Human Hepatoma G2 Research Articles

In vitro toxicological evaluation of ethyl carbamate in human HepG2 cells.

Ethyl carbamate (EC) is a multi-site carcinogen in experiment animals and probably carcinogenic to humans (IARC group 2A). The present study was designed to investigate the cytotoxicity effect of EC on human hepatoma G2 (HepG2) cells. The results revealed that EC inhibited the viability of HepG2 cells significantly in a dose-dependent manner. Further analysis indicated that high concentration of EC induced cell apoptosis, inhibited the G1 to S phase transition along with increased expression of p53 and p21 and decreased the expression of cyclin E and Cdk 2, but no significant change in p27 expression was observed, which were evidenced by both real time PCR and western blotting analyses. Moreover, the results of the DCFH-DA assay suggested that oxidative stress was involved in the cytotoxic effects of EC. Altogether, the present work indicated that p21, cyclin E and Cdk2, which were regulated by p53, might account for the effect of EC on cell viability and cell cycle arrest, but p27 was not involved in the pathway in HepG2 cells treated with EC.

Development of monoclonal antibodies and immunochromatographic lateral flow device for rapid test of alanine aminotransferase isoenzyme 1

BackgroundAlanine aminotransferase (ALT) has been used as a sensitive marker for liver injury in people and in preclinical toxicity studies. But measurement of ALT isoenzymes, ALT1 and ALT2, was reported to be of more diagnostic value. The aim of this study is to develop an ideal pair of anti-ALT1 monoclonal antibodies (MAbs) of high specificity and affinity, and subsequently prepare a Immunochromatographic lateral flow device (LFD) for rapid test of ALT1 in human serums. MethodsThe complete coding sequence of ALT1 gene (1500 bp) was cloned from human hepatoma G2 cells (HepG2) and inserted into the expression vector pET-32a(+). ALT1 recombinant protein was routinely prepared by E. coli BL21 (DE3) expression and Ni2+ affinity purification. Balb/c mice were immunized with purified ALT1 and the splenocytes were fused with Sp2/0 myeloma cells. The positive clones, verified by indirect enzyme-linked immunosorbent assay (ELISA) using purified ALT1, were subcloned to single clones by limiting dilution process. A MAb pair was selected from the obtained MAbs according the sandwich ELISA pairing results and then used for lateral flow device (LFD) production. After evaluation of the sensitivity and specificity, the LFD strips were employed to test human serum samples with known ALT activity levels. ResultsALT1 recombinant protein was expectedly prepared by expression and purification. A total of 8 stable clones that produced antibodies specifically recognizing ALT1 protein were developed. After sandwich ELISA pairing, an ideal pair of anti-ALT1 MAbs, designated as BD7 and DG3, were selected and proved to be of high specificity, titer and affinity. Based on the MAb pair, LFD strips specifically for ALT1 rapid test were subsequently prepared. The detection threshold of the LFD strips was 12 U/L. No cross reaction was found. ConclusionsThe ALT1 LFD with high sensitivity and specificity was successfully developed. It is valuable for testing ALT1 protein in human sera and can be a beneficial complement for traditional ALT test.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies withAspidosperma ramiflorum in vivo and in vitro.

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellow peroba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium berghei in mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.

The Effect of GADD45a on Furazolidone-Induced S-Phase Cell-Cycle Arrest in Human Hepatoma G2 Cells.

In this study, overexpression of GADD45a induced by furazolidone in HepG2 cells could arouse S-phase cell cycle arrest, suppress cell proliferation, and increase the activities of cyclin D1, cyclin D3, and cyclin-dependent kinase 6 (CDK6). To the opposite, GADD45a knockdown cells by RNAi could reduce furazolidone-induced S-phase cell cycle arrest, increase the cell viability, decrease the activities of cyclin D1, cyclin D3, and CDK6; however, cyclin-dependent kinase 4 (CDK4) showed no change. Moreover, data from our current studies show that cyclin D1, cyclin D3, and CDK6 are target genes functioning at the downstream of the GADD45a pathway induced by furazolidone. These results demonstrate that the GADD45a pathway is partially responsible for the furazolidone-induced S-phase cell cycle arrest. GADD45a influences furazolidone-induced S-phase cell cycle arrest in human hepatoma G2 cells via cyclin D1, cyclin D3, and CDK6, but not CDK4.

