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.

Andrographolide Ameliorates Beta-Naphthoflavone-Induced CYP1A Enzyme Activity and Lipid Peroxidation in Hamsters with Acute Opisthorchiasis.

BACKGROUND: Opisthorchis viverrini (OV) infection generates oxidative stress/free radicals and is considered as a primary cause ofcholangiocarcinoma since it primarily triggers sclerosing cholangitis. OBJECTIVE: In this study, the impacts of andrographolide on acute opisthorchaisis in ß-naphthoflavone (BNF)-exposed hamsters were investigated. MATERIAL AND METHOD: Ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities and Thiobarbituric acid reaction substances (TBARS) assay of andrographolide in acute opisthorchiasis in the BNF-exposed hamsters were assessed. RESULTS: The results showed that andrographolide ameliorated the hepatic CYP1A1 and CYP1A2 activities by decreases of the specific enzymatic reactions of EROD and MROD, respectively, in the BNF-exposed hamsters. Moreover, andrographolide lowered the formation of malondialdehyde in the livers and brains of the hamsters. CONCLUSION: These observations revealed the promising chemo-protective and antioxidant activities of andrographolide via suppression of the specific EROD and MROD reactions and lipid peroxidation against acute opisthorchiasis in the BNF-exposed hamsters.

Iron-Chelating and Anti-Hemolytic Properties of Ethanolic Extract of Lotus (Nelumbonucifera Gaertn) Leaves.

BACKGROUND: Iron overload is the major consequence of blood transfusion in ß-thalassemia patients. Redox iron plays a critical role in the formation of reactive oxygen species and subsequently leads to oxidative stress damage in many cells, especially red blood cells and hepatocytes. Iron deposition in hepatocytes is associated with fibrosis and cirrhosis. Polyphenolic compounds found in natural products are interesting iron chelators and antioxidants. OBJECTIVE: This study aims to evaluate the iron-chelating properties and free-radical scavenging activities of lotus leaf extract in iron-loaded HepG2 cells. MATERIAL AND METHOD: Lotus (Nelumbonucifera Gaertn) leaves were extracted with 80% (v/v) ethanol. The extract was examined for free-radical scavenging activity by using 2, 2-diphenyl-1-picrylhydrazyl assay (DPPH assay); iron-binding and anti-hemolytic activities using spectrophotometrical method. Iron-depriving activity of the extract was determined in iron loaded human hepatocellular (HepG2) cells using fluorescence technique. RESULTS: The lotus extract showed antioxidant and anti-hemolytic activities in a concentration-dependent manner. Furthermore, it was able to bind iron rapidly and was saturated within 10 minute. With 24-hour treatment, this extract dose dependently decreased the level of labile iron pool in iron loaded HepG2 cells. CONCLUSION: Lotus leaf extract had strong antioxidant activities, iron chelating properties on iron loaded HepG2 cells and anti-hemolytic activity.

Effects of Pueraria mirifica and miroestrol on the antioxidation-related enzymes in ovariectomized mice.

OBJECTIVES: The influences of Pueraria candollei var. mirifica (PM), a Thai medicinal plant with long tradition of medicinal consumption among menopausal women for rejuvenation and estrogen hormone replacement, on oxidative status in ovariectomized (OVX) mice were determined. METHODS: The crude extract of PM and its active phytoestrogen, miroestrol (MR), were given to OVX mice. The effect of them on antioxidation enzymes and glutathione (GSH) levels in livers and uteri were examined in OVX mice and compared with the synthetic estradiol hormone. KEY FINDINGS: Ovariectomy significantly decreased total GSH content, reduced GSH content, and the ratio of GSH to oxidized glutathione (GSSG) in both the livers and the uteri of mice. Moreover, an ovariectomy reduced the activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT). The crude extract of PM as well as MR significantly increased levels of GSH, levels of reduced GSH, and the ratio of GSH/GSSG in both the livers and the uteri, while estradiol did not. In addition, the potential of PM and MR to return the activities of GPx, SOD, and CAT to normal levels was noted. CONCLUSIONS: These observations support using PM and MR as promising alternative medicine candidates for hormone replacement therapy of estradiol because of their ability to improve GSH levels and the activities of antioxidative enzymes, especially in OVX mice.

Impact of Pueraria candollei var. mirifica and its potent phytoestrogen miroestrol on expression of bone-specific genes in ovariectomized mice.

