Human Hepatoma SMMC-7721 Cells Research Articles

Oleanolic acid derivatives inhibit the Wnt/β-catenin signaling pathway by promoting the phosphorylation of β-catenin in human SMMC-7721 cells.

Oleanolic acid, isolated from privet, has shown antitumor effects in several cancers. However, the underlying molecular mechanism associated with these effects is largely unknown. In this study, we explored the effect of oleanolic acid derivatives on the Wnt/β-catenin signaling pathway in human hepatocellular carcinoma SMMC-7721 cells. The mRNA and protein levels of related genes were determined by real-time quantitative PCR and Western blot, respectively. Treatment of SMMC-7721 cells with oleanolic acid derivatives led to the downregulation of the mRNA and protein levels of β-catenin, c-myc, and cyclin D1. Treatment with oleanolic acid derivatives decreased the levels of β-catenin in both the cytoplasm and the nucleus. Moreover, oleanolic acid derivatives promoted the phosphorylation of β-catenin (Ser33/37/Thr41) in the cytoplasm. Our results suggest that oleanolic acid derivatives inhibit the Wnt/β-catenin signaling pathway by stimulating the phosphorylation of β-catenin (Ser33/37/Thr41) in human SMMC-7721 cells.

Microcystin-LR induces a wide variety of biochemical changes in the A549 human non-small cell lung cancer cell line: Roles for protein phosphatase 2A and its substrates.

Our previous studies have described the toxic effects of microcystin-LR (MC-LR) in various normal cell lines and human hepatoma SMMC-7721 cells, but the specific effects of MC-LR in other types of cancer cells with respect to protein phosphatase 2A (PP2A) have not been fully elaborated. A549 human lung adenocarcinoma cells have been identified to express organic anion-transporting polypeptides (OATP) involved in cellular uptake of MC-LR, and thus probably make an appropriate in vitro model to assess MC-LR's cytotoxicity. Hence, in our present study, A549 cells were treated with various concentrations of MC-LR for 24 h. The presence of MC-LR in A549 cells was confirmed, and PP2A activity, PP2A substrates, cytoskeleton, apoptosis, and proliferation were subsequently explored. The results showed that 5-10 μM MC-LR inhibited PP2A activity significantly but 0.5-1 μM MC-LR did not change PP2A activity dramatically. The inhibition could result from the hyperphosphorylation of PP2A/C at Tyr307, an elevation in the total PP2A/C expression and the dissociation of α4/PP2A/C complexes. Moreover, MC-LR led to rearrangements of filamentous actin and microtubules, which might be correlated with the hyperphosphorylation of Ezrin, VASP and HSP27 due to PP2A inhibition and mitogen-activated protein kinase (MAPK) activation. However, exposure to MC-LR for 24 h failed to trigger either apoptosis or proliferation, which might be related to PP2A-inhibition-induced hyperphosphorylation of Bcl-2 and Bad and the activation status of Akt. In conclusion, our data indicated that MC-LR induced extensive molecular and cellular alterations in A549 cells through a PP2A-centered pathway, which differed in some respects from our previous study in SMMC-7721 cells. To our knowledge, this is the first report comprehensively demonstrating the effects of MC-LR in A549 cells, and our findings provide insights into the mechanism of MC-LR toxicity in cancer cells. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1065-1078, 2017.

Thermoresponsive diblock glycopolymer by RAFT polymerization for lectin recognition

