Decitabine, independent of apoptosis, exerts its cytotoxic effects on cell growth in melanoma cells.

Decitabine is a synthesized cytosine analog that is a potent inhibitor of DNA methylation. There have been a few reports on the in vitro anti-melanoma effect of decitabine or its functional mechanisms. We investigated the anti-proliferation effect of decitabine on the cultured murine melanoma cell line K1735M2. MTT assay showed that decitabine had strong inhibition on melanoma K1735M2 in a time- and dose-dependent manner in vitro. Morphological observation showed that decitabine could induce melanoma K1735M2 cells to produce dendrite-like structures with the increase of decitabine concentration and incubation time. Decitabine could effectively induce K1735M2 cells to differentiate in vitro. Additionally, decitabine could induce a dose-dependent G2/M cell cycle arrest in K1735M2 cells. We provided experimental evidences that the anti-proliferation effect of decitabine on murine K1735M2 melanoma cells was associated predominately with G2/M cell cycle arrest and the induction of differentiation rather than apopotosis.

MicroRNA-224 is upregulated in HepG2 cells and involved in cellular migration and invasion.

BACKGROUND AND AIM: MicroRNAs are a class of small non-coding RNAs that negatively regulate the expression of their target genes. The aim of the present study was to explore the effects of microRNA on biological behaviors of HepG2 cells and further analyze its characteristics. METHODS: We detected different expression profiles of miRNAs in HepG2 and L02 cell lines by microRNA microarray. Northern blot, quantitative real-time polymerase chain reaction, methylthiazolyl tetrazolium, fluorescence-activated cell sorting, scratch wound, transwell migration and Matrigel invasion assays and western blot were carried out to determine whether or not microRNA-224 (miR-224) can influence the biological behaviors of HepG2 cells. RESULTS: MiR-224 was significantly upregulated in HepG2 cells. Cell proliferation, migration and invasion, but not cell cycles, were altered after changing the expression of miR-224. Taking invasion and migration as a breakthrough, a close relationship between the expression of miR-224 and its proteins such as PAK4 and MMP9, which were involved in the invasion of tumor, was found. CONCLUSIONS: Overexpression of miR-224 was involved in the malignant phenotype of HepG2 cells, and it may be an important factor in regulating the migration and invasion of HepG2 cells.

Reactive oxygen species production and Bax/Bcl-2 regulation in honokiol-induced apoptosis in human hepatocellular carcinoma SMMC-7721 cells.

We investigated possible mechanism(s) where honokiol induces apoptosis in human hepatocellular carcinoma SMMC-7721 cells. MTT assay showed that honokiol has strong inhibition on SMMC-7721 cells in a dose-dependent manner. SMMC-7721 cells after honokiol treatment display morphological characteristics such as cell shrinkage, detachment from the culture plate, formation of apoptotic bodies, change to a round shape, and marked nuclear condensation and fragmentation after 32258 staining. Cell apoptosis was measured by Annexin-V/PI staining and alternatively, by the subG0/G1 percentage of the cell cycle analysis followed by FACS. An obvious loss of ΔΨ(m) and a quick burst of ROS was detected when honokiol reached 4µg/ml, which was coincident with the high apoptosis percentage in our previous research. Up-regulation of Bax and down-regulation of Bcl-2 were observed, suggesting that honokiol-induced apoptosis was associated with reactive oxygen species (ROS) production and an increase of Bax/Bcl-2 ratios.

[The expression of Tec and the level of its phosphorylation in primary hepatic carcinomas].

OBJECTIVES: To detect the expressions of Tec tyrosine kinase in hepatocellular carcinoma and the levels of phosphorylation of tyrosine kinase in liver cancer tissues, paracancerous tissues and normal liver tissues and to find the significance of their differences. METHODS: 200 specimens of tissues, including liver cancer tissues, surrounding liver tissues not more than 1.5 cm from the cancers, and normal liver tissues were investigated for Tec protein expression and Tec phosphorylation by tissue microarrays and immunohistochemistry (SP method). RESULTS: The positive immunohistochemical stainings of Tec in cancerous tissues and non-cancerous tissues showed no obvious differences, nevertheless, the immunostaining levels in liver cancer tissues were much higher than in non-cancerous tissues and they correlated with the grading of tumors (P < 0.05). The phosphorylation of Tec was significantly expressed in liver cancer tissues (73%) in comparison with other tissues (42%, 10% both P < 0.05), but it did not correlate with any clinicopathological characteristics. CONCLUSION: Overexpression of Tec is associated with the tumorigenesis and development of liver cancer; inhibiting Tec or degrading Tec phosphorylation directly might affect the progression of liver cancer.

Inhibition of cell growth and induction of G1-phase cell cycle arrest in hepatoma cells by steroid extract from Meretrix meretrix.

In this study, we report that the steroid extract 5alpha, 8alpha-epidioxycholest-6-ene-3beta-ol (MME) from Meretrix meretrix has the ability to inhibit growth of hepatoma cells and to induce G1-phase cell cycle arrest in two human hepatoma cell lines, HepG2 and Hep3B. HepG2 cells were more sensitive than Hep3B to MME. The extract markedly up-regulated the expression of p53 and p21WAF1/CIP1 in HepG2, suggesting that MME-induced G1 phase cell cycle arrest in HepG2 might be p53-dependent. Therefore, the up-regulation of p27KIP1and p16INK4A in both cell lines indicates that a p53-independent pathway might be involved in the mechanism of MME inducing cell cycle arrest. In conclusion, MME induces G1 phase cell cycle arrest via both p53-dependent and p53-independent pathways.

