腺病毒介导的p16基因对TJ899细胞系活性的影响

目的 研究五型腺病毒载体 (Ad5 )携带p16基因对恶性脑胶质瘤细胞系TJ899生长状态的影响。方法 免疫组化 (SP法 )测定p16蛋白表达 ,四甲基偶氮唑蓝 (MTT)法和克隆形成实验测定恶性脑胶质瘤细胞系生长状态。结果 重组体腺病毒能介导p16外源基因在恶性脑胶质瘤细胞系TJ899细胞中阳性表达 ,6d时肿瘤细胞生长抑制率为 93% ,并且能显著地抑制肿瘤细胞的克隆形成能力。结论 腺病毒介导p16基因能在肿瘤细胞中表达 。

Antitumor Activity of the Ganoderma Lucidum Spore Alcohol Extract in Vitro

Several cancer cell lines(epithelioma cells or leukemia cells)from human being or mouse were first used to study the antitumor activity of the Ganoderma lucidum spore alcohol extract(GLSAE)in vitro by the MTT test A comparision was made between the sporodermbroken(SB)and sporoderm nonbroken(SN)GLSAE It was showed that both GLSAE SB and GLSAE SN could inhibit the proliferation of these cancer cells,but the activity of GLSAE SB was much higher than that of GLSAE SN These results suggested that Ganoderma lucidum spore could probably be used for tumor treatment

Differential Expression of PKC Isoforms and Their Tumoricidal Activity in Two Macrophage Cell Lines: Involvement of Nitric Oxide-dependent Mechanisms

Objective: To investigate the role of PKC isoforms in the regulation of LPS-triggered tumoricidal activity in macrophages and further elucidate its signal mechanisms. Methods: Two macrophage cell lines (P388D1 and RAW264.7) were stimulated by LPS alone, or with long-term of PMA pretreatment. Then cytotoxicities to P815 cells (by MTT assay) and IL-1, TNF- (by ELISA) and nitric oxide (NO) production (by Griess reagent) in supernatants were measured. Western blot for PKC isoforms after long-term PMA pretreatment was analyzed. Results: RAW264.7 cells were stimulated with LPS to kill target tumor cells P815, whereas P388D1 cells failed to develop such an ability. Down-regulation of PKC isoforms by chronic treatment with PMA significantly inhibited the LPS-induced cytotoxicity in RAW264.7 cells. In unstimulated state, Western blotting with rabbit antiserum specific for the PKC, 1, 2, or showed all 5 isoforms were detected in P388D1 cells, while only PKC, PKC1 and PKC were detected in RAW264.7 cells. Exposure of the cells to long-term of PMA treatment significantly down-regulated the expression of PKC, PKC1 and PKC in RAW264.7 cells. But in P388D1 cells, although PKC, PKC and PKC were down-regulated, the expression of PKC1 and PKC2 could not be regulated. Comparing with LPS-induced IL-1, TNF- and NO production by the two macrophage cell lines, P388D1 failed to produce NO. In RAW264.7 cells, LPS-induced NO production and antitumor activity was attenuated by the addition of L-NAME, an iNOS inhibitor. Conclusion: The results indicated a critical role of PKC in LPS-induced antitumor activity and this cytotoxicity is mainly due to PKC- mediated NO production by RAW264.7 cells, but not a direct cytotoxic activity.

Effects of cisplatin on telomerase activity and telomere length in BEL-7404 human hepatoma cells

Telomerase activity was inhibited in a dose and time-dependent manner with the treatment of cisplatin for 24, 48, or 72 h in a concentration ranged from 0.8 to 50 1uM in BEL-7404 human hepatoma cells. There were no changes in expression pattern of three telomerase subunits, its catalytic reverse transcriptase subunit (hTERT), its RNA component (hTR) or the associated protein subunit (TP1), after cisplatin treated for 72 h with indicated concentrations. Mean telomere lengths were decreased by the cisplatin treatment. Cell growth inhibition and cell cycle accumulation in G2/M phase were found to be correlated with telomerase inhibition in the present study, but percentages of cell apoptosis did not change markedly during the process.

Biochemical mechanisms in drug-induced liver injury:Certainties and doubts

Drug-induced liver injury is a significant and still unresolved clinical problem. Limitations to knowledge about the mechanisms of toxicity render incomplete the detection of hepatotoxic potential during preclinical development. Several xenobiotics are lipophilic substances and their transformation into hydrophilic compounds by the cytochrome P-450 system results in production of toxic metabolites. Aging, preexisting liver disease, enzyme induction or inhibition, genetic variances, local 02 supply and, above all, the intrinsic molecular properties of the drug may affect this process. Necrotic death follows antioxidant consumption and oxidation of intracellular proteins, which determine increased permeability of mitochondrial membranes, loss of potential, decreased ATP synthesis, inhibition of Ca^2＋-dependent ATPase, reduced capability to sequester Ca^2＋ within mitochondria, and membrane bleb formation. Conversely, activation of nucleases and energetic participation of mitochondria are the main intracellular mechanisms that lead to apoptosis. Non-parenchymal hepatic cells are inducers of hepatocellular injury and targets for damage. Activation of the immune system promotes idiosyncratic reactions that result in hepatic necrosis or cholestasis, in which different HLA genotypes might play a major role. This review focuses on current knowledge of the mechanisms of drug-induced liver injury and recent advances on newly discovered mechanisms of liver damage. Future perspectives including new frontiers for research are discussed.

