The Apoptotic Differences Between MCF-7 and MDA-MB-231 Human Breast Cancer Cell lines After treatment withAllyl isothiocyanate

• Main Authors: Bo Peng, 彭勃
• Other Authors: 鍾景光
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/cd55b3

碩士 === 中國醫藥大學 === 生物科技學系碩士班 === 100 === Allyl isothiocyanate (AITC), which is a natural compound in cruciferous plants. According to the research, AITC caused G2/M arrest in bladder cancer UMUC- 3. Moreover AITC could inhibit the MMP-2 and 9 of liver cancer cells SK-Hep1, than inhibit cell adhesion, migration and invasion. Therefore, we want to find the difference between MCF-7 and MDA-MB-231 after the AITC treated. The cell viability of AITC influence on MCF-7 and MDA-MB-231 by MTT assay, after the MTT used camera to record the cell morphology. DNA damage and DNA break caused by AITC were checked on Comet assay and DAPI assay. DNA electrophoresis to prove the DNA fragmentation produced. Than we detected cell cycle arrest phase, decline in mitochondrial membrane potential, Reactive oxygen species (ROS) release conditions, release of calcium and activity of caspase-3, -9 by Flow cytometry analysis. Finally, western blotting is here proving the express of apoptosis associate protein. According to our experimental results, AITC induced the decrease of cell viability in dose-dependent manner in MCF-7 and MDA-MB-231 cells. AITC would induce cell cycle arrest in G0/G1 phase in MCF-7 but not in MDA-MB-231. This compound could make reduction of mitochondria membrane potential decrease in MCF-7, but not in MDA-MB-231. About ROS and Calcium release, AITC had some effect on two breast cancer cell lines. From the data of western blot, it shows that AIF, Endo G, Calpain 2, Caspase-3, GRP-78 and Caspase-9 were increase in MDA-MB-231, and in MCF-7 also show AIF and Caspase-9 were increasing in MCF-7. After treatment of AITC, MCF-7 would active Bax and Bak, than Cytochrome c combined with pro-Caspase-9 to apoptosome. Next would increased Caspase-9, Caspase-7 and PARP expression, finally cell produced apoptosis. At part of apoptosis of MDA-MB-231were increase expression of GADD153. After Ca2+ release, it were increase expression of Calpain 2, Caspase-12, Caspase-9 and Caspase-3. At the same time, Ca2+ were effect mitochondria released Cytochome c combined with pro-Caspase-9 form apoptosome, which actived Caspase-3 to produce apoptosis. Moreover, MDA-MB-231 would through expression of GRP-78 and JNK produced apoptosis. Immunofluorescence staining for confocal microscopy shows GADD153, AIF, and Endo G were increased expression after AITC treatment in two breast cancer cell lines. Based on the results, we suggest that the primary mechanism of AITC induced MDA-MB-231 apoptosis through increase the GADD153 expression, but mechanism of MCF-7 apoptosis by AITC may through mitochondria pathway.