| This paper discusses the apoptosis and its mechanism of human colorectal cancer (lovo) cells induced by the extract of Chloranthus japonicus Sieb.(CJ) and Chloranthus multistachys Pei (CM) which belong to the genus of Chloranthus in Chloranthaceae family. The chemical composition of the plants in this genus mainly include terpenoids, coumarone class, flavonoids, amide class, organic acids, steroid and steroidal saponins etc. As folklore medicines in China, the whole plants have been traditionally used for hundreds of years to treat boils, detumescence, snake bite, bone fractures, cough, and rheumatic pain. In recent years, research has shown that the extract of Chloranthus japonicus Sieb. can induce apoptosis, however, its molecular mechanism is unknown, furthermore, there is no report focus on apoptosis induced by the extract of Chloranthus multistachys Pei.In this experiment, we try to discuss the anti-tumor function of the extract of CJ and CM by the way of MTT, microscopic observation, flow cytometry, Western Blot etc., and improve our understanding of apoptosis mechanism induced by the two kinds of plant extracts. The main results are as follows:1. The inhibit effect of the extract of CJ and CM act on this three kinds of tumor cells in vitro proliferation. The extract of CJ and CM were put into Caco、Lovo、MCF-7cells respectively for72h, and then detection by MTT. Results show that these three kinds of cell proliferation have been inhibited in62.5-1000μg/ml concentration range, growth inhibition rate show the gradually strengthen trend with the increased extract dose, which clear prove the dependent relationship between dose and effect. With linear regression equation seek the IC50value, according to this, the two kinds of plant extracts were sensitive to Lovo cells, so we choose Lovo cells as a follow-up study.2. The extract of CJ and CM influence on Lovo cell proliferation. The different concentration of two extracts were put into Lovo cell for24h、48h and72h, with the MTT method to detect, the results show that the inhibition rate will increase with the concentration raised when they were in the same time. With the time prolonged, the inhibition rate raised gradually. So there is an obvious relationship between time and the concentration.3. When the two extract act on Lovo cell for48h, we observe a morphological change in microscope. Compared with the controls, the cells state became worse, the number of cells constantly reduce,the ability of stick wall and the connection between the cells will also reduce. The cells can’t connect and appear space. After high concentration extracts working, cell fragment and background particles will increase. It appear more cell suspension. The drug concentration is more increase, the cell state is worse. After the largest concentration extract act on cells, there will be a large number of cells death and disintegration.4. The extract of CJ and CM act in Lovo cells for48h by staining Hoechst33258. With the fluorescent microscope, we observe the cell unclear was reduced after the drug treatment. Cell unclear is bright blue and gather in the unclear surrounding or cracking into pieces.5. We detect the cell apoptosis rate by AV-PI staining after the extract of CJ and CM act in Lovo cells for24h、48h. Test and analysis by relevant software, when the cell treat by the extract of CJ for24h with the concentration of250μg/ml,500μg/ml,1000μg/ml, the cell apoptosis rate were10.97%,15.33%,18.77%respectively; For48h cell apoptosis rate were18.77%,21.20%,54.93%respectively. When the cell treat by the extract of CM for24h with the concentration of250μg/ml,500μg/ml,1000μg/ml, the cell apoptosis rate were10.67%,12.9%,15.57%respectively; For48h cell apoptosis rate were10.43%,16.97%,47.10%respectively.6. The test of DNA content in the cell cycle after the extract of CJ and CM were treated on Lovo cells for48h. The extract of CJ and CM with the different concentration were treated on Lovo cells for48h, the cell cycle phase distribution change dramatically, DNA content increased at the S phase, however, at the G2/M period DNA content decrease, which show that the cell cycle was blocked at the S phase.7.The detect of relative cell apoptosis protein after the extract of CJ and CM were treated on Lovo cells for48h. Western blot results show that procaspase-3,8,9were acted after this two extract treat Lovo cells for48h, Compared with the control, it has a significant difference (P<0.05). This proved preliminary it may be achieve cell apoptosis through the cells and extra cellular way work together when this two extract induce cell apoptosis. |
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