Antitumor Effect Of Human Trail On Adenoid Cystic Carcinoma Using A Novel Magnetic Nanoparticles Mediated Gene Expression

ACC is one of the most common malignant neoplasms in the human salivary gland,representing¹0%of salivary gland tumors. Adenoid cystic carcinoma is the second mostcommon malignant tumor in the salivary gland.The ACC grows relatively slowly and has a poorprognosis compared to other malignant tumors in salivary glands. However, the currenttreatments for ACC,including surgery, radiation therapy, chemotherapy and combinationsthereof,still could not improve overall mortality rates. To efficiently and successfully treatACC, new treatments are needed. To date, cancer is still one of major lethal diseases inhuman for which treatment can be problematic. Over decade ago, cancer gene therapy startedto develop with the gene therapy developing. This novel treatment has great potential, butmany issues need to be solved before cancer patients can be safely and efficiently treated. Thoseissues like specific, efficient transfection/transduction, toxicity and so on. Gene therapymeans transfer the extenal gene to cells,corret the heredity and get impairment of gene.After20-year research, there was great progression to treat the heredity disease,metabolism diseaseand malignant tumor using gene therapy,and some treatments have been used in clinical case.In the current study, we tried to use our novel developed nanoparticale system to addressspecific, efficient transfection/transduction and toxicity issues during cancer gene therapy.TRAIL belongs to the tumor necrosis factor (TNF)-cytokine superfamily. TRAIL inducesapoptosis in a wide variety of transformed cell lines. The killing activity of TRAIL iscancer-specific and has little or no effect on most normal cells and tissues. Although themechanism for this cancer-specific action of TRAIL is poorly understood, it is recognized thatTRAIL has the potential to become a promising antitumor therapy reagent and may provide anovel approach for cancer treatment.The human telomerase reverse transcriptase (hTERT) ishighly active in85–90%of human cancer, while its activity is lower or undetactable in mosthuman normal somatic cells. The hTERT promoter was considered as a tumor specific promoter.Many studies have used the hTERT promoter to drive antitumor gene expression selectively incancer cells, without or little affecting normal cells to restrict or limit unwanted side effects.Our previous study demonstrated that adenoviral vector encoded human TRAIL, driven by the hTERT promoter, could efficiently mediate apoptosis of an ACC tumor model in vivo. Weused the plasmid pACTERT-TRAIL.Recently medical nanotechnology has developed quickly and is already employed in manyclinical applications. These multifunctional nanoparticles become very promising for manyapplications. Nano-or micro-particles provide specific surface for many kind of molecules tobind, such as chemical compounds or DNA molecules (on the surface or in the center, bothmethods are exit). Magnetic nanoparticle are a special kind of nanoparticle, which has beenused in drug delivery and rapid biological separation. Several previous studies demonstrated thatDNA molecules could bind to magnetic nanoparticles by non-covalent bonds and used this toincrease transduction efficiency by applying a magnetic field around tumor.To overcome treatment limitations, we developed a novel treatment combining genetherapy and nanotechnology. In this study, we created a plasmid pACTERT-TRAIL, whichused the human telomerase reverse transcriptase promoter,a tumor specific promoter,to drivea TRAIL. A Fe₃O₄-PEI-plasmid complex (FPP) was generated, in which the Fe₃O₄nanoparticles modified by positive charge polyethylenimine to be able to carry the negativecharge plasmid.ï¼·e used polymer modified Fe₃O₄nanoparticles as vector,which is prepared byalkaline coprecipitation in the presence of the cationic polymer PEI. The amine groups on thesurface of nanoparticles can combine with the phosphate radical of plasmid DNA,pACTERT-TRAIL, by electrostatic interaction to form nanocomposite particles. This non-viraldelivery system could be more safe with less side effects. The magnetism of nanocompositeparticles could locally target the DNA delivery. The PEI polymer can protect the DNA fromdigesting in endosome due to their buffer capacity so-called―proton sponge mechanism‖. FPPinteract with the negative charges of the cell membrane facilitating entry into the cells. A studysuggested that positive molecules interact with cells strongly leading to faster cellular uptakecompared to neural and negative molecules.pACTERT-TRAIL transfected with the PEI modified iron oxide nanoparticles with aNd-Fe-B magnet into SACC-83cells,the proliferation and apoptosis of SACC-83weredetected by MTT,FCM techniques. In vivo, after FPP was injected into tumors of ACC innude mice,the tumor volumes,the morphology of organs and elements of blood in nude mousewere detected, in order to illuminate the function of the pACTERT-TRAIL.The grouptransfected with the PEI modified iron oxide nanoparticles with a Nd-Fe-B magnet had the highest transfection efficiency. The results from MTT assay and Annexin V-FITC assayindicated that the PEI modified iron oxide nanoparticles with a Nd-Fe-B magnet couldefficiently deliver functional plasmids into the target cells. The group of PEI modified ironoxide nanoparticles with a Nd-Fe-B magnet inhibit the tumor growth of adenoid cysticcarcinoma and there were no obviously change of morphology of organs and elements of bloodin the nude mice.Human TRAIL has a selective tumor-specific killing activity and little or no effect on mostnormal cells and tissues.Previous studies, however, showed that high concentration ofTRAIL could induce toxicity of hepatocytes and human brain cells in vitro. The TRAIL toxicitieshave potential ischemic and hemorrhagic reaction in normal tissues. To minimize the TRAILpotential side effects and restrict the expression of TRAIL in the cancer cells, we selected thehTERT promoter as a cancer specific promoter. Our previous study had shown that the hTERTpromoter selectively mediated the expression of TRAIL in SACC-83tumor cells resulting in thespecific killing of these cells in vitro. In present study, we used the PEI modified iron oxidenanoparticles to deliver therapeutic plasmid into target cells and employed a Nd-Fe-B magnetaround the target at the same time to further restrict the transfection occurred in the target side.Therefore, the PEI modified iron oxide nanoparticles with a Nd-Fe-B magnet provides asimple, specific, efficient and less toxic transfection method in vitro and in vivo compared tothe adenoviral vector.In summary, this study demonstrated that we were able to create a useful PEI modifiediron oxide nanoparticles, which could link plasmid DNA and efficiently transfected thetherapeutic plasmid into the ACC tumor cells, SACC-83, in vitro and in vivo in a specificmanner using a Nd-Fe-B magnet around the target site. This PEI modified iron oxidenanoparticles show the possible clinical potential to treat the ACC in near future.