Earticle

Genome editing is one of the most demanding tools in mammalian cells to unravel the role of genes and to implement gene therapy of genetic diseases. Zinc finger nuclease is an engineered restriction enzyme which is constructed by combining an array of zinc finger DNA-binding motifs in the N-terminus and the FokΙ endonuclease in C-terminus. Almost every ZFN recognizes three consecutive nucleotides starting with guanine in a context-dependent manner, thereby limiting the use of ZFN in genome editing. To address this challenge, we replaced ZFNs with a gene-specific nucleotides, which were connected to FokΙ nuclease by using (his)6-tag and GS linkers. We tested in vitro whether this engineered FokΙ shows a site-specific nuclease activity. For this, the engineered FokI and nucleotide complex was incubated with template DNA which was produced from 70-mer and 100-mer nucleotides base-paired each other. The results indicate that the complex showed a site-specific nuclease activity, which suggests that the nucleotide-based FokΙ can be used for the universal genome editing.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, particularly prevalent in Asia, with very poor prognosis. Although α-fetoprotein (AFP) is the most effective biomarker available to detect HCC, it has a low clinical sensitivity and specificity as biomarker. Therefore, there is a need for the development of more sensitive and specific assays that can supplement AFP for the early detection of liver cancer. Recently, autoantibodies, which are generated by immune system recognizing the presence of the abnormal tumor-associated antigens, are suggested as promising biomarkers for the early detection of tumors. In this study, to improve the early detection and diagnosis of HCC, determination of autoantibody responses to AFP in HCC patients was performed and analyzed its correlation with the progression of disease. The serum AFP levels were measured using sandwich ELSA commercially available and anti-AFP autoantibody levels were determined by ELISA using purified AFP from HepG2 cells as coating antigen. Assays were performed for 50 healthy controls, 50 patients with HCC, 36 patients with liver cirrhosis, and 35 patients with chronic hepatitis. Anti-AFP autoantibodies were detected in patients’ sera showing elevated AFP levels, which revealed that AFP secreted from tumor cells elicit immune response which induce tumor-associated autoantibodies. Even when the AFP level was very low anti-AFP responses were detectable, impling that autoantibody response to AFP can be used as an early detection biomarker. Several patient sera showing highly elevated AFP, however, did not show anti-AFP antibody response, which might be related to the intrinsic property of the AFP molecule as an immune suppressor. In addition, simultaneous detection of AFP and anti-AFP response in patients’ sera improved the diagnosis of HCC and we discuss about its usage as HCC diagnosis biomarker.

To metastasize, tumor cells must acquire abilities to penetrate surrounding normal tissues and overcome barriers of dissemination and distant growth such as invasion, anoikis resistance, evasion of immune surveillance, extravasation, colonization, and growth in new microenvironments. Recent evidence suggests that galectin-3, a galactose binding protein, can promote metastasis by regulating detachment of cancer cells. Moreover, highly metastatic human breast cancer cells show higher levels of expression of galectin-3 and overexpression of galectin-3 enhances cell motility and invasiveness in lung cancer cells, suggesting that galectin-3 regulates cancer metastasis. However, the mechanism by which galectin-3 promotes metastasis has not been fully understood. We demonstrated here that overexpression of galectin-3 induced Epitherial-to-Mesenchymal Transition (EMT) and EMT induced by galectin-3 overexpression was remarkable in serum deprivation condition. The cell–cell adhesion molecule E-cadherin is stabilized by linking intracellularly with the actin cytoskeleton but overexpression of galectin-3 weakened the interaction of E-cadherin with cytoskeleton. Additionally, we found that galectin-3 overexpressing HT-29 cells were more resistant to anoikis stress and serum deprivation than parental HT-29 cells. Based on these findings we propose that galectin-3 is an important molecule that promotes cancer metastasis through induction of EMT and anoikis resistance.

Cancer is often difficult to achieve early diagnosis, which is, however, a decisive requisite for favorable outcome in cancer treatments. Cancer biomarkers have been sought for several purposes preferably in blood and pinpointing cancer cells-derived aberrant glycoprteins would be a well-grounded approach to cancer biomarker discovery. One of the glycosyltransferases responsible for aberrant glycosylation in cancer is N-acetylglucosaminyltransferase V (GnT-V), which catalyzes an addition of b1,6-Nacetylglucosamine (GlcNAc) to the core N-glycan, and many lines of evidence have demonstrated the role of GnT-V in cancer development. Tissue inhibitor of metalloproteinase (TIMP-1) and protein tyrosine phosphatase kappa (PTPk) were suggested to be involved in cancer malignancy upon aberrantly glycosylation by GnT-V. In addition to GnT-V, several glycosyltransferase co-works to render the altered glycan structures in cancer cells including sialryl Lewis antigen and core-fucosylation, which prompted us to mine serological biomarker candidates in cancer sera. Immunodepleted on an immune-LC column, serological proteins were enriched by carbohydrate beads conjugated with lectins like L-PHA, DSA, E-selectin, AAL, Con-A. The fraction refractive to lectin enrichments was resolved on an SDS-PAGE gel, and fractionated by molecular mass. Both the captured glycoproteins and gel-seperated proteins were tryptic-digested for sequence determination in an LTQ-FTICR mass spectrometer. Candidate proteins showing high sesitivity and specificity during the discovery phase were selected and the panel of biomarker candidates is currently under in depth analyses for validation.