A thermoresponsive double-hydrophilic diblock glycopolymer, poly(diethyl- eneglycol methacrylate)-block-poly(6-O-vinyladipoyl-d-glucose) (PDEGMA-b-POVAG), was successfully prepared by a combination of enzymatic synthesis and reversible addition-fragment chain transfer (RAFT) polymerization protocols using poly(diethyl- eneglycol methacrylate) (PDEGMA) as macro-RAFT agent. The block glycopolymer was characterized by 1H NMR and GPC. UV–vis, DLS and TEM studies revealed that the glycopolymer PDEGMA-b-POVAG was thermoresponsive with LCST at 31.0°C, and was able to self-assemble into spherical micelles of various sizes in aqueous solution. The glucose pendants in the glycopolymer could interact with the lectin Concanavalin A (Con A), the average hydrodynamic diameters of glycopolymer micelles increased to 170nm from 110nm after recognizing Con A. The diblock glycopolymer micelles have excellent biocompatibility with pig iliac endothelial cells, as measured using the MTT assay, but micelles loaded with Con A could be used to induce apoptosis in human hepatoma SMMC-7721 cells.

Targeted nanobubbles in low-frequency ultrasound-mediated gene transfection and growth inhibition of hepatocellular carcinoma cells.

The use of SonoVue combined with ultrasound exposure increases the transfection efficiency of short interfering RNA (siRNA). The objective of this study was to prepare targeted nanobubbles (TNB) conjugated with NET-1 siRNA and an antibody GPC3 to direct nanobubbles to hepatocellular carcinoma cells. SMMC-7721 human hepatocellular carcinoma cells were treated with six different groups. The transfection efficiency and cellular apoptosis were measured by flow cytometry. The protein and messenger RNA (mRNA) expression were measured by Western blot and quantitative real-time PCR, respectively. The migration and invasion potential of the cells were determined by Transwell analysis. The results show that US-guided siRNA-TNB transfection effectively enhanced gene silencing. In summary, siRNA-TNB may be an effective delivery vector to mediate highly effective RNA interference in tumor treatment.

Inhibition of mTOR promotes hyperthermia sensitivity in SMMC-7721 human hepatocellular carcinoma cell line.

The mammalian target of rapamycin (mTOR) is a critical mediator of the phosphoinositide 3-kinase/protein kinase B/mTOR signaling pathway, and mTOR activity is induced following heat shock. Thermotherapy is used to treat hepatocellular carcinoma (HCC). However, the role of mTOR in modulating thermosensitivity in HCC has yet to be elucidated. In the present study, the antisense plasmid pEGFP-C1-mTOR was transfected into SMMC-7721 cells, and the expression levels of mTOR were analyzed by reverse transcription-polymerase chain reaction and western blot analysis. The thermal responses of the transfected cells were also examined. The results revealed that SMMC-7721 cells were sensitive to heat treatment, and cell viability was significantly inhibited following hyperthermia treatment (P<0.01). The mRNA and protein expression levels of mTOR decreased post-transfection. Cell proliferation, colony-forming ability and motility were all significantly decreased following hyperthermia treatment in the transfected cells. Flow cytometry analysis demonstrated that apoptosis was significantly increased following treatment (P<0.01). The number of cells in S phase was increased, and the cell cycle was arrested in S phase. In conclusion, inhibition of mTOR increased the thermosensitivity of SMMC-7721 cells by increasing cellular apoptosis and inducing S phase arrest.

MicroRNA-497 regulates cell proliferation in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver. MicroRNA-497 (miR-497) is known to be downregulated in several types of human cancer; however, the expression, function and underlying mechanisms of miR-497 in HCC remain unclear. Therefore, the present study investigated miR-497 expression in HCC samples and HCC-derived cell lines using reverse transcription-quantitative polymerase chain reaction. The protein expression of one of the predicted common targets of miR-497, insulin-like growth factor-1 receptor (IGF-1R), was assessed using western blot analyses and immunohistochemistry. The role of miR-497 in regulating the proliferation of HCC-derived cells was also investigated in vitro and in vivo. Of 60 paired specimens from HCC patients, miR-497 was downregulated in 42 cancer specimens compared with adjacent non-cancer tissues. Western blotting and immunohistochemical analyses revealed that IGF-1R expression was significantly increased in HCC compared to control tissues. In addition, overexpression of miR-497 was observed to inhibit colony formation and tumor growth in MHCC-97H human HCC cells. Conversely, SMMC-7721 human HCC cells transfected with a miR-497 inhibitor exhibited enhanced colony formation and tumor growth. Finally, IGF-1R protein, phosphoinositide 3-kinase/Akt signaling pathway-associated proteins and cyclin pathway-associated proteins were differentially expressed between miR-497-overexpressing cells and miR-497-silenced cells. These results indicate that miR-497 may be a potentially effective gene therapy target.

