Atit Silsirivanit

A novel serum carbohydrate marker on mucin 5AC: Values for diagnostic and prognostic indicators for cholangiocarcinoma

The incidence of cholangiocarcinoma (CCA) is increasing globally. Currently, there is no powerful marker for the diagnosis of CCA, which has led to late diagnosis and poor patient outcome. This study was designed to establish a new monoclonal antibody (MoAb) for detecting a serum marker associated with CCA.

CA-S27: a novel Lewis a associated carbohydrate epitope is diagnostic and prognostic for cholangiocarcinoma.

Early and specific diagnosis is critical for treatment of cholangiocarcinoma (CCA). In this study, a carbohydrate antigen‐S27 (CA‐S27) monoclonal antibody (mAb) was established using pooled CCA tissue‐extract as immunogen. The epitope recognized by CA‐S27‐mAb was a new Lewis‐a (Lea) associated modification of MUC5AC mucin. A Soybean agglutinin/CA‐S27‐mAb sandwich ELISA to determine CA‐S27 in serum was successfully developed. High level of CA‐S27 was detected in serum of CCA patients and could differentiate CCA patients from those of gastro‐intestinal cancers, hepatomas, benign hepatobiliary diseases and healthy subjects with high sensitivity (87.5%) and high negative predictive value (90.4%). The level of serum CA‐S27 was dramatically reduced after tumor removal, indicating tumor origin of CA‐S27. Patients with high serum CA‐S27 had significantly shorter survivals than those with low serum CA‐S27 regardless of serum MUC5AC levels. Fucosyltransferase‐III (FUT3) was shown to be a regulator of CA‐S27 expression. Suppression of CA‐S27 expression with siRNA‐FUT3 or neutralization with CA‐S27 mAb significantly reduced growth, adhesion, invasion and migration potentials of CCA cells in vitro. In summary, we demonstrate that serum CA‐S27, a novel carbohydrate antigen, has potential as diagnostic and prognostic markers for CCA patients. CA‐S27 involves in promoting cell growth, adhesion, migration and invasion of CCA cells.

Cancer biomarker discovery for cholangiocarcinoma: the high-throughput approaches

Cholangiocarcinoma (CCA) is difficult to diagnose at an early stage and most tumors are detected at late stage where surgery or other therapy is ineffective. Many advanced techniques are applied to diagnose CCA; however, most are expensive and have varying degrees of accuracy. A less invasive and simpler procedure such as serum markers would be of substantial clinical benefit for diagnosis, monitoring, and predicting outcome for CCA patients. Recent advances in “Omics” technologies offer remarkable opportunities for establishment of biomarker‐related to diseases. In this review, the potential biomarkers obtained from proteomics and glycomic studies are evaluated. Several protein markers were discovered from patient specimen, using two dimensional‐differential gel electrophoresis couple with liquid chromatography tandem mass spectrometry (2D‐DIGE/LC‐MS‐MS), matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry (MALDI‐TOF‐MS), surface enhanced laser desorption/ionization (SELDI)‐TOF‐MS and capillary electrophoresis (CE)‐MS, etc. Newly reported CCA‐associated glyco‐biomarkers were identified using lectin‐assisted, monoclonal antibody‐assisted or specific‐target strategies. The combination between carbohydrate binding‐lectin and core protein‐binding mAb significantly increased the values for detection of the glyco‐biomarkers for CCA. Searching for specific and sensitive molecular markers to be used for population screening is worth being evaluated. This could lead to earlier diagnosis and improve outcome. Further investigation of those biomarker functions is also of value in order to better understand the tumor biology and use them as targets for future therapeutic agents.

Serum α1β-glycoprotein and afamin ratio as potential diagnostic and prognostic markers in cholangiocarcinoma

Cholangiocarcinoma (CCA) affects the intra- and extrahepatic bile ducts and is commonly burdened by a late presentation and resulting high mortality rate. Accordingly, finding non-invasive biomarkers with adequate diagnostic/prognostic values is a priority in high-risk populations. In this study, we analyzed proteomes of serum samples from six CCA cases and ten healthy subjects using two-dimensional polyacrylamide gel electrophoresis to identify CCA-associated spots. Thirty-six CCA-associated proteins found in sera were identified by mass spectrometry. α1β-Glycoprotein (A1BG) and afamin (AFM) were detected consistently at different degrees in CCA sera compared with controls and were validated for their diagnostic and prognostic potential in a larger cohort of 64 patients with CCA, 4 with benign biliary diseases and 20 healthy subjects and compared between pre- and postsurgery serum samples from 26 CCA patients to ascertain a prognostic correlation. A single blot test developed to assess the serum A1BG/AFM ratio could detect CCA cases with 87.5% specificity, 84.4% sensitivity and the levels were significantly higher in CCA compared with controls. A high level of postoperative serum A1BG/AFM ratio was associated with worse outcomes and the infiltration of resection margins. The A1BG/AFM ratio may constitute a novel non-invasive candidate marker to diagnose CCA and its outcomes with high specificity and sensitivity. Prospective studies are awaited to demonstrate the clinical value of this observation.

