| Gastric cancer is the most common malignant tumor of digestive system with a poor overall prognosis, and chemotherapy is the main treatment method. However, existing chemotherapeutic agents have side effects, drug resistance and many other issues.To find effective drug for treating gastric cancer from natural drugs can provide a new approach for the treatment of gastric cancer. Tetramethylpyrazine (Tetramethylpyrazine, TMPZ) is an alkaloid extracted from the rhizome of chuanxiong whose traditional treatment effect is mainly manifested as improving microcirculation, anti-oxidative stress. In recent years, tetramethylpyrazine has become the focus in anti-tumor therapy research, and studies have shown that tetramethylpyrazine can reverse multiple drug resistance(MDR) in gastric cancer. However,whether tetramethylpyrazine itself has anti tumor effect and what is the mechanism, there is no relevant research reports so far.AMP activated protein kinase (AMPK AMP-activated protein kinase,) is an intracellular "energy regulator", which was activated when cell was in the energy shortage condition and was inhibited in energy excess condition. While the activation of AMPK can inhibit the synthesis of intracellular protein, fatty acid, polysaccharide, and the process is necessary for tumor cell proliferation and development. Previously we have found in the clinical samples that the expression of activity status of AMPK-phosphorylation status of AMPK (phosph-AMPK) in gastric cancer tissues were almost undetectable or very weak. Whether tetramethylpyrazine could inhibit the growth of gastric cancer through the activation of AMPK? So far, there is no report in the literature.This study foused on the research of the effection on gastric cancer cell growth and apoptosis of different concentrations of TMPZ,on different time points by using gastric cancer cell SGC7901as the research object. On the base of that TMPZ could inhibit the growth of gastric cancer cells and promote it’s apoptosis,we further explored the mechanism of TMPZ’s anti gastric cancer effect from the ROS/AMPK signal pathway, and the main results in this dissertation were summarized as follows:1. Expression of phosphorylated AMPK in pathologically confirmed gastric cancer tissues, paracancerous tissues and normal gastric mucosa tissue were detected using immunohistochemistry.The results showed that, the positive expression of phosph-AMPK was94.0%in normal gastric mucosa tissues, in paracancerous tissues was72.0%, and in carcinoma there was4.0%almost no expression. The expression of p-AMPK protein in normal gastric mucosa tissues was significantly higher than that in paracancerous tissues and gastric cancer tissues,while the expression of p-AMPK protein in paracancerous tissues was significantly higher than that in gastric cancer tissues.Further Western blot analysis showed that p-AMPK protein expression in normal tissues was0.93±0.14, in paracancerous tissues was0.43±0.08and in gastric cancer tissues was0.07±0.01, there was significant difference between the three group(P<0.05).2.Investigation of the effects of TMPZ on human gastric cancer cell SGC7901and the dose-effect, time-effect relationship. We treated the tumor cells with different concentrations of TMPZ (0,0.5,1,2,4,6,8mM) for6,12,24,48h, and methyl thiazolyl tetrazolium (MTT) assay was used to examine the growth of the tumor cells. Different concentrations of TMPZ treatment of the tumor cells for24h, cell apoptosis was detected by flow cytometry. The results showed that there was no significant effect of low concentration TMPZ on the growth of the tumor cells, while high concentration TMPZ (4,6,8mM) significantly inhibited the growth of the tumor cells after24h; and obviously induced the apoptosis of the tumor cells after24h treatment in a concentration-dependent manner. At the same time, we further confirmed that higher concentrations TMPZ had no significant effect on the survival rate and the apoptosis of human gastric epithelial cells GES-1, human umbilical vein endothelial cells HUVECs and human Myocardial cells H9c2.3.According to the apoptosis of tumor cells induced by the high concentration TMPZ in a dose-dependent manner, we used flow cytometry to detect how the high concentration of TMPZ effceted the ROS production and we found that the high concentration TMPZ could raise the concentration of ROS in tumor cells in a dose-dependent manner and inhibit the tumor cells growth. After administration of NAC,a ROS scavenger, ROS content in cells decreased significantly, and the high concentration TMPZ could not inhibit the growth of tumor cells anymore.4.Western blot detection the effect of high concentration TMPZ in phosphorylation of AMPK in human gastric cancer cell line SGC7901. Results showed that the high concentration TMPZ could activate AMPK in a dose-dependent manner and increase the phosphorylation level of AMPK, but after administration of NAC, phosphorylation of AMPK induced by high concentration TMPZ was abolished.5.Using AMPK specific inhibitor Compound C, we deeply study the high concentration TMPZ activation the mitochondrial apoptosis pathway through the ROS/AMPK signaling pathway. The results showed that, through the activation of AMPK,TMPZ promoted the migration of Bax from the cytosol to the mitochondria, and the collapse of mitochondrial membrane potential, then cytochrome C released from mitochondria to cytosol, and activated caspase-9, Caspase-3, resulting in apoptosis of gastric cancer cell line SGC7901.To sum up, inspired by the AMPK activity in gastric cancer tissues,we studied the effect of TMPZ against gastric cancer cell SGC7901and it’s mechanism.We found that high concentration TMPZ inhibited the growth of gastric cancer cells, and promoted the apoptosis of gastric cancer cells, but did not have significant effect on the other cells. The mechanism may be that the high concentration TMPZ induced the generatin of a large amount of ROS, leading to activation of AMPK, Bax migration from the cytosol to the mitochondria, leading to mitochondrial membrane potential collapse, then CytC was released into the cytoplasm, to activate caspase-9, caspase-3. finally induce the apoptosis of tumor cells. |
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