Sung-Hye Hong

Down-regulation of Mitochondrial F1F0-ATP Synthase in Human Colon Cancer Cells with Induced 5-Fluorouracil Resistance

5-Fluorouracil (5-FU) is widely used for treatment of advanced colorectal cancer. However, it is common for such patients to develop resistance to 5-FU, and this drug resistance becomes a critical problem for chemotherapy. The mechanisms underlying this resistance are largely unknown. To screen for proteins possibly responsible for 5-FU resistance, cells resistant to 5-FU were derived from human colon cancer cell lines and two-dimensional gel electrophoresis–based comparative proteomics was done. Two-dimensional gel electrophoresis data showed there was lower expression of the α subunit of mitochondrial F1F0-ATP synthase (ATP synthase) in 5-FU–resistant cells compared with parent cells. Western blotting showed that expression of other ATP synthase complex subunits was also lower in 5-FU–resistant cell lines and that these resistant cells also showed decreased ATP synthase activity and reduced intracellular ATP content. The ATP synthase inhibitor, oligomycin A, strongly antagonized 5-FU–induced suppression of cell proliferation. When 5-FU sensitivity was compared with ATP synthase activity in six different human colon cancer cell lines, a positive correlation has been found. Furthermore, suppressed ATP synthase d-subunit expression by siRNA transfection increased cell viability in the presence of 5-FU. Bioenergetic dysfunction of mitochondria has been reported as a hallmark of many types of cancers (i.e., down-regulation of ATP synthase β-subunit expression in liver, kidney, colon, squamous oesophageal, and lung carcinomas, as well as in breast and gastric adenocarcinomas). Our findings show that ATP synthase down-regulation may not only be a bioenergetic signature of colorectal carcinomas but may also lead to cellular events responsible for 5-FU resistance.

Promoter hypermethylation downregulates RUNX3 gene expression in colorectal cancer cell lines.

It was recently reported that RUNX3 gene expression is significantly downregulated in human gastric cancer cells due to hypermethylation of its promoter region or hemizygous deletion (Cell, 109, 2002). To verify the genetic alterations and methylation status of the RUNX3 gene in colorectal carcinogenesis, we analysed for mutations, loss of heterozygosity (LOH), and RUNX3 gene promoter hypermethylation, in 32 colorectal cancer cell lines. RT–PCR analysis showed undetectable or low RUNX3 expression in 16 cell lines, and no mutations were found in the RUNX3 gene by PCR-SSCP analysis. Of these 16 cell lines, hypermethylation of the RUNX3 promoter was confirmed in 12. The following observations were made: (i) RUNX3 was re-expressed after 5-aza-2′-deoxycytidine treatment, (ii) the RUNX3 promoter was found to be methylated by MS-PCR, and (iii) hypermethylation of the RUNX3 promoter was confirmed by direct sequencing analysis after sodium bisulfite modification in the above 12 cell lines. RUNX3 was neither methylated nor expressed in four cell lines. Of these four, microsatellite instability (MSI) at the RUNX3 locus was found in three, SNU-61 (D1S246), SNU-769A, and SNU-769B (D1S199). This study suggests that transcriptional repression of RUNX3 is caused by promoter hypermethylation of the RUNX3 CpG island in colorectal cancer cell lines, and the results of these experiments may contribute to an understanding of the role of RUNX3 inactivation in the pathogenesis of colorectal cancers.

Decreased pyruvate kinase M2 activity linked to cisplatin resistance in human gastric carcinoma cell lines

Resistance to anticancer drugs is a major obstacle preventing effective treatment of disseminated cancers. Understanding the molecular basis to chemoresistance is likely to provide better treatment. Cell lines resistant to cisplatin or 5‐fluorouracil (5‐FU) were established from human gastric carcinoma cell lines SNU‐638 and SNU‐620. Comparative proteomics involving 2‐dimensional gel electrophoresis (2‐DE) and matrix‐associated laser desorption ionization‐mass spectroscopy (MALDI‐MS) was performed on protein extracts from these parental and drug‐resistant derivative lines to screen drug resistance‐related proteins. Pyruvate kinase M2 (PK‐M2) was identified as a protein showing lower expression in cisplatin‐resistant cells compared to parental cells. Consistent with this finding, PK‐M2 activity was also lower in cisplatin‐resistant cells. Suppression of PK‐M2 expression by antisense oligonucleotide resulted in acquired cisplatin resistance in SNU‐638 cells. Furthermore, PK‐M2 activity in 11 individual human gastric carcinoma cell lines positively correlated with cisplatin sensitivity. Taken together, PK‐M2 protein and activity levels were lower in cisplatin‐resistant human gastric carcinoma cell lines compared to their parental cell lines. Furthermore, suppression of PK‐M2 expression using antisense oligonucleotides increased cisplatin resistance. These data clearly link PK‐M2 and cisplatin resistance mechanisms.

