Chun-Chieh Chen

Curcumin Suppresses Metastasis via Sp-1, FAK Inhibition, and E-Cadherin Upregulation in Colorectal Cancer

Colorectal cancer (CRC) is a serious public health problem that results due to changes of diet and various environmental stress factors in the world. Curcumin is a traditional medicine used for treatment of a wide variety of tumors. However, antimetastasis mechanism of curcumin on CRC has not yet been completely investigated. Here, we explored the underlying molecular mechanisms of curcumin on metastasis of CRC cells in vitro and in vivo. Curcumin significantly inhibits cell migration, invasion, and colony formation in vitro and reduces tumor growth and liver metastasis in vivo. We found that curcumin suppresses Sp-1 transcriptional activity and Sp-1 regulated genes including ADEM10, calmodulin, EPHB2, HDAC4, and SEPP1 in CRC cells. Curcumin inhibits focal adhesion kinase (FAK) phosphorylation and enhances the expressions of several extracellular matrix components which play a critical role in invasion and metastasis. Curcumin reduces CD24 expression in a dose-dependent manner in CRC cells. Moreover, E-cadherin expression is upregulated by curcumin and serves as an inhibitor of EMT. These results suggest that curcumin executes its antimetastasis function through downregulation of Sp-1, FAK, and CD24 and by promoting E-cadherin expression in CRC cells.

A genetic linkage map of Nicotiana plumbaginifolia/ Nicotiana longiflora based on RFLP and RAPD markers

Abstractu2008u2008We have constructed a genetic linkage map for Nicotiana plumbaginifolia/Nicotiana longiflora (2n= 2x=20), based on the segregation of 69 RFLP and 102 RAPD loci in 99 F2 plants from the cross N. plumbaginifolia×N. longiflora. The map consists of nine major linkage groups, each containing more than nine marker loci, and spans 1062 cM. Twenty of the RFLP markers were mapped previously to Nicotiana sylvestris (2n=2x=24) chromosomes using monosomic alien addition lines. Taxonomically, N. plumbaginifolia and N. sylvestris belong to the same section, namely the Alatae; however, cytogenetic evidence indicates that they are not closely related. Comparison of the distribution of markers common to both maps suggests that genome reorganization has occurred during the evolution of these two species. Evidence is also presented that genome reorganization may be accompanied by gain and loss of specific classes of DNA sequences in their genomes.

Protocadherin 10 suppresses tumorigenesis and metastasis in colorectal cancer and its genetic loss predicts adverse prognosis.

Protocadherin 10 (PCDH10), a novel tumor suppressor gene in human cancers, is located in a common deleted region at chromosome 4q28 in colorectal cancer (CRC). This study aimed to ascertain the genetic loss of PCDH10 and its clinical relevance in CRC and to explore the tumor suppressor function of PCDH10. The genetic deletion of PCDH10 was determined in 171 pairs of primary tumors and corresponding normal mucosae by loss of heterozygosity study. In total, 53 carcinomas were positive for allelic loss of PCDH10. The genetic aberration was significantly associated with tumor progression and distant metastasis (pu2009=u20090.021 and pu2009=u20090.018, respectively) and was an independent predictor of poor survival for CRC patients (pu2009=u20090.005). Expression of PCDH10 gene was silenced or markedly down‐regulated in all of 12 CRC cell lines tested and in 41 of 53 colorectal carcinomas compared with their matched normal mucosae. Ectopic expression of PCDH10 suppressed cancer cell proliferation, anchorage‐independent growth, migration and invasion in vitro. Subcutaneous injection of PCDH10‐expressing CRC cells into SCID mice revealed the reduction of tumor growth compared with that observed in mock‐inoculated mice. Furthermore, through intrasplenic implantation, the re‐expression of PCDH10 in silenced cells restrained liver metastasis and improved survival in SCID mice. In conclusion, PCDH10 is a pivotal tumor suppressor in CRC, and the loss of its function promotes not only tumor progression but also liver metastasis. In addition, the genetic deletion of PCDH10 represents an adverse prognostic marker for the survival of patients with CRC.

