The Effect On Telomerase In The EC9706 Cells Inhibited By Nanoparticles For Antisense Oligodeoxynucleotide Of HTERT In Vitro And In Vivo

Esophageal carcinoma is one of common malignant tumors in China. Celluar progression to malignancy is thought to involve multistep events. Activiation of telomerase and the stable of telomere length are essentially required for cellular immortalization and genetic alteratens.Telomerase, a ribonucleoprotein enzyme, synthesizes DNA-protein complexes which is called telomere at the end of eukaryotic chromosomes that protects chromosome ends from fusion and degradation, ensures the complete replication of telomeric DNA. Telomerase is thought to be necessary for cellular immortality and carcinogenesis consists of three subunits: human telomerase RNA (hTR), human telomerase reverse transcriptase (hTERT), and telomerase associated proteinI (TPI). hTERT is a catalytic subunit of telomerase and its expression is the rate-limiting factor of the enzymatic activity of human telomerase. According to studies, the most of examined rumors showed telomerase activity correlating with the expression of the activity-limiting component hTERT. hTERT was found in nearly all types of cancer but not in most normal, somatic cells. So it is thought the target of anticancer strategy. Esophageal carcinoma has been reported to have a high frequency of detectable telomerase activity and hTERT gene expression was consistent with detectable telomerase activity in 91% of esophageal tissues. This suggested a strong relationship between the expression of hTERT mRNA and telomerase activity in esophageal tissues.It was shown that transfection with antisense oligodeoxynucleotide (ASODN) specifically reduces the expression of target genes. By use of antisense strategy, antisense oligodeoxynucleotides (ASODN) complementary to hTERT mRNA and DNA could be used to inhibit hTERT gene expression and telomerase activity. But naked ODNs are easy to be degraded by nuclease biologically and weak intracellular penetration, in order to overcome the defect, the key is to find proper vehicles to carry and protect ODNs . Lots of researchers adopted various vehicles including viral or non-viral gene vehicles system. Among these carriers, cationic polymers have shown interesting potentialities due to their stability , their easy of preparation, as well as chemical modification to condense ODNs into nanoparticles,et al. Polyethylenimine (PEI) emerged as a potent polymer for clinical gene therapy, which includs L-PEI and B-PEI,and not only can adhere to and transfect cells by forming PEI/ODN but also equipe with target-oriented substance ,such as NGR, to enhance transfection efficiency in vivo.The purpose in the study was to investigate the effection on EC9706 cells inhibited by Line-polythenimine-based(L-PEI) antisense oligodeoxynucleotide of hTERT in vitro and in vivo, by observing hTERT protein levels and cell apoptosis. And NGR/PEI/ASODN polyplex was texted in vivo to prove its feasibility. This offers the theoretic and experiment basis as a novle gene vector for gene therapy on esophageal cancer.Methods:1. One ASODN complementary to hTERT and one sense oligodeoxynucleo tides( SODN) were designed and synthesized. Each ODNs were transfected into EC9706 cells by L-PEI.2. Methods in vitro2.1 EC9706 cells were maintained in RPMI 1640-10% fetal bovine serum(FBS), at 37℃in a humidified atmosphere of 50 mL/L CO₂ in air.2.2 Cells of the experiment were divided into four groups:cell group: RPMI-1640 and 10% FBS (control group)ASODN group: RPMI-1640 and 10% FBS and ASODNPEI/SODN group: RPMI-1640 and 10% FBS and PEI/SODNPEI/ASODN group: RPMI-1640 and 10% FBS and PEI/ASODN2.3 The complexation of ASODN and L-PEI was tested by agarose gel retardation assay with N/P=0, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0,9.0.2.4 The proliferation of EC9706 cells were observed by MTT assay. Cellular uptake of L-PEI/ ASODN complexes was detected by laser confocal microscope. The expression of hTERT protein were detected by immunohistochemictry assay kit. The expression of hTERT mRNA were determined by RT-PCR. Cell apoptosis were determined by flow cytometry (FCM).3. Methods in vivo3.1 The probability of constructing nude mice model carried tumors was explored by injecting human esophageal cancer cell line EC9706.3.2 Groups:3.2.1 Groups to inhibit the growth of tumor in nude miceNegative group 200μl saline was injected via tail vein of mice.SODN group 