Various forms of vitamin E have been under intensive study as chemopreventive and chemotherapeutic agents for a number of cancers.1 Many in vitro, animal, and epidemiological studies have presented evidence of an anti-cancer activity for vitamin E, but there are few studies of vitamin E in prostate cancer,2 and the mechanisms by which forms of vitamin E induce apoptosis in cancer cells remains largely unknown.3 Therefore, proteomics may help to understand the molecular events associated with the cytotoxic effects of vitamin E on cancer cells.
Figure 1: Analysis of 2-D gel by Dymension software showing proteins that are up or down-regulated three hours after delta-tocotrienol treatment.
Materials and methods
Proteins (15 µg per sample) extracted from a prostate cancer (LNCaP) cell line treated with delta-tocotrienol (20 µM) at three or six hours or LNCaP treated with ethanol as a control also at three or six hours were mixed with re-hydration solution (9 M urea, 2% [w/v] CHAPS, 0.5% [v/v] ZOOM carrier ampholytes, 20 mM DTT, and 0.002% of bromophenol blue). The proteins were loaded onto immobilized pH 310 non-linear gradient strips into each sample loading well of an IPG Runner Cassette. The strips were incubated for one hour at room temperature for re-hydration. Isoelectric focusing was performed under standard conditions. The focused strips were equilibrated and then loaded onto 4-20% SDS-PAGE gradient gels and run under standard 2-D gel electrophoresis conditions. The resulting gels were silver stained using a protocol compatible with LTQ XL linear quadrupole ion trap and matrix-assisted laser desorption/ionizationtime of flight mass spectrometry (MALDI-TOF MS). Images of the 2-D gels were generated with a Molecular Imager PharosFX scanner (Bio-Rad, Hercules, Calif.) using a 100 micron resolution in the dpi mode. Gel image analysis was performed with Dymension software. Dymension is suitable for determining the amount of protein present, before and after delta-tocotrienol induction because the software rapidly detects and assigns statistical confidence to each and every difference in spot normalized volume, to accurately highlight up or down-regulated proteins. The results of the analysis were displayed as a table and 3-D spot profile, making it easy to compare many expression profiles simultaneously and to detect proteins for future analysis.
The protein spots that showed significant changes in intensity compared to the controls were excised from separate gels and will be analysed by LTQ XL linear quadrupole ion trap LC/MS to identify those which are up or down-regulated by delta-tocotrienol treatment.
Figure 2: Analysis of 2-D gel by Dymension software showing proteins which are up or down-regulated six hours after delta-tocotrienol treatment.
The Dymension analysis of 2-D gels containing proteins from delta-tocotrienol treated cells identified more than 100 protein spots, which showed significant changes in intensity (Table 1, above, Figures 1 and 2, page 14). Changes in protein expression were most significant at three hours after delta-tocotrienol administration when spot intensities of seven proteins were increased or decreased by between 16-20 fold.
The results show that a specific set of proteins are regulated both at early and later times following delta-tocotrienol treatment and these proteins have been characterized by their apparent molecular weights and isoelectric points. The changes observed at early time points are particularly interesting because they are likely to reflect the underlying molecular mechanisms at the time when the delta-tocotrienol induces programmed cell dead (apoptosis) in LNCaP cells.
Using Dymension 2-D gel analysis software has enabled the detection of spots that are between 4 and 20 times up or down regulated post delta-tocotrienol treatment. The software's ability to perform complicated 2-D protein gel analysis demonstrates that Dymension can determine the proteomic mechanisms of delta-tocotrienol treatment. If the up and down-regulated proteins can be identified by MALDITOF MS analysis and database searches, they could provide critical information for the design of more effective drugs for the treatment and prevention of prostate cancer.
About the Authors Christian Muenyi is a Research Technician in the Department of Pediatrics at East Tennessee State University (ETSU), where he has spent four years successfully studying the proteomic effects of vitamin E on cancer cells. His research expertise is underpinned by a masters degree in organic chemistry from ETSU and a degree in chemistry from the University of Buea in Cameroon. Dr. William L. Stone is the Director of pediatrics research at East Tennessee State University. Dr. Hamid Kasmai is Professor Emeritus in the Department of Chemistry at East Tennessee State University. Hongsong Yang is a Research Assistant in the Department of Pediatrics at ETSU.
|Table 1: Number of Proteins Up or Down-regulated 3 and 6 Hours After Delta-tocotrienol Treatment|
|Difference in expression levels||Number of spots Up-regulated (3 hours post treatment)||Number of spots down-regulated (3 hours post treatment)||Number of spots up-regulated (6 hours post treatment)||Number of spots down-regulated(6 hours post treatment)|
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