Shisa3 Is Associated with Prolonged Survival through Promoting β-Catenin Degradation in Lung Cancer

RATIONALEnDespite advances in treatment and prognosis of non-small cell lung cancer (NSCLC), patient outcomes are still unsatisfactory.nnnOBJECTIVESnTo reduce the morbidity and mortality of patients with NSCLC, a more comprehensive understanding of mechanisms involved in cancer progression is urgently needed.nnnMETHODSnBy comparison of gene expression profiles in the cell line pair with differential invasion ability, CL1-0 and CL1-5, we found that Shisa3 was highly expressed in the low invasive cells. The effect of Shisa3 on invasion, migration, proliferation, apoptosis, epithelial-mesenchymal transition, and anchorage-independent growth activities in vitro and on tumor growth and metastasis in mice models were examined. The underlying mechanism of Shisa3 was explored by microarray and pathway analysis. Finally, the correlation of Shisa3 expression and clinical outcome was also calculated.nnnMEASUREMENTS AND MAIN RESULTSnWe identified Shisa3 as a novel tumor suppressor, which induces β-catenin degradation resulting in suppression of tumorigenesis and invasion in vitro. Shisa3 decreased the tumor growth in mice with subcutaneous implantation and reduced the number of metastatic nodules in mice with tail vein injection and orthotopic implantation. Shisa3 performs the tumor suppression activity through WNT signaling predicted by microarray analysis. Our data found that Shisa3 accelerates β-catenin degradation and was positively associated with overall survival and progression-free survival of NSCLC.nnnCONCLUSIONSnOur results reveal that Shisa3 acts as a tumor suppressor by acceleration of β-catenin degradation and provide new insight for cancer prognosis and therapy.

DNA Hypermethylation of SHISA3 in Colorectal Cancer: An Independent Predictor of Poor Prognosis.

BackgroundShisa3 is a novel tumor suppressor identified in lung cancer. However, its antitumor activity in other human cancers and the mechanism of gene inactivation remain unknown.MethodsSHISA3 expression was measured by reverse transcription-PCR (RT-PCR) and quantitative RT-PCR (RT-qPCR). DNA methylation was determined by bisulfite sequencing and pyrosequencing.ResultsDown-regulation of SHISA3 expression was observed in all of 11 colorectal cancer (CRC) cell lines and was further confirmed in 34 (65.4xa0%) of 52 colorectal carcinomas by RT-qPCR. Four of six CRC cell lines could restore SHISA3 expression after treatment with 5-aza-2′-deoxycytidine. Tumor-specific methylation of five CpG sites in the first intron of SHISA3 was identified by bisulfite sequencing, and their methylation levels were quantified in 127 pairs of primary CRC tissues by bisulfite pyrosequencing. The methylation levels of SHISA3 in tumors were noticeably higher than that in their matched normal mucosae. In addition, SHISA3 hypermethylation was significantly associated with an increased risk of disease recurrence in patients with stage II and III disease (Pxa0=xa00.007) and was an independent predictor of poor overall survival [hazard ratio (HR) 2.9, 95xa0% confidence interval (CI) 1.5–5.8; Pxa0=xa00.002] and disease-free survival (HR 4.0, 95xa0% CI 1.6–10.2; Pxa0=xa00.003) of CRC patients.ConclusionsSHISA3 gene is epigenetically inactivated in a substantial fraction of CRC, and its hypermethylation is of prognostic significance in predicting clinical outcome. The quantitative bisulfite pyrosequencing assay established could be a cost-effective tool for providing a potential biomarker of adverse prognosis in CRC.

Mapping of DNA markers to arms and sub-arm regions of Nicotiana sylvestris chromosomes using aberrant alien addition lines.

