Screening Of Anti-tumor Metastasis And Anti-inflammation Small Molecular Compound And Studying Of Their Underlying Mechanism

Malignant tumor is a serious threat to human health and a leading cause of death. As reported by World Health Organization, an estimated17,000,000new cases of cancer were diagnosed worldwide every year, with an estimated7,900,000deaths. Rates are rising as lifestyles change and more people live to an old age in the world. Gastric carcinoma is the second most common cause of death and the third most common cancer worldwide. The incidence is also high in our country,170,000deaths are reported every year (about1/4of all cancer deaths). Loss control of tumor cells invasion and metastasis to esophagus, lung, lymph node and liver is a main cause of death in gastric cancer. Tumorigenesis and metastasis were caused by abnormal expression of oncogene and tumor suppressor gene, which involved in regulating cell cycle, apoptosis, cell-cell interaction, cell-ECM connection, and recoding some important enzymes, such as uPA and MMPs. So remedying the expression of these genes may provide an available method to cancer therapy.Inflammation-related desease is another threat and clinical challenge to human being as well as cancer. Inflammation is usually a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process. Always, wounds and infections will be cleaned by the defense system of host. But in some pathological situation, chronic inflammation can also lead to a host of diseases, such as rheumatoid arthritis, hay fever, atherosclerosis, glomerulonephritis and gastroenteritis et al., sometimes even promote the progression of cancer. It is for that reason that inflammation is normally closely regulated by the body, including the regulation of inflammatory cytokines and mediator secretion. As those factors can significantly promote the progression of inflammation, it is wildly believed that down-regulated the secretion of factors could be useful for theraputic of inflammation-related desease.In here we establish a series of methods to screen compounds synthesized by our laboratory, so that to get some potential anti-tumor metastasis or anti-inflammatory drug candidate. And then identify and study their underlying mechanism. This paper was mainly composed of two parts:1、Screening of anti-tumor metastasis small molecular compounds and studying of their possible mechanism.Metastasis is the biggest character of malignant cancer and cause95%deaths in cancer patients. The formation of metastatic nodules is a multistep and complex process that includes cell proliferation, digestion of the ECM, cell migration to circulation system or lymph nodes, remigration and growth of tumors at metastatic sites. As matrix metalloproteinases are well known extracellular matrix-degrading enzymes, it is widely believed that the aberrant expressions of MMPs were involved in these processes. So inhibit the expression of MMP-9may block the metastasis of tumor cell. According the characteristic of tumor metastasis, we carried out the following research:(1) We established an anti-tumor metastasis drug screening platform with gelatin zymography assay, transwell assay, luciferase assay, WB, EMSA focus on MMP-9, with which enhanced expression is found in the progression and invasion of tumors.(2) By screening the comounds, we found an anti-tumor metastasis candidate CADPE and then studied its possible mechanism in gastric cancer. Results shown:a. CADPE could suppress the migration and invasion of gastric cancer cell with little effects on cell viability.b. CADPE could inhibit the expression and activity of MMP-9in both gastric cancer cell and breast cancer cell MD-MBA-231.c. MMP-9has a direct connection with cancer cell invasion, as MMP-9specific inhibitor I can significantly suppress the cell invasion by matrigel based transwell assay.d. CADPE could regulate the activation of transcription factor AP-1. The inhibition of AP-1was mediated by the blocking of FAK/MEK/ERK/AP-1signaling pathway.In this part, for the first time we reported that CADPE suppressed cancer cell invasion and migration by blocking PMA induced matrix metalloproteinase-9expression and FAK/MEK/ERK-mediated AP-1activation in gastric cancer. These results indicated that CADPE could be a new potential agent in therapy of gastric cancer by targeted on MMP-9and metastasis.2、Screening anti-inflammatory drug and studying of their possible mechanismIn the process of inflammation, a cascade of biochemical events propagates the inflammatory response, including the local vascular system, the immune system, and various cells in the injured tissue. With the secretion of inflammatory mediators, such as histamine, leukotriene, PGE, bradykinin, complement system and thrombin et.al, vascular system got marked changes, including vasodilation, increased permeability, and slowing of blood flow, finally the recruitment and estravasation of lerkocytes. Leukocytes, especially granulocytes secrets cytokines TNF-a, IL-6, IL-1promote the secretion of others inflammatory mediators and recruit the macrophage to inflammatory site, and then making disease worse. According the characteristic of inflammation, we carried out the following study:(1) Screen small molecular compounds that were synthsised by our library with Real-time PCR and ELISA, so that we can find some candidate drugs that could down-regulate the secretion of pro-inflammatory cytokines TNF-a and IL-6, and then block the process of inflammation.(2) By screening of anti-inflammation drugs, our attention were mainly focused on blocking cytokines TNF-a, IL-6, which are harmful to the host by producing fever, inflammation, tissue destruction, and sometimes shock and death. Finally, we got a potential candidate YF-449, and studied its possible mechanism in anti-inflammation. Our results shown:a. YF-449could suppress the secretion of TNF-a and IL-6in Raw264.7cells with no significant effects on cell viability.b. YF-449could down-regulate the expression of inflammatory-related enzymes, such as MMP-9, iNOS and COX-2, and then the synthesis of NO and PGE2.c. YF-449influences the nuclear translocation of NF-κB.d. YF-449inhibits the nuclear translocation of NF-κB by blocking the degradation of NF-κB inhibitor protein, IκBa, and related signaling pathway.e. YF-449could protect mice from death in LPS-Septic shock model, and suppress the blood level of TNF-a and IL-6.In this part, through the screening systems were established, anti-inflammatory active small molecular compound YF-449was got. We found that YF-449inhibit TNF-a, IL-6, MMP-9, iNOS and COX-2expression_by blocking TLR4/MyD88//IKKa/IκBa/NF-κB signaling pathway. YF-449also enhanced the survival rate of mice in LPS-Septic shock model. These results suggested that YF-449could be a new anti-inflammatory agent in futher clinical therapy and study.