Suppression of NAD(P)H-quinone oxidoreductase 1 enhanced the susceptibility of cholangiocarcinoma cells to chemotherapeutic agents

Background

Cholangiocarcinoma (CCA) is highly resistant to most of the known chemotherapeutic treatments. NAD(P)H-quinone oxidoreductase 1 (NQO1) is an antioxidant/detoxifying enzyme recently recognized as an important contributor to chemoresistance in some human cancers. However, the contribution of NQO1 to chemotherapy resistance in CCA is unknown.

Methods

Two CCA cell lines, KKU-100 and KKU-M214, with high and low NQO1 expression levels, respectively, were used to evaluate the sensitivity to chemotherapeutic agents; 5-fluorouracil (5-FU), doxorubicin (Doxo), and gemcitabine (Gem). NQO1 and/or p53 expression in KKU-100 cells were knocked down by siRNA. NQO1 was over-expressed in KKU-M214 cells by transfection with pCMV6-XL5-NQO1 expression vector. CCA cells with modulated NQO1 and/or p53 expression were treated with chemotherapeutic agents, and the cytotoxicity was assessed by SRB assay. The mechanism of enhanced chemosensitivity was evaluated by Western blot analysis.

Results

When NQO1 was knocked down, KKU-100 cells became more susceptible to all chemotherapeutic agents. Conversely, with over-expression of NQO1 made KKU-M214 cells more resistant to chemotherapeutic agents. Western blot analysis suggested that enhanced chemosensitivity was probably due to the activation of p53-mediated cell death. Enhanced susceptibility to chemotherapeutic agents by NQO1 silencing was abolished by knockdown of p53.

Conclusions

These results suggest that inhibition of NQO1 could enhance the susceptibility of CCA to an array of chemotherapeutic agents.     

Licochalcone A Induces Cholangiocarcinoma Cell Death Via Suppression of Nrf2 and NF-κB Signaling Pathways

Objective: To investigate the anti-tumor effect of licochalcone A (LCA) on proliferation and migration in cholangiocarcinoma (CCA) cells and to elucidate their underlying mechanisms. Methods: Human CCA cells, KKU-100, KKU-213, KKU-214, KKU-156, and KKU-452 were used to study effect of LCA on proliferation and migration by a cytotoxicity assay, wound healing assay. Reactive oxygen species levels were evaluated using DHE-fluorescent probes. Proteins associated with cancer survival and progression were analyzed by immune blotting assay. Results: LCA suppressed proliferation and induced cell death in CCA cells including KKU-100, KKU-213, KKU-214, KKU-156, and KKU-452. The CCAs cells were suppressed in association with LCA-induced accumulation of intracellular reactive oxygen species (ROS). Increased formation of ROS was causally related with suppression of Nrf2 and its down-stream antioxidant and cytoprotective enzymes. These effects may lead to the expression of Bax and release of cytochrome c and ensuring cell death.  Interestingly, LCA could also inhibit cell migration and cell cycle arrest at low concentrations. These effects were associated with down-regulation of NF-kB, STAT3 and their down-stream proteins, cyclin D1, VEGF, and ICAM-1. Conclusions: These results suggest that LCA has potential therapeutic activity in suppression of CCA cells.     

Increased activation of PI3K/AKT signaling pathway is associated with cholangiocarcinoma metastasis and PI3K/mTOR inhibition presents a possible therapeutic strategy