Abstract.Seven monosomic addition plants, each containing the full complement of Nicotiana plumbaginifolia (2n = 20, genome constitution PP) and an aberrant chromosome of Nicotiana sylvestris (2n = 24, SS), were produced from backcrosses of hyperdiploid derivatives of the sesquidiploid hybrid PPS to N. plumbaginifolia. The N. sylvestris chromosomes in these plants were characterized by karyotype analysis, Southern hybridization with DNA markers previously localized on N. sylvestris chromosomes and a 269-bp fragment from the 3′ end of 25S rDNA, and fluorescence in situ hybridization using 25S rDNA, 5S rDNA and telomere repeats (TTTAGGG)n as probes. The N. sylvestris chromosomes in these plants were identified to be telocentrics 6S, 7S and 8S, and deletions 7S, 10, 12S and 12L, respectively. The successful identification of aberrant chromosomes in these lines enabled us to assign DNA markers to arms and sub-arm regions of N. sylvestris chromosomes. All aberrant chromosomes in the addition lines could be transmitted through mitosis and meiosis. The potential applications of the addition lines in high-resolution physical mapping, the isolation of N. sylvestris chromosomes by flow cytometry, and an understanding of the chromosomal distribution of 45S rDNA in N. sylvestris are discussed.

Abstract 4024: Aberrant DNA methylation of Shisa3 is a novel prognostic biomarker for colorectal cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnShisa3, a newly identified tumor suppressor, inhibits invasion and metastasis via regulation of epithelial-mesenchymal transition in non-small cell lung cancer. We first screened 11 colorectal cancer (CRC) cell lines as well as 10 pairs of primary tumors and matched normal mucosas for Shisa3 expression by semi-quantitative RT-PCR. Shisa3 gene was silenced or down-regulated in 8 (72.7%) cell lines and 8 (80%) CRC tumors, suggesting that Shisa3 might be also involved in colorectal tumorigenesis. Accordingly, we further confirmed Shisa3 expression was significantly decreased in 59.6% (31/52) of colorectal carcinomas compared with corresponding normal mucosas by real-time RT-PCR (P < 0.001). Using gene copy number assay, we ruled out chromosomal instability pathway as the major mechanism resulting in Shisa3 silencing in CRC. While Shisa3 mRNA expression was restored in 66.7% (4/6) of CRC cell lines as exposure to DNA methylation inhibitor 5-Aza-2′-deoxycytidine. CpG islands spanning around Shisa3 gene were predicted via MethPrimer program. Aberrant methylation of 5 CpG loci in Shisa3 intron 1 was identified in CRC cell lines and primary tumors by bisulfite sequencing, and their methylation levels determined by pyrosequencing were inverse correlation with Shisa3 mRNA expression. In addition, among 127 pairs of CRC tissues, Shisa3 methylation levels in tumors were much higher than that in corresponding normal mucosas (P < 0.001), and Shisa3 hypermethylation was significantly associated with disease recurrence in stages B and C patients (P = 0.008). Kaplan-Meier survival analysis showed that patients with Shisa3 hypermethylation revealed significantly shortened overall survival (P < 0.001) and disease-free survival (P = 0.006). In conclusion, Shisa3 gene hypermethylation could serve as a molecular predictor of disease recurrence and poor prognosis in CRC.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4024. doi:1538-7445.AM2012-4024

Somatic Hybridization Between Nicotiana sylvestris Speg. & Comes and N. plumbaginifolia Viv.

Although Nicotiana sylvestris and N. plumbaginifolia belong to the same section, viz., Alatae of the subgenus Petunioides, they differ markedly in morphology, geographic distribution, and karyotype (Goodspeed 1954). N. sylvestris is the maternal parent of cultivated allotetraploid N. tabacum (Gray et al. 1974), which is in the subgenus Tabacum, and possesses some morphological characters not present in the section Alatae. N. sylvestris occurs exclusively in northwestern Argentina, while N. plumbaginifolia has a much wider distribution, extending from northwestern Argentina to Brazil, Peru, Guatemala, Cuba, and Mexico. The chromosomes of N. sylvestris 2n=24) are rather uniform in size and are clearly bi-armed, while those of N. plumbaginifolia (2n=20) differ significantly in size and are telocentric or acrocentric (Fig. 1A,B).