5-Aza-2'-deoxycytidine induced growth inhibition of leukemia cells through modulating endogenous cholesterol biosynthesis

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.

Fig. 1.

SILAC-based quantitative proteomic method for revealing the 5-Aza-CdR-induced perturbation of protein expression in the global proteome. A, Flowcharts of forward SILAC combined with LC-MS/MS for the comparative analysis of protein expression in Jurkat-T cells upon 5-Aza-CdR treatment. B, The distribution of expression ratios (treated/untreated) for the quantified proteins, including those quantified in only one set of SILAC labeling experiment.

Fig. 2.

Representative ESI-MS results revealing the 5-Aza-CdR-induced down-regulation of FDPS. Shown are the MS for the [M+2H]²⁺ ions of FDPS peptides EFWPQEVWSR and EFWPQEVWSR* (A), as well as TQNLPNCQLISR and TQNLPNCQLISR* (B) (“R*” designates the heavy arginine) from forward (left) and reverse (right) SILAC labeling experiments.

Fig. 3.

5-Aza-CdR perturbed de novo cholesterol synthesis in leukemia cells. Shown are the histograms of cholesterol levels in Jurkat-T (A), K562 (B), HL60 (C), and WM-266–4 (D) cells that are untreated, treated with 5 μm 5-Aza-CdR treatment for 24 h alone or together with cholesterol. The values represent mean ± S.D. of results obtained from three independent experiments. “*”, p < 0.05; “**”, p < 0.01; “***”, p < 0.001. The p values were calculated by using unpaired two-tailed t test.

Fig. 4.

5-Aza-CdR-induced growth inhibition of leukemia cells can be rescued by externally added cholesterol. The viability of Jurkat-T (A), K562 (B), and WM-266–4 (C) cells after 12 and 24 h of treatment with 0 (dotted line), 30 (dashed line) or 60 mg/L (solid line) cholesterol alone (left), or together with 5 μm 5-Aza-CdR (right).

Fig. 5.

DPP4 gene regulates negatively endogenous cholesterol biosynthesis in WM-266–4 cells. Displayed are histograms of DPP4 expression level (A) and cholesterol content (B) in WM-266–4 cells after siRNA knockdown of DPP4 gene. Shown in (C), and (D) are the Western blot image and quantification results for FDPS and FDFT1 protein levels in WM-266–4 cells after DPP4 siRNA knockdown.

Fig. 6.

A mechanistic model underlying the anti-leukemic effect of 5-Aza-CdR.