Metabotropic Glutamate Receptor 4-Mediated 5-Fluorouracil Resistance in a Human Colon Cancer Cell Line

Purpose: 5-Fluorouracil (5-FU) has been the mainstay treatment for colorectal cancer for the past few decades. However, as with other cancers, development of 5-FU resistance has been a major obstacle in colorectal cancer chemotherapy. The purpose of this study was to gain further understanding of the mechanisms underlying 5-FU resistance in colorectal cancer cells. Experimental Design: A 5-FU-resistant cell line was established from the human colon cancer cell line SNU-769A. Protein extracts from these two cell lines (parent and resistant) were analyzed using comparative proteomics to identify differentially expressed proteins. Results: 5-FU-resistant human colon cancer cells were found to overexpress metabotropic glutamate receptor 4 (mGluR4). Other experiments showed cellular resistance to 5-FU (i.e., cell survival) was altered by the mGluR4 agonist l-2-amino-4-phosphonobutyric acid (L-AP 4), and by the mGluR4 antagonist (S)-amino-2-methyl-4-phosphonobutanoic acid (MAP 4), in that L-AP 4 increased 5-FU resistance in SNU-769A cells, whereas MAP 4 ablated 5-FU resistance in 5-FU-resistant cells. However, there was no significant effect of L-AP 4 or MAP 4 on basal cAMP and thymidylate synthase levels. Interestingly, 5-FU down-regulated mGluR4 expression, and MAP 4 suppressed proliferation in both cell lines. Conclusions: We here report mGluR4 expression in human colon cancer cell line, which provides further evidence for extra-central nervous system expression of glutamate receptors. Overexpression of mGluR4 may tentatively be responsible for 5-FU resistance and, although activation by agonist promotes cell survival in the presence of 5-FU, decreased mGluR4 expression or inactivation by antagonist contributes to cell death.

Mutations of the Birt-Hogg-Dubé (BHD) gene in sporadic colorectal carcinomas and colorectal carcinoma cell lines with microsatellite instability

Birt-Hogg-Dube (BHD) syndrome, an inherited autosomal genodermatosis characterised by benign tumours of the hair follicle, is associated with renal neoplasia, lung cysts, and spontaneous pneumothorax.1 The novel causative gene, identified by linkage analysis in BHD families, is localised on chromosome 17p11.2.2 Protein truncating germinal mutations within a hypermutable (C)8 tract occur in patients with BHD syndrome and lead to an increased risk of kidney cancer.3 Microsatellite repeats are widely distributed throughout the genome. Owing to a defect in the DNA mismatch repair gene, a subset of tumours accumulates frequent deletion and insertion mutations in these repetitive DNA sequences.4–6 Most microsatellite instability (MSI) has so far been described in non-coding DNA within introns of intergenic regions in the genome. However, in some cancer related genes and mismatch repair genes, MSI has been identified in protein coding regions. The first target sequence identified within a coding region was a poly (A)10 nucleotide tract of the TGFBR2 gene.7 The other mutational targets of MSI have been found in repetitive sequences of IGF2R 8 and BAX 9 genes involved in the regulation of cell growth and in the promotion of apoptosis, respectively. Furthermore, frameshift mutations in repeat sequences of the DNA mismatch repair genes MSH3 and MSH6 have been reported.10 In tumours with MSI, the mechanism of tumorigenesis is believed to involve frameshift mutations of microsatellite repeats within the coding regions of genes, the inactivation of which is considered to contribute to tumorigenesis. Early reports suggested that BHD syndrome was associated with a predisposition to colon neoplasms,11–13 but Zbar et al 14 reported that colon cancer and colon polyps are not related to BHD syndrome. Recently, Khoo et al 15 reported that colorectal neoplasia is an associated feature of BHD in some families. …

