Time-dependent Inhibitory Effect And Subcellular Distribution Of Sequence-specific HER-2/neu Phosphorothioate Antisense Oligodeoxynucleotides

Aim: Antisense oligodeoxynucleotides (ODNs) ranging from 15 to 30 bases and specific to mRNA targets have been used to inhibit gene expression. The most common mechanism of antisense action is RNaseH-mediated RNA degradation subsequent to the binding of antisense to its complement in the mRNA target. Sequence-specific HER-2/neu antisense ODNs that interfere with HER-2/neu mRNA expression are potential therapeutic agents for breast carcinoma. Several studies have demonstrated that HER-2/neu antisense ODNs can specially down-regulate p185HER2/neu expression and inhibit breast cancer cells proliferation. In previous work, we optimized the antisense drug design against the HER-2/neu mRNA based on the simulated secondary structures of mRNA provided by software or professional website, phylogenic analysis of the mRNA and the quantitative structure-activity relationship analysis. Several sequence-specific HER-2/neu antisense ODNs with different inhibitory effect on breast carcinoma have been designed. In this study, several HER-2/neu antisense ODNs (HA-2741, 824, 193 and 1027) with different sequences were picked up to study the relationship between inhibitory effect and subcellular distribution in target cell SK-BR-3, which overexpress p185HER2/neu.Methods: Purified, single-stranded, phosphorothioated oligodeoxynucleotides (S-ODNs) or 6-carboxyfluorescein labeled S-ODNs (FAM-S-ODNs) were synthesized by Sangon Biotech Company. S-ODNs were transfected into target cells by using the Lipofectamine? 2000 (Invitrogen, USA). After transfection, TRNzol reagent was used to isolate total RNA according to the manufacturer's protocol. After treatment with DNAse I to remove the genomic DNA contamination, RNA was reverse transcribed to cDNA. Fluorescence Real-time quantitative PCR were carried out using SYBR Green I dye. Levels ofβ-actin mRNA were quantitated as an internal standard. The ratio of HER-2/neu mRNA expression relative to that of the internal standard mRNA (β-actin) was calculated using the semi-quantitative method of Pfaffl according to equation: Ratio= (EHER-2)△CtHER-2 (control-treated) /(Eβ-actin)△Ctβ-actin (control-treated)For western blotting, cells were lysed with sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) loading buffer. Total protein was separated on a 8% SDS-PAGE. For confocal microscopy analysis, cells were plated in glass bottom culture dishes at 3×105cells/dish and transfected with FAM-S-ODNs. After fixation, the cells were treated with RNase, and then were stained with propidium iodide (PI). The subcellular localizations of FAM-S-ODNs were assessed using a confocal laser scanning image system equipped with a Zeiss LSM 510 META microscope.Results: The ability of four sequence-specific HER-2/neu antisense S-ODNs to reduce the endogenous level of HER-2/neu mRNA are different in SK-BR-3 breast cancer cells. Expression levels of HER-2/neu mRNA were decreased in SK-BR-3 cells after transfection with antisense S-ODNs (HA-2741, HA-824, HA-193 and HA-1027) in a time-dependent manner. The four HER-2/neu antisense S-ODNs had significantly different abilities to alter the levels of the corresponding transcripts, ranging from 0.34 to 0.65 fold of the transcript levels in control cells at 72 h post-transfection. In this experiment, strong reduction of the HER-2/neu mRNA was achieved by transfection with HA-2741, and, to a lesser extent, with HA-824 and HA-193. HA-1027 exhibited only a slight reduction of HER-2/neu mRNA. Western blot analyses of p185HER2/neu levels in transfected SK-BR-3 cells showed decreased protein levels almost consistent with the results of real-time quantitative PCR analyses. A clear reduction of p185HER2/neu expression observed after transfection with HA-2741, and a weakest with HA-1027. The extremum of the inhibition emerged at 48 - 72 h after transfection, despite diverse sequences. Subcellular distribution of S-ODNs in SK-BR-3 cell was characterized by monitoring the FAM-labeled S-ODNs using LSM. After transfection using Lipofectamine, most of HA-2741 and HA-824 were rapidly distributed throughout the cell nucleus, bright spherical foci designated phosphorothiate bodies (PS bodies) and only a small percentage was present in the cytoplasm, particularly the perinuclear region. In contrast, green fluorescence of HA-193 and HA-1027 were found mainly within the cytoplasm in punctuate manner, and to a very low extent in the nucleus. At 72 h post-transfection, the green fluorescence has almost diminished.Conclusion: In this study, we found that S-ODNs transfected by using Lipofectamine distribute in both nucleus and cytoplasm. The subcellular distribution of S-ODNs with different inhibitory efficacy differed remarkably, and the distributional character of individual S-ODNs was correlated to its inhibitory efficacy. Within 24 h after transfection, most of the HA-2741 and HA-824 were rapidly accumulated in the cell nucleus uniformly and only a small percentage was in the cytoplasm as punctuate fluorescence. Meanwhile, majority of the HA-193 and HA-1027 presented in the cell cytoplasm as green fluorescence spots and to a very low extent in the nucleus. At 48 h after transfection, HA-2741 slightly decreased while HA-193 increased in the cell nucleus. At 72 h after transfection, there was little green fluorescence spots in cell cytoplasm, which showed that ODNs degraded. The green fluorescence spots in cytoplasm might be the fluorescent metabolites of the FAM-labeled ODNs transported into cytoplasmic lysosomes. All these results correlate to the inhibitory effects of ODNs on HER-2/neu expression that HA-2741 and HA-824 with high efficacy last for long time, while HA-3 and HA-4 with low efficacy last for shorter time. So, we have the hypothesis that S-ODNs with high efficacy has high binding affinity to target mRNA, and it is more stable in nucleus to perform for long time. In contrast, S-ODNs with low efficacy has weak mRNA binding affinity, and it's more sensitive to nuclease.