Evaluation and molecular docking study of two flavonoids from Oroxylum indicum (L.) Kurz and their semi-synthetic derivatives as histone deacetylase inhibitors.

Chrysin (5,7-dihydroxyflavone, 6) and galangin 3-methyl ether (5,7-dihydroxy-3-methoxy flavone, 7) were obtained from the leaves of Oroxylum indicum (L.) Kurz in 4% and 6% yields, respectively. Both compounds could act as pan-histone deacetylase (HDAC) inhibitors. Structural modification of these lead compounds provided thirty-eight derivatives which were further tested as HDAC inhibitors. Compounds 6b, 6c, and 6q were the most potent derivatives with the IC50 values of 97.29 ± 0.63 µM, 91.71 ± 0.27 µM, and 96.87 ± 0.45 µM, respectively. Molecular docking study indicated the selectivity of these three compounds toward HDAC8 and the test against HDAC8 showed IC50 values in the same micromolar range. All three compounds were further evaluated for the anti-proliferative activity against HeLa and A549 cell lines. Compound 6q exhibited the best activity against HeLa cell line with the IC50 value of 13.91 ± 0.34 µM. Moreover, 6q was able to increase the acetylation level of histone H3. These promising HDAC inhibitors deserve investigation as chemotherapeutic agents for treating cancer.

Tiliacora triandra Leaf Powder Ethanolic Extract in Combination with Cisplatin or Gemcitabine Synergistically Inhibits the Growth of Cholangiocarcinoma Cells In Vitro and in Nude Mouse Xenograft Models.

Background and Objectives: The treatments of cholangiocarcinoma (CCA) with Cisplatin (Cis) and Gemcitabine (Gem) often cause side effects and drug resistance. This study aimed to investigate the combined effects of Tiliacora triandra leaf powder ethanolic extract (TLPE) and Cis or Gem on CCA cells in vitro and in nude mouse xenografts. Materials and Methods: Antiproliferative activity was evaluated using MTT assay. Drug interaction was studied by Chou-Talalay method. Apoptosis induction and cell cycle arrest were analyzed by flow cytometry. Cell cycle and apoptosis regulating proteins were evaluated by western blot analysis. Results:Treatments with Cis or Gem in combination with TLPE significantly inhibited the growth of KKU-M213B and KKU-100 cells compared with single drug treatments. Synergistic drug interactions were observed with the dose reduction of Cis and Gem treatments. The safety of TLPE was demonstrated in vitro by the hemolytic assay. Synergistic combination treatments down-regulated Bcl2 and reduced the ratio of Bcl2/Bax in both CCA cells. TLPE enhanced tumor suppression of both Cis and Gem in nude mouse xenograft models. Combination treatments with Cis and TLPE reduced Cis toxicity, as demonstrated by the enhanced body weight change of the treated mice compared with the treatment with Cis alone. Furthermore, TLPE reduced hepatotoxicity caused by Gem treatment and reduced kidney and spleen toxicities caused by Cis treatment. Conclusion: These findings suggest that TLPE enhances the anticancer activity of Cis and Gem and reduces their toxicity both in vitro and in nude mouse xenograft models.

Ethanolic extract of Ya-nang (Tiliacora triandra) leaf powder induces apoptosis in cholangiocarcinoma cell lines via induction of hyperacetylation and inhibition of growth signaling.

