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인체 췌장암세포주(Capan-1)의 증식에 미치는 amiloride의 억제효과
임대관,김신,김유리,노지훈,이지현,김지연,박무인,정근옥,박건영,구자영 고신대학교 의학부 2004 高神大學校 醫學部 論文集 Vol.19 No.1
Background/Aims Cytoplasmic alkalinization induced by activation of the Na+/H+anti porter which is stimulated upon the addition of growth-promoting agents, such as insulin, epidermal growth factor, phorbol ester, plays an essential role in the initiation on cell proliferation. In the present study the effects of amiloride, a specific and reversible inhibitor of Na+/H+antiporter, on the growth of human pancreatic carcinoma cell line, Capan-1 cells was examined and the effects of 5-fluorouracil (5-FU) were also studied. Cell cycle analysis was done to examine the mechanisms for the inhibitory effects of amiloride. Materials/Methods The growth of Capan-1 cells were examined by counting cell number on two and four days treatment with 1 μM, 5 μM, 10 μM, 20 μM, 40 μM, 80 μM, 160 μM amiloride, and 0.1 ㎍/㎖, 0.3 ㎍/㎖ 5-FU, after plating Capan-1 cells into 35-mm2 plastic dishes at d density of 10x104 cells/dish. The reversibility of the effects of amiloride was examined on two day to eight days treatment with 20 μM amiloride after seeding 2×104 cells/dish. Cell cycle analysis was done on the sells after four days treatment with 20 μM amiloride. Results Amiloride significantly inhibited the growth of Capan-1 cells in a dose-dependent fashion (p<0.05). The inhibitary effect of amiloride on the growth of Capan -1 cells was firstly shown at the concentration of 5 μM, which is not so higher than the concentration of 0.1-0.2 μM attainable by administration of usual dose of amiloride (5-10㎎). Forty-eight percent inhibition of growth was found at an amiloride concentration of 20μM after 4 days treatment, and ninety-three percent inhibition of growth was found at an amiloride concentration of 160μM after 4 days treatment. The inhibitory effect of amiloride on growth of Capan-1 cells was reversible since removal of amiloride by a media change after 48 hours treatment lead to significantly more growth than amiloride treated group (p<0.05). The reversibility of growth inhibition suggests that amiloride in not a non-specific cytotoxin for Capan-1 cells. Amiloride combined with 5-FU significantly inhibited the growth of Capan-1 cells in a dose-dependent fashion compared to an amiloride or a 5-FU alone (p<0.05). After four days treatment with 20 μM amiloride, the faction of cells in G0-G1 phase, S phase and G2-M phase was 47.3%, 35.8%, 16.9% respectively in the amiloride group (20 μM), and 44.3%, 37.1%, 18.6% in the control group. showing no significant differences between the two groups. Conclusions Amiloride significantly inhibited the growth of Capan-1 cells in a dose-dependent fashion, which was reversible. The reversibility of growth inhibition suggests that amiloride is not a non-specific cytotoxin for Capan-1 cells. The concentration of 5 μM, which is not so higher than the concentration of 0.1-0.2 μM attainable by administration of usual dose of amiloride (5-10㎎), which suggests amiloride or its analogues may be used alone or in conjunction with 5-FU for the treatment of pancreatic carcinoma. Further study is needed to clarify the effects of more potent analogues of amiloride on the growth of human pancreatic carcinoma cells.