ROS-Mediated Anti-Tumor Effect of Coptidis Rhizoma against Human Hepatocellular Carcinoma Hep3B Cells and Xenografts

Coptidis Rhizoma is the dried rhizome from the Coptis chinensis Franch. that has been shown to have a number of beneficial pharmacological properties including antioxidant, anti-inflammatory, and anti-cancer effects. However, the anti-cancer effects of Coptidis Rhizoma on hepatocellular carcinoma (HCC) remain unclear. In this study, we investigated the anti-cancer properties of Coptidis Rhizoma ethanol extract (CR) in HCC Hep3B cells and in a xenograft mouse model. Our results showed that the CR significantly inhibited cell growth and induced apoptosis in Hep3B cells through increased expression of Bcl-2 associated x-protein (Bax) and cleavage of poly-ADP ribose polymerase (PARP), reduced expression of Bcl-2, and activated caspases. CR also increased the generation of intracellular reactive oxygen species (ROS), which caused a loss of mitochondrial membrane potential (MMP, ΔΨm) and activation of the mitochondria-mediated intrinsic apoptosis pathway. Moreover, N-acetylcysteine (NAC), a ROS inhibitor, markedly blocked the effects of CR on apoptotic pathways. CR also induced the expression of light chain 3 (LC3)-I/II, a key autophagy regulator, whereas CR-mediated autophagy was significantly suppressed by NAC. In addition, pre-treatment with NAC perfectly attenuated the inhibition of cell invasion and migration of CR-stimulated Hep3B cells. Furthermore, oral administration of CR suppressed Hep3B tumor growth in xenograft mice without toxicity, alterations to body weight, or changes in hematological and biochemical profiles. Taken together, our findings suggest that CR has anti-tumor effects that result from ROS generation, and may be a potential pharmacological intervention for HCC.     

Polyphenol contents of Pu-Erh teas and their abilities to inhibit cholesterol biosynthesis in Hep G2 cell line

Thirty samples of Pu-Erh tea (a microbial fermented Chinese tea) were collected and assayed for cholesterol synthesis inhibitory activity and polyphenol composition. All samples were able to inhibit the cholesterol biosynthesis in Hep G2 cell model and the inhibition ratios ranged from 7% to 35%. The inhibition abilities of tea polyphenol standards were in the order of gallocatechin gallate (GCG) > epigallocatechin gallate (EGCG) > epicatechin gallate (ECG) > gallic acid > epigallocatechin (EGC) > myricetin > quercetin > catechin (C) > epicatechin (EC). It appears that catechins with a galloyl structure on the B ring or a gallic acid moiety in the structure would have better inhibitory activity. In summary, tea polyphenol may play a role on the cholesterol biosynthesis inhibitory ability of Pu-Erh tea.     

Ethanolic extracts of Antrodia cinnamomea mycelia fermented at varied times and scales have differential effects on hepatoma cells and normal primary hepatocytes

Mycelia of Antrodia cinnamomea (AC), an edible fungus native to Taiwan, were produced by submerged fermentation with various fermentation times in 250 mL, 5 and 500 L fermentors and were evaluated for the effect of fermentation products on the viabilities of Hep3B and HepG2 hepatoma cells and normal primary rat hepatocytes. The results showed that the ethanolic extracts of AC mycelia (from 250 mL fermentation for 8 wk and 5 and 500 L fermentations for 4 wk) possessed high antihepatoma activity. The IC(50) of ethanolic extract of AC mycelia fermented for 8 wk in a 250 mL fermentor against Hep3B and HepG2 cells were 82.9 and 54.2 microg/mL, respectively. Furthermore, the IC(50) for Hep3B and HepG2, treated with ethanolic extract of AC mycelia fermented for 4 wk in the 5 L fermentor were 48.7 and 3.8 microg/mL, respectively. Those treated with ethanolic extract of AC mycelia fermented for 4 wk in the 500 L fermentor were 36.9 and 3.1 microg/mL, respectively. No adverse effects of all samples on normal primary rat hepatocytes were observed.     

