Vithoon Viyanant

Cytotoxic activity of Thai medicinal plants against human cholangiocarcinoma, laryngeal and hepatocarcinoma cells in vitro

BackgroundCholangiocarcinoma is a serious public health in Thailand with increasing incidence and mortality rates. The present study aimed to investigate cytotoxic activities of crude ethanol extracts of a total of 28 plants and 5 recipes used in Thai folklore medicine against human cholangiocarcinoma (CL-6), human laryngeal (Hep-2), and human hepatocarcinoma (HepG2) cell lines in vitro.MethodsCytotoxic activity of the plant extracts against the cancerous cell lines compared with normal cell line (renal epithelial cell: HRE) were assessed using MTT assay. 5-fluorouracil was used as a positive control. The IC50 (concentration that inhibits cell growth by 50%) and the selectivity index (SI) were calculated.ResultsThe extracts from seven plant species (Atractylodes lancea, Kaempferia galangal, Zingiber officinal, Piper chaba, Mesua ferrea, Ligusticum sinense, Mimusops elengi) and one folklore recipe (Pra-Sa-Prao-Yhai) exhibited promising activity against the cholangiocarcinoma CL-6 cell line with survival of less than 50% at the concentration of 50 μg/ml. Among these, the extracts from the five plants and one recipe (Atractylodes lancea, Kaempferia galangal, Zingiber officinal, Piper chaba, Mesua ferrea, and Pra-Sa-Prao-Yhai recipe) showed potent cytotoxic activity with mean IC50 values of 24.09, 37.36, 34.26, 40.74, 48.23 and 44.12 μg/ml, respectively. All possessed high activity against Hep-2 cell with mean IC50 ranging from 18.93 to 32.40 μg/ml. In contrast, activity against the hepatoma cell HepG2 varied markedly; mean IC50 ranged from 9.67 to 115.47 μg/ml. The only promising extract was from Zingiber officinal (IC50 = 9.67 μg/ml). The sensitivity of all the four cells to 5-FU also varied according to cell types, particularly with CL-6 cell (IC50 = 757 micromolar). The extract from Atractylodes lancea appears to be both the most potent and most selective against cholangiocarcinoma (IC50 = 24.09 μg/ml, SI = 8.6).ConclusionsThe ethanolic extracts from five plants and one folklore recipe showed potent cytotoxic activity against CL-6 cell. Sensitivity to other cancerous cell lines varied according to cell types and the hepatocarcinoma cell line. HepG2 appears to be the most resistant to the tested extracts.

Molecular cloning and characterization of cathepsin L encoding genes from Fasciola gigantica

In this study cDNAs encoding cathepsin L-like proteins of Fasciola gigantica were cloned by the reverse transcription polymerase chain reaction method (RT-PCR) from total RNA of adult specimens. DNA sequence analyses revealed that six different cathepsin L cDNA fragments were isolated, which have DNA sequence identities of 87-99% towards the homologous genes from F. hepatica. Gene expression was studied at the RNA level by Northern and RNA in situ hybridizations. Northern analysis showed the cathepsin L genes to be strongly expressed in adult parasites as a group of 1050 nt sized RNAs. RNA in situ hybridization localized cathepsin L RNA to the cecal epithelial cells. Southern hybridization was used to determine the number of cathepsin L genes and indicated the presence of a family of closely related cathepsin L genes in the genome of F. gigantica.

Anticancer activities against cholangiocarcinoma, toxicity and pharmacological activities of Thai medicinal plants in animal models