Protective effects of hepatic stellate cells against cisplatin-induced apoptosis in human hepatoma G2 cells.

The effects of hepatic stellate cells (HSCs) on tumorigenicity of HCC have been previously reported. However, the detailed mechanisms responsible for these effects remain unclear. In this study, we investigated the effects of HSCs on cisplatin-induced apoptosis in human hepatoma HepG2 cell lines. HepG2 cells were treated with cisplatin alone or co-cultured with LX-2 cells 3 days before incubation with cisplatin. Cisplatin causes apoptosis in HepG2 cells and LX-2 cells protect HepG2 cells from death. The protection of LX-2 cells against cisplatin-induced cytotoxicity in HepG2 cells appeared to be related to the inhibition of apoptosis, as determined by cytotoxicity assay and nuclear staining analysis. p53 and Bax mRNA levels were elevated, and cell cycle arrest was produced after cisplatin treatment. LX-2 cells suppressed this elevation of p53 and Bax as well as the cell cycle arrest induced by cisplatin, when compared with those of the treated cells with cisplatin alone. The LX-2 cells pretreatment inhibited the cisplatin-induced apoptosis, which was related with the incomplete blockage in p53 activation. In summary, the results of our present study demonstrate that HSCs protect HepG2 cells against cisplatin-induced apoptosis and its protective effects occur via inhibiting the activation of p53, which is of critical importance for enhanced understanding of fundamental cancer biology.

Genotoxic and oxidative stress effects of 2-amino-9H-pyrido[2,3-b]indole in human hepatoma G2 (HepG2) and human lung alveolar epithelial (A549) cells

2-Amino-9H-pyrido[2,3-b]indole (AαC), which is present in high quantities in cigarette smoke and also in fried food, has been reported to be a probable human carcinogen. However, few studies have reported on the genotoxicity and oxidative stress induced by AαC. This study investigated the genotoxic effects of AαC in human hepatoma G2 (HepG2) and human lung alveolar epithelial (A549) cells using the comet assay. Significant increases in DNA fragment migration indicated that AαC causes serious DNA damage in HepG2 and A549 cells. The role of oxidative stress in the mechanism of AαC-induced genotoxicity was clarified by measuring the level of intracellular reactive oxygen species (ROS), the GSH/GSSG ratio and the formation of 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage. The results showed that the levels of ROS and 8-OHdG increased, whereas the GSH/GSSG ratio decreased. The concentration of 8-OHdG was positively related to DNA damage. Taken together, these results indicate that AαC can induce genotoxicity and oxidative stress and that AαC likely exerts genotoxicity in HepG2 and A549 cells through ROS-induced oxidative DNA damage. This is the first report to describe AαC-induced genotoxic and oxidative stress in HepG2 and A549 cells.

Modulation of the DNA repair system and ATR-p53 mediated apoptosis is relevant for tributyltin-induced genotoxic effects in human hepatoma G2 cells

The toxic effects of tributyltin (TBT) have been extensively documented in several types of cells, but the molecular mechanisms related to the genotoxic effects of TBT have still not been fully elucidated. Our study showed that exposure of human hepatoma G2 cells to 1–4μmol/L TBT for 3hr caused severe DNA damage in a concentration-dependent manner. Moreover, the expression levels of key DNA damage sensor genes such as the replication factor C, proliferating cell nuclear antigen and poly (ADP-ribose) polymerase-1 were inhabited in a concentration-dependent manner. We further demonstrated that TBT induced cell apoptosis via the p53-mediated pathway, which was most likely activated by the ataxia telangiectasia mutated and rad-3 related (ATR) protein kinase. The results also showed that cytochrome c, caspase-3, caspase-8, caspase-9, and the B-cell lymphoma 2 were involved in this process. Taken together, we demonstrated for the first time that the inhibition of the DNA repair system might be more responsible for TBT-induced genotoxic effects in cells. Then the generated DNA damage induced by TBT initiated ATR-p53-mediated apoptosis.