TGF—β1对肝癌细胞系Hep3B中干性相关基因表达的影响

有研究表明TGF-β1可以诱导诸多上皮来源的癌细胞和正常细胞发生EMT并使其功能发生改变。实验就TGF-β1对肝癌细胞系Hep3B的作用展开研究：通过CCK8检测TGF-β1对Hep3B细胞增殖的影响,RT-PCR实验检测TGF-β1处理后细胞中EMT及干性相关基因的表达变化。结果表明：TGF-β1对Hep3B细胞的增殖无抑制作用;TGF-β1处理Hep3B细胞6d后EMT相关基因的mRNA表达水平并无显著改变,但TGF-β1可上调Hep3B细胞干性基因Oct-4,Klf-4,Nanog,C-myc的表达,并下调分化基因albumin的表达。结果提示TGF-β1一定程度上影响肝癌细胞系的干性基因表达,但并不一定是以发生EMT为前提的。     

Antiproliferative effects of sea buckthorn (Hippophae rhamnoides L.) extracts on human colon and liver cancer cell lines

Sea buckthorn berries contain many bioactive compounds that have anticancer properties. To investigate whether the antiproliferative effects could be associated with the presence of certain compounds, a sequential extraction was performed. The extraction started with heptane followed by ethyl acetate, ethanol, and water. A second protocol using ethanol:water (1:1) was also used. The contents of the extracts were determined and their effects on cell proliferation were investigated in both Caco-2 and Hep G2 cells. The ethyl acetate fraction was exclusively found to contain high levels of ursolic acid, together with low amounts of phenolics. The ethanol:water extracts contained high levels of phenolic compounds and proanthyocyanidin, but little ursolic acid. When the antiproliferative effects were examined, the strongest inhibitory effect was found in the ethyl acetate extract for the Caco-2 cells and in the ethanol:water extract for the Hep G2 cells. The antiproliferative effects were in both cases dose-dependent and were in the case of the ethyl acetate extract associated with an increase in apoptosis. The results obtained show that the choice of extraction solvent is of considerable importance and that ursolic acid might be more important than the polyphenols in inhibiting the cancer cell proliferation.     

凋亡蛋白对Hep2细胞影响的研究

从鸡贫血病毒(CAV———ch icken anem ia virus)总DNA中克隆凋亡蛋白(Apoptin)基因片段,并与pcDNA3.1/CT-GFP-TOPO载体构建融合载体,转化E.coli.DH5α,筛选氨苄抗性阳性克隆并鉴定.以该融合载体转染Hep2细胞后,在倒置荧光显微镜下观察到绿色荧光标记,同时观察到细胞核形态学呈凋亡特征变化,表明凋亡蛋白可以诱导Hep2细胞系发生凋亡.     

TGF-β1对肝癌细胞系Hep3B中干性相关基因表达的影响

有研究表明TGF-β1可以诱导诸多上皮来源的癌细胞和正常细胞发生EMT并使其功能发生改变。实验就TGF-β1对肝癌细胞系Hep3B的作用展开研究:通过CCK8检测TGF-β1对Hep3B细胞增殖的影响,RT-PCR实验检测TGF-β1处理后细胞中EMT及干性相关基因的表达变化。结果表明:TGF-β1对Hep3B细胞的增殖无抑制作用;TGF-β1处理Hep3B细胞6d后EMT相关基因的mRNA表达水平并无显著改变,但TGF-β1可上调Hep3B细胞干性基因Oct-4,Klf-4,Nanog,C-myc的表达,并下调分化基因albumin的表达。结果提示TGF-β1一定程度上影响肝癌细胞系的干性基因表达,但并不一定是以发生EMT为前提的。     

Large‐scale submerged fermentation of Antrodia cinnamomea for anti‐hepatoma activity

