1'-Acetoxychavicol acetate-induced cytotoxicity is accompanied by a rapid and drastic modulation of glutathione metabolism.

The effect of 1'-acetoxychavicol acetate (ACA), an anticarcinogenic compound naturally obtained from rhizomes and seeds of South East Asia plants, on the intracellular concentration of glutathione and the activities of enzymes related to glutathione metabolism was studied in Ehrlich ascites tumor cells. We showed in a previous study that ACA induced apoptosis in tumor cells and the cell death was reversed by the addition of N-acetlycysteine or glutathione ethylester. Here we found that ACA caused a rapid decrease in glutathione level in less than 10 min after ACA exposure. At the time, glutathione reductase activity was significantly inhibited and gamma-glutamyl cysteine increased by ACA exposure. These results show that ACA caused the decrease in the intracellular GSH levels in Ehrlich ascites tumor cells, suggesting that ACA-induced decrease of the cellular GSH levels can lead to growth arrest of cancer and enhancement of the efficacy other anticancer drugs.

Combination of Zizyphus jujuba and green tea extracts exerts excellent cytotoxic activity in HepG2 cells via reducing the expression of APRIL.

Hepatocellular carcinoma is a type of tumor highly resistant to available chemotherapeutic agents. The treatment of hepatocellular carcinoma remains a challenge that needs new approaches in the future. In a previous study, we demonstrated that the chloroform fraction (CHCl(3)-F) from Z. jujuba has anticancer activity in human liver cancer cells (HepG2), and that combining CHCl(3)-F with green tea extracts (GTE) results in enhanced effects of anticancer activity in the cells. To further understand the mechanism of the anticancer activity of combining CHCl(3)-F and GTE in HepG2 cells, we investigated whether the addition of a mixture of CHCl(3)-F and GTE would affect the expression of APRIL (a proliferation-inducing ligand), which was expressed in HepG2 cells from 4 hours of incubation in vitro. We have shown that CHCl(3)-F and GTE enhanced anti-cancer activity by reducing the expression of APRIL. We speculate that the CHCl(3)-F and GTE mixture might provide a lead to a new drug design to treat hepatocellular carcinoma in the future.

(1'S)-Acetoxychavicol acetate and its enantiomer inhibit tumor cells proliferation via different mechanisms.

Elucidation of the mechanisms underlying potential anticancer drugs continues and unraveling these mechanisms would not only provide a conceptual framework for drug design but also promote use of natural products for chemotherapy. The biological effects of (1'S)-acetoxychavicol acetate ((S)-ACA) have been widely investigated. However, in most cases, a natural product or synthetic racemic compound was used in the study. In this study, we prepared (S)-ACA and its enantiomer (R)-ACA by a lipase-catalyzed esterification method and sought to determine the mechanisms of action of (S)-ACA and (R)-ACA in the growth inhibitory effect in Ehrlich ascites tumor cells (EATC). (S)-ACA caused an accumulation of tumor cells in the G1 phase of the cell cycle, which was accompanied by a decrease in phosphorylated retinoblastoma protein (Rb), an increase in Rb and a decrease in the phosphorylation of p27kip1. However, (R)-ACA caused an accumulation of tumor cells in the G2 phase of the cell cycle, an increase in hyperphosphorylated Rb and an increase in the phosphorylation of p27kip1. The results obtained in the present study demonstrate for the first time, to the best of our knowledge, that both (S)-ACA and (R)-ACA caused the inhibition of tumor cells growth but the inhibition was caused via different mechanisms.

Mechanism of growth inhibitory effect of Blumea balsamifera extract in hepatocellular carcinoma.

Blumea balsamifera is known to improve physiological disorders such as rheumatism and hypertension, but its anticancer activity has not been well elucidated. In this study, we found that Blumea balsamifera MeOH extract (BME) induced growth-inhibitory activity in rat and human hepatocellular carcinoma cells without cytotoxicity in rat hepatocytes which were used as a normal cell model. BME induced cell cycle arrest at the G1 phase via decreases in the expression of cyclin-E and phosphorylation of retinoblastoma protein. Furthermore, BME reduced the level of a proliferation-inducing ligand, that stimulates tumor cell growth. These findings suggest that BME has possible therapeutic potential in hepatoma cancer patients and that depletion of cellular APRIL is an important mechanism in the growth-inhibitory effect of BME.