Cytotoxic Activities, SAR and Anti-Invasion Effects of Butylphthalide Derivatives on Human Hepatocellular Carcinoma SMMC7721 Cells.

A series of butylphthalide derivatives (BPDs) 1–8 were isolated from the extract of the dried rhizome of Ligusticum chuanxiong Hort. (Umbelliferae). The cytotoxic activities of BPDs 1–8 were evaluated using a panel of human cancer cell lines. In addition, the SAR analysis and potential anti-invasion activities were investigated. The sp2 carbons at C-7 and C-7a appeared to be essential for the cytotoxic activities of BPDs. BPDs 5 and 6 remarkably inhibited the migration and invasion of cancer cells. The anti-invasion activity of dimer 6 was demonstrated to be significantly higher than monomer 5.

Gallic acid as a selective anticancer agent that induces apoptosis in SMMC-7721 human hepatocellular carcinoma cells.

Gallic acid (3,4,5-trihydroxybenzoic acid; GA) is a naturally occurring plant polyphenol, isolated from water caltrop, which has been reported to exert anticancer effects. The present study investigated the antiproliferative effects of GA on the HepG2 and SMMC-7721 human hepatocellular carcinoma (HCC) cell lines using MTT and colony formation assays. In particular, the underlying mechanism of GA-induced apoptosis in SMMC-7721 cells was studied in vitro by flow cytometry and western blotting. The results of the present study indicated that GA was capable of inhibiting the proliferation of HepG2 and SMMC-7721 cells in a time- and dose-dependent manner, as well as inducing the apoptosis of SMMC-7721 cells. GA induced caspase-3, caspase-9 and reactive oxygen species activity, elevated the expression of apoptosis regulator Bcl-2-like protein 4 and reduced the mitochondrial membrane potential in SMMC-7721 cells. When compared with HL-7702 normal human hepatocytes, GA demonstrated selective toxicity for HCC cells. In conclusion, GA is able to induce apoptosis in SMMC-7721 cells in vitro via mitochondrial-mediated pathways, and may possess the potential to be a novel therapeutic compound for use in the treatment of HCC.

USP14 activation promotes tumor progression in hepatocellular carcinoma

To elucidate the molecular mechanisms underlying the pathogenesis and treatment of human primary hepatocellular carcinoma (HCC), it is important to explore novel HCC-associated genes. In the present study, we examined the expression of ubiquitin-specific peptidase 14 (USP14) in patients with HCC using quantitative PCR and immunohistochemical techniques. The expression of USP14 in tumor tissues of patients with HCC was significantly higher than that in adjacent non-cancerous and normal liver tissues. It was also determined whether the expression profile of USP14 was associated with the clinical characteristics of HCC. Increased USP14 expression was associated with some clinicopatho-logical variables, such as advancing tumor stage. A Kaplan-Meier curve analysis demonstrated that patients with HCC having a high USP14 expression had a significantly poorer prognosis after surgery than patients with lower USP14 expression levels. Knockdown of USP14 with the lentiviral vector delivery of shRNA in human hepatocarcinoma SMMC7721 cells suppressed cell proliferation, altered the cell cycle and induced cell apoptosis. Additionally, the Wnt/β-catenin pathway was activated in HCC patients with USP14 overexpression. These findings strongly suggested that USP14 activation plays an oncogenic role in promoting tumor progression in HCC. Thus, our findings suggested that USP14 is involved in the progression of HCC and may be a useful therapeutic target in HCC. These findings likely reflect the key role that USP14 plays in the pathogenesis of HCC. Therefore, the identification of USP14 and USP14-driven genes may promote the investigation of its functional role to develop more effective therapies for HCC, especially advanced HCC.

