| Background: Carcinoma is one of the leading causes of death from diseases in the world, hi addition to cytotoxic therapy, radiotherapy, surgery, gene therapy and immunotherapy are effective for malignant tumor. However, many patients show limited responses to these therapies or become unresponsive after treatment. The search for effective anti-tumor agents is of global importance. Recently, many investigators purified amounts of ribosome inactivating proteins (RIPs) from native Momordica charantia L, which were identified to have anti-tumor activity and exert their effects by induction of apoptosis of carcinoma cells. There are others active proteins in Momordica charantia L. whose bioactivities remain unknown.Objective: To purify proteins from Momordica charantia L that can inhibit the proliferation of tumor cells and to investigate their anti-tumor mechanisms.Methods: (1) The proteins from Momordica charantia seeds were purified by a procedure involving extract by acetones and a series of chromatographies on Hiprep 16/10 SOURCE SOS and HiLoad 26/60 Superdex 75. The protein fractions's purity were characterized by the SDS-PAGE and 2-D electrophoresis. The molecular weight of the proteins was measured by SDS-PAGE and the isoelectric point (pi) was measured by isoelectric focusing gel electrophoresis (IEF). (2) The anti-tumor effect of 14 protein fractions from Momordica charantia was measured by MTT assay onhuman myeloblastosis HL-60 cell line and mouse lymphadenoma P388 cell line and measured by SRB assay on human non-small cell lung carcinoma A549 cell line and human hepatocellular carcinoma BEL-7402 cell line. (3) The anti-tumor activity of HP2-751 and HP3-751, which exert higher anti-tumor activity on A549, BEL-7402, HL-60 and P388, was studied on several tumor cell lines, such as leukocythemia, lung carcinoma, hepatocarcinoma, colon cancer, breast cancer, oval carcinoma and gastrocarcinoma. (4) The BEL-7402 cells were treated with different concentrations of HP2-751, HP3-751 and HP5-751 at 12hr and were treated with the same dose (0.25mg/ml)of HP2-751, HP3-751 and HP5-751 at different times. The loss of DNA fragments, after plasma-membrane of treated cells, was revealed by propidium-iodide staining, followed by flow cytometer analysis. The percentage of apoptotic cells was calculated from the area of hypodiploid peak. The ultrastructure of BEL-7402 cells that were treated by proteins from Momordica charantia was surveyed by electron micrograph.Result: (1) 14 protein fractions were obtained after cation exchange chromatography and gel filtration chromatography, which were transiently called as HP1-751, HP1-753, HP2-751, HP2-752, HP3-751, HP3-752, HP3-753, HP4-751, HP5-751, HP5-752, HP5-753, HP6-751, HP6-7523 and HP6-753. They are all strong basic proteins, whose pi are between 9.0-9.6 and whose molecular weight are between 13-29KD. HP2-751 and HP3-751 appeared as a single point on the figure of 2-D eletrophoresis. (2)The anti-tumor effect of 14 protein fractions was evaluated in vitro on human myeloblastosis HL-60 cells, mouse lymphadenoma P388 cells, human non-small cell lung carcinoma AS49 cells and human hepatocellular carcinoma BEL-7402 cells, of which, HP2-751, HP3-751, HP3-753 and HP5-751 inhibit the proliferation of all the 4 tumor cell lines, the ICso value between 1.6 X10"6 and 2.4X10'5moI/L. Different tumor cell lines exhibit different sensitivities to the same protein fraction from Momordica charantia and so do the same tumor cell line to different protein fractions. The most sensitive tumor cell lines to HP2-751 were human non-small cell lung carcinoma A549 cells and human hepatocellular carcinoma BEL-7402 cells with IC50 value 7.6 X10"6 mol/L and 4.8 X KT6 mol/L respectively. The most sensitive tumor cell lines to HP5-751 were human myeloblastosis HL-60 cells with ICso value 4.3 X 10"6 mol/L. The most sensitive tumor cell lines to HP3-751 were mouse lymphadenoma P388 cells with IC50 value 1.6X10"6 mol/L. (3) The anti-tumor activity of HP2-751 and HP3-751 to several tumor cell l...
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