Acanthamoeba in Southeast Asia - Overview and Challenges.

Acanthamoeba, one of free-living amoebae (FLA), remains a high risk of direct contact with this protozoan parasite which is ubiquitous in nature and man-made environment. This pathogenic FLA can cause sight-threatening amoebic keratitis (AK) and fatal granulomatous amoebic encephalitis (GAE) though these cases may not commonly be reported in our clinical settings. Acanthamoeba has been detected from different environmental sources namely; soil, water, hot-spring, swimming pool, air-conditioner, or contact lens storage cases. The identification of Acanthamoeba is based on morphological appearance and molecular techniques using PCR and DNA sequencing for clinico-epidemiological purposes. Recent treatments have long been ineffective against Acanthamoeba cyst, novel anti-Acanthamoeba agents have therefore been extensively investigated. There are efforts to utilize synthetic chemicals, lead compounds from medicinal plant extracts, and animal products to combat Acanthamoeba infection. Applied nanotechnology, an advanced technology, has shown to enhance the anti-Acanthamoeba activity in the encapsulated nanoparticles leading to new therapeutic options. This review attempts to provide an overview of the available data and studies on the occurrence of pathogenic Acanthamoeba among the Association of Southeast Asian Nations (ASEAN) members with the aim of identifying some potential contributing factors such as distribution, demographic profile of the patients, possible source of the parasite, mode of transmission and treatment. Further, this review attempts to provide future direction for prevention and control of the Acanthamoeba infection.

Antioxidant, antimelanogenic, and skin-protective effect of sesamol.

Sesame contains high nutritional value and important bioactive lignans which are good for health-promoting effects including sesamol. Sesamol is found in trace amounts in sesame. The biological action from the trace amounts of sesamol found might indicate its efficacy. This paper presents a systematic study of the antimelanogenic and skin-protective effects (antioxidant) of sesamol and positive compounds. The results showed that sesamol had the most scavenging 2,2-Diphenyl-1-picrylhydrazyl hydrate (DPPH·) radical with an IC50 value < 14.48 µM. The antioxidant power (Ferric reducing antioxidant power value) of sesamol at a concentration of 0.1129 µM was 189.88 ± 17.56 µM FeSO4. Sesamol inhibited lipid peroxidation with an IC50 value of 6.15 ± 0.2 µM. Moreover, sesamol possessed a whitening effect by inhibition of mushroom tyrosinase at an IC50 value of 1.6 µM and an inhibition of cellular tyrosinase with 23.55 ± 8.25% inhibition at a concentration of 217.2 µM. Sesamol exhibited high antioxidant and anti-tyrosinase activity compared to the positive control, kojic acid and ß-arbutin. Sesamol from edible sesame seed could therefore have an alternative cosmeceutical purpose.

Torquetenovirus infection among northeastern Thai blood donors.

Semi-nested polymerase chain reaction technique was used to detect Torquetenovirus (TTV) DNA in 234 healthy blood donors in northeast Thailand. The incidence of TTV was 28% in 101 healthy blood donors negative for HBsAg and anti-HCV antibody, 25% in 71 HBsAg carriers and 29% among 62 with anti-HCV antibody. No association of TTV infection was found with gender, age, and HBV or HCV infection.

Sesamol induces mitochondrial apoptosis pathway in HCT116 human colon cancer cells via pro-oxidant effect.

AIMS: Sesamol lignan is a phenolic compound found in sesame seeds. We investigated the effect of different concentrations of sesamol on oxidative stress in colorectal carcinoma cells (HCT116). MAIN METHODS: Antioxidation in vitro was determined from elimination of the DPPH radical, ferric reducing-antioxidant power (FRAP), O2(-), and peroxyl radical scavenging activity. Intracellular O2(-), H2O2 and GSH levels were determined by DHE, DCFH-DA, and CMF-DA assay, respectively. Cell viability was detected by neutral red assay. Cell cycle proportion and mode of apoptotic HCT116 cells death was analyzed by flow cytometry. Apoptosis in sesamol-treated HCT116 cells was confirmed by morphological changes in the nuclei using DAPI staining and changes in mitochondrial membrane potential using the DiOC6(3) assay. KEY FINDINGS: Sesamol at both low (0.05 and 0.25mM) and high (0.5, 2, 5, and 10mM) concentrations concurrently reduced FRAP reagent and scavenged DPPH, and O2(-). Sesamol at low concentrations scavenged ROO, but ROO-scavenging was decreased at higher concentrations. Sesamol suppressed cell viability via disruption of cell cycle progression at high concentrations (0.5, 1, 2, and 5mM), thereby causing S-phase arrest and inducing apoptosis-through the production of intracellular O2(-), mitochondrial dysfunction, and DNA fragmentation. SIGNIFICANCE: High concentrations of sesamol induced the mitochondrial apoptosis pathway in human colon cancer HCT116 cells via a pro-oxidant effect.

