| Objective:To screen out the best inhibitory effects herbs from 531 kinds of herbs in three esophageal cancer cells TE-1,EC9706 and EC109, observe the effects of the formula of clearing heat and resolving phlegm (composed by n-butyl alcohol extracted fractions of four Chinese herbs Chuan Shanlong, Tu Beimu, Zao Jiao, Chong Lou) and its separated prescriptions on the proliferation inhibition, cell cycle, and EGFR-PLC-γ1-PKC-a-ERK and PLCε1-p53-NFkB signaling transduction pathway of human esophageal cancer cell TE-1,EC9706 and EC109; study the mechanism of esophageal cancer treated by the formula of clearing heat and resolving phlegm, explore the molecular pathogenesis of esophageal cancer, so as to reveal the generality of esophageal cancer morbidity and its molecular mechanism treated by TCM.Methods:Ethanol extracted method was used to prepare the alternative herbs, human esophageal cancer cell TE-1,EC9706 and EC109 were selected as the experimental objects, the growth of cells were observed by inversed fluorescent microscope, and calculated the inhibition rate of all alternative herbs on TE-1,EC9706 and EC109 cells with MTT method, screening out herbs(Chuan Shanlong, Tu Beimu, Zao Jiao, Chong Lou) with obviously inhibition effect to all three cells; the four screened herbs were extracted by chloroform, ethyl acetate, n-butyl alcohol and water respectively, and got their chloroform, ethyl acetate, n-butyl alcohol and water extracted fractions; then the effects of four differently extracted fractions of four herbs on three cells proliferation were detected with MTT methods, screening out the highest inhibition rate extracted fraction of four herbs to all three cells, composing formula of clearing heat and resolving phlegm, measuring the dose-effect relationship of the formula and its two separated prescriptions on all three cells; cells growth state were observed under inverted microscope; flow cytometry method was used to measure the cell cycle of TE-1,EC9706 and EC109 cells after effected by the formula of clearing heat and resolving phlegm and its two separated prescriptions; western blot method was used to check the protein expression in EGFR-PLC-γ1-PKC-a-ERK and PLCε1-p53-NFkB signal transduction pathway in three cells, and observed the effect of the formula of clearing heat and removing phlegm and its two separated prescriptions on the protein expression of TE-1, EC9706 and EC109 cells.Results: ① Four kinds of herbs Chuanshanlong, Chonglou, Zaojiao and Tubeimu with having the best inhibitory effects on three esophageal cells TE-1,EC9706 and EC109 was screened out from 531 kinds of herbs.②Four kinds of herbs Chuanshanlong, Chonglou, Zaojiao and Tubeimu were extracted by chloroform, ethyl acetate, n-butyl alcohol and water respectively, the n-butyl alcohol effective fraction of four kinds of herbs were screened out with the best inhibitory effects on three esophageal cells TE-1,EC9706 and EC109.③The n-butyl alcohol extracted fraction of four herbs (Chuan Shanlong, Tu Beimu, Zao Jiao, Chong Lou) and the formula of clearing heat and removing phlegm could significantly inhibit the growth of TE-1,EC9706 and EC109 cells, and the whole formula had the most obvious effects. ④Formula of clearing heat and removing phlegm and its two separate prescriptions could all inhibit TE-1,EC9706 and EC109 cells proliferation, and effect of the whole formula was the strongest, and presented dosage-effects and time-effects dependent relationship for the inhibition of TE-1,EC9706 and EC109 cells proliferation with the increasing of medicine concentration and time. ⑤ormula of clearing heat and removing phlegm and its two separate prescriptions all had obviously effects on TE-1,EC9706 and EC109 cell cycle. For TE-1 cell:compared with the normal group, cellular percent of G1 stage reduced in all treatment groups, and the whole formula group reduced most obviously, cellular percent of S stage increased in all treatment groups, and the whole formula group increased most obviously, and the inhibition rate from the strong to weak was the resolving phlegm group> the whole formula group> the clearing heat group, cellular percent of G2 stage rarely change; For EC9706 cell:compared with the normal group, cellular percent of G1 stage rarely change, cellular percent of S stage reduced in all treatment groups, and the whole formula group reduced most obviously, cellular percent of G2 stage increased in all treatment groups, and the whole formula group increased most obviously, and the inhibition rate from the strong to weak was the whole formula group> the clearing heat group> the resolving phlegm group; For EC109 cell:compared with the normal group, cellular percent of G1 stage reduced in all treatment groups, cellular percent of S stage increased in all treatment groups, the whole formula group increased most obviously, and the inhibition rate from the strong to weak was the whole formula group>the clearing heat group>the resolving phlegm group, cellular percent of G2 stage rarely change. ⑥EGFR, PLC-γ1, PKC-α, ERK, PLCε1, NFkB were over-expressed and p53 low-expressed in human esophageal cancer cell line TE-1,EC9706 and EC109.⑦When the cells were treated with IC50 dosage, the protein expression level of EGFR-PLC-γ1-PKC-α-ER K in the TE-1,EC9706 and EC109 cells decreased, and the PLCε1 and NFkB protein expression level of PLCε1-p53-NFkB in the EC9706, EC109 and TE-1 cells decreased, while p53 increased.Conclusions: ①Formula of clearing heat and removing phlegm and its two separated prescriptions all can significantly inhibit the proliferation of TE-1,EC9706 and EC109 cells, in which the whole formula has the strongest effect; the removing heat and removing phlegm have synergistic effects.②Formula of clearing heat and removing phlegm affected TE-1 cell: formula of clearing heat and removing phlegm and its separate components all had obviously effects on TE-1 cell and inhibited cell proliferation in the S stage; Formula of clearing heat and removing phlegm affected EC9706 cell:formula of clearing heat and removing phlegm and its separate components all had obviously effects on EC9706 cell and inhibited cell proliferation in the G2 stage; Formula of clearing heat and removing phlegm affected EC109 cell:formula of clearing heat and removing phlegm and its separate components all had obviously effects on EC109 cell and inhibited cell proliferation in the S stage. ③There is close relationship between the occurrence of esophageal cancer and the EGFR, PLC-yl, PKC-a, ERK, PLCε1 and NFkB protein over-expression, p53 protein low-expressed in the signal transduction pathway of EGFR-PLC-γ1-PKC-α-RK and PLCε1-p53-NFkB.④ormula of clearing heat and removing phlegm can down-regulate the EGFR、PLC-γ1、 PKC-a、ERK、PLCε1 and NFkB protein expression level and high-regulate the p53 protein expression level of EGFR-PLC-γ1-PKC-a-ERK and PLCε1-p53-NFkB signal transduction pathway. They may inhibit the growth of EC9706、EC109TE-1 cells through inhibiting the above two signal transduction pathways.⑤sophageal cancer pathogenesis of heat and phlegm have closely relationship withEGFR-PLC-γ1-PKC-a-ERK and PLCsl-p53-NFkB signal transduction. ⑥his research provides effectively traditional Chiese medicine formula for the clinical treatment ind research of esphogeal cancer. |
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