| There are over one million women would be diagnosed with breast cancer per year all around the world. It shares 25% within the women cancer. The normal treatments of breast cancer are not only traditional operation, radiotherapy, chemotherapy and endocrine therapy, using the Chinese medicine to do the multimodality therapy also is the hot field of searching recently. The Chinese medicine has quite good effect, no matter to reduce the clinical symptoms, improve the quality of survival, prevent recurrence and transfer, and improve the effect but decrease toxicity in the cooperation with radiation and chemotherapy. So to find the antitumor compounds from natural products has become the important direction of searching antitumor drugs. Draconis Sanguis is rare resinae medicine with the effects of promoting blood circulation to remove blood stasis, remove the necrotic and recover the muscle, stopping blood and relieving pain. It is called as ‘the magic medicine of activing blood’. The recent research shows that Draconis Sanguis has the character with cytotoxicity and antineoplastic active. It could activate signalling pathway of apoptosis, induce tumor cell apoptosis. Lourein A is one kind of flavonoid in the Draconis Sanguis, it should be a dihydrochlcone. Its molecular formula is 4- hydroxy – 2,6- dimethoxy dihydrogen. And the content is very abundant in the Draconis Sanguis. But now, the research of Lourein A in the antitumor activity is less than less.In view of this, the task is to via observing the effects and the possible mechanism that the Lourein A in the cell proliferation apoptosis and protein ER expressing, to find a new anti-cancer medicine with the quality of high activity, low toxicity side effect and antagonizing drug-resistance.This research applies activated monomer Loureirin A of Draconis Sangui, which to act on breast cancer MCF-7 cell line of positive estrogen receptor, and to observe the influence and possibile mechanism of Loureirin A on breast cancer MCF-7 cell proliferation apoptosis and expression of ER protein. The details of experiment including: 1. Cultivation of MCF- 7 cell in vitro, use different concentration of Loureirin A(15,30,45,60,75,90,105,120μmol/L) to role the cell with 24 h, 48 h or 72 h, apply MTT assay to assess this cells inhibiting effect growth of pharmacy, and calculate IC50. 2. From MTT to check how Loureirin A affects breast cancer MCF-7 cell growth inhibition, and from the detection of cell proliferation protein Ki67, to oberseve the proliferative effect of MCF-7 by Loureirin A. 3. From checking the expression of cell apoptosis-related protein Caspase-3, Bax, Bcl-XL/S, to discuss if Loureirin A induce breast cancer cell MCF-7 apoptosis in vitro and other possible things or not. 4. From checking the condition of ERα and ERβ of breast cancer cell after intervention by Loureirin A, to study the results of Loureirin A affects on ER expression.Methods:1. Detection the growth effect of MCF- 7 cell by Loureirin A and calculate IC50Using different concentration of Loureirin A(15,30,45,60,75,90,105,120μmol/L) to do with MCF-7 cell in vitro, after rolling the cell with 24 h, 48 h or 72 h, apply MTT assay to assess this cells inhibiting effect growth of pharmacy, and calculate cell inhibition rate and IC50. Then select the best experimental concentration Loureirin A for it.2. Experiment in vitro of MCF-7 cell effects by Loureirin AUsing Loureirin A to act on breast cancer MCF-7 cell, measure growth curve by MTT, detect cell proliferation symbolic protein Ki67 by immunohistochemistry, and observe the influence of proliferative effect of MCF-7 cell by Loureirin A; Using Hoechst33258 and flow cytometry, to assess the influence of apoptosis of MCF-7 cell which taken by Loureirin A; and detect the result of Loureirin A in expression change of apoptosis regulatory factor Caspase3, Bax and Bcl-XL/S Protein with immunofluorescence, immunohistochemical and western blot.Results:1. Loureirin A result in growth effect in MCF-7.(1) From MTT detection, Loureirin A can work to growth inhibition with the MCF-7 cell, and also it has time and dose dependent in certain range, data analysis obtains that after working with Loureirin A, IC50 is half maximal inhibitory concentration, which valued 53.13μmol/L, and the best experimental concentration is to get 60 μmol/L.(2) From using immunofluorescence to detect the expression of cell proliferative marker protein Ki67, it can be shown as that the expression strength of Ki67 in disposed group is obviously lower than by control group.2. Loureirin A result in apoptosis in MCF-7Through Hoechst33258 staining, viable cell can be dyed light blue fluorescence and nucleus roundness, it can be even seen that quite dark particle, the apoptotic cell should be dyed brilliant blue fluorescence, from using karyolobism or debris shape to analyze the apoptosis situation of each group of the cells: the apoptotic rates of MCF-7 which has been treated by Loureirin A or control culture medium 48 h are 39.47%(15/38),10.07%(14/139), respectively,These two results have significant differences; After working 72 h of MCF-7 cell by Loureirin A, the apoptosis rate is 52.3%, which is apparently higher than control group(15.41% 45/292); Compared with the working time, 72 h and 48 h of MCF-7 cell treated by Loureirin A, p<0.05, so there is statistics difference.3. The influence of apoptosis regulatory factor Caspase3, Bax and Bcl-XL/S by Loureirin A:(1) Immunofluorescence result shows that Bax is mainly positioned on cell cytoplasm, membrane or nuclear membrance, compared to two groups, the fluorescence intensity of disposed group is obviously stronger than control group.(2) Immunohistochemical result shows that Caspase-3 is mainly expressed in cytoplasm and karyon, the positive reaction is claybank, compared to two groups, the average optical value of disposed group(0.368±0.682)is obviously higher than control group(0.218±0.326,p<0.05 vs disposed group); Bax is mainly positioned on cellcytoplasm, cell membrane or karyotheca, the positive reaction is claybank; compared to two groups, the average optical value of disposed group(0.411±0.295) is obviously higher than control group(0.255±0.490,p<0.05 vs disposed group). The result of western blot for Bax and Bcl-XL/S shows that the relative expression level of Bax in disposed group is obviously higher than control group(p<0.05), however the relative expression level of Bcl-XL/S is obviously lower than control group(p<0.05).(3) Flow cytometry result shows that the cell apoptosis rate of breast cancer cell after working 72 h in disposed group is 34.6%, however which in control group is 7.23%, so these two have significant difference(P<0.05).4. The influence of Loureirin A to estrogen ERα and ERβ:Immunofluorescence result shows that ERα is expressed in cytoplasm and karyon or cell membrane, compared to two groups, the fluorescence intensity of ERα in disposed group is obviously weaker than control group;ERβ is mainly expressed in karyon and cytoplasm, compared to two groups, the fluorescence intensity of disposed group is obviously stronger than control group. The western blot test results of ERα and ERβ have been shown that ERα of disposed group, which expression level is obviously lower than the control group(p<0.05), however the relative expression level of ERβ is obviously higher than the control group(p<0.05).Conclusion:1. Loureirin A has inhibiting effect on breast cancer cell MCF-7 of people who express ER positively, and its growth inhibition by means of interferencing proliferative activity and inducing tumor cell apoptosis.2. Loureirin A may activate Bax/Bcl-xl mitochondria signal channel to induce breast cancer cell apoptosis from up-regulating caspase-3.3. Loureirin A may through up-regulating the expression of ERβ and down-regulating ERα to reduce the proportion of ERα/ERβ, and make S-DNA synthesis reducing. By controlling the speed of cell proliferation, it can regulate the growth differentiation of cancer cell. Its regulatory mechanism can not be detailed now, and should have further study and exploration. |
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