| Lung cancer is the most common cause of cancer death in both men and women,and in recent years,it has been the fastest growth malignant tumors.Histologically,lung cancer can be divided into two categories:small cell Lung Cancer(SCLC)and non-small cell Lung Cancer(NSCLC),with the latter comprising 85%of lung cancer cases.Compared with SCLC,NSCLC has a slower growth and division rate,a relatively late spread and metastasis.However,About 75%of patients are in the middle and late stages of diagnosis,resulting in poor therapeutic effect and a low 5-year survival rate.Although in recent 20years we have made great progress in the fields of advanced medical instruments,surgical techniques,radiotherapy and chemotherapy,and gene therapy,the overall therapeutic effect of non-small cell lung cancer is still poor.Cisplatin(DDP)based chemotherapy is still the most effective chemotherapy for lung cancer at present,but cisplatin-Resistance in most patients significantly reduces the therapeutic effect.Therefore,it is of great scientific significance and clinical value to find a solution to cisplatin resistance.Cisplatin resistance is the result of many mechanisms.It is generally acknowledged that there are three main aspects:(1)gene mutation and mitochondrial apoptosis;(2)enhanced gene repair;(3)cell apoptosis is blocked.In addition,drug delivery system,metabolic level and changes in tumor microenvironment,such as hypoxia,can also lead to cisplatin resistance in NSCLC.Many scholars have been devoted to reversing the cisplatin resistance of NSCLC at gene level for a long time.Many genes and signaling pathways related to cisplatin resistance of NSCLC have been found,such as FHIT,PI3K-NF?B signaling pathway,and gene silencing or overexpression techniques have been used to enhance the cisplatin sensitivity of NSCLC.Based on above all,our team has also been engaged in related research,and found that interleukin-6(IL-6)and its downstream signaling pathway have a certain relationship with cisplatin resistance in NSCLC as follows:Part I IL-6 and its signaling pathway induce cisplatin resistance by up-regulating DNA repair-related molecules and anti-apoptotic ability in non-small cell lung cancerObjectives:To investigate the role of IL-6 in the development of cisplatin resistance in non-small cell lung cancer.Methods:Combined with high efficiency transfection kit,We transfected A549 and H157 non-small cell lung cancer cell lines with lentivirus vector carrying IL-6 si RNA and SC si RNA as control,and detecting the expression level of IL-6 m RNA by Q-PCR.The cytotoxicity of cisplatin to different groups of cells was detected by MTT;Flow cytometry was used to detect the apoptosis of cells in different groups after cisplatin treatment.The expression levels of ATM,CHK1,Bcl-2,Bcl-x L,Mcl-1 and other apoptosis related genes were detected by Q-PCR and Western blot.In order to explore the possible signal pathway of IL-6 in cisplatin resistant process,we used different signal pathway inhibitors,and then detected the expression of related genes and proteins by Q-PCR and Western blot.We established nude mice models of subcutaneous transplanted tumor with different cells,and monitored and recorded the growth rate of subcutaneous transplanted tumor,tumor size and weight of nude mice before and after cisplatin treatment.Finally,we euthanized the nude mice to obtain tumor tissue,and performed HE staining and immunohistochemical staining to detect the expression level of the above-mentioned related genes and proteins in different treatment groups.Results:we obtained IL-6 knockout cells(A549 IL-6si and H157 IL-6si)and their corresponding scrambling control cell lines(A549sc and H157sc)using lentivirus mediated method.Both Q-PCR and ELISA analysis showed that IL-6si cell lines had high IL-6 knockdown efficiency(more than 90%).Using MTT method,we found that A549IL-6si and H157 IL-6si cells were more sensitive than sc cells,and we found that the growth of A549 il-6si cell-derived xenograft tumor was significantly slower than the tumor from A549sc cells after cisplatin treatment in animal experiments.We further analyzed the expression of various molecules in tumor tissue to determine the relationship between IL-6and cisplatin sensitivity.The knockdown state of IL-6 was confirmed by immunohistochemistry,and less Ki67 positive cells were detected in A549 IL-6si cell-derived tumors by immunohistochemical staining with the antibody of proliferation marker Ki67,which was consistent with the tumor growth data.The results of Annexinv flow cytometry showed that the apoptosis rate of IL-6si cells was higher than that of SC cells(8.7%and 2.8%for A549 cells,9.5%and 3.9%for H157 cells,respectively).The results of Q-PCR and Western blot showed that the expression of Bcl-2 and Mcl-1(not Bcl-XL and Bax)in cisplatin treated SC cells increased significantly,but not in Si cells.IHC staining of tumor tissue supported the results of the experiment in vitro,which showed that the number of Bcl-2 and Mcl-1 staining cells in tumor from SC cells was higher than that from IL-6 Si cells.We found that