Preliminary Study on Effects of Solamargine on the Growth and Apoptosis of Human Hepatocarcinoma Cells HepG 2 and Its Mechanism / 中国药房

OBJECTIVE：To ex plore the effects of solamargine on the growth and apoptosis of human hepatocarcinoma cells HepG2 and its underlying mechanism. METHODS ：The effects of 0（blank group ）-12 μmol/L solamargine treatment of 24，48 h on survival rate of HepG 2 cells were investigated. The effects of 0（blank group ），6 μmol/L solamargine treatment of 10 days on cell clone formation were also investigated. The effects of 0（blank group ），4，6，8 μmol/L solamargine for 24 h on the apoptotic rate of cells，mRNA expression of Bcl- 2，Bax and caspase- 3， protein expression of Bcl- 2 and cleaved caspase- 3 as well as ratio of p-AMPKα to AMPKα were all tested. The effects of AMPK inhibitor as compound C on the protein expression of AMPKα and Bcl- 2 in cells were investigated after treated with 6 μmol/L solamargine for 24 h. RESULTS ：Compared with 020-39318678。E-mail：wujingjing6028@gzucm.edu.cn blank group ，1-12 μ mol/L solamargine for 24，48 h could significantly decrease the survival rates of cells （P＜0.05）in a concentration-dependent manner ；IC50 of them were 8.310 and 7.996 μmol/L，respectively；the rate of cell clone formation was decreased significantly after treated with 6 μmol/L solamargine for 10 days（P＜0.05）. The apoptotic rate of HepG 2 cells，mRNA expression of Bax and caspase- 3，protein expression of cleaved caspase-3（except for 8 μmol/L）as well as ratio of p-AMPKα to AMPKα（except for 8 μmol/L）were all increased significantly after treated with 6，8 μmol/L solamargine（P＜0.05）；mRNA and protein expression of Bcl- 2 were decreased significantly （P＜ 0.05）；the changes of some indexes were in a concentration-dependent manner. The compound C could inhibit protein expression of AMPKα，and reverse the inhibitory effect of solamargine on Bcl- 2 protein. CONCLUSIONS ：Solamargine can inhibit the proliferation of HepG 2 cells and induce apoptosis ，the mechanism of which may be associated with activating AMPK signaling pathway.

Caesalpinia bonduc Linn Extracts Exhibit Hepatoprotective Effect on HepG2 Cells against Paracetamol by Up-regulating Glutathione Related Genes

Background: Caesalpinia bonduc (CB) is said to ownvarious pharmacological and therapeutic applicationagainst a number of diseases. It is used widely in folkmedicine to treat liver diseases. In the present study,we have made a sincere effort to evaluate thehepatoprotective activity of CB leaf extracts againstParacetamol (PCM) induced toxicity and theirmechanism of hepatoprotection in the humanHepatocarcinoma cells (HepG2 cells) therebyproviding scientific evidence for the same. Materialand Methods:The hepatoprotective activity of CB wasassessed in-vitro by the estimation of glutathione(GSH) and Malondialdehyde (MDA), anti-apoptoticassay/Annexin V and the expressions of genes such asGlutathione Reductase (GS-R) and GlutamateCysteine Ligase, Catalytic (GCLC). Results: Theobtained results suggest that the aqueous extract of CBpossess significant hepatoprotective activity. Thisactivity may be due to the possible antioxidantproperty and the free radical scavenging ability of theextracts, which might clear the toxic metabolites ofPCM. Conclusion: The present study suggests that theaqueous extract of CB have potential hepatoprotectiveactivity, which may prevent the lipid peroxidation ofthe cell membrane by its antioxidant properties.

