Transfer of siRNA against XIAP induces apoptosis and reduces tumor cells growth potential in human breast cancer in vitro and in vivo.

BACKGROUND: Gene targeting using short interfering RNA(siRNA) has become a common strategy to explore gene function because of its prominent efficacy and specificity. It is proven that the application of siRNA technology to gene therapy is effective. In this study, we constructed a siRNA expression plasmid against gene X-linked inhibitor of apoptosis (XIAP), and then used breast cancer cells MCF-7 to assess its functions. MATERIALS AND METHODS: XIAP siRNA plasmid was constructed using an U6pro vector contained U6 promoter, After the plasmid had been transfected into MCF-7 cells and effected on the cell cycle, the expression change of XIAP was examined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The apoptosis of the transfected cells was analyzed by flow cytometry, and TUNEL method. The in vitro cellular growth activities were assayed by MTT incorporation. Twenty-four nude mice were randomly divided into 3 equal groups and were inoculated with electroinjection of blank plasmid, scrambled nucleotide control (control siRNA), or siRNA against XIAP subcutaneously respectively, then the appearance and size of tumors were observed. Four weeks later the mice were killed and the volumes of tumor were calculated so as to evaluate the therapeutic effects of siRNA against XIAP. RESULTS: The successful construction of siRNA against XIAP plasmid was identified with sequencing. After the siRNA expression vector was transfected into the MCF-7 cells, the expression of XIAP gene was inhibited significantly (by 90%). The cellular growth activities in the MCF-7 cells transfected with siRNA against XIAP plasmid decreased obviously. The siRNA against XIAP plasmid knocked down XIAP expression in MCF-7 cells obviously, arrested the cell cycle in G1 phase, inhibited cell proliferation significantly, and promoted cell apoptosis in a tendency. TUNEL assay and flow cytometry showed that the classic apoptosis characters of the MCF-7 cells transfected with siRNA against XIAP plasmid manifested an apoptosis rate of 77.2%, significantly higher than those in the control siRNA group and in the blank plasmid group (both p < 0.01). The growth speed and formation rate of xenograft tumor in mice transfected with siRNA against XIAP transfected mice slowed down significantly. By HE staining, a lot of necrotic tissues could be observed in the siRNA against XIAP transfected group, however, there was no similar inhibitive effect in the control siRNA or blank plasmid group. CONCLUSION: This study represents that MCF-7 transfected cells with siRNA against XIAP remarkably suppress tumor growth and induces apoptosis, both in vitro and in vivo. This novel modality may be a promising tool for cancer therapy.

Expressions of inducible nitric oxide synthase and matrix metalloproteinase-9 and their effects on angiogenesis and progression of hepatocellular carcinoma.

AIM: To determine the expressions of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase-9 (MMP-9) in hepatocellular carcinoma (HCC) and to investigate the relationship between iNOS and MMP-9 expression and their effects on angiogenesis and progression of HCC. METHODS: In this study, we examined iNOS, MMP-9, and CD34 expression in specimens surgically removed from 32 HCC patients and 7 normal liver tissues by immunohistochemical staining. Meanwhile, microvessel density (MVD) was determined as a marker of angiogenesis by counting CD34-positive cells. RESULTS: The positive rates of iNOS and MMP-9 expression were 71.88% (23/32) and 78.13% (25/32) in HCC. MMP-9 expression was significantly correlated with tumor size, capsule status, TNM stage, and risk of HCC recurrence (P = 0.032, P = 0.033, P = 0.007, and P = 0.001, respectively). There was also a significant relationship between iNOS expression and capsule status and risk of HCC recurrence (P = 0.049 and P = 0.004, respectively), but no correlation between iNOS expression and tumor size and TNM stage. There was a positive association between MVD and TNM stage and risk of HCC recurrence (P = 0.037 and P = 0.000, respectively). The count of MVD was significantly different in different iNOS and MMP-9 immunoreactivity groups (F = 17.713 and 17.097, P = 0.000 and P = 0.000, respectively). The examination of Spearman's rank correlation coefficient showed that there was a significant positive correlation between MVD and iNOS, MMP-9 immunoreactivity (r = 0.754 and 0.751, P = 0.000 and P=0.000, respectively). There was also a significant association between MMP-9 and iNOS expression in HCC (P = 0.010). CONCLUSION: Nitric oxide (NO) produced by iNOS could modulate MMP-9 production and therefore contribute to tumor cell angiogenesis and invasion and metastasis in HCC. The strong expression of iNOS and MMP-9 in HCC may be helpful in evaluating the recurrence of HCC, predicting poor prognosis. For patients with strong expression of MMP-9 and iNOS, the optimal treatment scheme needs to be selected.

Expression of TIMP-3 gene by construction of a eukaryotic cell expression vector and its role in reduction of metastasis in a human breast cancer cell line.

The present study is aimed at studying the gene for TIMP-3, a mammalian tissue inhibitor, by constructing a recombinant eukaryotic cell vector for gene therapy in human breast cancer. We obtained the TIMP-3 gene from the human placent by RT-PCR. TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vector by means of gene cloning to construct pcDNA3.1 recombinant vector. Human breast cancer cell line MDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent. Then the expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined. The correct construction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis, PCR amplication and nucleotide sequencing. Western blotting showed that the transfected cells were able to express TIMP-3, indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructed successfully. Our experiments further indicated that the potential of metastasis was significantly reduced for the transfected cell line MDA-MB-453.

Clinical evaluation of serum interleukin 10 in patients with acute pancreatitis.

OBJECTIVES: To evaluate the behavior of serum interleukin 10 (IL-10) in patients with acute pancreatitis and to explore the relationship between this cytokine and the severity of the disease. METHODS: In 45 patients with acute pancreatitis, the serum concentrations of IL-10 was determined on days 1, 2, 3, 4, 5 after admission. Twelve healthy subjects were also studied as controls. These subjects were tested using a commercial ELISA kit. The severity of pancreatitis was determined according to APACHE II score and Balthazar CT criteria. RESULTS: Healthy subjects had no detectable serum levels of IL-10. In acute pancreatitis patients, the serum IL-10 levels were increased on the first day after the onset of the disease and then progressively decreased in the following days. On the first day after the onset of acute pancreatitis, the serum levels of IL-10 in patients with mild acute pancreatitis were significantly higher than in those with severe acute pancreatitis. In the following days, however, no statistically significant difference was observed between the two groups. CONCLUSIONS: Serum IL-10 concentration reflects the severity of acute pancreatitis. IL-10 is a useful variable for early prediction of the prognosis of acute pancreatitis. The low values of serum IL-10 in patients with severe acute pancreatitis suggests that there may be altered down-regulation of immune system response. An enhanced release of IL-10 may be a method for early treatment of acute pancreatitis.

Surgical mortality in patients with malignant obstructive jaundice: a multivariate discriminant analysis.

OBJECTIVE: To estimate the operative mortality in patients with malignant obstructive jaundice. METHODS: Twelve risk factors were analyzed using multivariate discriminant analysis in 90 patients who had been operated on. RESULTS: Operative mortality was significantly related to the following factors: age, duration of jaundice, packed RBC volume, white blood cell count and concentration of blood urine nitrogen; it was not significantly related to diseases and types of operation. The following formula was obtained: packed RBC volume x 0.09954-age x 0.04018- blood urine nitrogen x 0.23693-duration of jaundice x 2.07388-WBC count x 0.21118+ 5.26593. With this formula, an operative mortality of 77.8% was predicted. CONCLUSION: With a positive value from the formula, the patient should be operated on; otherwise non-operative treatment is advocated.