Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells. METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin (pSuNeo-SW), were constructed by ligating into the vector, pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sail and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin-biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi. RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SW, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited. CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.

Specific COX-2 inhibitor NS398 induces apoptosis in human liver cancer cell line HepG2 through BCL-2

AIM: To evaluate the effects of NS-398, a cyclooxygenase-2 (COX-2) inhibitor, on the proliferation and apoptosis of HepG2 cells. METHODS: The effects of NS-398 on the proliferation of HepG2 cells were evaluated by MTT. DNA fragmentation gel analysis was used to analyze the apoptotic cells. DNA ploidy and apoptotic cell percentage were calculated by flow cytornetry. The expression of COX-2 and Bcl-2 mRNA was identified by competitive RT-PCR. Furthermore, expression level of Bcl-2 was detected using Western blot in HepG2 after treated with NS-398. RESULTS: NS-398 inhibited cell proliferation and induced apoptosis of HepG2 cells in a concentration-dependent manner. DNA ploidy analysis showed that S phase cells were significantly decreased with increase of NS-398 concentration. The quiescent GO/G1 phase was accumulated with decrease of Bcl-2 mRNA. Whereas NS-398 had no effect on the expression of COX-2 mRNA, and no correlations were found between COX-2 mRNA and HepG2 cell proliferation and apoptosis induced by NS-398 (r=0.056 and r=0.119, respectively). Bcl-2 protein level was inhibited after treated with NS-398. CONCLUSION: NS-398 significantly inhibits the proliferation and induces apoptosis of HepG2 cells. Mechanisms involved may be accumulation of quiescent GO/G1 phase and decrease of Bcl-2 expression.

Mechanism of apoptotic effects induced selectively by ursodeoxycholic acid on human hepatoma cell lines

AIM: To investigate the effects of ursodeoxycholic acid (UDCA) on apoptosis and proliferation of hepatoma cell lines. METHODS: Human hepatoma cell lines HepG2 and BEL 7402 were cultured in medium supplemented with different concentrations of UDCA, normal human hepatic line L-02 was used as control. Cell proliferation, apoptosis and gene expression were detected using methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, Western blot, DNA ladder assay, electron microscopy, and immunocytochemistry. RESULTS: Ursodeoxycholic acid inhibited the proli- feration of HepG2 and BEL7402 cell lines in a dose- dependent manner. Ursodeoxycholic acid can change cell cycle distribution of HepG2 and BEL7402, the proportion of cells in G0-G1 phase increased whereas the proportion of S phase cells and G2-M phase cells decreased. Ursodeoxycholic acid arrested the cell cycle in G0-G1 phase by down-regulating the cell cycle related proteins cyclin D1, D3 and retinoblastoma protein (pRb). The apoptotic rates of HepG2 and BEL7402 treated with UDCA (1.0 mmol/L) were significantly higher than those of control. In the HepG2 and BEL7402 treated with UDCA, expression of bcl-2 decreased whereas expression of Bax increased, the nuclear fragmentation and chromosomal condensed, cells shrank and lost attachment, apoptotic bodies and DNA ladders appeared. UDCA had no effect in inducing apoptosis on L-02 cell lines. CONCLUSION: UDCA can selectively inhibit proliferation and induce apoptosis of HepG2 and BEL7402 cell lines by blocking cell cycle and regulating the expression of Bax/bcl-2 genes.

Survivin antisense compound inhibits proliferation and promotes apoptosis in liver cancer cells

