Effect of lentivirus-mediated shRNA targeting VEGFR-3 on proliferation, apoptosis and invasion of gastric cancer cells.

It has been reported that vascular endothelial growth factor receptor 3 (VEGFR-3) is highly expressed in most tumor tissues, including gastric cancer. However, the effects of VEGFR-3 knockdown on the proliferation, apoptosis and invasion of gastric cancer cells and downstream signaling molecules have not yet been well established. In the present study, four short hairpin RNA (shRNA) sequences targeting the VEGFR-3 gene (NM_002020) were designed and cloned into a lentiviral vector, pRNAT-U6.2/Lenti, to construct four recombinant lentiviral vectors. The vectors with the two highest interfering efficiencies were selected to be co-transfected with packaging vectors in HEK293T cells to assemble lentivirus particles. Results from Western blot analysis showed that the VEGFR-3 shRNA-4 lentivirus-infected group (sh#4) had the highest efficiency of gene silencing in the MKN45 cell line compared with the parental and control group. The sh#4 group significantly slowed cell proliferation, decreased the mean percentage of S-phase cells and increased the mean percentage of G1 phase cells, promoted cell apoptosis, and also significantly inhibited cell invasion of MKN45 compared with the other two groups. Furthermore, the expression of the anti-apoptotic factor Bcl-2 was significantly decreased in the sh#4 group compared to that of the other two groups. Moreover, results from qRT-PCR revealed that knockdown of VEGFR-3 with the shRNA lentiviral vector resulted in down-regulation of the downstream neural cell adhesion molecule contactin-1 (CNTN-1). In conclusion, the recombinant lentivirus particles were able to remarkably suppress VEGFR-3 expression, regulate the cell cycle, inhibit proliferation and induce apoptosis in the MKN45 cell lines.

[Therapeutic effect of liver transplantation on acute liver failure].

OBJECTIVE: To evaluate the short-term and long-term outcomes of emergent liver transplantation recipients with acute liver failure and to identify factors that influenced these outcomes. METHODS: 318 consecutive patients who underwent liver transplantations between January 2001 and December 2004 were analyzed retrospectively (all the cases were followed up to December 2005). According to UNOS grading scale, all recipients preoperative status were evaluated. 54 patients were acute liver failure (Group I, UNOS 1 and 2A), and the other 264 cases were chronic liver diseases (Group II, UNOS 2B and 3). The postoperative effects in different groups were compared, including the survival rates, incidences of complications, rates and causes of retransplantation, rates and causes of death. RESULTS: Comparing UNOS2B/3 to UNOS1/2A, the perioperative mortality were 3.7%; 22.6%, the rate of complications 16.7%; 55.6%, 1 year survival rate 91.3%; 74.1%, 3 year survival rate 86.4%; 68.5%, the retransplantation rate 1.1%; 18.5% respectively. CONCLUSION: Since the technique of liver transplant is very advanced, the effect of surgery is mainly depended on the function of liver and other organs in patients. The recipients with UNOS2B/3 have better short-term and long-term outcomes as comparing to UNOS1/2A. In addition, the recipients with UNOS1/2A are burdened with much higher mortality.

Chemoresponse to docetaxel correlates with expression of the survivin splicing variants in patients with gastric cancer.

BACKGROUND/AIMS: Survivin, a potential predictive marker to chemotherapeutic drugs, reduces the susceptibility of tumor cells to proapoptotic stimuli, thereby promoting tumor cell survival during tumor treatment with anticancer agents. In the present study, we examined the correlation between drug-response and expression of survivin in gastric cancer. METHODOLOGY: Drug-response was performed by histoculture drug-response assay (HDRA) in 42 patients with advanced gastric cancer. Survivin variants was studied by real-time RT-PCR at mRNA levels. RESULTS: In our data, twenty-one cases (50%) were sensitive to at least one of drugs tested in the HDRA and 80% (16/20) were positively confirmed clinically, with 100% sensitivity and 83.3% specificity. The diagnostic efficacy of the HDRA were calculated as the area under the receiver operating characteristic (AUC-ROC) curve and showed a value of 0.917. More importantly, significant statistical differences (p=0.012) in chemoresponse to docetaxel were revealed depending on the mRNA level of wild-type survivin. CONCLUSIONS: These clinical data demonstrated, for the first time, that wild-type survivin is useful for evaluating the docetaxel-response in patients with gastric cancer. Moreover, the HDRA is an excellent clinical useful drug-response assay for patients to individualize their chemotherapy.

