Endoplasmic Reticulum Stress-Mediated Apoptosis Induced by VR12684 Isolated from Mallotus spodocarpus in Cholangiocarcinoma Cell Line.

INTRODUCTION: Cholangiocarcinoma (CCA) is a poor prognosis of a malignant tumor that has been unresponsive to conventional chemotherapeutic agents. Effective and novel therapeutic agents are urgently needed. VR12684 (isolated from Mallotus spodocarpus) has been reported to exhibit growth inhibitory activities in cancer cell lines. The present study investigated the growth inhibitory mechanisms of this compound in a human CCA cell line (KKU-M156). METHODS: The effects of VR12684 on anti­proliferation, cell cycle arrest and apoptosis induction in CCA cells were demonstrated by SRB assay, flow cytometry, acridine orange/ethidium bromide (AO/EB) staining and western blot analysis. RESULTS: Treatment with VR12684 decreased cell proliferation in a dose- and time-dependent manner in the KKU-M156 cell line. VR12684 induced cell cycle arrest in the G2 phase in KKU-M156 through down-regulation of cyclin B1 and Cdk1 and up-regulation of p21, p27 and p53 levels. VR12684 induced mitochondria-mediated apoptosis by increasing DNA fragmentation, the Bax/BCL-2 ratio and AIF, and decreasing survivin with subsequent activation of caspase-9 and -3. This compound could induce apoptosis through the endoplasmic reticulum (ER) stress-mediated pathway by up-regulation of GRP78, IRE1α and GADD153 levels leading to down-regulation of Bcl-2 and activation of calpain-1, caspase-7 and -12. CONCLUSION: These results suggested that VR12684 inhibited KKU-M-156 cell growth by way of cell cycle arrest and induction of apoptosis, at least in part, through the mitochondria- and ER-associated intrinsic pathways. Such compounds warrant evaluation as a candidate for the treatment of human CCA.

Inhibitory effect of isomorellin on cholangiocarcinoma cells via suppression of NF-κB translocation, the phosphorylated p38 MAPK pathway and MMP-2 and uPA expression.

Evidence indicates that most cancer deaths are caused by tumor invasion and metastasis. Cholangiocarcinoma (CCA) is a tumor of the bile duct epithelium characterized by slow growth, rapid metastasis and poor prognosis. Caged xanthones are extracted from gamboge, a dry resin exuded by Garcinia hanbury. These compounds have been reported to be cytotoxic to several types of cancer cells, without affecting normal cells. The aim of the present study was to determine the effect of isomorellin on the inhibition of CCA cell (KKU-100) viability, migration, invasion and the expression of invasion-regulated proteins. Cytotoxicity of isomorellin was evaluated using a sulforhodamine B assay. The anti-migratory and anti-invasive effects of isomorellin on KKU-100 cells were assessed using wound healing and chamber invasion assays, respectively. Furthermore, the activities of matrix metalloproteinases (MMPs)-2 and -9, and urokinase-type plasminogen activator (uPA) were also investigated. The expression levels of proteins regulating invasion were determined via western blot analysis. The cell viability of KKU-100 cells was decreased following treatment with isomorellin in a dose-dependent manner, with IC50 values at 24, 48 and 72 h of 3.46±0.19, 3.78±0.02 and 4.01±0.01 µM, respectively. Wound healing and chamber invasion assays indicated that isomorellin significantly inhibited KKU-100 cell migration and invasion in a dose-dependent manner. In addition, isomorellin significantly inhibited cancer cell migration and invasion abilities via focal adhesion kinase (FAK), protein kinase C (PKC), the phosphorylated (p)-p38 mitogen-activated protein kinase (MAPK) pathway, and nuclear factor (NF)-κB expression and translocation to the nucleus, thus resulting in downregulation of MMP-2, uPA and cyclooxygenase-2 (COX-2) expression. Therefore, inhibition of MMP-2, uPA and COX-2 expression may result in decreased CCA cell invasion ability. These data demonstrated for the first time that the suppression of KKU-100 cell viability, invasion and migration, and downregulation of NF-κB, MMP-2, uPA and the p-p38 MAPK pathway, may result in isomorellin-mediated anti-invasiveness.

