Study on the inhibitory effects of cell proliferation by urinary proteins

• Main Author: 劉秋萍
• Other Authors: 施桂月
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/79849804156818253905

碩士 === 國立成功大學 === 生物化學研究所 === 87 === According to European history, people use urine as a kind of medicine four thousand years ago. Urine therapy is also popular in China and Japan for a very long time in the history. The scientific evidence of urine therapy is very limited. It is first reported in 1986 that normal human urine contains an antineoplastic protein. A protein fraction isolated from human urine exhibits antiproliferative activity against human tumor cell lines without affecting the growth of several normal diploid cell lines or tumor cells of mouse or hamster origin. The major protein of this fraction is characterized and designated antineoplastic urinary protein (ANUP). The urinary protein is a homo-dimeric protein. Each subunit has a molecular mass of 16300 Da. as determined by sedimentation equilibrium and by polyacrylamide - gel electrophoresis. The amino acid composition of ANUP exhibits some features similar to collagen. The isoelectric point of ANUP is 4.2 as determined by chromatofocusing technique. In the recent report, it is demonstrated that ANUP is a strong growth inhibitor for KS (Kaposi’s sarcoma) and endothelial cell lines. ANUP is thus a potential candidate agent in the treatment of KS and other diseases. In this study, the lyophilized human urine protein powder (ANUP-O) exhibited antiproliferative activity against human tumor cell lines. The antineoplastic activity was observed in the first fraction of Sephacryl S-200 chromatography. The fraction consisted of immunoglobulins and other proteins as analyzed by polyacrylamide - gel electrophoresis and Western blot. The proteins could be precipitated by 30% saturated ammonium sulfate followed by protein A affinity column. The active components contained immunoglobulins and immunoglobulin G (IgG) fragment CH3, and were further analyzed by HPLC (high performance liquid chromatography). However, the antiproliferative activity disappeared after HPLC purification. The immunoglobulins purified from human plasma could not inhibit cell growth. The fragment CH3 of IgG produced by pepsin digestion had no antiproliferative activity. A modified procedure was used to purify the ANUP-O. The lyophilized powder was dissolved in phosphate-buffered saline (PBS) and heated at 60oC for 10 h. Tris-glycine buffer (0.25 M) containing 4 mM SDS, pH 8.8 was added to the cooled solution. The resulting solution was incubated at 37oC for 18-24 h before filtration at room temperature through an UM30 Amicon Diaflo membrane. The filtrate (containing ANUP) was adjusted to pH 4.2 and set at 4oC for 24 h. The fraction with strong antiproliferative activity contained proteins of molecular masses smaller than 30 kDa. The IC50 (50% inhibitory concentration) of the active components of ANUP-O for cancer cell proliferation is 0.85 mg/ml, and for endothelial cell proliferation is 0.25 mg/ml. In conclusion, we demonstrated that the antiproliferative proteins with molecular masses less than 30 kDa existed in the urinary protein fractions. The protein might have a strong association with immunoglobulins.