[Prognostic significance of survivin splicing variants in expression gastric cancer].

OBJECTIVE: To study the expression and associations of three survivin splicing variants in gastric cancer and normal gastric mucosa, and to evaluate the prognostic significance. METHODS: Real time quantitative RT-PCR was used to detect the expression of three survivin splicing variants in tumor and matched normal gastric mucosa specimens from 77 cases with gastric cancer. RESULTS: The expression of three survivin splicing variants than upregulated significantly in gastric cancer than those in normal mucosas (P< 0.01). In cancer tissues, the expression rates of survivin, survivin-2B, survivin-deltaEx3 were 100%, 79.8% (61/77), 64.9% (50/77) respectively. The survival rate was significantly lower in the patients with high survivin expression than those with low survivin expression (P< 0.01). CONCLUSION: Among three survivin splicing variants, the expression level of wild-type survivin mRNA is an important predictor for prognosis.

[Effect of inhibiting survivin expression with antisense oligodeoxynucleotides on sensitivity of hepatocellular carcinoma cell lines HepG2 and HepG2/ADM to adriamycin].

BACKGROUND & OBJECTIVE: Survivin deserves attention as a selective target for cancer therapy because it is silenced in differentiated adult tissues, but is expressed in a variety of human tumors, and is involved in tumorigenesis and chemoresistance. Antisense oligodeoxynucleotides (ASODN) can be used to inhibit the expression of survivin to induce apoptosis or enhance chemosensitivity of tumor cells. This study was to investigate the effect of inhibiting survivin expression with ASODN on sensitivity of hepatocellular carcinoma cell lines HepG2 and HepG2/ADM to Adriamycin (ADM). METHODS: The expression of survivin in HepG2 and HepG2/ADM cells was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Sensitivities of HepG2 and HepG2/ADM cells to ASODN and ADM were detected by MTT assay. ASODN was transfected into HepG2 and HepG2/ADM cells; expression of survivin was detected by RT-PCR. Synergetic effects of low concentrations of ASODN and subtoxic concentrations of ADM on HepG2 and HepG2/ADM cells were detected by isobolography. The expression of active Caspase-3 and cell apoptosis were evaluated by flow cytometry. RESULTS: mRNA level of survivin in HepG2/ADM cells was 15 folds as that in HepG2 cells; protein level of Survivin in HepG2/ADM cells was 18 folds as that in HepG2 cells. Both HepG2 and HepG2/ADM cells were sensitive to ASODN-mediated cytotoxicity in a concentration-dependent manner. The 50% inhibitory concentration (IC(50)) of ASODN was 317.90 nmol/L for HepG2 cells, and 480.74 nmol/L for HepG2/ADM cells. The maximal cytotoxicity was observed at 500 nmol/L of ASODN; the inhibitory rate was 71.10% in HepG2 cells, and 53.67% in HepG2/ADM cells. The IC(50) of ADM was 0.36 microg/ml for HepG2 cells, and 2.12 microg/ml for HepG2/ADM cells; the resistance index was 6. ASODN efficiently down-regulated mRNA level of survivin in both cell lines in a concentration-dependent manner. For HepG2 cells, with the IC(50) of 271.93 nmol/L, the maximal effect of ASODN was achieved at a concentration of 400 nmol/L, at which mRNA level of survivin was down-regulated by 69.12%; for HepG2/ADM cells, with the IC(50) of 365.72 nmol/L, its maximal effect was achieved at a concentration of 400 nmol/L, at which mRNA level of survivin was down-regulated by 60.01%. ASODN in combination with ADM synergetically enhanced sensitivity of HepG2 cells to ADM by 6 folds, and HepG2/ADM cells by 4 folds. Combination treatment of ASODN and ADM gradually enhanced activity of Caspase-3 and cell apoptosis in a concentration-dependent manner, and resulted in caspase-dependent cell death in HepG2/ADM cells. CONCLUSION: Inhibiting survivin expression with ASODN could sensitize hepatocellular carcinoma cells to ADM, and the combination of ASODN and ADM may be a reasonable approach for clinical treatment of ADM-resistant hepatocellular carcinoma.