Anti-parasitic Drug Ivermectin Exhibits Potent Anticancer Activity Against Gemcitabine-resistant Cholangiocarcinoma In Vitro.

BACKGROUND/AIM: The antiparasitic drug, ivermectin (IVM), exerts anticancer activities in diverse cancer types. However, its anticancer activity against cholangiocarcinoma (CCA), especially the drug-resistant phenotype, has not yet been explored. MATERIALS AND METHODS: IVM was tested for its anticancer activity against gemcitabine-sensitive (KKU214) and gemcitabine-resistant (KKU214GemR) CCA cell lines in vitro using the sulforhodamine B and clonogenic assays as well as cell-cycle analysis. RESULTS: IVM treatment inhibited cell proliferation and colony formation of both KKU214 and KKU214GemR in a dose- and time-dependent manner. KKU214GemR cells were more sensitive than KKU214 to IVM treatment. IVM treatment caused S-phase cell-cycle arrest and also cell death as indicated by an increase of sub-G0/G1 population in KKU214GemR cells treated with IVM for 48 h. CONCLUSION: IVM exerts anti-CCA activities and gemcitabine-resistant KKU214GemR cells are more sensitive to IVM treatment. Thus, IVM might be useful as an alternative treatment for CCA, especially in patients who do not respond to gemcitabine.

Rhinacanthin-C Extracted from Rhinacanthus nasutus (L.) Inhibits Cholangiocarcinoma Cell Migration and Invasion by Decreasing MMP-2, uPA, FAK and MAPK Pathways

Cholangiocarcinoma is a malignant tumor with high metastatic and mortality rates. We investigated the effects of rhinacanthin-C on cell proliferation, migration, invasion and the expression of proteins regulating cancer cell invasion-regulated proteins in a cholangiocarcinoma (KKU-M156) cell line. Cytotoxicity of rhinacanthin-C was determined by the SRB assay. Using wound-migration, chamber-migration and chamber-invasion assays, we assessed the effects of rhinacanthin-C against KKU-M156 cells. The activities of matrix metalloproteinases 2 and 9 (MMP-2, MMP-9) and urokinase-type plasminogen activator (uPA) were determined using gelatinase and uPA zymography assays. The expression of invasion-regulated proteins was investigated using western-blot analysis. After treatment with rhinacanthin-C, KKU-M156 cells exhibited antiproliferative effects in a dose-dependent manner with greater efficacy than in Vero cells: IC50 values were 1.50 and 2.37 µM, respectively. Rhinacanthin-C significantly inhibited cell migration and invasion of KKU-M156 cells in a dose-dependent manner. Consistent with this observation, treatment with rhinacanthin-C was associated with a decrease in the expression levels of FAK, p-FAK, MMP-2, and a decrease in the levels of p38-, JNK1/2- and ERK1/2-MAPK pathways as well as inhibiting NF-κB/p65 expression and translocation of NF-κB/p65 to the nucleus. We have shown for the first time that the anti-metastatic effects of rhinacanthin-C on KKU-M156 cells are mediated via inhibition of the expression of invasion-regulated proteins. Rhinacanthin-C may deserve consideration as a potential agent for the treatment of cholangiocarcinoma.

Anthocyanin complex exerts anti-cholangiocarcinoma activities and improves the efficacy of drug treatment in a gemcitabine-resistant cell line.