Synergistic effects of melphalan and Pinus kesiya Royle ex Gordon (Simaosong) extracts on apoptosis induction in human cancer cells.

BACKGROUND: This study aims to determine the synergistic effects of the chemotherapeutic drug melphalan and the phytoconstituents extracted from Pinus kesiya Royle ex Gordon (Simaosong) in human cancer cells. METHODS: P. kesiya twigs extracted from 50 % ethanol-water were evaluated alone (6-500 µg/mL) and in combination with melphalan (0.75-15 µg/mL). The cytotoxic effects of single extract or extract and melphalan combination were examined by a neutral red assay to investigate their antiproliferative and apoptosis induction effects in the U937 and HepG2 cell lines. Nuclei morphological change and DNA fragmentation were examined by DNA nuclei staining with 4´6-diamidino-2-phenylindole (DAPI) and agarose gel electrophoresis, respectively. The chemical constituents of the P. kesiya extract were assessed using gas chromatography-mass spectrometry (GC-MS) analysis. The synergistic effects of different IC50 ratios of the P. kesiya extract and melphalan combination were analyzed in each cancer cell line. The dose reduction index (DRI) was calculated to determine the extent of concentration reduction in the combination treatment compared with the concentration of each single treatment. RESULTS: The IC50 ratios for melphalan to P. kesiya extract that caused 75 % antiproliferation could be reduced after combination. This response was greater in the U937 cells than in the HepG2 cells (all P < 0.001). Melphalan and P. kesiya extract had a similar effect on apoptosis induction both singly and in combination. P. kesiya extract synergized the antiproliferation and apoptosis induction effects of melphalan. CONCLUSIONS: Combining the P. kesiya extract with melphalan reduced toxicity while retaining the therapeutic efficacy of melphalan.

Apoptosis-inducing effects of jujube (Zǎo) seed extracts on human Jurkat leukemia T cells.

BACKGROUND: Jujube (Zǎo) seeds exhibited anticancer effects and used in Chinese medicine for many years. This study aims to investigate the apoptosis-inducing effects of seed extracts from eight different cultivated species ('Apple', 'Bombay', 'Jumbo', 'Kaew', 'Nomsod', 'Rianthong', 'Samros', and 'Taiwan') on human Jurkat leukemia T cells. METHODS: We evaluated the effects of seed extracts from eight jujube cultivated species on human Jurkat leukemia T cells. The crude seed extracts were prepared sequentially by using water, 95 % ethanol, dichloromethane, ethyl acetate, chloroform or hexane. The antiproliferative effects of the jujube seed extracts relative to that of melphalan were evaluated by neutral red assays. Apoptotic cell death induced by the ethanolic extracts at 1 × IC50 and 2 × IC50 concentrations was demonstrated by DAPI staining, gel electrophoresis, flow cytometry with Annexin V/propidium iodide staining, and caspase-3, -8, and -9 enzyme activities. RESULTS: Ethanolic extracts of 'Taiwan', 'Jumbo', 'Nomsod', 'Rianthong', 'Samros', and 'Bombay', significantly inhibited the proliferation of Jurkat cells compared with untreated cells (all P < 0.001), while the extracts of 'Kaew' and 'Apple' were inactive. The six active extracts preferentially induced apoptotic cell death in a concentration-dependent manner with DNA fragmentation (2 × IC50). Increased caspase-3 activity was detected after treatment with the six extracts. The 'Taiwan', 'Nomsod', 'Jumbo', and 'Rianthong' extracts (2 × IC50) induced both the extrinsic and intrinsic apoptosis pathways by increasing caspase-8 and caspase-9 activity, respectively. Alkaloids (Dragendorff's method) and reducing sugars (Fehling's test) were mainly identified in the apoptosis-inducing extracts. CONCLUSIONS: The tested of six active extracts ('Taiwan', 'Jumbo', 'Nomsod', 'Rianthong', 'Samros' and 'Bombay') contained alkaloids or reducing sugars, and induced caspase-dependent apoptosis in human Jurkat leukemia T cells.