Effect of Modified Lichongtang Combined with 5-Fluorouracil on Epithelial Mesenchymal Transition in H22 Tumor-bearing Mice / 中国实验方剂学杂志

Objective: To investigate the effect of modified Lichongtang combined with 5-fluorouracil (5-FU) on tumor epithelial-mesenchymal transition (EMT) in H22 tumor-bearing mice. Method: Mouse models of transplanted hepatoma were constructed. After tumor formation, they were randomly divided into 4 groups:blank group, 5-FU group(2.5 mg·kg-1 5-FU intraperitoneal injection), modified Lichongtang combined with 5-FU group (5-FU+Chinese medicine group), and modified Lichongtang group (Chinese medicine group,25 g·kg-1 gavage),n=10 in each group. The effect of modified Lichongtang combined with 5-FU on the tumor inhibition rate of subcutaneous transplanted tumor was observed. The gene expression levels of E-cadherin,N-cadherin,Snail,and Twist in transplanted tumor were observed by Real-time PCR. The protein expression levels of E-cadherin,N-cadherin,Snail,and Vimentin were detected by using Western blot. Result: The tumor inhibiting rate was 59.18%,84.42%,and 10.39% respectively in 5-FU group, 5-FU+Chinese medicine group,and Chinese medicine group. All of these can inhibit the growth of liver cancer transplantation tumor, and the tumor inhibiting rate of 5-FU+Chinese medicine group was significantly higher than that in 5-FU group (PPPPPPPPPPPConclusion: Modified Lichongtang, 5-FU and their combination have inhibitory effect on the growth of transplanted tumors of hepatocarcinoma mice, and the combination of the two drugs can enhance the effect of chemotherapy and to some extent inhibit the toxicity of 5-FU. The mechanism may be related to the inhibition of liver cancer EMT.

Effects of fractionated irradiation on the invasion and metastasis of residual HepG2 cells / 中华放射医学与防护杂志

Objective To investigate the changes of invasion and migration potential of residual hepatocarcinoma cells after fractionated X-ray irradiation and its underlying mechanism.Methods HepG2 cells were exposed to X-rays (2 Gy × 10) and recovered for 30 days after irradiation to generate residual cells.The changes of cellular invasion and migration potential were detected in the residual and its control cells using a Transwell assay.The expressions of epithelial-mesenchymal transition(EMT)-related proteins of N-cadherin and Snail were detected by Western blot.HepG2 subcutaneous tumor models were established using nude mice that were divided into control and radiation group.In radiation group,the tumors were locally irradiated with a dose of 2 Gy per fraction daily,5 days per week for 2 weeks until the cumulative dose reached to 20 Gy.The growth of the tumor was observed,and on the day 39 after cell implantation (i.e.day 14 after radiation),the liver metastasis and the expression of N-cadherin in tumor with or without radiation were detected.Results The invasions and migrations of the residual cells and xenograft tumor were significantly enhanced in comparison with the control group (t =5.126,7.714,P ＜0.05).The expressions of N-cadherin and Snail in the radiation group were significantly higher than that in the control group (t =7.509,7.184,P＜0.05).In the HepG2 subcutaneous tumor model,the weight and volume of tumor in nude mice of the radiation group was significantly smaller than that in the control group (t =2.396,3.170,P ＜0.05),and the number of liver metastases in nude mice and the expression of N-cadherin in tumor were significantly higher than those in the control group (t =2.994,5.695,P ＜0.05).Conclusions Fractionated irradiation enhances the abilities of invasion and migration by inducing EMT in hepatocarcinoma cells,which provides new insights of the recurrence and metastasis of hepatocarcinoma after radiotherapy.

Effects of 3 Extracts of Acanthopanax sessiliflorus Fruits on the Proliferation and Apoptosis of Human Hepatocarcinoma Cells SMMC-7721 / 中国药房

OBJECTIVE:To study the effects of 3 extracts of Acanthopanax sessiliflorus fruits on the proliferation and apoptosis of human hepatocarcinoma cells SMMC-7721,and to provide reference for confirming the mechanism of anti-tumor effect. METHODS:MTT assay was adopted to investigate the effects of low-mass concentration,medium-mass concentration and high-mass concentration of ethanol extract(0.92,1.84,3.68 mg/mL),crude polysaccharide extract(0.06,0.12,0.24 mg/mL)and refined polysaccharide extract (0.04, 0.08, 0.16 mg/mL) from A. sessiliflorus fruits on the proliferation and apoptosis of SMMC-7721 cells after treated for 24,36,48 h,respectively. Flow cytometry was used to investigate the effects of 1.84 mg/mL ethanol extract,0.24 mg/mL crude polysaccharide extract and 0.16 mg/mL refined polysaccharide extract on cell cycle and cell apoptosis after treated for 24 h. The above tests were all negative control(only adding cells without drugs). RESULTS:Compared with negative control,3 extracts of A. sessiliflorus fruits could significantly inhibit the proliferation of SMMC-7721 cells (P<0.01),could significantly decrease the percentage of SMMC-7721 cells in G0/G1 and G2/M phase(P<0.01),could significantly increase the percentage of SMMC-7721 cells in S phase (P<0.01) and the apoptosis rate of SMMC-7721 cells (P<0.05);especially the effects of ethanol extract from A. sessiliflorus fruits were the most obvious. CONCLUSIONS:Three extracts of A.sessiliflorus fruits can inhibit the proliferation of human hepatocarcinoma SMMC-7721 cells,block SMMC-7721 cells in S phase and induce the apoptosis of SMMC-7721 cells.

