Preliminary Study On The Antitumor Effect And Mechanism Of A New Compound Preparation(HTCM),Which Composed Of Different Active Components Of Traditional Chinese Medicine

Part 1 Screening of new Chinese medicine compound preparations （HTCM）Objective:To study the effects of different components of Chinese herbal medicine on proliferation of human lung cancer,liver cancer,breast cancer and normal human liver cells.Methods:Under the guidance of the theory of anti-tumor Chinese medicine,the research group used modern pharmaceutical technology prepared out R_X-2(R_1-2,R_2-2,R_3-2,R_4-2,R_5-2,R_6-2,R_7-2,R_8-2,R_9-2),which were extracted by 75%ethanol form new compound Chinese medicine preparations,and R_X-3(R_1-3,R_2-3,R_3-3,R_4-3,R_5-,R_6-3,R_7-3,R_8-3,R_9-3),which were extracted by 95%ethanol form new compound Chinese medicine preparations.The A549,H460,Calu-1,SMMC-7721,Bel-7402,HepG2,L02,MCF-7,T47D cells in the logarithmic growth phase were respectively treated with different concentrations of R_X-2and R_X-3（final concentration was 0.45mg/ml、0.3mg/ml、0.2mg/ml、0.13mg/ml、0.089mg/ml、0.059mg/ml）for 72h.The MTT assay was used to test the effect of cell proliferation of R_X-2-2 and R_X-3-3 on the nine kinds of cells.Calculating the IC₅₀0 values.All data were analyzed by SPSS 19.0 software.Results:R_X-2-2 and R_X-3-3 had different degrees inhibition on nine kinds of cell lines,and all dates analyzed by single factor analysis of variance and LSD-t test,P<0.05,the difference was statistically significant.Among them,in R_X-2,SMMC-7721 cells were the most sensitive to R_4-2(IC₅₀=0.077mg/ml),the IC₅₀values of R_4-2-2 on L02 cells was 0.176mg/ml;in R_X-3,SMMC-7721 cells were the most sensitive to R_2-3(IC₅₀=0.037mg/ml),the IC₅₀0 values of R_2-3-3 on L02cells was 0.168mg/ml.R_2-3-3 has a stronger inhibitory effect on the proliferation of SMMC-7721 cell,But R_4-2-2 and R_2-3-3 have little difference in toxicity to L02.So,it was chosen as the drug for further experiment.Conclusion:（1）With the increase of concentration,the inhibition of R_X-2and R_X-3-3 to A549、H460、Calu-1、SMMC-7721、Bel-7402、HepG2、L02、MCF-7、T47D cells proliferation enhanced;（2）R_2-3,a new compound preparation,has a strong inhibitory effect on the proliferation of SMMC-7721cells,but it has low toxicity to human normal hepatocytes at the same concentration.Therefore,it will be the next step drug.Part 2 Antitumor activity of R_2-3-3 in subcutaneous tumor model of SMMC-7721 cells in nude miceObjective:To explore the effect of R_2-3-3 on the growth of subcutaneous tumor in nude mice model of SMMC-7721 cells and its effect on the liver and kidney function in nude mice.Methods:To establish the subcutaneous tumor model in nude mice for human liver cells SMMC-7721:After 7 days,the nude mice,with tumor volume>50 mm³ were chosen.Based on the experiment scheme,nude mice were randomly divided into 4 groups:control group（daily saline）,low-dosage（100mg/kg daily administration）,medium-dosage（200mg/kg daily administration）and high-dosage（400mg/kg the next day administration）by intragastric administration.For a total of 18 days to medicine in every day or every other day given drug,tumor size and nude mice weight were measured every other day.Tumor size was calculated for tumor volume（TV）,relative tumor volume（RTV）and relative tumor proliferation rate（T/C%）at each time for detecting the influence of growth of subcutaneous tumor in nude mice for human liver cells SMMC-7721.After 18 days of administration,the mice were decapitated and their liver and kidney functions were detected.Tumor weighing.The HE stained paraffin sections were taken from the heart,liver,lung,kidney and other organs.All data were analyzed by SPSS 19.0 software.Results:In the control group,with the increase of tumor volume,the activity of nude mice was reduced,and the diet and water not change significantly.After 18 days later,in the control group the average volume of tumor was 202.67±39.55 mm³,the tumor weight was 0.107±0.004g.In low concentration group the average volume of tumor was 90.33±29.74 mm³,the inhibition rate was 73.56%;The tumor weight was 0.035±0.007g,the inhibition rate was 67.3%.In the medium concentration group the average volume of tumor was 54.33±6.03 mm³,the inhibition rate was 98.23%;The tumor weight was 0.018±0.007g,the inhibition rate was 83.2%.In the high concentration group the average volume of tumor was 70.33±30.11 mm³,the inhibition rate was 88.89%;The tumor weight was 0.027±0.014g,the inhibition rate was74.8%.Compared with the low concentration group,the tumor inhibition rate of the medium concentration group was increased.Compared with the medium concentration group,the tumor inhibition rate of the high concentration group was reduced.The relative tumor proliferation rate of the medium concentration group and the high concentration group after treatment the tenth days was<60%,in the low concentration group after treating drug for Twelfth days the relative tumor proliferation rate was<60%.HE staining showed that the control group in the liver showed large areas of inflammatory cell infiltration and edema of liver cells,liver cells to no drug group in special morphological changes;control group and drug group of kidney had no obvious abnormality.The ALT and AST values in the control group（2127 U/L,2766 U/L）were higher than those in the low concentration group（215.9 U/L,347.4 U/L）,the medium concentration group（314.7 U/L,542.5 U/L）and the high concentration group（137.4 U/L,398.5 U/L）.But there was no significant difference for Urea and Crea values.Conclusion:（1）R_2-3-3 could obviously inhibit human liver cells SMMC-7721proliferation and the growth of subcutaneous tumor in nude mice.It presented a good concentration-response relationship and revealed strong anti-tumor activity in vitro and in vivo;（2）When the dose was equal,the daily administration was stronger than that of the next day;（3）In a short time,R_2-3probably has no side effects on liver and kidney.Part 3 The anti hepatoma effect of R_2-3-3 relating to cell cycle and apoptosis Objective:Preliminary study on the mechanism of R_2-3-3 against liver cancer.Methods:Take SMMC-7721,Bel-7402 and L02 cells in the growing period of logarithm to be intervented by different concentration traditional Chinese medicine compound preparation（final concentration was 0.12 mg/ml,0.09mg/ml,0.06 mg/ml）for 72 hours.Flow cytometry（FCM）was used to analyze the relationship between its anti hepatoma effect and cell cycle or apoptosis.Results:Compared with the negative control group,in the new Chinese medicine compound group G1 phase cells increased,S phase,G2 phase cells decreased.the compound preparation of traditional Chinese medicine high dose group G1 phase cells most obviously increased;However,there was no obvious difference in G1,S and G2 phase cells when Bel-7402 group and L02 group compared with negative control group.Compared with the negative control group,there was no obvious difference in the apoptosis rate of the traditional Chinese medicine compound preparation group.Conclusion:It is speculated that the mechanism of R_2-3-3 inhibits human hepatocellular carcinoma cell line SMMC-7721 proliferation may be associated with Cell cycle arrest in G1 phase,the mechanism that R_2-3-3 inhibits the proliferation of Bel-7402 cells may not be associated with the cell cycle and apoptosis.