Induction of apoptosis in human hepatocellular carcinoma cells by extracts of Lannea coromandelica (Houtt.) Merr. and Diospyros castanea (Craib) Fletcher.

BACKGROUND: Herbal plants are a preferred source of anticancer agents. This study aims to screen the anticancer activity of a crude extract of twigs of (a) Bombax anceps Pierre var. anceps (BA); (b) Catunaregam tomentosa (Blume ex DC.) Tirveng. (CT); (c) Erythrophleum succirubrum Gagnep. (ES); (d) Lannea coromandelica (Houtt.) Merr. (LC); and (e) leaves and (f) twigs of Diospyros castanea (Craib) Fletcher (DC). METHODS: The 50 % ethanol-water extracts were prepared from each plant sample. In vitro anticancer effects of six extracts on the human hepatocellular carcinoma cell line (HepG2) in terms of cytotoxicity were investigated by neutral red assay, apoptosis induction by 4',6-diamidino-2-phenylindole (DAPI) staining, and DNA fragmentation by agarose gel electrophoresis. Normal Vero cells were tested for comparison and to determine cancer selectivity. Gas chromatography-mass spectrometry analysis was performed to identify the compounds in the extracts. RESULTS: The six crude extracts had different cytotoxicities and were classified into three groups based on their IC50 value and selectivity index (SI). DC (twig) crude extract had both a high cytotoxicity and SI toward HepG2 cells comparable to melphalan (P = 0.023). The crude extracts of DC (leaves), LC (twig), and BA (twig) had moderate cytotoxicity and a lower SI. Although all crude plant extracts induced apoptosis in more than 50 % of the DAPI-positive apoptotic HepG2 cells, only DC (twig) and LC (twig) showed laddering in the DNA fragmentation assay. 2-Palmitoylglycerol was the major compound common to both. Pyrogallol and lupeol were the major compounds in DC (twig) crude extract. Hexadecanoic acid and octadecenoic acid were the major compounds in LC (twig) crude extract, which had high toxicity but low selectivity. CONCLUSION: Ethanolic extracts from DC and LC twigs induced apoptosis in the HepG2 cell line. Pyrogallol and lupeol in DC (twig) might be responsible for the cytotoxicity toward the HepG2 cancer cells.

Epidemiology of hepatitis C virus genotypes in northeastern Thai blood samples.

BACKGROUND: Hepatitis C virus (HCV) infection is an important cause of liver cancer in Thailand. The highest prevalence of anti-HCV positive among Thai blood donors is found in the northeastern region. The present analysis of the genotype distribution among anti-HCV positive northeastern-Thai blood donors was conducted to provide a base for the epidemiological pattern of HCV infection in this region. MATERIALS AND METHODS: A total of 112 HCV seropositive healthy blood donors were randomly selected and tested for the presence of HCV-RNA by RT-PCR. HCV-RNA positive samples were genotyped by direct sequencing at core region genomes and confirmed by phylogenetic analysis. RESULTS: HCV viremia was found in 94.6% (106/112) of HCV seropositive blood donors. There were 3 major genotypes distributed among this population. HCV genotype 3a was the most prevalent (71.7%) followed by genotypes 1a (7.5%), 1b (7.5%), 6i (3.8%), 6f (2.8%) and 6n (1.9%). CONCLUSIONS: HCV genotype 3a in asymptomatic infections in northeastern Thailand is significantly higher than other previous reports. Subgenotype 6 prevalence is less than in neighboring countries and distribution patterns differ. The findings are relevant as predictors for using interferon therapy in this population.

