| Objective:Gastric cancer is a common human malignant tumor, and its mortality rate ranked the forefront in the malignant tumor in China, Chinese herbal medicine has a unique advantage of multi-effect, multi-target, low toxicity, slight drug resistance, immunity improvement and it has a wide source with low price, which plays an important role in cancer therapy. Applicating modern medical methods, to explore the anti-tumor effect of Chinese herbal medicine, and extracting the active ingredients to development anti-cancer drug with Chinese characteristics and independent intellectual property rights, has always been a focus for researchers.Until now in the research of old famous Chinese Prescriptions, there are few reports in the Separation of the anti-cancer active monomer to filter the best combination of drugs with pharmacodynamics as an index. In this study, by research some important monomer’s proliferation to human gastriccancercell MGC-803,such as kaempferol, agrimopholB, and its compatibility in Shan Zhaowei professor’s anti-cancer prescription,the Qizhu prescription, we can preliminarily investigate the mechanism of action of kaempferol and agrimopholB to the human gastric cancer cell MGC-803by detecting the telomerase activity of Caspase-9and telomerase. So lay the foundation for the development of new anti-cancer drugs, and provide an objective basis and theoretical support for the development and application of the clinical tumor treatment medication.Methods:Kaempferol, agrimopholB are soluble in ethanol, the initial concentration of2mg/mL, respectively1mg/mL. Experiment group were kaempferol A, B, C, D, agrimopholB, a, b,c,d group, according to the proportion of compounding concentration7.5,15,30,60μg/mL kaempferol, agrimopholB medium, and containing1%ethanol plus, volume3%calf serum1640medium negative solvent control group E125μg/mL5-FU as positive control group F. MTT method was used to detect7.5-60μg/mL of four different concentrations of kaempferol, agrimopholB effects on the cells of MGC-80324h,36h,48h cell proliferation inhibition rate; using transmission electron microscopic observation of kaempferol, agrimopholB MGC-803cell24h morphological changes of apoptosis by PI marker; flow cytometry kaempferol, agrimopholB MGC-803cell24h apoptosis rate; using spectrophotometric determination of kaempferol, agrimopholB role of MGC-803cells after24h Caspase-9protein activation degree, using fluorescence quantitative PCR assay for detection of kaempferol, agrimophol B treated MGC-803cells after24h telomerase activity.Result:1.MTT method for the detection of tip:7.5-60μg/mL of four different concentrations of kaempferol, agrimopholB may vary in the degree of inhibition of cell proliferation, kaempferol, agrimopholB inhibitory action on the proliferation of cells with increased drug concentration and prolonging exposure time and gradually strengthened,(the largest concentration of kaempferol, agrimopholB36h,48h and the positive control group no difference) dosing group inhibition rate compared with the control group, there was significant difference (P<0.01).30μg/mL concentrations of two drugs combined with the role of group and there was significant difference compared with monotherapy group (P<0.05).2.15μg/mL,30μg/mL concentration of kaempferol, agrimopholB and30μg/mL concentration of two drug treatment after the cells were observed under transmission electron microscope to apoptosis characteristic morphological changes, such as the cell size, cell membrane shrinkage, surface microvilli disappeared, and nuclear pyknosis, along the nuclear envelope distribution.3.Flow cytometry showed:15μg/mL,30μg/mL concentration of kaempferol, agrimopholB promotes MGC-803cell apoptosis, cell in24h, flow cytometry is visible on the apoptosis peak, kaempferol, agrimopholB and30μg/mL two drug addition to cell cycle arrest in the Go phase, apoptosis rate were61.17%,60.02%,29.5%,36.56%,65.88%。4.Spectrophotometric detection of Caspase-9results show:15μg/mL,30μg/mL concentration of kaempferol, agrimopholB can enhance MGC-803cell Caspase-9expression, kaempferol, treated MGC-803cells of24h, Caspase-9expression of the relative content of30μg/mL concentration is higher than that of15μg/mL concentration group. AgrimopholB treated MGC-803cells of24h, Caspase-9expression of the relative content of30μg/mL concentration is lower than that of15μg/mL medicine group and control group, the expression of Caspase-9relative content, there was significant difference P<0.05. Combination of the two groups have significant difference than the single drug group at P<0.05.5.Real time fluorescent quantitative PCR assay for detection of15μg/mL,30μg/mL concentration of kaempferol, agrimopholB treated MGC-803cells after24h telomerase activity,30μg/mL concentration were higher than that of15μg/mL concentration group, adding group compared with the control group with significant difference, two medicine additive group than the single drug groups there was significant difference P<0.05.Conclμsion:1. The concentration of7.5-60μg/mL in four different concentrations of kaempferol, agrimopholB treated MGC-803cells24h,36h,48h, can inhibit the proliferation of such inhibition of proliferation, probably by inducing apoptosis.2. The concentration of7.5-60μg/mL in four different concentrations of kaempferol, agrimopholB treated MGC-803cells24h,36h,48h, can induce the apoptosis.3. The concentration of15μg/mL30μg/mL concentration of kaempferol, agrimopholB treated MGC-803cells after24h by active Caspase-9, inhibits the activity of telomerase, which promotes the apoptosis.4.30μg/mL concentration of kaempferol, agrimopholB two drugs combined with inhibition of cell proliferation and telomerase activity is enhanced, the expression of Caspase-9kaempferol is higher than agrimopholB and the two drugs combined with weak monotherapy in the role of. |
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