| 1. Four sequences of HER-2 mRNA were obtained from Genbank. Among those sequences, the Aligned Scores for three (4441 bp, 4478 bp, 4530 bp) exceed 98, another sequence, although only 1316 bp of length, it's Aligned Score also reached 78. So except for the antisense designed against HER-2 mRNA of 4530 bp, which attracted more attentions of researchers, we emphasized our efforts on antisense design against HER-2 mRNA of 1316 bp on the basis of conservation of secondary structures. Secondary structure of 4530 bp was simulated via the Genebee website and one structure was obtained; Secondary structure of 1316 bp was predicted by application of the computer mRNA secondary structure prediction software RNAstructure (version 3.5, 1999) and finally got one optimal and two suboptimal secondary structures. Thirteen antisense S-ODNs targeting the secondary structural elements of 4530 bp were designed and some important parameters including free energy (△G °37) related to the target secondary structural elements were calculated according tothe nearest neighbor model. For 1316 bp HER-2 mRNA, we divided local secondary structural motifs into three sections: completely conservative local motifs (LMs), which were the common LMs in three simulant secondary structures; variable LMs, which were variable LMs among all three simulant secondary structures; relatively conservative LMs, which were the common LMs in two of three simulant secondary structures. We also analyzed characters of its secondary structures, calculated the parameters including AG°37 and finally designed nineteen S-ODNs respectively against three parts above. The lengths of all S-ODNs were 20-mer. Randomized oligos was set as random control sequence, and HA4 was set as positive control. Mean (n = 3 - 5) 50 % inhibitory effects on proliferation of SK-BR-3 cells (IC50) of S-ODNs were evaluated. The QSAR (quantitative structure-activity relationship) analysis through multiple regression was obtained by SPSS.2. The results indicated that to the antisense against 4530 bp, four S-ODNs had statistically significant lower IC50 (53±38, 43±20, 41±20, 58±28 nmol-L"1, respectively) than that of HA4 (97±27 nmol-L"1) (P < 0.05); To the antisense against 1316 bp, nine out of 11 S-ODNs against completely conservative LMs got lower or similar IC50 values compared with HA4, on the other hand, two out of 3 S-ODNs against relatively conservative LMs got lower or similar IC50 values compared with HA4, and only 2 out of 5 S-ODNs targeting variable LMs had acceptable activities. Average IC50 of S-ODNs against completely conservative LMs was significantly lower than that of S-ODNs against relatively conservative and diverse LMs (147±43 nmol-L"1 vs 466±165 nmol-L"1, P < 0.05). QSAR results indicated that as to the S-ODNs against 4530 bp, only the reaction free energy (AG "37/?) reached significance (R = 0.604, P = 0.029), other parameters such as the base number of internal loop, bulge loop, hairpin, AG°37T related to S-ODNs (AG°37S), the AG°37 of drug-target-formed duplex (AG°37d) and the target sequence AG°37 (AG°37t) were insignificant. QSARanalysis for S-ODNs against 1316 bp indicated that stability of the local secondary structure, base number of bulge loop and AG "375 were statistically significant, and stability was most significant (R = 0.741, P = 0.003); Anyway, when we only analyzed parameters of S-ODNs targeting completely conservative LMs, we found that in addition to significant parameters above, other parameters such as the number of internal loop, hairpin, AG°37s, AG^r were also significant (R=0.967, .P=0.005). Results indicated that antisense drug design based on the combination method of secondary structure prediction, phylogenetic analysis and QSAR analysis was helpful for obtaining better S-ODNs candidates than positive control HA4.3. Three oligomers including HA824, HA2741 and HA4136 were further investigated for anti-cancer activities. The cell lines used for evaluation were breast cancer cell lines SK-BR-3, MDA-MB-453, MDA-MB-231, and the normal fibroblast cell line NIH-3T3, the former two were HER-2 over-expressed, whereas the other two were lower or none expressed. Results showed that the inhibitory effects of the three sequences selected on breast cancer cells were correlated with the basic HER-2 expression levels of the cell lines, that is, the anti-proliferation effects of these three sequences on SK-BR-3 and MDA-MB-453 cells were remarkable and dose-dependent, whereas the effects on MDA-MB-231 or NIH-3T3 cells were not remarkable. Taken into consideration IC50 and percentage of inhibition on cell growth, HA2741 was superior to the other two candidates (IC50, SK-BR-3: 56±16 nmol-L"1 vs HA4: 128±47 nmol-L'1, P < 0.05, MDA-MB-453 39±8 nmol-L"1 vs HA4: 114±33 nmol-L"1, P < 0.05. Where the HA4 was the previously reported positive control). Further studies indicated that the anti-cancer effects of HA2741 were sequence-specific indicated by the facts that HA2741 was with superior activity to its control sequences in the HER-2 over-expressed cell lines, wheras with close activity to the controls in the lower expressed cell lines. In order to knowvvhether the anti-proliferation effects of HA2741 on breast cancer cells was related to the down-regulation of targeting mRNA and HER-2 receptor, we detected the expression of HER-2 mRNA and its receptor before and after treated with HA2741 by means of RT-PCR and immunocytochemistry (ICC). At the concentration of 200 nmol-L"' for 9 hours, HA2741 did down-regulate the expression of HER-2 mRNA in MDA-MB-453 cells dramatically, and HER-2 receptor also changed from strong positive before treatment to weak positive or negative after treatment in the meantime. Results indicated HA2741 achieved its goal by specifically inhibited the expression of HER-2 rnRNA and its receptor. Moreover, a lot of manifestation of apoptosis appeared in MDA-MB-453 cells after exposed to HA2741 for 24-72 hours observed by fluorescent microscope, whereas MDA-MB-231 cells, which were HER-2 lower expression, almost had no such changes even after incubation with the same concentration of HA2741 as long as 72 hours. Apoptosis appearances observed here including cresentoid pyknosis, karyorrhexis, apoptosis bodies, and so on. Consequently, a hypothesis might be made that HA2741 displayed the inhibitory effect on the proliferation of carcinoma cells via the potent mechanism of apoptosis inducement.4. In order to evaluate the anti-cancer effects of HA2741 on HER-2 over-expressed breast cancer, An animal xenograft model was successfully established in BALC/neu nude mice using the SK-BR-3 cells. In vivo effects of HA2741 and its control sequence alone or in combination with the chemotherapeutic agent docetaxel on tumor growth were investigated. The results showed that a multiple intravenous injection of HA2741 (q.d. X12) inhibited the growth of breast cancer in the nude mice in a dose-dependent manner, 5 weeks after the first administration, the inhibitory rate of the tumor growth reached a level of 76.3% ±8.5%. Further, HA2741 improved the sensitivity to the routine chemotherapeutic agent of the tumors, resulted in theelevation of the tumor growth inhibitory rate of docetaxol. At the dose rate of 5 mg-kg"'-d"1, HA2741 helped the lower dose of Docetaxol (7.5 mg-kg"'-d"') reach the inhibitory rate of tumor growth from lower than 50% to as high as 88.3%±4.5%, which was comparative with the inhibitory rate (88.7%±4.3%)obtained following a higher dose of docetaxol alone (15 mg-kg'l-d"1). On the other hand, at the same dose rate of 5 mg'kg"'-d"1, the control sequences plus docetaxel did not enhance the anti-cancer activity of the latter. Results of IHC indicated that the inhibitory effect of HA2741 on tumor growth was in accordance with the down-regulation of HER-2 expression.
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