Cratoxylum formosum (Jack) Dyer ssp. pruniflorum (Kurz) Gogel. (Hóng yá mù) extract induces apoptosis in human hepatocellular carcinoma HepG2 cells through caspase-dependent pathways.

BACKGROUND: Cratoxylum formosum (Jack) Dyer ssp. pruniflorum (Kurz) Gogel. (Hóng yá mù) (CF) has been used for treatment of fever, cough, and peptic ulcer. Previously, a 50% ethanol-water extract from twigs of CF was shown highly selective in cytotoxicity against cancer cells. This study aims to investigate the molecular mechanisms underlying the apoptosis-inducing effect of CF. METHODS: The cytotoxicity of CF was evaluated in the human hepatocellular carcinoma (HCC) HepG2 cell line in comparison with a non-cancerous African green monkey kidney epithelial cell line (Vero) by a neutral red assay. The apoptosis induction mechanisms were investigated through nuclear morphological changes, DNA fragmentation, mitochondrial membrane potential alterations, and caspase enzyme activities. RESULTS: CF selectively induced HepG2 cell death compared with non-cancerous Vero cells. A 1.5-fold higher apoptotic effect compared with melphalan was induced by 120 µg/mL of the 50% ethanol-water extract of CF. The apoptotic cell death in HepG2 cells occurred via extrinsic and intrinsic caspase-dependent pathways in dose- and time-dependent manners by significantly increasing the activities of caspase 3/7, 8, and 9, decreasing the mitochondrial membrane potential, and causing apoptotic body formation and DNA fragmentation. CONCLUSIONS: CF extract induced a caspase-dependent apoptosis in HepG2 cells.

Application of FTIR microspectroscopy for characterization of biomolecular changes in human melanoma cells treated by sesamol and kojic acid.

BACKGROUND: Hyperpigmentation is aesthetic undesirable. Sesamol and the standard antimelanogenic agent (kojic acid) were shown to hinder melanogenesis by blocking tyrosinase and reducing melanin content. OBJECTIVE: The FTIR microspectroscopy was used in an attempt to find a novel method to define biological alternation in a melanogenesis inhibition of sesamol and kojic acid. METHODS: Tyrosinase inhibition and melanin content of sesamol and kojic acid were evaluated. The FTIR microspectroscopy was adopted to define the vibrational characteristic involved with the melanogenesis in the untreated SK-MEL2 cells vs. the sesamol- and kojic-treated SK-MEL2 cells. RESULTS: Sesamol and kojic acid inhibited mushroom tyrosinase at IC50 of 0.33 µg/ml and 6.1±0.4 µg/ml, respectively. Moreover, 30 µg/ml sesamol inhibited 23.55±8.25% cellular tyrosinase activity in human SK-MEL2 cells, while 600 µg/ml kojic acid inhibited 33.9±1.4% cellular tyrosinase activity in the same cells. In the SK-MEL2-treated with two inhibitors, the FTIR spectra assigned to the lipid and nucleic acid bands were significantly depleted with the secondary protein structure shifted to a more ß-pleated secondary protein one. CONCLUSION: Both sesamol and kojic acid display a similar pattern of antimelanogenesis activity albeit to a different degree. The mechanism of their whitening effect may be via the alteration of (a) the enzyme conformation disallowing the ordinary enzyme-substrate interaction and maybe (b) the integrity of the lipid-containing melanosome. Our results support the alternative use of FTIR microspectroscopy as a simple and reagent-free method for characterization of biomolecular changes in human melanoma cells.

Anticancer effect of the extracts from Polyalthia evecta against human hepatoma cell line (HepG2).

