Study of Effect of pll-DCIONP-mediated Wild-type p53 Gene Delivery Resulted in Targeting Therapy and Efficacy of Reversing Resistance in Cisplatin-resistant Lung Adenocarcinoma
|School||Central South University|
|Keywords||lung cancer magnetic iron oxide nanoparticle wt-p53 gene targeting therapy multidrug resistance|
BackgroundAs one of the leading causes of death among all the malignant tumorsin the world, lung cancer will become the biggest health-killer in the 21 stcentury. Great improvements have made in the efficacy of treating lungcancer with chemotherapy. However, the overall 5-year survival rate ofpatients is less than 15％. A majority of patients with lung cancer rapidlydevelop resistance to cisplatin causing therapy failure. For this reason, itis of importance to search for an appropriate and effective way to reversecisplatin resistance in lung cancer.p53 mutations are the most frequently found of genetic change in thelung cancer.Mutations in the p53 gene have been identified with a highfrequency of 50％and 70％in NSCLC and SCLC respectively. It isthought that p53 mutations could stimulate angiogenesis and metastasisof neoplasm, make neoplasm more metastatic, transactivate promoter ofdrug resistance gene in a sequence-specific way and induce expression ofdrug resistance gene so as to show a poor treatment response tochemotherapy and radiation. Furthermore patients with mutated p53presented a poor survival. For this reason, replacing mutated p53 orfunctionally inactivating p53 will be one of the most important strategiesof therapeutic gene targeting and reversal of multidrug resistance.Efficient and nontoxic gene delivery and expression of deliveredexogenous gene are important determinant of gene therapy. It is also adraconic task for research of gene therapy. Nowadays gene deliverysystems are mainly divided into viral vectors and non-viral vectors. Theviral vectors have very strong capabilities of delivering genes so that it isused most extensively in gene therapy of tumor. But viral vectors areassociated with immunogenicity, toxicity, lack of tissue specificity,difficulty in large-scale production and potential risk of inducing tumorigenic mutations. Non-viral vectors have become an attractivealternative because it can be easily scaled up, produced at relatively lowcosts and with less toxicity. But most non-viral vectors are limited in lowefficiency of transfection, especially in vivo. Therefore it is verynecessary to search for efficient and non-toxic targeted gene deliverysystems.The development of nanotechnology has provided us new ways toovercome barrier of gene delivery vectors. The nanoparticles can beeffectively endocytosed by the cells resulting in a high cellular uptake ofthe entrapped DNA because of its subcellular size. There are manyadvantages of nanoparticles as gene delivery vectors: （1） They have ahigh efficiency for nucleotide delivery. （2） They can deliver nucleotide tothe target size. （3） They can protect nucleotide from degradation bydigestive enzyme and phagocytosis by reticuloendothelial system. （4）They have the abilities of killing cancer cells, but not normal cells.Magnetic nanoparticles have character of superparamagnetism besidesthose mentioned above. Magnetic nanoparticles can deliver the focusednucleotide to the target site/cells via high-field/high-gradient magnets.Thus the nucleotide is released slowly from the nanoparticles resulting insustained intracellular gene delivery and it plays a role of targeting genetherapy.The authors examined p53 mutations in cisplatin-resistant humanlung adenocarcinoma cells A549/CDDP. Poly-L-Lysine modified ironoxide magnetic nanoparticles（pll-DCIONP） was synthesized as wt-p53gene carriers, The potential adsorbing wt-p53 gene and resistingDNase-Ⅰand blood serum digestion of pll-DCIONP/wt-p53 