Screening of optimal siRNA interference sequence of CIT gene and its inhibition expression in hepatoma SK-Hep-1 / 中华肝脏病杂志

Objective@#To construct and screen optimal siRNA interference sequence of CIT gene and to detect its interference efficiency as well as proliferation effect in human hepatoma cell line SK-Hep-1.@*Methods@#Three siRNA target spots were designed and synthesized according to the CIT gene sequence. SK-Hep-1 HCC cells were transfected by liposome transfection. The knockdown efficiency of the target CIT gene was detected by real-time PCR and Western blot. Expressional change of CIT in SK-Hep-1 cells after 48 hours of siRNA interference were observed by immunohistochemistry and confocal microscopy. The proliferation of SK-Hep-1 cells after 48 hours of siRNA interference was detected by EdU cell proliferation assay. A t-test was used to compare the mean of two samples, and one-way ANOVA was used to compare the mean of multiple samples.@*Results@#Western blot results showed that the three interference sequences were targeted at different target spots. The expression level of CIT protein in KD-1,-2, and-3 groups were decreased (P < 0.01) than control, while the protein expression level of KD1 group was the lowest. Real-time PCR results showed that compared with the control group, the expression level of CIT mRNA in KD-1, -2, and -3 groups decreased (P < 0.01), while that in KD1 group was the lowest. Laser confocal microscopy also confirmed that the morphological expression of CIT attenuated significantly after transfection with siRNA. The results of EdU proliferation assay showed that siRNA transfected with CIT significantly attenuated the proliferation of SK-Hep-1 hepatoma cells (P < 0.05).@*Conclusion@#The successful construction and screening of siRNA fragments can effectively inhibit the expression and proliferation of CIT gene in hepatoma SK-Hep-1.

Effects of Betulinic Acid on Proliferation of Human Liver Cancer HepG2 Cells / 中国中医药信息杂志

Objective To observe the effects of betulinic acid (BA) on proliferation of human hepatoma stem cell;To discuss its anti-cancer mechanism from the aspects of cell cycle and cell apoptosis. Methods HepG2 stem cells were cultivated in vitro and testified the self-renewal capacity. The effects of BA in concentration of 40, 20, 10, 5, 2.5, 1.25μmol/L on the cell vitality of cultured human liver cancer stem cells for 24 and 48 hours were measured with CCK-8 method. The human hepatoma stem cell line HepG2 was administrated by BA at concentrations of 40, 20, 10, 5μmol/L for 48 hours, and cell cycle and apoptosis rate were measured by flow cytometry. Results BA could inhibit HepG2 stem cell proliferation obviously with dose-effect relationship. BA influenced cell cycle, and induced tumor stem cell apoptosis. 40μmol/L BA blocked cell cycle in S phase, and cell apoptosis rate reached 10.86%. Conclusion BA has obvious inhibitory effects on proliferation of HepG2 liver cancer stem cell, which probably plays a part in anti-cancer by influencing cell cycle and inducing cell apoptosis.

Inhibitory Effect of Total Alkaloids from Lotus Seed on Human Hepatoma Cells / 中国药师

Objective: To study the inhibitory effect of total alkaloids from lotus seed on human hepatoma HepG2 cells.Methods: The effect of total alkaloids from lotus seed on the growth of HepG2 cells was studied by CCK-8 kit.The apoptosis rate of HepG2 cells was detected by flow cytometry.Results: When the action time was the same, with the increase of drug concentration, the inhibitory rate of total alkaloids from lotus seed on HepG2 cells increased, in a dose-dependent manner.At 72 h, the half inhibitory concentration (IC50) of total alkaloids from lotus seed on HepG2 cells was 1.501 μg·ml-1.At the same concentration, the inhibitory rate of the total alkaloids from lotus seed on HepG2 cells increased with the extension of the action time.At 72 h, the inhibition rate of 10 μg·ml-1 total alkaloids from lotus seed reached 72%.After treated with the total alkaloids from lotus seed at different concentrations, the apoptosis rate of HepG2 cells significantly increased in a dose-dependent manner.Compared with the blank control group, the difference was statistically significant (P <0.05), and the apoptosis rate of HepG2 cells was 85.6% treated with 20 μg·ml-1 total alkaloids from lotus seed.Conclusion: The total alkaloids from lotus seed can induce cell apoptosis and inhibit the proliferation of human hepatoma HepG2 cells.