Miroestrol (MR) is a highly active phytoestrogen isolated from tuberous root of Pueraria candollei var. mirifica (PM). Modulatory effects of PM and MR on osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) mRNAs which are bone-specific genes were investigated in ovariectomized female ICR mice. After ovariectomy, expression of OPG mRNA was suppressed but that of RANKL was induced. Estradiol benzoate (E2) recovered OPG expression to the level comparable to the sham while that of RANKL was suppressed in ovariectomized mice. PM crude extract (PME) significantly down-regulated the expression of RANKL mRNA with no change in the OPG level whereas MR elevated the expression of OPG mRNA with lowering level of RANKL mRNA, resulting in the increased OPG/RANKL ratio, and consequently lead to lowering progression of osteoporosis at molecular level. These findings revealed potential of PME and MR on bone loss prevention via increasing the ratio of OPG to RANKL (osteoformation/osteoresorption) in liver of ovariectomized mice. Therefore, using PME and MR as alternative hormone replacement therapy of E2 might be beneficial recommended due to advantageous on regulation of osteoporosis related genes.

Suppression of BSEP and MRP2 in mouse liver by miroestrol and deoxymiroestrol isolated from Pueraria candollei.

Miroestrol and deoxymiroestrol are highly active phytoestrogens isolated from the tuberous root of Pueraria candollei var. mirifica (Leguminosae). Modulatory effects of miroestrol and deoxymiroestrol on the mRNAs of BSEP and MRP2 genes involved in bile salt transportation, in C57BL/6 mice were investigated. In contrast to estradiol, miroestrol and deoxymiroestrol suppressed the expression of BSEP and MRP2 mRNA in both male and female mice. The results suggest for the first time that the use of miroestrol and deoxymiroestrol-containing products as alternative medicines or health supplements should be concerned according to their effects on key genes that regulate the bile salt export pump, which could result in the risk of hepatotoxicity and intrahepatic cholestasis.

Different profiles of hepatic alkoxyresorufin O-dealkylase activities in small rodents.

The aims of the present study were to determine cytochrome P450 enzyme activity in six strains of experimental rodents (n = 5/sex/species): ICR, C57BL/6 and DBA/2 mice; Sprague Dawley and Wistar rats; and Dunkin Hartley guinea pigs. After animals were treated with the typical inducers ß-naphthoflavone (BNF), dexamethasone (DEX) and phenobarbital (PB), the levels of O-dealkylation of ethoxyresorufin (EROD), methoxyresorufin (MROD), pentoxyresorufin (PROD) and benzyloxyresorufin (BROD) activity were determined using responsive catalytic reactions to study CYP1A1, CYP1A2 and CYP2B, respectively. A maximal induction of EROD and MROD was found in BNF-treated animals from all strains (2.4- to 15.1-fold) except DBA/2 (0.9- to 1.8-fold). C57BL/6 mice had the strongest BNF-induced EROD (15.1-fold) and MROD (8.3-fold) activities. No differences in BNF-induced EROD and MROD activities were observed between males and females. However, the EROD activity of Wistar rats and the MROD activity of Sprague Dawley rats were higher in males than females. DEX induced PROD activity only in mice (1.3- to 7.1-fold), but not in rats and guinea pigs (0.2- to 1.1-fold). However, induction of BROD activity was found in DEX-treated mice and rats (1.5 to 12.5-fold), but not in guinea pigs (0.3 to 0.4-fold). PB caused a significant elevation of PROD (1.7- to 10.4-fold) and BROD (31- to 13.2-fold) activities in all the animals. PB-induced BROD activity was higher in females than males in Sprague Dawley rats. These observations strongly suggest that the choice of experimental animal strain, species and inducer is of critical importance for studies of drug metabolism and interaction.

Bimodal action of miroestrol and deoxymiroestrol, phytoestrogens from Pueraria candollei var. mirifica, on hepatic CYP2B9 and CYP1A2 expressions and antilipid peroxidation in mice.

Miroestrol and deoxymiroestrol are phytoestrogens isolated from Pueraria candollei var. mirifica. The influence of miroestrol and dexoymirosestrol on hepatic cytochrome P450 (P450) enzymes and antioxidative activity in brain was examined in C57BL/6 mice compared with that of a synthetic female sex hormone estradiol. We hypothesized that miroestrol and deoxymiroestrol would induce CYP2B9 expression, whereas CYP1A2 expression would be suppressed compared with estradiol. Miroestrol and deoxymiroestrol treatment significantly increased uterus weight and volume. In addition, both of these phytoestrogens induced the expression of CYP2B9 and suppressed the expression of CYP1A2, as expected. Hepatic P450 activities correspondingly showed that both compounds increased benzyloxyresorufin O-dealkylase activity, whereas methoxyresorufin O-dealkylase activity was reduced. These observations suggested that miroestrol and deoxymiroestrol might affect hepatic P450 enzymes, including the CYP2B9 and CYP1A2 P450 isoforms. Assessment of lipid peroxidation demonstrated that miroestrol and deoxymiroestrol markedly decreased levels of malondialdehyde formation in the mouse brain. This is the first report suggesting miroestrol and deoxymiroestrol as potential alternative medicines to estradiol because of their distinctive ability to regulate mouse hepatic P450 expression and their beneficial antioxidative activities.

Down regulation of gene related sex hormone synthesis pathway in mouse testes by miroestrol and deoxymiroestrol.

