Sensitive quantitative assay for point mutations in the rat H-ras gene based on single nucleotide primer extension.

Point mutations in oncogenes and tumor suppressor genes occur at early stages in the carcinogenic process. Point mutations in ras family oncogenes are the most common mutational events in several types of human cancer, and are available as molecular markers for the detection of cancer cells in carcinogenicity bioassay systems as well as in clinical samples. Although several techniques are utilized to detect point mutations in carcinogenicity bioassay systems, the sensitivity is too low to determine a small number of mutations. In order to overcome the disadvantage and to sensitively determine gene mutation rates for in vivo carcinogenicity bioassays of presumptive carcinogens, we established a Thermosequenase Cycle End Labeling (TCEL) method, a sensitive approach based on single nucleotide primer extension. One of the characteristics of the method is a high sensitivity of 1:100,000, ten times the sensitivity of the mutant allele-specific amplification now commonly employed. Using TCEL, we here quantified H-ras mutations in the livers of rats treated with a genotoxic carcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline. Our findings suggest that this method may be applied for many genetic targets as a component in vivo.

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.

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.

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.

[Inconsistency in quantitation of estrogen receptor and progesterone receptor with dextran charcoal method and enzyme immunoassay].

Six hundred and eighty-eight breast specimens including 442 breast cancers were studied to investigate the consistency and correlations between dextran charcoal assay (DCC) and enzyme immunoassay (EIA) for estrogen receptor (ER) and progesterone receptor (PgR). In DCC, ER was quantitated with competition of 16 alpha-125I-estradiol and diethyl stilbesterol, and PgR with 3H-R5020 and R5020. In EIA, kit from Abbott was used in ER and PgR quantitation. The mean age of the patients was 52 years and the mean age of patients with benign lesions was 39 years. The consistency rate was 95% for ER, and 83% for PgR, while the consistency coefficient kappa was 0.90 and 0.66, respectively. For the specimens in which the number of binding sites was calculated in both DCC (x) and EIA (y), the correlation coefficient was 0.787 and the linear regression formula was y = 0.5x + 58. For PgR, the correlation coefficient kappa was 0.612 and the regression formula was y = 2.6x + 91. In multiple regression analysis for consistency of DCC and EIA, for ER, there was an inconsistent trend for positive PgR and a consistent trend for patients in their fifties. For PgR, the trend was inconsistent for benign lesions and positive ER. In comparison with the efficacy of endocrine treatment, no responder was found in ER negative patients for both DCC and EIA. In PgR-negative patients a responder was found for both DCC and EIA. By Western blot analysis, anti-ER antibody provided in the ER.EIA kit showed affinity only for ER alpha and not for beta. In conclusion, in terms of the treatment efficacy for both ER and PgR, the current use of ER with EIA instead of DCC seems to give equivalent result, and old DCC data can be converted into the regression formula. However, PgR calculated by EIA was not equivalent with that of DCC.

Development of hepatocytes from ES cells after transfection with the HNF-3beta gene.

We have attempted to generate embryonic stem (ES) cell-derived hepatocytes expressing liver-specific functional properties by use of ES cell technology. It was found that ES cells are allowed to differentiate into hepatocytes possessing high metabolic activities when hepatocyte nuclear factor (HNF)-3beta-transfected ES cells are cultured in alpha-MEM medium supplemented with 10% fetal bovine serum (FBS) and fibroblast growth factor (FGF)-2 in the three-dimensional cell culture system at 5% CO2. The differentiated cells induced albumin, triacylglycerol, urea, and glycogen synthesis as well as further expression of metabolic proteins and serum factors as markers of hepatocytic differentiation for at least 4 months. The cells differentiated from HNF-3beta-transfected ES cells also had hepatocyte-like ultrastructural characteristics, including several endoplasmic reticula, mitochondrion, and glycogen. Our findings indicate that generation of hepatocytes maintaining high metabolic functions developed from mouse ES cells will facilitate the study of the basic mechanism for hepatogenesis and will certainly provide new opportunities for tissue transplantation.

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.

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.

Modulation of ornithine decarboxylase activity and mitogen-activated protein kinases in protocatechualdehyde-induced apoptosis of CTLL-2 cells.

Protocatechualdehyde (PA, a dihydroxybenzene derivative) has previously been shown to induce apoptotic cell death in cytotoxic T cells (CTLL-2). However, the molecular mechanisms by which PA regulates apoptosis are still unclear. In this study, the possible roles of ornithine decarboxylase activity (ODC) and mitogen-activated protein kinases (MAPKs) in the PA-induced apoptosis process were further investigated. We demonstrated that PA inhibited ODC activity induced by IL2 in a time- and dose-dependent manner. Furthermore, the expression of ODC mRNA stimulated by IL2 was also effectively suppressed. 0.12 mM PA inhibited the activation of ERK1/2 induced by IL2 and enhanced the activation of JNK, which was abrogated by IL2. No alteration in the effect of p38 MAPK on the apoptosis process was observed in the CTLL-2 cells. PD98059 (a specific ERK1/2 inhibitor) inhibited cell growth, led to cell apoptotic death and effectively decreased ODC activity and suppressed ERK1/2 activation induced by IL2. These data indicate that PA induced apoptosis in CTLL-2 cells by two mechanisms; either via inhibiting ODC induction or interfering with MAPK signaling pathways.