Hepatocellular Carcinoma Cell Line BEL-7402 Research Articles

Anti-tumor effect of germacrone on human hepatoma cell lines through inducing G2/M cell cycle arrest and promoting apoptosis

Germacrone is one of the main bioactive components in the traditional Chinese medicine Rhizoma curcuma. In this study, the anti-proliferative effect of germacrone on the human hepatoma cell lines and the molecular mechanism underlying the cytotoxicity of germacrone were investigated. Treatment of human hepatoma cell lines HepG2 and Bel7402 with germacrone resulted in cell cycle arrest and apoptosis in a dose-dependent manner as measured by MTT assay, flow cytometric and fluorescent microscopy analysis, while much lower effect on normal human liver cell L02 was observed. Flow cytometric analysis revealed that germacrone induced G2/M arrest in the cell cycle progression that was associated with an obvious decrease in the protein expression of cyclin B1 and its activating partner CDK1 with concomitant inductions of p21. Hoechst 33258 and Annexin V/PI staining results showed that the total cell number in apoptosis associated with a dose-dependent up-regulation of Bax and down-regulation of Bcl-2/Bcl-xl was increased. In the meantime, the up-regulation of p53 and reactive oxygen species increase were observed, which suggested that germacrone might be a new potent chemopreventive drug candidate for liver cancer via regulating the expression of proteins related to G2/M cell cycle and apoptosis, and p53 and oxidative damage may play important roles in the inhibition of human hepatoma cells growth by germacrone.

HLA-A2-restricted point-mutated Survivin peptide elicited specific cytotoxic T lymphocyte reaction against hepatocarcinoma

Objective To investigate the anti-hepatocarcinoma(anti-HCC) function of HLA-A2-restricted point-mutated Survivin peptide induced CTLs.Methods The HLA-A2-restricted Survivin nonapeptides were evaluated using bioinformatics software.The binding affinity of Survivin peptide to HLA-A2 molecular was determined with flow cytometry analysis.After peptide-induced CTLs were generated in vitro,flow cytometry and ELISA were performed to detect the levels of IFN-γ,which were secreted by reactive CTLs.Peptide-induced CTLs were co-cultured with hepatoma cell lines HepG2 and BEL-7402.The rates of tumor cells lysis were assayed using CytoTox 96(R) and the morphological changes of tumor cells were observed with inverted phase contrast microscope.Results Point-mutated Survivin nonapeptide Sur79M2 (KMSSGCAFL) was filtered out,which was shown higher scores compared with the wild-type peptide Sur79.Consistent with the results of software analysis,Sur79M2 showed higher binding ability in T2 binding assays.At the same time,Sur79M2-induced CTLs could release a large number of IFN-γ after incubated with target cells rather than Sur79.When co-cultured with HCC cell lines HepG2 and BEL-7402,Sur79M2-induced CTLs effectively lysis HepG2 on HLA-A2-restricted manner without killing effect on BEL-7402 that do not express HLA-A2 molecules.Conclusion Sur79M2 could elicited specific cytotoxic T lymphocytes in vitro,which were able to specifically kill HCC cell lines on HLA-A2-restricted manner. Key words: Survivin; Anti-hepatocarcinoma; Point-mutated peptide; HLA-A2-restricted

Mechanism of hydroxyapatite nanoparticles inducing apoptosis of human hepatoma cell line BEL7402

Objective To investigate the mechanism of apoptosis induction bu nano hydroxyapatite nano-particles (HAP).Methods Western blotting was used to detect the expression of anti-oncogene p53 and that of oncogene c-myc.The catalytic activity of the Caspases was measured by using a colofimetric assay.Telomeric repeat amplification protocol (TRAP) combined with enzyme-linked immunosorbent assay (ELISA) was used to detect the inhibitory rate of telomerase activity.Results The treatment of BEL-7402cells with nano-HAP decreased the expression of p53 protein,but increased the expression of c-myc protein.The Caspase-3 activity was increased during nano-HAP-induced apoptosis,while the telomerase activity was decreased by nano-HAP in a dose- and time-dependent manner.With the progressive increases in concentrations of nano-HAP (0,50,75,100,150,200 mg/L),the telomerase activity was down-regulated ( 1.94,1.26,1.21,1.14,0.86,0.59,respectively,all P ＜ 0.O1 ).At the concentration of 50 mg/L nano-HAP at different time points (0,12,24,36,48 h),the telomerase activity was down-regulated ( 1.94,1.73,1.57,1.43,1.26,respectively,all P ＜0.O1 ).Conclusion These results suggested that nano-HAP-induced apoptosis was mediated by altering the expression of anti-oncogene p53 and oncogene cmyc,activating Caspase-3 and inhibiting activity of telomerase. Key words: Hydroxyapatite nanoparticles; Carcinoma, hepatocellular; p53; Caspase-3; Telomerase