Phosphatidylinositol 3-kinase (PI3K) signaling plays a critical role in cholangiocarcinoma (CCA), as well as anti-cancer drug resistance and autophagy, the type II program cell death regulation. In this work, we aimed to: (1) determine the expression levels of several key components of PI3K signaling and (2) evaluate whether NVP-BEZ235, a novel dual PI3K/mTOR inhibitor, could inhibit CCA cell growth. Immunohistochemistry for p85α, p110α, AKT, p-AKT (T308), mTOR, p-mTOR (S2448), GSK-3β, p-GSK-3β (S9), PTEN, and p-PTEN (S380, T382/383) was performed in 30 CCA patients. Western blotting was used to analyze PTEN and p-PTEN expression in the cell lines (KKU-OCA17, KKU-100, KKU-M055, KKU-M139, KKU-M156, KKU-M213, and KKU-M214). The effects of NVP-BEZ235 on CCA cells were evaluated using a growth inhibition assay, flow cytometer and migration assay. Increased activation of PI3K/AKT signaling was reproducibly observed in the CCA tissues. The expression of p85α, mTOR, and GSK-3β was significantly correlated with metastasis. Interestingly, PTEN suppression by loss of expression or inactivation by phosphorylation was observed in the majority of patients. Furthermore, NVP-BEZ235 effectively inhibited CCA cell growth and migration through reduced AKT and mTOR phosphorylation and significantly induced G1 arrest without apoptosis induction, although increase autophagy response was observed. In conclusion, the constitutive activation of PI3K/AKT pathway in CCA is mainly due to PTEN inactivation by either loss of expression or phosphorylation along with an increased expression in its pathway components heralding a poor prognosis for CCA patients. This work also indicates that inhibition of PI3K and mTOR activity by the inhibitor NVP-BEZ235 has anti-cancer activity against CCA cells which might be further tested for CCA treatment.     

Luteolin Arrests Cell Cycling,Induces Apoptosis and Inhibits the JAK/STAT3 Pathway in Human Cholangiocarcinoma Cells

Cholangiocarcinoma (CCA) is one of the aggressive cancers with a very poor prognosis. Several efforts have been made to identify and develop new agents for prevention and treatment of this deadly disease. In the present study, we examined the anticancer effect of luteolin on human CCA, KKU-M156 cells. Sulforhodamine B assays showed that luteolin had potent cytotoxicity on CCA cells with IC50 values of 10.5±5.0 and 8.7±3.5 μM at 24 and 48 h, respectively. Treatment with luteolin also caused a concentration-dependent decline in colony forming ability. Consistent with growth inhibitory effects, luteolin arrested cell cycle progression at the G2/M phase in a dose-dependent manner as assessed by flow cytometry analysis. Protein expression of cyclin A and Cdc25A wasdown-regulated after luteolin treatment, supporting the arrest of cells at the G2/M boundary. Besides evident G2/M arrest, luteolin induced apoptosis of KKU-M156 cells, demonstrated by a distinct sub-G1 apoptotic peak and fluorescent dye staining. A decrease in the level of anti-apoptotic Bcl-2 protein was implicated in luteolininduced apoptosis. We further investigated the effect of luteolin on JAK/STAT3, which is an important pathway involved in the development of CCA. The results showed that interleukin-6 (IL-6)-induced JAK/STAT3 activationin KKU-M156 cells was suppressed by treatment with luteolin. Treatment with a specific JAK inhibitor, AG490, and luteolin diminished IL-6-stimulated CCA cell migration as assessed by wound healing assay. These data revealed anticancer activity of luteolin against CCA so the agent might have potential for CCA prevention and therapy.     

Cucurbitacin B Diminishes Metastatic Behavior of Cholangiocarcinoma Cells by Suppressing Focal Adhesion Kinase

Objective: Cholangiocarcinoma (CCA) is a malignant tumor with aggressive metastatic property resulted from dysregulation of metastasis-regulated signaling pathways. The aim of this study was to investigate the effect of cucurbitacin B on metastatic behavior of CCA cells through modulation of focal adhesion kinase (FAK) protein. Methods: KKU-452 cells were treated with a specific FAK inhibitor, FAK inhibitor-14, or cucurbitacin B at various concentrations for 24 h. Cell viability was assessed by sulforhodamine B assay. The migratory and invasive abilities of the cells were investigated using wound healing and transwell invasion assays, respectively. The fibronectin-coated plate was used for adhesion assay. The effects of the test compounds on FAK activation and the expression of metastasis-associated proteins were determined by Western blot analysis. The amount of MMP-9 was evaluated using a commercial ELISA Kit. Results: FAK inhibitor-14 and cucurbitacin B at concentrations which minimally affected KKU-452 cell viability could suppress FAK activation, evidently by decreased level of phospho-FAK protein after exposure to the compound. At these conditions, cucurbitacin B suppressed metastatic behavior including migration, invasion and adhesion abilities of CCA cells similar to FAK inhibitor-14. Further molecular studies demonstrated that FAK inhibitor-14 and cucurbitacin B downregulated the expression of metastasis-associated proteins including MMP-9, ICAM-1 and VEGF. Consequently, exposure to cucurbitacin B inhibited the production of MMP-9 enzyme in CCA cells similar to FAK inhibitor-14 treatment. Conclusion: FAK participated in regulation of metastatic behavior of KKU-452 CCA cells. Cucurbitacin B suppressed FAK activation in the cells which was associated with inhibition of metastasis essential steps and their related metastatic proteins. The compound may be developed as a novel therapeutic agent for CCA metastasis therapy.     

