Computational determination of hERG-related cardiotoxicity of drug candidates.

BACKGROUND: Drug candidates often cause an unwanted blockage of the potassium ion channel of the human ether-a-go-go-related gene (hERG). The blockage leads to long QT syndrome (LQTS), which is a severe life-threatening cardiac side effect. Therefore, a virtual screening method to predict drug-induced hERG-related cardiotoxicity could facilitate drug discovery by filtering out toxic drug candidates. RESULT: In this study, we generated a reliable hERG-related cardiotoxicity dataset composed of 2130 compounds, which were carried out under constant conditions. Based on our dataset, we developed a computational hERG-related cardiotoxicity prediction model. The neural network model achieved an area under the receiver operating characteristic curve (AUC) of 0.764, with an accuracy of 90.1%, a Matthews correlation coefficient (MCC) of 0.368, a sensitivity of 0.321, and a specificity of 0.967, when ten-fold cross-validation was performed. The model was further evaluated using ten drug compounds tested on guinea pigs and showed an accuracy of 80.0%, an MCC of 0.655, a sensitivity of 0.600, and a specificity of 1.000, which were better than the performances of existing hERG-toxicity prediction models. CONCLUSION: The neural network model can predict hERG-related cardiotoxicity of chemical compounds with a high accuracy. Therefore, the model can be applied to virtual high-throughput screening for drug candidates that do not cause cardiotoxicity. The prediction tool is available as a web-tool at http://ssbio.cau.ac.kr/CardPred .

Anti-Adipogenic Effects on 3T3-L1 Cells and Zebrafish by Tanshinone IIA.

Tanshinone IIA is a diterpene quinone isolated from the roots of Salviamiltiorrhiza bunge that has traditionally been used in China for the treatment of cardiovascular and cerebrovascular disorders. Although there is recent evidence showing that tanshinone IIA has an anti-obesity effect, its underlying mechanism of anti-obesity effect is poorly understood. Here, we investigated the effect of tanshinone IIA on lipid accumulation in 3T3-L1 preadipocytes and zebrafish. Notably, tanshinone IIA at 10 µM concentration greatly reduced lipid accumulation and triglyceride (TG) contents during 3T3-L1 preadipocyte differentiation, suggesting its anti-adipogenic effect. On mechanistic levels, tanshinone IIA reduced the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), and perilipin A but also the phosphorylation levels of signal transducer and activator of transcription-3/5 (STAT-3/5) in differentiating 3T3-L1 cells. In addition, tanshinone IIA strongly inhibited leptin and resistin mRNA expression in differentiating 3T3-L1 cells. Importantly, the tanshinone IIA's lipid-reducing effect was also seen in zebrafish. In sum, these findings demonstrate that tanshinone IIA has anti-adipogenic effects on 3T3-L1 cells and zebrafish, and its anti-adipogenic effect on 3T3-L1 cells is largely attributable to the reduced expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, perilipin A, and STAT-3/5.

Transport characteristics and transporter-based drug-drug interactions of TM-25659, a novel TAZ modulator.

The in vitro metabolic stability and transport mechanism of TM-25659, a novel TAZ modulator, was investigated in human hepatocytes and human liver microsomes (HLMs) based on the preferred hepatobiliary elimination in rats. In addition, the in vitro transport mechanism and transporter-mediated drug-drug interactions were evaluated using oocytes and MDCKII cells overexpressing clinically important drug transporters. After a 1 h incubation in HLMs, 92.9 ± 9.5% and 95.5 ± 11.6% of the initial TM-25659 remained in the presence of NADPH and UDPGA, respectively. Uptake of TM-25659 readily accumulated in human hepatocytes at 37 ºC (i.e. 6.7-fold greater than that at 4 ºC), in which drug transporters such as OATP1B1 and OATP1B3 were involved. TM-25659 had a significantly greater basal to apical transport rate (5.9-fold) than apical to basal transport rate in the Caco-2 cell monolayer, suggesting the involvement of an efflux transport system. Further studies using inhibitors of efflux transporters and overexpressing cells revealed that MRP2 was involved in the transport of TM-25659. These results, taken together, suggested that TM-25659 can be actively influxed into hepatocytes and undergo biliary excretion without substantial metabolism. Additionally, TM-25659 inhibited the transport activities of OATP1B1 and OATP1B3 with IC50 values of 36.3 and 25.9 µm, respectively. TM-25659 (100 µm) increased the accumulation of the probe substrate by 160% and 213%, respectively, through the inhibition of efflux function of P-gp and MRP2. In conclusion, OATP1B1, OATP1B3, P-gp and MRP2 might be major transporters responsible for the pharmacokinetics and drug-drug interaction of TM-25659, although their contribution to in vivo pharmacokinetics needs to be further investigated.