The Therapeutic Effect of Quercetin-loaded PLGA-TPGS Nanoparticles on the Hepatocarcinoma Ectopic Solid Tumor-bearing Mice / 中国医科大学学报

Objective To investigate the therapeutic effects of Quercetin (QT)-loaded PLGA-TPGS nanoparticles (QPTN) on solid tumor-bearing mice with HCa-F hepatocarcinoma in vivo.Methods The model of HCa-F hepatocarcinoma solid tumor-bearing mice was established by implanting HCa-F cells into 48 mice.The mice were divided into 6 groups randomly:the negative control,empty PLGA-TPGS nanoparticles,5-Fluorouracil solutions (FS),Quercetin solutions (QTS),QT-loaded PLGA nanoparticles (QPN),and QPTN groups.Each group was treated using tail vein twice a day for 20 days;then,all mice were sacrificed.The increment tumor volumes and tumor growth inhibition rate were counted.Then,tumor specimens were prepared for hematoxylin & eosin (HE) staining and observed under a microscope.Results The results showed that the increment tumor volumes of mice in the QPTN,QPN,and FS groups were significantly smaller than that in the negative control group (P ＜ 0.05 or P ＜ 0.01).The tumor growth inhibition rate of the QPTN group was 59.07％,which was much higher than that of the QTS group (23.94％),the FS group (35.14％),and the QPN group (46.14％).The results of the HE staining on the tumor sections also indicated that the QPTN group showed a better therapeutic outcome compared to the other groups.Conclusion The QPTN has a better therapeutic effect on the model of solid tumor using HCa-F cells-bearing mice than the QPN,QTS,and FS.

Ofloxacin rhodanine derivatives induces apoptosis of human hepatocarcinoma cells / 中国药理学通报

Aim To study the effect of 6-(3-Benzyl-4-oxo-2-thioxo-thiazolidin-5-ylidenemethyl)-9-fluoro-3-methyl-10-(4-methyl-piperazin-1-yl)-2,3-dihydro-7-oxo-7-hydro-pyrido[1,2,3-de][1,4]benzoxazine (R3) on apoptosis of the human hepatocarcinoma SMMC-7721 cells (in vitro).Methods With different concentrations of R3 used to treat SMMC-7721 cells, esophageal squamous cell carcinoma EC-9706 cells, human colon adenocarcinoma cell line Caco-2 cells and in human L-02 hepatocytes (in vitro), and the inhibition effects of R3 on cell proliferation were examined by MTT assay.Cell apoptosis was determined using DAPI fluorescence staining and TUNEL method.The cell cycle was detected using flow cytometry with PI staining.Protein expression of p53 and caspase-3 was detected with Western blot analysis.Results Treatment with R3 (2~20 μmol·L-1) potently inhibited the proliferation of the cancer cells (the IC50 value at 24 h in SMMC-7721 cells, EC-9706cells and CaCO-2 cells was 3.893, 4.181 and 3.408 μmol·L-1, respectively).In contrast, R3 had weak cytotoxicity against L-02 cells with IC50 value of 38.96 μmol·L-1.Ofloxacin had weak cytotoxicity against SMMC-7721 cells with IC50 value of 240.137 μmol·L-1.Sunitinib had cytotoxicity against SMMC-7721 cells with IC50 value of 8.075 μmol·L-1.Treatment of SMMC-7721cells with different concentrations of R3 for 24 h increased the percentage of the apoptosis cells (P<0.05) and caused insufficient preparation for G1/S transition.In addition, R3 increased protein expression of p53, caspase-3 and the cleaved activated forms of caspase-3 in SMMC-7721 cells.Conclusion R3 as a kind of ofloxacin rhodanine derivatives exerts potent and selectively anticancer activity through the induction of apoptosis of SMMC-7721 cells.