Synergistic effect of green tea polyphenols on their protection against FK506-induced cytotoxicity in renal cells.

FK506 (tacrolimus) is a widely used immunosuppressant first employed in the management of rejection in organ transplantation, but now used for autoimmune disease. However, the nephrotoxicity induced by FK506 remains a serious clinical problem. We previously demonstrated that FK506 caused a significant increase in apoptosis of LLC-PK1 cells, a porcine proximal tubule cell line, but the addition of green tea extract and its polyphenolic components suppressed the cell death. Here, we examined the synergistic effect of tea polyphenols on the protection of FK506-induced cell death. The combined treatment with 5 microM (-)-epigallocatechin-gallate (EGCG) and 5 microM of (+)-catechin (C), (-)-epicatechin (EC), (-)-epigallocatechin (EGC) or (-)-epicatechin-gallate (ECG) reduced FK506-induced cytotoxicity in LLC-PK1. Similarly, the combined treatment with 5 microM EGC and 5 microM of C, EC, EGCG or ECG also reduced the cytotoxicity. These results showed that the co-treatments with EGCG and EGC, EGCG or ECG, and EGC and ECG have stronger synergistic effects on the protection of FK506-induced cell death. Furthermore, the combined treatment of EGCG (5 microM) and EGC (5 microM) showed a significant time-dependent suppression of the increased intracellular ROS levels 15 min after the addition of FK506, as well as on caspase activation. The results of these synergistic effects of the constituents of green tea extract suggest that its protective effects may reside in more than just one of its constituent.

Green tea extract enhances the selective cytotoxic activity of Zizyphus jujuba extracts in HepG2 cells.

Anticarcinogenic effects attributed to phytochemicals may be based on synergistic, additive, or antagonistic interactions of many compounds. In our previous study, we demonstrated that the chloroform fraction (CHCl(3)-F) from Z. jujuba has anticancer activity in HepG2 cells. In China, many people drink jujuba tea and believe in the synergic effects of jujuba and tea for better health. We therefore investigated the effects of CHCl(3)-F and green tea extract (GTE), and their underlying mechanisms of action in HepG2 cells. Our results showed that GTE enhanced the effect of CHCl(3)-F on cell viability in HepG2 cells, without cytotoxicity in rat hepatocytes, which was used as a normal cell model. Furthermore, combination of CHCl(3)-F and GTE caused an effect on G1 phase arrest but not on apoptosis. Interestingly, the mechanism of the G1 arrest was associated, not with an increase in p27(Kip1) levels and the hypohosphorylation of Rb, which are pathways used by CHCl(3)-F on G1 arrest in HepG2 cells, but with increases in p53 and p21(Waf1/Cip1) levels, and a decrease in cyclin E levels. Collectively, our findings suggest that combination of CHCl(3)-F and GTE produces an enhanced cell growth inhibition effect, and that the resultant G1 arrest was caused via a different mechanism as that of CHCl(3)-F treatment alone.

Anticancer activities and mechanisms of Blumea balsamifera extract in hepatocellular carcinoma cells.

Blumea balsamifera (also known as sambong), a medicinal plant, is known to improve physiological disorders such as rheumatism and hypertension. However, its anticancer activity has not been well elucidated. In this study, we found that Blumea balsamifera MeOH extract (BME) induced growth inhibitory activity in rat and human hepatocellular carcinoma cells (McA-RH7777, HepG2, respectively) without cytotoxicity as in with rat hepatocytes used as a normal cell model. BME induced cell cycle arrest at G1 phase via decreases in expression of cyclin-E and phosphorylation of retinoblastoma (Rb) protein in both dose- and time-dependent manners. Furthermore, BME reduced the level of a proliferation related ligand (APRIL) which stimulates tumor cell growth. The anti-proliferative effect of BME was improved slightly but significantly by the treatment with recombinant human APRIL. These findings suggest that BME may have a possible therapeutic potential in hepatoma cancer patients and the depletion of cellular APRIL may be one of the important mechanisms on the growth inhibitory effect of BME.

