Identification Of LSD1 Inhibitor And Its Inhibits Invision And Metastasis In Human Gastric Cancer Cell Line MGC803

Gastric cancer is the fourth most common cancer and the second leading cause of cancer death in the world. The incidence is especially high in East Asia and is about twice as high in men than in woman. The 5-year survival rate is less than 30% in most countries. Hence, investigations which decipher molecular basis underlying development and invasion mechanism of gastric cancer may lay a foundation for the diagnosis and treatment of gastric cancer.Histone modifications play important roles in regulating gene expression in various cellular processes by altering the underlying chromatin structure and thus influencing related pathological conditions. Histone methylation is one such modification that was thought to be static and enzymatically irreversible until the recent discovery of histone demethylases. Lysine specific demethylase 1 (LSD1) is the first histone demethylase that removes methyl groups on lysine residues and mediates expression of many genes important in cancer progression. LSD1 is overexpressed in a variety of malignant tumor cells. Therefore, obtaining selective and potent LSD1 inhibitor is a promising way and hot point for cancer treatment. Invasion and metastasis of cancer cell are the major cause of death in cancer patients. Gastric cancer is characterized by high motility and invasion and distal metastasis. Hence, it is of great significance to explore the mechanism of invasion and metastasis of gastric carinoma cells. Recent research shows that histone methylation can regulate expression of genes associated with cancer invasion and metastasis. It is unclear how LSD1 regulate the invasion and metastasis of gastric cancer cells.A novel series of pyrimidine-thiourea-hybrids were synthesized and their LSD1 inhibitory effect was evaluated in order to have the potent and selective LSD1 inhibitors. The cancer migration and invasion inhibitory effect and the mechanism were investigated. The mechanism of LSD1 in regulation of TGFβ1 in gastric cancer cell line MGC803 was elucidated.1) The optimization of LSD1 inhibitor screening modelOur previous experiment use a fluorescence-based format and require no wash or liquid transfer steps, and the homogeneous "mix-and-measure" nature makes the assay simple and robust with relatively low cost. However, the assays usually tend to generate a high number of false positives because of fluorescent interference from labeled substrates, colored and fluorescent compounds, and fluorescent tracers. AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) is versatile assay technology developed to measuring analytes using a homogenous protocol. This technology is an example of a bead-based proximity assay. Here, singlet oxygen molecules, generated by high energy irradiation of Donor beads, travel over a constrained distance(approx.200 nm) to Acceptor beads. This results in excitation of a cascading series of chemical reactions, ultimately causing generation of a chemiluminescent signal.The activity of LSD1 and its substrate specificity are mainly regulated by association with a number of co-regulatory proteins. CoREST is one such important binding protein that endows LSD1 with the ability to associate with and demethylate nucleosomal substrates. So far, the expression and purification of CoREST in vitro has been completed.Meanwhile, a novel series of pyrimidine-thiourea-hybrids were synthesized, their activities against LSD1 were also evaluated. With the aid of SAR study, one group novel LSD1 inhibitors was identified. The most potent one is compound 6b, could inhibit LSD1 activity with IC50=0.65μM. Therefore, the following investigation was performed with compound 6b mainly.Compound 6b exhibits potent LSD1 inhibitory activity in vitro. We also found that compound 6b did not show any significant inhibitory effect on MAO-A and MAO-B. Our findings indicate that compound 6b is a highly selective LSD1 inhibitor. Time-dependent assay suggested that, within 8 min, the compound at different concentrations(0.5,2.0, and 8.0μM) performed time-dependent inhibitory effect against LSD1. Another experiment was also carried out with biolayer interferometry(BLI) to confirm whether there is a direct interaction between the small-molecule and the LSD1 recombinant or not. Compound 6b performed a direct interaction with LSD1 at Kd=3.7μM. Taken together, these findings demonstrate that compound 6b may tightly but reversibly bind to LSD1.2) Inhibitory effect of LSD1 inhibitor against cell migtation,invasion and the mechanism studyWound healing assay and transwell experiment indicated that compound 6b could inhibite the cell migration and invasion. Western blot experiment indicated that compound 6b could induce the overexpression of E-Cadherin and inhibit N-Cadherin expression. ELISA experiment also indicate that compound 6b can inhibit TGFβ1 expression in MGC803 cell. At the same time we constructed lentiviral-LSDl-shRNA vector and transfected it into MGC803. Then we used ELISA to characterize the change in TGFβ1 expression induced by LSD1 knockdown. The experiment results show that LSD1 knockdown inhibited the protein expression of TGFβ1 in gastric cancer cell line MGC803. ChIP assays revealed that the TGFβ1 gene was directly regulated by LSD1. We suppose that compound 6b could inhibit TGFβ1 expression to inhibit EMT by altering the LSD1 activity and thus resulted in the inhibition of cell migration and invasion.