In vitro antitumour activity of stearic acid-g-chitosan oligosaccharide polymeric micelles loading podophyllotoxin

Development of successful formulations for poorly water-soluble drugs remains a longstanding critical and challenging issue in cancer therapy. The stearic acid-g-chitosan oligosaccharide (CSO-SA) micelles have been presented as potential candidates for intracellular antitumour agent delivery carrier. Herein, podophyllotoxin (PPT) loaded CSO-SA micelles (CSO-SA/PPT) were prepared by a dialysis method. The drug encapsulation efficiency could reach a higher level, the micellar size and the zeta potential increased with increasing charged amounts of drug. The cumulative release percentage of PPT drug from micelles enhanced with decreasing PPT content in the micelles. The cytotoxicities of CSO-SA/PPT micelles against human breast carcinoma (MCF-7) cells, human lung cancer cells (A549) and human hepatoma cell line (Bel-7402) were higher than that of free PPT formulation. The higher cytotoxicities were due to the faster PPT transport into tumour cells mediated by CSO-SA micelles. Overall, CSO-SA micelles might be a promising carrier for PPT delivery in cancer therapy.

Biological effects based quality control of a traditional Chinese medicine

The main objective of this paper is to establish a quality control method based on lipid-lowering and anti-oxidation effect of Jiang-Zhi-Ning (JZN). Biological effects based quality standard of its active components was investigated. Two kinds of cells: oxidized low-density lipoprotein (Ox-LDL) injured human umbilicalvein endothelial cell (HUVEC) and human hepatoma cell line Bel-7402 were employed to investigate the biological effects of the standard product of JZN and 5 batches of its test samples. Calculation of the biological effect ratio (BER) = effect of test products of JZN / effect of active constituents of JZN×100%. By evaluation of the effect indexes of lipid-lowering and antioxidant activities of JZN, and by the computation of the biological effect ratio, the biological effect of the active constituents of JZN was confirmed and 11 indexes were assessed as standard biological effect indexes. Moreover, the biological effect ratio of the active constituents of JZN in low, middle and high concentrations of five test samples all ranges from 80 to 120%; as such, they were all judged as eligible products. The standard biological effect of active constituents of JZN could be assessed as standard effect. This research also provided evidence for quality standard evaluation system of Chinese herbal compound. Key words: Quality control, biological effects, antioxidant activities, traditional Chinese medicine.

Cucurbitacin B inhibits 12‐O‐tetradecanoylphorbol 13‐acetate‐induced invasion and migration of human hepatoma cells through inactivating mitogen‐activated protein kinase and PI3K/Akt signal transduction pathways

Cucurbitacin B (CuB) is an active component isolated from various plants used as folk medicine in Asian countries and has shown diverse antitumor activities. There is, however, no documented effect of CuB on the migration and invasion of human hepatoma cells yet. The purpose of this study was to assess the effect of CuB on the migration and invasion of hepatoma cells and to explore the possible mechanism. Human hepatoma cell lines HepG2 and BEL-7402 were used for the study. Effects of CuB on cancer cell migration and invasion were evaluated in vitro with wound healing and transwell assays. The effect of CuB on the expression of matrix metalloproteinase (MMP)-9, mitogen-activated protein kinases (MAPKs), Akt, nuclear factor-κB (NF-κB), c-Fos and c-Jun was investigated with gelatin zymography and/or western blotting. Cucurbitacin B has significantly suppressed 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cell invasion and migration in a concentration-dependent manner, which was accompanied with suppression of TPA-induced MMP-9 expression through inactivation of phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 and Akt. In the nucleus, it has also strongly suppressed TPA-stimulated expression of NF-κB, c-Jun and c-Fos. Cucurbitacin B has a potential value for suppressing metastasis of human hepatoma cells through suppressing the expression of MMP-9.

