Publications by authors named "Patcharee Jearanaikoon"

44 Publications

Monitoring the Progression of Liver Fluke-Induced Cholangiocarcinoma in a Hamster Model Using Synchrotron FTIR Microspectroscopy and Focal Plane Array Infrared Imaging.

Anal Chem 2020 12 10;92(23):15361-15369. Epub 2020 Nov 10.

Center for Biospectroscopy, School of Chemistry, Faculty of Science, Monash University, Victoria 3800, Australia.

Cholangiocarcinoma (CCA) is a bile duct cancer that originates in the bile duct epithelium. Northeastern Thailand has the highest incidence of CCA, and there is a direct correlation with liver fluke () infection. The high mortality rate of CCA is a consequence of delayed diagnosis. Fourier transform infrared (FTIR) spectroscopy is a powerful technique that detects the absorbance of molecular vibrations and is perfectly suited for the interrogation of biological samples. In this study, we applied synchrotron radiation-FTIR (SR-FTIR) microspectroscopy and focal plane array (FPA-FTIR) microspectroscopy to characterize periductal fibrosis and bile duct cells progressing to CCA induced by inoculating metacercariae into hamsters. SR-FTIR and FPA-FTIR measurements were performed in liver sections harvested from 1-, 2-, 3-, and 6-month post-infected hamsters compared to uninfected liver tissues. Principal component analysis (PCA) of the tissue samples showed a clear discrimination among uninfected and early-stage (1 and 2 months) and cancerous-stage (3 and 6 months) tissues. The discrimination is based on intensity changes in the phosphodiester band (1081 cm), amino acid residue (∼1396 cm), and C═O stretching carboxylic esters (1745 cm). Infected tissues also show definitive bands at ∼1280, 1234, and 1201 cm characteristic of the collagen triplet and indicative of fibrosis. Hierarchical cluster analysis (HCA) was performed on the FPA data and showed a classification into specific cell types. Hepatocyte, fibrotic lesion, and bile duct (cancer) were classified and HCA mapping showed similar cellular distribution pattern compared to Sirius red staining. This study was also extended to less invasive sample analysis using attenuated total reflectance-FTIR (ATR-FTIR) spectroscopy. Sera from -infected and uninfected hamsters were analyzed using multivariate analysis, including principal component analysis (PCA), and partial least squares-discriminant analysis (PLS-DA). PCA was able to classify spectra of normal, early-stage CCA, and CCA, while the PLS-DA gave 100% accuracy for the validation. The model was established from 17 samples (11 normal, 6 cancer) in the calibration set and 9 samples in the validation set (4 normal, 2 cancer, 3 precancerous). These results indicate that FTIR-based technology is a potential tool to detect the progression of CCA, especially in the early stages of the disease.
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http://dx.doi.org/10.1021/acs.analchem.0c02656DOI Listing
December 2020

Infrared spectroscopy coupled to cloud-based data management as a tool to diagnose malaria: a pilot study in a malaria-endemic country.

Malar J 2019 Oct 16;18(1):348. Epub 2019 Oct 16.

Centre for Biospectroscopy, School of Chemistry, Faculty of Science, Monash University, Wellington Road, Clayton, VIC, 3800, Australia.

Background: Widespread elimination of malaria requires an ultra-sensitive detection method that can detect low parasitaemia levels seen in asymptomatic carriers who act as reservoirs for further transmission of the disease, but is inexpensive and easy to deploy in the field in low income settings. It was hypothesized that a new method of malaria detection based on infrared spectroscopy, shown in the laboratory to have similar sensitivity to PCR based detection, could prove effective in detecting malaria in a field setting using cheap portable units with data management systems allowing them to be used by users inexpert in spectroscopy. This study was designed to determine whether the methodology developed in the laboratory could be translated to the field to diagnose the presence of Plasmodium in the blood of patients presenting at hospital with symptoms of malaria, as a precursor to trials testing the sensitivity of to detect asymptomatic carriers.

Methods: The field study tested 318 patients presenting with suspected malaria at four regional clinics in Thailand. Two portable infrared spectrometers were employed, operated from a laptop computer or a mobile telephone with in-built software that guided the user through the simple measurement steps. Diagnostic modelling and validation testing using linear and machine learning approaches was performed against the gold standard qPCR. Sample spectra from 318 patients were used for building calibration models (112 positive and 110 negative samples according to PCR testing) and independent validation testing (39 positive and 57 negatives samples by PCR).

Results: The machine learning classification (support vector machines; SVM) performed with 92% sensitivity (3 false negatives) and 97% specificity (2 false positives). The Area Under the Receiver Operation Curve (AUROC) for the SVM classification was 0.98. These results may be better than as stated as one of the spectroscopy false positives was infected by a Plasmodium species other than Plasmodium falciparum or Plasmodium vivax, not detected by the PCR primers employed.

Conclusions: In conclusion, it was demonstrated that ATR-FTIR spectroscopy could be used as an efficient and reliable malaria diagnostic tool and has the potential to be developed for use at point of care under tropical field conditions with spectra able to be analysed via a Cloud-based system, and the diagnostic results returned to the user's mobile telephone or computer. The combination of accessibility to mass screening, high sensitivity and selectivity, low logistics requirements and portability, makes this new approach a potentially outstanding tool in the context of malaria elimination programmes. The next step in the experimental programme now underway is to reduce the sample requirements to fingerprick volumes.
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http://dx.doi.org/10.1186/s12936-019-2945-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794904PMC
October 2019

The Upregulation of OCT4 in Acidic Extracellular pH is Associated with Gemcitabine Resistance in Cholangiocarcinoma Cell Lines.

Asian Pac J Cancer Prev 2019 09 1;20(9):2745-2748. Epub 2019 Sep 1.

Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen, Thailand.

Background: Cholangiocarcinoma (CCA), although is an uncommon liver cancer originating from bile duct epithelial cells, is one of the top 10 most fatal cancers. Chemoresistance is an unmet need always found in CCA patients. Tumor microenvironment conditions such as hypoxia, nutrient starvation and acidic extracellular pH play critical roles in chemoresistance and cancer progression. However, the effect of acidic extracellular pH on cellular response and chemoresistance in CCA has not been studied. Methods: Human CCA cell lines (KKU-M213, KKU-M055 and KKU-100) were cultured under acidic (pH 6.5) or non-acidic (pH 7.4) condition and were used for gene expression, doubling time and cytotoxicity assay. Results: The acidic extracellular pH (pH 6.5) significantly increased doubling times of CCA cell lines compared with non-acidic condition (pH 7.4). Interestingly, extracellular acid condition induced gemcitabine resistance in CCA cell lines. We showed that Octamer-binding transcription factor 4 (Oct4) was upregulated in these cell lines under extracellular acid condition. Conclusion: Our findings demonstrate that CCA cells can adapt to survive in acidic environment after which chemoresistance has been developed. Oct4 may be a key transcriptional regulator which mediates chemoresistance in response to acidic extracellular pH.
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http://dx.doi.org/10.31557/APJCP.2019.20.9.2745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6976837PMC
September 2019

The development of simultaneous measurement of viral load and physical status for human papillomavirus 16 and 18 co-infection using multiplex quantitative polymerase chain reaction.