Cholangiocarcinoma (CCA) is a deleterious bile duct tumor with poor prognosis and is relatively resistant to chemotherapy. Therefore, alternative or supplementary agents with anticancer and chemosensitizing activities may be useful for the treatment of CCA. A novel anthocyanin complex (AC) nanoparticle, developed from extracts of cobs of purple waxy corn and petals of blue butterfly pea, has exhibited chemopreventive potential in vivo. In the present study, the anti-CCA activities of AC and their underlying molecular mechanisms were investigated further in vitro using a CCA cell line (KKU213). The potential use of AC as a chemosensitizer was also evaluated in a gemcitabine-resistant CCA cell line (KKU214GemR). It was demonstrated that AC treatment suppressed proliferation of KKU213 CCA cells in dose- and time-dependent manners. AC treatment also induced apoptosis and mitochondrial superoxide production, decreased clonogenicity of CCA cells, and downregulated forkhead box protein M1 (FOXM1), nuclear factor-κB (NF-κB) and pro-survival protein B-cell lymphoma-2 (Bcl-2). The expression of endoplasmic reticulum (ER) stress-response proteins, including protein kinase RNA-like ER kinase, phosphorylated eIF2α, eukaryotic initiation factor 2α and activating transcription factor 4, also decreased following AC treatment. It was also identified that AC treatment inhibited KKU214GemR cell proliferation in dose- and time-dependent manners. Co-treatment of KKU214GemR cells with low doses of AC together with gemcitabine significantly enhanced efficacy of the latter against this cell line. Therefore, it is suggested that AC treatment is cytotoxic to KKU213 cells, possibly via downregulation of FOXM1, NF-κB, Bcl-2 and the ER stress response, and by induction of mitochondrial superoxide production. AC also sensitizes KKU214GemR to gemcitabine treatment, which may have potential for overcoming drug resistance of CCA.

Synergistic Effect of Forbesione From Garcinia hanburyi in Combination with 5-Fluorouracil on Cholangiocarcinoma

Background: Chemotherapy for advanced cholangiocarcinoma (CCA) is largely ineffective; thus innovative combinations of chemotherapeutic agents and natural compounds represent a promising strategy. This study aimed to investigate the synergistic effects of forbesione combined with 5-fluorouracil (5-FU) in hamster cholangiocarcinoma (Ham-1) cells both in vitro and in vivo. The anti-tumor effects of 5-FU combined with forbesione in vitro were determined using the Sulforhodamine B (SRB) assay and the effects in vivo were assessed in transplanted Ham-1 allograph models. Using ethidium bromide/acridine orange (EB/AO) staining, the morphological changes of apoptotic cells was investigated. The expressions of apoptosis-related molecules after combined treatment with forbesione and 5-FU were determined using real-time RT-PCR and western blot analysis. Forbesione or 5-FU alone inhibited proliferation of Ham-1 cells in a dose-dependent manner and their combination showed a synergistic proliferation inhibitory effect in vitro. In vivo studies, forbesione in combination with 5-FU exhibited greater inhibition of the tumor in the hamster model compared with treatment using either drug alone. Forbesione combined with 5-FU exerted stronger apoptotic induction in Ham-1 cells than did single drug treatment. The combination of drugs strongly suppressed the expression of B-cell lymphoma 2 (Bcl-2) and procaspase-3 while enhancing the expression of p53, Bcl-2-associated X protein (Bax), apoptotic protease activating factor-1 (Apaf-1), caspase-9 and caspase-3, compared with single drug treatments. These results explained the decreased expression of cytokeratin 19 (CK19) positive cells and proliferation cell nuclear antigen (PCNA) positive cells in Ham-1 cell tumor tissues of the treated hamsters. There was no apparent systemic toxicity observed in the treated animals compared with the control groups. Forbesione combined with 5-FU strongly induced apoptosis in Ham-1 cells. The growth inhibitory effect of combined treatment using these two drugs was much greater than treatment with either drug alone, both in vitro and in vivo.