Mechanism of phloretin induced the apoptosis of hepatoma carcinoma cell SMMC-7721 / 中国生化药物杂志

[Abstract ] Objective To investigate effect of phloretin on apoptotic of hepatoma carcinoma cells SMMC-7721, and explore its mechanisms.Methods Logarithmic phase of hepatoma carcinoma cells SMMC-7721 were cultured separately with 30, 60, 120 mg/L phloretin, morphological alterations of apoptotic were observed by phase contrast microscopy and AO/EB double fluorescence staining method was used to observe were low, medium and high concentration trentment group, respectively.the cells treated by phloretin.Apoptotic rates, cell cycle progression, mitochondrial trans-membrane potential and intracellular calcium homeostasis were detected by flow cytometry.Results Cells appeared typical apoptosis morphological alterations.Phloretin induced SMMC-7721 cell line apoptosis in a dosage and duration dependent manner.Cell cycle was arrested at G1 phase, mitochondrial trans-membrane potential decreased, intracellular free Ca2 +increased.Conclusion Phloretin induce apoptosis of SMMC-7721 by affecting cell cycle progression, reducing mitochondrial trans-membrane potential and changing intracellular calcium homeostasis.

Effects of proliferation and apoptosis of phloretin on the hepatoma carcinoma cells HepG-2 / 中国生化药物杂志

Objective To investigate proliferative and apoptotic effects of phloretin on hepatoma carcinoma cells, hepatoma carcinoma cells HepG-2 was used as research materials.Methods This research observed morphological alterations using phase contrast microscopy and electron microscopy, cell proliferation were detected by MTT assay, and using flow cytometry detected apoptotic rates, cell cycle progression, mitochondrial trans-membrane potential and intracellular calcium homeostasis.ResuIts Apoptotic cells appeared morphological alterations.Phloretin exerted a inhibitory the proliferation of HepG-2 cell line, and induced its apoptosis in a dosage and duration dependent manner.Cell cycle was arrested at G1 phase, mitochondrial trans-membrane potential dropped, intracellular free Ca2 + increased.ConcIusion Phloretin can induce apoptosis of HepG-2 via arresting cell cycle progression, reducing mitochondrial trans-membrane potential and disturbing intracellular calcium homeostasis.

The forecast of anticancer targets of cryptotanshinone based on reverse pharmacophore-based screening technology / 中国天然药物

Anticancer targets of cryptotanshinone were evaluated and rapidly forecasted with PharmMapper, a reverse pharmacophore-based screening platform, as well as drug target databases, including PDTD, DrugBank and TTD. The pathway analyses for the collection of anticancer targets screened were carried out based on the KEGG pathway database, followed by the forecast of potential pharmacological activities and pathways of the effects of cryptotanshinone, and verification of some of the targets screened using whole cell tests. The results showed that a total of eight targets with anticancer potential were screened, including MAP2K1, RARα, RXRα, PDK1, CHK1, AR, Ang-1 R, and Kif11. These targets are mainly related to four aspects of the cancer growth: the cell cycle, angiogenesis, apoptosis, and androgen receptor. The cell tests showed that cryptotanshinone can inhibit the viability of human hepatoma cells SMMC-7721, which is related to the reduction of expression of MAP2K1 mRNA. This method provides a strong clue for the study of the anticancer effects and mechanisms of action of cryptotanshinone in the future.

Adaptive response to ionizing radiation induced by low dose of gamma ray in human hepatoma cell lines

When cells are exposed to a low dose of a mutagenic or clastogenic agent, they often become less sensitive to the effects of a higher dose administered subsequently. Such adaptive responses were first described in Escherichia coli. Studies on mammalian cells have been limited to human lymphocytes exposed to low doses of an alkylating agent. In this study, the adaptive response to 1 cGy of gamma rays was investigated in human tumor cells using two human hepatoma cell lines, Hep G2 and Hep 3B. Experiments were carried out by delivering 1 cGy followed by 50 cGy of gamma radiation and chromatid breaks were scored as an endpoint. The results of this study indicate that prior exposure to 1 cGy of gamma rays reduces the number of chromatid breaks induced by subsequent higher doses (50 cGy). The time necessary for the expression of the adaptive response was determined by varying the time interval between the two doses from 1 hour to 72 hours. In G2 chromatids, the adaptive response was observed both at short time intervals, as early as 1 hour, and at long time intervals. In S chromatids, however, the adaptive response was shown only at long time intervals. When 3-aminobenzamide, an inhibitor of poly (ADP-ribose) polymerase, was added after 50 cGy, adaptive responses were abolished in all the experimental groups. Therefore, it is suggested that the adaptive response can be observed in human hepatoma cell lines, which is first documented through this study.