Oncol Lett 2018 Dec 4;16(6):6977-6987. Epub 2018 Oct 4.

Center for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.

Persistent infection with human papillomavirus (HPV) type 16 and 18 is known to be a major risk factor for cervical cancer. Increased prevalence of co-infection with these high-risk types has been observed in pre-cancerous and cancerous tissues. The determination of physical status and copy numbers of viruses is therefore useful in clinical settings. A simple multiplex quantitative polymerase chain reaction (qPCR) for HPV16/HPV18 co-infection in one tube reaction was established in the present study using TaqMan-based PCR for E2 and E6 viral DNA. The detection range was up to 10 copies with 100% specificity and high precision (CV of cycle time <0.5%). The analytical accuracy and robustness were verified by competitive assay using an unequal mixture of HPV16/HPV18 DNA. No significant effect was demonstrated when compared with the simplex qPCR. The detection of physical status was evaluated in cervical samples, including 5 pre-cancerous and 15 cancerous samples. No significant difference was observed between simplex and multiplex qPCR (P=0.372). In conclusion, the developed multiplex qPCR method successfully demonstrated the viral status of the common HPV types in one tube. This assay will facilitate viral assessment and monitoring of cervical cancer associated with HPV16 and HPV18 co-infection.
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http://dx.doi.org/10.3892/ol.2018.9549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6256370PMC
December 2018

Aberrant methylation of HTATIP2 and UCHL1 as a predictive biomarker for cholangiocarcinoma.

Mol Med Rep 2018 Mar 19;17(3):4145-4153. Epub 2017 Dec 19.

Department of Clinical Chemistry, Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.

Cholangiocarcinoma (CCA) is the most common primary liver cancer in Northeastern Thailand where liver fluke infection is highly endemic. Although aberrant DNA methylation in CCA has been reported by several investigators, little is known regarding the associations between them. In the present study, the results obtained from our previously published methylation array were analyzed and 10 candidate genes involved in DNA repair [protein phosphatase 4 catalytic subunit (PPP4C)], apoptosis [runt related transcription factor 3 (RUNX3), interferon regulatory factor 4 (IRF4), ubiquitin C‑terminal hydrolase L1 (UCHL1) and tumor protein p53 inducible protein 3 (TP53I3)], cell proliferation [cyclin D2 (CCND2) and Ras association domain family member 1 (RASSF1)], drug metabolism [aldehyde dehydrogenase 1 family member A3 (ALDH1A3) and solute carrier family 29 member 1 (SLC29A1)] and angiogenesis [human immunodeficiency virus‑1 tat interactive protein 2 (HTATIP2)] were selected for quantification of their methylation levels in 54 CCA and 19 adjacent normal tissues using methylation‑sensitive high‑resolution melting. The associations between the methylation status of the individual genes and clinical parameters were statistically analyzed. High methylation levels were observed in UCHL1, IRF4, CCND2, HTATIP2 and TP53I3. The median methylation level of UCHL1 was 57.3% (range, 3.15 to 88.7%) and HTATIP2 was 13.6% (range, 7.5 to 36.7%). By contrast, low methylation of HTATIP2 and UCHL1 was identified in adjacent normal tissues. The methylation status of HTATIP2 and UCHL1 was associated with patients' overall survival. CCA patients with high methylation of HTATIP2 and low methylation of UCHL1 exhibited longer overall survival. In addition, multivariate Cox regression analysis demonstrated that UCHL1 methylation was an independent factor for CCA with hazard ratio of 1.81 (95% confidence interval, 1.01‑3.25) in high methylation group. The combination of HTATIP2 and UCHL1 methylation status strongly supported their potential predictive biomarker in which patients with CCA who had high methylation of HTATIP2 and low methylation of UCHL1 showed longer overall survival than those with low HTATIP2 methylation and high UCHL1 methylation. In conclusion, the present study revealed the value of aberrant DNA methylation of HTATIP2 and UCHL1, which may serve as a potential predictive biomarker for CCA.
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http://dx.doi.org/10.3892/mmr.2017.8319DOI Listing
March 2018

High expression of CCDC25 in cholangiocarcinoma tissue samples.

Oncol Lett 2017 Aug 21;14(2):2566-2572. Epub 2017 Jun 21.

Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon Kaen 40002, Thailand.

Cholangiocarcinoma (CCA) is a malignant transformation of biliary epithelial cells. It is a slow growing tumor, but is also highly metastatic with a poor prognosis. Bile acids are known to transactivate the epidermal growth factor receptor (EGFR) in cholangiocytes and induce cyclooxygenase-2 expression. The protein expression profiles of bile acid-treated CCA cells were studied using a proteomic approach. To elucidate the possible mechanisms involved in the bile acid-mediated enhancement of CCA cell migration, the effects of six bile acids, including cholic, deoxycholic, taurocholic, taurodeoxycholic, glycocholic and glycodeoxycholic acid, on the migration of CCA cells were examined using wound healing assays. Subsequently, the possible proteins involved in enhanced CCA cell migration were investigated using a proteomic approach. Changes to the protein expression profiles of CCA cells following bile acid treatment was examined using two-dimensional electrophoresis and mass spectrometry. The results demonstrated that cholic and deoxycholic acid significantly enhanced the migration of CCA cells, compared with the treated MMNK-1 control cells. CCA cells had 77 overexpressed protein spots following cholic acid treatment, and 50 protein spots following deoxycholic acid treatment, compared with the treated MMNK-1 control cells. Liquid chromatography tandem-mass spectrometry analysis revealed that coiled-coil domain containing 25 (CCDC25) was significantly overexpressed in cholic acid-treated CCA cells compared with in cholic acid-treated control cells. When the expression levels of CCDC25 were investigated using western blot analysis, CCDC25 was demonstrated to be highly expressed in CCA tissues, but not in the adjacent non-cancerous tissue samples. The identified proteins were further analyzed for protein-chemical interactions using STITCH version 3.1 software. CCDC25 protein was identified to be associated with Son of sevenless homolog 1 and growth factor receptor-bound protein 2, which are involved in EGFR signaling. The results of the present study demonstrated that following cholic acid treatment, CCDC25 is overexpressed in CCA cells, which is associated with significantly enhanced cell migration. This suggests that CCDC25 is a potential therapeutic target for the treatment of patients with CCA.
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http://dx.doi.org/10.3892/ol.2017.6446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5529998PMC
August 2017

Detection assay for HPV16 and HPV18 by loop‑mediated isothermal amplification with lateral flow dipstick tests.