Effect of platycodin D on radiosensitivity of human hepatoma cell line and related mechanisms of action / 中华肝脏病杂志

Objective@#To investigate the effect of platycodin D on the radiosensitivity of human hepatoma cell lines HepG2 and SMMC-7721 and related mechanisms of action.@*Methods@#MTT assay was used to analyze the effect of different concentrations of platycodin D with different treatment times on cell viability. The cells were pretreated with 5 μg/ml platycodin D for 24 hours followed by X-ray irradiation at different radiation doses. Colony-forming assay was used to measure the radiosensitizing effect of platycodin D on cells. The quasi-threshold dose (Dq), mean lethal dose (Do), extrapolation number (N), sensitizer enhancement ratio (SER), and survival fraction (SF) at different radiation doses were calculated, and the multi-target single-hit model was used to fit the cell survival curve according to the formula SF = l-(l-e-D/D0)N. Flow cytometry was used to investigate the distribution of cell cycle, and Western blotting was used to measure the changes in the protein expression of phosphorylated phosphatidylinositol 3’-kinase (pPI3K), phosphorylated protein kinase (pAkt), nuclear factor-κB (NF-κB), and phosphorylated nuclear factor inhibiting protein (pIκBα). A one-way analysis of variance, the t-test, or the least significant difference test was used for statistical analysis based on the type of data.@*Results@#Platycodin D reduced the viability of HepG2 and SMMC-7721 cells in a dose-dependent manner; the IC50 value for HepG2 cells was 24.2 ± 0.61 μg/ml at 24 hours and 7.68 ± 0.46 μg/ml at 48 hours, and that for SMMC-7721 cells was 23.8 ± 0.57 μg/ml at 24 hours and 8.63 ± 0.86 μg/mL at 48 hours. After the combined treatment with platycodin D and irradiation, there were significant reductions in Dq (P = 0.002), Do (P = 0.002), and N value (P = 0.003), the survival curve markedly shifted to the left, and SER was 1.347 ± 0.04 in HepG2 cells and 1.418 ± 0.05 in SMMC-7721 cells. In addition, platycodin D significantly inhibited the increase in the proportion of cells in G2/M phase, the increases in the protein expression of pPI3k (P = 0.002), pAkt (P = 0.003), and NF-κB (P = 0.002), and the reduction in the protein expression of pIκBα (P = 0.003).@*Conclusion@#Platycodin D can increase the radiosensitivity of HepG2 or SMMC-7721 cells, possibly by enhancing the growth inhibition effect of irradiation and inhibiting the activation of the PI3k/Akt and NF-κB pathways.

Effect of Sonic Hedgehog signaling blockade on growth of hepatocarcino-ma cells / 中国病理生理杂志

AIM:To investigate the effect of Sonic Hedgehog ( Shh) signaling blockade on the growth of hema-tocarcinoma cells and underlying mechanisms.METHODS: The expression of Shh signaling molecules in hematocarci-noma cell lines BEL-7402, Huh7 and HepG2 was detected by RT-PCR.The cell viability was detected by MTT assay.The cell cycle and apoptosis were analyzed by flow cytometry.The expression of apoptosis-related proteins was determined by Western blot.RESULTS:Shh signaling molecules were all expressed in BEL-7402, Huh7 and HepG2 cells.The mRNA expression of Patched ( Ptch) , Gli1 and Gli2 was down-regulated by anti-Shh antibody.Blockade of Shh signaling pathway inhibited the proliferation of hepatocarcinoma cells with increasing cells in G0/G1 phase and induced the apoptosis of hepa-tocarcinoma cells.Treatment with anti-Shh antibody down-regulated the protein expression of pro-caspase-3, pro-caspase-8 and pro-caspase-9, while up-regulated the protein levels of cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 in BEL-7402 cells.CONCLUSION:Blockade of Shh signaling pathway inhibits the growth of hepatocarcinoma at different levels by cell cycle arrest and inducing apoptosis of hematocarcinoma cells.

