Young-Kyoung Shin

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.

Prognostic significance of microsatellite instability in sporadic colorectal cancer

Background and aimsColorectal cancers exhibiting microsatellite instability (MSI) appear to have unique biological behavior. The influence of MSI on the prognosis of sporadic colorectal cancers is controversial and requires further investigation. The aim of this study was to analyze the association between MSI status and clinicopathological features and prognosis in sporadic colorectal cancer patients.Patients and methodsOf the 322 consecutive colorectal cancer patients operated upon at the Seoul National University Hospital between January and December 1998, we examined the clinicopathological features and prognosis of 248 patients with sporadic primary colorectal cancer. The MSI status of these 248 patients has been reported in a previous study. Of the 248 patients, 23 (9.3%) had MSI+ tumors. The patients’ clinicopathological parameters were obtained from their medical records, and follow-up and survival data were obtained from medical records and phone calls.ResultsMSI+ sporadic colorectal cancers were found predominantly in the proximal colon (p<0.001) and were associated with poor differentiation (p=0.030), a lower preoperative serum carcinoembryonic antigen (CEA) level (p=0.012), and less frequent systemic metastasis (p=0.034) than MSI− tumors. Low tumor grade (p=0.022), low tumor T-stage (p=0.002), no lymph node metastasis (p<0.001), no systemic metastasis (p<0.001), adjuvant chemotherapy (p<0.001) and MSI+ status (p=0.038) were independent favorable prognostic factors for survival in sporadic colorectal cancer patients.ConclusionMSI status was an independent favorable prognostic factor for survival in sporadic primary colorectal cancer patients.

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.

Upregulation of glycolytic enzymes in proteins secreted from human colon cancer cells with 5-fluorouracil resistance

5‐Fluorouracil (5‐FU) is the most commonly used chemotherapeutic agent for colorectal cancer (CRC). However, resistance to this drug is a major obstacle in CRC chemotherapy. Accurate prediction of response to 5‐FU would avoid unnecessary chemotherapy and allow the selection of other effective drugs. To identify a candidate predictor of 5‐FU resistance, we isolated secreted proteins that were up‐ or downregulated in a 5‐FU‐resistant cancer cell line, compared with the parent cell line (SNU‐C4), using a stable isotope‐coded labeling protocol. For validating the clinical applicability of this method, levels of the identified proteins were determined in the sera of 46 patients treated with 5‐FU. In total, 238 proteins with molecular weights ranging from 50 to 75 kDa were identified. Among these, 45 and 35 secreted proteins were up‐ and downregulated in the 5‐FU‐resistant cell line, respectively. We observed significant upregulation of glycolytic enzymes, including glyceraldehyde‐3‐phosphate dehydrogenase, pyruvate kinase M2 (PK‐M2), transketolase, and NADP(+)‐dependent malic enzyme 1. In particular, the level of PK‐M2, a key enzyme in the glycolytic pathway, showed an increasing tendency in both sera and tissues from CRC patients displaying no response to 5‐FU‐based chemotherapy (progressive and stable disease cases), compared with that in complete or partial responders to 5‐FU‐based chemotherapy; however, it did not reach the statistical significance. In conclusion, increasing pattern of PK‐M2 observed with 5‐FU resistance induced in vitro and in sera and tissues from CRC patients displaying poor response to 5‐FU‐based chemotherapy suggest the relevance of dysregulated glycolysis and 5‐FU‐resistant CRC.

Microsatellite instability and mutations in DNA mismatch repair genes in sporadic colorectal cancers.

