Induction of apoptosis by Koelreuteria formosana trypsin inhibitor in human colorectal carcinoma cell (Colo320)
| Autor: | Chen-Ni Wu, 伍珍妮 |
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| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 101 Colorectal cancer has persistently been the third most common cancer related cause of death in Taiwan. Research today has suggested a strong association between protease inhibitor and the apoptosis of tumor cells, and identifying protease inhibitor for the application of tumor growth inhibition possesses great research value. The primary objective of this research focuses on the Koelreuteria formosana trypsin inhibitor (KFTI) extracted from Koelreuteria formosana and its ability to induce apoptosis in human colorectal cell (Colo320). KFTI was first extracted from Koelreuteria formosana and then purified using ammonia sulfate. Activity analysis revealed that protein with the desired activity is salted by ammonia sulfate at approximately 70-100%. The active region was then passed through a Sephadex G-50, DEAE-cellulose 52 and affinity chromatography column. The final protease inhibitor obtained from the purifying process was then analyzed using SDS-PAGE. The inhibitor is of Kunitz-type and its molecular weight is approximately 19 kDa, which comprises of a 12.3 kD and 6.7 kD subunit linked by disulfide bond. The protein properties of KFTI were further analyzed. We discovered that KFTI is heat resistant as it is able to retain 77% of its activity after 10 minutes of high temperature (100°C) processing. We also processed the KFTI protease inhibitor with pH value ranging from 3.0-10.0 and its activity is unaffected by change in pH value. Nevertheless, only 17% activity was retained after being processed in SDS of different concentration for 10 minutes, and we conclude that its structure is very unstable to SDS. The protein was also processed in DTT of different concentration for 10 minutes and less than 10% of its protease inhibitor activity was retained after the experiment. From this result, we can tell that the disulfide bond plays a very important role in maintaining its structural stability. We then used MTT assay to evaluate KFTI’s influence on the growth of colorectal cancer cells (Colo320). We discovered that the survival rate of the cancer cells decreases with an increase in KFTI concentration and exposure time. Cell survival rate decreased to 72.2%, and 70% +72h at a concentration of 20μM. Cell apoptosis was then evaluated using flow cytometry with annexin V and the result revealed that cells processed by KFTI at a concentration of 15μM for 48 and 72 hours have and increased rate of M2 (apoptosis) and we therefore conclude that KFTI is able to induce apoptosis of Colo320 cells with a mere 15μM concentration. Western blot was used to evaluate the association of apoptosis and the relative proteins such as caspase-3, caspase-9, Bcl-2. The test also revealed that the cells were driven towards apoptosis. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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