In Vitro and In Vivo Inhibitory Effects of α-Mangostin on Cholangiocarcinoma Cells and Allografts

We investigated the anti-cholangiocarcinoma effect of α-mangostin from Garcinia mangostana pericarp extract (GM) in a human cholangiocarcinoma (CCA) cell line and a hamster CCA allograft model. In vitro, human CCA cells were treated with GM at various concentrations and for different time periods; then cell-cycle arrest and apoptosis were evaluated using flow cytometry, and metastatic potential with wound healing assays. In vivo, hamster allografts were treated with GM, gemcitabine (positive control) and a placebo (negative control) for 1 month; tumor weight and volume were then determined. Histopathological features and immunostaining (CK19 and PCNA) characteristics were examined by microscopy. The present study found that α-mangostin could: inhibit CCA cell proliferation by inducing apoptosis through the mitochondrial pathway; induce G1 cell-cycle arrest; and inhibit metastasis. Moreover, α-mangostin could inhibit CCA growth, i.e. reduce tumor mass (weight and size) and alter CCA pathology, as evidenced by reduced positive staining for CK19 and PCNA. The present study thus suggested that α-mangostin is a promising anti-CCA compound whose ready availability in tropical countries might indicate use for prevention and treatment of CCA.

Benzoyltyramine Alkaloids Atalantums A-G from the Peels of Atalantia monophylla and Their Cytotoxicity against Cholangiocarcinoma Cell Lines.

Seven new benzoyltyramines, atalantums A-G (1-7), and five known compounds were isolated from the peels of Atalantia monophylla. All compounds were examined for cytotoxicity against the cholangiocarcinoma cell lines KKU-M214, KKU-M213, and KKU-M156. Compound 5 exhibited the strongest cytotoxicity against KKU-M156 cells, with an IC50 value of 1.97 ± 0.73 µM, an approximately 4.7-fold higher activity than that of the ellipticine standard. Compound 1 displayed strong cytotoxicity against KKU-M214 cells, with an IC50 value of 3.06 ± 0.51 µM, nearly equal to that of the 5-fluorouracil standard. In the case of the KKU-M213 cell line, compounds 2, 4, and 11 exhibited stronger cytotoxicity than the ellipticine standard, with IC50 values of 2.36 ± 0.20, 5.63 ± 0.22, and 2.71 ± 0.23 µM, respectively. Compounds 1, 5, and 7 displayed cytotoxicity against KKU-M214 cells, with IC50 values of 3.06 ± 0.51, 8.44 ± 0.47, and 7.37 ± 1.29 µM, respectively.

Genetic characterization of Helicobacter pylori vacA and cagA genes in Thai gastro-duodenal and hepatobiliary patients.

INTRODUCTION: H. pylori has been detected in patients with hepatobiliary diseases. It is currently unclear whether the H. pylori detected in hepatobiliary patients are genetically similar to those in gastro-duodenal patients. The aim of this study was to determine H. pylori vacA and cagA genotypes in Thai patients with gastro-duodenal and hepatobiliary diseases. METHODOLOGY: H. pylori DNA was extracted from samples from gastric biopsies of gastro-duodenal patients (n=100) and from bile samples of hepatobiliary patients (n=80). The vacA and cagA genotypes were performed via polymerase chain reaction (PCR) followed by DNA sequencing. RESULTS: The vacA m1 was found in Thai hepatobiliary patients (90%) at a higher rate compared with gastro-duodenal patients (50%).The combined vacA s1a+c/m1 were mostly found in Thai gastro-duodenal and hepatobiliary patients. The cagA gene was detected in 94% of patients with gastro-duodenal diseases compared with 28.8% in those with hepatobiliary diseases (p<0.05). On the other hand, the Western type cagA was more prominent among hepatobiliary patients (100%) than gastro-duodenal patients (57.4%), and this type was grouped into same cluster with Thai gastro-duodenal patients via phylogenetic analysis. CONCLUSIONS: Based on vacA and cagA analysis, we conclude that infection with H. pylori in gastro-duodenal and hepatobiliary patients may be caused by the different H. pylori strains.

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

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-1 ) 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 CH3 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.

Burkholderia pseudomallei Biofilm Promotes Adhesion, Internalization and Stimulates Proinflammatory Cytokines in Human Epithelial A549 Cells.

Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis. Inhalational exposure leading to pulmonary melioidosis is the most common clinical manifestation with significant mortality. However, the role of B. pseudomallei biofilm phenotype during bacterial-host interaction remains unclear. We hypothesize that biofilm phenotype may play a role in such interactions. In this study, B. pseudomallei H777 (biofilm wild type), B. pseudomallei M10 (biofilm mutant) and B. pseudomallei C17 (biofilm-complemented) strains were used to assess the contribution of biofilm to adhesion to human lung epithelial cells (A549), intracellular interactions, apoptosis/necrosis and impact on proinflammatory responses. Confocal laser scanning microscopy demonstrated that B. pseudomallei H777 and C17 produced biofilm, whereas M10 did not. To determine the role of biofilm in host interaction, we assessed the ability of each of the three strains to interact with the A549 cells at MOI 10. Strain H777 exhibited higher levels of attachment and invasion compared to strain M10 (p < 0.05). In addition, the biofilm-complemented strain, C17 exhibited restored bacterial invasion ability. Flow cytometry combined with a double-staining assay using annexin V and propidium iodide revealed significantly higher numbers of early apoptotic and late apoptotic A549 cells when these were infected with strain H777 (1.52%) and C17 (1.43%) compared to strain M10 (0.85%) (p < 0.05). Strains H777 and C17 were able to stimulate significant secretion of IL-6 and IL-8 compared with the biofilm mutant (p < 0.05). Together, these findings demonstrated the role of biofilm-associated phenotypes of B. pseudomallei in cellular pathogenesis of human lung epithelial cells with respect to initial attachment and invasion, apoptosis and proinflammatory responses.

Antitumor effect of forbesione isolated from Garcinia hanburyi on cholangiocarcinoma in vitro and in vivo.

Cholangiocarcinoma (CCA) is a malignancy with no effective therapy and poor prognosis. Forbesione, a caged xanthone isolated from Garcinia hanburyi, has been reported to inhibit proliferation and to induce apoptosis in human CCA cell lines. The present study aimed to further explore the potential anticancer properties of forbesione by testing its effects against the hamster CCA cell line Ham-1 in vitro and in vivo. It was observed that forbesione inhibited the growth of Ham-1 cells in vitro and suppressed Ham-1 growth as allograft in hamsters by inducing cell cycle arrest at the S phase. This was mediated by decreasing the protein expression of cyclin E, cyclin A and cyclin-dependent kinase 2. In addition, increased expression of p21 and p27 was detected, which could possibly explain the reduced expression of proliferating cell nuclear antigen and of the bile duct cell marker cytokeratin 19 observed in forbesione-treated Ham-1 cells in vitro and in tumor tissues of forbesione-treated hamsters. Furthermore, forbesione induced apoptosis through multiple pathways. The death receptor pathway was activated by increased expression of Fas, Fas-associated death domain and activated caspase-3, along with decreased expression of procaspase-8 and procaspase-3. The mitochondrial pathway was driven by increased expression of B-cell lymphoma (Bcl)-2-like protein 4, activated caspase-9 and inhibitor of κB-α, along with decreased expression of Bcl-2, survivin, procaspase-9 and nuclear factor-κB/p65. The endoplasmic reticulum pathway was stimulated by increased expression of activated caspase-12 and decreased expression of procaspase-12. No side effects or toxicity were observed in forbesione-treated hamsters. Thus, forbesione is a potential drug candidate for cancer therapy that deserves further investigation.

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

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.

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

To establish and characterize the gemcitabine-resistant cholangiocarcinoma (CCA) cell lines, CCA KKU­M139 and KKU­M214 cell lines were exposed stepwisely to increasing gemcitabine (GEM). The resultant drug-resistant cell lines, KKU­M139/GEM and KKU­M214/GEM, retained the resistant phenotype in drug-free medium at least for 2 months. Sulforhodamine B assay demonstrated that KKU­M139/GEM and KKU­M214/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 KKU­M214/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.