AbstractPURPOSE: This study was designed to investigate the frequency of mutations in DNA mismatch repair genes in sporadic colorectal cancers. METHODS: Genomic DNAs procured from paraffin blocks of the pathologic specimens from 230 consecutive patients with colorectal cancer were examined for their microsatellite instability status using a mononucleotide microsatellite marker, BAT-26, and also evaluated expressions of hMLH1, hMSH2, and hMSH6 proteins by immunohistochemical staining. Any of these 230 patients did not have family histories of hereditary nonpolyposis colorectal cancer, familial adenomatous polyposis, colorectal cancer, or hereditary nonpolyposis colorectalrelated cancers, such as endometrial, small bowel, and ureteral and renal pelvic cancers. When microsatellite instability was positive, mutations in the simple repeated sequences of TGF-βRII, BAX, IGF IIR, hMSH3, and hMSH6 genes were examined. In microsatellite instability–positive or staining-negative cases, polymerase chain reaction–single- strand conformation polymorphism and DNA sequencing detected mutations of hMLH1, hMSH2, and hMSH6 genes. If mutations were found in tumor tissue samples, we tested for a germline mutation with a microdissected corresponding normal tissue. RESULTS: Among 230 cases of sporadic colorectal cancer, 21 (9.1 percent) manifested microsatellite instability. In the immunohistochemical staining, 20 (8.6 percent) showed loss of expressions. All 20 staining-negative cases were microsatellite instability–positive. Only 1 of 21 (4.8 percent) microsatellite instability– positive cases showed intact staining for three proteins. The frame-shift mutations of the simple repetitive sequences were found in 17 cases (81.0 percent) in TGF-βRII, 11 (52.4 percent) in BAX, 5 (23.8 percent) in IGF IIR, 7 (33.3 percent) in hMSH3, and 8 (38.1 percent) in hMSH6 genes. Germline mutation was observed in only one case, which accounts for 4.8 percent among positive microsatellite instability and 0.4 percent of total patients, and was found in hMSH2. Five somatic mutations (2 in hMLH1, 2 in hMSH2, and 1 in hMSH6) also were found. CONCLUSION: The results indicated that a germline mutation of DNA mismatch repair gene was a rare event in sporadic colorectal cancers.

Mutational analysis of OGG1, MYH, MTH1 in FAP, HNPCC and sporadic colorectal cancer patients: R154H OGG1 polymorphism is associated with sporadic colorectal cancer patients

MYH, OGG1 and MTH1 are members of base excision repair (BER) families, and MYH germline mutations were recently identified in patients with multiple adenomas or familial adenomatous polyposis (FAP). A total of 20 APC-negative Korean FAP patients were analyzed for OGG1, MYH and MTH1 germline mutations. A total of 19 hereditary nonpolyposis colorectal cancer (HNPCC), 86 suspected HNPCC, and 246 sporadic colorectal cancer cases were investigated for OGG1 and MYH mutations. A total of 14 R154H OGG1 polymorphisms were identified in hereditary, sporadic colorectal cancers, and normal controls. For the case-control analysis of OGG1 R154H, a total of 625 hereditary or sporadic colorectal cancer patients and 527 normal controls were screened. R154H was a rare polymorphism associated with sporadic colorectal cancer patents (OR: 3.586, P= 0.053). R154H does not segregate with cancer phenotypes. Upon examining the possibility of recessive inheritance of R154H, we could not identify any complementary mutations in OGG1, MYH or MTH1. Samples with R154H were further screened for mutations of K-ras, β-catenin, APC, p53, BRAF and the microsatellite instability (MSI) status. Eight somatic mutations were identified in these genes and G:C to T:A transversion mutations were not dominant in samples harboring R154H. This result raises the possibility that OGG1 R154H may function as a low/moderate-penetrance modifier for colorectal cancer development.

A novel germline mutation in the MET extracellular domain in a Korean patient with the diffuse type of familial gastric cancer.

Gastric cancer is one of the most deadly cancers world wide. Although its incidence has declined in recent years, it is still the most prevalent cancer in Asian countries such as Korea and Japan.1 Germline mutations of the cell to cell adhesion molecule E-cadherin ( CDH1 ) have been reported in patients with the diffuse type of familial gastric cancer.2,3 However, the frequency of CDH1 mutations is low overall4 and the observed mutations differ between western and Asian patients.5 Truncating mutations (that is, nonsense, frameshift, and alternative splicing mutations) predominate in patients of western origin,2,3 whereas only a few missense mutations have been found in patients of Asian extraction.5,6 This suggested that CDH1 does not play a major role in gastric cancer development in Asian countries, and prompted researchers to investigate other gastric cancer causing genes. One such gene is that for the MET receptor tyrosine kinase. MET transduces motility, proliferation, and morphogenic signals of hepatocyte growth factor/scatter factor (HGF/SF) in epithelial cells.7 Similar to other receptor tyrosine kinase genes such as RET , the MET gene encodes a protein with an extracellular domain (exon 2–13), a transmembrane domain (exon 13), and a tyrosine kinase domain (exon 15–21).8,9 MET germline mutations have been reported in patients with hereditary papillary renal carcinoma (HPRC).7 Most of the MET mutations associated with HPRC or sporadic papillary renal carcinomas7 were missense mutations in the tyrosine kinase domain,10,11 and overexpression of MET has been reported in human diseases such as breast, prostate, gastric, and ovarian cancers.12,13 In the specific context of gastric cancers, a MET germline missense mutation was found in a Korean patient suffering from intestinal gastric cancer.11 The mutation, located at the juxtamembrane domain (exon 14), …

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. …