In vitro metabolism and transport of the new dipeptidyl peptidase 4 inhibitors, KR66222 and KR66223.

We investigated the in vitro metabolism and transport of KR66222 and KR66223, new inhibitors of dipeptidyl peptidase (DPP) 4, using human liver microsomes (HLMs) and a Caco-2 cell monolayer. Human liver microsomal incubation of KR66222 in the presence of the NADPH-generating system resulted in the formation of two metabolites, identified as S-oxidation (KR68334) and hydrolysis (KR66223) products using liquid chromatography/tandem mass spectrometry. The formation of KR66223 via an esterase and the formation of KR68334 via CYP3A5 and CYP3A4 seem to be major factors in the in vitro metabolism of KR66222 using HLMs. Additionally, KR66222 had a significantly greater basal to apical transport rate (2.5-fold) than apical to basal transport in the Caco-2 cell monolayer, suggesting the involvement of an efflux transport system. Further studies using inhibitors of efflux transporters and P-glycoprotein (P-gp) overexpressed cells revealed that P-gp was involved in the basal to apical transport of KR66222. These findings suggest that KR66222 undergoes a significant first pass effect, which may serve to decrease the bioavailability of KR66222. The active metabolite, KR66223, was stable for 1 h at 37°C in pooled HLMs (98.9 ± 2.6% of control) and did not undergo P-gp-mediated efflux in Caco-2 cells. Apparent permeability of KR66223 (4.96 × 10(-6) cm/s) was comparable to that of KR66222 (4.08 × 10(-6) cm/s). In conclusion, considering pharmacokinetic variability and the intestinal first-pass effect caused by the involvement of CYP3A and P-gp, KR66223 seems to have better in vitro metabolism and permeability characteristics than KR66222.

Inhibitory effects of ketoconazole and rifampin on OAT1 and OATP1B1 transport activities: considerations on drug-drug interactions.

Ketoconazole and rifampin are the most widely used compounds examined in recent drug-drug interaction (DDI) studies, and they have multiple roles in modulating drug metabolizing enzymes and transporters. To determine the underlying mechanisms of DDI, this study was performed to investigate the inhibitory effects of ketoconazole and rifampin on the functions of OAT1 and OATP1B1, and to evaluate the potential of ketoconazole and rifampin for DDI with substrate drugs for these transporters in a clinical setting. Ketoconazole inhibited OATP1B1-mediated transport activity, while rifampin inhibited OAT1 and OATP1B1. Inhibition by rifampin and ketoconazole of the uptake of olmesartan, a substrate for OAT1 and OATP1B1, was evaluated in oocytes overexpressing these transporters. The K(i) values for rifampin on OAT1 and OATP1B1-mediated olmesartan uptake were 62.2 and 4.42 µM, respectively, and the K(i) value for ketoconazole on OATP1B1-mediated olmesartan uptake was 66.1 µM. As measured plasma concentrations of rifampin and ketoconazole were 7.29 and 6.4-13.3 µM, respectively, the likelihood of an OATP1B1-mediated drug-drug interaction between rifampin and olmesartan is thought to be possible, whereas OAT1 or OATP1B1-mediated DDI between rifampin or ketoconazole and olmesartan appears unlikely in the clinical setting.

Sitagliptin attenuates metformin-mediated AMPK phosphorylation through inhibition of organic cation transporters.