The Effects of Ibuprofen,Naproxen and Diclofenac on cell Apoptosis,Cell Proliferation and Histology Changes in Human Cholangiocarcinoma Cell Lines

Objective: To examine the effects of ibuprofen, naproxen and diclofenac, non-steroidal anti-inflammatory drugs (NSAIDs) on cell proliferation activity of the human CCA cell lines. Methods: KKU-M139 and KKU-213B cell lines were used in this study. The cell viability was assessed by the MTT assay. Lipid synthesis determined by Oil red O staining and colorimetric assay. An inverted phase-contrast light microscope was used to investigate the histological change of the cells. Caspases 3/7 activity and AnnexinV/PI were used to assess apoptosis by multiple microplate reader. Results: The results showed that ibuprofen, naproxen and diclofenac suppressed the viability of the KKU-M139 and KKU-213B cells in a dose-dependent manner, as measured especially diclofenac. However, these three NSAIDs slightly decreased lipid synthesis determined by Oil red O staining and colorimetric assay. The histological change observations showed the shrinking cell and become star-shaped in high dose treated groups. Interestingly, these NSAIDs exhibited in both of KKU-M139 and KKU-213B cell lines, the diclofenac-treated cells had the most injury cells. The cells exhibited cell injury features. In addition, the detection of caspase 3/7 and AnnexinV/PI in this investigation revealed early cell apoptotic characteristics. Conclusion: These finding suggest that ibuprofen, naproxen and diclofenac suppress cell viability. The results reveal that ibuprofen, naproxen and diclofenac, which induce the histological change and apoptosis. This study indicates that these NSAIDs may be used as an anti-proliferation agent for the treatment of CCA in the future.     

Low Dose Berberine Suppresses Cholangiocarcinoma Cell Progression as a Multi-Kinase Inhibitor

Background: Berberine (BBR), a natural isoquinoline alkaloid, possesses diverse pharmacological properties and anti-cancer effects that have been demonstrated in many in vitro and in vivo studies. In this study, the inhibitory effects and molecular mechanism of low dose BBR on EMT-induced cell migration, and invasion capability of cholangiocarcinoma (CCA) cell lines were demonstrated. Methods: The commercially available BBR chloride powder with purity ≥ 95% was used in this study. Effects of BBR on cell growth of two human CCA cell lines, KKU-213A and KKU-213B were measured using MTT assay. The progressive phenotypes-cell adhesion, migration, and invasion were evaluated using cell adhesion, wound healing, and Boyden chamber assays. Molecular docking analysis was performed to assess the possible binding mode of BBR against EGFR, Erk, STAT3 and Akt. The effects of BBR on the activations of EGF/EGFR and its downstream effectors were demonstrated using Western blotting. Results: BBR inhibited growth of CCA cells in a dose dependent manner. At sub-cytotoxic dose, BBR significantly inhibited cell adhesion, migration, invasion and decreased expression of vimentin, slug, and VEGFA of both CCA cell lines. Molecular docking suggested the simultaneous inhibitory activity of BBR on EGFR, Erk, STAT3 and Akt. The Western blot analyses revealed that upon the EGF/EGFR activation, BBR considerably attenuated the activations of EGFR, Erk, STAT3 and Akt. Conclusion: Low dose of BBR suppresses EMT and thus aggressiveness of CCA cells, in part by its multi-kinase inhibitor property on EGFR and its downstream pathways.  BBR might be beneficial for therapy of human CCA.     

