Microarray Analysis

Anti-angiogenesis effect of 3′-sulfoquinovosyl-1′-monoacylglycerol (SQMG) via upregulation of thrombospondin 1.

Cancer Sci. 2012 May 16;

Authors: Matsuki K, Tanabe A, Hongo A, Sugawara F, Sakaguchi K, Takahashi N, Sato N, Sahara H

Abstract

We previously reported that 3′-sulfoquinovosyl-1′-monoacylglycerol (SQMG) effectively suppresses the growth of solid tumors, likely via its anti-angiogenic activity. To investigate how SQMG affects angiogenesis, we performed DNA microarray analysis and quantitative real-time PCR. Consequently, upregulation of thrombospondin 1 (TSP-1) in SQMG-treated tumors in vitro and in vivo was confirmed. To address the mechanisms of TSP-1 upregulation by SQMG, we established stable TSP-1-knockdown transformants (TSP1-KT) by short hairpin RNA induction and performed reporter assay and in vivo assessment of anti-tumor assay. On the reporter assay, transcriptional upregulation of TSP-1 in TSP1-KT could not be induced by SQMG, thus suggesting that TSP-1 upregulation by SQMG occurred via TSP-1 molecule. In addition, growth of TSP1-KT xenografted tumors in vivo was not inhibited by SQMG, thus suggesting that anti-angiogenesis via TSP-1 upregulation induced by SQMG did not occur, as the SQMG target molecule TSP-1 was knocked down in TSP1-KT transformants. These data provide that SQMG is a promising candidate for the treatment of tumor-induced angiogenesis via TSP-1 upregulation.

PMID: 22587436 [PubMed - as supplied by publisher]

Anti-angiogenesis effect of 3′-sulfoquinovosyl-1′-monoacylglycerol (SQMG) via upregulation of thrombospondin 1.

Cancer Sci. 2012 May 16;

Authors: Matsuki K, Tanabe A, Hongo A, Sugawara F, Sakaguchi K, Takahashi N, Sato N, Sahara H

Abstract

We previously reported that 3′-sulfoquinovosyl-1′-monoacylglycerol (SQMG) effectively suppresses the growth of solid tumors, likely via its anti-angiogenic activity. To investigate how SQMG affects angiogenesis, we performed DNA microarray analysis and quantitative real-time PCR. Consequently, upregulation of thrombospondin 1 (TSP-1) in SQMG-treated tumors in vitro and in vivo was confirmed. To address the mechanisms of TSP-1 upregulation by SQMG, we established stable TSP-1-knockdown transformants (TSP1-KT) by short hairpin RNA induction and performed reporter assay and in vivo assessment of anti-tumor assay. On the reporter assay, transcriptional upregulation of TSP-1 in TSP1-KT could not be induced by SQMG, thus suggesting that TSP-1 upregulation by SQMG occurred via TSP-1 molecule. In addition, growth of TSP1-KT xenografted tumors in vivo was not inhibited by SQMG, thus suggesting that anti-angiogenesis via TSP-1 upregulation induced by SQMG did not occur, as the SQMG target molecule TSP-1 was knocked down in TSP1-KT transformants. These data provide that SQMG is a promising candidate for the treatment of tumor-induced angiogenesis via TSP-1 upregulation.

PMID: 22587436 [PubMed - as supplied by publisher]

Early Detection of Lung Cancer by Molecular Markers in Endobronchial Epithelial-Lining Fluid.

J Thorac Oncol. 2012 Jun;7(6):1001-1008

Authors: Kahn N, Meister M, Eberhardt R, Muley T, Schnabel PA, Bender C, Johannes M, Keitel D, Sültmann H, Herth FJ, Kuner R

Abstract

INTRODUCTION:: Early detection of malignancies in the lung by less-invasive methods aims at achieving efficient intervention and subsequently a reduction of the high mortality rate. We investigated whether biomarker analysis in endobronchial epithelial-lining fluid (ELF) collected by bronchoscopic microsampling (BMS) may be useful for a definitive preoperative diagnosis. METHODS:: ELF was collected from subsegmental bronchi close to the indeterminate pulmonary nodule, which was detected by computed tomography, and from the contralateral lung. Diagnosis was confirmed by transbronchial biopsy or surgery. The study includes 142 ELF samples from 51 non-small-cell lung cancer patients and 20 benign cases. Microarray analysis was done with a patient subset (n = 15) to narrow down genes associated with a malignant phenotype. Thirteen potential biomarkers have been further analyzed by quantitative real-time polymerases chain reaction in an independent patient cohort (n = 56). RESULTS:: All patients underwent BMS without complications. Gene-expression analyses by microarrays and quantitative real-time polymerases chain reaction could be reliably applied to ELF samples, and resulted in potential biomarkers for malignant pulmonary nodules. Four genes (tenascin-C, [C-X-C motif] ligand 14, S100 calcium binding protein A9, and keratin 17) were found to be upregulated in ELF of non-small-cell lung cancer patients with adenocarcinoma or squamous cell carcinoma. Combined analysis of tenascin-C expression and the nodule size improved the prediction of malignancy in this patient cohort. CONCLUSIONS:: Our study suggests that the analysis of specific biomarkers in ELF collected by BMS could be a potentially useful adjunct to other diagnostic techniques aiming at the preoperative diagnosis of malignant pulmonary nodules.