Autophagic cell death of human hepatoma G2 cells mediated by procyanidins from Castanea mollissima Bl. Shell-induced reactive oxygen species generation

The autophagy of human hepatoma G2 (HepG2) cells induced by procyanidins from chestnut (Castanea mollissima Bl.) shell (CSPCs) was investigated, and the inherent relationship between autophagic levels and reactive oxygen species (ROS) generation was studied. The results showed that CSPCs induced HepG2 cell death in a time- and concentration-dependent manner, increased the accumulation of autophagolysosomes and microtubule-associated proteins light chain 3-II (LC3-II, a marker of autophagy). However, these phenomena were not observed in the group pretreated with the autophagy inhibitor 3-MA, suggesting that CSPCs induced HepG2 cell autophagy. Furthermore, we found that CSPCs triggered ROS generation in cells, while the levels of ROS decreased in the N-acetylcysteine (Nac) co-treatment, revealing that CSPCs-mediated autophagy was partly blocked by Nac. In addition, treatment with CSPCs decreased the mitochondrial membrane potential of HepG2 cells. These results suggested CSPCs could trigger autophagy via ROS generation, which may be associated with the mitochondria-dependent signaling way.

Cytotoxic effects of procyanidins from Castanea mollissima Bl. shell on human hepatoma G2 cells in vitro

Significant cytotoxic effects of procynadins from chestnut (Castanea mollissima Bl.) shell (CSPC) on human hepatoma G2 (HepG2) cells were found in vitro. CSPC could inbibit HepG2 proliferation in a dose-dependent manner (100–400μg/mL), arrest cell cycle in the G0/G1 phase, induce apoptosis and trigger necrosis of HepG2. Proapoptotic effect of CSPC was evidenced by nuclear condensation, internucleosomal DNA fragmentation. Treatment of HepG2 cells with CSPC caused a loss of mitochondrial membrane potential and stimulated reactive oxidative species (ROS) generation. These results suggested CSPC could trigger apoptosis and necrotic cell death in HepG2 cell, which might be associated with ROS generation through the mitochondria-dependent signaling way.

Involvement of the p38 MAPK signaling pathway in S‐phase cell‐cycle arrest induced by Furazolidone in human hepatoma G2 cells

Given the previously described essential role for the p38 mitogen-activation protein kinase (p38 MAPK) signaling pathway in human hepatoma G2 cells (HepG2), we undertook the present study to investigate the role of the p38 MAPK signaling pathway in cell-cycle arrest induced by Furazolidone (FZD). The aim of this study was to determine the effects of FZD on HepG2 cells by activating and inhibiting the p38 MAPK signaling pathway. The cell cycle and proliferation of HepG2 cells treated with FZD were detected by flow cytometry and MTT assay in the presence or absence of p38 MAPK inhibitors (SB203580), respectively. Cyclin D1, cyclin D3 and CDK6 were detected by quantitative real-time PCR and western blot analysis. Our data showed that p38 MAPK became phosphorylated after stimulation with FZD. Activation of p38 MAPK could arise S-phase cell-cycle arrest and suppress cell proliferation. Simultaneously, inhibition of the p38 MAPK signaling pathway significantly prevented S-phase cell-cycle arrest, increased the percentage of cell viability and decreased the expression of cyclin D1, cyclin D3 and CDK6. These results demonstrated that FZD arose S-phase cell-cycle arrest via activating the p38 MAPK signaling pathway in HepG2 cells. Cyclin D1, cyclin D3 and CDK6 are target genes functioning at the downstream of p38 MAPK in HepG2 cells induced by FZD.