Large Intervening Non-Coding RNA HOTAIR is Associated with Hepatocellular Carcinoma Progression

Increasing evidence suggests that large intervening non-coding RNAs (lincRNAs) regulate key pathways in cancer invasion and metastasis. In this observational retrospective study, the expression of the oncogenic lincRNA HOX transcript antisense RNA (HOTAIR) gene was measured in 63 patients with hepatocellular carcinoma (HCC) following hepatic resection. The HOTAIR gene was significantly overexpressed in HCC tissues compared with adjacent non-tumour tissues. Patients with high HOTAIR gene expression in their tumours had an increased risk of recurrence after hepatectomy. There was also a significant correlation between HOTAIR expression and lymph node metastasis. In vitro assays in the HCC cell line Bel7402 demonstrated that knockdown of HOTAIR lincRNA reduced cell proliferation and was associated with reductions in levels of matrix metalloproteinase-9 and vascular endothelial growth factor protein, which are important for cell motility and metastasis. In conclusion, HOTAIR lincRNA might be a potential biomarker for the existence of lymph node metastasis in HCC.

MiRNA-195 sensitizes human hepatocellular carcinoma cells to 5-FU by targeting BCL-w

The role of microRNA-195 in developing acquired drug resistance in hepatocellular carcinoma cells was investigated. Expression profiling of miRNAs revealed a limited set of miRNAs with altered expression in drug resistant hepatocellular carcinoma cell line BEL-7402/5-FU compared to its parental BEL-7402 cell line. Real-time PCR confirmed down-regulation of miRNA-195 in BEL-7402/5-FU cells. Overexpression of miRNA-195 sensitized BEL-7402/5-FU cells to anticancer drugs. Consistent with these findings, miR-195 antisense oligonucleotide induced drug resistance in BEL-7402/5-FU cells. Also, the basal levels of the anti-apoptotic protein Bcl-w were high in BEL-7402/5-FU cells and miR-195 overexpression repressed Bcl-w protein level and inhibited the luciferase activity of a Bcl-w 3' untranslated region-based reporter construct in both BEL-7402/5-FU and BEL-7402 cells. These results indicate that miR-195 could improve the drug sensitivity at least in part by targeting Bcl-w to increase cell apoptosis in hepatocellular carcinoma cells.

The effect of 5-fluorouracil on enriching cancer stem cells of hepatoma cell line BEL-7402

To investigate the effect of 5-FU (5-fluorouracil) on enriching cancer stem cells of HCC cell line BEL-7402 and the biological characteristics of enriched cells. The enriching concentration of 5-FU was determined by CCK-8 (cell counting kit-8). Flow Cytometry was used to determine the changes in cell cycle and positive expression ratio of surface marker CD56, CD54, EpCAM and CD133. The self-renewal and differentiation of positive cells were tested by colony formation assay, and were compared with the control group. Enriching concentration of 5-FU was determined as 10 μg/ml with 48 h incubation. After enrichment, G0/G1 phase cells increased from 57.50 %+/-0.98% to 68.70%+/-3.41% (P<0.05). Whereas S phase cells decreased from 40.26%+/-4.12% to 31.80%+/-4.15% (P<0.01); G2/M phase cells disappeared in experimental group, and was 5.80%+/-1.87% in control group (P<0.01). The proportion of the cell cycle changed with significant statistical differences. Meanwhile, positive rate of cell surface makers CD56, CD54, EpCAM and CD133 increased from 0.57%+/-0.12%, 8.10%+/-6.79%, 0.3%+/-0.01% and 3.20%+/-0.99% to 4.13%+/-0.06%, 50.08%+/-1.69%, 0.55%+/-0.07% and 10.51%+/-1.13%, respectively. The difference was significant (P<0.05). The colony forming ratio of CD56, CD54, EpCAM and CD133 negative cells and positive cells were 2.11%+/-0.21%, 3.32%+/-0.31%; 0.86%+/-0.101%, 2.40%+/-0.52 %; 7.19%+/-0.56%, 7.73%+/-0.71%; 2.70%+/-0.26%, 5.75%+/-0.81%, respectively, and significant differences were found between (P<0.05). 5-fluorouracil enriched the cancer stem cell population in HCC cell line BEL-7402. CD56 and CD54 can be used as important surface markers in research of liver cancer stem cells.

