musical requirements of players at every level of proficiency in every genre. Yamaha mouthpieces are designed in consideration of an appropriate throat size, chamber size and shape, baffle shape and angle, bore size, window size, and facing curve depth. An inspection after every step of the manufacturing process ensures that the mouthpiece can be relied upon with confidence.


text:
Four Scottish Estates Honoured at Special WES Reception
Bell Ingram sponsors WES Awards reception at Game Fair
Kilmun Farmhouse has stunning views over Loch Awe
Delightful Sutherland farm within easy reach of the NC500
Forestry Manager and Assistant Forester based in Perth and Inverness
Visit Bell Ingram's stand at the Royal Highland Show
Farm Machinery Dispersal Sale – 29th June 2019
Mull over island life in one of the 'UK's best places to live'
Buyers can bag a beautiful B&B in the shadow of Ben Nevis
Top property qualification for Oban letting agent Jackie
Major Appointments at Bell Ingram Boost Business
ONE of Scotland's leading independent land agents has advanced its operations with the appointment of three experts, including one of the sector's most experienced professionals.
Rob Whitson is to join Bell Ingram as Partner and will take responsibility for running the company's offices in Inverness and Bonar Bridge.
Rob, who was formally with Strutt & Parker in Inverness where he headed their Rural Department, will lead on Bell Ingram's estate management and professional work in the north of Scotland.
Graham Lumby, Managing Partner at Bell Ingram, said: "Land reform legislation, Brexit, and many other issues affecting the rural economy are continuing to challenge anyone with an interest or involvement in land or estate management. Having experienced professionals who can provide expert advice and be readily accessible in the locality is essential."
The appointment of Rob Whitson means that our clients have a senior and highly skilled professional to support them during these challenging times."
Since moving to Inverness, the former army officer has had extensive experience in Highland estate management, including deer and fisheries management, and brings with him expertise in valuations, renewables and forestry.
Rob said: "My background in surveying and the military has equipped me with a whole host of transferrable skills that I'm looking forward to applying to my work at Bell Ingram. I hope I can give clients the support required to optimise their rural businesses in the north of Scotland.
"Bell Ingram is a highly trusted and respected firm of land and property agents. It gives a national service which is delivered locally, so they're very aware of the localised issues in their area. "Our industry faces a varied and broad spectrum of political and economic issues such as land reform and Brexit. The role of the land agent is also changing as people's expectations change, so we have to adapt and continue to provide a first-class service to clients."
Bell Ingram is also advancing its operations in different parts of the country with two further appointments.
Andrew Fuller joins the Oban office as an Associate Estate Agent following almost 15 years managing high-level private and corporate development projects in the United Arab Emirates. With substantial experience in property management and investment, Andrew is now focused on making the Oban office the premium rural Estate Agency in the Western Isles and wider mainland region.
Andrew said: "The range of expertise that Bell Ingram provides and the variety of sectors that it covers is remarkable so I'm looking forward to applying myself to a company that encompasses a great many of my key interests. Working in Oban is never dull. The west coast is a buoyant market, and despite Brexit approaching, the mind patterns of buyers have not changed. People aren't holding back when it comes to island properties, especially with the increasingly popular buy-to-let property market and the rise of services such as Airbnb. My commute from the Isle of Mull to Oban alone makes it all worth it. With stunning scenery and ever-changing conditions – there really is no place like it."
Thomas Rust also joins the expanding company as an Assistant Land Agent after graduating with a degree in Rural Business Management from Scotland's Rural College (SRUC) earlier this year. His interest in land management was realised during his time working in the forestry sector in the North East of Scotland. Thomas said: "What draws me to rural land management is the opportunity to work outside and the wide scope of things you get to deal with on a day-to-day basis. From agriculture and forestry to renewables and pipeline management, the variety of work is exciting and it is fulfilling to be in a position of being able to provide valuable professional advice to land owners. I'm excited to put my head down, learn from the experienced colleagues around me and develop my skills."
Established 117 years ago, Bell Ingram has 130 professional staff across 11 UK offices including: farm, estate and forestry managers; chartered surveyors, estate agents, architects, planners, and building surveyors; and tourism, GIS mapping, and renewable energy specialists.
Article also featured in the following publications:
Insider.co.uk
Scottish Business News Network
Published on 14th August 2018


text:
I'm Not Dead Tour è il terzo tour della cantante statunitense Pink, a supporto dell'album I'm Not Dead (2006).