Objective: To develop alternative medicine for reducing undesired side effects of chemotherapy in CCA patients, the anticancer activity of Tiliacora triandra leaf powder ethanolic (TLPE) extract against cholangiocarcinoma cell lines was investigated. Methods: Antiproliferation was studied using the MTT assay while apoptosis induction and cell cycle arrest were analyzed by flow cytometry. The levels of key proteins and phenolic acid content were analyzed by western blotting and reversed-phase HPLC, respectively. Results: TLPE extract inhibited CCA cell growth in a dose- and time-dependent manner, with IC50 values of 7.86 ± 0.05 µg/ml for KKU-M213B cells and 8.59 ± 0.36 µg/ml for KKU-100 cells at an exposure time of 72 h. TLPE extract inhibited the growth of CCA cell lines by inducing apoptosis of both cell lines and causing an increased population of KKU-100 cells at G0/G1 phase. TLPE extract up-regulated Ac-H3 but down-regulated p-ERK, p53, Bax, CDK4 and Bcl2 expressions in KKU-M213B cells. TLPE extract up-regulated Ac-H3, p21 and Bax but down-regulated p-ERK, p53, CDK4 and Bcl2 expressions in KKU-100 cells. Additionally, phenolic acids including p-hydroxybenzoic, vanillic, syringic, p-coumaric, ferulic and sinapinic acids were identified. Conclusion: These results suggest the possibility of developing T. triandra leaf powder ethanolic extract as a chemotherapeutic or chemoprevention agent for cholangiocarcinoma.

HDAC Inhibitory and Anti-Cancer Activities of Curcumin and Curcumin Derivative CU17 against Human Lung Cancer A549 Cells.

Previous research reported that the curcumin derivative (CU17) inhibited several cancer cell growths in vitro. However, its anticancer potential against human lung cancer cells (A549 cell lines) has not yet been evaluated. The purpose of this research was to examine the HDAC inhibitory and anti-cancer activities of CU17 compared to curcumin (CU) in A549 cells. An in vitro study showed that CU17 had greater HDAC inhibitory activity than CU. CU17 inhibited HDAC activity in a dose dependent manner with the half-maximal inhibitory concentration (IC50) value of 0.30 ± 0.086 µg/mL against HDAC enzymes from HeLa nuclear extract. In addition, CU17 could bind at the active pockets of both human class I HDACs (HDAC1, 2, 3, and 8) and class II HDACs (HDAC4, 6, and 7) demonstrated by molecular docking studies, and caused hyperacetylation of histone H3 (Ac-H3) in A549 cells shown by Western blot analysis. MTT assay indicated that both CU and CU17 suppressed A549 cell growth in a dose- and time-dependent manner. Besides, CU and CU17 induced G2/M phase cell cycle arrest and p53-independent apoptosis in A549 cells. Both CU and CU17 down-regulated the expression of p53, p21, Bcl-2, and pERK1/2, but up-regulated Bax expression in this cell line. Although CU17 inhibited the growth of lung cancer cells less effectively than CU, it showed less toxicity than CU for non-cancer cells. Accordingly, CU17 is a promising agent for lung cancer treatment. Additionally, CU17 synergized the antiproliferative activity of Gem in A549 cells, indicating the possibility of employing CU17 as an adjuvant treatment to enhance the chemotherapeutic effect of Gem in lung cancer.

Histone Deacetylase Inhibitory Activity and Antiproliferative Potential of New [6]-Shogaol Derivatives.

Twenty newly synthesized derivatives of [6]-shogaol (4) were tested for inhibitory activity against histone deacetylases. All derivatives showed moderate to good histone deacetylase inhibition at 100 µM with a slightly lower potency than the lead compound. Most potent inhibitors among the derivatives were the pyrazole products, 5j and 5k, and the Michael adduct with pyridine 4c and benzothiazole 4d, with IC50 values of 51, 65, 61 and 60 µM, respectively. They were further evaluated for isoform selectivity via a molecular docking study. Compound 4d showed the best selectivity towards HDAC3, whereas compound 5k showed the best selectivity towards HDAC2. The potential derivatives were tested on five cancer cell lines, including human cervical cancer (HeLa), human colon cancer (HCT116), human breast adenocarcinoma cancer (MCF-7), and cholangiocarcinoma (KKU100 and KKU-M213B) cells with MTT-based assay. The most active histone deacetylase inhibitor 5j exhibited the best antiproliferative activity against HeLa, HCT116, and MCF-7, with IC50 values of 8.09, 9.65 and 11.57 µM, respectively, and a selective binding to HDAC1 based on molecular docking experiments. The results suggest that these compounds can be putative candidates for the development of anticancer drugs via inhibiting HDACs.