Anti-Proliferative Effects of Compound A and Its Effect in Combination with Cisplatin in Cholangiocarcinoma Cells

Background: Cholangiocarcinoma (CCA) is a fatal cancer with high resistance to anticancer drugs.  The development of new drugs or compounds to be used alone or in combination with currently available chemotherapeutic agents to improve the treatment of CCA is needed. Compound A (CpdA), which is a small plant-derived glucocorticoid receptor modulator, strongly inhibited the growth and survival of several cancers.  However, the effect of CpdA on cholangiocarcinoma has not been elucidated. The aim of this study was to investigate the effect of CpdA on CCA. Methods: Cytotoxicity of CpdA was tested in primary cells including peripheral blood mononuclear cells (PBMCs), fibroblasts, and human umbilical vein endothelial cells (HUVECs), as well as on CCA cell lines (KKU-100, KKU-055, and KKU-213) was examined. Cell cycle distribution and IL-6 expression was assessed by flow cytometry and real-time polymerase chain reaction, respectively.  The effect of combination CpdA and cisplatin was evaluated by cell viability assay. Results: CpdA significantly inhibited cell cycle at G1 phase in CCA cell lines, and reduced IL-6 mRNA expression.  However, combination CpdA and cisplatin did not enhance the inhibitory effect. TGFβR-II expression was increased in CCA cells after the combination treatment. Conclusions: These results indicate the potential of CpdA for CCA treatment. However, combination treatment with CpdA and cisplatin increased CCA cell survival. The molecular mechanism is likely attributable to promotes cell survival via the TGFβR-II signaling pathway. The combination of CpdA with other anticancer drugs for CCA treatment should be further examined.     

吉非替尼对胃癌细胞株放射增敏的作用

背景与目的:表皮生长因子受体(epidermal growth factor receptor,EGFR)在绝大部分人类上皮肿瘤中都有表达,其表达高低与放疗抗拒有关.我们检测EGFR酪氨酸激酶抑制剂吉非替尼(gefitinib)对高表达胃癌细胞株放射增敏的作用,并初步探讨其机制.方法:Western blot法测定7株人胃癌细胞株(MKN45、SGC7901、SNU-1、N87、AGS、SNU-16、KATO-Ⅲ)中EGFR蛋白的表达,选取2株EGFR相对高表达的胃癌细胞用于后续实验.采用MTT法测定吉非替尼的半数抑制浓度(50%inhibition concentration,IC50),克隆形成实验计算细胞存活率,拟合生存曲线并计算放射生物学参数,流式细胞仪检测吉非替尼联合放疗的凋亡率及细胞周期分布.结果:选取7株胃癌细胞中EGFR表达最高的MKN45和SGC7901细胞,发现其存活率均随吉非替尼浓度或放射剂量的增加而明显下降(P＜0.05).MTT法检测吉非替尼对MKN45及SGC7901细胞的IC50分别为0.4mmol/L和0.8 mmol/L.MKN45细胞在0.1×IC50及0.2×IC50剂量下的增敏比(SER)分别为1.102和1.154,SGC7901则为1.092和1.176.吉非替尼或照射均可增加凋亡率,减少S期细胞比例及增加G2/M期细胞比例(P＜0.01).结论:吉非替尼序贯照射应用可提高EGFR高表达胃癌细胞的放射敏感性并阻碍细胞增殖、促进凋亡和干扰细胞周期分布.吉非替尼有望成为EGFR高表达胃癌的放射增敏剂.     