Combined pluronic P85- and ultrasound contrast agents-mediated gene transfection to HepG2 cells

This study examined the effect of P85 (a pluronic block copolymer) and microbubble (MB) ultrasound contrast agents under ultrasound irradiation on gene transfection and expression. The pEGFP plasmids that can encode enhanced green fluorescent protein (pEGFP) served as a report gene and were mixed with different concentrations of MB/0.05% (w/v) P85. Then the plasmids were transfected into human hepatoma G2 (HepG2) cells. The HepG2 cells treated with MB/P85 or without treatment were exposed to ultrasound (US parameters: 1 MHz, 1.0 W/cm(2), 20 s, 20% duty cycle). Twenty-four hours later, the transfection efficiency was assessed by fluorescence microscopy and fluorescence activated cell sorting (FACS) analysis. The cell viability was evaluated by Trypan blue exclusion test. The results showed that the gene transfection efficiency in HepG2 cells under ultrasound irradiation was significantly higher than that without ultrasound irradiation. HepG2 cells in the MB or P85 group in the absence of ultrasound expressed less amount of green fluorescent protein. The expression efficiency reached (22.14 ± 3.06)% and the survival rate was as high as (55.73 ± 3.32)% in the 30% MB plus P85 group. It was concluded that MB and P85 in the presence of ultrasound can enhance gene transfection and expression.

C-Myc influences olaquindox-induced apoptosis in human hepatoma G2 cells

Olaquindox, a synthetic antimicrobial compound, was banned as feed additives in the U.S. and the EU. In China, the use of olaquindox is banned in poultry and aquaculture feed, restricted in livestock feed for growth promotion. Olaquindox's safety is the object of increasing attention. The present study was undertaken to investigate whether and how olaquindox elevates expression of c-Myc, which influences olaquindox-induced apoptosis in HepG2 cells. For a better understanding of c-Myc's role in susceptibility of human hepatoma G2 cells to olaquindox-induced apoptosis, two vectors (the pSilencer-cmyc(Si-cmyc) and the control vector) were transfected to HepG2 cells. The cells were pretreated with Si-cmyc, which expressed only 35-65% c-Myc protein levels compared to those of the parental cells and the control cells. We examined effects of olaquindox on reactive oxygen species (ROS) production in these c-Myc low-expressing cells, and on apoptosis. Our data revealed that ROS production induced by olaquindox treatment was partially blocked by Si-cmyc transfection and partly inhibited olaquindox-induced apoptosis through decreased ROS generation. Further data showed that olaquindox induced decreased ROS by Si-cmyc transfection through decreased cytochrome c release to cytosol, which inhibited apoptosis of the cells. These results suggest that c-Myc might be important during olaquindox-induced apoptosis in human hepatoma G2 cells.

Curcumin enhanced adriamycin-induced human liver-derived Hepatoma G2 cell death through activation of mitochondria-mediated apoptosis and autophagy

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cancer killer in the world. Adriamycin (ADM) is a widely used anti-cancer drug. However, the efficacy is low since high dose of ADM causes toxicity to normal tissues. Curcumin, derived from turmeric ( Curcumin longa), has shown its therapeutic potential against HCC. Here, we aim to investigate the effects of curcumin combined with ADM on human liver-derived Hepatoma G2 (HepG2) cell death. We found that combination treatment of curcumin with ADM significantly decreased the number of viable cells as compared to single agent treatment. Hoechst staining demonstrated that apoptotic cell death occurred upon curcumin and ADM treatment. The decreased Bcl-2/Bax protein ratio and the activation of caspase-3 were also detected. In addition, curcumin plus ADM led to mitochondrial fragmentation, the reduction of mitochondrial membrane potential, as well as the activation of autophagy. These findings suggest the combined treatment of curcumin with ADM might be an optional chemotherapeutic method for HCC.

