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Cbl‑associated protein (CAP) is encoded by the sorbin and SH3 domain‑containing 1 (SORBS1) gene. CAP has been reported to be associated with the actin cytoskeleton, receptor tyrosine kinase signaling and cell adhesion through interactions with various proteins. It may be hypothesized that SORBS1 has numerous unknown functions, which may include providing a favorable condition for metastasis. Although CAP has been demonstrated to possess a number of functions, the role of this protein has only been reported in metabolic signaling pathways and its function in cancer remains to be elucidated. In the present study, SORBS1 expression was detected in colorectal cancer cell lines divided into the primary group and the metastatic group by reverse transcription‑quantitative PCR and western blot analysis. In addition, SORBS1 expression was manipulated by vector transfection and lentivirus transduction. The metastatic role of SORBS1, as determined by assessing its effects on cell proliferation and migration, was determined by colony formation assay, cell cycle analysis and Boyden chamber assay. To elucidate the SORBS1‑binding protein, immunoprecipitation was performed. Co‑localization of SORBS1 and AHNAK nucleoprotein (AHNAK) was identified by confocal microscopy. Notably, the protein expression levels of CAP were higher in SNU‑769A and SW480 cells than in SNU‑769B and SW620 cells. In addition, the number of colonies in the SORBS1‑overexpressing group was significantly increased compared with that of the control group, as determined using the colony formation assay; the SORBS1 overexpression group formed >8‑fold more colonies than the control group. The proliferative ability of the SORBS1 overexpression group was also significantly increased compared with the control group over the entire incubation period. Cell migration assays revealed that the number of migrated SORBS1‑knockdown cells was reduced compared with the control in both HCT‑116 and SNU‑C4 cell lines; migration area was decreased to 31 and 26% in HCT‑116 and SNU‑C4 cell lines, respectively. Consequently, it was confirmed that SORBS1 could form a complex with AHNAK, which functions as a tumor suppressor through inhibition of phosphorylated‑ERK and Rho‑associated coiled‑coil containing protein kinase 1. In conclusion, SORBS1 may serve a crucial role in cancer growth and migration via inhibition of AHNAK expression.

Resistance to chemotherapeutic agents has been considered as a major reason for the high incidence rate of recurrence and metastasis suffered by colorectal cancer (CRC) patients. ATP-binding cassette sub-family G member 2 (ABCG2) is involved in drug resistance. DNA methylation of the ABCG2 promoter site has a significant influence on the regulation of epigenetic gene expression. In the present study, we investigated whether the methylation status of the ABCG2 promoter is related to drug sensitivity in CRC cell lines. In order to examine the ABCG2 expression level and identify the methylation status, RT-PCR, qRT-PCR analysis, MS-PCR and bisulfite sequencing were conducted on 32 CRC cell lines. SNU-C4, LS174T and NCI-H716 were selected as low ABCG2-expressing and high promoter methylated cell lines. The cell proliferation assay for 5-fluorouracil, oxaliplatin and irinotecan was performed after 5-aza-2'-deoxycytidine (5-aza) treatment in these cell lines. In the 32 CRC cell lines, 25% of the cell lines expressed low or no ABCG2 expression. Of these cell lines, SNU-C4, LS174T and NCI-H716 were hypermethylated at the promoter region, ~20%. Demethylation of ABCG2 was induced by 5-aza, which enhanced the ABCG2 expression level and influenced the cell proliferation similar to treatment with the anticancer agents. Our data suggest that the ABCG2 expression level regulated by methylation is related to anticancer drug sensitivity. Based on these results, it can be applied to predict the anticancer drug response.