AIM: To evaluate the effects of survivin on cell proliferation and apoptosis in liver cancer. METHODS: MTT assay was used to generate and optimize phosphorothioate antisense oligonucleotides (ODNs)-LipofectamineTM2000 (LiP) compound by varying ODNs (μg): LiP (μL) ratios from 1:0.5 to 1:5. Then, liver cancer cells (HepG2) were transfected with the compound. By using RT-PCR and Western blot, the expression levels of survivin mRNA and proteins were detected in HepG2 cells treated with antisense compounds (ODNs:LiP=1:4), and compared with those treated with sense compounds (1:4) as control. MTT assay was applied to the determination of cell proliferation in HepG2 cells. Active caspase-3 was evaluated by flow cytometric analysis. The morphological changes were assessed by electron microscopy. Laser scanning confocal microscopy was performed to detect the subcellular localization of survivin proteins in treated and untreated cells. RESULTS: Antisense compounds (1:4) down-regulated survivin expression (mRNA and protein) in a dose-dependent manner with an IC50 of 250 nmol/L. Its maximum effect was achieved at a concentration of 500 nmol/L, at which mRNA and protein levels were down-regulated by 80%. The similar results were found in MTT assay. Antisense compound (l:4)-treated cells revealed increased caspase-3-like protease activity compared with untreated cells. Untreated cells as control were primarily negative for the presence of active-caspase-3. As shown by transmission electron microscopy, treated cells with antisense compounds (1:4) resulted in morphological changes such as blebbing and loss of microvilli, vacuolization in the cytoplasm, condensation of the cytoplasm and nuclei, and fragmented chromatin. Immunofluorescence analysis confirmed the presence of survivin protein pool inside the cytoplasm in untreated cells. Labeled-FITC immunofluorescence staining of survivin clearly showed that survivin was distributed mainly in a spotted form inside the cytoplasm. Whereas cells treated with antisense compounds were rare and weak inside the cytoplasm. CONCLUSION: Down-regulation of survivin expression induced by the antisense compounds reduces tumor growth potential, promotes apoptosis and affects the localization of survivin proteins in HepG2 cells. Furthermore, survivin protein is a key molecule associated with proliferation and apoptosis, and antisense oligonucleotides targeting survivin have a bright prospect in the therapy of liver cancer.

Effects of thalidomide on angiogenesis and tumor growth and metastasis of human hepatocellular carcinoma in nude mice

AIM: To investigate the effects of thalidomide on angiogenesis, tumor growth and metastasis of hepatocellular carcinoma in nude mice.METHODS: Twenty-four nude mice were randomly divided into therapy group and control group, 12 mice in each group. Thalidomide dissolved in 0.5% sodium carboxyl methyl cellulose (CMC) suspension was administered intraperitoneally once a day at the dose of 200 mg/kg in therapy group, and an equivalent volume of 0.5% CMC in control group. Mice were sacrificed on the 30th d, tumor size and weight and metastases in liver and lungs were measured. CD34 and VEGF mRNA in tumor tissue were detected by immunohistochemistry and semi-quantitative RT-PCR respectively and microvessel density (MVD) was counted. Serum concentrations of TNF-α and ALT and AFP were also tested.RESULTS: MVD and VEGF mRNA in therapy group were less than those in control group (31.08±16.23 vessels/HP vs 80.00±26.27 vessels/HP, 0.0538±0.0165 vs 0.7373±0.1297,respectively, P＜0.05). No statistical difference was observed in tumor size and weight and metastases in liver and lungs.TNF-α was significantly lower in therapy group than in control group (28.64±4.64 ng/L vs42.69±6.99 ng/L, P＜0.05). No statistical difference in ALT and AFP was observed between groups.CONCLUSION: Thalidomide can significantly inhibitangiogenesis and metastasis of hepatocellular carcinoma.Italso has inhibitory effects on circulating TNF-α.

Clinical significance of the expression of isoform 165 vascular endothelial growth factor mRNA in noncancerous liver remnants of patients with hepatocellular carcinoma

