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

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 (Opisthorchis viverrini) 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 O. viverrini 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-1), amino acid residue (∼1396 cm-1), and C═O stretching carboxylic esters (1745 cm-1). Infected tissues also show definitive bands at ∼1280, 1234, and 1201 cm-1 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 O. viverrini-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.

Antitumor Effect of Shikonin, a PKM2 Inhibitor, in Cholangiocarcinoma Cell Lines.

BACKGROUND/AIM: Pyruvate kinase M2 (PKM2) is an enzyme that is predominantly overexpressed in various types of cancer. The role of PKM2 in liver fluke-associated cholangiocarcinoma (CCA) remains unclear. This study aimed to investigate the antitumor activity of shikonin, a PKM2 inhibitor, in CCA cells. MATERIALS AND METHODS: Immunohistochemistry and immunoblotting were used to determine PKM2 expression in CCA tissues and cells. Antiproliferative effects of shikonin were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony-formation and trypan blue exclusion assays. The anti-metastatic activity of shikonin was determined using the Boyden chamber assay. Mechanisms by which shikonin inhibited CCA progression were determined. RESULTS: PKM2 was overexpressed in CCA compared to normal bile duct epithelial cells. Shikonin significantly inhibited growth, and migration of CCA cells while inducing their death. A mechanistic study revealed that antitumor effects of shikonin in CCA cells depended on increased production of reactive oxygen species. CONCLUSION: Shikonin may be a novel therapeutic agent for patients with CCA.

Curative effect of xanthohumol supplementation during liver fluke-associated cholangiocarcinogenesis: Potential involvement of autophagy.

Xanthohumol (XH), a plant flavonoid, was shown to attenuate cholangiocarcinoma (CCA) development induced by the liver fluke Opisthorchis viverrini (Ov) and N-dinitrosomethylamine (NDMA) in the hamster model. We investigated the possible involvement of autophagy, a self-degrading process dysregulated in cancer, in XH chemotherapeutic effect. During cholangiocarcinogenesis, the expression of LC3 (an autophagic marker) was increased in the precancerous stage and decreased in the cancerous stage. The XH-treated ON (Ov plus NDMA) group showed retarded progression of CCA along with increased expression of LC3. The possible relation between autophagy and cell death was investigated in cultured human CCA cells. XH induced apoptosis associated with reduced expression of BCL-2 and increased expression of BAX. In parallel, XH induced the autophagy flux, as testified by increased LC3-II and decreased p62, along with induction of BECLIN1 and Vps34. Inhibition of BECLIN1-dependent autophagy greatly limited XH toxicity in CCA cells. These data suggest that XH attenuates the development of CCA through overstimulation of autophagy which then precipitates apoptosis.

Ecological Niche Model based on Maximum Entropy for mapping distribution of Bithynia siamensis goniomphalos, first intermediate host snail of Opisthorchis viverrini in Thailand.

The snail Bithynia siamensis goniomphalos acts as first intermediate host of the liver fluke, Opisthorchis viverrini, which causes opisthorchiasis in humans. In this study, we used a geographic information system (GIS), remote sensing (RS) and software using the maximum entropy (MaxEnt) algorithm to predict the distribution of B. s. goniomphalos in Thailand on the basis of environmental and climatic factors. The MaxEnt model for B. s. goniomphalos was excellent, with average test AUC values of 0.89. The predicted distribution of B. s. goniomphalos was affected by altitude, land cover, normalized difference vegetation index (NDVI), precipitation in the driest month (BIO 14), land surface temperature (LST) and soil pH. The areas suitable for B. s. goniomphalos were mostly in Northeast Thailand and some northern parts of the country. The presence of B. s. goniomphalos decreases with increasing altitude and increasing NDVI value. Bithynia s. goniomphalos is most likely to occur in paddy fields and cropland. Opisthorchiasis prevalence was directly proportional to the likelihood of snail occurrence as predicted by the model. This is the first time this ecological niche model has been used to predict Bithynia snail distribution and hence to provide a basis for future work of opisthorchiasis prevention in opisthorchiasis-endemic countries.

