| Objective: Phytoestrogen (PE) is a group of natural non-steroid compound which displays estrogen-like activity because of their structural similarity to human estrogens and exhibits high affinity binding to estrogen receptor beta. Because of its specific property: estrogen activity and anti-estrogen activity, compared with HRT, PE provided a more effective and safer method. We were devoted to the research for PE in the long term. This paper has further studied the characteristic and mechanism of action of Osthole and Resveratrol in MCF-7 cells.Methods:1 The growth inhibition or proliferation of osthole or resveratrol in MCF-7 cells and other tumor cells was analyzed by MTT and SRB assay.Before the beginning of experiments, MCF-7 cells were seeded in phenol redfree RMPI1640 medium containing 5% charcoal dextran-treated FBS, then inoculate MCF-7 cells and other tumor cells in exponential phase of growth by using 96-hole cultivation plate , adding osthole or resveratrol after 24 hours of cultivating. Measure the growth inhibiting/proliferating rate IC50 or GI50 in vitro towards tumors cells by MTT and SRB method after 48 hours of cultivating.2 Microscopic structure changes was observed by inverted microscope and nuclear morphological assessment of apoptotic cells and dead cells were observed by fluorescence microscopy. We assessed the morphological change of effects osthole or resveratrol in MCF-7 cells by inverted microscope (×200) and fluorescence microscope with adding Giemsa or Hoechst33342, then reserved the result.3 The DNA ladder was revealed by agarose gel electrophoresis. We extract DNA from the MCF-7 cells, treated with osthole or resveratrol for 48h, for agarose gel electrophoresis.4 The apoptotic rate, Proliferation Index and the cell cycle state were examined by flow cytometry analysis (FCM).We collected MCF-7 cells which seeded in phenol redfree RMPI1640 medium containing 5% charcoal dextran-treated FBS in exponential phase of growth, adding osthole or resveratrol, then keeping on cultivating the Hela for 24h and 48h, then detected by flow cytometry by turns.5 The protein expression of PCNA was detected by immunohistochemistry.6 The mRNA expression of PCNA,Bcl-2 and bax in MCF-7 cells was semi-quantified by reverse transcription PCR(RT-PCR).7 The protein expression of P21,Caspase-3,CDK2 in MCF-7 cells was semi-quantified by Western-blot. Results:1 Osthole or resveratrol effects the proliferation of MCF-7 cells. MTT assay showed that osthole or resveratrol with lower concentration could make a proliferative effect in MCF-7 cells, the highest proliferative concentration separately was 1.6×10-7mol/L and 1.0×10-7 mol/L. Osthole or resveratrol with higher concentration inhibited the growth of MCF-7 cells, IC50 separately was 7.47×10-5mol/L and 8.70×10-5mol/L. Resu1ts of MTT and SRB assay also showed the cytostatic effect to many other tumor cells. Further more, the resu1ts of cell growth curve matched showed the effect has the anti-tumor activity.2 Osthole or resveratrol effects cell cycle. Flow cytometry studies revealed that after treatment with different concentrations of osthole or resveratrol for 24h and 48h, osthole or resveratrol with lower concentration can both increase DNA percentage in S+G2M phase of cell cycle. After treatment with higher concentration of osthole the number of cells in S phase was higher than that in untreated cells; and the number of cells in G1 phase was higher than that in untreated cells after treatment with higher concentration of resveratrol. The percentage of cells arrested in S or G1 phase increased with the increasing of concentration.3 Osthole or resveratrol with higher concentration induced tumor cells apoptosis.Osthole or resveratrol with 0.5×10-4mol/L1.0×10-4mol/L induced morphological changes by Giemsa and Hoechest staining, which were characteristics of apoptosis. Contrast cells displayed excellent growth characteristics-polygonal shape with round large nucleus featuring prominent mu1tiple nucleoli, and well spread on the growth surface. Osthole or resveratrol with 0.5×10-4mol/L1.0×10-4mol/L evoked typical apoptotic features such as membrane blebbing, cell shrinkage and detachment, and chromosome condensation and fragmentation.Agarose gel electrophoresis showed typical DNA fragmentation pattern and confirmed the apoptosis induced by osthole or resveratrol with 0.5×10-4mol/L～1.0×10-4mol/L. DNA fragmentation caused by osthole or resveratrol was dose- dependent.Finally, flow cytometric analysis of MCF-7 cells exposed to osthole or resveratrol with 0.5×10-4mol/L～1.0×10-4mol/L confirmed the morphological observations above. The DNA fluorescence histograms of PI-stained cells showed the low DNA stainability of the treated apoptotic cells, which resu1ted in a distinct, quantifiable region A0 peak. In contrast, the G1 peak predominated in control cells. Quantification of dose-dependency was done by monitoring the amount of nuclei with subdiploid DNA content with flow cytometry. The effect was also time-dependent. The proportions of apoptotic cells incubated in both osthole and resveratrol for 48h were higher than in the same concentration for 24h.4 Protein expression of PCNA by immunohistochemistry. Osthole at the concentration of 1.6×10-7mol/L and resveratrol at the concentration of 1.0×10-7mol/L could significantly up-regulate PCNA protein expression. 5 Osthole or resveratrol regu1ated expression of PCNA mRNA, Bax mRNA and Bcl-2 mRNA.Expression of PCNA mRNA, Bax mRNA and Bcl-2 mRNA in MCF-7 cells exposed to osthole or resveratrol was investigated by RT-PCR. The resu1ts showed that PCNA mRNA and Bcl-2 mRNA levels increased after treatment with osthole or resveratrol of 0.8×10-7mol/L3.2×10-7mol/L or 0.5×10-7mol/L2.0×10-7mol/L, meanwhile the expression of Bax mRNA decreased for 24h cu1turing Hela cell in the present of PB-LY. The effects with the concentration of 0.5×10-4mol/L～1.0×10-4mol/L indicated an opposite results. 6 Regulating expression of P21 protein, Caspase-3 protein and CDK2 protein by osthole or resveratrol.Western blotting experiments showed apoptosis induced by osthole or resveratrol of 0.5×10-4mol/L1.0×10-4mol/L was associated with the increased of P21 protein and Caspase-3 protein ,and decreased of CDK2 protein.Conclusion: Osthole or resveratrol showed two aspects of the effect in MCF-7 cells: lower concentration could make a proliferative effect in MCF-7 cells; higher concentration inhibited the growth and induced apoptosis of MCF-7 cells. Osthole or resveratrol with lower concentration obvious increased DNA percentage in S+G2M phase of cell cycle. Osthole or resveratrol with higher concentration respectively caused cell cycle arrest to S or G1 phase in the dose-dependent manner. Osthole or resveratrol with different concentration can also influence the cell proliferation and apoptosis related gene and protein: PCNA, Bcl-2, Bax, P21, Caspase-3 and CDK2.They significantly showed the phytoestrogen activity, and wou1d be a prospective naturally occurring drugs for the prevention and treatment of perimenopausal syndrome. |
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