Immunomodulatory Potential of the Industrialized Houttuynia cordata Fermentation Product In Vitro and in Wistar Rats.

Houttuynia cordata fermentation products (HCFPs) are produced and widely used as dietary supplements for health and immune support. However, the effect on immune function for these products has not been clearly demonstrated. In this study, soluble fractions of the selected HCFP were used for determination of the immunomodulatory potential, both in vitro and in animal models. Viability and proliferation of rat splenocytes and phagocytic activity of human neutrophils were evaluated. Studies on immunomodulatory effects, including hematological parameters, mitogen-driven lymphocyte proliferation and hemagglutination, were performed in both healthy and immunosuppressed rats. Soluble fraction of the selected HCFP significantly enhanced phagocytic activity of human neutrophils and tended to stimulate splenocyte viability and proliferation. There was no morbidity or mortality for administration of a 14-day regimen of the selected HCFP in both male and female rats. The healthy rats treated with HCFP gained body weight less than the control group, suggesting a reduction in calorie intake. Moreover, low dose of HCFP caused an increased B cell proliferation in ex-vivo, which was related to the increased antibody titer against SRBC in immunosuppressed rats. Our results indicate that the selected HCFP enhances the phagocytic activity of the neutrophils and augments the antibody production in immunosuppressed rats.

Anticancer effects of the combined Thai noni juice ethanolic extracts and 5-fluorouracil against cholangiocarcinoma cells in vitro and in vivo.

Application of 5-fluorouracil (5-FU) in cholangiocarcinoma (CCA) is limited by adverse side effects and chemoresistance. Therefore, the combination therapy of 5-FU with other substances, especially natural products may provide a new strategy for CCA treatment. The aim of this study was to evaluate the combination effects of 5-FU and two ethanolic extracts of Thai noni juice (TNJ) products on CCA cell lines and nude mice xenografts. The results of antiproliferative assay showed the combination treatment of 5-FU and each TNJ ethanolic extract exerted more cytotoxicity on CCA cells than either single agent treatment. Synergistic effects of drug combinations can enable the dose reduction of 5-FU. The mechanism underlying a combination treatment was apoptosis induction through an activation of p53 and Bax proteins. In the nude mouse xenograft model, combination treatments of 5-FU with each TNJ ethanolic extract suppressed the growth of CCA cells implanted mice more than single agent treatments with no effects on mouse body weight, kidney, and spleen. Moreover, low doses of TNJ ethanolic extracts reduced the hepatotoxicity of 5-FU in nude mice. Taken together, these data suggested that the ethanolic extracts of TNJ products can enhance the anti-CCA effect and reduce toxicity of 5-FU.

Influence of side-chain changes on histone deacetylase inhibitory and cytotoxicity activities of curcuminoid derivatives.

Using curcuminoids as lead compounds, fifty-nine curcuminoid derivatives with different side chains at the phenolic moiety were synthesized. All compounds were investigated for their histone deacetylase (HDAC) inhibitory activities. The potent pan-HDAC inhibitors were further tested against three human cancer cell lines including Hela, HCT116 and MCF-7 with MTT-based assay. The bisethylamide 4z and the mono-sec-butyl derivative 5j manifested good antiproliferative activities against HCT116 cancer cells with the IC50 values as 14.60 ± 1.19 µg/mL and 7.33 ± 0.98 µg/mL, respectively. Molecular docking study of both compounds with Class I HDACs revealed that the compounds might bind tightly to the binding pocket of HDAC2. These findings suggested that these compounds can be putative candidates for the development of anticancer drugs via inhibiting HDACs.