To assess potential interactions between sitagliptin and metformin, we sought to characterize the in vitro inhibitory potency of sitagliptin on the uptake of MPP(+) and metformin, representative substrates for OCTs, and to evaluate the pharmacological pathways that may be affected by the combination of metformin and sitagliptin. Among the OATs and OCTs screened, OAT3-mediated salicylate uptake and OCT1- and OCT2-mediated MPP(+) uptake were inhibited by sitagliptin. The K(i) values of sitagliptin for OCT1- and OCT2-mediated metformin uptake were 34.9 and 40.8 µM, respectively. As OCT1 is the gate protein for metformin action in the liver, we investigated whether sitagliptin-mediated OCT1 inhibition affected metformin-induced activation of AMPK signalling. Treatment with sitagliptin in MDCK-OCT1 and HepG2 cells resulted in a reduced level of phosphorylated AMPK, with K(i) values of 38.8 and 43.3 µM, respectively. These results suggest that the inhibitory potential of sitagliptin on OCT1 may attenuate the first step of metformin action, that is, the phosphorylation of AMPK. Nevertheless, the likelihood of a drug-drug interaction between sitagliptin and metformin is believed to be remote in usual clinical setting.

Potential Moracin M Prodrugs Strongly Attenuate Airway Inflammation In Vivo.

This study aims to develop new potential therapeutic moracin M prodrugs acting on lung inflammatory disorders. Potential moracin M prodrugs (KW01-KW07) were chemically synthesized to obtain potent orally active derivatives, and their pharmacological activities against lung inflammation were, for the first time, examined in vivo using lipopolysaccharide (LPS)-induced acute lung injury model. In addition, the metabolism of KW02 was also investigated using microsomal stability test and pharmacokinetic study in rats. When orally administered, some of these compounds (30 mg/kg) showed higher inhibitory action against LPSinduced lung inflammation in mice compared to moracin M. Of them, 2-(3,5-bis((dimethylcarbamoyl)oxy)phenyl)benzofuran-6-yl acetate (KW02) showed potent and dose-dependent inhibitory effect on the same animal model of lung inflammation at 1, 3, and 10 mg/kg. This compound at 10 mg/kg also significantly reduced IL-1ß concentration in the bronchoalveolar lavage fluid of the inflamed-lungs. KW02 was rapidly metabolized to 5-(6-hydroxybenzofuran-2-yl)-1,3-phenylene bis(dimethylcarbamate) (KW06) and moracin M when it was incubated with rat serum and liver microsome as expected. When KW02 was administered to rats via intravenous or oral route, KW06 was detected in the serum as a metabolite. Thus, it is concluded that KW02 has potent inhibitory action against LPS-induced lung inflammation. It could behave as a potential prodrug of moracin M to effectively treat lung inflammatory disorders.

A novel PPARgamma agonist, KR62776, suppresses RANKL-induced osteoclast differentiation and activity by inhibiting MAP kinase pathways.

We investigated the effects of a novel peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, KR62776, on osteoclast differentiation and function, and on the underlying signaling pathways. KR62776 markedly suppressed differentiation into osteoclasts in various osteoclast model systems, including bone marrow mononuclear (BMM) cells and a co-culture of calvarial osteoblasts and BMM cells. KR62776 suppressed the activation of tartrate-resistant acid phosphatase (TRAP) and the expression of genes associated with osteoclast differentiation, such as TRAP, dendritic cell-specific transmembrane protein (DC-STAMP), and osteoclast-associated receptor (OSCAR). Furthermore, KR62776 reduced resorption pit formation in osteoclasts, and down-regulated genes essential for osteoclast activity, such as Src and alphavbeta3 integrin. An analysis of a signaling pathway showed that KR62776 inhibited the receptor activator of nuclear factor-kappaB ligand (RANKL)-induced activation of p38 mitogen-activated protein kinase (p38MAPK), extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and nuclear factor-kappaB (NF-kappaB). Together, these results demonstrate that KR62776 negatively affects osteoclast differentiation and activity by inhibiting the RANKL-induced activation of MAP kinases and NF-kappaB.

Association of IL-15 polymorphisms with bone mineral density in postmenopausal Korean women.

