Study On Mechanism Of Tetraethylpyrazine Against Angiogenesis Induced By Gastric Cancer Cells In Vitro

BackgroundStatistical data shows the mortality of gastric cancer was the highest amongall tumors in China, surgery and neoadjuvant treatment failed to improve the survival rate mainly because of recurrence and metastasis. Anti-angiogenesis was considered as an new effective therapy to prevent tumor growth and metastasis, and many researcher were looking for the drugs which can against tumor angiogenesis. Tetraethylpyrazine(TMP), a drug isolated from rhizome of Ligusticum walliichi which was considered have a blood-activating stasis -removing effect, was found in our previous animal experiment of TMP that after the treatment with it, the expression of Microvessel counts (MVC) , vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF) decrease. To invest the mechanism of TMP against angiogenesis induced by gastric cancer, we based our experiment in vitro on the animal experiment which described above.Object1. To examine the effects of TMP to proliferation of human umbilicalvascular endothelial cells(HUVEC) cultured in angiogenic factor free media.2. To examine the effects of TMP to the HUVEC proliferation induced by culture supernatants harvested from gastric adenocarcinoma cells .3. To examine the effects of TMP to the secretion of VEGF and bFGF by gastric adenocarcinoma cells in vitro.4. To examine the effects of TMP to the mRNA expression level of angiogenic factor which secretion inhibited by TMP from gastric adenocarcinoma cells in vitro.Method1. Experiment I: HUVEC was obtained and cultivated by modified Jaffesmothod and cells properties is identified by detection of Factor VIII-relatedantigen immunofluorescence.2. Experiment II:After 24 hour cultured in 96-well plates, HUVEC were divided into groups:normal control group (only SFM complete media was added), five TMP groups (the SFM complete media contained TMP at a concentration of 1, 5, 25, 125, 625 ug/ml was added in turns) . The effect of TMP to proliferation of HUVEC at following 24 hour , 48 hour, 72 hour was evaluated by MTT assay.3. Experiment III:including three steps.Step I:The culture supernatants harvested from gastric adenocarcinoma cell line SGC-7901 at 48 hour was store at -20'C for the next step;Step II:After 24 hour of HUVEC cultured in 96-well plates, conditioned media with or without TMP was prepared before the plate was took out of the incubator. The supernatant harvested in step I was mixed with SFM complete media at a raito of 1:1 as conditioned media without TMP;The supernatant harvested in step I was mixed with SFM complete media contained TMP at a ratio of 1:1 as conditioned media with TMP, the mixture contained TMP at a final concentration of 1, 5, 25, 125, 625ug/ml in turns.Step III:After conditioned media with or without TMP was prepared, the HUVEC cultured in 96-well plates for 24 hours were divided into groups: normal control group ( only SFM complete media was added );conditioned control group (conditioned media without TMP was added);five TMP groups (conditioned media with TMP described in step II was added in turns). The effect of TMP to proliferation of HUVEC at following 24 hour, 48 hour, 72 hour was evaluated by MTT assay.4. Experiment IV: After 24 hour culture in 6-well plates, SGC-7901 was divided into groups: normal control group ( only SFM complete media was added );five TMP groups (the SFM complete media with TMP at a concentration of 1, 5, 25, 125, 625 u g/ml was added in turns ). Enzyme linked immunosorbent assay ELISA were used to assess the VEGF and bFGF protein concentration in the supernatants harvested at following 12 hour, 24 hour, 48 hour.5. Experiment V: After 24 hour cultured in 6 well plates, SGC-7901 in every plate was divided into groups: normal control group ( only SFM complete media was added );TMP groups (the sum of groups and concentration in this experiment was according to the Experiment IV positive findings). Cells was collected at the following 12 hour, 24 hour, 48hour. The angiogenic factor mRNA level was determined by RT-PCR.Results1. Experiment I: Most of cells were positive cells with the greenfluorescence in cytoplasm and the negative cells without.2. Experiment II: All groups absorbance means gradually became higher with the time going on;There was no sinificant difference of absorbance means between every TMP group and normal control group(P>0. 05).3. Experiment III: all groups absorbance means gradually became higher with the time going on;The absorbance means of conditioned group and every TMP group was significantly higher than that of control groups(P<0.05);The absorbance means of TMP group 5 at following 24 hourN 48hour, 72hour was significantly lower than that of conditioned group at the same time (P<0. 05);The absorbance means of TMP group 5 at 48 hour and 72 hour was significant ly lower than that of conditioned groups at the same time (P<0. 05),too;The difference of absorbance means was not significant when the other TMP groups compare with conditioned group at the same time. The inhibition rate of TMP group 4 and group 5 absorbance means gradually became higher with the time going on and group 5 inhibition rate higher than that of group 4 at the same time.4. Experiment IV:The concentration of VEGF and bFGF in all groups gradually became higher with the time going on;There was no significant difference of VEGF between every TMP group and control group (P>0. 05);The concentration of bFGF at 48 hour in TMP group 4 and TMP group 5 was lower than that in normal control group at the same time (P<0. 05);There was no sinificant difference of bFGF between the other TMP goup and nomal conditioned group.5. Experiment V: The expression of bFGF mRNA from SGO7901 treated with TMP at concentration of 125 u g/ml and 625 u g/ml was determined , and there was no sinificant difference of it between TMP groups and control group (P<0. 05).Conclusion1. TMP may not have no significant effect to proliferation of HUVEC culturedin media without angiogenic factors, but it can inhibited the proliferation of HUVEC induced by gastric adenocarcinoma cells culture supernatant in a time-and doze-dependent manner.2. TMP have not significant effect to the secretion of VEGF from gastric adenocarcinoma cells in vitro.3. TMP can inhibited the secretion of bFGF from gastric adenocarcinoma invitro, but it is still to be discussed that whether TMP can affect the mRNA expression level of bFGF.