Melittin induces PTCH1 expression by down-regulating MeCP2 in human hepatocellular carcinoma SMMC-7721 cells

Hepatocellular carcinoma (HCC) has a high mortality rate worldwide and still remains to be a noticeable public health problem. Therefore, new remedies are urgently needed. Melittin, a major component of bee venom, is known to suppress cell growth in various cancers including HCC. However, the mechanism of the anticancer effect of melittin on HCC has not been fully elucidated. It has been reported that Methyl-CpG binding protein 2 (MeCP2) plays a key role in tumor proliferation, apoptosis, migration and invasion. In the present study, we found the high expression of MeCP2 in human HCC tissues and in the SMMC-7721 cell line. MeCP2 silencing inhibited cell proliferation, while over-expression of MeCP2 promoted cell growth in SMMC-7721 cells. It indicates that MeCP2 may be an attractive target for human HCC. We further found that melittin could inhibit cell proliferation by reducing MeCP2 expression in vitro. Interestingly, the inhibitory effect of melittin on cell proliferation was due to a delay in G0/G1 cell cycle progression, without influencing cell apoptosis. Next, we investigated the potential molecular mechanisms and found that MeCP2 could modulate Shh signaling in SMMC-7721 cells. Further study indicates that melittin may induce the demethylation of PTCH1 promoter, resulting in the increased expression of PTCH1. Furthermore, the expression of Shh and GLI1 was significantly lowered upon treatment of melittin. These results suggest that melittin can block Shh signaling in vitro. In short, these results indicate that melittin inhibits cell proliferation by down-regulating MeCP2 through Shh signaling in SMMC-7721 cells.

Effects of Oxymatrine on the Proliferation and Apoptosis of Human Hepatoma Carcinoma Cells.

Oxymatrine, one of the main active components of extracts from the dry roots of Sophora flavescens, has been reported to possess anticancer activities in vitro and in vivo However, the precise mechanism involved remains largely unknown. The present study is conducted to investigate the anticancer activity and the underlying mechanisms of oxymatrine in human hepatoma cells (Hep-G2 and SMMC-7721) in vitro and in vivo Hep-G2 and SMMC-7721 cells were treated by oxymatrine and subjected to methyl thiazolyl tetrazolium analysis, Hoechst 33342 staining, annexin V/propidium iodide double staining, reverse transcription polymerase chain reaction, and Western blot analysis. In addition, SMMC-7721 xenograft tumors were established in male nude BALB/c mice, and oxymatrine was intravenously administered to evaluate the anticancer capacity in vivo Our results showed that oxymatrine inhibited the proliferation and induced apoptosis of Hep-G2 and SMMC-7721 cells in a dose-dependent manner in vitro Furthermore, the RNA and protein expression of Bax and caspase 3 levels were significantly upregulated, whereas the expression of Bcl-2 was downregulated. These protein interactions may play a pivotal role in the regulation of proliferation and apoptosis. More importantly, our in vivo studies showed that administration of oxymatrine decreased tumor growth in a dose-dependent manner. Immunohistochemistry analysis demonstrated an increase of Bax and caspase 3 and a decrease of Bcl-2 in tumor tissues following oxymatrine treatment which are consistent with the in vitro results. Taken together, our findings indicated that oxymatrine can inhibit cell proliferation and induce apoptosis of human hepatoma Hep-G2 and SMMC-7721 cells and might offer a therapeutic potential advantage for human hepatoma chemoprevention or chemotherapy.