High glucose levels boost the aggressiveness of highly metastatic cholangiocarcinoma cells via O-GlcNAcylation

Increased glucose utilization is a feature of cancer cells to support cell survival, proliferation, and metastasis. An association between diabetes mellitus and cancer progression was previously demonstrated in cancers including cholangiocarcinoma (CCA). This study was aimed to determine the effects of high glucose on protein O-GlcNAcylation and metastatic potentials of CCA cells. Two pairs each of the parental low metastatic and highly metastatic CCA sublines were cultured in normal (5.6 mM) or high (25 mM) glucose media. The migration and invasion abilities were determined and underlying mechanisms were explored. Results revealed that high glucose promoted migration and invasion of CCA cells that were more pronounced in the highly metastatic sublines. Concomitantly, high glucose increased global O-GlcNAcylated proteins, the expressions of vimentin, hexokinase, glucosamine-fructose-6-phosphate amidotransferase (GFAT) and O-GlcNAc transferase of CCA cells. The glucose level that promoted migration/invasion was shown to be potentiated by the induction of GFAT, O-GlcNAcylation and an increase of O-GlcNAcylated vimentin and vimentin expression. Treatment with a GFAT inhibitor reduced global O-GlcNAcylated proteins, vimentin expression, and alleviated cell migration. Altogether, these results suggested the role of high glucose enhanced CCA metastasis via modulation of O-GlcNAcylation, through the expressions of GFAT and vimentin.

Mechanistic insights of O-GlcNAcylation that promote progression of cholangiocarcinoma cells via nuclear translocation of NF-κB.

O-GlcNAcylation, an O-linked protein glycosylation with a single molecule of N-acetylglucosamine (GlcNAc), is reversibly controlled by O-GlcNAc transferase (OGT) and N-acetyl D-glucosaminidase (OGA). Aberrant O-GlcNAcylation contributes an important role in initiation and progression of many human cancers. Elevation of O-GlcNAcylation in tumor tissues and poor prognosis of cholangiocarcinoma (CCA) patients have been reported. In this study, the role of O-GlcNAcylation in promoting tumor progression was further investigated in CCA cell lines. Suppression of O-GlcNAcylation using small interfering RNAs of OGT (siOGT) significantly reduced cell migration and invasion of CCA cells whereas siOGA treated cells exhibited opposite effects. Manipulating levels of O-GlcNAcylation did affect the nuclear translocation of NF-κB and Akt-phosphorylation together with expression of matrix-metalloproteinases (MMPs). O-GlcNAcylation and nuclear translocation of NF-κB, the upstream signaling cascade of MMP activation were shown to be important for MMP activation. Immunoprecipitation revealed the elevation of O-GlcNAc-modified NF-κB with increased cellular O-GlcNAcylation. Involvement of O-GlcNAcylation in MMP-mediated migration and invasion of CCA cells was shown to be via O-GlcNAcylation and nuclear translocation of NF-κB. This information indicates the significance of O-GlcNAcylation in controlling the metastatic ability of CCA cells, hence, O-GlcNAcylation and its products may be new targets for treatment of metastatic CCA.

Improve discrimination power of serum markers for diagnosis of cholangiocarcinoma using data mining-based approach

OBJECTIVE Cholangiocarcinoma (CCA) is usually fatal because of the absence of tests for early detection and lack of effective therapy. Tumor markers with adequate diagnostic values are of clinical significance. This study is aimed to improve the diagnostic power of serum markers using the computational data mining technique to develop a combined diagnostic model that yielded the best diagnostic values for CCA. DESIGN AND METHODS Eight CCA-associated markers-carcinoembryonic antigen, carbohydrate antigen 19-9, alkaline phosphatase (ALP), and gamma glutamyl transferase, biliary-ALP, mucin5AC, CCA-associated carbohydrate antigen (CCA-CA) and CA-S27-were used as the inputs for the C4.5 decision tree classification model and the selected model was confirmed by ANN analyses. Eight serum markers for CCA were determined in the training set of 85 histologically proven-CCA patients and 82 control subjects. The chosen set of combined markers that gave the best diagnostic values for CCA was then validated in the testing set of 22 CCA patients and 60 controls. RESULTS A decision tree diagram built by the C4.5 algorithm suggested the serial analysis of CCA-CA and ALP for distinguishing CCA patients from non-CCA subjects with all diagnostic parameters ≥95%. The combined tests showed a precise diagnosis in the testing set. CONCLUSIONS The C4.5 model indicates the combined markers of CCA-CA and ALP that produced the more precise diagnosis for CCA.

