The Proliferation Inhibition And Differentiation Induction Effects Of Lutein On Esophageal Carcinoma 9706 Cell Line And Their Relevant Molecular Mechanism

Background and Objective:Cancer has become the major killer of human life, which responsible for nearly 7 million deaths. Esophageal cancer (EC), which was generated from epithelial cells, isone of them in the world. It is not easily to detect and its development is fast mostly with unlucky prognosis. EC shows remarkable variation in its geographic distribution in the world. Esophageal cancer is the sixth leading cause of cancer happened worldwide, the fourth in China. So China is the area with high morbidity and mortality of EC, where 70% of the cases worldwide are diagnosed. Developing strategies for protection and effective means for its treatment are important for the continued improvement of the nation's health and continued economic development. Despite extensive research and aggressive clinical treatment for many years, the survival rate of esophageal cancer patients is still low and it threatens people's life and health. Therefore, there is tremendous interest in finding a new anti-cancer drug that is effective and has low toxicity. Epidemiological data indicate diet and alcohol intake are major risk factors for developing esphogeal cancer, and it is proved that there are containing both cancerogenic and anti-cancer ingredients in the food. Finding some sort of an 'anti-cancer' ingredient for protection and more effective treatment for esophageal cancer has long been a goal of researchers. Esophageal cancer patients, who are immunosuppressed and weakened from, are often not able to tolerate chemotherapy after surgery. Therefore, some moderate medicine should be adopted, which can kill cancer cells and improve the immunity of patients. Currently, many scholars are trying to develop drugs from the natural products.Phytochemicals are anti-mutation proteins in plants that might have anti-cancer qualities. In recent years the study of these proteins has becomes a hot topic in the area of food chemistry and nutrition research. Lutein is one natural phytochemical extraction, classified into oxygen-containing carotenoids. Numerous epidemiological studies have demonstrated that lutein has extensive biological capabilities in protecting age-related macular degeneration, preventing the development of cancer, cardiovascular disease and enhancing immune functions. But in China, the application of lutein is limited to the forage and its biological capability has not been appreciated. The investigations of professor Wang mingchen show that lutein has obvious biological capacity of antioxidant, anti-teratogenic and anti-mutagenicity. This study seeks to elucidate the effects of lutein on proliferation and differentiation of EC9706 cell line and its relevant mechanism in order to explore a new clue and method forprevention and treatment to esophageal cancer.Materials and Methods:1. EC9706 cell lines are routinely cultured in plate. Detecting and drawing its growthcurve and standard growth curve.2. Proliferation activity was evaluated by MTT chromometry. The inhibition rate of EC9706 cells was calculated after treated with different concentration of lutein.3. HE staining method was used to observe the change of cancer cells after the EC9706 cells were treated with lutein.4. Methyl green-pyronine staining was applied to observe the proportion of cells between differentiation and proliferation so as to confirm the effect of lutein on the differentiation of esophageal cancer cells.5. Flow cytometry was conducted to elucidate the change of EC9706 cell cycle and apoptosis after the EC9706 cells were treated with lutein.6. Immunochemistry was utilized to test the effect of lutein on the expression of Bcl-2, Bax, and on Cyclin D1 and Cyclin E after the EC9706 cells were treatedwith lutein.7. SPSS 10.0 statistical package was used for statistical analysis. One-way ANOVAand LSD were used to analyze all experimental data. Comparison of rates wasdetermined using chi-square test. A values of p<0.05 were considered statisticallysignificant.Results:1. The inhibition effect of lutein on the proliferation of EC9706 cell1.1 Cells were observed using a inverted microscope. The density of EC9706 cells were decreased in lutein group (high concentration). Some morphological alterations of the cells are shown: cell shrunk with nuclei, cytotoxicitic changes with the increase of lutein concentration and lutein-treated time.1.2 From HE staining, EC9706 cells appear like typical malignant tumor cell characters in that the cell is round, the nucleus is purple-blue, the endochylema is light purple-blue and the ratio of nucleus and endochylema is increased; but the cells of lutein-treated group appears ellipitical or irregular in shape. The colour of most cells are lighter stained and appeared lighter purple. The nucleus is small and the ratio of nucleus and endochylema is decreased. Several cells have vacuolar denaturation. EC9706 cells appear obvious morphological changes in lutein-treated group indicating that lutein can decrease the aggressiveness of the cancer cell via inducing EC9706 cell maturation and differentiation.1.3 The growth curve of the negative control group presents obviously elevated tendency with time dependent manner. All cell growth curves of lutein-treated group are lower than the control group after a period (p<0.05). The survival rate of group 80μg/ml (lutein) and group 160μg/ml (lutein) are 57.77% and 45.57%, respectively.1.4 The PI of the lutein treated groups is lower than that of negative control group after 96h, in a dose-dependent and time-dependent manner.2. The induction effect of lutein on EC9706 cell differentiationMethyl green-pyronine staining indicates that the number of proliferative cells in lutein-treated group decreased significantly compared with negative control group cells after 72h and 96h. The ratio of cell proliferation and differentiation appears to decrease directly with the increase of lutein concentration.3. The inhibition effect of lutein on EC9706 cell cycle3.1 From the distribution of different phases, the proportion of G₀/G₁ phase increased and S phase decreased in lutein treated group. Moreover the G₂/M phase has a tendency to decrease. Statistical analysis data indicate that all lutein treated groups have an effect on the cell cycle (p<0.05).3.2 The expression of Cyclin D1 and Cyclin E protein were detected in all groups.After 96h, the average integral optical densities (AIOD) of Cyclin D1 and Cyclin E in lutein treated groups are lower than those of control group (p<0.05).4. The induction effect of lutein on EC9706 cell apoptosis4.1 Characteristic sub-G₁ peaks (apoptosis peaks) appear in DNA histogram in lutein treated groups, but it is rare in the control group.4.2 The expression of Bcl-2 and Bax protein were detected in all groups. After 96h, the average integral optical density (AIOD) of Bcl-2 in lutein treated groups is lower than those in control group (p<0.05), whereas AIOD of Bax are higher than those in control group.Conclusions:1. MTT experiment indicates that lutein has inhibitory effects on cell proliferation in the EC9706 cell line, in a doest and time-dependent manner.2. The observation of HE staining and Methyl green-pyronine staining demonstrates that lutein can induce differentiation of EC9706 cell line.3. Flow cytometry indicates that lutein can arrest G₀/G₁ phase of EC9706 in all cell cycle. The decrease expression of EC cell cycle regulation protein Cyclin D1 and Cyclin E might be the mechanism of this phenomenon.4. Flow cytometry demonstrates that lutein can induce apoptosis of EC9706 cells. The decrease expression of bcl-2 and the increase expression of bax might beinvolved in this mechanism.