The role of death receptor 3 in the biological behavior of hepatocellular carcinoma cells.

Death receptor 3 (DR3) belongs to the tumor necrosis factor (TNF) receptor superfamily, primarily found in lymphoid tissues. Reports have determined that DR3 may also be distributed in numerous types of tumors. Therefore, it is thought that DR3 may have an important role in the process of tumorigenesis. The aim of the present study was to observe the effect of silencing DR3 expression on hepatocarcinoma cell growth, apoptosis and invasion in order to elucidate the role of DR3 in tumor development. The hepatocarcinoma cell lines (HepG2, Huh7, SMMC7721 and Bel­7402) and normal human liver cells (HL­7702) were transfected with three stealth RNA interference (RNAi) sequences that target the DR3 gene. Reverse transcription quantitative polymerase chain reaction was used to detect the expression levels of DR3 in hepatocarcinoma cell lines and normal liver HL­7702 cells. MTT assay and flow cytometry (FCM) were used to determine the rates of cell proliferation and apoptosis, respectively. Following silencing of the DR3 gene, western blot analysis was used to determine the protein expression of P53, Fas, Caspase8, nuclear factor kappa­light­chain­enhancer of activated B cells (NF­κB) and Caspase3. DR3 messenger RNA (mRNA) expression in hepatocarcinoma cell lines was significantly increased compared with that in the normal liver cell line. Three targeted DR3 gene small interfering RNAs significantly inhibited DR3 gene expression in Bel­7402 cells at the nucleic acid level. AF02670.1_stealth_883 and cocktail demonstrated the most efficient inhibition of DR3 gene expression at 48 and 72 h following transfection, with mRNA inhibition rates of 89.46 and 92.75%, and 90.53 and 94.25% (P<0.01), respectively. Cell viability was significantly reduced by AF02670.1_stealth_883 and RNAi cocktail at 24, 48 and 72 h following transfection. The inhibition rates of cell proliferation were 50.76 and 61.76% (P<0.05) at 72 h following transfection. FCM revealed that AF02670.1_stealth_883 and RNAi cocktail also induced apoptosis in Bel­7402 cells at 72 h following transfection. Reduction of NF­κB and P53 levels was observed (P<0.05) in Bel­7402 cells following DR3 silencing, whereas levels of Fas, Caspase3 and Caspase8 were markedly elevated (P<0.05). DR3 expression levels in hepatocellular carcinoma cells were significantly higher than those in normal cells. DR3 silencing effectively inhibited proliferation and invasion of hepatocellular carcinoma cells in vitro. However, silencing of the DR3 gene affect levels of apoptosis antigen­3 ligand in cells, therefore indicating that it may be involved with other pathways that regulate apoptosis in HCCs. In conclusion, the results of the present study indicated that DR3 may be a promising therapeutic target molecule for further study of hepatocellular carcinoma gene therapy.

Anticancer potential of Euphorbia helioscopia L extracts against human cancer cells.

Euphorbia helioscopia L is considered a traditional Chinese herb which is widely distributed in China. The active anticancer fractions and anticancer mechanism of the herb are unclear. In this study, we evaluated the growth inhibitory effects of Euphorbia helioscopia L extracts on five different human cancer cell lines for screening the main active fraction with antitumor effect. In this regard, the ethyl acetate extract (EAE) was found to markedly inhibit the proliferation of SMMC-7721 cells in a time- and dose-dependent manner. EAE treatment arrested cell cycle in G-1 phase and EAE used at the concentration range of 100-200 µg/mL induced a marked increase of subdiploid peak. After EAE treatment at the concentrations of 150 and 200 µg/mL, the percentage of apoptotic cells was increased. At the EAE concentration of 200 µg/mL, the typical morphology of early apoptotic change was observed in SMMC-7721 cells. Since tumorigenesis is often defined by an uncontrolled proliferation and transplantability, we also determined the anti-invasive effects of EAE. The EAE treatment displayed a dose-dependent inhibitory effect on tumor cell invasion and MMP-9 expression. Also, the major active fraction was assayed using high-performance liquid chromatography (HPLC). The data showed that the flavonoids could be the main constituents of EAE. Based on the evidence from these data, we inferred that the EAE of Euphorbia helioscopia L could have chemopreventive potential against the human cancer.