ATM and CHK-1 expression levels were significantly up-regulated after cisplatin treatment in A549sc and H157sc cells,but not in A549 IL-6si and H157 IL-6si cells.In addition,we also found that the expression of p53,p73 and ERCC1 were significantly up-regulated in sc cells but not in IL-6 si cells.IHC staining data obtained from tumor tissue also showed that the number of ATM and CHK-1staining cells in A549sc xenografts was higher than that in A549 IL-6si xenografts.In the study of signaling pathway,we found that STAT3,ERK,MAPK and Akt signaling pathways,whether or not treated with cisplatin,were highly activated in A549sc and H157sc cells,but not significantly increased in A549 IL-6si and H157 IL-6si cells.In A549sc and H157sc cells,inhibitors(SB203850,LY294002,AG490 and U0126)were added to inhibit MAPK,Akt,JAK/STAT3 and MEK/ERK signal pathways respectively.The results showed that MAPK,Akt and MEK/ERK pathway inhibitors almost completely blocked the up regulation of Bcl-2 and ERCC1 molecules,while the group using JAK/STAT3 pathway inhibitors showed a lower effect.Conclusions:The sensitivity of NSCLC cells to cisplatin depends on the level of IL-6in cells.NSCLC cells with high expression of IL-6 are more likely to develop cisplatin resistance and have a higher chance of survival than those with low or no IL-6 in cells.The overexpressed IL-6 up-regulated the expression of apoptosis inhibition(Bcl-2,Mcl-1)and DNA repair related molecules(ATM,CHK-1,ERCC1,p53,tp73)after cisplatin treatment.In addition,IL-6 up-regulated the genes related to the anti-apoptosis and DNA repair by activating signal pathway including MEK/ERK signal pathway,p38 MAPK,EGFR-STAT3 signal pathway and Akt signal transduction pathway.Now that we have identified the relationship between IL-6 and cisplatin resistance in NSCLC,we believe that with the expression of IL-6 in NSCLC effectively reduced,we can enhance the sensitivity to cisplatin,thereby preventing or reversing cisplatin resistance.However,the use of viral vectors in basic research is limited by their immunogenicity and lack of specificity.The development of gene vectors is the most important in gene therapy,and in recent years,researchers have developed a variety of gene delivery materials,including viral and non-viral vectors.Viral gene vectors have high transfection efficiency,but the lack of specificity and immunogenicity limits their further application.Non-viral gene vectors have broad application prospects in clinical transformation because of their simple synthesis,low immunogenicity and high transfection efficiency.Branched poly(?-amino ester)is a new non-viral vector with effective gene delivery function.However,the increase of cytotoxicity caused by high molecular weight and high density positive charge limited its application.Therefore,many researchers envisage whether nano-carriers with high gene condensed ability and high molecular weight can be degraded and expelled from the body through external stimulation after gene delivery,so as to reduce the toxicity of materials.More and more new responsive nano-carriers have been developed,such as light sensitivity,temperature sensitivity,p H response,ROS response and so on,which has made remarkable achievements in basic research and laid a solid theoretical foundation for further clinical application,and consolidated the theoretical basis for the future clinical use.Based on the close combination of clinical and basic research,our research team and Professor Yin Lichen group of Functional Nano&Soft Materials of Soochow University designed a stimulus-responsive polymer gene delivery system and carried out preliminary research as follows:Part II Photo-responsive and rapidly degradable Branched Poly(?-Amino Ester)s for gene delivery.Objectives:To design and synthesize stable and UV responsive hyper-branched poly(?-amino ester)(BPAE-NB)for gene targeted delivery,and to verify its efficiency,controllability and safety.Methods:We synthesized branched BPAE-NB by Michael addition reaction of A2(amine)+B3(triacrylate)+C2(diacrylate),in which the branched monomer(B3)was used to form a highly branched structure,and the nitrobenzene containing monomer(NPBMDA,A2)was selected as a UV sensitive group.Furthermore,1-(3-aminopropyl)-4-methylpiperazine(MPZ)was used to seal the polymer to improve the cation charge density,so as to obtain branched poly(?-aminoester)(BPAE-NB)with UV response.In the same way,the UV in-sensitive BPAE-CC was synthesized for experimental comparison,and their gene carrying effect was compared with the straight chain poly(?-aminoester)(LPAE-NB).Three different materials were characterized by 1H NMR.Combined with DNA and si RNA,we detected the size and potential changes of the three polyplexes after UV irradiation.The polymers were characterized by gel permeation chromatography(GPC),dynamic light scattering(DLS),agarose gel electrophoresis and heparin sodium displacement test.Flow cytometry and fluorescence microscopy were used to analyze the transfection of different polymers carrying fluorescent EGFP,and confocal scanning microscopy(CLSM)was used to detect the kinetics of different polymers entering the cells.MTT test was used to detect the toxicity of the materials and the polymer/DNA polyplexes.Results:we designed and synthesized stable UV response hyper-branched poly(?