A BODIPY-based NIR Probe for Detecting Mercury(II) in Solution and in Living Cells

Abstract A new BODIPY-based near-infrared (NIR) fluorescent probe, based on the deprotection of dithioacetal, has been developed, with good selectivity for Hg2+. The proposed sensing mechanism was supported by 1H NMR studies. Moreover, the probe was successfully applied to the fluorescence imaging of Hg2+ in Human Hepatoma SMMC-7721 cells under physiological conditions.

DNA methyltransferase 3b silencing affects locus-specific DNA methylation and inhibits proliferation, migration and invasion in human hepatocellular carcinoma SMMC-7721and BEL-7402cells.

DNA methylation is an important regulator of gene transcription, and its role in carcinogenesis has been a topic of considerable interest in previous years. The present study examined the influence of DNA methyltransferase 3b (DNMT3b) on cell proliferation, migration and invasion, and the methylation status of identified tumor suppressor genes in hepatoma SMMC-7721 and BEL-7402 cells. DNMT3b was silenced by small interfering RNA (siRNA) in human hepatocellular carcinoma cell lines. Transfection efficiency was verified using a fluorescent imaging system, reverse transcription polymerase chain reaction (RT-PCR) and western blotting. A cell proliferation assay was performed to evaluate cell viability. Cell cycle distribution and apoptosis were analyzed by flow cytometry. The migratory and invasive ability of cells was measured using a Transwell assay. Methylation-specific PCR (MSP) was performed to assess methylation in the promoter region of genes. The present data revealed that DNMT3b siRNA successfully inhibited expression of the DNMT3b gene in these two liver cancer cell lines and therefore inhibited the proliferation of the transfected cells, stimulated apoptosis in the cells, led to an accumulation of cells in the G2/M phase and decreased cell migration and invasion. It was also found that silencing DNMT3b expression results in hypomethylation of specific sets of gene promoters and increases the expression of distinct set of genes in HCC cell lines. The present study is therefore useful for assessing the specificity of emerging action based on the altered expression of associated regulatory genes, particularly in methylation-silenced genes.

Inhibitory effect of emodin on human hepatoma cell line SMMC-7721 and its mechanism.

Da Huang (Radix et Rhizoma Rhei) is the dried root or rhizome of Rheum palmatum L., Rheum tanguticum Maxim ex Balf. or Rheum officinale Braill of family Polygonaceae. It has heat clearing, damp drying, fire purging and toxin removing effects. Because of its definite curative efficacy, it has been widely applied in clinical settings. To study the inhibitory effect of emodin on human hepatoma cell line SMMC-7721 and its mechanism. MTT assay, flow cytometry and electron microscopy were used to investigate the inhibitory effect of different concentrations of emodin on human hepatoma cell line SMMC-7721. 12 h, 24 h and 48 h after the action of 20, 40 and 80 umol/L emodin on SMMC-7721 cells, the proliferation of human hepatoma SMMC-7721 cells was inhibited; the inhibitory effects showed time-and concentration-dependence. 48 h after the action of different concentrations of emodin on SMMC-7721 cells, cells in G2/M phase increased significantly, while the proportion of S phase cells gradually declined. Emodin can inhibit human hepatoma cell line SMMC-7721.

Insufficient radiofrequency ablation promotes human hepatoma SMMC7721 cell proliferation by stimulating vascular endothelial growth factor overexpression.

The aims of the current study were to investigate the influence of insufficient radiofrequency ablation (RFA) on the cell proliferation of the human hepatocellular carcinoma (HCC) cells, SMMC7721, and to elucidate the underlying mechanism. SMMC7721 cells were subjected to a 47°C treatment regimen to simulate insufficient RFA, in the presence or absence of KN93 [a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII)], PD98059 [a specific inhibitor of extracellular signal-regulated kinase (ERK)], or axitinib (a specific inhibitor of vascular endothelial growth factor (VEGF) receptor]. Cell proliferation was determined using a thiazolyl terazolium assay (MTT). The levels of CaMKII, phospho-CaMKII, ERK, phospho-ERK and VEGF were observed by western blot analysis. The results demonstrated that the 47°C treatment regimen: i) Triggered upregulation of VEGF expression in the SMMC7721 cells, which was reduced by CaMKII or ERK inhibition; ii) induced ERK activation was prevented by KN93; and iii) promoted SMMC7721 cell proliferation, which was greatly inhibited by axitinib, KN93 and PD98059. In conclusion, the results indicated that insufficient RFA promotes SMMC7721 cell proliferation by activating CaMKII/ERK-dependent VEGF overexpression.

