| The Hedgehog signaling pathway which has long been known to control embryonic development is essential for numerous processes in cell proliferation and differentiation during and after embryogenesis. Recent research suggests that uncontrolled activation of the pathway results in specific types of cancer. Mutation or overexpression of Hedgehog signaling pathway genes, such as Ptch1,Smo,Shh,Gli1 and Gli2, is implicated in the development of several different types of cancer particularly of basal cell carcinoma. As mammary gland is the organ that stems from skin, there must be some relations between deregulation of Hedgehog signaling pathway and breast cancer formation and maintenance.Cyclopamine which is the specific inhibitor of Hedgehog signaling pathway can inhibit the Hedgehog signaling pathway through direct interaction with SMO. The past results show that inhibition of Hedgehog signaling by cyclopamine decreases survival of different types of cancer cells, suggesting a role for Hedgehog pathway antagonists in breast cancer therapy. The aim of our study is to investigate the effect of cyclopamine on growth, proliferation and apoptosis of human mammary adenocarcinoma cell line Bcap-37 and MDA-MB-231.We used the following methods: The cells were incubated with cyclopamine, which is the specific inhibitor of Hedgehog signaling pathway. Then the inverted microscope was used to observe the morphology changes of the cells and MTT assay, BrdU incorporation assay and flow cytometry (FCM) were used to examine the influence of cyclopamine on growth, DNA synthesization, cell cycle and apoptosis of the cells.The main results were as follows:1. Compared with the control group, after the cells were incubated with cyclopamine, obvious morphology changes of the cells were observed: the third dimension of the cells disappeared, the number of vacuoles in the cells was increased, some of the cells became small and round, the interval between the cells was obscure which all suggested that the growth of the cells was inhibited.2. The results of MTT assay showed that cyclopamine (10-7～10-5mol/L) inhibited the proliferation of the cells in a dose dependent manner and the inhibitory effect of cyclopamine (5×10-6mol/L) became more significant with action time extended.3. The results of BrdU incorporation assay showed that the number of BrdU positive labeling cells was decreased in the cyclopamine (5×10-6mol/L) treat group which suggested that the DNA synthesization of the cells was obviously inhibited by cyclopamine. 4. We used flow cytometry to study the effect of cyclopamine on the cell cycle of the breast cancer cells. The results showed that, after treatment with cyclopamine(5×10-6mol/L) for 72h, the proportion of Bcap-37 cells in S phase decreased from 24.2% to 19.2% and the proportion of the cells in G2 phase increased from 7.3% to 14.7%, but the proportion change of the cells in G1 phase was not significant which indicated that cyclopamine(5×10-6mol/L) inhibited the proliferation of Bcap-37 cells and the cells were blocked in G2 phase; the proportion of MDA-MB-231 cells in S phase decreased from 42.1% to 28.8%, the proportion of MDA-MB-231 cells in G1 phase increased from 46.1% to 65.5% and the proportion of MDA-MB-231 cells in G2 phase decreased from 11.9% to 5.7% which indicated that MDA-MB-231 cells were blocked in G1 phase and cyclopamine (5×10-6mol/L) inhibited the proliferation of the cells.5. The results of apoptosis detection by flow cytometry showed that cyclopamine induced the apoptosis of Bcap-37 and MDA-MB-231 cells. After treatment with cyclopamine (5×10-6mol/L) for 72h, the apoptotic index of Bcap-37 cells increased from 1.0% to 5.3% and the apoptotic index of MDA-MB-231 cells increased from 4.5% to 20.1%.All these results suggest that cylopamine inhibit the growth and proliferation of breast cancer cells effectually and induce apoptosis of the cells which indicates Hedgehog signaling pathway may be inappropriately activated in breast cancer and inhibiting Hedgehog signaling pathway can be a useful method in breast cancer treatment. |
PDF Full Text Request