Identification of apoptosis sensitivity regulating molecules in gastrointestinal cancer cells

• Project/Area Number: 18590747
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Gastroenterology
• Research Institution: Keio University
• Principal Investigator: TAKAISHI Hiromasa Keio University, School of Medicine, Instructor (80286468)
• Co-Investigator(Kenkyū-buntansha): HIGUCHI Hajime Keio University, School of Medicine, Instructor (20306682)
• Project Period (FY): 2006 – 2007
• Project Status: Completed (Fiscal Year 2007)
• Budget Amount: ¥3,590,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥390,000); Fiscal Year 2007: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
• Keywords: siRNA / Mcl-1 / side population

Research Abstract

Mcl-1(Myeloid cell leukemia-1) is an anti-apoptotic protein that regulates apoptosis sensitivity particularly in hematological and gastrointestinal malignancies. Here, we formulated the hypothesis that suppression of Mcl-1 would be an attractive strategy to overcome apoptosis resistance in Mcl-1 overexspressing gastric cancer. The AIMs was ; i) to examine Mcl-1 expression profiling among gastric cancer cell lines, and ii) to evaluate if Mcl-1 depletion sensitizes apoptosis by anti-cancer drugs. METHOD : 7 gastric cancer cell lines were used. Expression of Mcl-1 was assessed by Western blot analysis. Cells were exposed to anti-cancer drugs such as 5-fluorouracil and cisplatin. Apoptosis was quantitated by a morphological observation and caspase activity measurement. Adenovirus-mediated RNA interference (RNAi) technology was used to knock down the expression of Mcl-1. Releasing cytochrome C was evaluated by subcellular fractionation and immunoblot analysis. To evaluate if Mcl-l expressio n levels in cancer stem cells and non-stem cancer cells, side population (SP) cells, a cancer stem cell-enriched fraction, were identified and isolated by Hoechst 33342 staining and flowcytometry. RESULTS : 6 of 7 gastric cancer cell lines overexpressed Mcl-1 protein. There was correlation between Mcl-1 protein level and chemotherapy resistance. Depletion of Mcl-1 protein by RNAi technology effectively sensitizes the Mcl-1 expressing cells to anti-cancer drug-induced apoptosis. Mcl-1 knockdowned apoptosis was associated with mitochondrial depolarization, cytochrome c release from mitochondria and caspase activation. In addition, vast amounts of Mcl-1 mRNA were expressed in SP cells, implying that Mcl-1 mediates chemotherapy resistance in cancer stem cells. IN CONCLUSION : These results suggest that Mcl-1 mediates the resistance of apoptosis in gastric cancer cells by blocking the mitochondrial pathway of cell death. Mcl-1 depletion appears to be an attractive strategy to overcome chemotherapy resistance in gastric cancer cells.

Research Products

• [Presentation] Transforming growth factor(TGF)-bate induces pancreatic cancer cell invasion and epithelial to mesenchymal(EMT)in side-population(SP)cells but not in non SP cells (2007)
  • Author(s): Kabashima A, Higuchi H, Matsuzaki Y, Hasegawa G, Kuriyama N, Takaishi H, Izumiya M, Iizuka H, Sakai G, and Hibi T.
  • Organizer: 2007 AACR Annual Meeting
  • Place of Presentation: Los Angeles,CA
  • Description: 「研究成果報告書概要(和文)」より
• [Presentation] Mcl-1 depletion sensitizes the apoptosis of gastrointestinal cancer cells mediated by anti-cancer drugs (2007)
  • Author(s): Iizuka H, Higuchi H, Sumimoto H, Kabashima A, Kuriyama N, Sakai G, Izumiya M, Ymagishi Y, Takaishi H, Kawakami Y, Mizoguchi H, and Hibi T.
  • Organizer: 2007 AACR Annual Meeting
  • Place of Presentation: Los Angeles,CA
  • Description: 「研究成果報告書概要(和文)」より
• [Presentation] Transforming growth factor (TGF)-bate induces pancreatic cancer cell invasion and epithelial to mesenchymal (EMT) in side-population (SP) cells but not in non SP cells. (2007)
  • Author(s): Kabashima, A., Higuchi, H., Matsuzaki, Y., Hasegawa, G., Kuriyama, N., Takaishi, H., Izumiya, M., Iizuka, H., Sakai, G., Hibi, T
  • Organizer: 2007 AACR Annual Meeting
  • Place of Presentation: Los Angeles, CA
  • Description: 「研究成果報告書概要(欧文)」より
• [Presentation] Mcl-1 depletion sensitizes the apoptosis of gastrointestinal cancer cells mediated by anti-cancer drugs. (2007)
  • Author(s): Iizuka, H., Higuchi, H., Sumimoto, H., Kabashima, A., Kuriyama, N., Sakai, G., Izumiya, M., Ymagishi, Y., Takaishi, H., Kawakami, Y., Mizoguchi, H., Hibi, T
  • Organizer: 2007 AACR Annual Meeting
  • Place of Presentation: Los Angeles, CA
  • Description: 「研究成果報告書概要(欧文)」より