Cytotoxic Activities of Salvia. of the Labiatae Family

ABSTRACT

Eight crude extracts of five Salvia. species were evaluated for cytotoxic activities against brine shrimps and four human cancer cell lines [human colon adenocarcinoma (HCA), HepG2, MCF-7, and human pancreatic carcinoma (HPC) along with a normal mouse cell line (areolar cells)] as a control. In the brine shrimp lethality test, all samples, except S. fruticosa. L. (Sifnos collection) and S. verbenaca. L. (Zante collection), were found to be highly active with ED₅₀ values less than 300?μg/ml. In the case of human cancer cell lines, S. fruticosa., collected from Kalymnos and Crete, were active against HCA cells with LC₅₀?=?60.4 and 40.1?μg/ml respectively. Interestingly, only one sample, S. fruticosa. collected from Kalymnos, was active against HepG2 cells with LC₅₀?=?68.1?μg/ml. In the case of MCF-7 cells, S. fruticosa. collected from three different locations (Kalymnos, Rhodos, and Crete) showed similar activity with LC₅₀?=?43.1, 41.1, and 42.3?µg/ml, respectively. All S. fruticosa. samples were found to be cytotoxic toward a normal mouse cell line when tested at 0.1?mg/ml. All the other samples had LC₅₀ values greater than 75?µg/ml,and were considered to be inactive.     

Effects of indirubin and isatin on cell viability,mutagenicity, genotoxicity and BAX/ERCC1 gene expression

Context: Indigofera suffruticosa Miller (Fabaceae) and I. truxillensis Kunth produce compounds, such as isatin (ISA) and indirubin (IRN), which possess antitumour properties. Their effects in mammalian cells are still not very well understood.

Objective: We evaluated the activities of ISA and/or IRN on cell viability and apoptosis in vitro, their genotoxic potentials in vitro and in vivo, and the IRN- and ISA-induced expression of ERCC1 or BAX genes.

Materials and methods: HeLa and/or CHO-K1 cell lines were tested (3 or 24?h) in the MTT, Trypan blue exclusion, acridine orange/ethidium bromide, cytokinesis-blocked micronucleus (CBMN) and comet (36, 24 and 72?h) tests after treatment with IRN (0.1 to 200?μM) or ISA (0.5 to 50?μM). Gene expression was measured by RT-qPCR in HeLa cells. Swiss albino mice received IRN (3, 4 or 24?h) by gavage (50, 100 and 150?mg/kg determined from the LD₅₀ – 1?g/kg b.w.) and submitted to comet assay in vivo.

Results: IRN reduced the viability of CHO-K1 (24?h; 5 to 200?μM) and HeLa cells (10 to 200?μM), and was antiproliferative in the CBMN test (CHO-K1: 0.5 to 10?μM; HeLa: 5 and 10?μM). The drug did not induce apoptosis, micronucleus neither altered gene expression. IRN and ISA were genotoxic for HeLa cells (3 and 24?h) at all doses tested. IRN (100 and 150?mg/kg) also induced genotoxicity in vivo (4?h).

Conclusion: IRN and ISA have properties that make them candidates as chemotherapeutics for further pharmacological investigations.     

Bovine leukemia virus inhibition in vitro by ribavirin

Ribavirin treatment of an established monolayer of fetal lamb kidney (FLK) cells chronically infected with bovine leukemia virus (BLV) resulted in inhibition of the extra- and intracellular internal viral polypeptide antigen, as measured by CF test, at concentrations of 10 and 32 μg/ml, respectively. Similar treatment of F-81 cells newly infected with BLV caused significant reduction in viral syncytia formation at ribavirin levels as low as 3.2 μg/ml. Attempts to eliminate or reduce the BLV infection in FLK cells by 8 passages of the cells in the continual presence of 3.2 or 1.0 μg/ml of ribavirin were unsuccessful. Multiple passages of FLK cells in the presence of higher concentrations of ribavirin substantially retarded cell growth, although short-term treatment of established cell monolayers appeared to be well tolerated as evidenced by cell appearance. Biochemical cytostatic studies of resting F-81 cell monolayers showed cytostatic effects at the same dosage levels where antiviral effects occurred.     