BackgroundChemotherapy of cholangiocarcinoma (CCA), a devastating cancer with increasing worldwide incidence and mortality rates, is largely ineffective. The discovery and development of effective chemotherapeutics is urgently needed.Methods/DesignThe study aimed at evaluating anticancer activities, toxicity, and pharmacological activities of the curcumin compound (CUR), the crude ethanolic extracts of rhizomes of Zingiber officinale Roscoe (Ginger: ZO) and Atractylodes lancea thung. DC (Khod-Kha-Mao: AL), fruits of Piper chaba Hunt. (De-Plee: PC), and Pra-Sa-Prao-Yhai formulation (a mixture of parts of 18 Thai medicinal plants: PPF) were investigated in animal models. Anti-cholangiocarcinoma (anti-CCA) was assessed using CCA-xenograft nude mouse model. The antihypertensive, analgesic, anti-inflammatory, antipyretic, and anti-ulcer activities and effects on motor coordination were investigated using Rota-rod test, CODA tail-cuff system, writhing and hot plate tests, carrageenan-induced paw edema test, brewers yeast test, and alcohol-induced gastric ulcer test, respectively. Acute and subacute toxicity tests were performed according to the OECD guideline for testing of chemicals with modification.ResultsPromising anticancer activity against CCA in nude mouse xenograft model was shown for the ethanolic extract of AL at all oral dose levels (1000, 3000, and 5000 mg/kg body weight) as well as the extracts of ZO, PPF, and CUR compound at the highest dose level (5000, 4000, and 5000 mg/kg body weight, respectively). PC produced no significant anti-CCA activity. Results from acute and subacute toxicity tests both in mice and rats indicate safety profiles of all the test materials in a broad range of dose levels. No significant toxicity except stomach irritation and general CNS depressant signs were observed. Investigation of pharmacological activities of the test materials revealed promising anti-inflammatory (ZO, PPF, and AL), analgesic (CUR and PPF), antipyretic (CUR and AL), antihypertensive (ZO and AL), and anti-ulcer (CUR, ZO, and AL) activities.ConclusionPlants used in Thai traditional medicine for the treatment of various ailments may provide reservoirs of promising candidate chemotherapeutics for the treatment of CCA.

Type I cystatin (stefin) is a major component of Fasciola gigantica excretion/secretion product.

In the present study we describe type 1 cystatin, a cysteine protease inhibitor, as a major released antigen of the tropical liver fluke Fasciola gigantica (FgStefin-1). Immunohistochemical analysis showed that FgStefin-1 is abundant in (a) tissue of tegumental type, including oral and ventral sucker, pharynx, genital atrium, metraterm, cirrus and (b) the intestinal epithelium. Faint staining was observed in the epithelia of ovary and proximal uterus. Immunoblots showed the presence of FgStefin-1 in the parasites excretion/secretion (ES) product and immunodepletion demonstrated that FgStefin-1 herein is partially complexed with cathepsin L. Furthermore, quantitation of FgStefin-1 in comparison to cathepsin L in ES product and crude worm extract of adults supports a major external function of FgStefin-1 with an estimated 50% being released in at least equimolar amounts to cathepsin L. Sera of an experimentally infected rabbit reacted with recombinant FgStefin-1 starting 8 weeks postinfection. Activity analyses of recombinant FgStefin-1 showed nanomolar inhibition constants for mammalian cathepsin B, L, and S cysteine proteases and released cysteine proteases of the parasite. The protein is active over a wide pH range and is heat stable. Our results suggest protective functions of FgStefin-1, regulating intracellular cysteine protease activity, and possibly protection against extracellular proteolytic damage to the parasites intestinal and tegumental surface proteins. Considering inhibition kinetics and previously demonstrated immunomodulatory properties of cystatin in parasitic nematodes a comparable function of FgStefin-1 is suggested and is at present under investigation.

Production and characterization of a monoclonal antibody against 28.5 kDa tegument antigen of Fasciola gigantica.

A monoclonal antibody (MoAb) against the 28.5 kDa tegumental antigen of Fasciola gigantica was produced by the hybridoma technique using spleen cells from BALB/c mice immunized with the tegumental extract from adult F. gigantica. This MoAb was found to be of the isotype IgG(1), kappa-light chain, and shown by immunoblotting to specifically react with the 28.5 kDa antigen present in the tegument, excretion-secretion material of the adult, whole-body extracts of newly excysted juveniles, 5-week-old juvenile and adult parasites. It did not cross-react with antigens from other trematode parasites, including Schistosoma mansoni, Eurytrema pancreaticum and Paramphistomum spp. Immunolocalization of this antigen by indirect immunofluorescence indicated that it was present as a major component of the adult tegument, particularly in its outer rim, tegumental cells, and their processes. Furthermore, the epithelium linings of the oral sucker, buccal tube, pharynx, caecal bifurcation, both male and female genital canals, which were the continuation of the tegumental-type epithelium, were also positively stained with this MoAb. A similar pattern of immunolocalization, but with weaker staining intensity, was observed in newly excysted, 5- and 7-week-old juveniles. Thus this antigen is expressed in all developmental stages of the parasite, and it could be a strong candidate for immunodiagnosis and vaccine development.

