Analysis Of Differentially Expressed Proteins In Rapamycin Treated Hela Cells By Two-dimensional Electrophoresis

BackgroundThe evolutionarily conserved checkpoint protein kinase, TOR(target of rapamycin), has emerged as a major effecter on cell growth and proliferation via the regulation of protein synthesis in recent years. The mammalian target of rapamycin was first identified and cloned by Brown in 1994. The mTOR is involved in a series of pathological and pathophysiological processes, such as mRNA translation, membrane transportation, protein degrading, protein kinase C signal transduction and ribosome synthesis. Although the direct relationship between mTOR and cancer has not been erected, many cancer cell lines can control the mRNA translation and protein synthesis through a stunning number of downstream targets of mTOR.Rapamycin was originally isolated from a strain of the soil bacterium, Sereptomyces hygroscopicus. It acts by forming an inhibitory complex with its intracellular receptor, the FK506-binding protein, which binds a region in the C terminus of mTOR and destroys the interaction between mTOR and Raptor(regulatory associated protein of mTOR), thereby inhibiting mTOR activity.Rapamycin and its analog CCI-779 can partly induce the growth retardation ofcancer cells, reduce the tumor angiogenesis. Rapamycin shows an excellent effect against the tumor. However, the way how inhibition of mTOR causes the growth stagnation of tumor cells still remains controversial.Two-dimension electrophoresis is a classic and widely used method for the proteomics analysis. The immobilized pH gradients and Immobiline? reagents brought superior resolution and reproducibility to the first dimension isoelectric focusing. The comparison between the drug-treated and non-treated groups can help us find the protein spots associated the variation of mTOR activity. It can help the further researchs on mTOR function.In the present study, the IC50 of rapamycin on Hela cells was assessed by MTT assay. Then we studied the variation of protein maps between the IC50 treated and non-treated cells based on the method of 2D electrophoresis and ImageMaster? Platinum Software 5.0. We tried to find out the proteins associated with the variation of mTOR activity.Method1. Cell culture and the calculation of IC50Human Hela cells were incubated in RPMI1640 supplemented with 10% fetal bovine serum. We calculated the inhibition rate of Rapamycin on Hela cells by measuring the absorbency of groups treated by different rapamycin concentrations. The growth inhibition curve was drew and IC50 was calculated.2. Immobilized pH gradients isolectric focusingThe rapamycin treated and non-treated cells were incubated under the same conditions. Total protein was extracted at the same time from the two groups. After 2D electrophoresis, gel maps were gained by Umax scanner and Labscan? software. The maps were corrected with a series of methods, such as revising, spot analysis, background clearing up, emendation, measuring.3. statistical analysisSPSS 13.0 program package for windows was used for data analysis. The criterion for statistical significance was a=0.05.Results1. The inhibition degree of rapamycin on Hela cells depended on the drug concentration and duration. The increase of concentration and duration can enhance the rapamycin inhibition rate. The IC50 of rapamycin was 38.924nM. Rapamycin showed an outstanding inhibitory effect on Hela cells.2. Clear background, well-resolved and reproducible 2D maps of Hela cells were acquired due to the optimization of the electrophoresis conditions. Three same experiments were performed on the drug treated and non-treated groups. 1256±37 and 1312±25 spots were gained respectively in treated and non-treated groups. The match rate of treated groups was 89.5%, while 92.3% in the non-treated groups. The reproduction of the gels was assessed following the method reported by Corbett. Fifty matching, clear protein spots existed in the three gels were selected. We found the maps overlapped well. The average deviations of protein positions in treated groups were 1.07±0.12mm in IEF direction and 1.22±0.35mm in SDS-PAGE direction, while 0.89±0.23mm and 1.02±0.26mm in non-treated groups.3. Three same experiments were repeated on the two groups under the same conditions. The protein distribution of the two groups was approximately the same pattern. Most of the protein spots fell into the range of PI 4.0-4.2, MW 20.2~70.0kD. The horizontal strips were distributed in the bottom of the map. Twenty-one different protein spots were displayed between the two groups, of which eleven decreased in abundance and six showed higher expression in rapamycin treated groups, four only expressed in non-treated groups.