[Biological behaviors and chemosensitivity of NSCLC A549 cells after IGF-IR gene silencing by targeting RNAi in vitro].

OBJECTIVE: To investigate the biological behaviors and chemosensitivity of non-small cell lung cancer (NSCLC) cell line A549 after IGF-IR gene silencing by RNA interference (RNAi) in vitro. METHODS: Two plasmids siRNA 1 and 2 expressing IGF-IR siRNA with human U6 promoter were constructed,and an unrelated siRNA was used as negative control. NSCLC A549 cells were transfected with sequence-specific siRNA or unrelated siRNA as control. Quantitative RT-PCR and Western blot were used to detect the expression of IGF-IR. NSCLC A549 cells were transfected with siRNA and treated with DDP. MTT assay and flow cytometry were used to assess the effects of IGF-IR silencing on tumor cell proliferation and chemosensitivity. RESULT: Transfection of NSCLC cells with siRNA resulted in reduction of IGF-IR mRNA expression by 78.9 % and protein production by 89.8%. The decrease in IGF-IR levels caused significant growth inhibition of A549 cells both at 48 h and at 72 h, and decrease of the IC50 of DDP at 24 h, 48 h and at 72 h. Flow cytometry showed that 77.5% of A549 cells retained in G0/G1 phase. CONCLUSION: The sequence specific suppression of IGF-IR gene expression by RNAi enhances sensitivity to DDP in NSCLC cell.

[shRNA-mediated insulin-like growth factor I receptor gene silencing inhibits cell proliferation, induces cell apoptosis, and suppresses tumor growth in non-small cell lung cancer: in vitro and in vivo experiments].

OBJECTIVE: To study the effects of RNA interference (RNAi)-mediated insulin-like growth factor I receptor (IGF-IR) gene silencing on human lung cancer cells. METHODS: Plasmids expressing IGF-IR shRNA1 and IGF-IR shRNA2 were constructed. Human non-small cell lung cancer cells of the line A549 were cultured and transfected with sequence-specific shRNA. RT-PCR was used to monitor the IGF-IR mRNA expression. Western blotting was used to detect the expression of IGF-IR, bcl-2 and caspase-3, associated with apoptosis, and IGF-IR signaling pathways-associated proteins, total and phospho-ERK1/2 and Akt. MTT assay and flow cytometry were used to examine the cell activity and cell cycle. Twelve nude mice were injected subcutaneously with A549 cells, 20 days later the mice were randomly divided into 3 groups to be injected into the tumor with IGF-IR, PBS, or blank plasmid respectively 4 times with the interval of 5 days. Five days after the 4th injection the mice were killed and the tumors were taken out. TUNNEL assay was used to detect the apoptotic cell in the tumor. RESULTS: RT-PCR showed that the IGF-IR mRNA expression level of the A549 cells transfected with IGF-IR shRNA1 was only 24% +/- 4% that of the A549 cells transfected with blank plasmid (P < 0.05); however, the IGF-IR mRNA expression level of the A549 cells transfected with IGF-IR shRNA2 was 78% +/- 5% that of the A549 cells transfected with blank plasmid (P > 0.05). The IGF-IR protein expression level of the A549 cells of the IGF-IR shRNA1 group was only 10.2% +/- 2.8% that of the A549 cells of the blank plasmid group (P < 0.05). Western blotting showed that the protein expression levels of bcl-2 and caspase-3p20 of the A549 cells of the IGF-IR shRNA1 group were 46% +/- 6% and 156% +/- 8% those of the negative controls (both P < 0.05); however, the protein expression levels of bcl-2 and caspase-3p20 of the A549 cells of the IGF-IR shRNA2 group were not different from those of the negative control cells. The Akt kinase and ERK phosphorylation levels of the A549 cells of the IGF-IR shRNA1 group were 10% and 36% +/- 3% those of the negative control cells respectively (both P < 0.05). Since 48h after the transfection the active cell number of the IGF-IR shRNA1 group was 64% +/- 7% that of the negative group (P < 0.05), and this decrease effect lasted to 72 h after (67% +/- 6% that of the negative cells, P < 0.05). 48 h after the transfection the percentage of cells at G(0)/G(1) phase of the IGF-IR shRNA1 group was 77.5%, significantly higher than that of the negative control group, and the percentages of the cells at S and G(2)/M phases of the IGF-IR shRNA1 group were 15.7% and 7.3% respectively, both significantly lower than those of the negative control group (23.0% and 29.9% respectively). Since the second injection the tumor size of the mice of IGF-IR shRNA group was 40% - 50% that of the PBS group (P < 0.05), and the tumor size of the mice of the PBS group was 90% that of the control group. TUNNEL assay showed that the number of apoptotic cells in the tumors of the IGF-IR shRNA1 group mice was 118 +/- 8/high power, significantly higher than that of the control group (70 +/- 9, P < 0.05). CONCLUSION: RNAi technique effectively inhibits the expression of IGF-IR, thus decreasing the NSCLC cell proliferation inducing apoptosis and inhibiting the tumor growth.

