Diagnostic power of DNA methylation markers suggestive of cholangiocarcinoma in ERCP-based brush cytology.

BACKGROUND AND AIMS: Accurate differentiation between cholangiocarcinoma (CCA) and benign biliary stricture is of paramount importance. Biliary brush cytology is a simple and safe diagnostic approach that provides relatively high specificity; however, sensitivity is limited. Previous reports indicated the aberrations of DNA methylation in CCA. This study aimed to investigate the diagnostic performance of the methylation index (MI) of HOXA1 and NEUROG1 gene promoters in CCA. METHODS: Patients with biliary stricture who underwent ERCP with brush cytology in Siriraj Hospital from September 2016 to December 2019 were prospectively enrolled. The MI of HOXA1 (MI_H) and MI of NEUROG1 (MI_N) were determined by quantitative methylation-specific polymerase chain reaction. The diagnostic power for CCA was tested for MI from both genes and serum carbohydrate antigen 19-9 (CA19-9). RESULTS: Sixty-seven patients were included in the study; 41 patients had a final diagnosis of CCA, and 26 patients were determined to have a benign biliary stricture. The results showed that both MI_H and MI_N had higher sensitivity and accuracy (95.1% and 82.3% and 90.2% and 89.5%, respectively) than brush cytology (61.5% and 78.1%) and CA19-9 (69.4% and 77.8%). The combination of brush cytology, both methylation markers, and CA19-9 increased the sensitivity and accuracy to 97.4% and 91.0%. Methylation markers were positive in 5 of 6 patients with confirmed CCA whose cytology and CA19-9 were negative. CONCLUSIONS: DNA methylation increased the sensitivity for the diagnosis of CCA; therefore, the use of DNA methylation is promising for diagnosis of CCA in patients with biliary strictures. A future validation study is warranted to assess its role in clinical practice. (Clinical trial registration number: NCT04568512.).

Evaluation of the safety and healing potential of a fibroin-aloe gel film for the treatment of diabetic foot ulcers.

OBJECTIVE: This study aimed to develop a wound dressing prepared from the blending of silkworm fibroin and aloe gel extract for use in the treatment of diabetic foot ulcers (DFUs). METHODS: Fibroin extracted from silkworm cocoons and aloe gel extract were dissolved in deionised water. pH levels were then adjusted with lactic acid solution. A simple casting technique was used to obtain the fibroin-aloe gel film. The surface morphology, hardness, flexibility and infrared spectrum of the sterilised film were tested. Swelling ratio was measured from changes in weight. The cytocompatibility of the film to human dermal fibroblast was determined using XTT assay. Hard-to-heal DFUs (grade I Wagner score) were treated with the film for four weeks. The application site was assessed for allergic reactions and/or sensitisation. Wound size was measured using standardised digital photography. RESULTS: A total of five hard-to-heal DFUs were treated. The obtained film sterilised with ozonation showed a non-porous structure. The elongation at break and tensile strength of the wet film were 9.00±0.95% and 6.89±1.21N, respectively. Fourier-transform infrared spectroscopy data indicated the presence of amides I, II and III, of peptide linkage, which are the chemical characteristics of the fibroin. Functional groups relating to healing activity of the aloe gel extract were also found. The swelling ratio of the film immersed in water for 24 hours was 0.8±0.01. In three DFUs (40-50mm2 in size), a wound area reduction of 0.4-0.8mm2/day was observed and were healed in 2-3 weeks. The remaining two SFUs (500mm2 in size) showed a wound area reduction of 4mm2/day and were almost closed at four weeks. No allergic reaction or infection was observed in any of the wounds. CONCLUSION: The obtained film showed a non-porous structure, and its strength and flexibility were adequate for storage and handling. The film tended to increase the proliferation of fibroblasts. The wound dressing showed potential for accelerating the healing rate of DFUs.

Development of an engineered peptide antagonist against periostin to overcome doxorubicin resistance in breast cancer.

BACKGROUND: Chemoresistance is one of the main problems in treatment of cancer. Periostin (PN) is a stromal protein which is mostly secreted from cancer associated fibroblasts in the tumor microenvironment and can promote cancer progression including cell survival, metastasis, and chemoresistance. The main objective of this study was to develop an anti-PN peptide from the bacteriophage library to overcome PN effects in breast cancer (BCA) cells. METHODS: A twelve amino acids bacteriophage display library was used for biopanning against the PN active site. A selected clone was sequenced and analyzed for peptide primary structure. A peptide was synthesized and tested for the binding affinity to PN. PN effects including a proliferation, migration and a drug sensitivity test were performed using PN overexpression BCA cells or PN treatment and inhibited by an anti-PN peptide. An intracellular signaling mechanism of inhibition was studied by western blot analysis. Lastly, PN expressions in BCA patients were analyzed along with clinical data. RESULTS: The results showed that a candidate anti-PN peptide was synthesized and showed affinity binding to PN. PN could increase proliferation and migration of BCA cells and these effects could be inhibited by an anti-PN peptide. There was significant resistance to doxorubicin in PN-overexpressed BCA cells and this effect could be reversed by an anti-PN peptide in associations with phosphorylation of AKT and expression of survivin. In BCA patients, serum PN showed a correlation with tissue PN expression but there was no significant correlation with clinical data. CONCLUSIONS: This finding supports that anti-PN peptide is expected to be used in the development of peptide therapy to reduce PN-induced chemoresistance in BCA.

Periostin induces epithelial­to­mesenchymal transition via the integrin α5ß1/TWIST­2 axis in cholangiocarcinoma.

Periostin (PN) (also known as osteoblast­specific factor OSF­2) is a protein that in humans is encoded by the POSTN gene and has been correlated with a reduced survival of cholangiocarcinoma (CCA) patients, with the well­known effect of inducing epithelial­to­mesenchymal transition (EMT). The present study investigated the effect of PN, through integrin (ITG)α5ß1, in EMT­mediated CCA aggressiveness. The alterations in EMT­related gene and protein expression were investigated by real­time PCR, western blot analysis and zymogram. The effects of PN on migration and the level of TWIST­2 were assessed in CCA cells with and without siITGα5 transfection. PN was found to induce CCA cell migration and EMT features, including increments in Twist­related protein 2 (TWIST­2), zinc finger protein SNAI1 (SNAIL­1), α-smooth muscle actin (ASMA), vimentin (VIM) and matrix metallopeptidase 9 (MMP­9), and a reduction in cytokeratin 19 (CK­19) together with cytoplasmic translocation of E-cadherin (CDH­1). Additionally, PN markedly induced MMP­9 activity. TWIST­2 was significantly induced in PN­treated CCA cells; this effect was attenuated in the ITGα5ß1­knockdown cells and corresponded to reduced migration of the cancer cells. These results indicated that PN induced CCA migration through ITGα5ß1/TWIST-2­mediated EMT. Moreover, clinical samples from CCA patients showed that higher levels of TWIST­2 were significantly correlated with shorter survival time. In conclusion, the ITGα5ß1­mediated TWIST­2 signaling pathway regulates PN­induced EMT in CCA progression, and TWIST­2 is a prognostic marker of poor survival in CCA patients.