Targeting therapy with epirubicin polybutylcyanoacrylate magnetic nanoparticles for transplanted hepatoma in nude mice

Objective To observe the effect of epirubicin polybutylcyanoacrylate magnetic nanoparticles (EPI-PBCA-MNPS) on the transplanted hepatoma in nude mice.Methods Human hepatoma cell line Bel-7402 nude mice models were established and divided into 5 groups with each of 10 once gross tumor volume was up to 20-30 mm3:normal saline group (NS),epirubicin group (EPI),epirubicin polybutylcyanoacrylate nanoparticles group (EPI-PBCA-NPS),epirubicin polybutylcyanoacrylate magnetic nanoparticles group (EPI-PBCA-MNPS) and epirubicin polybutylcyanoacrylate magnetic nanoparticles+magnetic field group(EPI-PBCA-MNPS+MF).Except for NS group, the dosage of epirubicin was 0.002 mg/g body weight or 40 μg for intravenous injection.After two-weeks therapy,all nude mice models were executed and the tumor volume and weight were measured so as to calculate the volume inhibition ratio and the weight inhibition ratio.Every tumor specimen was made into pathological section and labelled with serial number for comparing neorobiosis degree in different groups before and after therapy.Results There were statistically difference among the other groups (P 0.05).The two ratios in the EPI-PBCA-MNP+MF group (94.26%,80.82%) were significantly more than those in other groups (P<0.01).The pathological sections demonstrated the most severe tumour neorobiosis in the EPI-PBCA-MNP+MF group.Conclusion With the additional magnetic field,EPI-PBCA-MNPS has significant anticancer effect on human hepatoma nude mice models due to its favourable magnetic targeting. Key words: Epirubicin; Polybutylcyanoacrylate; Magnetic nanoparticles; Hepatocellular carcinoma; Transplanted tumor

Reversal effect of 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone on multi-drug resistance in resistant human hepatocellular carcinoma cell line BEL-7402/5-FU

Multi drug resistance (MDR) is a major obstacle in the chemotherapeutic treatment of many human cancers. 2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), a chalcone, isolated from the buds of Cleistocalyx operculatus, has been shown to have antitumor effects on human carcinoma SMMC-7721 cells in vitro and in vivo. In this paper, we studied the reversal effect and the mechanism of DMC on human hepatocellular carcinoma drug-resistant cells BEL-7402/5-FU in vitro. Administration of DMC reversed the multi-drug resistance of human hepatocellular carcinoma BEL-7402/5-FU cells significantly. DMC enhanced the sensitivity of BEL-7402/5-FU cells to 5-fluorouracil (5-FU) and doxorubicin (DOX). Staining with Hoechst 33258 and flow cytometric analysis showed that DMC has apoptosis-inducing effect on BEL-7402/5-FU cells. It could also increase the concentration of 5-FU in the resistant multi-drug-resistant cells. We also observed that over-expression of the multi-drug resistance-associated protein (MRP1) and of the glutathione S-transferase π (GST-π) contributed to MDR in BEL-7402/5-FU cells. The mRNA expressions of MRP1 and GST-π and the protein expression of MRP1 were decreased by DMC. These data demonstrated that DMC could effectively reverse MDR in BEL-7402/5-FU cells.

Decreased expression of XPO4 is associated with poor prognosis in hepatocellular carcinoma