complexswas analyzed, magnetic field used to direct movement of pll-DCIONPand A549/CDDP cells was transfected with wt-p53 DNA-loadednanoparticles. The efficiency of transfection was analyzed to study effectof magnetic iron oxide nanoparticle-mediated wild-type p53 genedelivery on sustained antiproliferative activity and apoptosis in human lung adeno-carcinoma cell lines A549/CDDE The authors further treatedA549/CDDP xenograft in nude mice with an intravenous injection ofwt-p53 DNA-loaded nanoparticles so as to study effect of highly efficientand targeted tumor-killing, reversal of cisplatin resistance and in vitro andvivo toxicity of pll-DCIONP delivery systems.PartⅠUse of magnetic iron oxide nanoparticles as wt-p53 genecarrier and transfeetion with it in cisplatin-resistant lungadenocarcinoma cellsObjective To evaluate the feasibility of using iron oxidenanoparticles as gene carder and transfecting with it in cisplatin-resistantlung adenocarcinoma cells. Methods 1.The dextran coated iron oxidenanoparticles （DCIONP） was synthesized with deposition. The surface ofDCIONP was modified by self-assembled poly-L-lysine to form particlecomplexes （pll-DCIONP）.The configuration of pll-DCIONP was detectedby SEM. The diameter and Zeta surface potential of pll-DCIONP weredetected by Zetasizer. 2. The potential adsorbing wt-p53 gene andresisting DNase-Ⅰand blood serum digestion of pll-DCIONP wasanalyzed by spectrophotometer and agarose gel electrophoresis. 3. To testthe ability of pll-DCIONP to transfer gene in vitro, assays of delivery ofreporter plasmid DNA into A549/CDDP cells, The efficiency oftransfection was analyzed by fluorescent microscope and flow cytometry.4. The pll-DCIONP was evaluated as a kind of wt-p53 gene carrier bytransfecting human lung adenocarcinoma cell line A549/DDP in vitro.The expression of intracellular p53 gene was analyzed by RT-PCR andWestern blot. 5. The toxicity of delivery systems of pll-DCIONP in vitrowas measured by MTT. Results 1. The diameter of the pll-DCIONP is60～80nm with a satisfactory dispersed status, the Zeta surface potentialof pll-DCIONP is positively charged at different pH. 2. Under thedifferent condition of pH, the pll-DCIONP have the potential to adsorbwt-p53 gene and the potential of adsorbing was the strongest aspll-DCIONP/wt-p53 complexes prepared at w/w ratio of 1:1, pll-DCIONP/wt-p53 complex have the potential to resist DNase-Ⅰandblood serum digestion. 3. Magnetic field was used to direct movement ofpll-DCIONP capable of transferring reporter plasmid DNA into cells evenmore efficiently than the lipids examined in vitro（P＜0.01）. 4. Cellstransfected with wt-p53 DNA-loaded nanoparticles pll-DCIONPdemonstrated a sustained and increased p53 gene levels with incubationtime as opposed to a decrease in gene levels in the cells transfected withthe wt-p53 DNA-lipofectamine complex. 5. There was no significanttoxicity of delivery systems of pll-DCIONP. Conclusion pll-DCIONPwill become one of favored gene carriers for wt-p53 gene delivery and ithave the potential of transfecting the wt-p53 gene in cisplatin-resistantlung adenocarcinoma cells with a sustained and significantly efficientgene expression.PartⅡMagnetic iron oxide nanoparticle-mediated wild-type p53gene delivery resulted in sustained antiproliferative activity,apoptosis and its efficacy of reversing resistance in cisplatin-resistanthuman lung adenocarcinoma cellsObjective 1. To examine p53 mutations in cisplatin resistant humanlung adeno-carcinoma cells lines A549/CDDP. 2. To study effect ofmagnetic iron oxide nanoparticle-mediated wild-type p53 gene deliveryon sustained antiproliferative activity, apoptosis and its efficacy ofreversing resistance in cisplatin-resistant human lung adenocarcinomacell lines A549/CDDP. Methods 1. Mutation of p53 gene was screenedby