Composition of Lycium barbarum polysaccharides and their apoptosis-inducing effect on human hepatoma SMMC-7721 cells

BackgroundLycium barbarum polysaccharide (LBP) is a natural functional component that has a variety of biological activities. The molecular structures and apoptosis-inducing activities on human hepatoma SMMC-7721 cells of two LBP fractions, LBP-d and LBP-e, were investigated.ResultsThe results showed that LBP-d and LBP-e both consist of protein, uronic acid, and neutral sugars in different proportions. The structure of LBP was characterized by gas chromatography, periodate oxidation, and Smith degradation. LBP-d was composed of eight kinds of monosaccharides (fucose, ribose, rhamnose, arabinose, xylose, mannose, galactose, and glucose), while LBP-e was composed of six kinds of monosaccharides (fucose, rhamnose, arabinose, mannose, galactose, and glucose). LBP-d and LBP-e blocked SMMC-7721 cells at the G0/G1 and S phases with an inhibition ratio of 26.70 and 45.13%, respectively, and enhanced the concentration of Ca2 + in the cytoplasm of SMMC-7721.ConclusionThe contents of protein, uronic acid, and galactose in LBP-e were much higher than those in LBP-d, which might responsible for their different bioactivities. The results showed that LBP can be provided as a potential chemotherapeutic agent drug to treat cancer.

Nutlin-3-induced redistribution of chromatin-bound IFI16 in human hepatocellular carcinoma cells in vitro is associated with p53 activation.

Interferon-γ inducible protein 16 (IFI16), a DNA sensor for DNA double-strand break (DSB), is expressed in most human hepatocellular carcinoma cell (HCC) lines. In this study we investigated the re-localization of chromatin-bound IFI16 by Nutlin-3, a DNA damage agent, in HCC cells in vitro, and the potential mechanisms. Human HCC SMMC-7721 (wild-type TP53), Huh-7 (mutant TP53), Hep3B (null TP53) and normal fetal liver L02 cell lines were examined. DSB damage in HCC cells was detected via γH2AX expression and foci formation assay. The expression of IFI16 and IFNB mRNA was measured using RT-PCR, and subcellular localization and expression of the IFI16 protein were detected using chromatin fractionation, Western blot analysis, and fluorescence microscopy. Treatment of SMMC-7721 cells with Nutlin-3 (10 μmol/L) or etoposide (40 μmol/L) induced significant DSB damage. In SMMC-7721 cells, Nutlin-3 significantly increased the expression levels of IFI16 and IFNB mRNA, and partially redistributed chromatin-bound IFI16 protein to the cytoplasm. These effects were blocked by pretreatment with pifithrin-α, a p53 inhibitor. Furthermore, Nutlin-3 did not induce ectopic expression of IFI16 protein in Huh-7 and Hep3B cells. Moreover, the association of IFI16 with chromatin and Nutlin-3-induced changes in localization were not detected in L02 cells. Nutlin-3 regulates the subcellular localization of IFI16 in HCC cells in vitro in a p53-dependent manner.

Increased ARPP-19 expression is associated with hepatocellular carcinoma.