Exportin 4 (XPO4) is a recently-discovered candidate tumor-suppressor gene identified in a liver cancer mouse model. To investigate the role of XPO4 in hepatocellular carcinoma (HCC) pathogenesis, we determined XPO4 expression and its correlation to prognosis in human primary HCC. The XPO4 mRNA transcription level in HCC cell lines and tissue samples were detected by real-time quantitative polymerase chain reaction (PCR). XPO4 protein expression in 123 primary HCC clinical surgical specimens were analyzed by immunohistochemical detection. Real-time quantitative PCR showed a decrease in XPO4 expression in HCC cell lines BEL-7402, Hep-G2, and SK-hep1 compared to the normal liver cell line LO2. Decreased XPO4 mRNA was also found in the majority of tumor tissues compared with matched non-tumor liver tissues (P = 0.004). Immunohistochemical detection revealed that XPO4 expression was reduced in 51 of 123 (41.5%) tumor resection samples compared with adjunct non-tumor tissues. We also found XPO4 expression to be significantly correlated with tumor size (P = 0.045) and histopathological classification (P = 0.004). Kaplan-Meier survival curves showed that the downregulation of XPO4 resulted in a significantly poor prognosis (P = 0.008, log-rank test), and multivariate Cox's analysis showed that XPO4 expression was an independent prognostic factor for overall survival of HCC patients (P = 0.013). Our data suggest that XPO4 could be involved in the progression of human HCC and could serve as a potential target for gene therapy in the treatment of HCC.

Lipid-lowering and antioxidant activities of Jiang-Zhi-Ning in Traditional Chinese Medicine

Jiang-Zhi-Ning (JZN) is composed of four Chinese herbs, i.e., Fleeceflower Root, Fructus Crataegi, Folium Nelumbinis and Semen Cassiae. It was used to strengthen blood circulation of coronary artery, arrhythmia and hyperlipidemia. The main objective of this paper is to evaluate lipid-lowering and antioxidant activities of extract and effective fraction of JZN by using in vitro experiments on hyperlipidemic rats. Moreover, in vivo experiments on cells were performed to investigate lipid-lowering and antioxidant activities of effective fraction and active constituents of JZN. Wistar rats with high fat diet-induced hyperlipidemia were used as in vitro models to study biological effects of lipid-lowering and antioxidant activities of extract and effective fraction of JZN. Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), Coronary Index and Atherogenic Index were investigated to evaluate lipid-lowering effects of extract and effective fraction of JZN. Serum total nitric oxide synthase (NOS), nitric oxide (NO), endothelin-1 (ET-1), malondialdehyde (MDA), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) were detected to measure antioxidant effects of extract and effective fraction of JZN. Furthermore, oxidized low-density lipoprotein (Ox-LDL) injured human umbilical vein endothelial cell (HUVEC) model was employed as in vivo experiment to study lipid-lowering and antioxidant effects of effective fraction and active constituents of JZN. NO, ET-1, MDA SOD and T-AOC in HUVECs or culture media were investigated to evaluate antioxidant activity of effective fraction and active constituents of JZN. Using human hepatoma cell line Bel-7402, reverse transcription polymerase chain reaction (RT-PCR) technology was performed to investigate cholesterol metabolism effects of effective fraction and active constituents of JZN. Expressions of low density lipoprotein receptor (LDL-R), 3-hydroxy-3-methyl-HMG-coenzyme A reductase (HMG-CoAR), and cholesterol 7α-hydroxylase (CYP7A1) mRNA of the liver cells were investigated to evaluate JZN on associated receptor and enzymes of cholesterol metabolism. High-performance liquid chromatography (HPLC) and spectrophotometry were used to study the impact of effective fraction and active constituents of JZN on synthesis and translation of cholesterol during the process of metabolism by measuring inside and extracellular contents of total bile acid (TBA) of Bel-7402. Extract and effective fraction of JZN significantly reduced contents of TC, TG and LDL-C, CRI and AI in hyperlipidemic rats as well as significantly increased contents of HDL-C in the rats. Moreover, they significantly enhanced the activity of NOS and increased contents of NO. They also caused significant reductions in contents of ET-1 and MDA as well as significant increase in SOD activity and T-AOC in the hyperlipidemic rats. Several indicators were found to be concentration-dependent. As far as in vivo experiments to investigate biological activities of effective fraction and active constituents of JZN were concerned, it was found that they restored and enhanced the vitality of HUVECs with a concentration-dependent manner as well as content of NO in the culture media of HUVEC. They caused reductions in the contents of ET-1 in the culture media of HUVEC and contents of MDA in HUVECs. They also caused an increase in the vitality of SOD and T-AOC in HUVECs. Furthermore, they enhanced LDL-RmRNA expression, with a concentration-dependent manner. Low and medium concentrations of effective fraction and active constituents of JZN could inhibit expression of HMG-CoAR mRNA. High concentration counterpart could enhance expression of the HMG-CoAR mRNA. They enhanced expression of CYP7A1 mRNA in a concentration-dependent manner. Finally, they caused reductions in the contents of cholesterol in Bel-7402. They also increased intercellular content of total bile acid as well as lowered extracellular contents of TBA in the cells in a concentration-dependent manner. We demonstrated for the first time lipid-lowering and antioxidant activities of extract and effective fractions as well as active constituents of JZN. Active constituents of JZN had the same biological effects with effective fraction and extract of JZN. Therefore, this study supports its ethnopharmacological use in Traditional Chinese Medicine to manage hyperlipidemia and paves a basis for establishing quality control method of Chinese medicine.

