Research On The Antiproliferative And Apoptosic Action Of Fufangkushen Injection Alone And When Combined With Oxaliplatin And Endostar In Human Hepatoma Cell

Objective: Observe the effects of Fufangkushen injection alone and the effects when it is combined with oxaliplatin and human recombinant endostatin (endostar) on human hepatoma cell lines SMMC-7721 in vitro, and investigate the mechanism, to provide the theoretical foundation for the combination of chemotherapy with traditional medicine and antiangiogenic therapy to cure hepatocarcinoma.Methods: Use various methods to detect the growth condition of human hepatoma cell lines SMMC-7721 in vitro when they are affected by different concentration of Fufangkushen injection, oxaliplatin and endostar alone and by the combinative use of the drugs above: (1) Estimating the inhibition of proliferation by MTT-test; (2) Observing the morphologic changes by light microscope; (3) Observing the morphologic characteristics of apoptosis by fluorescence microscope; (4) Analyzing the distribution of cell cycle by flow cytometry; (5) Observing the dyed nucleolus of apoptosis cell by TUNEL method; (6) Detecting the expressions of apoptosis-associated gene proteins: Survivin, Bcl-2 and Caspase-3 by immunohistochemical technique.Results: (1) Fufangkushen injection could inhibit the proliferation of SMMC-7721 cell lines, and the inhibition depends on the exposure time and dose; there were synergistic effects when combine Fufangkushen injection of 50μl/ml with oxaliplatin of 1μg/ml, while no obvious effects when combined with endostar; (2) Observed by light microscope, the Cells shrank and turned round, and the contacts among cells became loose; (3) Observed by fluorescence microscope, the early and late stage apoptosis took place in cells; (4) The analysis of cell cycle showed that drugs blocked cells at G₀/G₁ phase, the proportion of cells increased at G₀/G₁ phase while it decreased at S phase; (5) The TUNEL method detected that the nucleuses of apoptotic cells were dyed deep brown, the apoptosis index (AI) increased when combined with oxaliplatin, while there was no obvious difference when combined with endostar; (6) The expressions of Survivin and Bcl-2 were up-regulated, while the expressions of Caspase-3 were down-regulated; The expressions of Survivin and Bcl-2 was increased while Caspase-3 was decreased when combined with oxaliplatin, no obvious difference was observed when combined with endostar.Conclusion: Fufangkushen injection could inhibit the proliferation of SMMC-7721 cell lines, the effects were achieved by apoptosis related to the blocking of cell cycle at G₀/G₁ phase, down-regulation of the expressions of Caspase-3 and up-regulation of the expressions of Survivin and Bcl-2. When combined with oxaliplatin, it strengthened the inhibition of proliferation and inducement of apoptosis, while no obvious strengthening effects were observed when combined with endastar. This research provides the experimental foundation for the combination of TCM with western medicine to cure hepatocarcinoma.