Anti-inflammatory activity of the dietary supplement Houttuynia cordata fermentation product in RAW264.7 cells and Wistar rats.

Houttuynia cordata Thunb. has been used as a traditional medicine to treat a variety of ailments in Asian countries such as China, Japan, South Korea, and Thailand. In Thailand, H. cordata fermentation products (HCFPs) are commercially produced and popularly consumed throughout the country without experimental validation. Anti-inflammatory activity of H. cordata fresh leaves or aerial parts has previously been reported, however, the anti-inflammatory activity of the commercially available HCFPs produced by the industrialized process has not yet been investigated. The aim of this study was to evaluate in vitro and in vivo anti-inflammatory potential of the selected industrialized HCFP. LPS-induced RAW264.7 and carrageenan-induced paw edema models were used to evaluate the anti-inflammatory activity of HCFP. The phenolic acid components of HCFP aqueous and methanolic extracts were investigated using HPLC analysis. In RAW264.7 cells, the HCFP aqueous and methanolic extracts reduced NO production and suppressed LPS-stimulated expression of PGE2, iNOS, IL-1ß, TNF-α and IL-6 levels in a concentration-dependent manner, however, less effect on COX-2 level was observed. In Wistar rats, 3.08 and 6.16 mL/kg HCFP reduced paw edema after 2 h carrageenan stimulation, suggesting the second phase anti-edematous effect similar to diclofenac (150 mg/kg). Whereas, 6.16 mL/kg HCFP also reduced paw edema after 1 h carrageenan stimulation, suggesting the first phase anti-edematous effect. Quantitative HPLC revealed the active phenolic compounds including syringic, vanillic, p-hydroxybenzoic and ferulic acids, which possess anti-inflammatory activity. Our results demonstrated for the first time the anti-inflammatory activity of the industrialized HCFP both in vitro and in vivo, thus validating its promising anti-inflammation potential.

Antiproliferative activities and phenolic acid content of water and ethanolic extracts of the powdered formula of Houttuynia cordata Thunb. fermented broth and Phyllanthus emblica Linn. fruit.

BACKGROUND: Houttuynia cordata Thunb. and Phyllanthus emblica Linn. are native plants with medicinal and nutritive significance in Asia. The present study was aimed at evaluating antiproliferative effects on human cancer cell lines and identifying the phenolic acid composition of water and ethanolic extracts of the powdered formula of H. cordata fermented broth and P. emblica fruit. METHODS: Anticancer activity of the extracts was evaluated against HeLa, HT29, HCT116, MCF7 and Jurkat cells using an MTT assay and flow cytometric analysis of apoptosis induction and cell cycle arrest. Reverse phase HPLC was exploited for identification and quantification of some phenolic acids. RESULTS: MTT assay showed that both water and ethanolic extracts significantly decreased the viability of cancer cells in a dose- and time-dependent fashion. Based on the IC50 values, ethanolic extract (IC50 values = 0.12-0.65 mg/mL) was more cytotoxic than water extract (IC50 values = 0.22-0.85 mg/mL) and Jurkat cells were the most sensitive to both extracts (IC50 values = 0.12-0.69 mg/mL). The underlying mechanism for antiproliferative activity was apoptosis induction, especially in HT29, HCT116, MCF7 and Jurkat cells. HT29 cells were the most sensitive to extract-induced apoptosis. Ethanolic extract was more effective at inducing apoptosis than water extract. Moreover, cell cycle arrest was found to be another mechanism behind growth inhibition in Jurkat and HCT116 cells. However, these extracts were relatively less toxic to non-cancer Vero cells. HPLC analysis demonstrated that the powder mix extracts contained seven identified phenolic acids namely gallic, p-hydroxybenzoic, vanillic, syringic, p-coumaric, ferulic and sinapinic acids, where p-coumaric acid was detected in the highest concentration followed by ferulic acid. CONCLUSION: Overall, the results of this study suggest the powdered formula of H. cordata fermented broth and P. emblica fruit as an alternative medicine for cancer prevention and treatment.