Terpenoids from the root bark of Pterolobium macropterum.

Five new compounds, including pteroloterins A-C (1, 3, and 4), 1ß-acetoxytaepeenin C (2), and 8aα-hydroxycadinenal (5), and 11 known compounds were isolated from the root bark of Pterolobium macropterum. All compounds were evaluated for cytotoxicity against the cholangiocarcinoma cell lines. Compound 9 showed weak cytotoxicity against the KKU-M139 cell line with an IC50 value of 23.24 ± 0.18 µM and showed no activity against normal cells.

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.

ROLE OF HLYA-POSITIVE VIBRIO CHOLERAE NON-O1/NON-O139 ON APOPTOSIS AND CYTOTOXICITY IN A CHINESE HAMSTER OVARY CELL LINE.

Vibrio cholerae non-O1/non-O139 is capable of producing sporadic outbreaks of cholera-like diarrhea; however, the pathogenic mechanisms of this bacterium remain unclear. The objectives of this study were to: 1) compare the apoptosis induction and cytotoxicity between hlyA-positive and hlyA-negative strains of V. cholerae non-O1/non-O139; 2) clarify the molecular mechanisms by which these strains induce apoptosis; and 3) compare clinical and environmental V. cholerae non-O1/non-O139 isolates with respect to cytotoxicity and ability to induce apoptosis. Using cytotoxicity and apoptosis assays, it was shown that hlyA-positive strains of V. cholerae non-O1/non-O139 had significantly higher cytotoxic activity (70.6%) and levels of apoptosis induction (59.6%) than hlyA- negative strains (37.0% and 37.5%, respectively). Western blot analyses revealed that hlyA-positive strains had significantly increased expression of Bax; active caspase-3 and -9; and significantly decreased expression of NF-κB and Bcl-2 relative to hlyA-negative strains. Expression of BID did not differ significantly between hlyA-positive and negative strains. The truncated BID was not found, indicating that V. cholerae non-O1/non-O139 induces apoptosis through a mitochondria- dependent apoptosis pathway and not an extrinsic pathway. V. cholerae non-O1/ non-O139 isolated from clinical sources exhibited significantly higher cytotoxic activity (79%) and levels of apoptosis induction (65.2%) than bacteria isolated from environmental sources (63% and 54.6%, respectively), suggesting that the clini- cal isolates may have other virulence-associated genes besides hlyA. Our results indicate that hlyA products play a role in cytotoxicity and apoptosis induction and that a mitochondria-dependent apoptosis pathway is involved.

Synergistic effects of isomorellin and forbesione with doxorubicin on apoptosis induction in human cholangiocarcinoma cell lines.

BACKGROUND: Chemotherapy for advanced cholangiocarcinoma (CCA) is largely ineffective, but innovative combinations of chemotherapeutic agents and natural compounds represent a promising strategy. In our previous studies, isomorellin and forbesione, caged xanthones isolated from Garcinia hanburyi, were found to induce cell cycle arrest and apoptosis in CCA cell lines. The subject of our inquiry is the synergistic effect(s) of these caged xanthones with doxorubicin on growth inhibition and apoptosis induction in human CCA cell lines. METHODS: KKU-100, KKU-M139 and KKU-M156 cell lines and Chang cells were treated with either isomorellin or forbesione alone or in combination with doxorubicin. Cell viability was determined using the sulforhodamine B assay. The combined effects of plant compounds with doxorubicin were analyzed using the isobologram and combination index method of Chou-Talalay. Apoptosis was determined by ethidium bromide/acridine orange staining. Protein expressions were determined by Western blot analysis. RESULTS: Isomorellin or forbesione alone inhibited the growth of these CCA cell lines in a dose-dependent manner and showed selective cytotoxicity against CCA cells but not against Chang cells. Isomorellin/doxorubicin combination showed a synergistic growth inhibitory effect on KKU-M139 and KKU-M156 cells, while the forbesione/doxorubicin combination showed a synergistic growth inhibitory effect on KKU-100 and KKU-M139 cells. The percentages of apoptotic cells were significantly higher in the combined treatments than in the respective single drug treatments. The combined treatments strongly enhanced the expression of Bax/Bcl-2, activated caspase-9 and caspase-3, while suppressing the expression of survivin, procaspase-9 and procaspase-3, compared with single drug treatments. The degree of suppression of NF-κB activation mediated by a decrease in the expression of NF-κB/p65, a reduction of the pIκB-α level and an increase in the IκB-α protein level, was significantly higher in the combined treatment groups than in the single drug treatment groups. The degree of suppression of MRP1 protein expression was also significantly higher in the combined treatment than in the single drug treatment groups. CONCLUSION: The combinations of isomorellin/doxorubicin and forbesione/doxorubicin showed significant synergistic effects on the growth inhibition and apoptosis induction in KKU-M156 and KKU-100 cells. Caged xanthones may be useful adjunct treatments with chemotherapy for Opisthorchis viverrini (OV)-associated CCA.