Synergistic anti-cancer effect of baicalein and silymarin on human hepatoma HepG2 Cells

This study investigated the effect of baicalein, silymarin, and their combination, on two human liver-derived cell lines, HepG2 (hepatocellular carcinoma) and Chang liver (non-tumor liver cells). It was found that 6.75 μg/ml baicalein or 100 μg/ml silymarin alone significantly inhibited the growth of HepG2. When baicalein was used in combination with silymarin on HepG2, an additive effect at 24 h and a synergistic effect at 48 h were observed. The viability at 48 h was 85.62% from 6.75 μg/ml baicalein treatment; but the viability reduced to 49.67%, 38.56%, and 19.61% when 25, 50, and 100 μg/ml silymarin respectively, was added to the treatment. By contrast, each treatment had little or no effect on Chang liver. Compared to treatment of baicalein or silymarin alone on HepG2, combination of both drugs synergistically increased the percentages of cells in G0/G1 phase and decreased those in S-phase, which were associated with up-regulation of Rb, p53, p21^Cip1 and p27^Kip1 and down-regulation of cyclin D1, cyclin E, CDK4 and phospho-Rb. The results indicate that the combination of baicalein and silymarin eradicates tumor cells efficiently, has minimal deleterious effects to the surrounding normal cells, and offers mechanistic insight for further exploitation of HCC treatment.     

Cytotoxicity of Fusarium mycotoxins to mammalian cell cultures as determined by the MTT bioassay.

Fusarium mycotoxins occur worldwide in cereal grains and animal feeds and cause outbreaks of Fusarium mycotoxicoses in humans and animals. In this study mammalian cell cultures were used to screen the cytotoxicity of the most common Fusarium mycotoxins; deoxynivalenol (DON), zearalenone (ZEN), fumonisin B(1) (FB(1)) and moniliformin (MON). The most sensitive cell line for each Fusarium mycotoxin was determined for further toxicological investigations as an alternative to whole animal testing. Chinese hamster ovary cells (CHO-K1) were found to be the most sensitive for DON and FB(1) with IC(50) values of 0.27 and 85.5 microg/ml, respectively, after 48-h exposure. The hepatocellular carcinoma cells (HepG2) showed the highest sensitivity to MON with IC(50) values of 39.5 for 48 h and 26.8 microg/ml for 72-h exposure. Balb/c mice keratinocyte cell line (C5-O) was found to be the most sensitive to ZEN with IC(50) of 24.1 microg/ml after 72-h exposure. DON was found the most cytotoxic to the cell cultures of all the mycotoxins tested, followed by MON, ZEN, and FB(1). The results indicated that CHO-K1, C5-O, and HepG2 cells were found to be the sensitive cell lines for preliminary screening of DON, ZEN and MON contaminated feed and food extracts, respectively.     

Evaluation of the genotoxic potential and acute oral toxicity of standardized extract of Andrographis paniculata (KalmCold™)

Andrographis paniculata is used in the traditional medicine for cold and influenza remedy. The main endeavor in this study was to assess the genotoxicity of the standardized extract of A. paniculata (KalmCold™) through three different in vitro tests: Ames, chromosome aberration (CA), and micronucleus (MN). Ames test was performed at 5000 μg/ml, 1581 μg/ml, 500 μg/ml, 158 μg/ml, 50 μg/ml, 16 μg/ml, while the clastogenicity tests were performed at 80 μg/ml, 26.6 μg/ml, 8.8 μg/ml for short-term treatment without S9; 345 μg/ml, 115 μg/ml, 38.3 μg/ml for short-term treatment with S9; and 46 μg/ml, 15.3 μg/ml, 5.1 μg/ml for long-term without S9 using DMSO as a vehicle control. Results of Ames test confirmed that KalmCold™ did not induce mutations both in the presence and absence of S9 in Salmonella typhimurium mutant strains TA98 and TAMix. In CA and MN, KalmCold™ did not induce clastogenicity in CHO-K1 cells in vitro. Based on our results, it is evident that KalmCold™ is genotoxically safe.     