Peanut testa extracts possessing histone deacetylase inhibitory activity induce apoptosis in cholangiocarcinoma cells.

Previous studies demonstrated that peanut testa extracts (KK4 and ICG15042) containing natural histone deacetylase (HDAC) inhibitors inhibited the growth of several human cancer cell lines via apoptosis induction. The aims of this study were to investigate the anti-proliferative effects and the mechanism(s) responsible for apoptosis induction mediated by these peanut testa extracts in human cholangiocarcinoma cell lines (KKU-M214 and KKU-100). The anti-proliferative effects were assessed by MTT assay. Apoptotic cell death and cell cycle arrest were analyzed by flow cytometry. The caspase activities were studied using colorimetric caspase activity assay and western blot analysis. Our results revealed that KK4 and ICG15042 extracts inhibited cell proliferation of both KKU-M214 and KKU-100 cells in a dose- and time-dependent manner, with IC50 values of 38.28 ±â€¯0.29 (KK4), 43.91 ±â€¯1.94 (ICG15042) µg/mL for KKU-M214 and 78.40 ±â€¯1.74 (KK4), 82.77 ±â€¯0.94 (ICG15042) µg/mL for KKU-100 at 72 h. Apoptosis induction by these peanut testa extracts were observed in both KKU-M214 and KKU-100 cells in a concentration-dependent manner. Moreover, the percentage of cells in the sub-G1 phase was significantly increased in both KKU-M214 and KKU-100 cells. Cell cycle arrest was not observed in other cell cycle phases. Activation of caspases 8 and 3 were apparent integral parts of apoptosis induction in both cells. Both peanut testa extracts also caused down-regulation of p53, p21, Bcl-2 and pERK1/2 protein expression in these cells. These results suggest that peanut testa extracts may be potential anti-cancer agents for cholangiocarcinoma chemoprevention or chemotherapy.

Characterization and localization of Opisthorchis viverrini fructose-1,6-bisphosphate aldolase.

Opisthorchis viverrini (Ov) infection is a long-time public health problem in Thailand that can lead to bile duct cancer, cholangiocarcinoma (CCA). Characterization of the Ov proteins at a molecular level will increase our knowledge of host-parasite interaction that can be applied to new drug, vaccine, or immunodiagnostic development. In this study, an important enzyme in the Ov glycolytic pathway, fructose-1,6-bisphosphate aldolase (FBPA), that had been obtained from a previous study was characterized and immunolocalized. The full-length sequence of OvFBPA gene is 1089bp and encodes 362 amino acids with a predicted molecular weight and isoelectric point of 39.54kDa and 7.61, respectively. Additionally, three OvFBPA isoforms were identified by sequence analysis. The amino acid sequence of OvFBPA-1 characterized in this study shared 98% identity to FBPA isoform 1 of Clonorchis sinensis that was classified based on highly conserved active residues to class-I FBPA. The recombinant OvFBPA-1 protein was expressed as a soluble form in Escherichia coli at 25°C with N-terminal His-tagged fusion protein and the purified OvFBPA-1 protein was used to generate polyclonal antibody in mice. Antibody against rOvFBPA-1 protein was able to detect the native OvFBPA-1 protein in both Ov infected hamster liver section and Ov excretory-secretory (ES) products by immunohistochemistry and western blotting, respectively.

Synergistic anticancer effects of cisplatin and histone deacetylase inhibitors (SAHA and TSA) on cholangiocarcinoma cell lines.

