Molecular Mechanism Of Matrine Induced Apoptosis In Gastric Cancer MKN45 Cells

Background:Matrine, one of the main active components from the dry roots of Sophora flavescence, was known to induce apoptosis in a variety of tumor cells in vitro. However, the molecular mechanism of cell apoptosis induced by Matrine remains elusive. Nowadays, it is of particular significance to use molecular biological methods to study the pharmacologic effects of traditional Chinese herbs to reiterate the underlying pharmacological mechanism and to do research in finding new anti-cancer drugs, which has also become the trend in exploiting the Chinese medicine. In such a circumference, a deeper study on the molecular mechanism of Matrine in anti-tumour effect is promising and valuable.Apoptosis is an accurately regulated process and the modulating molecules are as follows: caspase family, Bcl-2 family, IAP family, p53 and NF-kB. Nowadays, there is no serial report on the herbal morphon inducing cell apoptosis via the above molecules and the accurate signal transduction pathway. Our study detected the protein level of the above molecules by Western-blot in Matrine treated MKN45 cells to explore the molecular mechanism of Matrine inducing apoptosis. The generalization of the method itself has a deep theorical and practicalsignificance in exploitation the pharmacological effects of herbal morphon and their molcecular target on cancr.Objects:To evaluate wether Matrine could induce MKN45 cells apoptosis in vitro. To investigate the molecular mechanism of Matrine induced MKN45 cells apoptosis. To investigate the effect of Matrine on the protein level of modulating molecules of apoptosis, i.e, caspase family, Bcl-2 family protein, p53 and NF-kB. To analyze the signal transduction pathway of Matrine induced MKN45 cells apoptosis in vitro.Methods:1. MTT colorimetric assay was used to determine cells proliferation inhibition of MKN45 cells after Matrine intervention.2. Flow cytometric analysis was used to detecte the percentage of apoptotic cells and cell cycle changing after Matrine intervention.3. DNA ladder electrophoresis was used to detect the apoptotic DNA.4. Immunoblotting was used to detect the protein level of cleaved caspases, Bcl-2 family member, p53, IkB family and the subunit of NF-kB.Results:The main results were divided into some sections as follows:1. Matrine inhibited MKN45 cells proliferation and induced apoptosis1.1 Matrine inhibited MKN45 cells proliferationThe proliferation inhibition effect of Matrine on MKN45 cells was determined with the MTT colorimetric assay. MTT results showed that after treated with Matrine for 48 h, the proliferation inhibition rate of MKN45 cells was 6.25±2.34%, 9.04±1.52%, 12.56±4.34%, 49.91±2.78%, 94.14±0.62%, respectively at variousconcentrations (0.05, 0.10, 0.25, 0.50, 1.0 mg/ml) (P < 0.0001, bilateral ANOVA), and at the dosage of 0.50 mg/ml, the inhibition rate sharply rised to 49.91%. The 1C50 value was 0.53 mg/ml (95% CI, 0.49-0.58 mg/ml). The results showed exposure of MKN45 cells to Matrine caused significant growth inhibition in a dose-dependent manner.1.2 Matrine induced cell cycle block and apoptosis in MKN45 cellsWe further analyzed cell cycle and apoptosis by the flow cytometry analysis. The results of flow cytometry analysis revealed that with the dose increased, the percentage of the S phase cells declined, and a decline tendency of G2/M phase in Matrine-treated cells. As for the G0/G1 phase, only cells at 0.50 mg/ml group dropped remarkably. In cell apoptosis, the percentages of apoptotic MKN45 cells observed after 0, 0.05, 0.10, 0.25, 0.50 mg/ml of Matrine for 48 h were as follows: 0.52%, 1.44%, 1.63%, 6.22%, and 26.88%, respectively. The results showed exposure of MKN45 cells to Matrine caused cell cycle arrest at G0/G1 phase, and induced apoptosis in a dose-dependent manner.1.3 DNA laddering assay analyzed the apoptotic DNA in Matrine treated MKN45 cellsTo further confirm the apoptosis, we conducted DNA laddering assay in Matrine-treated cells. The results showed that DNA fragmentation ladder only appeared in Matrine-treated groups.1.4 Matrine activated caspase-3 and -7 in a time-independent manner in MKN45 cellsThe protein levels of cleaved-caspases were detected using western blot in MKN45 cells at different time points, the result showed that only after 48h interation, the cleaved caspase-3 and caspase-7 were detected. It meant that the caspase activation of MKN45 cells by Matrine was time-independent. 2. Effects of Matrine on Bcl-2 family member and p53 in MKN45 cells2.1 Effects of Matrine on pro-survival sub-family of Bcl-2 family in MKN45 cellsWe analyzed the protein level of Bcl-2 and Bcl-xL in Matrine treated MKN45 cells by western blot. The results showed that there were no significant changes in the protein levels of the pro-survival proteins like Bcl-2 and Bcl-xL in Matrine at 0.05 and 0.10 mg/ml doses, but at the 0.25 and 0.50 mg/ml doses, the protein levels declined compared with controls.2.2 Effects of Matrine on pro-apoptotic molecules of Bcl-2 family in MKN45 cellsApoptotic protein levels were analyzed using western blot in Matrine treated MKN45 cells. In the tested seven pro-apoptotic proteins (Bak, Bok, Bax, Bim, Puma, Bad, and Bik), there were significant changes exhibiting much higher at 0.05 and 0.10 mg/ml doses group and a declination tendency at 0.25 and 0.50 mg/ml, except Bad and Bik, whose protein levels had little difference in various groups.2.3 Effects of Matrine on Bcl-2/Bad/Bax pathwayTo investigate the Bcl-2/Bad/Bax pathway, we analyzed the protein level of phosphorylated Bad, phosphorylated Bcl-2, 14-3-3. The results showed little difference within various doses groups exsited.2.4 Effects of Matrine on the protein level of p53P53 was the up-stream modulator of pro-apoptotic molecules, Bax and Puma. We further investigated the protein level of p53, which showed up-regulated at low dosages and down-regulated at higher dosages. It had same tendency with thepro-apoptotic proteins.3. Effects of Matrine on IkBs and NF-kB3.1 Effects of Matrine on the protein level of IkBsWestern blot analysis of IkBs protein level in Matrine treated MKN45 cell revealed that the protein level of IKKa, phosphor IkB MKN45 cells-> p65, p52-> p53->Bax, Puma-> caspases activtion->apoptosis...