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.

Molecular analysis of Helicobacter pylori virulent-associated genes in hepatobiliary patients.

OBJECTIVES: The Helicobacter pylori virulence-associated genes in hepatobiliary patients, including vacA, iceA, babA2, cagA and cagE, have not been reported. The aim of this study was to investigate these genes and the association of those and the clinical outcomes in hepatobiliary diseases. METHODS: Eighty H. pylori-PCR-positive cases were obtained from hepatobiliary patients, representing both cholangiocarcinoma (CCA) (n= 58) and cholelithiasis (n= 22). The diversity of virulence genes was examined by polymerase chain reaction and DNA sequencing. Phylogenetic analysis of cagA was determined using the maximum parsimony method. RESULTS: The vacAs1a + c/m1, iceA1 and babA2 genes were the most predominant genotypes in both CCA and cholelithiasis patients. The cagA and cagE genes were found significantly more frequently in patients with CCA than those with cholelithiasis (P < 0.05). The cagA positive samples were the Western-type cagA and showed that almost all of the detected sequences in Thai hepatobiliary and Thai gastric cancer patients were classified in the same cluster but separated from the cluster of Japan and other countries. CONCLUSIONS: The cagA and cagE genes may be associated in the pathogenesis of hepatobiliary diseases, especially of CCA. Besides the bacterial variation, other host factors may be involved in the pathogenesis of hepatobiliary cancer.

Periostin activates integrin α5ß1 through a PI3K/AKT­dependent pathway in invasion of cholangiocarcinoma.

Periostin (PN) is mainly produced from stromal fibroblasts in cholangiocarcinoma (CCA) and shows strong impact in cancer promotion. This work aimed to investigate the mechanism that PN uses to drive CCA invasion. It was found that ITGα5ß1 and α6ß4 showed high expression in non-tumorigenic biliary epithelial cells and in almost all CCA cell lines. PN had preferential binding to CCA cells via ITGα5ß1 and blocking this receptor by either neutralizing antibody or siITGα5 could attenuate PN-induced invasion. After PN-ITGα5ß1 binding, intracellular pAKT was upregulated whereas there was no change in pERK. Moreover, PN could not activate AKT in condition of treatment with a PI3K inhibitor. These data provide evidence that PN-activated invasion of CCA cells is through the ITGα5ß1/PI3K/AKT pathway. Strategies aimed to inhibit this pathway may, thus, provide therapeutic benefits.

Estrogen is increased in male cholangiocarcinoma patients' serum and stimulates invasion in cholangiocarcinoma cell lines in vitro.

PURPOSE: Cholangiocarcinoma is defined as a chronic liver disease with altered estrogen metabolism and could result in estrogen retention. Estrogenic response was known as a promoting factor in progression of some cancer. In this study, we determined the significant increase of estrogen level in cholangiocarcinoma patients' sera. METHODS: The estrogen levels in cholangiocarcinoma patients' sera were measured and correlated with clinical presentations. Estrogen receptor-α expressions in cholangiocarcinoma tissues were detected by immunohistochemistry method. KKU-100 and KKU-M213 cholangiocarcinoma cell lines were treated with 17ß-estradiol and tested the proliferative and invasive effects. RESULTS: The estrogen levels showed positive correlations with serum bilirubin and alkaline phosphatase and a negative correlation with albumin. This study also showed an association with shorter survival times when patients with low and high serum estrogen levels were compared. In vitro studies demonstrated the effect of estrogen on cell proliferation and invasion in dose-dependent manners, which could be inhibited by tamoxifen, a clinical used estrogen antagonist. Invasion showed an association with the TFF1 gene expression and could be inhibited by small interfering RNA against TFF1 gene. Estrogen receptor-α was the main estrogen receptor that response to 17ß-estradiol stimulation. CONCLUSIONS: TFF1 trefoil protein could be one of the effectors for estrogen-induced invasion in cholangiocarcinoma via the estrogen receptor-α. These findings could lead to an understanding of the mechanism of cholangiocarcinoma progression.

NQO1 expression correlates with cholangiocarcinoma prognosis.