The Applications of Hepatitis C Virus (HCV) Replication System in Developing Anti-HCV Reagents

Hepatitis C virus (HCV) is known to be a major cause of chronic hepatitis, liver cirrhosis and hepatocarcinoma. Therapeutic reagents are improving, but are still limited, and the protective vaccine against HCV is not available yet. However, the research of HCV life cycle and pathogenesis has been difficult due to obstacles, which are the lack of effective cell culture systems and small-animal models. Recently, breathtaking progress in terms of HCV replication system has been made using various forms of HCV clones and human hepatocarcinoma 7 cell lines (huh 7). The establishment of complete cell-culture system for HCV replication gave researchers opportunities to study the entire viral life cycle including entry, assembly, release of viral particles and the interaction with host cells. In fact, these efforts now appear to move into the identification and the development of innovative anti-HCV reagents. In this review, we go over the biological characters of HCV, a variety of in vitro cell culture, in vivo animal models of HCV infection, HCV immune-pathogenesis and the application of HCVcc system in terms of developing anti-HCV reagents.

Therapeutic mechanism of treating SMMC-7721 liver cancer cells with magnetic fluid hyperthermia using Fe2O3 nanoparticles

This study aimed to investigate the therapeutic mechanism of treating SMMC-7721 liver cancer cells with magnetic fluid hyperthermia (MFH) using Fe2O3 nanoparticles. Hepatocarcinoma SMMC-7721 cells cultured in vitro were treated with ferrofluid containing Fe2O3 nanoparticles and irradiated with an alternating radio frequency magnetic field. The influence of the treatment on the cells was examined by inverted microscopy, MTT and flow cytometry. To study the therapeutic mechanism of the Fe2O3 MFH, Hsp70, Bax, Bcl-2 and p53 were detected by immunocytochemistry and reverse transcription polymerase chain reaction (RT-PCR). It was shown that Fe2O3 MFH could cause cellular necrosis, induce cellular apoptosis, and significantly inhibit cellular growth, all of which appeared to be dependent on the concentration of the Fe2O3 nanoparticles. Immunocytochemistry results showed that MFH could induce high expression of Hsp70 and Bax, decrease the expression of mutant p53, and had little effect on Bcl-2. RT-PCR indicated that Hsp70 expression was high in the early stage of MFH (<24 h) and became low or absent after 24 h of MFH treatment. It can be concluded that Fe2O3 MFH significantly inhibited the proliferation of in vitro cultured liver cancer cells (SMMC-7721), induced cell apoptosis and arrested the cell cycle at the G2/M phase. Fe2O3 MFH can induce high Hsp70 expression at an early stage, enhance the expression of Bax, and decrease the expression of mutant p53, which promotes the apoptosis of tumor cells.

Co-culturing cytokine-induced killer cells with dendritic cells enhances the cytotoxic activities of CIKs against hepatocarcinoma cells / 中华微生物学和免疫学杂志

Objective To explore in vitro cytotoxic activities of DCIKs against hepatocarcinoma cells by co-culturing cytokine-induced killer cells (CIKs) with dendritic cells (DCs) derived from peripheral blood of patients with hepatocellular carcinoma (HCC).Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 23 patients with HCC and cultured with cytokines to induce DCs and CIKs.DCIKs were induced by co-culturing CIKs with DCs.After 14 days of co-culture,the phenotypes of DCIKs and CIKs were analyzed by flow cytometry,and their in vitro cytotoxic activities against SMCC-7721 and HepG2 hepatocarcinoma cells were measured by MTT assay.Levels of IL-12,IL-4 and IFN-γ in the supernatants of cell culture were detected by enzyme-linked immunosorbent assay (ELISA).Results High expressions of CD3+CD8+ and CD3+CD56+ were observed on DCIKs.The percentages of effector cells,cytotoxic activity and cytokine secretion were all significantly increased with DCIKs as compared with those CIKs without DC co-culture (P＜0.05).Conclusion Co-culture of CIKs with DCs can enhance the differentiation of effector cells and the cytolytic activities of CIKs against hepatocarcinoma cells in vitro.Immunotherapy with DCIKs may be a promising strategy for the treatment of patients with HCC.