Effects of formula composed of active monomers of removing heat and phlegm prescription on malignant phenotypes and growth signaling in esophageal carcinoma cells.

Abstract

e16051 Background: Esophageal carcinoma (EC) ranks seventh in the incidence of malignant tumors and the sixth mortality worldwide. Removing heat and phlegm prescription(RHPP, composed of Rhizoma Dioscoreae Nipponicae, Rhizoma Paridis, Saponin, Rhizoma Bolbostemmae) was an effective formula screened from 531 Chinese herbs for treating EC in our previous study. The purpose of this study is to further track, isolate, purify and identify the active monomers of RHPP, study the effect of each active monomer on Eca109, EC9706, EC-1, TE-1 four EC cells and the signaling mechanism, then optimize and compose new active monomers formula and study its mechanism on EC cells. Methods: The active monomer of RHPP was extracted, separated and identified by ethanol extractioncolumn, chromatography, recrystallization, HPLC, spectroscopic method,etc. The effects of four kinds of active monomers and their composed formula on the proliferation, migration, clone formation, cell cycle and signal pathway proteins expression of Eca109 cell, EC9706 cell and TE-1 cell were investigated by MTT assay, RTCA assay, soft agar assay, flow cytometry and western blot analysis. Results: Four active monomers were identified from Rhizoma Dioscoreae Nipponicae (CSL), Saponin (ZJ), Rhizoma Bolbostemmae (TBM), Rhizoma Paridis (CL) respectively with molecular weights of 868, 1003, 1365, 854.5, and corresponding molecular formulas of C45H72O16, C50H82O20, C64H100O31 and C44H70O16(China patient No. ZL202010136431.6). Four active monomers significantly affected the cell morphology, proliferation, migration, cloning ability, cell cycle of four EC cells. The IC50 values of the active monomer of CSL, ZJ, TBM and CL for Eca109 were 1.29±0.12, 2.42±0.09, 1.93±0.09, 3.04±0.28μg/ml respectively; for EC9706 were 1.41±0.02, 5.94±0.45, 1.97±0.09, 0.63±0.04μg/ml respectively; for TE-1 were 3.72±0.28, 35.58±0.58, 7.90±0.41, 1.85±0.09μg/ml respectively; for EC-1 were 1.11±0.14, 3.56±0.28, 1.23±0.02, 0.61±0.02μg/ml respectively. Four active monomers could downregulate EGFR, PLC-γ1, and PKCα protein expression. Using the baseline proportional increase and decrease design method to compose new removing heat prescription(RH), removing phlegm prescription(RP) and removing heat and phlegm prescription (RHPP), the ratio of each drug monomer in RH, RP and RHPP were mCSL: mCL = 6: 4, mZJ: mTBM = 3: 7, mCSL: mCL: mZJ: mTBM = 48: 32: 6: 14. RH, RP and RHPP all could inhibit the proliferation, migration, cloning ability, cell cycle of four EC cells. Conclusions: The four active monomers and new composed RHPP all can inhibit the proliferation of EC9706, Eca109, TE-1, EC-1 four EC cells, which have close relationship with EGFR, PLC-γ1, and PKCαproteins expression. This study provides new formula and basis for the development of anti-esophageal carcinoma drugs in traditional Chinese medicine.