The Extrasomatic Proliferate Inhibition Of Yiliuning On Tumor Cells Outside Of Digestive System

Background and ObjectiveCancer, with an increasingly high incidence recent years, is a terrible threat of people's health and life. The treatment to it has always been a subject with worldwide attention. Currently, surgery, radiotherapy and chemotherapy are dominant means of curing it. However, due to the harmful side effects accompanying with those treatments and the range of their applicably, there are still considerable limitations of them. On the other hand, Chinese medication and medicine have special effects on tumors, either on restraining the diffusion of cancer cells or on killing them. Furthermore, Chinese medication and medicine can also help with the recovery after surgery, relieve discomforts coming with radiotherapy or chemotherapy, and change beneficially the symptoms and physical reactions which improve patients' life quality. Therefore, a larger and larger number of scholars and patients show interests in them and gradually accept the Chinese treatment. Because of its noticeable functions at improving immunity of body, reducing poisonous effects of traditional therapies, increasing positive effects, preventing relapse, stopping pathological changes and gentle harmful side effects, researches of and experiments on Chinese medicine have increased in recent years. Yiliuning is a clinical prescription for tumor of the digestive system composed of Rab,Duc,Sol,Aca. It has the functions of antitumor, clearing heat and detoxication, detumescence and removing stasis. It is a good treatment for advanced esophageal, gastric cardia-gastric fundus cancer, liver and pancreatic cancer with almost no toxicity. Rab,Duc,Sol in the recipe are drugs used to clearing heat and detoxication. Aca can enhance immune function and have antitumor effect. Preliminary experimental shows combination of Rab and Duc two drugs or Rab,Aca,Sol,Duc four drugs can significantly inhibit the growth of tumor cells in vitro, the combination of four drugs can arrest the Eca-109 cells during S-phase and induce cell apoptosis. There is no research about Yiliuning's effect on tumor cells out of the digestive system. This subject is based on the early experiment to study on human breast cancer MDA-MB-231 cells, lung cancer NCI-H520 cells, lung cancer A549 cells, and cervical carcinoma Hela cells. By observing the changes in cell morphology, determining growth inhibition rate and detecting cell cycle and apoptosis with flow cytometry, we can explore the growth inhibition of Yiliuning on tumor cells outside of digestive system, pick out the best prescription and dosage. These can provide experiment basis for clinical application in the treatment outside of digestive system solid tumor.Method1 Preparation of drug is in accordance to the relevant literature extraction method for extracting the components of crude extract. Crude extract is diluted by culture fluid in the experiment. Medicamentous concentration = Weight of medical herbs/ Volume culture fluid.2 Cell culture MDA-MB-231, NCI-H520, A549, Hela cells were inoculated with culture medium L15, RPMI-1640, F12, High glucose DMEM medium which have the volume fraction of 10% inactivated fetal calf serum ,and conventional cultured ?in incubator with 37℃,5%CO₂,95% relative humidity.3 Determination of cell growth inhibition rate The MDA-MB-231,NCI-H520,A549,Hela cells in the logarithmic phase are inoculated in the board with 96 holes with density of 1×10⁵/ml, 5×10⁴/ml, 5×10⁴/ml, 5×10⁴/ml and are taken incubation after 24 hours, adding Yiliuning's different combination as the experimental group , every concentration is set in 6 holes, setting comparison. After cultivated in incubator with 37℃, 5%CO₂,95%RH for 48h, cell growth inhibiting is measured by MTT assay, then test the growth inhibition ratios. And repeat it 3 times.4 Observation of cell morphology Compare the experimental group with the blank group to find the difference in cell morphology with inverted microscope.5 Detection of cycle and apoptosis rate. Flow cytometry detected cell cycle and apoptosis rate.6 With SPSS10.0 software package, one-way ANOVA and Chi-Square test were used to analyze all experimental data, size of the test was P<0.05.Result1 The extrasomatic proliferate inhibition of Yiliuning on tumor cells outside of digestive system.1.1 The proliferate inhibition on MDA-MB-231 when Yiliuning "Rab (2mg/ml) + Duc (0.4mg/ml) + Sol (0.4mg/ml) + Aca (0.4mg/ml)" act onMDA-MB-231 cells 48h the growth inhibitory rate is 99.74%.1.2 The proliferate inhibition on NCI-H520 When Yiliuning "Rab (2mg/ml) + Duc (1mg/ml) + Sol (1mg/ml) + Aca (1mg/ml)" act on NCI-H520 cells for 48h the growth inhibitory rate is 96.41%.1.3 The proliferate inhibition on A549 When Yiliuning "Rab ( 3mg/ml) + Duc (0.8mg/ml) +Sol (0.8mg/ml) +Aca (0.8mg/ml)" act on A549 cells for 48h thegrowth inhibitory rate is 80.62%.1.4 The proliferate inhibition on Hela When Yiliuning "Rab (2mg/ml) + Duc (1mg/ml) + Sol (1mg/ml) + Aca (1mg/ml) " act on Hela cells for 48h the growthinhibitory rate is 97.98%.2 Control group: MDA-MB-231 cells are spindle, growing inherent to the culture flask, the growth is in good condition. The experimental group with four medicines combinated: Cells are separating mutually, those inherent to the culture flask reduce obviously, cells become crimple and grow downwards, lots of cells disintegrate to fragment. 3 By means of flow cytometry to test the cell cycle and apoptosis rates of MDA-MB-231 cells.After acting on cells for 48 hours, the S phase cells percentage in every experimental group is obviously increased and the G₀/G₁ phase cells percentage is obviously decreased. Each experimental group presents the sub-G₁-summit which was the sign of apoptosis, the apoptosis rate is increased. Compared with blank group, the difference has statistical significance (P<0.05) .Conclusions1 The combination of Rab, Duc, Sol, Aca can significantly inhibit the growth of the MDA-MB-231, A549, NCI-H520, Hela cells.2 The combination of Rab, Duc, Sol, Aca can make MDA-MB-231 cells arrest in S phase and induce apoptosis.3 When the four medicines combinated, they have more cytotoxic with little dose, they can inhibit proliferation of Tumor Cells synergeticly.4 Yiliuning, in the best prescription is composed of four Rab+Duc+Sol+Aca drug combination.