Enhancement of Apo2L/TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis in non-small cell lung cancer cell lines by chemotherapeutic agents without correlation to the expression level of cellular protease caspase-8 inhibitory protein.

OBJECTIVE: Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anticancer drug that promotes apoptosis specifically in tumor cells. Because not all cancer cells are susceptible to Apo2L/TRAIL, the aim of our study was to determine whether non-small cell lung cancer cells can be sensitized by chemotherapeutic agents for Apo2L/TRAIL-induced apoptosis. In addition, endogenous expression levels of the caspase-inhibiting cellular protease caspase-8 inhibitory protein (C-FLIP) were measured to investigate partial resistance to Apo2L/TRAIL. METHODS: Six human lung cancer cell lines (A549, NCI-H358, Calu1, Calu6, SkMes1, and SkLu1) were incubated with soluble Apo2L/TRAIL and two different concentrations each of cisplatin, paclitaxel, doxorubicin, 5-fluorouracil, and camptothecin. After 24 hours the rate of apoptosis was measured by annexin V/propidium iodide staining followed by FACScan analysis. Expression levels of C-FLIP in cell lines and lung cancer biopsy specimens were determined by Western blotting. RESULTS: Treatment of lung cancer cells with Apo2L/TRAIL alone resulted in apoptotic cell death in four cell lines (P <.001). Combining Apo2L/TRAIL and chemotherapeutic agents enhanced the rate of apoptosis significantly. Statistical analysis revealed a synergistic effect of Apo2L/TRAIL in combination with 1.8 mmol/L camptothecin and 100 micromol/L cisplatin, each in four of the six cell lines (P <.002). Western blot analysis showed that sensitization to Apo2L/TRAIL did not correlate with the expression of cellular protease caspase-8 inhibitory protein. Furthermore, no increased cellular protease caspase-8 inhibitory protein levels relative to those in normal lung tissue could be found in non-small cell lung cancer specimens from 12 patients. CONCLUSION: Apo2L/TRAIL-induced apoptosis in non-small cell lung cancer cell lines is significantly enhanced by chemotherapeutic agents. Resistance and sensitization to Apo2L/TRAIL are not correlated with the endogenous expression level of cellular protease caspase-8 inhibitory protein, implying that in non-small cell lung cancer other mechanisms are responsible for inhibition of the Apo2L/TRAIL pathway. Even though the molecular mechanism remains unclear, the combination of Apo2L/TRAIL with chemotherapy may be a promising treatment modality for non-small cell lung cancer.

Non-viral gene delivery to atelectatic and ventilated lungs.

BACKGROUND: Three different nonviral vectors and naked DNA were evaluated for in vivo transfer of plasmid DNA to rat lungs through airways in either atelectatic or ventilated lungs. METHODS: The F344 rats underwent instillation of 300 microg DNA (pCIluc, luciferase) to the left lung. Naked DNA, linear polyethylenimine, branched polyethylenimine, and lipid GL-67 (in either atelectatic or ventilated lungs) were assessed (n = 5 per group). After 24 hours, left lung PaO2 (mm Hg) and luciferase activity (RLU/mg) were measured. The median (range) was given, and the analysis of variance was applied, followed by the planned comparison on log-transformed data. RESULTS: In atelectatic lungs, lipid GL-67 was best (927 [330 to 4112] RLU/mg; p < 0.001 versus other groups of atelectatic lung; p < 0.001 versus all other groups), but highest luciferase activity in all groups was measured in ventilated lungs using linear polyethylenimine (1,240 [922 to 2519] RLU/mg; p < 0.001 versus other groups of ventilated lung; p < 0.001 versus all other groups). In comparison with naked DNA, all nonviral vector systems significantly impaired PaO2 24 hours after airway transfection (p < 0.001; naked DNA versus all other groups). Regardless of transfection technique, PaO2 was worst in lungs transfected by linear polyethylenimine. CONCLUSIONS: Highest transfection was achieved with GL-67 in atelectatic lungs and with linear polyethylenimine in ventilated lungs. All gene delivery systems impaired gas exchange of the transduced lung in comparison with naked DNA.