Helicobacter pylori cag pathogenicity island (cagPAI) involved in bacterial internalization and IL-8 induced responses via NOD1- and MyD88-dependent mechanisms in human biliary epithelial cells.

Helicobacter pylori infection has been proposed to be associated with various diseases of the hepatobiliary tract, including cancer of the bile duct epithelial cells (cholangiocarcinoma, CCA). The ability of H. pylori bacteria to cause pathogenic effects in these cells has, however, yet to be investigated. Given that the cag pathogenicity island (cagPAI) is required for H. pylori pathogenesis in gastric epithelial cells, we investigated wild-type and cag mutant strains for their ability to adhere, be internalized and induce pro-inflammatory responses in two bile duct epithelial cell lines derived from cases of CCA. The findings from these experiments were compared to results obtained with the well-characterized AGS gastric cancer cell line. We showed that the cagPAI encodes factors involved in H. pylori internalization in CCA cells, but not for adhesion to these cells. Consistent with previous studies in hepatocytes, actin polymerization and α5ß1 integrin may be involved in H. pylori internalization in CCA cells. As for AGS cells, we observed significantly reduced levels of NF-κB activation and IL-8 production in CCA cells stimulated with either cagA, cagL or cagPAI bacteria, when compared with wild-type bacteria. Importantly, these IL-8 responses could be inhibited via either pre-treatment of cells with antibodies to α5ß1 integrins, or via siRNA-mediated knockdown of the innate immune signaling molecules, nucleotide oligomerization domain 1 (NOD1) and myeloid differentiation response gene 88 (MyD88). Taken together, the data demonstrate that the cagPAI is critical for H. pylori pathogenesis in bile duct cells, thus providing a potential causal link for H. pylori in biliary tract disease.

A combination of praziquantel and the traditional medicinal plant Thunbergia laurifolia on Opisthorchis viverrini infection and cholangiocarcinoma in a hamster model.

Cholangiocarcinoma (CCA) associated by Opisthorchis viverrini remains a health problem in Southeast Asia including Thailand. At present, there is still no efficient treatment for CCA. Thunbergia laurifolia is a traditionally used medicinal plant; its aqueous leave extract possesses the antioxidant activity and anti-inflammatory on hamster opisthorchiasis had been reported previously. Here, we demonstrate the combined effects of the T. laurifolia extract plus antihelminthic drug, praziquantel (PZ) on hamsters with opisthorchiasis and hamsters with opisthorchiasis related-cholangiocarcinoma through light microscopic observations of histopathological changes, as well as liver function tests for alanine transaminase (ALT) and alkaline phosphatase, and kidney function tests for blood urea nitrogen and creatinine. Results showed T. laurifolia extract combined with praziquantel reduced inflammatory cell aggregation and inhibiting CCA development, which were correlated to the serum ALT level. These present studies suggest that administration of T. laurifolia after praziquantel treatment clearly improve the hepatobiliary system and could reduce the risk of subsequent CCA development in human.