Comparison of the Effects of All-Trans-Retinoic Acid in HepG2 and MCF7 Cells Using Semi-Quantitative Shotgun Proteomics

Decreased proliferation in a number of cell lines treated with all-trans Retinoic Acid (atRA) has been well documented[1], but the inhibition in proliferation is not seen in all cell lines, and the mechanism is not fully understood. It has been proposed that different expression profiles of nuclear receptors (RAR, PPAR) and soluble binding proteins (CRABP-II, FABP5) are responsible for the different effects[2] via specific channeling of atRA between binding proteins and nuclear receptors. Therefore it was hypothesized atRA treatment would be associated with different proteomic profiles in different cell lines due to different nuclear receptor activation. To test this hypothesis, the proteomic signatures of MCF7 and HepG2 cells were characterized after atRA treatment. MCF7 cells were chosen based on the well documented effects of atRA on their proliferation. HepG2 cells were used as based on the observation the proliferation of the cell line is resistant to atRA treatment. The effect of atRA in cell proliferation was determined by MTT assays, and metabolism of atRA was monitored by the formation of known atRA metabolites, which also inhibit proliferation[1]. For proteomic analysis, the cells were treated with 10 µM atRA or vehicle DMSO control for 72 hours. All samples were analyzed on a LTQ Velos (Thermo) using the data-independent-acquisition method (PAcIFIC) described by Panchaud et al. [3]. Protein identifications were made using SEQUEST requiring a false discovery rate <1% and identification of 2 unique peptides per protein. 73 proteins whose expression was either increased >20% or decreased >20%, were analyzed using DAVID[4, 5] to identify atRA associated signaling pathways. A distinct difference in the enriched pathways up-regulated and down-regulated between the cell lines was detected and included anti-apoptosis, glycolysis/gluconeogenesis, and pentose-phosphate clusters. While down-regulated in atRA treated MCF7 cells, the pathways were up-regulated in HepG2 cells. These responses were associated with a higher rate of atRA metabolism in HepG2 cells compared to MCF7 cells suggesting a potential role of the metabolites in regulation of cellular effect. This data demonstrates that multiplexed shotgun proteomic methods can be used to characterize cell line specific effects of xenobiotics, and will be useful for quantitative comparisons of tissue specific pharmacological of toxicological effects of therapeutic entities.

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2.         Schug, T.T., et al., Opposing effects of retinoic acid on cell growth result from alternate activation of two different nuclear receptors. Cell, 2007. 129(4): p. 723-33.

3.         Panchaud, A., et al., Precursor acquisition independent from ion count: how to dive deeper into the proteomics ocean. Anal Chem, 2009. 81(15): p. 6481-8.

4.         Huang da, W., B.T. Sherman, and R.A. Lempicki, Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc, 2009. 4(1): p. 44-57.

5.         Huang da, W., B.T. Sherman, and R.A. Lempicki, Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res, 2009. 37(1): p. 1-13.