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 effectsof rhinacanthin-C on cell proliferation, migration, invasion and the expression of proteins regulating cancer cellinvasion-regulated proteins in a cholangiocarcinoma (KKU-M156) cell line. Cytotoxicity of rhinacanthin-C wasdetermined by the SRB assay. Using wound-migration, chamber-migration and chamber-invasion assays, we assessedthe 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 zymographyassays. The expression of invasion-regulated proteins was investigated using western-blot analysis. After treatmentwith rhinacanthin-C, KKU-M156 cells exhibited antiproliferative effects in a dose-dependent manner with greaterefficacy than in Vero cells: IC50 values were 1.50 and 2.37 μM, respectively. Rhinacanthin-C significantly inhibited cellmigration and invasion of KKU-M156 cells in a dose-dependent manner. Consistent with this observation, treatmentwith rhinacanthin-C was associated with a decrease in the expression levels of FAK, p-FAK, MMP-2, and a decrease inthe levels of p38-, JNK1/2- and ERK1/2-MAPK pathways as well as inhibiting NF-κB/p65 expression and translocationof NF-κB/p65 to the nucleus. We have shown for the first time that the anti-metastatic effects of rhinacanthin-C onKKU-M156 cells are mediated via inhibition of the expression of invasion-regulated proteins. Rhinacanthin-C maydeserve consideration as a potential agent for the treatment of cholangiocarcinoma.     

PLC and PI3K Pathways are Important in the Inhibition of EGF-lnduced Cell Migration by Gefitinib (‘lressa’, ZD1839)

BACKGROUND: Expression of epidermal growth factor receptor (EGFR) by human breast cancer tissues is associated with poor clinical response. The EGFR tyrosine kinase inhibitor (EGFR-TKI), gefitinib ('Iressa', ZD1839), is a leading example of a molecular targeted agent, and has an anti-proliferative effect on various cancer cells. But the details of the anti-cancer effect and mechanism have not been elucidated. We studied the anti-cancer effect of gefitinib in breast cancer cell lines and the intracellular pathway downstream of EGFR associated with cell migration. METHODS: In this study, we analysed the anti-proliferative and anti-migratory effect of gefitinib in EGFR (+) breast cancer cell lines by WST-1 analysis and chemotaxis chamber analysis. We analyzed several intracellular phosphorylated pathways which are activated by mitogen activated kinases (extracellular signal-regulated protein kinase 1 and 2: MEK), phosphatidylinositol 3'-kinase (PI3K) and phpspholipase C (PLC), by blocking those pathways using inhibitors of each kinase, and also investigated the effects on the phosphorylation of myosin light chain (MLC). RESULTS: Gefitinib inhibited proliferation in most of these cell lines. MDA-MB231 was shown to be resistant. Furthermore, proliferation of MDA-MB231 cells was not affected by EGF stimulation, but migration of MDA-MB231 cells was significantly inhibited. PI3K and PLC inhibitors blocked EGF-stimulated cell migration and MLC phosphorylation, but the MEK inhibitor did not influence cell migration. CONCLUSIONS: Gefitinib has an anti-migratory effect on MDA-MB231 that results in an anti-proliferative effect. PI3K and PLC are important for the migration of MDA-MB231 cells, and gefitinib may inhibit migration by blocking these signalling pathways.     

The Importance of CYP19A1 in Estrogen Receptor-Positive Cholangiocarcinoma

CYP19A1, also called aromatase, is a key enzyme for converting androgens to estrogens of estrogen synthesis. Elevated serum estrogen and high expression levels of estrogen-related proteins are found in cholangiocarcinoma (CCA; bile duct cancer). However, the expression of CYP19A1 in relation to estrogen-related proteins, including estrogen receptors (ERα, ERβ, and GPR30) and an estrogen response protein (TFF1), has never been explored in CCA. In this study, we investigated the expressions of CYP19A1 and estrogen-related proteins in CCA tissues (n?=?74; 51 males and 23 females) using immunohistochemistry. The results showed that CYP19A1 was overexpressed in CCA cells compared with that in normal bile duct cells in the adjacent tissues. High expression of CYP19A1 was correlated with the metastatic status of the patients. High CYP19A1 expression was also positively correlated with GPR30 expression. Correlation between high CYP19A1 expression in the tumor tissues and shorter survival time was more prominent in male than in female CCA patients. To elucidate further, the effect of CYP19A1 knockdown on a CCA cell line was examined using a specific siRNA. When CYP19A1 gene expression was suppressed, migration and proliferation activities of CCA cells were significantly reduced. Moreover, the cell proliferation of high CYP19A1-expressing KKU-213 cells was more profoundly suppressed by CYP19A1 inhibitors (exemestane and letrozole) than low CYP19A1-expressing KKU-100 cells. Thus, CYP19A1 promotes CCA progression with aggressive clinical outcomes via increased migration and proliferation activities of cancer cells. CYP19A1 can be a potential chemotherapeutic target for CCA, especially in male patients.     

