Human Esophageal Cancer Eca-109 Cell Apoptosis Induced By 8-Br-cAMP And Quercetin

The extracellular signals, including hormone, cytokine, drug, etc., which can transmit into the cell through cascade transduction of intracellular signaling molecules lead to the biological effect. The signals for cell growth, differentiation and apoptosis possess their certain transduction pathways. The apoptotic agents also can transmit into the cell through cascade transduction of intracellular signaling molecules, resulting in degradation of DNA and cell apoptosis. Thus, the study on signal transduction pathway of cell apoptosis may provide data for elucidating the mechanism of cell apoptosis, tumouregenesis and may have significance for tumour therapy.8-Bromo-cyclic 3', 5'-adenosine monophosphate (8-Br-cAMP) can induce the cell differentiation, apoptosis and inhibition ofmalignant cell growth through selective binding with the receptor of cAMP, proteinkinase A (PKA). Quercetin is one kind of plant flavonoid. It was reported that the quercetin could inhibit the tumor cell growth through repressing activity of proteinkinase C (PKC) or tyrosine kinase (TPK). In this experiment the common apoptosis pathway of human esophageal cancer Eca-109 cells induced by both 8-Br-cAMP and quercetin, the alteration of intracellular Ca2+ during apoptosis process and detection of related genes' amplification and expression ( wild type p53 (wp53), c-myc, bcl-2, iNOS) were studied.The subcultured Eca-109 cells were divided randomly into 3 groups: (1) the control group was cultured only with DMEM medium; (2) 8-Br-cAMP (Br) group was cocultured with final concentration, 2 X 10'5 mol/L of 8-Br-cAMP for 48h; (3) Quercetin (Q) group was cocultured with 43umol/L final concentration of quercetin for 48 h. The cell suspension with concentration of 2 X 106 /ml was dropped onto the treated slides for each group. The intracellular DNA and RNA were extracted from Eca-109 cells respectively for microarray detection and the DNA ladder electrophoresis was performed in 1.5% agarose gel.The experiments were carried out by cytoflowmetry fordetection of cell cycle, by TUNEL method for cell apoptosis, detection of intracellular Ca2+ with confocal laser microscope. The immunoreactivity (IR) of Caspase-3 and VEGF by immunocytochemistry and detection of related genes' amplification and expression (wild type p53 (wp53), c-myc, bcl-2, iNOS) by microarray technique were performed. Results:1. Cell apoptosis by TUNEL method: the condensed nucleus was purple-stained and located marginally in the apoptic cell. The cell apoptosis rate was 2% in the control group, 45% in the Br group and 42% in the Q group. The difference between the control and Br or Q groups was significant, P<0.01, withX 2 statistical methed; whereas there was no difference between Br and Q groups, P>0.05.2. Immunoreactivity (IR): The Caspase-IR in brownish color was located in the cytoplasm of Br and Q group cells. There was a very weak Caspase-IR in the control group cells. The difference between the control and Br or Q groups was significant, PO.01 with Ridit statistical methed; whereas there was difference between Br and Q groups, P<0.01. The VEGF- IR in brownish color appeared in the cytoplasm of the control group cells, whileonly a very weak VEGF-IR was present in the Br or Q Group cells. The difference between the control and Br or Q groups was significant, P<0.01 with Ridit statistical methed; whereas there was no difference between Br and Q groups, P>0.05.3. Detection of cell cycle by cytoflowmetry: the percentage of G2-M phase was 23.9% in the Br group, 25.6% in the Q group and 12.75% in the control group.4. Detection of DNA ladder in agarose electrophoresis: The DNA ladder pattern was exhibited in both Br and Q groups, while there was no ladder found in the control group.5. DNA amplification by microarray: The DNA signals of wp53 and iNOS were stronger in Br and Q groups than that in the control group; The DNA signals of c-myc and bcl-2 were weaker than that in the control gro...