The Effect of Nano-apatite on the Expression of Telomerase Gene of Human Hepatocellular Carcinoma Cells

To investigate the effect of nano-apatite on the expression of the telomerase gene of human hepatocellular carcinoma cell lines and further explore the mechanism of the nano-apatite inhibiting cancer cells. Using the hybridization in situ method to detect the expression of the telomerase gene of human hepatocellular carcinoma cells treated with the nano-apatite for 4 h at 37 ℃ . The hybridization in situ showed that the cytoplasm of the positive cells was stained in nigger- brown. The positive cell rate of the control group was 88.49% , the cisplatin group was 25.6% , the nano-apatite group was 63.6% . The activity of telomerase gene was both obviously dedined comparing with the control group and the difference had significance （ p 〈 0. 05, p 〈 0.01 ）. The nanoapatite obviously inhabit the expression of the telomerase gene of human hepatocellular carcinoma cells.

Cyclin L2, a novel RNA polymerase Ⅱ-associated cyclin, is involved in pre-mRNA splicing and induces apoptosis of human hepatocellular carcinoma cells

We report the cloning and functional characterization of human cyclin L2, a novel member of the cyclin family. Human cyclin L2 shares significant homology to cyclin L1, K, T1, T2, and C, which are involved in transcriptional regulation via phosphorylation of the C-terminal domain of RNA polymerase Ⅱ. The cyclin L2 protein contains an N-terminal "cyclin box" and C-terminal dipeptide repeats of alternating arginines and serines, a hallmark of the SR family of splicing factors. A new isoform and the mouse homologue of human cyclin L2 have also been cloned in this study. Human cyclin L2 is expressed ubiquitously in normal human tissues and tumor cells. We show here that cyclin L2 co-localizes with splicing factors SC-35 and 9G8 within nuclear speckles and that it associates with hyperphosphorylated, but not hypophosphorylated, RNA polymerase Ⅱ and CDK p110 PITSLRE kinase via its N-terminal cyclin domains. It can also associate with the SC-35 and 9G8 through its RS repeat region. Recombinant cyclin L2 protein can stimulate in vitro mRNA splicing. Overexpression of human cyclin L2 suppresses the growth of human hepatocellular carcinoma SMMC 7721 cells both in vitro and in vivo, inducing cellular apoptosis. This process involves up-regulation of p53 and Bax and decreased expression of Bcl-2. The data suggest that cyclin L2 represents a new member of the cyclin family, which might regulate the transcription and RNA processing of certain apoptosis-related factors, resulting in tumor cell growth inhibition and apoptosis.

Holotransferrin Enhances Selective Anticancer Activity of Artemisinin against Human Hepatocellular Carcinoma Cells

Artemisinin, also termed qinghaosu, is extracted from the traditional Chinese medicine ar- temesia annua L. （the blue-green herb） in the early 1970s, which has been confirmed for effectively treating malaria, Additionally, emerging data prove that artemisinin exhibits anti-cancer effects against many types of cancers such as leukemia, melanoma, etc. Artemisinin becomes cytotoxic in the presence of ferrous iron. Since iron influx is high in cancer cells, artemisinin and its analogs selectively kill can- cer cells with increased intracellular iron concentrations. This study is aimed to investigate the selective inhibitory effects of artemisinin on SMMC-7721 cells in vitro and determine the effect of holotransfer- fin, which increases the concentration of ferrous iron in cancer cells, combined with artemisinin on the anticancer activity. MTT assay was used for assessing the proliferation of SMMC-7721 cells treated with artemisinin. The induction of apoptosis and inhibition of colony formation in SMMC-7721 cells treated with artemisinin were determined by TdT-mediated dUTP nick end labeling （TUNEL） and col- ony formation assay, respectively. The results showed that artemisinin at various concentrations signifi- cantly inhibited growth, colony formation and cell viability of SMMC-7721 cells （P〈0.05）, likely due to induction of apoptosis of SMMC-7721 cells. Of interest, it was found that incubation of artemisinin combined with holotransferrin sensitized the growth inhibitory effect of artemisinin on SMMC-7721 cells （P〈0.01）. Our data suggest that treatment with artemisinin leads to inhibition of viability and pro- liferation, and apoptosis of SMMC-7721 ceils. Furthermore, we observed that holotransferrin signifi- cantly enhanced the anti-cancer activity of artemisinin. This study may provide a potential therapeutic choice for liver cancer.

