RESUMO
The antisense strategy has been applied to regulate gene expression in a sequence specific manner, which enables suppression of the proliferation of cancer cells and exploration of the functions of unknown genes. In order to generalize and to enhance the ability of the strategy, functionalization of antisense DNAs was done using a photo-crosslinking reagent, 4,5',8-trimethylpsoralen, and the possibility of photodynamic antisense regulation of gene expression was examined. Psoralen-conjugated oligo(nucleoside phosphorothioate)s (Ps-S-oligo) were prepared and used to inhibit the proliferation of human cervical carcinoma cells. Upon UVA irradiation of Ps-S-oligo treated cells, Ps-S-oligo complementary to the initiation codon region (Ps-P-As) of HPV18-E6*-mRNA of human cervical carcinoma cells inhibited drastically the cell growth (IC(50)=16 nM). In contrast, Ps-S-oligo with mismatched sequences and scrambled one showed lesser inhibitory effects than Ps-P-As. These results showed that the inhibition by Ps-S-oligo was dependent on (a) sequence, (b) UVA irradiation, (c) concentration and (d) cell line. The amount of intact HPV18-E6*-mRNA was decreased in a sequence dependent manner, indicating that the antiproliferative effect of Ps-P-As was an antisense manner. The psoralen-conjugated antisense DNA has significant potential to regulate gene expression, which may provide useful information to explore the novel gene regulating reagents.
Assuntos
Reagentes de Ligações Cruzadas/uso terapêutico , Expressão Gênica/efeitos dos fármacos , Oligonucleotídeos Antissenso/uso terapêutico , Fármacos Fotossensibilizantes/uso terapêutico , Trioxsaleno/uso terapêutico , Neoplasias do Colo do Útero/tratamento farmacológico , DNA/farmacologia , DNA/efeitos da radiação , Feminino , Ficusina/farmacologia , Ficusina/uso terapêutico , Humanos , Oligonucleotídeos Antissenso/farmacologia , Fármacos Fotossensibilizantes/farmacologia , Trioxsaleno/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos da radiaçãoRESUMO
To increase the antisense regulatory effect of oligo(nucleoside phosphorothioate)s (S-Oligo), a photo-crosslinking reagent, 4, 5', 8-trimethylpsoralen, was used in this study. Psoralen-conjugated oligo(nucleoside phosphorothioate) (Ps-S-Oligo) complementary to the human papillomavirus type 18 (HPV18) mRNA drastically inhibited the cellular proliferation of cervical cancer cells only upon UVA-irradiation. In contrast, Ps-S-Oligos with mismatched sequences and scrambled one showed lesser inhibitory effects than that with matched one. These results suggest that psoralen-conjugated antisense S-Oligo has significant potential to regulate gene expression upon UVA-irradiation.
