The Anticancer Activity And Markers Screening Of Apple Extracts And Lutein On Esophageal Cancer Cells

Background and objective:Esophageal cancer is one of the most frequently cancers in the world. It is very important to find the pathway to cure and prevent esophageal cancer or others, and to decrease threat of cancers. Phytochemical from natural plant food with antioxidant activity, and can inhibit cancer cell proliferation, decrease lipid superoxidation, lower cholesterol and so on. Therefore it is significantly important to find anticancer components of plants for prevention and therapy esophageal cancer.Epidemiologic studies suggests that consumption of fruits and vegetables has been associated with reduced risk of chronic diseases such as cardiovascular disease and cancer. Phytochemicals, especially phenolics, in fruits and vegetables are thought to be the major bioactive compounds for the health benefits. Flavonoids belong to phenolics from fruits, vegetables and tea with antioxidant activity, antianaphylaxis, bacteriostasis and inhibitting cancer cell proliferation which may play a key role in reducing chronic disease risk. In recent years investigation results indicated that people intake many flavonoid from food, the amount of intake even are more than that of micronutrients.Apple is abundant with phytochemicals like phenolics and flavonoids, In the laboratory, it has been founded that apple extracts(AE) have very strong antioxidant activity, can inhibit cancer cell proliferation, decrease lipid oxidation, and lower cholesterol. Animal experiments indicated that apple extracts can prevent breast tumor. some reports supported the effect of apple extracts anticancer. But the mechanism of it was not still clear. In order to better prevent and control cancers, further study on mechanism of anticancer of apple phytochemicals is very necessary.Lutein belongs to carotenoid, epidemiological studies have shown that lutein was associated with risk of reducing many chronic diseases, including age-related macular degeneration (AMD), cancer, cardiovascular diseases and improvement immunity. The synergistic effects of phytochemicals in fruits and vegetables are responsible for their potential antioxidant and anticancer activities. it is important significance to elucidate anticancer mechanisms of vegetable and fruit for development of health food in theoretic and practical by detection effects of AE on anti-proliferative, trigger apoptosis and molecular events.In this study, effect of apple extracts and lutein on the potential of anti-proliferative and induction apoptosis have been surveyed in human esophageal cancer EC9706 cells, at the same time, difference protein expression associated with cells proliferation ,cell cycle by proteomic technique in EC9706 cells.The objective of this study was to detect the effects of prevention and therapy of apple extracts on the human esophageal cancer in vitro, and to investigate the anticancer mechanism, especially to screen proteins that may be as the molecular targets of apple flavonoid extracts, and to provid clue for finding efficient and low toxic drugsfor esophageal cancer therapy.Methods:The research is divided into 5 parts, methods and contents are as follows:Part 1: Preparation and detection of apple extracts. This part was designed to investigate the total soluble flavonoids in apples, total flavonoids from apple specimen was extracted with solvent extraction, and detected by aluminiumion colouration.Part 2: The effects of apple extracts and lutein on the cycle and differentiation of esophageal cancer cells. EC9706 cells grow in PRMI 1640 medium containing 10% bovine serum and treated with apple extracts and lutein. The proliferation of EC9706 was analyzed by MTT assay, Flow cytometry was employed to investigate proportion of cycle distribution and immunohistochemistry was performed to determine apoptosis related protein cyclin D1 expresion.Part 3: The effects of apple extracts and lutein on the apoptosis of esophageal cancer cells. EC9706 cells were grown in PRMI 1640 medium containing 10% bovine serum and were treated with apple extracts and lutein. Flow cytometry was employed to investigate proportion of cycle distribution, immunohistochemistry and western blot was performed to determine apoptosis related protein Bcl-2 and Bax expresion.Part 4: To screen and identify the differentially expressed genes of apple extracts in esophageal cancer EC9706 cells. The effects of AE was explored with the technique of mRNA differential display.Part 5: Applying proteomics technology in screening biomarker. difference protein were screened by two dimensional gel electrophoresis in EC9706 cells. Results:The results are as follows:1. Total flavonoids contents in nine kinds fresh fruit extracts were in the range from 40.11 mg/100g to 122.88 mg/100g, Total flavonoids contents are highest in Red Fuji apple, followed by Sand pear, Gold pear, Fragrant pear, Fengshui pear, Reddish orange, Dangshan pear, Shuijing pear and kiwi fruit.2. The results of antiproliferation activities of apple extracts showed that apple extracts could inhibite cell proliferation and DNA synthesis, induce G2/M phase arrest, but for Lutein ,it was in G₀/G₁ phase.3. Apple extracts and lutein could induce apoptosis by downregulation bcl- 2 mRNA expression and upregulation the bax mRNA expression.4. Differential expression finger printing and gene sequence was obtained by DDRT -PCR. The results suggested that differential expression were relative to SET and PDZ structural domain, cell cycle, growth regulation, tumor D52 and methionine adenosyltransferase II.5. the data from two dimensional gel electrophoresis implied that eighteen peptide were associated with cell metabolism, signal transduction, cytoskeleton, oxidative stress, immunization, cell proliferation, apoptosis and molecular chaperones.Conclusions:1. Apples and other fruits contain flavonoids and the contents are abundance but the contents of flavonoid varies greatly among of fruit. Compard with other fruits, flavonoid contents is highest in apple.2. Apples and lutein play anticancer effect by inhibiting cell proliferation. Lutein regulates cell cycle by downregulation Cyclin D1 expression,. In addition, Lutein could promote cell differentiation, which can delay cell growth and reduce malignancy degree.3. Apple and lutein inhibited EC9706 cell proliferation by inducing apoptosis and cell cycle block by mediating Bax and Bcl-2 express, which may be one of the mechanism of apple and lutein inducing apoptosis in esophageal cancer cells.4. Differential expression finger printing was obtained by DDRT -PCR. differential expression of gene sequence were obtained by clone and sequencing. The data showed the gene sequence of differential expression involve SET and PDZ structural domain,which suggested that effects of AE on genes involve transmembrane receptor or ionic channel proteins ,and intracellular signal regulation as well as interaction of proteins, which would further affect locus of proteins, signal transmition and protein complex formation. Our results showed that apple extracts inhibited cell proliferation, induced G2/M phase arrest in EC9706 cells. The inhibition may be relative to mitosis arrest induced by downregulation expression of D52. Furthermore, AE anticancer activities was associated with downregulation expression of methionine adenosyltransferase II which promote cell growth and dedifferentiation. These results from the part provided the clue for further exploreing molecular event on mechanism of AE, prevention and cure of esophageal cancer.5. This study indicated that proteins expression were changed by apple extracts, these proteins are associated with cell growth cycle , apoptosis and cell morphous and phenotype,which help us to further understand the mechanism of apple extracts.furthermore these protein may be as targets of drug action.6. The present results showed that apple extracts inhibited cell proliferation, induced apoptosis and changed cell morphous and phenotype, which are associated with some proteins and gene expression. The synergistic effects of flavonoids in apple are responsible for their potential anticancer activity by affecting cell metabolism, signal transduction, oxidative stress, regulating immunity.