Autophagy inhibition enhances etoposide-induced cell death in human hepatoma G2 cells

Induction of autophagy usually acts as a survival mechanism of cancer cells in response to chemotherapy. However, the function and molecular mechanism of autophagy in human hepatoma cells under drug treatment is still not clear. To address this issue, we established an experimental model in which HepG2 cells were treated with etoposide, a widely used anticancer agent. We demonstrate the etoposide-induced accumulation of GFP-LC3 dots by fluorescent microscopy, the up-regulation of LC3-II protein expression by Western blotting and the increased number of autophagic vacuoles by electron microscopy, confirming the activation of autophagy by etoposide in HepG2 cells. Inhibition of autophagy by either 3-methyladenine (3MA) or beclin-1 small interfering RNA enhanced etoposide-induced cell death. Furthermore, activation of p53 and AMPK was detected in etoposide-treated cells and inhibition of AMPK triggered apoptosis through suppression of autophagy. On the other hand, inactivation of p53 promoted cell survival through augmentation of autophagy. Collectively, these findings indicate that etoposide-induced autophagy promotes hepatoma cell adaptation and survival, and that autophagy inhibition improves the chemotherapeutic effect of etoposide. Moreover, AMPK activation is clearly associated with etoposide-induced autophagy. We conclude that manipulation of AMPK may be a promising approach of adjuvant chemotherapy for hepatocellular carcinoma.

Furazolidone induced oxidative DNA damage via up-regulating ROS that caused cell cycle arrest in human hepatoma G2 cells

Furazolidone (FZD) is an antimicrobial agent that has been shown to have mutagenic, genotoxic and potentially carcinogenic properties when tested in a variety of systems in vitro and in vivo. In this study, we investigated FZD's DNA damaging effect in human hepatoma cells aiming at further defining the molecular mechanism of FZD's cytotoxicity. Addition of FZD resulted in cell growth suppression and cell cycle arrest in S phase accompanied by remarkable DNA strand breaks with increased levels of intracellular reactive oxygen species (ROS) and 8-hydroxydeoxyguanosine. Activities of antioxidases were down-regulated following FZD treatment and antioxidant agent catalase and superoxide dismutase ameliorated FZD's DNA damaging effects. Moreover, FZD caused much more extensive damage to mitochondrial DNA (mtDNA) than to nuclear DNA for which the decrease in mtDNA content correlated with FZD usage in a dose-dependent manner. However, there was no evidence of FZD induced mtDNA mutation in the mitochondrial DNA displacement loop. These results demonstrate that FZD up-regulates the production of intracellular ROS to cause oxidative DNA damage with mtDNA being the most vulnerable targets. Oxidative stress and the injury of mtDNA could be early indicators of FZD-induced cytotoxicity, with the resulting abnormal progression of the cell cycle.