Mechanism of growth inhibitory effect of cape aloe extract in ehrlich ascites tumor cells.

Cape aloe (Aloe ferox Miller) has been a herb well known for its cathartic properties and has also been used popularly as a health drink (juice, tea and tonic) in the United States and in Europe. Cape aloe extract also has been reported to possess several pharmacological effects, such as anti-inflammatory, anti-bacterial, anti-fungal and protective effect against liver injury. However, the investigations on an anti-tumor activity in cape aloe extract are very few and subsequent mechanisms have not been well elucidated. In this study, we examined the effect of the selective growth inhibitory activity of cape aloe extract and found that the cape aloe extract, especially the dichloromethane (CH(2)Cl(2)) extract, caused a dose-dependent growth inhibitory effect in Ehrlich ascites tumor cells (EATC), but not in mouse embryo fibroblast (NIH3T3) cells, which was used as a normal cell model. Furthermore, the CH(2)Cl(2) extract caused an accumulation of cells in the G1 phase and a decrease of cells in the S and G2/M phase of the cell cycle and inhibited DNA synthesis in a dose-dependent manner. In addition, other results suggest that cell cycle arrest and inhibition of proliferation in EATC by the CH(2)Cl(2 )extract are associated with decreased retinoblastoma protein (Rb) phosphorylation.

Mechanism of the anti-cancer activity of Zizyphus jujuba in HepG2 cells.

The Zizyphus jujuba fruit has been used as a traditional Chinese medicinal herb and considered to affect various physiological functions in the body for thousands of years. However, its anti-cancer activity and mechanism of action remain to be elucidated. We investigated the anti-cancer activity of Zizyphus jujuba Mill and its underlining mechanisms of action in human hepatoma cells (HepG2) and found that the extract of Z. jujuba decreased the viability of the cells. Further extraction of the initial Z. jujuba extract with organic solvents revealed that the chloroform fraction (CHCl(3)-F) was the most effective. Interestingly, the CHCl(3)-F induced not only apoptosis but also G1 arrest at a low concentration (100 mug/ml) and G2/M arrest at a higher concentration (200 mug/ml) by cell cycle assay. Apoptosis, an increase in intracellular ROS (reactive oxygen species) level, a decline of mitochondrial membrane potential at low Z. jujuba concentrations, and a ROS-independent mitochondrial dysfunction pathway at high concentrations were all observed. CHCl(3)-F-induced G1 arrest in HepG2 cells was associated with an increase in hypohosphorylation of Rb and p27(Kip1), and a decrease of phosphorylated Rb. However, CHCl(3)-F-induced G2/M arrest in HepG2 cells correlated with a decrease of the p27(Kip1) levels and generation of the phosphorylation of p27(Kip1), however the hypohosphorylation of Rb protein remained. Collectively, our findings suggest that the CHCl(3)-F extract of Z. jujuba extract induced a concentration dependent effect on apoptosis and a differential cell cycle arrest in HepG2 cells.

Protective effect of green tea extract and tea polyphenols against FK506-induced cytotoxicity in renal cells.

