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The Mechanism that NF-κB Influence the Process of Benzo［a］Pyrene-mediated COX-2 Up-regulation

Author: PuHao
Tutor: WeiLiXin
School: Second Military Medical University
Course: Oncology
Keywords: Hepatocytes Benzo [ alpha ] pyrene NF - kappa B COX-2 Signal Transduction
CLC: R735.7
Type: Master's thesis
Year: 2010
Downloads: 49
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Abstract

Background: liver cancer (hepatocellular carcinoma, HCC) is a common malignant tumor in the world, and showed a rising trend year by year in China, especially in the south-east, is the high incidence of liver cancer. On the other hand, environmental factors also play an important role in the occurrence of liver cancer and genetic. Benzo [alpha] pyrene (Benzo [alpha] pyrene, B [alpha] P) is the first environmental carcinogens found in humans, widely present in the smoke from burning coal, oil, etc. as well as cigarette smoke, automobile exhaust. As atmosphere carcinogens representatives, before B [alpha] P carcinogenicity studies mainly focused on the B [alpha] P and lung cancer relationship above. However, studies have shown that the growth of grains, fruits and vegetables in the vicinity of cities and large factories in the B [a] P was significantly higher than the content of the corresponding crop in rural and remote mountainous areas, with the grain of the region made from vegetable oils and food animals in this region grain-fed meat and dairy products have high B [alpha] P content. Given that the liver is the body's most important B [a] P metabolic organ for B [alpha] P specific mechanisms that affect liver function is necessary to carry out in-depth discussion. Cyclooxygenase (cyclooxygenase) is the rate-limiting enzyme of the arachidonic acid synthesis of prostaglandins. Mammals are at least two COX isoenzymes, namely COX-1 and COX-2. COX-1 sustained expression in many normal tissues and maintain normal physiological function of COX-2 in most organizations do not express or low expression, can be growth factors, endotoxin and tumor-promoting factor induced. The research data show that B [alpha] P treatment can up-regulate the expression of COX-2 in a variety of cells. Upregulation of COX-2 is one of the B [alpha] P cells most commonly affects. In addition, many studies have shown that high expression of COX-2 in many liver disease in the early and mid-, and inhibition of COX-2 expression, you can reduce the proliferation of liver cancer cell growth. In view of the above, it is necessary to affect the normal liver cells instead of liver cancer cells, COX-2 expression investigate the mechanism of B [alpha] P. Contents and results: The first part: benzo [a] pyrene on COX-2 expression in the liver cells in this part of the experiment, we first moved into human hepatocytes Chang Liver containing COX-2 promoter The Luciferase plasmid vector. Stably transfected cell lines were successfully constructed, the B [alpha] P added to the cell culture medium to stimulate the cells. After a certain time to a certain dose of stimulation, cell lysis was observed wherein the luciferase activity changes. Confirmed the results of this part of the experiment, the up-regulation of COX-2 expression in Chang liver cells exhibit B [alpha] P treatment dose and double time-dependent, so as to clarify the B [alpha] P Chang liver cells of COX-2 expression has raised the role. , We by Western blot experiments we observed results. Part II: Chang Liver cells benzo [a] pyrene-induced COX-2 increase in the role of NF-kappaB in this part of the experiment, we Chang Liver cells B [alpha] P treatment before cells added to the culture medium of NF-kappa B inhibitor PDTC upon inhibitor effective, then B [alpha] P-treatment stimulate the cells. Thereafter, with the first part to detect changes in luciferase activity of COX-2. The experimental results show that the B [alpha] P-induced COX-2 up-regulated dependent on the upregulation of NF-kappaB. In subsequent experiments, we used Western blot experiments prove Chang Liver cells in the NF-kappaB p65 subunit does occur in B [alpha] P under the obvious nuclear transfer, resulting in the upregulation of NF-kappa B activity. Part III: Benzo [alpha] pyrene via PI-3K. ERKs pathway induced upregulation of NF-kappaB expression in liver cells in this part of the experiment, we first containing the luciferase reporter gene plasmid DNA binding sequences of NF-kappaB transferred Chang Liver cells, pending the establishment of stable transfected cell lines, we first added in the cell culture medium of the MAPK and PI-3K inhibitor of the signal transduction pathway and, after the cells of the B [alpha] P -treatment. Processing after a certain time to a certain dose, we observed changes in the case of the luciferase reporter gene activity of NF-kappaB in cells. The experimental results show that the B [alpha] P treatment effect can be blocked by the ERKs and a specific inhibitor of PI-3K increase of NF-kappaB activity. This suggests that the B [alpha] P mainly through the two signal transduction pathway mediated by the intracellular activity of NF-kappa B raised. Conclusions: (1) the stimulation of benzo [a] pyrene can significantly promote the COX-2 activity increase in Chang Liver cells. Chang liver cells of COX-2 reporter gene activity in benzo [a] pyrene stimulated increase mainly depends on the upregulation of NF-kappa B activity. Chang liver cells, NF-kappaB activity increase is mainly dependent on the PI-3K and ERKs pathway.

The Mechanism that NF-κB Influence the Process of Benzo［a］Pyrene-mediated COX-2 Up-regulation

Abstract

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