È iniziato a San Francisco il 27 giugno 2006 e si è concluso, dopo 138 date in svariate parti del mondo, a Città del Capo l'11 settembre 2007. La tournée permise a Pink di esibirsi per la prima volta negli Emirati Arabi e in Sudafrica.

Storia
Il tour partì da San Francisco, il 27 giugno 2006, visitando inizialmente teatri e club. Venne descritto da Pink come "intimo". Successivamente, il tour approdò in Europa, dove venne rivisitato e adattato per gli show che si sarebbero svolti nelle arene e negli stadi.

Da gennaio del 2007 fino a marzo dello stesso anno, l'artista portò la tournée nuovamente in Nord America, come artista d'apertura del Future Sex/LoveShow
di Justin Timberlake.

Ad aprile del 2007, la tournée riprese visitando l'Oceania, con un totale di 36 spettacoli e incassando ben 42 milioni di dollari, per poi tornare in Europa e svolgersi in aree all'aperto e come parte di alcuni festival musicali.

Le ultime date si svolsero a Dubai e in Sudafrica, dove il tour terminò l'11 settembre 2007, a Città del Capo. Insieme al successivo Funhouse Tour (2009), lI'm Not Dead Tour è il tour di maggior successo di Pink.

Lo show

Il palco

Durante il tour, vennero usati due differenti palchi. Il primo, usato in Nord America, nella seconda leg europea, in Africa e in Asia, era composto semplicemente da un main stage, dove stavano la cantante con la band, e un manifesto con la scritta "PINK". Durante lo spettacolo, non c'erano ne ballerini ne cambi d'abito. Il secondo palco invece, usato in Oceania e in molte date della prima leg europea, era più grande e simile a quello di un concerto pop, con un main stage, un grande passerella e tre grandi schermi. Durante lo show, diviso in quattro atti, erano presenti alcuni ballerini e la cantante effettuava vari cambi d'abito.

Sinossi dello show
La maggior parte degli show che si svolsero nelle arene avevano inizio con una grande tenda che cadeva a terra, rivelando l'intero palco. Successivamente le coriste iniziavano a intonare una intro simile ad una marcia militare. Pink entrava in scena con indosso un abito nero, un cappello da ammiraglio e degli occhiali da sole, per eseguire 'Cuz I Can. In seguito la cantante scendeva più in basso,  avvicinandosi al pubblico, ed eseguiva Trouble. Seguivano poi i brani Just like a Pill, Who Knew, la title track I'm Not Dead e la sua celebre cover di Lady Marmalade (che le fece guadagnare un Grammy Award nel 2001) con i quali si spogliava di parte del suo primo costume.

Il secondo atto si apriva con il numero più provocatorio dello show, ossia Stupid Girls. Durante quest'ultima infatti, l'artista era vestita come nel video musicale e presentava vari stereotipi di "ragazze stupide", interpretate da delle ballerine. Tra queste c'era anche una ragazza con un finto seno eccessivamente grande, che la cantante faceva scoppiare come dei palloncini. Una volta eseguito il brano, l'atmosfera cambiava immediatamente con la performance di There You Go, messa in scena con uno stile spanish e degli abiti maschili. Seguiva una breve coreografia fatta con delle sedie, durante la quale Pink cantava il ritornello di God Is a DJ e spariva poi dietro le quinte.
Il brano successivo era Fingers, che l'artista cantava da dentro una grande rete azzurra sospespesa in aria, con soltanto della biancheria intima leopardata addosso. Un cambio d'abito e la cantante faceva il suo ritorno in scena, con l'esecuzione di Family Portrait, che resta uno dei suoi brani più famosi.

