Mechanistic Study of the Phytocompound, 2- β -D-Glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne in Human T-Cell Acute Lymphocytic Leukemia Cells by Using Combined Differential Proteomics and Bioinformatics Approaches

Abstract

Bidens pilosa, a medicinal herb worldwide, is rich in bioactive polyynes. In this study, by using high resolution 2-dimensional gel electrophoresis coupled with mass spectrometry analysis, as many as 2000 protein spots could be detected and those whose expression was specifically up- or downregulated in Jurkat T cells responsive to the treatment with 2-β-D-glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne (GHTT) can be identified. GHTT treatment can upregulate thirteen proteins involved in signal transduction, detoxification, metabolism, energy pathways, and channel transport in Jurkat cells. Nine proteins, that is, thioredoxin-like proteins, BH3 interacting domain death agonist (BID protein involving apoptosis), methylcrotonoyl-CoA carboxylase beta chain, and NADH-ubiquinone oxidoreductase, were downregulated in GHTT-treated Jurkat cells. Further, bioinformatics tool, Ingenuity software, was used to predict signaling pathways based on the data obtained from the differential proteomics approach. Two matched pathways, relevant to mitochondrial dysfunction and apoptosis, in Jurkat cells were inferred from the proteomics data. Biochemical analysis further verified both pathways involving GHTT in Jurkat cells. These findings do not merely prove the feasibility of combining proteomics and bioinformatics methods to identify cellular proteins as key players in response to the phytocompound in Jurkat cells but also establish the pathways of the proteins as the potential therapeutic targets of leukemia.

Figure 1

2DE gels of cellular proteins from the GHTT-treated (a) and DMSO-treated Jurkat T cells (b). Upregulated proteins (red on gel (a)) and downregulated proteins (blue on gel (b)) were identified by MS or MS/MS analysis. Identity of these spots is listed in Table 1.

Figure 2

Enlarged view of the spots up- and downregulated by treatment with DMSO and GHTT. Red and blue circles indicate the increase fold and decrease fold of the proteins listed in Table 1, respectively.

Figure 3

Pathways analysis of the proteins listed in Table 1 by means of the Ingenuity software. Mitochondrial dysfunction pathway (a) and granzyme B (GZB) signaling pathway (b) are proposed according to the structured network knowledge-based strategy. In (a), increase in the permeability of VDVC proteins in the outer mitochondrial membrane is assumed to allow for CYTC and, therefore, relevant to mitochondrial dysfunction and apoptosis. The VDVC-mediated apoptosis involves the formation of apoptosome and an activation of the caspase cascade. PRDX3 acts as an antioxidant protein to catalyze the degradation/reduction of hydrogen peroxide to water. Oxidative stress promotes the formation of ROS in mitochondrial complexes 1 to 4 and can cause mitochondrial damage. PARK7 acts as an antioxidant player and antagonizes the loss of mitochondrial function. In (b), GZB exerts its apoptotic function via the BID-dependent and BID-independent pathways. In BID-dependent route, GZB degrades BID to generate its active truncated BID (tBID), thereby inducing cell death via the formation of apoptosome and activation of caspases. In BID-independent route, GZB can activate different caspases and caspase substrates (e.g., LMNB1, etc.) independent of BID cleavage, leading to apoptosis. Red and blue indicate the proteins which are up- or downregulated by the plant compound, GHTT, in Jurkat cells. Translocation (—▹), activation (⟶), inhibition (⊣), and catalysis (—⋄) are indicated.

Figure 4

Effect of GHTT on mitochondrial membrane potential (a) and apoptosis (b) in Jurkat cells. (a) Jurkat cells were incubated with TMRM. After washing, the cells were treated with GHTT, DMSO, a negative control, and tunicamycin (Tm, 10 μg/mL), a positive control, followed by flow cytometry analysis. (b) Jurkat cells were incubated with GHTT and DMSO. After washing, the cells were stained with PI and annexin V and subjected to flow cytometry analysis. (c) Total lysates of Jurkat cells treated with GHTT and DMSO underwent SDS-PAGE. Following protein transfer, the membrane was blotted with the indicated antibodies.