Interleukin-15 (IL-15) has been suggested to participate in bone metabolism by stimulating osteoclast differentiation and mediating inflammatory bone loss. This study investigated the effect of IL-15 gene polymorphisms on the bone mineral density (BMD) and bone fracture rates of postmenopausal women. Sequencing of the IL-15 gene in 24 Koreans revealed 16 single-nucleotide polymorphisms (SNPs), of which five were selected for further study. Postmenopausal Korean women (n = 844) were genotyped for these SNPs, and their BMDs and risk of fractures were assessed. It was found that the +20A > G, +13467C > A, +13653A > T, and +13815A > T IL-15 gene polymorphisms were significantly associated with the BMD of the lumbar spine and femoral neck and that their effects were gene-dose dependent. BMD was reduced when the minor allele of +13467A and +13653T or the common allele of +20A and +13815A was present. Haplotype (ht) analyses revealed that ht1 (GCAT) and ht2 (AATA) were associated with BMD of the lumbar spine and femoral neck. However, there was no association between the risk of fracture and IL-15 SNPs or hts. These results suggest that the +20A > G, +13467C > A, +13653A > T, and +13815A > T SNPs in the IL-15 gene affect BMD and, thus, could be genetic markers of osteoporosis.

Synthesis of aristolactam analogues and evaluation of their antitumor activity.

A series of natural aristolactams and their analogues have been prepared and evaluated for antitumor activity against human cancer cells, including multi-drug resistant cell lines. Naturally occurring aristolactams, such as aristolactam BII (cepharanone B), aristolactam BIII, aristolactam FI (piperolactam A), N-methyl piperolactam A, and sauristolactam showed moderate antitumor activities in selected cell lines. However, several synthetic aristolactam derivatives exhibited potent antitumor activities against a broad array of cancer cell lines with GI(50) values in the submicromolar range.

Cuban Sugar Cane Wax Acid and Policosanol Showed Similar Atheroprotective Effects with Inhibition of LDL Oxidation and Cholesteryl Ester Transfer via Enhancement of High-Density Lipoproteins Functionality.

BACKGROUND: Cuban sugarcane wax acids (SCWA) and policosanol (PCO) are mixtures of higher aliphatic acids and alcohols, respectively, purified from sugarcane wax with different chief components. Although it has been known that they have antioxidant and anti-inflammatory activities, physiological properties on molecular mechanism of SCWA have been less studied than PCO. METHODS: In this study, we compared antiatherogenic activities of SCWA and PCO via encapsulation with reconstituted high-density lipoproteins (rHDL). RESULTS: After reconstitution, SCWA-rHDL showed smaller particle size than PCO-rHDL with increase of content. PCO-rHDL or SCWA-rHDL showed distinct inhibition of glycation with similar extent in the presence of fructose. PCO-rHDL or SCWA-rHDL showed strong antioxidant activity against cupric ion-mediated oxidation of low-density lipoproteins (LDL), and inhibition of oxLDL uptake into macrophages. Although PCO-rHDL showed 1.2-fold stronger inhibition against cholesteryl ester transfer protein (CETP) activity than SCWA-rHDL, SCWA-rHDL enhanced 15% more brain cell (BV-2) growth and 23% more regeneration of tail fin in zebrafish. CONCLUSION: PCO and SCWA both enhance the beneficial functions of HDL to maximize its antioxidant, antiglycation, and antiatherosclerotic activities and the inhibition of CETP. These enhancements of HDL functionality by PCO and SCWA could exert antiaging and rejuvenation activity.

Short-Term Consumption of Cuban Policosanol Lowers Aortic and Peripheral Blood Pressure and Ameliorates Serum Lipid Parameters in Healthy Korean Participants: Randomized, Double-Blinded, and Placebo-Controlled Study.

The current study was designed to investigate the short-term effects of policosanol consumption on blood pressure (BP) and the lipid parameters in healthy Korean participants with prehypertension. A total of 84 healthy participants were randomly allocated to three groups receiving placebo, 10 mg of policosanol, or 20 mg of policosanol for 12 weeks. Based on an average of three measurements of peripheral BP, the policosanol 20 mg group exhibited the most significant reduction, that is, up to 7.7% reduction of average systolic BP (SBP) from 136.3 ± 6.1 mmHg (week 0) to 125.9 ± 8.6 mmHg (week 12, p < 0.001). Between group comparisons using repeated measures ANOVA showed that the policosanol 20 mg group had a significant reduction of SBP at 12 weeks (p = 0.020) and a reduction of diastolic BP (DBP) at 8 weeks (p = 0.041) and 12 weeks (p = 0.035). The policosanol 10 mg and 20 mg groups showed significant reductions in aortic SBP of 7.4% and 8.3%, respectively. The policosanol groups showed significant reductions of total cholesterol (TC) of 9.6% and 8.6% and low-density lipoproteins (LDL-C) of 21% and 18% for 10 mg and 20 mg of policosanol, respectively. Between group comparisons using repeated measures ANOVA showed that the policosanol (10 mg and 20 mg) groups at 12 weeks had a significant reduction of TC (p = 0.0004 and p = 0.001) and LDL-C (p = 0.00005 and p = 0.0001) and elevation of %HDL-C (p = 0.048 and p = 0.014). In conclusion, 12-week consumption of policosanol resulted in significant reductions of peripheral SBP and DBP, aortic SBP and DBP, mean arterial pressure (MAP), and serum TC and LDL-C with elevation of % HDL-C.