Clinical application of cisplatin against cholangiocarcinoma is often associated with resistance and toxicity posing urgent demand for combination therapy. In this study, we evaluated the combined anticancer effect of cisplatin and histone deacetylase inhibitors (HDACIs), suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), on the cholangiocarcinoma KKU-100 and KKU-M214 cell lines. Antiproliferative activity was evaluated using MTT assay. Apoptosis induction and cell cycle arrest were analyzed by flow cytometry. Cell cycle and apoptosis regulating proteins were evaluated by western blot analysis. MTT assay showed that cisplatin, SAHA and TSA dose-dependently reduced the viability of KKU-100 and KKU-M214 cells. The combination of cisplatin and HDACIs exerted significantly more cytotoxicity than the single drugs. Combination indices below 1.0 reflect synergism between cisplatin and HDACIs, leading to positive dose reductions of cisplatin and HDACIs. Cisplatin and HDACIs alone induced G0/G1 phase arrest in KKU-100 cells, but the drug combinations increased sub-G1 percent more than either drug. However, cisplatin and HDACIs alone or in combination increased only the sub-G1 percent in KKU-M214 cells. Annexin V-FITC staining revealed that cisplatin and HDACIs combinations induced more apoptotic cell death of both KKU-100 and KKU-M214 cells than the single drug. In KKU-100 cells, growth inhibition was accompanied by upregulation of p53 and p21 and downregulation of CDK4 and Bcl-2 due to exposure to cisplatin, SAHA and TSA alone or in combination. Moreover, combination of agents exerted higher impacts on protein expression. Single agents or combination did not affect p53 expression, however, combination of cisplatin and HDACIs increased the expression of p21 in KKU-M214 cells. Taken together, cisplatin and HDACIs combination may improve the therapeutic outcome in cholangiocarcinoma patients.

Anticancer Effects of Curcuma C20-Dialdehyde against Colon and Cervical Cancer Cell Lines.

BACKGROUND: Recent attention on chemotherapeutic intervention against cancer has been focused on discovering and developing phytochemicals as anticancer agents with improved efficacy, low drug resistance and toxicity, low cost and limited adverse side effects. In this study, we investigated the effects of Curcuma C20-dialdehyde on growth, apoptosis and cell cycle arrest in colon and cervical cancer cell lines. MATERIALS AND METHODS: Antiproliferative, apoptosis induction, and cell cycle arrest activities of Curcuma C20-dialdehyde were determined by WST cell proliferation assay, flow cytometric Alexa fluor 488-annexin V/propidium iodide (PI) staining and PI staining, respectively. RESULTS: Curcuma C20 dialdehyde suppressed the proliferation of HCT116, HT29 and HeLa cells, with IC50 values of 65.4±1.74 µg/ml, 58.4±5.20 µg/ml and 72.0±0.03 µg/ml, respectively, with 72 h exposure. Flow cytometric analysis revealed that percentages of early apoptotic cells increased in a dose-dependent manner upon exposure to Curcuma C20-dialdehyde. Furthermore, exposure to lower concentrations of this compound significantly induced cell cycle arrest at G1 phase for both HCT116 and HT29 cells, while higher concentrations increased sub-G1 populations. However, the concentrations used in this study could not induce cell cycle arrest but rather induced apoptotic cell death in HeLa cells. CONCLUSIONS: Our findings suggest that the phytochemical Curcuma C20-dialdehyde may be a potential antineoplastic agent for colon and cervical cancer chemotherapy and/or chemoprevention. Further studies are needed to characterize the drug target or mode of action of the Curcuma C20-dialdehyde as an anticancer agent.

Identification of peanut seed prolamins with an antifungal role by 2D-GE and drought treatment.