The nephrotoxicity induced by the immunosuppressive drug FK506 (tacrolimus or fujimycin), limits its usefulness in widespread application, and the underlying mechanism has not been completely understood. The primary targets of FK506 in the kidney are the proximal tubular epithelial cells. In this study, the protection of green tea extract against FK506-induced cell death of LLC-PK1 cells was investigated. FK506 caused a significant decrease in survival of the cells, but the addition of green tea extract reduced this effect in a dose-dependent manner. Treatment of the cells with 50 microM (41.1 microg/ml) FK506 induced a significant increase in annexin V-positive/propidium iodide-negative cells from 2.68 to 14.5%, whereas the addition of 6.25, 12.5, and 25 microg/ml of green tea extract caused a significant protective effect in apoptotic cells from 14.5 to 6.51, 3.20 and 3.02%, respectively. The effect of five different constituent tea polyphenols was also examined. Epigallocatechin-gallate and epigallocatechin significantly reduced FK506-induced cytotoxicity but epicatechin and catechin had no effect on cell viability. Furthermore, changes in cytochrome c release and caspase activation, which characterize apoptosis, were studied. Epigallocatechin-gallate and epigallocatechin suppressed a significant release of cytochrome c and activation of caspase-3 in FK506-treated LLC-PK1 cells.

Reactive oxygen species-independent G1 arrest induced by evening primrose extract in Ehrlich ascites tumor cells.

We previously demonstrated that evening primrose extract (EPE) induced apoptosis in Ehrlich ascites tumor cells (EATC), and this effect was specific on tumor cells. Furthermore, our results demonstrated that EPE exposure elicited a rapid increase in the activity of superoxide dismutase and intracellular peroxides levels. These changes caused translocation of Bax to mitochondria and a subsequent release of mitochondrial cytochrome c. However, no activation of caspase-3 was observed in EPE-treated EATC. On the other hand, apoptosis-inducing factor (AIF) was translocated from mitochondria to nuclei. The EPE-induced translocation of AIF was suppressed with the addition of catalase, suggesting that the rapid intracellular peroxide levels after addition of EPE triggers off induction of apoptosis, which is AIF-mediated and caspase-independent. In this study, we have shown that EPE elicited a dose-dependent accumulation of cells in the G1 phase and inhibited DNA synthesis. Our results also demonstrated that cell cycle arrest and inhibition of proliferation in EATC by EPE are associated with decreased Rb phosphorylation. Furthermore, inhibitions of Rb phosphorylation and DNA synthesis by EPE were not suppressed with the addition of catalase. The present study suggests that intracellular peroxides, which trigger off induction of apoptosis, are not the trigger of EPE-induced G1 arrest in cell cycle.

Caspase-independent apoptosis induced by evening primrose extract in Ehrlich ascites tumor cells.

We previously demonstrated that evening primrose extract (EPE) induced apoptosis in Ehrlich ascites tumor cells, while mouse embryo fibroblast cells (NIH3T3) used as a normal cell model, showed no effect of cell viability by treatment of EPE. Furthermore, our results demonstrated the rapid increase in intracellular peroxides levels, loss of mitochondrial membrane potential and the release of cytochrome c to cytosol, suggesting that the rapid increase in intracellular peroxides levels after addition of EPE triggers off induction of apoptosis. In this study, we identified that EPE elicited the translocation of Bax to mitochondria and apoptosis-inducing factor (AIF) to nuclei, but no activation of caspase-3-like protease. We also demonstrated that the rapid EPE-induced increase in hydrogen peroxide levels caused the translocation of Bax to mitochondria, and then mitochondrial cytochrome c was released. One of the main consequences of mitochondrial cytochrome c release is the activation of caspase-3. However, no caspase-3 activation was observed. On the other hand, AIF was translocated from mitochondria to nuclei. The EPE-induced translocation of AIF was suppressed with the addition of catalase, suggesting that the rapid intracellular peroxide levels after addition of EPE triggers off induction of apoptosis, which is AIF-mediated and caspase-independent.

Cellular thiol status-dependent inhibition of tumor cell growth via modulation of retinoblastoma protein phosphorylation by (-)-epigallocatechin.

