Wunchana Seubwai

Cepharanthine exerts antitumor activity on cholangiocarcinoma by inhibiting NF-κB

Cholangiocarcinoma (CCA) is a major cause of cancer deaths in northeast Thailand. It is aggressive, highly metastatic, and responds poorly to traditional chemotherapy. We demonstrated the potential for Cepharanthine (CEP), a biscoclaurine alkaloid extracted from Stephania cepharantha, to treat CCA. CEP significantly inhibited growth of human CCA cell lines in a dose‐ and time‐dependent manner, regardless of the histologic type of tumor origin. Increasing cell apoptosis via caspase‐3 and capase‐9 activation was demonstrated in CEP‐treated cells. We found that CEP controlled the growth of CCA cells through nuclear factor‐kappa B (NF‐κB) inactivation by inhibiting nuclear translocation. CEP treatment effectively reduced tumor size in CCA‐inoculated mice without serious side effects. CEP also increased cell apoptosis in primary histocultures of CCA patients’ tissues; this was demonstrated by immunohistochemistry using TUNEL staining. Our results suggest that CEP possesses therapeutic potential against human CCA. (Cancer Sci 2010)

Overexpression of vitamin D receptor indicates a good prognosis for cholangiocarcinoma: implications for therapeutics.

Up‐regulation of vitamin D receptor (VDR) expression has been shown in several tumors and is thought to represent an important endogenous response to tumor progression. The authors aimed to verify the expression of VDR and its clinical significance in histologically proven cholangiocarcinoma (CCA).

22-Oxa-1,25-dihydroxyvitamin D3 efficiently inhibits tumor growth in inoculated mice and primary histoculutre of cholangiocarcinoma.

It is well known that 1α,25‐Dihydroxyvitamin D3(1,25[OH]2D3) restrains cell proliferation and induces differentiation and apoptosis in normal and tumor cells. The authors of this report recently demonstrated that 1,25(OH)2D3 effectively inhibits the proliferation of cholangiocarcinoma (CCA) cell lines. The antitumor activity and the underlying mechanism of 22‐oxa‐D3, an analog of vitamin D, in mice and in tissue cultures from patients with CCA were further explored in the current study.

Orotate phosphoribosyl transferase mRNA expression and the response of cholangiocarcinoma to 5-fluorouracil

AIM To determine whether expression of certain enzymes related to 5-fluorouracil (5-FU) metabolism predicts 5-FU chemosensitivity in cholangiocarcinoma (CCA). METHODS The histoculture drug response assay (HDRA) was performed using surgically resected CCA tissues. Tumor cell viability was determined morphologically with hematoxylin and eosin- and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-stained tissues. The mRNA expression of thymidine phosphorylase (TP), orotate phosphoribosyl transferase (OPRT), thymidylate synthase (TS), and dihydropyrimidine dehydrogenase (DPD) was determined with real-time reverse transcriptase-polymerase chain reaction. The levels of gene expression and the sensitivity to 5-FU were evaluated. RESULTS Twenty-three CCA tissues were obtained from patients who had been diagnosed with intrahepatic CCA and who underwent surgical resection at Srinagarind Hospital, Khon Kaen University from 2007 to 2009. HDRA was used to determine the response of these CCA tissues to 5-FU. Based on the dose-response curve, 200 μg/mL 5-FU was selected as the test concentration. The percentage of inhibition index at the median point was selected as the cut-off point to differentiate the responding and non-responding tumors to 5-FU. When the relationship between TP, OPRT, TS and DPD mRNA expression levels and the sensitivity of CCA tissues to 5-FU was examined, only OPRT mRNA expression was significantly correlated with the response to 5-FU. The mean expression level of OPRT was significantly higher in the responder group compared to the non-responder group (0.41 ± 0.25 vs 0.22 ± 0.12, P < 0.05). CONCLUSION OPRT mRNA expression may be a useful predictor of 5-FU chemosensitivity of CCA. Whether OPRT mRNA could be used to predict the success of 5-FU chemotherapy in CCA patients requires confirmation in patients.

High glucose enhances progression of cholangiocarcinoma cells via STAT3 activation

Epidemiological studies have indicated diabetes mellitus (DM) as a risk of cholangiocarcinoma (CCA), however, the effects and mechanisms of high glucose on progression of CCA remain unclear. This study reports for the first time of the enhancing effects of high glucose on aggressive phenotypes of CCA cells via STAT3 activation. CCA cells cultured in high glucose media exerted significantly higher rates of cell proliferation, adhesion, migration and invasion than those cultured in normal glucose. The phosphokinase array revealed STAT3 as the dominant signal activated in response to high glucose. Increased nuclear STAT3, p-STAT3 and its downstream target proteins, cyclin D1, vimentin and MMP2, were shown to be underling mechanisms of high glucose stimulation. The link of high glucose and STAT3 activation was confirmed in tumor tissues from CCA patients with DM that exhibited higher STAT3 activation than those without DM. Moreover, the levels of STAT3 activation were correlated with the levels of blood glucose. Finally, decreasing the level of glucose or using a STAT3 inhibitor could reduce the effects of high glucose. These findings suggest that controlling blood glucose or using a STAT3 inhibitor as an alternative approach may improve the therapeutic outcome of CCA patients with DM.

