| Object: Ovarian cancer is the most lethal tumor of the femal genital tract. The conventional principles of treatment are surgical treatment and chemotherapy. 5 -year survival in patients with ovarian cancer continues to be 28%~35%. It is necessary to find a new treatment to prolong survival time of the patients with ovarian cancer. Amplification and overexpression of the c-erbB2 proto-oncogene has been observed in 20% to 40% of human epithelial ovarian cancers, where it characterizes a group of patients with unfavorable tumor biology and poor prognosis. Experimental evidence indicates that c-erbB2 overexpression contributes to transformation and tumor progression. In normal tissues, P185 is only expressed at low levels in some epithelial cell types. Therefore, the p185 protein represents an attractive target for antisense therapy.This study was designed to investigate the effect of antisense oligonucleotides (AS-ODN) of c-erbB2 gene on SKOV3 ovarian cancer cell line.Methods: 1. HPLC-purified or polyacrylamide gel-purfied 15-mer phosphorothioate oligodeoxynucleotides targeted against the c-erbB2 mRNA and complementary-sense control oligonucleotides were synthesized fromShanghai Sangon. 2. SKOV3 ovarian cancer cells were seeded in 24-well microtiter plates at 5×104 cells/well and allowed to adhere for 24 hr. We divided the treatments into three groups: AS-ODN group; S-ODN group; untreated control group. Each group cells were transfected with 5uM of AS-ODN, S-ODN or distilled water. The proliferation of cells were observed through living cell count. Morphological changes of apoptotic cells were investigated by light microscopy. DNA fragmentation was analysed by agarose gel electrophoresis. Kinetics of induction of apoptosis , cell cycle and c-erbB2 gene expression were analysed by flow cytometry.Result: 1. The growth of SKOV3 cells could be suppressed time dependently by 5uM AS-ODN treatments after transfection. 2. C-erbB2 antisense oligonucleotides treated SKOV3 cells showed characteristic morphological changes of apoptosis. 3. A ladder-like patter of DNA fragments was demonstrated on agarose gel electrophoresis by AS-ODN treatments. 4. The apoptosis rate of SKOV3 ovarian cancer cells in AS-ODN, S-ODN and untreated control groups were 9.02%, 2.67% and 2.98% respectively at 24hr, while 15.92%, 3.94% and 4.02% respectively at 72hr by flow cytometry. The apoptosis - inducting effect of c-erbB2 antisense oligouncleotideds was in a time-depent manner. The apoptosis rate in AS-ODN group was significantly higher than that of S-ODN and untreated controlgroups(p<0.01). There was no significant difference between S-ODN group and untreated control group(p>0.05). Cell cycle analysed by flow cytometry showed the cells of AS-ODN group increased in G0~G1 phase in 73.77% and decreased in both S and G2~M phases in 13.83%, 12.68%. The PI(proliferation index) in AS-ODN, S-ODN and untreated control groups was 26.48%, 32.86% and 33.34% respectively. The PI in AS-ODN group was significantly lower than that of S-ODN and untreated control groups(P<0.01). There was no significant diference between S-ODN group and untreated control group(P>0.05). It indicates that c-erbB2 antisense oligonuclotides induced apoptosis of ovarian cancer cells mainly in G0~G1 phase. 5. The p185 protein expression in AS-ODN, S-ODN and untreated control groups was 6.27, 8.42 and 8.37 respectively at 24 hour while 5.12, 7.47 and 7.53 respectively at 72 hour. There was significant difference between AS-ODN group and control groups. It indicates that 5uM dose of antisense oligonucleotides distinctly reduced the 185-kDa level of the c-erbB2 gene produt in SKOV3 cells with 24 hr. The complementary-sense and untreated , used as control , had no effect.Conclusions: C-erbb2 antisense phosphorothioate oligonucleotides could not only inhibit the proliferation and the expression of the p185 protein but also induce the apoptosis in SKOV3 ovarin cancer cells. Antisense effects onp185 expression and cancer cell proliferation are sequ... |
PDF Full Text Request