OBJECTIVE: To investigate the anticancer activity of Polyalthia evecta (P. evecta) (Pierre) Finet & Gagnep against human hepatoma cell line (HepG2). METHODS: The anticancer activity was based on (a) the cytotoxicity against human hepatoma cells (HepG2) assessed using a neutral red assay and (b) apoptosis induction determined by evaluation of nuclei morphological changes after DAPI staining. Preliminary phytochemical analysis of the crude extract was assessed by HPLC analysis. RESULTS: The 50% ethanol-water crude leaf extract of P. evecta (EW-L) showed greater potential anticancer activity with high cytotoxicity [IC50 = (62.8 ± 7.3)µg/mL] and higher selectivity in HepG2 cells than normal Vero cells [selective index (SI) = 7.9]. The SI of EW-L was higher than the positive control, melphalan (SI = 1.6) and the apoptotic cells (46.4 ± 2.6) % induced by EW-L was higher than the melphalan (41.6 ± 2.1)% (P<0.05). The HPLC chromatogram of the EW-L revealed the presence of various kinds of polyphenolics and flavonoids in it. CONCLUSIONS: P. evecta is a potential plant with anticancer activity. The isolation of pure compounds and determination of the bioactivity of individual compounds will be further performed.

Viral hepatitis B, C infection and genotype distribution among cholangiocarcinoma patients in northeast Thailand.

The prevalence of HBV and HCV infection among 295 cholangiocarcinoma (CCA) patients in northeast Thailand was analyzed. Hepatitis B surface antigen (HBsAg) was detected in 8.8% (26/295 cases) and antibodies to HCV (anti-HCV) in 2.7% (8/295 cases) of CCA cases. Screening for HBV DNA was performed in 15 of 26 HBV seropositive cases and genotypes could be determined in all 15. HBV genotypes C and B were detected in 73.3% (11/15 cases) and 26.7% (4/15 cases), respectively. HCV RNA was detected in 87.5% (7/8 cases) of anti-HCV positive cases. Specifically, 57.1% (4/7 cases) were HCV genotype 1a and 42.9% (3/7 cases) were HCV genotype 3a. The prevalence of infection and genotype distribution of both HCV and HBV among CCA in northeast Thailand is comparable to that in the general population, suggesting that HCV and HBV infections are, if at all, not serious risk factors for CCA.

Synergistic anticancer effect of the extracts from Polyalthia evecta caused apoptosis in human hepatoma (HepG2) cells.

OBJECTIVE: To evaluate the anticancer activity of the extract fraction of Polyalthia evecta (P. evecta) (Pierre) Finet & Gagnep and the synergistic anticancer effect of the extracts from P. evecta by using the ATR/FT-IR spectroscopy. METHODS: The 50% ethanol-water crude leaf extract of P. evecta (EW-L) was prepared and was further fractionated to isolate various fractions. The anticancer activity was investigated from cytotoxicity against HepG2 using a neutral red assay and apoptosis induction by evaluation of nuclei morphological changes after DAPI staining. Synergistic anticancer effects of the extracts from P. evecta were performed using the ATR/FT-IR spectroscopy. RESULTS: The result showed that the EW-L showed higher cytotoxicity and apoptosis induction in HepG2 cells than its fractionated extracts. The hexane extract exhibited higher cytotoxicity and apoptosis induction than the water extracts, but less than the EW-L. The combined water and hexane extracts apparently increased cytotoxicity and apoptosis induction. The %apoptotic cells induced by the extract mixture were increased about 2-fold compared to the single hexane extract. CONCLUSIONS: The polar extract fraction is necessary for the anticancer activity of the non-polar extract fraction. The ATR/FT-IR spectra illustrates the physical interaction among the constituents in the extract mixture and reveals the presence of polyphenolic constituents in the EW-L, which might play a role for the synergistic anticancer effect.

Evaluation of the anticancer potential of six herbs against a hepatoma cell line.