Oxidative stress contributes to gold nanoparticle-induced cytotoxicity in human tumor cells

Due to their exceptional properties, gold nanoparticles (AuNPs) have shown promising medical and technological applications in the treatment of cancer and the development of antimicrobial packaging and time–temperature indicators in the food sector. However, little is known about their cytotoxicity when they come into contact with biological systems. The aim of this work was to compare the effects of three commercially available AuNPs of different sizes (30, 50 and 90?nm) on human leukemia (HL-60) and hepatoma (HepG2) cell lines. AuNP-induced cytotoxicity was dose and time-dependent, with IC₅₀ values higher than 15?μg/mL. Nanoparticle (NP) size and cell line slightly influenced on the cytotoxicity of AuNPs, although HL-60 cells proved to be more sensitive to the cytotoxic response than HepG2. N-Acetyl-l-cysteine (NAC) protected HL-60 and HepG2 cells only against treatment with 30?nm AuNPs. In both cell types, glutathione (GSH) content was drastically depleted after 72?h of incubation with the three AuNPs (less than 30% in all cases), while the reduction of superoxide dismutase activity (SOD) activity depended on cell line. HepG2, but not HL-60 cells, exhibited a decrease of SOD activity (～45% of activity). The three AuNPs also caused a two-fold elevation of reactive oxygen species (ROS) production in both cell lines. Thus, protective effect of NAC, depletion of GSH and increase of ROS appear to be determined by NP size and indicate that oxidative stress contributes to cytotoxicity of AuNPs.     

Comparative analysis of phase I and II enzyme activities in 5 hepatic cell lines identifies Huh-7 and HCC-T cells with the highest potential to study drug metabolism

Primary human hepatocytes (hHeps) are still gold standard to perform human drug metabolism studies, but their availability is limited by donor organ scarcity. Therefore, hepatoma cell lines are widely used as alternatives, although their phases I and II drug-metabolizing activities are substantially lower compared with hHeps. The major advantage of these cell lines is immediate availability, standardized culture conditions and unlimited life span. Therefore, the aim of this study was to investigate the drug-metabolizing profile of five human hepatoma cell lines (HepG2, Hep3B, HCC-T, HCC-M and Huh-7) over a culture period of 10 passages. Fluorescent-based assays for seven different cytochrome P450 (CYP) isoforms and seven different phase II enzymes were performed and compared with enzymatic activities of hHeps. CYP activities were much lower in the cell lines (5?C15% of hHeps), whereas phase II enzyme activities that are involved in buffering oxidative stress (e.g., Glutathione-S-transferase) reached levels comparable with hHeps. Furthermore, phases I and II enzyme activities in hepatoma cell lines vary strongly during culture time. Interestingly, the most constant results were obtained with Huh-7 cells. Huh-7 cells as well as HCC-T cells exhibited a drug-metabolizing profile closest to hHeps between passages two and four. Toxicity studies with Diclofenac, Paracetamol and Verapamil in both cell lines show dose?Cresponse characteristics and EC₅₀ values similar to hHeps. Therefore, we propose that due to the more consistent results throughout the passages, Huh-7 could be an alternative system to the limitedly available hHeps and frequently used HepG2 cell line in the study of drug metabolism.     

Induction of apoptosis of malignant gliomas cells by a prenylated chalcone

Context: Malignant gliomas are the most commonly diagnosed brain tumors in adults. Chalcone and its derivatives have shown potential against glioblastoma and malignant gliomas.

Objective: The inhibitory activity of geranyl prenylated chalcone was investigated in four glioma cell lines: C6, U87?MB, CNS-1 and 13-06?MB. Cell death caused by the prenylated chalcone was determined to be necrosis or apoptosis.

Materials and methods: The inhibitory activity of geranyl prenylated chalcone with 5, 10, 15, 20, 25, 30 and 40?μg/ml (treatment time: 24, 48 and 72?h) was investigated in C6, U87 MB, CNS-1 and 13-06?MB. Cell cycle distribution, DNA fragmentation, chromatin condensation and protein expression were used as indicators of apoptosis. The migration ability of glioma cells with 30?μg/ml prenylated chalcone after 24 and 36?h incubation was also studied by the scratch wound assay.

