Abstract
5-Aza-2'-deoxycytidine (5-Aza-CdR), a nucleoside analog that can inhibit DNA cytosine methylation, possesses potent antitumorigenic activities for myeloid disorders. Although 5-Aza-CdR is known to be incorporated into DNA and inhibit DNA (cytosine-5)-methyltransferases, the precise mechanisms underlying the drug's antineoplastic activity remain unclear. Here we utilized a mass spectrometry-based quantitative proteomic method to analyze the 5-Aza-CdR-induced perturbation of protein expression in Jurkat-T cells at the global proteome scale. Among the ≈ 2780 quantified proteins, 188 exhibited significant alteration in expression levels upon a 24-hr treatment with 5 μm 5-Aza-CdR. In particular, we found that drug treatment led to substantially reduced expression of farnesyl diphosphate synthase (FDPS) and farnesyl diphosphate farnesyltransferase (FDFT1), two important enzymes involved in de novo cholesterol synthesis. Consistent with this finding, 5-Aza-CdR treatment of leukemia (Jurkat-T, K562 and HL60) and melanoma (WM-266-4) cells led to a marked decrease in cellular cholesterol content and pronounced growth inhibition, which could be rescued by externally added cholesterol. Exposure of these cells to 5-Aza-CdR also led to epigenetic reactivation of dipeptidyl peptidase 4 (DPP4) gene. Additionally, suppression of DPP4 expression with siRNA induced elevated protein levels of FDPS and FDFT1, and increased cholesterol biosynthesis in WM-266-4 cells. Together, the results from the present study revealed, for the first time, that 5-Aza-CdR exerts its cytotoxic effects in leukemia and melanoma cells through epigenetic reactivation of DPP4 gene and the resultant inhibition of cholesterol biosynthesis in these cells.
Highlights
Epigenetic events, defined as mitotically and meiotically heritable changes in gene expression that are not due to alteration in primary DNA sequence [1], play important roles in carcinogenesis and tumor progression [2]
farnesyl diphosphate synthase (FDPS) and FDFT1, two important enzymes involved in cholesterol biosynthesis, were decreased significantly upon 5-Aza-CdR treatment
We observed that 5-Aza-CdR-induced growth inhibition of Jurkat-T, HL60 and K562 cells could be abrogated by externally added cholesterol, supporting the conclusion that inhibition in endogenous cholesterol biosynthesis constitutes the major pathway leading to the growth inhibition of leukemia cells
Summary
Epigenetic events, defined as mitotically and meiotically heritable changes in gene expression that are not due to alteration in primary DNA sequence [1], play important roles in carcinogenesis and tumor progression [2]. To exploit the molecular mechanisms contributing to the anticancer activity of 5-Aza-CdR in leukemia cells, we employed liquid chromatography-tandem MS (LC-MS/MS) together with stable isotope labeling by amino acid in cell culture (SILAC) to assess, at the global proteome scale, the perturbation in protein expression of Jurkat-T human leukemia cells upon 5-Aza-CdR treatment. In this context, SILAC is a simple and efficient metabolic isotope-labeling method; when combined with LC-MS/MS analysis, the method can afford accurate quantification of subtle changes of protein abundance in the whole proteome [9]. This finding, along with follow-up studies allowed us to discover, for the first time, that
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