Mol Med Rep 2017 May 23;15(5):3203-3209. Epub 2017 Mar 23.

Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.

Cervical cancer is the third highest cause of death in developing countries and most commonly results from high‑risk human papillomavirus (HR‑HPV) infection. Among HR‑HPV genotypes, HPV16 and HPV18 are the most prevalent in cervical cancers. Therefore, the present study aimed to develop a detection assay for HPV16 and HPV18 infection using loop‑mediated isothermal amplification (LAMP) with lateral flow dipstick (LFD) tests. This assay is a simplified, user‑friendly method for the visual detection of HPV genotypes. DNA was extracted from clinical tissue samples, and HPV genotyping was performed using nested polymerase chain reaction (PCR). The clinical samples were demonstrated to include 44 HPV16‑positive, 18 HPV18‑positive and 80 HPV‑negative samples. All DNA samples were also used as templates for a LAMP reaction (30 min at 65˚C), and subsequently, a fluorescein isothiocyanate‑labelled probe was hybridized with the reaction product. Finally, the LFD test was performed. The sensitivity of the LAMP‑LFD test was higher than LAMP‑turbidity, exhibiting up to 100‑fold higher sensitivity for HPV16 and 10‑fold higher sensitivity for HPV18. All HPV16 and HPV18‑positive samples generated positive results in both assays; however, 22 samples detected as HPV‑negative by LAMP‑turbidity exhibited positive results by LAMP‑LFD test (22 of 80 samples). Therefore, these samples were further examined using quantitative (q)PCR. The results demonstrated that 20 out of the 22 samples designated positive by LAMP‑LFD, but negative by LAMP turbidity, gave a positive result with qPCR, while the remaining 2 samples were negative by qPCR. The present results suggested that LAMP‑LFD provided higher sensitivity than LAMP‑turbidity and nested PCR. Thus, the LAMP‑LFD test developed in the present study might be useful for the detection of HPV16 and HPV18 in local hospitals.
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http://dx.doi.org/10.3892/mmr.2017.6370DOI Listing
May 2017

 Thymosin β10 as a predictive biomarker of response to 5-fluorouracil chemotherapy in cholangiocarcinoma.

Ann Hepatol 2016 Jul-Aug;15(4):577-85

Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.

Unlabelled:  Introduction and aim. 5-Fluorouracil (5-FU) is the most commonly used chemotherapeutic drug in the treatment of cholangiocarcinoma (CCA). Since development of drug resistance to 5-FU in CCA patients is the primary cause of treatment failure, a better understanding of the mechanism of drug resistance of this cancer is essential to improve the efficacy of 5-FU in CCA therapy.

Material And Methods: A 5-FU resistant CCA cell line (M214-5FUR) for a comparative chemo-resistance study was established. Real time RT-PCR was used to determine gene expression levels. Cell cytotoxicity was measured by the MTT assay. Protein expression levels were detected by the immunofluorescene method.

Results: It was found that 5-FU resistance was associated with the overexpression of T?10 in CCA cell lines. 5-FU treatment at various concentrations induced the expressions of T?10 and ABC transporters (ABCB1, ABCG2 ABCA3) in two CCA cell lines, KKU-M055 and KKU-M214. M214-5FUR, a 5-FU-resistant cell line, exhibited a 5-FU resistant phenotype with a 16-fold extremely high expression of T?10 and ABC transporters, as compared to the parental cells, KKU-M214. siRNA targeted to T?10 significantly reduced expression of ABC transporters tested in the M214-5FUR cells (P < 0.05).

Conclusions: The present novel findingsof T?10 connected with drug resistance as shown in this study provides a new insight for the therapeutic value of T?10 as a predictive biomarker of 5-FU chemoresistance. Inhibiting T?10 may be a valuable adjunct for suppression of ABC transporters and sensitizing chemotherapy treatment, especially 5-FU in CCA patients.
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February 2017

Classification of Gemcitabine resistant Cholangiocarcinoma cell lines using synchrotron FTIR microspectroscopy.

J Biophotonics 2017 Mar 21;10(3):367-376. Epub 2016 Mar 21.

Department of Anatomy and Developmental Biology, Monash University, Victoria, Australia.

Cholangiocarcinoma (CCA), a cancer of bile duct epithelium, is a major health problem in Thailand especially in the northeast. Overall treatment outcomes have not shown much improvement because the disease is usually detected at an advanced stage and often shows chemotherapeutic resistance. High-throughput Fourier Transform Infrared (FTIR) microspectroscopy can be used for cell classification and has the potential to diagnose cancer and possibly predict chemo-response. This study was aimed to differentiate gemcitabine-sensitive and gemcitabine-resistant induction in two CCA cell lines (KKU-M139 and KKU-M214) and xenograft tissues using synchrotron-FTIR microspectroscopy. Partial Least Squares Discriminant Analysis (PLS-DA) could discriminate between chemo-sensitive and chemo-resistant cells in the FTIR fingerprint spectral region (1800-1000 cm ) with more than 90% sensitivity and specificity. The chemo-resistant and chemo-sensitive phenotypes were different in protein (amide I, amide II), lipids (carbonyl group and CH deformation) and phosphodiester from nucleic acids. Additionally, spectra from xenograft tissues showed similar results to the cell line study with marked differences between chemo-resistant and chemo-sensitive CCA tissues, and PLS-DA could discriminate the chemotherapeutic response with 98% sensitivity and specificity. This is the first study to demonstrate the use of FTIR microspectroscopy to assess chemo-response both in vitro and in vivo.
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http://dx.doi.org/10.1002/jbio.201500253DOI Listing
March 2017

Comparison of Automated and Conventional IHC Visual Scoring Analysis for MHC Class I and Tapasin Expression in Cervical Carcinoma.

J Med Assoc Thai 2016 Jan;99 Suppl 1:S67-75

Background: Cervical cancer (CXCA) is the second most common cancer among women in Thailand and worldwide. Immune evasion caused by down-regulation of host immune responsive genes, such as MHC class I and loss of antigen processing machinery (APM), presents a capability leading to cancer development. Immunohistochemical staining (HC) is regarded as a common technique for protein marker detection in clinical laboratories. At present, IHC automation has been launched to facilitate the speed and feasibility to replace conventional IHC. However, evaluation of its use is still limited.

Objective: This study aimed to evaluate IHC scoring by automated visual analysis compared to conventional IHC analysis.

