| As one of the most common malignant tumors, breast carcinoma has high prevalence and mortality rate all over the world. Nowadays breast cancer common therapy has still high reoccurrence and metastasis rate. The emergence of gene therapy has brought new hope to human beings for conqouring human tumor completely.Being with high specificity and low toxicity, antisense therapy has been paid more and more attention in cancerous therapeutics researches developed greatly. Recently, antisense oligodeoxynucleotides has become a smart tool for exploring gene function and gene expression regulating in basic and clinical researches.As reverse transcriptase, telomerase can synthesize the repeated sequences of telomere DNA and add hexanucleotides to the end of replicating chromosomes by using its own RNA template. It was recognized as one of the most important tumor markers, found in nearly 90% cancer cells but no in normal somatic cells. Human telomerase has three subunits: human telomerase RNA (hTR), human telomerase reverse transcriptase (hTERT) and telomerase associated protein I (TPI). Activation of telomerase is essentially needed for cellular immortalization. hTERT is the catalytic subunit of telomerase and its expression is the rate-limiting factor of activity of human telomerase. So it is considered as an ideal target of anticancer strategy. ASODN complementary to hTERT DNA could inhibit hTERT gene expression and telomerase activity, and induce tumor cells apoptosis finally. But naked oligo -deoxynucleotides will be degraded by nuclease easily leading to weak intracellular penetration. In order to conquer this defect, researchers adopted various gene vehicles including viral and non-viral gene vehicles to transport ASODN. PEI, a kind of novel non-viral gene delivery, which can form PEI/DNA to enhance transfection efficiency in vitro. But its transfection efficiency is decreasing obviously caused by clearance of MPS in vivo. Furthermore, PEI/DNA has cell cytotoxicity with concentration of free PEI. Liposome is a kind of vesicle that consists of an aqueous compartment enclosed in phospholipid bilayer. Researchers try to achieve an ideal result through the association with liposome and PEI/DNA. Then we equipped LPD with target-oriented CNGRC peptides in order to make LPD can enter into tumor effectively following the blood circulation.In this research, the tumor-targeting and anti-tumor activity of NGR/LPD was investigated in BALB/c-nude mice bearing human breast tumors transplanted subcutaneously. Compared with LPD, PEI/ASODN and free ASODN, NGR/LPD could enter into tumor tissue efficiently after administering via veins, and the quantity of NGR/LPD enter into tumor tissue increased continuously with time increment, but PEI/ASODN condensates and LPD couldn't enter into tumor tissue efficiently. It was also found that NGR/LPD could significantly inhibit the growth of tumor efficiently, compared with LPD, PEI/ASODN and other control groups. Furthermore, tumors transplanted subcutaneously in the nude mice being administered NGR/LPD grew much slowly compared with the control groups, and its inhibiting tumor growth efficiency was correlated with dose in vivo.This experiment study demonstrated that NGR/LPD was a kind of stable, tumor-targeted and easy-prepare vector for gene delivery, which can protect ASODN better than PEI/ASODN, and has perfect tumor-targeting function. It may be a promising candidate for non-viral gene delivery system.Methods:1. The preparation of NGR/LPD,PEI/ASODN and LPD.2. Investigate the characteristics and capsulate efficiency of NGR/LPD. 3. Human breast carcinoma cell line MCF-7 that grew logarithmically were traslplanted subcutaneously to BALB/C-nu/nu mice.3.1 groups to inhibit the growth of tumor in nude mice:Free ASODN group: 200μl free ASODN was injected via vein.Negative group: 200μl Isaline was injected via vein.NGR/ Lipo/PEI-SODN group: 200μl NGR/Lipo/B-PEI-SODN was injected via vein.LPD high dose group: 200μl LPD was injected via vein.LPD low dose group: 100μl LPD was injected via vein.NGR/ LPD high dose group: 200μl NGR/ LPD was injected via vein.NGR/ LPD low dose group: 100μl NGR/ LPD was injected via vein.PEI/ASODN