Effects Of β-sitosterol On Proliferation Inhibition Of Human Cervical Carcinoma SiHa Cells And Associated Mechanisms

Cervical cancer is one of the most frequently gynecological neoplasm. In recent years the prevalence of cervical HPV infection has raised, the incidence of cervical cancer increased. More and more young women were suffered with cervical cancer. Cervical cancer becomes one of severe diseases that could be harmful to women's health.β-sitosterol is a phytosterol. Several in vitro and animal studies suggest that β-sitosterol offer protection from the most common cancers including colon, prostate and breast. In 1970's, β-sitosterol suppository and β-sitosterol tablets were used on cervical os in vaginal to treat 115 cases of cervical cancer, the effective rate was 75.65%. The exact pharmacological mechanism by which β-sitosterol offers this treatment has not been determined. In this study, we have investigated the effect of β-sitosterol on the growth inhibition of cervical cancer cells and the associated mechanisms.There are three parts in the study: Part Ⅰ Effects of β-sitosterol on the cellular biological behavior in the cervical cancer cell lines. Part Ⅱ Effects of β-sitosterol on microtubular systems in cervical cancer SiHa cells. Part Ⅲ Effects of β-sitosterol on IFN and its downstream genes in cervical cancer SiHa cells.Part Ⅰ Effects of β-sitosterol on the biological behavior in the cervical cancer cell lines.Objective To study the effects of β-sitosterol on morphology, proliferation, cell cycle and apoptosis of human cervical cancer cell lines. Methods Theβ-sitosterol was incubated at different concentrations with cervical cancer cell line SiHa and HeLa. The effects of β-sitosterol on cell proliferation were tested by MTT assay. The effects of β-sitosterol on morphology, ultrastructure and cell cycle were studied by phase-contrast microscope, electron microscope and flowcytometry(FCM). Results β-sitosterol could obviously inhibit the proliferation of SiHa / HeLa cells in a time and dose dependent manner. Better results of growth inhibitory effects were got in SiHa cells than that in HeLa cells. The total protein of pinella has no effects on SiHa cells proliferation. 20 μmol/L β-sitosterol induced the accumulation of SiHa cells in S phase in the cell cycle. And the percent of apoptosis and necrosis increased. The morphology and the ultrastructure changed significantly after treated with 20 μmol/L β-sitosterol. Conclusions β-sitosterol can suppress proliferation of cervical cancer cells. Better results of growth inhibition were got in cervical squamous carcinoma than that in cervical adenocarcinoma. β-sitosterol induces the accumulation of cells in S phase.Part Ⅱ Effects of β-sitosterol on microtubule systems in cervical cancer cellsObjective To investigate β-sitosterol's effects on microtubular system in SiHa cell line. Methods The expression and distribution of microtubule and microtubule associated protein 2 in SiHa cells were investigated by confocal microscopy. Immunoblotting analysis was used to determine tubulin α, microtubule associated protein 2, and the proportion of polymerization of tubulin. Results Confocal microscopy showed an abnormal microtubular network in SiHa cell treated with β-sitosterol for 5 days, and the expression of microtubule associated protein 2 was markedly down-regulated. Further analysis by immunoblotting confirmed the down-regulation of β-sitosterol on the expression of both microtubule associated protein 2 and tubulin α. Moreover, β-sitosterol reduced the proportion of polymerization of microtubule in a time-dependent manner. Conclusion β-sitosterol could down-regulate the expression of tubulin α and microtubule associated protein 2 in SiHa cells, and inhibit the microtubular polymerization. Our results suggested ananti-microtubule characteristic of β-sitosterol which might contribute to the proliferation inhibition of SiHa cells.Part Ⅲ Effects of β-sitosterol on IFN and its downstream genes in cervical cancer SiHa cellsObjective To study the effects of β-sitosterol on IFN, its downstream genes, and HPV E6/E7 protein in cervical cancer SiHa cells. Methods RT-PCR was used to estimate the relative mRNA amounts of IFNα, IFNβ, ISG15, OAS(p40/46), OAS(p69/71), RNase L, MxA, PKR in SiHa cells exposed to β-sitosterol. Western blot were used to measure the STAT1, E6 and E7 protein levels before and after β-sitosterol's treatment. Results after treated with 20 μmol/L β-sitosterol, the mRNA's expression of IFNβ, ISG15, OAS(p39/41), OAS(p69/71) and MxA increased significantly in a time-dependent manner. But the RNase L and PKR's mRNA expression showed no change before and after β-sitosterol's exposure. Through exposure to β-sitosterol also led to an increase in STAT1 and a decrease in E7 protein. Conclusion β-sitosterol could induce IFNβ and its downstream gene's mRNA expression. The products of these genes could contribute to β-sitosterol's effect on SiHa cells directly or indirectly, and then caused growth inhibition. β-sitosterol could also decrease HPV E7 protein directly of through IFN's anti-virus pathway indirectly.