Determination of Antioxidant and Cytotoxic Activities of Red Seaweed, （Gracilaria manilaensis） against Different Cancer Cell Lines

Antioxidants play an important role in inhibiting and scavenging free radicals in human, providing protection against cellular damage in relation to cancer initiation. Seaweeds have been proved to have high antioxidant activity. Thus, this research was carried out to determine the antioxidant and anticancer properties of edible red seaweed, Gracilaria manilaensis （Gracilariales, Rhodophyta）. The extracts were prepared by Soxhlet extraction using organic solvents with different polarities. The antioxidant activities of extracts were determined in terms of their flee radical scavenging activity （RSA IC50） and total phenolic content （TPC）. The cytotoxic activity of extracts were tested against human ovarian cancer cell line （Caov-3）, human breast cancer cell line （MDA-MB-231 and MCF-7）, human cervical cell line （HeLa）, mouse fibroblast cell line （L929） and Madin-Darby canine kidney （MDCK） and the cell viability after 72 h incubation was determined by methylene blue assay. The findings showed that acetone extract has the lowest DPPH IC50 value followed by ethyl acetate extract. Both extracts also showed high values of TPC. Dichloromethane extract had the strongest cytotoxic on MDA-MB-231 （53.90 μg/mL ± 5.59 μg/mL） and HeLa （95.50 μg/mL）. While, acetone and ethyl acetate extracts were cytotoxic on MCF-7 （66.07 μg/mL） and Caov-3 （69.67 μg/mL ± 13.94 μg/mL）. It could be concluded that the antioxidant and cytotoxic activities of G. manilaensis were influenced by the types of solvents used and thus had a potential to develop as a cancer chemoprevention or anticancer agent against selected cancer.

Silencing Sp1 suppresses telomerase activity and promotes apoptosis of SW480 cells line in colorectal carcinoma

Objective:The aim of the study was to examine the effect of Sp1 on the expression of the human telomerase reverse transcriptase(hTERT) gene in human colorectal carcinoma SW480 cells.Methods:The Sp1 shRNA plasmid was transfected into colorectal carcinoma SW480 cells line by liposome mediation for transient expression.After Sp1 shRNA plasmid transfected SW480 cells,the exogenous Sp1 protein expression was determined by the method of Western blot.At same time,hTERT mRNA expression was detected by RT-PCR,telomerase activity was determined by the telomeric repeat amplification protocol(TRAP) assay,and the apoptotic rate of cells was also tested by flow cytometry.Results:The protein expressions of Sp1 gene could be reduce by transfecting of pGenesil-1-Sp1(+) recombinant plasmid into SW480 cells.The apoptotic rate was increased compared with pGenesil-1-Sp1(-)/SW480 and SW480(P < 0.05),which indicated that lowexpression of Sp1 gene could lead to low level of telomerase activity and induce apoptosis.Conclusion:Silencing Sp1 may suppress the activity of telomerase by inhabiting hTERT gene expression.

Antiproliferative Activity of Kappaphycus Alvarezii Extract on Three Cancer Cell Lines （NCIH 460, HCT 116 and U 251）

The methanolic extract of red seaweed Kappaphycus alvarezii was evaluated against three different cancer cell lines to study for its antiproliferative effect. Lung cancer cell line （NCIH 460）, Colon cancer cell line （HCT 116） and Glial cell carcinoma （U 251） are the three selected cell lines investigated in this study. Different concentrations of methanol extract （0.01, 0.10, 1.00, 10.00 and 100.00 lag/mL respectively） of Kappaphycus alvarezii were prepared and screened by quantitative MTT （Microculture Tetrazolium） （3-（4, 5-dimethylthiazol）-2, 5-diphenyltetrazolium bromide） assay. MTT assay are the colorimetric assay which was applied to assess the viability and proliferation of cancer cells to determine the cytotoxicity of methanol extract ofK. alvarezii. The MTT test is based on the enzymatic reduction of the tetrazolium salt （MTT） to formazon crystals exclusively in living metabolically active cells developed purple color complex which was directly proportional to the viability of cells. To elucidate the in-vitro anticancer activity the Lethal Concentration （LC50）, Growth Inhibition （GI50） and Total Growth Inhibition （TGI） of the extract were investigated individually for each cancer celt line. Analysis of the extract has shown good cytotoxicity in all tested cancer cell lines, with an IC50 of 55 μg/mL against NCIH460 and U251, 65 μg/mL for HCT116 respectively. GI50 was found to be 5 μg/mL for NCIH 460 and 10 μg/mL for HCT 116 and U251 cell lines. TGI was 19 μg/mL for NCIH 460, 29 μg/mL for HCT 116 and 25 μg/mL for U 251 cell lines. The cytotoxicity of the extract were significantly high in Lung Cancer Cell line （NCIH 460） when compared to Colon Cancer Cell line （HCT 116） and Glial Cell Carcinoma （U251） in the following order NCIH 460 〉 HCT 116 〉 U251.