Fasciola gigantica: The in vitro effects of artesunate as compared to triclabendazole on the 3-weeks-old juvenile

The in vitro effect of artesunate (ATS) on the 3-week-old juveniles of Fasciola gigantica was compared with triclabendazole (TCZ) by incubating the parasites in M-199 medium containing the drugs at concentrations of 20, 40, and 80 μg/ml for 1, 3, 6, 12, and 24h. The anthelmintic activities of these drugs were evaluated based on the relative motility value (RM) and the alterations of the tegument as observed by scanning (SEM) and transmission (TEM) electron microscopy. The RM values of TCZ-treated flukes decreased significantly from 6 to 24h for all dosages. For ATS-treated flukes, RM value decreased markedly from 12 to 24h, but the rates of decline were less than TCZ at the same doses. When observed by SEM, the tegument showed similar sequence of morphological changes after treatments with both drugs, comprising of swelling of tegumental ridges, followed by blebbing and later rupturing of the blebs, leading to erosion and lesion, and disruption of the tegument. When examined by TEM, ultrastructural changes in the tegument and associated structures after treatments with TCZ and ATS were similar which comprised of swelling, blebbing of the tegument, dilation of basal infoldings, and depolymerization of the microtrabecular network. After a longer incubation time, the tegument was completely sloughed off and the tegument cell bodies became necrotic. Additionally, in ATS-treated flukes, mitochondria showed severe swelling, rupturing of outer membrane, and their interior filled with flocculent materials.

Cytotoxicity, Toxicity, and Anticancer Activity of Zingiber Officinale Roscoe Against Cholangiocarcinoma

Cholangiocarcinoma (CCA) is an uncommon adenocarcinoma which arises from the epithelial cells of the bile ducts. The aim of the study was to investigate the cytotoxicity, toxicity, and anticancer activity of a crude ethanolic extract of ginger (Zingiber officinale Roscoe) against CCA. Cytotoxic activity against a CCA cell line (CL-6) was assessed by calcein-AM and Hoechst 33342 assays and anti-oxidant activity was evaluated using the DPPH assay. Investigation of apoptotic activity was performed by DNA fragmentation assay and induction of genes that may be involved in the resistance of CCA to anticancer drugs (MDR1, MRP1, MRP2, and MRP3) was examined by real-time PCR. To investigate anti-CCA activity in vivo, a total of 80 OV and nitrosamine (OV/ DMN)-induced CCA hamsters were fed with the ginger extract at doses of 1000, 3000, and 5000 mg/kg body weight daily or every alternate day for 30 days. Control groups consisting of 10 hamsters for each group were fed with 5-fluorouracil (positive control) or distilled water (untreated control). Median IC50 (concentration that inhibits cell growth by 50%) values for cytotoxicity and anti-oxidant activities of the crude ethanolic extract of ginger were 10.95, 53.15, and 27.86 μg/ml, respectively. More than ten DNA fragments were visualized and up to 7-9 fold up-regulation of MDR1 and MRP3 genes was observed following exposure to the ethanolic extract of ginger. Acute and subacute toxicity tests indicated absence of any significant toxicity at the maximum dose of 5,000 mg/kg body weight given by intragastric gavage. The survival time and survival rate of the CCA-bearing hamsters were significantly prolonged compared to the control group (median of 54 vs 17 weeks). Results from these in vitro and in vivo studies thus indicate promising anticancer activity of the crude ethanolic extract of ginger against CCA with the absence of any significant toxicity. Moreover, MDR1 and MRP3 may be involved in conferring resistance of CCA to the ginger extract.

Comparative analysis of two fatty acid binding proteins from Fasciola gigantica.