high dose group: 200μl PEI/ASODN was injected via vein.PEI/ASODN low dose group: 100μl PEI/ASODN was injected via vein.3.2 Groups to test the distribution of drug in nude miceGroupⅠ200μl ASODN-FAM was injected via vein.GroupⅡ200μl PEI/ASODN-FAM was injected via vein.GroupⅢ200μl LPD-FAM was injected via vein.GroupⅣ200μl NGR/LPD-FAM was injected via vein.3.3 After the human breast carcinoma xenografts of athymic mice were established. Drugs were injected every other day during 3 weeks, and growth of tumors were observed every other day.3.4 Distribution of free ASODN,PEI/ASODN, LPD and the NGR/LPD complex 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.3.6 The tumor tissue hTERT mRNA expression was detected by RT-PCR.3.7 The expression level of hTERT protein, C-myc and apoptosis-related protein Bcl-2 was measured through immunohistochemistry staining.3.8 Apoptotic cells in tumors were evaluated with TUNEL assey.4. Statistical analysis: The SPSS 11.0 Ststistical Pachage program was used for all analyses. The data were expressed as mean±S.D, and analyzed using the t-test for two groups and ANOVA for more than two groups. P-value of <0.05 was considered to be statistically singficant.Results:1. NGR/LPD complex with an average size of 178.2 nm, which is discrete, spherical and well capsulated. NGR/LPD is polyplex with the reasonable zeta potential which can be conserved stably in common condition.2. The fluorescence intensity in the carcinoma xenografts tissue which transfected by NGR/LPD was much higher than those transfected by LPD, PEI/ASODN and free ASODN. In NGR/LPD group, which fluorescent emission was time-dependent increasedly. The distribution of free ASODN, PEI/ASODN and LPD was so fractional compared with NGR/LPD.3. It was found that the human breast carcinoma xenografts on BALB/C nude mice grew much slowly after administering NGR/LPD, compared with LPD, PEI/ASODN condensates and the control groups, and there was significant difference between the low and high dose groups.4. The expression of hTERT mRNA decreased obviously in NGR/LPD compared with LPD, PEI/ASODN condensates and the negative control(P<0.05). There was still significant difference between the low and high dose groups(P<0.05). There was no significant difference between ASODN, NGR/Lipo/B-PEI-SODN ,LPD, PEI/ASODN condensates and the negative control (P>0.05) .5. In NGR/LPD high dose group, the expression of hTERT and bcl-2 protein was decreased, and C-myc protein was increased (P<0.05). There was still significant difference between the low and high dose groups(P<0.05). There was no significant difference between ASODN, NGR/Lipo/B-PEI-SODN, LPD, PEI/ASODN condensates and the negative control (P>0.05) .6. The apoptotic index of NGR/LPD group was obviously higher than the negative group(P<0.05, P<0.01). There was significant difference between the low and high dose groups(P<0.05). There was no significant difference between ASODN, NGR/Lipo/B-PEI-SODN ,LPD, PEI/ASODN condensates and the negative control (P >0.05) .7. Observing pathological sections of tumors transplanted subcutaneously each group, we find that The growth of tumor cells in the control groups is active, tumor giant cells can be seen easily, a bit scattered, patchy necrotic region can be seen. Interstitial fibrosis is obvious in tumor organization of NGR/LPD high-dose group. Necrosis is more serious than that in the control groups. A large number of serous effusion in the alveolar cavity and widened alveolar septum can be seen in PEI/ASODN high-dose group. PEI/ASODN low-dose group and other groups are not abnormal.Conclusion:1. NGR/LPD was a kind of stable tumor-targeted vector which has no cell cytotoxicity and easy to be prepared for gene delivery.2. ASODN which complementary to hTERT DNA can inhibit hTERT gene expression and telomerase activity, induce tumor cells apoptosis finally.3. NGR/LPD could enter into tumor intentionally following the blood circulation to inhibit the tumor growth efficiency, and the effects were concentration-dependent.4. NGR/LPD showed perfect tumor-targeting and ideal anti-tumor activity in vivo. It will be a promising tumor gene therapy application. |
PDF Full Text Request