Induction of apoptosis with diallyl disulfide (DADS) in AGS gastric cancer cell line

Abstract

마늘에서 추출한 diallyl disulfide(DADS) 는 세포사멸 과정을 통해 여러 악성 종양의 성장에서 증식 억제 효과를 나타내는 것이 실험적으로 밝혀졌다. 이 연구를 통해 인체 위암세포주(human adenocarcinoma gastric cell line, AGS cell line)의 증식과정에서 DADS 의 효과를 알아보고 세포사멸과 연관된 분자생물학적 과정에서 어떤 영향을 나타내는지 알아보고자 하였다. 기본 배양액에서 배양된 위암세포주(AGS cell line)에 다양한 농도의 DADS를 추가하여 48시간동안 배양한 다음 MTT assay를 이용하여 생존한 세포의 양을 측정하였다. DADS가 세포사멸 과정에 미치는 영향을 알기 위해서 Annexin Ⅴ 염색을 이용하여 세포사멸이 유도된 세포를 측정하였고 ROS 의 생성은 형광물질에 염색되는 정도를 측정하여 알아보았다. 세포주기의 측정을 위해 형광 염색을 이용하여 저배수체의 DNA 용량을 측정하였고 세포사멸이 일어나는 세포내 기전을 밝히기 위해 Fas, caspase-3, Bax, Bcl-2 의 활성변화를 웨스턴 블럿 방법을 사용하여 측정하였다. DADS를 첨가하여 배양한 후 위암세포주의 증식이 억제되었으며 Annexin V 에 염색되는 정도도 증가하였고 특히 400 μM 의 고농도의 DADS를 첨가하였을 때 그 효과가 크게 나타났다. 활성산소종에 반응하는 형광물질의 증가는 DADS의 농도가 증가할수록 더 증가하였으나 농도에 따른 차이는 뚜렷하지 않았다. DADS를 첨가한 후 Fas, caspase-3, Bax 의 활성이 증가하고 Bcl-2의 활성이 감소하여 세포내 신호전달물질이 DADS 에 의한 세포사멸유도과정에 관여할 것으로 생각된다. 결론적으로 이 논문을 통해 DADS는 위암세포주의 증식을 억제하고 세포사멸을 유도하는 효과가 있으며 이는 DADS의 농도가 증가할수록 더 뚜렷하게 나타났다. 이러한 효과는 DADS 투여 후 활성산소종의 생성이 증가하고 세포주기상의 변화나 세포내 Fas, caspase-3, Bax 의 활성증가, Bcl-2의 활성감소 현상과 연관이 있을 것으로 생각되었다. 그러나 DADS의 농도증가에 따른 효과가 일정한 상관관계를 나타내지는 않고 있어 DADS의 농도변화에 따른 위암세포주의 증식억제 효과의 기전을 밝히기 위한 연구가 더 필요하리라 생각된다.;In recent years, the control of cancer has been focused on chemoprevention that inhibits the development of malignancies from precancerous lesions. Epidemiological studies revealed that high consumption of certain vegetables reduces the risk of variable cancer development including colorectal, stomach, lung and esophageal cancer. Allium vegetables such as garlic, onions, leeks, scallions, chives and shallots are famous for their anti-cancer effect. Among them, garlic is a plant commonly used for seasoning food in many different cultures of the world, especially in Asian countries. Diallyl disulfide (DADS) is a major organosulfur compound derived from garlic and reported to be comprised of approximately 60% garlic oil. The property of DADS is divided into anti-oxidation properties and induction of apoptosis, which is thought to be dependant on the cell type and DADS concentration. Moreover, it has been reported that DADS is able to inhibit the proliferation of several tumor cells. In gastric cancer, the definite anti-cancer effect of DADS and the mechanism of property has not been established. In this study, the effect of DADS was investigated in terms of the proliferation of AGS, gastric adenocarcinoma cell line at various concentrations from low to high levels (0-400 μM). The effect of DADS on induction of apoptosis and production of reactive oxygen species (ROS) was investigated to discover the molecular mechanism of DADS associated with oxidative stress. AGS cells were grown and treated with different amounts of DADS ranging from 0 to 400 μM (0, 50, 100, 200, and 400 μM) at 37℃ in a Dulbecco's Modified Eagle Medium (DMEM) supplemented with serum. The viability of cultured cells was determined by MTT assay. To detect the induction of apoptosis, Annexin V-FITC/PI staining assay was performed. These samples were processed for analysis of ROS by usual flow cytometric techniques. The distribution of cells in the cell cycle was measured by a flow cytometer. And using the Western blot analysis, the change of Fas, caspase-3, Bax, Bcl-2 activity was measured. The viability of AGS cells was reduced in a dose dependent manner. The percentage of live AGS cells was decreased to the 23% of that in the control group after 400 μM DADS treatment for 48 hrs. The Annexin V positive/ PI negative (apoptosis portion) area increased gradually from low concentration of DADS to high concentration. The percentage of apoptotic cell increased approximately 2.4% in the control group to 9.66% in 400 μM DADS treatment group. The level of ROS production increased according to the concentration of DADS. The amount of ROS production of control group and 50 μM DADS treatment group did not show any significant difference. However, when comparing among the DADS treatment groups, the amount of ROS production increased in a dose dependent manner. The mean level of ROS production increased from 586.65 in 50 μM DADS treatment group to 1235.69 in 400 μM DADS treatment group. In the cell cycle evaluation with flow cytometric analysis, the percentage of sub-diploid DNA content increased from 8.71% at 50 μM to 25.74% at 400 μM DADS treatment group. When intracellular proteins associated apoptosis were evaluated by using western blot, the expressions of Fas, caspase-3, Bax were increased and that of Bcl-2 was decreased in dose dependent manner. These results indicated that DADS-induced apoptosis might be related such events. In conclusion, DADS decreased the viability of AGS cells and induced apoptosis in a dose-dependent manner. This effects might be associated with the increase of ROS production and change of cell cycle regulation and some cellular molecular activity change such as up-regulation of Fas, caspase-3, Bax activity and down-regulation of Bcl-2. But relationship of the anti-proliferative effect of DADS and related molecular changes was not clearly proportional to the concentration of DADS, so further study to reveal the distinct mechanism will be needed.