The MicroRNA miR-139 Suppresses Metastasis and Progression of Hepatocellular Carcinoma by Down-regulating Rho-Kinase 2

We investigated mechanisms of hepatocellular carcinoma (HCC) metastasis and identified an antimetastatic microRNA (miRNA), miR-139, that is down-regulated in human HCC samples. Effects of stable and transient expression of miRNA-139 and its inhibitors were studied in the human HCC cell lines SMMC-7721 and BEL7402; cells were analyzed for migration and invasion. Liver samples from patients with metastatic HCC were analyzed for levels of miRNA-139; data were compared with survival data using the Kaplan-Meier method and compared between groups by the log-rank test. Tumor formation and metastasis from human HCC MHCC97L cells that did or did not express miR-139 were analyzed in mice. Down-regulation of miR-139 in HCC was associated significantly with poor prognosis of patients and features of metastatic tumors, including venous invasion, microsatellite formation, absence of tumor encapsulation, and reduced differentiation. miR-139 expression was reduced in metastatic HCC tumors compared with primary tumors. Overexpression of miR-139 in HCC cells significantly reduced cell migration and invasion in vitro and the incidence and severity of lung metastasis from orthotopic liver tumors in mice. miR-139 interacted with the 3' untranslated region of Rho-kinase 2 (ROCK2) and reduced its expression in HCC cells. Levels of miR-139 were correlated inversely with ROCK2 protein in human HCC samples. Overexpression of miR-139 did not inhibit HCC cell motility when ROCK2 was knocked down. The microRNA miR-139 interacts with ROCK2 and reduces its expression in HCC cells. Down-regulation of miR-139 increased the invasive abilities of HCC cells in vitro and HCC metastasis in vivo. Expression of miR-139 is reduced in human metastatic HCC samples and correlates with prognosis.

Comparison of the multi-drug resistant human hepatocellular carcinoma cell line Bel-7402/ADM model established by three methods

BackgroundTo compare the biological characteristics of three types of human hepatocellular carcinoma multi-drug resistant cell sub-lines Bel-7402/ADM models established by three methods.MethodsEstablished human hepatocellular carcinoma adriamycin (ADM) multi-drug resistant cell sub-lines models Bel-7402/ADMV, Bel-7402/ADML and Bel-7402/ADMS by three methods of in vitro concentration gradient increased induction, nude mice liver-implanted induction and subcutaneous-implanted induction respectively. Phase contrast microscopy was used to observe the cells and the MTT (methyl thiazolyl tetrazolium) method was used to detect drug resistance of the three different sub-lines of cells.ResultsThe three groups of drug resistant cells, Bel-7402/ADMV, Bel-7402/ADML and Bel-7402/ADMS generated cross-resistance to ADM and CDDP (cis-Diaminedichloroplatinum), but showed a significant difference in resistance to Bel-7402 IC50 value (P < 0.01). The doubling times were significantly extended compared to the parent cell line (39 h) and were 65 h (Bel-7402/ADMV), 46 h (Bel-7402/ADML), and 45 h (Bel-7402/ADMS). The excretion rates of ADM were significantly increased compared with the parent cell (34.14%) line and were 81.06% (Bel-7402/ADMV), 66.56% (Bel-7402/ADML) and 61.56% (Bel-7402/ADMS). Expression of P-gp and MRP in the three groups of resistant cells was significantly enhanced (P < 0.01). There was no significant variation in the expression of GSH/GST (P > 0.05).ConclusionsStable resistance was involved in the resistant cell line model established by the above three methods. Liver implantation was a good simulation of human hepatocellular and proved to be an ideal model with characteristics similar to human hepatocellular biology and the pharmacokinetics of anticancer drugs.