Cholangiocarcinoma (CCA) is a rare type of liver cancer with a very poor prognosis. The prevalence of CCA is markedly variable with the highest incidence in the northeast Thailand, followed by other parts of Southeast Asia and China. Currently, there is still no reliable biomarker for diagnosis or treatment. NADPH-quinone oxidoreductase 1 (NQO1) is a xenobiotic metabolizing enzyme detoxifying chemical stressors and antioxidants, thereby providing cytoprotection in normal tissues. However, NQO1 is over-expressed in some cancers, suggesting roles in carcinogenesis and tumor progression. In this study, we examined NQO1 activity in surgical specimens from CCA patients and found much higher values than in the adjacent normal tissues. Immunohistochemical analysis revealed strong staining in tumor epithelial elements, whereas the non-tumor bile ducts and liver parenchyma were weakly stained. NQO1 mRNA expression in tumor tissues was widely varied among 43 patients. A significant association was observed between high level of NQO1 expression and short overall survival time by the Cox proportional hazard ratio of 2.40, p<0.05. By histological classification, non-papillary adenocarcinoma was an independent predictor for poor prognosis with the hazard ratio of 2.79, p<0.05. NQO1 expression may serve as a prognostic biomarker for the CCA.

Quality of life after donor nephrectomy for living donor kidney transplantation at Srinagarind Hospital.

OBJECTIVE: To determine the quality of life (QoL) of donors who have undergone nephrectomy for living donor kidney transplantation at Srinagarind Hospital, using the Thai version of the Short-Form, 36-item, health survey (SF-36). MATERIAL AND METHOD: The SF-36 questionnaires were sent by mail to 93 living donors who underwent nephrectomy between Jan 1, 1990 and Dec 31, 2008. The first part collected demographic data and the donor/recipient relationship, the second surveyed QoL, and the third asked about decision-making, donation-related stress and feedback. RESULTS: Forty-nine questionnaires were returned completed (30 women; 19 men: mean age 44.2 +/- 9.5 (range, 28-65) years). Thirty-one participants (61%) were siblings of the recipients. The QoL scores were not significantly different from the general Thai population; albeit nominally higher for mental health and social function. Upon reflection, only 5 (10%) had second thoughts. CONCLUSION: Donor nephrectomy did not affect QoL; thus, from that perspective living kidney transplantation is a suitable procedure for donors.

Gene expression profiling of cholangiocarcinoma-derived fibroblast reveals alterations related to tumor progression and indicates periostin as a poor prognostic marker.

BACKGROUND: Fibroblasts play important roles in several cancers. It was hypothesized that cholangiocarcinoma (CCA)-associated fibroblasts (Cfs) differ from non-tumorigenic liver fibroblasts (Lfs) in their gene expression profiles resulting in the capability to promote cancer. Periostin (PN) is a multi-functional protein and has emerged as a promising marker for tumor progression. The role of PN in CCA, however, has not yet been explored. RESULTS: In this study, the gene expression profile of Cfs in comparison to Lfs was performed using oligonucleotide microarrays. The common- and unique-expressed genes in Cfs and the promising roles in cancer promotion and progression were determined. PN was markedly over-expressed in Cfs confirmed by real time RT-PCR and western blot analysis. Immunohistochemistry examination of a number of patients with intrahepatic CCA showed the expression of PN solely in stromal fibroblasts, but was expressed neither in cancer cells nor immune cells. Low to no expression of PN was observed in tissues of benign liver disease and hepatocellular carcinoma. CCA patients with high levels of PN had significantly shorter survival time than those with low levels (P = 0.026). Multivariate analysis revealed high levels of PN (P = 0.045) and presence of lymph node metastasis (P = 0.002) as independent poor prognostic factors. The in vitro study revealed that recombinant PN induced CCA cell proliferation and invasion. Interestingly, interference RNA against integrin alpha 5 significantly reduced the cellular response to PN-stimulated proliferation and invasion. CONCLUSION: The gene expression profile of fibroblasts in CCA is apparently explored for the first time and has determined the genes involving in induction of this cancer progression. High PN can be used to distinguish CCA from other related liver diseases and is proposed as a prognostic factor of poor survival. Regulation of fibroblast-derived PN in CCA proliferation and invasion may be considered as an alternative therapeutic approach.

Alpha-smooth muscle actin-positive fibroblasts promote biliary cell proliferation and correlate with poor survival in cholangiocarcinoma.