Effects of GUASHA on Heart Rate Variability in Healthy Male Volunteers under Normal Condition and Weightlifters after Weightlifting Training Sessions

Objectives. This paper aims at exploring the effects of GUASHA on heart rate variability between healthy volunteers under normal condition and weightlifters after training sessions. Methods. Ten healthy male volunteers under normal condition and 15 male weightlifters after weightlifting training sessions were recruited into two groups. Electrocardiography was recorded before and immediately after 20-minute GUASHA. HRV was calculated in both the time domain and the frequency domain. Results. Stress index was reduced, while standard deviation of N-N intervals (SDNN), proportion derived by dividing the number of interval differences of successive N-N intervals greater than 50 ms, and root mean square of successive differences (RMSSD) were enhanced after GUASHA therapy in the two groups. The changes in SDNN and RMSSD were higher in the healthy men group than in the weightlifters group. In addition, low frequency was decreased whereas high frequency was significantly increased in healthy men after the GUASHA session. Conclusions. GUASHA therapy facilitates the parasympathetic nervous activity and modulates the balance between parasympathetic and sympathetic activities in both healthy men under normal condition and weightlifters after training sessions as indicated. Although the changes of the HRV parameters were similar in both groups, the responsiveness was more pronounced in healthy men than in male weightlifters.

Novel mutations in cholangiocarcinoma with low frequencies revealed by whole mitochondrial genome sequencing.

BACKGROUND Mitochondrial DNA (mtDNA) mutations have been shown to be associated with cancer. This study explored whether mtDNA mutations enhance cholangiocarcinoma (CCA) development in individuals. MATERIALS AND METHODS The whole mitochondrial genome sequences of 25 CCA patient tissues were determined and compared to those of white blood cells from the corresponding individuals and 12 healthy controls. The mitochondrial genome was amplified using primers from Mitoseq and compared with the Cambridge Reference Sequence. RESULTS A total of 161 mutations were identified in CCA tissues and the corresponding white blood cells, indicating germline origins. Sixty-five (40%) were new. Nine mutations, representing those most frequently observed in CCA were tested on the larger cohort of 60 CCA patients and 55 controls. Similar occurrence frequencies were observed in both groups. CONCLUSIONS While the correspondence between the cancer and mitochondrial genome mutation was low, it is of interest to explore the functions of the missense mutations in a larger cohort, given the possibility of targeting mitochondria for cancer markers and therapy in the future.

Tai chi improves oxidative stress response and DNA damage/repair in young sedentary females.

[Purpose] This study was to examine the effects of 12 weeks of Tai Chi (TC) exercise on antioxidant capacity, and DNA damage/repair in young females who did not perform regular physical exercise. [Subjects and Methods] Ten female students from a Chinese university voluntarily participated in this program. All of them practiced the 24-form simplified Tai Chi, 5 times weekly, for 12 weeks. Plasma levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA), glutathione (GSH), hydroxyl radical inhibiting capacity (OH·-IC), 8-hydroxy-2’-deoxyguanosine (8-OHdG), and 8-oxoguanine DNA glycosylase (OGG1) were measured at 0, 8, and 12 weeks. Heart rate (HR) was monitored during the last set of the training session at 4, 8, and 12 weeks. [Results] Plasma SOD and OH·-IC levels were increased at 8 and 12 weeks compared to the baseline (0 weeks). Gpx and GSH levels did not change significantly throughout the study period. The plasma MDA level was decreased significantly at 8 weeks but not at 12 weeks compared to the baseline value. While the plasma 8-OHdG level did not change throughout the study period, the plasma OGG1 level was significantly increased at 8 and 12 weeks compared to the baseline value. [Conclusion] TC practice for 12 weeks efficiently improved the oxidative stress response in young females who did not perform regular physical exercise. The TC exercise also increased the DNA repairing capacity.