| Object:Malignant cancer which as a serious disease had great threat to human health,it accounted for 23%of all deaths,and had no downward trend in the past 50 years. According to the latest statistics of American Cancer Association in 2008,estimated new lung cancer cases accounted for 15%of cancer diagnoses,but it was the most cancer related deaths in both men and women,accounting for 29%of total cancer deaths.Meanwhile,in 2005,New England Journal of Medicine reported cancer has become the first in male and the third in female causes of death in China,and lung cancer was the first reason in cancer mortality.Lung cancer is classified clinically as small cell(13%)or non-small cell(87%)for the purposes of treatment.Although there are many treatment methods,such as surgery,radiotherapy,chemotherapy, targeted biological therapy,as well as combination treatment,the 1-year survival rate of lung cancer is about 41%,and 5-year survival rate only 15%.It was considered to be the most malignant tumor and had the greatest threat to the life.Radiotherapy and chemotherapy were widely used in clinical treatment of lung cancer,but they had no targeting and gave great side effects to patients.In recent years,many effective cancer gene therapy targets were discovered and provided new idea of lung cancer gene therapy.Signal Transducers and Activators of Transcription (STAT)family are potential cytoplasmic transcription factors,which played dual functions of transferring signal and initiating transcription.Especially,STAT3 plays important roles in embryo development and cell proliferation.The activation of STAT3 is instantaneous and strictly regulated in normal tissue.However,it was constitutively activated in many hematopoietic and solid tumors including leukemia, multiple myelomas,as well as prostate,breast,lung,ovarian,gastric cancers.The activated STAT3 could regulate the expression of many genes including negative factors for immune response(IL-10,VEGF and TGF-β)and genes associated with proliferation,apoptosis and cell cycle(bcl-xl,cyclinD1,mcl-1,surviving,cyclin E).It could enhance proliferation,inhibit apoptosis,induce angiogenesis and invade immune surveillance.STAT3 was therefore being considered as oncogene.Some cytokines such as IL-6 and EGF can bind to their receptor and activate tyrosine kinase, and then activate STAT3,two p-STAT3 molecules dimmerized and translocated to nucleus,and then bindeded to specific DNA response element in the promoter region and initiated gene transcription.It was one of the effective methods to inhibit a cluster of genes by blocking the binding of transcription factor with their DNA response element,of which, transcription factor decoy(TFD)was mostly used.The transcription factor decoy oligodeoxynucleotide(decoy-ODN)which had the same sequence with DNA response element and had high affinity to transcription factor was synthesized,and the decoy ODN were transfected into cells and competited binding to transcription factor with their own response element.They were used as decoy molecular to alter gene transcription.Compared with other methods,"Decoy" technology has unique advantages and more application potential,such as cluster control,short sequence, easy to transfection,high specificity,et al.By now,decoy ODN method was widely used as a tool to investigate the gene function,and more and more clinical study were done to evaluate their potential value in clinic.Chemotherapy is the common method to treat tumor in clinic,resistance to chemotherapy which termed multidrug resistance(MDR)is considered one of the major obstacles to successful cancer chemotherapy.The overexpression of P-glycoprotein(P-gp)is the main reason in MDR.By now,many MDR modulators were investigated;however,the use of efflux-pump modulators in clinical cancer treatments had proved disappointing.Thus,it is of great importance to develop new compounds or strategies that are capable of circumventing P-gp-mediated MDR with improved clinical characteristics.The mechanisms to induce the expression of P-gp and to acquire drug resistance were unclear.Identification of the mechanisms and the inducing factors were a high priority.Constitutive STAT3 has been shown to confer resistance to chemotherapy-induced apoptosis in some malignancies.Inhibition of STAT3 activity enhances chemo-sensitivity in carcinoma.Meanwhile,it was found that STAT3 mRNA was overexpression in drug-resistant cancer cells than in sensitive cells,STAT3 activity was also specifically elevated in drug-resistant cells.In clinic, STAT3 is highly activated in advanced tumors which present non-sensitivity to chemotherapy.All the results suggested that STAT3 activity might be associated with drug resistance and there might