PMID: 22588153 [PubMed - as supplied by publisher]

Early Detection of Lung Cancer by Molecular Markers in Endobronchial Epithelial-Lining Fluid.

J Thorac Oncol. 2012 Jun;7(6):1001-1008

Authors: Kahn N, Meister M, Eberhardt R, Muley T, Schnabel PA, Bender C, Johannes M, Keitel D, Sültmann H, Herth FJ, Kuner R

Abstract

INTRODUCTION:: Early detection of malignancies in the lung by less-invasive methods aims at achieving efficient intervention and subsequently a reduction of the high mortality rate. We investigated whether biomarker analysis in endobronchial epithelial-lining fluid (ELF) collected by bronchoscopic microsampling (BMS) may be useful for a definitive preoperative diagnosis. METHODS:: ELF was collected from subsegmental bronchi close to the indeterminate pulmonary nodule, which was detected by computed tomography, and from the contralateral lung. Diagnosis was confirmed by transbronchial biopsy or surgery. The study includes 142 ELF samples from 51 non-small-cell lung cancer patients and 20 benign cases. Microarray analysis was done with a patient subset (n = 15) to narrow down genes associated with a malignant phenotype. Thirteen potential biomarkers have been further analyzed by quantitative real-time polymerases chain reaction in an independent patient cohort (n = 56). RESULTS:: All patients underwent BMS without complications. Gene-expression analyses by microarrays and quantitative real-time polymerases chain reaction could be reliably applied to ELF samples, and resulted in potential biomarkers for malignant pulmonary nodules. Four genes (tenascin-C, [C-X-C motif] ligand 14, S100 calcium binding protein A9, and keratin 17) were found to be upregulated in ELF of non-small-cell lung cancer patients with adenocarcinoma or squamous cell carcinoma. Combined analysis of tenascin-C expression and the nodule size improved the prediction of malignancy in this patient cohort. CONCLUSIONS:: Our study suggests that the analysis of specific biomarkers in ELF collected by BMS could be a potentially useful adjunct to other diagnostic techniques aiming at the preoperative diagnosis of malignant pulmonary nodules.

PMID: 22588153 [PubMed - as supplied by publisher]

Global methylation profiling for risk prediction of prostate cancer.

Clin Cancer Res. 2012 May 15;18(10):2882-95

Authors: Mahapatra S, Klee EW, Young CY, Sun Z, Jimenez RE, Klee GG, Tindall DJ, Donkena KV

Abstract

PURPOSE: The aim of this study was to investigate the promoter hypermethylation as diagnostic markers to detect malignant prostate cells and as prognostic markers to predict the clinical recurrence of prostate cancer.

EXPERIMENTAL DESIGN: DNA was isolated from prostate cancer and normal adjacent tissues. After bisulfite conversion, methylation of 14,495 genes was evaluated using the Methylation27 microarrays in 238 prostate tissues. We analyzed methylation profiles in four different groups: (i) tumor (n = 198) versus matched normal tissues (n = 40), (ii) recurrence (n = 123) versus nonrecurrence (n = 75), (iii) clinical recurrence (n = 80) versus biochemical recurrence (n = 43), and (iv) systemic recurrence (n = 36) versus local recurrence (n = 44). Group 1, 2, 3, and 4 genes signifying biomarkers for diagnosis, prediction of recurrence, clinical recurrence, and systemic progression were determined. Univariate and multivariate analyses were conducted to predict risk of recurrence. We validated the methylation of genes in 20 independent tissues representing each group by pyrosequencing.

RESULTS: Microarray analysis revealed significant methylation of genes in four different groups of prostate cancer tissues. The sensitivity and specificity of methylation for 25 genes from 1, 2, and 4 groups and 7 from group 3 were shown. Validation of genes by pyrosequencing from group 1 (GSTP1, HIF3A, HAAO, and RARβ), group 2 (CRIP1, FLNC, RASGRF2, RUNX3, and HS3ST2), group 3 (PHLDA3, RASGRF2, and TNFRSF10D), and group 4 (BCL11B, POU3F3, and RASGRF2) confirmed the microarray results.