Germ Line Mutations of Mismatch Repair Genes in Hereditary Nonpolyposis Colorectal Cancer Patients with Small Bowel Cancer: International Society for Gastrointestinal Hereditary Tumours Collaborative Study

Purpose: The aim of study was to determine the clinical characteristics and mutational profiles of the mismatch repair genes in hereditary nonpolyposis colorectal cancer (HNPCC) patients with small bowel cancer (SBC). Experimental Design: A questionnaire was mailed to 55 members of the International Society for Gastrointestinal Hereditary Tumours, requesting information regarding patients with HNPCC-associated SBC and germ line mismatch repair gene mutations. Results: The study population consisted of 85 HNPCC patients with identified mismatch repair gene mutations and SBCs. SBC was the first HNPCC-associated malignancy in 14 of 41 (34.1%) patients for whom a personal history of HNPCC-associated cancers was available. The study population harbored 69 different germ line mismatch repair gene mutations, including 31 mutations in MLH1, 34 in MSH2, 3 in MSH6, and 1 in PMS2. We compared the distribution of the mismatch repair mutations in our study population with that in a control group, including all pathogenic mismatch repair mutations of the International Society for Gastrointestinal Hereditary Tumours database (excluding those in our study population). In patients with MSH2 mutations, patients with HNPCC-associated SBCs had fewer mutations in the MutL homologue interaction domain (2.9% versus 19.9%, P = 0.019) but an increased frequency of mutations in codons 626 to 733, a domain that has not previously been associated with a known function, versus the control group (26.5% versus 2.8%, P < 0.001). Conclusions: In HNPCC patients, SBC can be the first and only cancer and may develop as soon as the early teens. The distribution of MSH2 mutations found in patients with HNPCC-associated SBCs significantly differed from that found in the control group (P < 0.001).

Establishment and characterization of 13 human colorectal carcinoma cell lines: mutations of genes and expressions of drug-sensitivity genes and cancer stem cell markers.

Thirteen human colorectal cancer (CRC) cell lines were established from 10 primary tumors and 3 metastatic tumors obtained from 13 Korean patients. Characteristics of the cell lines including morphology in vivo and in vitro; mutations of the K-ras, p53, APC and MMR genes and microsatellite instability (MSI) status in vitro were determined. Expression of drug-sensitivity genes including MDR1, MXR, MRP1 and COX2 was also analyzed. The cell lines were unique as judged by DNA fingerprinting using 16 short tandem repeats. Eleven of the cell lines grew as adherent populations and the remaining two as floating aggregates. None of the cell lines were contaminated with Mycoplasma or bacteria. All cell lines showed high viability with relatively long doubling times. Six cell lines contained mutations at K-ras. Seven cell lines displayed p53 gene missense, nonsense and frameshift mutations. MSI was found in three cell lines and two cell lines with an MSI-high phenotype-possessed hMLH1 mutations. Nine cell lines had an APC mutation. MRP1 was highly expressed in all cell lines, and high expression of MDR1, MXR and COX2 evident in eight, six and six cell lines, respectively. Embryonal stem cell markers (MELK, SOX4 and OCT4) were expressed in most of cell lines. The cancer stem cell biomarkers CD133, CD44 and Lgr5 were expressed in 12, 13 and 13 cell lines, respectively. The presently well-characterized CRC cell lines should be useful in investigations of the biological characteristics of CRC, particularly for investigations related to gene alterations associated with CRC and biology of cancer stem cells.