be some relation between STAT3 and MDR.In this study,we used STAT3 decoy ODN to inhibit the activity of STAT3 in lung cancer cells in vitro and in vivo,and observed their effect on proliferation and apoptosis,and then investigated their mechanisms.We also studied inhibition of STAT3 enhanced the sensitivity of drug-resistant leukemia cells to chemotherapy,and the roles and mechanisms of STAT3 in reversing multidrug resistance were also investigated.It provided new idea and method for cancer gene therapy.Methods:Western blotting and EMSA assays were used to detect the expression and activity of STAT3 and its DNA binding ability in pulmonary giant cell carcinoma cell line PG and non-small-cell lung cancer A549 cell line.Cell counting method was used to observe the effect of AG490,a specific inhibitor of Jak2,on the proliferation of the cells.The ODN transfection rate and its location in cells were assayed by flow cytometry and fluorescence microscopy after transfected with lipofectamine.The inhibition effect of STAT3 decoy ODN on STAT3 activity in lung cancer cells was analyzed by luciferase reporter construct assay.Cell counting and 3H incorporation methods were used to detect the influence of decoy ODN on cell growth.The apoptosis was assayed using Annexin V and PI by flow cytometry.The expression of STAT3 target genes related cell cycle and anti-apoptosis were detected using RT-PCR and western blotting.We established the A549 nude mice xenograft and intratumorally injected decoy or scramble ODN and vehicle control.The tumor growth was observed by volume calculated using the length and wide.The tumor was weighed after the final injection,and froze it quickly to do frozen section.The apoptosis induced by decoy ODN in vivo were detected using TUNEL.The expression of STAT3 target genes related cell cycle and anti-apoptosis were detected by immunohistochemistry after decoy ODN treatment in vivo.The activity of STAT3 and expression of its target genes were analyzed by western blotting and luciferase reporter plasmid assay in multidrug resistance K562/A02 cells and drug sensitive K562 cells.The inhibition of STAT3 activity by JSI-124 was detected by western blotting.CCK-8 kit and cell counting were used to detect whether inhibition of STAT3 reversed MDR of K562/A02 to adfiamycin.The apoptosis was assayed by flow cytometry.The accumulation of intracellular adriamycin was detected by flow cytometry after treated with JSI-124.The transcripts of MDR1 and other MDR associated genes were detected by real time PCR in K562/A02 cells which treated with JSI-124 or not,and the expression of P-gp was assayed using western blotting.Meanwhile,another STAT3 signal pathway inhibitor, AG490(Jak2 specific inhibitor),was chosen.The phosphorylation of STAT3 was assayed by western after treated with AG490.CCK-8 kit was used to detect whether inhibition STAT3 by AG490 increased the sensitivity of K562/A02 cells to adriamycin.The transcripts of MDR1 and other MDR associated genes were detected by real time PCR in K562/A02 cells after treated with AG490 or not,and the expression of P-gp was assayed using western blotting.The possible STAT3 binding sites in the promoter of MDR1 were searched by computer assisted bioinformatics analysis.Result:part 11.Bioinformatics analysis suggested that the designed decoy ODN had strong binding ability with STAT3.According to the obvious reports and the database of transcription factor binding sequence,we designed the decoy ODN sequence which had high affinity with STAT3.2.The constitutively activated STAT3 played important roles in the proliferation of lung cancer cells.Western blotting indicated that STAT3 was highly expressed and was constant phosphorylation(Try705,Ser727)in pulmonary giant cell carcinoma cell line PG and non-small-cell lung cancer A549 cell line.The results of EMSA demonstrated that 32P-labeled STAT3 decoy ODN could bind with activated STAT3,but scramble ODN could not.The results further suggested STAT3 in lung cancer cells was activated and had DNA binding ability,and our designed decoy ODN sequence could bind with activated STAT3.Blocking signal pathway using Jak2 specific inhibitor AG490 could significantly inhibit the cell growth;all the results demonstrated the critical role of Jak2/STAT3 signal pathway in the proliferation of PG and A549 cells.3.Decoy ODN could be transfected into cells efficiently and most of them located in nucleus.To detect the transfection rate,FITC labeled ODN were transfected with lipofectamine 2000 and detected by flow cytometry,the results demonstrated that ODN could be dose dependently transfected into cells.After cell nucleus stained with DAPI and observed with fluorescence microscope,we found most of ODN located