1,25-Dihydroxycholecalciferol with low-calcium diet reduces acute rejection in rat lung allotransplantation.

OBJECTIVES: The effect of 1,25-dihydroxycholecalciferol (calcitriol, vitamin D3) with a low-calcium diet on the acute lung allograft rejection in a rat unilateral left lung transplantation model was evaluated. METHODS: Three transplantation groups were studied (n = 5, male Brown-Norway to Fischer F344, 235 ± 15 g body weight): calcitriol and low-calcium diet, low-calcium diet and normal diet. Calcitriol (4 µg/kg/day) was injected intraperitoneally for 5 days, starting from the day of transplantation. In addition, two non-transplantation groups were compared: (n = 3, Brown-Norway) to measure the level of cytokines, and Fischer F344 receiving calcitriol and a low-calcium diet to measure the serum calcium level. The recipients of transplantation were killed on Day 5 post-transplant. The contralateral right main bronchus and the pulmonary artery were occluded for 5 min and blood was drawn for the blood gas analysis, and the grafts were assessed for histology (International Society for Heart and Lung Transplantation 1996/rank scale). Lung levels of interleukin (IL)-2, IL-6, IL-12 and tumour necrosis factor-α (TNF-α) were assessed within the calcitriol and low-calcium diet, low-calcium diet and Brown-Norway groups. The serum calcium level was assessed in the Fischer F344 group. An analysis of variance with Tukey's post hoc test was used to compare the arterial blood oxygen pressure and the lung cytokine expression between groups. A non-parametric Kruskal-Wallis test followed by the Siegel and Castellan post hoc test was used to assess the differences between the groups according to the lung graft rejection grading. Student's paired t-test was used to compare the serum calcium level. RESULTS: The arterial PaO(2) was significantly higher in the calcitriol and the low-calcium diet groups when compared with low-calcium diet or normal diet groups (356 ± 72 mmHg; P < 0.05 vs other groups). The arterial and bronchial rejection observed in calcitriol and low-calcium diet group was significantly milder than in the low-calcium diet or normal diet groups (A1-2, B1-2; P < 0.05 vs other groups). IL-2 and IL-6 levels were significantly higher in low-calcium diet vs calcitriol and low-calcium diet and Brown-Norway groups. IL-12 and TNF-α did not differ among the groups. There was no significant difference in serum calcium level before and after the treatment in the Fischer F344 group. CONCLUSIONS: Calcitriol with a low-calcium diet treatment improves lung function, reduces lung allograft acute rejection, decreases IL-2 and IL-6 allograft expression and does not change the serum calcium level significantly.

Enhancement of integrin-mediated transfection of haematopoietic cells with a synthetic vector system.

The aim of the present study was to develop and assess an integrin-targeted synthetic vector system for the transfection of haematopoietic cell lines and dendritic cells. The vector consists of a cationic liposome, Lipofectin (L), a peptide that both targets integrins and binds to DNA (I) and plasmid DNA (D). These components interact electrostatically to form the LID vector complex. Transfection conditions were optimized for the ratio of vector components, the amount of DNA and transfection incubation time. The kinetic analysis of transgene expression revealed a peak of activity at about 24 h, followed by a rapid decline over the next 48 h. Targeted gene delivery was demonstrated by comparing transfected luciferase reporter gene levels using LID complexes containing integrin-targeting peptide sequences with a control peptide. In addition, transfection levels of integrin-targeted LID complexes were significantly enhanced by treatment of cells with PMA, which was also shown to activate integrin receptors and enhance binding to fibronectin. Under optimized conditions transfection efficiencies of 19% for TF-1 cells, 28% for Jurkat cells and 10% for primary dendritic cells were achieved. The LID vector may thus find application for gene-transfer experiments in haematopoietic cell lines and for the development of genetic vaccines using transfected dendritic cells.