Results: After 24?h, treatment with 20?μg/ml prenylated chalcone reduced the proliferation (approximately 50%) of all four glioma cell lines (half maximal inhibitory concentration (IC₅₀)?=?20?μg/ml). Glioma cell death was verified by the fluorescence-activated cell sorter as prenylated chalcone-induced apoptosis. After running the analysis of protein expression, apoptotic activity induced by the prenylated chalcone was caspase independent for the C6 and U87?MB cell lines, but caspase dependent for the 13-06?MB and CNS-1 cell lines. In addition, prenylated chalcone treatment (30?μg/ml) resulted in the inhibition of glioma cell migration after 24 and 36?h treatment.

Discussion and conclusion: Because prenylated chalcone-induced apoptosis inhibited the proliferation and reduced the invasiveness of glioma cells, the prenylated chalcone has potential as a new chemotherapeutic reagent in the treatment of malignant gliomas. The ultimate goal was to develop a novel potential multi-therapy for treating gliomas.     

表观遗传学药物FK228治疗肝癌的实验研究

目的观察表观遗传学药物FK228对人肝癌细胞HepG2的体内外抑制作用,并初步探讨其机制。方法采用不同浓度的FK228和氟尿嘧啶(5-FU)处理HepG2细胞48 h,采用CCK-8法观察比较FK228对人肝癌细胞HepG2的生长抑制作用。将5μg/L FK228和25μg/ml 5-FU分别单独或联合作用于2种肝癌细胞48 h,使用流式细胞仪检测细胞凋亡率。体内实验构建BALB/c裸鼠皮下移植瘤模型,通过腹腔注射FK228 (1 mg/kg)和5-FU (20 mg/kg),比较FK228对HepG2裸鼠移植瘤生长抑制作用。结果 FK228对人肝癌细胞HepG2具有明显的生长抑制作用,且呈剂量依赖性,其48 h的半数抑制浓度(IC50)为(4.20±0.24)μg/L,而5-FU的IC50值为(117.50±8.40)μg/ml;FK228联合5-FU处理组与相应浓度的单药组相比,联合用药组的细胞存活率明显降低(P<0.05)。流式细胞仪检测结果显示,FK228可以显著诱导肝癌细胞HepG2凋亡,且联合用药后细胞凋亡率明显增加(P<0.05)。给药20 d后,FK228组、5-FU组、联合给药组及对照组的肿瘤体积分别为(344.71±118.87)、(351.97±73.46)、(220.36±72.12)、(639.76±241.47)mm3。FK228组、5-FU组、联合给药组的裸鼠肿瘤体积均小于对照组(P<0.05)。免疫组化结果显示,与对照组相比,FK228对肝癌细胞的体内促凋亡作用并不明显(P>0.05),但与5-FU联合应用后,促凋亡作用明显增强(P<0.05);与对照组和5-FU组相比,FK228可以明显抑制肿瘤内的血管生成(P<0.05)。结论 FK228对人肝癌细胞HepG2具有明显的体内外生长抑制作用,且与5-FU联合应用后,抑制作用更加明显。FK228与5-FU联合应用,可以明显促进HepG2肝癌细胞的凋亡和抑制肿瘤的血管生成。     

晚期糖基化终产物对人肝癌细胞HepG2增殖的影响

目的探讨糖尿病晚期糖基化终产物(AGEs)对肝癌细胞HepG2增殖的影响及其机制。方法体外培养人肝癌细胞HepG2,以终浓度分别为100、200和400μg/ml的AGEs处理细胞24h,并设正常对照组进行比较。运用细胞计数试剂盒8研究AGEs对HepG2增殖的影响,流式细胞术检测细胞周期的改变,Western blot检测肝癌细胞抗凋亡基因表达。结果 AGEs呈浓度依赖性显著促进HepG2细胞增殖(P<0.05)。与对照组比较,200μg/ml AGEs干预24h后可以减少HepG2细胞G1期百分率,同时增加S期百分率(P<0.05)。AGEs可致细胞抗凋亡基因B细胞淋巴瘤-白血病2(Bcl-2)相关蛋白表达增加。结论 AGEs能促进HepG2细胞的增殖,其机制可能与上调Bcl-2相关蛋白表达,加速细胞G1期向S期转换相关。     

Upgrading HepG2 cells with adenoviral vectors that encode drug-metabolizing enzymes: application for drug hepatotoxicity testing

Introduction: Drug attrition rates due to hepatotoxicity are an important safety issue considered in drug development. The HepG2 hepatoma cell line is currently being used for drug-induced hepatotoxicity evaluations, but its expression of drug-metabolizing enzymes is poor compared with hepatocytes. Different approaches have been proposed to upgrade HepG2 cells for more reliable drug-induced liver injury predictions.

