The Role And Mechanism Of MS-275 On P53 Gene Mutation Type Gastric Cancer SGC–7901 Cells

Background:Gastric cancer is the fourth most common cancer in the world, and its mortality is in the second place of cancer-related deaths. In our country, both the incidence and the mortality caused by gastric cancer are all in the world’s top three. At present, the available treatment means of gastric cancer for patients mainly include surgery, adjuvant systemic chemotherapy, etc. Traditional treatments are fallen well short of their goals for the patients with intermediate and advanced carcinoma of stomach, especially the recurrent and refractory ones. And traditional treatments are also liable to causing a variety of complications. In order to achieve better treatment effects and less side effects, increasing researches are committed to seeking a molecular targeted therapy drugs for patients with GC.Recent studies have found that histone acetylation and deacetylation play an important role in changing structure of chromosome and regulating gene transcription. And the excessive acetylation of histone is related to the occurrence of tumor closely. Histone deacetylase inhibitors(HDACIs) can inhibit the excessive acetylation of histone, suppress the proliferation of human tumor cells and induce apoptosis. As one of the benzamide class HDACIs, MS-275 can kill a variety of solid tumor cells and hematological malignant tumor cells.Our research at the early stage has indicated that MS-275 can selectively kill gastric cancer cells MKN-45, which contain wild-type p53 gene. However, as much as 50% of gastric cancer cells contain mutational p53 gene. The accurate role and mechanism of MS-275 on p53 mutation type gastric cancer cells still remain obscure. Objectives:1. To investigate the selectively killing effect of MS-275 on p53 gene mutation type gastric cancer cells SGC-7901;2. To further explore the related molecular mechanism of MS-275’s selectively killing effect in vitro. Methods:1. Cell viability determination by WST-1: The SGC-7901 and GES-1 cells weretreated with different concentrations of MS-275. After that, the cell viability wasdetected by WST-1.2. Cytotoxicity Assay: The lactate dehydrogenase(LDH) Cytotoxicity Assay Kit wasused to detect the level of LDH, which was released by SGC-7901 and GES-1 afterdifferent concentrations of MS-275 treatment.3. Mitochondria membrane potential determination by JC-1: JC-1 was used to detectthe mitochondria membrane potential of SGC-7901 cells.4. Release of reactive oxygen species(ROS) determination by Flow cytometry: Flocytometry was used to detect the ROS mass of SGC-7901 cells, which were treatedwith different concentrations of MS-275 and/or the combination of MS-275 andcatalase.5. The expression of Trx in SGC-7901 cells was assessed by Western blotting.6. The expression of cell cycle-related gene(p21, p27, p57, Cyclin B1 and Cyclin D1)in SGC-7901 cells were estimated by Western blotting and q PCR. Results:1. After they were treated with different concentrations of MS-275, the survival rateof SGC-7901 cells decreased significantly in a dose-dependent manner. At the sametime, the level of LDH release increased obviously. But there were no obviouschanges of survival rate and LDH release observed in GES-1 cells.2. MS-275 treatment could decrease the mitochondrial membrane potential andincrease the ROS mass of SGC-7901 cells in a dose-dependent manner. The level ofthe ROS mass could be reduced by catalase obviously.3. MS-275 treatment could decrease the protein level of Trx in SGC-7901 cells in adose-dependent manner.4. The results of Western Blotting and q PCR indicated that the relative contents ofcell cycle proteins p21, p27 and p57 could be up-regulated by MS-275, at the sametime, Cyclin B1 and Cyclin D1 could be down-regulated. Conclusions:MS-275 treatment could selectively kill p53 mutation type gastric cancer cells SGC-7901 in vitro, but had no obvious effect on normal gastric mucosa cells. The mechanism of MS-275 included that, inducing tumor cells’ apoptosis through the way of oxidative stress(increased the ROS mass and decreased the expression of Trx, which could damage the mitochondrial inner membrane, reduce the mitochondrial membrane potential and induce the tumor cells’ apoptosis), and regulating the expression levels of cell cycle-related factors p21, p27, p57, Cyclin B1 and Cyclin D1, which could arrest the process of cell cycle and inhibit the proliferation of tumor cells.