Expression Of Death Associated Protein 3 (DAP3) In Human Gastric Cancer And Its Significance

The incidence of gastric cancer is still high in recent years. In China, the morbidity and mortality of gastric cancer are most high amomg malignant tumors.To study the apoptosis mechanism of gastric cancer do better to know the biological characters of occurrence, devolepment and ending of gastric cancer. Death Associated Proteins (DAP) are members of a apoptosis protein family among which DAP-kinase and cathepsin-D were comprehensively studied. Recently, more studies are carried on Death Associated Proteins 3(DAP3) in light of its invovlvement in TNF induced apoptosis.This paper aims:1.to observe the expression of Death Associated Protein 3 in gastric cancer in vitro and vivo;2. To observe the influence of TRAIL, rh-TNFa and 5-FU on DAP3 in vitro and vivo. 3. to study the mechanism of DAP3 involved in the treatment of TRAIL for human gastric cancer. 4. to study the expression of DAP3 in human gastric cancer tissues and its relation with DR₄ and DR₅, the particular receptors of TRAIL in order to know the clinical value of DAP3.Phase I The expression of Death Associated Protein 3 (DAP3)in two human gastric cancer cell lines and the influence ofTRAIL, rh-TNFα and 5-FU on DAP3[Objectives] To observe the expression of Death Associated Protein 3 (DAP3) in two human gastric cancer cell lines BGC-823 and HGC-27 and to observe the influence of TRAIL, rh-TNFα and 5-FU on DAP3. [Methods] Two human gastric cancer cell lines: BGC-823 and HGC-27 were treated with TRAIL, rh-TNFa and 5-FU at different concentration, 0, 50ng/ml. 100ng/ml, 200ng/ml for TRAIL, 0, 50ng/ml, 100ng/ml. 200ng/mlfor rh-TNFa, and 0, 10^"M, 10"5M. 1O¹M for 5-FU respectively. The OD numbers were measured by acid phosphatase method after 48 hours. The expression of DAP3 before and after treatment with TRAIL, rh-TNFa and 5-FU were determined by Western blot method.[Results] 1, Two human gastric cancer cell lines BGC-823 and HGC-27 were treated with TRAIL at different concetrations of 0. 50ng/ml, lOOng/ml, 200ng/ml respectively.The OD numbers of BGC-823 after 48 hours were 0.565 + 0.039, 0.555 + 0.049, 0.339 + 0.044 and 0.214 + 0.051 for each concetration , while the 0D numbers of HGC-27 were 0.771+0.039, 0.751+0.043, 0.419 + 0. 047 |n 0.231 + 0. 059 respectively. There was no difference between dose 0 and 50ng/ml (P>0. 05) while presented difference between dose 0 and lOOng/ml (P<0. 05). And there still presented difference between dose lOOng/ml and 200ng/ml(P<0.05). So the dosage lOOng/ml was chosen as the treatment concentration of TRAIL for human gastric cancer cell lines. By the way, lOOng/ml was chosen as the treatment concentration of rh-TNFa for human gastric cancer cell lines, 10nM for 5-FU. 2, No expression of DAP3 could be detected in either BGC-823 or HGC-27 before treatment. When treated with TRAIL, rh-TNFa or 5-FU, the expression of DAP3 could be detected after 48 hours.Phase II The influence of TRAIL, rh-TNFa and 5-FUon DAP3 in vivo[Objective] To observe the influence of TRAIL, rh-TNFa and 5-FU on DAP3 in vivo(nude mice implanted with human gastric cancer cell lines BGC-823 and HGC-27 sub-cutaneously).[Methods] Sixty nude mice were randomly divided to two groups: group A(BGC-823) and group B(HGC-27), 30 for each group. 5X107ml cancer cells were implanted sub-cutaneously. When tumor nodules reached 150-250mm' in size, 50 nude mice were chosen to be randomly divided to four groups while the nude mice with too small or big tumors were excluded: (l)Ten for TRAIL group while 5 for either group A or group B; (2)Ten for rh-TNFa group while5 for either group A or group B;(3)Ten for 5-FU group while 5 for either group A or group B; (4)Twenty for control group while 10 for either group A or group B. The dose for TRAIL and rh-TNFa was 5ug/kg weight equally while 15mg/kg weight for 5-FU, the same quantity of natrii sodium for control group. Each drug was administrated intraperitoneally 3 times one week. The rate of tumor inhibition was measured after three weeks. The expressions of DAP3 were determined by Western blot method. [Result] No expression of DAP3 could be detected in metastased tumor nodules of either BGC-823 or HGC-27 before treatment. After treated with TRAIL, rh-TNFa or 5-FU, the expression of DAP3 could be detected among which TRAIL and rh-TNF a have more influence than 5-FU in metastased tumor nodules of BGO823.Phase HI The mechanism of DAP3 involved in the treatment of TRAIL for human gastric cancer[Objective]To compare the expression level of caspase 8 in human gastric cancer cell lines BGC-823 before and after treatment with TRAIL. The mechanism of DAP3 involved in the treatment of human gastric cancer was. studied.[Methods]Human gastric cancer cell line BGC-823 were treated with TRAIL. The expression of caspase 8 was determined by RT-PCR after 48 hours. [Result]When BGC-823 was treated with TRAIL, the expression of DAP3 could be detected. Meanwhile, the expression level of caspase 8 mRNA became stronger compared with control group.Phase IV The expression of DAP3 in human gastric cancer tissue and its relation with DR* and DR5[Objectives] To study the expression of DAP3 in human gastric cancer tissues and its relation with DR4 and DR3, the particular receptors of TRAIL. [Methods] Forty-seven human gastric cancer tissues were collected from Huashan hospital. The expression of DAP3, DR, and DR5, the particular...