| Ovarian cancer is one of the most gynecological malignant tumors. Because most patients with the disease were diagnosed in a late stage, the prognosis is still poor so far. Although treated with intensive chemotherapy and anti-angiogenesis therapy, some patients would still develop reoccurrence some years later. However, tumor metastasis and recurrence have not been well defined so far. Therefore, it is urgent to investigate the mechanism of tumor metastasis and reoccurrence, discover new predictor of early metastasis and find new treatment.According to the recent researches, the phenomenon of epithelial-mesenchymal transition (EMT) is closely related to the tumor metastasis, which is characteristic of two distinct features:lose of epithelial phenotype and acquisition of mesenchymal phenotype. When tumor cells undergone EMT, they lose the cell-cell adhesion, migrate more easily, and become more invasive.In addition, owing to the similarity between cancer cells and stem cells in terms of proliferation pattern and molecular phenotypes, theory of cancer stem cells was put forward recently. According to this theory, there are a thimbleful proportion of cancer cells within tumor tissues which act as stem cells, bearing the ability of self-renewal and differentiation in tumorigenesis. More and more researches suggest that cancer stem cells exist in some kinds of tumors which serve as the root of cancer development, invasion, metastasis and recurrence.However, neither EMT nor cancer stem cells can explain precisly the mechanism of tumor metastasis. Whether they have some inner relations druing tumor development remains largely unknown. Our previous study on human ovarian cancer have showed that there is indeed a minor proportion of side-population (SP) cells effluxing the fluorescent dye Hoechst 33342 possessing stem-like pluoripotent properties. Compared with non-side-population (non-SP) cells, SP cells were shown to have stronger ability of proliferation in vitro and higher potential of tumor formation in vivo. Microarray assay showed that expressions of epithelial-related genes in SP cells are higher, whereas the mesenchymal-related genes are relatively lower comparing to those in non-SP cells, suggesting that differentiation of SP cells into non-SP cells undergoes the process of EMT to become more migratory which leads to local invasion and engraftment.In addition to our previous finding, this study took a further step to investigate the biological behavior of SP cells in ovarian cancer when the process of EMT was regulated intrinsically and extrinsically. The investigations on tumor cells pathological behavior will provide new strategy for basic research of tumor metastasis.1. The regulation of TGF-β1-induced EMT to human ovarian cancer SP cellsSP cells and non-SP cells were isolated by FACS based on the ability to efflux the fluorescence dye Hoechst33342 and cultured in vitro in the presence of TGF-β1 at a concentration of lOng/ml. The morphology changes of SP cells and non-SP cells were observed under phase contrast inverted microscope. Majority of SP cells have round shape whereas non-SP cells are fibroblast-like presence. After cultured with TGF-β1, some of the SP cells gradually extend and develop protuberances white the protuberances of non-SPs elongated, of which are characterized as EMT. RNA samples from different time points were collected by adding Trizol. EMT-related and stem cells pluripotency-related genes were analyzed by RT-PCR. The expressions of EMT-related genes were increased both in SP cells and non-SP cells and, SP cells respond more dramatically to the activation of TGF-β1. However, stem cell pluripotency-related genes were decreased in SP cells after TGF-β1 stimulation, suggesting the pluripoency of SPs was decreased during the process of EMT. Finally, the percentage of HO-8910PM-SP cells after culturing with TGF-β1 was detected to be decreased by FCM. Hence, the results show that TGF-β1 can efficiently induce epithelial SP cells undergoing mesenchymal transition concomitant pluripotency decreasing and differentiation promotion.2. The regulation of Snaill-SiRNA to human ovarian cancer SP cellsPrevious study showed that the expressions of mesenchymal phenotype markers, including Snail1, Snail2, Smad2 and Smad3 were higher in ovarian cancer SP cells than those in non-SPs. Among them, expression of Snail 1 was significantly decreased at about 5 folds lower in SP cells compared to non-SP cells. Thus wechose Snail 1 as the target gene to be downregulated by using small interfering RNA encoded in lentiviral vector, Ninety-nine% of HO8910PM cells were stably transduced by Snail1-SiRNA detected by FCM. The silence effect of Snail1 and other EMT-related markers were examined by RT-PCR and Western Blots. The proportion of SP cells declined after SiRNA inhibition. Thus we doubt that whether the cells encoded with SiRNA underwent apoptosis more than vector control cells. To clarify this hypothesis, we further investigated cellular apoptotic potential after SiRNA inhibition. The nucleus were stained by Hoechst33342/PI and apoptosis cells were identified as nuclei condensation and nuclei fragmentation. The results showed that SiRNA cells have more apoptosis than vector control cells, indicating silence of Snaill can enhance cell death. With the view of cell membrane location, the ABCG2 pump in apoptotic cells after Snaill down-regulation would be damaged and the capability of effluxing the dye will decline. SP cells percentage was therefore decreased. Furthermore, the population of SP cells could be further decreased after cultured with TGF-β1. Hence, these results indicated that inhibition of EMT by silence of Snail1 decrease stem-like cell percentage by promoting cell apoptosis.In conclusion, our study found that the percentage of SP cells can be regulated. (1) TGF-β1 can induce epithelial-mesenchymal transmission of SP cells, decrease the pluripotency of stem-like SP cells, and promote stem cells differentiation. (2) The process of EMT can be efficiently blocked by Snaill-SiRNA concomitant cell apoptosis enhancement. (3) Either TGF-β1 stimulation or Snaill-SiRNA blockage can decrease the percentage of SP cells. However, the function they exerted on tumorigenesis and the governing mechanism remains to be investigated further.
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