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I. 서론 10
II. 실험 재료 및 방법 14
1) 재료 및 항체 14
2) 세포배양 14
3) MTT 실험법 14
4) Flow cytometry를 이용한 세포주기 분석 15
5) Flow cytometry를 이용한 세포사멸 분석 15
6) DAPI 염색 15
7) Western blot 분석 16
8) Xenograft model 16
9) 자료 분석 16
III. 결과 17
1) 5-OH-NIO가 MDA-MB-231 세포 증식에 미치는 영향 17
2) 5-OH-NIO가 MDA-MB-231 세포주기에 미치는 영향 20
3) 5-OH-NIO가 MDA-MB-231 세포사멸에 미치는 영향 24
4) 5-OH-NIO가 MDA-MB-231 세포괴사에 미치는 영향 28
5) 5-OH-NIO가 MDA-MB-231 xenograft model에서 종양 억제에 미치는 영향 33
IV. 고찰 36
V. 참고문헌 40
Figure 1. The effect of 5-OH-NIO on cell proliferation of MDA-MB-231 cells. 19
Figure 2. Cell cycle analysis by flow cytometry. 22
Figure 3. G2/M phase cell cycle regulators expression in MDA-MB-231 cells. 23
Figure 4. Apoptosis analysis of 5-OH-NIO treated cells by flow cytometry and DAPI stain. 26
Figure 5. Apoptosis analysis of 5-OH-NIO treated cells by immunoblot. 27
Figure 6. Induction of necrosis by 5-OH-NIO in MDA-MB-231 cells using LDH release and PI stain. 30
Figure 7. Necrosis analysis of 5-OH-NIO treated cells by PI stain and immunoblot. 32
Figure 8. Antitumor effect of 5-OH-NIO in xenograft model. 35
Purpose: The novel indirubin derivatives 5-nitro-5'-hydroxy- indirubinoxime (5-OH-NIO) was designed and tested for antitumor activity both in vitro and in vivo using breast cancer cell lines, MDA-MB-231 cells.
METHODS: Cell viability was examined by MTT assay. To examine apoptotic cell death, Annexin V/PI staining for flow cytometry and Western blot analysis and DAPI staining were performed. Necrotic cell death was determined by leakage of lactate dehydrogenase (LDH), HMGB1, and PI staining in 5-OH-NIO-treated MDA-MB-231 cells.
Six-week-old male nude mice were inoculated s.c. on the right flank with MDA-MB-231 cells. Indirubin derivatives were directly injected into the tumor every other day. Animals were monitored daily and tumor volume was measured by caliper.
Results: Indirubin derivatives showed potent antiproliferative activity on various human cancer cells. 5-OH-NIO inhibited cells proliferation in a dose dependent manner. The ratio of annexin V-positive cells and cleaved caspase-3/7 was increased by 5-OH-NIO treatment in MDA-MB-231 cells, suggesting that 5-OH-NIO induced cell death in MDA-MB-231 cells may be owing to apoptosis. In addition, LDH secretion and PI staining were detected in MDA-MB-231 cells treated with 5-OH-NIO, showing that 5-OH-NIO also induced necrotic cell death in the MDA-MB-231 cells. The inhibitory effect of 5-OH-NIO on tumor growth was confirmed by in vivo tumor xenograft model. 5-OH-NIO induced significant inhibition of tumor growth in nude mice bearing MDA-MB-231-induced tumors.
Conclusions: These data showed that novel indirubin derivatives 5-OH-NIO arrested the tumor growth by inhibiting cell proliferation and inducing apoptosis and necrosis. These findings provide the potential value of indirubin derivatives as novel candidates for antitumor agents.
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