Areas covered: We describe the advantages and limitations of HepG2 cells transduced with adenoviral vectors that encode drug-metabolizing enzymes for safety risk assessments of bioactivable compounds. Adenoviral transduction facilitates efficient and controlled delivery of multiple drug-metabolizing activities to HepG2 cells at comparable levels to primary human hepatocytes by generating an ‘artificial hepatocyte’. Furthermore, adenoviral transduction enables the design of tailored cells expressing particular metabolic capacities.

Expert opinion: Upgraded HepG2 cells that recreate known inter-individual variations in hepatic CYP and conjugating activities due to both genetic (e.g., polymorphisms) or environmental (e.g., induction, inhibition) factors seems a suitable model to identify bioactivable drug and conduct hepatotoxicity risk assessments. This strategy should enable the generation of customized cells by reproducing human pheno- and genotypic CYP variability to represent a valuable human hepatic cell model to develop new safer drugs and to improve existing predictive toxicity assays.     

Multi-platform genotoxicity analysis of silver nanoparticles in the model cell line CHO-K1

Investigation of the genotoxic potential of nanomaterials is essential to evaluate if they pose a cancer risk for exposed workers and consumers. The Chinese hamster ovary cell line CHO-K1 is recommended by the OECD for use in the micronucleus assay and is commonly used for genotoxicity testing. However, studies investigating if this cell line is suitable for the genotoxic evaluation of nanomaterials, including induction of DNA adduct and micronuclei formation, are rare and for silver nanoparticles (Ag NPs) missing. Therefore, we here systematically investigated DNA and chromosomal damage induced by BSA coated Ag NPs (15.9 ± 7.6 nm) in CHO-K1 cells in relation to cellular uptake and intracellular localization, their effects on mitochondrial activity and production of reactive oxygen species (ROS), cell cycle, apoptosis and necrosis. Ag NPs are taken up by CHO-K1 cells and are presumably translocated into endosomes/lysosomes. Our cytotoxicity studies demonstrated a concentration-dependent decrease of mitochondrial activity and increase of intracellular reactive oxygen species (ROS) in CHO-K1 cells following exposure to Ag NPs and Ag⁺ (0–20 μg/ml) for 24 h. Annexin V/propidium iodide assay showed that Ag NPs and Ag⁺ induced apoptosis and necrosis, which is in agreement with an increased fraction of cells in subG1 phase of the cell cycle. Genotoxicity studies showed that Ag NPs but also silver ions (Ag⁺) induced bulky-DNA adducts, 8-oxodG and micronuclei formation in a concentration-dependent manner, however, there were quantitative and qualitative differences between the particulate and ionic form of silver. Taken together, our multi-platform genotoxicity and cytotoxicity analysis demonstrates that CHO-K1 cells are suitable for the investigation of genotoxicity of nanoparticles like Ag NPs.     

In vivo study of effects of artesunate nanoliposomes on human hepatocellular carcinoma xenografts in nude mice

Abstract

To investigate the effect of artesunate nanoliposomes on cultured cells in vitro and hepatocellular carcinoma xenografts in BALB/c-nu mice. Fluorescence polarization was applied for measurement of mitochondrial membrane fluidities; inhibition test of tumor cell proliferation in vitro was performed and nude mice xenograft model from human hepatocellular carcinoma (HCC) was established. Cytotoxicity of these compounds was evaluated by MTT assay on hepatocellular carcinoma xenografts in nude mice. Anisotropy (r-value) of blank nanoliposomes didn’t change, it had no statistically significance between the blank nanoliposomes group and the control group, it indicated that artesunate had no obvious effect on L-O2 human normal liver cells. IC₅₀ values of artesunate nanoliposomes and artesunate API (active pharmaceutical ingredient) against HepG-2 cells were 15.997 and 19.706?μg/ml; IC₅₀ values of the same drugs against L-O2 normal human liver cells were 100.23 and 105.54?μg/ml, respectively. Tumor growth inhibitory effect of artesunate nanoliposomes was 32.7%, and artesunate API was 20.5%, respectively. HepG-2 cells treated with artesunate nanoliposomes showed dose-dependent apoptosis. The antitumor effect of artesunate nanoliposomes on human hepatoma HepG2 cells were stronger than that of artesunate API at the same concentration.     