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)-Lipofectamine2000 (LiP) compound by varying ODNs (mug):LiP (muL) 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 (1:4)-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.

[The effect of antisense survivin-liposome complex on cell growth, apoptosis and cell cycle in hepatocellular carcinoma cells].

OBJECTIVE: To investigate the effects and the mechanisms of cell growth inhibition in hepatocellular carcinoma cells after induction with antisense survivin-liposome (LIP) complex, and to provide evidence in treatment for hepatocellular carcinoma and tumors expressing survivin. METHODS: Survivin ODNs was transfected into HepG2 cells mediated by LiP reagent. The expression of survivin mRNA and protein was detected by RT-PCR and Western blot. MTT assay was applied to determine cell proliferation in HepG2 cells. Active caspase-3 and apoptosis rate were evaluated by flow cytometric analysis. The morphological changes were assessed by electron microscopy. Cell cycle was analyzed by flow cytometry in the cell cycle-synchronized hepatocellular carcinoma cells treated with the antisense compound. RESULTS: Antisense compound efficiently down-regulated survivin expression (mRNA and protein) in a dose-dependent manner with an IC(50) of 250 nmol/L. Its maximal effect was achieved at a concentration of 600 nmol/L, when expression levels were down-regulated by 80%, as revealed by gradually increase of caspase-3-like protease activity and apoptosis rate in a time-dependent manner. Morphological apoptotic changes such as membrane blebbing, loss of microvilli, cytoplasmic vasculization, condensation of cytoplasm and nucleus, chromatin fragmentation, and apoptosis and cell growth inhibition were observed. In the cell cycle-synchronized hepatocellular carcinoma cells, antisense compound induced cell cycle arrest followed by apoptosis. After treated with low concentration of compound, the cell cycle was arrested at S phase or G2/M phase; while at high concentration, the cell cycle was mainly arrested at S phase. Apoptosis was obviously observed and the rate of apoptosis was increased in a time and concentration-dependent manner. CONCLUSION: Antisense survivin has significant inhibitory effect on growth of hepatocellular carcinoma cells in vitro. This is associated with cell cycle arrest and apoptosis.

Expression level of wild-type survivin in gastric cancer is an independent predictor of survival.

AIM: Survivin is a novel antiapoptotic gene in which three splicing variants have been recently cloned and characterized. Survivin has been found to be abundantly expressed in a wide variety of human malignancies, whereas it is undetectable in normal adult tissues. We aimed to study the expression of three survivin splicing variants in gastric cancer, and to evaluate the prognostic significance of the expression of survivin variants in gastric cancer. METHODS: Real time quantitative RT-PCR was performed to analyze the expression of survivin variants in 79 paired tumors and normal gastric mucosa samples at the mRNA level. Proliferative and apoptotic activity was measured using Ki-67 immunohistochemical analysis and the TUNEL method, respectively. RESULTS: All the cases tested expressed wild-type survivin mRNA, which was not only the dominant transcript, but also a poor prognostic biomarker (P = 0.003). Non-antiapoptostic survivin-2B mRNA was correlated with tumor stage (P = 0.001), histological type (P = 0.004), and depth of tumor invasion (P = 0.041), while survivin-DeltaEx3 mRNA showed a significant association with apoptosis (P = 0.02). CONCLUSION: Wild-type survivin mRNA expression levels are of important prognostic value and significant participation of survivin-2B and survivin-DeltaEx3 is suggested in gastric cancer development.

Effects of daidzein on estrogen-receptor-positive and negative pancreatic cancer cells in vitro.