Synthesis and Bioactivity of N-Benzoyl-N'-[5-(2'-substituted phenyl)-2-furoyl] Semicarbazide Derivatives

In order to find novel chitin synthesis inhibitors (CSIs) with good activity, benzoylphenylurea, a typical kind of CSIs, was chosen as the lead compound and 15 novel derivatives containing furan moieties were designed by converting the urea linkage of benzoylphenylureas into a semicarbazide and changing the aniline part into furoyl groups. The title compounds were synthesized by the reaction of substituted benzoyl isocyanates with 5-(substituted phenyl)-2-furoyl hydrazine, and the structures were confirmed by IR, 1H-NMR, elemental analysis and single crystal X-ray diffraction analyses (compound E2). The bioassay results indicated that the title compounds exhibit good insecticidal activity, especially towards Plutella xylostella L., but had lower fungicidal activity. Inspiringly, the title compounds possessed obvious anticancer activity against human promyelocytic leukemic cell line (HL-60), and some of the title compounds also had activity against human hepatocellular carcinoma cell line (Bel-7402), human gastric carcinoma cell line (BGC-823), and human nasopharyngeal carcinoma cell line (KB). The results indicated that the linkage in the lead compounds was important to the bioactivity and spectra. The modification on the urea linkage is an effective strategy to discover new pesticide and drug candidates.

Syntheses, structural characterizations and properties of 12-MC-4 organotin(IV) metallacrowns: [12-MC RSn(IV)N(shi)-4] and [12-MC RSn(IV)N(Clshi)-4] (R = Et, Bu, Ph; H 3shi = salicylhydroxamic acid; H 3Clshi = 5-chlorosalicylhydroxamic acid)

Five 12-MC-4 organotin(IV) metallacrowns(MCs) with the types of [12-MC RSn(IV)N(shi)-4] (R = Et ( 1), Bu ( 2), Ph ( 3); H 3Shi = salicylhydroxamic acid) and [12-MC RSn(IV)N(Clshi)-4] (R = Et ( 4), Bu ( 5), H 3Clshi = 5-chlorosalicylhydroxamic acid) have been synthesized and characterized by elemental analyses, IR and TGA. X-ray single-crystal diffraction analyses were also carried out and showed that all complexes 1– 5 contain a neutral 12-membered metallacrown ring which is formed by the succession of four repeating units of –[Sn–N–O]–, indicating the substituents on the tin(IV) atom are uninfluential in coordination of organotin(IV) centers with hydroxamic acid. Fluorescence properties of complexes 1– 5 have been investigated, where complex 3 displays strong fluorescence emissions in the blue region. In addition, antitumor activities of complexes 4 and 5 have also been tested, and both the complexes exhibit weak activity towards human hepatocellular carcinoma cell line (Bel-7402) and Hela cell line.

Apoptosis-inducing activity of the antimicrobial peptide cecropin of &amp;lt;italic&amp;gt;Musca domestica&amp;lt;/italic&amp;gt; in human hepatocellular carcinoma cell line BEL-7402 and the possible mechanism

We studied the apoptosis-inducing properties of the antimicrobial peptide cecropin of Musca domestica in human hepatocellular carcinoma cell line BEL-7402 and its underlying mechanism. Proliferation inhibition of the human hepatocellular carcinoma BEL-7402 cells and the human normal liver cells were determined by the MTT assay, and the cell viability was determined by trypan blue dye exclusion assay. The apoptotic tumor cells treated with cecropin were examined by transmission electron microscopy and terminal-deoxynucleotidyl transferase mediated nick end labeling. The apoptosis rate was measured by flow cytometry (FCM) with PI/Annexin-V double staining. Western blot analysis and RT-PCR were used to determine the expression levels of proteins involved in apoptosis, such as Fas, Fas-L, caspase-8, and caspase-3. The experimental results showed that Musca domestica cecropin inhibited the proliferation of human hepatocellular carcinoma BEL-7402 cells in dosedependent and time-dependent manners, without affecting the proliferation of normal liver cells. FCM showed that the cell apoptosis rates were 5.1+/-0.11%, 8.1+/-0.04%, and 10.9+/-0.15% after the treating with 100 mM cecropin for 24, 48, and 72 h, respectively. The rates of apoptosis were 5.4+/-0.14% and 8.0+/-0.13% after the treating with 25 and 50 microM cecropin for 72 h, respectively. Western blot analysis and RT-PCR showed that the apoptosisrelated molecules including Fas, Fas-L, caspase-8 and caspase-3 were activated. This study showed that the antimicrobial peptide cecropin-inducing apoptosis in human hepatocellular carcinoma BEL-7402 cells, which might be associated with upregulation of Fas, Fas-L, and caspase-8 and caspase-3 and triggering extrinsic apoptotic pathway.