Ambient particulate matter on DNA damage in HepG2 cells

Ambient particulate matter (PM) has been reported to be associated with increased respiratory, cardiovascular, and malignant lung diseases. The aim of the present study was to investigate the variability of the DNA-damage induced by thoracic particles (PM( 10)) sampled in different locations and seasons (2006) in Dalian, China, in human hepatoma G2 (HepG2) cells. Significant differences in percentage of tail DNA induced by the extractable organic matter of PM(10) were revealed between summer and winter seasons and among monitoring sites in single cell gel electrophoresis (SCGE) assay. The percentage of tail DNA in HepG2 cells significantly increased in a dose-dependent manner after exposure to 7.5 and 30 μg/mL extractable organic matter of PM(10) for 1 hour. In order to clarify the underlying mechanisms, we evaluated the level of reactive oxygen species (ROS) production with the 2, 7-dichloro-fluorescein diacetate (DCFH-DA) assay. Significantly increased level of ROS was observed in HepG2 cells at higher concentrations (15 and 30 μg/mL). Significantly increased levels of 8-hydroxydeoxyguanosine (8-OHdG) were also shown in HepG2 cells. In this study, the accumulation of nuclear factor kappa B (NF-κB) p65 protein induced by the extractable organic matter of PM(10) was detected by western blotting in HepG2 cells, and the protein expression of NF-κB p65 significantly increased after the treatment with 30 μg/mL extractable organic matter of PM(10) for 24 hours. These results indicate that the extractable organic matter of PM(10) causes DNA strand breaks in HepG2 cells, and significant differences in percentage of tail DNA in dependence on locality and season are revealed. The extractable organic matter of PM(10) exerts DNA damage effects in HepG2 cells, probably through oxidative DNA damage induced by intracellular ROS, increase of 8-OHdG formation, and protein expression of NF-κB p65.

Three New Homoisoflavanones from the Ophiopogon japonicusKer‐Gawler (Liliaceae)

AbstractThree new homoisoflavanones, 1–3, together with a known one, 4, were obtained from the AcOEt extract of the tuberous roots of Ophiopogon japonicus (Liliaceae). They were identified as (3R)‐2,3‐dihydro‐7‐hydroxy‐5‐methoxy‐3‐(4‐methoxybenzyl)‐6,8‐dimethyl‐4H‐chromen‐4‐one (1), (3R)‐3‐(1,3‐benzodioxol‐5‐ylmethyl)‐2,3‐dihydro‐7‐hydroxy‐5‐methoxy‐6,8‐dimethyl‐4H‐chromen‐4‐one (2), (3R)‐3‐(1,3‐benzodioxol‐5‐ylmethyl)‐2,3‐dihydro‐7‐hydroxy‐5‐methoxy‐6‐methyl‐4H‐chromen‐4‐one (3), and ophiopogonanone A (4). Their structures were determined on the basis of extensive NMR‐spectroscopic and mass‐spectrometric analyses. The three new compounds are rare homoisoflavanones which contain a MeO group at C(5). Compounds 1 and 2 showed weak cytotoxicity against the HepG2 (human hepatoma G2), KB (human oral epidermoid carcinoma), and MCF‐7 (human breast adenocarcinoma) cell lines in an MTT assay. Compound 3 exhibited weak cytotoxicity against HepG2 and MCF‐7, and moderate cytotoxicity against KB cell lines. Compound 4 showed moderate cytotoxicity against HepG2, KB, and MCF‐7 cell lines.

Genotoxic effect of 6-gingerol on human hepatoma G2 cells

6-gingerol, a major component of ginger, has antioxidant, anti-apoptotic, and anti-inflammatory activities. However, some dietary phytochemicals possess pro-oxidant effects as well, and the risk of adverse effects is increased by raising the use of doses. The aim of this study was to assess the genotoxic effects of 6-gingerol and to clarify the mechanisms, using human hepatoma G2 (HepG2) cells. Exposure of the cells to 6-gingerol caused significant increase of DNA migration in comet assay, increase of micronuclei frequencies at high concentrations at 20-80 and 20-40 microM, respectively. These results indicate that 6-gingerol caused DNA strand breaks and chromosome damage. To further elucidate the underlying mechanisms, we tested lysosomal membrane stability, mitochondrial membrane potential, the intracellular generation of reactive oxygen species (ROS) and reduced glutathione (GSH). In addition, the level of oxidative DNA damage was evaluated by immunocytochemical analysis on 8-hydroxydeoxyguanosine (8-OHdG). Results showed that lysosomal membrane stability was reduced after treatment by 6-gingerol (20-80 microM) for 40 min, mitochondrial membrane potential decreased after treatment for 50 min, GSH and ROS levels were significantly increased after treatment for 60 min. These suggest 6-gingerol induces genotoxicity probably by oxidative stress; lysosomal and mitochondrial damage were observed in 6-gingerol-induced toxicity.