Miroestrol and deoxymiroestrol are phytoestrogens isolated from tuberous root of Pueraria candollei var. mirifica. Modulatory effects of miroestrol and deoxymiroestrol on enzymes involved in sex-hormone synthesis pathway in male C57BL/6 mice were investigated using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Miroestrol and deoxymiroestrol suppressed the expressions of 3ß-HSD, 17ß-HSD1, and CYP17 while CYP19 mRNA expression was slightly decreased. In addition, the expression of 17ß-HSD2 was induced in correlation with those did by estradiol. These observations supported that miroestrol and deoxymiroestrol could exhibit the same effect as estradiol regarding regulation of testicular gene related sex hormone synthesis pathway.

Modified expression of aryl hydrocarbon receptor-related genes by deoxymiroestrol, a phytoestrogen, in mouse hepatocytes in primary culture.

ETHNOPHARMACOLOGICAL RELEVANCE: Deoxymiroestrol (DM), a strong phytoestrogen from Pueraria candollei Wall. ex Benth. var. mirifica (family Leguminosae). This plant has long been used in traditional medicine for rejuvenation. MATERIALS AND METHODS: The expression of aryl hydrocarbon receptor-related genes in mouse hepatocytes in primary culture was quantified by real-time RT-PCR and hepatic microsomal P450 activity was assessed by using ethoxyresorufin O-dealkylation. RESULTS: The mRNA expression of the aryl hydrocarbon receptor (AhR), AhR nuclear translocator, and CYP1A1 was suppressed, whereas that of CYP1B1, estrogen receptor α (ERα), CYP2B9, and glutathione-S-transferase a2 (GSTa2) was increased. The effects of DM on the gene expression depended on treatment period and concentrations, and were similar to those of ß-estradiol (E2). DM and E2 at pharmacological concentrations had a marked synergistic effect on CYP1A1 expression after combined treatment with a typical CYP1 inducer, ß-naphthoflavone (ßNF), at the level of both transcription and enzymatic activity. DM enhanced the inducible mRNA expression of CYP1A1 and CYP1B1 similar to E2. Meanwhile, the expression of ERα mRNA was not affected by ßNF, which, on the contrary, completely eliminated the DM-induced mRNA expression of ERα, CYP2B9, and GSTa2. CONCLUSION: The findings that DM modified the expression of several metabolism-related genes suggest the need for caution when using health supplements having phytoestrogenic activity.

Improved isoflavonoid production in Pueraria candollei hairy root cultures using elicitation.

The effect of abiotic and biotic elicitors (methyl jasmonate, chitosan, salicylic acid, Agrobacterium, and yeast extract) at various concentrations on total isoflavonoid accumulation was studied in the hairy root cultures of Pueraria candollei. All elicitors stimulated isoflavonoid production. Yeast extract (0.5 mg/ml) was the most efficient giving total isoflavonoids at 60 ± 1 mg/g dry wt, which was 4.5-fold higher than control hairy roots on day 3 of elicitation.

Isoflavonoid production in a hairy roots culture of Pueraria candollei.

A hairy roots culture of Pueraria candollei was established using Agrobacterium rhizogenes ATCC15834 and grown in half-strength Murashige and Skoog (MS) medium. The highest production of total isoflavonoids was found to be (36.48 +/- 4.09) mg/g dry wt [(3.39 +/- 0.20) mg/g dry wt puerarin, (29.91 +/- 3.74) mg/g dry wt daidzin, (1.65 +/- 0.09) mg/g dry wt genistin, (0.76 +/- 0.03) mg/g dry wt daidzein, and (0.76 +/- 0.03) mg/g dry wt genistein, respectively]. The total isoflavonoid content in hairy roots of P. candollei was 5.18-fold higher than that of the native tuber. Effects of sucrose content and medium type on growth and isoflavonoid production were investigated. 5% (w/v) Sucrose was an optimum content for the growth and isoflavonoid accumulation in P. candollei hairy roots. Half-strength MS medium had the highest effect for biomass production whereas woody plant medium had mostly stimulated isoflavonoid content in hairy roots.

Production of isoflavonoids in callus cultures of Pueraria candollei var. mirifica.

Pueraria candollei Wall. ex Benth. var. mirifica (Airy Shaw & Suvat.) Niyomdham was investigated for callus induction using Murashige and Skoog (MS) medium containing different plant growth regulators. After 8 weeks of culture, 66-100% of leaf or stem explants formed calli. Calli from stem explants cultured on MS medium supplemented with 0.5 mg/l thidiazuron (TDZ) gave the maximum of shoot induction (16%) and the highest level of total isoflavonoids [(50.39 +/- 7.06) mg/g dry wt], which was 7-fold higher than that of the native tuber [(7.04 +/- 0.29) mg/g dry wt]. These results suggest that addition of TDZ to the culture medium markedly enhances the production of isoflavonoids in calli induced from stem explants of P. candollei var. mirifica.