Modulation of DNA repair in vitro after treatment with chemotherapeutic agents by the epidermal growth factor receptor inhibitor gefitinib (ZD1839).

PURPOSE: The epidermal growth factor receptor (EGFR) is commonly expressed in human tumors and provides a target for therapy. Gefitinib (Iressa, ZD1839) is a quinazoline derivative that inhibits EGFR tyrosine kinase activity. Gefitinib demonstrated anticancer efficacy in vivo, and although experiments in vitro have suggested that inhibition of EGFR modulates the activity of chemotherapeutic agents, the mechanism of this interaction is unclear. We investigated mechanisms for this modulation. EXPERIMENTAL DESIGN: The antiproliferative effect of gefitinib alone or combined with cisplatin, melphalan, and etoposide was determined in a human breast (MCF-7) cancer cell line. Using the alkaline single-cell gel electrophoresis (comet) assay, we investigated kinetics of DNA damage and repair after treatment with the chemotherapeutic drugs combined with gefitinib. To investigate whether the phosphatidylinositol 3'-kinase pathway was contributing to repair-inhibition produced by gefitinib, cells were exposed to chemotherapy in combination with the phosphatidylinositol 3'-kinase inhibitor LY294002. RESULTS: A superadditive (synergistic) increase in growth inhibition for combined treatment with gefitinib was found for cisplatin and etoposide, but not with melphalan. There was delayed repair of DNA strand breaks after treatment with etoposide combined with gefitinib, and repair of DNA interstrand cross-links produced by cisplatin is delayed in combination with gefitinib. Inhibition of cell proliferation and DNA repair was identical in cells treated with LY294002. Immunoprecipitation of cell extracts demonstrated that after exposure to gefitinib, there was an association between EGFR and DNA-PK(CS). CONCLUSION: Gefitinib acts through inhibition of repair of cisplatin and etoposide-induced DNA damage; this effect is mimicked by inhibitors of the phosphatidylinositol 3'-kinase suggesting similar mechanisms of action.     

Antitumor activity of gefitinib in malignant rhabdoid tumor cells in vitro and in vivo.

PURPOSE: Malignant rhabdoid tumor (MRT) is a rare and highly aggressive neoplasm of young children. Current treatments have had only limited success. Epidermal growth factor receptor (EGFR) was found recently to be expressed on MRT cell lines. Gefitinib (trade name Iressa) is an oral and selective EGFR-tyrosine kinase inhibitor and has been demonstrated to be effective in inhibiting the proliferation of cancer cells in vivo as well as in clinical trials. This encouraged us to examine the antitumor effects of gefitinib on MRT cells in vitro and in vivo. EXPERIMENTAL DESIGN: The expression of EGFR in two MRT tumors and two MRT cell lines (MP-MRT-AN and KP-MRT-NS), established from these two tumor tissues, was examined by immunohistochemistry, immunofluorescence, and immunoblot. The effect of gefitinib on EGFR phosphorylation was examined by immunoblot. The effects of gefitinib on cell growth and apoptosis were examined by cell growth assay and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. The in vivo effect of gefitinib was assessed in athymic mice that had been xenografted with MRT cells. RESULTS: The expression of EGFR was detected in both tumor tissues and cell lines. Gefitinib inhibited EGFR-phosphorylation (IC(50) < 0.1 micromol/L) and in vitro cell growth (IC(50) = approximately 10-12 micromol/L), and a high concentration of gefitinib (20 micromol/L) induced apoptosis in vitro (MP-MRT-AN, 42.9% and KP-MRT-NS, 47.2%). Furthermore, gefitinib at 150 mg/kg had a cytostatic effect on established MRT xenografts (MP-MRT-AN, P = 0.039 and 0.0014; and KP-MRT-NS, P = 0.048 and 0.0086). CONCLUSIONS: Our results demonstrate that gefitinib has antitumor effects in MRT cells in vitro and in vivo and, thus, has promise as a novel and therapeutic strategy for MRT.     