Influence of Recombinant Human Growth Hormone (rhGH) on Proliferation of Hepatocellular Carcinoma Cells with Positive and Negative Growth Hormone Receptorsin Vitro

Recombinant human growth hormone (rhGH) is increasingly used in the clinic because it promotes the synthesis of proteins. However, rhGH is able to increase malignant transformation and tumor recurrence. The aim of this study was to investigate the effects of rhGH on hepatocellular carcinoma (HCC) cells with positive and negative growth hormone receptors (GHR) in order to guide its clinical application. Cells of the human HCC cell lines Bel-7402 (GHR+) and SMMC-7721 (GHR-) as well as human umbilical vein endothelial cell line ECV304 cells in the exponential growth phase were harvested and divided into experimental and control groups. After the human HCC cells were cultured alone or co-cultured with ECV304 cells under the different treatments, cell cycle phase, proliferation index, and expression levels of vascular endothelial growth factor (VEGF) mRNA and proteins were determined. In the Bel-7402 GHR+ cells treated with rhGH, both the percentage of cell in G2-M phase and the proliferation index were higher than those of controls (P < 0.05); this was not the case in the SMMC-7721 GHR- cells treated with rhGH (P > 0.05). Although there was no difference in the cell doubling times between ECV304 cells co-incubated with Bel-7721 GHR-cells treated with rhGH and without rhGH, the doubling times of ECV304 cells co-incubated with Bel-7402 GHR+ cells, when treated with rhGH, were significantly shortened compared to those of controls (P < 0.05). The cell doubling times of ECV304 cells co-incubated with Bel-7721 GHR- or Bel-7402 GHR+ cells which were treated with bevacizumab were longer than those of controls and of cells with rhGH (P < 0.05). The VEGF mRNA and protein expression levels were higher in Bel-7402 GHR+ cells treated with different doses of rhGH than controls (P < 0.05 or P < 0.01); however, there was no statistically significant difference in the expression levels of VEGF mRNA and proteins between SMMC-7721 GHR- cells treated with rhGH and controls. rhGH can induce VEGF secretion and stimulate proliferation of Bel-7402 GHR+ cells in vitro, but has little effect on the proliferation of SMMC-7721 GHR-cells, suggesting that rhGH may be applied safely to treatment for the catabolic state in patients with GHR-negative HCC.

Melatonin and Doxorubicin synergistically induce cell apoptosis in human hepatoma cell lines

To investigate whether melatonin has synergistic effects with doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402. The synergism of melatonin and doxorubicin inhibited the cell growth and induced cell apoptosis in human hepatoma cell lines HepG2 and Bel-7402. Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Cell apoptosis was evaluated using TUNEL method and flow cytometry. Apoptosis-related protein Bax, Bcl-2 and caspase-3 expressions were measured by immunohistochemical staining. Treatment with melatonin (10(-8)-10(-5) mol/L) alone had a dose-related inhibitory effect on cell proliferation but no cytotoxic effect on hepatoma cell lines HepG2 and Bel-7402. Interestingly, when combined with doxorubicin, melatonin significantly increased the effects of cell growth inhibition and cell apoptosis. Furthermore, TUNEL staining and flow cytometry revealed that cooperative apoptosis induction was associated with decreased expression of Bcl-2 as well as increased expression of Bax and Caspase3. The synergism of melatonin and doxorubicin inhibits hepatoma cell growth and induces cell apoptosis.