Permanently growing cell lines can be invaluable because of their usefulness in a variety of experimental situations. We report the characteristics of seven cell lines designated, SNU-306, SNU-334, SNU-1528, SNU-1553, SNU-1581, SNU-1958 and SNU-2372, which were established from three primary carcinomas, two pleural effusion, one pericardial effusion and one ascitic fluid samples obtained from seven Korean breast carcinoma patients. The histopathology of the primary tumors and their in vitro growth characteristics are described. DNA fingerprinting analysis and genetic alterations in the p53 and EGFR genes were conducted. The expression levels of the ER-α, PR, C-erbB2, E-cadherin, COX-2, MDR and MXR genes were investigated and sensitivity to anticancer drugs was screened. Growth was as adherent cells (four cell lines), floating aggregates (one cell line) and both (two cell lines). All lines were free of mycoplasma or bacteria and were proven unique by DNA fingerprinting analysis using 18 microsatellite markers. Estrogen receptor (ER) mRNA was highly expressed in five cell lines and low or undetectable in SNU-1958 and SNU-2372. Progesterone receptor (PR) mRNA was expressed only in the SNU-306. SNU-1958 and SNU-2372 were hormone receptor-negative and C-erbB2-negative (triple-negative). SNU-1528 had an in-frame deletion of 42 base pairs of p53 gene and showed over 20-fold resistance for taxol compared to the other cell lines. There were no mutation in the EGFR gene; COX-2 was expressed in four cell lines and MXR was expressed in two cell lines. These well-characterized seven breast cancer cell lines, which include two triple-negative cell lines, will be useful for the study of breast cancer biology.

Metastasis of cancer cells is a major cause of death in cancer patients. The process of cancer metastasis includes the proliferation of primary cancer cells, local invasion, intravasation and cancer cell survival in blood flow, extravasation and attachment to secondary organs and metastatic growth in a new environment. In these mechanisms of cancer metastasis, CXC chemokine receptor 4 (CXCR4) and its ligand play an important role. Stromal cell-derived factor-1α (SDF-1α, also known as CXCL12) is well known as a ligand of CXCR4, and macrophage migration-inhibitory factor (MIF) has recently become known as a ligand of CXCR4. In many types of cancers including breast, pancreatic and colorectal cancer (CRC), CXCR4/SDF-1α has been investigated in metastasis-related cancer behavior, which include cell proliferation, adhesion, migration and invasion. However, CXCR4/MIF has rarely been investigated in the metastatic behavior of colon cancer cells. In this report, the effect of SDF-1α or MIF was studied on cell cycle, cell proliferation, adhesion and migration of the CXCR4-expressing colon cancer cell line SW480. SDF-1α or MIF caused a decrease in the number of cells in G0/G1 phase and an increase in the numbers of cells in S and G2/M phases. In addition, SDF-1α or MIF caused an increase in cell proliferation, cell adhesion to fibronectin and migration. AMD3100, a CXCR4 antagonist, attenuated these effects, which included increased cell proliferation, adhesion and migration due to treatment of CXCR4-expressing colon cancer cells with SDF-1α or MIF. In conclusion, SDF-1α or MIF affects the metastasis-related behaviors of CXCR4-expressing colon cancer cells.

Recently, the leucine-rich repeat G protein-coupled receptor 5 (LGR5/GPR49) was identified as a potential marker of intestinal stem cells in human. The LGR5 is known as a Wnt signaling target gene, and its expression pattern is related with β-catenin mutation. H2O2 is a member of reactive oxygen species (ROS) and regulates metabolism, aging, apoptosis and the intensity of growth factor signaling. In addition, it acts as a negative or positive regulator of Wnt signaling. However, the effect of H2O2 on Wnt signaling and its target gene LGR5 is not clear. In this study, we investigated the effects of ROS on cancer stem cells, in colorectal cancer cells. Colorectal cancer cells were treated with exogenous H2O2, after which cellular responses and the expression of LGR5 were examined. In SNU-C2A cells, proliferation increased following treatment with 50-300 µM of H2O2, whereas cell viability significantly decreased after treatment with 600-900 µM of H2O2. Expression of heme oxygenase (HO)-1 and jun, which aid in the reduction of oxidative stress, were induced in the low dose H2O2-treated SNU-C2A cells. The LGR5 expression level was significantly increased following 50-300 µM H2O2 treatment; in addition, β-catenin was increased in H2O2-treated colon cancer cells. However, the increased β-catenin was detected not in the nucleus but in the cytoplasm, which means that β-catenin was stabilized in the cytoplasm and not translocated into the nucleus where it could function as a transcription factor for the expression of LGR5. In addition, there was no direct interaction between LGR5 and β-catenin. In this study, we found that LGR5 expression increased when cancer cells were treated with a low dose of H2O2. Our results indicate that the LGR5 increase resulted via activation of the JNK signaling pathway. The induction of LGR5 expression influenced cell proliferation in colorectal cancer cells.