Cancer-associated fibroblasts have been proposed to play a role in promoting carcinogenesis and tumor progression. To our knowledge, no direct evidence concerning fibroblasts in the genesis of cholangiocarcinoma (CCA) has previously been presented. This study aims to assess the value of activated fibroblasts with high alpha-smooth muscle actin (alpha-SMA) expression as an indicator for survival in CCA patients. The immunohistochemistry results indicated a high expression of alpha-SMA in CCA fibroblasts which had a statistically significant correlation with larger tumor size (P=0.009) and shorter survival time (P=0.013). The effect of CCA-associated fibroblasts (Cfs) on non-tumorigenic biliary epithelial cells (H-69) and CCA cell lines was investigated in vitro and compared to the effect of non-tumorigenic liver fibroblasts (Lfs). The increased proliferation effect of Cfs having high alpha-SMA on H-69 and 4 CCA cell lines compared to Lfs that expressed low alpha-SMA was observed. Cell cycle analysis indicated that Cf-derived conditioned-medium and direct Cf-epithelial cell contaction could drive epithelial cells into S+G2/M phases. These results indicate that fibroblasts in CCA stroma express high alpha-SMA and can be a prognostic indicator for poor patient survival. CCA fibroblasts have proliferative effects which may directly effect tumor promotion and progression of biliary epithelial cells. This warrants further investigation of fibroblasts as alternative therapeutic targets in CCA patients.

Enhanced expression of mucin 6 glycoprotein in cholangiocarcinoma tissue from patients in Thailand as a prognostic marker for survival.

BACKGROUND AND AIM: Cholangiocarcinoma (CCA) is a mucin-producing cancer that has poor prognosis. Mucin 6 (MUC6) is a mucin that is normally co-expressed with the trefoil factor family-2 (TFF2) trefoil peptide. Both MUC6 and TFF2 have been reported to be involved in the progression of many types of cancers. The aim of this study was to determine the expression of MUC6 and TFF2 in CCA tissues and associate these results with clinical data. METHODS: MUC6 and TFF2 were detected in CCA tissues by immunohistochemistry. The correlations of MUC6 and TFF2 expressions with clinical data were analyzed. RESULTS: We determined the significant co-expression of both proteins in serial CCA tissues. The high expressions of MUC6 and TFF2 were demonstrated in 37% and 31% of patients, respectively. The expression levels decreased in the advanced stage of CCA when clinical metastasis was exhibited. The high expression of either protein showed a correlation with prolonged postoperative survival time, but only a high expression of MUC6 is significantly correlated with a 5-year survival rate. A multivariate Cox regression analysis revealed that a low expression of MUC6, high expression of TFF2, age of patients >56 years, tumor size >5 cm, and poorly-differentiated histological type were independent, poor prognostic indicators for CCA. CONCLUSION: MUC6 showed a good correlation with the survival of CCA patients. It may be of value to propose that MUC6 is a good prognostic marker for CCA management.

Increased TFF1 trefoil protein expression in Opisthorchis viverrini-associated cholangiocarcinoma is important for invasive promotion.

AIMS: Cholangiocarcinoma (CCA) is a poor prognosis cancer that presents with metastatic disease. This cancer expresses MUC5AC, a mucin which normally co-expresses with trefoil factor family 1 (TFF1) protein. TFF1 is a signalling protein that can activate epithelial cell invasion and has been considered as a metastasis stimulating agent. The aim of this study was to determine the co-expression of TFF1 and MUC5AC in CCA tissues and examine the activity of TFF1 for stimulating the invasive property of CCA cell lines. METHODS: In this study, TFF1 and MUC5AC were detected in CCA tissues by using immunohistochemistry. The correlations of both proteins expression with clinical data were analyzed. The activity of TFF1 was investigated using an in vitro invasion assay with established CCA cell lines KKU-100 and KKU-M213. RESULTS: We demonstrated a high level of expression of TFF1 in 91.80% of CCA that is associated with a high level of co-expression with MUC5AC in 80.33% of cases. In vitro invasion assay showed that both cell lines have similar responses to TFF1 that could act as both a chemokinetic and chemotactic agent. The dose-response curves were bell-shaped. CONCLUSION: TFF1 showed co-expression with MUC5AC in CCA tissues and invasive stimulating activity in vitro. These results may indicate a role for TFF1 in promoting tumor invasion in CCA.

Detection of H. pylori in dyspeptic patients and correlation with clinical outcomes.