AIM: To study the effects of daidzein on human pancreatic cancer cells in vitro. METHODS: Human estrogen-receptor (ER)-positive pancreatic cancer cells MiaPaCa-2 and ER-negative pancreatic cancer cells PANC-1 were treated by 0.1 micromol/L, 1 micromol/L, 10 microL, 25 microL, 50 microL, 75 microL and 100 microL of daidzein, respectively. Its antiproliferative effect was studied by MTT assay. RESULTS: Daidzein inhibited the growth of MiaPaCa-2 and PANC-1 cells at the concentrations from 0.1 microL to 100 microL. A dose- and time-dependent manner was found. The IC(50) of daidzein on MiaPaCa-2 and PANC-1 cells was 45 microL and 75 micro, respectively. After MiaPaCa-2 cells were treated by daidzein for 3 d and at the concentrations more than IC(50), the inhibitory manner was identical and the inhibition appeared a saturation phenomenon, but the inhibitory manner of daidzein on PANC-1 cells was different from that of MiaPaCa-2 cells. CONCLUSION: Daidzein has antiproliferative effects on human estrogen-receptor-positive and negative pancreatic cancer cells, but their mechanisms may be different.

[Elevated expression level of wild-type survivin in gastric cancer promotes in vitro docetaxel-resistance].

OBJECTIVE: To study the expression of three survivin splicing variants in gastric cancer and to evaluate the significant correlation between survivin variants' expression and chemoresistance in gastric cancer. METHODS: Real time quantitative RT-PCR was used to analyze the mRNA expression of survivin variants in 39 gastric tumor specimens resected during operation. The clinical resistance to anticancer agents [CDDP, MMC, 5-Fu, docetaxel (Taxotere TXT), and GEM] was analyzed by histoculture drug-response assay (HDRA). RESULTS: Among the 39 tumor samples, survivin expression was detected in all tumor samples (39/39); 79.5% (31/39) of the samples demonstrated survivin-2B expression and 66.7% (26/39) of the samples had survivin-Delta Ex3 expression. HDRA showed that the in vitro efficacy rates of CDDP, MMC, 5Fu, TXT, and GEM were 36.8% (14/38), 31.2% (10/32), 23.1% (9/39), 20.5% (8/39), and 12.5% (4/32) respectively, equivalent to the previous HDRA studies and historical clinical studies in gastric cancer patients. The expression rate of wild-type survivin was significantly higher in the group of chemoresistance to TXT than in the group sensitive to docetaxel (P = 0.021). CONCLUSION: Elevated expression level of wild-type survivin promotes docetaxel-resistance in patients with gastric cancer.

[Effect of survivin targeting on cell proliferation and apoptosis in pancreatic cancer cells].

OBJECTIVE: To investigate the effects of antisense survivin-Lip compound on pancreatic cancer cell proliferation and apoptosis and its mechanism. METHODS: Pancreatic cancer cells of the line PANC-1 were cultured. Survivin oligonucleotide (ODN) was transfected into the PANC-1 cells mediated by Lip reagent. The expression of survivin mRNA and that of surviving protein were detected by RT-PCR and Western blotting. MTT assay was applied to determine the proliferation of PANC-1 cells. Active caspase-3 and apoptosis rate were evaluated by flow cytometric analysis. The morphological changes were assessed by electron microscope. Lascar scanning confocal microscope immunofluorescence analysis was performed to detect the subcellular localization of survivin protein on treated cells and untreated cells. RESULTS: Antisense compound efficiently down-regulated the survivin expression (mRNA and protein) in dose-dependent manner with an IC50 of 300 nmol/L. Its maximum effect was achieved at the concentration of 500 nM, at which the expression level was down-regulated by 80%. The similar results were found in MTT assay. As revealed by gradually increased caspase-3-like protease activity and apoptosis rate in a time-dependent manner, and the morphological changes of apoptosis such as blebbing and loss of microvilli, vacualization in cytoplasm, condensation of cytoplasm and nucleus, and fragmented chromatin, treatment with antisense compound induced apoptosis and inhibited cell growth. Fluororescein isothiocyanate (FITC)-labeled immunofluorescence staining of survivin clearly showed that survivin was expressed mainly in the formation of a spotted distribution inside the cytoplasm of untreated cells. Survivin protein molecules were clearly seen in the cytoplasm of the untransfected cells and distributed like spots and almost disappeared in the transfected cells with morphological changes conforming to the changes of apoptosis. CONCLUSION: Survivin protein is a key molecular connecting proliferation with apoptosis and antisense oligonucleotides targeting survivin have a bright prospect in therapy of pancreatic cancer cells.