Current Perspectives on Opisthorchiasis Control and Cholangiocarcinoma Detection in Southeast Asia.

Similar to bile duct cancer or cholangiocarcinoma (CCA) in the western world, opisthorchiasis-associated CCA in Southeast Asia is an aggressive cancer with high mortality rates. It is known to cause a significant health burden in the opisthorchiasis region in Thailand and possibly throughout mainland Southeast. To reduce this health burden, a comprehensive prevention and control program for opisthorchiasis, as well as CCA, is required. In this review, our aim is to provide a brief update of the current situation regarding the natural history of opisthorchiasis and health burden of CCA in Southeast Asia. A comprehensive approach to tackling these issues being implemented in Thailand under the "Cholangiocarcinoma Screening and Care Program" is described. This comprehensive program consists of a three stage prevention and patient care program. The primary prevention component involves opisthorchiasis screening using a new and sensitive urine assay. The secondary prevention component involves screening for CCA and periductal fibrosis, with suspected CCA patients following the protocol for confirmation and appropriate treatment. Due to the eco-epidemiology of opisthorchiasis-induced CCA, the anticipated impacts and outcomes of the program include short-, medium-, and the long-term goals for the reduction of CCA incidence. To achieve long-term sustainable impacts, concerted efforts to raise social awareness and participating action by general public, non-government organizations, and government agencies are necessary. The strategic plans developed for this program can be expanded for use in other endemic areas as well as being a model for use in other chronic diseases.

Targeting hexokinase II as a possible therapy for cholangiocarcinoma.

Overexpression of hexokinase 2 (HKII) has been demonstrated in various cancers. A number of in vitro and in vivo studies in several cancers show the significance of HKII in many cellular processes including proliferation, metastasis and apoptosis. However, the role of HKII in Opisthorchis viverrini (Ov) associated cholangiocarcinoma (CCA) is still unknown. In the present study, the expression and roles of HKII were determined in Ov associated CCA. The expression of HKII was investigated in 82 patients with histologically proven CCAs by immunohistochemistry. HKII was distinctively expressed in CCA tissues. It was rarely expressed in normal bile duct epithelium, but was expressed in hyperplastic/dysplastic and in 82% of CCA bile ducts. The observation was confirmed in the Ov associated hamster model. Suppression of HKII expression using siRNA significantly decreased cell proliferation, migration and invasion of CCA cell lines. Similar results were obtained using lonidamine (LND), an inhibitor of HK. LND significantly inhibited growth of 4 CCA cell lines tested in dose and time dependent fashion. Comparison the cytotoxic effects of LND and siRNA-HKII suggests the off target of LND above 100 µM. In addition, LND in non-cytotoxic doses could suppress migration and invasion of CCA cells. These results indicate the association of HKII in cholangiocarcinogenesis and progression and suggest the possibility of HKII as a therapeutic target for CCA.

Overexpression of lactate dehydrogenase A in cholangiocarcinoma is correlated with poor prognosis.

Lactate dehydrogenase A (LDHA), a key metabolic enzyme, plays a crucial role in the final step of anaerobic glycolysis. Overexpression of LDHA is observed in many human malignancies in association with tumor progression. The purpose of this study was to investigate LDHA expression pattern during carcinogenesis, its clinico-pathological association, and evaluate the prognostic value of LDHA in CCA patients. LDHA expression was investigated using immunohistochemistry technique in both hamster- (n=60) and human-CCA tissues (n=82). Plasma LDH from healthy control (n=40) and CCA patients (n=29) were determined using an enzymatic based assay. The association of LDHA expression with clinico-pathological findings and prognostic value were evaluated by statistical analysis. In the CCA hamster model, an increase of LDHA expression was associated with the progression of CCA-genesis. Higher LDHA overexpression was associated with shorter survival of CCA patients. Multivariate analysis indicated that LDHA expression including histological type and N stage of tumor were independent prognostic risk factor of patient's survival. However, there was no difference in plasma LDH level between CCA patients and healthy controls. LDHA expression is involved in cholangiocarcinogenesis. Overexpression of LDHA can be a marker of poor prognosis in CCA patients and it might be a potential target for CCA treatment.