200μl NGR/PEI/SODN was injected via tail vein of mice.Vein group 200μl NGR/PEI/ASODN was injected via tail vein of miceSubcutaneous group 200μl NGR/PEI/ASODN was injected beside tumor.3.2.2 Groups to test the distribution of drug in nude miceGroupâ… 100μl NGR/PEI/ASODN was injected via tail vein of mice.Groupâ…¡100μl NGR/PEI/ASODN was injected beside tumor.Groupâ…¢100μl PEI/ASODN was injected via tail vein of mice.3.3 After nude mice had been injected with esophageal cancer cell EC9706 for 2 weeks,drugs were injected every other day during 2 weeks,and the growth of tumors were observed.3.4 Distribution of L-PEI/ ASODN complexes in nude mice was detected by laser confocal microscope after injected for 1h,3h,6h.3.5 After treatment,the tumor tissues were fixed by 10% formalin to make ordinary pathological section,HE staining,and were observed by microscope.3.6 Apoptosis was quantified with electron microscopy in tumor tissues.3.7 The expression of hTERT protein were detected by immunohistochemictry assay kit.4. Statistical analysis: The SPSS Statistical Pachage program was used for all analyses. The data were expressed as mean±S.D., and analyzed by one-way ANOVA. P-value of <0.05 was considered to be statistically significant.Results:1. When N/P=10, and the dose of ASODN was 40μg/ml, the average diameter for L-PEI/ASODN was 194±11nm , zeta potential were 16.8±3.9mv. When N/P=6.8, the average diameter for NGR/L-PEI/ASODN was 128±5nm , zeta potential were 26.2±1.2mv.2. Results in vitro :2.1 Agraose gel electrophoresis retardation assay showed that L-PEIcompletely retarded ASODN at N/P=6.2.2 After 24h of transfecting,the inhibitory effects on EC9706 cells were tested and were time-dependant. The significant differences were found between ASODN and control group(p<0.05),no difference appeared among L-PEI/SODN and control group(p >0.05).2.3 The result from confocal microscope showed the ability of uptake of ASODN by L-PEI was enhanced.2.4 hTERT protein expression in EC9706 cells were detected by immunohistochemictry: negative control group and L-PEI/ASODN group cells were negative staining, blank control group cells were positive staining with blue nuclei and cytoplasm in brown granules.2.5 After transfecting L-PEI/ASODN for 48 h, compared with control group, the hTERT mRNA expression decreased observably (p < 0.05).2.6 FCM showed that the early and later apoptosis rates in L-PEI/ASODN group,which is 38.03% and 52.17% respectively. 3. Result in vivo3.1 After treating for one week in the nude mice, the mean volumes of tumors in the vein and subcutaneous groups were obviously smaller than blank control group and SODN group, and there were significant difference (p<0.05 ) .3.2 The result from confocal microscope showed that the distribution of drug in the twe NGR/PEI/ASODN groups were in the liver ,kidney,lung and tumor, which their fluorescent emission were time-dependent increasedly. The distribution of drug in the PEI/ASODN group was not been seen in the tumor.3.3 hTERT protein expression in the rumors were detected by immunohistochemictry: L-PEI/ASODN group were poor positive staining with blue nuclei and cytoplasm in light brown granules, blank control group and SODN group were strong positive staining with blue nuclei and cytoplasm in brown granules.3.4 Electronmicroscopy showed that there were apoptosis in the tumors of L-PEI/ASODN group and were not classical apoptosis in the control group.Conclusions:1. L-PEI/ASODN and NGR/L-PEI/ASODN are polyplexes with the reasonable diameter and zeta potential.2. When N/P=10,and the dose of ASODN was 40μg/ml, L-PEI can condence ASODN to form nanoparticles with lower cytotoxity in vitro,which enter into tumor cells , protect the degradation of ASODN and can make the proliferation of EC9706 cells inhibited by hTERT ASODN effectively.3. When N/P=6.8, NGR/L-PEI/ASODN nanoparticles was not any cytotoxity in nude mice.,which can reach tumor cell in vivo directedly. The test suggested NGR can be applied in clinical geneic therapy.4. EC9706 cells apoptosis appeared after transfecting ASODN in vitro and in vivo, The results presented that there was a positive correlativity between the telomerase activity and the apoptotic rate.5. After transfecting, the expression of hTERT mRNA was decreased. This verifed that ASODN against hTERT have sequence specialization. 6. After transfering, the expression of hTERT protein was inhibited. This showed that hTERT mRNA controlled the expression of hTERT protein.