AIM: To investigate the prognostic role of isoform 165 vascular endothelial growth factor messenger RNA (VEGF165 mRNA)in noncancerous liver tissues from patients with primary hepatocellular carcinoma (HCC).METHODS: Using a reverse-transcription polymerase chain reaction (RT-PCR)-based assay, VEGF mRNA was determined prospectively in noncancerous liver tissues from 60 consecutive patients with HCC undergoing curative resection. We categorized the patients with VEGF165 mRNA over 0.500 in noncancerous liver tissues as group A, and those below 0.500 as group B.RESULTS: Among the isoforms of VEGF mRNA by multivariate analysis, a higher level of VEGF165 mRNA in noncancerous liver tissue correlated significantly with a higher risk of HCC recurrence (P = 0.039) and recurrence-related mortality (P= 0.048), but VEGF121 did not. The other significant predictors of recurrence consisted of vascular permeation (P = 0.022),daughter nodules (P = 0.033), cellular dedifferentiation (P = 0.033), an absent or incomplete capsule (P = 0.037).A significant variable of recurrence-related mortality was Vascular permeation (P= 0.012). As to the clinical manifestations of 16 patients who developed recurrence,the recurrent tumor number over 2, recurrent extent over two-liver segments, and the median survival after recurrence,all significantly correlated with group A patients (P = 0.043,0.043, and 0.048, respectively). However, the presence of extrahepatic metastasis was not (P＞0.05). The difference in recurrence after treatment between the two groups had no statistical significance (P＞0.05).CONCLUSION: The higher expression of isoform VEGF165mRNA in noncancerous liver remnant of patients with HCC may be a significant biological indicator of the invasiveness of postoperative recurrence.

Differential expression of βig-h3 in human hepatoma cell lines

Objective：To observe the differential expression of TGF-β-induced gene human clone 3 （βig- h3） in human hepatoma cell lines. Methods：Human hepatoma cells HHCC, 7721, T7721 constructed by stably transfectiug HAb18G/CD147 cDNA into 7721 and normal human liver cell QZG were cultured as previously. RT-PCR and western blot were used to investigate the differential expression of βig-h3 in human hepatoma cell lines and normal liver cell. Results ：The results of RT-PCR suggested that the expression of βig-h3 mRNA in human hepatoma cells was higher than that in normal human liver cell QZG（P〈0.01）, and its expression level in human hepatoma cells in turn was HHCC〉T7721〉7721. Moreover, the similar results of βig-h3 protein expression were testified by western blot. Conclusion：This study demonstrates that the expression of βig-h3 in hepatoma cell lines is higher than that in normal liver cell QZG, which provides a sound basis for exploring the function of βig-h3 in processes of adhesion and metastasis of human hepatoma cells.

Antisense EGFR sequence enhances apoptosis in a human hepatoma cell line BEL-7404

Effects of antisense epidermal growth factor receptor (EGFR) sequence on apoptotic cell death were examined in a human hepatoma cell line BEL-7404 cells. In the cells of JX-1, a sub clone of BEL-7404 stably transfected with antisense EGFR vector (Cell Research, 3:75, 1993), an enhanced rate (9.5%) of spontaneous apoptosis was detected by flow cytometry, whereas the rates of spontaneous apoptosis in JX-0 cells, a sub-clone of BEL-7404 transfected by control vector, and the parellt BEL-7404 cells were almost equal and about 1.7%. Serum-starvation for 72 h increased the rate of apoptosis of JX-1cells up to 33.7%, while JX-0 and BEL-7404 cells, under the same condition, produced less than 5% of apoptotic cells. Observation with electron microscope demonstrated that condensation and fragmentation of chromatin and formation of apoptotic bodies of ten occurred in JX-1 cells, especially during serumstarvation. These results, combined with the data of DNA fragmentation Elisa test, suggested that antisense EGFR sequence enhances apoptosis in the human hepatoma cells.Comparison of intracellular Ca2+ level and the responsiveness of JX-1 cells to the induced action of EGF and tharpsigargin (TG) treatment with that of control JX-0cells indicated that antisense EGFR might interrupt the EGF/EGFR signaling pathway resulting in the decreass of intracellular Ca2+ pool content as well as the responsiveness of these cells to the extracellular signals. These findings suggest that antisense EGFR either directly or indirectly reglllates Ca2+ storage in endoplasmic reticulum,thereby enhances apoptosis in the hnman hepatoma cells.