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Rapamycin, mTOR Inhibitor, Inhibits the Cell Growth and Proliferation by Mitotic Arrest in Human Gastric Cancer Cells. Kyung-Hee Chun, Ph.D Center for Uterine Cancer, National Cancer Center, 809-Madu-1-dong, Ilsan-gu, Goyang, Geonggi-do, 411-769, Korea Although decreasing mortality by the improved prognosis, early diagnosis, surgery and adjuvant therapy, gastric cancer is still the most common and the second leading cause of cancer-related deaths in northeast Asia, including Korea and Japan. The only available treatment to cure gastric cancer is detection of early stage disease and surgical resection. Therefore, improvement of survival rates and treatment is needed in gastric cancer patients, one of the promising approaches is the development of novel therapeutic drugs that specifically target molecular regulators of cancer growth and proliferation. Interestingly, the highly activation of phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) signaling pathway was reported in tissues of gastric cancer patients, the inhibition of PI3K/Akt signaling pathway is a good candidate in gastric cancer treatment. The mammalian target of rapamycin (mTOR), a downstream effecter of the PI3K/Akt signaling pathway plays the important roles to mediate cell survival and proliferation, and one of potent mTOR inhibitor, rapamycin has been underwent clinical trials. However, the exact role of rapamycin in mediating growth inhibitory effects in gastric cancers is not yet clear. In this study, it was demonstrated mTOR inhibition by rapamycin on gastric cancer proliferation with focus on its cell cycle arrest activity. After treatment with rapamycin in 6 gastric cancer cell lines, AGS, SNU216, and SNU638 cells were most sensitive by dose- and time- dependent manner. Rapamycin inhibited gastric cancer cell growth significantly, compared to other cell lines derived cancers that undergo clinical trial. To elucidate the mechanism by rapamycin in gastric cancers, cell cycle arrest was detected by PI staining. AGS cells showed G2/M arrest after 1 to 3 day treatment with rapamycin. However, rapamycin sensitive MCF7 (breast adenocarcinoma) or H549 (lung adenocarcinoma) was detected G1 arrest and/or apoptosis instead of G2/M arrest, it was suggested that G2/M arrest by rapamycin is cell type and/or organ specific manner in gastric cancer. Rapamycin also changed the expression levels of cell cycle related proteins. Interestingly, p21 expression increased and sustained until 48 hr after treatment in AGS. The protein expression of p27 and p53 was not detected in change. Cdc2 and cyclin B1 increased and cdc25c decreased after treatment. Increasing levels of polo like kinase 1 (PLK1) and aurora A, and phosphorylated histone H3, which was known mitotic arrest index, also were detected after rapamycin treatment. It supposed that rapamycin arrests cell cycle in late G2 and/or in mitotic phase. We confirmed that mitotic arrest by rapamycin in AGS cells, mitotic arresting shaped cells increased almost 25% compared to control, and phosphorylated histone H3 staining cells were found in rapamycin treated or in nocodazole treated gastric cancer cells, but not in control cells. Finally, it was detected by confocal microscope that rapamycin treatment prevented centrosome maturation and mitotic spindle bypolarity, and resulted in accumulation of aberrant prometaphase cells. In summary, it was demonstrated that rapamycin inhibits gastric cancer cell growth by mitotic arrest to accumulate abnormal prometaphase cells. Our results suggest that rapamycin, clinically useful mTOR inhibitor is promising in the treatment of gastric cancer and further clinical studies are needed to validate the therapeutic potential of rapamycin in gastric cancer patients. Kyung-Hee Chun 1994 Faculty of Pharmacy, Duksung Women’s University Seoul, Korea. 1996 Master, Graduate School of Pharmacy, Duksung Women’s University Seoul, Korea. 2000 Ph.D., Graduated School of Pharmaceutical Science, Functional biology, Tokyo university, Tokyo, Japan. 2003 Postdoctoral Fellow, Thoracic Head&Neck Medical Oncology, MD Anderson Cancer Center, Houston, TX, USA. 2006 Instructor, Thoracic Head&Neck Medical Oncology, MD Anderson Cancer Center, Houston, TX, USA. Present Senior Scientist, Center for gastric cancer, National Cancer Center, Koyang, Kyonggi, Korea. Specialty and Present Interest: Developing new drugs for cell cycle arrest and apoptosis induction. Phone: 82-31-920-2385; FAX: 82-31-920-2316; E-mail: [email protected]