Material And Method: The paraffin-embedded tissues of 96 invasive CXCA were processed using a tissue microarray (TMA) platform followed by automated IHC staining of the anti-MHC class I (heavy chain, β2M) and an APM-Tapasin expression. Conventional IHC and automated slide scanning with scoring visual analysis were compared.

Results: The results showed significant association between conventional and automated IHC evaluation (p-value > 0.05, Chi-square) for MHC class I and Tapasin stated in percentage of positive cancer cells, whereas intensity was found (p-value < 0.05, Chi-square) with moderate agreement (p-value < 0.001, kappa) 0.434-0.615 and 0.353-0.554, respectively. After calculated values, the results showed significant association between conventional and automated IHC evaluation (p-value > 0.05, Chi-square) for MHC class I and Tapasin with the highest agreement level (p-value < 0.001, kappa) of summation 0.595-0.755 and multiply scoring 0.633-0.689, respectively.

Conclusion And Discussion: The automation softwarefor IHC scoring and interpretation can be used for the determination of MHC class I and Tapasin in CXCA. In addition, an antigen presentation pattern must be included to allow an accurate result for MHC class I in clinical use. An appropriate sample size and design of staging coverage as well as clinical prognosis outcomes of progression should be used infurther investigation.
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January 2016

The evaluation of loop-mediated isothermal amplification-quartz crystal microbalance (LAMP-QCM) biosensor as a real-time measurement of HPV16 DNA.

J Virol Methods 2016 Mar 13;229:8-11. Epub 2015 Dec 13.

Department of Biomedical Engineering, Faculty of Engineering, Mahidol University, Nakhon Pathom 73170, Thailand.

We have previously developed quartz crystal microbalance biosensor integrated with loop-mediated isothermal amplification (LAMP-QCM) for human papillomavirus (HPV) type58 DNA detection. Infection with HPV, particularly HPV16, remains a serious health problem due to its major risk factor contributing to cervical cancer. In the present study, LAMP-QCM biosensor was evaluated in terms of a quantitative assay for copy number of HPV16 DNA in cervical samples compared to quantitative PCR using TaqMan assay (TaqMan-qPCR). The detection limit of LAMP-QCM was found to be 10 fold more sensitive than TaqMan-qPCR with 100% specificity and 7.6% imprecision. Different plot of HPV16 DNA copy number using Bland-Altman analysis revealed 94% correlation between LAMP-QCM and qPCR. We therefore concluded that the developed LAMP-QCM biosensor provides a possible rapid and sensitive assay for HPV16 DNA quantification in a routine laboratory.
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http://dx.doi.org/10.1016/j.jviromet.2015.12.005DOI Listing
March 2016

Targeting the ∆133p53 isoform can restore chemosensitivity in 5-fluorouracil-resistant cholangiocarcinoma cells.

Int J Oncol 2015 Dec 5;47(6):2153-64. Epub 2015 Oct 5.

Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.

Lack of the normal p53 transactivation domain, ∆133p53 isoform exhibits anti-p53 function. Many studies report the correlation between ∆133p53 expression and poor survival in various cancers, including cholangiocarcinoma (CCA), which is a cancer of the bile ducts. CCA almost always results in short survival times. The relevance of ∆133p53 to drug resistance in CCA is not yet well understood. This study aimed to demonstrate the association between ∆133p53 and 5-fluorouracil (5-FU) resistance in CCA. ∆133p53 protein was highly expressed in CCA patients with poor outcome compared to favorable outcome but was not statistically significant. However, a significant correlation was found between normalized ∆133p53 levels and 5-FU resistance which was defined by an ex vivo histoculture drug response assay (P=0.019). Two stable 5-FU-resistant CCA cell lines, KKU-M139R (IC50 38.8 µM) and KKU-M214R (IC50 39.5 µM), were used as a model to evaluate the role of ∆133p53. Increased ∆133p53 was correlated with 5-FU in a dose-dependent manner. The transient knockdown of ∆133p53 expression can restore drug sensitivity in both resistant CCA cells with 11- to 45-fold reduction of IC50 compared to control. Upon ∆133p53 silencing, apoptotic signaling was enhanced by the upregulation of Bax and downregulation of Bcl-2. Additionally, p21 and p27 were upregulated, resulting in cell cycle arrest at G2. Inhibition of colony formation and prolong doubling time were also observed. Our findings demonstrated that chemosensitivity can be modulated via targeting of ∆133p53 suggesting the potential use of ∆133p53 as a candidate for targeting therapy in CCA.
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http://dx.doi.org/10.3892/ijo.2015.3188DOI Listing
December 2015

Establishment and characterization of gemcitabine-resistant human cholangiocarcinoma cell lines with multidrug resistance and enhanced invasiveness.

Int J Oncol 2015 Jul 22;47(1):398-410. Epub 2015 May 22.

Department of Forensic Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.

To establish and characterize the gemcitabine-resistant cholangiocarcinoma (CCA) cell lines, CCA KKU‑M139 and KKU‑M214 cell lines were exposed stepwisely to increasing gemcitabine (GEM). The resultant drug-resistant cell lines, KKU‑M139/GEM and KKU‑M214/GEM, retained the resistant phenotype in drug-free medium at least for 2 months. Sulforhodamine B assay demonstrated that KKU‑M139/GEM and KKU‑M214/GEM were 25.88- and 62.31-fold more resistant to gemcitabine than their parental cells. Both gemcitabine-resistant cell lines were cross-resistant to 5-fluorouracil (5-FU), doxorubicin and paclitaxel indicating their multidrug-resistant nature. Using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR and western blot analyses, gemcitabine-resistant cells showed upregulation of RRM1 and downregulation of hENT1 and dCK. In relation to multidrug resistance, these cell lines showed upregulation of multidrug resistance protein 1 (MRP1) leading to an increase of drug efflux. Using cell adhesion and Boyden chamber transwell assays, these cell lines also showed higher cell adhesion, migration and invasion capabilities via the activations of protein kinase C (PKC), focal adhesion kinase (FAK), extracellular signal-regulated kinase-1/2 (ERK1/2) and nuclear factor-κB (NF-κB). Higher activity of matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (uPA) was also observed by a gelatin zymography assay and a casein-plasminogen zymography assay. Flow cytometry analysis indicated the G2/M arrest regulated by downregulation of cyclin B1 and cyclin-dependent kinase 1 (Cdk1) resulted in an extended population doubling time. Using Annexin V/propidium iodide staining, evasion of apoptosis via an intrinsic pathway was observed in both cell lines in association with upregulation of Bcl-2 and downregulation of Bax. Interestingly, Fas was additionally downregulated in KKU‑M214/GEM supporting the view of its higher GEM resistant characteristics. These findings indicate that long-term exposure of CCA cell lines to gemcitabine induce not only multidrug resistance but also enhance their invasiveness.
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http://dx.doi.org/10.3892/ijo.2015.3019DOI Listing
July 2015

Silk fibroin/gelatin-chondroitin sulfate-hyaluronic acid effectively enhances in vitro chondrogenesis of bone marrow mesenchymal stem cells.