The cAMP-regulated phosphoprotein 19 (ARPP-19) plays a key role in cell mitotic G2/M transition. Expression of ARPP-19 was increased in human hepatocellular carcinoma (HCC) compared to adjacent non-tumorous liver tissues in 36 paired liver samples, and the level of ARPP-19 in HCC tissues was positively correlated with the tumor size. To determine the interrelationship between ARPP-19 expression and HCC, we silenced ARPP-19 expression in the human hepatocarcinoma HepG2 and SMMC-7721 cells using lentivirus encoding ARPP-19 siRNA. HepG2 and SMMC-7721 cells with ARPP-19 knockdown displayed lowered cell growth rate, retarded colony formation and increased arrest at the G2/M phase transition. Silencing ARPP-19 in HCC cells resulted in decreased protein levels of phospho-(Ser) CDKs substrates and increased levels of inactivated cyclin division cycle 2 (Cdc2). Therefore, ARPP-19 may play a role in HCC pathogenesis through regulating cell proliferation.

2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone, from buds of Cleistocalyx operculatus, induces apoptosis in human hepatoma SMMC-7721 cells through a reactive oxygen species-dependent mechanism.

Nowadays, much effort is being devoted to detect new substances that not only significantly induce the death of tumor cells, but also have little side effect on normal cells. Our previous study showed that 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) exhibited significant cytotoxic potential with an IC50 value of 32.3±1.13μM against SMMC-7721 cells and could induce SMMC-7721 cells apoptosis. In the present study, we found that DMC was almost nontoxic to human normal liver L-02 and human normal fetal lung fibroblast HFL-1 cells as their IC50 values (111.0±4.57 and 152.0±4.83µM for L-02 and HFL-1 cells, respectively) were much higher. To further explore the apoptotic mechanism of DMC, we investigated the role of the reactive oxygen species (ROS) in the apoptosis induced by DMC in SMMC-7721 cells. Our results suggested that the cytotoxicity and the generation of intracellular ROS were inhibited by N-acetylcysteine (NAC). Reversal of apoptosis in NAC pretreated cells indicated the involvement of ROS in DMC-induced apoptosis. The loss of mitochondrial membrane potential (ΔΨm) induced by DMC was significantly blocked by NAC. NAC also prevented the decrease of Caspase-3 and -9 activities, the increase of Bcl-2 protein expression and the decrease of p53 and PUMA protein expressions. Together, these results indicated that ROS played a key role in the apoptosis induced by DMC in human hepatoma SMMC-7721 cells.

Puerarin inhibits growth and induces apoptosis in SMMC-7721 hepatocellular carcinoma cells.

Puerarin, a predominant isoflavonoid compound extracted from the Chinese medicinal herb Radix Puerariae, is considered to exhibit an antitumor effect. In the present study, the effects of puerarin on SMMC-7721 human hepatocellular carcinoma cells were investigated. Cell viability was assessed by MTT assay. Apoptosis was detected by flow cytometry with Annexin V-fluorescein isothiocyante staining and morphological observation of nuclear changes by Hoechst staining. The mitochondrial membrane potential (MMP) was monitored using rhodamine123. The generation of reactive oxygen species (ROS) was quantified using dichloro‑dihydro‑fluorescein diacetate. Polymerase chain reaction and western blot analysis were used to detect the expression levels of apoptosis‑associated genes. The results revealed that high concentrations of puerarin (500, 1,000 and 1,500µg/ml) significantly inhibited the proliferation of SMMC-7721 cells in a time- and dose-dependent manner. Simultaneously, apoptotic rates were increased and cell morphology was changed following puerarin treatment. Furthermore, puerarin‑induced apoptosis of SMMC-7721 cells was associated with loss of MMP and generation of ROS. Puerarin treatment increased caspase‑3,8,9 and apoptosis‑inducing factor (AIF) mRNA expression levels in SMMC‑7721 cells, while the phosphorylation levels of P38, extracellular signal‑regulated kinase (ERK1) and c-Jun N‑terminal kinase were also increased. Furthermore, caspase-9 and AIF protein expression was upregulated. In conclusion, puerarin inhibited proliferation and induced apoptosis in SMMC‑7721 cells via the mitochondria‑dependent pathway; however, the specific mechanisms require further investigation.