Annexin A1 Is a Potential Prognostic Marker for,and Enhances the Metastasis of,Cholangiocarcinoma

Objective: Annexin A1 (ANXA1) is a calcium-dependent phospholipid-binding protein which contributes to proliferation, cancer progression and metastasis. Overexpression of ANXA1 is closely associated with metastasis in numerous types of cancer. Cholangiocarcinoma (CCA) is a bile-duct cancer which has high rates of metastasis. Previously, we demonstrated up-regulation of ANXA1 in a highly metastatic CCA cell line (KKU-213AL5). Here, we investigated the functions of ANXA1 in the progression of CCA cell lines and evaluated its clinical impacts in human CCA tissues.  Methods: Effects of ANXA1 on metastatic potential of CCA cell lines were evaluated using cell-proliferation, clonogenic, migration and invasion assays. The expression of ANXA1 in 44 intrahepatic human CCA tissues was investigated using immunohistochemistry (IHC). The association of ANXA1 with clinicopathological features of CCA patients was analyzed. Results: Silencing of ANXA1 expression using siRNA significantly decreased cell proliferation, colony formation, cell migration and invasion in the KKU-213AL5 cell line. IHC results showed low expression of ANXA1 in normal bile ducts in the non-tumor area. In contrast, high expression of ANXA1 in human CCA tissues was associated with advanced tumor stage, tumor size and presence of lymph-node metastasis. Conclusion: These findings strongly imply that ANXA1 contributes to the progression of CCA. ANXA1 can serve as a potential prognostic marker for CCA. Ablation of ANXA1 action may be an alternative strategy to prevent metastasis of CCA.     

High levels of HER-2 expression alter the ability of epidermal growth factor receptor (EGFR) family tyrosine kinase inhibitors to inhibit EGFR phosphorylation in vivo.

The epidermal growth factor receptor (EGFR) and HER-2 tyrosine kinases have been implicated in the development, progression, and severity of several human cancers and are attractive targets for therapeutic intervention. SU11925 was developed as a small molecule inhibitor of the tyrosine kinase activity of both EGFR and HER-2. In cellular assays, SU11925 exhibited similar potency against EGFR and HER-2, inhibiting EGF-stimulated EGFR autophosphorylation in A431 (human epidermoid carcinoma) cells with an IC(50) of 30 nM and HER-2 phosphorylation in SK-OV-3TP5 (human ovarian carcinoma) cells with an IC(50) of 38 nM. In contrast to its similar activity against the two targets in cellular assays, approximately 10-fold higher plasma concentrations of SU11925 were required to inhibit HER-2 phosphorylation in HER-2-overexpressing tumors compared with EGFR phosphorylation in EGFR-overexpressing tumors in vivo. Consistent with the proposed mechanism of action of this inhibitor, SU11925 inhibited the s.c. growth of EGFR- and HER-2-dependent tumors in athymic mice at doses that produced substantial inhibition of target receptor phosphorylation in vivo. An unexpected finding from these studies was that higher plasma concentrations of SU11925 were required to inhibit EGFR phosphorylation in vivo in tumors that also express high levels of HER-2 than in tumors that express EGFR alone. This observation, which suggests that it is more difficult to inhibit EGFR phosphorylation in vivo in cells that express high levels of HER-2, was confirmed with ZD1839 (Iressa), a selective EGFR inhibitor that also targets the tyrosine kinase catalytic site. The potential clinical implications of this observation are discussed.