The Experimental Study Of Therapy Orbital Rhabdomyosarcoma By C-FLIP Antisense Oligonucleotides Loaded Nanoparticles

Objective1. To investigate the expression of c-FLIP (cellular FLICE-like inhibitory protein) in orbital rhabdomyosarcoma tissues and the human rhabdomyosarcoma cell line RD in mRNA levels and its roles in the pathogenesis of rhabdomyosarcoma.2. To explore the effects of c-FLIP anti-sense oligodeoxynucleotides (ASODN) on the expression of c-FLIP in RD cell line and subsequent effect on apoptosis.3. To prepare the PLGA nanoparticles carrying c-FLIP ASODN and explore their effects on apoptosis in RD cell line.Methods1. RT-PCR was employed to detect the expression of c-FLIP in human rhabdomyosarcoma RD cell line and 18 specimens of orbital rhabdomyosarcoma and matched normal tissue.2. c-FLIP-ASODN and control sequence (Control-ODN) were transfected into RD Cells with Oligofectamin. c-FLIP protein expression was examined by Western Blotting and the expression of c-FLIP mRNA was detected by RT-PCR. The apoptosis rates of RD Cells were assessed quantificationally by flow cytometric analysis with Annexin V.3. By a double-emulsion evaporation technique, PLGA nanoparticleswere used to cover c-FLIP-ASODN and were used as a carrier of transf ection. Characteristics of the nanoparticles were further evaluated.4. RD Cells were treated by PLGA nanoparticles carrying c-FLIP-ASODN, the effects on the expression of c-FLIP and apoptosis were further investigated.Results1. c-FLIP mRNA was positively expressed in RD cell line and all the 22 specimens of orbital rhabdomyosarcoma and matched normal tissue. The expression levels of c-FLIP in the 18 specimens of orbital rhabdomyosarcoma were higher than those in matched normal tissue.2. c-FLIP-ASODN could down-regulate the expression of c-FLIP, and after treating by c-FLIP-ASODN, the expression level of c-FLIPs and c-FLIPL in RD cells were decreased to 43±3% and 48 + 5% respectively, comparing with the untreated ones, and the apoptosis rate was increased to 29. 3 ±6. 3%.3. The prepared c-FLIP-ASODN-PLGA nanoparticles had regular spherical surface. The mean diameter of the particles is 95. 5nm and the mean encapsulation ratio is 48%. The percentage of c-FLIP-ASODN covering by the nanoparticles is 0.579 + 0.016%, and the releasing duration in vitro is 15 days. After covering by PLGA nanoparticles, the c-FLIP-ASODN was protected from being degrading by nucleases.4. PLGA nanoparticles carrying c-FLIP-ASODN could down-regulate the expression of c-FLIP and increase the apoptosis rate of RD cells. After treating by c-FLIP-ASODN nanoparticles, the expression level of c~FLIPs and c-FLIPl in RD cells were decreased to 35 + 2% and 46 + 5% respectively, comparing with the untreated ones, and the apoptosis rate was increased to 32. 6+5. 3%. The trasnfection effect of PLGA nanoparticles was higher than Oligofectamin with statistically significance.Conclusion1. c-FLIP was positively expressed in RD cell line and the specimens of orbital rhabdomyosarcoma and matched normal tissue. The expression level of c-FLIP in rhabdomyosarcoma was much higher than that in paired normal tissue. Overexpression of c-FLIP was a tumor-specific phenomenon, which can provide a selective growth advantage for rhabdomyosarcoma cells to escape death receptor-mediated apoptosis.2. The method of preparing c-FLIP-ASODN PLGA nanoparticles is simple and characteristics of the nanoparticles can be reached requirement. It' 11 be a good choice to use c-FLIP-ASODN PLGA nanoparticles as gene therapy carrier.3. c-FLIP-ASODN-PLGA nanoparticles could effectively transfect RD Cells and down-regulate c-FLIP mRNA expression, and further recover the sensibility of RD Cells to apoptosis signals. c-FLIP-ASODN would become a new target of gene therapy for orbital rhabdomyosarcoma.