Down-regulation of IGF-IR using small, interfering, hairpin RNA (siRNA) inhibits growth of human lung cancer cell line A549 in vitro and in nude mice.

Type I insulin-like growth factor receptor (IGF-IR), which is frequently overexpressed in a variety of human cancers including lung cancer, mediates cancer cell proliferation and tumor growth. In this study, we used a human U6 promoter-driven DNA-template approach to induce hairpin RNA (hpRNA)-triggered RNAi to silence IGF-IR gene expression in the human lung cancer cell line A549, and then evaluate its effects on apoptosis, apoptosis-related gene expression, and the growth of tumor cells in vitro and in nude mice. IGF-IR expression levels were found to markedly decrease in cells transfected with a plasmid expressing hairpin siRNA for IGF-IR (by more than 78.9%). Down-regulation of IGR-IR concomitantly accompanied reduction of bcl-2 as well as pERK and pAkt levels, activation of caspase-3, apoptosis and growth inhibition of A549 cells in vitro. Direct intratumoral injections of plasmid DNA expressing hpRNA for IGF-IR significantly regressed pre-established tumors in nude mice. Our results support the therapeutic potential of RNAi as a method for gene therapy in treating lung cancer.

Pretreatment with aminophylline reduces release of Troponin I and neutrophil activation in the myocardium of patients undergoing cardioplegic arrest.

OBJECTIVE: Cardioplegic arrest and subsequent reperfusion results in myocardial injury partly related to local inflammation in the heart. It has been proven that aminophylline has numerous anti-inflammatory effects. This study has been designed to evaluate the effects of aminophylline used as a cardioprotective agent for patients undergoing cardiopulmonary bypass (CPB) for valve replacement. METHODS: Thirty patients undergoing elective valve replacement were randomized to receive either aminophylline (n=15), or normal saline (control n=15). Administration of aminophylline (5mg/kg) was injected intravenously after induction of anesthesia. The cardiac Troponin I (cTnI), myocardial myeloperoxidase (MPO) activity, atrial cyclic AMP, and a coronary sinus neutrophil count were measured before and after cardioplegic arrest. RESULTS: There were no differences between the two groups with regard to clinical variables. The cTnI concentration increased significantly after aortic declamping in both groups. However, it was significantly lower, 8h after aortic declamping, in aminophylline group (1.00+/-0.41 vs 2.37+/-1.35 ng/ml p=0.038). The atrial cAMP was significantly higher before aortic cross-clamping in aminophylline group (42.5+/-6.7 pmol/g tissue vs 30.6+/-12.4 pmol/g tissue p=0.04). In addition, we found that the aminophylline group had a significantly lower MPO after reperfusion (1.50+/-0.58 U/g tissue vs 0.86+/-0.24 U/g tissue p=0.003), and a significantly lower neutrophil count 30 min after aortic declamping (0.68+/-0.11x10(3) cell/ml vs 0.32+/-0.16x10(3) cell/ml, p=0.023). CONCLUSIONS: Pretreatment with intravenous aminophylline reduces the subclinical myocardial injury and neutrophil activation in patients undergoing CPB for valve replacement.