Mater Sci Eng C Mater Biol Appl 2015 24;52:90-6. Epub 2015 Mar 24.

Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand. Electronic address:

Tissue engineering is becoming promising for cartilage repair due to the limited self-repair capacity of cartilage tissue. We previously fabricated and characterized a three-dimensional silk fibroin/gelatin-chondroitin sulfate-hyaluronic acid (SF-GCH) scaffold and showed that it could promote proliferation of human bone marrow mesenchymal stem cells (BM-MSCs). This study aimed to evaluate its biological performance as a new biomimetic material for chondrogenic induction of BM-MSCs in comparison to an SF scaffold and conventional pellet culture. We found that the SF-GCH scaffold significantly enhanced the proliferation and chondrogenic differentiation of BM-MSCs compared to the SF scaffold and pellet culture in which the production of sulfated glycoaminoglycan was increased in concordance with the up-regulation of chondrogenic-specific gene markers. Our findings indicate the significant role of SF-GCH by providing a supportive structure and the mimetic cartilage environment for chondrogenesis which enables cartilage regeneration. Thus, our fabricated SF-GCH scaffold may serve as a potential biomimetic material for cartilage tissue engineering.
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http://dx.doi.org/10.1016/j.msec.2015.03.043DOI Listing
February 2016

DNA methylation level of OPCML and SFRP1: a potential diagnostic biomarker of cholangiocarcinoma.

Tumour Biol 2015 Jul 5;36(7):4973-8. Epub 2015 Feb 5.

Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand.

Cholangiocarcinoma (CCA) is a malignancy of the bile duct epithelium which is caused by liver fluke infection. The clinical symptoms of CCA were revealed as the disease progresses to advanced stage. Thus, specific diagnostic biomarkers are important for this fatal disease. We applied methylation-sensitive high-resolution melting (MS-HRM) to quantify DNA methylation levels of opioid binding protein/cell adhesion molecule-like gene (OPCML) and Secreted frizzled-related protein 1 (SFRP1) in 73 primary CCA and 10 adjacent normal tissues and evaluated the sensitivity, specificity, and accuracy of the assay. The median methylation level of OPCML in CCA was 38.7 % (ranged from 0 to 82.2 %) and of SFRP1 was 31.5 % (ranged from 0 to 86.2 %). Methylation cutoff values of OPCML and SFRP1 derived from adjacent normal tissue were 6.90 and 10.44 %, respectively. With these cutoff values, the area under curve (AUC) of OPCML was 0.932 (95 % CI 0.878-0.986) and of SFRP1 was 0.951 (95 % CI 0.905-0.996). The sensitivity, specificity, and accuracy of OPCML were 89.04, 100, and 90.36 %, respectively, and of SFRP1 were 83.56, 100, and 85.54 %, respectively. In conclusion, the DNA methylation levels of OPCML and SFRP1 could be potential biomarkers for diagnosis of CCA with high specificity, sensitivity, and accuracy, in particular for biopsy specimens. Further validation in noninvasive samples such as serum or plasma is warranted for clinical applicability, especially as early diagnostic biomarkers.
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http://dx.doi.org/10.1007/s13277-015-3147-2DOI Listing
July 2015

Discrimination of micromass-induced chondrocytes from human mesenchymal stem cells by focal plane array-Fourier transform infrared microspectroscopy.

Talanta 2014 Dec 27;130:39-48. Epub 2014 Jun 27.

Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand. Electronic address:

Rapid and sensitive methods for identifying stem cell differentiation state are required for facilitating future stem cell therapies. We aimed to evaluate the capability of focal plane array-Fourier transform infrared (FPA-FTIR) microspectroscopy for characterising the differentiation of chondrocytes from human mesenchymal stem cells (hMSCs). Successful induction was validated by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis for collagen and aggrecan expression as chondrocyte markers in parallel with the spectroscopy. Spectra derived from chondrocyte-induced cells revealed strong IR absorbance bands attributed to collagen near 1338 and 1234 cm(-1) and proteoglycan at 1245 and 1175-960 cm(-1) compared to the non-induced cells. In addition, spectra from control and induced cells are segregated into separate clusters in partial least squares discriminant analysis score plots at the very early stages of induction and discrimination of an independent set of validation spectra with 100% accuracy. The predominant bands responsible for this discrimination were associated with collagen and aggrecan protein concordant with those obtained from RT-PCR and Western blot techniques. Our findings support the capability of FPA-FTIR microspectroscopy as a label-free tool for stem cell characterization allowing rapid and sensitive detection of macromolecular changes during chondrogenic differentiation.
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http://dx.doi.org/10.1016/j.talanta.2014.05.037DOI Listing
December 2014

Loss of E-cadherin promotes migration and invasion of cholangiocarcinoma cells and serves as a potential marker of metastasis.

Tumour Biol 2014 Sep 28;35(9):8645-52. Epub 2014 May 28.

Center for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand.

Tumor progression is characterized by loss of cell adhesion and increase of invasion and metastasis. E-cadherin, a cell adhesion molecule, is frequently downregulated and has been proposed as an important mediator in epithelial-mesenchymal transition (EMT) in tumors. In this study, we investigated the expression of E-cadherin and its association with cancer invasion and prognosis in cholangiocarcinoma (CCA). Immunohistochemistry results demonstrated a statistically significant association between the positive metastasis status with low E-cadherin protein expression in human CCA tissues (P = 0.04). Statistical trends were identified for low E-cadherin level and shorter survival time (P = 0.08). Targeting the E-cadherin expression in CCA cells with siRNA caused upregulation of vimentin, a mesenchymal marker, and disappearance of the E-cadherin/β-catenin adhesion complex from cell membranes. Moreover, migration and invasion abilities of the cells were increased under this condition. These findings suggest that reduction of E-cadherin contributes to CCA progression by attenuating the strength of cellular adhesion, which affects motility as well as regulating the expression of EMT-related genes during CCA invasion and metastasis. Thus, E-cadherin can act as a central modulator of tumor cell phenotype and is a potential metastasis marker in CCA.
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http://dx.doi.org/10.1007/s13277-014-2087-6DOI Listing
September 2014

Chitinase 3 like 1 is associated with tumor angiogenesis in cervical cancer.

Int J Biochem Cell Biol 2014 Jun 29;51:45-52. Epub 2014 Mar 29.

Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand. Electronic address:

Elevated serum levels of a secreted glycoprotein chitinase 3 like 1 (CHI3L1) are associated with poor prognosis and short survival time of patients with cervical cancer (CxCa). Our previous microarray data showed the increased expression of CHI3L1 in invasive CxCa compared to normal tissue, implicating a potential role of CHI3L1 in CxCa. To establish the pathological role of CHI3L1 in the development of CxCa, this study focused on its expression in CxCa and angiogenic impacts in tumor vessel formation. CHI3L1 activated angiogenesis by promoting endothelial cell migration and tube formation in vitro but failed to protect CxCa cell lines, CaSki and HeLa against apoptosis induced by γ-irradiation. In addition, the capability of CHI3L1 to induce proliferation and migration of CaSki and HeLa cells was cell type specific. In an analysis of 103 specimens from CxCa patients, increased expression levels of CHI3L1 mRNA and protein in invasive CxCa were 4-fold (P<0.05) and 2-fold (P<0.01), respectively, stronger than those in normal subjects. The immunostaining of CHI3L1 was positively correlated with VEGF expression (P=0.0019) and microvessel density (P=0.0110). Moreover, CHI3L1 expression was also positively associated with cancer metastasis (P=0.011). The data suggest the crucial role of CHI3L1 by promoting angiogenesis, which may contribute to the development and progression of CxCa. The findings help establish CHI3L1 as a prognostic biomarker and therapeutic target for CxCa patients.
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http://dx.doi.org/10.1016/j.biocel.2014.03.021DOI Listing
June 2014

Ratio disruption of the ∆133p53 and TAp53 isoform equilibrium correlates with poor clinical outcome in intrahepatic cholangiocarcinoma.

Int J Oncol 2013 Apr 8;42(4):1181-8. Epub 2013 Feb 8.

Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences; Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.

All p53 family members are expressed in several isoforms through alternative promoters and alternative splicing. However, the significance of these isoforms is not yet well understood in cholangiocarcinoma (CCA). In this study, we investigated the expression of p53, p63, p73 and their isoforms at the mRNA and protein levels in CCA. The overexpression of ∆133p53 was observed in the CCA cell lines and clinical specimens. Moreover, the high expression of ∆133p53/TAp53 correlated with short overall survival (p<0.001). Defective p53, including mutant and ∆Np53, was associated with poor prognosis (p<0.024). Multivariate analysis demonstrated that ∆133p53/TAp53 and mutant p53 protein may be used as independent prognostic factors for CCA. To our knowledge, this is the first report of the use of ∆133p53/TAp53 as a potential biomarker in CCA.
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http://dx.doi.org/10.3892/ijo.2013.1818DOI Listing
April 2013

Activated charcoal enhanced the antigen-expression and dendritic cell maturation of the vaccine using Listeria-platform.

Kobe J Med Sci 2012 Sep 13;58(3):E63-71. Epub 2012 Sep 13.

Department of Pathology, College of Medicine and Public Health, Ubon Ratchathani University, Ubon Ratchathani, Thailand.

Background: Listeria monocytogenes (LM) has been used as a vaccine vector based upon its ability to induce a strong cell-mediated immune response. LM inactivated with γ-irradiation retains immunogenic properties and is an attractive platform for clinical use since it would have improved safety concerns compared to live vectors. Activated charcoal has been shown to enhance expression of LM proteins such as PrfA.

Aim: To investigate the effect of various growth conditions supplemented with activated charcoal on recombinant antigen expression.

Methods: We prepared γ-irradiated ovalbumin-expressing LM (LM-OVA) after growth under various culture conditions. We cultured LM-OVA at various temperatures including 25°C, 37°C and 37°C with activated charcoal and compared OVA expression by western blot analysis, dendritic cells maturation and OVA-specific T cells.

Results: The OVA expression was highest in γ-irradiated LM-OVA grown with activated charcoal at 37°C. Compared to other growth conditions, γ-irradiated LM-OVA grown with activated charcoal at 37°C induce better DC maturation as well as production of the highest number of antigen-specific IFN γ-secreting T cells.

Conclusion: The further study should be demonstrated the potential to alter growth conditions to enhance OVA expression resulting for vaccine vectors, thereby improving their safety and efficacy.
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September 2012

Contribution of RIZ1 to regulation of proliferation and migration of a liver fluke-related cholangiocarcinoma cell.

Asian Pac J Cancer Prev 2012 ;13(8):4007-11

Department of Biomedical Sciences, Graduate School, Khon Kaen University, Khon Kaen, Thailand.

Purpose: Retinoblastoma-interacting zinc finger gene (RIZ1) is a tumor suppressor gene which is highly inactivated by promoter hypermethylation in patients with liver fluke-related cholangiocarcinoma (CCA). Epigenetic aberration of this gene might withdraw the ability to restrain tumor cell proliferation and migration. We aimed to define the role of RIZ1 on cell proliferation and migration in CCA cell line.

Materials And Methods: Small interference RNA (siRNA) was used to knock down the expression of RIZ1 in a CCA-derived cell line in which cell proliferation and cell migration were performed.

Results: A predominant nuclear localization of RIZ1 was observed. Reduction of RIZ1 by siRNA augmented cell proliferation and migration.

Conclusion: The result suggested that RIZ1 might play a role in regulating cell proliferation and migration in CCA. Reduction of RIZ1 expression may aggravate the progression of CCA.
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http://dx.doi.org/10.7314/apjcp.2012.13.8.4007DOI Listing
April 2013

The development of DNA-based quartz crystal microbalance integrated with isothermal DNA amplification system for human papillomavirus type 58 detection.

Biosens Bioelectron 2013 Feb 17;40(1):252-7. Epub 2012 Aug 17.

Graduate School, Khon Kaen University, Khon Kaen 40002, Thailand.

To address the effect of dramatic change in temperature and viscosity during PCR process on quartz crystal microbalance (QCM) sensor and to increase the sensitivity, isothermal amplification was employed in the system. We combined loop-mediated isothermal amplification (LAMP) technique with QCM, called as LAMP-QCM, for detection of high-risk human papillomavirus viral DNA type 58 (HPV-58) which is commonly found in Asian women. The liquid-phase LAMP-QCM prototype comprised the frequency counter, a temperature control device and housing of the quartz crystal with polished gold electrodes on both sides. QCM detection signal was monitored in real-time based on an avidin-biotin binding between avidin coated QCM surface and specific biotinylated LAMP products. Analytical performance was evaluated for precision, sensitivity and specificity. A plasmid clone containing the HPV-58 sequence was diluted from 10(6) to 1 copy and used for detection limit. Cut-off value was estimated at 28.8 Hz from negative viral template. The system could detect 100 copies with Δf at 34.0±3.6 Hz compared to 1000 copies detected by conventional LAMP. No cross-reaction was observed with other HPV types. The HPV-58 detection was compared among LAMP-QCM, conventional LAMP and nested PCR in 50 cervical cancer tissues. The positive rate of LAMP-QCM was higher than that of conventional LAMP with 100% sensitivity and 90.5% specificity. The integrated LAMP-QCM system has improved the detection limit up to ten times compared to conventional LAMP with less-time consuming.
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http://dx.doi.org/10.1016/j.bios.2012.07.033DOI Listing
February 2013

Tumor necrosis factor-α (TNF-α) stimulates the epithelial-mesenchymal transition regulator Snail in cholangiocarcinoma.

