Effects Of Peptide Yy And Vitamin E Succinate On Proliferation And Apoptosis In Human Esophageal Adenocarcinoma Cell Line SEG-1

Epidemiological data showed that the incidence of esophageal adenocarcinoma rose rapidly in the world especially in several western developed countries. The majority of these cancers arise from a background of premalignant Barrett esophagus. The prognosis of esophageal adenocarcinoma is poor, so it's of great significance to strengthen in-depth study of medical intervention and prevention measures. In recent years, both at home and abroad study found that gastrointestinal peptide hormone-peptide YY (PYY) has an important role in regulating the biological behavior on gastrointestinal cancer.PYY is a 36 amino acid, straight chain polypeptide, which is found mostly within L-type endocrine cells of the gastrointestinal (GI) tract mucosa. PYY first described by Tatemoto in 1982, is a member of the peptide family of gut hormones that includes pancreatic polypeptide and neuropeptide Y. PYY has many effects in the gastrointestinal tract, all of which appear inhibitory. Its actions include decreasing pancreatic exocrine and gastric secretion, slowing of gastric emptying and small intestine motility, reduction of blood flow to the gastrointestinal tract and pancreas and suppression of cholecystokinin- stimulated pancreatic growth. PYY circulates in two active forms: PYY(1-36) and PYY(3-36). PYY(1-36) acts through Y1- and Y5-receptors in the central nervous system to stimulate appetite and to promote weight increase, while PYY(3-36) binds Y2-receptors, which inhibit appetite and promote weight loss. One risk factor that has emerged as being strongly associated with the risk of adenocarcinoma of the esophagus is obesity. PYY has been demonstrated as a potential antiobesity hormone. It has been found that PYY exists in many carcinoma tissues, and has inhibitory effect on pancreatic cancer, colon cancer, Barrett's esophageal adenocarcinoma, breast cancer, prostate cancer and dyscrasia resulted from cancer. Treatment with PYY decreases growth in pancreatic and breast tumors, most likely through a reduction in intracellular cAMP. But the exact mechanism(s) of action are still not fully understood.Vitamin E Succinate (VES) is the derivate of vitamin E (VE). Compared with VE, VES is more stable. VES has been shown to be a widespread potent inhibitor of human cancer cells in vivo and vitro. It can induce human B lymphoma, prostate, colon, lung, gastric, and endometrial tumor cells in culture to undergo apoptosis but not normal human epithelial cells. Thus,VES epitomises a group of novel compounds that hold subs tantial promise as future anticancer drugs. Adenocarcinomas of the esophagus have not been tested with PYY and VES for their effects on cell growth and survival. Therefore, using in vitro cell culture technique, we discussed the effects of PYY and VES on proliferation and apoptosis of Barrett's esophageal adenocarcinoma SEG-1 cell line.Objective: To investigate the effects of PYY and VES on the proliferation and apoptosis of Barrett's esophageal adenocarcinoma SEG-1 cell line and examine whether they have synergistic anticancer role; to investigate the influcence of PYY on the concentrations of intracellular cAMP. So as to establish the experimental or theoretical bases for application of PYY associating with VES in treating esophageal adenocarcinoma.Methods: SEG-1 cells were cultured in DMEM with 10% fetal bovine serum at 37℃in 5% CO2 gas incubator with saturation humidity with five groups:blank control, ethanol control(final volume concentration: 0.2%), PYY(500 pmol·ml-1),VES(20 mg·L-1),PYY+VES (500 pmol·ml-1 PYY + 20 mg·L-1 VES).1 The proliferation of SEG-1 cells was detected by MTT assay. The cells were seeded in 96 well plates. After being treated with PYY,VES for 12,24,48 hours, 20μl MTT and 150μl DMSO were added into the plates. The absorbance at 570 nm of the cell lysate was measured by enzyme labeling instrument and the inhibition rate of PYY and VES on tumor cells was calculated.2 Flow cytometric analysis: SEG-1 cells were treated with PYY,VES and combination of the two agents for 24 hour, respectively. Cells in experimental groups and control group were harvested and fixed with ice cold 70% ethanol at 4℃. Precipitates were digested with 0.5% pepsin after centrifugation. And then cells were resuspended in 0.5 ml propidium iodide (PI)/RNase A solution. Cells were incubated in the dark at room temperature for 15 min. The fluorescence emission of stained cells was measured with a flow cytometer. Data were analyzed with Multipcycle software.3 The apoptosis of SEG-1 cells was detected by TUNEL staining. After being treated with PYY,VES for 24 hours on the slide, then added relative reagents which were needed as the instruction of TUNEL kit.4 After treatment with different concentrations of PYY (10-5,10-6,10-7,10-8 mol·L-1), the concentrations of intracellular cAMP with treatment of different concentration of PYY (10-5,10-6,10-7,10-8 mol·L-1) were detected by immunoradiation assay according to the instruction of cAMP detected kit.Results: 1 The effect of PYY,VES on the proliferation of SEG-1 cells. As MTT assay displayed, compared with control groups, both of PYY and VES inhibited SEG-1 cell proliferation in a time-dependent manner(12~48h). Combination with PYY and VES inhibited the proliferation more remarkably than the two agents applied alone (P<0.01).2 The effect of PYY,VES on the apoptosis of SEG-1 cells. (1) The apoptosis rates were (29.94±0.36)%,(12.71±1.45)% and (35.30±2.38)% in SEG-1 cells treated with PYY,VES and combination of the two agents, respectively, which were significantly higher than that in control group (1.74±0.08)% (P<0.01). The apoptosis rates in the cells treated with PYY and VES were much greater than that treated with applied singly(P<0.01). (2) Compared with control groups, the apoptotic indexs were significantly increased by TUNEL assay. The apoptotic indexs were (24.54±1.10)%,(14.62±2.96)% and (39.75±3.43)%, respectively.3 The detection of expression of intracellular cAMP in SEG-1 cells. The concentration of intracellular cAMP were (313.0±8.2) pmol·L-1,(282.3±10.2) pmol·L-1,(239.7±8.0) pmol·L-1,(174.2±5.6) pmol·L-1 with treatmeat of different concentration of PYY(10-5,10-6,10-7,10-8 mol·L-1) respectively. The decrease of concentrations of intracellular cAMP was dose-dependent.Conclusion : Both of PYY and VES can inhibit cell proliferation and induce apoptosis in SEG-1 cells. Co-treatment with PYY and VES may have synergistic anticancer role. PYY can inhibit the growth of SEG-1 cells probably by reducing the expression of intracellular cAMP.