CONCLUSIONS: Our study provides a global assessment of DNA methylation in prostate cancer and identifies the significance of genes as diagnostic and progression biomarkers of prostate cancer. Clin Cancer Res; 18(10); 2882-95. ©2012 AACR.

PMID: 22589488 [PubMed - in process]

Global methylation profiling for risk prediction of prostate cancer.

Clin Cancer Res. 2012 May 15;18(10):2882-95

Authors: Mahapatra S, Klee EW, Young CY, Sun Z, Jimenez RE, Klee GG, Tindall DJ, Donkena KV

Abstract

PURPOSE: The aim of this study was to investigate the promoter hypermethylation as diagnostic markers to detect malignant prostate cells and as prognostic markers to predict the clinical recurrence of prostate cancer.

EXPERIMENTAL DESIGN: DNA was isolated from prostate cancer and normal adjacent tissues. After bisulfite conversion, methylation of 14,495 genes was evaluated using the Methylation27 microarrays in 238 prostate tissues. We analyzed methylation profiles in four different groups: (i) tumor (n = 198) versus matched normal tissues (n = 40), (ii) recurrence (n = 123) versus nonrecurrence (n = 75), (iii) clinical recurrence (n = 80) versus biochemical recurrence (n = 43), and (iv) systemic recurrence (n = 36) versus local recurrence (n = 44). Group 1, 2, 3, and 4 genes signifying biomarkers for diagnosis, prediction of recurrence, clinical recurrence, and systemic progression were determined. Univariate and multivariate analyses were conducted to predict risk of recurrence. We validated the methylation of genes in 20 independent tissues representing each group by pyrosequencing.

RESULTS: Microarray analysis revealed significant methylation of genes in four different groups of prostate cancer tissues. The sensitivity and specificity of methylation for 25 genes from 1, 2, and 4 groups and 7 from group 3 were shown. Validation of genes by pyrosequencing from group 1 (GSTP1, HIF3A, HAAO, and RARβ), group 2 (CRIP1, FLNC, RASGRF2, RUNX3, and HS3ST2), group 3 (PHLDA3, RASGRF2, and TNFRSF10D), and group 4 (BCL11B, POU3F3, and RASGRF2) confirmed the microarray results.

CONCLUSIONS: Our study provides a global assessment of DNA methylation in prostate cancer and identifies the significance of genes as diagnostic and progression biomarkers of prostate cancer. Clin Cancer Res; 18(10); 2882-95. ©2012 AACR.

PMID: 22589488 [PubMed - in process]

Screening of genes related to sulfide metabolism in Urechis unicinctus (Echiura, Urechidae) using suppression subtractive hybridization and cDNA microarray analysis.

Comp Biochem Physiol Part D Genomics Proteomics. 2012 Apr 28;

Authors: Shi X, Shao M, Zhang L, Ma Y, Zhang Z

Abstract

Exogenous sulfide can generally induce metabolic injuries in most organisms and even cause death. However, organisms inhabiting intertidal zones, hydrothermal vents, and cold seeps, can tolerate, metabolize, and utilize sulfide. In this study, both suppression subtractive hybridization and cDNA microarray analysis were employed to screen sulfide metabolism-related genes from the body wall in echiuran worm Urechis unicinctus, a marine sediment species. A total of 3456 monoclones were isolated and 82 were identified as differentially expressed genes in worms exposed to 50μM sulfide for 24h, compared to controls. The identified genes encoded proteins with multiple processes, including metabolism, cellular process, biological regulation, response to stimulus, multicellular organismal process, localization, development, and cellular component organization. Eight genes, serase, vacuolar protein, src tyrosine kinase, sulfide oxidase-like oxidoreductase, aprataxin, SN-RNP, aminopeptidase, and predicted protein, were selected to verify expression in the worm using qRT-PCR. The agreement of gene expression evaluation was 62.5% between the results of microarray analysis and qRT-PCR. These new data will provide clues for further probing of the molecular mechanism of sulfide metabolism.

PMID: 22591583 [PubMed - as supplied by publisher]

Screening of genes related to sulfide metabolism in Urechis unicinctus (Echiura, Urechidae) using suppression subtractive hybridization and cDNA microarray analysis.

