Quantitative Proteomic Analysis Reveals the Perturbation of Multiple Cellular Pathways in Jurkat-T Cells Induced by Doxorubicin

Abstract

Doxorubicin remains an important part of chemotherapy regimens in the clinic and is considered as an effective agent in the treatment of acute lymphoblastic leukemia (ALL). Although the cellular responses induced by doxorubicin treatment have been investigated for years, the precise mechanisms underlying its cytotoxicity and therapeutic activity remain unclear. Here we utilized mass spectrometry, together with stable isotope labeling by amino acids in cell culture (SILAC), to analyze comparatively the protein expression in Jurkat-T cells before and after treatment with a clinically relevant concentration of doxorubicin. We were able to quantify 1066 proteins in Jurkat-T cells with both forward and reverse SILAC measurements, among which 62 were with significantly altered levels of expression induced by doxorubicin treatment. These included the upregulation of core histones, heterogeneous nuclear ribonucleoproteins and superoxide dismutase 2 as well as the down-regulation of hydroxymethylglutaryl-CoA synthase and farnesyl diphosphate synthase. The latter two are essential enzymes for cholesterol biosynthesis. We further demonstrated that the doxorubicin-induced growth inhibition of Jurkat-T cells could be rescued by treatment with cholesterol, supporting that doxorubicin exerts its cytotoxic effect, in part, via suppressing the expression of hydroxymethylglutaryl-CoA synthase and farnesyl diphosphate synthase, thereby inhibiting the endogenous production of cholesterol. The results from the present study provided important new knowledge for gaining insights into the molecular mechanisms of action of doxorubicin.

Quantitative proteomic analysis of cyclosporine-induced toxicity in a human kidney cell line and comparison with tacrolimus

“…As previously described, Histone proteins present many sites of post-translational modifications (PTMs) that may lead to inaccurate quantification, potentially introduced by CsA-induced PTMs [33,34,48]. Therefore, Histone H2A expression following CsA exposure was determined using only peptides that do not contain any known PTMs, confirming its down-regulation (SILAC ratio = 0.50 ± 0.22, Table 1 and Table S2 -Supporting Information).…”

“…The use of SILAC together with a long 180 min nanoHPLC separation gradient coupled off-line with MALDI-TOF/TOF, although without preliminary SDS-PAGE fractionation, allowed us to identify 650 proteins in cell lysates of both forward and reverse experiments after CsA exposure. In accordance with recently published SILAC reports, only the 495 proteins whose exposed/non-exposed ratio could be estimated in at least two SILAC sets including the reverse were finally considered in this study [33,34] (Fig. 2B).…”

“…Protein identification and SILAC ratios upon cyclosporine and tacrolimus exposure Recently, the SILAC method has been successfully applied to examine the slight differential protein expression induced by other drugs and toxicants [33,34]. Coupling this approach with SDS-PAGE as a first dimension protein separation and reverse phase nanoHPLC as a second dimension one, directly coupled to mass spectrometry analysis, Xiong and Dong identified more than 1000 proteins in HL-60 or Jurkat-T cells exposed to arsenite and doxorubicin, respectively [33,34].…”

“…Coupling this approach with SDS-PAGE as a first dimension protein separation and reverse phase nanoHPLC as a second dimension one, directly coupled to mass spectrometry analysis, Xiong and Dong identified more than 1000 proteins in HL-60 or Jurkat-T cells exposed to arsenite and doxorubicin, respectively [33,34]. In the present study, SILAC was employed for the first time to gain insight into the molecular mechanisms of CsA-induced toxicity at the protein level and evaluate if the proteins perturbed by CsA were also modulated by TAC.…”

5-Aza-2′-deoxycytidine Induced Growth Inhibition of Leukemia Cells through Modulating Endogenous Cholesterol Biosynthesis

“…The cholesterol level was measured by HPLC using methanol as mobile phase, and the effluent from the column was monitored with a UV detector at a wavelength of 210 nm. The cellular level of cholesterol was determined using a calibration curve constructed from HPLC analyses of different amounts of cholesterol, as described recently (15).…”