Effects of Synergistic Inhibition on α-glucosidase by Phytoalexins in Soybeans.

To determine the mechanism of action of the effects of phytoalexins in soybeans, we analyzed α-glucosidase inhibition kinetics using Michaelis-Menten plots and Lineweaver-Burk plots. The results showed that the type of inhibition with glyceollin was competitive, that of genistein was noncompetitive, that of daidzein was uncompetitive, and luteolin showed a mixed mode of action. The Ki values were determined using a Dixon plot as glyceollin, 18.99 µM; genistein, 15.42 µM; luteolin, 16.81 µM; and daidzein, 9.99 µM. Furthermore, potential synergistic effects between glyceollin and the three polyphenols were investigated. A combination of glyceollin and luteolin at a ratio of 3:7 exhibited synergistic effects on α-glucosidase inhibition, having a combination index (CI) of 0.64244, according to the CI-isobologram equation. Collectively, these results showed that a combination of glyceollin and luteolin has the potential to inhibit α-glucosidase activity via a synergistic mode of inhibition.

Docosahexaenoic acid induces apoptosis in CYP2E1-containing HepG2 cells by activating the c-Jun N-terminal protein kinase related mitochondrial damage.

Docosahexaenoic acid (DHA) causes apoptosis of various cancer cells, but the mechanism of DHA-induced cell death is still unclear. We hypothesized that the early signaling of apoptosis may be important in causing cell death as well as the production of free radical metabolites. DHA caused time- and dose-dependent cell death in human HepG2 hepatoma cells transduced with CYP2E1 (E47) but not in C34 (without CYP2E1), suggesting an important role of CYP2E1 in the DHA-mediated damage. DHA increased the c-Jun N-terminal protein kinase (JNK) activity until 8 h without activating other mitogen-activated protein kinases. The contents of proapoptotic Bad and FasL at 4 h and cytochrome c and caspase 3 activity at 8 h were increased and accompanied by the JNK activation in a successive manner. In contrast, Bax and Bcl-2 were not changed. Levels of lipid peroxides (LPOs) were elevated three- and fivefold at 8 and 24 h, respectively, in DHA-induced E47 cells. However, pretreatment with chlormethiazole (CMZ), a specific inhibitor of CYP2E1, significantly reduced the levels of LPO, CYP2E1, JNK activity and the rate of cell death. In addition, pretreatment with quercetin (one as a JNK inhibitor and one as an antioxidant) significantly reduced the cell death rate and JNK and SEK-1 activities. Our results indicated that DHA-mediated apoptosis in E47 cells was induced through the activation of the JNK-related cell death pathway, which may be involved in the production of LPO or reactive oxygen species during the CYP2E1 catalytic cycle, followed by mitochondrial injury and apoptosis.

Corrigendum to "Expression of miRNA-122 Induced by Liver Toxicants in Zebrafish".

[This corrects the article DOI: 10.1155/2016/1473578.].

PPARγ agonists induce adipocyte differentiation by modulating the expression of Lipin-1, which acts as a PPARγ phosphatase.