Tea polyphenols have been shown to inhibit tumor cell growth, but there is limited information on their effects on cell signaling and cell cycle control pathways. We have shown the involvement of such mechanisms as activation of mitogenic activated protein kinases, decreases in ornithine decarboxylase activity and in cellular thiol levels, elicitation of mitochondrial cytochrome c release, and activation of caspases by the green tea galloyl polyphenol, epigallocatechin (EGC). In the current study, we sought to determine how EGC alters cell cycle and its related control factors in its growth inhibitory effect in Ehrlich ascites tumor cells. The significant finding here is that EGC caused a dose-dependent accumulation of cells in the G1 phase and a decrease in the phosphorylation of the retinoblastoma (Rb) protein, which was also in a cellular thiol-dependent manner. The involvement of a cellular thiol-dependent modulation in Rb phosphorylation leading to the regulation of tumor cell growth by a green tea polyphenol is a novel observation, to the best of our knowledge.

Green tea extract inhibits angiogenesis of human umbilical vein endothelial cells through reduction of expression of VEGF receptors.

Epidemiological and animal studies have indicated that consumption of green tea is associated with a reduced risk of developing certain forms of cancer. However, the inhibitory mechanism of green tea in angiogenesis, an important process in tumor growth, has not been well established. In the present study, green tea extract (GTE) was tested for its ability to inhibit cell viability, cell proliferation, cell cycle dynamics, vascular endothelial growth factor (VEGF) and expression of VEGF receptors fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/Kinase insert domain containing receptor (Flk-1/KDR) in vitro using human umbilical vein endothelial cells (HUVECs). GTE in culture media did not affect cell viability but significantly reduced cell proliferation dose-dependently and caused a dose-dependent accumulation of cells in the G1 phase. The decrease of the expression of Flt-1 and KDR/Flk-1 in HUVEC by GTE was detected with immunohistochemical and Western blotting methods. These results suggest that GTE may have preventive effects on tumor angiogenesis and metastasis through reduction of expression of VEGF receptors.

Role of intracellular reactive oxygen species and mitochondrial dysfunction in evening primrose extract-induced apoptosis in Ehrlich ascites tumor cells.

Herbal medicines are increasingly being utilized to treat a wide variety of disease processes. Evening primrose extract (EPE) is extracted from Oenothera biennis L., one species of evening primroses, which has been shown to have several pharmacological effects. However, anti-tumor activity in the extract of defatted seeds of O. biennis L. has not been defined thus far. In this study, we identified the major biochemical changes upon EPE treatment and investigated the functional relationship between these changes. We found that EPE-induced apoptosis in Ehrlich ascites tumor cells as evidenced by morphological changes. Furthermore, our results demonstrated rapid increase of intracellular peroxides levels, loss of mitochondrial membrane potential and the release of cytochrome c from mitochondria to cytosol. These results suggest that the rapid increase of intracellular peroxides levels after addition of EPE triggers off induction of apoptosis.

Involvement of protein tyrosine phosphorylation and reduction of cellular sulfhydryl groups in cell death induced by 1' -acetoxychavicol acetate in Ehrlich ascites tumor cells.

Elucidation of the mechanisms underlying potential anticancer drugs continues and unraveling these mechanisms would not only provide a conceptual framework for drug design but also promote use of natural products for chemotherapy. To further evaluate the efficacy of the anticancer activity of 1'-acetoxychavicol acetate (ACA), this study investigates the underlying mechanisms by which ACA induces death of Ehrlich ascites tumor cells. ACA treatment induced loss of cell viability, and Western blotting analysis revealed that the compound stimulated tyrosine phosphorylation of several proteins with 27 and 70 kDa proteins being regulated in both dose- and time-dependent manner prior to loss of viability. Protein tyrosine kinase inhibitor herbimycin A moderately protected cells from ACA-induced toxicity. In addition, cellular glutathione and protein sulfydryl groups were also significantly reduced both dose- and time-dependently during evidence of cell death. Replenishing thiol levels by antioxidant, N-acetylcysteine (NAC), an excellent supplier of glutathione and precursor of glutathione, substantially recovered the viability loss, but the recovery being time-dependent, as late addition of NAC (at least 30 min after ACA addition to cultures) was, however, ineffective. Addition of NAC to ACA treated cultures also abolished tyrosine phosphorylation of the 27 kDa protein. These results, at least partly, identify cellular sulfhydryl groups and protein tyrosine phosphorylation as targets of ACA cytotoxicity in tumor cells.