This work revealed peanut seed prolamins likely displaying a defensive role besides the known nitrogen storage. Drought stress and proteomic approaches were used in varieties of peanuts to explore the prolamin member in association with a test against Aspergillus flavus spore germination. The stress effect was showed by aerial biomass, leaf content of malondialdehyde, and seed contamination by A. flavus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles were not informative for the antifungal polypeptides. From two-dimensional gel electrophoresis, the suspected polypeptides were those with pI 5.45-5.75 and sizes of 22.0-30.5 kDa specifically in Spanish-type peanuts. Regarding to the drought effect in most of these peanuts, the spot peak volume analysis deduced three novel prolamin-related antifungal polypeptides at pI 5.75-5.8 with 30.5, 27.5-28.5, and 22.0-22.5 kDa, which was confirmed after isoelectric purification at pH 5.60. The data could not yet conclude their correlation with resistance to drought and to seed infection by A. flavus.

The first trimeric Galanthus nivalis agglutinin-related lectin of Orchidaceae was found in Dendrobium pendulum: purification, characterization, and effects of stress factors.

KEY MESSAGE: Trimeric Galanthus nivalis agglutinin-related lectin of Orchidaceae with two conformational forms was first studied in Dendrobium pendulum . It was highly expressed by stress factors. Using mannan-agarose column chromatography, a mannose-binding protein was purified from Dendrobium pendulum Roxb. pseudobulb. After heating in the presence of sodium dodecyl sulfate (SDS) with or without 2-mercaptoethanol, the protein showed one band with molecular mass of 14.0 kDa on SDS-polyacrylamide gel electrophoresis (PAGE). Without heating, three bands were found at positions of 14.0, 39.4, and 41.5 kDa, but a higher amount of 39.4 and 41.5 kDa protein bands were seen in the presence of 2-mercaptoethanol. Liquid chromatography-tandem mass spectrometry and database search indicated that the 14.0 kDa protein band contained three peptide fragments identical to parts of a lectin precursor from Dendrobiu m findleyanum Parish & Rchb.f. Native-PAGE and Ferguson plot showed that the purified protein had two native forms with molecular masses of 44.2 and 45.3 kDa, indicating three 14.0 kDa polypeptide subunits. The purified protein exhibited the agglutination activity with trypsinized chicken erythrocytes. It was then recognized as a Galanthus nivalis agglutinin-related lectin and named D. pendulum agglutinin (DPA). Using reverse transcription-polymerase chain reaction and DNA sequencing, the deduced amino acid sequence of DPA precursor showed the highest homology (96.4%) with a lectin precursor of D. findleyanum and contained three mannose-binding sites. Greater amounts of DPA were found when the pseudobulbs were treated with stress factors including ultraviolet light, abscisic acid, hydrogen peroxide, and acetylene gas.

Cloning, expression, and characterization of a novel Opisthorchis viverrini calcium-binding EF-hand protein.

A novel 22.8 kDa of Opisthorchis viverrini (Ov) calcium-binding EF-hand protein (Ov CaBP) was identified and isolated from an immunoscreening of the adult stage Ov cDNA library by using a human cholangiocarcinoma (CCA) serum. This protein was related to other calcium-binding proteins and conserved among the trematodes. Ov CaBP shared 98% amino acid identity to 22.8 kDa of Clonorchis sinensis CaBP and both were classified as a new group of CaBP EF-hand protein by multiple sequence alignment and phylogenetic tree analysis. The open reading frame of Ov CaBP was 585 bp which encoded for 194 amino acids. The N-terminal part is composed of two calcium-binding EF-hand motifs whereas the C-terminal part contains a dynein light chain motif (DLC). In addition, transcription analysis by RT-PCR revealed that it was constitutively transcribed in all stages, including metacercariae, juvenile, and adult. Furthermore, recombinant Ov CaBP protein (rOv CaBP) was expressed as a soluble protein and antibody generated against this rOv CaBP protein was capable of detecting Ov CaBP in the Ov somatic extracts but not in Ov ES products. This anti-rOv CaBP serum was also used to localize Ov CaBP in Ov infected hamster's liver sections which the distribution of Ov CaBP was located in gut epithelium, miracidia in eggs and slightly in parenchyma. Moreover, rOv CaBP protein showed a calcium-binding property in non-denaturing gel mobility shift assay.