DNA direct sequencing in cell lines A549 and A549/CDDP. 2.Theanti-proliferative effect of magnetic iron oxide nanoparticle-mediatedwild-type p53 gene delivery against A549/CDDP cells was tested by MTT.3. The apoptosis of cells transfected with wt-p53 DNA-loadedpll-DCIONP was detected by fluorescent microscope. The apoptosis ratioof tumor cells transfected with wt-p53 DNA-loaded pll-DCIONP wasmeasured with flow cytometry. The variation of expression ofpro-apoptosis gene Bax mRNA of cells transfected with wt-p53 DNA-loaded pll-DCIONP was determined by RT-PCR. The activity ofCaspase-3 of cells transfected with wt-p53 DNA-loaded pll-DCIONP wasmeasured by colorimetric assay. 4. The resistance to cisplatin of cellsA549/CDDP was evaluated by MTT and the resistance index of cellscalculated respectively before and after transfection with wt-p53DNA-loaded pll-DCIONP. 5. The expression of mRNA and protein oflung resistance protein （LRP） of cells transfected with wt-p53DNA-loaded pll-DCIONP was measured by RT-PCR and IHC SP assayrespectively. Results 1. There was C to A transversion in 82nd nucleotideof 5th exon of cells A549/CDDP. 2. The anti-proliferative effect was moresustained and greater in cells transfected with wt-p53 DNA-loadedpll-DCIONP than the DNA-lipofectamine complex. The anti-proliferativeeffects became stronger with incubation time and with an increase in thecase of pll-DCIONP. The anti-proliferative effect became stronger withwt-p53 DNA-loaded pll-DCIONP and low-does cisplatin together,whereas the effect was transient and lasted for only 1 day when the cellswere transfected with DNA-lipofectamine complex and the inhibitoryeffect with DNA-lipofectamine complex did not extend beyond 3 dayspost-transfection. There was no significant difference in theanti-proliferative effect of cells cultivated with low-does cisplatin withcontrols（P＞0.05）. 3. As compared with the controls, cells transfected withwt-p53 DNA-loaded pll-DCIONP caused typical apoptotic changes,including distinct morphological changes characterized by chromatincondensation, nuclear shrinkage and nuclear cleavage. And the cells grewmore rounded, smaller and some floated. The apoptosis ratio and activityof Caspase-3 were more sustained and higher in cells transfected withwt-p53 DNA-loaded pll-DCIONP than the DNA-lipofectamine complex.The apoptosis ratio grew higher with incubation time in the case ofpll-DCIONP, The apoptosis ratio and activity of Caspase-3 became higherwith wt-p53 DNA-loaded pll-DCIONP and low-does cisplatin together,whereas the effect was transient and lasted for only 1 day when the cells were transfected with DNA-lipofectamine complex and the apoptosiseffect and activity of Caspase-3 with DNA-lipofectamine complex didnot extend beyond 3 days post-transfection. There was no significantdifference in the apoptosis effect and activity of Caspase-3 of cellscultivated with low-does cisplatin with control（P＞0.05）.pll-DCIONP-mediated wild-type p53 gene delivery could up-regulatelevels of expression of Bax mRNA of A549/CDDP cell. Meanwhile itdemonstrated a higher Bax mRNA level compared to cells transfectedwith wt-p53 DNA-lipofectamine complex（P＜0.05）. 4. The IC50 andresistance index to cisplatin were down-regulated in cells transfected withwt-p53 DNA-loaded pll-DCIONP and the DNA-lipofectamine complex,but the effect of down-regulate was more significant in cells transfectedwith wt-p53 DNA-loaded pll-DCIONP than the DNA-lipofectaminecomplex（P＜0.01）. 5. Cells transfected with wt-p53 DNA-loadedpll-DCIONP demonstrated a relatively lower level of LRP mRNA andprotein than the wt-p53 DNA-lipofectamine complex on 3rd and 5th dayin this study. Conclusion 1. Genetic and protein pattern changes werefound in p53 mutations. 