Med Oncol 2012 Dec 19;29(5):3083-91. Epub 2012 Aug 19.

Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand.

Epithelial-mesenchymal transition (EMT) is a series of events during which epithelial cells lose many of their epithelial characteristics and take on properties that are typical of mesenchymal cells that lack cell-cell adhesion properties. EMT may be activated by various types of growth factors or inflammatory cytokines. In many types of epithelial cancers, the EMT-derived tumor cells are susceptible to metastasis. During tumor progression, epithelial cells acquire a gene expression pattern closely resembling that of mesenchymal cells. This study aimed to investigate the expression of the EMT-associated transcription factor Snail and an adhesion molecule E-cadherin in cholangiocarcinoma (CCA) tissues. The effect of TNF-α on EMT activation in CCA cells was also demonstrated. The qRT-PCR analysis revealed that Snail expression significantly increased in CCA (P = 0.01) and was correlated with tumor metastasis (P = 0.02). The expression of Snail was inversely associated with E-cadherin (P = 0.004). The stimulation of TNF-α enhances migration behavior and showed significantly induced expression of Snail in CCA cell lines, whereas expression of E-cadherin and CK-19 (the epithelial marker) was reduced. Immunofluorescence analysis revealed that TNF-α-treated CCA cell lines increased nuclear translocation of Snail, whereas E-cadherin was dramatically decreased. Our findings suggest that the changes in the expression of Snail or E-cadherin might regulate EMT development in CCA resulting in promoting tumor progression. Overexpression of Snail could be used as a prognostic marker for monitoring the treatment efficiency of CCA patients.
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http://dx.doi.org/10.1007/s12032-012-0305-xDOI Listing
December 2012

Serum adhesion molecule-1 (ICAM-1) as a potential prognostic marker for cholangiocarcinoma patients.

Asian Pac J Cancer Prev 2012 ;13 Suppl:107-14

Centre Research and Development Medical Diagnostic, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand.

Cholangiocarcinoma (CCA) is a malignancy of bile ducts with a high incidence of invasion and metastasis. This disease is often detected in advanced stages because of the difficulties of early diagnosis, leading to a high mortality rate. However, biomarkers for early CCA detection are still lacking. In this study, to identify potential biomarker proteins, differential secretome analysis by the GeLC-MS/MS approach was applied with four CCA cell lines and a control immortalized cholangiocyte cell line. Among 78 up-regulated proteins, 53 including ICAM- 1 were exclusively expressed in four CCA secretomes but not in MMNK1. Based on this result, we measured ICAM-1 levels in serum samples of CCA patients and healthy controls and found significantly higher values in CCA patients' sera. Receiver operating characteristic curve analysis suggested that serum ICAM-1 level could be a discriminatory diagnostic marker for CCA and healthy controls (area under curve=0.829) with a sensitivity of 77% and a specificity of 70% at a cut off value of 167 ng/ml. Moreover, the serum ICAM-1 showed positive correlations with alkaline phosphatase and carcinoembryonic antigen levels. Comparison of ICAM-1 levels of paired pre- and post-operative sera of 12 cases revealed significant decrease after tumor resection. However, serum ICAM-1 levels were not significantly different between CCA and benign biliary diseases with mainly inflammatory features.
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April 2016

A novel TFF2 splice variant (∆EX2TFF2) correlates with longer overall survival time in cholangiocarcinoma.

Oncol Rep 2012 Apr 7;27(4):1207-12. Epub 2011 Dec 7.

Graduate School, Khon Kaen University, Khon Kaen 40002, Thailand.

Trefoil factor 2 (TFF2) is a member of trefoil factor family found to be overexpressed in many cancers including cholangiocarcinoma (CCA). The majority of studies have focused on wild-type TFF2 (wtTFF2) expression, but information regarding alternative splicing variants of TFF2 mRNA has not been reported. In this study, we aimed to identify and quantify a novel TFF2 splice variant in cholangiocarcinoma (CCA). Seventy-eight tumors and 15 normal adjacent tissues were quantified for the expression of the TFF2 splice variant relative to wild-type (wt) TFF2 mRNA using quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). The ratio of TFF2 splice variant against wtTFF2 was analyzed for associations with clinical parameters. We found a novel TFF2 splice variant, exon 2 skipping (∆EX2TFF2), resulting in a stop codon (TAG) at exon 1. The ∆EX2TFF2/wtTFF2 ratio in tumors was significantly higher than in normal tissue (P<0.01). Interestingly, high ∆EX2TFF2/wtTFF2 ratio was significantly associated with good prognosis compared with low ratio (P=0.017). In contrast, the presence of wtTFF2 protein was associated with poor survival of CCA patients (P=0.034). This is the first report of a trefoil factor splice variant and its potential application as a prognostic biomarker in CCA.
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http://dx.doi.org/10.3892/or.2011.1583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3583483PMC
April 2012

Rapid detection of the most common high-risk human papillomaviruses by loop-mediated isothermal amplification.

J Virol Methods 2011 Dec 27;178(1-2):22-30. Epub 2011 Aug 27.

Department of Clinical Chemistry, Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.

Persistent infection with high-risk human papillomavirus (HPV) is a major risk factor for development of cervical cancer. At present, polymerase chain reaction (PCR)-based methods, the most widely molecular tools used for HPV detection, are time-consuming and require expensive instruments. In this study, loop-mediated isothermal amplification (LAMP) was established for detection of HPV types 16, 18, 45 and 58 which are frequently found in Thailand. The optimal condition for detection of these high risk HPVs was 63°C for 60min. Since a white magnesium pyrophosphate precipitate is a characteristic by product of the LAMP reaction which can be visualized directly by the naked eye, the entire assay time of LAMP is 1h compared to 6-8h of for a nested PCR detection. The detection limit of LAMP assay was shown to be equivalent to nested PCR that could amplify 10(2) copies of HPV-18 and 10(3) copies of HPV 16, 45 and 58, as determined by either turbidity detection or agarose gel electrophoresis. No cross-reaction was observed, indicating that LAMP assay has high type-specificity. The assay showed successful detection of HPV in 56 clinical specimens. Using nested PCR as the gold standard, the sensitivity, specificity, negative predictive values and positive predictive values of LAMP assay were 100%. In conclusion, LAMP assay is a high efficiency, low cost diagnostic tool, useful for rapid, accurate, direct detection of HPV for clinical diagnosis.
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http://dx.doi.org/10.1016/j.jviromet.2011.08.007DOI Listing
December 2011

Characterisation of chondrogenic differentiation of human mesenchymal stem cells using synchrotron FTIR microspectroscopy.