Comp Biochem Physiol Part D Genomics Proteomics. 2012 Apr 28;

Authors: Shi X, Shao M, Zhang L, Ma Y, Zhang Z

Abstract

Exogenous sulfide can generally induce metabolic injuries in most organisms and even cause death. However, organisms inhabiting intertidal zones, hydrothermal vents, and cold seeps, can tolerate, metabolize, and utilize sulfide. In this study, both suppression subtractive hybridization and cDNA microarray analysis were employed to screen sulfide metabolism-related genes from the body wall in echiuran worm Urechis unicinctus, a marine sediment species. A total of 3456 monoclones were isolated and 82 were identified as differentially expressed genes in worms exposed to 50μM sulfide for 24h, compared to controls. The identified genes encoded proteins with multiple processes, including metabolism, cellular process, biological regulation, response to stimulus, multicellular organismal process, localization, development, and cellular component organization. Eight genes, serase, vacuolar protein, src tyrosine kinase, sulfide oxidase-like oxidoreductase, aprataxin, SN-RNP, aminopeptidase, and predicted protein, were selected to verify expression in the worm using qRT-PCR. The agreement of gene expression evaluation was 62.5% between the results of microarray analysis and qRT-PCR. These new data will provide clues for further probing of the molecular mechanism of sulfide metabolism.

PMID: 22591583 [PubMed - as supplied by publisher]

The CRKL gene encoding an adaptor protein is amplified, overexpressed, and a possible therapeutic target in gastric cancer.

J Transl Med. 2012 May 16;10(1):97

Authors: Natsume H, Shinmura K, Tao H, Igarashi H, Suzuki M, Nagura K, Goto M, Yamada H, Maeda M, Konno H, Nakamura S, Sugimura H

Abstract

ABSTRACT: BACKGROUND: Genomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as ERBB2, are highly amplified in gastric cancer. We searched for the possible amplification of other genes in gastric cancer. Methods and Results A genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers, and 22 genes (including ERBB2) in five highly amplified chromosome regions (with a copy number of more than 6) were identified. Particular attention was paid to the CRKL gene, the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3) domains. An extremely high CRKL copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence in situ hybridization (FISH), and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360) of the primary gastric cancers that were analyzed. The CRKL copy number was also examined in 360 primary gastric cancers using a FISH analysis, and CRKL amplification was found to be associated with CRKL overexpression. Finally, we showed that MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide. CONCLUSION: These results suggested that CRKL protein is overexpressed in a subset of gastric cancers and is associated with CRKL amplification in gastric cancer. Furthermore, our results suggested that CRKL protein has the ability to regulate gastric cell proliferation and has the potential to serve as a molecular therapy target for gastric cancer.

PMID: 22591714 [PubMed - as supplied by publisher]

The CRKL gene encoding an adaptor protein is amplified, overexpressed, and a possible therapeutic target in gastric cancer.

J Transl Med. 2012 May 16;10(1):97

Authors: Natsume H, Shinmura K, Tao H, Igarashi H, Suzuki M, Nagura K, Goto M, Yamada H, Maeda M, Konno H, Nakamura S, Sugimura H

Abstract

ABSTRACT: BACKGROUND: Genomic DNA amplification is a genetic factor involved in cancer, and some oncogenes, such as ERBB2, are highly amplified in gastric cancer. We searched for the possible amplification of other genes in gastric cancer. Methods and Results A genome-wide single nucleotide polymorphism microarray analysis was performed using three cell lines of differentiated gastric cancers, and 22 genes (including ERBB2) in five highly amplified chromosome regions (with a copy number of more than 6) were identified. Particular attention was paid to the CRKL gene, the product of which is an adaptor protein containing Src homology 2 and 3 (SH2/SH3) domains. An extremely high CRKL copy number was confirmed in the MKN74 gastric cancer cell line using fluorescence in situ hybridization (FISH), and a high level of CRKL expression was also observed in the cells. The RNA-interference-mediated knockdown of CRKL in MKN74 disclosed the ability of CRKL to upregulate gastric cell proliferation. An immunohistochemical analysis revealed that CRKL protein was overexpressed in 24.4% (88/360) of the primary gastric cancers that were analyzed. The CRKL copy number was also examined in 360 primary gastric cancers using a FISH analysis, and CRKL amplification was found to be associated with CRKL overexpression. Finally, we showed that MKN74 cells with CRKL amplification were responsive to the dual Src/BCR-ABL kinase inhibitor BMS354825, likely via the inhibition of CRKL phosphorylation, and that the proliferation of MKN74 cells was suppressed by treatment with a CRKL-targeting peptide. CONCLUSION: These results suggested that CRKL protein is overexpressed in a subset of gastric cancers and is associated with CRKL amplification in gastric cancer. Furthermore, our results suggested that CRKL protein has the ability to regulate gastric cell proliferation and has the potential to serve as a molecular therapy target for gastric cancer.

PMID: 22591714 [PubMed - as supplied by publisher]