in nucleus.4.STAT3 decoy ODN specifically inhibited the transcription activity of STAT3. STAT3 decoy ODN dose dependently decreased the expression of luciferase induced by STAT3.The more STAT3 decoy ODN were transfected,the lower activity of luciferase were induce by STAT3.However,STAT3 scramble ODN had no effect on the expression of luciferase.The transcription activity of STAT3 was significantly decreased when 50nmol/L decoy ODN were transfected into PG and A549 cells (p＜0.01),but scramble ODN didn't show detectable effect(p＞0.05).In a word, STAT3 decoy ODN could effectively and specifically inhibit the transcription activity of STAT3.5.STAT3 decoy ODN could effectively inhibit the proliferation of PG and A549 cells.To investigate the effect of decoy ODN on cell growth,different concentration of decoy ODN were transfected,and incubated for 24h,48h and 72h respectively, then cell number were counted.The results demonstrated that STAT3 decoy ODN could significantly inhibit the proliferation of cells,the inhibition rate was 69%for PG cells when treated with 50nM decoy ODN,and inhibition rate was 64%for A549 cells when treated with 25nM decoy ODN.To further establish the inhibition effect, 3HTdR incorporation method was used.The results suggested that decoy ODN could also significantly inhibit the incorporation of 3HTdR,with the inhibition rate 61%and 66%for PG and A549 cells respectively(p＜0.01).However,STAT3 scramble ODN didn't decrease the proliferation compared with vehicle control.Meanwhile,STAT3 decoy ODN had no obviously effect on proliferation of NIH/3T3 cells which had no activated STAT3.6.STAT3 decoy ODN induced cell apoptosis.To investigate the effect of decoy ODN on apoptosis,annexin-V/PI were used to detect the apoptosis by flow cytometry when transfected with ODN for 12h.The results demonstrated that apoptosis was significantly increased when A549 cells were treated with 25nM decoy ODN,the apoptosis rate rose from 6.41%to 29%,but scramble ODN had no obviously changes on cell apoptosis.7.STAT3 decoy ODN down-regulated the expression of STAT3 targeted genes related with cell cycle and anti-apoptosis.To investigate the mechanism of STAT3 decoy ODN on cell proliferation,the expression of STAT3 target genes were detected after transfected with 50nM and 25nM decoy ODN for PG and A549 cells respectively.Reverse transcription PCR results demonstrated that the mRNA levels of bcl-xl,cyclinD1,mcl-1,survivin and cyclinE were down regulated after transfected with STAT3 decoy ODN compared with vehicle control.The expression of bcl-xl and cyclinD1 were also significantly inhibited when detected by western blotting. However,STAT3 scramble ODN had no obvious effect on the expression of the target genes including mRNA levels and protein levels.8.STAT3 decoy ODN inhibited growth and induced apoptosis of A549 cells in xenograft.A549 nude mice xenograft model were established by inoculating A549 cells in nude mice subcutaneously,and ODN or vehicle control PBS were intratumor injected.The results showed that STAT3 decoy ODN effectively inhibited the growth of A549 cells in vivo,and the inhibition rate reached 69.5%after thirty days treatment. The tumor weight was also decreased,and inhibition rate reached to 52.7%.The tumor tissues were sectioned,and TUNEL was used to detect the apoptosis.The results showed that the apoptosis positive cells were significantly higher in STAT3 decoy ODN treatment group than that in PBS treatment control group,which increased about 20 folds(p＜0.001).However,STAT3 scramble ODN treatment group had similar apoptosis positive cells with control group.9.STAT3 decoy ODN inhibited the proliferation of lung cancer cells in vivo via down regulating its target genes.The expression of bcl-xl and cyclinD1 were assayed using frozen section by immunohistochemistry.The results demonstrated that the expression of bcl-xl and cyclinD1 in STAT3 decoy ODN treatment group were much lower than that in scramble ODN treatment group or vehicle control group (p＜0.05).Result:part 21.STAT3 is more active in adriamycin-resistant K562/A02 cells than its parental K562 cells.The expression and activity of STAT3 were higher in drug resistant K562/A02 ceils than that in drug sensitive K562 cells which detected by western blotting.The P-gp which is related with MDR was highly expressed in K562/A02 cells but not in K562 cells.The luciferase reporter assay also showed that the transcription activity of STAT3 was higher in K562/A02 cells than that in K562 cells.2.JSI-124(Cucurbitacin I)inhibited the STAT3 activated in K562/A02 cells dose and time dependently.JSI-124 was highly selective inhibitor for JAK/STAT3 and did not inhibit other oncogenic and tumor survival pathways.The