Identification of mitochondrial FoF1‐ATP synthase involved in liver metastasis of colorectal cancer

Liver metastasis is a major cause of poor survival of colorectal cancer patients. In order to identify the proteins associated with liver metastasis in colorectal cancer, we carried out two‐dimensional gel electrophoresis‐based comparative proteomic analysis of normal colon mucosa, primary colon cancer tissue and corresponding metastatic tumor tissue in liver. The proteins identified were further validated by immunohistochemical analysis of 67 quadruplet samples of normal colon primary colorectal cancer and normal liver‐synchronous liver metastasis, and 251 colorectal cancers as well as in vitro invasion assay of the human colon cancer cell line, SNU‐81. From proteomic assessment, the mitochondrial FoF1‐ATP synthase (ATP synthase) α‐subunit was identified as a protein that is upregulated in liver metastasis compared with the primary tumor. Immunohistochemical analyses confirmed a significant increase in the expression of ATP synthase α‐ and d‐subunits in synchronous liver metastasis compared with primary tumor and normal mucosa, respectively. ATP synthase α‐ and d‐subunits were overexpressed in 197 (78.5%) and 190 (75.7%), respectively, of the 251 colorectal cancers. The α‐ and d‐subunits were significantly associated with liver metastasis (P < 0.05) as well as low histological grade (P < 0.0001). The d‐subunit also correlated with venous invasion (P = 0.026) and distant metastasis (P = 0.032). In stage III cancers, d‐subunit expression was independently associated with poor survival (P = 0.017). Furthermore, transfection of small interfering RNA targeted to ATP synthase α‐ and d‐subunits resulted in decreased in vitro invasiveness of the human colon cancer cell line. Our overall findings demonstrate that increased ATP synthase is associated with liver metastasis of colorectal cancer. (Cancer Sci 2007; 98: 1184–1191)

Galectin-3 stabilizes heterogeneous nuclear ribonucleoprotein Q to maintain proliferation of human colon cancer cells

Abstract.Comparative analysis of proteomes using 5-fluorouracil (5-FU)-resistant human colon cancer cell line revealed that decreased galectin-3 expression was significantly associated with retarded proliferation. However, in the presence of 5-FU proliferation rate of cells with suppressed galectin-3 expression did not differ from that of cells with normal galectin-3 expression, even galectin-3 suppression augmented apoptosis. Mechanism by which galectin-3 regulates cancer cell proliferation has been identified in immunoprecipitates of the anti-galectin-3 antibody. Heterogeneous nuclear ribonucleoprotein Q (hnRNP Q) was identified as a protein interacting with galectin-3. Interestingly, while galectin-3 protein was not affected by the hnRNP Q level, its suppression was accompanied by a decrease in hnRNP Q expression. The present study demonstrates that galectin-3 stabilizes hnRNP Q via complex formation, and reduction in the hnRNP Q level leads to slow proliferation and less susceptibility to 5-FU.

Identification of mitochondrial F1F0‐ATP synthase interacting with galectin‐3 in colon cancer cells

To evaluate the effect of galectin‐3 in cell cycle regulation of colon cancer cells, we looked for binding molecules interacting with galectin‐3 and examined the changes in cell cycle by suppressing galectin‐3 and the binding molecule. To identify target molecules interacting with galectin‐3, we analyzed immunoprecipitate of the anti‐galectin‐3 antibody obtained from human colon cancer cell line, using matrix‐assisted laser desorption ionization‐mass spectrometry. We validated subcellular localization of galectin‐3 and ATP synthase identified, and ATP synthase activity was determined in the presence of galectin‐3. Cell cycle regulation was monitored after galectin‐3 siRNA transfection. ATP synthase b‐subunit was identified in immunoprecipitate of the anti‐galectin‐3 antibody. Galectin‐3 and ATP synthase were co‐isolated in the inner membrane vesicles of mitochondria. Galectin‐3 has an inhibitory activity against ATP synthase, and intracellular ATP content showed increasing tendency after galectin‐3 suppression. Suppression of galectin‐3 resulted in G0/G1 progression of human colon cancer cells arrested at S, S/G2 and G2/M phase in the presence of doxorubicin, and etoposide or nocodazole, respectively. Compared to cells in which ATP synthase d‐subunit was suppressed alone, sub‐G1 fraction caused by etoposide or nocodazole was decreased in cells with galectin‐3 suppression alone. In conclusion, galectin‐3 co‐localized with ATP synthase in the inner membrane of mitochondria and has an inhibitory effect on ATP synthase in human colon cancer cells. In the presence of cell cycle synchronizing drugs, doxorubicin, etoposide, or nocodazole, suppression of galectin‐3 induced cell cycle progression to G0/G1 phase. (Cancer Sci 2008; 99: 1884–1891)