Aberrant expression of NF-κB in liver fluke associated cholangiocarcinoma: implications for targeted therapy.

Background Up-regulation and association of nuclear factor kappa B (NF-κB) with carcinogenesis and tumor progression has been reported in several malignancies. In the current study, expression of NF-κB in cholangiocarcinoma (CCA) patient tissues and its clinical significance were determined. The possibility of using NF-κB as the therapeutic target of CCA was demonstrated. Methodology Expression of NF-κB in CCA patient tissues was determined using immunohistochemistry. Dehydroxymethylepoxyquinomicin (DHMEQ), a specific NF-κB inhibitor, was used to inhibit NF-κB action. Cell growth was determined using an MTT assay, and cell apoptosis was shown by DNA fragmentation, flow cytometry and immunocytofluorescent staining. Effects of DHMEQ on growth and apoptosis were demonstrated in CCA cell lines and CCA-inoculated mice. DHMEQ-induced apoptosis in patient tissues using a histoculture drug response assay was quantified by TUNEL assay. Principal Findings Normal bile duct epithelia rarely expressed NF-κB (subunits p50, p52 and p65), whereas all CCA patient tissues (n  =  48) over-expressed all NF-κB subunits. Inhibiting NF-κB action by DHMEQ significantly inhibited growth of human CCA cell lines in a dose- and time-dependent manner. DHMEQ increased cell apoptosis by decreasing the anti-apoptotic protein expressions–Bcl-2, XIAP–and activating caspase pathway. DHMEQ effectively reduced tumor size in CCA-inoculated mice and induced cell apoptosis in primary histocultures of CCA patient tissues. Conclusions NF-κB was over-expressed in CCA tissues. Inhibition of NF-κB action significantly reduced cell growth and enhanced cell apoptosis. This study highlights NF-κB as a molecular target for CCA therapy.

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.

Diabetes mellitus: Possible risk and promoting factors of cholangiocarcinoma: Association of diabetes mellitus and cholangiocarcinoma

The highest incidence of Cholangiocarcinoma (CCA), a malignancy of bile duct epithelia, is in the Northeast of Thailand. The liver fluke, Opisthorchis viverrini, is the known risk factor for CCA development in this region. Approximately 1% of O. viverrini infected individuals develop CCA. There could be other factors that influence the cholangiocarcinogenesis particularly in the O. viverrini infected individuals. The global epidemiological studies of risk factors for CCA in non-O. viverrini related patients indicated diabetes mellitus (DM) as a risk factor of CCA. The molecular studies in many cancers indicated that high levels of glucose, insulin and an obese condition directly and indirectly enhanced growth of cancers. For O. viverrini associated CCA, there is limited information related to DM and CCA development. High mortality rates of CCA and DM, however, were reported in the same geographical areas of northeastern Thailand. Whether DM is a factor that enhances CCA development in O. viverrini infected individuals or promotes CCA progression are discussed in a perspective of epidemiological and molecular studies.

Targeted delivery of 5-fluorouracil to cholangiocarcinoma cells using folic acid as a targeting agent

There are limits to the standard treatment for cholangiocarcinoma (CCA) including drug resistance and side effects. The objective of this study was to develop a new technique for carrying drugs by conjugation with gold nanoparticles and using folic acid as a targeting agent in order to increase drug sensitivity. Gold nanoparticles (AuNPs) were functionalized with 5-fluorouracil (5FU) and folic acid (FA) using polyethylene glycol (PEG) shell as a linker (AuNPs-PEG-5FU-FA). Its cytotoxicity was tested in CCA cell lines (M139 and M213) which express folic acid receptor (FA receptor). The results showed that AuNPs-PEG-5FU-FA increased the cytotoxic effects in the M139 and M213 cells by 4.76% and 7.95%, respectively compared to those treated with free 5FU+FA. It is found that the cytotoxicity of the AuNPs-PEG-5FU-FA correlates with FA receptor expression suggested the use of FA as a targeted therapy. The mechanism of cytotoxicity was mediated via mitochondrial apoptotic pathway as determined by apoptosis array. In conclusion, our findings shed some light on the use of gold nanoparticles for conjugation with potential compounds and FA as targeted therapy which contribute to the improvement of anti-cancer drug efficacy. In vivo study should be warranted for its effectiveness of stability, biosafety and side effect reduction.