-amino ester),BPAE-NB,and UV insensitive BPAE-CC and linear poly(?-amino ester,LPAE-NB).The chemical structure of the polymer was characterized by 1H NMR.The degree of branching(DB)of bpae-nb is 0.55.Gel permeation chromatography(GPC)was used to determine the Mw of BPAE-NB.Its Mn was 18.1 k Da and PDI was 1.83.Compared with LPAE,it has higher molecular weight and helicity.UV visible spectroscopy and GPC were used to study the photoinitiated polymer degradation.When the DMF solution of BPAE-NB was exposed to UV light(?=365 nm,20 m W/cm~2)for 2min,the absorbance at 280 nm(OD280)was significantly increased.GPC analysis further confirmed the degradation of BPAE-NB.The MWS of BPAE-NB decreased significantly under UV irradiation(?=365 nm,20 m W/cm~2,10 min),and the peak almost disappeared.In contrast,the insensitive BPAE-CC could not be degraded by UV light,so its molecular weight remains unchanged,and its chromatographic peak has no obvious change.Gel retardation test showed that in 1%agarose gel,when the weight ratio of polymer/DNA was higher than 0.5,DNA migration was completely blocked,indicating that DNA was completely condensed with branched BPAE-NB.In contrast,LPAE-NB shows weak DNA condensation ability,which requires a higher polymer/DNA weight ratio,and can fully condense DNA at 15:1.The quantitative EB resistance test further verified these results,in which more than 85%of DNA condensed when the weight ratio of polymer/DNA was greater than 1 by BPAE-NB,while LPAE-NB could achieve 85%of DNA concentration efficiency when the weight ratio of polymer/DNA was greater than 10.The measurement results of DLS also showed that when the weight ratio of BPAE/DNA was?5,a polymer with a diameter of about 180 nm and a surface positive charge of about 30 m V was formed.Using p Luc as the tracer plasmid,we detected the transfection efficiency of various polymers in He La cells.When the optimal weight ratio was 15:1,the transfection efficiency of BPAE-NB/DNA or BPAE-CC/DNA polyplexes was significantly higher than that of LPAE-NB/DNA polyplexes.Compared with PEI,the transfection efficiency of BPAE-NB was increased by 15 times,and UV irradiation significantly promoted the transfection efficiency of BPAE-NB/DNA polyplexes,but did not promote the transfection efficiency of BPAE-CC/DNA polyplexes.We studied the cellular uptake and internalization mechanism of polymers formed by different polymers and YOYO-1-DNA.The results showed that BPAE-NB/DNA(ratio of 10-30)significantly increased DNA uptake in He La cells,which was 2-4 times higher than LPAE-NB and PEI.The DNA uptake level of BPAE-CC was similar to that of BPAE-NB due to its similar cation charge density and molecular structure,which was significantly higher than that of LPAE.Similarly,CLSM also showed that the cell uptake level of BPAE-NB/DNA polyplexes was higher than that of LPAE-NB/DNA polyplexes.In the study of cell uptake mechanism,the results of uptake experiments showed that:at 4?,cell uptake was significantly reduced by more than 70%,CPZ,GNT and m?CD also significantly inhibited cell uptake,while WTM did not significantly inhibit cell uptake.The results of MTT assay showed that the cytotoxicity of BPAE-NB/DNA polyplexes was almost no toxicity at the concentration for effective transfection,and UV irradiation significantly reduced the cytotoxicity of BPAE-NB polymer,and it was time-dependent in a certain time range,while the cytotoxicity of BPAE-CC,which was not sensitive to the UV light,did not decrease.And we found that more polymers are needed to fully condense si RNA compared with DNA.Compared with the high ratio of 150:1(LPAE:si RNA,w/w)needed to condense si RNA,BPAE had a more obvious advantage of lower ratio of 15:1(BPAE:si RNA,w/w),which could overcome the toxicity caused by the use of high-dose materials.We selected survivin as the target gene,and successfully transfected He La cells with BPAE-NB combined with survivin si RNA.The Q-PCR result showed that the level of survivin m RNA of transfected cells was significantly decreased,and the MTT assay result showed that the cell viability was significantly decreased,which was due to the knockdown of survivin.Conclusions:We designed and developed BPAE-NB with high degree of branching and UV triggered degradation,which has multivalent structures,strong DNA/si RNA condensation ability,and high gene transfected efficiency.With the UV light,BPAE-NB can degrade into small fragments,which can trigger the release of DNA/si RNA in cells,enhance the efficiency of gene transfection,and reduce the self toxicity of polymers.It provided an effective way to solve the contradiction between efficiency and toxicity of gene carrier based on polycation,and provided practical methods and new ideas and strategies for the rational design of non viral gene delivery materials. |
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