BACKGROUND: Submerged cultivation of Antrodia cinnamomea was carried out for manufacturing the fermentation product with anti‐hepatoma activity. The fermentation process was optimized for different parameters at shake flask level to obtain products with high inhibition potency against Hep G2 hepatoma cells. Scale‐up of the fermentation process was then achieved from 250 mL shake flask to 5 L, 500 L and 5‐ton fermenters. RESULTS: Glucose and malt extract were found to be the best carbon and nitrogen sources, respectively. The initial pH of 5.0 and an operating temperature of 22 °C were the best for a product with lowest IC₅₀ value. A shorter cultivation time was required when the scale of fermentation increased from 5 L to 5 tons. The reducing sugar and solids contents of the broth filtrate were correlated exponentially with the IC₅₀ of the ethanolic extract of mycelium for hepatoma cells, and the level of ergosterol in the mycelium extract follows the same profile as the increase in Hep G2 cells inhibition. CONCLUSION: When Antrodia cinnamomea is cultured in a 5‐ton fermenter, 4 weeks are required for the fermentation product to reach the highest anti‐hepatoma activity. The solid and reducing sugar contents of the broth filtrate as well as the ergosterol content in the ethanol extract of mycelium can serve as the marker during fermentation for manufacturing product with anti‐hepatoma activity. Copyright © 2008 Society of Chemical Industry     

Flavonoids from Rabdosia rubescens exert anti-inflammatory and growth inhibitory effect against human leukemia HL-60 cells

Eight flavonoids, 5,8,4′-trihydroxy-6,7,3′-trimethoxyflavone (1), 5,4′-dihydroxy-6,7,8,3′-tetramethoxyflavone (2), nodifloretin (3), pedalitin, (4), penduletin (5), cirsiliol (6), luteolin (7), and quercetin (8), were isolated from the aerial parts of Rabdosia rubescens. Structures were established on the basis of 1D and 2D NMR and HRMS data. All flavonoids were tested for cytotoxicity in a small panel of cancer cell lines (Hep G2, COLO 205, MCF-7, and HL-60) and anti-inflammatory activity in LPS-treated RAW264.7 macrophage. Among them, compounds (1) and (4) were modestly active for inhibiting nitrite production in macrophage. Compound (2) was demonstrated to be selectively active against HL-60 cells with an IC₅₀ of 7.55 μM. This value is comparable to the IC₅₀ 4.64 μM of doxorubicin, the positive control used in this investigation.     

Cold Atmospheric Pressure Plasma-Activated Medium Induces Selective Cell Death in Human Hepatocellular Carcinoma Cells Independently of Singlet Oxygen,Hydrogen Peroxide,Nitric Oxide and Nitrite/Nitrate

Cold atmospheric pressure plasma (CAP) and plasma-activated medium (PAM) induce cell death in diverse cancer cells and may function as powerful anti-cancer agents. The main components responsible for the selective anti-cancer effects of CAP and PAM remain elusive. CAP or PAM induces selective cell death in hepatocellular carcinoma cell lines Hep3B and Huh7 containing populations with cancer stem cell markers. Here, we investigated the major component(s) of CAP and PAM for mediating the selective anti-proliferative effect on Hep3B and Huh7 cells. The anti-proliferative effect of CAP was mediated through the medium; however, the reactive oxygen species scavenger N-acetyl cysteine did not suppress PAM-induced cell death. Neither high concentrations of nitrite or nitrite/nitrate nor a low concentration of H₂O₂ present in the PAM containing sodium pyruvate affected the viability of Hep3B and Huh7 cells. Inhibitors of singlet oxygen, superoxide anions, and nitric oxide retained the capacity of PAM to induce anti-cancer effects. The anti-cancer effect was largely blocked in the PAM prepared by placing an aluminum metal mesh, but not a dielectric PVC mesh, between the plasma source and the medium. Hence, singlet oxygen, hydrogen peroxide, nitric oxide, and nitrite/nitrate are not the main factors responsible for PAM-mediated selective death in Hep3B and Huh7 cells. Other factors, such as charged particles including various ions in CAP and PAM, may induce selective anti-cancer effects in certain cancer cells.