Zinc deficiency enhances sensitivity to carbon tetrachloride-induced hepatotoxicity in rats.

Zinc (Zn) is an essential nutrient that is required in humans and animals for many physiological functions, including immune and antioxidant functions, growth and reproduction. The present study was conducted to examine the influence of Zn deficiency on the protective action against mild oxidative stress induced by a low dose of carbon tetrachloride (CCl(4)) in rats. Male Wistar rats were administered 125 or 250 µL/kg body weight CCl(4), which caused mild or no elevation of serum LDH, AST and ALT enzyme levels in rats fed a diet with adequate Zn. Treatment with CCl(4) (125 µL/kg) caused a significant release of these enzymes into the serum of rats fed a Zn-deficient diet but not in those given a diet with adequate Zn. Furthermore, no histological abnormalities were observed in CCl(4)-untreated rats fed either a diet with adequate Zn or a Zn-deficient diet or in CCl(4) (125 µL/kg)-treated rats fed a diet with adequate Zn. In CCl(4) (125 µL/kg)-treated rats fed a Zn-deficient diet, however, we observed associated collagen accumulation in the liver and hepatic necrosis. The degree of fibrosis was also more severe in CCl(4) (250 µL/kg)-treated rats fed a Zn-deficient diet. These results show that zinc deficiency during an oxidative stress injury negates the protective actions of certain treatments that normally block oxidative damage. The present study suggests that Zn plays an important role in regulating the antioxidative defense system under mild CCl(4) toxic conditions.

Comparison of glutathione reductase activity and the intracellular glutathione reducing effects of 13 derivatives of 1'-acetoxychavicol acetate in Ehrlich ascites tumor cells.

In a previous study, we showed that (1'S)-acetoxychavicol acetate ((S)-ACA) caused a rapid decrease in glutathione (GSH) levels less than 15 min after exposure. (S)-ACA-induced cell death was reversed by the addition of N-acetylcysteine. In the current study, we investigated the inhibitory activities of 13 derivatives of (S)-ACA on tumor cell viability, intracellular GSH level and GR activity. Correlations were found among a decrease in cell viability, intracellular GSH levels and the activity of GR in Ehrlich ascites tumor cells treated with the various ACA analogues. A test of the 13 derivatives revealed that the structural factors regulating activity were as follows: (1) the para or 1'-position of acetoxyl group (or other acyl group) was essential, (2) the presence of a C2'-C3' double or triple bond was essential, and (3) the S configuration of the 1'-acetoxyl group was preferable.

Chemical constituents of cape aloe and their synergistic growth-inhibiting effect on Ehrlich ascites tumor cells.

The constituents of cape aloe were investigated after a preliminary screening of the growth-inhibiting effect on Ehrlich ascites tumor cells (EATC) of several extracts of this plant. Ten compounds were isolated from the dichloromethane (CH(2)Cl(2)) extract that showed the strongest activity, and their structures were elucidated as aloe-emodin (1), p-hydroxybenzaldehyde (2), p-hydroxyacetophenone (3), pyrocatechol (4), 10-oxooctadecanoic acid (5), 10-hydroxyoctadecanoic acid (6), methyl 10-hydroxyoctadecanoate (7), 7-hydroxy-2,5-dimethylchromone (8), furoaloesone (9), and 2-acetonyl-8-(2-furoylmethyl)-7-hydroxy-5-methylchromone (10) based on MS and various NMR spectroscopic techniques. Compounds 2-7 were isolated for the first time from cape aloe. Compounds 4-7 and 10 showed a significant growth-inhibiting effect, and compound 1 exhibited a remarkable synergistic effect on compounds 8-10, which was not observed with the treatment by each compound alone on EATC. These results suggest that the strong growth-inhibiting effect of the CH(2)Cl(2) extract was dependent not on one compound alone, but on the synergistic effect from the combination of compound 1 and the other compounds.