The objectives of this study were to evaluate the methods used to diagnose Helicobacter pylon infection in gastric biopsies, and to evaluate the correlation between H. pylori infection and clinical outcomes. Gastric biopsies, obtained from 210 patients, were evaluated for H. pylori by culture, a commercial rapid urease test (RUT, Pronto Dry) and histological examination. A true positive result was either the culture or both the RUT and histological examination were positive. The results showed a H. pylori infection rate of 44.3% (93/210). The sensitivities, specificities, positive predictive values and negative predictive values were 88.2, 100, 100, and 91.4 % by the culture; 95.7, 98.3, 97.8, and 96.6% by RUT; and 96.8, 59.8, 59.8, and 65.7% by histological examination, respectively. The prevalences of H. pylori in non-ulcer dyspepsia (NUD), peptic ulcer dyspepsia (PUD) and gastric cancer (GCA) patients were 41.2, 57.9 and 70.6%, respectively. The chi-squared-test showed that GCA patients were significantly more frequent infected with H. pylori than NUD patients (p<0.05). Our study indicates that the RUT method was highly sensitive, specific and appropriate for routine clinical use.

Anti-p53 antibodies and p53 protein expression in cholangiocarcinoma.

BACKGROUND/AIMS: Mutations of p53 are found in the majority of human malignancies. The accumulated mutant p53 can be detected in tumor sections by immunohistochemical methods. The abnormal accumulation of the defective p53 protein can induce the host to develop anti-p53 antibodies in sera of cancer patients. This study aimed to investigate the presence of anti-p53 antibodies in sera of patients with cholangiocarcinoma and to evaluate the correlation between such antibodies and p53 protein accumulation. METHODOLOGY: The presence of serum anti-p53 antibodies in 49 patients with cholangiocarcinoma was determined by ELISA kit (Pharma Cell, France). Immunohistochemical detection of p53 protein expression was examined in available tissue samples of 36 patients. RESULTS: Serum anti-p53 antibodies were detected in 6 of 49 patients with cholangiocarcinoma (12.2%). Immunostaining of p53 was found in 15 of 36 patients (41.6%) and 4 of these 15 patients (26.7%) were positive for anti-p53 antibodies. The association between anti-p53 antibodies and p53 protein expression was statistically significant (P=0.023). No correlation was found between the presence of anti-p53 antibodies and sex, age, histological grade, site and stage of tumor (P>0.05). CONCLUSIONS: The majority of serum anti-p53 antibodies detected in cholangiocarcinoma were specifically associated with the accumulation of p53 protein in tumor tissues. However, antibody generation against the p53 protein is a relatively uncommon event in cholangiocarcinoma.

Clinical significance of serum total sialic acid in cholangiocarcinoma.

BACKGROUND: High levels of serum total sialic acid (TSA) have been reported in cholangiocarcinoma (CCA) patients. In this study, the clinical value and possible cause of increased total sialic acid in the serum in cholangiocarcinoma patients were examined. METHODS: Total sialic acid was determined in 172 serum and 25 tumor tissue samples taken from cholangiocarcinoma patients using the periodate thiobarbituric acid method. RESULTS: The total sialic acid content of the tumor tissue was significantly greater than that of the serum and not related to the concentration found in the serum. The serum total sialic acid was not correlated with age, sex, body mass index, blood group, tumor location, tumor stage, metastatic condition, histological types and survival of the patients. The increased total sialic acid in the serum had a significant correlation with serum MUC5AC mucin, alkaline phosphatase and the CA19-9, and the numbers of white blood cell and neutrophils. CONCLUSIONS: The concentration of serum sialic acid was not associated with clinicopathologic features or the tumor burden. The glycoproteins secreted from the tumor and inflammatory cells might be responsible for the increased total sialic acid in the serum in these patients.

Microsatellite alterations in liver fluke related cholangiocarcinoma are associated with poor prognosis.

We have characterized the role of genetic alterations in the development of liver fluke related cholangiocarcinoma. We analyzed the loss of heterozygosity (LOH) and microsatellite instability (MSI) of hMSH2, hMLH1, and p53 genes in 55 patients with intrahepatic cholangiocarcinoma by using polymerase chain reaction based microsatellite markers D2S119, D3S1611, and TP53, respectively and determined the association between microsatellite alterations and patient survival. A total of 27 (49.1%) out of 55 cases exhibited microsatellite alterations in one locus or more. Of 55 samples, 11 (20%) demonstrated MSI at D2S119 and four (7%) showed MSI at D3S1611. LOH was shown in seven out of 36 (19%) informative cases for D3S1611 and 16 out of 50 (32%) for TP53. Microsatellite alterations at loci studied were significantly associated with poor survival (P=0.0098). This study suggests that genetic alterations of DNA mismatch repair genes and tumor suppressor gene p53 may be involved in cholangiocarcinogenesis and these alterations may be of value as prognostic indicators for liver fluke related cholangiocarcinoma.