A questo punto aveva inizio la parte acustica. Durante questo atto, Pink indossava un lungo abito bianco e una bandana avvolta in testa. I primi brani ad essere eseguiti erano una cover di Redemption Song e il singolo The One That Got Away, seguiti da Dear Mr. President. Con questa canzone l'artista si rivolgeva direttamente all'allora presidente degli Stati Uniti, George W. Bush, criticando il suo governo e accusandolo di non essere attento al fatto che molte persone si trovino in condizioni di vita disagiate. Questa sezione veniva chiusa con una cover del celebre brano What's Up dei 4 Non Blondes, brano che Pink riproporrà anche negli anni successivi.

L'ultima parte dello show iniziava con la controversa U + Ur Hand, eseguita con una giacca nera e una grande motocicletta presente sul palcoscenico. Dopo essersi tolta la giacca, la cantante rivelava una maglietta e un gonnellino grigio, per eseguire 18 Wheeler e Don't Let Me Get Me. Il brano seguente era Leave Me Alone (I'm Lonely), con il quale Pink ringraziava il pubblico e i ballerini facevano un inchino conclusivo. Poco dopo la cantante tornava sul palco con indosso un body nero e dei pantaloni lunghi, per eseguire Nobody Knows. L'ultima canzone dello spettacolo era la celebre Get the Party Started, che per l'occasione venne mixata con Sweet Dreams (Are Made of This) degli Eurythmics. Durante il brano Pink, con solo il body indosso, volteggiava in aria con le ballerine tramite dei tessuti aerei, per poi scatenare una pioggia di coriandoli e sparire dietro le quinte.

Scaletta

Prima leg europea, Oceania, Asia, Africa
 'Cuz I Can
 Trouble
 Just like a Pill
 Who Knew
 I'm Not Dead
 Lady Marmalade
 Stupid Girls (contiene elementi di Most Girls)
 There You Go
 God Is a DJ (dance break; eseguito solo il ritornello)
 Fingers
 Family Portrait
 Redemption Song
 The One that Got Away
 Dear Mr. President
 What's Up
 U + Ur Hand
 18 Wheeler
 Don't Let Me Get Me
 Leave Me Alone (I'm Lonely)
 Nobody Knows
 Get the Party Started (contiene elementi di Sweet Dreams (Are Made of This) degli Eurythmics)

Nord America
'Cuz I Can
Trouble
Just like a Pill
Stupid Girls
Who Knew
Lady Marmalade
Family Portrait
18 Wheeler
Lonely Girl
I'm Not Dead
Don't Let Me Get Me
There You Go
What's Up
Leave Me Alone (I'm Lonely)
Mercedes Benz
The One that Got Away
Dear Mr.President
Get the Party Started

Seconda leg europea
Cuz I Can
Trouble
Just like a Pill
Who Knew
Stupid Girls
I'm Not Dead
Family Portrait
My Vietnam
Last to Know
Try Too Hard
Me and Bobby McGee / Mercedes Benz / Piece of My Heart
Lonely Girl
Don't Let Me Get Me
18 Wheeler
Dear Mr. President
What's Up
Leave Me Alone (I'm Lonely)
U + Ur Hand
Get the Party Started

Registrazioni

La data di Londra, il 4 dicembre 2006, venne registrata e distribuita in formato DVD, con il nome di Live from Wembley Arena, London, England. La prima pubblicazione avvenne il 22 marzo 2007.

Nel DVD viene mostrato l'intero show (ad eccezione delle performance di Lady Marmalade e Redemption Song) più una galleria fotografica e alcuni momenti della vita di Pink durante il tour.