PPARγ agonists induced obesity in animal models as a side effect. Microarray experiments reveal that PPARγ agonist upregulates the expression of lipin-1 and this upregulation is correlated with the activity of the agonists. Lipin-1 induced by PPARγ agonists decreased the levels of PPARγ and ERK1/2 phosphorylation through direct interaction with these proteins in 3T3-L1 cells. In PPARγ agonist-treated 3T3-L1 preadipocytes, the knockdown of lipin-1 expression by small interfering RNA inhibited the adipogenesis that was induced by PPARγ agonists. In contrast, PPARγ2 expression was increased, and lipid droplets were accumulated in lipin-1-overexpressing 3T3-L1 adipocytes. Rosiglitazone (RGZ), a strong PPARγ agonist, synergistically promoted PPARγ dephosphorylation and adipogenesis in lipin-1-overexpressing 3T3-L1 preadipocytes. Therefore, lipin-1 has dual functions as a transcriptional cofactor and phosphatidate phosphatase (PAP) in the differentiation of preadipocyte cells induced by strong PPARγ agonists. In addition, the adipogenesis of 3T3-L1 cells was markedly upregulated by diacylglycerol (DAG), which was produced by lipin-1. Therefore, lipin-1 induction by PPARγ agonists might be an important factor in understanding the biological mechanism of the agonists' adverse effects, and this information may be valuable in the development of type-2 diabetes mellitus (T2DM) therapeutics with reduced adverse effects and greater tolerability.

Expression of miRNA-122 Induced by Liver Toxicants in Zebrafish.

MicroRNA-122 (miRNA-122), also known as liver-specific miRNA, has recently been shown to be a potent biomarker in response to liver injury in mammals. The objective of this study was to examine its expression in response to toxicant treatment and acute liver damage, using the zebrafish system as an alternative model organism. For the hepatotoxicity assay, larval zebrafish were arrayed in 24-well plates. Adult zebrafish were also tested and arrayed in 200 mL cages. Animals were exposed to liver toxicants (tamoxifen or acetaminophen) at various doses, and miRNA-122 expression levels were analyzed using qRT-PCR in dissected liver, brain, heart, and intestine, separately. Our results showed no significant changes in miRNA-122 expression level in tamoxifen-treated larvae; however, miRNA-122 expression was highly induced in tamoxifen-treated adults in a tissue-specific manner. In addition, we observed a histological change in adult liver (0.5 µM) and cell death in larval liver (5 µM) at different doses of tamoxifen. These results indicated that miRNA-122 may be utilized as a liver-specific biomarker for acute liver toxicity in zebrafish.

Acetoaminophen-induced accumulation of 8-oxodeoxyguanosine through reduction of Ogg1 DNA repair enzyme in C6 glioma cells.

Large doses of acetaminophen (APAP) could cause oxidative stress and tissue damage through production of reactive oxygen/nitrogen (ROS/RNS) species and quinone metabolites of APAP. Although ROS/RNS are known to modify DNA, the effect of APAP on DNA modifications has not been studied systematically. In this study, we investigate whether large doses of APAP can modify the nuclear DNA in C6 glioma cells used as a model system, because these cells contain cytochrome p450-related enzymes responsible for APAP metabolism and subsequent toxicity (Geng and Strobel, 1995). Our results revealed that APAP produced ROS and significantly elevated the 8-oxo- deoxyguanosine (8-oxodG) levels in the nucleus of C6 glioma cells in a time and concentration dependent manner. APAP significantly reduced the 8- oxodG incision activity in the nucleus by decreasing the activity and content of a DNA repair enzyme, Ogg1. These results indicate that APAP in large doses can increase the 8-oxodG level partly through significant reduction of Ogg1 DNA repair enzyme.

Troglitazone but not rosiglitazone induces G1 cell cycle arrest and apoptosis in human and rat hepatoma cell lines.

Rosiglitazone (RSG), an agonist of peroxisome proliferator-activated receptor gamma (PPARgamma), induces minor toxicity in humans relative to another PPARgamma agonist, troglitazone (TRO). In contrast, recent reports suggest that RSG causes growth arrest and apoptosis of normal and cancerous cells. Therefore, in this study, we investigated the relative toxicities of TRO and RSG on three different hepatoma cell lines, and observed that TRO, but not RSG, was cytotoxic. Additionally, we studied the mechanism by which TRO induced damage to HepG2 hepatoma cells. Our results indicated that TRO increased the levels of p53, p27, and p21, while it reduced the levels of cyclin D1 and phospho-Rb in a time-dependent manner. Increased p27 and p21 levels coincided with reduced activities of cell cycle dependent kinases (cdk) such as cdk2- and cyclin A-protein kinases 24 h after TRO treatment. These results demonstrate that TRO, but not RSG, causes G1 arrest of hepatoma cells, most likely through changing the levels of cell cycle regulators. Furthermore, because RSG did not affect the levels of cell cycle regulators, TRO-mediated growth inhibition appears independent of PPARgamma activation.