western blot results demonstrated that JSI-124 could inhibit the activity of STAT3 dose and time dependently,and with significant inhibition at 1μM for 24h.3.Blocking STAT3 increased the sensitivity of K562/A02 cells to adriamycin and promoted its apoptosis under the treatment of adriamycin.JSI-124 inhibited the proliferation of K562/A02 cells in dose and time dependent manner,which were normally cultured in medium containing 1μg/ml adriamycin.To further investigate whether JSI-124 could reverse the adriamycin resistance in K562/A02 cells, K562/A02 and K562 cells were treated with different concentrations of adriamycin lonely or combined with 1μM JSI-124,after 48h,the proliferation were observed. JSI-124 significantly increased the sensitivity of K562/A02 cells to adriamycin. Blocking STAT3 made about 8-fold reduction in adriamycin resistance as determined by fold-change in IC50.However,this effect was not seen in drug sensitivity K562 cells.Treatment of K562/A02 with both drugs resulted in significantly greater cell apoptosis than either drug alone.Combination treatment of both drugs increased the apoptosis from 2.72±0.53%to 11.67±2.8%.4.JSI-124 promoted apoptosis through increasing the up-take of adriamyein in K562/A02 cells partly.Compared with their control,JSI-124 dose dependently increased accumulation of adriamycin in drug resistant cells(p＜0.05).But this effect was not obviously in drug sensitivity cells.The fluorescence of rhodamine-123 was higher when treatment with JSI-124,which indicated that JSI-124 decreased rhodamine-123 efflux medicated by P-gp.5.MDR1 and other MDR associated genes transcripted in K562/A02 cells were down regulated by JSI-124.The real-time PCR results showed that the transcript of MDR1 was decreased by JSI-124 dose dependently.There were only～38%of control when treated with 1.25μM JSI-124(p＜0.01),and only～25%when treated with 2.5μM(p＜0.01).Other MDR associated genes such as MRP1 and BCRP1 were also decreased.JSI-124 decreased the abundance of P-gp in a concentration-dependent manner.P-gp was reduced to～47%and～29%of the control level after treated with 1.25μM and 2.5μM JSI-124 for 36h,respectively.6.Jak2 specific inhibitor AG490 enhanced the drug sensitivity of K562/A02 via down-regulating P-gp.The activity of STAT3 was inhibited by AG490 dose dependently,while the sensitivity of K562/A02 cells to adriamycin was significantly increased.About 8～16-fold in adriamycin resistance was reduced at 100μM AG490 as determined by fold-change in IC50.The transcription of MDR1 was down-regulated by AG490,and inhibition ratio reached to 57%at 100μM(p＜0.01).The amount of P-gp was reduced to 58%of the control level when treating with AG490.7.STAT3 takes part in the regulation of P-gp.Computer assisted searches of potential STAT3 binding sites within the entire P-gp promoter region by TFSEARCH and TRANSFAC database revealed only one potential site which containing putative STAT3 DNA binding elements.This region of the P-gp promoter is located approximately 1014 base pairs upstream of the initiation coding for P-gp,and has strong binding activity with STAT3. Conclusion:In this study,we demonstrated that STAT3 was highly constitutively activated in human lung cancer cells,and played important roles in cell proliferation.STAT3 decoy ODN could specificily block the activity of STAT3 in lung cancer cells,which resulted in proliferation inhibition and apoptosis promotion in vitro and in vivo.Their mechanisms were also investigated that it was related with the down-regulating of STAT3 targeted anti-apoptosis and cell cycle genes.These results demonstrated that it is feasibility for lung cancer gene therapy by using STAT3 decoy ODN to inhibit STAT3 signal pathway.We next studied the feasibility of reversing multidrug resistance by inhibiting STAT3 activity,and investigated their mechanisms.We found that the activity of STAT3 in MDR cells was higher than that in sensitivity cells. Inhibition of STAT3 by JSI-124 reversed multidrug resistance of MDR cells to chemotherapy and increased the apoptosis induced by chemotherapy,which were associated with the down-regulation of MDR1 and P-gp levels.At the same time,the accumulation of adriamycin in cells treated with JSI-124 could be increased which inhibited the cell proliferation further.It provided the feasibility of reversing multidrug resistance by blocking STAT3 and the foundation for combination STAT3 inhibitor with chemotherapy drug in treatment of multidrug resistance.Therefore, targeting activated STAT3 in cancer cells not only inhibits tumor growth but also reverses multidrug resistance.It provided an excellent new target and method for cancer gene therapy. |
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