Modulating effects of survivin antisense oligonucleotide on changes of apoptosis and cell cycle of human hepatocellular carcinoma cell line SMMC-7721

To investigate the modulating effects of survivn antisense oligonucletode (ASODN) on the cell cycle and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and explore its mechanism.Methods Survivin ASODN was transfected into SMMC-7721 cells mediated by DOTAP liposomal reagent.Electron microscopy,flow cytometry and RT-PCR were used to detect the changes in cell ultrastructure,apoptosis,cell cycle and the expression of cyclinB1 mRNA,respectively.Results After transfection of survivin ASODN,the expression of cyclinB1 mRNA in the cells significantly increased and increase in G2-M arrest and apoptosis appeared.Meanwhile,the cell ultrastructure had apoptotic changes such as chromatin condensation and apoptotic body formation.Conclusion Survivin ASODN can induce the expression of cyclinB1 that may result in G2-M arrest.Consequently,apoptosis is triggered.Survivin ASODN transfection might be an improtant new treatment for HCC.14 refs,2 figs,1 tab.

The receptor for β2GPⅠon membrane of hepatocellular carcinoma cell line SMMC-7721 is annexin Ⅱ

AIM: To evaluate the receptor protein which can specifically bind to β2GPⅠon the membrane of hepatocellular carcinoma (HCC) cell line SMMC-7721, and to study the biological function of the receptor.METHODS: Through β2GPⅠ-affinity chromatography column, the peptid-polysome-mRNA complex, which can specially bind to β2GPⅠ, stayed with the column and was separated from the whole polysome of liver cells, and then eluted and collected. Using cDNA synthesis kit and cDNA PCR kit, the corresponding cDNA was obtained and sequenced. RT-PCR was used to amplify annexinⅡ, and flow cytometry was used to study the competitive binding of annexinⅡ with β2GPⅠto SMMC-7721.RESULTS: A total of 1.1 kb of the cDNA fragment of the specific binding protein of β2GPⅠon liver cell membrane was obtained. The sequence of cDNA shared high homology with human annexinⅡ (98%). AnnexinⅡ was expressed on the membrane of SMMC-7721, and could compete with β2GPⅠfor combining with SMMC-7721.CONCLUSION: The receptor for β2GPⅠon membrane of SMMC-7721 cells is annexinⅡ, which might bridge HBV to infect hepatocytes.

Inhibitory effect of metformin on the proliferation of human hepatoma HepG2 cells and its potential mechanism

Objective： This work aimed to study the inhibitory effect and the related mechanism of metformin （MET） on the proliferation of human hepatoma HepG2 cells. Methods： Human hepatoma HepG2 cells were treated with MET （0, 2, 10, and 50 mM）. The inhibitory effect of MET on the proliferation of HepG2 cells was determined by MTT method. The apoptosis of HepG2 cells was detected by flow cytornetry. The expression of cyclin D1 in HepG2 cells was examined by Western blot. ROS-DHE fluorescence probe was used to stain the reactive oxygen species （ROS） generated by HepG2 cells after treat- ment. Results： MET could inhibit the proliferation of HepG2 cells in a dose and time dependent manner. MET promoted the apoptosis of HepG2 cells. In addition, MET suppressed the expression of cell cycle protein cyclin D1 and induced the produc- tion of ROS in HepG2 cells. Conclusion： MET can inhibit the proliferation of human hepatoma HepG2 cells and induce cell apoptosis. Meanwhile, MET has the ability to decrease the expression of cyclin D1 and induce ROS generation, which may be involved in the mechanism of inhibiting hepatoma cells proliferation.

Optimization of the microwave-assisted extraction of phlorotannins from Saccharina japonica Aresch and evaluation of the inhibitory effects of phlorotannin-containing extracts on HepG2 cancer cells

The use of a microwave-assisted extraction （MAE） method for the extraction ofphlorotannins from Saccharinajaponica Aresch （S.japonica） has been evaluated with particular emphasis on the influential parameters, including the ethanol concentration, solid/liquid ratio, extraction time, extraction temperature, and microwave power. The MAE procedure was optimized using single-factor design and orthogonal array design （OAD）. The content of total phlorotannins in S. japonica was determined using a Folin-Ciocalteu （FC） assay. A maximum total phlorotannin content of 0.644 mg of phloroglucinol equivalent per gram of dry weight plant （mg PGE/g DW） was obtained using the optimized model, which included an ethanol concentration of 55%, solid/liquid ratio of 1：8, extraction time of 25 min, irradiation power of 400 W, and temperature of 60~C. Under similar conditions, the application of a conventional extraction method led to a lower phlorotarmin yield of 0.585 mg PGE/g WD. These results demonstrated that the MAE approach provided better results for the extraction ofphlorotarmins from S.japonica and was a promising technique for the extraction of phenolic compounds from S. japonica and other materials. In addition, screening tests for the inhibitory activity showed that the phlorotannin-containing extracts significantly inhibited the growth of human hepatocellular carcinoma cells （HepG2） by inducing their apoptosis. The morphological changes that occurred during cell apoptosis were characterized using Hoechst33258 staining.