BACKGROUND: Six herbs in the Plant Genetics Conservation Project that have been used as complementary medicines were chosen on the basis of their medicinal value, namely Terminalia mucronata, Diospyros winitii, Bridelia insulana, Artabotrys harmandii, Terminallia triptera, and Croton oblongifolius. This study aims to evaluate the potential anticancer activity of 50% ethanol-water extracts of these six herbs. METHODS: Fifty percent ethanol-water crude extracts of the six herbs were prepared. The cytotoxicity of the herbal extracts relative to that of melphalan was evaluated using a hepatoma cell line (HepG2), and examined by neutral red assays and apoptosis induction by gel electrophoresis and flow cytometry after 24 h. RESULTS: A significant difference was found between the cytotoxicity of the 50% ethanol-water crude extracts and melphalan (P = 0.000). The 50% ethanol-water crude extracts of all six herbs exhibited cytotoxicity against HepG2 cells, with IC50 values ranging from 100 to 500 µg/mL. The extract of T. triptera showed the highest cytotoxicity with an IC50 of 148.7 ± 12.3 µg/mL, while melphalan had an IC50 of 39.79 ± 7.62 µg/mL. The 50% ethanol-water crude extracts of D. winitii and T. triptera, but not A. harmandii, produced a DNA ladder. The 50% ethanol-water crude extracts of D. winitii, T. triptera, and A. harmandii induced apoptosis detected by flow cytometry. CONCLUSION: The 50% ethanol-water crude extracts of D. winitii, T. triptera, and A. harmandii showed anticancer activity in vitro.

FTIR microspectroscopy discriminates anticancer action on human leukemic cells by extracts of Pinus kesiya; Cratoxylum formosum ssp. pruniflorum and melphalan.

Apoptosis is the principal molecular goal of chemotherapeutics for effective anticancer action. We studied the effect of 50% ethanolic-water extracts of Pinus kesiya, Cratoxylum formosum ssp. pruniflorum and melphalan on cytotoxicity and apoptosis induction for human leukemic U937 cells, and explored the mode of action using FTIR microspectroscopy. The number of viable U937 cells in vitro was decreased in a concentration-dependent manner by all tested compounds, although potency differed between the U937 and Vero cells. Melphalan and the extract of C. formosum exhibited relatively lower IC(50) values (15.0 ± 1.0 and 82.7 ± 3.2 µg/mL respectively) and higher selectivity (selective index>3) than the extract of P. kesiya (299.0 ± 5.2 µg/mL; selective index<3) on the U937 cells. All three compounds significantly induced apoptosis through the late stage - seen by the indicative DNA ladder - with the most effective being melphalan, then the P. kesiya and C. formosum extracts. FTIR microspectroscopy revealed that all three compounds raised the intensity of the ß-pleated sheet - higher than that of the untreated U937 cells - corresponding to a shift in the α-helix band associated with an alteration in the secondary structure of the protein band, confirming induction of apoptosis via pro-apoptotic proteins. The differences in intensity of the FTIR bands associated with lipids, proteins and nucleic acids were responsible for discrimination of the anticancer mode of action of each of the three compounds. The FTIR data suggest that the two plant extracts possessed anticancer activity with a different mode of action than melphalan.

Cytotoxic and apoptotic effects of six herbal plants against the human hepatocarcinoma (HepG2) cell line.

BACKGROUND: Six plants from Thailand were evaluated for their cytotoxicity and apoptosis induction in human hepatocarcinoma (HepG2) as compared to normal African green monkey kidney epithelial cell lines. METHODS: Ethanol-water crude extracts of the six plants were tested with neutral red assay for their cytotoxicity after 24 hours of exposure to the cells. Apoptotic induction was tested in the HepG2 cells with diamidino-2-phenylindole staining. DNA fragmentation, indicative of apoptosis, was analyzed with agarose gel electrophoresis. Alkylation, indicative of DNA damage, was also evaluated in vitro by 4-(4'-nitrobenzyl) pyridine assay. RESULTS: The extract of Pinus kesiya showed the highest selectivity (selectivity index = 9.6) and potent cytotoxicity in the HepG2 cell line, with an IC50 value of 52.0 ± 5.8 µg/ml (mean ± standard deviation). Extract of Catimbium speciosum exerted cytotoxicity with an IC50 value of 55.7 ± 8.1 µg/ml. Crude extracts from Glochidion daltonii, Cladogynos orientalis, Acorus tatarinowii and Amomum villosum exhibited cytotoxicity with IC50 values ranging 100-500 µg/ml. All crude extracts showed different alkylating abilities in vitro. Extracts of P. kesiya, C. speciosum and C. orientalis caused nuclei morphological changes and DNA laddering. CONCLUSION: The extracts of C. speciosum, C. orientalis and P. kesiya induced apoptosis. Among the three plants, P. kesiya possessed the most robust anticancer activity, with specific selectivity against HepG2 cells.