Analyst 2011 Jun 28;136(12):2542-51. Epub 2011 Apr 28.

Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Thailand.

A major limiting factor in stem cell therapy is the accurate identification of the differentiation state of cells destined for transplantation. This study aimed to evaluate the potential of synchrotron radiation Fourier transform infrared (SR-FTIR) microspectroscopy as a novel technique to probe the differentiation state of human mesenchymal stem cells (hMSCs) to chondrocytes over a period of 7, 14 and 21 days of induction. The chondrogenic markers were determined using reverse transcription polymerase chain reaction, histology and immunohistochemistry. The changes of average spectra located near 1338-1230 and 1175-960 cm(-1) indicated increased levels of collagen and aggrecan, respectively, in chondrocyte-induced hMSCs compared with control cells. Classification of independent test spectra using partial least squares discriminant analysis (PLS-DA) could distinguish control and chondrocyte-induced cells with 100% accuracy. We conclude that the SR-FTIR microspectroscopy technique is sensitive for monitoring the differentiation state of stem cells under chondrogenic induction particularly at an early stage. It provides biochemical information that is complimentary to that obtained from conventional techniques, and may give more unambiguous results particularly at the very early stage of cellular differentiation. In addition, the spectroscopic approach is more straightforward, non-destructive and requires less sample preparation compared with the conventional methodologies.
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http://dx.doi.org/10.1039/c1an15182gDOI Listing
June 2011

Trefoil factors: tumor progression markers and mitogens via EGFR/MAPK activation in cholangiocarcinoma.

World J Gastroenterol 2011 Mar;17(12):1631-41

Graduate School, Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.

Aim: To investigate trefoil factor (TFF) gene copy number, mRNA and protein expression as potential biomarkers in cholangiocarcinoma (CCA).

Methods: TFF mRNA levels, gene copy number and protein expression were determined respectively by quantitative reverse transcription polymerase chain reaction (PCR), quantitative PCR and immunohistochemistry in bile duct epithelium biopsies collected from individuals with CCA, precancerous bile duct dysplasia and from disease-free controls. The functional impact of recombinant human (rh)TFF2 peptide treatment on proliferation and epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) signaling was assessed in the CCA cell line, KMBC, by viable cell counting and immunoblotting, respectively.

Results: TFF1, TFF2 and TFF3 mRNA expression was significantly increased in CCA tissue compared to disease-free controls, and was unrelated to gene copy number. TFF1 immunoreactivity was strongly increased in both dysplasia and CCA, whereas TFF2 immunoreactivity was increased only in CCA compared to disease-free controls. By contrast, TFF3 immunoreactivity was moderately decreased in dysplasia and further decreased in CCA. Kaplan-Meier analysis found no association of TFF mRNA, protein and copy number with age, gender, histological subtype, and patient survival time. Treatment of KMBC cells with rhTFF2 stimulated proliferation, triggered phosphorylation of EGFR and downstream extracellular signal related kinase (ERK), whereas co-incubation with the EGFR tyrosine kinase inhibitor, PD153035, blocked rhTFF2-dependent proliferation and EGFR/ERK responses.

Conclusion: TFF mRNA/protein expression is indicative of CCA tumor progression, but not predictive for histological sub-type or survival time. TFF2 is mitogenic in CCA via EGFR/MAPK activation.
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http://dx.doi.org/10.3748/wjg.v17.i12.1631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3070136PMC
March 2011

Effects of thymidine phosphorylase on tumor aggressiveness and 5-fluorouracil sensitivity in cholangiocarcinoma.

World J Gastroenterol 2010 Apr;16(13):1631-8

Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.

Aim: To evaluate the role of thymidine phosphorylase (TP) in cholangiocarcinoma using small interfering RNA (siRNA).

Methods: A human cholangiocarcinoma-derived cell line KKU-M139, which has a naturally high level of endogenous TP, had TP expression transiently knocked down using siRNA. Cell growth, migration, in vitro angiogenesis, apoptosis, and cytotoxicity were assayed in TP knockdown and wild-type cell lines.

Results: TP mRNA and protein expression were decreased by 87.1% + or - 0.49% and 72.5% + or - 3.2%, respectively, compared with control cells. Inhibition of TP significantly decreased migration of KKU-M139, and suppressed migration and tube formation of human umbilical vein endothelial cells. siRNA also reduced the ability of TP to resist hypoxia-induced apoptosis, while suppression of TP reduced the sensitivity of KKU-M139 to 5-fluorouracil.

Conclusion: Inhibition of TP may be beneficial in decreasing angiogenesis-dependent growth and migration of cholangiocarcinoma but may diminish the response to 5-fluorouracil chemotherapy.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2848371PMC
http://dx.doi.org/10.3748/wjg.v16.i13.1631DOI Listing
April 2010

Genetic and epigenetic alterations of RIZ1 and the correlation to clinicopathological parameters in liver fluke-related cholangiocarcinoma.

Exp Ther Med 2010 Mar 1;1(2):385-390. Epub 2010 Mar 1.

Department of Biomedical Sciences, Graduate School ; Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, ; Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.

The retinoblastoma interacting zinc finger (RIZ1) gene is adjacent to D1S228 where microsatellite instability has been associated with poor patient survival in liver fluke-associated cholangiocarcinoma (CCA). An understanding of the molecular mechanisms underlying the carcinogenesis and pathogenesis of CCA is necessary to improve patient survival. Therefore, we determined the genetic and epigenetic alterations of RIZ1 in 81 CCA samples and 69 matched non-tumor tissues. Methylation was found in 31 of 81 (38%) tumor samples and in 5 of 69 (7%) matched non-tumor tissues. Frameshift mutations (2 of 81) and loss of heterozygosity (LOH) (14 of 81) were not common. Statistical analysis found no significant correlation between RIZ1 alterations and clinicopathological features, but RIZPro704 LOH was associated with patient survival in the multivariate analysis. RIZ1 hypermethylation may be one of the crucial molecular events contributing to cholangiocarcinogenesis, and RIZPro704 LOH may adversely impact patient survival. The biological function of RIZ1 in CCA should be further investigated in order to verify its potential role in regulating this cancer.
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http://dx.doi.org/10.3892/etm_00000060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3445879PMC
March 2010