Date

Tour di Pink
Tour musicali del 2006
Tour musicali del 2007


text:
Q: Using PL/SQL how do you I get a file's contents in to a blob? I have a file. I want to get its contents into a blob column in my oracle database or into a blob variable in my PL/SQL program. What is the best way to do that?

A: To do it entirely in PL/SQL, the file would need to be on the server, located in a directory which you'd need to define in the database. Create the following objects:
CREATE OR REPLACE DIRECTORY
    BLOB_DIR
    AS
    '/oracle/base/lobs'
/

CREATE OR REPLACE PROCEDURE BLOB_LOAD
AS

    lBlob  BLOB;
    lFile  BFILE := BFILENAME('BLOB_DIR', 'filename');

BEGIN

    INSERT INTO table (id, your_blob)
        VALUES (xxx, empty_blob())
        RETURNING your_blob INTO lBlob;

    DBMS_LOB.OPEN(lFile, DBMS_LOB.LOB_READONLY);

    DBMS_LOB.OPEN(lBlob, DBMS_LOB.LOB_READWRITE);

    DBMS_LOB.LOADFROMFILE(DEST_LOB => lBlob,
                          SRC_LOB  => lFile,
                          AMOUNT   => DBMS_LOB.GETLENGTH(lFile));

    DBMS_LOB.CLOSE(lFile);
    DBMS_LOB.CLOSE(lBlob);

    COMMIT;

END;
/

A: Depends a bit on your environment.  In Java you could do it something like this...
// Need as OracleConnection in mConnection

// Set an EMPTY_BLOB()
String update = "UPDATE tablename"+
                " SET   blob_column = EMPTY_BLOB()"+
                " WHERE  ID = "+id;
CallableStatement stmt = mConnection.prepareCall(update);
stmt.executeUpdate();

// Lock the row FOR UPDATE
String select    = "BEGIN " +
                        "  SELECT " + blob_column
                        "  INTO ? " +
                        "  FROM " + tablename +
                        "  WHERE  ID = '" + id + "'" +
                        "  FOR UPDATE; " +
                        "END;";

stmt = mConnection.prepareCall(select);
stmt.registerOutParameter(1, java.sql.Types.BLOB);
stmt.executeUpdate();

BLOB blob = (BLOB) stmt.getBlob(1);
OutputStream bos = blob.setBinaryStream(0L);
FileInputStream fis = new FileInputStream(file);
//  Code needed here to copy one stream to the other
fis.close();
bos.close();
stmt.close();

mConnection.commit();

But it really depends what environment / tools you're using.  More info needed.

redpajama_set_name: RedPajamaStackExchange


I didn't want to post this because it doesn't look very good but I'm trying to be open and honest about this ankle recovery. I was just ok'd to walk without the air cast yesterday. I was SO happy to finally be able to walk, with a brace, and resume somewhat normal activities. . However, today was very frustrating for the simple fact that my body was just not moving the way it used to. I know I was just cleared to walk and I know I had a significant injury but I didn't honestly think my body just wouldn't move into what was a natural position/reaction. . I know it will get better, but for now it's very frustrating. In other news, my scars are looking really good and I'm finally able to drive again.
Cutest. Acroyoga. Ever 😍😍😅 Prepare your body so you can play and express yourself pain/inhibition free 👉To learn the foundational poses of yoga, along with their modifications and progressions, check out the ebook (link in bio) 🙏🏻❤ don't forget to share this with someone who would enjoy it! @acromillissa ・・・ BEST FLYER EVER!! 🤘🤘🤘🤘🤘🤘🤘 Having a blast playing 💪 . . . .
🍗 But how do I get more protein? @bodysmartfitness . 🆙 Yes, protein powder is always an easy go-to for upping that protein macro in your daily diet but how else can you increase it without going over on your fat and carb macros? . ⬆ Here are a few examples from our British fridges and store cupboards that help you hit your calorie and macro targets. From snacks to lean meats, fish and dairy products each of these foods can be incorporated easily into your diet. 👌👏💛 . . . .


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