Antitumor effect of matrine in human hepatoma G2 cells by inducing apoptosis and autophagy

To study the antitumor effect of matrine in human hepatoma G2 (HepG2) cells and its molecular mechanism involved in antineoplastic activities. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect viability of HepG2 cells. The effect of matrine on cell cycle was detected by fl ow cytometry. Annexin-V-FITC/PI double staining assay was used to detect cellular apoptosis. Cellular morphological changes were observed under an inverted phase contrast microscope. Transmission electron microscopy was performed to further examine ultrastructural structure of the cells treated with matrine. Monodansylcadaverine (MDC) staining was used to detect autophagy. Whether autophagy is blocked by 3-methyladenine (3-MA), an autophagy inhibitor, was evaluated. Expression levels of Bax and Beclin 1 in HepG2 cells were measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Matrine significantly inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner, and induced G1-phase cell cycle arrest and apoptosis of HepG2 cells in a dose-dependent manner. The total apoptosis rate was 0.14% for HepG2 cells not treated with matrine. In contrast, the apoptosis rate was 28.91%, 34.36% and 38.80%, respectively, for HepG2 cells treated with matrine at the concentration of 0.5, 1.0 and 2.0 mg/mL. The remarkable morphological changes were observed under an inverted phase contrast microscope. Abundant cytoplasmic vacuoles with varying sizes were observed in HepG2 cells treated with matrine. Furthermore, vacuolization in cytoplasm progressively became larger and denser when the concentration of matrine was increased. Electron microscopy demonstrated formation of abundant autophagic vacuoles in HepG2 cells after matrine treatment. When the specific autophagic inhibitor, 3-MA, was applied, the number of autophagic vacuoles greatly decreased. MDC staining showed that the fluorescent density was higher and the number of MDC-labeled particles in HepG2 cells was greater in matrine treatment group than in control group. Fewer autophagic vacuoles were observed in the combined 3-MA and matrine treatment group when 3-MA was added before matrine treatment, indicating that both autophagy and apoptosis are activated when matrine-induced death of hepatoma G2 cells occurs. Real-time quantitative RT-PCR revealed that the expression levels of Bax gene, an apoptosis-related molecule, and Beclin 1 gene which plays a key role in autophagy were higher in matrine treatment group than in control group, indicating that Beclin 1 is involved in matrine-induced autophagy and the pro-apoptotic mechanism of matrine may be related to its upregulation of Bax expression. Matrine has potent antitumor activities in HepG2 cells and may be used as a novel effective reagent in treatment of hepatocellular carcinoma.

Alterations of cellular organelles in human liver-derived hepatoma G2 cells induced by adriamycin

Adriamycin (ADM) is a commonly used chemotherapeutic drug in the treatment of hepatocellular carcinoma. However, the mechanisms involved in ADM-induced cell death and the molecular basis of ADM resistance are still unclear. To observe the early events that occurred in hepatoma cells in response to ADM, we investigated the alterations of morphology and subcellular distributions of cellular organelles in human liver-derived hepatoma G2 (HepG2) cells after ADM treatment. HepG2 cells were exposed to different doses of ADM for up to 60 h. Cytotoxicity occurred 24 h after 0.05 microg/ml ADM application, and remaining living cells showed irregular shapes but continued to multiply. Some cellular organelles altered their subcellular distribution or morphology after ADM treatment, including mitochondria, autophagic vacuoles, and Golgi apparatus. Immunoblotting with anti-LC3 antibody showed the upregulation of LC3-II protein, confirming that ADM leads to the induction of autophagy in HepG2 cells. Our findings suggest that among most of the cellular organelles, mitochondria and autophagic vacuoles were involved in the early ADM response, and may contribute to ADM-induced HepG2 cell death.