Fatty acid binding proteins are considered to be promising vaccine candidates against trematodiasis. In order to provide additional information about their function in Fasciola gigantica we performed a comparative analysis of FgFABP1 and FgFABP3, two isoforms with quite different isoelectric points of 4.9 and 9.9 and 67% sequence identity. Both are expressed in the juvenile and adult parasite but differ in their tissue-specific distribution. In addition, the sequence of FABP3 is identical in F. hepatica and F. gigantica indicating the proteins functional importance in this genus. Immune sera produced against soluble recombinant FgFABPs reacted with 14 kDa antigens in crude worm, soluble egg, cirrus sac extracts, and excretion/secretion product. Both FgFABPs were located in the parenchyma of the parasite but in addition, FgFABP1 was abundant in testes and spermatozoa while FgFABP3 was abundant in vitelline cells, eggs, and caecal epithelium. Mass spectrometry identified FgFABP1 and FgFABP3 in the ES product whereas only FgFABP3 was identified in egg extract. Serum samples of an experimentally infected rabbit reacted from week 6 post-infection with FgFABP3 and from week 12 with FgFABP1 while sera of infected sheep were not reactive. The results suggest differences in the biological functions of these 2 isoforms and differences in the host/parasite interaction that should be considered for their potential as vaccines against fascioliasis.

Molecular and immunological characterization of encoding gene and 14-3-3 protein 1 in Fasciola gigantica

A cDNA encoding Fg14-3-3 protein 1 was cloned by immunoscreening of an adult-stage Fasciola gigantica cDNA library using a rabbit antiserum against tegumental antigens of the parasite. The protein has a deduced amino acid sequence of 252 residues and a calculated molecular weight of 28.7 kDa. It shows sequence identity values between 57.6 and 58.1% to the human 14-3-3 beta, zeta, theta, and eta proteins and is in a phylogenetic cluster with the 14-3-3 protein 1 of Schistosoma spp. Nucleic acid analyses indicate that the Fg14-3-3 protein 1 is encoded by a single copy gene and that this gene is expressed as a transcript of 1250 nucleotides. In adult and 4-week-old parasites the genes transcriptional and translational products were localized in the gut epithelium, parenchyma, tegument cells, and in the reproductive organs. An antiserum against recombinant Fg14-3-3 protein 1 detected a slightly smaller 14-3-3 protein in the parasites excretion/secretion material and showed cross-reactivity with 14-3-3 proteins in extracts of other trematodes and mouse. Antibodies against Fg14-3-3 protein were detected in the sera of rabbits as early as 2 weeks after infection with metacercariae of F. gigantica and the antibody titre increased continuously over a 10-week observation period.

Functional analysis of novel aquaporins from Fasciola gigantica

Fascioliasis, caused by liver flukes of the genus Fasciola, is an important disease of ruminants. In order to identify a potential new drug target we have studied aquaporin (AQP) in Fasciola gigantica. AQPs facilitate the transport of water, glycerol and other small solutes across biological membranes. The structure, function, and pathology of AQPs have been extensively studied in mammals but data for AQPs from trematodes is still limited. In the present study, we have functionally characterized two closely related AQP isoforms, FgAQP-1 and FgAQP-2, from the trematode F. gigantica. Immunohistochemical analysis located the FgAQPs in the tegumental cells, their processes and the tegument itself. In addition, they were present in the epithelial linings of testes and ovary. Expression in Xenopus oocytes of these FgAQPs increased osmotic water permeability 3-4-fold but failed to increase glycerol and urea permeability. AQPs have two highly conserved NPA motifs that are important for the function of the channel pore. In FgAQP-1 and FgAQP-2 the first NPA motif is changed to TAA. Substitution of Thr with Asn in the TAA motif of FgAQP-1 increased its water permeability twofold but did not affect urea and glycerol impermeability while the substitution at the pore mouth of Cys204 by Tyr caused loss of water permeability. In addition, the FgAQPs did not increase methylamine and ammonia permeability after expression in yeast. In comparison to rat AQP-1 the described FgAQPs showed low water permeability and further in vivo analyses are necessary to determine their contribution to osmoregulation in Fasciola.