Error-prone replication of West Nile virus caused by ribavirin

Ribavirin has been reported to cause error-prone replication and viral extinction in RNA viruses. The antiviral activity of ribavirin against West Nile virus (WNV) was evaluated in various cell lines to select a model in which mutagenic effects could be studied. The antiviral activity was greatest in HeLa cells as compared to CV-1, L929, Vero, or MA-104 cells. WNV was also passaged sequentially in cell monolayers treated with ribavirin to determine whether cumulative mutations could lead to viral extinction in these cell lines. The virus was abrogated in HeLa cells after 4 passages, while high viral titers persisted after many passages in other cells. A molecular clone of WNV was propagated in HeLa cells treated with 15 microg/mL ribavirin, and sequencing of viral genome segments revealed significant increases in transition mutations, demonstrating that ribavirin induced error-prone replication. The relative infectivity of viral RNA synthesized in the presence of ribavirin was shown to be reduced compared with untreated controls. These data support the hypothesis that error catastrophe is one of the modes of action for ribavirin against WNV.     

Osthole induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells

Context: Osthole [7-methoxy-8-(3-methyl-2-butenyl) coumarin] isolated from the fruit of Cnidium monnieri (L.) Cuss, one of the commonly used Chinese medicines listed in the Shennong’s Classic of Materia Medica in the Han Dynasty, had remarkable antiproliferative activity against human hepatocellular carcinoma HepG2 cells in culture.

Objectives: This study evaluated the effects of osthole on cell growth, nuclear morphology, cell cycle distribution, and expression of apoptosis-related proteins in HepG2 cells.

Materials and methods: Cytotoxic activity of osthole was determined by the MTT assay at various concentrations ranging from 0.004 to 1.0?µmol/ml in HepG2 cells. Cell morphology was assessed by Hoechst staining and fluorescence microscopy. Apoptosis and cell-cycle distribution was determined by annexin V staining and flow cytometry. Apoptotic protein levels were assessed by Western blot.

Results: Osthole exhibited significant inhibition of the survival of HepG2 cells and the half inhibitory concentration (IC₅₀) values were 0.186, 0.158 and 0.123?µmol/ml at 24, 48 and 72?h, respectively. Cells treated with osthole at concentrations of 0, 0.004, 0.02, 0.1 and 0.5?μmol/ml showed a statistically significant increase in the G2/M fraction accompanied by a decrease in the G0/G1 fraction. The increase of apoptosis induced by osthole was correlated with down-regulation expression of anti-apoptotic Bcl-2 protein and up-regulation expression of pro-apoptotic Bax and p53 proteins.

Conclusion: Osthole had significant growth inhibitory activity and the pro-apoptotic effect of osthole is mediated through the activation of caspases and mitochondria in HepG2 cells. Results suggest that osthole has promising therapeutic potential against hepatocellular carcinoma.     

Cytotoxic effects of Eryngium kotschyi and Eryngium maritimum on Hep2, HepG2, Vero and U138 MG cell lines

Abstract

Context: Eryngium maritimum L. and the endemic Eryngium kotschyi Boiss. of the Apiaceae family are used for antiinflammatory, antivenom, antinociceptive and diuretic purposes in folk medicine in Turkey.

Objective: This study investigated the cytotoxic effects of the plant extracts belonging to Eryngium L. genus on various cell lines.

Materials and methods: Cytotoxic activites of the lyophilized aqueous aereal and root parts of the plant extracts on human hepatocellular carcinoma (HepG2), human laryngeal epidermoid carcinoma (Hep2), human glioma (U138-MG) and African green monkey kidney epithelial (Vero) cell lines at 8.33–266.62?µg/ml concentrations were analyzed by lactate dehydrogenase (LDH) leakage and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) cell viability assays.