Assistant radiotherapeutic effect of Jiaqi Mixture on tumor-bearing mice

Objective:The aim was to study the assistant radiotherapeutic effect of Jiaqi Mixture (JQM) on Ehrlich's ascites carcinoma (EAC) mice.Methods:The EAC-cancer model was made up with Kunming mice.The tumor-bearing mice were treated with whole body exposure (8 Gy) and intragastric administration of JQM,and the changes of tumor weight,the total number of white blood cells (WBC) and immune system were observed.Results:The average tumor weight,WBC,spleen coefficient,the stimulation index (SI) of Con A and LPS and the natural killing (NK) cell activity of mice decreased in some degree after radiotherapy,but the average tumor weight decreased more obviously in radiotherapy + medicine groups (compared with tumor control group,P < 0.05);and the other above indexes were much higher in radiotherapy + medicine groups than those in radiotherapy groups (P < 0.05-0.01).Conclusion:It was suggested that JQM can enhance the effect of radiation therapy and protect the normal immune system caused by radiation therapy.

Chromosomal localization of ribosomal DNA sequences in an apple rootstock using a digoxygenin detection system

A 6kb rDNA probe comprising the 18S coding plusspacer sequences has been hybridized to the metaphase chromosomes of apple rootstock cultivar MM106 demon-strating the localization of ribosomal gene arrays in the vicinity of the telomeric regions of the short arms of chro-mosomes 6 and 14. The in situ results using digoxygenin labelling coupled to an alkaline phosphatase immunoassay were confirmed by silver staining for NORs and nucleoli. This study demonstrates the feasibility of molecular cyto-genetic analysis of very small chromosomes (1.0-2.7μm)of apple.

Anti-inflammatory activity of ginsenosides in LPS-stimulated RAW 264.7 cells

Ginsenosides, the main active constituents of Panax ginseng Meyer （P. ginseng）, have potential therapeutic effects. All tested ginsenosides except gin- senoside F1 have previously been reported in inflammation studies using the RAW 264.7 macrophage cell line. We ex- amined the anti-inflammatory effects of single sugar moiety ginsenosides such as compound K （CK）, Rh2, Rhl, and F1 that were isolated from P. ginseng through in silico docking studies. We investigated their biological activity predictions, including absorption, distribution, metabolism, excretion, and toxicity and PASS properties, on the suppression of NF- κB, followed by in vitro validation in lipopolysaccharide （LPS）-stimulated RAW 264.7 macrophage cells. The molecular docking results of our study showed that all treated ginsenosides are non-toxic and may be drug-like molecules. The molecular binding interactions of these ginsenosides with the active residues of NF-κB noticeably support their anti-inflammatory activity. CK and Rhl sig- nificantly reduced the production of nitric oxide, cyclooxy- genase-2 （COX-2）, and pro-inflammatory cytokines such as prostaglandin E2 and tumor necrosis factor alpha （TNF-α） in a dose-dependent manner. Real-time PCR and Western blot analyses further confirmed that protopanaxadiols （PPDs） and protopanaxatriols （PPTs） inhibitory effects may have been due to the down-regulation of TNF-α, inducible nitric oxide synthase, COX2, nuclear factor kappa B （NF-κB）, and I kappa B kinase. The expression of co-stimulatory molecules such as ROS was also inhibited by CK and Rhl in a dose- dependent manner. Furthermore, activation of NF-κB in LPS-stimulated RAW 264.7 macrophages was significantly suppressed by CK and Rhl. Taken together, these results provide evidence that PPD- and PPT-type ginsenosides in- cluding CK and Rhl may exhibit strong anti-inflammatory effects by inhibiting pro-inflammatory mediators through down-regulation of NF-κB.

Design, synthesis and antitumor activity evaluation of novel 2,6-dichloro-3,5-dinitrotoluene derivatives

A series of novel 2,6-dichloro-3,5-dinitrotoluene derivatives were designed, synthesized in the present study, and their antitumor activities against five cell lines（A431, HepG2, A549, HT-29 and HEK-293） were tested. Most of the compounds exhibited moderate-to-significant cytotoxicity and high selectivity against one or more cell lines in comparison with cisplatin. Studies on their preliminary structure-activity relationships（SARs） indicated that compounds containing phenyl（piperazin-1-yl） methanone groups, especially chlorine atom at 4-position of the phenyl ring, were more effective. Compound 4g was found to be the most potent derivative with IC_（50） values of 1.04, 3.20, 6.93, 4.10 and 20.15 μmol/L against A431, HepG2, A549, HT-29 and HEK-293 cell lines, respectively, which was better than positive control cisplatin, one of the most clinically used chemotherapeutic drugs.