Expression And Biological Significance Of Leptin, BDNF And Its Receptor (Trk B) In Human Colorectal Carcinoma Cells

Objectives:Colorectal carcinoma is a kind of"modern disease"-correlated with lifestyle and diet. A large number of epidemiological studies, particularly in the epidemiological studies of immigrants, showed that the incidence of colorectal carcinoma was correlated with energy intake, obesity and physical inactivity. The predominant factor of colorectal carcinoma is the environmental factor rather than genetic factor. Colorectal carcinoma is a kind of carcinoma correlated with lifestyle. The high incidences are most likely attributed to the increasing risk factors associated with"westernization", such as obesity and physical inactivity. The morbidity and mortality of colorectal carcinoma will be increasing steadily, and it should be the most common malignant tumors in the future for a long period in our country. Adipose tissues secrete estrogen, insulin, insulin-like growth factors, and also secrete various of bioactive substances called adipokines, including leptin and adiponectin. Brain-derived neurotrophic factor (BDNF) secreted by hypothalamic targets on white adipose tissue controlling the synthesis and secretion of leptin, and it is known as BDNF/Leptin axis. Tyrosine kinase receptor B (Trk B) is the BDNF specific receptor. The concentration of serum leptin is directly correlated with the mass of adipose and body weight. In our previous experiments, we have studied the expression and biological significance of leptin and leptin receptor in colorectal carcinoma tissues, paired para-carcinoma and normal colorectal tissues, and found that leptin and its receptor were closely correlated with colorectal cancinoma. In this study, we studied the expression of leptin and BDNF in DLD-1 colon cancinoma cells by western blot assay, and the proliferation of DLD-1 colon cancinoma cells in response to leptin and BDNF by MTT assay. We are trying to discover the factors influencing the incidence and development of colorectal cancinoma, and trying to provide new methods and theoretical basis for the diagnosis and treatment of it.Methods:1 DLD-1 colorectal cancinoma cells and normal human intestinal epithelial crypt (HIEC) cells are grown in Dulbecco's modified Eagle medium(DMEM) containing 10% fetal bovine serum(FBS) medium ,incubatede in 5% CO2, at 37°C, use logarithmic growth phase cells for experiment.2 Assess the expression of leptin, BDNF and Trk B in DLD-1 and HIEC cells by western blot. Firstly, extracted the total protein from cells. Secondly, the target protein may be separated by electrophoresis based on its molecular weight (SDS/PAGE), size and charge. Thirdly, the proteins are transferred from the gel to a PVDF membrane. After"blocked", the membrane antibodies (first antibodies) can be used to probe for the presence of particular protein because of the specifically binding of antigen with against it. The second antibody is purchased already conjugated to a labeling agent such as the enzyme horseradish peroxidase. This marker is then visualized by a colorimetric reaction catalyzed by the enzyme which yields a colored product that remains fixed to the membrane. Finally, the x-ray film.3 Take logarithmie growth DLD-1cells in 96-well plates 103 per well were cultured in 10% FBS containing medium for 24h. Cells were treated with different concentrations of leptin, BDNF and/or fluorouracil (5-FU). Then, cells were cultured for different times. The home microplate reader measured OD values at 570nm wavelength of the hole, analysed cells proliferation ability.4 Dates are presented as mean±S.E.M and analyzed by one-way ANOVA.Results:1 DLD-1 cells higher express leptin and Trk B, do not express BDNF. HIEC cells do not express leptin and BDNF, lower express Trk B. 2 The tumer proliferation rates of leptin are gradually increased with concentration increased. The tumer proliferation rates of the concentration less than 10^-9 mol/L are increased not significant compared with the control group(p﹥0.05),when equal to or greater than 10^-9 mol/L, they are significant higher(p<0.05). The same concentration with the time increased, the tumer proliferation rates are gradually increased. When equal to or greater than 10^-9mol/L, the tumer proliferation rates at 48h are significant higher than 24h(p<0.05). But it is no significant difference between 48h and 72h(p﹥0.05). Leptin can reduce the inhibition of 5-Fu in tumer cells, decrease the tumer inhibiton rates significantly(p<0.05).3 The tumer proliferation rates of BDNF are gradually increased with concentration increased. The tumer proliferation rates of the concentration equal to or less than 0.05μg/mL are increased not significant compared with the control group(p﹥0.05),when greater than 0.05μg/mL, they are significant higher(p<0.05). The same concentration with the time increased, the tumer proliferation rates are gradually increased. The tumor proliferation rates at 72h are significant higher than 24h(p<0.05). But it is not significant difference between 24h and 48h(p﹥0.05). BDNF can also reduce the inhibition of 5-Fu in tumer cells, decrease the tumer inhibiton rates significantly(p<0.05).Conclusions:1 Leptin can promote the DLD-1 colorectal cancinoma cells proliferation. The proliferation is more intense by the higher concentration and the longer time.2 DLD-1 colorectal cancinoma cells higher express leptin, indicating that colorectal cancinoma cells can synthesize and secrete leptin to proliferate themselves.3 BDNF can promote the DLD-1 colorectal cancinoma cells proliferation. The proliferation is more intense by the higher concentration and the longer time.4 DLD-1 colorectal cancinoma cells do not express BDNF, but higher express Trk B, indicate that colorectal carcinoma cells can not synthesize BDNF, but they can be proliferated by the BDNF from serum.