Results: Inhibitory concentration 50 (IC₅₀) values were found <100?µg/ml in most cases varying around 16.33–125.66?µg/ml. IC₅₀ values for E. kostchyi and E. maritimum root parts on Hep2 cells (32.86 and 30.25?µg/ml, respectively), E. kotschyi aereal, E. maritimum aereal and root parts on HepG2 cells (31.75, 32.42 and 35.01?µg/ml, respectively) by MTT assay were found to be close to the US National Cancer Institute (NCI) recommendations (IC₅₀?<?30?µg/ml) to define the antivity aganist cancer cells. The lowest IC₅₀ values according to the LDH method were observed in Hep2 cells and the highest in U138-MG cells. Root parts were found to be more toxic than aereal parts for both plants in both methods in general.

Discussion and conclusion: Both plant extracts exerted cytotoxic activity aganist Hep2 and HepG2 cells, with low IC₅₀ values defining their promising anticancer property according to NCI; however, further analysis are needed to confirm their activity.     

Hepatoprotective effect of ganoderma triterpenoids against oxidative damage induced by tert-butyl hydroperoxide in human hepatic HepG2 cells

Context: Ganoderma triterpenoids (GTs) have been recognised as an important bioactive ingredient in Ganoderma Lucidum (Leyss. ex Fr.) Karst. (Polyporaceae), widely used for treating and preventing chronic hepatopathy of various etiologies.

Objective: The objective of this study is to better understand the hepatoprotective effect of GTs and to enhance their use in food supplement pharmaceutical and medical industries.

Materials and methods: HepG2 cells were pretreated in the presence or absence of GTs (50, 100 and 200?μg/ml) for 4?h, then exposed with 60?μmol/L of t-BHP for an additional 4?h. The cell viability was evaluated by MTT method. ALT, AST and LDH production in culture medium and intracellular MDA, GSH and SOD levels were determined. Moreover, the total triterpenoid content and chemical constituents in GTs were detected by ultraviolet spectrophotometry and HPLC/Q-TOF-MS, respectively.

Results: GTs (50, 100 and 200?μg/ml) significantly increased the relative cell viability by 4.66, 7.78 and 13.46%, respectively, and reduced the level of ALT by 11.44%, 33.41% and 51.24%, AST by 10.05%, 15.63% and 33.64%, and LDH by 16.03%, 23.4% and 24.07% in culture medium, respectively. GTs could also remarkably decrease the level of MDA and increase the content of GSH and SOD in HepG2 cells. Furthermore, the total triterpenoid content in GTs was 438?mg GAAEs/g GTs. And 16 triterpenoids in GTs were identified or tentatively characterised.

Discussion and conclusion: Our results showed that GTs had potent cytoprotective effect against oxidative damage induced by t-BHP in HepG2 cells, thus suggesting their potential use as liver protectant.     

Anticancer activity of a low immunogenic protein toxin (BMP1) from Indian toad (Bufo melanostictus, Schneider) skin extract

Earlier, a protein (BMP1, MW-79kDa) had been isolated from Indian toad (Bufo melanostictus) skin aqueous extract possessed anticancer activity against EAC bearing mice (Bhattacharjee et al., 2011). In the present study, the anti-proliferative and apoptogenic activities of BMP1 have been evaluated in leukemic (U937 and K562) and hepatoma (HepG2) cells. BMP1 dose dependently inhibited U937 and K562 cell growth having IC₅₀ values of 49 μg/ml and 30 μg/ml respectively. The anti-proliferative activity of BMP1 was observed in MTT assay, proliferating cell nuclear antigen (PCNA) expression and cell cycle arrest study. Flow-cytometric data revealed that BMP1 arrested cell cycle in U937 and K562 cells at Sub-G1 and G1 phases. The BMP1-induced dose dependent expressions of CDKIs (p21^cip1 and p27^kip1) and inhibition of CDK2 and PCNA expression in HepG2 cells support the inhibition of cell proliferation due to G1 arrest. BMP1-induced apoptosis analyzed by annexin-V binding study and the DNA fragmentation by comet assay were correlated with the sub-G1 arrest. The parallel induction of bax and p53 expression in HepG2 cells and the up-regulation of caspase 3 and caspase 9 due to BMP1 treatment indicated the involvement of p53-dependent intrinsic pathway of apoptosis. BMP1 was found to be low immunogenic in nature.