CHFR is a recently identified mitotic stress check-point gene. CHFR is ubiquitously expressed in normal human tissues, whereas loss of CHFR expression has been observed in human tumors. Silencing of CHFR has been associated with aberrant promoter methylation and histone deacetylation in several cancer types. In this study, we investigated epigenetic CHFR inactivation in human gastric cancers by examining CHFR expression and methylation status in gastric cancer cell lines with RT-PCR analysis, bisulfite PCR and sequencing. A series of primary gastric tumors were also analyzed for CHFR methylation. Eight of 12 (66.7%) gastric cancer cell lines and 19/43 (44.2%) primary gastric tumors showed CHFR methylation. In addition, CpG methylation status correlated well with CHFR expression in the human gastric cancer cell lines, in which treatment with 5-aza-dC resulted in de novo or enhanced expression of CHFR. Combination treatment of 5-aza-dC with trichostatin A showed a synergistic effect on CHFR expression in some cases. Our results indicate that aberrant promoter methylation of the CHFR gene was observed in a significant proportion of human gastric cancers and was responsible for the inactivation of the CHFR gene in gastric cancers.

Hereditary non-polyposis colorectal cancer (HNPCC), an inherited cancer predisposition syndrome, has been associated with germline mutations in DNA mismatch repair (MMR) genes. However, because all mutation carriers of hMLH1/hMSH2 do not account for CRC susceptibility, modifying genes may play a role in the variation of disease expression. In this study, we determined the GSTM1 and GSTT1 genotypes in 104 family members representing 19 Korean HNPCCs carrying hMLH1/hMSH2 mutation, and investigated the influence of GSTM1 and GSTT1 geno-/phenotype status on both age at diagnosis of CRC and cancer occurrence. The overall frequency of the GSTM1 and GSTT1 geno-/phenotype in 55 non-carriers, compared with that in mutation carriers (n=49), was not significantly different, and no significant correlation was found between mean age at diagnosis and the allelomorphs encoding the GSTM1 or GSTT1 enzymes. However, a comparison of the allele frequencies of GSTM1 in affected (n=30) and unaffected (n=19) mutation carriers revealed a significant difference, as the null allele was more prevalent in individuals with cancer (p=0.03; odds ratio, 3.7; 95% confidence interval, 1.1-12.7). Our results suggest that the genotypes of GSTM1 are associated with cancer occurrence in Korean HNPCC family members carrying the hMLH1/hMSH2 mutation. However, a bias due to the small sample size of this study cannot be rule out. Although evidence that GST genotypes are associated with increased cancer risk has often been controversial, the genotyping of GSTM1 could have implications for genetic counseling and the management of MMR gene mutation carriers.

As telomeres play a role in protecting DNA, there is the possibility that telomerase activity is involved with cellular response to DNA-damaging agents. This study was designed to investigate the association between telomerase and the doxorubicin altered cell cycle in drug resistant gastric carcinoma cell lines. Three doxorubicin resistant gastric carcinoma cell lines and their parent cell lines (SNU-1, SNU-16 and SNU-620) were incubated with doxorubicin at the final concentration induced resistance and ten times final concentration for 24 h. Telomerase activity and hTERT mRNA expression were lowered by doxorubicin treatment in parent cell lines, but in drug resistant cell lines, telomerase activity and hTERT mRNA expression were not repressed by doxorubicin treatment. Bcl-2 protein expression, which is known to regulate telomerase activity, did not change in doxorubicin resistant cell lines but decreased in parent cell lines by doxorubicin treatment. Cell cycle analysis revealed that the parent cell lines had an increased fraction of cells in G2/M phase after doxorubicin treatment and doxorubicin resistant cell lines had maintained fractions in G0/G1 phase. Doxorubicin treatment did not alter cyclin B or cdc2 protein level, which is known as the essential component of G2/M transition. G2/M arrest in the parent cell lines was associated with an increase in inhibitory phosphorylation of Tyr15 on cdc2. In summary, the parent cell lines showed G2/M arrest and a reduction of telomerase activity after doxorubicin treatment. In contrast, reduced telomerase activity, Bcl-2 expression and G2/M arrest after doxorubicin treatment did not appear in resistant cell lines. Therefore, relative resistance to doxorubicin may be related to high levels of bcl-2 or intact cell cycle and consequently high telomerase activity.