2. Magnetic iron oxide nanoparticle-mediatedwild-type p53 gene delivery resulted in sustained antiproliferative activityand apoptotic effects on cisplatin-resistant human lung adenocarcinomacell lines A549/CDDP. 3. Magnetic iron oxide nanoparticle-mediatedwild-type p53 gene delivery resulted in a reversal effect of cisplatinresistance in cisplatin-resistant human lung adenocarcinoma cell linesA549/CDDP, Down-regulating the levels of cellular expression of LRPmay be one of mechanism of resistance reversal.PartⅢMagnetic iron oxide nanoparticle-mediated wild-type p53gene delivery resulted in targeting gene treatment with cisplatin-resistant human lung adenocarcinoma xenograft in nude miceObjective To explore the feasibility of magnetic iron oxidenanoparticle-mediated wild-type p53 gene delivery results in targetinggene treatment with cisplatin-resistant human lung adeno-carcinoma xenograff in nude mice. Methods 1. Cisplatin-resistant cell linesA549/CDDP were cultured routinely and A549/CDDP cells implanted tosubcutaneous in nude mice to establish cisplatin-resistant xenograftanimal model. 2. Magnetic field was used to direct movement ofpll-DCIONP. The wt-p53 DNA-loaded pll-DCIONP was injectedintravenously and the tumor growth was then observed. Volumes andweight of tumor mass were detected respectively. Then tumor growthindex was thereby calculated. 3. Then specimens were fixed byparaformaldehyde and then paraffin section, Hematoxylin and eosin （HE）staining and histological examination performed. 4. Apoptotic index intumor tissues was measured by flow cytomety. 5. Expression of lungresistance protein （LRP） mRNA was measured through real-time PCRand expression of LRP protein detected by IHC SP assay. Results 1.There was no obvious side-effect for treatment following the wt-p53DNA-loaded pll-DCIONP to be injected intravenously and liver, kidneyand spleen tissues was proved no distinct injured by HE staining assay. 2.Treatment with single wt-p53 DNA-loaded pU-DCIONP, The growth ofxenograft was slow even part of them shrank. The weight of tumor massand growth index of tumor were 0.698±0.226g and 1.153±0.657respectively and it was significantly less than control groups andCDDP groups treated with low-does cisplatin（P＜0.001） and also lessthan Lip groups treated with the wt-p53 DNA-lipofectaminecomplex（P＜0.01）. The effect became stronger with wt-p53 DNA-loadedpll-DCIONP and low-does cisplatin to be co-administrated. 3. Treatmentwith single wt-p53 DNA-loaded pll-DCIONP, The apoptotic index oftumor tissues was 42.49±11.76％and it was significantly more thancontrol groups （10.62±3.89％）and CDDP groups （10.47±4.12％）treated with low-does cisplatin（P＜0.001）. And it was also more thanLip groups（18.63±5.26％） treated with the wt-p53 DNA-lipofectaminecomplex（P＜0.01）. The effect became stronger with wt-p53 DNA-loadedpll-DCIONP and low-does cisplatin to be co-administrated. 4. Treatment with single wt-p53 DNA-loaded pll-DCIONP, The expression of LRP oftumor tissues was down-regulated and it was significantly less thancontrol groups and CDDP groups treated with low-doescisplatin（P＜0.001）. And it was also less than Lip groups treated withthe wt-p53 DNA-lipofectamine complex（P＜0.01）. The levels ofexpression of LRP dropped with wt-p53 DNA-loaded pll-DCIONP andlow-does cisplatin to be co-administrated. Conclusion 1. Magnetic ironoxide nanoparticle-mediated wild-type p53 gene delivery resulted intargeting anti-tumor with cisplatin resistant human lung adeno-carcinomaxenograft in vivo and reversed the resistance in cisplatin resistant humanlung adenocarcinoma xenograft in vivo. The effect grew stronger thanxenograft treated with the wt-p53 DNA-lipofectamine complex. 2.Down-regulating the expression levels of of LRP in tumor tissues may beone of mechanism of resistance reversal. 3. There was no significant invivo toxicity of delivery systems of pll-DCIONP.