| Breast cancer is the most common cancer among women greatly threatened physical and psychological health of them. Every year, more than one million women were diagnosed as breast cancer around the world. At the same time, four hundred thousand women die of it. The rate of incidence in occident is higher. And the incidence of the disease is upgrading in china. Now, Surgery is the main treatment for breast cancer cooperated with chemoradiotherapy. However, chemoradiotherapy also cause serious side effects. Hence searching and exploiting antineoplastic agents with little side effect and good therapeutic effect is a clinical imperative.Trollius chinensis, the dried flowers and flower bud of Ranunculaceae plants, have heat-clearing and detoxifying effect. In the folk, the drug is used to cure tonsillitis, swollen throat and mouth and acute enteritis. The modern pharmacological study indicates that total flavonoids, the important effective constituents of trollius chinensis, have good effect on antioxidant, antiviral, anti-inflammatory, antibiosis, lower blood pressure and spasmolysis effect.Previous research of team revealed that trollius flavonoids were able to inhibit the proliferation of MCF-7 cells. However, the action mechanism have not been fully clarified. In this experiment, this topic verified the apoptosis of MCF-7 cells by multiple way including HE staining, fluorescence microscope, DIC, scanning electron microscope, laser confocal microscopy. The protein expression was detected using western blot method, which elucidated the biochemical mechanisms to provide power for clinical anti-tumor drug development. Experimental methodThe experiment was divided into four groups: black control group, solvent control, drug group(0, 0.0991, 0.1982, 0.3964, 0.7928, 1.5856 mg·m L-1), time in this case was fixed at 24 h, 48 h and 72 h. MTT method was exploited for detecting the inhibition rate. The exploited of differential-interference(DIC) software verified cell apoptosis from three-dimensional angle. HE stain was exploited for observing the nuclear and cytoplasmic changes of MCF-7 cells. Hoechst33258 fluorescence method was developed for viewing the morphological changes of cell nucleus during cell apoptosis. Scanning electron microscope was used for observing the changes of cell surface. Laser co-focus light microscopy combined with the labeling of nucleus and phospholipids on the surface of cells mirrored the apoptosis of MCF-7 cells. Western method was used for detecting the expression of bcl-2, NF-κB, p53, caspase-3, 9 and Fas L. Experimental results 1.The inhibition effect of total flavonoids from trollius chinensis on breast cancer MCF-7 cells.Total flavonoids of trollius chinensis at different concentrations had inhibition effect on breast cancer MCF-7 cells. Compared with black control group, the maximum inhibition ratio of DMSO in solvent group was below 1%. DMSO below 1% have no effect on the proliferation and activity of cells. So there was no solvent group in the following experiments. In the same time phase point, inhibition ratio of total flavonoids of trollius chinensis at different concentrations increased as concentration increased, there were significant differences between normal control and drug group(P < 0.05); Inhibition ratio shows significant differences between different time groups(P < 0.05). At different time phase, inhibition ratio of drug group with same concentration increased as time goes on. There were significant differences between different time groups(P < 0.05). 2.The morphological observation of apoptotic MCF-7 cell induced by total flavonoids from trollius chinensis 2.1 Differential interference(DIC) was used for detecting the cellular morphologyIn black control group, normal cells of size uniformity was closely packed. After treated with drugs, cells shrank to round and cell junction became loose. The adherent cells decreased, bubbly and gemmiformly “Membrane bubble” appeared on cell surface. Cell nucleus swelled and cytoplasm condensed, cells shows typical morphological features. As concentration increased, apoptotic cells increased and characteristic morphological changes of apoptosis becomes more and more obvious. 2.2 The result of HE stainingIn black control group, cells in the shape of fusiform grew adhering to the wall and the cells were intact. The nucleoplasm were homogeneous stained. Nucleolus is distinct and nucleoplasm makes up a high proportion. The cells were arranged compactly, cell in divisions could be seen. Compared with normal group, MCF-7 cells treated with drugs(0.0991, 0.1982, 0.3964 mg·m L-1) shrunk and were gradually deformed for dehydration, cell in divisions disappeared, cell bodies became smaller and cell nuclear condensation. Intercellular spaces dilatation, condensed chromatin was dyed into blue-black. The cytomembrane of MCF-7 cells treated with drugs(0.7964, 1.5856 mg·m L-1) was disrupted, the adherent cell decreased as drug concentration increase, which was in consistent with that of scanning electron microscope and differential interference. 2.3 Hoechst dying to observe cellular morphologyIn black control group, the nucleus of MCF-7 cells, showing uniform dispersion of low-density fluorescence, had regular shape. Treated with drugs at different concentrations, cell apoptosis and the number of adherent cells decreased, cells nuclei present darker nuclei than the normal cells. The proportion of darker nuclei increased. Fragmented nuclei appeared, as concentration increased, characteristic morphological changes of apoptosis became more and more obvious. 2.4 The result of scanning electron microscopeThe MCF-7 cells in black control group, cling to holder, was covered with abundant developed microvilli. Cells having the alberto aquilani’s shape were intact. The cells connected with the neighbors firmly and stretched in all directions. Compared with control group, microvilli on the surface of cells in different drug groups atrophy and scattered around cells. cells shrinked and turned smaller, junction between cells fractured. As concentration increased, apoptotic cells increased, characteristics of apoptosis became obvious. In different phases, cells show different apoptosis characteristics. In high concentration(1.5856 mg·m L-1) group, even cell membrane collapse appearing a hole-like structure. 2.5 The observe result of laser co-focus light microscope(Annexin V-FITC double labeled method)In black control group, MCF-7 cells were not readily stained by Annexin V or PI. Compared with control group, MCF-7 cells in drug group apoptosis. When cells were in the early stage, phosphatidylserine(PS) were transferred to the surface membranes of cells, cells were dyed by Annexin V showing green fluorescent. The late apoptotic cells, membrane of which were no longer integrity, cell nucleus were dyed red also. Cells were dyed by Annexin V and PI showing typical morphological features. In brief, cells presented the characteristically morphological changes of apoptosis, apoptotic morphological characteristics obviously appeared. 3. The function mechanism of MCF-7 cell apoptosis induced by total flavonoids of trollius chinensis.The results of Western blot indicated that compared with protein in black control group,the expression of Fas L and p53 in drug group have no obvious changes, verifying that total flavonoids have no effect on the expression of Fas L and p53. However, total flavonoids could down-regulate the expression of NF- κ B and Bcl-2, meantime up-regulate the expression of caspase-3 and caspase-9. Conclusion 1. Total flavonoids of trollius chinensis could inhibit the proliferation of MCF-7 cells inducing the apoptosis of cells. 2. Total flavonoids of trollius chinensis could down-regulate the expression of NF-κB and bcl-2 activating caspase-3, 9 to induce the apoptosis of cells. |
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