The effects of oridonin on cell growth, cell cycle, cell migration and differentiation in melanoma cells.

Oridonin, a diterpenoid isolated from Rabdosia rubescens (Hemsl.) Hara, is currently one of the most important traditional Chinese herbal medicines. We investigated the anti-proliferation effect of oridonin on the cultured murine melanoma cell line K1735M2. The growth inhibition activity of oridonin for K1735M2 cells occurred in a time- and dose-dependent manner (IC50 was 7.4+/-0.6 microM). Further studies showed that these inhibition effects were associated with dose-dependent G2/M phase arrest and differentiation induction. Detection of morphological observation showed that oridonin could induce K1735M2 cells to produce dendrite-like structures. The results of the migration indicated that oridonin affected motility of K1735M2 cells in a dose-dependent manner. These results suggested that oridonin is a potential candidate for melanoma cancer therapy.

[Preparation of inclusion complex of daidzein and hydropropyl-beta-cyclodextrin].

OBJECTIVE: To prepare an inclusion complex of daidzein and hydropropyl-beta-cyclodextrin to enhance the solubility of daidzein. METHOD: The inclusion complex of daidzein was prepared by the solution stirring method. The binary system of daidzein and HP-beta-CD was confirmed by differential thermal, thermogravimetry analysis, infrared spectroscopy and X-ray diffractometry. RESULT: The drug content in the inclusion complex was 6. 76% and the solubility was 13.68 mg x mL(-1). The identification results showed that the inclusion complex was formed. CONCLUSION: The preparation method of the inclusion complex of daidzein and hydropropyl-beta-cyclodextrin is simple and available, with a increased solubility of daidzein.

Kakispyrol, a new biphenyl derivative from the leaves of Diospyros kaki.

A new biphenyl derivative, 4',5-dimethoxy-3-beta-D-glucopyranosyloxy-4-hydroxy-biphenyl, named kakispyrol (1), has been isolated from the leaves of Diospyros kaki, together with three known compounds, vitexin (2), 2'-O-rhamnosyl vitexin (3) and isorhamnetin-3-O-beta-D-glucopyranoside (4). The structure of compound 1 has been determined on the basis of spectroscopic evidence.

Inhibitory effects of some flavonoids on the activity of mushroom tyrosinase.

Mushroom tyrosinase (EC 1.14.18.1) is a copper containing oxidase that catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones, and then forms brown or black pigments. In the present study, the effects of some flavonoids on the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) have been studied. The results show that flavonoids can lead to reversible inhibition of the enzyme. A kinetic analysis showed that the flavonols are competitive inhibitors, whereas luteolin is an uncompetitive inhibitor. The rank order of inhibition was: quercetin > galangin > morin; fisetin > 3,7,4;-trihydroxyflavone; luteolin > apigenin > chrysin.

Daidzein enhances osteoblast growth that may be mediated by increased bone morphogenetic protein (BMP) production.

Daidzein, a natural isoflavonoid found in Leguminosae, has received increasing attention because of its possible role in the prevention of osteoporosis. In the present investigation, primary osteoblastic cells isolated from newborn Wistar rats were used to investigate the effect of this isoflavonoid on osteoblasts. Daidzein (2-50 microM) increased the viability (P<0.05) of osteoblasts by about 1.4-fold. In addition, daidzein (2-100 microM) increased the alkaline phosphatase activity and osteocalcin synthesis (P<0.05) of osteoblasts by about 1.4- and 2.0-fold, respectively. Alkaline phosphatase and osteocalcin are phenotypic markers for early-stage differentiated osteoblasts and terminally differentiated osteoblasts, respectively. Our results indicated that daidzein stimulated osteoblast differentiation at various stages (from osteoprogenitors to terminally differentiated osteoblasts). We also investigated the effect of daidzein on bone morphogenetic protein (BMP) production in osteoblasts that display the mature osteoblast phenotype. The results indicated that BMP2 synthesis was elevated significantly in response to daidzein (the mRNA increased 5.0-fold, and the protein increased 7.0-fold), suggesting that some of the effects of daidzein on the cell may be mediated by the increased production of BMPs by the osteoblasts. In conclusion, daidzein has a direct stimulatory effect on bone formation in cultured osteoblastic cells in vitro, which may be mediated by increased production of BMPs in osteoblasts.

Effects of genistein and daidzein on the cell growth, cell cycle, and differentiation of human and murine melanoma cells(1).

Genistein and daidzein are two major isoflavonoids in dietary soybean that have inhibition effect on the cell growth of different tumor cell lines. We previously reported the anti-tumor activities of genistein and daidzein in human co1on tumor (HCT) cells and their different ability to enhance the activation of murine lymphocytes. In the present study, the effect of genistein and daidzein on the cell growth, cell cycle progression, and differentiation of murine K1735M2 and human WM451 cel1s was investigated. It was found that genistein could inhibit the cell growth of two metastatic melanoma cell lines, murine Kl735M2 and human WM45l in a dose-dependent manner. Flow cytometry showed that genistein could cause arrest of both Kl735M2 and WM45l at G(2)/M phase, while daidzein increased the cell numbers at S phase, decreased the cell numbers at G(1) phase. Detection of melanin and morphological observation showed that genistein can induce Kl735M2 and WM45l to produce dendrite-like structure and produce more melanin by 80%. In contrast, daidzein only retarded the growth of K1735M2 and did not induce differentiation in either K1735M2 or WM451. These results suggest that genistein and daidzein in soybean can inhibit certain malignant phenotype of melanoma via different mechanisms and be potential medical candidates for melanoma cancer therapy.