Influence Of Huangqin-Tang To Proliferation Of Gastric Cancer Stem Cells In Vitro

ObjectiveIn order to study the effect of Huangqin-Tang on proliferation of gastric cancer stem cells in vitro, we separated and enriched gastric cancer stem cells from gastric cancer cell line SGC-7901.Method1、HPLC test:The contents of9kinds of main active ingredient in Huangqin-Tang were analyzed by HPLC.2、Gastric cancer stem cell morphology:We separated and enriched gastric cancer stem cells in gastric cancer cell line SGC-7901through serum-free culture method.3、Identification:The capabilities of tumor-sphere formation, chemotherapy resistance, nude mice xenograft formation have been tested to identifying whether the separated tumor cells are stem-like cells.4、MTT test:We used different concentrations of Huangqin-Tang to treat gastric cancer stem cells for24hours, and then tested each group’s OD value.5、Cell apoptosis rate test:We detected each group’s cell apoptosis rate in flowcytometry.6、Cell cycle test:We tested each group’s cell cycle in flow cytometry.Result1、HPLC test:Huangqin-Tang contains9kinds of main active ingredient: Albiflorin, Paeoniflorin, Glycyrrhizic acid, Ononin, Baicalin, Wogonoside, Baicalein, Wogonin and Liquiritin, their contents and stabilities are in line with requirements.2、Gastric cancer stem cell morphology:SGC-7901gastric cancer cells were grown in serum-free stem cell medium (EGF, bFGF, B27, N-2), in about4to7days cells were grown into tumor-spheres. After one week, the shape and cell numbers of tumor-spheres significantly increased. Cells could be passaged every14or15days, so there are sufficient cells for the subsequent experiments.3、Identification:After tumor-sphere formation, chemotherapy resistance, nude mice xenograft formation tests,we identified that cells obtained from SGC-7901gastric cancer cell lines have stem-like properties, they are gastric cancer stem cells. 4、Measurement of cell proliferation inhibition rate:0.5mg/ml-3.0mg/ml Huangqin-Tang esposure induced a dose-and time-dependent reduction of cell proliferation inhibition rate in24hours.5、Cell apoptosis rate:Compared with the blank control group and the negative control group, Huangqin-Tang markedly upregulated the levels of cell apoptosis rate.6、Cell cycle:Compared with the blank control group and the negative control group, the proportion of cells in Go/G1phase in each Huangqin-Tang group had no significant change, while in S phase decreased and in G2/M phase increased.Conclusion1、Through serum-free culture methoed,gastric cancer stem cells were successfully separated and enriched from gastric cancer cell line SGC-7901.2、Huangqin-Tang can significantly inhibite gastric cancer stem cell’s proliferation in vitro, and it may be related to induction of cell apoptosis and cell cycle arrest.